Biomedical subjects
Publications and source records attributed to C H Nash.
A new species of a new genus of the Actinomycetales was discovered, Kibdelosporangium aridum. This strain produces a new family of glycopeptide antibiotics designated aridicins, that contain an unusual glycolipid constituent. They inhibit Gram-positive bacteria, including staphylococci, enterococci and Clostridium sp.
Plasmids were isolated from two species of Streptosporangium by CsCl-ethidium bromide equilibrium density gradient centrifugation. A plasmid isolated from S. brasiliense, designated pSgB-1, was characterized by electron microscopy and agarose gel electrophoresis. The pSgB-1 plasmid is a closed circular DNA molecule of 9.4 X 10(6) Da. A restriction endonuclease map was generated and unique cleavage sites were found for EcoRI, ClaI, XbaI, and MstII. Another plasmid, pSgV-1, isolated from S. viridognriseum, has an estimated Mr of 54 X 10(6). The pSgB-1 plasmid is phenotypically cryptic but an unusual phenotypic trait, resembling phage plaques, may be associated with the S. viridogriseum plasmid pSgV-1.
The antifungal agent Aculeacin A at subinhibitory levels induced lytic plaques in lawns of Candida albicans. Electron microscopic examination of plaque lysates suspended in phosphotungstic acid revealed the presence of spherical particles 12, 18, and 28 to 30 nm in size. Particles were also found in ultrathin sections of treated C. albicans cells. The plaque lysate lost infectivity after treatment with UV light, heat treatment at 80 degrees C for 10 min, or being held at pH 2 for 30 min.
Mutants of Candida albicans resistant to aculeacin A, a yeast cell-wall inhibitor, were isolated after mutagenesis with ultraviolet light. The parental strain was sensitive to 0.1 approximately 0.5 microgram/ml of the antibiotic. In contrast, the minimum inhibitory concentration for the mutants ranged from 50 to 200 microgram/ml. Except for papulocandin, another cell-wall inhibitor, the antibiotic susceptibility of the mutants was similar to the parental strain. The parent strain and the aculeacin resistant mutants exhibited similar morphological changes at subinhibitory levels of aculeacin and had comparable growth rates on complex media. The lipid and sterol content of the parent and the mutants were significantly different. For example, the total lipid content was two-fold higher in the mutant strains. Drug resistance in the mutants was specific for aculeacin and papulocandin and appeared to be associated with alteration in the lipid composition of membranes.
Spores of Streptosporangium brasiliense aggregated at acidic pH's and were dissociated into free spores by changing to neutral or alkaline pH's. This finding was utilized in preparing uniform spore suspension, in which simple filtrations gave rise to consistent preparation of free spores at alkaline pH.
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The stability of cefonicid (SK&F 75073) towards representatives of six major classes of beta-lactamases was determined using a spectrophotometric assay. Cefonicid was stable to hydrolysis by the Type I enzyme from Enterobacter cloacae and by the enzyme from the anaerobe, Bacteroides fragilis. It was 6 to 7 times more stable than cefamandole to the Type IIIA and B enzymes from Escherichia coli, a little less stable than this antibiotic to the Type V enzyme from E. coli, and of equal stability to the Type IV enzyme from Klebsiella aerogenes. Cefonicid was a non-competitive inhibitor (Ki of 0.8 x 10(-6)M) of cephalothin hydrolysis by the Type I enzyme.
Univariate and multivariate regression methods were used to analyze 17 potential clinical prognostic factors among 138 patients with advanced breast cancer who received Adriamycin-cyclophosphamide combination chemotherapy between 1973 and 1977 at the University of Arizona. Follow-up of patients was through September 1979, and survival data were nearly complete. Different factors varied in the relationship to outcome, but age, treatment, and response were important. Selecting the three most strongly related factors, predictive regression equations were developed, which described three types of possible outcome: 1) objective response (age, treatment, and liver involvement), 2) freedom from relapse (age, lung involvement, and response), and 3) survival (age, the number of involved sites [less than or equal to 2 or > 2], and treatment). Since use of the regression equations is cumbersome for clinical practice, three simplified tables were constructed to readily predict response, duration of response, and survival before the initiation of treatment.
Fluorescent staining procedures were developed for elucidating the nucleoid region in Streptosporangium albidum and Streptosporangium brasiliense. In these procedures, plugs of nutrient agar were inoculated with the microorganims and then covered with a sterile glass slide. The growing cells adhered to the surface of the slide and remained attached throughout the staining procedures. Two separate staining methods were utilized, one with bisbenzimid H33258 and the other with auramine O. Fluorescent microscopy revealed intensely stained nucleoid regions within mycelia, spores, and sporangia.
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The susceptibility of Cephalosporium acremonium to selected amino acid analogues was markedly influenced by the carbon source used in the test media. Lysine hydroxamate, beta-hydroxy norvaline, and hexafluorovaline were toxic when tested with ribose, ribose or fructose, and ribose or galactose, respectively. In contrast, thialysine and thiaisoleucine inhibited C. acremonium with glucose, fructose, galactose, sucrose, mannitol, sorbitol, and soluble starch. Neither of these analogues was toxic at levels tested when glycerol was used as a carbon source. The minimal inhibitory concentrations (MIC) of thialysine, homoserine, and alpha-methylserine were greater than 1000, greater than 1000, and 250 microgram/mL, respectively, with glycerol. In contrast, the MIC values for the same three analogues were 31, 62, and 125 microgram/mL, respectively, with mannitol. The matching of the carbon sources with the specific amino acid analogues expands the number of analogues useful for selecting derepressed mutants. Thialysine-resistant mutants (tlysR) of C. acremonium which excrete lysine were isolated on a medium containing mannitol.
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Mannitol kinase and mannitol-1-phosphate dehydrogenase activities were detected in two Micromonospora isolates. The presence of these enzyme activities indicates that mannitol is catabolized first to mannitol-1-phosphate and then to fructose-6-phosphate. Mannitol-oxidizing enzymes were also surveyed in representative species of four other genera of actinomycetes. Mannitol-1-phosphate dehydrogenase was detected in cell-free extracts of Streptomyces lactamdurans. In contrast, cell-free extracts of Mycobacterium smegmatis, Nocardia erythrophila, Streptomyces lavendulae, and Actinoplanes missouriensis contained mannitol dehydrogenase activity but no detectable mannitol-1-phosphate dehydrogenase activity. The mannitol dehydrogenase activities in the latter species support the operation of a pathway for catabolism of mannitol that involves the oxidation of mannitol to fructose, followed by phosphorylation to fructose-6-phosphate.
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The final step in the biosynthesis of mycophenolic acid involves the transfer of a methyl group from S-adenosylmethionine to demethylmycophenolic acid. The enzyme, S-adenosylmethionine:demethylmycophenolic acid O-methyltransferase, was isolated from Penicillium stoloniferum and purified 2,700-fold by ammonium sulfate fractionation and diethylaminoethyl-cellulose and Sephadex G-200 chromatography. Maximum enzyme activity was achieved at pH 7.5 and a temperature of 27 to 28 C. The apparent K(m) for demethylmycophenolic acid was 3.1 x 10(-6) M. The enzyme preparation was 50% inactivated when exposed to 33 C for 15 min. Mycophenolic acid, homocystine, S-adenosyl-homocysteine, ethanol, and Mg(2+) inhibited the methyltransferase. This enzyme appears to be subject to end product inhibition which may regulate the synthesis of mycophenolic acid. The methyltransferase activity was highest during the early phases of the fermentation.