[leish-l] Fwd: Articles found by RefScout 09/02/2005 5/2005 part 2

[jeffreyj at usp.br]

    Date: Wed, 9 Feb 2005 12:01:20
    From: info at refscout.com


New!

Have a look at our new tool, the RefScout‘s PDF-Manager (PDFM)! The RefScout‘s
PDFM will revolutionize your life with PDF files!

Simply let your PDF files be organized by the RefScout‘s PDFM in a table and
get
direct link to your local copy. In addition, the RefScout‘s PDFM will alert you
each time the NLM PubMed updates information concerning your specific
reference!
Get your free 2 months trial version now at RefScout’s PDF-Manager.





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.
100 articles are sent in previous email(s))



********************************************************************************************************************

 The following references are revised files and are brought to you in
accordance
to license agreement with the NLM.

********************************************************************************************************************






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 Física, 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 Bioquímica 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 Clínicas, 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.




********************************************************************************************************************

 The following references are revised files and are brought to you in
accordance
to license agreement with the NLM.

********************************************************************************************************************


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