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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.

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)-β1 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-β1 was biologically active, suggesting thatLeishmania cathepsin B can cleave latent TGF-β1 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-β1.

Antioxidants have been implicated in protecting cells from oxygen radicals produced as a result of aerobic metabolism and in response to foreign pathogens by phagocytic cells. The mechanisms allowing pathogens to withstand the toxic prooxidant environment within the phagolysosome are poorly understood. We have cloned and characterized three antioxidant genes belonging to the 2-Cys family of peroxidoxins from Leishmania chagasi that may prove to provide these parasites with an enhanced defense mechanism against toxic oxidants. The 5′-untranslated regions and coding regions of each gene are highly conserved, whereas the 3′-untranslated regions have diverged significantly. L. chagasi peroxidoxin 1 (LcPxn1) is predominantly expressed in the amastigote stage, whereas LcPxn2 and LcPxn3 are expressed mainly in the promastigote stage, with LcPxn3 being far less abundant than LcPxn2. LcPxn2 and LcPxn3 possess a nine-amino acid extension at the carboxyl terminus, which LcPxn1 lacks. LcPxn1 appears to exist as high molecular weight multimers in vivo, and recombinant LcPxn1 was shown to detoxify hydrogen peroxide and alkyl hydroperoxides. We also present strong evidence that recombinant LcPxn1 can enzymatically detoxify hydroxyl radicals, an activity never before clearly demonstrated for a protein.

We have analyzed the human (h) metallothionein (MT)-IG proximal promoter region (−174 to +5) using a TATA box mutation (TATCA) and four trinucleotide mutants of the proximal MREa. Transient transfection of HepG2 cells was complemented by in vitro transcription with rat liver nuclear extracts. In both systems, mutations of the TATA box and conserved core of metal responsive element (MRE)a were detrimental to hMT-IG promoter activity suggesting that both elements make significant contributions to hMT-IG transcription. Although MRE binding factors were active in vitro, further metal activation of MT promoter activity was accomplished only by in vivo metal treatment rather than addition of zinc in vitro. Southwestern blotting identified nuclear proteins in rat liver and HepG2 cells which physically interact with MREa in a zinc-dependent manner and could be responsible for MREa function in each system. In addition, the functional effects of the TATCA mutation correlate with altered physical interaction with TATA box-binding protein as observed using DNase I protection.

In this study, the contributions of the two metal-responsive elements (MREs) of the rainbow trout (Salmo gairdnerii) metallothionein (tMT)-B gene promoter (-137 to +5) were analyzed. The effect of MRE mutations on the basal and zinc-induced activities of tMT-B promoter-reporter gene fusions were determined by transfection of a rainbow trout hepatoma (RTH-149) cell line. Together, MREa and MREb cooperate to elicit a significant response to zinc but exhibit differential basal and metal-induced activity. The MREa sequence (-62 to -51) is important for basal promoter activity and can function independently, whereas the more distal MREb (-89 to -100) mainly contributes to metal induction through cooperative interactions with MREa. The degree of basal character of the MREs is partially determined by nucleotide differences at the flexible position N of the MRE consensus TGC(G/A)CNC. In mouse L and HepG2 cells, MREa activity is conserved, but the contributions of the MREb region differ, including reduced cooperativity with MREa. There are also differences in the apparent molecular masses of the rainbow trout and mammalian nuclear factors that bind to the tMT-B promoter and MREa sequence.

In this report, we have measured the cadmium (Cd2+)-induced expression of all known metallothionein I (MT-I) mRNAs in a human hepatoma cell line, Hep G2. Among the human MT-I gene family promoters, marked sequence conservation exists; despite this, the mRNA accumulation level for each species was found to be quite unique. This differential Cd2+ induction of MT-I family members provides an ideal opportunity to assess whether the characteristic response results from subtle isoform-specific variations in promoter structure. Accordingly, we have examined the mechanism for differential expression of two isoforms, MT-IG and MT-IF, by transient transfection into Hep G2 cells. In the presence of Cd2+, MT-IG promoter activity and endogenous mRNA level were, respectively, 4.7- and 3-fold greater than those of MT-IF. This close correlation between promoter activity and mRNA accumulation strongly suggests that differential expression occurs at the level of transcription. The difference in Cd(2+)-stimulated activity was found to be conferred by 240- and 243-base pair promoter fragments spanning nucleotides -174 to +66 and -172 to +71 of the MT-IG and MT-IF genes, respectively. One of the most striking nonhomologies between the promoters is a single A (TATAAA) to C (TATCAA) transversion in the TATA motifs of MT-IG and MT-IF genes, respectively. To determine whether such a subtle change in the TATA motif could account for the marked differences in promoter function, we constructed MT-IG-TATCA and MT-IF-TATAA promoters and measured their activities in transient transfection and cell-free transcription assays. Results of both assays showed a profound difference between the two motifs that paralleled the difference in Cd(2+)-stimulated MT-IG and MT-IF mRNA levels. In summary, we have shown that differential regulation of two MT-I promoters is primarily due to a single base alteration in their TATA motifs.

The sequences responsible for heavy metal-inducible expression are situated within the proximal 437 and 160 base pairs (bp) of MT-IF and MT-IG 5'-flanking sequence, respectively. Only 105 bp of proximal MT-IG 5'-flanking sequence containing a TATA box, two metal responsive elements (MREs), and three GC motifs and 147 bp of proximal MT-IF 5'-flanking sequence containing a TATCA box, four MREs, and two GC motifs were required for heavy metal-inducible expression. However, the proximal 111 bp of MT-IF 5'-flanking sequences (a TATCA box, two MREs, and two GC motifs) was not responsive to heavy metals and competes less efficiently than the 105-bp MT-IG fragment in a competition transfection analysis. The MT-IF promoter fragment containing MREc and MREd is substantially stronger and a more efficient competitor than the MT-IG promoter fragment containing MREc and MREd. Furthermore, the proximal 160 bp of MT-IG 5'-flanking sequence functions as a strong metal-inducible promoter but not as a metal-inducible enhancer. Mobility shift analysis of MT-IF and MT-IG promoter subregions suggests a correlation between protein binding to MRE sequences and MT gene expression. These data illustrate that the overall structural and functional organization of the MT-IF and MT-IG promoters are very different and that the molecular mechanisms governing differential expression levels of human MT genes are quite complex.

The expression of three human metallothionein genes, MT-IIA, MT-IF, and MT-IG was studied in the human hepatoblastoma (HepG2), the hepatocarcinoma (Hep3B2), the embryonic kidney (Hek 293), and the lymphoblastoid-derived (Wi-L2) cell lines. The pattern of expression of each specific MT gene in response to various heavy metals was different among the four cell lines studied indicating differential regulation of MT gene expression. The MT-IF or MT-IG and the MT-IIA genes were regulated in a cell-type specific manner in response to heavy metals and dexamethasone, respectively. DNA methylation was shown to be correlated to cell-type specific regulation of MT gene expression since 5-azacytidine treatment resulted in the expression of the MT-IF and MT-IG genes in response to cadmium and zinc in Wi-L2 cells, of the MT-IIA gene in response to dexamethasone in Wi-L2 cells, and of the MT-IG in response to zinc and copper in Hek 293 cells. Furthermore, transfection studies indicated that all the trans-acting factors necessary for the expression of these genes were present and functional in Wi-L2 and Hek 293 cells. The differential level of expression of the MT-IF and MT-IG genes in response to heavy metals in the Hek 293 cell line was shown to be correlated to their chromatin structure.

At least two classes of protamine mRNA are present in both trout testis polysomal RNA and RNA from the postribosomal supernatant fraction of trout testis hormogenate both of which direct the synthesis of protamine in a Krebs II ascites S-30. One contains poly(A) tracts and the other is devoid of poly(A). Sucrose gradient analyses showed that the poly(A) containing protamine mRNA (poly(A) (+)) sedimented IN THE 6 S region with a shoulder in the 4 S region while the protamine mRNA devoid of poly(A) (poly(A) (-)) appeared to sediment at about 4 S and could not be resolved from tRNA. Analysis of the poly(A) (+) protamine mRNA by boundary sedimentation in an analytical ultracentrifuge showed a sedimentation coefficient of 5.7 S, a value which gives rise to an estimate of 165 to 170 nucleotides per molecule. The poly(A) (+) protamine mRNA migrated as a single species in formamide-containing polyacrylamide gels and its mobility in relation to markers of tRNA (4 S) and 5 S RNA was consistent with its sedimentation velocity of 6 S. The RNA present in the major band on an aqueous polyacrylamide gel was extracted and shown to code for protamine in a wheat germ cell-free system.

We have analyzed the pattern of expression of three human metallothionein (MT) genes (MTI-F, MTI-G, and MTII-A) in the hepatoblastoma cell line, HepG2, in response to the metal ion inducers cadmium, copper, and zinc. The absolute number of transcripts of each of the three genes were measured, and the data clearly suggest differential regulation of these members of the MT gene family by the different inducers both in terms of the rate and the extent of transcript accumulation. The lowest levels of transcript accumulation was observed for MTI-F gene (maximum 4,000 molecules of mRNA per cell) and copper was shown to be its poorest inducer (up to 2,000 molecules per cell). Cadmium is the poorest inducer of MTI-G gene even though transcripts of this gene accumulated at comparatively higher levels than those of MTI-F. Copper- and zinc-induced MTI-G transcript accumulation was up to 12,000 molecules per cell whereas the corresponding value for cadmium was only 4,500. MTII-A was the only gene expressed in the absence of any externally added inducer. Also, in contrast to the MTI genes, the MTII-A gene was equally responsive to all the metal ions tested and the induced levels of accumulation were much higher (up to 75,000 molecules of MTII-A mRNA per cell).

Elevation of the incubation temperature of Chinook salmon embryo cells from 20 to 24 degrees C or exposure to heavy metals such as CdCl2 (5 microM) or ZnCl2 (100 to 500 microM) induces the reversible expression of a set of heat shock or stress proteins. Continuous exposure of the cells to either metal ions or heat shock results in recovery of protein synthesis to a control-like pattern. Treatment of these cells with either ZnCl2 or CdCl2 also induces the protein metallothionein. Heat shock, however, does not induce metallothionein, suggesting that it does not belong to the common group of heat shock or stress proteins. The induction of these stress proteins can be inhibited by pretreatment with actinomycin D, suggesting that their expression is regulated at the transcriptional level. The major stress proteins are detectable in the products of an in vitro translation system programmed with RNA isolated from heat shock- or metal ion-treated cells. A recombinant DNA probe complementary to Drosophila mRNA coding for the 70,000-dalton heat shock protein was found to hybridize to RNA isolated from heat shock-or metal ion-treated cells but not from control cells. The fish mRNA coding for the heat shock protein with a molecular weight of 70,000 appears to be of similar size to the corresponding Drosophila mRNA.

The human metallothionein (MT)-IG gene (hMT-IG) is tandemly linked in a head-to-head fashion with the hMT-IF gene. The hMT-IG gene encodes a MT-I polypeptide and has a tripartite structure. The 5'-flanking region of the hMT-IG gene has a TATAA box, four GC motifs, and at least four metal responsive elements. The 3'-untranslated region has a variation of the polyadenylation signal, AATTAA, and the 3'-flanking region a YGTGTTYY RNA processing signal. This gene is expressed in hepatoma-derived cell lines (Hep G2 and Hep3B2) in response to the heavy metals (cadmium, copper, and zinc) but not to the glucocorticoid analogue dexamethasone. In contrast, the lymphoblastoid cell line (Wi-L2) does not express the hMT-IG gene. These results suggest that the hMT-IG gene is regulated differentially and in a cell type-specific manner. Transient expression studies of the chloramphenicol acetyltransferase (CAT) gene under the transcriptional control of either the hMT-IG or hMT-IF promoter in Hep G2 cells has demonstrated that both promoters contain all the necessary cis-acting elements to elicit a similar pattern of heavy metal inducibility. However, the hMT-IG promoter in all instances is five times more active than the hMT-IF promoter. The differences in promoter activity of these genes could possibly be due to inherent differences in their basal level regulatory sequences. The expression of MT-IGcat in transfected Wi-L2 cells demonstrates that the hMT-IG promoter is not cell type-specific.