[leish-l] Fwd: Articles found by RefScout 09/02/2005 5/2005 part 2
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Date: Wed, 9 Feb 2005 12:01:20
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This is RefScout-Newsletter 5/2005
Due to more than 100 requests your newsletter has been split. This is part 2.
REQUEST: [ leishmaniasis ]
(151 articles match this request. 31 articles matching other requests removed.
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PMID: 12149396
TITLE: Enzyme-linked immunosorbent assays for diagnosis of leishmaniasis in
patients coinfected with human immunodeficiency virus.
REFERENCE: J Clin Microbiol 2002 Aug 40(8):3110
PMID: 11880434
TITLE: Enzyme-linked immunosorbent assay based on soluble promastigote antigen
detects immunoglobulin M (IgM) and IgG antibodies in sera from cases of
visceral and cutaneous leishmaniasis.
AFFILIATION: Entomology Dept., Division of Communicable Diseases and Immunology,
Walter Reed Army Institute of Research, Silver Spring, Maryland 20910-7500, USA.
Jeffrey.Ryan at na.amedd.army.mil
REFERENCE: J Clin Microbiol 2002 Mar 40(3):1037-43
Leishmaniasis causes significant morbidity and mortality in areas where
it is endemic. In areas where it is nonendemic, global travel and
increased incidence of the disease in human immunodeficiency virus and
intravenous-drug user populations are also causes for concern. The
unavailability of rapid and reliable tests for diagnosis of the various
leishmaniases makes patient management difficult. We have developed an
enzyme-linked immunosorbent assay (ELISA) that can detect immunoglobulin
M (IgM) and IgG antibodies in patients with visceral and cutaneous
leishmaniasis. These practical assays are based on soluble antigens from
promastigotes cultivated in a protein-free medium. In preliminary
studies, 129 visceral (Brazil, Italy, North Africa, and Nepal) and 143
cutaneous (Brazil) leishmaniasis patients with controls were tested.
Overall, the tests showed a sensitivity of 95.1%. In addition, the ELISA
correctly identified 42 sera from Brazilian dogs with canine
leishmaniasis and 10 healthy controls. Serological tests for the various
clinical manifestations of leishmaniasis could be useful
epidemiological and patient management tools in populations of areas of
endemicity and nonendemicity.
PMID: 11520798
TITLE: Regulation of interferon-gamma gene expression by nuclear factor of
activated T cells.
AFFILIATION: Department of Pathology, Harvard Medical School, and The Center for
Blood Research, Boston, MA 02115, USA.
REFERENCE: Blood 2001 Sep 98(5):1480-8
Transcription factors of the nuclear factor of activated T cells (NFAT)
family are thought to regulate the expression of a variety of inducible
genes such as interleukin-2 (IL-2), IL-4, and tumor necrosis factor-
alpha. However, it remains unresolved whether NFAT proteins play a role
in regulating transcription of the interferon- gamma (IFN-gamma) gene.
Here it is shown that the transcription factor NFAT1 (NFATc2) is a major
regulator of IFN-gamma production in vivo. Compared with T cells
expressing NFAT1, T cells lacking NFAT1 display a substantial IL-4-
independent defect in expression of IFN-gamma mRNA and protein. Reduced
IFN-gamma production by NFAT1(-/-)x IL-4(-/-) T cells is observed after
primary in vitro stimulation of naive CD4+ T cells, is conserved through
at least 2 rounds of T-helper cell differentiation, and occurs by a
cell-intrinsic mechanism that does not depend on overexpression of the
Th2-specific factors GATA-3 and c-Maf. Concomitantly, NFAT1(-/-)x IL-4
(-/-) mice show increased susceptibility to infection with the
intracellular parasite Leishmania major. Moreover, IFN-gamma production
in a murine T-cell clone is sensitive to the selective peptide inhibitor
of NFAT, VIVIT. These results suggest that IFN-gamma production by T
cells is regulated by NFAT1, most likely at the level of gene
transcription.
PMID: 11544330
TITLE: Sodium stibogluconate is a potent inhibitor of protein tyrosine
phosphatases and augments cytokine responses in hemopoietic cell lines.
AFFILIATION: Department of Cancer Biology, Lerner Research Institute, Cleveland
Clinic Foundation, Cleveland, OH 44195, USA.
REFERENCE: J Immunol 2001 Sep 167(6):3391-7
Using in vitro protein tyrosine phosphatase (PTPase) assays, we found
that sodium stibogluconate, a drug used in treatment of leishmaniasis,
is a potent inhibitor of PTPases Src homology PTPase1 (SHP-1), SHP-2,
and PTP1B but not the dual-specificity phosphatase mitogen-activated
protein kinase phosphatase 1. Sodium stibogluconate inhibited 99% of SHP
-1 activity at 10 micrograms/ml, a therapeutic concentration of the drug
for leishmaniasis. Similar degrees of inhibition of SHP-2 and PTP1B
required 100 micrograms/ml sodium stibogluconate, demonstrating
differential sensitivities of PTPases to the inhibitor. The drug
appeared to target the SHP-1 domain because it showed similar in vitro
inhibition of SHP-1 and a mutant protein containing the SHP-1 PTPase
domain alone. Moreover, it forms a stable complex with the PTPase: in
vitro inhibition of SHP-1 by the drug was not removed by a washing
process effective in relieving the inhibition of SHP-1 by the reversible
inhibitor suramin. The inhibition of cellular PTPases by the drug was
suggested by its rapid induction of tyrosine phosphorylation of cellular
proteins in Baf3 cells and its augmentation of IL-3-induced Janus
family kinase 2/Stat5 tyrosine phosphorylation and proliferation of Baf3
cells. The augmentation of the opposite effects of GM-CSF and IFN-alpha
on TF-1 cell growth by the drug indicated its broad activities in the
signaling of various cytokines. These data represent the first evidence
that sodium stibogluconate inhibits PTPases and augments cytokine
responses. Our results provide novel insights into the pharmacological
effects of the drug and suggest potential new therapeutic applications.
PMID: 11488977
TITLE: Susceptibility to Leishmania mexicana infection is due to the inability
to produce IL-12 rather than lack of IL-12 responsiveness.
AFFILIATION: Department of Immunology and Infectious Diseases, Harvard School of
Public Health, Boston, Massachusetts, USA.
REFERENCE: Immunol Cell Biol 2001 Aug 79(4):320-2
Almost all inbred mice are highly susceptible to parasites of the
Leishmania mexicana complex that includes L. amazonensis and L. mexicana
. Recent studies have reported that T cells from L. amazonensis-infected
mice fail to respond to IL-12 due to impaired IL-12R expression. Here,
we demonstrate that lymph node cells from L. mexicana-infected C57BL/6
and 129Sv/Ev mice respond efficiently to exogenous IL-12 in vitro and
produce IFN-gamma. Moreover, we also show that deletion of signal
transducer and activator of transcription (STAT)4 gene in resistant
STAT6-/- mice renders them susceptible to L. mexicana. These findings
indicate that an inability to produce IL-12 rather than unresponsiveness
to this cytokine is responsible for susceptibility to L. mexicana.
Moreover, the data also demonstrate that the STAT4-mediated pathway is
critical for the development of protective immunity against cutaneous
leishmaniasis, regardless of the species of Leishmania and/or genetic
background of the mice.
PMID: 11349082
TITLE: Protection against cutaneous leishmaniasis induced by recombinant
antigens in murine and nonhuman primate models of the human disease.
AFFILIATION: Infectious Disease Research Institute, Seattle, Washington 98104,
USA. acampos at idri.org
REFERENCE: Infect Immun 2001 Jun 69(6):4103-8
Leishmaniasis affects approximately 2 million people each year
throughout the world. This high incidence is due in part to the lack of
an efficacious vaccine. We present evidence that the recombinant
leishmanial antigens LmSTI1 and TSA, which we identified and
characterized previously, induce excellent protection in both murine and
nonhuman primate (rhesus monkey) models of human cutaneous
leishmaniasis. The remarkable protection induced by LmSTI1 and TSA in an
animal model that is evolutionarily close to humans qualifies this
antigen combination as a promising candidate subunit vaccine against
human leishmaniasis.
PMID: 11334939
TITLE: Cellular trafficking in trypanosomatids: a new target for therapies?
AFFILIATION: Department of Biochemistry and Molecular Biology, Oswaldo Cruz
Institute, FIOCRUZ, Av. Brasil 4365, RJ 21045-900, Rio de Janeiro, Brazil.
REFERENCE: Int J Parasitol 2001 May 31(5-6):536-43
Pathogenic trypanosomatids cause a plethora of diseases marked by the
lack of efficient vaccines and therapies. As a consequence, studies are
being conducted that are geared towards the understanding of basic
mechanisms and various biological aspects of these parasites that might
be used as targets for new developments in these areas. One such aspect
is the understanding of specific cellular trafficking mechanisms that
might be attacked with the intention of disease control. In this paper,
we give an overview of our current knowledge of cellular targeting
mechanisms in trypanosomatids, with special emphasis on our data related
to lysosomal targeting of cysteine proteinases in Leishmania.
PMID: 10779601
TITLE: Targeted integration into a rRNA locus results in uniform and high level
expression of transgenes in Leishmania amastigotes.
AFFILIATION: Max-Planck Institut für Biologie, Abteilung Membranbiochemie,
Tübingen, Germany.
REFERENCE: Mol Biochem Parasitol 2000 Apr 107(2):251-61
This report describes the construction of a DNA cassette for integration
into a genomic small sub-unit rRNA locus of Leishmania mexicana by
homologous recombination. Reporter genes encoding beta-galactosidase or
green fluorescent protein and the gene conferring hygromycin resistance
were integrated downstream of a RNA polymerase I-driven rRNA promoter.
To ensure high expression of the marker proteins in the intracellular,
amastigote stage, transgene coding sequences were followed by the
intergenic region of the L. mexicana cysteine proteinase B 2.8 gene
which provides processing signals required for high level expression in
this life-cycle stage. Integration of the DNA cassette was also
efficiently obtained in L. major. We show that either beta-galactosidase
or the green fluorescent protein were abundantly, stably and uniformly
expressed in promastigotes and amastigotes of both Leishmania sp. The
transgenic lines allow parasite detection at high sensitivity in the
tissues of infected mice and will be useful to follow infections in
macrophages in culture and in animal hosts.
PMID: 11227767
TITLE: A simple colorimetric method to screen drug cytotoxicity against
Leishmania using the dye Alamar Blue.
AFFILIATION: Abteilung Parasitologie, Hygiene-Institut der
Ruprecht-Karls-Universität, Im Neuenheimer Feld 324, D-69120 Heidelberg,
Germany.
REFERENCE: Parasitol Int 2000 Jan 48(3):265-9
A quantitative colorimetric assay using the oxidation-reduction
indicator Alamar Blue was developed to measure cytotoxicity of compounds
against the protozoan parasite Leishmania. Absorbance increased
linearly with the plating density of promastigotes of L. major MRHO/IR/
76 vaccine strain up to at least 2.5 x 10(6) cells/ml when parasites
were incubated for 72 h in the presence of 10% Alamar Blue. The 50%
effective dose values of common drugs (amphotericin B, pentostam and
paromomycin) obtained by this assay were in the same range as previously
determined by other methods. The Alamar Blue assay permits a simple,
reproducible and reliable method for screening antileishmanial drugs.
PMID: 10549622
TITLE: Impaired NFATc translocation and failure of Th2 development in
Itk-deficient CD4+ T cells.
AFFILIATION: Department of Medicine, University of California San Francisco
94143, USA.
REFERENCE: Immunity 1999 Oct 11(4):399-409
Naive Itk-deficient CD4+ T cells were unable to establish stable IL-4
production, even when primed in Th2-inducing conditions. In contrast,
IFNgamma production was little affected. Failure to express IL-4
occurred even among cells that had gone through multiple cell divisions
and was associated with a delay in the kinetics and magnitude of NFATc
nuclear localization. IL-4 production was restored genetically by
retroviral reconstitution of Itk or biochemically by augmenting the
calcium flux with ionomycin. In vivo, Itk-deficient mice were unable to
establish functional Th2 cells. Development of protective Th1 cells was
unimpeded. These data define a nonredundant role for Itk in modulating
signals from the TCR/CD28 pathways that are specific for the
establishment of stable IL-4 but not IFNgamma expression.
PMID: 10490099
TITLE: SOCS1 is a critical inhibitor of interferon gamma signaling and prevents
the potentially fatal neonatal actions of this cytokine.
AFFILIATION: The Walter and Eliza Hall Institute of Medical Research, Victoria,
Australia.
REFERENCE: Cell 1999 Sep 98(5):597-608
Mice lacking suppressor of cytokine signaling-1 (SOCS1) develop a
complex fatal neonatal disease. In this study, SOCS1-/- mice were shown
to exhibit excessive responses typical of those induced by interferon
gamma (IFNgamma), were hyperresponsive to viral infection, and yielded
macrophages with an enhanced IFNgamma-dependent capacity to kill L.
major parasites. The complex disease in SOCS1-/- mice was prevented by
administration of anti-IFNgamma antibodies and did not occur in SOCS1
-/- mice also lacking the IFNgamma gene. Although IFNgamma is essential
for resistance to a variety of infections, the potential toxic action of
IFNgamma, particularly in neonatal mice, appears to require regulation
. Our data indicate that SOCS1 is a key modulator of IFNgamma action,
allowing the protective effects of this cytokine to occur without the
risk of associated pathological responses.
PMID: 10438949
TITLE: BCL-6-deficient mice reveal an IL-4-independent, STAT6-dependent pathway
that controls susceptibility to infection by Leishmania major.
AFFILIATION: Metabolism Branch, National Cancer Institute, Laboratory of
Parasitic Diseases, National Institutes of Health, Bethesda, MD, USA.
REFERENCE: J Immunol 1999 Aug 163(4):2098-103
The BCL-6 gene negatively regulates Th2 responses as shown by the
finding that BCL-6-deficient (BCL-6-/-) mice develop a lethal Th2-type
inflammatory disease. The response of inbred mouse strains to infection
with Leishmania major is under genetic control; BALB/c mice are
susceptible and develop a progressive parasite burden, whereas most
other common laboratory strains of mice are resistant to infection. We
found that BCL-6-/- mice on a resistant genetic background (C57BL/6 x
129 intercrossed mice) were highly susceptible to L. major infection;
they resembled BALB/c mice in terms of lesion size, parasite load, and
the production of Th2 cytokines. BCL-6-/-IL-4-/- double-mutant mice were
also susceptible to L. major infection and produced 10-fold higher
levels of the Th2 cytokine IL-13 than IL-4-/- littermate controls. By
contrast, BCL-6-/-STAT6-/- double-mutant mice were resistant to L. major
infection despite also producing elevated levels of IL-13. These
results show that STAT6 is required for susceptibility to L. major
infection and suggest that IL-13 signaling through STAT6 may contribute
to a nonhealing, exacerbated L. major infection.
PMID: 10458767
TITLE: STAT-4 mediated IL-12 signaling pathway is critical for the development
of protective immunity in cutaneous leishmaniasis.
AFFILIATION: Department of Immunology and Infectious Diseases, Harvard School of
Public Health, Boston, USA.
REFERENCE: Eur J Immunol 1999 Aug 29(8):2524-9
Recent studies have demonstrated that two IL-12 signaling pathways, a
STAT 4 - dependent and STAT4 - independent, are involved in the
development of a Th1-like response. To determine their roles in the
development of protective immunity against Leishmania major, we
monitored progression of cutaneous Leishmania major infection in STAT4-
deficient mice (STAT4-/-) compared to similarly infected wild-type (
STAT4+/+) mice. Although the onset of lesion growth was delayed in STAT4
-/- mice during the early phase of infection, these mice eventually
developed large, non-healing lesions, whereas STAT4+/+ mice resolved
their lesions. As infection progressed, both STAT4+/+ and STAT4-/- mice
infected with L. major displayed similar titers of Leishmania-specific
IgG1 and IgE but later produced lower IgG2a. On days 20 and 40 post-
infection, Leishmania antigen-stimulated lymphnode cells from STAT4-/-
mice produced significantly lower amounts of IFN-gamma than those from
STAT4+/+ mice as measured by enzyme-linked immunosorbent assay. There
was no significant difference, however, in IL-4 and IL-12 production
between the two groups. These results indicate that STAT4-mediated IL-12
signaling is critical for the development of protective Th1 response
following L. major infection in genetically resistant mice. Additionally
, they demonstrate that, although genetically resistant mice lacking
STAT4 signaling pathway develop large, non-healing lesions, they do not
default towards a Th2-like response.
PMID: 10338536
TITLE: Early gene expression of NK cell-activating chemokines in mice resistant
to Leishmania major.
AFFILIATION: Institute for Clinical Microbiology and Immunology, University of
Erlangen-Nürnberg, Erlangen, Germany.
REFERENCE: Infect Immun 1999 Jun 67(6):3155-9
Susceptibility of mice to Leishmania major is associated with an
insufficient NK cell-mediated innate immune response. We analyzed the
expression of NK cell-activating chemokines in vivo during the first
days of infection in resistant and susceptible mice. The mRNA expression
of gamma interferon-inducible protein 10 (IP-10), monocyte
chemoattractant protein 1 (MCP-1), and lymphotactin was upregulated 1
day after infection in the draining lymph nodes of resistant C57BL/6
mice but not in those of susceptible BALB/c mice. In vivo local
treatment of BALB/c mice with recombinant IP-10 shortly after infection
resulted in an enhanced NK cell activity in the draining lymph node. The
data suggest that although the recruitment of NK cells is normal in
susceptible mice, the lack of NK cell-activating chemokines is a factor
resulting in a suboptimal NK cell-mediated defense.
PMID: 10320373
TITLE: Requirement for type 2 NO synthase for IL-12 signaling in innate
immunity.
AFFILIATION: Institut für Klinische Mikrobiologie, Immunologie und Hygiene,
Universität Erlangen, Wasserturmstrasse 3, D-91054 Erlangen, Germany.
REFERENCE: Science 1999 May 284(5416):951-5
Interleukin-12 (IL-12) and type 2 NO synthase (NOS2) are crucial for
defense against bacterial and parasitic pathogens, but their
relationship in innate immunity is unknown. In the absence of NOS2
activity, IL-12 was unable to prevent spreading of Leishmania parasites
, did not stimulate natural killer (NK) cells for cytotoxicity or
interferon-gamma (IFN-gamma) release, and failed to activate Tyk2 kinase
and to tyrosine phosphorylate Stat4 (the central signal transducer of
IL-12) in NK cells. Activation of Tyk2 in NK cells by IFN-alpha/beta
also required NOS2. Thus, NOS2-derived NO is a prerequisite for cytokine
signaling and function in innate immunity.
PMID: 9834104
TITLE: Mice with STAT6-targeted gene disruption develop a Th1 response and
control cutaneous leishmaniasis.
AFFILIATION: Department of Immunology and Infectious Diseases, Harvard School of
Public Health, Boston, MA 02115, USA.
REFERENCE: J Immunol 1998 Dec 161(11):6180-8
The cutaneous growth of Leishmania mexicana was measured in STAT6-
deficient mice (STAT6-/-) and compared with that in similarly infected
wild-type (STAT6+/+) mice. Following s.c. inoculation with 5 x 10(6)
amastigotes of L. mexicana into the shaven rump, STAT6+/+ mice developed
large, nonhealing cutaneous lesions, while STAT6-/- mice failed to
develop detectable lesions during most of the course of study. As
infection progressed, STAT6+/+ mice infected with L. mexicana displayed
significantly higher titers of Leishmania-specific IgG1 and IgE compared
with STAT6-/- mice, which conversely produced significantly higher
titers of Leishmania-specific IgG2a, indicating development of a Th1-
like response in the latter group. At 12 wk postinfection, Leishmania Ag
-stimulated lymph node cells from STAT6-/- mice produced significantly
higher amounts of IL-12 and IFN-gamma than those from STAT6+/+ mice as
measured by ELISA. However, there was no significant difference in IL-4
production between the two groups. Semiquantitative RT-PCR of transcript
levels in intact draining lymph nodes and skin from inoculation sites
confirmed a similar pattern of cytokines in vivo as that observed in
stimulated lymph node cells in vitro. These results indicate that STAT6-
mediated IL-4 signaling is critical for progression of L. mexicana
infection in genetically susceptible mice and demonstrate that in the
absence of STAT6, susceptible mice default toward a Th1-like response
and control cutaneous L. mexicana infection.
PMID: 7591091
TITLE: Attenuation of gamma interferon-induced tyrosine phosphorylation in
mononuclear phagocytes infected with Leishmania donovani: selective inhibition
of signaling through Janus kinases and Stat1.
AFFILIATION: Department of Medicine (Division of Infectious Diseases, University
of British Columbia Faculty of Medicine, Vancouver, Canada.
REFERENCE: Infect Immun 1995 Nov 63(11):4495-500
The induction of gene transcription in response to gamma interferon is
impaired in mononuclear phagocytes infected with Leishmania donovani,
and the mechanisms involved are not fully understood. The changes in
gene expression brought about by gamma interferon are thought to involve
transient increases in the activities of cellular protein tyrosine
kinases, including the Janus kinases Jak1 and Jak2, leading to tyrosine
phosphorylation of the transcription factor Stat1. To investigate the
mechanisms accounting for the impaired responses to gamma interferon, a
model system for examining overall changes in protein tyrosine
phosphorylation, activation of Jak1 and Jak2 and phosphorylation of
Stat1 was developed in phorbol 12-myristate 13-acetate-differentiated U-
937 cells. Analysis of whole-cell lysates by antiphosphotyrosine
immunoblotting showed that incubation with gamma interferon brought
about specific increases in phosphotyrosine labeling of several proteins
. Increased labeling of these proteins occurred to similar extents in
control cells and in cells that had been infected with L. donovani for
16 h. Jak1, Jak2, and Stat1 were immunoprecipitated from control and
interferon-treated cells, and tyrosine phosphorylation of these proteins
, detected by antiphosphotyrosine immunoblotting was used to measured
their activation. Tyrosine phosphorylation of Jak1, Jak2, and Stat1
increased markedly, in a dose-dependent manner, in U-937 cells incubated
with gamma interferon. In contrast, in cells infected with L. donovani
, tyrosine phosphorylation of Jak1, Jak2, and Stat1 was markedly
impaired. This effect was dependent upon the duration of exposure to L.
donovani and was maximal and complete at 16 h. Results similar to those
observed with U-937 cells were also obtained with human peripheral blood
monocytes. These findings indicate that infection of human mononuclear
phagocytes with L. donovani leads to impaired gamma interferon-mediated
tyrosine phosphorylation and selective effects on the Jak-Stat1 pathway
. Unresponsiveness to gamma interferon for activation of this pathway
may explain impaired transcriptional responses in leishmania-infected
cells.
PMID: 7768775
TITLE: Effect of iron chelation on the in-vitro growth of Leishmania
promastigotes.
AFFILIATION: Laboratory of Biochemistry, Hellenic Pasteur Institute, Athens,
Greece.
REFERENCE: J Antimicrob Chemother 1995 Jan 35(1):23-9
The development of vaccines and drugs to control leishmaniasis is
urgently needed. The presence of a leishmania transferrin receptor on
the parasite suggests that an adequate supply of iron is needed for the
life cycle of leishmania. We have investigated the effect of iron
deprivation on the growth of leishmania promastigotes in vitro using an
iron chelation approach. All chelators tested reduced the rate of
promastigote multiplication in a dose-dependent fashion, whereas
referrated ones did not. The hydroxypyridin-4-one chelators CP94 and L1
were found to be more efficient than desferrioxamine. We suggest that
iron depletion may be an effective mechanism against leishmania
infection.
PMID: 7849584
TITLE: Protein crystallography and infectious diseases.
AFFILIATION: Department of Biological Structure, University of Washington,
Seattle 98195.
REFERENCE: Protein Sci 1994 Oct 3(10):1670-86
The current rapid growth in the number of known 3-dimensional protein
structures is producing a database of structures that is increasingly
useful as a starting point for the development of new medically relevant
molecules such as drugs, therapeutic proteins, and vaccines. This
development is beautifully illustrated in the recent book, Protein
structure: New approaches to disease and therapy (Perutz, 1992). There
is a great and growing promise for the design of molecules for the
treatment or prevention of a wide variety of diseases, an endeavor made
possible by the insights derived from the structure and function of
crucial proteins from pathogenic organisms and from man. We present here
2 illustrations of structure-based drug design. The first is the
prospect of developing antitrypanosomal drugs based on crystallographic
, ligand-binding, and molecular modeling studies of glycolytic
glycosomal enzymes from Trypanosomatidae. These unicellular organisms
are responsible for several tropical diseases, including African and
American trypanosomiases, as well as various forms of leishmaniasis.
Because the target enzymes are also present in the human host, this
project is a pioneering study in selective design. The second
illustrative case is the prospect of designing anti-cholera drugs based
on detailed analysis of the structure of cholera toxin and the closely
related Escherichia coli heat-labile enterotoxin. Such potential drugs
can be targeted either at inhibiting the toxin's receptor binding site
or at blocking the toxin's intracellular catalytic activity. Study of
the Vibrio cholerae and E. coli toxins serves at the same time as an
example of a general approach to structure-based vaccine design. These
toxins exhibit a remarkable ability to stimulate the mucosal immune
system, and early results have suggested that this property can be
maintained by engineered fusion proteins based on the native toxin
structure. The challenge is thus to incorporate selected epitopes from
foreign pathogens into the native framework of the toxin such that
crucial features of both the epitope and the toxin are maintained. That
is, the modified toxin must continue to evoke a strong mucosal immune
response, and this response must be directed against an epitope
conformation characteristic of the original pathogen.
PMID: 2717947
TITLE: Receptor-mediated drug delivery to macrophages in chemotherapy of
leishmaniasis.
AFFILIATION: Institute of Microbial Technology, Chandigarh, India.
REFERENCE: Science 1989 May 244(4905):705-7
Methotrexate coupled to maleylated bovine serum albumin was taken up
efficiently through the "scavenger" receptors present on
macrophages and led to selective killing of intracellular Leishmania
mexicana amazonensis amastigotes in cultured hamster peritoneal
macrophages. The drug conjugate was nearly 100 times as effective as
free methotrexate in eliminating the intracellular parasites.
Furthermore, in a model of experimental cutaneous leishmaniasis in
hamsters, the drug conjugate brought about more than 90% reduction in
the size of footpad lesions within 11 days. In contrast, the free drug
at a similar concentration did not significantly affect lesion size.
These studies demonstrate the potential of receptor-mediated drug
delivery in the therapy of macrophage-associated diseases.
REQUEST: [ leishmania ]
(200 articles match this request. 108 articles matching other requests removed)
PMID: 15652336
TITLE: Real-time measurements of membrane surface dynamics on macrophages and
the phagocytosis of Leishmania parasites.
AFFILIATION: Departamento de FÃsica, ICEx, Universidade Federal de Minas
Gerais, CEP 30123-970, Belo Horizonte, Minas Gerais, Brasil.
REFERENCE: Exp Cell Res 2005 Feb 303(2):207-17
Defocusing microscopy was used for real-time observation and
quantification of membrane surface dynamics in murine bone marrow
macrophages. Small random membrane fluctuations (SRMF), possibly
metabolic driven, were detected uniformly over all membrane surface.
Morphological and dynamical parameters of ruffles, such as shape,
dimensions, and velocity of propagation, were analyzed. Optical tweezers
were used to promote phagocytosis of single Leishmania amazonensis
amastigotes by selected macrophages. Analysis of ruffling activity on
the macrophages before and during phagocytosis of the parasites
indicated that increased ruffling response near forming phagosomes, most
likely induced by the parasite, accelerates phagocytosis. The effects
of temperature decrease on the dynamics of membrane surface fluctuations
and on the phagocytosis of parasites were used to determine the overall
activation energies involved in these processes. The values obtained
support the existence of strong correlation between membrane motility
and phagocytic capacity.
PMID: 15596060
TITLE: Expression of hypoxia-inducible factor-1alpha in the cutaneous lesions of
BALB/c mice infected with Leishmania amazonensis.
AFFILIATION: Departamento de Parasitologia, Instituto de Biologia, Universidade
Estadual de Campinas, 13083-970, Campinas, São Paulo, Brazil.
REFERENCE: Exp Mol Pathol 2005 Feb 78(1):49-54
The hypoxia-inducible factor-1alpha (HIF-1alpha) is expressed in
response to hypoxia and has been recently demonstrated in a variety of
cells such as tumor cells and tumor-associated macrophages. Several
characteristics of leishmanial lesions in humans and in animal models,
such as microcirculation impairment, metabolic demand for leukocyte
infiltration into infected tissue, parasite proliferation, and secondary
bacterial infection, are strong indications of a hypoxic
microenvironment in the lesions. We evaluated HIF-1alpha expression in
the cutaneous lesions of BALB/c mice during Leishmania amazonensis
infection. Immunohistochemical analyses of the lesions demonstrated,
only in the later stages of infection when the lesion size is maximal
and parasite burden is enormous and massive numbers of recruited
macrophages and ulcers are observed, positive HIF-1alpha-infected cells
throughout the lesions. HIF-1alpha is expressed mainly in the cytoplasm
and around parasites inside the parasitophorous vacuoles of macrophages
. This is the first evidence that macrophages in the microenvironment of
lesions caused by a parasite produce a hypoxia-inducible factor.
PMID: 15610825
TITLE: Trypanothione biosynthesis in Leishmania major.
AFFILIATION: Division of Biological Chemistry and Molecular Microbiology, School
of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1
5EH, Scotland, UK.
REFERENCE: Mol Biochem Parasitol 2005 Jan 139(1):107-16
Trypanothione plays a crucial role in regulation of intracellular thiol
redox balance and in defence against chemical and oxidant stress.
Crithidia fasciculata requires two enzymes for the formation of
trypanothione, namely glutathionylspermidine synthetase (GspS; EC 6.3.1.
8) and a glutathionylspermidine-dependent trypanothione synthetase (TryS
; EC 6.3.1.9), whereas Trypanosoma cruzi and Trypanosoma brucei use a
broad-specificity trypanothione synthetase to make trypanothione from
glutathione (GSH) and spermidine. Here, we report the identification of
two genes in Leishmania major with similarity to previously identified
GSPS and TRYS. GSPS is an apparent pseudogene containing two frame shift
mutations and two stop codons, whereas TRYS is in a single open-reading
frame. The enzyme encoded by TRYS was expressed and found to catalyse
formation of trypanothione with GSH and either spermidine or
glutathionylspermidine. When GSH is varied as substrate the enzyme
displays substrate inhibition (apparent K(m)=89muM, K(i)(s)=1mM, k(cat)=
2s(-1)). At a fixed GSH concentration, the enzyme obeys simple
hyperbolic kinetics with the other substrates with apparent K(m) values
for spermidine, glutathionylspermidine and MgATP of 940, 40 and 63muM,
respectively. Immunofluorescence and sub-cellular fractionation studies
indicate that TryS localises to the cytosol of L. major promastigotes.
Phylogenetic analysis of the GspS and TryS amino acid sequences suggest
that in the trypanosomatids, TryS has evolved to replace the GspS/TryS
complex in C. fasciculata. It also appears that the L. major still
harbours a redundant GSPS pseudogene that may be currently in the
process of being lost from its genome.
PMID: 15639138
TITLE: Leishmania amazonensis: early proteinase activities during
promastigote-amastigote differentiation in vitro.
AFFILIATION: Departamento BioquÃmica e Biologia Molecular, Instituto Oswaldo
Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
REFERENCE: Exp Parasitol 2005 Jan 109(1):38-48
Leishmania proteinase activity is known as parasite differentiation
marker, and has been considered relevant for leishmanial survival and
virulence. These properties suggest that Leishmania proteinases can be
promising targets for development of anti-leishmania drugs. Here, we
analyze the activities of four proteinases during the early phase of the
Leishmania amazonensis promastigotes differentiation into amastigotes
induced by heat shock. We have examined activities of cysteine-, metallo
-, serine-, and aspartic-proteinase by hydrolysis of specific
chromogenic substrates at pH 5.0 and at the optimal pH for each enzyme.
Our results show that metallo-, serine-, and aspartic-proteinases
activities were down-regulated during the shock-induced transformation
of promastigotes into amastigotes. In contrast, cysteine-proteinase
activity increased concomitantly with the promastigote differentiation.
Immunocytochemical localization using two anti-cysteine-proteinase
monospecific rabbit antibodies detected the enzyme in several cell
compartments of both parasite stages. Our results show different
proteinase activity modulation and expression during the early phases of
the shock-induced parasite transformation.
PMID: 15639137
TITLE: Leishmania major: clathrin and adaptin complexes of an intra-cellular
parasite.
AFFILIATION: Department of Biological Sciences, Centre for Molecular
Microbiology and Infection, Imperial College, London SW7 2AY, UK.
REFERENCE: Exp Parasitol 2005 Jan 109(1):33-7
To investigate the role of clathrin-mediated trafficking during the
Leishmania lifecycle, open reading frames encoding clathrin heavy chain
and the beta-adaptins, major components of the adaptor complexes, have
been analysed both in silico and experimentally. The Leishmania genome
encodes three beta-adaptins, which arose at a time predating speciation
of these divergent trypanosomatids. Unlike Trypanosoma brucei, both
clathrin heavy chain and beta-adaptin1 are constitutively expressed
throughout the Leishmania life cycle. Clathrin relocalises in
amastigotes relative to promastigotes, consistent with developmental
alterations to the morphology of the endo-membrane system.
PMID: 15619518
TITLE: Leishmania inhibits STAT1-mediated IFN-gamma signaling in macrophages:
increased tyrosine phosphorylation of dominant negative STAT1beta by Leishmania
mexicana.
AFFILIATION: Department of Microbiology, The Ohio State University, 484 West
12th Avenue, Columbus, OH 43210, USA.
REFERENCE: Int J Parasitol 2005 Jan 35(1):75-82
Previous studies have demonstrated that Leishmania donovani attenuates
STAT1-mediated signaling in macrophages; however it is not clear whether
other species of Leishmania, which cause cutaneous disease, also
interfere with macrophage IFN-gamma signaling. Therefore, we determined
the effect of Leishmania major and Leishmania mexicana infection on
STAT1-mediated IFN-gamma signaling pathway in J774A.1 and RAW264.7
macrophages. We found that both L. major and L. mexicana suppressed
IFNgammaRalpha (alpha subunit of interferon gamma receptor) and IFN-
gammaRbeta (beta subunit of interferon gamma receptor) expression,
reduced levels of total Jak1 and Jak2, and down-regulated IFN-gamma-
induced Jak1, Jak2 and STAT1 activation. The effect of L. mexicana
infection on Jak1, Jak2 and STAT1 activation was more profound when
compared with L. major. Although tyrosine phosphorylation of STAT1alpha
was decreased in IFN-gamma stimulated macrophages infected with L. major
or L. mexicana, those infected with L. mexicana showed a significant
increase in phosphorylation of the dominant negative STAT1beta. These
findings indicate that L. major and L. mexicana attenuate STAT1-mediated
IFN-gamma signaling in macrophages. Furthermore, they also demonstrate
that L. mexicana preferentially enhances tyrosine phosphorylation of
dominant negative STAT1beta, which may be one of the several survival
mechanisms used by this parasite to evade the host defense mechanisms.
PMID: 15619580
TITLE: Screening of New Caledonian and Vanuatu medicinal plants for
antiprotozoal activity.
AFFILIATION: Laboratoire de Pharmacognosie (associé au CNRS-BioCIS), Faculté
de Pharmacie, Université Paris-Sud, rue J.B. Clément, 92296 Châtenay-Malabry
Cedex, France.
REFERENCE: J Ethnopharmacol 2005 Jan 96(3):569-75
Sixty-seven extracts of 30 medicinal plants traditionally used in New
Caledonia or Vanuatu by healers to treat inflammation, fever and in
cicatrizing remedies were evaluated in vitro for their antiprotozoal
activity against Leishmania donovani, Leishmania amazonensis and
Trypanosoma cruzi. Among the selected plants, Pagiantha cerifera was the
most active against both Leishmania species; four extracts were active
against promastigotes of Leishmania donovani at EC(50) values inferior
to 5mug/ml. Garcinia pedicillata extract had an EC(50) value of 12.5mug/
ml against intracellular amastigotes of Leishmania amazonensis. Alone
Amborella trichopoda reduced by more of 80% the trypomastigotes of
Trypanosoma cruzi in the blood.
PMID: 15621448
TITLE: Characterisation of a developmentally regulated amino acid transporter
gene from Leishmania amazonensis.
AFFILIATION: Departamento de Parasitologia, Instituto de Ciências Biomédicas,
Universidade de São Paulo, São Paulo, Brazil.
REFERENCE: FEMS Microbiol Lett 2005 Jan 242(2):275-80
The metabolism of protozoan parasites of the Leishmania genus is
strongly based on amino acid consumption, but little is known about
amino acid uptake in these organisms. In the present work, we identified
a Leishmania amazonensis gene (La-PAT1) encoding a putative amino acid
transporter that belongs to the amino acid/auxin permease family, a
group of H(+)/amino acid symporters. This single copy gene is
upregulated in amastigotes, the life cycle stage found in the mammalian
host. La-PAT1 putative orthologous sequences were identified in
Leishmania infantum, Leishmania donovani, Leishmania major and
Trypanosoma.
PMID: 15501825
TITLE: Second-site Suppression of a Nonfunctional Mutation within the Leishmania
donovani Inosine-Guanosine Transporter.
AFFILIATION: Department of Biochemistry and Molecular Biology, Oregon Health and
Science University, Portland, Oregon 97239.
REFERENCE: J Biol Chem 2005 Jan 280(3):2213-9
LdNT2 is a member of the equilibrative nucleoside transporter family,
which possesses several conserved residues located mainly within
transmembrane domains. One of these residues, Asp(389) within LdNT2, was
shown previously to be critical for transporter function without
affecting ligand affinity or plasma membrane targeting. To further
delineate the role of Asp(389) in LdNT2 function, second-site
suppressors of the ldnt2-D389N null mutation were selected in yeast
deficient in purine nucleoside transport and incapable of purine
biosynthesis. A library of random mutants within the ldnt2-D389N
background was screened in yeast for restoration of growth on inosine.
Twelve different clones were obtained, each containing secondary
mutations enabling inosine transport. One mutation, N175I, occurred in
four clones and conferred augmented inosine transport capability
compared with LdNT2 in yeast. N175I was subsequently introduced into an
ldnt2-D389N construct tagged with green fluorescent protein and
transfected into a Deltaldnt1/Deltaldnt2 Leishmania donovani knockout.
GFP-N175I/D389N significantly suppressed the D389N phenotype and
targeted properly to the plasma membrane and flagellum. Most
interestingly, N175I increased the inosine K(m) by 10-fold within the
D389N background relative to wild type GFP-LdNT2. Additional
substitutions introduced at Asn(175) established that only large,
nonpolar amino acids suppressed the D389N phenotype, indicating that
suppression by Asn(175) has a specific size and charge requirement.
Because multiple suppressor mutations alleviate the constraint imparted
by the D389N mutation, these data suggest that Asp(389) is a
conformationally sensitive residue. To impart spatial information to the
clustering of second-site mutations, a three-dimensional model was
constructed based upon members of the major facilitator superfamily
using threading analysis. The model indicates that Asn(175) and Asp(389
) lie in close proximity and that the second-site suppressor mutations
cluster to one region of the transporter.
PMID: 15542612
TITLE: Reconstitution of GDP-mannose Transport Activity with Purified Leishmania
LPG2 Protein in Liposomes.
AFFILIATION: Department of Molecular and Cellular Biochemistry, University of
Kentucky Medical Center, College of Medicine, Lexington, Kentucky 40536.
REFERENCE: J Biol Chem 2005 Jan 280(3):2028-35
Activated nucleotide sugars required for the synthesis of
glycoconjugates within the secretory pathway of eukaryotes are provided
by the action of nucleotide sugar transporters (NSTs). Typically, NSTs
are studied in microsomal preparations from wild-type or mutant lines;
however, in this setting it can be difficult to assess NST properties
because of the presence of glycosyltransferases and other interfering
activities. Here we have engineered Leishmania donovani to express high
levels of an active LPG2 Golgi GDP-Man transporter bearing a C-terminal
polyhistidine tag. The functional LPG2-HIS was solubilized, purified by
metal affinity chromatography, and reconstituted into
phosphatidylcholine-containing liposomes using polystyrene SM-2 beads.
The proteoliposomes exhibited robust GDP-Man transport activity with an
apparent K(m) of 6.6 mum. Transport activity was enhanced by preloading
of GMP and showed specificity for multiple substrates (GDP-Ara and GDP-
Fuc). In contrast to the activity in crude microsomes, transport was not
dependent on the presence of divalent cations. Thus, reconstitution of
transport activity using purified LPG2 protein in liposomes provides
firm experimental evidence that a single polypeptide is solely required
for NST activity and is able to mediate the uptake of multiple
substrates. These studies are relevant to the study of NST structure and
function in both protozoan parasites as well as their higher eukaryotic
hosts.
PMID: 15626463
TITLE: Immunoglobulin G and E responses in various stages of canine
leishmaniosis.
AFFILIATION: Laboratori de Parasitologia, Facultat de Farmà cia, Universitat de
Barcelona, Avda. Diagonal s/n, Barcelona, E-08028, Spain.
REFERENCE: Vet Immunol Immunopathol 2005 Jan 103(1-2):77-81
Pathogenesis in visceral leishmaniosis is associated with depressed
cellular immunity and a significant rise of antileishmanial antibodies.
We assessed the relative levels of immunoglobulin E anti-Leishmania
infantum, together with those of IgG, IgG1 and IgG2, using the enzyme-
linked immunosorbent assay (ELISA) test, in non-infected and infected
dogs with or without symptoms, and their association with symptoms to
differentiate the stages of the infection. The expression of all
immunoglobulins (IgG, IgG1, IgG2 and IgE) was higher in symptomatic dogs
than in all other categories. IgG and IgG2 expression was higher in the
infected asymptomatic group than in the non-infected group, whereas
IgG1 and IgE expression was only higher in symptomatic animals. This
correlation between the expression of IgG1 and IgE and the pathology of
leishmaniosis points to their potential role as markers of the active
disease.
PMID: 15629360
TITLE: Development of a recombinant Leishmania major strain sensitive to
ganciclovir and 5-fluorocytosine for use as a live vaccine challenge in
clinical trials.
AFFILIATION: Department of Biotechnology, Pasteur Institute of Tehran, Pasteur
Square, Tehran, Iran.
REFERENCE: Vaccine 2005 Jan 23(9):1170-7
To provide a safer live challenge strain for use in clinical vaccine
trials, a double drug sensitive strain of Leishmania major was derived
using advances in gene targeting technology by stably introducing into
the chromosome a modified HSV-1 thymidine kinase gene (tk), conferring
increased sensitivity to ganciclovir (GCV), and a Saccharomyces
cerevisiae cytosine deaminase gene (cd), conferring sensitivity to 5-
fluorocytosine (5-FC). In vitro studies showed that the homozygous L.
major (tk-cd(+/+)) promastigotes were killed by either drug alone, and
together the drugs acted synergistically. In vivo infection studies
showed that progressively growing lesions in BALB/c mice, caused by L.
major (tk-cd(+/+)), were completely cured by 2 weeks of treatment with
either drug alone or in combination. Treated animals showed no signs of
reoccurrence of infection for at least 4 months when the experiments
were terminated.
PMID: 15610826
TITLE: Leishmania lipophosphoglycan activates the transcription factor
activating protein 1 in J774A.1 macrophages through the extracellular
signal-related kinase (ERK) and p38 mitogen-activated protein kinase.
AFFILIATION: School of Life Sciences, Jawaharlal Nehru University, New Delhi,
110067 India.
REFERENCE: Mol Biochem Parasitol 2005 Jan 139(1):117-27
Leishmania donovani is an obligatory intracellular pathogen that resides
and multiplies in the phagolysosomes of macrophages. The outcome of
this infection depends on the balance between the host ability to
activate macrophage killing and the parasite ability to suppress or
evade this host immune response. Lipophosphoglycan (LPG) glycoconjugate
, the surface molecule of the protozoan parasite is a virulence
determinant and a major parasite molecule involved in this process. In
this study, we examined the ability of Leishmania and its surface
molecule, lipophosphoglycan to activate activating protein 1 (AP-1)
through the mitogen-activated protein kinase (MAPK) cascade. We report
here that the Leishmania surface molecule, lipophosphoglycan stimulates
the simultaneous activation of all three classes of MAP kinases,
extracellular signal-related kinases (ERKs), the c-jun amino-terminal
kinase (JNK) and the p38 MAP kinase with differential kinetics in J774A.
1 macrophage cell line. Furthermore, both L. donovani and its surface
molecule lipophosphoglycan resulted in a dose- and time-dependent
induction of AP-1 DNA-binding activity. We have also shown a dose-
dependent increase of AP-1 binding activity in both low and high
virulent strains of parasite. The use of inhibitors selective for ERK (
PD98059) and p38 (SB203580) pathway showed that pre-incubation of cells
with either SB203580 or PD98059 affected the binding activity of AP-1
suggesting that both p38 and ERK MAP kinase activation appear to be
necessary for AP-1 activation by LPG. Lipophosphoglycan induced IL-12
production and generation of nitric oxide in murine macrophages. These
results demonstrate that L. donovani LPG activates pro-inflammatory,
endotoxin-like response pathway in J774A.1 macrophages and the
interaction may play a pivotal role in the elimination of the parasite.
PMID: 15452362
TITLE: Therapeutic Potential of New Pt(II) and Ru(III) Triazole-Pyrimidine
Complexes against Leishmania donovani.
AFFILIATION: Institute of Biotechnology, Faculty of Sciences, University of
Granada, Campus Universitario Fuentenueva s/n, Granada, Spain.
REFERENCE: Pharmacology 2005 Jan 73(1):41-8
We have already established an in vitro culture system using murine
macrophages infected with Leishmania donovani in which the time course
of parasite growth is determined quantitatively. We adopted this system
for the screening of three triazole-pyrimidine derivatives that would
ideally prove to be effective against L. donovani with no toxicity to
the host cell. Amphotericin B deoxycholate was used as the standard drug
and gave a IC(50) value of 3.89 mug/ml. The three triazole-pyrimidine
compounds assayed have been reported to be potent growth inhibitors of L
. donovani promastigote and amastigote stages. Compounds SPIV and SPVI
exhibited the highest toxicity for extracellular forms of parasites,
with IC(50) values of 19.95 and 21.61 mug/ml, respectively. The triazole
-pyrimidine SPV, although to a lower degree, also showed pronounced
effects against promastigote forms with IC(50) of 33.14 mug/ml. Drug
activity was higher against amastigote than against promastigote stages
. The compounds SPIV and SPVI interfered with the synthesis of
macromolecules, affecting primarily DNA at the lower concentration
tested (5 mug/ml), while SPV also showed interference, though to a
lesser extent, and at a higher concentration (15 mug/ml) the percentage
of inhibition rose considerably. The synthesis or RNA and proteins was
also depressed significantly by these compounds at administration rates
of 15 mug/ml. Ultrastructural alterations were evident in the main
organelles of L. donovani (nucleus, kinetoplast, mitochondria), after
the addition of the three compounds at a concentration of 5 mug/ml, to
the in vitro culture. The in vitro promastigote forms of L. donovani can
degrade glucose to carbon dioxide, and part of the carbon skeleton of
the glucose is excreted as end metabolites. The excretion of these
metabolites, mainly acetate, was also inhibited by the three compounds
assayed, suggesting that this could be due to a direct effect on some of
the enzymes related to this fermentation pathway or to the inhibition
exerted by the compounds on enzyme synthesis. Copyright (c) 2005 S.
Karger AG, Basel.
PMID: 15637336
TITLE: The Expression of Mannose Receptors in Skin Fibroblast and Their
Involvement in Leishmania (L.) amazonensis Invasion.
AFFILIATION: Lab. Biologia Celular, Departamento de Ultra-estrutura e Biologia
Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Avenida Brasil 4365,
Manguinhos, Rio de Janeiro, RJ, 21045-900, Brasil. soeiro at ioc.fiocruz.br.
REFERENCE: J Histochem Cytochem 2005 Jan 53(1):35-44
Leishmania are protozoa that invade mononuclear phagocytes with the
involvement of different ligand-receptor systems, including mannose
receptors. Until now, scant data are available concerning the mechanisms
that govern the infection of Leishmania in other host cell types such
as fibroblasts. Our aim was to analyze the expression of mannose
receptors in primary cultures of skin fibroblasts (SF) further
characterizing their role during the invasion of promastigotes of
Leishmania (L.) amazonensis. Both fluorescent, light, and electron
microscopy assays revealed that SF have mannose receptors since they
bound and internalized mannosylated ligands in addition to being
positively labeled by fuc-BSA-FITC probes. d-mannose competition assays
revealed the participation of mannose receptors during the parasite
association with SF presenting upregulated receptor expression during
the initial steps of the infection. After longer periods of Leishmania:
fibroblasts contact, the modulation noted in the host mannose receptors
was reverted concomitantly to the infection control, suggesting that the
parasites were required for the alteration maintenance and providing
evidences that the SF may display microbicidal mechanisms to control the
Leishmania infection.
PMID: 15639739
TITLE: Sabotage and exploitation in macrophages parasitized by intracellular
protozoans.
AFFILIATION: Department of Microbiology and Immunology, College of Veterinary
Medicine, Cornell University, Ithaca, NY 14853-6401, USA.
REFERENCE: Trends Parasitol 2005 Jan 21(1):35-41
Macrophages are crucial in immunity to infection. They possess potent
antimicrobial function, and efficiently process and present peptide
antigens for T-cell activation. Despite this, the intracellular
protozoan parasites Toxoplasma gondii, Trypanosoma cruzi and Leishmania
spp. target macrophages for infection. Each has adopted unique
strategies to subvert macrophage antimicrobial functions. The parasites
sabotage killing activities through sophisticated manipulation of
intracellular macrophage signaling pathways. These subversive activities
are probably dictated by the need to evade microbicidal effector
function, as well as to avoid proinflammatory pathology that can
destabilize the host-parasite interaction. The molecular details of how
intracellular protozoans manipulate macrophage signal transduction
pathways for their own ends are beginning to emerge.
PMID: 15616293
TITLE: Antileishmanial Activity of Parthenolide, a Sesquiterpene Lactone
Isolated from Tanacetum parthenium.
AFFILIATION: Departamento de Análises ClÃnicas, Universidade Estadual de
Maringá, Bloco I-90 Sala 123 CCS, Av. Colombo 5790, BR-87020-900, Maringá,
Paraná, Brazil. cvnakamura at uem.br.
REFERENCE: Antimicrob Agents Chemother 2005 Jan 49(1):176-82
The in vitro activity of parthenolide against Leishmania amazonensis was
investigated. Parthenolide is a sesquiterpene lactone purified from the
hydroalcoholic extract of aerial parts of Tanacetum parthenium. This
isolated compound was identified through spectral analyses by UV,
infrared, (1)H and (13)C nuclear magnetic resonance imaging, DEPT (
distortionless enhancement by polarization transfer), COSY (correlated
spectroscopy), HMQC (heteronuclear multiple-quantum coherence), and
electron spray ionization-mass spectrometry. Parthenolide showed
significant activity against the promastigote form of L. amazonensis,
with 50% inhibition of cell growth at a concentration of 0.37 mug/ml.
For the intracellular amastigote form, parthenolide reduced by 50% the
survival index of parasites in macrophages when it was used at 0.81 mug/
ml. The purified compound showed no cytotoxic effects against J774G8
macrophages in culture and did not cause lysis in sheep blood when it
was used at higher concentrations that inhibited promastigote forms.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with gelatin
as the substrate showed that the enzymatic activity of the enzyme
cysteine protease increased following treatment of the promastigotes
with the isolated compound. This finding was correlated with marked
morphological changes induced by parthenolide, such as the appearance of
structures similar to large lysosomes and intense exocytic activity in
the region of the flagellar pocket, as seen by electron microscopy.
These results provide new perspectives on the development of novel drugs
with leishmanicidal activities obtained from natural products.
PMID: 15613100
TITLE: Soluble hemoglobin-haptoglobin scavenger receptor CD163 as a
lineage-specific marker in the reactive hemophagocytic syndrome.
AFFILIATION: Department of Medicine, University Hospital, Zurich, Switzerland.
REFERENCE: Eur J Haematol 2005 Jan 74(1):6-10
Schaer DJ, Schleiffenbaum B, Kurrer M, Imhof A, Bachli E, Fehr J, Moller
HJ, Moestrup SK, Schaffner A. Soluble hemoglobin-haptoglobin scavenger
receptor CD163 as a lineage-specific marker in the reactive
hemophagocytic syndrome. Eur J Haematol 2005: 74: 6-10. (c) Blackwell
Munksgaard 2005.Abstract: Reactive hemophagocytic syndrome (RHS) is a
disease of overwhelming macrophage activity triggered by infection,
malignancy or autoimmune disorders. Currently used laboratory markers
for the quantitative assessment of monocyte/macrophage activation lack
lineage-restricted expression patterns and thus specificity. Serum
levels of the macrophage specific scavenger receptor CD163 were
detemined by enzyme-linked immunosorbent assay (ELISA) and were found to
be highly increased in patients with RHS (median 39.0 mg/L).
Significantly lower levels were determined in patients with sepsis (
median 9.1 mg/L), acute mononucleosis (median 8.2 mg/L), Leishmania
infection (median 6.7 mg/L) and healthy controls (median 1.8 mg/L).
Follow-up of patients with a relapsing course of the disease revealed
close correlations of sCD163 with clinical disease activity, serum
ferritin and other markers of macrophage activity. Large sinusoidal
accumulations of CD163 expressing macrophages actively engaged in
phagocytosis of blood cells were detected in spleen sections of RHS
patients. Our data suggests sCD163 to be a macrophage-specific marker in
patients with disorders of inappropriate macrophage activation.
PMID: 15549728
TITLE: Development of an anti-IL-12 p40 auto-vaccine: protection in experimental
autoimmune encephalomyelitis at the expense of increased sensitivity to
infection.
AFFILIATION: Ludwig Institute for Cancer Research, Brussels Branch, Brussels,
Belgium.
REFERENCE: Eur J Immunol 2004 Dec 34(12):3572-81
IL-12 and IL-23, which share the IL-12 p40 subunit, have been ascribed
central roles in many autoimmune disorders. We describe here an anti-IL-
12 (alphaIL-12) auto-vaccine that potentially blocks both factors in
vivo. Immunization of mice with mouse IL-12 coupled to OVA or Pan DR
epitope (PADRE) peptide induced Ab directed against the IL-12 p40
subunit, which prevented IFN-gamma production in response to IL-12
administration in vivo. Experimental autoimmune encephalomyelitis, an IL
-23-dependent disease model, induced in SJL mice with a proteolipid
protein (PLP) peptide was almost undetectable after alphaIL-12
vaccination. Myelin oligodendrocyte glycoprotein (MOG)-induced disease
in C57BL/6 mice was also significantly inhibited. This protection
correlated with inhibited Th1 cytokine responses in vitro and with an
increase in the IgG1/IgG2a anti-PLP Ab balance. Detrimental consequences
of alphaIL-12 vaccination were evaluated in C57BL/6 mice infected with
Leishmania major (L.m.). While delayed-type hypersensitivity (DTH)
suppression and immunoglobulin as well as interleukin production
patterns reflected a major shift toward a Th2-type response, L.m. growth
was still significantly retarded as compared to that seen in
susceptible BALB/c mice. However, vaccinated animals ultimately failed
to control parasite expansion. These results suggest that some chronic
autoimmune diseases may benefit from alphaIL-12 vaccination at the
expense of reduced, but not completely abrogated, cell-mediated immunity.
PMID: 15584972
TITLE: Interleukin-15, as Interferon-gamma, Induces the Killing of Leishmania
infantum in Phorbol-Myristate-Acetate-Activated Macrophages Increasing
Interleukin-12.
AFFILIATION: Department of Immuno-Hematology and Transfusion Medicine,
University of Palermo, Palermo, Italy.
REFERENCE: Scand J Immunol 2004 Dec 60(6):609-14
Abstract The potential leishmanicidal activity of interleukin-15 (IL-15
) was examined while priming with the cytokine phorbol-myristate-acetate
(PMA)-activated macrophages and infecting them with Leishmania infantum
parasites. The activation of macrophage cultures with IL-15 determined
a significant anti-leishmanial activity, comparable with that induced by
interferon-gamma (IFN-gamma). The killing of Leishmania in macrophages
primed with IL-15, as well as with IFN-gamma, was followed by an
increase in the IL-12 synthesis. The neutralization of IL-15 or IFN-
gamma, by specific monoclonal antibodies (MoAb) caused a significant
reduction in leishmanicidal activity. Furthermore, in PMA-activated
macrophages, the neutralization of IL-12 production by a specific anti-
IL-12 MoAb reduced leishmanicidal activity induced by IL-15 and IFN-
gamma. Data indicate that IL-15 could have a role as an activator of
leishmanicidal activity, directly or indirectly, by inducing IL-12
production.
PMID: 15588704
TITLE: Inactivation of parasite cysteine proteinases by the NO-donor
4-(phenylsulfonyl)-3-((2-(dimethylamino)ethyl)thio)-furoxan oxalate.
AFFILIATION: Dipartimento di Biologia and Laboratorio Interdipartimentale di
Microscopia Elettronica, Università Roma Tre, Viale Guglielmo Marconi 446,
I-00146 Roma, Italy. ascenzi at uniroma3.it
REFERENCE: Biochim Biophys Acta 2004 Dec 1703(1):69-77
NO-donors block Plasmodium, Trypanosoma, and Leishmania life cycle by
inactivating parasite enzymes, e.g., cysteine proteinases. In this study
, the inactivation of falcipain, cruzipain, and Leishmania infantum
cysteine proteinase by the NO-donor 4-(phenylsulfonyl)-3-((2-(
dimethylamino)ethyl)thio)-furoxan oxalate (SNO-102) is reported. SNO-102
inactivates dose- and time-dependently parasite cysteine proteinases;
one equivalent of NO, released from SNO-102, inactivates one equivalent
of L. infantum cysteine proteinase. With SNO-102 in excess over the
parasite cysteine proteinase, the time course of enzyme inhibition
corresponds to a pseudo-first-order reaction for more than 90% of its
course. The concentration dependence of the pseudo-first-order rate
constant is second-order at low SNO-102 concentration but tends to first
-order at high NO-donor concentration. This behavior may be explained by
a relatively fast pre-equilibrium followed by a limiting pseudo-first
order process. Kinetic parameters of L. infantum cysteine proteinase
inactivation by SNO-102 are affected by the acidic pK shift of one
apparent ionizing group (from pK(unl)=5.8 to pK(lig)=4.7) upon enzyme
inhibition. Falcipain, cruzipain and L. infantum cysteine proteinase
inactivation is prevented and reversed by dithiothreitol and L-ascorbic
acid. Furthermore, the fluorogenic substrate N-alpha-benzyloxycarbonyl-
Phe-Arg-(7-amino-4-methylcoumarin) protects parasite cysteine
proteinases from inactivation by SNO-102. The absorption spectrum of the
inactive S-nitrosylated SNO-102-treated L. infantum cysteine proteinase
displays a maximum at about 340 nm. These results indicate that the
parasite cysteine proteinase inactivation by SNO-102 occurs via the NO-
mediated S-nitrosylation of the Cys25 catalytic residue.
PMID: 15596523
TITLE: A Leishmania major Response Locus Identified by Interval-specific
Congenic Mapping of a T Helper Type 2 Cell Bias-controlling Quantitative Trait
Locus.
AFFILIATION: Dept. of Immunology, University of Washington, 1959 N.E. Pacific
St., HSC I607I, Seattle, WA 98195. mbix at u.washington.edu.
REFERENCE: J Exp Med 2004 Dec 200(12):1605-12
The propensity of naive CD4 T cells to become T helper (Th) type 2 cells
correlates with susceptibility to infection by the protozoal parasite
Leishmania major. Using genetic linkage analysis, we earlier identified
Dice1 as a Th2 cell bias-controlling quantitative trait locus on
chromosome 16. Using interval-specific congenic mapping, we now resolve
Dice1 into two independent genetic loci, Dice1.1 and Dice1.2, which
control Il4 expression from naive Th cells and thereby indirectly
control Th2 cell bias. Interestingly, only one of the two congenic
intervals containing Dice1.1 and Dice1.2, respectively, also contained
an L. major response locus, indicating that L. major responsiveness can
be insensitive to determinants that influence Th2 cell bias by
controlling naive T cell Il4 expression. These results lay the
groundwork for identifying the Dice1.1 and Dice1.2 genes controlling
naive T cell Il4 expression and L. major responses, and for testing
whether these control other Th2 cell-dependent processes such as worm
expulsion, allergic asthma, and dermatitis.
PMID: 15466466
TITLE: Growth Phase Regulation of the Main Folate Transporter of Leishmania
infantum and Its Role in Methotrexate Resistance.
AFFILIATION: Centre de Recherche en Infectiologie du Centre de Recherche du
Centre Hospitalier and Division de Microbiologie, Faculté de Médecine,
Université Laval, Québec G1V 4G2, Canada.
REFERENCE: J Biol Chem 2004 Dec 279(52):54494-501
The protozoan parasite Leishmania relies on the uptake of folate and
pterin from the environment to meet its nutritional requirements. We
show here that a novel gene (folate transporter 1 (FT1)) deleted in a
Leishmania infantum methotrexate-resistant mutant corresponds to the
main folate transporter (K(m), 410 nm). FT1 was established as the main
folate transporter by both gene transfection and by targeted gene
deletion. Modulation of the expression of FT1 by these manipulations
altered the susceptibility of Leishmania cells to methotrexate. Folate
transport was stage-regulated with higher activity in the logarithmic
phase and less in the stationary phase. FT1 fused to green fluorescent
protein led to the observation that FT1 was located in the plasma
membrane in the logarithmic phase but was retargeted to an intracellular
organelle followed by a degradation of the protein in stationary phase
. Leishmania has several folate transporters with different
characteristics, and the growth stage-related activity of at least one
transporter is regulated post-translationally.
PMID: 15569786
TITLE: Characterization of the early cellular immune response to leishmania
major using peripheral blood mononuclear cells from leishmania-naive humans.
AFFILIATION: Department of Microbiology, Immunology and Pathology, College of
Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort
Collins, Colorado.
REFERENCE: Am J Trop Med Hyg 2004 Nov 71(5):568-76
While the response to Leishmania major is well characterized in mice,
there is much less known about the human immune response, particularly
early after exposure to the parasite. Therefore, we developed a primary
in vitro (PIV) system that allowed us to address these questions. We co-
cultured peripheral blood mononuclear cells from Leishmania-naive donors
with L. major parasites and found that the responding PIV cells
produced interferon-gamma and interleukin-12 (IL-12). When restimulated
, these PIV cells also occasionally produced IL-5. Both CD4 and CD8
cells and both HLA class I and II cell activation pathways appeared to
play a role in the PIV system, and cell activation was dependent upon
the presence of antigen-presenting cells. Moreover, PIV cells generated
with L. major showed considerable cross-reactivity with other species of
Leishmania. Finally, the PIV cells augmented intracellular killing of L
. major when they were co-cultured with macrophages infected with the
parasite.
PMID: 15636182
TITLE: Evaluation of the trypanocidal and leishmanicidal in vitro activity of
the crude hydroalcoholic extract of PFAFFIA glomerata (Amarathanceae) roots.
AFFILIATION: Mestrando do Curso de Pós-Graduação Strictu Sensu em Promoção
de Saúde, Universidade de Franca, Franca, SP, Brazil.
REFERENCE: Phytomedicine 2004 Nov 11(7-8):662-5
Three different concentrations (1, 10 and 50 microg/ml) of lyophilized
hydroalcoholic crude extract of Pfaffia glomerata roots were assayed in
vitro against strains of Trypanosoma cruzi (Y) and Leishmania
braziliensis. It was observed that P. glomerata hydroalcoholic extract
was relatively active within the tested concentrations for L. (V)
braziliensis, but inactive against T. cruzi. Despite the fact that both
protozoans belong to the Trypanosomatidae family, we suggest that the
difference observed for activity should be related to the biological
differences between the two parasite species.
PMID: 15504880
TITLE: Antimicrobial and antileishmanial activities of hypocrellins A and B.
AFFILIATION: The National Center for Natural Products Research, School of
Pharmacy, The University of Mississippi, University, MS 38677, USA.
REFERENCE: Antimicrob Agents Chemother 2004 Nov 48(11):4450-2
Hypocrellins A and B were evaluated for in vitro antimicrobial and
antileishmanial activities. Hypocrellin A exhibited promising activity
against Candida albicans and moderate activity against Staphylococcus
aureus, methicillin-resistant S. aureus, Pseudomonas aeruginosa, and
Mycobacterium intracellulare. Hypocrellin B showed weak antimicrobial
activities. Hypocrellin A exhibited potent antileishmanial activity,
while hypocrellin B was only moderately active. These results of
promising antifungal and antileishmanial activity of hypocrellin A may
be useful for further structure-activity relationship and in vivo
studies.
PMID: 15648659
TITLE: An effective diaryl derivative against Leishmania amazonensis and its
influence on the parasite X macrophage interaction.
AFFILIATION: Department of Immunology, Oswaldo Cruz Institute, FIOCRUZ, Rio de
Janeiro, Brazil.
REFERENCE: J Enzyme Inhib Med Chem 2004 Oct 19(5):437-9
The activity of several diarylheptanoid derivatives (curcuminoids) was
previously evaluated against Leishmania amazonensis promastigotes and
among them the most active compound was 5-hydroxy-7- (4-hydroxy-3-
methoxyphenyl)-1-(4-methoxyphenyl)-1,4,6-heptatrien-3-one. This study
was carried out to investigate the influence of this diaryl derivative
on the infective promastigotes and Balb/c mice peritoneal macrophage
interaction. The potential in vitro toxicity was also evaluated.
Promastigotes pretreated for 24 hours with the compound had their
infective capacity significantly decreased. When the infection of Balb/c
macrophage by L. amazonensis promastigotes was already installed,
addition of the drug resulted in a diminishing of the infection rate. It
was demonstrated that the compound was not toxic to the host macrophage
in a concentration equivalent to the LD50/24h from the previous in
vitro experiment.
PMID: 15487964
TITLE: Visceral leishmaniasis: a trip to the Greek Islands is not always
idyllic.
AFFILIATION: Infectious Diseases, Queen Elizabeth Hospital, Woodville Road,
Woodville, SA 5111, Australia. Oui.Ju at fmc.sa.gov.au
REFERENCE: Med J Aust 2004 Oct 181(8):446-7
Although cutaneous leishmaniasis is occasionally seen in Australia in
overseas travellers and migrants, visceral leishmaniasis has been
reported rarely and only in people who were immunocompromised. We
describe an 18-year-old immunocompetent man who presented with
pancytopenia and a 2-week history of fever and lethargy a year after
visiting the Greek Islands. Visceral leishmaniasis was diagnosed after a
bone marrow biopsy showed protozoa, and the patient responded well to
treatment with liposomal amphotericin. To our knowledge, this is the
first case of visceral leishmaniasis in an immunocompetent patient in
Australia.
PMID: 15579321
TITLE: Leishmania parasites (Kinetoplastida: Trypanosomatidae) reversibly
inhibit visceral muscle contractions in hemimetabolous and holometabolous
insects.
AFFILIATION: Department of Parasitology, Hadassah Medical School, Hebrew
University, Ein Kerem, P.O. Box 12272, Jerusalem 91120, Israel.
REFERENCE: J Invertebr Pathol 2004 Oct 87(2-3):123-8
Female sand flies can acquire protozoan parasites in the genus
Leishmania when feeding on an infected vertebrate host. The parasites
complete a complex growth cycle in the sand fly gut until they are
transmitted by bite to another host. Recently, a myoinhibitory peptide
was isolated from Leishmania major promastigotes. This peptide caused
significant gut distension and reversible, dose-dependent inhibition of
spontaneous hindgut contractions in the enzootic sand fly vector,
Phlebotomus papatasi. The current study further characterizes
myoinhibitory activity in L. major and other kinetoplastid parasites,
using the P. papatasi hindgut and other insect organ preparations.
Myoinhibitory activity was greatest in cultured promastigotes and in
culture medium in late log-phase and early stationary-phase, coinciding
with development of infective Leishmania morphotypes in the sand fly
midgut. L. major promastigote lysates inhibited spontaneous contractions
of visceral muscle preparations from hemimetabolous (Blattaria and
Hemiptera) and holometabolous (Diptera) insects. Inhibition of visceral
muscle contractions in three insect orders indicates a conserved mode of
action. Myoinhibitory activity was detected also in Leishmania
braziliensis braziliensis, a Sudanese strain of Leishmania donovani, and
the kinetoplastid parasite Leptomonas seymouri. Protozoan-induced
myoinhibition mimics the effect of insect myotropins. Inhibiting host
gut contractions protects Leishmania parasites from being excreted after
blood meal and peritrophic matrix digestion, allowing development and
transmission of infective forms.
PMID: 15603761
TITLE: Leishmania spp.: on the interactions they establish with
antigen-presenting cells of their mammalian hosts.
AFFILIATION: Unité d'Immunophysiologie et Parasitisme Intracellulaire, Institut
Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France. jantoine at pasteur.fr
REFERENCE: Adv Parasitol 2004 58():1-68
Identification of macrophages as host cells for the mammalian stage of
Leishmania spp. traces back to about 40 years ago, but many questions
concerning the ways these parasites establish themselves in these cells
, which are endowed with potent innate microbicidal mechanisms, are
still unanswered. It is known that microbicidal activities of
macrophages can be enhanced or induced by effector T lymphocytes
following the presentation of antigens via MHC class I or class II
molecules expressed at the macrophage plasma membrane. However,
Leishmania spp. have evolved mechanisms to evade or to interfere with
antigen presentation processes, allowing parasites to partially resist
these T cell-mediated immune responses. Recently, the presence of
Leishmania amastigotes within dendritic cells has been reported
suggesting that they could also be host cells for these parasites.
Dendritic cells have been described as the only cells able to induce the
activation of naive T lymphocytes. However, certain Leishmania species
infect dendritic cells without inducing their maturation and impair the
migration of these cells, which could delay the onset of the adaptive
immune responses as both processes are required for naive T cell
activation. This review examines how Leishmania spp. interact with these
two cell types, macrophages and dendritic cells, and describes some of
the strategies used by Leishmania spp. to survive in these inducible or
constitutive antigen-presenting cells.
PMID: 15591782
TITLE: Hypoxia Modulates Expression of the 70-kD Heat Shock Protein and Reduces
Leishmania Infection in Macrophages.
AFFILIATION: Department of Parasitology, Biology Institute, Universidade
Estadual de Campinas, Campinas, Brazil.
REFERENCE: J Biomed Sci 2004 Nov-Dec 11(6):847-54
Hypoxia, a microenvironmental factor present in diseased tissues, has
been recognized as a specific metabolic stimulus or a signal of cellular
response. Experimental hypoxia has been reported to induce adaptation
in macrophages such as differential migration, elevation of
proinflammatory cytokines and glycolytic enzyme activities, and
decreased phagocytosis of inert particles. In this study we demonstrate
that although exposure to hypoxia (5% O(2), 5% CO(2), and balanced N(2
)) did not change macrophage viability, or 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) cleavage and proliferation, it
significantly reduced expression of the 70-kD heat shock protein (HSP70
), which was restored to prehypoxia levels after reoxygenation. The
influence of low oxygen tension on macrophage functional activity was
also studied, i.e. the ability of these cells to maintain or resist
infection by a microorganism. We demonstrate that macrophages from two
different sources (a murine cell line and primary cells) exposed to
hypoxia were efficiently infected with Leishmania amazonensis, but after
24 h showed a reduction in the percentage of infected cells and of the
number of intracellular parasites per macrophage, indicating that
hypoxia induced macrophages to kill the intracellular parasites. These
results support the notion that hypoxia, a microenvironmental factor,
can modulate macrophage protein expression and functional activity.
Copyright (c) 2004 National Science Council, ROC and S. Karger AG, Basel.
PMID: 15544717
TITLE: Itraconazole can be effective in the treatment of sporotrichoid
leishmaniasis.
AFFILIATION: Department of Infectious and Tropical Diseases, University of
Milan, L. Sacco Hospital, Milan, Italy.
REFERENCE: J Travel Med 2004 Sep-Oct 11(5):328-30
PMID: 15582507
TITLE: Specificity of modified Drosophila mariner transposons in the
identification of Leishmania genes.
AFFILIATION: Departamento de Biologia Celular e Molecular e Bioagentes
Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São
Paulo. Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto-SP, Brazil.
REFERENCE: Exp Parasitol 2004 Nov-Dec 108(3-4):109-13
Genetic manipulation of the protozoan Leishmania has led to a better
understanding of the survival and development of these pathogens within
their hosts. The association of the Leishmania genome sequencing
information with the ability of transposons to introduce or destroy
phenotypes allows a global perspective on the role and importance of
genes in cellular pathways. Herein we report the construction and
testing of mariner transposable elements carrying the neomycin
phosphotransferase, green fluorescent protein, or beta-glucuronidase
genes as reporters for translational fusion events. We demonstrate that
the expression of the reporter genes will occur only when the genes are
inserted in-frame within predicted genes. Our results not only add to
the mariner toolkit for gene manipulation but also strengthen the
evidence that the mariner system is a reliable means for the study of
gene expression in Leishmania.
PMID: 15582509
TITLE: Trypanosomatid flagellum biogenesis: ARL-3A is involved in several
species.
AFFILIATION: Laboratoire de Génomique Fonctionnelle des Trypanosomatides, UMR
CNRS 5162, Université Bordeaux 2, 146 Rue Léo Saignat, 33 000 Bordeaux,
France.
REFERENCE: Exp Parasitol 2004 Nov-Dec 108(3-4):126-33
Overexpression in Leishmania amazonensis promastigotes of the GTPase-
deficient small G protein LdARL-3A-Q70L specifically provokes the loss
of the flagella [J. Cell Sci. 113 (2000) 2065] without affecting cell
viability and body size. However, motility is lost and, remarkably,
cells do not survive in the insect vector Lutzomyia longipalpis gut,
leading to interruption of parasite transmission [Cell Microbiol. 5 (
2003) 717]. We report here that overexpression of the same protein in
Leishmania major, Leishmania donovani, and Crithidia fasciculata also
led to significant alterations of the flagella. Surprisingly, ablation
of TbARL-3A expression by RNAi in Trypanosoma brucei brucei also
provoked flagella shortening, revealing that overexpression of the
GTPase-deficient protein seems functionally equivalent to a drastic
reduction in its native counterpart abundance. This renders possible
complementary studies of an essential pathway in related organisms.
Potential significance for the protein function is discussed as well as
future strategies for stopping the transmission of several neglected
parasitic diseases.
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PMID: 15326547
TITLE: Inhibiting activities of the secondary metabolites of Phlomis
brunneogaleata against parasitic protozoa and plasmodial enoyl-ACP Reductase, a
crucial enzyme in fatty acid biosynthesis.
AFFILIATION: Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe
University, Ankara, Turkey.
REFERENCE: Planta Med 2004 Aug 70(8):711-7
Anti-plasmodial activity-guided fractionation of Phlomis brunneogaleata
(Lamiaceae) led to the isolation of two new metabolites, the iridoid
glycoside, brunneogaleatoside and a new pyrrolidinium derivative (2 S,4
R)-2-carboxy-4-( E)- p-coumaroyloxy-1,1-dimethylpyrrolidinium inner salt
[(2 S,4 R)-1,1-dimethyl-4-( E)- p-coumaroyloxyproline inner salt].
Moreover, a known iridoid glycoside, ipolamiide, six known
phenylethanoid glycosides, verbascoside, isoverbascoside, forsythoside B
, echinacoside, glucopyranosyl-(1-->G (i)-6)-martynoside and
integrifolioside B, two flavone glycosides, luteolin 7- O-beta- D-
glucopyranoside ( 10) and chrysoeriol 7- O-beta- D-glucopyranoside ( 11
), a lignan glycoside liriodendrin, an acetophenone glycoside 4-
hydroxyacetophenone 4- O-(6'- O-beta- D-apiofuranosyl)-beta- D-
glucopyranoside and three caffeic acid esters, chlorogenic acid, 3-O-
caffeoylquinic acid methyl ester and 5- O-caffeoylshikimic acid were
isolated. The structures of the pure compounds were elucidated by means
of spectroscopic methods (UV, IR, MS, 1D and 2D NMR, [alpha] (D)) and X-
ray crystallography. Compounds 10 and 11 were determined to be the major
anti-malarial principles of the crude extract (IC (50) values of 2.4
and 5.9 micrograms/mL, respectively). They also exhibited significant
leishmanicidal activity (IC (50) = 1.1 and 4.1 micrograms/mL,
respectively). The inhibitory potential of the pure metabolites against
plasmodial enoyl-ACP reductase (FabI), which is the key regulator of
type II fatty acid synthases (FAS-II) in P. falciparum, was also
assessed. Compound 10 showed promising FabI inhibiting effect (IC (50
) = 10 micrograms/mL) and appears to be the first anti-malarial natural
product targeting FabI of P. falciparum.
PMID: 15163652
TITLE: Targeting Leishmania (L.) chagasi amastigotes through macrophage
scavenger receptors: the use of drugs entrapped in liposomes containing
phosphatidylserine.
AFFILIATION: Instituto de Ciências Biomédicas, Dept. Parasitologia -
Universidade de São Paulo, Brazil.
REFERENCE: J Antimicrob Chemother 2004 Jul 54(1):60-8
OBJECTIVES: We devised liposome-entrapped antimony with the negatively
charged lipid phosphatidylserine-liposome-entrapped antimony (Sb-LP)-in
order to improve their targeting to infected macrophages through the
interaction with scavenger receptors (SRs). METHODS: SR production was
indirectly evaluated by its mRNA synthesis in infected and uninfected
peritoneal macrophages using RT-PCR. The interaction and cytotoxicity of
Sb-LP with SRs and their metabolism were determined by incubation with
macrophages in the presence of cytochalasin B, chloroquine or different
competitive ligands, with determination of the 50% inhibitory
concentration (IC50) in vitro in infected macrophages. The intracellular
trafficking of Sb-LP was evaluated by confocal microscopy using trapped
fluorescent dyes. RESULTS: Our results showed an up-regulation of
macrophage SR mRNA during the initial steps of Leishmania (L.) chagasi
infection. By competitive ligand assays, we demonstrated the
preferential uptake of Sb-LP by macrophage SRs. Sb-LP was 16-fold more
effective (IC50=14.11 microM) than the free drug (IC50=225.9 microM)
against L. (L.) chagasi-infected macrophages. The binding and uptake of
Sb-LP in macrophages were shown to be energy-dependent and were reduced
in the presence of cytochalasin B, showing the dependency of the cell
microfilament system. Confocal analysis using trapped fluorescent dyes
showed fluorescence of parasites or in their close proximity, compatible
with the localized delivery of the liposomes. CONCLUSIONS: The uptake
of Sb-LP was reduced in infected macrophages, despite their
effectiveness and targeting ability, suggesting a low metabolic rate in
infected macrophages that could be overcome by the higher efficiency of
the liposomal formulation. These in vitro results suggest that liposomes
could improve the therapeutic index of old drugs, such as pentavalent
antimony, via targeted delivery to Leishmania-infected cells.
PMID: 15066023
TITLE: Sphingolipid-free Leishmania are defective in membrane trafficking,
differentiation and infectivity.
AFFILIATION: Wellcome Trust Laboratories for Molecular Parasitology, Centre for
Molecular Microbiology and Infection, Department of Biological Sciences,
Imperial College London, London SW7 2AZ, UK. p.w.denny at imperial.ac.uk
REFERENCE: Mol Microbiol 2004 Apr 52(2):313-27
Sphingolipids are structural components of the eukaryotic plasma
membrane that are involved, together with cholesterol, in the formation
of lipid microdomains (rafts). Additionally, sphingolipid metabolites
have been shown to modulate a wide variety of cellular events, including
differentiation and apoptosis. To investigate the role of de novo
sphingolipid biosynthesis in Leishmania, we have focused on serine
palmitoyltransferase (SPT), which catalyses the first, rate-limiting
step in the synthetic pathway. Genetic ablation of one SPT subunit,
LmLCB2, yields viable null parasites that can no longer synthesize
ceramide and sphingolipids de novo. Unexpectedly, LmLCB2 expression (and
sphingolipid biosynthesis) is stage regulated in Leishmania, being
undetectable in intramacrophage parasites. As expected from this
observation, the LmLCB2 null mutants maintain infectivity in vivo.
However, they are compromised in their ability to form infective
extracellular parasites, correlating with a defect in association of the
virulence factor, leishmanolysin or GP63, with lipid rafts during
exocytosis and an observed relocalization of a second virulence factor,
lipophosphogycan, during differentiation. Thus, de novo sphingolipid
biosynthesis is critical for membrane trafficking events in
extracellular Leishmania but has at best a minor role in intracellular
pathogenesis.
PMID: 14698430
TITLE: Tests of cytoplasmic RNA interference (RNAi) and construction of a
tetracycline-inducible T7 promoter system in Trypanosoma cruzi.
AFFILIATION: Departamento de Bioquimica e Imunologia, Universidade Federal de
Minas Gerais, Caixa Postal 486, Belo Horizonte, Brazil.
REFERENCE: Mol Biochem Parasitol 2004 Feb 133(2):175-86
The technique of RNA interference (RNAi) is exceedingly useful for
knocking down the expression of a specific mRNA in African trypanosomes
and other organisms for the purpose of examining the function of its
gene. However, when we attempted to apply RNAi in the Latin American
trypanosome, Trypanosoma cruzi, to diminish expression of mRNA encoding
the surface protein amastin, we found that the amastin double-stranded
RNA (dsRNA) was not efficiently degraded in either epimastigotes or
amastigotes, and the level of amastin mRNA remained unchanged. We
generated a strain of T. cruzi CL-Brener in which the T7 promoter and
tetracycline operator could be used to maximize tetracycline-regulated
dsRNA synthesis and constructed plasmids that direct dsRNA against four
different T. cruzi endogenous genes (encoding beta-tubulin, GP72 (
flagellar adhesion protein), ribosomal protein P0 and amastin) and an
exogenously added gene (GFP; green fluorescent protein). After either
stable or transient transfection of these plasmids into T. cruzi, the
expected RNAi phenotype was not observed for any of the five genes,
although the T. cruzi beta-tubulin RNAi plasmid did give the expected
FAT cell phenotype in the African trypanosome, Trypanosoma brucei. These
data indicate that, similar to Leishmania, T. cruzi lacks one or more
components necessary for the RNAi pathway and that these components will
need to be engineered into T. cruzi, or compensated for, before RNAi
can be used to study gene function in this organism.
PMID: 14711591
TITLE: The amino terminal domain of a novel WD repeat protein from Trypanosoma
cruzi contains a non-canonical mitochondrial targeting signal.
AFFILIATION: Department of Infectious and Tropical Diseases, London School of
Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
REFERENCE: Int J Parasitol 2004 Jan 34(1):63-71
WD (tryptophan/aspartic acid) repeat proteins perform a wide variety of
functions in eukaryotic cells. They are characterised by the presence of
a number of conserved repeat motifs that contribute to the beta-
propeller structures which are the common feature of this large group of
proteins. We report here the properties of the first characterised
member of this family in the American trypanosome, Trypanosoma cruzi (
TcBPP1). In the CL Brener clone the protein is 482 amino acids long and
is predicted to contain four WD repeat motifs, flanked by amino and
carboxyl terminal extensions. TcBPP1 is a single copy gene present on a
1.0/1.6 Mb pair of homologous chromosomes in a locus that is syntenic
with the corresponding regions of Trypanosoma brucei and Leishmania
major chromosomes. Consistent with the proposed hybrid nature of the CL
Brener clone, the proteins encoded by the two different alleles share
only 97% identity at the amino acid level. To determine subcellular
location, we examined transfected parasites for the distribution of
green fluorescent protein (GFP) fused with different regions of TcBPP1.
These studies demonstrated that a 115 amino acid peptide derived from
the amino terminal domain of TcBPP1 is able to target GFP to the
mitochondrion. Interestingly this region lacks a typical amino terminal
presequence suggesting that mitochondrial import is mediated by an
alternative targeting signal.
PMID: 14609948
TITLE: Sphingolipids are essential for differentiation but not growth in
Leishmania.
AFFILIATION: Department of Molecular Microbiology, Box 8230, Washington
University School of Medicine, 660 S. Euclid Ave, St Louis, MO 63110, USA.
REFERENCE: EMBO J 2003 Nov 22(22):6016-26
Sphingolipids (SLs) play critical roles in eukaryotic cells in the
formation of lipid rafts, membrane trafficking, and signal transduction
. Here we created a SL null mutant in the protozoan parasite Leishmania
major through targeted deletion of the key de novo biosynthetic enzyme
serine palmitoyltransferase subunit 2 (SPT2). Although SLs are typically
essential, spt2- Leishmania were viable, yet were completely deficient
in de novo sphingolipid synthesis, and lacked inositol
phosphorylceramides and other SLs. Remarkably, spt2- parasites
maintained 'lipid rafts' as defined by Triton X-100 detergent resistant
membrane formation. Upon entry to stationary phase spt2- failed to
differentiate to infective metacyclic parasites and died instead. Death
occurred not by apoptosis or changes in metacyclic gene expression, but
from catastrophic problems leading to accumulation of small vesicles
characteristic of the multivesicular body/multivesicular tubule network
. Stage specificity may reflect changes in membrane structure as well as
elevated demands in vesicular trafficking required for parasite
remodeling during differentiation. We suggest that SL-deficient
Leishmania provide a useful biological setting for tests of essential SL
enzymes in other organisms where SL perturbation is lethal.
PMID: 14500512
TITLE: Iron superoxide dismutases targeted to the glycosomes of Leishmania
chagasi are important for survival.
AFFILIATION: Department of Biological Sciences, University of Calgary, 2500
University Drive NW, Calgary, Alberta, Canada T2N 1N4.
REFERENCE: Infect Immun 2003 Oct 71(10):5910-20
Kinetoplastid glycosomes contain a variety of metabolic activities, such
as glycolysis, beta-oxidation of fatty acids, lipid biosynthesis, and
purine salvage. One advantage of sequestering metabolic activities is
the avoidance of cellular oxidative damage by reactive oxygen species
produced as a by-product of metabolism. Little is known about how
glycosomes themselves withstand these toxic metabolites. We previously
isolated an iron superoxide dismutase from Leishmania chagasi that is
expressed at low levels in the early logarithmic promastigote stage and
increases toward the stationary promastigote and amastigote stages. We
have since identified a second highly homologous Lcfesodb gene that is
expressed at high levels in the early logarithmic promastigote stage and
decreases toward the stationary promastigote and amastigote stages.
Localization studies using green fluorescent protein fusions have
revealed that LcFeSODB1 and LcFeSODB2 are localized within the
glycosomes by the last three amino acids of their carboxyl termini. To
better understand the specific role that FeSODB plays in parasite growth
and survival, a single-allele knockout of the Lcfesodb1 gene was
generated. The parasites with these genes exhibited a significant
reduction in growth when endogenous superoxide levels were increased
with paraquat in culture. Furthermore, the FeSODB1-deficient parasites
exhibited a significant reduction in survival within human macrophages.
Our results suggest that LcFeSODB plays an important role in parasite
growth and survival by protecting glycosomes from superoxide toxicity.
PMID: 12807872
TITLE: Functional analysis of an inosine-guanosine transporter from Leishmania
donovani. The role of conserved residues, aspartate 389 and arginine 393.
AFFILIATION: Department of Biochemistry and Molecular Biology, Oregon Health and
Science University, Portland, Oregon 97239, USA.
REFERENCE: J Biol Chem 2003 Aug 278(35):33327-33
Equilibrative nucleoside transporters encompass two conserved, charged
residues that occur within predicted transmembrane domain 8. To assess
the role of these "signature" residues in transporter function
, the Asp389 and Arg393 residues within the LdNT2 nucleoside transporter
from Leishmania donovani were mutated and the resultant phenotypes
evaluated after transfection into Delta ldnt2 parasites. Whereas an
R393K mutant retained transporter activity similar to that of wild type
LdNT2, the R393L, D389E, and D389N mutations resulted in dramatic losses
of transport capability. Tagging the wild type and mutant ldnt2
proteins with green fluorescent protein demonstrated that the D389N and
D389E mutants targeted properly to the parasite cell surface and
flagellum, whereas the expression of R393L at the cell surface was
profoundly compromised. To test whether Asp389 and Arg393 interact, a
series of mutants was generated, D389R/R393R, D389D/R393D, and D389R/
R393D, within the green fluorescent protein-tagged LdNT2 construct.
Although all of these ldnt2 mutants were transport-deficient, D389R/
R393D localized properly to the plasma membrane, while neither D389R/
R393R nor D389D/R393D could be detected. Moreover, a transport-
incompetent D389N/R393N double ldnt2 mutant also localized to the
parasite membrane, whereas a D389L/R393L ldnt2 mutant did not,
suggesting that an interaction between residues 389 and 393 may be
involved in LdNT2 membrane targeting. These studies establish
genetically that Asp389 is critical for optimal transporter function and
that a positively charged or polar residue at Arg393 is essential for
proper expression of LdNT2 at the plasma membrane.
PMID: 12742588
TITLE: Improvements in transfection efficiency and tests of RNA interference
(RNAi) approaches in the protozoan parasite Leishmania.
AFFILIATION: Department of Molecular Microbiology, Washington University Medical
School, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.
REFERENCE: Mol Biochem Parasitol 2003 May 128(2):217-28
Approaches which eliminate mRNA expression directly are ideally suited
for reverse genetics applications in eukaryotic microbes which are
asexual diploids, such as the protozoan parasite Leishmania. RNA
interference (RNAi) approaches have been successful in many species,
including the related parasite Trypanosoma brucei. For RNAi tests in
Leishmania, we developed improved protocols for transient and stable DNA
transfection, attaining efficiencies of up to 25 and 3%, respectively.
This facilitated RNAi tests at the alpha-tubulin locus, whose inhibition
gives a strong lethal phenotype in trypanosomatids. However, transient
or stable transfection of DNAs encoding mRNAs for an alpha-tubulin stem-
loop construct and GFP to monitor transfection resulted in no effect on
parasite morphology, growth or tubulin expression in Leishmania major or
L. donovani. Transient transfection of a 24-nucleotide double-stranded
alpha-tubulin siRNA also had no effect. Similar results were obtained in
studies targeting an introduced GFP gene with a GFP stem-loop construct
. These data suggest that typical RNAi strategies may not work
effectively in Leishmania, and raise the possibility that Leishmania is
naturally deficient for RNAi activity, like Saccharomyces cerevisae. The
implications to parasite biology, gene amplification, and genetic
analysis are discussed.
PMID: 12744545
TITLE: Primer on medical genomics. Part VII: The evolving concept of the gene.
AFFILIATION: Department of Biochemistry and Molecular Biology, Mayo Clinic,
Rochester, Minn 55905, USA.
REFERENCE: Mayo Clin Proc 2003 May 78(5):580-7
The draft sequence of the human genome was reported 2 years ago, and the
task of filling gaps and polishing the sequence is nearing completion.
However, despite this remarkable achievement, there is still no
definitive assessment of the number of genes contained in the human
genome. In part, this uncertainty reflects our growing understanding of
the complexity and diversity of gene structure. Examples of complex gene
structure are considered in the context of a discussion about the
evolution of our understanding of gene structure and function.
PMID: 12632163
TITLE: A microplate assay for Leishmania amazonensis promastigotes expressing
multimeric green fluorescent protein.
AFFILIATION: Department of Microbiology and Immunology, Temple University School
of Medicine, 3400 North Broad Street, Room 509 OMS, Philadelphia, PA 19140, USA.
marionc at astro.temple.edu
REFERENCE: Parasitol Res 2003 Mar 89(4):266-71
Convenient and economical assays capable of screening many compounds are
vital to advance the development of drug therapy. This is particularly
important for many of the infections that occur mainly in the Third
World. The development of such a spectrofluorometric assay for the
protozoan parasite Leishmania is presented here. Using multimeric (four
monomers) green fluorescent protein (GFP), Leishmania amazonensis
promastigotes were generated with brightness measurable in 96-well
microtiter plates. The promastigotes maintained the parental
characteristics, were infective to murine macrophages and to mice, and
the level of GFP fluorescence corresponded to the number of inoculated
cells. The feasibility of using this assay for testing drugs kinetically
and in a concentration-dependent manner, under microplate culture
condition, was demonstrated with amphotericin B and the herbicide
oryzalin, respectively. This assay is the first to allow a real-time
analysis of antileishmanial agents with live promastigotes. The method
of expressing multimeric GFP for in vitro drug screening is likely to be
extendable to many species of parasitic protozoa.
PMID: 12356675
TITLE: Differential regulation of CD36 expression in antigen-presenting cells:
Oct-2 dependence in B lymphocytes but not dendritic cells or macrophages.
AFFILIATION: The Walter and Eliza Hall Institute of Medical Research, PO Royal
Melbourne Hospital, Victoria 3050, Australia. corcoran at wehi.edu.au
REFERENCE: Int Immunol 2002 Oct 14(10):1099-104
In mice, three antigen-presenting cell types [B lymphocytes, macrophages
and dendritic cells (DC)] express the scavenger receptor CD36. This
molecule has been implicated in many important functions, including DC
maturation and antigen presentation. In murine B cells, the CD36 gene
requires the Oct-2 transcription factor for its expression. We
previously found that B cells from Oct-2-null mice display defects in
maturation, survival and proliferation. Here we have looked for a
possible role for CD36 in B cells, but found that CD36 is dispensable
for all responses tested. Although loss of CD36 did not directly affect
B cell function, it did modulate slightly the isotype and level of IgG
produced in vivo in naive mice, and IgM in Leishmania-infected mice. We
also show that in DC and macrophages, CD36 expression is independent of
Oct-2. We conclude that CD36 does not play a major role in B cell
function, but that CD36 may contribute indirectly to humoral immunity
through cells of the innate immune system.
PMID: 12403168
TITLE: Bioactivity of crude extracts and some constituents of Blutaparon
portulacoides (Amaranthaceae).
AFFILIATION: Faculdade de Ciências Farmacêuticas de Ribeirão Preto -
Universidade de São Paulo, Brasil. mjsalvador at bol.com.br
REFERENCE: Phytomedicine 2002 Sep 9(6):566-71
Crude extracts (aerial parts and roots, both dried),
methylenedioxyflavonol, and a mixture of acyl steryl glycosides isolated
from Blutaparon portulacoides, were assayed for their toxicity against
Trypanosoma cruzi trypomastigotes and Leishmania amazonensis amastigotes
from axenic cultures. The antimicrobial activity was also investigated
, in a screening conducted using fifteen strains of Gram-positive and
Gram-negative bacteria, along with the yeasts, Candida albicans and
Candida tropicalis. To assess the antibacterial activity of the isolated
compounds, the minimum inhibitory concentrations (MICs) were determined
. There are no reports of acyl steryl glycosides in the genus Blutaparon
and their biological activities are being evaluated for the first time.
PMID: 11882658
TITLE: Molecular dissection of the functional domains of a unique,
tartrate-resistant, surface membrane acid phosphatase in the primitive human
pathogen Leishmania donovani.
AFFILIATION: Cell Biology Section, Laboratory of Parasitic Diseases, NIAID,
National Institutes of Health, Bethesda, Maryland 20892-0425, USA.
REFERENCE: J Biol Chem 2002 May 277(20):17994-8001
The primitive trypanosomatid pathogen of humans, Leishmania donovani,
constitutively expresses a unique externally oriented, tartrate-
resistant, acid phosphatase on its surface membrane. This is of interest
because these organisms are obligate intracellular protozoan parasites
that reside and multiply within the hydrolytic milieu of mammalian
macrophage phago-lysosomes. Here we report the identification of the
gene encoding this novel L. donovani enzyme. In addition, we
characterized its structure, demonstrated its constitutive expression in
both parasite developmental forms, and determined the cell surface
membrane localization of its translated protein product. Further, we
used a variety of green fluorescent protein chimeric constructs as
reporters in a homologous leishmanial expression system to dissect the
functional domains of this unique, tartrate-resistant, surface membrane
enzyme.
PMID: 11897125
TITLE: A new expression vector for Crithidia fasciculata and Leishmania.
AFFILIATION: Division of Biological Chemistry and Molecular Microbiology, School
of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1
5EH, Scotland, UK. etetaud at hippocrate.u-bordeaux2.fr
REFERENCE: Mol Biochem Parasitol 2002 Apr 120(2):195-204
Crithidia fasciculata is a monogenetic parasite of insects. It grows in
fully defined media without requiring serum, which facilitates
biochemical analysis. We have constructed a series of expression systems
that allows expression of transfected genes in the kinetoplastid
protozoa Crithidia and Leishmania. These cells can be readily
transfected with plasmid DNA by electroporation and transformants
selected with various antibiotic resistance markers. 5'-Trans-splicing
signals and poorly defined regions within the 3'-untranslated regions of
genes are required for optimal expression of genes in trypanosomatids.
We, therefore, inserted the intergenic region of the C. fasciculata
phosphoglycerate kinase (PGK) genes A and B, which allows
polyadenylation of the target gene and spliced leader addition to the
selectable marker gene. Part of the intergenic region of the PGK locus
was added upstream of the target gene to permit its trans-splicing. A 3
'-untranslated sequence from the Crithidia glutathionylspermidine
synthetase (GSPS) was also added to allow the polyadenylation of the
selectable marker gene. Genes can be readily inserted using a multiple
cloning site and can be expressed as a fusion protein with a poly-
histidine sequence at either the N or C-terminus or fused with green
fluorescent protein. Biologically active proteins can be expressed in C
. fasciculata or L. amazonensis promastigotes and purified by affinity
chromatography using a metal chelating column.
PMID: 11755199
TITLE: RNA polymerase I-mediated transcription of a reporter gene integrated
into different loci of Leishmania.
AFFILIATION: Faculté de Médecine, Centre de Recherche en Infectiologie, Centre
Hospitalier de l'Université Laval, Université Laval, Pavillon CHUL, 2705 boul.
Laurier, Ste-Foy, G1V 4G2, Québec, Canada.
REFERENCE: Mol Biochem Parasitol 2002 Jan 119(1):153-8
PMID: 11738822
TITLE: New Mos1 mariner transposons suitable for the recovery of gene fusions in
vivo and in vitro.
AFFILIATION: Department of Molecular Microbiology, Washington University Medical
School, Box 8230, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
REFERENCE: Gene 2001 Dec 280(1-2):97-105
The Drosophila Mos1 element can be mobilized in species ranging from
prokaryotes to protozoans and vertebrates, and the purified transposase
can be used for in vitro transposition assays. In this report we
developed a 'mini-Mos1' element and describe a number of useful
derivatives suitable for transposon mutagenesis in vivo or in vitro.
Several of these allow the creation and/or selection of tripartite
protein fusions to a green fluorescent protein-phleomycin resistance (
GFP-PHLEO) reporter/selectable marker. Such X-GFP-PHLEO-X fusions have
the advantage of retaining 5' and 3' regulatory information and N- and C
-terminal protein targeting domains. A Mos1 derivative suitable for use
in transposon-insertion mediated linker insertion (TIMLI) mutagenesis is
described, and transposons bearing selectable markers suitable for use
in the protozoan parasite Leishmania were made and tested. A novel '
negative selection' approach was developed which permits in vitro assays
of transposons lacking bacterial selectable markers. Application of
this assay to several Mos1 elements developed for use in insects
suggests that the large mariner pM[cn] element used previously in vivo
is poorly active in vitro, while the Mos1-Act-EGFP transposon is highly
active.
PMID: 11598129
TITLE: Developmental regulation of heat shock protein 83 in Leishmania. 3'
processing and mRNA stability control transcript abundance, and translation id
directed by a determinant in the 3'-untranslated region.
AFFILIATION: Department of Life Sciences, Ben Gurion University of the Negev,
Beer-Sheva 84105, Israel.
REFERENCE: J Biol Chem 2001 Dec 276(51):47922-9
Developmental gene regulation in trypanosomatids proceeds exclusively by
post-transcriptional mechanisms. Stability and abundance of heat shock
protein (HSP)70 and HSP83 transcripts in Leishmania increase at
mammalian-like temperatures, and their translation is enhanced. Here we
report that the 3'-untranslated region (UTR) of HSP83 (886 nucleotides)
confers the temperature-dependent pattern of regulation on a
chloramphenicol acetyltransferase (CAT) reporter transcript. We also
show that the majority of the 3'-UTR sequences are required for
increasing mRNA stability during heat shock. Processing of the HSP70 and
HSP83 primary transcripts to poly(A)(+) mRNA was more efficient during
heat shock; therefore, even when stability at 33 degrees C was reduced
by deletions in the 3'-UTR, transcripts still accumulated to comparable
and even higher levels. Translation of heat shock transcripts in
Leishmania increases dramatically upon temperature elevation. Unlike in
other eukaryotes in which the 5'-UTR confers preferential translation on
heat shock transcripts, we show that translational control of HSP83 in
Leishmania originates from its 3'-UTR. The 5'-UTR alone cannot induce
translation during heat shock, but it has a minor contribution when
combined with the HSP83 3'-UTR. We identified an element located between
positions 201 and 472 of the 3'-UTR which is essential for increasing
translation of the CAT-HSP83 reporter RNA at 33-37 degrees C. This
region confers preferential translation during heat shock even in
transcripts that were less stable. Thus, investigating the traditionally
conserved heat shock response reveals that Leishmania parasites use
unique pathways for translational control.
PMID: 11709363
TITLE: Expression of green fluorescent protein as a marker for effects of
antileishmanial compounds in vitro.
AFFILIATION: Institute of Parasitology, University of Zürich, CH-8057 Zürich,
Switzerland.
REFERENCE: Antimicrob Agents Chemother 2001 Dec 45(12):3654-6
Transgenic Leishmania infantum promastigotes, which constitutively
express green fluorescent protein (GFP) in their cytoplasm, were used to
monitor the effects of antileishmanial compounds in real time. The GFP-
based assay provided a reliable measure of drug-induced inhibitory
effects on protein expression, resulting in a dynamic picture of the
responses of leishmanial promastigotes to the compounds tested.
PMID: 11731148
TITLE: The effect of co-overproduction of DnaK/DnaJ/GrpE and ClpB proteins on
the removal of heat-aggregated proteins from Escherichia coli DeltaclpB mutant
cells--new insight into the role of Hsp70 in a functional cooperation with
Hsp100.
AFFILIATION: Department of Biochemistry, University of Gdansk, Kladki 24,
80-822, Gdansk, Poland. kedzie at biotech.univ.gda.pl
REFERENCE: FEMS Microbiol Lett 2001 Nov 204(2):355-60
The effect of overproduction of the Hsp70 system proteins (DnaK, DnaJ,
GrpE) and/or ClpB (Hsp100) from plasmids on the process of formation and
removal of heat-aggregated proteins from Escherichia coli cells (the S
fraction) was investigated by sucrose density gradient centrifugation.
Two plasmids were employed: pKJE7 carrying the dnaK/dnaJ/grpE genes
under the control of the araB promoter and pClpB carrying the clpB gene
under the control of its own promoter (sigma(32)-dependent). In the wild
-type cells the S fraction after 15 min of heat shock amounted to 21% of
cellular insoluble proteins (IP), and disappeared 10 min after transfer
of the culture to 37 degrees C. In contrast to this, in the clpB mutant
the S fraction was larger (35% IP) and its elimination was retarded,
nearly 60% of the aggregated proteins remained stable 30 min after heat
shock. This result points to the importance of ClpB in removal of the
heat-aggregated proteins from cells. Overproduction of the Hsp70 system
proteins (exceeding by about 1.5-fold that of wild-type) in wild-type
and DeltaclpB cells completely prevented the formation of the S fraction
during heat shock. Overproduction of ClpB (exceeding by about eight-
fold that of wild-type) in the same background did not prevent protein
aggregation after heat shock and only partly compensated for the effect
of the mutation in the clpB gene. Monitoring the S fraction during co-
production of DnaK/DnaJ/GrpE and ClpB in the DeltaclpB mutant revealed
that both the levels of expression and the ratios of ClpB to Hsp70
system proteins had a significant effect on the formation and removal of
protein aggregates in heat-shocked E. coli cells. In the presence of
excess ClpB, an increase in the levels of DnaK, DnaJ and GrpE was
required to prevent aggregate formation upon heat shock or to
efficiently remove protein aggregates after heat shock. Therefore, it is
supposed that a high level of ClpB under some conditions, especially at
insufficient levels of Hsp70 system proteins, may support protein
aggregation resulting from heat shock and may lead to stabilization of
hydrophobic aggregates.
PMID: 11770103
TITLE: Post transcriptional control of gene expression in Leishmania.
AFFILIATION: Department of Life Sciences, Ben Gurion University of the Negev,
Beer-Sheva, Israel. shapiram at bgumail.bgu.ac.il
REFERENCE: Med Microbiol Immunol (Berl) 2001 Nov 190(1-2):23-6
Leishmania parasites are ancient eukaryotes, characterized by unusual
molecular mechanisms. We have used the gene encoding for Hsp83 as a
model system for studying regulatory mechanisms that control
developmental gene regulation. We previously showed that protein coding
genes are regulated exclusively by post-transcriptional mechanisms,
while no transcriptional activation could be observed even for the
conserved Hsp83 gene. We now show that processing and maturation of the
Hsp83 polycistronic primary transcripts is more efficient at elevated
temperatures. The mature transcripts are more stable during heat shock,
with regulation conferred by 3' UTRs. Poly(A) tails of Hsp83 are
approximately 30 nucleotides long, as common for other low eukaryotes.
The mechanism that signals differential degradation is still unclear,
since it was not possible to detect differences in deadenylation of
Hsp83 transcripts at varying temperatures. Heat shock transcripts are
preferentially translated at 33-37 degrees C, but unlike Drosophila,
translational regulation is controlled by a region within the 3' UTR.
Using this traditionally conserved system emphasizes that regulatory
mechanisms in Leishmania differ from those prevailing in other
eukaryotes.
PMID: 11481434
TITLE: Trypanosoma cruzi trans-sialidase: a potent and specific survival factor
for human Schwann cells by means of phosphatidylinositol 3-kinase/Akt
signaling.
AFFILIATION: Parasitology Research Center, Department of Pathology, Tufts
University School of Medicine, Boston, MA 02111, USA.
REFERENCE: Proc Natl Acad Sci U S A 2001 Aug 98(17):9936-41
Patients infected with Trypanosoma cruzi may remain asymptomatic for
decades and show signs of neuroregeneration in the peripheral nervous
system (PNS). In the absence of such neuroregeneration, patients may die
in part by extensive neuronal destruction in the gastrointestinal tract
. Thus, T. cruzi may, despite their invasion of the PNS, directly
prevent cell death to keep nerve destruction in check. Indeed, T. cruzi
invasion of Schwann cells, their prime target in PNS, suppressed host-
cell apoptosis caused by growth-factor deprivation. The trans-sialidase
(TS) of T. cruzi and the Cys-rich domain of TS reproduced the
antiapoptotic activity of the parasites at doses (> or =3.0 nM)
comparable or lower than those of bona fide mammalian growth factors.
This effect was blocked by LY294002, an inhibitor of
phosphatidylinositol 3-kinase (PI3K). TS also activated Akt, a
downstream effector of PI3K. Ectopic expression of TS in an unrelated
parasite, Leishmania major, turned those parasites into activators of
Akt in Schwann cells. In contrast, the Cys-rich domain of TS did not
block apoptosis in Schwann cells overexpressing dominant-negative Akt or
constitutively active PTEN, a negative regulator of PI3K/Akt signaling
. The results demonstrate that T. cruzi, through its TS, triggers the
survival of host Schwann cells via the PI3K/Akt pathway, suggesting a
role for PI3K/Akt in the pathogenesis of Chagas' disease.
PMID: 11420107
TITLE: Identification and characterisation of a RAD51 gene from Leishmania
major.
AFFILIATION: Department of Biochemistry, Imperial College of Science, Technology
and Medicine, South Kensington, SW7 2AZ, London, UK. paul.g.mckean at man.ac.uk
REFERENCE: Mol Biochem Parasitol 2001 Jul 115(2):209-16
The RAD51 gene is a homologue of Escherichia coli recA which plays a
central role in homologous recombination and DNA repair. This paper
describes the identification of the RAD51 gene from the trypanosomatid
parasite Leishmania major. The LmRAD51 gene codes for a 377 amino acid
polypeptide with a predicted molecular mass of 41259 Da that is highly
homologous to the Rad51 family of proteins. Recombinant L. major Rad51
protein (LmRad51) was over-expressed in a bacterial expression system,
purified to homogeneity and shown to bind DNA and exhibit DNA-stimulated
ATPase activity, consistent with previously reported biochemical
characteristics of Rad51 protein. Although LmRad51 expression is below
the level of detection in exponentially growing cultures of Leishmania,
high levels of LmRad51 mRNA and protein expression can be detected
following exposure to the DNA-damaging agent phleomycin. LmRAD51 is one
of the first examples of a DNA damage-inducible gene to be characterised
in Leishmania, and will be invaluable in studying the contribution of
homologous recombination to Leishmania virulence.
PMID: 11353834
TITLE: Point mutations in a nucleoside transporter gene from Leishmania donovani
confer drug resistance and alter substrate selectivity.
AFFILIATION: Department of Molecular Microbiology, Oregon Health Sciences
University, Portland, OR 97201, USA.
REFERENCE: Proc Natl Acad Sci U S A 2001 May 98(11):6092-7
Leishmania parasites lack a purine biosynthetic pathway and depend on
surface nucleoside and nucleobase transporters to provide them with host
purines. Leishmania donovani possess two closely related genes that
encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.
1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin
in addition to the natural substrates. In this study, we have
characterized a drug-resistant clonal mutant of L. donovani (TUBA5) that
is deficient in LdNT1 transport and consequently resistant to
tubercidin. In TUBA5 cells, the LdNT1.2 genes had the same sequence as
wild-type cells. However, because LdNT1.2 mRNA is not detectable in
either wild-type or TUBA5 promastigotes, LdNT1.2 does not contribute to
nucleoside transport in this stage of the life cycle. In contrast, the
TUBA5 cells were compound heterozygotes at the LdNT1.1 locus containing
two mutant alleles that encompassed distinct point mutations, each of
which impaired transport function. One of the mutant LdNT1.1 alleles
encoded a G183D substitution in predicted TM 5, and the other allele
contained a C337Y change in predicted TM 7. Whereas G183D and C337Y
mutants had only slightly elevated adenosine K(m) values, the severe
impairment in transport resulted from drastically ( approximately 20-
fold) reduced V(max) values. Because these transporters were correctly
targeted to the plasma membrane, the reduction in V(max) apparently
resulted from a defect in translocation. Strikingly, G183 was essential
for pyrimidine nucleoside but not adenosine transport. A mutant
transporter with a G183A substitution had an altered substrate
specificity, exhibiting robust adenosine transport but undetectable
uridine uptake. These results suggest that TM 5 is likely to form part
of the nucleoside translocation pathway in LdNT1.1
PMID: 11274130
TITLE: Characterization of Brucella suis clpB and clpAB mutants and
participation of the genes in stress responses.
AFFILIATION: Institut National de la Santé et de la Recherche Médicale U-431,
Université Montpellier II, F-34095 Montpellier, France.
REFERENCE: J Bacteriol 2001 Apr 183(8):2677-81
Pathogens often encounter stressful conditions inside their hosts. In
the attempt to characterize the stress response in Brucella suis, a gene
highly homologous to Escherichia coli clpB was isolated from Brucella
suis, and the deduced amino acid sequence showed features typical of the
ClpB ATPase family of stress response proteins. Under high-temperature
stress conditions, ClpB of B. suis was induced, and an isogenic B. suis
clpB mutant showed increased sensitivity to high temperature, but also
to ethanol stress and acid pH. The effects were reversible by
complementation. Simultaneous inactivation of clpA and clpB resulted in
a mutant that was sensitive to oxidative stress. In B. suis expressing
gfp, ClpA but not ClpB participated in degradation of the green
fluorescent protein at 42 degrees C. We concluded that ClpB was
responsible for tolerance to several stresses and that the lethality
caused by harsh environmental conditions may have similar molecular
origins.
PMID: 11274727
TITLE: Analysis of the adjuvant effect of recombinant Leishmania infantum Hsp83
protein as a tool for vaccination.
AFFILIATION: Instituto Nacional de ParasitologÃa Dr. Mario F. Chaben/ANLIS Dr.
Carlos G. Malbran, Departamento de ParasitologÃa Sanitaria, Av. Velez
Sarsfield 563, 1281, Buenos Aires, Argentina.
REFERENCE: Immunol Lett 2001 Mar 76(2):107-10
The properties of Leishmania infantum hsp83 (LiHsp83) to elicit an
immune response against a fused reporter antigen, maltose binding
protein (MBP), was studied. CF1 mice were immunized with different
purified recombinant proteins: MBP, LiHsp83 and MBP fused to LiHsp83 (
MBP-LiHsp83). Serum samples were obtained at days 0, 21, 28, 60, 90, 120
and 150 post-immunization. MBP-LiHsp83 fusion protein elicited a strong
humoral response against MBP, higher than that one obtained in mice
immunized with MBP alone or MBP mixed with LiHsp83, showing the
secretion of both anti-MBP IgG2a and IgG1 isotypes (IgG2a/IgG1 ratio: 2:
1). This response was specific for recombinant proteins and was
maintained for at least 150 days, whereas the reactivity in mice
immunized with MBP alone dissapeared at day 90. After in vitro
stimulation with MBP, spleen cells from MBP-LiHsp83 immunized mice
showed higher proliferation indices and produced higher secretion of IFN
-gamma than spleen cells from either control or MBP-immunized mice. In
all groups of mice IL-4 was undetectable. Thus we consider that LiHsp83
may be a promising candidate to be used as carrier of fused antigens for
adjuvant-free vaccination.
PMID: 11224567
TITLE: Molecular determinants of complex formation between Clp/Hsp100 ATPases
and the ClpP peptidase.
AFFILIATION: Department of Biology, Massachusetts Institute of Technology, 77
Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
REFERENCE: Nat Struct Biol 2001 Mar 8(3):230-3
The Clp/Hsp100 ATPases are hexameric protein machines that catalyze the
unfolding, disassembly and disaggregation of specific protein substrates
in bacteria, plants and animals. Many family members also interact with
peptidases to form ATP-dependent proteases. In Escherichia coli, for
instance, the ClpXP protease is assembled from the ClpX ATPase and the
ClpP peptidase. Here, we have used multiple sequence alignments to
identify a tripeptide 'IGF' in E. coli ClpX that is essential for ClpP
recognition. Mutations in this IGF sequence, which appears to be part of
a surface loop, disrupt ClpXP complex formation and prevent protease
function but have no effect on other ClpX activities. Homologous
tripeptides are found only in a subset of Clp/Hsp100 ATPases and are a
good predictor of family members that have a ClpP partner. Mapping of
the IGF loop onto a homolog of known structure suggests a model for ClpX
-ClpP docking.
PMID: 11260523
TITLE: Secretory and endocytic pathways converge in a dynamic endosomal system
in a primitive protozoan.
AFFILIATION: Cell Biology Section, Laboratory of Parasitic Diseases, Division of
Intramural Research, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, MD 20892-0425, USA.
REFERENCE: Traffic 2001 Mar 2(3):175-88
Leishmania are a group of primitive eukaryotic trypanosomatid protozoa
that are apically polarized with a flagellum at their anterior end.
Surrounding the base of the flagellum is the flagellar reservoir that
constitutes the site for endocytosis and exocytosis in these organisms.
In the present study, we define a novel multivesicular tubular
compartment involved in the intracellular trafficking of macromolecules
in Leishmania. This dynamic structure appears to subtend the flagellar
reservoir and extends towards the posterior end of the cell. Functional
domains of several surface-expressed proteins, such as the gp63 glycosyl
phosphatidyl inositol anchor and the 3'nucleotidase/nuclease
transmembrane domain were fused to green fluorescent protein. These
chimeric proteins were found to traffic through the secretory pathway
and, while reaching their intended destinations, also accumulated within
the intracellular tubular compartment. Using various compounds that are
efficient fluid-phase markers used to track endocytosis in higher
eukaryotes, we showed that this tubular compartment constitutes an
important station in the endocytic pathway of these cells. Based on our
functional observations of its role in the trafficking of expressed
proteins and endocytosed markers, this compartment appears to have
properties similar to endosomes of higher eukaryotes.
PMID: 11268689
TITLE: Green fluorescent protein-tagged Leishmania in phlebotomine sand flies.
AFFILIATION: Instituto de BiologÃa Experimental, Facultad de Ciencias,
Universidad Central de Venezuela, Caracas 1041-A, Venezuela.
REFERENCE: J Med Entomol 2001 Jan 38(1):39-43
In this work we have used for the first time green fluorescent protein (
GFP) tagged cells of the human parasite Leishmania donovani to observe
its development in the gut of phlebotomine sand flies. Low numbers of
GFP-tagged L. donovani were more easily detected than nontagged
Leishmania, suggesting that GFP-tagged Leishmania could be used to
efficiently study the biology of Leishmania in their vectors, and open
the possibility of using nonaxenic flies. Using this method, we found
that GFP-tagged L. donovani, the ethiological agent of Old World Kala-
azar, were able to establish an infection within the gut of Lutzomyia
species, which are vectors of New World Leishmania. The GFP-tagged
parasites divide successfully in the gut of colonized and in wild caught
Lu. longipalpis (Lutz & Neiva, 1912), Lu. ovallesis (Ortiz, 1952),
and Lu. youngi (Feliciangeli & Murillo, 1985). In the case of
Lulongipalpis the labeled parasite exhibited a normal anterior
development as the one observed in its natural vector.
PMID: 11121557
TITLE: A stable marker for specific T-cells: a TCR alpha/green fluorescent
protein (GFP) fusionprotein reconstitutes a functionally active TCR complex.
AFFILIATION: Clinical Microbiology, Immunology and Hygiene, University of
Erlangen-Nürnberg, Wasserturmstrasse 3, 91054, Erlangen, Germany.
REFERENCE: J Immunol Methods 2000 Dec 246(1-2):165-74
The detection of antigen specific clonal T-cell populations in vivo
during T-cell selection and an immune responses is often hampered due to
the lack of suitable clonotype specific monoclonal antibodies. In order
to determine the potential usefulness of green fluorescent protein (GFP
) to follow specific T-cells in vivo, we decided to express and analyze
the function of a T-cell receptor (TCR) alpha chain-GFP fusionprotein.
The TCRalpha and beta chain cDNAs of a Leishmania major-specific murine
T helper 2 cell clone were cloned and inserted into the pHSE3'
expression vector. Simultaneously, a TCRalpha expression vector was
constructed containing a C-terminal in frame fusion with the open
reading frame of the enhanced GFP (EGFP). TCRalpha/TCRbeta or TCRalpha-
EGFP/TCRbeta constructs were expressed in T-cell hybridoma cells 58alpha
(-)beta(-) which lack an endogenous TCR but still express CD3 components
. The TCRalpha-EGFP fusionprotein was detected with the expected
molecular weight by immunoprecipitation and Western Blot analysis.
Surface staining of TCR components was detected in transfectants
expressing the wild type TCR heterodimer and, with only a slight
reduction in intensity, also in those expressing the TCR-EGFP complex.
Hence, expression and transport to the outer cell membrane is possible
despite the 27 kD C-terminal extension of the TCRalpha. Most importantly
, the EGFP-tagged TCR was functional since the transfectants produced IL
-2 in response to stimulation via their TCR. Thus, TCR-EGFP constructs
represent attractive tools to study posttranslational regulation of TCR
expression and ligand-induced TCR clustering as well as the fate of
antigen specific T-cells during tolerance induction and immunity in
transgenic mouse models.
PMID: 11087917
TITLE: Double-stranded RNA interference in Trypanosoma brucei using head-to-head
promoters.
AFFILIATION: Department of Biochemistry, College of Medicine, 4-403 Bowen
Science Building, 51 Newton Road, University of Iowa, Iowa City, IA 52242,
USA.
REFERENCE: Mol Biochem Parasitol 2000 Nov 111(1):67-76
The discovery of double-stranded RNA interference (dsRNAi) in
Trypanosoma brucei provides a convenient method to generate knockout
phenotypes in this protozoan parasite [Ngo H, Tschudi C, Gull K, Ullu E
. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei.
Proc Natl Acad Sci USA 1998;95:14687-14692]. The presence of double-
stranded RNA (dsRNA) dominantly silences gene expression in a sequence-
specific manner by causing the corresponding endogenous RNA to be
degraded. To simplify the generation of knockout phenotypes in T. brucei
via dsRNAi, we used two promoters arranged as an inverted repeat on a
plasmid. This promoter arrangement generates transcripts of both strands
of DNA inserted between the promoters, which then form dsRNA. We have
used plasmids encoding either two T. brucei ribosomal RNA promoters or
two bacteriophage T7 promoters to interfere with expression of alpha-
tubulin (TUB), green fluorescent protein (GFP), paraflagellar rod
protein A (PFRA), flagellum-adhesion glycoprotein 1 (FLA1), and histone
2B (H2B) in T. brucei. We show here that FLA1 is required for flagellar
attachment in T. brucei and that H2B is required for parasite growth.
Thus, the two-promoter approach efficiently generates dsRNAi in T.
brucei and can be used to produce both specific and random knockout
phenotypes in T. brucei. This approach should be useful in generating
knockout phenotypes in other kinetoplastid parasites including
Trypanosoma cruzi and Leishmania.
PMID: 10942757
TITLE: Isolation and characterization of gomesin, an 18-residue cysteine-rich
defense peptide from the spider Acanthoscurria gomesiana hemocytes with
sequence similarities to horseshoe crab antimicrobial peptides of the
tachyplesin family.
AFFILIATION: Laboratório de Artrópodes, Instituto Butantan, Avenue Vital
Brazil, 1500, CEP 05503-900, São Paulo, Brazil.
REFERENCE: J Biol Chem 2000 Oct 275(43):33464-70
We have purified a small size antimicrobial peptide, named gomesin, from
the hemocytes of the unchallenged tarantula spider Acanthoscurria
gomesiana. Gomesin has a molecular mass of 2270.4 Da, with 18 amino
acids, including a pyroglutamic acid as the N terminus, a C-terminal
arginine alpha-amide, and four cysteine residues forming two disulfide
bridges. This peptide shows marked sequence similarities to
antimicrobial peptides from other arthropods such as tachyplesin and
polyphemusin from horseshoe crabs and androctonin from scorpions.
Interestingly, it also shows sequence similarities to protegrins,
antimicrobial peptides from porcine leukocytes. Gomesin strongly affects
bacterial growth, as well as the development of filamentous fungi and
yeast. In addition, we showed that gomesin affects the viability of the
parasite Leishmania amazonensis.
PMID: 10940917
TITLE: Regulation of type 2 nitric oxide synthase by type 1 interferons in
macrophages infected with Leishmania major.
AFFILIATION: Institute of Clinical Microbiology, Immunology, and Hygiene,
University of Erlangen, Erlangen, Germany.
REFERENCE: Eur J Immunol 2000 Aug 30(8):2257-67
We recently reported that the infection of macrophages with Leishmania
major led to the release of type 1 interferons (IFN-alpha /beta ).
Moreover, at day 1 of infection of mice with L. major, IFN-alpha /beta
was required for the expression of type 2 (inducible) NO synthase (NOS2
or iNOS) which, however, was restricted to a few macrophages in the
dermis. Here, we further characterized the regulation of NOS2 by IFN-
alpha /beta. Macrophages that were either simultaneously or sequentially
exposed to L. major promastigotes and IFN-alpha /beta expressed NOS2
and anti-leishmanial activity. In contrast, when high amounts of IFN-
alpha /beta were used or when IFN-alpha /beta was added to the
macrophages 2 h prior to the parasites, almost no induction of NOS2 was
observed. After pretreatment with IFN-alpha /beta, tyrosine
phosphorylation and nuclear DNA binding of Stat1alpha, the degradation
of the NF-kappaB inhibitor (IkappaBalpha and beta), and the nuclear
translocation of NF-kappaB were strongly impaired compared with
macrophages exposed to IFN-alpha /beta and L. major simultaneously. Thus
, IFN-alpha /beta exerts agonistic or antagonistic effects on the
expression of NOS2 in macrophages infected with a microbial pathogen,
depending on the sequence of the stimuli and the amount of IFN-alpha /
beta added. The limited number of NOS2-positive macrophages at day 1 of
infection in vivo might result from a blockage of non-infected
macrophages by IFN-alpha /beta that is released by neighboring infected
cells.
PMID: 10924759
TITLE: Expression of the AM gene locus in infective stages of Leishmania.
AFFILIATION: Wellcome Trust Laboratories for Molecular Parasitology, Department
of Biochemistry, Imperial College of Science, Technology and Medicine, London,
UK.
REFERENCE: Mol Biochem Parasitol 2000 Jun 109(1):73-9
PMID: 10748102
TITLE: Dissection of the functional domains of the Leishmania surface membrane
3'-nucleotidase/nuclease, a unique member of the class I nuclease family.
AFFILIATION: Cell Biology Section, Laboratory of Parasitic Diseases, Division of
Intramural Research, NIAID, National Institutes of Health, Bethesda, Maryland
20892-0425, USA.
REFERENCE: J Biol Chem 2000 May 275(21):16366-72
Class I nucleases are a family of enzymes that specifically hydrolyze
single-stranded nucleic acids. Recently, we characterized the gene
encoding a new member of this family, the 3'-nucleotidase/nuclease (Ld3'
NT/NU) of the parasitic protozoan Leishmania donovani. The Ld3'NT/NU is
unique as it is the only class I nuclease that is a cell surface
membrane-anchored protein. Currently, we used a homologous episomal
expression system to dissect the functional domains of the Ld3'NT/NU.
Our results showed that its N-terminal signal peptide targeted this
protein into the endoplasmic reticulum. Using Ld3'NT/NU-green
fluorescent protein chimeras, we showed that the C-terminal domain of
the Ld3'NT/NU functioned to anchor this protein into the parasite cell
surface membrane. Further, removal of the Ld3'NT/NU C-terminal domain
resulted in its release/secretion as a fully active enzyme. Moreover,
deletion of its single N-linked glycosylation site showed that such
glycosylation was not required for the enzymatic functions of the Ld3'NT
/NU. Thus, using the fidelity of a homologous expression system, we have
defined some of the functional domains of this unique member of the
class I nuclease family.
PMID: 10720539
TITLE: Inhibition of interferon-gamma signaling by Leishmania donovani.
AFFILIATION: Laboratory of Parasitic Biology and Biochemistry, Center for
Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD
20852-1448, USA.
REFERENCE: J Infect Dis 2000 Mar 181(3):1121-8
Leishmania infection causes marked down-regulation of interferon (IFN)-
gamma-induced gene activity in macrophages, but the mechanism of the
blockade has not been fully defined. The IFN-gamma signal transduction
pathway was analyzed in Leishmania donovani-infected phorbol-
differentiated U937 human promonocytic cells. IFN-gamma stimulation
induced marked phosphorylation of its own receptor (IFN-gammaR)-alpha
chain. Phosphorylation of the receptor subunit was significantly
inhibited after 24 h of infection with the parasite, apparently because
of decreased amounts of the receptor subunit. Formation of the IFN-
gammaR complex, as assessed by tyrosine phosphorylation and association
of Jak2, was strongly inhibited in cells infected for 24 h. Inhibition
of the IFN-gammaR complex formation correlated with inhibition of
STAT1alpha binding to the IFN-gamma response region. Pretreatment with
purified parasite lipophosphoglycan before IFN-gamma stimulation had no
effect on tyrosine phosphorylation. Thus, inhibition of tyrosine
phosphorylation of the IFN-gammaR-alpha chain and subsequent signal
transduction are most likely due to the decreased amount of IFN-gammaR-
alpha protein after infection.
PMID: 10490990
TITLE: Mycobacterium tuberculosis inhibits IFN-gamma transcriptional responses
without inhibiting activation of STAT1.
AFFILIATION: Division of Infectious Diseases, University of California, San
Francisco 94143, USA.
REFERENCE: J Immunol 1999 Oct 163(7):3898-906
IFN-gamma activates macrophages to kill diverse intracellular pathogens
, but does not activate human macrophages to kill virulent Mycobacterium
tuberculosis. We tested the hypothesis that this is due to inhibition
of IFN-gamma signaling by M. tuberculosis and found that M. tuberculosis
infection of human macrophages blocks several responses to IFN-gamma,
including killing of Toxoplasma gondii and induction of FcgammaRI. The
inhibitory effect of M. tuberculosis is directed at transcription of IFN
-gamma-responsive genes, but does not affect proximal steps in the Janus
kinase-STAT pathway, as STAT1alpha tyrosine and serine phosphorylation
, dimerization, nuclear translocation, and DNA binding are intact in M.
tuberculosis-infected cells. In contrast, there is a marked decrease in
IFN-gamma-induced association of STAT1 with the transcriptional
coactivators CREB binding protein and p300 in M. tuberculosis-infected
macrophages, indicating that M. tuberculosis directly or indirectly
disrupts this protein-protein interaction that is essential for
transcriptional responses to IFN-gamma. Gamma-irradiated M. tuberculosis
and isolated cell walls reproduce the effects of live bacteria,
indicating that the bacterial component(s) that initiates inhibition of
IFN-gamma responses is constitutively expressed. Although
lipoarabinomannan has been found to exert effects on macrophages, it
does not account for the inhibitory effects of cell walls. These results
indicate that one mechanism for M. tuberculosis to evade the human
immune response is to inhibit the IFN-gamma signaling pathway, and that
the mechanism of inhibition is distinct from that reported for
Leishmania donovani or CMV, in that it targets the interaction of STAT1
with the basal transcriptional apparatus.
PMID: 10347181
TITLE: Protease trafficking in two primitive eukaryotes is mediated by a
prodomain protein motif.
AFFILIATION: Department of Pathology, University of California, San Francisco,
California 94121, USA.
REFERENCE: J Biol Chem 1999 Jun 274(23):16249-56
Trypanosome protozoa, an early lineage of eukaryotic cells, have
proteases homologous to mammalian lysosomal cathepsins, but the
precursor proteins lack mannose 6-phosphate. Utilizing green fluorescent
protein as a reporter, we demonstrate that the carbohydrate-free
prodomain of a trypanosome cathepsin L is necessary and sufficient for
directing green fluorescent protein to the lysosome/endosome compartment
. A proper prodomain/catalytic domain processing site sequence is also
required to free the mature protease for delivery to the lysosome/
endosome compartment. A nine-amino acid prodomain loop motif, implicated
in prodomain-receptor interactions in mammalian cells, is conserved in
the protozoa. Site-directed mutagenesis now confirms the importance of
this loop to protease trafficking and suggests that a protein motif
targeting signal for lysosomal proteases arose early in eukaryotic cell
evolution.
PMID: 10376993
TITLE: Inhibition of succinyl CoA synthetase histidine-phosphorylation in
Trypanosoma brucei by an inhibitor of bacterial two-component systems.
AFFILIATION: Institut für Allgemeine Mikrobiologie, University of Bern,
Switzerland.
REFERENCE: Mol Biochem Parasitol 1999 May 100(1):53-9
Recent drug screenings for new antibacterial drugs directed against
histidine phospho-relay signalling pathways in bacteria have resulted in
compounds which potently inhibit the histidine kinase activity of
bacterial two-component systems. The present study demonstrates that one
of these compounds, LY266500, is also a potent inhibitor of histidine
phosphorylation in the unicellular eukaryotic parasite Trypanosoma
brucei, both in vitro and in whole cells. In vitro, it inhibits
histidine phosphorylation of mitochondrial succinyl CoA synthetase.
LY26650 does not interfere with the phosphotransfer from the histidine-
phosphorylated protein to ADP. In standardized cell culture tests,
LY266500 potently inhibits the proliferation of the human pathogens T.
brucei rhodesiense and Leishmania donovani. Since the inhibitory
activity in vivo is life-cycle stage specific and correlates well with
the mitochondrial activity in the different stages, the effect of
LY266500 is most likely due to its specific inhibition of the
mitochondrial succinyl CoA synthetase.
PMID: 9804987
TITLE: Characterization of a GTP-binding protein in the ADP-ribosylation factor
subfamily from Leishmania tarentolae.
AFFILIATION: Department of Microbiology and Immunology, UCLA School of Medicine,
10833 Le Conte Avenue, Los Angeles, CA 90095-1747, USA.
REFERENCE: Biochim Biophys Acta 1998 Nov 1442(2-3):347-52
We report the cloning and characterization of a gene, LtARL, which
encodes a small GTP-binding protein, from the protozoan Leishmania
tarentolae. Hybridization analysis of genomic DNA under high stringency
conditions indicates the single-copy nature of LtARL. LtARL is
transcribed and yields a approximately 0.9 kb mRNA that is processed at
the 5' end by trans-splicing. When expressed in Escherichia coli, LtArl
binds GTP with a low stoichiometry and in a phospholipid-independent
manner. Based on the greatest sequence identity with Homo sapiens Arl3
and lipid-independent binding of guanine nucleotides we designate this
gene LtARL and the encoded protein LtArl.
PMID: 9430230
TITLE: Down-regulation of IL-4 gene transcription and control of Th2 cell
differentiation by a mechanism involving NFAT1.
AFFILIATION: The Center For Blood Research and Department of Pathology, Harvard
Medical School, Boston, Massachusetts 02115, USA.
REFERENCE: Immunity 1997 Dec 7(6):849-60
Transcription factors of the NFAT family play a critical role in the
immune response by activating the expression of cytokines and other
inducible genes in antigen-stimulated cells. Here we show that a member
of this family, NFAT1, is involved in down-regulating the late phase of
IL-4 gene transcription, thus inhibiting T helper 2 responses. Whereas
stimulated T cells from wild-type mice show a transient increase and
then a rapid decline in the steady-state levels of IL-4 mRNA in vitro,
the levels of IL-4 gene transcripts in NFAT1-deficient T cells are
maintained at high levels under the same conditions. Consistent with
this observation, NFAT1-/- mice are more susceptible to infection with
Leishmania major. This report provides evidence that NFAT proteins
regulate not only the initiation but also the termination of gene
transcription.
PMID: 9476797
TITLE: Identification of a transcription factor like protein at the TOR locus in
Leishmania mexicana amazonensis.
AFFILIATION: University of North Dakota School of Medicine, Department of
Biochemistry and Molecular Biology, Grand Forks 58202, USA.
sdetke at mail.med.und.nodak.edu
REFERENCE: Mol Biochem Parasitol 1997 Dec 90(2):505-11
The TOR gene (TOxic nucleoside Resistance gene) was mapped to a 2.3 kb
fragment on the amplified DNA from tubercidin resistant Leishmania (TUB
). This DNA fragment conferred upon wild type cells resistance to
tubercidin, inosine dialdehyde, formycin A and B and allopurinol
riboside and a reduced ability to accumulate purine nucleobases and
nucleosides. These properties were characteristic of the parental TUB
cells which carried the intact amplified DNA and have been hypothesized
to be caused by a reduction in the activity of the multiple purine
transporters within this organism. The TOR gene was found to be
partially homologous to the rodent and human Oct-6/SCIP/Tst-1 gene. It
lacked, however, the POU specific domain of this class of transcription
factors and contained only the first two helices of the POU homeodomain
. This truncated homeodomain was not required to confer resistance upon
wild type cells to toxic nucleosides, suggesting that TOR was not a
repressor with independent DNA binding capability.
PMID: 9297701
TITLE: The dihydroxyacetonephosphate pathway for biosynthesis of ether lipids in
Leishmania mexicana promastigotes.
AFFILIATION: Research Unit for Tropical Diseases, Catholic University of
Louvain, Brussels, Belgium.
REFERENCE: Mol Biochem Parasitol 1997 Oct 89(1):61-72
Biosynthetic studies using both [14C]- and [32P]-labelled substrates and
a cell-free system to synthesise 1-O-alkyl moieties in glycerolipids,
have shown that the three initial steps in ether-lipid biosynthesis in
Leishmania mexicana promastigotes resemble those described for mammals
and are associated with glycosomes. Purified glycosomes were able to
sequentially synthesise the first intermediates of the ether-lipid
biosynthetic pathway [acyl-dihydroxyacetonephosphate (DHAP), alkyl-DHAP
and acyl/alkyl-glycerol-3-phosphate (G3P)] when incubated in the
presence of radiolabelled DHAP, palmitoyl-CoA, hexadecanol and NADPH.
However, when glycosomes were incubated under the same conditions in the
presence of radiolabelled G3P, a rapid synthesis of acyl-G3P and
phosphatidic acid was observed without any formation of alkyl-G3P,
suggesting that the enzyme alkyl-synthase recognises only acyl-DHAP as
substrate. Both the DHAP acyltransferase (DHAP-AT) and alkyl-DHAP
synthase activities were located inside glycosomes whereas the alkyl/
acyl-DHAP oxidoreductase activity was associated with the cytoplasmic
face of the glycosomal membrane. The G3P acyltransferase (G3P-AT) and
lyso-phosphatidic acid acyltransferase activities were not found inside
glycosomes. The results suggest that the DHAP-AT and G3P-AT activities
are catalysed by two distinct enzymes associated with different sub-
cellular compartments.
PMID: 9310377
TITLE: Molecular cloning of the hamster CMP-sialic acid transporter.
AFFILIATION: Institut für Medizinische Mikrobiologie, Medizinische Hochschule
Hannover, Germany.
REFERENCE: Eur J Biochem 1997 Aug 248(1):187-92
Chinese hamster ovary (CHO) glycosylation mutants of the Lec2
complementation group are unable to express sialylated glycoproteins and
glycolipids due to a defect in the Golgi CMP-sialic acid transporter (
CMP-Sia-Tr). Using an expression cloning strategy, we isolated a cDNA
encoding the hamster CMP-Sia-Tr which complements the Lec2 phenotype.
The deduced amino acid sequence of the cloned cDNA shows 95% identity to
the recently cloned murine CMP-Sia-Tr. The expression of a hamster CMP-
Sia-Tr fusion protein with an N-terminal MDYKDDDDK (FLAG) sequence
revealed Golgi localisation of the transporter. Amino acid sequence
comparison revealed strong similarity (44.6% identity and 19.3%
similarity) of CMP-Sia-Tr to the recently cloned human UDP-galactose
transporter (UDP-Gal-Tr). In contrast, sequence similarities to the
yeast UDP-N-acetylglucosamine transporter (UDP-GlcNAc-Tr) and the GDP-
mannose transporter (GDP-Man-Tr) of Leishmania donovani are restricted
to a region encoding the two most C-terminally located transmembrane
helices. A computer-based structural analysis of CMP-Sia-Tr proposes an
eight transmembrane helix model with the N- and C-termini located on the
cytosolic side of the Golgi membrane.
PMID: 9065742
TITLE: Scavenger receptor-mediated delivery of antisense mini-exon
phosphorothioate oligonucleotide to Leishmania-infected macrophages. Selective
and efficient elimination of the parasite.
AFFILIATION: Division of Biomedical Sciences, Meharry Medical College,
Nashville, TN 37208, U.S.A. chaudh45 at ccvax.mmc.edu
REFERENCE: Biochem Pharmacol 1997 Feb 53(3):385-91
Targeted delivery of a 17-mer antisense phosphorothioate
oligodeoxyribonucleotide, complementary to the common 5'-end of every
mRNA of the parasite cells, to the phagolysosomes of cultured murine
macrophages infected with Leishmania mexicana amazonensis selectively
and efficiently eliminated the parasite cells without causing any
detectable harm to the host cells. The antisense mini-exon
oligonucleotide (ASM) was encapsulated into liposomes coated with
maleylated bovine serum albumin (MBSA), the artificial ligand for
macrophage scavenger receptors. MBSA-coating of the liposomes allowed
specific binding of the liposomes to the macrophages, their receptor-
mediated uptake, and subsequent degradation of the liposomes inside
macrophage phagolysosomes to release ASM. When incubated with Leishmania
-infected macrophages, MBSA-liposome-encapsulated ASM (10 microM) was
able to kill >90% of the parasites within 5 hr as compared with 20%
killing within this time period by free ASM. Oligonucleotides with
complementary nucleotide sequence or with the same base composition as
ASM but scrambled sequence had no antileishmanial effect under the
conditions of the assay. This study reflects the efficacy of scavenger-
receptor-mediated delivery of antisense phosphorothioate oligos in
killing intraphagolysosomal pathogens.
PMID: 9050240
TITLE: Function of metal-ion homeostasis in the cell division cycle,
mitochondrial protein processing, sensitivity to mycobacterial infection and
brain function.
AFFILIATION: Department of Biochemistry, Tel Aviv University, Ramat Aviv,
Israel.
REFERENCE: J Exp Biol 1997 Jan 200(Pt 2):321-30
A novel Saccharomyces cerevisiae mutant, unable to grow in the presence
of 12.5 mmol l-1 EGTA, was isolated. The phenotype of the mutant is
caused by a single amino acid change (Gly149 to Arg) in the essential
yeast cell division cycle gene CDC1. The mutant could be suppressed by
overexpression of the SMF1 gene, which codes for a plasma membrane Mn2+
transporter. We observed that the yeast SMF1 gene shares homology with
the mouse Nramp gene. Nramp (Bcg) was cloned as a gene responsible for
mouse resistance to infection with mycobacteria and is identical with
the Ity and the Lsh genes conferring resistance to infection by
Salmonella typhimurium and Leishmania donovani, respectively. Although
the cloning of Nramp identified the gene responsible for the resistance
of mice to mycobacteria, its function is unknown. We propose that the
mammalian protein, like the yeast transporter, is a Mn2+ and/or Zn2+
transporter. Following the phagocytosis of a parasite into the phagosome
, the macrophage produces reactive oxygen and/or nitrogen intermediates
that are toxic for the internalized bacteria. The survival of the
pathogen during the burst of macrophage respiratory activity is thought
to be partly mediated by microbial superoxide dismutase (SOD), which
contains Mn2+ or Fe2+ in its active centre. Nramp may transport Mn2+
from the extracellular milieu into the cytoplasm of a macrophage and,
after the generation of the phagosome, remove Mn2+ from the organelle.
Thus, the Mn(2+)-depletion of the phagosome microenvironment by the
Nramp gene product may be a rate-limiting step in the metalloenzyme's
production by the engulfed bacteria. This limitation will restrict the
mycobacterial ability to produce active enzymes such as SOD and prevent
the propagation of the ingested microorganisms. Conversely, an increased
concentration of Mn2+ in the phagosome caused by a defective Nramp
transporter (Bcgs) may promote the growth of the mycobacteria and render
the organism sensitive to the pathogen. We use a similar approach to
identify, clone and study other metal-ion transporters.
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