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Biomedical subjects

I Diamond

Publications and source records attributed to I Diamond.

At least 19 recordsLinked to original sources
PubMed2001

Cloning of a novel isoform of the mouse NBMPR-sensitive equilibrative nucleoside transporter (ENT1) lacking a putative phosphorylation site.

We have isolated a mouse cDNA clone corresponding to a novel isoform of the NBMPR-sensitive equilibrative nucleoside transporter (ENT1). The cDNA contains a 6 bp deletion in the open reading frame that changes the amino acid composition in a consensus casein kinase II (CKII) phosphorylation site at Ser-254. The clone containing Ser-254 is termed mENT1.1 and the clone lacking the serine termed mENT1.2. The deduced amino acid sequence of mENT1.1 corresponds to the previously cloned human and rat ENT1 proteins at Ser-254. Tissue distribution studies show that mRNA for both ENT1 isoforms are ubiquitously co-expressed in mouse. Analysis of genomic DNA corresponding to mouse ENT1 indicates the isoforms can be produced by alternative splicing at the end of exon 7. CEM/C19 cells stably expressing mENT1.1 and mENT1.2 show similar dose response curves for NBMPR and dipyridamole inhibition of [(3)H]adenosine uptake as well as exhibiting comparable selectivity for both purine and pyrimidine nucleosides but not the corresponding nucleobases.

Amino Acid Sequence↗
PubMed2001

Ethanol acts synergistically with a D2 dopamine agonist to cause translocation of protein kinase C.

Ethanol and other drugs of abuse increase synaptic dopamine levels; however, little is known about how ethanol alters dopaminergic signaling. We have reported that ethanol induces translocation of delta and epsilon protein kinase C (PKC) in neural cells in culture. Using NG108-15 and Chinese hamster ovary cell lines that express the dopamine D2 receptor (D2R), we show here that the D2R agonist R(-)-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide (NPA) also causes translocation of delta and epsilon PKC to the same sites as ethanol-induced translocation. D2R agonist and ethanol-induced translocation of delta and epsilon PKC share a common pathway that is blocked by pertussis toxin and requires phospholipase C (PLC) activity. These data suggest that both D2R agonists and ethanol activate PLC via a trimeric G protein leading to production of diacylglycerol with subsequent activation and translocation of delta and epsilon PKC. Moreover, ethanol and NPA, when present together at low concentrations that alone are ineffective, act synergistically to cause translocation of delta and epsilon PKC. Our data suggest that ethanol causes translocation of delta and epsilon PKC but cells expressing the D2R, such as neurons in the nucleus accumbens, may be particularly sensitive to low concentrations of ethanol.

Alcoholism↗
PubMed2001

A multilevel analysis of the effects of rurality and social deprivation on premature limiting long term illness.

STUDY OBJECTIVE: To examine the geographical variation in self perceived morbidity in the south west of England, and assess the associations with rurality and social deprivation. DESIGN: A geographically based cross sectional study using 1991 census data on premature Limiting Long Term Illness (LLTI). The urban-rural and intra-rural variation in standardised premature LLTI ratios is described, and correlation and regression analyses explore how well this is explained by generic deprivation indices. Multilevel Poisson modelling investigates whether Customized Deprivation Profiles (CDPs) and area characteristics improve upon the generic indices. SETTING: Nine counties in the south west of England PARTICIPANTS: The population of the south west enumerated in the 1991 census. MAIN RESULTS: Intra-rural variation is apparent, with higher rates of premature LLTI in remoter areas. Together with high rates in urban areas and lower rates in the semi-rural areas this indicates the existence of a U shaped relation with rurality. The generic deprivation indices have strong positive relations with premature LLTI in urban areas, but these are a lot weaker in semi-rural and rural locations. CDPs improve upon the generic indices, especially in the rural settings. A substantial reduction in unexplained variation in rural areas is seen after controlling for the level of local isolation, with higher isolation, at the wider geographical scale, being related to higher levels of LLTI. CONCLUSIONS: This study highlights the need to treat rural areas as heterogeneous, although this has not been the tendency in health research. Generic deprivation indices are unlikely to be a true reflection of levels of deprivation in rural environments. The importance of CDPs that are specific to the area type and health outcome is emphasised. The significance of physical isolation suggests that accessibility to public and health services may be an important issue, and requires further research.

Adolescent↗
PubMed2000

Genomic organization and expression of the mouse equilibrative, nitrobenzylthioinosine-sensitive nucleoside transporter 1 (ENT1) gene.

We have cloned and characterized the genomic structure of the mouse gene for the NBMPR-sensitive equilibrative nucleoside transporter (mENT1), which is located on chromosome 17C. About 12-kb of genomic DNA was sequenced including the promoter region, 12 exons, 11 introns, and the 3'-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. Primer extension analysis demonstrated a transcription initiation site located 252 bp upstream of the translation start site. Analysis of the 2.5-kb 5'-flanking sequence shows putative binding sites for several transcription factors, including GATA-1, IRF-2, Pit-1, myogenin, CREB, Sp-1, Ap-2, MAZ, and GR. We demonstrated that mouse ENT1 mRNA was highly expressed in heart, spleen, lung, liver, and testis. Lower levels of expression were detected in brain and kidney. Functional analysis of the 5'-flanking region showed that the nucleotide sequence from -652 to -111 contains cis-regulatory elements that promote gene expression. We found two Sp-1 binding sites (-296/-303, -307/-313) and two MAZ binding sites (-353/-359, -522/-528) in this region. Luciferase assay results suggest that MAZ and Sp-1 transcription factors are important positive regulators of transcription for the ENT1 gene in NG108-15 cells.

3' Untranslated Regions↗
PubMed2000

Conditional gene expression in the epidermis of transgenic mice using the tetracycline-regulated transactivators tTA and rTA linked to the keratin 5 promoter.

To produce conditional expression of genes in the mouse epidermis we have generated transgenic mouse lines in which the tetracycline-regulated transcriptional transactivators, tTA and rTA, are linked to the bovine keratin 5 promoter. The transactivator lines were crossed with the tetOlacZ indicator line to test for transactivation in vivo. In the absence of doxycycline, the K5/tTA line induced beta-galactosidase enzyme activity in the epidermis at a level 500-fold higher than controls, and oral and topical doxycycline caused a dose- and time-dependent suppression of beta-galactosidase mRNA levels and enzyme activity. In the K5/rTA lines, doxycycline induced beta-galactosidase activity between 3- and 50-fold higher depending on the founder line, and this occurred within 24-48 h after dosing. Histochemical analysis of all lines localized beta-galactosidase expression to the basal layer of the epidermis and the outer root sheath of the hair follicle, as well as other keratin 5 positive tissues. In several K5/rTA lines, skin-specific transactivation was restricted to the hair follicle. Treatment of these double transgenic mice with 12-O-tetradecanoyl-phorbol-13-acetate caused rapid migration of beta-galactosidase marked cells from the hair follicle through the interfollicular epidermis, demonstrating the usefulness of this specific double transgenic for fate mapping cells in the epidermis. These results show that the tetracycline regulatory system produces effective conditional gene expression in the mouse epidermis, and suggest that it should be amenable to suppression and activation of foreign genes during development and specific pathologic conditions relevant to the epidermis.

Animals↗
PubMed2000

Uncoupling of betaIIPKC from its targeting protein RACK1 in response to ethanol in cultured cells and mouse brain.

Protein kinase C (PKC) is involved in many neuroadaptive responses to ethanol in the nervous system. PKC activation results in translocation of the enzyme from one intracellular site to another. Compartmentalization of PKC isozymes is regulated by targeting proteins such as receptors for activated C kinase (RACKs). It is possible, therefore, that ethanol-induced changes in the function and compartmentalization of PKC isozymes could be due to changes in PKC targeting proteins. Here we study the response of the targeting protein RACK1 and its corresponding kinase betaIIPKC to ethanol, and propose a novel mechanism to explain how ethanol modulates signaling cascades. In cultured cells, ethanol induces movement of RACK1 to the nucleus without affecting the compartmentalization of betaIIPKC. Ethanol also inhibits betaIIPKC translocation in response to activation. These results suggest that ethanol inhibition of betaIIPKC translocation is due to miscompartmentalization of the targeting protein RACK1. Similar events occurred in mouse brain. In vivo exposure to ethanol caused RACK1 to localize to nuclei in specific brain regions, but did not affect the compartmentalization of betaIIPKC. Thus, some of the cellular and neuroadaptive responses to ethanol may be related to ethanol-induced movement of RACK1 to the nucleus, thereby preventing the translocation and corresponding function of betaIIPKC.

Animals↗
PubMed1999

Ethanol-induced translocation of cAMP-dependent protein kinase to the nucleus. Mechanism and functional consequences.

Ethanol induces translocation of the catalytic subunit (Calpha) of cAMP-dependent protein kinase (PKA) from the Golgi area to the nucleus in NG108-15 cells. Ethanol also induces translocation of the RIIbeta regulatory subunit of PKA to the nucleus; RI and Cbeta are not translocated. Nuclear PKA activity in ethanol-treated cells is no longer regulated by cAMP. Gel filtration and immunoprecipitation analysis confirm that ethanol blocks the reassociation of Calpha with RII but does not induce dissociation of these subunits. Ethanol also reduces inhibition of Calpha by the PKA inhibitor PKI. Pre-incubation of Calpha with ethanol decreases phosphorylation of Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and casein but has no effect on the phosphorylation of highly charged molecules such as histone H1 or protamine. cAMP-response element-binding protein (CREB) phosphorylation by Calpha is also increased in ethanol-treated cells. This increase in CREB phosphorylation is inhibited by the PKA antagonist (R(p))-cAMPS and by an adenosine receptor antagonist. These results suggest that ethanol affects a cascade of events allowing for sustained nuclear localization of Calpha and prolonged CREB phosphorylation. These events may account for ethanol-induced changes in cAMP-dependent gene expression.

Animals↗
PubMed1999

Coordinated movement of RACK1 with activated betaIIPKC.

Protein kinase C (PKC) isozymes move upon activation from one intracellular site to another. PKC-binding proteins, such as receptors for activated C kinase (RACKs), play an important role in regulating the localization and diverse functions of PKC isozymes. RACK1, the receptor for activated betaIIPKC, determines the localization and functional activity of betaIIPKC. However, the mechanism by which RACK1 localizes activated betaIIPKC is not known. Here, we provide evidence that the intracellular localization of RACK1 changes in response to PKC activation. In Chinese hamster ovary cells transfected with the dopamine D2L receptor and in NG108-15 cells, PKC activation by either phorbol ester or a dopamine D2 receptor agonist caused the movement of RACK1. Moreover, PKC activation resulted in the in situ association and movement of RACK1 and betaIIPKC to the same intracellular sites. Time course studies indicate that PKC activation induces the association of the two proteins prior to their co-movement. We further show that association of RACK1 and betaIIPKC is required for the movement of both proteins. Our results suggest that RACK1 is a PKC shuttling protein that moves betaIIPKC from one intracellular site to another.

Animals↗
PubMed1999

Contraceptive use dynamics of Asian women in Britain.

In-depth interviews were conducted with married Asian women from Indian, Pakistani and Bangladeshi backgrounds, to investigate patterns of contraceptive use and influences on contraceptive decision making. The results show two distinctively different contraceptive 'lifecycles'. Non-professional women typically have little knowledge about contraception until after their marriage or first birth. Their patterns of contraceptive behaviour show low levels of contraceptive use until after their first birth, when condom use is most prevalent. Non-professional women are influenced by their extended family, religion and cultural expectations on their fertility and family planning decisions. Professional women show an entirely different pattern of contraceptive behaviour. They are more likely to have knowledge about contraception before marriage, use some method of contraception throughout their childbearing years (typically the pill) and cite personal, practical or economic considerations in their fertility decisions rather than religious, cultural or extended family influences.

Adult↗
PubMed1999

Contraceptive switching in Bangladesh.

Bangladesh has experienced a substantial decline in fertility that has been achieved by means of a large increase in the use of modern methods of contraception. As contraceptive prevalence increases, aspects of contraceptive-use dynamics, including reasons for discontinuation and behavior after discontinuation, become important influences on fertility. This report uses calendar data from the 1993-94 Bangladesh Demographic and Health Survey to examine contraceptive behavior following discontinuation of modern-method use. The individual-level characteristics found to influence switching behavior include the method used, method-related difficulties with previous contraceptive use, and education. A large amount of unexplained variation in switching rates remains, however, largely at the individual level, but also at the community level for certain types of transition.

Adolescent↗
PubMed1999

Chronic dipyridamole therapy produces sustained protection against cardiac ischemia-reperfusion injury.

Sustained protection against ischemia-reperfusion injury is not available for patients at risk for myocardial infarction who may require emergent reperfusion therapy. Whereas ischemic preconditioning and adenosinergic agents reduce myocardial injury, they are only effective when given immediately before ischemia or reperfusion. We recently found chronic ethanol exposure, an adenosine uptake inhibitor, produced sustained cardioprotection against ischemia-reperfusion injury. We now ask whether chronic dipyridamole therapy, a clinically usable nucleoside transport inhibitor, induces similar cardioprotection. Perfused hearts from guinea pigs, given dipyridamole (4 mg. kg(-1). day(-1)) in their water for 2-6 wk (n = 10 for each group), underwent ischemia-reperfusion. Injury was assessed by recovery of left ventricular developed (LVDP) and end-diastolic (LVEDP) pressures and creatine kinase release. During reperfusion, hearts from dipyridamole-treated animals (6 wk) had 74% higher LVDP, 28% lower LVEDP, and 61% lower creatine kinase release versus controls. Adenosine A(1)-receptor antagonism (8-cyclopentyl-1, 3-dipropylxanthine; 200 nM) abolished the protection of dipyridamole but A(2) antagonism (3,7-dimethyl-1-propargylxanthine; 10 mM) did not. Dipyridamole therapy produces sustained protection against ischemia-reperfusion injury in guinea pigs. This cardioprotection requires adenosine A(1) receptor signaling at the time of ischemia.

Animals↗
PubMed1999

What determines geographical variation in rates of acceptance onto renal replacement therapy in England?

OBJECTIVE: To determine the independent effects of need and supply factors on the known geographical variation in acceptance rates onto renal replacement therapy (RRT) in England. METHODS: Data were obtained from all renal units in England on the characteristics of all cases aged 16 years and over, resident in England, who were accepted onto RRT in 1991 and 1992. Of these, 5715 (94.5%) had a valid postcode that could be matched to a census ward. Multilevel modelling using Poisson regression was used. The number of acceptances in each census ward within age bands 16-34, 35-64 and 65+ was the dependent variable. Independent effects modelled were: (1) individual factors (age, sex); (2) census ward need factors--ethnicity (expressed as the percentage of the ward population that was Asian or African-Caribbean), socio-economic deprivation--and supply factors--'access' to the nearest renal unit using crowfly and road travel time and distance, and services available to each ward expressed as number of haemodialysis stations per 100,000 catchment population of the nearest renal unit; (3) district health authority level effects. RESULTS: Age was a major determinant of acceptance, with a 7-fold higher rate in males aged over 64 years compared with younger men. Acceptance rates were lower in females, with a negative age-sex interaction in females aged over 64 years. The percentage of both Asian and African-Caribbean populations per ward was a highly significant positive determinant. Deprivation was also a significant determinant, best represented by a customised index. There was an inverse relation of acceptance with distance, especially road travel time. Other supply side variables had a significant effect though there was no independent district effect. There was some variation in the strength of these relationships by type of area (Greater London, urban and non-urban). CONCLUSIONS: Need and supply factors influence service use as expressed as acceptance onto RRT. Pressure to expand RRT services needs to be aimed at areas with large minority ethnic populations and those living far from existing units.

Adolescent↗
PubMed1999

Safer sex at holiday centres: providing contraceptive services to seasonal workers.

Many seasonal workers experience an increase in sexual activity whilst employed at a holiday centre. Evidence of sexual risk-taking while at a holiday centre has important social and health implications for purchasers and providers of sexual health services in areas which experience an annual influx of seasonal workers. This research investigates the contraceptive behaviour of seasonal workers and focuses on their access to contraception and sexual health services. In-depth interviews were conducted with seasonal workers at holiday centres along the south coast of England. Respondents were, interviewed at the beginning of the season and again, five months later at the end of the season. This longitudinal methodology enabled changes in contraceptive behaviour to be identified as well as the strategies for seeking contraception and sexual health services throughout the season. The results of this study show that there are a range of different motivations which influence seasonal worker' use of contraception and sexual risk-taking while at a holiday centre. Categories of contraceptive protection are developed to assist purchasers and providers to identify the variety of sexual health needs of workers at holiday centres and determine the most effective strategies for delivering contraceptive and sexual health services to these workers. The paper describes the motivations which influence contraceptive use and sexual risk-taking amongst seasonal workers, identifies the contraceptive and sexual health needs of these workers, and discusses the difficulties workers experienced in meeting these needs while at a holiday centre.

Adolescent↗
PubMed1998

Activation of epsilon protein kinase C correlates with a cardioprotective effect of regular ethanol consumption.

In addition to decreasing the incidence of myocardial infarction, recent epidemiological data suggest that regular alcohol consumption improves survival after myocardial infarction. We recently found that chronic ethanol exposure induces long-term protection against cardiac ischemia-reperfusion injury, which improves myocardial recovery after infarction. Furthermore, this cardioprotection by ethanol is mediated through myocyte adenosine A1 receptors. We now determine the role of protein kinase C (PKC) in ethanol's protective effect against ischemia-reperfusion injury. Using perfused hearts of ethanol-fed guinea pigs, we find that improved contractile recovery and creatine kinase release after ischemia-reperfusion are abolished by PKC inhibition with chelerythrine. Western blot analysis and immunofluorescence localization demonstrate that regular ethanol consumption causes sustained translocation (activation) of epsilonPKC, but not delta or alphaPKC. This same isozyme is directly implicated in ischemic preconditioning's protection against ischemia-reperfusion injury. Our findings suggest (i) that regular ethanol consumption induces long-term cardioprotection through sustained translocation of epsilonPKC and (ii) that PKC activity is necessary at the time of ischemia to mediate ethanol's protective effect against ischemia-reperfusion injury. Studying this selective effect of ethanol on epsilonPKC activation may lead to new therapies to protect against ischemia-reperfusion injury in the heart and other organ systems.

Alcohol Drinking↗
PubMed1998

Alcohol consumption reduces ischemia-reperfusion injury by species-specific signaling in guinea pigs and rats.

We recently discovered that regular alcohol consumption reduces ischemia-reperfusion injury to the same degree as ischemic preconditioning in guinea pig hearts. Ischemic preconditioning, like this cardioprotective effect of alcohol, is mediated by adenosine signaling in guinea pigs. In rats, ischemic preconditioning may be mediated predominantly by alpha1-adrenergic signaling. To be certain that this protective effect of alcohol is a general biological response, we searched for alcohol's cardioprotection in rat and identified a potential signaling mechanism. Hearts isolated from alcohol-fed guinea pigs and rats were subjected to ischemia-reperfusion. Hearts from alcohol-fed animals showed greater recovery of left ventricular developed pressure than controls (guinea pigs, 46 vs. 29%; rats, 50 vs. 31%) and decreased myocyte necrosis assessed by creatine kinase release (guinea pigs, 204 +/- 42 vs. 440 +/- 70 U . ml-1 . g dry wt-1; rats 158 +/- 13 vs. 328 +/- 31 U . ml-1 . g dry wt-1). Adenosine receptor blockade [8-(p-sulfophenyl)theophylline] abolished alcohol's protection in guinea pig but not rat hearts. By contrast, alpha1-adrenergic blockade (prazosin) abolished alcohol's protection in rat but not guinea pig hearts. We conclude that regular alcohol consumption reduces ischemia-reperfusion injury and is mediated by species-specific signaling mechanisms. A major goal of cardiovascular research is to find a pharmacologically induced chronic state of preconditioning. Understanding the mechanisms of alcohol's cardioprotection against ischemia-reperfusion injury may aid in reaching this goal.

Alcohol Drinking↗
PubMed1998

Varying effects of ethanol on transfected cell lines.

The use of transfected cell lines has provided a powerful approach to study the functions of transfected proteins. This approach has also proven useful to determine the effects of acute and chronic ethanol exposure on particular proteins. We show here, however, that the effects of ethanol on transfected proteins vary between transiently transfected cells and stably transfected cell lines. Moreover, we found that the effect of ethanol on a particular protein depends on the plasmid vectors used for the transfection.

Animals↗