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A simple technique to enhance the humoral immune response to intracellular protein antigens in genetic immunizations is demonstrated in mice. In this approach, the intracellular protein is intentionally secreted from expressing cells as a chimeric protein, comprising an N-terminal secreted protein fused to the intracellular protein antigen. Using the Leishmania chagasi Ldccys1 cysteine protease (411CP) as an example of an intracellular protein antigen and both human and murine granulocyte colony stimulating factor (GMCSF) as examples of N-terminal secretion competent fusion partners in chimeric proteins, a humoral response in plasmid DNA immunized mice could only be detected by Western blotting when the expressed 411CP was secreted.

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.

In this study, we tested the protective efficacy of recombinant Leishmania donovani iron superoxide dismutase B1 (SODB1) against Leishmania major infection in BALB/c mice. Mice were challenged with L. major 3 weeks after the second boost immunization with rSODB1 alone or in the presence of adjuvants. Injection of BALB/c mice with rSODB1 alone elicited both humoral and cellular immune responses. Administration of rSODB1 with CpG ODN or GLA-SE (a synthetic toll-like receptor 4 agonist) adjuvant resulted in the induction of anti-SODB1 IgG1, and more importantly of significantly high levels of IgG2a isotype. Immunization of mice with rSODB1 alone or with adjuvant induced the production of IFN-γ by splenocytes in response to stimulation with L. major soluble leishmanial antigens (SLA). Moreover, immunization protocols involving rSODB1 resulted in a significant decrease in IL-10 as compared to controls. The presence of CpG ODN or GLA-SE adjuvant in the immunization protocols resulted in a relative increase in IFN-γ in response to stimulation with rSODB1 in comparison to immunization with rSODB1 alone. Mice immunized with rSODB1 plus CpG ODN or GLA-SE, were able to partially control their Leishmania infections, as indicated by the reduction in footpad swelling and parasite numbers, compared to controls. These results suggest that immunization with recombinant SODB1 protein together with CpG ODN or GLA-SE can be potential vaccine candidate against leishmaniasis.

Cutaneous leishmaniasis (CL) is an increasing public health problem in Ethiopia. There is a concern that it is spreading with increased incidence. In this study, we used isoenzyme electrophoresis and internal transcribed spacer one (ITS1) PCR-RFLP techniques to identify Leishmania species from CL patients in Ethiopia. We obtained isolates from 55 localized cutaneous leishmaniasis (LCL), 3 diffused cutaneous leishmaniasis (DCL) and 36 biopsy samples from 34 LCL and 2 DCL cases from All Africa Leprosy and Tuberculosis Rehabilitation and Training Center (ALERT) and clinically diagnosed CL cases from Ochollo village. Both isoenzyme and ITS1 PCR-RFLP techniques showed that Leishmania aethiopica (L. aethiopica) was the aetiologic agent in all cases. Our study also showed that ITS1 PCR-RFLP could identify Leishmania species from biopsy samples and suggests the method could be used for epidemiological surveillance of leishmaniasis in Ethiopia and for species-specific diagnosis.

There is limited information on the biology and pathogenesis of Leishmania aethiopica, causative agent of cutaneous leishmaniasis (CL) in Ethiopia. In this study we have identified and characterized two cathepsin L-like cysteine protease genes, Laecpa and Laecpb, from L. aethiopica. The predicted amino acid sequence of Laecpa and Laecpb is more than 75% identical with homologous cathepsin L-like cysteine protease genes of other Leishmania species and less than 50% identical with human cathepsin L. Laecpa is expressed predominantly in the stationary, and to a lower level, during the amastigote stage while Laecpb is specifically expressed in the stationary stage of L. aethiopica development. Phylogenetic analysis showed that the two genes are grouped into separate clades which are the result of gene duplication. The isolation of these genes will be useful in developing Leishmania species specific diagnostics for molecular epidemiological studies and serves as a first step to study the role of cysteine proteases in L. aethiopica pathogenesis.

Cutaneous leishmaniasis (CL) is a painful, disfiguring and debilitating disease prevalent in Ethiopia and other countries around the world. In Ethiopia, CL is primarily caused by Leishmania aethiopica and less often by L. tropica and L. major. The intracellular survival mechanisms of Leishmania parasites are still not well understood. Recently a new family of antioxidant enzymes called peroxidoxins have been identified that play an important role in parasite survival. In this study, we have identified two distinct peroxidoxin genes (Pxn1 and Pxn2) that are part of a multi-gene family in L. aethiopica. Protein sequence analysis showed that Pxn1 and Pxn2 are highly homologous to peroxidoxins from other Leishmania species. We have found that L. aethiopica Pxn1 is predominantly expressed in amastigotes and stationary phase promastigotes, whereas Pxn2 is constitutively expressed in the different stages of the parasite. This pattern of RNA expression is consistent with patterns seen in some Leishmania species, but not all. Data from this study will be helpful in enhancing vaccine strategies and drug studies targeted towards peroxidoxins.

This study was performed to characterize the genes that code for superoxide dismutase (SOD) in Leishmania aethiopica. It involved three main steps: specimen collection and parasite isolation, species identification, and molecular characterization of the SOD genes. Out of 20 skin slit specimens cultured and processed from suspected cutaneous leishmaniasis patients enrolled in the study, five (25%) were found to be positive for motile promastigotes. Isoenzyme electrophoresis and PCR-RFLP results confirmed that the isolates were L. aethiopica. Superoxide dismutase-B (SODB) genes were identified from L. aethiopica for the first time. Iron superoxide dismutase-B genes amplified from promastigotes of L. aethiopica (LaeFeSODB) were similar in size to the SODB genes of other Leishmania species. Nucleotide sequences of LaeFeSODB1 showed 95.4, 93.5, and 97.3% identity with L. donovani SODB1 (LdFeSODB1) L. major SODB1 (LmFeSODB1) and L. tropica SODB1 (LtrFeSODB1), respectively. Similarly, LaeFeSODB2 showed 95.9 and 94.1 and 97.6% identity with LdFeSODB2 and LmFeSODB2 and LtrFeSODB2, respectively. On the other hand, predicted amino acid sequence comparison indicated that LaeFeSODB1 had 91.3, 89.8, and 93.9% identity with LdFeSODB1, LmFeSODB1, and LtrFeSODB1, respectively. The difference in nucleic acid sequence of LaeFeSODB from that of LmFeSODB and LtrFeSODB can be utilized to develop specific molecular methods that help differentiate these species in places where there is an overlap in the distribution of these species. In addition, the data provide information about the situation of L. aethiopica with respect to SODB genes.

The mechanisms by which Leishmaniaparasites survive exposure to highly reactive oxygen (ROS) and nitrogen (RNS) species within phagosomes of macrophages are not well known. Recently it has been shown that RNS alone is sufficient and necessary to control Leishmania donovani infection in mice (Murray, H. W., and Nathan, C. F. (1999) J. Exp. Med. 189, 741–746). No enzymatic defense against RNS has been discovered inLeishmania to date. We have previously isolated two peroxidoxins (LcPxn1 and LcPxn2) from Leishmania chagasiand showed that recombinant LcPxn1 protein was capable of detoxifying hydrogen peroxide, hydroperoxide, and hydroxyl radicals (Barr, S. D., and Gedamu, L. (2001) J. Biol. Chem. 276, 34279–34287). In further characterizing the physiological role of peroxidoxins in Leishmania survival, we show here that recombinant LcPxn1 protein can detoxify RNS in addition to ROS, whereas recombinant LcPxn2 protein can only detoxify hydrogen peroxide. LcPxn1 and LcPxn2 are localized to the cytoplasm, and overexpression of LcPxn1 in L. chagasi parasites enhanced survival when exposed to exogenous ROS and RNS and enhanced survival within U937 macrophage cells. Site-directed mutagenesis studies revealed that the conserved Cys-52 residue is essential for detoxifying hydrogen peroxide, t-butyl hydroperoxide, and hydroxyl radicals, whereas the conserved Cys-173 residue is essential for detoxifying t-butyl hydroperoxide and peroxynitrite. This is the first report of an enzymatic defense against RNS in Leishmania.

For the first time, we have reported the establishment and serial propagation of an axenic culture of Leishmania chagasi amastigote-like forms. Parasites were characterized by microscopic evaluation and by the expression of two stage-specific genes, A2 and Ldccys2 amastigote-specific cysteine protease. The differentiated amastigote-like forms were maintained by serial cultivation.

2,b Significant levels of IgG3 and IgG4 and high levels of IgG1 leishmania-specific antibody differentiated the immune states in 10 patients with visceral leishmaniasis from those of vir- tually all 20 drug-cured and 18 subclinically infected subjects, whereas the level of IgG2 antibody was nondiscriminating. The most extreme "subclinically infected" outlier subse- quently developed disease. Overall, the immune states in subclinically infected and drug-cured persons were mutually indistinguishable but were readily distinguished from those of patients. These findings may have implications for the immunologic mechanism underlying drug cure in visceral leishmaniasis.

Transgenic tobacco plants and calli bearing the bacterialuid A gene under the transcriptional control ofrbcS, mas and CaMV35S promoter(s) were exposed to different concentrations of cadmium. The transcriptional activity of the promoters was monitored using p-nitrophenyl β-D-g1ucuronide as a substrate for the β-glucuronidase (uidA) reporter enzyme. Therbc S promoter was repressed by high concentrations of cadmium. An induction of themas promoter was seen after cadmium treatment of seedlings but not calli. The activity of the CaMV35S promoter was unaffected by cadmium in both seedlings and calli.

Recently we have demonstrated that the human hepatocellular carcinoma-derived cell lines, HepG2 and HuH7, contain gonadotropin-releasing hormone (gonadoliberin) receptors and respond to various molecular forms of gonadoliberin in terms of suppressed proliferation in vitro. This study provides the first demonstration that gonadoliberin inhibits the zinc-induced production of metallothionein mRNA in HepG2 and HuH7 cells. Administration of gonadoliberin agonist (gonadoliberin-A) inhibited the Zn-induced metallothionein mRNA level in a time-related and dose-related manner. The effect of gonadoliberin-A was found to be specific, because concomitant treatment with a gonadoliberin antagonist (gonadoliberin-ANT) blocked gonadoliberin-A inhibition of metallothionein mRNA accumulation. Furthermore, the gonadoliberin-A-induced inhibition of Zn-mediated metallothionein accumulation was found to correlate closely with suppresion of cell proliferation and [3H] thymidine uptake in these cells. It is known that the metal-binding protein metallothionein plays an important role in tumor cell pathobiology and resistance to chemotherapeutic drugs. The present findings may have important implications in the development of an effective chemotherapy for treatment of human liver cancer, in part, by improving the sensitivity of tumor cells through suppression of metallothionein production by gonadoliberin peptides.

In order to study the temporal and spatial regulation of a rat testis-specific heat-shock-like hst70 gene, an 0.8-kb fragment of its upstream DNA was fused to the lacZ gene and microinjected into one-cell murine embryos. Independent tgHST1 and tgHST2 transgenic mice strains were established, containing about 5-7 and 40-60 transgene copies/haploid genome, respectively. Enzyme assays in various tissues showed that transgene-encoded beta-galactosidase accumulates exclusively in testes of transgenic animals and cannot be detected until 16-17 days after birth. In-situ assays revealed that the enzyme accumulates mainly in pachytene primary spermatocytes. Our data complement previous studies on the endogenous rat hst70 and suggest that its 0.8-kb upstream region contains sufficient information to function as an active spermatogenesis-specific promoter.

A rainbow trout major heat-shock-protein-like gene (hsp 70) and corresponding cDNA clones were isolated by hybridization to heterologous hsp70 probes. DNA sequencing revealed that this gene is structurally similar to a mammalian heat-shock-cognate hsc70 gene and consists of eight introns. Northern blot and primer extension analyses showed that the corresponding mRNA is constitutively abundant in different trout tissues and salmonid cell lines. Fragments of the isolated gene containing the -900 - +30 and -217 - +58 sequence were linked to a bacterial chloramphenicol acetyltransferase reporter gene and transiently transfected into salmonid cells. The expression pattern of these constructs supports our conclusion that the isolated genomic and cDNA clones correspond to a trout heat-shock-cognate hsc70 gene.

The metallothionein (MT) gene expression profile was followed in rainbow trout during early embryo development and in liver and gonads during the period of sexual maturation. The hepatic MT mRNA levels increase at the end of sexual maturation in both male and female rainbow trout. Although both isoforms of MT mRNA accumulate in the liver, there is a preferential increase in MT-A in the female liver. Concomitantly with this increase in MT there is a redistribution of zinc and copper to MT. In the juvenile female there is an abundance of MT mRNA in the ovaries. This is correlated to high levels of zinc in the MT fraction upon Sephadex G-75 chromatography. During ovary development the MT mRNA levels and the MT-bound zinc levels drop, with an increase in zinc being bound to high-molecular-mass proteins. At ovulation most of the zinc is found in the membrane portion upon centrifugation. In contrast to the ovaries, there are no apparent changes in either trace metal distribution or MT mRNA levels during testis development. In the developing embryo there is an increase in MT-bound copper at gastrulation. This is accompanied by an increase in both isoforms of MT mRNA. At hatch both the copper and zinc levels increase in the MT fraction, with a concomitant increase in mainly MT-A mRNA. These findings indicate that the variations in MT mRNA levels during development are closely associated with metal regulation.

A lambda phage recombinant clone, 25 S, which contains a 15.5-kb EcoRI human genomic DNA fragment, has been characterized. Restriction mapping and Southern blot hybridization indicated a 3.0-kb HindIII fragment containing metallothionein (MT)-like sequences. Several interesting features were found upon comparison of this nucleotide sequence with that of other human MT genes: (1) sequences representing the 5' regulatory region, the 5' untranslated region, and the first exon are not contained in the 3.0-kb HindIII fragment; (2) the coding sequence of the second exon (amino acids 10-31 encoding a portion of the beta-domain of the MT protein) has 11 amino acid changes out of a total of 21, whereas, the third exon (amino acids 32-61, representing the complete alpha-domain of the MT protein) has only 4 amino acid substitutions; however, all cysteine residues are conserved; (3) this MT-like gene retains intron sequences and processing signals; (4) Southern blot analysis of human genomic DNA indicated this MT-like gene is located on a 10.5-kb EcoRI genomic DNA fragment; and (5) unusual AG/CT-rich repetitive elements are located within the second intron and upstream of the second exon of this MT-like gene. This gene is not expressed in response to metal induction in two human cell lines, as shown by northern blot analyses. Based on these observations, this MT-like gene represents a unique nonprocessed pseudogene of the human MT multigene family.

Two isoforms of metallothionein (MT) have been isolated from rainbow trout livers following CdCl2 injections. These MTs have been identified by standard procedures and appear to be similar to mammalian MTs. Total RNA from such induced livers was shown to contain high levels of MT-mRNA activity when translated in cell free systems. This activity was demonstrated to be in the 8 to 10S region of a sucrose gradient. The RNA fractions also showed homology to a mouse MT-I cDNA probe. The exposure of rainbow trout hepatoma (RTH) cells to various concentrations of CdCl2 and ZnCl2 induced the expression of MT and MT-mRNA. Exposure of Chinook salmon embryonic (CHSE) cells to these metals, however, did not result in MT synthesis, suggesting that the MT genes have not become committed to transcription. Instead, an unknown low molecular weight (MW = 14 kDa) protein was induced. This metal-inducible protein (MIP) was capable of binding 109Cd and was stable to heating, while the binding of the metal to this protein was not. These characteristics have been reported for a protein induced in rainbow trout liver following environmental exposure to cadmium. We suggest that both MT and MIP may function in detoxification of heavy metals. Images FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 7.

Poly(A)+ protamine mRNA's were isolated from rainbow trout testes and deadenylated by treatment with calf thymus RNase H. Four subcomponents of deadenylated PmRNA (PmRNA1-4) were purified by electrophoresis on a 6% polyacrylamide gel in 8 M urea. Translation of each PmRNA subcomponent in the wheat germ S-30 cell-free system showed that all subcomponents are biologically active but each codes for two or more protamine polypeptides suggesting molecular heterogeneity. However, the deadenylated mRNA's can be categorized into two groups based on the spectrum of protamines whose synthesis they stimulate.

We have synthesized a family of double-stranded cDNAs (ds cDNAs) using as a template the family of highly purified protamine mRNAs from rainbow trout testis. Individual pure protamine cDNA components were isolated by cloning this family of protamine ds cDNAs in a plasmid vector (pMB9). Clones containing protamine sequences were characterized by restriction mapping and by a positive hybrid-selected translation assay, which allowed us to correlate particular cDNAs with particular protein components. To allow more detailed comparisons, complete nucleotide sequences were determined for selected protamine clones. We have detected at least 5 distinctly different coding sequences, which nevertheless show at least 82% homology, and which have probably arisen by repeated gene duplication. These very highly conserved coding sequences do however contain a distinctly variable region near the 5'-end of the mRNA (N-terminus of the protein), corresponding to the major sites of serine phosphorylation. Since the amino acid sequences predicted by our DNA sequences were slightly different from those previously published (1), we have independently determined the amino acid sequences of protamine components CI, CII, CIII from our own source of trout testis. These new peptide sequences are completely consistent with those predicted by our nucleotide sequences. The 3'-untranslated regions of the protamine mRNAs are, surprisingly almost as highly conserved as the coding regions. Both coding and 3'-noncoding portions appear to be under a similar degree of selective pressure and evolutionary constraint to remain constant.

A variety of genes have been shown to change copy number during development, including rRNA genes in amphibians and chorion proteins in insects. Dihydrofolate reductase and metallothionein-1 (MT-1) genes are present in high copy number in cultured mammalian cells subjected to low levels of agents that will select for cells with amplified copies of specific genes. Recent studies have shown that the metallothionein-1 gene in mouse liver is regulated at the transcriptional level by treatment with heavy metals. We report here that, at cadmium concentrations 5 to 10-fold higher than that required to induce maximal transcription of the MT-1 gene, there is a 2 to 3-fold increase in MT-1 gene concentration in liver nuclear DNA by 6 hours after induction, and extra copies persist up to 3 weeks in the absence of further heavy metal treatment. The extra MT-1 gene copies that appear 6 hours after cadmium treatment are in a conformation that renders them relatively nuclease insensitive.

Micrococcal nuclease has been shown to preferentially cleave chromatin in the region of genes actively engaged in transcription. We have used this preferential cleavage to show that the metallothionein (MT) gene in adult mouse liver, when induced to produce mRNA by injection of cadmium, becomes more susceptible to nuclease cleavage. However, the MT gene in uninduced liver, and the alphafoetal protein (AFP) gene in both induced and uninduced liver, remain relatively resistant to nuclease cleavage. The AFP gene is not normally expressed in cadmium induced or uninduced liver. Thus, susceptibility of genes to nuclease cleavage appears to rise with increasing transcription of the gene.

We have isolated, using nick-translated cloned protamine cDNA's as probes, several genomic clones containing protamine gene sequences from a Charon 4A library of Eco R1 digested rainbow trout (Salmo gairdnerii) DNA. One clone was chosen for detailed study and the 2.5 kbp Bam HI-Eco R1 restriction fragment containing the gene was subcloned in the plasmid pBR322. A 920 bp Bg1 II - Bam HI restriction fragment contains a sequence coding for protamine component CII as well as regions 5' and 3' to the mRNA coding portion. Present in the region 5' to the mRNA coding sequence are the promoter associated signals "TATA" box and "CAAT" box. The 5' untranslated region of the mRNA whose length and sequence were not established from the cDNA clones (1) was determined by nuclease mapping and starts within a sequence similar to the "capping signal" found in other genes. The protamine gene for CII contains no introns, a situation common to most histone genes, but, unlike the histone genes does not occur close to other protamine genes in a "cluster".

Human metallothioneins (MT) are represented by a multigene family. Many of these genes are expressed differentially and in a cell type/tissute specific manner in response to a variety of inducers including heavy metals (eg. Cd, Zn, Cu) and dexamethasone (Dex) (1). One of the MT genes, MT-IC (2) is expressed in a hepatoblastoma cell line (Hep-G2) in response to heavy metals but not in a lymphoblastoid cell line (Wi-L2). This is shown in Fig.l (panel A) where a Northern blot of total RNA (5Ag) from these cell lines was probed with a nick translated MT-I0 specific DNA probe derived from its 3'-terminus. When a similar blot was hybridized to an RNA probe derived from the same region of the DNA, hybridization to the Wi-L2 RNA was also seen (Fig.l, panel B). This was demonstrated to be due to nonspecific hybridization of the MT-I RNA probe to the MT-IIA mRNA because of a region of 34 nucleotides (&70% GC) which is partially homologous within these genes (3). Since RNA/RNA hybrids of high GC contents are very stable it was difficult to melt this region even under stringent conditions of hybridization (3xSSC; 650C; 50% formamide) and washings (up to O.lxSSC; 650C). To eliminate this nonspecific hybridization due to high GC contents, the RNA probe was sythesized (4) in the presence of inosine 5'-triphosphate (ITP replacing GTP). When this probe was used, nonspecific hybridization was completely eliminated (Fig.l, panel C) even under less stringent conditions of hybridization (3xSSC; 65'C) and washings (up to lxSSC; 65°C). Figure 2 shows that even though the size of the RNA synthesized in the ITP containing reaction (I) is the same as that from the reaction containing GTP (G) (suggesting specific transcription initiation), the extent of its synthesis is reduced. It can , however, be increased by incubating the ITP containing reaction for up to 3 h with additional supplements of ITP and RNA3 olymerase. Under these conditions the extent of radioactive incorporation6(a P-UTP) 6 was increased up to -30% of the GTP containing reaction (12x10 versus 37x10 cpm from reactigns containing 0.5 Ag of the linearized DNA template). Since only about lxlO cpm/filter were used in hybridization, the reduced extent of RNA synthesis is not a limitation.