Biomedical subjects

J H Parish

Publications and source records attributed to J H Parish.

14 recordsLinked to original sources

PubMed1992

Strand scission in DNA induced by dietary flavonoids: role of Cu(I) and oxygen free radicals and biological consequences of scission.

The naturally occurring flavonoid, quercetin, in the presence of Cu(II) and molecular oxygen caused breakage of calf thymus DNA, supercoiled pBR322 plasmid DNA and single stranded M13 phage DNA. In the case of the plasmid, the product(s) were relaxed circles or a mixture of these and linear molecules depending upon the conditions. For the breakage reaction, Cu(II) could be replaced by Fe(III) but not by other ions tested [Fe(II), Co(II), Ni(II), Mn(II) and Ca(II)]. Structurally related flavonoids, rutin, galangin, apigenin and fisetin were effective or less effective than quercetin in causing DNA breakage. In the case of the quercetin-Cu(II) reaction, Cu(I) was shown to be essential intermediate by using the Cu(I)-sequestering reagent, bathocuproine. By using Job plots we established that, in the absence of DNA, five Cu(II) ions were reduced by one quercetin molecule; in contrast two ions were reduced per quercetin molecule in the DNA breakage reaction. Equally neocuproine inhibited the DNA breakage reaction. The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, iodide, mannitol, formate and catalase (the inhibition was complete in the last case). The strand scission reaction was shown to account for the biological activity of quercetin as assayed by bacteriophage inactivation. From these data we propose a mechanism for the DNA strand scission reaction of quercetin and related flavonoids.

Animals↗

PubMed1992

Activities of flavonoids for the cleavage of DNA in the presence of Cu(II): correlation with generation of active oxygen species.

The activities of several flavonoids and the related nonflavonoid compound epicatechin were compared with respect to Cu(II)-induced strand scission of DNA by using two different assays. The same series of compounds was used to study the stoichiometry of Cu(II) reduction in the absence of DNA. The compounds were compared for their ability to generate superoxide, hydrogen peroxide and the Cu(II)-dependent production of hydroxyl radicals. Flavonoids were examined to assess the production of a charge-transfer complex with Cu and the rate of decay of the complexes were compared. All the compounds tested had some ability to cause DNA strand scission in the presence of Cu(II), with myricetin being the most active and galangin the least active. The ability to cause such scission correlated with the rate of decay of the charge-transfer complex, the ability to generate active oxygen species and with the stoichiometry of Cu(II) binding. Analysis of the data in the light of the structural differences between the flavonoids led to a discussion of alternative Cu(II)-sequestering mechanisms.

Copper↗

PubMed1978

Transduction of Myxococcus virescens by coliphage P1CM: generation of plasmids containing both phage and Myxococcus genes.

Chloramphenicol-resistant Myxococcus virescens were obtained by infecting myxococci with Escherichia coli specialized transducing phage P1CM. The drug-resistant myxococci were phenotypically unstable. They contained more than one type of plasmid; these plasmids were not found in the parent strain. Chloramphenicol-resistant E. coli were obtained by transformation with either a fraction of myxococcal DNA containing the plasmids or with P1CM prophage DNA. These transformants contained plasmids. Escherichia coli transformed by DNA from the myxococci contained both P1CM and myxococcal genes. Individual transformant clones differed in the genetic make-up of their plasmids. Among the myxococcal genes expressed in these plasmid-harbouring E. coli strains were a capacity for self-transmissibility and a pattern of phage sensitivity characteristic of R factor incompatibility group W. Escherichia coli transformed with P1CM prophage contained incomplete P1CM genomes; none of the chloramphenicol-resistant transformants produced P1CM phage particles. The significance of these findings for an understanding of mechanisms for the generation of R factors is discussed.

Chloramphenicol↗

PubMed1977

Pigmentation phenotype instability in Myxococcus xanthus.

Cells of Myxococcus xanthus FB2 produce tan or yellow colonies. Subcultures of tan colonies yielded tan and yellow colonies and subcultures of most yellow colonies yielded only yellow colonies. Strain FB2 variants in which the color type is more stable were obtained. Yellow cells were distinguishable from tan by the presence of pigment(s) with an absorption maximum at 379 nm. Fluctuation Test experiments and the presence of this pigment(s) in liquid cultures of FB2 indicated that tan phenotype cells spontaneously became or segregated yellow cells in liquid culture. The frequency of appearance of yellow cells was increased in low density cultures (less than 10(6)/ml). The increase cannot be explained by differences in growth rates of the two phenotypes. No evidence that cell-cell contact or culture medium constituents affect the appearance of the yellow phenotype was found. Ultraviolet irradiation of FB2 resulted in an increased proportion of cells producing yellow colonies among the survivors. Greater UV resistance of yellow cells and UV-induced conversion of tan to yellow accounts for this increase. Low level photoreactivation of viability and of the tan phenotype occurred. Incubation of FB2 in medium containing mitomycin C, nalidixic acid, phenethyl alcohol, or at 36.5 degrees C also resulted in conversion of tan to yellow cells.

Myxococcales↗

PubMed1976

DNA of Myxococcus phage MX-1. Pyrimidine isostichs and the recognition of a minor pyrimidine.

DNA was isolated from bacteriophage MX-1 and was hydrolysed with acids. Hydrolysis with 90% formic acid produced several interconvertable fluorescent artefacts. These substances were produced in greater quantity following hydrolysis with H2SO4. In the course of the course of the study it was found that DNA extracted from phage by 4-aminosalicylate and phenol contained 4-aminosalicylate and breakdown products derived from 4-aminosalicylate following acid hydrolysis. Pyrimidine oligonucleotides released by formic acid-diphenylamine hydrolysis were fractionated by two-dimensional paper chromatography and by ion-exchange chromatography. In this way, the pyrimidine isostichs were obtained and further fractionated. The distribution and with isostich patterns from a variety of DNA sources. MX-1 DNA was found to contain an unusually high frequency of cytosine-rich isostichs 5 and 6 and very low occurrence of the sequence R-Y-R. Based on its absorption spectrum and the fact that it can be labelled in vivo with [14C] orotic acid, we suggest that H-base is a pyrimidine.

Aminosalicylic Acids↗

PubMed1976

Morphogenesis in Myxococcus xanthus and Myxococcus virescens Myxobacterales.

1. Myxococcus xanthus B and M. virescens V2 were compared with a view to establishing the control of their morphogenetic cycles. Both organisms are typical myxococci and on solid media with low concentrations of nutrient they form fruiting bodies, within which vegetative cells convert to myxospores. Ultrathin sections of vegetative M. virescens resembled those of M. xanthus and contained prominent heavily stained bodies, presumed to be polyphosphate granules. Shadowed preparations showed fimbriae associated with M. xanthus but not with M. virescens. 2. M. xanthus B converted to myxospores in liquid medium in response to certain alcohols. M. virescens V2 produced phase-refractile spheres, which were not viable and had an unusual ultrastructure. 3. The distributions of fruiting bodies on solid media containing 0.02% Casitone were recorded for the two species and were compared with a Poisson distribution. Cells responded to differences in cell density in a manner suggestive of a response to a chemotactic attractant. Cells growing vegetatively and also cells forming fruiting bodies produced 3',5'-cyclic adenosine monophosphate (cAMP) as measured by the incorporation of exogeneous [3H] adenosine into cAMP. 4. The significance of these findings for theories of fruiting body formation are discussed.

Chemotaxis↗

PubMed1976

DNA of Myxococcus bacteriophage MX-1: macromolecular properties and restriction fragments.

1. Bacteriophage MX-1 is a virulent DNA phage whose hosts include strains of Myxococcus xanthus, M. fulvus and M. virescens. DNA was extracted from purified phage preparations. The molecular weight of phage DNA was measured by sedimentation-velocity and by rate-zonal ultracentrifugation. The apparent molecular weight was found to vary for reasons discussed in the text. From rate-zonal ultracentrifugation, using calibrated sucrose gradients, the molecular weight was calculated to be 149 (+/-22) X 10(6) daltons. The base composition of the DNA was estimated by different methods and was found to be 50-52% (G + C). The DNA demonstrated an anomalous thermal denaturation profile in dilute buffer. Denatured DNA was fractionated by ion-exchange chromatography and by buoyant-density centrifugation. No significant strand separation was obained and it was concluded that overall base compositions of the two strands are very similar. 2. DNA from bacteriophage MX-1 was hydrolysed with restriction endonucleases R. EcoRI, R.EcoRII and R. HindIII. The restriction fragments were catelogued and their apparent molecular weights calculated from electrophoresis gels calibrated with fragments from the DNA of coliphage lambda. From the total fragments obtained with nuclease R.EcoRI, the minimum apparent molecular weight of MX-1 DNA was found to be 130 X 10(6) daltons.

Bacteriophages↗

PubMed1976

Restriction in Myxococcus virescens.

1. The plating efficiency of bacteriophage MX-1 on Myxococcus xanthus strains A and B and M. virescens V2 were compared. Comparison of strains V2 and A suggest that V2 is restrictive and A is not (restriction coefficient was approximately 8). A derivative of M. virescens V2 (strain V2-9) was obtained by repeated exposure of strain V2 to ultraviolet radiation. Strain V2-9 was also unrestrictive. Strain B is apparently unrestrictive too but analysis of phenotypic changes in phage derived from hosts V2, B and A suggested that some of the host-cell processes differ from orthodox restriction and modification. 2. Cell-free extracts from M. virescens V2 were fractionated by ion-exchange chromatography and two restriction endonucleases, R. MviV2I and R. MviV2II were identified. Nuclease I was found to hydrolyse coliphage lambdaDNA at apparently one site only and MX-1 DNA at approximatley 10 sites; nuclease II was found to hydrolyse MX-1 DNA at a very large number of sites and its restriction sequence was of comparable frequency with that of R. EcoRII. "Modified MX-1 DNA", obtained from phage whose last host was M. virescens V2 was hydrolysed by nuclease II but not by nuclease I. The significance of these findings for restriction in myxococci is discussed.

Bacteriophages↗

PubMed1976

Phage and defective phage of strains of Myxococcus.

1. Phage-like particles were found in the supernatants of cultures of strains of Myxococcus xanthus, M. virescens and M. fulvus. The largest number of such particles was associated with M. virescens V2. Most of the particles were similar in morphology to the virulent Myxococcus phage, MX-1. 2. Several new phages were isolated from soil and animal droppings. A new phage was isolated from cultures of M. virescens V2. All resembled phage MX-1 in morphology and were related to phage MX-1 serologically. One of these phage, om, was characterized by fractionation of its proteins by SDS-polyacrylamide gel electrophoresis and by analysis of restriction fragments of its DNA. The very close relatedness with MX-1 was confirmed by these techniques. Phage om, was found to exist in a state of pseudolysogeny with strains of M. virescens and M. fulvus. 3. Two types of bacteriocin-like activity were found associated with Myxococcus strains. In one case, the activity was extracted from chloroformkilled or from sonicated cells. In the second case it was associated with extracellular material. Strains of Salmonella and Cytophaga were found to be good indicators for this latter activity. These strains were found to be killed by phage MX-1. 4. The significance of these data for origin of the phages of myxococci are discussed and it is proposed that MX-1 and the newly isolated phages may be virulent mutants of a family of lysogenic phages.

Bacteriocins↗

PubMed1976

Bacteriophage MX-1: properties of the phage and its structural proteins.

Bacteriophage MX-1 is a virulent DNA phage for Myxococcus. The host range includes strains of Myxococcus xanthus, M. fulvus and M. virescens. The phage has a sedimentation coefficient (S degrees 20,w) of 1145S and a density of 1-531 g/ml. By using SDS-polyacrylamide gel electrophoresis, 23 phage proteins with apparent mol. wt. between 10000 and 150000 were resolved. Gel filtration in the presence of non-ionic detergent partially resolved the proteins. The fraction excluded from Sephadex G-100, fraction 1, contains two glycoproteins. Fraction 1 was resolved into three fractions (1-1, 1-2 and 1-3) by chromatography on Sephadex G-200. The glycoproteins were present in fraction 1-2; all the proteins from this fraction were derived from the phage tail. Comparison of the amino-acid, hexosamine and neutral-sugar compositions of the two glycoproteins showed that they are distinct molecular species; the smaller molecule is not a subunit of the larger. The significance of these findings is discussed and compared with the proteins of the tails of T-even phage of Escherichia coli.

Amino Acids↗

PubMed1976

Extrachromosomal DNA in chloramphenicol resistant myxococcus strains.

The presence of extrachromosomal DNA in strains of Myxococcus xanthus and M fulvus was examined by rate-zonal centrifugation of radioactively-labelled DNA in 'cleared lysates'. All the strains examined contained extrachromosomal DNA, with the exception of M. xanthus FBt. Chloramphenicol resistance is inducible in M. xanthus FBt. A peak of extrachromosomal DNA, containing covalently closed molecules, was found in one of the induced strains, implying that induction of chloramphenicol resistance is associated with the production of a plasmid. By incubating R+ strains of Escherichia coli with myxococci, R factor-mediated chloramphenicol resistance can be introduced into the latter. Evidence of extra chromosomal DNA in a derivative of M. xanthus with chloramphenicol resistance from R factor RI. 19 unique to the chloramphenicol strain, was obtained. By using a double-labelling technique, several chloramphenicol-resistant strains of M. fulvus M were examined. Evidence for a peak, unique for the chloramphenicol-resistant strain, was found in a strain with resistance derived from the R factor, S-a, but not from comparable strains with resistance derived from R factors R57b, R1. 19 and R478.

Chloramphenicol↗

PubMed1975

Mutants of Myxococcus xanthus insensitive to glycerol-induced myxospore formation.

Mutants of Myxococcus xanthus FBt unable to form myxospores in response to 0.5 M glycerol arise spontaneously with a frequency of 1--3 X 10(-5). These mutants are designated glc. Ultraviolet mutagenesis increases the frequency to a maximum of 7% of the survivors. The reversion frequency following ultraviolet irradiation of spontaneous glc mutants is less than 10(-3). Of four glc mutants examined, none form myxospores in response to the alternative inducers, ethylene glycol and dimethyl sulphoxide. One glc mutant is induced by 1.5 M glycerol; strain FBt responds to this glycerol concentration with low efficiency myxospore formation. Strain FBt and glc mutants all produce myxospores with low efficiency in response to phenyl ethanol. Of 117 glc mutants tested, 109 form fruiting bodies containing mature myxospores; thus, mutations to the glc phenotype do not normally block myxospore formation within the fruiting cycle of the organism.

Dimethyl Sulfoxide↗

PubMed1975

Transfer of drug resistance to myxococcus from bacteria carrying drug-resistance factors.

Resistance to chloramphenicol was successfully transferred from strains of Escherichia coli carrying R factors representative of compatibility groups F, W, S and N to strains of Myxococcus xanthus and M. fulvus. Resistance to kanamycin was transferred from an R factor in group S, and to neomycin from an R factor of group P. Myxobacterial strains differed in their capacity to take up the resistances and also in the stability of the resistance character. strains of M. fulvus were obtained that acquired resistance to chloramphenicol without exposure to R plus eubacterial strains. Cell-free preparations of all the chloramphenicol-resistant strains catalysed the acetylation of the drug. Cholramphenicol resistance was successfully transferred from the presumed R plus strains of Myxococcus and also from the spontaneously occurring chloramphenicol-resistant M. fulvus to other Myxococcus strains. Moreover, recombinants resistant to both rifampicin and 5-fluorouracil were obtained, though infrequently, by mixing Myococcus strains resistant to rifampicin and chloramphenicol with other myxococci resistant to 5-fluorouracil, both when the chloramphenicol resistance was derived from S-a (group W) and when it was the endogenous M fulvus resistance. Thus it appears that S-a and a new chloramphenicol resistance factor from M. fulvus will mobilize a chromosomal genetic marker in Myxococcus.

Acetylation↗

PubMed1975

Chloramphenicol resistance in Myxococcus xanthus.

Derivatives of Myxococcus xanthus FB(t) resistant to chloramphenicol (25 mug/ml) arose spontaneously with a frequency of approximately 10(-7). One of these organisms (FB(t)Cam(1) (r)) was characterized. FB(t)Cam(1) (r) showed a unique type of phenotypic instability. After transfer from medium containing chloramphenicol to medium lacking the drug, resistance was lost after approximately one generation. The loss resulted in a sharp drop in the total number of chloramphenicol-resistant organisms and was not due to segregation of chloramphenicol-susceptible organisms during growth. Cell-free extracts of strain FB(t)Cam(1) (r) converted chloramphenicol to acetyl chloramphenicols in a fashion implicating activity of chloramphenicol acetyltransferase. This activity was lost simultaneously with the loss of chloramphenicol resistance after removal of the drug from cultures. Organisms with a similar phenotype to FB(t)Cam(1) (r) could be produced at high frequencies when strain FB(t) was exposed to low concentrations of chloramphenicol (2 to 5 mug/ml), to 3-acetylchloramphenicol (25 mug/ml), or to 1,3-diacetylchloramphenicol (25 mug/ml). Since strain FB(t) is capable of deacetylating acetyl chloramphenicols, these effects are probably all due to low concentrations of chloramphenicol. In the presence of chloramphenicol, FB(t)Cam(1) (r) produced fruiting bodies and myxospores on fruiting agar; however, glycerol-induced myxospore formation was inhibited. In the absence of the antibiotic, chloramphenicol resistance was maintained by glycerol-induced myxospores.

Chloramphenicol↗