Biomedical subjects
Publications and source records attributed to S Silver.
A comparison of two distinct 11 year time periods at our institution demonstrated a change not only in the cause of intrahepatic abscess but also in the procedures used to diagnose this condition. Significant improvement in the methods of detection of intrahepatic abscess permits earlier diagnosis and therapy and thus a significantly improved prognosis.
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The level of resistance to Hg2+ determined by the inducible mer operon of plasmid NR1 was essentially the same for three gene copy number variants in Escherichia coli, less in Proteus mirabilis, and intermediate in P. mirabilis "transitioned" to a high r-determinant gene copy number. Cell-free volatilization rates of radioactive mercury indicated increasing levels of intracellular mercuric reductase enzyme from low- to high-gene copy number forms in P. mirabilis and from low- to high-copy number forms in E. coli, but the additional enzyme in E. coli was effectively cryptic.
The region of plasmid NR1 concerned with resistance to Hg2+ and organomercurials consists of sequences found on restriction endonuclease fragments EcoRI-H and EcoRI-I. When both fragments were cloned together into a derivative of plasmid ColE1, the hybrid plasmid conferred properties indistinguishable from those of the parental plasmid, NR1: resistance to Hg2+ and to the organomercurials merbromin and fluoresceinmercuric acetate and the inducible synthesis of the enzyme mercuric reductase. When fragment EcoRI-I was cloned into plasmid ColE1, cells containing the plasmid was as sensitive to Hg2+ and organomercurials as plasmidless strains. When fragment EcoRI-H was cloned into ColE1, cells with the hybrid plasmid were hypersensitive to Hg2+ and organomercurials. This hypersensitivity was inducible by prior exposure to low, subtoxic Hg2+ or merbromin levels. It was associated with an inducible hyperbinding activity attributed to a gene governing Hg2+ uptake and found on fragment EcoRI-H (which contains the proximal portion of a mercuric resistance [mer] operon).
A series of 23 transposon 801(Tn801)-induced mutations of plasmid R100-1 from mercuric salts resistance to sensitivity was studied. Although Tn801 transposed frequently into the mer region of the plasmid, fine structural analysis showed that the site of insertion within mer varied. About one-half of the Tn801 insertion events also caused a deletion of greater than 1 megadalton. Genetic and restriction endonuclease EcoRI and BamHI analysis of the mutant plasmid deoxyribonucleic acid elucidated the organization of the mer operon and suggested the existence of a trans-acting regulatory factor governing resistance to mercuric salts. Tn801 insertions leading to mercuric sensitivity occurred in the restriction endonuclease fragments EcoRI-H and EcoRI-I. Regulatory mutations leading to a 50-fold-reduced synthesis of mercuric reductase enzyme occurred in two complementation classes thought to represent the gene for a trans-acting inducer molecule and a cis-acting operator-promoter sequence. Mutations leading to total loss of the enzyme mercuric reductase occurred on both the EcoRI-H and EcoRI-I fragments, showing that the structural gene for this enzyme (merA) bridges the EcoRI cleavage site separating the segments. Hypersensitivity to mercuric salts resulted when Tn801 insertion occurred in the reductase gene in the operatordistal portion of the operon. Hypersensitive cells inducibly bound three to five times more Hg2+ at low concentrations than did sensitive (plasmidless) cells. This finding led to the proposal that another gene (merT) controls uptake of Hg2+ by the cells. Transcription of the operon was deduced to start in the EcoRI-H fragment and to move into the EcoRI-I fragment of the plasmid genome.
Transcription of the Marek's disease virus (MDV) genome in tumor tissues from MDV-infected chickens has been studied by analyzing the hybridization kinetics of (3)H-labeled MDV DNA with unlabeled RNA extracted from these tissues. Lymphoid tumors of ovary, spleen, liver, and kidney contained MDV genomes, but the virus-specific RNA sequences were transcribed from less than 15% of the viral DNA. A virus nonproductive lymphoblastoid cell line, designated MKT-1, has been established from a kidney lymphoma and contains 15 MDV genomes per cell. In these cells, 12 to 14% of the viral DNA was transcribed. Thus transcription of the MDV genome was restricted both in tumor tissues and MKT-1 cells. A hybridization experiment where RNA extracted from MKT-1 cells and RNA extracted from a spleen tumor were mixed and hybridized to (3)H-labeled MDV DNA indicated that the virus-specific RNAs from the two sources were encoded by the same DNA sequences. The polyribosomal fractions of MKT-1 cells and this spleen tumor contained only a portion of the virus-specific RNA sequences found in whole-cell extracts, indicating the existence of a posttranscriptional control mechanism which prevents the transfer of certain viral RNA transcripts to the polyribosomes. The data suggest that the repressed expression of the viral genome in lymphoid tumor tissues and MKT-1 cells may be the result of precise controls within the cell at the transcriptional and posttranscriptional levels.
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Plasmid-determined resistance to cadmium has only been found with plasmids from Staphylococcus aureus. Resistance to cadmium was associated with a lower accumulation of Cd(2+) ions by the plasmid-bearing resistant cells. Cadmium accumulation by susceptible cells was energy dependent and had those characteristics usually associated with a transmembrane active transport system. There was a specific interrelationship between cadmium accumulation and manganese accumulation and retention. Cd(2+) inhibited the uptake of Mn(2+) and accelerated the loss of intracellular Mn(2+) by the susceptible cells, but was without effect on Mn(2+) transport in resistant S. aureus cells. Under similar conditions, there was no differential effect of Cd(2+) on Mg(2+), Zn(2+), Co(2+), Ni(2+), or Rb(+) accumulation or exchange between the susceptible and the resistant strains.
Separate divalent cation transport systems for energy-dependent uptake of Mg2+ and Mn2+ were found both with aerobically and heterotrophically grown and with photosynthetically grown cells of Rhodopseudomonas capsulata. The maximum rate of Mg2+ uptake differed between photosynthetic and aerobic cells, while the Km for the Mg2+ transport system was constant. Photosynthetic midlog-phase cells exhibited Km's for uptake of about 55 micrometer Mg2+ and 0.5 micrometer Mn2+. The Vmax's also differed between the two systems: 0.6 to 1.8 mumol/min per g (dry weight) of cells for Mg2+, but only 0.020 mumol/min per g for Mn2+, making the distinction between a "macro-requirement" system and a system functioning at trace nutrient levels. Calcium was not normally taken up by intact cells of R. capsulata. However, chromatophore membranes isolated from photosynthetic cells took up Ca2+ by an energy-dependent process.
Three lambdamer (resistance to Hg2+ and mercurials) transducing phages were prepared from three independent cointegrate isolates of bacteriophage lambda and plasmid R100. DNA heteroduplex and restriction nuclease analyses of the lambdamer DNA showed that all three phages had resulted from lambda insertion at kilobase coordinate 8.6 of plasmid R100, followed by loss of different lengths of lambda DNA and replacement with different lengths of R100 DNA. Two of the lambdamer phages were defective, containing deletions from lambdaatt through the lambdaN gene and into the lambdarex gene; the third, VAlambda14, was an N+ Spi- plaque-forming phage. With VAlambda14, N-dependent transcription of R100 mer from the lambdapL promoter suggested that transcription of mer proceeded in the direction from IS1b toward the sulfonamide resistance determinant (i.e., from a plasmid promoter in restriction nuclease fragment EcoRI-H toward fragment EcoRI-I). Phage-directed protein synthesis in a UV-irradiated lambdaind- lysogen showed the Hg2+-inducible synthesis of three major polypeptides of molecular weights 68,000, 11,500, and 8,500 and three minor ones of molecular weights 54,000, 33,000, and 13,500. The largest of the major polypeptides is identified as the subunit of the mercuric reductase enzyme. The functions of the smaller polypeptides are not known. Hg2+ reductase enzyme assays confirmed the regulation of mer synthesis during phage infection.
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Expression of latent Epstein-Barr virus genomes in lymphoblastoid cells and somatic-cell hybrid of Burkitt's lymphoblastoid cells has been studied. IUDR treatment induced the formation of early antigen (EA), virus capsid antigen (VCA) and virus DNA replication in D98/Raji and D98/HR-1 cells whereas only EA was induced in Raji cells. HR-1 clone No. 9 did not respond to IUDR treatment. The pattern of transcription of virus genomes in these cell lines without IUDR treatment was uniform with 20--25% of virus DNA transcribed. IUDR treatment enhanced the transcription of virus DNA to 50% in D98/Raji, D98/HR-1 and Raji cells but no enhancement of virus genome transcription was observed in HR-1 clone No. 9 The amount of virus RNA in the cells calculated from DNA-RNA hybridization kinetics was found to be proportional to the number of virus genomes per cell indicating that every copy of virus DNA in these cells is actively transcribed.
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Of the 787 isolates, 99.8% were metal resistant, with most (99.5%) showing multiple resistance. Fifty-three percent of the isolates were both metal and drug resistant, whereas only 19% were metal resistant and drug sensitive.
Mercury and organomercurial resistance determined by genes on ten Pseudomonas aeruginosa plasmids and one Pseudomonas putida plasmid have been studied with regard to the range of substrates and the range of inducers. The plasmidless strains were sensitive to growth inhibition by Hg(2+) and did not volatilize Hg(0) from Hg(2+). A strain with plasmid RP1 (which does not confer resistance to Hg(2+)) similarly did not volatilize mercury. All 10 plasmids determine mercury resistance by way of an inducible enzyme system. Hg(2+) was reduced to Hg(0), which is insoluble in water and rapidly volatilizes from the growth medium. Plasmids pMG1, pMG2, R26, R933, R93-1, and pVS1 in P. aeruginosa and MER in P. putida conferred resistance to and the ability to volatilize mercury from Hg(2+), but strains with these plasmids were sensitive to and could not volatilize mercury from the organomercurials methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids, in addition, conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate. The other plasmids, FP2, R38, R3108, and pVS2, determined resistance to and decomposition of a range of organomercurials, including methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids also conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate by a mechanism not involving degradation. In all cases, organomercurial decomposition and mercury volatilization were induced by exposure to Hg(2+) or organomercurials. The plasmids differed in the relative efficacy of inducers. Hg(2+) resistance with strains that are organomercurial sensitive appeared to be induced preferentially by Hg(2+) and only poorly by organomercurials to which the cells are sensitive. However, the organomercurials p-hydroxymercuribenzoate, merbromin, and fluorescein mercuric acetate were strong gratuitous inducers but not substrates for the Hg(2+) volatilization system. With strains resistant to phenylmercury and thimerosal, these organomercurials were both inducers and substrates.