Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

A cholesterol switch controls phospholipid scrambling by G protein-coupled receptors.

Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.

A cholesterol switch controls phospholipid scrambling by G protein-coupled receptors.

Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs, yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.

Comparative HPTLC fingerprinting profile and GC-MS analysis of raw and purified Guggulu (Commiphora wightii. Arn. Bhand) by Ayurvedic purification method.

BACKGROUND: Guggulu is an oleo gum resin obtained from the plant Commiphora wightii (Arn.) Bhand., used in Ayurved ic medicines for various ailments like anti-inflammatory conditions, hyperlipidemia, thyroid disorders etc. Guggulsterones E & Z are responsible for these broad ranges of pharmacological actions. It is recommended to do Shodhana (purification) before incorporating it into medicinal formulations. Sahasrayoga, an Ayurvedic text, emphasizes the purification of Guggulu in a particular media, which is a long run practice in Kerala. OBJECTIVES: To compare the physicochemical and phytochemical parameters, quantitative estimation of Guggulsterone E & Z using high performance thin layer chromatography (HPTLC) and qualitative gas chromatography-mass spectrometry (GC-MS) analysis of Guggulu before and after purification. METHODS: Shodhana of Guggulu was performed in Water boiled with crushed fresh leaves of Neem (Azadirachta indica) and fresh rhizome of turmeric (Curcuma longa) using a special equipment (Dolayantra). Preliminary physicochemical and phytochemical evaluation, quantification of Guggulsterones E & Z using HPTLC and GC-MS analysis of raw and purified Guggulu were performed. RESULTS: Phytochemical evaluation of metabolites revealed marked variations. The mean concentrations of Guggulsterone E & Z showed significant differences before and after purification (p<0.01). On GC-MS analysis, it was found that few new compounds were added in the purified Guggulu. CONCLUSIONS: Therapeutic efficacy of Guggulu might have enhanced after traditional purification.

Management of perioperative tumour necrosis factor α inhibitors in rheumatoid arthritis patients undergoing arthroplasty: a systematic review and meta-analysis.

OBJECTIVE: Tumour necrosis factor α inhibitors (TNFis) are widely used in RA patients who undergo surgery, and optimal perioperative management must balance the risk of infection with the risk of post-operative flare. The purpose of this study is to examine the impact of TNFi exposure on surgical site infections (SSIs) in RA patients undergoing elective orthopaedic surgery by systematic review and meta-analysis. METHODS: A systematic review of the literature and meta-analysis were performed using PUBMED, EMBASE and the Cochrane Central Register of Controlled Trials, through May 2014. Two independent reviewers screened titles and abstracts, and analysed selected papers in detail. Included studies assessed RA patients with or without TNFi exposure prior to orthopaedic surgery, and described post-operative infections. Study quality was assessed using the Oxford Centre for Evidence-based Medicine Levels of Evidence. Meta-analyses of the individual study odds ratios (ORs) were conducted, and each pooled OR was calculated using a random effects model. RESULTS: Eight observational studies and three case control studies met inclusion criteria; risk of bias was low in eight studies and moderate in three. Publication bias was not apparent. These studies represent 3681 patients with recent exposure to TNFis (TNFi+) and 4310 with no recent exposure to TNFis (TNFi-) at the time of surgery. The TNFi+ group had higher risk of developing SSI compared with patients in the TNFi- group (random effects model: OR 2.47 (95% CI 1.66, 3.68); P < 0.0001). CONCLUSION: Data from the available literature suggest that there is an increased risk of SSIs in RA patients who use or have recently used TNFis at the time of elective orthopaedic surgery. Prospective studies to confirm these findings and establish the optimal withhold and restart time of TNFis, in the context of other risk factors for infection in RA patients such as higher disease activity, corticosteroid use, smoking and diabetes, are needed.

Constitutive phospholipid scramblase activity of a G protein-coupled receptor.

Opsin, the rhodopsin apoprotein, was recently shown to be an ATP-independent flippase (or scramblase) that equilibrates phospholipids across photoreceptor disc membranes in mammalian retina, a process required for disc homoeostasis. Here we show that scrambling is a constitutive activity of rhodopsin, distinct from its light-sensing function. Upon reconstitution into vesicles, discrete conformational states of the protein (rhodopsin, a metarhodopsin II-mimic, and two forms of opsin) facilitated rapid (>10,000 phospholipids per protein per second) scrambling of phospholipid probes. Our results indicate that the large conformational changes involved in converting rhodopsin to metarhodopsin II are not required for scrambling, and that the lipid translocation pathway either lies near the protein surface or involves membrane packing defects in the vicinity of the protein. In addition, we demonstrate that ß2-adrenergic and adenosine A2A receptors scramble lipids, suggesting that rhodopsin-like G protein-coupled receptors may play an unexpected moonlighting role in re-modelling cell membranes.

Social networking sites: an adjunctive treatment modality for psychological problems.

BACKGROUND: Social networking is seen as a way to enhance social support and feeling of well-being. The present work explores the potentials of social networking sites as an adjunctive treatment modality for initiating treatment contact as well as for managing psychological problems. MATERIALS AND METHODS: Interview schedule, Facebook intensity questionnaire were administered on 28 subjects with a combination of 18 males and 10 females. They were taken from the in-patient and out-patient psychiatry setting of the hospital. RESULTS: Facebook was the most popular sites and used to seek emotional support on the basis of the frequent updates of emotional content that users put in their profile; reconciliations, escape from the problems or to manage the loneliness; getting information about illness and its treatment and interaction with experts and also manifested as problematic use. CONCLUSIONS: It has implications for developing social networking based adjunctive treatment modality for psychological problems.

Synaptic vesicles position complexin to block spontaneous fusion.

Synapses continually replenish their synaptic vesicle (SV) pools while suppressing spontaneous fusion events, thus maintaining a high dynamic range in response to physiological stimuli. The presynaptic protein complexin can both promote and inhibit fusion through interactions between its α-helical domain and the SNARE complex. In addition, complexin's C-terminal half is required for the inhibition of spontaneous fusion in worm, fly, and mouse, although the molecular mechanism remains unexplained. We show here that complexin's C-terminal domain binds lipids through a novel protein motif, permitting complexin to inhibit spontaneous exocytosis in vivo by targeting complexin to SVs. We propose that the SV pool serves as a platform to sequester and position complexin where it can intercept the rapidly assembling SNAREs and control the rate of spontaneous fusion.

Reconstitution of glucosylceramide flip-flop across endoplasmic reticulum: implications for mechanism of glycosphingolipid biosynthesis.

Most glycosphingolipids are synthesized by the sequential addition of monosaccharides to glucosylceramide (GlcCer) in the lumen of the Golgi apparatus. Because GlcCer is synthesized on the cytoplasmic face of Golgi membranes, it must be flipped to the non-cytoplasmic face by a lipid flippase in order to nucleate glycosphingolipid synthesis. Halter et al. (Halter, D., Neumann, S., van Dijk, S. M., Wolthoorn, J., de Mazière, A. M., Vieira, O. V., Mattjus, P., Klumperman, J., van Meer, G., and Sprong, H. (2007) Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J. Cell Biol. 179, 101-115) proposed that this essential flipping step is accomplished via a complex trafficking itinerary; GlcCer is moved from the cytoplasmic face of the Golgi to the endoplasmic reticulum (ER) by FAPP2, a cytoplasmic lipid transfer protein, flipped across the ER membrane, then delivered to the lumen of the Golgi complex by vesicular transport. We now report biochemical reconstitution studies to analyze GlcCer flipping at the ER. Using proteoliposomes reconstituted from Triton X-100-solubilized rat liver ER membrane proteins, we demonstrate rapid (t(½) < 20 s), ATP-independent flip-flop of N-(6-((7-nitro-2-1,3-benzoxadiazol-4-yl)amino)hexanoyl)-D-glucosyl-ß1-1'-sphingosine, a fluorescent GlcCer analog. Further studies involving protein modification, biochemical fractionation, and analyses of flip-flop in proteoliposomes reconstituted with ER membrane proteins from yeast indicate that GlcCer translocation is facilitated by well characterized ER phospholipid flippases that remain to be identified at the molecular level. By reason of their abundance and membrane bending activity, we considered that the ER reticulons and the related Yop1 protein could function as phospholipid-GlcCer flippases. Direct tests showed that these proteins have no flippase activity.

Opsin is a phospholipid flippase.

Polar lipids must flip-flop rapidly across biological membranes to sustain cellular life [1, 2], but flipping is energetically costly [3] and its intrinsic rate is low. To overcome this problem, cells have membrane proteins that function as lipid transporters (flippases) to accelerate flipping to a physiologically relevant rate. Flippases that operate at the plasma membrane of eukaryotes, coupling ATP hydrolysis to unidirectional lipid flipping, have been defined at a molecular level [2]. On the other hand, ATP-independent bidirectional flippases that translocate lipids in biogenic compartments, e.g., the endoplasmic reticulum, and specialized membranes, e.g., photoreceptor discs [4, 5], have not been identified even though their activity has been recognized for more than 30 years [1]. Here, we demonstrate that opsin is the ATP-independent phospholipid flippase of photoreceptor discs. We show that reconstitution of opsin into large unilamellar vesicles promotes rapid (τ<10 s) flipping of phospholipid probes across the vesicle membrane. This is the first molecular identification of an ATP-independent phospholipid flippase in any system. It reveals an unexpected activity for opsin and, in conjunction with recently available structural information on this G protein-coupled receptor [6, 7], significantly advances our understanding of the mechanism of ATP-independent lipid flip-flop.

Unmet need for contraception in Kuwait: issues for health care providers.

Based on a nationally representive household survey of Kuwaiti women held in 1999 (n = 1502) unmet need for contraception was analyzed in Kuwait, an oil-rich Muslim country. It was found that 9.7% currently married women had an unmet need for contraception. Of those, 6.1% wanted to stop child bearing, while 3.6% wanted to space their children. A bivariate comparison of the women with unmet need and current contraceptors showed that the unmet need group comprised of relatively older women with a significantly higher level of parity and ones where husband or wife disapproved of contraception. Also, larger percentages of the unmet need group belonged to relatively lower socio-economic status and were Bedouins. Among the reasons for current non-use, two-third believed that they had a low risk of pregnancy due to infrequent sexual activity or sub-fecundity, and 22% were not using a method because of health concerns. A significantly larger percentage of the unmet need group disapproved of contraception, and believed that Islam forbids family planning, compared to current users (30% and 15%, respectively). The logistic regression analysis showed that the wife's perception of the husband's disapproval of contraceptive use had the strongest negative association with unmet need. We conclude that the contraceptive needs of about 90% of all non-pregnant currently married women who wanted to delay or limit children were being met adequately despite the absence of a formal family planning program, while about 10% women had an unmet need. Issues for health care providers are discussed and family planning counseling is recommended for higher risk older women with unmet need.

Effects of inflammatory cytokines on the permeability of human lung microvascular endothelial cell monolayers and differential eosinophil transmigration.

BACKGROUND: Rhinovirus (RV) infections can result in asthma exacerbations in both adults and children. Respiratory epithelium, the primary site of RV replication, responds to the viral infection by generating a variety of cytokines and chemokines capable of promoting airway inflammation and hence might increase asthma severity. Some of these mediators might also affect the permeability of underlying vascular endothelium. OBJECTIVE: We hypothesized that RV infections can promote airway inflammation and thus asthma by enhancing local vascular permeability. METHODS: Confluent human lung microvascular endothelial cell (HMVEC-L) monolayers were used as an in vitro model of vascular endothelium to determine whether cytokines associated with RV-induced infections are capable of modulating endothelial cell permeability as measured by means of transendothelial electrical resistance. Recombinant cytokines and chemokines were added to confluent HMVEC-L monolayers cultured on Transwell filters, and permeability was measured as decreased electrical resistance over time. Eosinophil transendothelial migration was assessed under the same experimental conditions. RESULTS: TNF- alpha, IL-1 beta, and IFN- gamma significantly increased HMVEC-L permeability. In contrast, GM-CSF, G-CSF, IL-8, IL-6, and RANTES had no effect. Although incubation of HMVEC-L monolayers with either TNF-alpha or IL-1beta promoted eosinophil migration, IFN-gamma had no effect, indicating that enhanced permeability alone was not sufficient for eosinophil infiltration. CONCLUSION: Select cytokines, generated in response to RV infection, can increase vascular permeability and might provide a mechanism by which RV infection can lead to edema, cellular infiltration, and inflammation and thus compromised airflow.

Living arrangements of older women and men in Kuwait.

The main objectives of this article are to analyze the correlates of living arrangements of persons aged 60 or above in the oil-rich, Muslim country of Kuwait and to examine whether or not patterns of co-residence differ by gender. Data were obtained from a nationally representative survey of households of Kuwaiti nationals, and this paper is based on the 687 older Kuwaiti residents of these households. Living arrangements were generally similar for women and men. Eighty nine percent of women and 94 percent of men co-reside in households with at least one son or daughter. Only 0.3 percent of men and 1.9 percent of women live alone. Socio-demographic characteristics of women and men differed significantly; 58 percent of women were widowed compared with 5 percent of men. Logistic regression analysis showed that women had two times higher odds than men of living without their children. The odds of residing without children also increased with the respondent's age and education but decreased with increasing wealth. Continued rapid demographic, socioeconomic, and cultural change in Kuwait foretells continued decline in co-residence with children, and the implications of such change in a small city-state merits further research.