[leish-l] Fwd: Articles found by RefScout 11/05/2005 - 19/2005
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Date: Wed, 11 May 2005 13:55:00
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REQUEST: [ leishmaniasis ]
(15 articles match this request. 3 articles matching other requests removed)
PMID: 15866473
TITLE: Development of a modified MTT assay for screening antimonial resistant
field isolates of Indian visceral leishmaniasis.
AUTHORS: Avijit Dutta, Samiran Bandyopadhyay, Chitra Mandal, Mitali Chatterjee
AFFILIATION: Dept. of Immunobiology, Indian Institute of Chemical Biology, 4
Raja S.C. Mullick Road, Kolkata-700 032, India.
REFERENCE: Parasitol Int 2005 Jun 54(2):119-22
The semi-automated MTT colorimetric assay has previously been applied on
Leishmania promastigotes based on the ability of viable parasites to
reduce the tetrazolium salt to an insoluble formazan product. As
promastigotes are non-adherent, application of the MTT assay in its
original form has a major drawback of a high and variable background
absorbance due to incomplete removal of phenol red, a component of most
media. We have accordingly optimised a modified MTT assay wherein the
absorbance linearity was maintained for cells ranging from 1x10(4) to
1x10(7) being 0.04+/-0.003-2.38+/-0.04. In contrast, the original MTT
assay had a narrower linearity range of 1x10(6)-1x10(7) cells,
absorbances being 0.05+/-0.005-1.54+/-0.005. The modified MTT assay was
effectively applied to study growth kinetics and identification of
antimonial resistant field isolates. Considering the growing problem of
antimonial unresponsiveness in the Indian subcontinent, this modified
MTT assay is a useful tool for Leishmania research.
PMID: 15866476
TITLE: Function of CD8(+) T lymphocytes in a self-curing mouse model of visceral
leishmaniasis.
AUTHORS: Panagiotis Tsagozis, Evdokia Karagouni, Eleni Dotsika
AFFILIATION: Laboratory of Cellular Immunology, Hellenic Pasteur Institute, 127
Vass. Sofias Ave., 11521 Athens, Greece.
REFERENCE: Parasitol Int 2005 Jun 54(2):139-46
CD8(+) T lymphocytes play an important role in the control of visceral
leishmaniasis in non self-cure mice (e.g. BALB/c). In the present study
, the mode of action of CD8(+) T cells and their in vivo contribution to
immunity was addressed in self-curing C57BL/6 mice. During the course
of the experimental infection, CD8(+) T cells specific for Leishmania
infantum (L. infantum) developed and apoptotic cell death subsequently
followed. They exhibited perforin-dependent cytotoxicity and a T(C)1
profile characterized by secretion of IFN-gamma and CC chemokines.
Despite evidence for activation of CD8(+) T lymphocytes, both
intravenous and intradermal infection of beta(2)-microglobulin deficient
C57BL/6 mice with L. infantum showed that these knockout animals had
similar parasite loads to their wild-type counterpart. Lymphocytes from
the beta(2)-microglobulin deficient mice produced high levels of IFN-
gamma, reflecting a T(H)1 response to the parasite, which was apparently
sufficient for the immunologic control of the pathogen. Thus, despite
their functional activation, CD8(+) T lymphocytes do not appear to play
a primary role in parasite restraint in the self-curing mouse model of
visceral leishmaniasis, as shown using beta(2)-microglobulin deficient
mice which do not produce functional CD8(+) T lymphocytes.
PMID: 15872214
TITLE: Case 4-2005: sodium stibogluconate for cutaneous leishmaniasis.
AUTHORS: Joshua D Hartzell, Naomi Aronson
REFERENCE: N Engl J Med 2005 May 352(18):1929
PMID: 15855481
TITLE: Antileishmanial activity of the terpene nerolidol.
AUTHORS: Denise C Arruda, Fabio Luiz D'Alexandri, Alejandro M Katzin, Silvia R B
Uliana
AFFILIATION: Departamento de Parasitologia, Instituto de Ciências Biomédicas,
Universidade de São Paulo, Av. Professor Lineu Prestes, 1374, CEP 05508-900,
São Paulo, SP, Brazil.
REFERENCE: Antimicrob Agents Chemother 2005 May 49(5):1679-87
The activity of nerolidol, a sesquiterpene used as a food-flavoring
agent and currently under testing as a skin penetration enhancer for the
transdermal delivery of therapeutic drugs, was evaluated against
Leishmania species. Nerolidol inhibited the growth of Leishmania
amazonensis, L. braziliensis, and L. chagasi promastigotes and L.
amazonensis amastigotes with in vitro 50% inhibitory concentrations of
85, 74, 75, and 67 microM, respectively. The treatment of L. amazonensis
-infected macrophages with 100 microM nerolidol resulted in 95%
reduction in infection rates. Inhibition of isoprenoid biosynthesis, as
shown by reduced incorporation of [2-(14)C]mevalonic acid (MVA) or [1-(
14)C]acetic acid precursors into dolichol, ergosterol, and ubiquinone,
was observed in nerolidol-treated promastigotes. This drug effect can be
attributed to the blockage of an early step in the mevalonate pathway,
since incorporation of the precursor [1(n)-(3)H]farnesyl pyrophosphate
in polyisoprenoids is not inhibited by nerolidol. L. amazonensis-
infected BALB/c mice were treated with intraperitoneal doses of 100 mg/
kg/day for 12 days or topically with 5 or 10% ointments for 4 weeks.
Significant reduction of lesion sizes in nerolidol treated mice was
observed for both treatment routes. However, long-term follow up
indicated that the disease was not cured in this highly susceptible
animal model. Nonetheless, the in vitro activity of nerolidol against
these parasites may prove a useful tool for the development of new drugs
for the treatment of leishmaniasis. In addition, biosynthesis of
dolichols with 11 and 12 isoprene units was identified in Leishmania, as
described for other trypanosomatids and Apicomplexa.
PMID: 15879027
TITLE: Development of Recombinant Chimeric Antigen Expressing Immunodominant B
Epitopes of Leishmania infantum for Serodiagnosis of Visceral Leishmaniasis.
AUTHORS: A Boarino, A Scalone, L Gradoni, E Ferroglio, F Vitale, R Zanatta, M G
Giuffrida, S Rosati
AFFILIATION: Dipartimento di Produzioni Animali, Epidemiologia ed Ecologia,
Facoltà di Medicina Veterinaria, Via Leonardo da Vinci 44, 10095 Grugliasco
(TO) Italy. sergio.rosati at unito.it.
REFERENCE: Clin Diagn Lab Immunol 2005 May 12(5):647-53
Wild canids and domestic dogs are the main reservoir of zoonotic
visceral leishmaniasis (VL) caused by Leishmania infantum (syn.:
Leishmania chagasi). Serological diagnosis of VL is therefore important
in both human and dog leishmaniasis from a clinical and epidemiological
point of view. Routine diagnosis of VL is traditionally carried out by
immunofluorescent antibody test (IFAT), which is laborious and difficult
to standardize and to interpret. In the last decade, however, several
specific antigens of Leishmania infantum have been characterized,
allowing the development of a recombinant-based immunoassay. Among them
, the whole open reading frame encoding K9 antigen, the gene fragment
encoding the repetitive sequence of K26, and the 3'-terminal gene
fragment of the kinesin-related protein (K39sub) were previously
evaluated as diagnostic markers for canine leishmaniasis and proved to
be independent in their antibody reactivity. Since sensitivity of
serological test is usually higher in multiple-epitope format, in this
study the relevant epitopes of K9, K26, and K39 antigens were joined by
PCR strategy to produce the chimeric recombinant protein. The resulting
mosaic antigen was found highly expressed in Escherichia coli and
efficiently purified by affinity chromatography. Antigenic properties of
this recombinant antigen were evaluated by indirect enzyme-linked
immunosorbent assay (ELISA) using a panel of human and dog sera
previously characterized by parasitological and/or serological
techniques. Chimeric ELISA showed 99% specificity in both human (n = 180
) and canine (n = 343) control groups, while sensitivity was higher in
canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly,
concordance between IFAT and canine chimeric ELISA (k = 0.95, 95%
confidence interval = 0.93 to 0.98) was higher than between IFAT and
human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87
). Results suggest the potential use of this new antigen for routine
serodiagnosis of VL in both human and canine hosts.
PMID: 15867961
TITLE: Moonlight and blood-feeding behaviour of Lutzomyia intermedia and
Lutzomyia whitmani (Diptera:Psychodidae:Phlebotominae), vectors of American
cutaneous leishmaniasis in Brazil.
AUTHORS: Nataly A Souza, Cláudia A Andrade-Coelho, Vanderlei C Silva, Alexandre
A Peixoto, Elizabeth F Rangel
AFFILIATION: Departamento de BioquÃmica e Biologia Molecular, Instituto Oswaldo
Cruz-Fiocruz, Rio de Janeiro, RJ, Brasil.
REFERENCE: Mem Inst Oswaldo Cruz 2005 Feb 100(1):39-42
Lutzomyia intermedia (Lutz & Neiva, 1912) and L. whitmani (Antunes &
amp; Coutinho, 1939) (Diptera: Psychodidae: Phlebotominae), two
important vectors of American cutaneous leishmaniasis in Brazil, occur
in sympatry in the locality of Posse county, Petropolis municipality,
state of Rio de Janeiro, Brazil. We investigated the influence of the
lunar cycle on the frequency of specimens of the two species caught
while attempting to bite the collectors and in CDC light traps. Analysis
of the numbers of sand flies captured in different lunar phases for two
consecutive years in the peridomestic site and forest shows that there
is a significant positive correlation between moonlight intensity and
the numbers of L. intermedia and L. whitmani females collected while
blood-feeding, whereas the opposite was observed for the CDC traps.
PMID: 15867962
TITLE: Multiplex-PCR for detection of natural Leishmania infection in Lutzomyia
spp. captured in an endemic region for cutaneous leishmaniasis in state of
Sucre, Venezuela.
AUTHORS: Alicia Jorquera, Ricardo González, Edgar Marchán-Marcano, Milagros
Oviedo, Mercedes Matos
AFFILIATION: Centro de Investigaciones en Ciencias de la Salud, Universidad de
OrienteVenezuela.
REFERENCE: Mem Inst Oswaldo Cruz 2005 Feb 100(1):45-8
We studied the natural infection of Lutzomyia (Lutzomyia) sp. with
Leishmania in endemic foci of cutaneous leishmaniasis in the Paria
peninsula, state of Sucre, Venezuela. Sand flies were collected between
March 2001 and June 2003, using Shannon light-traps and human bait. Of
the 1291 insects captured, only two species of phlebotomines were
identified: L. ovallesi (82.75%) and L. gomezi (17.42%). A sample of the
collected sand flies (51 pools of 2-12 individuals) were analyzed by
using a multiplex-PCR assay for simultaneous detection of New Word
Leishmaniaand Viannia subgenera. The results showed a total of 8 pools (
15.68%) infected; of these, 7 were L. ovallesi naturally infected with L
. braziliensis (2 pools) and L. mexicana (5 pools) and 1 pool of L.
gomezi infected by L. braziliensis.
PMID: 15856912
TITLE: Late cutaneous metastases in C3H SCID mice infected with Leishmania
amazonensis.
AUTHORS: Y Vanloubbeeck, M R Ackermann, D E Jones
AFFILIATION: Department of Veterinary Pathology, College of Veterinary Medicine,
Iowa State University, Ames, Iowa 50011, USA.
REFERENCE: J Parasitol 2005 Feb 91(1):226-8
The biological behavior of Leishmania amazonensis in the mammalian host
is highly variable, resulting in local to diffuse cutaneous lesions that
sometimes metastasize. Inflammation and, more specifically, CD4+ T
cells have been shown to enhance metastases in mice infected with L.
amazonensis, suggesting that the process may be lymphocyte mediated.
However, we document, in this study, the development of multiple
cutaneous metastases in C3H SCID mice infected with L. amazonensis. This
shows that functional T and B cells are not required for metastases to
occur.
PMID: 15757164
TITLE: Trench foot, jungle rot, and now, Baghdad boil.
AUTHORS: Brian F Mandell
REFERENCE: Cleve Clin J Med 2005 Feb 72(2):81
PMID: 15880235
TITLE: Identification of Leishmania (Leishmania) chagasi isolated from healthy
skin of symptomatic and asymptomatic dogs seropositive for leishmaniasis in the
Municipality of Rio de Janeiro, Brazil.
AUTHORS: Maria de Fátima Madeira, Armando de Oliveira Schubach, Tânia Maria
Pacheco Schubach, Cristianni Antunes Leal, Mauro Célio de Almeida Marzochi
AFFILIATION: Department of Micro-Imuno-Parasitology, Institute of Clinical
Research Evandro Chagas, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil.
REFERENCE: Braz J Infect Dis 2004 Dec 8(6):440-4
Euthanasia of seropositive dogs has been one of the principal measures
adopted by the Program for the Control of Leishmaniasis in Brazil for
many years. However, its efficacy is currently being questioned. We
obtained intact skin samples from 20 Leishmania-reactive dogs from the
municipality of Rio de Janeiro that had been referred for euthanasia.
The promastigote forms of Leishmania were isolated in culture from 18 of
these animals. Fourteen of these isolates were identified as Leishmania
(Leishmania) chagasi by isoenzyme electrophoresis; seven of these were
from asymptomatic dogs and seven were from symptomatic animals with
visceral leishmaniasis. In conclusion, cutaneous parasitism is found in
the intact skin of dogs naturally infected with L. (L.) chagasi,
irrespective of the presence or absence of clinical signs suggestive of
visceral leishmaniasis.
PMID: 15864860
TITLE: [Proceedings and abstracts of the XIX Annual Meeting of Applied Research
in Chagas Disease and the VII Annual Meeting of Applied Research in
Leishmaniasis. Uberaba, Brazil, 24-26 October 2003]
REFERENCE: Rev Soc Bras Med Trop 2003 36 Suppl 2():5-118
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The following references are revised files and are brought to you in accordance
to license agreement with the NLM.
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PMID: 15522675
TITLE: Mucosal leishmaniasis in an Indian AIDS patient.
AUTHORS: Sarman Singh
AFFILIATION: Division of Clinical Microbiology, All India Institute of Medical
Sciences, New Delhi, India. sarman_singh at yahoo.com <sarman_singh at yahoo.com>
REFERENCE: Lancet Infect Dis 2004 Nov 4(11):660-1
REQUEST: [ leishmania ]
(19 articles match this request. 8 articles matching other requests removed)
PMID: 15866472
TITLE: The involvement of neutrophils in the resistance to Leishmania major
infection in susceptible but not in resistant mice.
AUTHORS: Lin Chen, Zhi-Hui Zhang, Tadashi Watanabe, Takao Yamashita, Takatoshi
Kobayakawa, Akira Kaneko, Hiromi Fujiwara, Fujiro Sendo
AFFILIATION: Department of Immunology and Parasitology, Yamagata University
School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan.
REFERENCE: Parasitol Int 2005 Jun 54(2):109-18
To understand the immunomodulatory roles of neutrophils in Leishmania
major infection, we examined the expression of cytokine and chemokine
mRNAs from neutrophils of the infected resistant C3H/HeJ and susceptible
BALB/c mice. We also examined the effects of neutrophil depletion on
the expression of cytokine by peritoneal macrophages and draining lymph
node cells and on the footpad lesions and parasite burdens in these mice
. Neutrophils from resistant C3H/HeJ but not from susceptible BALB/c
mice expressed mRNAs for IL-12p40, IFN-gamma,TNF-alpha and monokine
induced by IFN-gamma(MIG). Neutrophil depletion of the resistant mice
reduced the expression of IFN-gammaandTNF-alpha in peritoneal
macrophages but did not affect the expression of IL-12p40 and IFN-gamma
in draining lymph node cells and the growth of footpad lesions. On the
other hand, neutrophil depletion of susceptible BALB/c mice did not
affect the expression of TNF-alpha and monocyte-derived chemokine (MDC)
in peritoneal macrophages but induced the early stage expression of IL-4
in draining lymph node cells and exacerbated the footpad lesions and
increased the parasite burden. The exacerbation of footpad lesions
induced by neutrophil depletion was abolished by rIL-12 treatment. Our
results suggest that even in susceptible BALB/c but not in C3H/HeJ mice
there is a certain resistance requiring neutrophils at the early stage
of infection.
PMID: 15879084
TITLE: Cutting Edge: The SLAM Family Receptor Ly108 Controls T Cell and
Neutrophil Functions.
AUTHORS: Duncan Howie, F Stephen Laroux, Massimo Morra, Abhay R Satoskar, Lucia
E Rosas, William A Faubion, Aimee Julien, Svend Rietdijk, Anthony J Coyle,
Christopher Fraser, Cox Terhorst
AFFILIATION: Division of Immunology, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, MA 02215. id="AFF2"> Department of
Microbiology, Ohio State University, Cleveland, OH 43210; MedImmune Inc.,
Gaithersberg, MD 20878.
REFERENCE: J Immunol 2005 May 174(10):5931-5
Ly108, a glycoprotein of the signaling lymphocytic activation molecule
family of cell surface receptors expressed by T, B, NK, and APCs has
been shown to have a role in NK cell cytotoxicity and T cell cytokine
responses. In this study, we describe that CD4(+) T cells from mice with
a targeted disruption of exons 2 and 3 of Ly108 (Ly108(DeltaE2+3))
produce significantly less IL-4 than wild-type CD4(+) cells, as judged
by in vitro assays and by in vivo responses to cutaneous infection with
Leishmania mexicana. Surprisingly, neutrophil functions are controlled
by Ly108. Ly108(DeltaE2+3) mice are highly susceptible to infection with
Salmonella typhimurium, bactericidal activity of Ly108(DeltaE2+3)
neutrophils is defective, and their production of IL-6, IL-12, and TNF-
alpha is increased. The aberrant bactericidal activity by Ly108(DeltaE2+
3) neutrophils is a consequence of severely reduced production of
reactive oxygen species following phagocytosis of bacteria. Thus, Ly108
serves as a regulator of both innate and adaptive immune responses.
PMID: 15855523
TITLE: Role of the ABC transporter MRPA (PGPA) in antimony resistance in
Leishmania infantum axenic and intracellular amastigotes.
AUTHORS: Karima El Fadili, Nadine Messier, Philippe Leprohon, Gaétan Roy,
Chantal Guimond, Nathalie Trudel, Nancy G Saravia, Barbara Papadopoulou,
Danielle Légaré, Marc Ouellette
AFFILIATION: Centre de Recherche en Infectiologie du Centre de Recherche du
CHUL, Iniversité Laval, Québec, Canada G1V 4G2.
REFERENCE: Antimicrob Agents Chemother 2005 May 49(5):1988-93
Antimonial compounds are the mainstay for the treatment of infections
with the protozoan parasite Leishmania. We present our studies on
Leishmania infantum amastigote parasites selected for resistance to
potassium antimonyl tartrate [Sb(III)]. Inside macrophages, the Sb(III)-
selected cells are cross-resistant to sodium stibogluconate (Pentostam
), the main drug used against Leishmania. Putative alterations in the
level of expression of more than 40 genes were compared between
susceptible and resistant axenic amastigotes using customized DNA
microarrays. The expression of three genes coding for the ABC
transporter MRPA (PGPA), S-adenosylhomocysteine hydrolase, and
folylpolyglutamate synthase was found to be consistently increased. The
levels of cysteine were found to be increased in the mutant.
Transfection of the MRPA gene was shown to confer sodium stibogluconate
resistance in intracellular parasites. This MRPA-mediated resistance
could be reverted by using the glutathione biosynthesis-specific
inhibitor buthionine sulfoximine. These results highlight for the first
time the role of MRPA in antimony resistance in the amastigote stage of
the parasite and suggest a strategy for reversing resistance.
PMID: 15879134
TITLE: Control of Infection with Leishmania major in Susceptible BALB/c Mice
Lacking the Common {gamma}-Chain for FcR Is Associated with Reduced Production
of IL-10 and TGF-{beta} by Parasitized Cells.
AUTHORS: Udaikumar M Padigel, Jay P Farrell
AFFILIATION: Department of Pathobiology, School of Veterinary Medicine,
University of Pennsylvania, Philadelphia, PA 19104.
REFERENCE: J Immunol 2005 May 174(10):6340-5
Previous studies have shown that the in vitro ligation of FcgammaRs with
IgG-opsonized Leishmania amastigotes promotes IL-10 production by
macrophages. In addition, infection of either BALB/c mice lacking the
common gamma-chain of Fc receptors (FcgammaR(-/-)) or mice genetically
altered to lack circulating Ab (J(H)D) with Leishmania pifanoi results
in reduced and delayed lesion development and a deficit in the
recruitment of inflammatory cells into infected lesions. We show in this
study that FcgammaR(-/-) mice can control infection with Leishmania
major and totally resolve cutaneous lesions. The ability to eventually
control infection is not associated with a reduction in lesion
inflammation or a reduction in the ability of Leishmania to parasitize
cells through week 6 of infection. The immune response in healing
FcgammaR(-/-) mice is associated with a reduction in numbers of cells
producing Th2-type cytokines, including IL-4 and IL-10, but not an
increase in numbers of IFN-gamma-producing cells characteristic of a
dominant Th1-type response. Instead, we observe a reduction in levels of
IL-10 and TGF-beta within infected lesions, including reduced levels of
these cytokines within parasitized macrophages. Together, these results
suggest that uptake of opsonized parasites via FcgammaRs may be a
strong in vivo stimulus for the production of anti-inflammatory
cytokines that play a role in susceptibility to infection.
PMID: 15849344
TITLE: Two cases of feline leishmaniosis in Switzerland.
AUTHORS: S Rüfenacht, H Sager, N Müller, V Schaerer, A Heier, M M Welle, P J
Roosje
AFFILIATION: Dermatology Unit, Department of Clinical Veterinary Medicine,
University of Berne, Switzerland.
REFERENCE: Vet Rec 2005 Apr 156(17):542-5
Two cats with Leishmania species infections were investigated. The first
had been imported from Spain with a non-healing, ulcerated nodule on a
hindleg. The presence of Leishmania species was detected by
histopathology and pcr on samples of skin. The lesion was unresponsive
to treatment with allopurinol for three months but the cat was treated
successfully by removing the lesion surgically. The second cat had lived
in both Spain and Switzerland, and had a history of recurrent skin
lesions on its head and neck. A diagnosis of pemphigus foliaceus was
made on the basis of histopathology, but Leishmania species serology (
elisa) and pcr of skin were positive, leading to a diagnosis of a
Leishmania species infection combined with pemphigus foliaceus.
PMID: 15856892
TITLE: The effect of amphotericin b derivatives on Leishmania and immune
functions.
AUTHORS: Tirtsa Ehrenfreund-Kleinman, Abraham J Domb, Charles L Jaffe, Abed
Nasereddin, Benni Leshem, Jacob Golenser
AFFILIATION: Department of Medicinal Chemistry, School of Pharmacy, Hebrew
University of Jerusalem, Jerusalem 91120, Israel.
REFERENCE: J Parasitol 2005 Feb 91(1):158-63
The effects of a water-soluble amphotericin B (AmB)-arabinogalactan (AG
) conjugate on several immune functions were investigated. The
experiments measured the effects of AmB-AG on (1) release of tumor
necrosis factor-alpha (TNF-alpha), nitric oxide (NO), and interferon-
gamma (IFN-gamma) from phagocytic cells and (2) cell-mediated immune
responses. AmB-AG increased TNF-alpha release from mouse peritoneal
macrophages and human monocytes but had no effect on IFN-gamma and NO
release. A commercial preparation of nonconjugated AmB (Fungizone) also
increased TNF-alpha production, but to a lesser extent than AmB-AG. AG
alone had no effect on TNF-alpha production, proving that AmB caused the
increased TNF-alpha production. AmB-AG and Fungizone were also tested
for their effect on B- and T-cell proliferation. Neither compound
altered T-lymphocyte responses to concanavalin A, but both inhibited the
stimulation of B lymphocytes by lipopolysaccharides. However, Fungizone
showed a stronger inhibitory effect on B cells. Allocytotoxicity was
also inhibited by AmB-AG and more strongly by Fungizone. The increased
production of TNF-alpha by cells treated with AmB-AG and the lower
inhibitory effect of AmB-AG on lymphocyte stimulation and
allocytotoxicity, as compared with Fungizone, explain the better
therapeutic efficacy of the AmB-polysaccharide conjugate. AmB is active
because of its preferential binding to ergosterol rather than
cholesterol, the former sterol preferentially present in parasite
surface membranes. This is also valid for the axenic amastigotes, which
were sensitive to the AmB-AG. Overall, our results suggest that the
antileishmanial activity of AmB-AG is mediated both directly and via
modulation of immune functions.
PMID: 15860773
TITLE: Characterization of the DNA-binding domain and identification of the
active site residue in the 'Gyr A' half of Leishmania donovani topoisomerase
II.
AUTHORS: Tanushri Sengupta, Mandira Mukherjee, Rakhee Das, Aditi Das, Hemanta K
Majumder
AFFILIATION: Molecular Parasitology Laboratory, Indian Institute of Chemical
Biology 4 Raja S.C. Mullick Road, Kolkata-700032, India.
REFERENCE: Nucleic Acids Res 2005 33(8):2364-73
DNA topoisomerase II is a multidomain homodimeric enzyme that changes
DNA topology by coupling ATP hydrolysis to the transport of one DNA
helix through a transient double-stranded break in another. To
investigate the biochemical properties of the individual domains of
Leishmania donovani topoisomerase II, four truncation mutants were
generated. Deletion of 178 aminoacids from the C-terminus (core and
LdDeltaC1058) had no apparent effect on the DNA-binding or cleavage
activities of the enzymes. However, when 429 aminoacids from the N-
terminus and 451 aminoacids from the C-terminus were removed (
LdDeltaNDeltaC), the enzyme was no longer active. Moreover, the removal
of 429 aminoacids from the N-terminus (LdDeltaNDeltaC, core and
LdDeltaN429) render the mutant proteins incapable of performing ATP
hydrolysis. The mutant proteins show cleavage activities at wide range
of KCl concentrations (25-350 mM). In addition, the mutant proteins,
excepting LdDeltaNDeltaC, can also act on kDNA and linearize the
minicircles. Surprisingly, the mutant proteins fail to show the
formation of the enhanced cleavable complex in the presence of etoposide
. Our findings suggest that the conformation required for interaction
with the drug is absent in the mutant proteins. Here, we have also
identified Tyr(775) through direct sequencing of the DNA linked peptide
as the catalytic residue implicated in DNA-breakage and rejoining. Taken
together, our results demonstrate that topoisomerase II are
functionally and mechanistically conserved enzymes and the variations in
activity seem to reflect functional optimization for its physiological
role during parasite genome replication.
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The following references are revised files and are brought to you in accordance
to license agreement with the NLM.
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PMID: 14698430
TITLE: Tests of cytoplasmic RNA interference (RNAi) and construction of a
tetracycline-inducible T7 promoter system in Trypanosoma cruzi.
AUTHORS: Wanderson D DaRocha, Keiko Otsu, Santuza M R Teixeira, John E Donelson
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: 10551367
TITLE: T7 RNA polymerase-driven transcription in mitochondria of Leishmania
tarentolae and Trypanosoma brucei.
AUTHORS: A M Estévez, O H Thiemann, J D Alfonzo, L Simpson
AFFILIATION: Howard Hughes Medical Institute, UCLA School of Medicine, Los
Angeles, CA 90095-1662, USA.
REFERENCE: Mol Biochem Parasitol 1999 Oct 103(2):251-9
The study of RNA editing and other molecular processes in the
trypanosome mitochondrion would benefit greatly from the ability to
insert and express exogenous DNA in the organelle. However, even with a
method to introduce DNA, the current lack of knowledge about
mitochondrial transcription would hinder efforts to obtain expression.
To circumvent this problem, Leishmania tarentolae promastigotes and
Trypanosoma brucei procyclic cells have been transfected with
bacteriophage T7 RNA polymerase targeted to the mitochondrion.
Mitochondria isolated from the transfectants contained active T7 RNA
polymerase, as shown by a comigration in density gradients of
mitochondrial marker enzymes and T7 polymerase activity. A DNA cassette
under T7 control was introduced into isolated mitochondria from the
transfectants by electroporation and the DNA was shown to be transcribed
. This system should allow the transcription of foreign genes of choice
within the mitochondrial matrix either in a transient assay using
electroporation of DNA into isolated mitochondria, or in a stable assay
using cells transfected with DNA by the biolistic gun method.
PMID: 8054844
TITLE: pJC20 and pJC40--two high-copy-number vectors for T7 RNA
polymerase-dependent expression of recombinant genes in Escherichia coli.
AUTHORS: J Clos, S Brandau
AFFILIATION: Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
REFERENCE: Protein Expr Purif 1994 Apr 5(2):133-7
We report the construction of two plasmid vectors, pJC20 and pJC40, for
the expression of recombinant genes in Escherichia coli under the
control of T7 RNA polymerase. Their small sizes of ca. 2.4 kb ease the
subcloning of large inserts and the high copy numbers obtained result in
satisfactory yields in all plasmid preparations. A multiple-cloning
site offers sites for directional cloning and nested deletions. In
addition, pJC40 encodes a cleavable amino-terminal histidine tail of 10
residues which is added to the gene product, thus allowing purification
by metal chelate chromatography. Observed expression yields are in the
range of 10% of total bacterial protein for all genes tested in our
laboratory.
PMID: 1448081
TITLE: Unstable amplification of two extrachromosomal elements in
alpha-difluoromethylornithine-resistant Leishmania donovani.
AUTHORS: S Hanson, S M Beverley, W Wagner, B Ullman
AFFILIATION: Department of Biochemistry and Molecular Biology, Oregon Health
Sciences University, Portland 97201-3098.
REFERENCE: Mol Cell Biol 1992 Dec 12(12):5499-507
We describe the first example of unstable gene amplification consisting
of linear extrachromosomal DNAs in drug-resistant eukaryotic cells.
alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with
an amplified ornithine decarboxylase (ODC) gene copy number contained
two new extrachromosomal DNAs, both present in 10 to 20 copies. One of
these was a 140-kb linear DNA (ODC140-L) on which all of the amplified
copies of the odc gene were located. The second was a 70-kb circular DNA
(ODC70-C) containing an inverted repeat but lacking the odc gene. Both
ODC140-L and ODC70-C were derived from a preexisting wild-type
chromosome, probably by a conservative amplification mechanism. Both
elements were unstable in the absence of DFMO, and their disappearance
coincided with a decrease in ODC activity and an increase in DFMO growth
sensitivity. These results suggest the possibility that ODC70-C may
play a role in DFMO resistance. These data expand the diversity of known
amplification mechanisms in eukaryotes to include the simultaneous
unstable amplification of both linear and circular DNAs. Further
characterization of these molecules will provide insights into the
molecular mechanisms underlying gene amplification, including the
ability of linear amplified DNAs to acquire telomeres and the
determinants of chromosomal stability.
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