Mercury resistance in bacterial strains isolated from hospitals and clinics.
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Bacteria↗
Biomedical subjects
J Chaudhuri
Publications and source records attributed to J Chaudhuri.
Studies on mercury resistance in yeasts isolated from natural sources.
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Food Analysis↗
Estrogen treatment improves spatial learning in APP + PS1 mice but does not affect beta amyloid accumulation and plaque formation.
We investigated the effects of ovariectomy (OVX) and 17 beta-estradiol (0.18 mg per pellet) treatment on spatial learning and memory, hippocampal beta amyloid (A beta) levels, and amyloid plaque counts in double transgenic mice (A/P) carrying mutated amyloid precursor protein (APPswe) and presenilin-1 (PS1-A246E). After OVX at 3 months of age, the mice received estrogen treatment for the last 3 months of their lifetime before they were killed at 6, 9, or 12 months of age. Estrogen treatment in A/P OVX mice increased the number of correct choices in a position discrimination task in the T-maze, and slightly improved their performance in a win-stay task (1/8 arms baited) in the radial arm maze (RAM). However, estrogen treatment did not reverse the A beta-dependent cognitive deficits of A/P mice in the water maze (WM) spatial navigation task. Furthermore, ovariectomy or estrogen treatment in OVX and sham-operated A/P mice had no effect on hippocampal amyloid accumulation. These results show that the estrogen treatment in a transgenic mouse model of Alzheimer's disease (AD) improves performance in the same learning and memory tasks as in the normal C57BL/6J mice. However, the estrogen effects in these mice appeared to be unrelated to A beta-induced cognitive deficits. Our results do not support the idea that estrogen treatment decreases the risk or alleviates the symptoms of Alzheimer's disease by inhibiting the accumulation of A beta or formation of amyloid plaques.
Age Factors↗
Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development.
XRCC4 is a non-homologous end-joining protein employed in DNA double strand break repair and in V(D)J recombination. In mice, XRCC4-deficiency causes a pleiotropic phenotype, which includes embryonic lethality and massive neuronal apoptosis. When DNA damage is not repaired, activation of the cell cycle checkpoint protein p53 can lead to apoptosis. Here we show that p53-deficiency rescues several aspects of the XRCC4-deficient phenotype, including embryonic lethality, neuronal apoptosis, and impaired cellular proliferation. However, there was no significant rescue of impaired V(D)J recombination or lymphocyte development. Although p53-deficiency allowed postnatal survival of XRCC4-deficient mice, they routinely succumbed to pro-B-cell lymphomas which had chromosomal translocations linking amplified c-myc oncogene and IgH locus sequences. Moreover, even XRCC4-deficient embryonic fibroblasts exhibited marked genomic instability including chromosomal translocations. Our findings support a crucial role for the non-homologous end-joining pathway as a caretaker of the mammalian genome, a role required both for normal development and for suppression of tumours.
Animals↗
Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation.
Telomeres are specialized nucleoprotein complexes that serve as protective caps of linear eukaryotic chromosomes. Loss of telomere function is associated with rampant genetic instability and loss of cellular viability and renewal potential. The telomere also participates in processes of chromosomal repair, as evidenced by the 'capture' or de novo synthesis of telomere repeats at double-stranded breaks and by the capacity of yeast telomeres to serve as repositories of essential components of the DNA repair machinery, particularly those involved in non-homologous end-joining (NHEJ). Here we used the telomerase-deficient mouse, null for the essential telomerase RNA gene (Terc), to assess the role of telomerase and telomere function on the cellular and organismal response to ionizing radiation. Although the loss of telomerase activity per se had no discernable impact on the response to ionizing radiation, the emergence of telomere dysfunction in late-generation Terc-/- mice imparted a radiosensitivity syndrome associated with accelerated mortality. On the cellular level, the gastrointestinal crypt stem cells and primary thymocytes showed increased rates of apoptosis, and mouse embryonic fibroblasts (MEFs) showed diminished dose-dependent clonogenic survival. The radiosensitivity of telomere dysfunctional cells correlated with delayed DNA break repair kinetics, persistent chromosomal breaks and cytogenetic profiles characterized by complex chromosomal aberrations and massive fragmentation. Our findings establish a intimate relationship between functionally intact telomeres and the genomic, cellular and organismal response to ionizing radiation.
Animals↗
Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40 S ribosomal preinitiation complex.
We have used an in vitro translation initiation assay to investigate the requirements for the efficient transfer of Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA (or an AUG codon) to form the 40 S preinitiation complex. We observed that the 17-kDa initiation factor eIF1A is necessary and sufficient to mediate nearly quantitative transfer of Met-tRNAf to isolated 40 S ribosomal subunits. However, the addition of 60 S ribosomal subunits to the 40 S preinitiation complex formed under these conditions disrupted the 40 S complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction was carried out with 40 S ribosomal subunits that had been preincubated with eIF3, the 40 S preinitiation complex formed included bound eIF3 (40 S.eIF3. Met-tRNAf.eIF2.GTP). In contrast to the complex lacking eIF3, this complex was not disrupted by the addition of 60 S ribosomal subunits. These results suggest that in vivo, both eIF1A and eIF3 are required to form a stable 40 S preinitiation complex, eIF1A catalyzing the transfer of Met-tRNAf.eIF2.GTP to 40 S subunits, and eIF3 stabilizing the resulting complex and preventing its disruption by 60 S ribosomal subunits.
Animals↗
Nonhomologous end-joining proteins are required for V(D)J recombination, normal growth, and neurogenesis.
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Animals↗
A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis.
XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.
Animals↗
Medical prophylaxis and treatment of cystoid macular edema after cataract surgery. The results of a meta-analysis.
OBJECTIVE: The study aimed to determine the effectiveness of prophylactic medical intervention in reducing the incidence of cystoid macular edema (CME) and the effectiveness of medical treatment for chronic CME after cataract surgery. DESIGN: The study design was a systematic review and meta-analysis of published reports of randomized clinical trials (RCTs). PARTICIPANTS: Sixteen RCTs involving 2898 eyes examining the effectiveness of medical prophylaxis of CME and 4 RCTs involving 187 eyes testing the effectiveness of medical treatment of chronic CME were used in the study. INTERVENTIONS: Medical prophylaxis of treatment (cyclo-oxygenase inhibitors or corticosteroids) versus control (placebo or active treatment) was performed. MAIN OUTCOME MEASURES: Incidence of angiographically diagnosed CME, incidence of clinically significant CME, and vision were measured. RESULTS: Thirty-six articles reported testing a prophylactic medical intervention for CME after cataract surgery. The incidence of CME varied extensively across studies and was related to the study design used. Summary odds ratios (OR) indicated that prophylactic intervention was effective in reducing the incidence of both angiographic CME (OR = 0.36; 95% confidence interval [CI] = 0.28-0.45) and clinically relevant CME (OR = 0.49; 95% CI = 0.33-0.73). There also was a statistically significant positive effect on improving vision (OR = 1.97; 95% CI = 1.14-3.41). A combination of the results of the four RCTs testing medical therapy for chronic CME indicated a treatment benefit in terms of improving final visual acuity by two or more Snellen lines (OR = 2.67; 95% CI = 1.35-5.30). Assessment of the quality of the 20 RCTs included in the meta-analyses indicated problems in the design, execution, and reporting of a number of trials. CONCLUSION: A combination of the results from RCTs indicates that medical prophylaxis for aphakic and pseudophakic CME and medical treatment for chronic CME are beneficial. Because most of the RCTs performed to date have problems related to quality, a well-designed RCT is needed to confirm this result, using clinical CME and vision as outcomes.
Aphakia, Postcataract↗
A targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombination.
The DNA-dependent protein kinase (DNA-PK) consists of Ku70, Ku80, and a large catalytic subunit, DNA-PKcs. Targeted inactivation of the Ku70 or Ku80 genes results in elevated ionizing radiation (IR) sensitivity and inability to perform both V(D)J coding-end and signal (RS)-end joining in cells, with severe growth retardation plus immunodeficiency in mice. In contrast, we now demonstrate that DNA-PKcs-null mice generated by gene-targeted mutation, while also severely immunodeficient, exhibit no growth retardation. Furthermore, DNA-PKcs-null cells are blocked for V(D)J coding-end joining, but retain normal RS-end joining. Finally, while DNA-PK-null fibroblasts exhibited increased IR sensitivity, DNA-PKcs-deficient ES cells did not. We conclude that Ku70 and Ku80 may have functions in V(D)J recombination and DNA repair that are independent of DNA-PKcs.
Animals↗
Molecular cloning and functional expression of a human cDNA encoding translation initiation factor 6.
Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. In this paper, we devised a procedure for purifying eIF6 from rabbit reticulocyte lysates and immunochemically characterized the protein by using antibodies isolated from egg yolks of laying hens immunized with rabbit eIF6. By using these monospecific antibodies, a 1.096-kb human cDNA that encodes an eIF6 of 245 amino acids (calculated Mr 26,558) has been cloned and expressed in Escherichia coli. The purified recombinant human protein exhibits biochemical properties that are similar to eIF6 isolated from mammalian cell extracts. Database searches identified amino acid sequences from Saccharomyces cerevisiae, Drosophila, and the nematode Caenorhabditis elegans with significant identity to the deduced amino acid sequence of human eIF6, suggesting the presence of homologues of human eIF6 in these organisms.
Amino Acid Sequence↗
Biochemical characterization of mammalian translation initiation factor 3 (eIF3). Molecular cloning reveals that p110 subunit is the mammalian homologue of Saccharomyces cerevisiae protein Prt1.
Eukaryotic translation initiation factor 3 (eIF3), which plays an essential role in initiation of protein synthesis, was purified from rabbit reticulocyte lysates using an assay that specifically measures its ability to stimulate the binding of Met-tRNAf (as a Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits. Purified eIF3 consisted of six major polypeptides of molecular masses 110, 67, 42, 40, 36, and 35 kDa but lacked the 170-kDa polypeptide reported to be a constituent of other eIF3 preparations. Characterization of purified eIF3 lacking the 170-kDa polypeptide showed that the eIF3-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex. eIF3, which was originally bound to the 40 S initiation complex, was released from the 40 S subunit during the subunit joining reaction. Additionally, chicken antibodies raised against rabbit reticulocyte eIF3 were used to immunochemically characterize eIF3 subunits and to isolate a 3.1-kilobase pair human cDNA that encodes the p110 subunit of mammalian eIF3. The derived amino acid sequence (calculated Mr 95,214) shows that the p110 subunit is the mammalian homologue of Saccharomyces cerevisiae protein Prt1p, a subunit of yeast eIF3.
Amino Acid Sequence↗
Function of eukaryotic translation initiation factor 1A (eIF1A) (formerly called eIF-4C) in initiation of protein synthesis.
We have used an efficient in vitro translation initiation system to show that the mammalian 17-kDa eukaryotic initiation factor, eIF1A (formerly designated eIF-4C), is essential for transfer of the initiator Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA to form the 40 S preinitiation complex (40 S.Met-tRNAf.eIF2.GTP). Furthermore, eIF1A acted catalytically in this reaction to mediate highly efficient transfer of the Met-tRNAf.eIF2.GTP ternary complex to 40 S ribosomal subunits. The 40 S complex formed was free of eIF1A indicating that its role in 40 S preinitiation complex formation is not to stabilize the binding of Met-tRNAf to 40 S ribosomes. Additionally, the eIF1A-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex. In contrast to other reports, we found that eIF1A plays no role either in the subunit joining reaction or in the generation of ribosomal subunits from 80 S ribosomes. Our results indicate that the major function of eIF1A is to mediate the transfer of Met-tRNAf to 40 S ribosomal subunits to form the 40 S preinitiation complex.
Animals↗
Studies on mercury-detoxicating enzymes from a broad-spectrum mercury-resistant strain of Flavobacterium rigense.
Flavobacterium rigense strain PR2, a broad-spectrum mercury-resistant bacterium abundantly present in soil exhibited multiple metal resistance properties. Mercury resistance was due to the sequential action of two mercury-detoxicating enzymes, organomercurial lyase and mercuric reductase. The levels of these enzyme activities were determined using different mercury compounds as inducers and substrates. Mercuric reductase was partially purified from the bacterium and the physicochemical properties of the enzyme were studied. The effect of several enzyme inhibitors and heavy metal ions on the enzyme activity was also studied.
Bacterial Proteins↗
Elimination of mercury and organomercurials by nitrogen-fixing bacteria.
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Azotobacter↗
Mercury and organomercurial resistance in bacteria isolated from freshwater fish of wetland fisheries around Calcutta.
Mercury-resistant bacteria belonging to the genera Bacillus, Escherichia, Klebsiella, Micrococcus, Pseudomonas, Salmonella, Sarcina, Shigella, Staphylococcus and Streptococcus were isolated from gills and guts of fresh water fish collected from wetland fisheries around Calcutta, India, contaminated with mercury compounds. The total number of bacteria, as well as Hg-resistant bacteria, were always higher in guts than gills. Bottom-dwelling fish contained higher number of bacteria, including Hg-resistant bacteria, than surface and middle water dwelling fish. They belonged either to narrow-spectrum or to broad-spectrum Hg-resistant groups and they also possessed other heavy metal and antibiotic resistant properties. In the presence of toxic levels of HgCl(2), phenylmercuric acetate (PMA) and methylmercuric chloride (MMC), the lag in growth of the bacterial strains gradually increased with increasing concentration of Hg-compounds. Narrow-spectrum Hg-resistant bacterial strains volatilized only HgCl(2) from the liquid medium in the range of 64-89%, whereas the broad-spectrum group exhibited a high level of HgCl(2) (80-94%), PMA (72-84%) and MMC (64-80%) volatilizing capacity with inducible mercuric reductase and organomercurial lyase enzyme activities in their cell-free extracts. Cell-free extracts prepared from narrow-spectrum Hg-resistant bacterial strains induced by HgCl(2) exhibited Hg(+2)-dependent NADPH oxidation, indicating the presence of only mercuric reductase enzyme.
Journal Article↗
Volatilization of mercury by resting mercury-resistant bacterial cells.
The mercuric ion reduction system encoded by the Hg2+ inducible mer operon confers bacterial resistance to mercuric ion. The mer A gene product which is a FAD-containing enzyme catalyzes the reduction of Hg2+ to volatile elemental mercury with the help of intracellular thiols and NADPH as a cofactor (Schottel 1974; Summers and Silver 1978; Fox and Walsh 1982; Misra 1992). Our earlier studies have shown that growing cells of different mercury-resistant bacteria reduce Hg2+ compounds to Hg(O) (Ray et al. 1989; Pahan et al. 1990a; Gachhui et al. 1989). We have also shown the effect of thiol compounds and flavins on mercury-degrading enzyme activities in mercury-resistant bacteria (Pahan et al. 1990b). Here we report that resting cells of mercury-resistant bacteria survive in a buffer system for several hours, synthesize inducible mercury-degrading enzymes and volatilize mercury from a mercury-containing buffer system. We know of no information regarding studies of mercury-degrading enzymes in resting mercury-resistant bacterial cells.
Azotobacter↗
The A1 x U72 base pair conserved in eukaryotic initiator tRNAs is important specifically for binding to the eukaryotic translation initiation factor eIF2.
The formation of a specific ternary complex between eukaryotic initiation factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic protein-synthesizing system of eukaryotes. We show that the A1 x U72 base pair conserved at the end of the acceptor stem in eukaryotic and archaebacterial initiator methionine tRNAs plays an important role in this interaction. We changed the A1 x U72 base pair of the human initiator tRNA to G1 x C72 and expressed the wild-type and mutant tRNA genes in the yeast Saccharomyces cerevisiae by using constructs previously developed in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type and mutant human initiator tRNAs in substantially pure form, free of the yeast initiator tRNA, and have analyzed their properties in vitro. The G1 x C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.
Base Sequence↗