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Biomedical subjects

J H Parish

Publications and source records attributed to J H Parish.

At least 37 records · Page 2Linked to original sources
PubMed1990

Strand scission in DNA by quercetin and Cu(II): identification of free radical intermediates and biological consequences of scission.

Quercetin was shown to reduce oxygen to superoxide. In the presence of Cu(II), the hydroxyl radical was formed. The strand scission of DNA was shown to occur under conditions in which Cu(II), quercetin and either hydrogen peroxide or oxygen were present and superoxide was not a necessary intermediate. Strand scission involved the hydroxyl radical and a radical DNA intermediate. The strand scission reaction was shown to account for the biological activity of quercetin as assayed by bacteriophage inactivation.

Copper↗
PubMed1989

Effects of 5-fluorouracil on cytotoxicity and RNA metabolism in human colonic carcinoma cells.

The cytotoxicity of 5-fluorouracil (5-FU) is due in part to the incorporation of the base into RNA molecules. We assessed the cytotoxicity of 5-FU in human colonic carcinoma HT-29 cells and examined mRNA activity (measured by protein biosynthesis in vivo and in vitro) and the maturation of rRNA precursors as two possible modes of action of 5-FU. The rRNA processing pathways were studied using rDNA sequences as probes in blot hybridisation protocols and were specific for both the precursors and mature rRNA species of the maturation pathways. The conclusion from the studies was that although differences in mRNA activity were detected in vivo and in vitro, the significance of these changes are as yet unknown. In contrast, the effects on the pre-rRNA processing pathways proved to be highly significant cytotoxic consequences of 5-FU administration. We discuss the implications of this finding for an understanding of the mode of action of the drug and for the future monitoring of tumour sensitivity to 5-FU.

Carcinoma↗
PubMed1989

Strand scission in DNA induced by quercetin and Cu(II): role of Cu(I) and oxygen free radicals.

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) and Ca(II)]. Structurally related flavonoids, rutin, galangin, apigenin and fisetin, were ineffective or less effective than quercetin in causing DNA breakage. In the case of the quercetin--Cu(II) reaction, Cu(I) was shown to be an essential intermediate by using the Cu(I)-sequestering reagents, neocuproine and bathocuproine. By using Job plots we established that, in the absence of DNA, five Cu(II) ions can be 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). From these data we propose a mechanism for the DNA strand scission reaction of quercetin and related flavonoids.

Copper↗
PubMed1987

Cloning, characterization and expression in Escherichia coli of a leucine biosynthetic gene from Streptomyces rochei.

Leucine and histidine biosynthetic genes from Streptomyces rochei HP1 that complemented auxotrophic mutations in S. lividans TK54 were cloned in pIJ61. DNA from one leucine recombinant plasmid was subcloned into pBR322. From the latter, a recombinant plasmid was obtained that complemented the leuA mutation in Escherichia coli CV512 but not other leucine markers in E. coli. Analysis of this and several subclones, including mutant plasmids constructed in vitro, established that the cloned S. rochei gene was expressed in E. coli from the tetracycline promoter of pBR322 to produce a polypeptide of 67 kDa; the corresponding coding region was shown to be within a 1.7 kbp DNA fragment. Blot hybridization revealed corresponding homologous genes in several other streptomycetes.

Cloning, Molecular↗
PubMed1984

Assay of cytotoxicity and mutagenicity of alkylating agents by using Neurospora spheroplasts.

A system relying on the use of Neurospora crassa spheroplasts has been developed for the assay of cytotoxicity and mutagenicity of chemical compounds. Mutagenicity was assayed by using reversion of alleles in the am gene selected to recognize certain specified transitions and also undefined point mutations. Cytotoxicity was quantified by measuring a 'cytotoxicity parameter', m, which appears in the exponential function that fits the survival/dose curve for each compound (under standard incubation conditions). Of the compounds tested, nitrogen mustard (Cl(CH2)2 NMe(CH2)2Cl) was cytotoxic and non-mutagenic, and ethyl nitrosourea was highly mutagenic but not cytotoxic. Of the remaining compounds tested, methyl nitrosourea, butadiene diepoxide, and cis platin (cis diammonia platinum II chloride) all showed comparable mutagenicity per survivor, although the values of m covered a wide range. Differences were found between the different compounds in the effects of the uvs-2 allele on survival and on the preponderance of G to A transitions.

Alkylating Agents↗
PubMed1984

Cytotoxicity and mutagenicity of cis platin assayed in neurospora.

A Neurospora spheroplast assay was used to study the cytotoxicity and mutagenicity of cis platin (cis dichlorodiammine platinum II) in a eukaryote. Mutagenicity was measured by using reversion of several alleles of the am (NH3 assimilatory glutamate dehydrogenase) gene. A mutation (uvs-2) that disables a DNA repair pathway in Neurospora resulted in reduced sensitivity to and elimination of mutability by cis platin. The implications for other eukaryotic systems (including human cells) are discussed.

Alleles↗
PubMed1982

Inactivation of bacteriophage lambda and lambda DNA by nitrogen mustard.

Bacteriophage lambda and lambda DNA were treated with alkylating agents. The survival of phage was assayed by infectivity and that of DNA by infectivity of phage particles assembled from the DNA in vitro. Phage lambda were more sensitive to nitrogen mustard (C1(CH2)2NMe(CH2)2C1; HN2) than was lambda DNA. The inactivation of lambda DNA was biphasic; the second component of the inactivation was sensitive to mutations allelic for recA, polA and uvrB. This behaviour was not shown by pBR322 plasmid DNA treated with HN2 nor by lambda DNA treated with monofunctional alkylating agents (or HN2 if the second alkylation reaction was stopped by addition of a mercaptan). From Arrhenius plots, the activation energy for the reactions with DNA and interact phage were found to be different. The activation energy for the inactivation of intact phage was the same as that (measured independently) for the predominant reaction (or class of reactions) in which HN2 cross-links DNA to protein in lambda particles. From these data we conclude that the inactivation of lambda by HN2 is due, primarily, to DNA-protein cross-linking. The implications for the mode of action of DNA-reactive bifunctional anti-viral and cytotoxic compounds are discussed.

Antiviral Agents↗
PubMed1982

Condensed DNA structures derived from bacteriophage heads.

Bacteriophage lambda particles were rendered osmotically fragile by incubation, spread over hypophase and examined by electron microscopy. When water was used as hypophase, condensed structures were released from the phage heads and treatment of these with cytochrome c or several alternative proteins resulted in the release of free, relaxed DNA. Phage were pretreated with nitrogen mustard, a bifunctional alkylating agent; when the condensed structures from such phage particles were treated with protein, DNA was released in small supercoiled domains. This confirmed a previous finding that bacteriophage DNA has a supercoiled topology and suggests that the winding pattern of DNA in the phage might involve small domains of coiled DNA analogous to nucleosomes. Such a conformation could be consistent with other studies on the arrangement of DNA in phage heads if the domains have parallel axes.

Bacteriophage lambda↗
PubMed1981

Liposome-mediated genetic transformation of Neurospora crassa.

Transformation of Neurospora crassa spheroplasts is reported for three different genes, using uncloned Neurospora DNA, both naked and encapsulated in synthetic phosphatidylserine liposomes. Whereas transformation by naked DNA is DNase-sensitive, that by liposomes is not. Per unit of transforming DNA, liposome transformation is significantly more efficient than that with naked DNA, ranging from 19x for the am gene to 41x for pyr-3. Levels of activity of pyr-3 and am transformants, and segregation data on pyr-3 transformation are given.

DNA, Fungal↗
PubMed1981

beta-Lactamase activity and resistance to penicillins in Myxococcus xanthus.

Several mutants and other variants of Myxococcus xanthus HP100 were obtained with differences in their sensitivity to carbenicillin and other penicillin derivatives. The specific activities of beta-lactamase in different resistant organisms varied from strain to strain but were consistently higher than in HP100. The relative molecular mass (Mr) of the enzyme in M. xanthus HP100 was found to be 22,300. In certain carbenicillin resistant strains a second fraction of beta-lactamase activity of molecular weight 186,000 presumed to be an octamer of the other form was present. The enzyme was found in cell free extracts and also in culture supernatants of all carbenicillin resistant mutants but not in culture supernatants of strain HP100. In all the carbenicillin resistant mutants a part of the intracellular enzyme activity was released by osmotic shock and this activity may be periplasmic. The forms of the enzyme present in the culture supernatants and released by osmotic shock were monomeric. Carbenicillin resistance was not transferable between strains by conjugation. One resistance allele inhibited the transfer of the R factor Sa between myxococci.

Carbenicillin↗
PubMed1980

Bacteriophage K7, a double stranded DNA phage that infects strains of Escherichia coli harbouring drug resistance factors of incompatability group W.

Bacteriophage K7 is specific for Escherichia coli strains harbouring R factors of incompatability group W, including hybrid coliphage P1-Myxococcus virescens plasmids. The phage has an unusual morphology with an isometric head and long tail of variable length. The tail lengths appear to fall into classes corresoonsing to simple multimers of a unit length. Partially purified lysates of the phage include material that may represent phage particles in the process of biogenesis and other material demonstrating attachment of phage to cell envelope. Newly released phage DNA contains single standed ends. In the course of work. E. coli strains that harbour R factor Sa were found to be apparently restrictive.

Coliphages↗
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↗