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R Barber

Publications and source records attributed to R Barber.

At least 55 records · Page 3Linked to original sources
PubMed1998

Apoptosis and expression of DNA repair proteins in ischaemic brain injury in man.

We examined the timing of apoptosis and the expression of the DNA repair proteins poly(ADP-ribose) polymerase (PARP) and Ku80 in sections of frontal and temporal lobes from patients who had suffered severe brain ischaemia due to a cardiac arrent. In situ end-labelling (ISEL) was used to detect apoptotic cells, and immunohistochemistry to assess PARP and Ku80. ISEL of scattered neurons and glia was demonstrable predominantly during the first 24 h after ischaemia. PARP and Ku80 immunoreactivity increased markedly after cerebral ischaemia, PARP particularly in the regions of greatest susceptibility to hypoxic injury: the CA1 field of the hippocampus and the depths of neocortical sulci. The up-regulation of PARP is in keeping with experimental observations concerning the key role of this enzyme in mediating ischaemic cell death.

Adult↗
PubMed1998

Salmeterol-induced desensitization, internalization and phosphorylation of the human beta2-adrenoceptor.

1. Partial agonists of the beta2-adrenoceptor which activate adenylyl cyclase are widely used as bronchodilators for the relief of bronchoconstriction accompanying many disease conditions, including bronchial asthma. The bronchodilator salmeterol has both a prolonged duration of action in bronchial tissue and the ability to reassert this activity following the temporary blockade of human beta2-adrenoceptors with antagonist. 2. We have compared the activation and desensitization of human beta2-adrenoceptor stimulation of adenylyl cyclase induced by salmeterol, adrenaline and salbutamol in a human lung epithelial line, BEAS-2B, expressing beta2-adrenoceptor levels of 40-70 fmol mg(-1), and in human embryonic kidney (HEK) 293 cell lines expressing 2-10 pmol mg(-1). The efficacy observed for the stimulation of adenylyl cyclase by salmeterol was only approximately 10% of that observed for adrenaline in BEAS-2B cells expressing low levels of beta2-adrenoceptor, but similar to adrenaline in HEK 293 cells expressing very high levels of receptors. Salmeterol pretreatment of these cells induced a rapid and stable activation of adenylyl cyclase activity which resisted extensive washing and beta2-adrenoceptor antagonist blockade, consistent with binding to a receptor exosite and/or to partitioning into membrane lipid. 3. The desensitization and internalization of beta2-adrenoceptors induced by the partial agonists salmeterol and salbutamol were considerably reduced relative to the action of adrenaline. Consistent with these observations, the initial rate of phosphorylation of the receptor induced by salmeterol and salbutamol was much reduced in comparison to adrenaline. 4. Our data suggest that the reduction in the rapid phase of desensitization of beta2-adrenoceptors after treatment with salmeterol or salbutamol is caused by a decrease in the rate of beta2-adrenoceptor kinase (betaARK) phosphorylation and internalization. In contrast, the rate of cyclic AMP-dependent protein kinase (PKA)-mediated phosphorylation by these partial agonists appears to be similar to adrenaline.

Adenylyl Cyclases↗
PubMed1998

Ultraviolet B induced suppression of induction of contact sensitivity in human skin is not associated with tumour necrosis factor-alpha-308 or interleukin-10 genetic polymorphisms.

Low doses of ultraviolet B (UVB) can induce localized immunosuppression in skin. This effect may be important in the induction of skin cancers and is thought to be mediated by tumour necrosis factor (TNF) alpha and interleukin (IL) 10 in conjunction with other factors. In humans a transition polymorphism in the TNF-alpha gene may affect TNF-alpha secretion and the promoter region of the IL-10 gene contains a CA repeat polymorphism which may affect gene function. We have therefore investigated the association of these polymorphisms with UVB-induced immunosuppression in humans. Volunteers (n = 42) were irradiated with UVB then sensitized on irradiated skin with diphenylcyclopropanone (DPCP) and subsequently antigen challenged with DPCP. DNA was extracted from blood samples and volunteers genotyped for the TNF-alpha polymorphism by polymerase chain reaction (PCR) and restriction digestion. The CA repeat polymorphism was amplified by PCR and sized by gel electrophoresis. Twenty-four volunteers were susceptible to UVB-induced immunosuppression and 18 were resistant. The association of allele frequencies and phenotype was statistically tested using a chi2-test. For both the TNF-alpha and IL-10 polymorphisms, there was no statistically significant association between allele types and response to UVB. These results indicate that variation in the immune response to UVB in humans is not associated with the TNF-alpha-308 transition or IL-10 CA repeat polymorphisms, although other as yet undetected DNA sequence variants of these genes may be involved.

Alleles↗
PubMed1998

Accumulation of p53 and Ki-67 expression do not predict survival in patients with fibrillary astrocytomas or the response of these tumors to radiotherapy.

OBJECTIVE: Although radiotherapy is often used in the treatment of patients with low-grade astrocytomas, its value is still uncertain. Radiotherapy carries a risk of morbidity for patients and has time and cost implications for health services. We have assessed the value of two histological variables, p53 accumulation and Ki-67 expression, in predicting the response of astrocytomas to radiotherapy. The former antigen was assessed because many astrocytic tumors show mutations in the p53 gene, the function of which is crucial for mediating cell death after radiotherapy, and the latter was assessed because it is expressed only in proliferating tumor cells, which may show greater radiosensitivity than nonproliferating cells. METHODS: Immunohistochemistry was used to detect the accumulation of p53 and expression of Ki-67 in a retrospective series of 96 patients with supratentorial fibrillary astrocytomas, 58 of whom had received postoperative radiotherapy. The immunohistochemical data were correlated with survival after radiotherapy. RESULTS: There was no significant difference in survival between the patients who did and those who did not receive radiotherapy. The p53 and Ki-67 labeling indices did not correlate with survival in either the irradiated or the nonirradiated cohort, nor with overall survival in the series as a whole. CONCLUSION: Immunohistochemical assessment of p53 accumulation and Ki-67 expression does not help in predicting the survival of patients with supratentorial fibrillary astrocytomas or in predicting whether particular patients are likely to benefit from radiotherapy.

Adolescent↗
PubMed1997

beta2-adrenergic receptor desensitization, internalization, and phosphorylation in response to full and partial agonists.

Previous studies indicated that partial agonists cause less desensitization of the beta2-adrenergic receptor (betaAR) than full agonists; however, the molecular basis for this in intact cells has not been investigated. In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of betaAR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the betaAR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified betaAR behaved identically to the wild type receptor with regard to agonist Kd, coupling efficiency, and desensitization. The coupling efficiencies for betaAR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0-30 min) of agonist-induced desensitization of betaAR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine >/= fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the betaAR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, betaAR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11-13-fold increases in the level of betaAR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3-4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of betaAR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 +/- 0.82) was not appreciably different from ephedrine (5.0 +/- 0.96) and significantly less than that caused by albuterol (10.4 +/- 1.7). In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of betaARK phosphorylation of the betaAR.

Adenylyl Cyclases↗
PubMed1997

Examination of the effects of increasing Gs protein on beta2-adrenergic receptor, Gs, and adenylyl cyclase interactions.

We have examined the effect of increased Gs protein levels on the abilities of three different beta2-agonists to induce GTP shifts and stimulate adenylyl cyclase response in an effort to investigate the kinetic association between the beta2-adrenergic receptor Gs and adenylyl cyclase. Agonist competition binding analysis and adenylyl cyclase concentration-response assays revealed that increases in Gs protein resulted in proportional increases in the areas of the GTP shift and adenylyl cyclase activity. Changes in the magnitude of the GTP shift were evaluated with a novel and straightforward approach for analyzing the GTP shift data that allowed us to determine the proportion of high agonist affinity binding receptor population and the apparent dissociation constant between the agonist bound receptor and Gs, regardless of the Gs protein level or the type of beta2-agonist. Using this method, we concluded that increased Gs results in the accumulation of the receptor population displaying high affinity towards agonist (HRGs) by increasing the number of receptor-Gs complexes (to a receptor:Gs protein ratio of about 0.7 at maximal Gs expression) without affecting the affinity between hormone bound receptor and Gs. Using the Gs protein levels determined with our novel analysis, we ran simulations using the theoretical shuttle model equation that relates the EC50 to available Gs. Fitting the simulations to experimental data required a receptor to catalytic unit ratio of 0.45 and revealed at least two distinct stages for beta2-agonist-stimulated adenylyl cyclase activity, namely, the activation of Gs by the beta2-adrenergic receptor (a step whose rate is dependent on the type of agonist used to stimulate activity), and the activation of adenylyl cyclase by active Gs (a step whose rate is independent of the type of agonist).

Adenylyl Cyclases↗
PubMed1997

Evidence for the shuttle model for Gs alpha activation of adenylyl cyclase.

Knowledge of the nature of the interaction between the stimulatory G protein (Gs) and the adenylyl cyclase catalytic unit (C) is essential for interpreting the effects of Gs mutations and expression levels on cellular response to a wide variety of hormones, drugs, and neurotransmitters. It has been proposed that beta-adrenergic receptor activation of adenylyl cyclase occurs either by a two-step "shuttle" mechanism where the receptor activates Gs independently of cyclase followed by Gs alpha activation of cyclase independent of the receptor; or the receptor activates a "precoupled" Gs-C complex in a single step. Simulations of the two models revealed that the two forms of activation are distinguishable by the effect of Gs levels on epinephrine-stimulated EC50 values for cyclase activation; specifically, the shuttle model predicts an increased potency of epinephrine stimulation as levels of Gs alpha increase. To address this problem, S49 cyc- cells were stably transfected with the gene for Gs alpha(long) regulated by the MMTV LTR promoter, which allowed for an induction of Gs alpha(long) expression levels over a 40-fold range by incubation of the cells for various times with 5 microM dexamethasone. Expression of Gs alpha was strongly correlated to the appearance of GTP shifts in the competitive binding of epinephrine with [125I]iodocyanopindolol to the beta-adrenergic receptors and epinephrine-stimulated adenylyl cyclase activity. Most importantly, high expression of Gs alpha resulted in lower EC50 values for epinephrine and prostaglandin E1 stimulation of adenylyl cyclase activity. The decrease in EC50 did not occur as a result of a change in beta2-adrenergic receptor, Gi alpha, G betagamma, or adenylyl cyclase levels. These novel findings demonstrate that a change in the level of a protein downstream of a plasma membrane receptor can influence hormone potency. We explain these results by using kinetic arguments to suggest that some fraction of hormone-activated adenylyl cyclase occurs via a shuttle mechanism, and not a purely precoupled mechanism.

Adenylyl Cyclases↗
PubMed1997

Identification of a major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis.

Psoriasis is a common chronic inflammatory disorder of the skin. To further understand the pathogenesis of psoriasis we have chosen to investigate the molecular genetic basis of the disorder. We have used a two-stage approach to search the human genome for the location of genes conferring susceptibility to psoriasis, using a total of 106 affected sibling pairs identified from 68 independent families. As over a third of the extended kindreds included affected relatives besides siblings, in addition to an analysis of allele sharing between affected sibling pairs, a novel linkage strategy was applied that extracts full non-parametric information. Four principal regions of possible linkage were identified on chromosomes 2, 8, 20 (p <0.005) and markers from the MHC region at 6p21 (p <0.0000006) for which significant evidence of linkage disequilibrium was also observed (p <0.00002). Whilst data from limited case control associations exist to implicate the MHC, the results of this genome wide analysis demonstrate that, at least in the population studied, a gene or genes located within the MHC and close to the class 1 HLA loci, represent the major determinant of the genetic basis of psoriasis.

Chromosomes, Human, Pair 6↗
PubMed1997

Bridging the care continuum for open heart surgery patients.

A pilot program followed patients who were recovering from coronary artery bypass graft procedures. A cardiac surgery nurse visits patients in their homes to provide physical assessments, reinforce discharge teaching and answer questions. Because of the pilot's success, the program was integrated with the transitional open heart unit.

Aftercare↗
PubMed1997

Increased endothelial expression of transglutaminase in glioblastomas.

Transglutaminases are a family of calcium-dependent enzymes that catalyse the formation of covalent crosslinks between proteins. They have several diverse functions and are thought to be involved in cell differentiation, apoptosis and blood coagulation. We have investigated the expression of tissue transglutaminase in five fibrillary astrocytomas, five anaplastic astrocytomas and seven glioblastomas by immunohistochemistry. Strongly labelled tumour cells were seen in most of the fibrillary and anaplastic astrocytomas and all of the glioblastomas. Labelling was particularly prominent in the pseudopalisading tumour cells that surrounded foci of necrosis and apoptosis in glioblastomas. Most of the immunostained cells did not themselves show morphological features of apoptosis. In addition, apoptotic cells were demonstrated using in situ end-labelling and by in situ hybridization with digoxigenin-labelled poly(A) oligonucleotide probes. Apoptotic cells demonstrated by both of these methods were most numerous in anaplastic astrocytomas and glioblastomas. However, their distribution did not correlate with that of the tumour cells showing transglutaminase labelling. Strong transglutaminase labelling was also observed in the endothelial cells of vessels showing microvascular proliferation in all of the glioblastomas studied. In contrast, endothelial transglutaminase labelling was weak or absent in lower grade astrocytic tumours. Enhanced expression of transglutaminase by endothelial cells in glioblastomas may contribute to the high prevalence of vascular thrombosis and necrosis in these tumours.

Adult↗
PubMed1997

The stability of the agonist beta2-adrenergic receptor-Gs complex: evidence for agonist-specific states.

A restricted version of the ternary complex model for receptor-G protein complex formation has recently been proposed. Known as the two-state model, this model proposes that in the context of agonist and G protein interactions, only two thermodynamic states exist for the receptor: active (R*) and inactive (R). One form of this model suggests that only the R* state of the receptor is capable of interacting with and subsequently activating G proteins. We directly tested the kinetic aspects of a strict two-state receptor model in a cell line containing the native beta2-adrenergic receptor that is capable of inducing Gs expression. We examined adenylyl cyclase activity in the presence of limiting GTP levels and concluded that there exists a different rate of heterotrimer dissociation (i.e., HR*G yields HR* + G*) for different beta2-agonists. This finding is inconsistent with a strict two-state model in which R* is a characteristic of the receptor that is independent of the identity of the agonist. It implies that agonist activation of adenylyl cyclase is more complicated than a simple two-state model.

Adenylyl Cyclases↗
PubMed1997

Demonstration of apoptotic cells in tissue sections by in situ hybridization using digoxigenin-labeled poly(A) oligonucleotide probes to detect thymidine-rich DNA sequences.

The recognition of apoptotic cells by morphological appearance alone may be difficult. We have investigated the use of in situ hybridization (ISH) with digoxigenin-labeled poly(A) probes to detect apoptotic cells in tissue sections. This method was compared to conventional morphologic assessment and in situ end-labelling (ISEL) in a range of tissues in which apoptosis is known to occur. ISH with poly(A) probes detected apoptotic nuclei in all tissues in which there was evidence of apoptosis as judged by conventional histology. ISH and, to a lesser extent, ISEL preferentially labeled shrunken but still intact nuclei with margination of chromatin, presumably at an early stage of apoptosis. The poly(A) hybridization was abolished by pretreatment of tissue sections with DNAse. After denaturation of DNA, poly(A) hybridized to nuclei in all cells. No convincing hybridization signal was detected in alcohol-fixed or fresh-frozen sections. Both ISEL and ISH labeled some of the nuclei in ischemic tissues. ISH with poly(A) oligonucleotide probes offers a simple alternative to ISEL for detection of cells in early stages of apoptosis. These probes hybridize to thymidine-rich sequences of DNA in the highly repeated Alu sequences within the nuclear genome. These sequences are believed to become available for hybridization after formalin fixation and paraffin embedding as a result of the apoptosis-related increase in the susceptibility of nuclear DNA to denaturation.

Apoptosis↗
PubMed1997

A survey of services for younger people with dementia.

OBJECTIVE: To identify the provision of services for younger people with dementia by trusts in England and to examine their attitudes towards specialization. DESIGN: Postal survey. PARTICIPANTS: Hospital and community trusts in England. Of 354 trusts, 304 responded (84%). MAIN OUTCOME MEASURES: Provision of general adult and old age psychiatry services: type of services provided for younger people with dementia and specialists providing these services; provision of specialists services for younger people with dementia; attitudes towards developing specialists services; assessment of need. RESULTS: The majority of services were provided by trusts with existing psychiatric services, in particular old age psychiatry services. Specialization was uncommon and practised by only 12 trusts. In contrast, 101 trusts saw the development of specialists services as 'necessary', especially those trusts who had investigated the needs of younger patients with dementia (p = 0.001). CONCLUSIONS: This survey supports the notion that trusts and purchasers should undertake to assess the needs of younger patients with dementia. Role of specialization is discussed.

Adult↗
PubMed1996

Collisions and encounters in simulations of receptor/GTP-binding protein interactions via simple diffusion.

In two intact cell systems in which GTP-binding protein (G) activity is initiated by the presence of agonist-bound receptors (R), it has been demonstrated that the rate of G activation is influenced by the rate of turnover of agonist occupancy among the receptor population. G activity is reduced when a low concentration of agonist-occupied receptors comprised by low fractional occupancy of a large receptor population is replaced by the presence of the same concentration of 100%-occupied receptors. This effect has been proposed to be due to a time interval of interaction between R and G (an encounter) that is long compared to the time of a single collection between R and G and long compared to the lifetime of an agonist-receptor complex. In a recent simulation study of R-G interaction via diffusion, the effect of agonist occupancy turnover was observed but it was assumed that long encounters were not operative. In this study, encounter intervals in simulations of R-G interaction by simple diffusion were measured in order to address that difference. The results demonstrate that relatively long encounters comprised of multiple, separate collisions are an inherent part of R-G interaction as modelled by diffusion. The implications for further implementation of simulation studies of R-G interaction are discussed.

Diffusion↗
PubMed1996

Psychiatrists working in primary care: a survey of general practitioners' attitudes.

OBJECTIVES: To collect information on current working arrangements between general practitioners (GPs) and mental health professionals and to assess GPs' attitudes towards developing closer working practices with psychiatrists in the primary care setting. METHOD: Six hundred and three GPs from South Australia were surveyed with questionnaires. Main outcome measures included information about existing primary care links between GPs and mental health professionals, GPs' preferred working arrangements with psychiatrists in the primary care setting and their attitude towards developing these practices, including perceived obstacles, advantages and disadvantages. RESULTS: One hundred and eighty-one completed questionnaires were returned. One in 11 GPs returning the questionnaire (RGPs) had established primary care links with a psychiatrist, 1 in 6 with clinical psychologists and 1 in 17 with psychiatric nurses and social workers. RGPs held positive attitudes towards developing closer links at their work settings with psychiatrists when it le8ads to improved collaboration and access to psychiatrists. Reservations were expressed about the public weakening of the GPs' primary care role. CONCLUSIONS: The joint needs of clinical care and GPs' further training in psychiatry could be addressed by further development of schemes to attract psychiatrists to work in primary care settings. This is mostly viewed very positively by GPs, although the percentage of GPs responding make these conclusions tentative. It is more likely to occur with changes to current funding of both private psychiatric care and GP remuneration, with a recognition of time spent in liaison.

Adult↗