PubMed Health⌕ Search

Biomedical subjects

L Gedamu

Publications and source records attributed to L Gedamu.

At least 37 records · Page 2Linked to original sources
PubMed1994

Functional specificity for salmon gonadotropin-releasing hormone (GnRH) and chicken GnRH-II coupled to the gonadotropin release and subunit messenger ribonucleic acid level in the goldfish pituitary.

GnRH is the key regulator of reproduction in the vertebrates. In this study, we investigated the release and synthesis of maturational gonadotropin hormone (GTH-II) stimulated by native GnRH forms, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), in the goldfish pituitary. The experimental approach was to study the differences between desensitization induced by sGnRH and cGnRH-II administered in homologous and heterologous fashion. Pulsatile alternate treatments with sGnRH and cGnRH-II (i.e. sGnRH/cGnRH-II or cGnRH-II/sGnRH) at 10(-8) M (every 30 min) resulted in a lower degree of desensitization compared with homologous treatments with either sGnRH or cGnRH-II (sGnRH/sGnRH or cGnRH-II/cGnRH-II), or when combined together (sGnRH+cGNRH-II). We also investigated the effects of continuous treatments with sGnRH and cGnRH-II, administered in a homologous or heterologous fashion. Increasing concentrations of either sGnRH or cGnRH-II (10(-8)-10(-6) M) administered continuously (60 min) in a homologous fashion resulted in significant desensitization of the pituitary GTH-II release. Alternate continuous treatments with sGnRH and cGnRH-II (i.e. sGnRH/cGnRH-II/sGnRH or cGnRH-II/sGnRH/cGnRH-II) resulted in lower degree of desensitization compared to homologous treatments, particularly at lower doses. We further investigated the effects of sGnRH and cGnRH-II on GTH-II beta and GTH-II alpha subunit messenger RNA (mRNA) levels in the goldfish pituitary. In sexually regressed animals, sGnRH treatment (4 micrograms/fish) increased the accumulation of GTH-II beta and GTH-II alpha mRNA, whereas cGnRH-II treatment was without effect. In sexually mature animals, however, both cGnRH-II and sGnRH stimulated accumulation of GTH-II beta and GTH-II alpha mRNA, with cGnRH-II exerting a greater effect on GTH-II subunit mRNA production. These results suggest a differential control of GTH-II subunit gene expression or mRNA stabilization by sGnRH and cGnRH-II in the goldfish pituitary based on the stage of gonadal recrudescence. In general, the present findings support the hypothesis that sGnRH and cGnRH-II regulate the release and synthesis of GTH-II through different receptor-effector mechanisms in the goldfish pituitary.

Animals↗
PubMed1993

Distinct TATA motifs regulate differential expression of human metallothionein I genes MT-IF and MT-IG.

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.

Base Sequence↗
PubMed1993

An upstream region of the rat spermatogenesis-specific heat-shock-like Hst70 gene confers testis-specific expression in transgenic mice.

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.

Animals↗
PubMed1993

Basal and inducible expression of metallothionein and heat shock protein 70 genes in bovine articular chondrocytes.

Human metallothionein (MT) and heat shock protein 70 (hsp70) cRNA probes cross-hybridized with the respective bovine mRNAs under high-stringency conditions, suggesting DNA sequence conservation of the two genes. Released primary bovine articular chondrocytes expressed MT and hsp70 mRNAs constitutively at variable levels, suggesting a possible physiological role of these proteins in cartilage. In first-passage chondrocytes both CdCl2 and ZnCl2 induced MT and hsp70 mRNAs. However, CdCl2 and ZnCl2 were better inducers of hsp70 and MT mRNAs relative to CuCl2. Serum, interleukin-1, and dexamethasone also induced MT but not hsp70 mRNAs. Actinomycin D severely reduced the basal and metal-induced expression of MT and hsp70 mRNAs, suggesting transcriptional control. Inclusion of cycloheximide, an inhibitor of protein synthesis, along with the metal inducers did not influence hsp70 induction but resulted in superinduction of MT mRNA, possibly due to the post-transcriptional stabilization of polysomes. MT and hsp70 induction by metals is therefore independent of de novo protein synthesis. These results demonstrate the potential of articular chondrocytes to express mRNAs for the two stress proteins in response to various physiologically relevant agents by transcriptional and post-transcriptional mechanisms. MTs and hsp70 are likely to have important functions in cartilage metabolism under normal and pathological conditions.

Animals↗
PubMed1993

Distinct expression pattern of early- and late-response genes in normal and osteoarthritic human synovial membranes.

The significance of activating proteins (AP-1), c-fos, c-jun and jun B relative to the AP-1 responsive metallothionein, collagenase and stromelysin gene expression in the pathophysiology of osteoarthritis (OA) was investigated. The 'early' c-fos, c-jun and jun-B mRNAs were ubiquitously expressed in normal and OA human knee synovial membranes. There was no strict correlation between expression of these and the AP-1 responsive, collagenase and stromelysin gene expression. Interestingly, the total metallothionein (MT) and the AP-1 responsive, MT-IIA gene-specific mRNAs were greatly diminished in OA compared with normal synovial membranes. The possible role of reduced expression of MT and trace metals in OA pathophysiology is discussed. Collectively, these data demonstrate a discoordinate expression of AP-1 encoding and their target genes in synovium.

Aged↗
PubMed1992

Expression of c-fos, c-jun, jun-B, metallothionein and metalloproteinase genes in human chondrocyte.

Normal and osteoarthritic (OA) human articular cartilage chondrocytes, released enzymatically in the presence of 0.5% fetal calf serum, display constitutive expression of early response activating protein (AP-1) genes; c-fos, c-jun and jun-B. Among the late AP-1 responsive genes, total metallothionein (MT) and stromelysin mRNAs were expressed at high levels in both normal and OA chondrocytes, while collagenase and hMT-IIA mRNA levels were elevated only in OA individuals. Despite the common AP-1 sequences present in their promoter regions, the three late genes were differentially expressed.

Aged↗
PubMed1992

Molecular cloning and characterization of a constitutively expressed heat-shock-cognate hsc71 gene from rainbow trout.

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.

Amino Acid Sequence↗
PubMed1991

Functional analyses of promoter elements responsible for the differential expression of the human metallothionein (MT)-IG and MT-IF genes.

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.

Base Sequence↗
PubMed1991

5-Azacytidine increases the total cellular copper content and basal level metallothionein mRNA accumulation of human Hep G2 cells.

In this study we have demonstrated the ability of 5-azacytidine to elevate the basal level expression of the metallothionein (MT)-IF and MT-IG genes and increase the basal level expression of the MT-IIA gene in Hep G2 cells, a cell line which exhibits heavy metal inducible MT gene expression. Atomic absorption analysis of 5-azacytidine treated Hep G2 cells detected a 2-fold increase in the total cellular copper content. Pretreatment of 5-azacytidine exposed cells with hydroxyurea and cycloheximide indicated that the increase in total cellular copper content was a direct response to 5-azacytidine treatment. S1 nuclease analysis illustrated that pretreatment of Hep G2 cells with KCN, a copper specific chelator and uptake inhibitor, suppressed 5-azacytidine- and copper-inducible MT-IG gene expression. Thus, the increase in MT gene expression in response to 5-azacytidine treatment can be correlated to an increase in the total cellular copper content. Possible mechanisms on how 5-azacytidine could alter the influx/efflux of copper in Hep G2 cells are discussed.

Animals↗
PubMed1990

Developmental regulation of metallothionein mRNA, zinc and copper levels in rainbow trout, Salmo gairdneri.

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.

Animals↗
PubMed1990

Differential regulation of metallothionein genes in rainbow trout fibroblasts, RTG-2.

Exposure of a trout gonadal fibroblast (RTG-2) cell line to ZnCl2, CdCl2 and CuCl2 resulted in differential levels of accumulation of metallothionein (MT) mRNA. ZnCl2 being the most effective agent induced MT mRNA in 3 h, with 172-fold induction after 48 h and continued accumulation up to 144 h. Following CdCl2 treatment, mRNA could be detected after 24 h, reaching peak levels at 72 h. Furthermore, trout MT mRNA could be detected up to 8 days after withdrawal of extraneous ZnCl2. Using a novel technique of primer extension and DNA sequencing with total RNA as template, specificity of the trout MTa and MTb gene-specific primers was established. Primer extension studies revealed a higher response of MTa to ZnCl2 and CdCl2 compared to MTb. Insensitivity of MT mRNA induction to cycloheximide suggested that induction by the metals was independent of de novo protein synthesis. However, simultaneous exposure of cells to actinomycin D and metals completely inhibited MT mRNA synthesis implying control at the transcriptional level.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

Cell-type specific and differential regulation of the human metallothionein genes. Correlation with DNA methylation and chromatin structure.

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.

Azacitidine↗
PubMed1990

A human metallothionein pseudogene containing AG/CT repetitive elements.

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.

Amino Acid Sequence↗
PubMed1989

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.

Animals↗
PubMed1989

Analysis of stress-induced gene expression in fish cell lines exposed to heavy meals 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.

Animals↗
PubMed1989

A role of metallothionein in zinc regulation after oestradiol induction of vitellogenin synthesis in rainbow trout, Salmo gairdneri.

The regulation of metallothionein (MT) biosynthesis in rainbow-trout liver was studied after a single intraperitoneal injection of oestradiol-17 beta. Sampling was performed after 2, 7, 14, 21, 28 and 35 days. Following induction of vitellogenin synthesis in the liver, liver somatic index (LSI) rose from 1.25 to 2.00 in 14 days. Associated with the increase in LSI was an elevation of hepatic vitellogenin mRNA and zinc concentrations. The vitellogenin mRNA concentrations peaked at 7 days after treatment. The zinc concentrations increased to a peak at day 14. MT was analysed by using differential pulse polarography and a rainbow-trout MT RNA probe. The MT mRNA concentrations rose after 14 days and remained elevated at 21 and 28 days. The MT concentrations increased after 14 days and remained elevated throughout the experimental period. The concentrations of MT-bound zinc increased in association with the elevation in MT concentrations in the oestradiol-treated rainbow trout. These findings indicate that MT is involved in the regulation of zinc during the period of vitellogenin induction and that MT may function by maintaining the pool of available zinc at an appropriate concentration.

Animals↗