Lashitew Gedamu

Analysis of stress-induced gene expression in fish cell lines exposed to heavy metals and heat shock

We have examined the effect of heavy metals on the expression of two major groups of stress-induced proteins in fish cell lines: the 70 kDa heat-shock proteins (hsp70) and metallothioneins (MTs). The rainbow trout hepatoma (RTH) cell line synthesized the hsp70 protein in response to zinc and heat shock, while chinook salmon embryonic (CHSE) cells synthesized this protein in response to these inducers, as well as cadmium. The synthesis of this 70 kDa protein was correlated with the accumulation of hsp70 mRNA as measured by hybridization to a trout hsp70 gene probe. Heavy metals also induced the synthesis of MT in RTH cells. However, heat shock did not result in induction of MT and its mRNA. Unlike RTH cells, CHSE cells did not synthesize MT following exposure to cadmium or zinc. When these cells were treated with 5-azacytidine prior to heavy metal treatment, accumulation of MT mRNA was observed. Northern blot analysis of total RNA from 5-azacytidine treated CHSE cells, using a trout MT (tMT-B) cDNA probe, indicated that the time-course of induction and the maximal level of MT mRNA accumulation in response to cadmium and zinc paralleled that observed in RTH cells. Copper and dexamethasone were ineffective in inducing MT mRNA in 5-azacytidine-treated CHSE cells. These results indicate that MT is specifically induced in response to heavy metal treatment, whereas the synthesis of hsp70 appears to be a general stress response. Furthermore, MT is differentially regulated by heavy metals and dexamethasone in these cell lines and the expression of MT is cell-type-specific.

The ontogeny of expression of murine metallothionein: comparison with the α-fetoprotein gene

The ontogeny of expression of mouse metallothionein was studied by RNA dot and Northern blot hybridization using a cloned cDNA probe. In some instances the synthesis of metallothionein was analyzed by cell-free translation of RNA as well as pulse-labeling of proteins in short-term organ cultures followed by polyacrylamide gel electrophoresis. Interesting parallels between metallothionein and alpha-fetoprotein gene expression during development were noted. Like alpha-fetoprotein mRNA ( Dziadek and Andrews, 1983), metallothionein mRNA was found to be abundant in developing liver as well as in visceral yolk sac endoderm. In addition, metallothionein mRNA was abundant in parietal yolk sac. During liver development metallothionein and alpha-fetoprotein mRNAs were abundant by Day 12 of gestation, increasing to maximal levels on Day 16 and decreasing during late fetal and neonatal life to basal levels in adult. Metallothionein mRNA increased in maternal liver and was also abundant in certain hepatomas. Synthesis of metallothionein and levels of metallothionein mRNA in visceral yolk sac increased from Day 9 of gestation to maximal levels on Days 11-12 and then decreased abruptly after Day 15. RNA from differentiated teratocarcinoma cells with primitive, parietal or visceral endoderm characteristics each contained high levels of metallothionein mRNA, whereas, levels of this mRNA varied widely among embryonal carcinoma stem cell lines. alpha-Fetoprotein mRNA was not detected in embryonal carcinoma cells but was expressed in visceral endoderm-like differentiated cells. These results indicate that parietal and visceral endoderm cells actively express the metallothionein gene and further suggest that expression may be initiated at the earlier stage of primitive endoderm.

Functional analysis of cathepsin B-like cysteine proteases from Leishmania donovani complex. Evidence for the activation of latent transforming growth factor beta.

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.

Cloning, characterization and overexpression of two iron superoxide dismutase cDNAs from Leishmania chagasi: role in pathogenesis.

We have isolated and characterized two superoxide dismutase (SOD) cDNAs from a Leishmania chagasi promastigote cDNA library using degenerate primers derived from conserved amino acid residues of previously isolated manganese and iron SODs. Comparison of these two L. chagasi SOD deduced amino acid sequences with previously isolated MnSOD and FeSOD amino acid sequences revealed that they have higher homology to, and complete conservation of, invariant residues found in iron-containing SODs. Southern blot analysis showed that one gene, L.c.FeSODA, is a single copy gene, whereas the other gene, L.c.FeSODB, belongs to a multi-gene family. Transcript levels and enzyme activities of L.c.FeSODA and L.c.FeSODB show differential stage expression, with higher levels present in the amastigote stage of the parasite compared to the promastigote stage. Expression of the L.c.FeSODs in an E. coli SOD null strain protected the bacteria against free radical generating agents. Overexpression of these FeSODs in L. chagasi parasites also showed enhanced protection against the free radical generating agents, paraquat and nitroprusside. The cloning, characterization and overexpression of the L.c.FeSODA and L.c.FeSODB genes and proteins demonstrates the possible role of SODs in Leishmania pathogenesis.

Induction of metallothionein synthesis in rainbow trout,Salmo gairdneri, during long-term exposure to waterborne cadmium

Rainbow trout were exposed to 200 μg cadmium/l in the water during four months at 6–10°C. The liver, kidney and gills were analyzed for cadmium, copper, zinc, metallothionein and metallothionein mRNA. Cadmium accumulated in all three organs and reached the highest concentration in the kidney. The tissue zine and copper concentrations showed no major alterations during the experiment. The cytosolic distribution of cadmium, copper and zinc was followed during four months of exposure by Sephadex G-75 chromatography. It was found that cadmium was predominantly associated with proteins of an apparent molecular vieght of 10,000 daltons. These proteins were further identified as metallothioneins after fast protein liquid chromatography on a Mono-Q column. The metallothionein concentration was significantly higher in liver of exposed fish than in control fish after only one month. The kidneys reached significantly elevated levels of metallothionein in the exposed group after three months. In the gills, elevated metallothionein concentrations were observed after four months of exposure. After four months of exposure, the metallothionein mRNA content of liver and kidney was analyzed using a rainbow trout anti-sense RNA probe. Elevated MT mRNA levels were observed in both kidney and liver. These results demonstrate thatde novo synthesis of metallothionein is induced by cadmium in rainbow trout after exposure to the metalvia water.

Endogenous and heavy-metal-ion-induced metallothionein gene expression in salmonid tissues and cell lines.

Endogenous levels of metallothionein (MT) mRNA were detected by RNA probes in several somatic and germ-line tissues of rainbow trout, such as eggs, ovaries and immature testis. These levels may be related to metal-ion homeostasis in the observed tissues. The induction kinetics of trout MT isoform B (MT-B) mRNA were studied after single intraperitoneal injections of CdCl2, CuCl2 and ZnCl2. MT-B mRNA was induced within 12 h in liver, kidney, spleen and gills. However, over the 48-h experimental period, the kinetics of MT-B mRNA accumulation differed in response to the three metal salts, possibly due to differential handling of the salts by these tissues. Multiple metal-salt injections induced high levels of MT-B mRNA in the four tissues studied. In the rainbow trout hepatoma cell line, ZnCl2 was a better inducer of the MT-B gene, as compared to CdCl2 and CuCl2. The expression of the exogenous trout MT-B promoter in Chinook salmon embryonic cell line indicates the presence of MT regulatory factors. In contrast, the endogenous MT genes in these cells are quiescent, possibly due to the methylation of their promoter region.

Iron Superoxide Dismutases Targeted to the Glycosomes of Leishmania chagasi Are Important for Survival

ABSTRACT Kinetoplastid glycosomes contain a variety of metabolic activities, such as glycolysis, β-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.

Metallothionein gene expression in fish cell lines: Its activation in embryonic cells by 5-azacytidine

We have investigated the regulation of metallothionein gene expression in two fish cell lines. Rainbow trout hepatoma (RTH) cells synthesized metallothionein in response to heavy metal exposure. The maximum level of metallothionein synthesis detected during zinc exposure was much greater than during cadmium exposure. The time-courses of metallothionein synthesis were different for the different metal inducers, suggesting that metallothionein may be differentially regulated by cadmium and zinc in these cells. The metal-induced synthesis of metallothionein was correlated with increased translational activity and accumulation of metallothionein-mRNA, suggesting that metallothionein may be regulated at the transcriptional and post-transcriptional levels in RTH cells. Chinook salmon embryo (CHSE) cells, unlike RTH cells, did not synthesize metallothionein or metallothionein-mRNA in response to heavy metal exposure. However, when these cells were treated with 5-azacytidine prior to heavy metal exposure, the synthesis of metallothionein was induced, suggesting that DNA methylation may play a role in metallothionein gene expression in fish.

Molecular cloning and characterization of two iron superoxide dismutase cDNAs from Trypanosoma cruzi.

Two cDNAs (FeSODA and FeSODB cDNAs) corresponding to superoxide dismutase (1.15.1.1., SOD) were isolated from a Trypanosoma cruzi cDNA library. Comparison of the deduced amino acid sequences with previously reported SOD protein sequences revealed that the T. cruzi open reading frames had considerable homology with FeSODs. The coding region of the T. cruzi FeSODB cDNA has been expressed in fusion with glutathione-S-transferase using an Escherichia coli mutant QC779, lacking both MnSOD and FeSOD genes (sodA sodB). Staining of native polyacrylamide gels for SOD activity of T cruzi crude lysate and the recombinant SOD suggests that this protein is an FeSOD. The recombinant enzyme also protected the E. coli mutant QC779 from paraquat toxicity. Northern blot analysis showed that FeSODB is differentially expressed, showing a higher level at the epimastigote stage of T. cruzi development; whereas, FeSODA is constitutively expressed at a lower level in all developmental stages. Furthermore, Southern hybridization shows that both FeSODA and FeSODB genes appear to be present in the T. cruzi genome as multiple repeating units (multi-copy gene family).

Differences in metallothionein gene expression in primary cultures of rainbow trout hepatocytes and the RTH-149 cell line

Primary cultures of rainbow trout, Salmo gairdneri, hepatocytes were used to study the expression of metallothionein (MT) genes in response to steroid hormone treatment. The expression pattern was compared to that of an immortal cell line (RTH-149). MT mRNA accumulated in both cell cultures after exposure to zinc while 17 beta-oestradiol had no effect in either system. Treatment with cortisol and corticosterone resulted in a 2-fold increase of metallothionein mRNA levels in the primary cultures but had no effect in the RTH-149 cell culture. Primary cultures that were exposed to zinc or cortisol showed a high temporal correlation (r = 0.974) between MT mRNA and MT protein levels. The basal level expression was 3-4-fold higher in primary cultures than in RTH-149 cells. The present study demonstrates the inducibility of rainbow trout MT genes in response to glucocorticoids. It further indicates that primary cultures are to be preferred to immortal cell lines when investigating the inducibility of MT mRNA.