Evolution of an ion-translocating ATPase.
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Adenosine Triphosphatases↗
Biomedical subjects
S Silver
Publications and source records attributed to S Silver.
Reduction of arsenate to arsenite by the ArsC protein of the arsenic resistance operon of Staphylococcus aureus plasmid pI258.
The arsenic resistance operon of Staphylococcus aureus plasmid pI258 consists of three genes, arsR (encoding the repressor regulatory protein), arsB (the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells), and arsC (the small gene whose protein product is required for arsenate resistance only, not for arsenite resistance). ArsC has now been shown to be an arsenate reductase, converting intracellular arsenate [As(V)] to arsenite [As(III)], which is then exported from the cells by an energy-dependent efflux process. The arsenate reductase activity was found in the soluble cytoplasmic fraction in Escherichia coli (and not associated with the periplasmic fraction or the sedimentable cell envelope). Purified ArsC protein coupled in vitro with thioredoxin plus dithiothreitol (but not 2-mercaptoethanol or reduced glutathione) to reduce arsenate to arsenite.
Adenosine Triphosphatases↗
Effects of gold(I) antiarthritic drugs and related compounds on Pseudomonas putida.
The effects of the antiarthritic drugs aurothiomalate (AuTm), aurothioglucose (AuTg), auranofin, its metabolite triethylphosphinegold(I)thioglucose (Et3PAuTg), and several related complexes on the growth of Pseudomonas putida were studied. Two strains were used, one of which (BK135) was more sensitive to Et3PAuTg (tolerant up to 4 microM) than the other (BK403; tolerant to at least 500 microM). Gold thiolate complexes and thiolate ligands alone had little effect on growth. Gold phosphine complexes increased the length of the lag phase of growth and reduced oxygen uptake. Marked changes in cellular morphology were determined by electron microscopy. Copper(II) compounds and aurothiomalate were synergistic in their growth inhibitory effects towards these bacteria. Experiments with 195Au suggested that a mechanism does not exist for the short term (minutes) uptake of gold by sensitive or resistant bacteria, but the resistant strain appeared to limit gold uptake over a longer term (hours).
Antirheumatic Agents↗
Crystallization, sequence, and preliminary crystallographic data for an antipeptide Fab 50.1 and peptide complexes with the principal neutralizing determinant of HIV-1 gp120.
X-ray quality crystals of an Fab fragment from an antipeptide monoclonal antibody (R/V3-50.1) that recognizes the principal neutralizing determinant (PND) of the gp120 glycoprotein of human immunodeficiency virus type 1 (HIV-1) (MN isolate) were grown as uncomplexed and peptide complexed forms. Crystals of the free Fab grew from high salt in orthorhombic space groups P2(1)2(1)2(1) and I222 and from polyethylene glycol in space groups P1 and P2(1). Seeds from either the P1 and P2(1) native (uncomplexed) Fab crystals induced nucleation of crystals of the Fab complexed to a 16-residue synthetic peptide corresponding to the PND when streak seeded into preequilibrated solutions of this complex. Data were collected from these complex crystals and from each of the four native Fab forms to at least 2.8 A resolution. The genes for the variable domain of the Fab were cloned and sequenced and the primary amino acid sequence was deduced from this information. Knowledge of the three-dimensional structure of this Fab-peptide complex will be important in the understanding of the PND of HIV-1 and its recognition by neutralizing monoclonal antibodies.
Amino Acid Sequence↗
Plasmid-determined metal resistance mechanisms: range and overview.
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Bacteria↗
Plasmid chromate resistance and chromate reduction.
Compounds of hexavalent chromium (chromates and dichromates) are highly toxic. Plasmid genetic determinants for chromate resistance have been described in several bacterial genera, most notably in Pseudomonas. Resistance to chromate is associated with decreased chromate transport by the resistant cells. The genes for a hydrophobic polypeptide, ChrA, were identified in chromate resistance plasmids of Pseudomonas aeruginosa and Alcaligenes eutrophus. ChrA is postulated to be responsible for the outward membrane translocation of chromate anions. Widespread bacterial reduction of hexavalent chromate to the less toxic trivalent chromic ions is also known. Chromate reduction determinants have not, however, been found on bacterial plasmids or transposons. In different bacteria, chromate reduction is either an aerobic or an anaerobic process (but not both) and is carried out either by soluble proteins or by cell membranes. Chromate reduction may also be a mechanism of resistance to chromate, but this has not been unequivocally shown.
Amino Acid Sequence↗
Regulation and expression of the arsenic resistance operon from Staphylococcus aureus plasmid pI258.
The arsenic resistance operon from Staphylococcus aureus plasmid pI258 was cloned and sequenced. The DNA sequence contains three genes in the order arsR, arsB, and arsC. The predicted amino acid sequences of the gene products are homologous with those of the products of the ars operons of plasmids pSX267 from Staphylococcus xylosus and R773 from Escherichia coli. The cloned staphylococcal ars operon confers resistances to arsenate, arsenite, and antimonite in S. aureus and Bacillus subtilis. The same operon was also expressed in E. coli and conferred resistance to arsenite but less resistance to arsenate and antimonite. Regulation of the pI258 ars operon was studied by using a translational arsB-blaZ fusion in S. aureus and a transcriptional arsB-luxAB fusion in E. coli. The ars operon was induced by arsenate [As(V)], arsenite [As(III)], and antimonite [Sb(III)], to which the strains were resistant, plus Bi(III) in S. aureus. Only arsenate and arsenite induced the operon in E. coli. Northern (RNA) blot DNA-RNA hybridization analysis showed inducible synthesis of a full-length ars mRNA, about 2.1 kb in size, both in S. aureus and in E. coli. S. aureus ars proteins were expressed in E. coli from the T7 phage promoter under the control of the T7 RNA polymerase. Primer extension (reverse transcriptase) analysis showed that the ars mRNA started at the same position (nucleotides 17 and 18 upstream from the arsR ATG) both in S. aureus and in E. coli. An internal deletion mutation in arsB resulted in decreased resistance to arsenate and total loss of arsenite and antimonite resistances. Partial deletion of 56 bp from the 3' end of the arsC gene resulted in loss of resistance to arsenate; the determinant retained arsenite and antimonite resistances.
Adenosine Triphosphatases↗
Gene regulation of plasmid- and chromosome-determined inorganic ion transport in bacteria.
Regulation of chromosomally determined nutrient cation and anion uptake systems shows important similarities to regulation of plasmid-determined toxic ion resistance systems that mediate the outward transport of deleterious ions. Chromosomally determined transport systems result in accumulation of K+, Mg2+, Fe3+, Mn2+, PO4(3-), SO4(2-), and additional trace nutrients, while bacterial plasmids harbor highly specific resistance systems for AsO2-, AsO4(3-), CrO4(2-), Cd2+, Co2+, Cu2+, Hg2+, Ni2+, SbO2-, TeO3(2-), Zn2+, and other toxic ions. To study the regulation of these systems, we need to define both the trans-acting regulatory proteins and the cis-acting target operator DNA regions for the proteins. The regulation of gene expression for K+ and PO4(3-) transport systems involves two-component sensor-effector pairs of proteins. The first protein responds to an extracellular ionic (or related) signal and then transmits the signal to an intracellular DNA-binding protein. Regulation of Fe3+ transport utilizes the single iron-binding and DNA-binding protein Fur. The MerR regulatory protein for mercury resistance both represses and activates transcription. The ArsR regulatory protein functions as a repressor for the arsenic and antimony(III) efflux system. Although the predicted cadR regulatory gene has not been identified, cadmium, lead, bismuth, zinc, and cobalt induce this system in a carefully regulated manner from a single mRNA start site. The cadA Cd2+ resistance determinant encodes an E1(1)-1E2-class efflux ATPase (consisting of two polypeptides, rather than the one earlier identified). Cadmium resistance is also conferred by the czc system (which confers resistances to zinc and cobalt in Alcaligenes species) via a complex efflux pump consisting of four polypeptides. These two cadmium efflux systems are not otherwise related. For chromate resistance, reduced cellular accumulation is again the resistance mechanism, but the regulatory components are not identified. For other toxic heavy metals (with few exceptions), there exist specific plasmid resistances that remain relatively terra incognita for future exploration of bioinorganic molecular genetics and gene regulation.
Base Sequence↗
The effect of stimulus repetition rate on the diagnostic efficacy of the auditory nerve-brain-stem evoked response.
This study investigates the hypothesis that an increase in the click presentation rate during diagnostic testing with the auditory nerve-brain-stem response (ABR) will increase the efficiency with which lesions may be detected in the nervous system. Cats were exposed to conditions of hypoxia, hypercapnia and acidemia, and hypoglycemia was induced in rats. ABR was recorded using the standard 10/sec click rate and also a higher (55/sec) rate during both the control state and experimental state. Various parameters of the ABR were compared at the two click rates in the control and experimental states to see if the higher click rate was more effective in detecting pathology in the nervous system. It was found that in only a very few cases was the higher stimulus presentation rate more effective, and that in general ABR recordings at one stimulus rate only is quite sufficient for work in a clinical setting.
Acidosis↗
A second gene in the Staphylococcus aureus cadA cadmium resistance determinant of plasmid pI258.
Two open reading frames on a 3.7-kb BglII-XbaI fragment which encodes the Staphylococcus aureus cadA cadmium (and zinc) resistance determinant of plasmid pI258 were identified (G. Nucifora, L. Chu, T. K. Misra, and S. Silver, Proc. Natl. Acad. Sci. USA 86:3544-3548, 1989). The [35S]methionine-labelled protein products of the 727-amino-acid CadA ATPase and of the 122-amino-acid CadC polypeptide in Escherichia coli were identified by using the T7 RNA polymerase-promoter expression system. A truncated CadA polypeptide (402 amino acids) did not confer resistance in S. aureus but was expressed in E. coli under control of the T7 RNA polymerase-promoter. Removal of 678 nucleotides from the 5' end of the published sequence (which includes the cadA promoter) abolished resistance to cadmium, whereas a 146-nucleotide-shorter deletion was without effect. The cadC gene is needed in addition to cadA for full resistance to cadmium in S. aureus and Bacillus subtilis. cadC functions both in cis and in trans.
Bacillus subtilis↗
Regulation of the cadA cadmium resistance determinant of Staphylococcus aureus plasmid pI258.
Regulation of the cadA cadmium and zinc resistance determinant of Staphylococcus aureus plasmid pI258 was demonstrated by using gene fusions and direct measurements of transcription. In growth experiments, cells harboring the intact cadA operon were induced with different cations and challenged by an inhibitory concentration of ZnCl2, a substrate of the CadA resistance system. Uninduced cells did not grow for 8 h after Zn2+ addition, whereas induced cells grew in the presence Zn2+. Cd2+ was a strong inducer, and Bi3+ and Pb2+ also induced well; Co2+ and Zn2+ were weak inducers. A translational beta-lactamase fusion to the cadA gene showed the same induction specificity as that seen with growth experiments with the intact cadA operon. A short beta-lactamase transcriptional fusion to the cadC gene also showed the same pattern of induction, establishing that the cadC gene was not involved in regulation. In Northern (RNA) blot hybridization experiments, a cadmium-inducible, 2.6-kb, operon-length transcript was detected. Primer extension experiments determined that Cd(2+)-inducible transcription of the cadA operon begins at nucleotides 676 and 677 of the published sequence (G. Nucifora, L. Chu, T. K. Misra, and S. Silver, Proc. Natl. Acad. Sci. USA 86: 3544-3548, 1989).
Base Sequence↗
Postnatal development of flash visual evoked potentials in the jaundiced Gunn rat.
In this study, the postnatal development of flash visual evoked potential (VEP) has been monitored in the jaundiced (jj) Gunn rat model of neonatal hyperbilirubinemia to determine whether this evoked response is affected by bilirubin-induced neurotoxicity. VEP could first be recorded at 16 d of age. The jj rats exhibited prolonged wave latencies and lower wave amplitudes during the 3rd wk of postnatal life, when compared with their nonjaundiced littermates. There was no correlation at 21 d of age between VEP parameters and either bilirubin levels or body weight. About one third of the jj animals died between 21 and 28 d of age. The average VEP wave latencies at 21 d of age of the rats who were to die was prolonged compared with those of rats who survived till at least 28 d of age. Thus, the latency of VEP waves at the age of 21 d appears to be related to the further outcome of jj Gunn rats. Although wave amplitudes were lower in jj as compared with nonjaundiced 21-d-old animals, there were no amplitude differences between the jj rats who would die and those who would survive during the 4th wk of life. These findings may contribute to the understanding of the pathogenesis of bilirubin encephalopathy in the neonatal period.
Age Factors↗
Nucleotide sequence and expression of a plasmid-encoded chromate resistance determinant from Alcaligenes eutrophus.
The nucleotide sequence of the 2.6-kilobase pair (kb) EcoRI fragment encoding chromate resistance (Chrr) on plasmid pMOL28 in Alcaligenes eutrophus was determined. Three open reading frames were assigned to three polypeptides which were expressed from this determinant in Escherichia coli under the control of a phage T7 transcription promoter. When the roles of the polypeptides and open reading frames were analyzed with deletion derivatives of the 2.6-kilobase fragment, the membrane-bound ChrA (401 amino acids) and ChrB (196 amino acids) polypeptides were essential for inducible chromate resistance and reduced accumulation of chromate, while the third open reading frame was not needed.
Alcaligenes↗
Human monoclonal antibody that recognizes the V3 region of human immunodeficiency virus gp120 and neutralizes the human T-lymphotropic virus type IIIMN strain.
We describe a human IgG1 monoclonal antibody (N701.9b) derived by Epstein-Barr virus transformation of B cells from a human immunodeficiency virus-seropositive asymptomatic donor. This antibody was shown to recognize the principal neutralizing domain contained within the V3 region of gp120 of the MN strain of human immunodeficiency virus and MN-like strains, as determined by binding to the PB-1 fragment of MN gp120 and to synthetic peptides corresponding to the V3 region of MN and related virus strains. The epitope identified by monoclonal antibody N701.9b was mapped to a segment of V3 containing at least 7 amino acids (amino acids 316-322), which is located in the "tip" and "right" side of the V3 loop of the MN strain. Furthermore, this antibody manifested potent type-specific fusion-inhibitory activity against the MN strain but not against the IIIB or RF virus strains. This antibody also neutralized four virus isolates that had MN-like V3 region sequences and failed to neutralize three other strains containing unrelated V3 region sequences. Our findings confirm that the V3 region stimulates type-specific neutralizing antibody during natural human immunodeficiency virus infection in humans. The potential clinical use of this antibody is discussed.
Amino Acid Sequence↗
Chromium reduction in Pseudomonas putida.
Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000. Chromate reductase activity was associated with soluble protein and not with the membrane fraction. The crude enzyme activity was heat labile and showed a Km of 40 microM CrO4(2-). Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells.
Chromates↗
Cloning, nucleotide sequence, and expression of the chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505.
The chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505 was cloned into broad-host-range vector pSUP104. The hybrid plasmid containing an 11.1-kilobase insert conferred chromate resistance and reduced uptake of chromate in P. aeruginosa PAO1. Resistance to chromate was not expressed in Escherichia coli. Contiguous 1.6- and 6.3-kilobase HindIII fragments from this plasmid hybridized to pUM505 but not to P. aeruginosa chromosomal DNA and only weakly to chromate resistance plasmids pLHB1 and pMG6. Further subcloning produced a plasmid with an insert of 2,145 base pairs, which was sequenced. Analysis of deletions revealed that a single open reading frame was sufficient to determine chromate resistance. This open reading frame encodes a highly hydrophobic polypeptide, ChrA, of 416 amino acid residues that appeared to be expressed in E. coli under control of the T7 promoter. No significant homology was found between ChrA and proteins in the amino acid sequence libraries, but 29% amino acid identity was found with the ChrA amino acid sequence for another chromate resistance determinant sequenced in this laboratory from an Alcaligenes eutrophus plasmid (A. Nies, D. Nies, and S. Silver, submitted for publication).
Amino Acid Sequence↗
Constitutive synthesis of a transport function encoded by the Thiobacillus ferrooxidans merC gene cloned in Escherichia coli.
Mercuric reductase activity determined by the Thiobacillus ferrooxidans merA gene (cloned and expressed constitutively in Escherichia coli) was measured by volatilization of 203Hg2+. (The absence of a merR regulatory gene in the cloned Thiobacillus mer determinant provides a basis for the constitutive synthesis of this system.) In the absence of the Thiobacillus merC transport gene, the mercury volatilization activity was cryptic and was not seen with whole cells but only with sonication-disrupted cells. The Thiobacillus merC transport function was compared with transport via the merT-merP system of plasmid pDU1358. Both systems, cloned and expressed in E. coli, governed enhanced uptake of 203Hg2+ in a temperature- and concentration-dependent fashion. Uptake via MerT-MerP was greater and conferred greater hypersensitivity to Hg2+ than did uptake with MerC. Mercury uptake was inhibited by N-ethylmaleimide but not by EDTA. Ag+ salts inhibited mercury uptake by the MerT-MerP system but did not inhibit uptake via MerC. Radioactive mercury accumulated by the MerT-MerP and by the MerC systems was exchangeable with nonradioactive Hg2+.
Biological Transport↗
Novel technique for creation of the subcutaneous peritoneal dialysis catheter tunnel.
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Catheterization↗