Hypercompliant Apical Membranes of Bladder Umbrella Cells

Urinary bladder undergoes dramatic volume changes during filling and voiding cycles. In the bladder the luminal surface of terminally differentiated urothelial umbrella cells is almost completely covered by plaques. These plaques (500 to 1000 nm) are made of a family of proteins called uroplakins that are known to form a tight barrier to prevent leakage of water and solutes. Electron micrographs from previous studies show these plaques to be interconnected by hinge regions to form structures that appear rigid, but these same structures must accommodate large changes in cell shape during voiding and filling cycles. To resolve this paradox, we measured the stiffness of the intact, living urothelial apical membrane and found it to be highly deformable, even more so than the red blood cell membrane. The intermediate cells underlying the umbrella cells do not have uroplakins but their membranes are an order of magnitude stiffer. Using uroplakin knockout mouse models we show that cell compliance is conferred by uroplakins. This hypercompliance may be essential for the maintenance of barrier function under dramatic cell deformation during filling and voiding of the bladder.     

Resequencing the susceptibility gene,ITGAM, identifies two functionally deleterious rare variants in systemic lupus erythematosus cases

Introduction

The majority of the genetic variance of systemic lupus erythematosus (SLE) remains unexplained by the common disease-common variant hypothesis. Rare variants, which are not detectable by genome-wide association studies because of their low frequencies, are predicted to explain part of this ”missing heritability.” However, recent studies identifying rare variants within known disease-susceptibility loci have failed to show genetic associations because of their extremely low frequencies, leading to the questioning of the contribution of rare variants to disease susceptibility. A common (minor allele frequency = 17.4% in cases) nonsynonymous coding variant rs1143679 (R77H) in ITGAM (CD11b), which forms half of the heterodimeric integrin receptor, complement receptor 3 (CR3), is robustly associated with SLE and has been shown to impair CR3-mediated phagocytosis.

Methods

We resequenced ITGAM in 73 SLE cases and identified two previously unidentified, case-specific nonsynonymous variants, F941V and G1145S. Both variants were genotyped in 2,107 and 949 additional SLE cases, respectively, to estimate their frequencies in a disease population. An in vitro model was used to assess the impact of F941V and G1145S, together with two nonsynonymous ITGAM polymorphisms, A858V (rs1143683) and M441T (rs11861251), on CR3-mediated phagocytosis. A paired two-tailed t test was used to compare the phagocytic capabilities of each variant with that of wild-type CR3.

Results

Both rare variants, F941V and G1145S, significantly impair CR3-mediated phagocytosis in an in vitro model (61% reduction, P = 0.006; 26% reduction, P = 0.0232). However, neither of the common variants, M441T and A858V, had an effect on phagocytosis. Neither rare variant was observed again in the genotyping of additional SLE cases, suggesting that there frequencies are extremely low.

Conclusions

Our results add further evidence to the functional importance of ITGAM in SLE pathogenesis through impaired phagocytosis. Additionally, this study provides a new example of the identification of rare variants in common-allele-associated loci, which, because of their extremely low frequencies, are not statistically associated. However, the demonstration of their functional effects adds support to their contribution to disease risk, and questions the current notion of dismissing the contribution of very rare variants on purely statistical analyses.     

Status of Emergency Obstetric Care in Six Developing Countries Five Years before the MDG Targets for Maternal and Newborn Health

Background

Ensuring women have access to good quality Emergency Obstetric Care (EOC) is a key strategy to reducing maternal and newborn deaths. Minimum coverage rates are expected to be 1 Comprehensive (CEOC) and 4 Basic EOC (BEOC) facilities per 500,000 population.

Methods and Findings

A cross-sectional survey of 378 health facilities was conducted in Kenya, Malawi, Sierra Leone, Nigeria, Bangladesh and India between 2009 and 2011. This included 160 facilities designated to provide CEOC and 218 designated to provide BEOC. Fewer than 1 in 4 facilities aiming to provide CEOC were able to offer the nine required signal functions of CEOC (23.1%) and only 2.3% of health facilities expected to provide BEOC provided all seven signal functions. The two signal functions least likely to be provided included assisted delivery (17.5%) and manual vacuum aspiration (42.3%). Population indicators were assessed for 31 districts (total population = 15.7 million). The total number of available facilities (283) designated to provide EOC for this population exceeded the number required (158) a ratio of 1.8. However, none of the districts assessed met minimum UN coverage rates for EOC. The population based Caesarean Section rate was estimated to be <2%, the maternal Case Fatality Rate (CFR) for obstetric complications ranged from 2.0–9.3% and still birth (SB) rates ranged from 1.9–6.8%.

Conclusions

Availability of EOC is well below minimum UN target coverage levels. Health facilities in the surveyed countries do not currently have the capacity to adequately respond to and manage women with obstetric complications. To achieve MDG 5 by 2015, there is a need to ensure that the full range of signal functions are available in health facilities designated to provide CEOC or BEOC and improve the quality of services provided so that CFR and SB rates decline.     

Analysis of a genome-wide association study-linked locus (CCR6) in Asian rheumatoid arthritis

A genome-wide association study in Japan identified the C-C chemokine receptor type 6 gene (CCR6) as associated with rheumatoid arthritis (RA). This finding has not been validated in other Asian populations. A case-control study involving 996 subjects, comprising 440 controls and 556 RA patients, was done to determine their anticyclic citrullinated peptide (anti-CCP) antibody status and CCR6 polymorphism (rs3093024) genotype. Three hundred eighty-seven patients were anti-CCP positive and 153 anti-CCP negative. Logistic regression showed that allele A was likely to increase the risk of developing RA among females via a recessive model (odds ratio [OR]=1.55, 95% confidence interval [CI]=1.01, 2.39), whereas the risk effect appeared to be reduced among males via an additive model (OR=0.60, 95% CI=0.42, 0.85). Considering only subjects who are anti-CCP positive, allele A increased RA risk among females via a recessive model (OR=1.68, 95% CI=1.07, 2.64) but decreased the risk among males via an additive model (OR=0.59, 95% CI=0.39, 0.89). We showed that CCR6 polymorphism was a risk factor among females but a protective factor among males. Functional studies are warranted to unravel the pathophysiological relevance of the gene variant and other linked variants with RA.     

Omega-3 polyunsaturated fatty acid supplementation reduces hypertension in TGR(mRen-2)27 rats

To establish the effect of dietary omega-3 PUFA on angiotensin II (ANG II)-mediated hypertension, male TGR (mRen-2)27 (Ren-2) rats (animals with high ANG II activity) were maintained on a diet either deficient or sufficient in omega-3 PUFA from conception. Half the animals on each diet were treated with the angiotensin-converting enzyme inhibitor, perindopril, from birth. Ren-2 rats fed the omega-3 PUFA deficient diet were significantly more hypertensive than those fed the omega-3 PUFA sufficient diet. Perindopril reduced the blood pressure of both omega-3 PUFA-deficient and omega-3 PUFA-sufficient diet-fed Ren-2 rats. Body weight, body fat and plasma leptin were reduced by perindopril treatment but not affected by omega-3 PUFA supply. Given that the elevated blood pressure of the Ren-2 rat is mediated by ANG II, the data suggest that omega-3 PUFA may reduce hypertension via the renin-angiotensin system.     

Central blockade of nitric oxide synthesis induces hyperthermia that is prevented by indomethacin in rats

The effect of blocking brain nitric oxide (NO) synthesis on body temperature regulation was tested in conscious rats. NO synthase was inhibited by administration of equivalent doses of N^G-nitro-L-arginine methyl ester (L-NAME) or N^G-monomethyl L-arginine monoacetate (L-NMMA) into a lateral cerebral ventricle (ICV) and core temperature was monitored. An ICV injection of 300 μg L-NAME increased colonic temperature in rats (n=8) by 1.9±0.1 °C (P<0.001). The increase in temperature in response to blockade of NO synthesis was significant by 1 h after injection and sustained for more than 3 h. The hyperthermic response to central NO blockade (using L-NMMA) was found to be dose-dependent between 2.8 to 282 μg. Intravenous administration of L-NAME at the highest dose used in the study (300 μg) had no effect on temperature, indicating that the mechanism was mediated by the brain. Pre-treatment with indomethacin (300 μg) blocked hyperthermic responses to ICV L-NAME (300 μg) administration. We conclude that, blockade of nitric oxide induces a cyclooxygenase-dependent hyperthermia in conscious rats that is mediated by the brain.     

Does Perinatal ω‐3 Polyunsaturated Fatty Acid Deficiency Increase Appetite Signaling?

Objective: To investigate the effect of maternal dietary ω‐3 polyunsaturated fatty acid (PUFA) deficiency and repletion on food appetite signaling. Research Methods and Procedures: Sprague‐Dawley rat dams were maintained on diets either supplemented with (CON) or deficient in (DEF) ω‐3 PUFA. All offspring were raised on the maternal diet until weaning. After weaning, two groups remained on the respective maternal diet (CON and DEF groups), whereas a third group, born of dams fed the DEF diet, were switched to the CON diet (REC). Experiments on food intake began when the male rats reached 16 weeks of age. Food intake was stimulated either by a period of food restriction, by blocking glucose utilization (by 2‐deoxyglucose injection), or by blocking β‐oxidation of fatty acids (by β‐mercaptoacetate injection). Results: DEF animals consumed more than CON animals in response to all stimuli, with the greatest difference (1.9‐fold) demonstrated following administration of 2‐deoxyglucose. REC animals also consumed more than CON animals in response to food restriction and 2‐deoxyglucose but not to β‐mercaptoacetate. Discussion: These findings indicate that supply of ω‐3 PUFA, particularly during the perinatal period, plays a role in the normal development of mechanisms controlling food intake, especially glucoprivic (i.e. reduced glucose availability) appetite signaling. Dietary repletion of ω‐3 PUFA from 3 weeks of age restored intake responses to fatty acid metabolite signaling but did not reverse those in response to food restriction or glucoprivic stimuli.     

Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway

3H-l-Phenylalanine is incorporated into a range of phenylpropanoid compounds when fed to tobacco cell cultures. A significant proportion of (3)H-trans-cinnamic acid formed from (3)H-l-phenylalanine did not equilibrate with exogenous trans-cinnamic acid and therefore may be rapidly channeled through the cinnamate 4-hydroxylase (C4H) reaction to 4-coumaric acid. Such compartmentalization of trans-cinnamic acid was not observed after elicitation or in cell cultures constitutively expressing a bean phenylalanine ammonia-lyase (PAL) transgene. Channeling between PAL and C4H was confirmed in vitro in isolated microsomes from tobacco stems or cell suspension cultures. This channeling was strongly reduced in microsomes from stems or cell cultures of transgenic PAL-overexpressing plants or after elicitation of wild-type cell cultures. Protein gel blot analysis showed that tobacco PAL1 and bean PAL were localized in both soluble and microsomal fractions, whereas tobacco PAL2 was found only in the soluble fraction. We propose that metabolic channeling of trans-cinnamic acid requires the close association of specific forms of PAL with C4H on microsomal membranes.     

Omega 6 to omega 3 fatty acid imbalance early in life leads to persistent reductions in DHA levels in glycerophospholipids in rat hypothalamus even after long-term omega 3 fatty acid repletion

Failure to provide omega 3 fatty acids in the perinatal period results in alterations in nerve growth factor levels, dopamine production and permanent elevations in blood pressure. The present study investigated whether changes in brain (i.e., hypothalamus) glycerophospholipid fatty acid profiles induced by a diet rich in omega 6 fatty acids and very low in alpha-linolenic acid (ALA) during pregnancy and the perinatal period could be reversed by subsequent feeding of a diet containing ALA. Female rats (6 per group) were mated and fed either a low ALA diet or a control diet containing ALA throughout pregnancy and until weaning of the pups at 3 weeks. At weaning, the pups (20 per group) remained on the diet of their mothers until 9 weeks, when half the pups were switched onto the other diet, thus generating four groups of animals. At 33 weeks, pups were killed, the hypothalamus dissected from the male rats and analysed for glycerophospholipid fatty acids. In the animals fed the diet with very little ALA and then re-fed the control diet containing high levels of ALA for 24 weeks, the DHA levels were still significantly less than the control values in PE, PS and PI fractions, by 9%, 18% and 34%, respectively. In this group, but not in the other dietary groups, ALA was detected in all glycerophospholipid classes at 0.2-1.7% of the total fatty acids. The results suggest that omega 6-3 PUFA imbalance early in life leads to irreversible changes in hypothalamic composition. The increased ALA and reduced DHA proportions in the animals re-fed ALA in later life are consistent with a dysfunction or down-regulation of the conversion of ALA to 18:4n-3 by the delta-6 desaturase.