We examined the DNA binding activity of mouse and human MTF-1 in whole cell extracts from cells cultured in medium containing zinc or cadmium and from untreated cells after the in vitro addition of zinc or cadmium, as well as using recombinant MTF-1 transcribed and translated in vitro and treated with various transition metals. Incubation of human (HeLa) or mouse (Hepa) cells in medium containing cadmium (5-15 M) did not lead to a significant increase (<2-fold) in the amount of MTF-1 DNA binding activity, whereas zinc (100 M) led to a 6 -15-fold increase within 1 h. MTF-1 binding activity was low, but detectable, in control whole cell extracts and was increased (>10-fold) after the in vitro addition of zinc (30 M) and incubation at 37°C for 15 min. In contrast, addition of cadmium (6 or 60 M) did not activate MTF-1 binding activity. Recombinant mouse and human MTF-1 were also dependent on exogenous zinc for DNA binding activity. Cadmium did not facilitate activation of recombinant MTF-1, but instead inhibited the activation of the recombinant protein by zinc. Interestingly, glutathione (1 mM) protected recombinant MTF-1 from inactivation by cadmium, and allowed for activation by zinc. It was also noted that zinc-activated recombinant MTF-1 was protected from cadmium only when bound to DNA. These results suggest that cadmium interacts with the zinc fingers of MTF-1 and forms an inactive complex. Of the several transition metals (zinc, cadmium, nickel, silver, copper, and cobalt) examined, only zinc facilitated activation of the DNA binding activity of recombinant MTF-1. These data suggest that transition metals, other than zinc, that activate MT gene expression may do so by mechanisms independent of an increase in the DNA binding activity of MTF-1. Metallothioneins (MT)1 constitute a conserved family of cysteine-rich heavy metal-binding proteins (1). In the mouse, MT-I and MT-II display a wide tissue distribution and have been demonstrated to participate in detoxification of transition metals such as cadmium (2, 3), zinc homeostasis (4), and protection against oxidative stress (5). MT-I and MT-II gene transcription is induced dramatically by heavy metals (especially zinc and cadmium) (6). Metal response elements (MRE) are essential for this induction, and these elements are present in multiple copies in the proximal promoters of MT genes. MREs were initially shown to mediate transcriptional response of MT genes to zinc and cadmium (7-9), and more recently to oxidative stress (10, 11).A protein that binds specifically to MREs and that transactivates MT gene expression has been cloned from mouse and human, and is termed MTF-1 (MRE-binding transcription factor-1) (12, 13). MTF-1 is a zinc finger transcription factor in the Cys 2 His 2 family. The DNA binding activity of MTF-1 is reversibly regulated by zinc interactions with the zinc finger domain (14). In contrast with some zinc finger proteins, including zinc finger transcription factors, which can bind zinc with picomolar to nanomolar disassociation constants (15...

An outbreak of skin lesions was reported in June 2005 in the district of Silti woreda, 150 km south of Addis Ababa, by the Christian Children's Fund (CCF) and confirmed to be cutaneous leishmaniasis (CL) by our group from the Armauer Hansen Research Institute in July 2005. A house-to-house survey of 1907 residents in three kebeles of Silti woreda conducted in April 2006 showed a prevalence of 4.8%. RFLP analysis of the internal transcribed spacer RNA (ITS1) showed that Leishmania aethiopica was the causative agent. In the survey, it was found that the age group 11-20 years was the most affected. Environmental factors such as proximity of the house to the gorge where hyraxes reside, presence of the plants Adhatoda schimperiana and Acacia spp. in the compound and sharing the same room with domestic animals were significantly associated with developing CL. The prevalence of active disease was higher in Kibet town (10.4%) compared to the rural kebeles. The identified risk factors of CL in the area need further study. The appearance of leishmaniasis in Silti, which was not known to be endemic for the disease, underlines the need to initiate a leishmaniasis control program in Ethiopia to limit its expansion.

Kinetoplastid glycosomes contain a variety of metabolic activities, such as glycolysis, ␤-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.Despite the evolution of a complex mammalian immune response against foreign pathogens, Leishmania continues to plague humans, causing death and disease worldwide. This places an emphasis on elucidating the molecular mechanisms employed by Leishmania to establish a successful infection. Leishmania is an intracellular protozoan parasite that infects mammalian macrophages. These parasites possess a digenic life cycle, consisting of an extracellular promastigote form that multiplies and develops within the alimentary tract of the sand fly vector and an intracellular amastigote form that resides and multiplies within the phagosome of the mammalian host macrophage.Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are toxic metabolites that damage all living cells. The superoxide anion (O 2˙Ϫ ) is produced by the reduction of oxygen and is the fundamental oxidant among a battery of reactive intermediates, as it is involved in numerous reactions generating a plethora of increasingly toxic intermediates, including hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (˙OH), and peroxynitrite (ONOO Ϫ ). Collectively, these prooxidants are toxic to DNA and can cause peroxidation of lipids and the inactivation of iron-sulfur enzymatic centers of dehydratases (15 order to adapt to their new environment. Numerous genes have been shown to be differentially expressed in the promastigote and amastigote stages, and many morphological and metabolic chan...

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Cutaneous leishmaniasis in Ethiopia is caused mainly by Leishmania aethiopica. In this study, the response of L. aethiopica to sodium stibogluconate (SSG) and liquid nitrogen in Silti has been investigated. Patients were divided into two groups by the treating physician and were treated with either liquid nitrogen or SSG. Punch biopsy samples were collected from 54 patients with mean age of 20.61 (± 9.87 SD) years for histological characterization. The histological spectrum found to be type-1, type-2, type-3 and type-4 were 37.0%, 3.7%, 37.0% and 22.2% respectively. One hundred and three patients with a mean age of 18.4 (± 11.7 SD) years were treated with liquid nitrogen. The mean duration of the lesions before the onset of treatment was 8.5 months (± 6.7 SD). Of the 103 patients 80.6% (83/103) were cured, 13.6% (14/103) were dropouts and 5.8% (6/103) did not respond. Twenty patients with a mean age of 19.55 (+1.64 SD) years were treated with Pentostam on conventional dose. Of the 20 patients 85.0% (17/20) were cured, 10.0% (2/20) were unresponsive and 5.0% (1/20) were dropouts. The per protocol cure rate for cryotherapy and Pentostam was 93.3% and 89.5% respectively. Hence, liquid nitrogen can be used as one of the treatment options especially in resource poor settings.

A chimeric gene containing a cloned human metallothionein-II (MT-II) processed gene was introduced into Brassica napus and Nicotiana tabacum cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens. Transformants expressed MT protein as a Mendelian trait and in a constitutive manner. Seeds from self-fertilized transgenic plants were germinated on media containing toxic levels of cadmium and scored for tolerance/ susceptibility to this heavy metal. The growth of root and shoot of transformed seedlings was unaffected by up to 100 μM CdCl2, whereas control seedlings showed severe inhibition of root and shoot growth and chlorosis of leaves. The results of these experiments indicate that agriculturally important plants such as B. napus can be genetically engineered for heavy metal tolerance/sequestration and eventually for partitioning of heavy metals in non-consumed plant tissues.

Cathepsin B-like genes from Leishmania donovani and Leishmania chagasi have been isolated and characterized. It is a single gene, which is constitutively expressed in all the life cycle stages of the parasite. Studies using cathepsin B-specific inhibitor treatment suggested that cathepsin B does not seem to play a role in the promastigote stages of the parasite, however it aids in the parasite survival within the host macrophages. Antisense mRNA inhibition of cathepsin B gene also revealed that it plays an important role in the parasite survival within the host macrophages. Furthermore, for the first time, we have shown that Leishmania whole cell lysates as well as the recombinant cathepsin B protein cleaved human recombinant latent transforming growth factor (TGF)-␤1 into a mature peptide releasing the latency associated protein, in a cell-free incubation system. Mink lung epithelial cell growth inhibition assay revealed that the cleaved TGF-␤1 was biologically active, suggesting that Leishmania cathepsin B can cleave latent TGF-␤1 into mature and active form. These results suggest that cathepsin B plays an important role in Leishmania survival within the host macrophages by activating latent TGF-␤1.