Mechanism of activation of photoreceptor phosphodiesterase by transducin and by low molecular weight proteins

Rod photoreceptor phosphodiesterase (PDE) is the central enzyme of visual transduction. PDE is a heterodimer (Palphabeta) with two associated inhibitory gamma subunits, and is bound to the disk membrane by isoprenyl groups. Each catalytic subunit contains a catalytic site and a high-affinity cGMP binding site. In this research, two independent mechanisms of PDE regulation are addressed. The first part examines transducin activation of PDE, focusing on the interactions between the gamma subunits and Palphabeta. The second part examines the 17 kDa delta protein believed to regulate bovine PDE. We found that transducin activation of PDE induces heterogeneity at the active sites as well as the cGMP binding sites, and that there is a strong correlation between cGMP binding and gamma binding to PDE. The PDE activation induced by transducin is approximately half of the maximum rate of Palphabeta lacking bound gamma inhibitory subunits. This research supports a model in which transducin interacts with gamma bound to only one of the catalytic subunits. The relationship between cGMP and gamma binding to PDE may play a role in light adaptation of rod photoreceptor cells. The delta protein is hypothesized to regulate adaptation of rod photoreceptor cells by delta binding to the prenyl groups on PDE and releasing PDE from the disk membrane and thereby preventing activation by transducin. The frog homologue of the bovine 17 kDa delta protein was identified in frog retina, but the amount of the delta in rod cells was low (0.03 +/- 0.01 mol delta per mol PDE). This is consistent with frog PDE being \u3e95% membrane-associated. Recombinant frog delta bound to PDE and was able to release PDE from the membrane. The majority of the delta in frog ROS is soluble, and the remainder does not co-purify with PDE when extracted from the membranes. Immunocytochemical evidence indicates that delta localizes to the cone cells. We hypothesize that delta may serve to direct the intracellular membrane trafficking for various prenylated proteins

SA cardiovascular laboratories mining diverse, yet relevant research topics

This issue of SA Heart reports on some ongoing work performed in South Africa that engages with more fundamental issues of the mechanisms of cardiovascular disease. This special edition of the journal highlights some of the high quality, internationally competitive, clinically relevant, basic cardiovascular science presently being undertaken by a number of groups around the country in this regard. Although the topics covered in the present issue are diverse, all have an important clinical context and in this editorial we will attempt to describe this context

Evaluation of the 17 kda prenyl binding protein as a regulatory protein for phototransduction in retinal photoreceptors

Journal ArticleThe mammalian rod photoreceptor phosphodiesterase (PDE6) holoenzyme is isolated in both a membrane-associated and a soluble form. Membrane binding is a consequence of prenylation of PDE6 catalytic subunits, whereas soluble PDE6 is purified with a 17-kDa prenyl-binding protein (PDEdelta) tightly bound. This protein, here termed PrBP/delta, has been hypothesized to reduce activation of PDE6 by transducin, thereby desensitizing the photoresponse. To test the potential role of PrBP/delta in regulating phototransduction, we examined the abundance, localization, and potential binding partners of PrBP/delta in retina and in purified rod outer segment (ROS) suspensions whose physiological and biochemical properties are well characterized. The amphibian homologue of PrBP/delta was cloned and sequenced and found to have 82% amino acid sequence identity with mammalian PrBP/delta. In contrast to bovine ROS, all of the PDE6 in purified frog ROS is membrane-associated. However, addition of recombinant frog PrBP/delta can solubilize PDE6 and prevent its activation by transducin. PrBP/delta also binds other prenylated photoreceptor proteins in vitro, including opsin kinase (GRK1/GRK7) and rab8. Quantitative immunoblot analysis of the PrBP/delta content of purified ROS reveals insufficient amounts of PrBP/delta (<0.1 PrBP/delta per PDE6) to serve as a subunit of PDE6 in either mammalian or amphibian photoreceptors. The immunolocalization of PrBP/delta in frog and bovine retina shows greatest PrBP/delta immunolabeling outside the photoreceptor cell layer. Within photoreceptors, only the inner segments of frog double cones are strongly labeled, whereas bovine photoreceptors reveal more PrBP/delta labeling near the junction of the inner and outer segments (connecting cilium) of photoreceptors. Together, these results rule out PrBP/delta as a PDE6 subunit and implicate PrBP/delta in the transport and membrane targeting of prenylated proteins (including PDE6) from their site of synthesis in the inner segment to their final destination in the outer segment of rods and cones

Hypertension in Africa: Redressing the burden of cardiovascular disease using cost-effective nonpharmacological approaches

Hypertension may affect approximately one fifth or more of all adult South Africans. Despite the considerable evidence derived from economically developed countries to indicate the extent to which hypertension contributes to cardiovascular disease (CVD), it is only more recently that data has emerged from the African continent to support a contention that hypertension is the principal risk factor for CVD in African populations and that CVD accounts for a major proportion of deaths in the elderly and in younger adults in rural Africa. Active engagement in the harsh realities of managing this complex clinical trait should therefore be foremost on the minds of the healthcare sector in Africa. In this regard there are unique challenges. In the present personal review we synthesise the evidence for or against the view that at a public health level, the answer to significantly reducing the burden of CVD produced by hypertension in African populations, may lie in something as simple as generating a healthier lifestyle. In this regard, we place recent evidence obtained from South African studies of the importance of modifiable cardiovascular risk factors related to hypertension, including salt intake and obesity, in the context of previously published evidence. We highlight the very recent and the first substantive evidence derived from an African community to show that salt intake indeed contributes to a significant portion of blood pressure (BP) variability in African populations, but this effect may be hidden because the impact is largely on central (aortic) rather than brachial BP. We also discuss the increasing evidence to show that in African populations, the adverse effects of the epidemic of obesity that faces emerging communities is likely to account for a substantial proportion of cardiovascular risk not through marked effects on brachial BP, but through indirect effects by promoting the adverse effects of BP on the heart. In the present review we therefore argue that despite limited absolute effects of salt intake and obesity on brachial BP, a marked benefit could be gained by the BP effects of salt restriction and body weight reduction in African communities

Rheumatoid arthritis is associated with reduced adiposity but not with unfavorable major cardiovascular risk factor profiles and enhanced carotid atherosclerosis in black Africans from a developing population: a cross-sectional study

INTRODUCTION: Rheumatoid arthritis (RA) is characterized by inflamed joint-derived cytokine-mediated high-grade systemic inflammation that enhances cardiovascular metabolic risk and disease in developed populations. We investigated the potential impact of RA on cardiovascular risk factors including systemic inflammation and atherosclerosis, and their relationships in black Africans from a developing population. METHODS: We evaluated demographic features, adiposity indices, major traditional cardiovascular risk factors, circulating C-reactive protein and interleukin-6 concentrations and ultrasound determined carotid intima-media thickness (cIMT) in 274 black Africans; 115 had established RA. Data were analyzed in confounder-adjusted mixed regression models. RESULTS: The body mass index and waist-height ratio were lower in RA compared to non-RA subjects (29.2 (6.6) versus 33.7 (8.0), P < 0.0001 and 0.58 (0.09) versus 0.62 (0.1), P = 0.0003, respectively). Dyslipidemia was less prevalent in patients with RA (odds ratio (OR) (95% confidence interval (CI) = 0.54 (0.30 to1.00)); this disparity was no longer significant after further adjustment for reduced adiposity and chloroquine use. RA was also not associated with hypertension, current smoking and diabetes. The number of major traditional risk factors did not differ by RA status (1.1 (0.8) versus 1.2 (0.9), P = 0.7). Circulating C-reactive protein concentrations were similar and serum interleukin-6 concentrations reduced in RA (7.2 (3.1) versus 6.7 (3.1) mg/l, P = 0.7 and 3.9 (1.9) versus 6.3 (1.9) pg/ml, P < 0.0001, respectively). The cIMT was 0.700 (0.085) and 0.701 (0.111) mm in RA and non-RA subjects, respectively (P = 0.7). RA disease activity and severity parameters were consistently unrelated to systemic inflammation, despite the presence of clinically active disease in 82.6% of patients. In all participants, adiposity indices, smoking and converting angiotensin inhibitor non-use were associated with increased systemic inflammation, which related to more atherogenic lipid profiles, and circulating low density lipoprotein concentrations were associated with cIMT (partial R = 0.153, P = 0.032); RA did not impact on these relationships (interaction P ≥0.1). CONCLUSIONS: Among black Africans, patients with established RA experience reduced overall and abdominal adiposity but no enhanced major traditional risk factor and atherosclerosis burden. This study further suggests that an absent interleukin-6 release by inflamed RA joints into the circulation may account for this unaltered cardiovascular disease risk

Impact of dietary-induced obesity on adrenergic-induced cardiomyocyte damage in rats

Although obesity is an independent risk factor for heart failure and even mild-to-moderate forms of obesity are associated with myocardial systolic dysfunction the mechanisms of the myocardial dysfunction have not been identifi ed. We assessed whether dietary-induced obesity is associated with an increased sensitivity of the myocardium to ß-adrenergic-induced cardiomyocyte apoptosis or fibrosis. To induce obesity, rats were fed a diet that promotes an increased caloric intake. Adrenergic-induced cardiomyocyte apoptosis was determined by injecting rats for 5 days with isoproterenol (0.01 mg/kg/day for 3 days and 0.02 mg/kg/day for 2 days) and then studying the degree of cardiomyocyte damage using a TUNEL assay and assessing the pathological score. Five months of feeding rats a diet that promoted the development of an increased body weight (Control=481±4.3 g, Diet=550±7.8 g, p‹0.001) and visceral fat content (Control=19.6±0.8 g, Diet=33.0±1.2 g, p‹0.0001), did not alter baseline cardiomyocyte apoptosis. However, 5 days of ß-adrenergic activation resulted in an enhanced cardiomyocyte apoptosis in rats receiving the experimental diet as compared to rats receiving a normal diet (p‹0.01). No changes in the myocardial pathological score (fibrosis) were noted. The enhanced adrenergic-induced cardiomyocyte apoptosis in obese rats could not be explained by dietary-induced increases in baseline left ventricular internal diameters, decreases in systolic function (endocardial or midwall fractional shortening) or differences in the response of the heart to adrenergic-induced increases in inotropic or chronotropic function. In conclusion, the present study suggests that obesity may contribute to myocardial dysfunction by increasing the sensitivity of the myocardium to adrenergic-induced cardiomyocyte damage

The relationship between the nitric oxide synthase gene and the risk of hypertension defi ned according to ambulatory blood pressures

Although nitric oxide (NO) plays an important role in blood pressure (BP) control, whether variation of genes involved in regulating the synthesis of NO infl uences BP is uncertain. As the heritability of BP is stronger for ambulatory than it is for conventional BP, we assessed the independent association of the well described functional exon 7 Glu298Asp variant of the eNOS gene with the presence of hypertension in 511 randomly selected normotensive control participants and 503 hypertensives with a diagnosis of hypertension confi rmed with 24-hour ambulatory BP profiles whilst off therapy. We also assessed the relationship between eNOS genotype and 24 hour ambulatory BP. Comparisons of genotype and allele frequencies indicated a lack of association of the exon 7 Glu298Asp gene variant with hypertension (Odds ratio of genotype predicting the presence of hypertension=0.97, confidence interval=0.70-1.30, p=0.92). However, patients with the Glu/Glu genotype of the Glu298Asp variant (n=424) had increased 24-hour systolic and diastolic blood pressures (152±1/97±1 mm Hg) in comparison to patients heterozygous for the Glu298Asp variant or homozygous for the 298Asp allele (n=79) (145±1/94±1 mm Hg, p‹0.005 for systolic BP and p‹0.001 for diastolic BP after multiple adjustments including age, gender, body mass index and the presence of diabetes mellitus). Differences in systolic and diastolic BP between genotype groups were noted during the day as well as at night. The association of eNOS genotype with ambulatory BP translated into an increased risk of more severe grades of hypertension in patients with the Glu/Glu genotype (grade II and III vs. grade I, Odds ratio=2.20, confidence interval=1.34-3.59, p‹0.0002). In conclusion, a functional gene variant (Glu298Asp) at the eNOS locus contributes ~1.4-2.5% to the variation in ambulatory blood pressure within hypertensives, but is not associated with the presence of hypertension in patients in whom the diagnosis has been confirmed by 24-hour ambulatory BP values. The relationship between eNOS genotype and 24-hour ambulatory BP and the severity of hypertension warrants further study

What changes in the transition to learning at university?

This paper reviews evidence about factors affecting student transitions to learning at university. We first review theoretical models of student transitions, and consider their different emphases and end-points. We then examine evidence about academic factors (eg approaches to learning and beliefs about knowledge), social factors (eg engagement and integration) and pedagogic factors (eg teaching methods) as potential influences on student learning transitions. We then attempt a synthesis of the findings and theory, and propose a transitions model in which quality of learning is the key central factor.Work on this article was supported by the Flying Start project (http://www.hope.ac.uk/flyingstart), funded by a National Teaching Fellowship Project grant awarded to Lin Norton and James Elander