Biomedical subjects

S Silver

Publications and source records attributed to S Silver.

At least 127 records · Page 7Linked to original sources

PubMed1985

Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport.

Cd2+ and Mn2+ accumulation was studied with wild-type Bacillus subtilis 168 and a Cd2+-resistant mutant. After 5 min of incubation in the presence of 0.1 microM 109Cd2+ or 54Mn2+, both strains accumulated comparable amounts of 54Mn2+, while the sensitive cells accumulated three times more 109Cd2+ than the Cd2+-resistant cells did. Both 54Mn2+ and 109Cd2+ uptake, which apparently occur by the same transport system, demonstrated cation specificity; 20 microM Mn2+ or Cd2+ (but not Zn2+) inhibited the uptake of 0.1 microM 109Cd2+ or 54Mn2+. 54Mn2+ and 109Cd2+ uptake was energy dependent and temperature sensitive, but 109Cd2+ uptake in the Cd2+-resistant strain was only partially inhibited by an uncoupler or by a decrease in temperature. 109Cd2+ uptake in the sensitive strain followed Michaelis-Menten kinetics with a Km of 1.8 microM Cd2+ and a Vmax of 1.5 mumol/min X g (dry weight); 109Cd2+ uptake in the Cd2+-resistant strain was not saturable. The apparent Km value for the saturable component of 109Cd2+ uptake by the Cd2+-resistant strain was very similar to that of the sensitive strain, but the Vmax was 25 times lower than the Vmax for the sensitive strain. The Km and Vmax for 54Mn2+ uptake by both strains were very similar. Cd2+ inhibition of 54Mn2+ uptake had an apparent Ki of 3.4 and 21.5 microM Cd2+ for the sensitive and Cd2+-resistant strains, respectively. Mn2+ had an apparent Ki of 1.2 microM Mn2+ for inhibition of 109Cd2+ uptake by the sensitive strain, but the Cd2+-resistant strain had no defined Ki value for inhibition of Cd2+ uptake by Mn2+.

Bacillus subtilis↗

PubMed1985

Cloning and DNA sequence of a plasmid-determined citrate utilization system in Escherichia coli.

The citrate utilization determinant from a large 200-kilobase (kb) naturally occurring plasmid was previously cloned into the PstI site of plasmid vector pBR325 creating the Cit+ tetracycline resistance plasmid pWR61 (15 kb). Tn5 insertion mutagenesis analysis of plasmid pWR61 limited the segment responsible for citrate utilization to a 4.8-kb region bordered by EcoRI and PstI restriction nuclease sites. The 4.8-kb fragment was cloned into phage M13, and the DNA sequence was determined by the dideoxyribonucleotide method. Within this sequence was a 1,296-base-pair open reading frame with a preceding ribosomal binding site. The 431-amino-acid polypeptide that could be translated from this open reading frame would be highly hydrophobic. A second long open reading frame with the potential of encoding a 379-amino-acid polypeptide preceded the larger open reading frame. Portions of the 4.8-kb fragment were further subcloned with restriction endonucleases BglII and BamHI, reducing the minimum size needed for a citrate-positive phenotype to a 1.9-kb BamHI-BglII fragment (which includes the coding region for the 431-amino-acid polypeptide, but only the distal 2/3 of the reading frame for the 379-amino-acid polypeptide). Citrate utilization results from a citrate transport activity encoded by the plasmid. With the 4.8-kb fragment (as with larger fragments) the citrate transport activity was inducible by growth on citrate. On transfer from glucose, succinate, malate, or glycerol medium to citrate medium, the Cit+ Escherichia coli strains showed a delay of 36 to 48 h before growth.

Amino Acid Sequence↗

PubMed1985

Herpes simplex virus 1 mutant deleted in the alpha 22 gene: growth and gene expression in permissive and restrictive cells and establishment of latency in mice.

R325-beta TK+, a herpes simplex virus 1 mutant carrying a 500-base-pair deletion in the alpha 22 gene and the wild-type (beta) thymidine kinase (TK) gene, was previously shown to grow efficiently in HEp-2 and Vero cell lines. We report that in rodent cell lines exemplified by the Rat-1 line, plating efficiency was reduced and growth was multiplicity dependent. A similar multiplicity dependence for growth and lack of virus spread at low multiplicity was seen in resting, confluent human embryonic lung (HEL) cells. The shutoff of synthesis of beta proteins was delayed and the duration of synthesis of gamma proteins was extended in R325-beta TK+-infected HEL cells relative to cells infected with the wild-type parent, but no significant differences were seen in the total accumulation of viral DNA. To quantify the effect on late (gamma 2) gene expression, a recombinant carrying the deletion in the alpha 22 gene and a gamma 2-TK gene (R325-gamma 2 TK) was constructed and compared with a wild-type virus (R3112) carrying a chimeric gamma 2-TK gene. In Vero cells, the gamma 2-TK gene of R325-gamma 2TK was expressed earlier than and at the same level as the gamma 2-TK gene of R3112. In the confluent resting HEL cells, the expression of the gamma 2-TK gene of the alpha 22- virus was grossly reduced relative to that of the alpha 22+ virus. Electron microscopic studies indicated that the number of intranuclear capsids of R325-beta TK+ virus was reduced relative to that of the parent virus in resting confluent HEL cells, but the number of DNA-containing capsids was higher. Notwithstanding the grossly reduced neurovirulence on intracerebral inoculation in mice, R325-beta TK+ virus was able to establish latency in mice. We conclude that (i) the alpha 22 gene affects late (gamma 2) gene expression, and (ii) a host cell factor complements that function of the alpha 22 gene to a greater extent in HEp-2 and Vero cells than in confluent, resting HEL cells.

Animals↗

PubMed1985

gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes.

True gamma or gamma 2 genes, unlike alpha, beta, and gamma 1 (beta gamma) genes of herpes simplex virus 1 (HSV-1), stringently require viral DNA synthesis for their expression. We report that gamma 2 genes resident in cells were induced in trans by infection with HSV-1 but that the induction did not require amplification of either the resident gene or the infecting viral genome. Specifically, to test the hypothesis that expression of these genes is amplification dependent, we constructed two sets of gamma 2-thymidine kinase (TK) chimeric genes. The first (pRB3038) consisted of the promoter-regulatory region and a portion of 5'-transcribed noncoding region of the domain of a gamma 2 gene identified by Hall et al. (J. Virol. 43:594-607) in the HSV-1(F) BamHI fragment D' to the 5'-transcribed noncoding and coding regions of the TK gene. The second (pRB3048) contained, in addition, an origin of HSV-1 DNA replication. Cells transfected with either the first or second construct and selected for the TK+ phenotype were then tested for TK induction after superinfection with HSV-1(F) delta 305, containing a deletion in the coding sequences of the TK gene, and viruses containing, in addition, a ts lesion in the alpha 4 regulatory protein (ts502 delta 305) or in the beta 8 major DNA-binding protein (tsHA1 delta 305). The results were as follows: induction by infection with TK- virus of chimeric TK genes with or without an origin of DNA replication was dependent on functional alpha 4 protein but not on viral DNA synthesis; the resident chimeric gene in cells selected for G418 (neomycin) resistance was regulated in the same fashion; the chimeric gene recombined into the viral DNA was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis; the gamma 2-chimeric genes resident in the host and in viral genomes were transcribed from the donor BamHI fragment D' containing the promoter-regulatory domain of the gamma 2 gene. The significance of the differential regulation of gamma 2 genes in the environments of host and viral genomes by viral trans-acting factors is discussed.

Animals↗

PubMed1984

Bacterial transformations of and resistances to heavy metals.

Bacteria carry out chemical transformations of heavy metals. These transformations (including oxidation, reduction, methylation, and demethylation) are sometimes byproducts of normal metabolism and confer no known advantage upon the organism responsible. Sometimes, however, the transformations constitute a mechanism of resistance. Many species of bacteria have genes that control resistances to specific toxic heavy metals. These resistances often are determined by extrachromosomal DNA molecules (plasmids). The same mechanisms of resistance occur in bacteria from soil, water, industrial waste, and clinical sources. The mechanism of mercury and organomercurial resistance is the enzymatic detoxification of the mercurials into volatile species (methane, ethane, metallic HgO) which are rapidly lost from the environment. Cadmium and arsenate resistances are due to reduced net accumulation of these toxic materials. Efficient efflux pumps cause the rapid excretion of Cd2+ and AsO4(3-). The mechanisms of arsenite and of antimony resistance, usually found associated with arsenate resistance, are not known. Silver resistance is due to lowered affinity of the cells for Ag+, which can be complexed with extracellular halides, thiols, or organic compounds. Sensitivity is due to binding of Ag+ more effectively to cells than to Cl-.

Antimony↗

PubMed1984

Mercuric ion-resistance operons of plasmid R100 and transposon Tn501: the beginning of the operon including the regulatory region and the first two structural genes.

The mercuric ion-resistance operons of plasmid R100 (originally from Shigella) and transposon Tn501 (originally from a plasmid isolated in Pseudomonas) have been compared by DNA sequence analysis. The sequences for the first 1340 base pairs of Tn501 are given with the best alignment with the comparable 1319 base pairs of R100. The homology between the two sequences starts at base 58 after the end of the insertion sequence IS-1 of R100. The sequences include the transcriptional regulatory region, and the homology is particularly strong in regions just upstream from potential transcriptional initiation sites. The trans-acting regulatory gene merR consists of 180 base pairs in both cases and codes for a highly basic polypeptide of 60 amino acids, which is also rich in serine. The Tn501 and R100 merR genes differ in 25 of the 180 base positions, and the resulting polypeptides differ in seven amino acids. The regulatory region before the major transcription initiation site contains potential -35 and -10 sequences and dyad symmetrical sequences, which may be the merR binding sites for transcriptional regulation. The first structural gene, merT, encodes a highly hydrophobic polypeptide of 116 amino acids. The R100 and Tn501 merT genes differ in 17% of their positions, leading to 14 (12%) amino acid changes. This region had previously been shown to encode a protein governing membrane transport of mercuric ions. The second structural gene, merC, would give a 91 amino acid polypeptide with a hydrophobic amino-terminal segment. The Tn501 and R100 merC genes differ at 37 base positions, leading to 10 amino acid changes.

Amino Acid Sequence↗

PubMed1984

Homology among arsenate resistance determinants of R factors in Escherichia coli.

Escherichia coli bearing R factors R773 or R46 or hybrid recombinant plasmids carrying the arsenic resistance determinants derived from these plasmids synthesized inducible polypeptides of similar apparent molecular weights when exposed to arsenite salts (R773 derivative, 64,000 and 16,000; R46 derivative, 62,000, 16,500, and 13,500). In addition, both plasmids encoded energy-dependent arsenate efflux systems and demonstrated DNA sequence homology by filter blot hybridization. Human isolates of arsenate- and arsenite-resistant enterobacteria were tested for homology with the arsenate operon of R773 by colony blot hybridization. Approximately one-third of the isolates hybridized strongly, and two-thirds showed little or no evidence of homology, suggesting the presence of two or more genetically distinct arsenate resistant determinants.

Arsenates↗

PubMed1983

Cloning and expression of R-factor mediated arsenate resistance in Escherichia coli.

The resistance transfer factor R773 confers inducible arsenate, arsenite and antimony resistance on Escherichia coli. The genes for these resistances were cloned into the EcoRI site of plasmid pBR322 to produce a 33 kilobase plasmid. pUM1. Bacterial strains transformed with pUM1 synthesized a polypeptide of the apparent molecular weight 64,000 daltons when induced with arsenite. This polypeptide could be visualized on sodium dodecyl sulfate polyacrylamide gels stained with Coomassie blue. It was observed both in the membrane and cytosol fractions but not among the periplasmic proteins present in osmotic shock fluid. Minicells isolated from strain JR410(pUM1) incorporated [35S]methionine into an inducible 64,000 dalton polypeptide, as demonstrated on autoradiographs of electrophoresed [35S]-labeled minicell lysates, confirming that this polypeptide is a plasmid gene product. A 4.3 kilobase HindIII fragment of pUM1 was subcloned into the HindIII site of pBR322, producing recombinant plasmid pUM3. This plasmid conferred constitutive resistance ot arsenite and arsenate. Extensive synthesis of two polypeptides of 64,000 and 16,000 daltons was observed both in Coomassie stained gels of whole cells and autoradiographs of gels of [35S]methionine-labeled minicells. Synthesis of both polypeptides was constitutive.

Arsenates↗

PubMed1983

Tn5 insertion mutations in the mercuric ion resistance genes derived from plasmid R100.

The mercuric resistance (mer) genes of plasmid R100 were cloned into plasmid pBR322. A series of transposon Tn5 insertion mutations in the mer genes were isolated and mapped. The mutants were characterized phenotypically by their sensitivity to Hg2+ and by binding and volatilization of 203Hg2+. Dominance and complementation tests were also performed. Mutations affecting the previously described mer genes merR (regulation), merT (transport), and merA (reductase) were characterized. Evidence was obtained for two new mer genes, which have been called merC and merD. A restriction enzyme map of the mer region was drawn with the gene order merRTCAD. Transcriptional merR-lac and merA-lac fusions were generated by insertion of phage Mu d amp lac into plasmid R100-1. These were used to study regulation of mer gene expression. The merR gene product appears to regulate negatively its own expression as well as acting as both a negative and a positive regulator of the merTCA genes.

DNA Transposable Elements↗

PubMed1983

Citrate utilization by Escherichia coli: plasmid- and chromosome-encoded systems.

Citrate utilization plasmids have previously been identified in atypical Escherichia coli isolates. A different citrate-utilizing (Cit+) variant of E. coli K-12 arose as a consequence of two chromosomal mutations (B. G. Hall, J. Bacteriol. 151:269-273, 1982). The processes controlling the transport of citrate in both a Cit+ chromosomal mutant and a Cit+ plasmid system were studied. Both systems were found to be inducible in growth experiments. In transport assays with whole cells, citrate-grown cells accumulated [1,5-14C]citrate at two to three times the rate of uninduced cells. Only the Vmax was affected by induction, and the Km for whole cells remained at 67 microM citrate for the chromosomal strain and 120 microM citrate for the plasmid-conferred system. There was no detectable accumulation of radioactivity with [6-14C]citrate, because of rapid metabolism and the release of 14CO2. Energy-dependent citrate transport was found with membrane vesicles obtained from both the chromosome-conferred and the plasmid Cit+ systems. The vesicle systems were inhibited by valinomycin and carbonyl cyanide m-chloro-phenylhydrazone but not by nigericin and monensin. In contrast to whole cells, the vesicle systems were resistant to Hg2+ and showed identical kinetics with [1,5-14C]citrate and [6-14C]citrate. H+ appeared to be important for citrate transport in whole cells and membranes. Monovalent cations such as Na+ and K+, divalent cations such as Mg2+ and Mn2+, and anions such as PO4(3-), SO4(2-), and NO3- were not required. The two systems differed in inhibition by citrate analogs.

Aconitic Acid↗

PubMed1982

Alternate innervations of facial musculature.

There have been several case reports in which spontaneous return of facial function has occurred without grafting or other surgical intervention. This implies that there may be alternate pathways to explain this phenomenon. Using the technique of retrograde axonal transport with chromogens tagged to the marker enzyme horseradish peroxidase, selected facial muscles of the monkey were injected to demonstrate CNS representation. The facial nucleus was identified, exhibiting topographic arrangement in the CNS. More importantly, in several specimens, nuclear cells of the mesencephalic tract also were stained. This is part of the trigeminal system, normally associated with proprioception, whose axons travel with other fifth cranial nerve branches. The trigeminal motor nucleus did not exhibit any pickup of the enzyme marker, indicating that there was no contamination from facial musculature innervated by the fifth cranial nerve. This finding suggests that the facial musculature, normally innervated by the seventh cranial nerve, has an additional nerve supply.

Animals↗

PubMed1982

Energy-dependent arsenate efflux: the mechanism of plasmid-mediated resistance.

Plasmid-mediated resistance to arsenate, arsenite, and antimony(III) is coordinately induced by arsenate, arsenite, antimony(III), and bismuth(III). Resistance to arsenate was recently shown [Silver, S., Budd, K., Leahy, K.M., Shaw, W.V., Hammond, D., Novick, R.P., Willsky, G.R., Malamy, M.H. & Rosenberg, H. (1981) J. Bacteriol. 146, 983-996] to be due to decreased accumulation of arsenate by the induced resistant cells. We report here that decreased net uptake results from accelerated efflux of arsenate by induced plasmid-containing cells of Staphylococcus aureus and Escherichia coli. The efflux system in S. aureus was inhibited by nigericin, monensin, and proton-mobilizing uncouplers; efflux was unaffected by valinomycin. The mechanism of arsenate efflux in S. aureus was apparently not by chemiosmotic coupling to the membrane electrical potential or pH gradient. The intracellular efflux system was inhibited by low pH and mercurials (reversible by mercaptoethanol). The efflux rate was relatively independent of external pH or phosphate level and showed a sigmoidal pattern of concentration dependence.

Arsenates↗

PubMed1982

Selection for mercurial resistance in hospital settings.

The frequency of resistance to Hg2+ in 1980 to 1981 collections from Barnes Hospital, St. Louis, Mo., was only 2% for Staphylococcus aureus and 9% for Escherichia coli. The frequency of Hg2+ resistance in E. coli isolates from Jikei University Hospital, Tokyo, Japan, was 57% during 1972 to 1977 and decreased to 29% in 1979 to 1982; for S. aureus the frequency of Hg2+ resistance dropped from 36% in 1972 to 1977 to 10% in 1979 to 1982. Frequencies of resistances to cadmium (S. aureus) and arsenic (S. aureus and E. coli) remained approximately constant during this time. The decrease in frequency of mercurial resistance is attributed to the termination of the use of organomercurials (largely phenylmercury and thimerosal) in hospital liquid detergents and disinfectants. It is proposed that selection for mercurial resistance occurred within the hospital setting when there was widespread use of mercurials. The resistance patterns and phage types for each of four new mercurial-resistant S. aureus isolates from St. Louis were distinct, indicating that no single type of "hospital staph" predominates. Furthermore, resistance to thimerosal, merbromin, and methylmercury and the ability to volatilize 14C from [14C]methylmercury were found with the new isolates and never with previously known mercurial resistance plasmids in S. aureus.

Bacteria↗

PubMed1982

Cadmium and manganese transport in Staphylococcus aureus membrane vesicles.

The presence of plasmid gene cadB did not affect Cd2+ accumulation, whereas plasmid gene cadA reduced Cd2+ accumulation by whole cells but not by membrane vesicles. Membrane vesicle studies indicated that Cd2+ uptake occurred via the Mn2+ transport system which was energized by the membrane electrical potential. Mn2+ and Cd2+ were competitive inhibitors of each other's transport, with Km's of 0.95 microM Mn2+ and 0.2 microM Cd2+. The kinetic parameters were nearly identical with vesicles prepared from sensitive and resistant cells, indicating that the cadA-encoded Cd2+ efflux system was inoperative in membrane vesicle preparations. Experiments with energy-inhibited cells indicated that the cadB gene product may bind Cd2+.

Biological Transport, Active↗

PubMed1982

Mercuric reductase enzyme from a mercury-volatilizing strain of Thiobacillus ferrooxidans.

Cell-free mercury volatilization activity (mercuric reductase) was obtained from a mercury-volatilizing Thiobacillus ferrooxidans strain, and the properties of intact-cell and cell-free activities were compared with those determined by plasmid R100 in Escherichia coli. Intact cells of T. ferrooxidans volatilized mercury at pH 2.5, whereas cells of E. coli did not. Cell-free enzyme preparations from both bacteria functioned best at or above neutral pH and not at all at pH 2.5. The T. ferrooxidans mercuric reductase was a soluble enzyme that was dependent upon added NAD(P)H. The enzyme activity was stable at 80 degrees C, required an added thiol compound, and was stimulated by EDTA. Antisera against purified mercuric reductases from transposon Tn501 and plasmid R831 (which inactivated mercuric reductases from a wide range of enteric and pseudomonad strains) did not inactivate the enzyme from T. ferrooxidans.

DNA Transposable Elements↗

PubMed1981

Inducible plasmid-determined resistance to arsenate, arsenite, and antimony (III) in escherichia coli and Staphylococcus aureus.

Plasmids in both Escherichia coli and Staphylococcus aureus contain an "operon" that confers resistance to arsenate, arsenite, and antimony(III) salts. The systems were always inducible. All three salts, arsenate, arsenite, and antimony(III), were inducers. Mutants and a cloned deoxyribonucleic acid fragment from plasmid pI258 in S. aureus have lost arsenate resistance but retained resistances to arsenite and antimony, demonstrating that separate genes are involved. Arsenate-resistant arsenite-sensitive S. aureus plasmid mutants were also isolated. In E. coli, plasmid-determined arsenate resistance and reduced uptake were additive to that found with chromosomal arsenate resistance mutants. Arsenate resistance was due to reduced uptake of arsenate by the induced plasmid-containing cells. Under conditions of high arsenate, when some uptake could be demonstrated with the induced resistant cells, the arsenate was rapidly lost by the cells in the absence of extracellular phosphate. Sensitive cells retained arsenate under these conditions. When phosphate was added, phosphate-arsenate exchange occurred. High phosphate in the growth medium protected cells from arsenate, but not from arsenite or antimony(III) toxicity. We do not know the mechanisms of arsenite or antimony resistance. However, arsenite was not oxidized to less toxic arsenate. Since cell-free medium "conditioned" by prior growth to induced resistant cells with toxic levels of arsenite or antimony(III) retained the ability to inhibit the growth of sensitive cells, the mechanism of arsenite and antimony resistance does not involve conversion of AsO2- or SbO+ to less toxic forms or binding by soluble thiols excreted by resistant cells.

Antimony↗

PubMed1981

Genetic analysis of mutants affected in the Pst inorganic phosphate transport system.

A number of mutant alleles affecting the Pst phosphate transport system have been divided into three complementation groups on the basis of constitutive alkaline phosphatase activity in appropriate partial diploid strains. The three complementation groups were represented by the alleles pstA2 and phoT32 and the newly described allele pstB401. The two alleles phoS28 and phoS21 appeared to be polar. The phoS28 allele affected both the phoT and pstB genes but not the pstA gene, whereas the phoS21 allele appeared to be a mutation in the pstA gene exerting polar effects on both the pstB and phoT genes. It was concluded that the three genes pstA, pstB, and phoT were part of an operon and that the phosphate-binding protein was not coded for by any of these genes. The phoS gene, defined as the structural gene for the phosphate-binding protein, is also part of the operon, but the phoS28 and phoS21 alleles are not mutations in the phoS gene and were reclassified as pho-28 and pho-21 alleles. The gene order was concluded to be pstA-(pstB-phoT)-phoS, with the pstA gene promotor proximal and the direction of transcription opposite to that of the nearby unc operon.

Alkaline Phosphatase↗

PubMed1981

Evaluation of a hospice program: effects on terminally ill patients and their families.

Hospice care in the United States has grown rapidly since its introduction here from England in the mid-1970s. Surprisingly little evaluation of services has been made public, particularly with regard not merely to demographics but also the actual effect of hospice caregiving on patients and families. This article describes an attempt to identify the life dimensions that hospice addresses and the levels of discomfort or well-being of patients and families achieved in a hospice home care program. After assessing the changes in medical, psychosocial, and spiritual status for each of a group of hospice home care patients, the trend toward greater wellbeing of patients in the program can be delineated.

Evaluation Studies as Topic↗