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Biomedical subjects

J Brennand

Publications and source records attributed to J Brennand.

At least 19 recordsLinked to original sources
PubMed2000

Alternative role for prolactin-releasing peptide in the regulation of food intake.

Prolactin-releasing peptide (PrRP) is a peptide ligand for the human orphan G-protein-coupled receptor hGR3/GPR10 and causes the secretion of prolactin from anterior pituitary cells. However, the lack of immunoreactive staining for PrRP in the external layer of the median eminence seems to rule out this peptide as a classical hypophysiotropic hormone and, furthermore, PrRP is less effective than another inducer of prolactin secretion, thyrotropin-releasing hormone, both in vitro and in vivo. Here we show a reduction in the expression of PrRP mRNA during lactation and fasting and an acute effect of PrRP on food intake and body weight, supporting the hypothesis of an alternative role for the peptide.

Animals↗
PubMed1999

Cloning and characterization of corticotropin-releasing factor and urocortin in Syrian hamster (Mesocricetus auratus).

Corticotropin-releasing factor and urocortin belong to a superfamily of neuropeptides that includes the urotensins-I in fishes and the insect diuretic peptides. Sequence analysis suggests that urocortin is the mammalian ortholog of urotensin-I, although the physiological role for this peptide in mammals is not known. Within the Rodentia, hamsters belong to a phylogenetically older lineage than that of mice and rats and possess significant differences in hypothalamic organization. We have, therefore, cloned the coding region of the Syrian hamster (Mesocricetus auratus) corticotropin-releasing factor and urocortin mature peptide by polymerase chain reaction. Hamster urocortin was prepared by solid-phase synthesis, and its pharmacological actions on human corticotropin-releasing factor R1 and R2 receptors were investigated. The deduced hamster corticotropin-releasing factor amino acid sequence and cleavage site is identical to that in rat, whereas the urocortin sequence is unique among the urocortin/urotensin-I/sauvagine family in possessing asparagine and alanine in positions 38 and 39, respectively. The hamster urocortin carboxy terminus sequence bears greater structural similarity to the insect diuretic peptide family, suggesting either retrogressive mutational changes within the mature peptide or convergent sequence evolution. Despite these changes, human and hamster urocortin are generally equipotent at cAMP activation, neuronal acidification rate, and R1/R2 receptor affinities.

Amino Acid Sequence↗
PubMed1999

Management of fetal alloimmune thrombocytopenia-a less invasive option?

Fetal alloimmune thrombocytopenia (FAITP) is a condition associated with significant infant morbidity and mortality. We report on the West of Scotland experience of 30 pregnancies complicated by FAITP over a 17-year period (1982-98). Management options included serial cordocentesis together with platelet transfusion, and maternal intravenous gammaglobulin (IVIgG) therapy. Of those pregnancies managed by serial cordocentesis all had poor outcomes. Weekly IVIgG was administered to the remaining pregnancies, all of which had a good outcome although four infants were thrombocytopenic at birth. None of these cases had previously been complicated by intracranial haemorrhage. In the milder end of the spectrum of FAITP we would suggest that IVIgG is an alternative treatment option.

Journal Article↗
PubMed1998

Direct regulation of GnRH transcription by CRF-like peptides in an immortalized neuronal cell line.

The existence of a CRF-dependent inhibition of GnRH transcription was investigated using a neuronal GnRH-expressing cell line (Gn11) stably transfected with mouse (-611 bp) or chicken (-3000 bp) GnRH promoter/luciferase reporter constructs. The presence of the CRF-R1 receptor was established using a specific CRF-R1 antiserum. After 7 h of incubation, urotensin-I and sauvagine increased the mouse GnRH-reporter bioluminescence by 1.3- and 1.2-fold, respectively, compared with control cells. Subsequently, CRF, urotensin-I and sauvagine decreased luciferase reporter activity to about 60% of the control values after 14 h. Similar trends occurred with the chicken GnRH promoter with UI increasing reporter gene activity 2.4-fold over the controls after 14 h incubation. These data provide additional evidence for the direct regulation of GnRH transcription by CRF-like peptides.

Amphibian Proteins↗
PubMed1998

Induction of labour: new horizons.

Induction of labour may be indicated in the maternal or the fetal interest, and is more likely to be successful if physiological mechanisms are replicated. Induction of labour in the presence of an unfavourable cervix presents the greatest challenge and pharmacological techniques must encourage cervical ripening if we are to advance our management of this common obstetric intervention.

Abortifacient Agents↗
PubMed1998

The influence of amniotic fluid on prostaglandin synthesis and metabolism in human fetal membranes.

OBJECTIVE: To investigate the effect of amniotic fluid on prostaglandin synthesis and metabolism in the fetal membranes. DESIGN: A cell culture study of amnion and chorion obtained at elective cesarean section incubated with amniotic fluid collected following either spontaneous labor and delivery, or elective cesarean section. SUBJECTS: Forty-eight pregnant women at 3742 weeks gestation: 24 in spontaneous labor and 24 delivered by elective cesarean section. RESULTS: Significantly more PGE2 and PGF2alpha were produced by amnion and chorion treated with amniotic fluid from spontaneous labor compared with elective cesarean section. Spontaneous labor amniotic fluid favors PGE2 and PGFM production by amnion and chorion respectively; while elective section fluid stimulates PGE2 synthesis by both tissues (reflected as PGEM in chorion). Amniotic fluid, from either spontaneous labor or elective section, had no effect on the metabolism of exogenous PGE2 or PGF2alpha by chorion cells. CONCLUSION: Spontaneous labor is associated with the presence of a substance in amniotic fluid which facilitates prostaglandin synthesis in the fetal membranes, but which is without effect on prostaglandin metabolism.

Amnion↗
PubMed1998

Activated protein C sensitivity, protein C, protein S and coagulation in normal pregnancy.

A prospective study of activated protein C sensitivity, protein C, protein S, and other coagulation factors in 239 women during normal pregnancy was carried out. Protein C activity appeared unaffected by gestation, although an elevation of protein C activity was observed in the early puerperium. A fall in total and free protein S with increasing gestation was observed. Activated protein C sensitivity ratio (APC:SR) showed a progressive fall through pregnancy. This fall correlated with changes in factor VIIIc, factor Vc and protein S. 38% of subjects, with no evidence of Factor V Leiden or anticardiolipin antibodies, showed a low APC:SR (APC:SR <2.6) in the third trimester of pregnancy. Aside from a significant reduction in birth weight, no difference in pregnancy outcome was observed between these subjects and those with a normal APC:SR. Activated protein C sensitivity ratio, modified by pre-dilution of patient samples with factor V depleted plasma, showed no consistent trend with gestation.

Birth Weight↗
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↗