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Biomedical subjects

J Shine

Publications and source records attributed to J Shine.

At least 73 records · Page 4Linked to original sources
PubMed1992

Effects of human, rat and porcine galanins on cardiac vagal action and blood pressure in the anaesthetised cat.

Galanin (GAL) is distributed in sympathetic nerves in the cat, and exogenous GAL inhibits cardiac vagal action and lowers blood pressure in this species. This study on anaesthetised cats compares the effects on cardiac vagal action and blood pressure of human, rat and porcine GAL. Human GAL has only recently been sequenced. It is of particular interest as it is not C-terminally amidated, unlike porcine and rat GAL. Many regulatory peptides require a C-terminal amide group for their action. However, human GAL showed similar biological activity to the other (amidated) GALs here. Omission of a single amino acid (Ser6) from rat GAL significantly attenuated both cardiovascular actions studied here.

Amino Acid Sequence↗
PubMed1992

Exclusion of close linkage of bipolar disorder to dopamine D1 and D2 receptor gene markers.

A potential role of dopamine in bipolar disorder has been suggested by several strands of evidence, namely the ability of dopaminergic agonists to induce mania and the effects of lithium, carbamazepine and the antipsychotics on central dopamine receptors and/or turnover. We therefore aimed to determine if bipolar disorder in two large bipolar pedigrees was linked to the recently cloned dopamine D1 (DRD1) and D2 (DRD2) receptors. (These have been mapped to chromosomal regions 5q35.1 and 11q22.3-q23, respectively). Linkage of bipolar disorder and recurrent depression to DRD1 and DRD2 was tested using a series of genetic models with varying penetrance levels. Additionally, linkage was examined using a series of levels of definitions of affective status (ranging from bipolar I alone to all affective illnesses). Close linkage to these markers was strongly excluded using each model and definition. The findings for DRD1 also persisted when a wide range of rates of 'sporadic' (non-genetic) presentations of illness were incorporated in the analysis, but the DRD2 results did not remain statistically significant at high sporadic rates. The exclusion of linkage to DRD2 is consistent with other recent reports.

Adolescent↗
PubMed1992

Dependence of hypotensive effect of neuropeptide-Y 18-36 fragment on intact vagus nerves.

Effects of neuropeptide Y (NPY 2.35-4.7 nmol) and the fragment NPY 18-36 (3.6-36 nmol) were studied in anaesthetized rats before and after vagotomy for actions on blood pressure (a postjunctional action) and on the change in pulse interval evoked by stimulation of the cut, peripheral vagus nerve (a prejunctional action). In intact rats NPY increased blood pressure. Low doses of NPY 18-36 (2.3-4.7 nmol) also increased blood pressure slightly but higher doses only transiently increased blood pressure and this was followed by a prolonged decrease in blood pressure. In vagotomised rats NPY increased blood pressure and attenuated cardiac vagal action. In higher doses the presynaptic effect of NPY 18-36 was more marked than its pressor action. NPY 18-36 (3.6-36 nmol) also increased blood pressure and attenuated cardiac vagal action. Thus, we conclude that the hypotensive action of NPY 18-36 depends on the intact vagus nerves: in the vagotomised rat NPY 18-36 has similar biological activity to NPY, but with reduced potency. To examine further the mechanism of hypotensive action of NPY 18-36 arterial rings from the central artery of the rabbit ear were also tested for direct actions of the peptides. This preparation is also suitable for testing any potentiation of contraction evoked by injection of noradrenaline. Neither NPY nor NPY 18-36 caused direct constrictor action in concentrations up to 10 microM. NPY 18-36 had no relaxing effect on constricted arterial rings.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Novel G protein-coupled receptors: a gene family of putative human olfactory receptor sequences.

We have taken advantage of the sequence conservation in the G protein-coupled receptor superfamily to isolate a fragment of a novel G protein-coupled receptor sequence using polymerase chain reaction (PCR) amplification of human genomic DNA. Screening of human genomic and hippocampal cDNA libraries with this amplified receptor fragment revealed a number of related sequences. Sequence analysis of four genomic clones and one cDNA clone clearly identifies these as related members of the G protein-coupled receptor family, as the deduced amino acid sequence reveals putative transmembrane domains and conserved amino acid residues. Southern blot analysis of restriction digests of human genomic DNA indicates that these receptor subtypes are likely to belong to a family of related genes. One of the proposed receptor sequences indicates the presence of pseudogenes in this family. Based on the homology of these sequences to a family of recently described receptors expressed exclusively in rat olfactory epithelium, it is suggested that these receptors represent a family of human odorant receptors.

Amino Acid Sequence↗
PubMed1992

Molecular characterization of a human brain adenosine A2 receptor.

A cDNA encoding a G protein-coupled receptor of unknown ligand specificity was isolated from a human hippocampal cDNA library by virtue of the high degree of structural homology between members of this receptor family. The cloned receptor DNA was transfected into human embryonic kidney 293 cells. Stably transfected cell lines bound a variety of adenosine agonists and antagonists with affinities characteristic of a brain adenosine A2a receptor. The A2a specific agonist CGS21680 stimulated cAMP production but did not alter intracellular calcium concentrations in transfected 293 cells.

Adenylyl Cyclases↗
PubMed1992

Molecular cloning and characterisation of a human brain A1 adenosine receptor cDNA.

Using the sequence conservation in the G protein-coupled receptor superfamily, we have isolated an adenosine A1 receptor cDNA from a human hippocampal cDNA library by homology screening. When expressed in mammalian CHO.K1 cells, the protein encoded by this cDNA binds the A1-specific antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) with high affinity (Kd = 0.56 +/- 0.11 nM) and, functionally, is able to inhibit cAMP production upon receptor activation with the A1-specific agonist N6-cyclopentyladenosine (CPA) (> 80% inhibition at 10(-7) M CPA). The binding and functional characteristics of the expressed cDNA demonstrate that we have isolated a human brain adenosine receptor cDNA of the A1 subtype.

Amino Acid Sequence↗
PubMed1992

G protein-coupled receptors.

The diversity of the G protein-coupled receptor superfamily is now being realised with the molecular cloning of DNA encoding many new receptors and receptor subfamilies. The existing pharmacological definitions of receptor subtypes have been extended dramatically with identification of additional subtypes at the molecular level. Functional analysis of cloned receptors by expression in heterologous cell types has demonstrated that individual receptor subtypes can couple to a variety of different effector systems.

Animals↗
PubMed1992

Structural subtypes of the dopamine D2 receptor are functionally distinct: expression of the cloned D2A and D2B subtypes in a heterologous cell line.

Dopamine, a major neurotransmitter in the mammalian nervous system, exerts its physiological effects through receptors of the G-protein-coupled receptor superfamily. Two major classes of dopamine receptor, D1 and D2, are distinguishable by both biochemical and pharmacological criteria. D1 receptors activate adenylyl cyclase, whereas the D2 class of receptors inhibits this second messenger system. Two subtypes of the human dopamine D2 receptor are generated by alternate splicing of the RNA transcript of a single gene. These two forms, termed D2A (long) and D2B (short), differ by the insertion of 29 amino acids within the putative third cytoplasmic loop, an intracellular domain thought to have a role in coupling this class of receptors to particular second messenger systems. We report here that the D2A and D2B structural subtypes are also functionally distinct. Expression of the two subtypes in a fibroblast cell line revealed that while occupation of both receptors leads to an increase in cytosolic free calcium concentration, they differ in their capacity to inhibit cAMP production. At physiological dopamine concentrations, the D2B-mediated inhibition of calcitonin gene-related peptide-stimulated cAMP accumulation is almost double the response mediated by the D2A subtype. Furthermore, the D2B subtype can maximally attenuate cAMP accumulation by up to 85%, whereas the D2A subtype is less effective, maximally inhibiting cAMP accumulation by only 64%. The D2A and D2B subtypes, thus, constitute functionally distinct forms of the dopamine receptor that can couple to multiple intracellular signalling pathways.

Animals↗
PubMed1991

Close linkage of bipolar disorder to chromosome 11 markers is excluded in two large Australian pedigrees.

The relationship between bipolar disorder and chromosome 11 markers remains uncertain. Whilst re-analysis of the Amish pedigree weakened previous evidence for close linkage (but could not exclude the possibility of genetic heterogeneity), a recent French study has found a significant association between this condition and tyrosine hydroxylase polymorphisms. We aimed to determine if bipolar disorder in two large Australian pedigrees (of Irish and English extraction respectively) was linked to these markers. Of the 84 family members available for testing, nine were diagnosed as bipolar I, one as bipolar II and six had recurrent unipolar depression. Linkage of bipolar disorder and recurrent depression to the chromosome 11p15 markers c-Harvey ras, insulin and tyrosine hydroxylase was tested using a series of genetic models with varying penetrance levels. Additionally, linkage was examined using a series of levels of definitions of affective status (ranging from bipolar I alone to all affective illnesses). Close linkage to these markers was strongly excluded using each model and definition. The findings also persisted when a wide range of rates of 'sporadic' (non-genetic) presentations of illness were incorporated in the analysis. These results are consistent with other recent studies indicating that bipolar disorder is not linked to chromosomal region 11p15.

Adolescent↗
PubMed1991

High density lipoproteins, genetic polymorphism for apo A-I and coronary artery disease.

HDL cholesterol and apolipoprotein A-I are associated with the development of coronary artery disease (CAD). The presence of a PstI site polymorphism adjacent to the gene encoding apo A-I (known as P2) has also been shown to be associated with CAD but this relationship is controversial. A case control study was conducted in an Australian population to re-examine whether the rare P2 allele is associated with CAD. Data were derived from 159 cases of angiographically confirmed CAD and 99 healthy controls. The proportion of CAD cases carrying the P2 allele did not differ significantly from controls (11% versus 9%). In a multiple logistic regression model controlling for the effects of age, country of birth, hypertension and hypotensive drugs, body mass index and lipid variables, the P2 allele failed to predict significantly the presence of CAD (odds ratio 1.83; 95% confidence interval 0.65-5.19).

Alleles↗
PubMed1991

Human galanin: molecular cloning reveals a unique structure.

Galanin, a novel neuropeptide/hypothalamic hormone originally identified and isolated by virtue of its carboxy-terminal amide group, has recently been shown to have a diverse range of biological activities, including potent effects on the secretion of insulin and growth hormone. The physiological role of galanin remains unclear, with different effects being observed when porcine and rat galanin have been used in various animal model systems and in human studies. Molecular cloning of cDNA encoding human galanin and galanin mRNA associated peptide (GMAP) from both pituitary and neuroblastoma sources has revealed a unique and unexpected structure. In contrast to porcine, bovine and rodent galanin, human galanin lacks a carboxy-terminal amide. By analogy to other neurohormones, the absence of carboxy-terminal amidation would be expected to have significant effects on functional properties such as affinity for different receptor subtypes and physiological half life, and may be responsible for the species specificity observed in the action of galanin.

Amino Acid Sequence↗
PubMed1991

Effects of exercise training and dietary manipulation on insulin-regulatable glucose-transporter mRNA in rat muscle.

Both exercise training and dietary manipulation (increasing omega-3/omega-6 fat ratio) can ameliorate insulin resistance caused by a high-fat diet in rats. We determined whether alterations in the expression of the insulin-regulatable (IR) and/or HepG2 glucose-transporter (GT) mRNAs were similarly affected. There was a significantly higher level of IRGT mRNA in skeletal muscle from exercise-trained versus sedentary high-fat-fed rats (27% increase, P less than 0.01). This difference is consistent with previously reported increases in muscle insulin-mediated glucose uptake. Skeletal muscle HepG2GT mRNA was too low to detect any training effect, but there was a tendency toward higher levels with training in cardiac muscle. In contrast, dietary manipulation, previously shown to lead to a much greater increase (100-300%) in muscle insulin-mediated glucose uptake, did not change IRGT or HepG2GT mRNA in skeletal muscle or heart. Thus, both dietary manipulation and exercise training increase insulin-stimulated glucose uptake in skeletal muscle, but only exercise training increases IRGT mRNA. Therefore, exercise training apparently increases GT production, whereas dietary manipulation improves glucose transport in skeletal muscle by other mechanisms.

Animals↗
PubMed1991

The noncemented porous-coated hip prosthesis. A three-year clinical follow-up study and roentgenographic analysis.

This was a retrospective study reviewing 68 hip arthroplasties performed with noncemented, porous-coated components at three institutions in the period between December 1983 and June 1987. Fifty of these were available for follow-up study. The mean follow-up period was three years (range, 18-56 months). Thirty-seven patients were treated by total hip arthroplasty (THA) with porous-coated components, and thirteen were implanted with porous-coated femoral stems with bipolar endoprostheses using a porous-coated femoral stem and a universal head. The mean Harris hip score at follow-up evaluation for the entire group was 85.7 points. Eighty percent of the patients had either a good or excellent result. Those patients implanted with a porous femoral stem with bipolar acetabular components had only 54% good or excellent results compared with 90% good or excellent results for those having THA. Roentgenographic findings included a 59% rate of femoral neck cortex osteopenia, an 80% rate of medial or lateral distal cortical hypertrophy, and an 80% rate of distal bone bridging. All of these roentgenographic changes were secondary to changes in stress transference to the proximal femur. There was no correlation between thigh pain or the degree of bone-prosthesis radiolucencies and the fit of the femoral stem.

Adult↗
PubMed1990

Steroid hormone receptor gene expression in human breast cancer cells: inverse relationship between oestrogen and glucocorticoid receptor messenger RNA levels.

The relative expression in human breast cancer cells of messenger ribonucleic acids (mRNA) encoding different steroid hormone receptors is unknown. Accordingly, mRNA levels in total RNA extracted from 13 human breast cancer cell lines were measured by Northern analysis employing complementary DNA probes for the human oestrogen (ER), progesterone (PR), androgen (AR), vitamin D3 (VDR) and glucocorticoid receptors (GR). The 7 ER+ lines expressed a single 6.4 kilobases (kb) ER mRNA. Interestingly, low concentrations of ER mRNA were detected in the ER- cell lines, MDA-MB-330 and BT 20. PR mRNA, predominantly a 13.5 kb species, was expressed in the 6 lines known to be ER+, PR+ by radioligand binding; however, one ER+ cell line, MDA-MB-134, failed to express PR mRNA. A 10.5 kb AR mRNA was expressed at significantly higher levels in ER+ than ER- cell lines. All cell lines expressed a single 4.6 kb mRNA for VDR and a single 7.4 kb mRNA for GR. ER and PR mRNA levels were positively correlated (p = 0.011) and each was positively correlated with androgen receptor (AR) mRNA levels (p less than or equal to 0.009). ER, PR and AR mRNAs were negatively associated with GR levels (p less than or equal to 0.012), while ER and AR mRNA levels were negatively correlated with mRNA for the epidermal growth factor receptor. In contrast, levels of VDR mRNA were unrelated to the concentration of any other steroid receptor mRNA. Our data demonstrate the coordinate expression of ER, PR and AR genes, and an inverse relationship between sex steroid hormone receptor and GR gene expression in human breast cancer cell lines.

Blotting, Northern↗
PubMed1990

Construction of cell lines that express high levels of the human estrogen receptor and are killed by estrogens.

To prepare large amounts of the human estrogen receptor (ER) for biochemical and biophysical studies we have employed the cloned ER sequences to construct Chinese hamster ovary (CHO) cell line derivatives that overexpress the receptor. We have employed an efficient expression vector (SV40 enhancer, human metallothionein IIA promoter) and a new system of gene amplification based on the human metallothionein IIA gene and stepwise selection in cadmium. Cells from the initial transfected pools, before gene amplification, had as much or more ER than human MCF7 cells and responded to the subsequent stepwise cadmium selection and amplification with increases in ER levels to about 2 million receptors/cell. Cell lines isolated from these pools are stable for human ER expression and display up to 6 million receptors/cell, or about 0.4% of the total cell protein. The CHO receptor activates a transfected reporter gene in responses to estrogen, is down-regulated in response to estrogens, displays the same electrophoretic mobility as the MCF7 receptor, and is free of degradation as initially extracted. CHO cells displaying 3 million or more human ER/cell (but not cells with lower levels) flatten and stop growing within the first 24 h after exposure to physiological estrogen concentrations. After several days in estrogen the majority of the cells lyse. The antiestrogen 4-hydroxytamoxifen also causes cell death, but another antiestrogen, ICI 164,384, is without toxic effect. The basis for these phenomena are unknown, but mutants isolated for survival of estrogen treatment have lost receptor expression, thereby confirming the role of receptor in cell death.

Animals↗
PubMed1990

Progestin regulation of estrogen receptor messenger RNA in human breast cancer cells.

Progestin antagonism of estrogen action is thought to be due, at least in part, to progestin down-regulation of the estrogen receptor (ER). The molecular mechanisms subserving this effect, and the functional consequences in terms of target cell sensitivity to estrogens, are poorly understood. The present study was undertaken to address these issues with particular emphasis on progestin regulation of ER gene expression at the mRNA level. The T-47D human breast cancer cell line was treated with the synthetic progestin, ORG 2058, and the resultant changes in ER mRNA and ER levels determined by Northern analysis and radioligand binding, respectively. Treatment of T-47D cells with ORG 2058 resulted in rapid down-regulation of ER mRNA levels to a nadir of 35-40% of control by 6 h. This fall in ER mRNA levels was accompanied by a slower but more sustained fall in ER binding to a nadir of 20% of control at 24 h. Between 12 and 24 h ER mRNA levels recovered partially while ER ligand binding continued to fall. At 48 h both ER mRNA and ER concentrations remained depressed, although the latter to a greater extent. ER mRNA half-life was determined by [3H]uridine incorporation to be approximately 60 min and was unaffected by progestin treatment during the early rapid phase of ER mRNA down-regulation. These data demonstrate that progestins cause rapid down-regulation of the ER mRNA and suggest that during the early rapid phase of this effect, reduced transcription of the ER gene rather than altered ER mRNA half-life mediate this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed1990

Inverse relationship between estrogen receptor and epidermal growth factor receptor mRNA levels in human breast cancer cell lines.

Epidermal growth factor receptors (EGF-R) are present in a number of human breast cancer cell lines and tumor biopsies. Furthermore, it has been suggested that EGF-R levels are higher in estrogen receptor negative (ER-) than in ER+ human breast tumors and that EGF-R status may be a prognostic indicator in breast cancer. The present study was undertaken to establish whether there is a quantitative relationship between EGF-R and ER mRNA concentrations in a series of 10 well-characterized human breast cancer cell lines. All cell lines expressed detectable quantities of EGF-R mRNA by Northern analysis but the relative abundance of EGF-R mRNA varied more than 50-fold. Two transcripts corresponding to the 10.5- and 5.8-kb mRNAs described in other cell types were present but in different relative proportions in different cell lines. When these lines were divided into an ER+ and an ER- group based on their ability to bind estradiol, ER- cell lines were shown to express significantly higher concentrations of EGF-R mRNA than did ER+ cell lines (p less than 0.005). Furthermore, linear-regression analysis revealed a significant inverse relationship between ER and EGF-R mRNA concentrations both within the group of 10 human breast cancer cell lines as a whole (r = 0.66) and within the 6 functionally ER + lines (r = 0.77). This demonstration of a significant (p less than 0.005) inverse relationship between the concentrations of ER and EGF-R mRNAs in ER + cell lines raises the possibility of reciprocal regulation of the expression of these genes in human breast cancer.

Blotting, Northern↗