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J Brennand

Publications and source records attributed to J Brennand.

At least 19 recordsLinked to original sources
PubMed1995

The application of the human beta-globin gene locus control region and murine erythroleukemia cell system to the expression and pharmacological characterization of human endothelin receptor subtypes.

The cDNAs encoding both A and B subtypes of the human endothelin receptor have been inserted into mammalian cell expression vectors that utilize the human globin gene, locus control region. These constructs have been introduced into murine erythroleukemia cells and inducible high level expression of the receptors has been achieved (approximately 1.5-pM/mg membrane protein and approximately 13,500 binding sites/cell for both receptor subtypes). Cell lines expressing these receptors were obtained on a rapid time scale (3-4 weeks), facilitated by the need for the analysis of only small numbers of cell clones/receptor (approximately 6). Competitive binding assays with endothelin-1 gave IC50s of 130 +/- 30 pM for endothelin-A receptor and 160 +/- 30 pM for endothelin-B receptor. Similar studies with the different isoforms of endothelin, sarafatoxin-S6b and -S6c, BQ123 and BQ3020, all gave the expected selectivity profiles. The IC50s for all compounds were in close agreement with those reported for native receptors. Thus, this expression system, which has several advantages over other described expression systems, is capable of rapidly providing large quantities of receptor for detailed pharmacological analyses or drug screening. In addition, the expressed receptors display the expected pharmacological profiles in the absence of any complicating, competing interactions from other subtypes or binding sites.

Binding, Competitive↗
PubMed1995

Fetal osteocalcin levels are related to placental 11 beta-hydroxysteroid dehydrogenase activity in humans.

OBJECTIVE: Overexposure to glucocorticoids in utero reduces birth weight and, in animals, leads to persistent hypertension in the offspring. The fetus is normally protected from maternal glucocorticoids by placental 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) which catalyses the conversion of cortisol to inert cortisone. In adult humans, osteocalcin is a sensitive marker of glucocorticoid exposure. The aim of this study was to determine whether cord blood osteocalcin levels were related to the ability of placental 11 beta-HSD to inactivate maternal cortisol. DESIGN: Cross-sectional study examining the relation between cord blood levels of osteocalcin and placental glucocorticoid metabolism at term. PATIENTS: Twenty-one women attending for delivery at the Simpson Memorial Maternity Pavilion in Edinburgh had cord venous and arterial blood samples collected at delivery. MEASUREMENTS: Cord plasma levels of osteocalcin, cortisol and cortisone were measured by radioimmunoassay and indices of placental 11 beta-HSD activity were calculated. RESULTS: All indices of placental 11 beta-hydroxysteroid dehydrogenase activity correlated directly and significantly with cord blood osteocalcin levels. For cord blood osteocalcin and the placental 11 beta-HSD Activity Index, Pearson's r was +0.58, r2 = 0.33 and P < 0.02. CONCLUSION: We conclude that term cord blood osteocalcin level reflects the effectiveness of placental glucocorticoid inactivation, and may be a marker for the development of adult hypertension.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1990

Regional expression of a MCD-peptide and dendrotoxin I-sensitive voltage-dependent potassium channel in rat brain.

In situ hybridization histochemistry has been used to analyze the regional expression of a class of voltage-dependent K+ channel that is sensitive to two polypeptide toxins (MCD peptide and dendrotoxin I) that produce spectacular effects on brain function. A heterogeneous expression of this K+ channel was observed throughout the brain. High mRNA contents were observed in the granule cells of the gyrus dentatus as well as in pyramidal cells of the Ammon horn (CA3 greater than CA1) and in the cerebellum. Conversely, low levels of expression were found in basal ganglia (caudate putamen, globus pallidus, and ventral pallidum).

Animals↗
PubMed1988

Different mechanisms of reversion of HPRT-deficient V79 Chinese hamster cells.

The revertibility of three spontaneous hypoxanthine phosphoribosyl transferase (HPRT)-deficient V79 cell lines has been determined after exposure to a number of alkylating agents. TG11 and 19 reverted at frequencies ranging from 1 X 10(-5) to 1 X 10(-4) after exposure to doses of ethylmethane sulphonate (EMS) N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) resulting in surviving fractions between 1.0 and 0.1. Reversion frequencies in TG15 ranged from 10(-7) to 5 x 10(-6) over a similar dose range. The relative efficiencies of different monofunctional alkylating agents in causing reversion of TG11 at equitoxic doses were ENU greater than EMS greater than N-ethyl-N-nitroso-guanidine greater than MNU greater than N-methyl-N-nitrosoguanidine greater than methylmethane sulphonate. Revertant frequencies for all three cell lines were maximal immediately after treatment and declined thereafter at a rate inversely proportional to dose. Such kinetics are explicable if reversion is due to miscoding opposite alkylated guanines. Reversion frequencies after N-butyl-N-nitrosourea exposure were 100-fold lower than after MNU and kinetics of expression of revertant colonies differed. Frequencies were low immediately after treatment, increased between 0 and 24 h then remained at a plateau. Similar kinetics were observed after chlorozotocin and bis-chloroethylnitrosourea exposure. This difference in expression kinetics suggests that reversion in this case is not the result of direct miscoding but of errors in excision repair. TG11, 15 and 19 had low spontaneous mutant frequencies which were either unaffected or only marginally increased by treatment with 5-azacytidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗
PubMed1987

Characterisation and nucleotide sequence of ogt, the O6-alkylguanine-DNA-alkyltransferase gene of E. coli.

The plasmid pO61 that was isolated from an E. coli genomic DNA library and codes for O6-alkylguanine (O6AG) DNA alkyltransferase (ATase) activity (1) has been further characterised. Subclones of the 9 Kb insert of pO61 showed that the ATase activity was encoded in a 2Kb Pst1 fragment but a partial restriction endonuclease map of this was different to that of the E. coli ada gene that codes for O6-AG and alkylphosphotriester dual ATase protein. Fluorographic analyses confirmed that the molecular weight of the pO61-encoded ATase was 19KDa i.e. similar to that of the O6AG ATase function that is cleaved from the 39KDa ada protein but rabbit polyclonal antibodies to the latter reacted only very weakly with the pO61-encoded protein. A different set of hybridisation signals was produced when E. coli DNA, which had been digested with a variety of restriction endonucleases was probed with 2Kb Pst 1 fragment or the ada gene. These results provided evidence for the existence of a second ATase gene in E. coli. The 2Kb Pst-1 fragment of pO61 was therefore sequenced and an open reading frame (ORF) that would give rise to a 19KDa protein was identified. The derived amino acid sequence of this showed a 93 residue region with 49% homology with the O6AG ATase region of the ada protein and had a pentamer and a heptamer of identical sequence separated by 34 amino acids in both proteins. The pentamer included the alkyl accepting cysteine residue of the ada O6AG ATase. The hydrophobic domains were similarly distributed in both proteins. Shine-Dalgarno, -10 and -35 sequences were identified and the origin of transcription was located by primer extension and S1 nuclease mapping. The amino-terminal amino acid sequence of the protein was as predicted from the ORF.

Amino Acid Sequence↗
PubMed1987

Protection of Chinese hamster cells against the cytotoxic and mutagenic effects of alkylating agents by transfection of the Escherichia coli alkyltransferase gene and a truncated derivative.

The cytotoxic and mutagenic effects of various monofunctional and bifunctional alkylating agents have been assessed in V79 Chinese hamster cells that express either the entire O6-alkylguanine (O6AG) and alkylphosphotriester alkyltransferase (ATase) gene (clone 8 cells) or a truncated form that codes only for O6AG ATase activity (clone SB cells). Protection ratios, as determined by D37 values, were greater for clone 8 cells than for SB cells. Significant protection against the mutagenic effects of N-methyl-N-nitrosourea and ethylmethanesulphonate at the hypoxanthine phosphoribosyltransferase (HPRT) locus was observed in clone 8 and SB cells. Streptozotocin and the haloethyl nitrosoureas, chlorozotocin and bis-chloroethylnitrosourea were less efficient in inducing HPRT-deficient mutants and a smaller degree of protection was afforded by the transfected genes. This is possibly due to the propensity of these compounds to induce multi-locus deletions. Southern analysis of DNA from clone 8 and SB cells indicated the presence of multiple copies of the plasmid integrated into clone 8 cells but few copies in clone SB cells. The copy number did not change but ATase levels fell when cells were grown in the absence of G418.

Alkylating Agents↗
PubMed1987

Functional expression of the Escherichia coli alkyltransferase gene in mammalian cells.

Alkylating agents can produce a variety of biological effects in mammalian cells and organisms including toxicity, mutagenicity and malignant transformation. These agents react with oxygen and nitrogen atoms in DNA resulting in 12 products some of which are known to be eliminated from DNA by repair systems. One method of assessing the relative importance of a specific product in any of the biological effects of DNA alkylation would be to convert a cell line that is deficient in a particular repair function into a repair-proficient cell line and to determine whether this influences the magnitude of the effect. The cloning and expression in mammalian cells of the Escherichia coli DNA repair gene coding for the O6-alkylguanine-alkylphosphotriester dual alkyltransferase will be described. The E. coli gene product acts on damage produced in host cell DNA by treatment with methylnitrosourea, and reduces the toxicity and mutagenicity of this agent. The effects on the toxicity of a variety of other mono and bifunctional alkylating agents have also been assessed.

Alkylating Agents↗
PubMed1986

Reduction of the toxicity and mutagenicity of alkylating agents in mammalian cells harboring the Escherichia coli alkyltransferase gene.

The toxic, mutagenic, and carcinogenic effects of alkylating agents have been attributed to their ability to damage DNA. Reaction at the O6 position of guanine results in miscoding during DNA replication, has been shown to be mutagenic in both bacteriophage and bacteria, and may be responsible for malignant transformation. In common with many other prokaryotes and eukaryotes the Escherichia coli B strain contains a protein that repairs O6-alkylation damage in DNA by transferring the alkyl group to one of its own cysteine residues. We have recently cloned the E. coli O6-alkylguanine alkyltransferase gene and shown it to encode a 37-kDa protein containing an additional activity that removes alkyl groups from alkylphosphotriesters in DNA. To examine the biological effects of this gene in mammalian cells, we have now inserted the coding sequence into a retrovirus-based selectable expression vector and transfected it into Chinese hamster V79 cells that lack endogenous alkyltransferase activity. A clone expressing high levels of the bacterial protein was selected and shown to produce a 37-kDa alkyltransferase protein and to rapidly repair O6-methylguanine produced in the host genome following exposure to N-methyl-N-nitrosourea. In comparison with a control population, this clone is considerably more resistant to the toxic and mutagenic effects of alkylating agents that react extensively with oxygen atoms in DNA. The usefulness of these clones in examining the role of DNA alkylation and other biological effects of alkylating agents is discussed.

Alkylating Agents↗
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↗