Can the gap in Aboriginal outcomes be explained by DOHaD.

In Australia, there are two distinct populations, each with vastly disparate health outcomes: Aboriginal and Torres Strait Islander People and non-Aboriginal Australians. Aboriginal Australians have significantly higher rates of health and socioeconomic disadvantage, and Aboriginal babies are also more likely to be born low birth weight or growth restricted. The Developmental Origins of Health and Disease (DOHaD) hypothesis advocates that a sub-optimal intrauterine environment, often manifested as diminished foetal growth, during critical periods of foetal development has the potential to alter the risk of non-communicable disease in the offspring. A better understanding of the role of the intrauterine environment and subsequent developmental programming, in response to both transgenerational and immediate stimuli, in Aboriginal Australians remains a relatively unexplored field and may provide insights into the prevailing health disparities between Aboriginal and non-Aboriginal children. This narrative review explores the role of DOHaD in explaining the ongoing disadvantage experienced by Aboriginal People in today's society through a detailed discussion of the literature on the association between foetal growth, as a proxy for the quality of the intrauterine environment, and outcomes in the offspring including perinatal health, early life development and childhood education. The literature largely supports this hypothesis and this review therefore has potential implications for policy makers not only in Australia but also in other countries that have minority and Indigenous populations who suffer disproportionate disadvantage such as the United States, Canada and New Zealand.

Translational control is required for the unfolded protein response and in vivo glucose homeostasis.

The accumulation of unfolded protein in the endoplasmic reticulum (ER) attenuates protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) at Ser51. Subsequently, transcription of genes encoding adaptive functions including the glucose-regulated proteins is induced. We show that eIF2alpha phosphorylation is required for translation attenuation, transcriptional induction, and survival in response to ER stress. Mice with a homozygous mutation at the eIF2alpha phosphorylation site (Ser51Ala) died within 18 hr after birth due to hypoglycemia associated with defective gluconeogenesis. In addition, homozygous mutant embryos and neonates displayed a deficiency in pancreatic beta cells. The results demonstrate that regulation of translation through eIF2alpha phosphorylation is essential for the ER stress response and in vivo glucose homeostasis.

Inhibition of beta(2)-adrenergic and muscarinic cholinergic receptor endocytosis after depletion of phosphatidylinositol bisphosphate.

Recent evidence supporting a role for phosphoinositides in the endocytosis of phospholipase C-coupled receptors has prompted an investigation of whether there exists a similar requirement for the internalization of adenylyl cyclase-linked receptors. When 1321N1 astrocytoma cells, which possess both muscarinic cholinergic receptors (mAChRs) that couple to phospholipase C and beta-adrenergic receptors (beta(2)-ARs) linked to adenylyl cyclase, were pretreated with wortmannin (WT) at a concentration known to inhibit phosphatidylinositol 4-kinase activity, the labeling of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)) was reduced. Stimulation of phosphoinositide breakdown by activation of mAChRs in WT-pretreated cells led to a further depletion of PIP(2). As previously demonstrated for SH-SY5Y neuroblastoma, inclusion of WT inhibited the endocytosis of mAChRs in 1321N1 cells by >85%. In contrast, the internalization of beta(2)-ARs was only partially ( approximately 30%) prevented. However, when the concentration of PIP(2) was further reduced by exposure of WT-pretreated 1321N1 cells to a muscarinic agonist, the endocytosis of beta(2)-ARs was substantially inhibited (>70%). Lower concentrations of WT (100 nM) that were sufficient to fully inhibit phosphatidylinositol 3-kinase activity had no effect on either phosphoinositide synthesis or receptor endocytosis. The results indicate that the agonist-induced endocytosis of an adenylyl cyclase-linked receptor such as the beta(2)-AR, like that of the phospholipase C-coupled mAChR, is dependent on the synthesis of phosphoinositides and, in particular, that of PIP(2).

A role for a wortmannin-sensitive phosphatidylinositol-4-kinase in the endocytosis of muscarinic cholinergic receptors.

A role for phosphoinositides in the endocytosis of muscarinic cholinergic receptors (mAChRs) has been investigated via inhibition of the activity of phosphatidylinositol-4-kinase (PI4K). Pretreatment of SH-SY5Y neuroblastoma cells with micromolar concentrations of wortmannin (WT), LY-294002, or phenylarsine oxide (PAO), three chemically distinct agents known to inhibit PI4K, resulted in both an inhibition of agonist-induced endocytosis of mAChRs and a selective reduction in the 32P-labeling of phosphatidylinositol-4-phosphate. PAO-mediated inhibition of both receptor endocytosis and phosphoinositide synthesis could be fully reversed by inclusion of the bifunctional thiol 2, 3-dimercaptopropanol. The requirement for phosphoinositide synthesis in mAChR endocytosis was independent of a role for these lipids in the maintenance of the cytoskeleton because disruption of the latter with cytochalasin D, ML-7, or colchicine failed to inhibit receptor internalization. Determination of PI4K activity in subcellular fractions of SH-SY5Y cells indicated that enzyme activity in fractions enriched in endocytic vesicles and cytosol was preferentially inhibited by WT, LY-294002, and PAO, a profile consistent with the subcellular distribution of the 110-kDa beta isoform of PI4K, as determined by Western blot analysis. Activity of PI4Kbeta present in immunoprecipitated cell lysates was inhibited >75% by inclusion of each of the three inhibitors. These results indicate that ongoing synthesis of phosphoinositides is necessary for mAChR endocytosis and that the activity of a WT-sensitive form of PI4K, such as PI4Kbeta, is required.

Cytoskeletal and phosphoinositide requirements for muscarinic receptor signaling to focal adhesion kinase and paxillin.

The mechanism whereby agonist occupancy of muscarinic cholinergic receptors elicits an increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin has been examined. Addition of oxotremorine-M to SH-SY5Y neuroblastoma cells resulted in rapid increases in the phosphorylation of FAK (t(1/2) = 2 min) and paxillin that were independent of integrin-extracellular matrix interactions, cell attachment, and the production of phosphoinositide-derived second messengers. In contrast, the increased tyrosine phosphorylations of FAK and paxillin were inhibited by inclusion of either cytochalasin D or mevastatin, agents that disrupt the cytoskeleton. Furthermore, phosphorylation of FAK and paxillin could be prevented by addition of either wortmannin or LY-294002, under conditions in which the synthesis of phosphatidylinositol 4-phosphate was markedly attenuated. These results indicate that muscarinic receptor-mediated increases in the tyrosine phosphorylation of FAK and paxillin in SH-SY5Y neuroblastoma cells depend on both the maintenance of an actin cytoskeleton and the ability of these cells to synthesize phosphoinositides.

Rural attachments for students in the health professions: are they worthwhile?

In the period 1991-96, 156 undergraduates from 14 health disciplines at the University of Sydney completed rural attachments in rural and remote areas of Australia as part of the Rural Careers Project. On return from their attachment, students were encouraged to write a brief report of their experiences. Ninety-two available reports were analysed as one means of assessing the success of the attachments with respect to informing students about rural health issues and stimulating their interest in rural careers after graduation. A content analysis of the students' written comments about their perceptions and experiences was completed. Students were extremely positive about the value of the attachments and expressed more positive than negative comments regarding their perceptions of rural life and work. The results show that rural attachments are indeed worthwhile learning opportunities.

Agonist-induced endocytosis of muscarinic cholinergic receptors: relationship to stimulated phosphoinositide turnover.

The ability of muscarinic cholinergic receptors to activate phosphoinositide turnover following agonist-induced internalization has been investigated. Incubation of SH-SY5Y neuroblastoma cells with oxotremorine-M resulted in a time-dependent endocytosis of both muscarinic receptors and alpha subunits of G0 and G11, but not of isoforms of phosphoinositide-specific phospholipase C, into a subfraction of smooth endoplasmic reticulum (V1). Agonist-induced increases in diacylglycerol mass and in 32P-phosphatidate labeling, much of which was of the tetraenoic species, were also observed in the V1 fraction, but these increases persisted when the agonist-induced translocation of receptors into the V1 fraction was blocked. All enzymes of the phosphoinositide cycle were detectable in the V1 fraction. However, with the exception of phosphatidylinositol 4-kinase, none was enriched when compared with cell lysates. Both 32P-labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinositol 4,5-bisphosphate, whereas the synthesis of phosphatidylinositol 4-phosphate is robust. These results indicate that endocytosed receptors do not appear to retain their ability to activate phosphoinositide turnover. The availability of the substrate for phospholipase C, phosphatidylinositol 4,5-bisphosphate, may be one factor that limits the activity of muscarinic receptors in this subcellular compartment.

Intra- and intercellular calcium signaling in human neuroepithelioma cells.

The characteristics of intra- and intercellular Ca2+ signaling in human SK-N-MCIXC neuroepithelioma cells have been examined by means of Fura-2 digital imaging microfluorimetry. When cells were exposed to maximally effective concentrations of either endothelin-1, ATP, norepinephrine or oxotremorine-M, the Ca2+ signals that accompany an increase in phosphoinositide turnover could be differentiated on the basis of their magnitude, shape and duration. When individual cells were microinjected with inositol 1,4,5-trisphosphate, a rise in [Ca2+]i was observed not only in the target cell, but also in neighboring cells. This intercellular propagation of Ca2+ signals was found to be mediated via the release of nucleotide di- and triphosphates which subsequently activate purinergic receptors linked to Ca2+ homeostasis on neighboring cells. These results indicate: (1) that agonist-specific Ca2+ 'signatures' are generated in SK-N-MCIXC cells; and (2) that an intercellular propagation of Ca2+ signals is triggered by a rise in [Ca2+]i.

Permeability of the blood brain barrier by the bradykinin agonist, RMP-7: evidence for a sensitive, auto-regulated, receptor-mediated system.

The novel bradykinin (BK) analog, RMP-7, was characterized in a series of in vitro tests to establish its selectivity as a B2 agonist. It was then used to study bradykinin's role in permeabilizing the blood brain barrier (BBB) and blood brain-tumor barrier (BTB), using an RG2 rat glioma model. These studies demonstrated that: (1) B2 receptor stimulation permeabilizes both the BBB and BTB in a dose-related fashion with greater effects observed in brain tumor-associated tissue, (2) the increased permeability is sensitive, rapid and transient, and (3) tachyphylaxis occurs with continuous agonist administration, suggesting autoregulation of the system's effects. These data therefore support the existence of a sophisticated, responsive and tightly regulated BK system whose activity modulates the permeability of the BBB.

The significance and trend of hypertension related deaths in urban Sierra Leonean Africans.

In order to determine the impact and trend of hypertension related deaths in the overall mortality of urban Sierra Leoneans, a review of death certificate records in the capital Freetown over the period 1983-1992 was undertaken. A total of 25119 consecutive records were examined to identify those with hypertension as a major or contributory cause of death. For the purpose of this study, hypertensive stroke, cardiac and renal deaths were selected as the main hypertension related disorders. Hypertension related deaths accounted for an average of 7.5% of all deaths and 13.7% of deaths in those aged 40 years and above between 1983 and 1992. For the latter group, stroke deaths accounted for 5.2%, cardiac deaths for 4.7%, and renal deaths for 0.8% of total mortality. There were more male hypertensive stroke deaths but when expressed as a percentage of the total deaths, no sex difference was noted. Hypertensive stroke deaths accounted for 7% of all deaths in the age group 50-69 years in females, while hypertensive cardiac deaths, caused 6% of deaths in the 60-69 year age group in both sexes. Hypertensive renal death was infrequently recorded being present mainly in the 40-49 year age group predominantly in males. When all hypertension related deaths were considered together, their major impact was in the seventh decade in females and in the eighth decade in males accounting for approximately 20% and 16% of all deaths respectively. Hypertension related deaths appeared to show a steady increase over the 10 year study period. Hypertension is a significant cause of death particularly in elderly urban Sierra Leoneans in Freetown and deaths from hypertension may be increasing.

Muscarinic receptor sequestration in SH-SY5Y neuroblastoma cells is inhibited when clathrin distribution is perturbed.

The possibility that clathrin plays a role in the agonist-mediated sequestration of muscarinic cholinergic receptors in human SH-SY5Y neuroblastoma cells has been investigated by the application of experimental paradigms previously established to perturb clathrin distribution and receptor cycling events. Preincubation of SH-SY5Y cells under hypertonic conditions resulted in a pronounced inhibition of agonist-induced muscarinic receptor sequestration (70-80% at 550 mOsm), which was reversed when cells were returned to isotonic medium. Depletion of intracellular K+ or acidification of the cytosol also resulted in > 80% inhibition of muscarinic receptor sequestration. Under conditions of hypertonicity, depletion of intracellular K+, or acidification of cytosol, muscarinic receptor-stimulated phosphoinositide hydrolysis and Ca2+ signaling events were either unaffected or markedly less inhibited than receptor sequestration. That these same experimental conditions did perturb clathrin distribution was verified by immunofluorescence studies. Hypertonicity and depletion of intracellular K+ resulted in a pronounced accumulation of clathrin in the perinuclear region, whereas acidification of the cytosol resulted in the appearance of microaggregates of clathrin throughout the cytoplasm and at the plasma membrane. The results are consistent with the possibility that muscarinic receptors in SH-SY5Y cells are endocytosed via a clathrin-dependent mechanism.

Agonist-specific calcium signaling and phosphoinositide hydrolysis in human SK-N-MCIXC neuroepithelioma cells.

Fura-2 digital imaging microfluorimetry was used to evaluate the Ca2+ signals generated in single clonal human neuroepithelioma cells (SK-N-MCIXC) in response to agonists that stimulate phosphoinositide hydrolysis. Addition of optimal concentrations of either endothelin-1 (ET-1), ATP, oxotremorine-M (Oxo-M), or norepinephrine (NE) all resulted in an increase in the concentration of cytosolic calcium (Ca2+i) but of different magnitudes (ET-1 = ATP > Oxo-M > NE). The Ca2+ signals elicited by the individual agonists also differed from each other in terms of their latency of onset, rate of rise and decay, and prevalence of a sustained phase of Ca2+ influx. The Ca2+ signals that occurred in response to ATP had a shorter latency and more rapid rates of rise and decay than those observed for the other three agonists. Furthermore, a sustained plateau phase of the Ca2+ signal, which was characteristic of the response to Oxo-M, was observed in < 40% of cells stimulated with ET-1 and absent from Ca2+ signals elicited after NE addition. Removal of extracellular Ca2+ enhanced the rate of decay of Ca2+ signals generated by ATP, ET-1, or Oxo-M and, when evident, abolished the sustained phase of Ca2+ influx. In the absence of extracellular Ca2+, NE elicited asynchronous multiple Ca2+ transients. In either the absence or presence of extracellular Ca2+-, > 94% of cells responded to ET-1 or ATP, whereas corresponding values for Oxo-M and NE were approximately 74 and approximately 48%.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid attenuation of muscarinic agonist stimulated phosphoinositide hydrolysis precedes receptor sequestration in human SH-SY-5Y neuroblastoma cells.

Agonist occupancy of muscarinic cholinergic receptors in human SH-SY-5Y neuroblastoma cells elicited two kinetically distinct phases of phosphoinositide hydrolysis when monitored by either an increased mass of inositol 1,4,5-trisphosphate, or the accumulation of a total inositol phosphate fraction. Within 5s of the addition of the muscarinic agonist, oxotremorine-M, the phosphoinositide pool was hydrolyzed at a maximal rate of 9.5%/min. This initial phase of phosphoinositide hydrolysis was short-lived (t1/2 = 14s) and after 60s of agonist exposure, the rate of inositol lipid breakdown had declined to a steady state level of 3.4%/min which was then maintained for at least 5-10 min. This rapid, but partial, attenuation of muscarinic receptor stimulated phosphoinositide hydrolysis occurred prior to the agonist-induced internalization of muscarinic receptors.

Contribution of G protein activation to fluoride stimulation of phosphoinositide hydrolysis in human neuroblastoma cells.

To examine the possibility that NaF enhances phosphoinositide-specific phospholipase C (PIC) activity in neural tissues by a mechanism independent of a guanine nucleotide binding protein (Gp), we have evaluated the contribution of Gp activation to NaF-stimulated phosphoinositide hydrolysis in human SK-N-SH neuroblastoma cells. Addition of NaF to intact cells resulted in an increase in the release of inositol phosphates (450% of control values; EC50 of approximately 8 mM). Inclusion of U-73122, an aminosteroid inhibitor of guanine nucleotide-regulated PIC activity in these cells, resulted in a dose-dependent inhibition of NaF-stimulated inositol lipid hydrolysis (IC50 of approximately 3.5 microM). When added to digitonin-permeabilized cells, NaF or guanosine-5'-O-thiotriphosphate (GTP gamma S) resulted in a three- and sevenfold enhancement, respectively, of inositol phosphate release. In the combined presence of optimal concentrations of NaF and GTP gamma S, inositol phosphate release was less than additive, indicative of a common site of action. Inclusion of 2-5 mM concentrations of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) fully blocked phosphoinositide hydrolysis elicited by GTP gamma S, whereas that induced by NaF was partially inhibited (65%). However, preincubation of the cells with GDP beta S resulted in a greater reduction in the ability of NaF to stimulate inositol phosphate release (87% inhibition). Both GTP gamma S and NaF-stimulated inositol phosphate release were inhibited by inclusion of 10 microM U-73122 (54-71%). The presence of either NaF or GTP gamma S also resulted in a marked lowering of the Ca2+ requirement for activation of PIC in permeabilized cells. These results indicate that in SK-N-SH cells, little evidence exists for direct stimulation of PIC by NaF and that the majority of inositol phosphate release that occurs in the presence of NaF can be attributed to activation of Gp.

Cyclic AMP potentiates receptor-stimulated phosphoinositide hydrolysis in human neuroepithelioma cells.

A stimulatory role for cAMP in the regulation of receptor-activated phosphoinositide hydrolysis has been examined in human SK-N-MCIXC and SK-N-MCIIE neuroepithelioma cells. The addition of optimal concentrations of oxotremorine-M, norepinephrine, endothelin-1, and ATP enhanced the release of inositol phosphates by 2-9-fold after activation of muscarinic, alpha 1-adrenergic, endothelin, and P2 nucleotide receptors, respectively. All combinations of these agonists elicited a release of inositol phosphates that was at least additive. However, the combined presence of oxotremorine-M and norepinephrine resulted in a phosphoinositide hydrolysis that was 30% greater than additive. This potentiation of inositol lipid hydrolysis resulted from an increased activity of the muscarinic receptor after the addition or norepinephrine and persisted after alpha 1-adrenergic receptor blockade. The enhancement of muscarinic receptor-stimulated inositol phosphate release could be quantitatively mimicked by inclusion of the beta-adrenergic agonist isoproterenol (EC50 approximately 0.1 microM), but not by alpha 1- or alpha 2-adrenergic agonists. Potentiation of oxotremorine-M-stimulated inositol lipid hydrolysis observed in the presence of either norepinephrine or isoproterenol was reduced in the absence of added Ca2+. Addition of either norepinephrine or isoproterenol to SK-N-MCIXC cells also resulted in a 16-fold increase in cAMP concentration. Although the cell-permeant 8-chloro-4-phenylthio-cAMP had a small inhibitory effect on basal inositol phosphate release, its inclusion resulted in a 19-31% enhancement of muscarinic, endothelin, ATP, and alpha 1-adrenergic receptor-stimulated phosphoinositide hydrolysis. We conclude 1) that, in SK-N-MCIXC cells, the addition of beta-adrenergic agonists selectively enhances muscarinic receptor-stimulated phosphoinositide hydrolysis through a cAMP-dependent process and 2) that the ability of exogenously added cAMP to enhance the activation of all four inositol lipid-linked receptors indicates that the effects of cAMP on inositol lipid hydrolysis are compartmentalized in these cells.

Purification of PKC-I, an endogenous protein kinase C inhibitor, and types II and III protein kinase C isoenzymes from human neutrophils.

Human neutrophil protein kinase C (PKC) activity is inhibited by an endogenous protein found primarily in the pellet fraction from homogenized specific granules, which was both heat- and proteinase-sensitive [Balazovich, Smolen & Boxer (1986) J. Immunol. 137, 1665-1673]. We now report that two PKC isoenzymes and the endogenous PKC inhibitor, which we named PKC-I, were purified from human neutrophils. A neutrophil soluble fraction that was subjected to DEAE-Sephacel chromatography yielded highly enriched PKC because, by definition, enzymic activity was strictly dependent on Ca2+ and phosphatidylserine. Hydroxyapatite chromatography resolved two peaks of PKC activity. Type II and Type III PKC isoenzymes were each identified on Western blots by using isoenzyme-specific monoclonal antibodies. Unlike rat brain, from which PKC isoenzymes were also purified, Type I PKC was not detected in human neutrophils. Western blots indicated that both Type II and Type III PKC isoenzymes had molecular masses near 80 kDa. In agreement with other reports, PKC was autophosphorylated in vitro. PKC-I, an endogenous neutrophil inhibitor of PKC, was purified to apparent homogeneity by DEAE-Sephacel and S-400 Sephacel chromatography. PKC-I had a molecular mass of 41 kDa. PKC-I inhibited purified PKC activity stimulated by 1,2-diacylglycerols in a concentration-dependent manner, and inhibited PKC-dependent phosphorylation of proteins present in neutrophil cytosol.

Taking time out: a study of women dentists who have experienced career breaks.

Data on the careers and work patterns of women dentists were established in 1975. Social and economic changes, as well as change within the profession, such as the fact that almost half the students entering dental schools are now female, pointed to the necessity of a further survey in 1985. Questionnaires were sent off to 4513 women dentists resident in England and Wales, with a resultant response of 76 per cent. Part of the questionnaire concentrated on the length of career breaks experienced by/taken by women dentists in order to determine whether there has been any change in the pattern of taking time out in dentistry over the decade. At the time of the survey, 388 women (12 per cent) were retired, in addition, just over half of the women who replied had experienced a break at some stage during their career. These breaks from practice are described and discussed.