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Biomedical subjects

V L Clark

Publications and source records attributed to V L Clark.

At least 55 records · Page 3Linked to original sources
PubMed1984

Anaerobic growth of Neisseria gonorrhoeae coupled to nitrite reduction.

A total of 204 Neisseria gonorrhoeae strains, including 39 penicillinase-producing strains, representing 64 distinct auxotype and serovar classes were tested for their ability to grow anaerobically with nitrite as a terminal electron acceptor. All strains grew anaerobically with subtoxic concentrations of nitrite, and all penicillinase-producing strains produced beta-lactamase when grown anaerobically. Nitrite reductase was produced constitutively under aerobic conditions in the absence of nitrite, and cytochrome oxidase was produced constitutively under anaerobic conditions. Strains could not grow anaerobically with sulfite as a terminal electron acceptor. Strain NRL 905 grew anaerobically in broth medium containing nitrite at a rate comparable to its growth rate under aerobic conditions. The feasibility and significance of in vivo anaerobic growth of N. gonorrhoeae is discussed.

Anaerobiosis↗
PubMed1984

Cloning genes for proline biosynthesis from Neisseria gonorrhoeae: identification by interspecific complementation of Escherichia coli mutants.

DNA from Neisseria gonorrhoeae KH45 was partially digested with Sau3A and inserted into the BamHI site of the cloning vector pLES2 . After introduction into Escherichia coli JM83 by transformation, two different size classes of plasmids were isolated that could complement the proAB deletion of JM83 . These plasmids ( pLES4 and pLES7 ) were characterized by restriction endonuclease digestion. Southern hybridization demonstrated that the inserts had sequence homology. Various deletions of these plasmids were constructed that had lost the ability to complement the proA lesion of chi 463, the proB lesion of chi 340, or both (plasmids pLES9 , pLES8 , and pLES10 , respectively). These deleted plasmids were introduced into a proline-requiring strain of N. gonorrhoeae, F62, with plasmids pLES4 , pLES7 , and pLES8 possessing the ability to correct the proline requirement of F62. Further analysis indicated that the hybrid plasmids were stably maintained as plasmids in N. gonorrhoeae.

Aldehyde Oxidoreductases↗
PubMed1984

Hypomagnesemia after cisplatin combination chemotherapy.

Sixty-six patients receiving a five-drug combination chemotherapy regimen containing low-dose cisplatin were studied for the presence of hypomagnesemia. Thirty-eight (76%) of 50 patients receiving treatment every four weeks became hypomagnesemic during treatment. The incidence increased with the cumulative cisplatin dose, ranging from 41% after a single course to 100% of patients receiving six cycles of therapy. The incidence seemed lower in patients receiving the combination with a greater interval (eight weeks v four weeks) between cycles. We report the incidence and severity of hypomagnesemia to be dose dependent. The cause of the higher incidence of hypomagnesemia observed in this series compared with others is unknown but may be related to an interaction of cisplatin with another drug contained in this regimen.

Adult↗
PubMed1983

A low-Mr factor isolated from Escherichia coli inhibits eukaryotic in vitro protein synthesis.

The effect of a low-Mr factor, partially purified from E. coli B, was investigated in E. coli, reticulocyte, and wheat germ lysate in vitro protein synthesis systems. Equal concentrations of factor were needed to inhibit protein synthesis in the eukaryotic system as compared to the prokaryotic system. Experiments suggested that the factor inhibits the initiation step in the eukaryotic systems.

Animals↗
PubMed1983

Characterization of a chimeric beta-lactamase plasmid of Neisseria gonorrhoeae which can function in Escherichia coli.

A chimeric beta-lactamase encoding plasmid, containing the 4.4 Mdal beta-lactamase plasmid and the 2.6 Mdal cryptic plasmid of Neisseria gonorrhoeae has been characterized by physical and biological methods. Digestion with restriction enzymes indicates the presence of the following restriction sites: 1 site: AccI, AvaI, HgiAI, HincII, MstI, PstI, PvuII, XbaI and XorI; 2 sites: HindIII and BamHI; 3 sites: BclI, Sau96I and AvaII; 6 sites: HinfI; greater than 8 sites: AluI, BbvI, DdeI, HgaI, HhaI, HpaII, MspI, Sau3A, TacI and TaqI. No restriction sites were found for the following: BglI, BglII, BstEII, EcoRI, EcoRII, HpaI, HphII, KpnI, SacI, SalI, SmaI, SstI, SstII, and XhoI. Five plasmid specific proteins have been identified by DNA directed in vitro protein synthesis (43K, 41K, 30K, 16K and 14K). The location on the physical map of the coding regions for each of these proteins has been determined by the following methods: using plasmid DNA restricted by various enzymes in an in vitro protein synthesis system; identifying promoter-containing regions by digesting plasmid DNA with DdeI, adding RNA polymerase and then determining which fragments are retained by nitrocellulose. This plasmid contains both parental phenotypes in that it encodes penicillin resistance and possesses the sequence necessary for uptake in the gonococcus. Transformation data indicate that this plasmid can function in both E. coli and N. gonorrhoeae and that growth in E. coli has no effect on the plasmid's ability to transform the gonococcus.

Bacterial Proteins↗
PubMed1983

Identification of three homology classes of small, cryptic plasmids in intestinal Bacteroides species.

A systematic analysis of the plasmid content of intestinal Bacteroides spp. was made. Eight of fifteen clinical isolates and seven of nineteen normal rectal flora isolates examined contained small plasmids (less than 5 Mda). The majority of these small plasmids could be assigned to three homology classes by Southern hybridization. Further investigation demonstrated the presence of subclasses within one of the homologous classes. Class I plasmids were 1.8 Mda, Class III were 3.7 Mda, and Class II plasmids were of three different molecular weights: 2.6 Mda (IIA), 3.2 Mda (IIB), and 4.0 Mda (IIC). Among representative plasmids from each class there was remarkable sequence similarity based upon digestion patterns obtained using the restriction endonucleases, AluI and DdeI. In addition, similar polypeptide products were observed using purified Class I plasmids as template in an Escherichia coli in vitro coupled transcription-translation system. Small plasmids were found in seven of the ten recognized species of intestinal bacteroides, indicating that no species barrier exists for these plasmids. In addition, since plasmids from all three classes were found together in a single isolate, it was concluded that these plasmids are not incompatible.

Bacteroides↗
PubMed1983

Construction and characterization of a new shuttle vector, pLES2, capable of functioning in Escherichia coli and Neisseria gonorrhoeae.

In vitro recombination techniques were used to construct a bifunctional shuttle vector capable of functioning in Neisseria gonorrhoeae and Escherichia coli. This 6-kb plasmid contains a selectable phenotype, beta-lactamase production, which functions in both organisms. It also contains the lac region from pUC9 that allows for the direct selection of hybrid plasmids in the appropriate E. coli hosts by disruption of beta-galactosidase alpha complementation. The lac region contains several unique restriction sites useful for cloning: EcoRI, SmaI, BamHI and SalI.

DNA Transposable Elements↗
PubMed1982

High correlation of the presence of methyladenine in Neisseria gonorrhoeae DNA with the AHU auxotype.

Strains of Neisseria gonorrhoeae were tested for the presence of methyladenine in the DNA sequence GATC by using the site-specific restriction endonucleases MboI and DpnI. It was found that 43 of 83 strains tested contained methylated DNA. When methylation was compared with the auxotype of the organism, 35 of 35 strains with the AHU (arginine-, hypoxanthine-, and uracil-requiring) auxotype and 8 of 48 strains with other auxotypes contained methyladenine. When the incidence of methylation in strains isolated from patients suffering from disseminated gonococcal infection was compared with that in strains isolated from patients suffering from uncomplicated gonococcal infection, no correlation with methylation and disseminated gonococcal infection was observed.

Adenine↗
PubMed1982

Anaerobic survival of clinical isolates and laboratory strains of Neisseria gonorrhoea: use in transfer and storage.

Eleven laboratory strains and 67 clinical isolates of Neisseria gonorrhoeae were tested for the ability to survive during anaerobic incubation. The survival of the laboratory strains was dependent on auxotype, temperature, and cell density on agar plates. For both the laboratory strains and the clinical isolates, anaerobic survival was better at lower temperatures. We concluded that anaerobic incubation, for as long as 7 days, is useful when transporting or storing N. gonorrhoeae.

Anaerobiosis↗
PubMed1980

Inhibition of the initiation of translation by a factor isolated from Escherichia coli cells.

A low molecular weight factor isolated from Escherichia coli cells was found to inhibit protein synthesis in vitro directed by RNA of bacteriophage R-17. The factor also inhibited poly(U)-directed translation, but higher concentrations were required. When the factor was added after R-17 RNA-directed translation was initiated, the onset of inhibition was delayed. Initiation-independent translation of polysomes was not inhibited. The factor inhibited the binding of N-formylmethionyl-tRNAfMet to 70S ribosomes to form the 70S initiation complex, and it released the N-flrmylmethionyl-tRNAfMet from preformed complexes. The factor did not prevent the formation of N-formylmethionylpuromycin. It was concluded that the factor inhibits specifically the initiation of translation.

Cell-Free System↗
PubMed1979

Isolation and characterization of an endogenous inhibitor of protein synthesis in Escherichia coli K-12.

A low-molecular-weight factor was isolated from cell extracts of Escherichia coli K-12. The concentration of the factor in cells was dependent upon nutritional conditions, the concentration being higher in faster growing cells. Treatment of cells with colicin K caused an increase in concentration of the factor. The factor inhibited protein synthesis in E. coli. This inhibition was reversible, apparently because of metabolism of the factor. The inhibition of synthesis of beta-galactosidase lasted longer than the inhibition of protein synthesis; cyclic AMP eliminated this difference. The factor inhibited the synthesis of beta-galactosidase from preformed lac mRNA, indicating an inhibition of translation. Kinetic studies of the onset of inhibition of beta-galactosidase synthesis by the factor suggested that the factor may inhibit protein synthesis at the initiation of translation.

Bacterial Proteins↗
PubMed1978

D-alanine incorporation into macromolecules and effects of D-alanine deprivation on active transport in Bacillus subtilis.

An auxotroph of Bacillus subtilis 168 unable to synthesize D-alanine loses the ability to support endogenously energized transport when deprived of D-alanine. Revertants of the mutant retain transport activity. The loss of transport is specific for substrates taken up by active transport; substrates taken up by group translocation are transported at normal rates. The loss of transport can be retarded by pretreatment of the cells with inhibitors of protein synthesis. Since the loss of transport could be due to an alteration in a D-alanine-containing polymer, we investigated the incorporation of D-[14C]alanine into macromolecules. The major D-alanine-containing polymers in B. subtilis are peptidoglycan and teichoic acid, with 4 to 6% of the D-[14C]alanine label found in trypsin-soluble material. Whereas the peptidoglycan and teichoic acid undergo turnover, the trypsin-soluble material does not. Treatment of the trypsin-soluble material with Pronase releases free D-alanine. Analysis of acid-hydrolyzed trypsin-soluble material indicated that approximately 75% of the radioactivity is present as D-alanine, with the remainder present as L-alanine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified D-[14C]alanine-labeled membranes indicated the presence of two peaks of radioactivity (molecular weights, 230,000 and 80,000) that could be digested by trypsin. The results suggest that D-alanine may be covalently bound to cellular proteins.

Alanine↗
PubMed1977

Inducible resistance to D-cycloserine in Bacillus subtilis 168.

Resistance to d-cycloserine could be induced in Bacillus subtilis 168 by sublethal concentrations of d-cycloserine. Sensitivity to the antibiotic could be regained by growth in the absence of d-cycloserine. The bactericidal activity of d-cycloserine apparently was not altered by resistant cells, and peptidoglycan synthesis was still inhibited by d-cycloserine in resistant cells. The d-cycloserine resistance apparently resulted from a decreased uptake of the antibiotic. The decrease in d-cycloserine transport could be prevented by simultaneous treatment of the cells with rifampin and d-cycloserine. d-Cycloserine was transported by the same system as glycine in B. subtilis. d-Cycloserine was able to exchange for intracellular glycine in both sensitive and resistant cells, suggesting that d-cycloserine is not excluded from the cell in resistant cultures.

Bacillus subtilis↗
PubMed1977

D-Cycloserine-induced alterations in the transport of D-alanine and glycine in Bacillus subtilis 168.

d-Alanine, l-alanine, and glycine transport was investigated in Bacillus subtilis 168 cells that were phenotypically resistant to d-cycloserine. These cells showed enhanced rates of uptake as compared with that observed in sensitive cells. The usual enhancement in d-alanine and glycine transport resulting from treatment of the cells with d-cycloserine could be prevented by the addition of rifampin. Kinetic analyses of the initial rate of glycine transport indicated an increase in the V(max) for transport in resistant cells, with no alteration in the K(m) for glycine. Investigations of the net transport of glycine revealed that resistant cells maintained a higher gradient of glycine than did sensitive cells. Kinetic analyses of the net transport of glycine suggested that a new system for the accumulation of glycine was present in d-cycloserine-resistant cells.

Alanine↗
PubMed1974

Active transport of D-alanine and related amino acids by whole cells of Bacillus subtilis.

Whole cells of Bacillus subtilis transported d-alanine and l-alanine by two different systems. The high-affinity system (K(m) of 1 muM and V(max) of 0.6 to 0.8 nmol/min per mg of protein) was specific for the two stereoisomers of alanine. The low-affinity system (K(m) of 10 muM for l-alanine and 20 muM for d-alanine and glycine) had a V(max) of 5 to 12 nmol/min per mg of protein. This system transported glycine, d-cycloserine, and d-serine, in addition to d- and l-alanine. Azide inhibited the uptake of these amino acids and caused the efflux of d-alanine from preloaded cells. These data suggest that transport of these amino acids is energized by the electron transport chain.

Alanine↗