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Biomedical subjects

J Brennand

Publications and source records attributed to J Brennand.

At least 37 records · Page 2Linked to original sources
PubMed1986

Expression of the E. coli O6-methylguanine-methylphosphotriester methyltransferase gene in mammalian cells.

Many prokaryotic and eukaryotic cells contain enzymes that repair damage introduced into their DNA following exposure to chemical, physical and biological agents. One such lesion that has received considerable attention is the potentially miscoding and mutagenic base O6-alkylguanine which is produced in varying amounts in DNA following reaction with monofunctional alkylating agents. As part of a study to assess the role of this lesion and its repair in the processes of cytotoxicity, mutagenicity and transformation, we have recently cloned the Escherichia coli gene which codes for the protein responsible for the repair of such damage in DNA. In the present study we describe the construction of a plasmid which allows the efficient expression of the bacterial gene in mammalian cells.

Animals↗
PubMed1986

Chinese hamster cells harbouring the Escherichia coli O6-alkylguanine alkyltransferase gene are less susceptible to sister chromatid exchange induction and chromosome damage by methylating agents.

Clones of Chinese hamster V79 cells harbouring the Escherichia coli O6-alkylguanine (O6-AG) alkylphosphotriester (AP) alkyltransferase (ATase) gene (clone 8) or a subclone of it that codes only for O6-AG ATase activity (clone SB) have been exposed to increasing doses of N-methyl-N-nitrosourea (MNU) or methylmethanesulphonate (MMS) and the frequencies of induced sister chromatid exchanges (SCEs) measured. In control (clone 2) cells, SCE induction was almost linearly proportional to dose of MNU or MMS and at the highest doses used (15 or 80 micrograms/ml) SCE frequencies were 6 or 8 times background levels, respectively. Slightly lower levels of MMS-induced SCEs were seen in clone 8 and clone SB cells whilst, in contrast, MNU-induced SCE levels in these two clones were drastically reduced being less than twice background levels at 15 micrograms/ml. After treatment with N-butyl-N-nitrosourea, SCE frequency was similar in all three clones. At higher doses, MNU treatment produced less chromatid aberrations and micronuclei in clone SB than in clone 2 cells. These results suggest that ATase-repairable damage is involved in the induction of SCE, chromosome aberrations and micronuclei in V79 cells.

Alkylating Agents↗
PubMed1986

Expression in mammalian cells of a truncated Escherichia coli gene coding for O6-alkylguanine alkyltransferase reduces the toxic effects of alkylating agents.

The lesion O6-alkylguanine (O6-AG) is produced in cellular DNA following exposure to monofunctional alkylating agents and its miscoding and mutagenic properties have been demonstrated in specific in vitro systems. In order to examine whether this lesion could be responsible for any of the biological effects of alkylating agents in mammalian cells, we have constructed a plasmid containing the O6-AG alkyltransferase (ATase) region of the gene from Escherichia coli, the product of which normally repairs both O6-AG and alkylphosphotriesters in DNA. We have transfected the construction into Chinese hamster fibroblasts which are deficient in endogenous ATase activity and selected a clone that expresses the truncated repair gene. We demonstrate that this protein is functional, acts on damage in host cell DNA and protects the cells from the toxic effects of those alkylating agents that react extensively at oxygen atom positions.

Alkylating Agents↗
PubMed1986

A comparison of the clinico-pathological features with stool pathogens in patients hospitalised with the symptom of diarrhoea.

The clinico-pathological features of 515 adult patients admitted to a major Regional Infectious Diseases Unit in United Kingdom with the symptom complex of diarrhoea were compared to the pathogens detected in their stool specimens. Routine clinical examination supported by basic pathological and laboratory investigations identified 138 (28%) in whom the cause of diarrhoea was extragastrointestinal or non-infectious gastrointestinal. Of the 351 patients (72%) with infectious gastroenteritis 72 (21%) had campylobacter, 59 (17%) had salmonella (22% bacteraemic) and 16 (5%) shigella. Clostridium difficile toxin accounted for a further 15 (4%)--antibiotics had been the antecedent cause in only one half of these. Routine microscopical examination of the faeces for red and white cells distinguished many with "culture positive" diarrhoea from those with "culture negative" infectious diarrhoea. Although there are no clinico-pathological features which are unique to a particular pathogen and unequivocally suggest a particular pathogen, certain features did tend to present more often in association with particular microorganisms, and this knowledge may suggest a bacterial diagnosis whilst awaiting the definitive results of stool microbiology. These features include prior antimicrobial therapy with positive sigmoidoscopical/histological features: Cl. difficile; protracted diarrhoea in elderly severely dehydrated patients: salmonellosis; foreign travel in males with bloody diarrhoea: shigellosis; abdominal pain in younger patients with a small degree of vomiting: campylobacteriosis. Early diagnosis may then prove useful in rationalizing initial therapy, particularly the appropriate use of antimicrobials.

Acute Disease↗
PubMed1985

Cloning of the E. coli O6-methylguanine and methylphosphotriester methyltransferase gene using a functional DNA repair assay.

Alkylating agents react with various nitrogen and oxygen atoms in DNA and many of the products are substrates for repair processes. Oxygen atom derivatives such as O6-methylguanine (O6-meG) O4-methylthymine and methylphosphotriesters (MP) have been shown to undergo repair by methyl group removal. The proteins involved in the latter reaction can be considered to be methyltransferases (MT) because their action results in the transfer of the methyl group to a cysteine residue within a polypeptide. A rapid and sensitive assay for MT activity has been developed and used to screen extracts of bacteria harbouring an E. coli genomic DNA library carried in a plasmid vector. We report here the cloning of an E. coli gene coding for O6-meG and MP MT repair functions. These two activities reside on a 37Kd protein that can undergo a host-dependent cleavage to produce an 18Kd protein which contains only O6-meG MT and a 13Kd protein which contains only MP MT.

Cloning, Molecular↗
PubMed1984

Structure, expression, and mutation of the hypoxanthine phosphoribosyltransferase gene.

The wild-type mouse hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) gene has been isolated from genomic libraries and its structure has been determined. This X chromosome-linked gene is greater than 33 kilobases long and is split into nine exons. All the exon sequences have been determined, and a single-base substitution in the HPRT cDNA coding sequence from a mouse neuroblastoma cell line that overproduces a mutant HPRT protein has been identified. The 5' end of the gene has been defined, both by nuclease S1 protection and primer extension studies and by a functional assay in which an HPRT minigene, capable of expression in cultured cells, was created by ligating the 5' end of the gene onto wild-type human HPRT cDNA. Sequences normally associated with eukaryotic promoters are not present in the immediate 5'-flanking region of the HPRT gene, which is instead highly G+C rich. This observation is discussed in relation to the possible link between DNA methylation and X-chromosome inactivation.

Animals↗
PubMed1983

Expression of human and Chinese hamster hypoxanthine-guanine phosphoribosyltransferase cDNA recombinants in cultured Lesch-Nyhan and Chinese hamster fibroblasts.

The results presented in this communication demonstrate that hypoxanthine-guanine phosphoribosyltransferase (HPRT) cDNA can be expressed in both Chinese hamster and human fibroblasts deficient in the endogenous gene product at levels permitting normal growth of the transformants. All the elements necessary for this expression are present in a pBR322-derived plasmid containing HPRT cDNA coding sequence and a retroviral long terminal repeat. These molecules function in both species investigated and, at least in the case of the Chinese hamster transformants, are efficient at the single copy level. Although the effects of the presence of intron sequences and a polyadenylation signal within the plasmids have yet to be evaluated, these studies demonstrate that neither is an absolute requirement for expression of HPRT cDNA sequences in cultured mammalian cells. We describe the construction of recombinant plasmids containing wild type human or Chinese hamster HPRT cDNA sequences in tandem with a retroviral LTR which confer the HPRT+ phenotype in HPRT-deficient V79 and Lesch-Nyhan fibroblasts. Both stable and unstable transformants, that expressed HPRT mRNA and protein, were isolated at high frequency.

Animals↗
PubMed1982

Hypoxanthine-guanine phosphoribosyltransferase genes of mouse and Chinese hamster: construction and sequence analysis of cDNA recombinants.

Recombinant plasmids containing DNA inserts complementary to mRNA coding for hypoxanthine-guanine phosphoribosyltransferase (HPRT) from mouse and Chinese hamster cell lines have been isolated from cDNA libraries and characterized by DNA sequence analysis. A total of 1292 nucleotides of the mouse cDNA sequence and 1301 nucleotides of the Chinese hamster cDNA sequence has been determined. Each of these sequences includes an open reading frame of 654 nucleotides (218 amino acids) corresponding to the HPRT protein coding region. The deduced amino acid sequences for the mouse and Chinese hamster enzymes are presented and compared to that of human HPRT. At least 95% of the amino acids are conserved in the three species. In addition, we present evidence that two species of HPRT mRNA, which differ in the site of polyadenylation that is utilized during processing of the RNA transcripts, exist in Chinese hamster cells.

Animals↗
PubMed1982

Cloned cDNA sequences of the hypoxanthine/guanine phosphoribosyltransferase gene from a mouse neuroblastoma cell line found to have amplified genomic sequences.

Cloned cDNA sequences of the murine hypoxanthine/guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8) gene have been isolated by using a mouse neuroblastoma cell line containing increased levels of a variant HPRT protein. We have used these sequences as probes to demonstrate that protein overproduction in this cell line is a consequence of at least a 20-fold increase in HPRT mRNA levels resulting from approximately 50-fold amplification of HPRT genomic sequences. The largest cDNA insert so far characterized represents about 70% of the HPRT mRNA sequence. This cDNA is shown to possess regions of homology with mRNA and DNA from Chinese hamster, baboon, and human, thus facilitating detailed analysis of this locus in these four species.

Animals↗
PubMed1982

The effects of methylated thymidines upon cultures of V79 cells and the mechanism of incorporation of O4-methylthymidine into their DNA.

High concentrations of O2-methylthymidine, 3-methylthymidine and O4-methylthymidine (O4-MedT) have little effect on the colony forming ability of cultured Chinese hamster (V79) cells. However, the methylated derivatives enhance the toxic effects of thymidine (dT) at concentrations at which the modified nucleosides alone show no toxicity. O4-MedT was the only modified nucleoside which is incorporated into V79 cell DNA at a detectable level. In the presence of added dT there is an increase in the amount of O4-MedT incorporated. O4-MedT did not induce mutation, either forward or reverse, in V79 cells. The results indicate that O4-MedT is incorporated into DNA as an analogue of deoxycytidine and once incorporated behaves as cytidine during DNA synthesis, in contrast to O4-MedT produced in cellular DNA synthesis, in contrast to O4-MedT produced in cellular DNA by methylating carcinogens which is miscoding and potentially mutagenic.

Animals↗
PubMed1981

Effects of metabolic inhibitors on cel lethality and mutation induction in Chinese hamster cells. II. The effect of posttreatment with non-toxic concentrations of thymidine.

The ability of posttreatment exposure to non-toxic concentrations of thymidine (TdR) to enhance the lethal effects of a number of alkylating agents, X-rays and UV and the lethal and mutagenic effects of N'-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) has been examined in V79 cell lines. TdR posttreatment enhanced the cytotoxic effects of ethyl methanesulphonate (EMS), MNU and ENU but not of UV or X-rays and increased both the spontaneous and MNU- and ENU-induced frequencies of azaguanine resistant (AZR) mutants. No significant effect of TdR on the spontaneous frequency of thioguanine resistant (TGR) mutants was demonstrated but the frequency of MNU-induced mutants to TGR premutagenic was enhanced. The effects on expression of both potentially lethal and premutagenic damage were reversed by addition of an equimolar concentration of deoxycytidine (dCdR). The enhancement in spontaneous and induced mutant frequency (IMF) at the HGPRT locus appears to be due to an alteration in the selective efficiency of urine analogous due to alteration in growth kinetics of cells exposed to TdR or treated with alkylated agents or posttreated with thymidine after alkylation damage and not to an alteration in the miscoding potential of alkylated bases.

Animals↗
PubMed1980

Relative mutagenicity of antineoplastic drugs and other alkylating agents in V79 Chinese hamster cells, independence of cytotoxic and mutagenic responses.

The mutagenic and cytotoxic effects of 4 antineoplastic drugs, vinblastine, vincristine, adriamycin and nitrogen mustard and of several monofunctional alkylating agents have been assayed in V79 Chinese hamster cells. Vincristine, vinblastine and nitrogen mustard did not significantly increase the frequency of TGRHGPRT- mutants but were were all highly cytotoxic. Adriamycin and the monofunctional alkylating agents were all significantly mutagenic even at the lowest doses tested (approx. 70% survival level). Induced mutant frequency increased linearly with increasing dose whereas dose-response curves for cytotoxicity for these effective mutagens invariably showed a shoulder followed by an exponential decline. At equitoxic doses the relative mutagenic effectiveness was MNU > ENU > EMS > MNNG > MMS greater than or equal to DMS. MNU was approx. 20 times more effective than MMS and DMS. Measurement of the total amount of alkylation and the relative amounts of reaction with individual DNA bases at approx. equitoxic doses of MNU and DMS indicated a significantly higher O6/N7 ratio after MNU (0.15) than after DMS (0.005). However, approx. equal numbers of mutants/10(5) cells/microM O6-Meguanine were induced by these 2 agents. These results support previous conclusions, that mutagenic and cytotoxic responses are independent in V79 cells.

Alkanesulfonates↗
PubMed1980

Excess thymidine is not mutagenic in Chinese hamster V79 fibroblasts.

The ability of thymidine to enhance the frequency of HGPRT-, 6-thioguanine resistant V79 cell mutants has been investigated as a function of post treatment growth interval. We have also studied the relative sensitivities towards exogenous thymidine of wild type and mutant cell lines. Our data imply that increases in mutant frequency following exposure of V79 cells to thymidine can be explained on the basis of a greater sensitivity of wild type cells compared with HGPRT deficient cells.

Animals↗
PubMed1980

Evidence for the involvement of lesions other than O6-alkylguanine in mammalian cell mutagenesis.

Marked differences between the mutagenic efficiency of N-methyl-N-nitrosourea (MNU), a potent carcinogen, methyl methane sulphonate (MMS) and dimethyl sulphate (DMS), both weak carcinogens, have been reported at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and ouabain loci in V79 cells. Differences in levels of O6-guanine methylation produced by these alkylating agents, has been interpreted as indicating that O6-methylguanine is the DNA lesion specifically responsible for their mutagenic and carcinogenic effects. Because of the heterogeneity of molecular events which can result in forward mutation this conclusion seems unjustified. The development and characterisation of a reverse assay from 6-thioguanine resistance and HAT medium sensitivity (TG(R) and HAT(S)), to 6-thioguanine sensitivity and HAT medium resistance (TG(S) and HAT(R)) HGPRT(-)-->HGPRT+ in V79 cells, has allowed us to test the above hypothesis in a more specific way. Ethyl methane sulphonate, a weak carcinogen and MNU, both of which produce significant levels of O atom alkylation, were similarly effective mutagens in the reverse direction. At equitoxic doses, DMS was 40-60 fold less efficient. There was however, no quantitative correlation between numbers of revertants induced and measured levels of O6-alkylguanine. From these and other observations it is concluded that O6-alkylguanine is not the only potentially mutagenic lesion in mammalian cells.

Adenine↗
PubMed1979

Mecillinam in enteric fever.

Twelve consecutive patients with enteric fever entered a trial of 14 days' treatment with mecillinam. Only three patients became afebrile within three days; four continued unimproved with fever and toxaemia for seven to nine days, when treatment was changed to chloramphenicol with good results. In one case the fever did not settle until the 13th day, and five days later the patient had a clinical relapse. Although all organisms recovered were fully sensitive to mecillinam, this drug is not an effective or consistent treatment for enteric fever.

Administration, Oral↗
PubMed1974

Invasive illness with Salmonella virchow infection.

Salmonella virchow infection in man is usually sporadic and is little known in Britain. We report an outbreak of 21 cases in the Manchester area. The source of infection was believed to be chickens bought at different shops. Symptoms were typhoidal or septicaemic.

Abdomen↗