Spherical-supported membranes as platforms for screening against membrane protein targets.

Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes ("SSBLM"). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment. To provide proof-of-concept, SSBLMs embedding the polytopic bacterial nucleoside transporter NupC were assembled on 100- and 200 nm silica particles. To test specific binding of antibodies, NupC was tagged with a poly-histidine epitope in one of its central loops between two transmembrane helices. Fluorescent labelling, small angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) were used to monitor formation of the SSBLMs. Specific binding of an anti-his antibody and a gold-nitrilotriacetic acid (NTA) conjugate probe was confirmed with ELISAs and cryo-EM. SSBLMs for screening could be made with purified and lipid reconstituted NupC, as well as crude bacterial membrane extracts. We conclude that SSBLMs are a promising new means of presenting membrane protein targets for (biomimetic) antibody screening in a native-like lipid environment.

Social support and religion: mental health service use and treatment of schizophrenia.

The perceptions and religious beliefs held by family members, mental health and health care professionals, and the community may affect the treatment of individuals with schizophrenia. To better identify and understand the influence of families, professionals and community members on individual's treatment for schizophrenia, this review paper examines: (1) the religious perceptions of families, professionals, and the public towards schizophrenia; (2) religious perceptions of the etiology of schizophrenia; (3) how others perceive religion as a coping mechanism; and (4) how religion influences treatment engagement and help-seeking behaviors. MEDLINE and PsycInfo databases were systematically searched from 1980 to 2010 using the terms schizophrenia, schizoaffective, schizophreniform, psychotic disorder not otherwise specified and religion, religiosity, spirituality, and faith. Forty-three (n = 43) original research studies met the inclusion criteria. This study found that religious beliefs influence the treatment of schizophrenia in the following ways: Religious themes were positively associated with coping, treatment engagement and help-seeking behavior. Evidence of religious underpinnings was found in perceptions of etiology. The findings also indicate that there is often both a preference among family members and caregivers to utilize religious-based professionals and caution toward mental health professionals. Researchers and professionals may find avenues for improving treatment through examining the interaction of religious and schizophrenia at the social support level.

Toxicity-structure activity evaluation of limonoids from Swietenia species on Artemia salina.

CONTEXT: Many plant extracts and compounds are being investigated for their cytotoxicity and hence their medicinal or therapeutic properties. Reports of toxicity studies with limonoid analogs have been sparse and have involved mainly crude extracts. In this study, individual natural limonoids have been isolated and their toxicity manipulated via semisynthesis. OBJECTIVE: The lethality of limonoid analogs from Swietenia macrophylla King and Swietenia aubrevilleana Stehlé & Cusin (Meliaceae) against Artemia salina Leach was determined. MATERIALS AND METHODS: Four known natural limonoids were isolated from the dry ground seeds of S. macrophylla and S. aubrevilleana, modified using acylation and hydrolysis reactions and tested in A. salina lethality assays at 1-400 ppm. A 50% lethal concentration (LC(50)) was determined by probit analysis. RESULTS: Higher levels of toxicity were achieved in most of the prepared analogs compared with the parent natural limonoids. The compound showing the highest toxicity with LC(50) 3.9 ppm was 3-O-benzoyl-3-detigloylisoswietenine (20). Other analogs with high toxicity were 6-O-benzoylswietenolide (7), 6-O-benzoylswietenine (17), and 3,6-O,O-dipropionylswietenolide (9), which showed LC(50) values of 4.3, 7.5, and 28.5 ppm, respectively. DISCUSSION AND CONCLUSIONS: Toxicity can be improved via semisynthesis. The compounds exhibiting high toxicity (low LC(50)) may be good candidates for cytotoxicity studies.

A process evaluation of Promotional Guides used by health visitors to support men's transition to fatherhood: a qualitative study.

BACKGROUND: Health visitors play an important role in supporting new parents in their transition to parenthood. A programme known as the Promotional Guide system is used by many health visitors in England with mothers and fathers to support this transition, but there is little known about health visitors' views of the Promotional Guides, how they are used in practice or barriers to effective implementation with fathers. AIMS: The aim of this study was to explore the following: (1) health visitors' use of Promotional Guides with fathers, (2) health visitors' assessment of father's mental health and wellbeing and (3) facilitators and barriers to using Promotional Guides in practice. METHODS: A prospective observational cohort study and a process evaluation informed by the Medical Research Council guidance were conducted. A purposive sample of 11 health visitors was interviewed, and an additional seven were observed using the Promotional Guides in practice. Data were analysed using framework analysis. RESULTS: Five main themes were identified from interview and observational data as follows: (1) Enquiry into fathers' mental health, (2) Promotional Guides in practice, (3) health visitors' perceptions of the Promotional Guides system, (4) barriers to using Promotional Guides with fathers and (5) facilitators and recommendations for using Promotional Guides with fathers. This study identified a number of barriers and facilitators to the use of Promotional Guides with fathers. Recommendations were made for improving services for first-time fathers, implementing the Promotional Guide system with fathers and highlighting areas for future research. CONCLUSION: This study considered the acceptability, feasibility and fidelity of using the Promotional Guide programme with fathers from the health visitor's perspective. The findings provided an insight into health visitors' experiences of working with fathers, inquiring about men's mental health needs and their use of the Promotional Guides with men during the perinatal period.

Membrane transport proteins: implications of sequence comparisons.

Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.

Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs.

In most mammalian cells nucleoside uptake occurs primarily via broad-specificity, es (e, equilibrative; 5, sensitive to NBMPR inhibition) transporters that are potently inhibited by nitrobenzylthioinosine (NBMPR). These transporters are essential for nucleotide synthesis by salvage pathways in hemopoietic and other cells that lack de novo pathways and are the route of cellular uptake for many cytotoxic nucleosides used in cancer and viral chemotherapy. They play an important role in adenosine-mediated regulation of many physiological processes, including neurotransmission and platelet aggregation, and are a target for coronary vasodilator drugs. We have previously reported the purification of the prototypic es transporter from human erythrocytes and have shown that this glycoprotein of apparent M, 55,000 is immunologically related to nucleoside transporters from several other species and tissues, including human placenta. Here we report the isolation of a human placental cDNA encoding a 456-residue glycoprotein with functional characteristics typical of an es-type transporter. It is predicted to possess 11 membrane-spanning regions and is homologous to several proteins of unknown function in yeast, nematodes, plants and mammals. Because of its central role in the uptake both of adenosine and of chemotherapeutic nucleosides, study of this protein should not only provide insights into the physiological roles of nucleoside transport but also open the way to improved therapies.

Immunogenicity and protective efficacy of a tuberculosis DNA vaccine.

Tuberculosis is the most widespread and lethal infectious disease affecting humans. Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 (Ag85), induced substantial humoral and cell-mediated immune responses and conferred significant protection against challenge with live M. tuberculosis and M. bovis bacille Calmette-Guérin (BCG). These results indicate that immunization with DNA encoding a mycobacterial antigen provides an efficient and simple method for generating protective immunity and that this technique may be useful for defining the protective antigens of M. tuberculosis, leading to the development of a more effective vaccine.

Apoptosis is regulated by the rate of glucose transport in an interleukin 3 dependent cell line.

In the absence of a survival stimulus, the interleukin 3 (IL-3)-dependent IC.DP cell line undergoes a process termed programmed cell death or apoptosis. Survival can be induced by IL-3, which can also stimulate proliferation of IC.DP cells. IC.DP cells have been stably transfected with the p160v-abl protein tyrosine kinase, activation of the kinase at the permissive temperature permits cell survival in the absence of IL-3 by suppression of apoptosis, although the growth factor is still required for proliferation. Both IL-3 and activation of the v-ABL tyrosine kinase stimulated glucose transport, which may in part be due to a translocation of transporters to the cell surface. Inhibition of glucose uptake markedly increased the rate of apoptosis in these cells, an effect that could be reversed by the provision of alternative energy sources such as glutamine. Growth factor- or oncogene-mediated increases in glucose uptake may therefore represent an important regulatory point in the suppression of apoptosis.

Sequence and structure of a human glucose transporter.

The amino acid sequence of the glucose transport protein from human HepG2 hepatoma cells was deduced from analysis of a complementary DNA clone. Structural analysis of the purified human erythrocyte glucose transporter by fast atom bombardment mapping and gas phase Edman degradation confirmed the identity of the clone and demonstrated that the HepG2 and erythrocyte transporters are highly homologous and may be identical. The protein lacks a cleavable amino-terminal signal sequence. Analysis of the primary structure suggests the presence of 12 membrane-spanning domains. Several of these may form amphipathic alpha helices and contain abundant hydroxyl and amide side chains that could participate in glucose binding or line a transmembrane pore through which the sugar moves. The amino terminus, carboxyl terminus, and a highly hydrophilic domain in the center of the protein are all predicted to lie on the cytoplasmic face. Messenger RNA species homologous to HepG2 glucose transporter messenger RNA were detected in K562 leukemic cells, HT29 colon adenocarcinoma cells, and human kidney tissue.

The bacterial community of Quesnel Lake sediments impacted by a catastrophic mine tailings spill differ in composition from those at undisturbed locations - two years post-spill.

The West Basin of Quesnel Lake (British Columbia, Canada) suffered a catastrophic disturbance event in August 2014 when mine tailings and scoured natural material were deposited into the lake's West Basin due to an impoundment failure at the adjacent Mount Polley copper-gold mine. The deposit covered a significant portion of the West Basin floor with a thick layer of material. Since lake sediments host bacterial communities that play key roles in the geochemical cycling in lacustrine environments, it is important to understand which groups inhabit the newly deposited material and what this implies for the ecological function of the West Basin. Here we report a study conducted two years post-spill, comparing the bacterial communities from sediments of both disturbed and undisturbed sites. Our results show that sediments from disturbed sites differed in physical and chemical properties than those in undisturbed sites (e.g. higher pH, particle size and Cu concentration). Furthermore, bacterial communities from the disturbed sites appeared to be legacy communities from the tailings impoundment, with metabolic potential revolving mainly around the cycling of S and metals, whereas the ones from the undisturbed sites were associated with the cycling of N.

Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.

1. The human (h) SLC29 family of integral membrane proteins is represented by four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterized family member, hENT1. They belong to the widely distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporter proteins. 2. A predicted topology of eleven transmembrane helices has been experimentally confirmed for hENT1. The best-characterized members of the family, hENT1 and hENT2, possess similar broad permeant selectivities for purine and pyrimidine nucleosides, but hENT2 also efficiently transports nucleobases. hENT3 has a similar broad permeant selectivity for nucleosides and nucleobases and appears to function in intracellular membranes, including lysosomes. 3. hENT4 is uniquely selective for adenosine, and also transports a variety of organic cations. hENT3 and hENT4 are pH sensitive, and optimally active under acidic conditions. ENTs, including those in parasitic protozoa, function in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis and, in humans, are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. 4. By regulating the concentration of adenosine available to cell surface receptors, mammalian ENTs additionally influence physiological processes ranging from cardiovascular activity to neurotransmission.

Properties of an entropy-based signal receiver with an application to ultrasonic molecular imaging.

Qualitative and quantitative properties of the finite part, H(f), of the Shannon entropy of a continuous waveform f(t) in the continuum limit are derived in order to illuminate its use for waveform characterization. Simple upper and lower bounds on H(f), based on features of f(t), are defined. Quantitative criteria for a priori estimation of the average-case variation of H(f) and log E(f), where E(f) is the signal energy of f(t) are also derived. These provide relative sensitivity estimates that could be used to prospectively choose optimal imaging strategies in real-time ultrasonic imaging machines, where system bandwidth is often pushed to its limits. To demonstrate the utility of these sensitivity relations for this application, a study designed to assess the feasibility of identification of angiogenic neovasculature targeted with perfluorocarbon nanoparticles that specifically bind to alpha(v)beta3-integrin expression in tumors was performed. The outcome of this study agrees with the prospective sensitivity estimates that were used for the two receivers. Moreover, these data demonstrate the ability of entropy-based signal receivers when used in conjunction with targeted nanoparticles to elucidate the presence of alpha(v)beta3 integrins in primordial neovasculature, particularly in acoustically unfavorable environments.

Treatment of log yard runoff using a r-circulating sand filtration process.

A re-circulating filtration process using oxide-coated sand successfully removed COD and turbidity from log yard runoff. After passing only one pore volume of the runoff through the sand column, 72% COD was removed. The 2.4% Fe and Al oxide coating on the sand contributed to better COD removal than was obtained when the sand was stripped of oxide coating (86% versus 52%, respectively), at least initially before saturation of adsorption sites on the oxide coating occurred. The best COD removal performance came from conditioned sand. This sand, from the same original source and identical to the oxide-coated sand used in all experiments, came from an existing experimental sand column that had been treating log yard runoff for 1 year. The "conditioning" resulted in the sand having a higher TOC content (0.26% wt) and smaller particle sizes. This sand was able to consistently remove 80% COD from repeated batches of log yard runoff with strengths up to 3690 mg l(-1).

NDRG4, an early detection marker for colorectal cancer, is specifically expressed in enteric neurons.

BACKGROUND: Promoter methylation of N-myc Downstream-Regulated Gene 4 (NDRG4) in fecal DNA is an established early detection marker for colorectal cancer (CRC). Despite its connection to CRC, NDRG4 is predominantly studied in brain and heart, with little to no knowledge about its expression or role in other organs. In this study, we aimed to determine the whole-body expression of NDRG4, with a focus on the intestinal tract. METHODS: We investigated NDRG4 expression throughout the body by immunohistochemistry, Western Blotting and in situ mRNA hybridization using tissues from NDRG4 wild-type, heterozygous and knockout mice and humans. In addition, we explored cell-specific expression of NDRG4 in murine whole-mount gut preparations using immunofluorescence and confocal microscopy. KEY RESULTS: NDRG4 is specifically expressed within nervous system structures throughout the body. In the intestinal tract of both mouse and man, NDRG4 immunoreactivity was restricted to the enteric nervous system (ENS), where it labeled cell bodies of the myenteric and submucosal plexuses and interconnecting nerve fibers. More precisely, NDRG4 expression was limited to neurons, as NDRG4 always co-localized with HuC/D (pan-neuronal marker) but never with GFAP (an enteric glial cell marker). Furthermore, NDRG4 was expressed in various neuropeptide Y positive neurons, but was only found in a minority (~10%) of neurons expressing neuronal nitric oxide synthase. CONCLUSIONS AND INFERENCES: NDRG4 is exclusively expressed by central, peripheral and enteric neurons/nerves, suggesting a neuronal-specific role of this protein. Our findings raise the question whether NDRG4, via the ENS, an understudied component of the tumor microenvironment, supports CRC development and/or progression.

Gemcitabine transport in xenopus oocytes expressing recombinant plasma membrane mammalian nucleoside transporters.

BACKGROUND: Gemcitabine, a pyrimidine analogue of deoxycytidine, is an anticancer nucleoside drug that requires functional plasma membrane nucleoside transporter proteins to reach its intracellular targets and cause cytotoxicity. Because of technical difficulties inherent in studying nucleoside transport in human cells, we rigorously defined gemcitabine membrane transportability by producing each of the available human (h) and rat (r) recombinant nucleoside transporters (NTs) individually in Xenopus laevis oocytes. METHODS: Oocytes were microinjected with in vitro-transcribed RNAs derived from complementary DNAs encoding (C = concentrative) rCNT1, rCNT2, hCNT1, hCNT2, (E = equilibrative) rENT1, rENT2, hENT1, and hENT2. Uptake of [(3)H]gemcitabine and [(14)C] uridine was measured 3 days after microinjection to determine kinetic constants. We also used the two-electrode, voltage-clamp technique to investigate the electrophysiology of hCNT1-mediated gemcitabine transport. RESULTS: Gemcitabine was transported by most of the tested proteins (the exceptions being the purine-selective rCNT2 and hCNT2), with the greatest uptake occurring in oocytes producing recombinant rCNT1 and hCNT1. Influxes of gemcitabine mediated by hCNT1, hENT1, and hENT2 were saturable and conformed to Michaelis-Menten kinetics with apparent K(m) values of 24, 160, and 740 microM, respectively. Gemcitabine had a limited ability to cross the lipid bilayer of oocyte membranes by simple diffusion. External application of gemcitabine to oocytes producing recombinant hCNT1 induced an inward current, which demonstrated that hCNT1 functions as a Na(+)/nucleoside co-transport protein and confirmed the transporter's ability to transport gemcitabine. CONCLUSIONS: Mammalian nucleoside transporters vary widely in their affinity and capacity to transport gemcitabine. Variation in the tumor and tissue distribution of plasma membrane nucleoside transporter proteins may contribute to the solid tumor activities and schedule-dependent toxic effects of gemcitabine.

Synthesis and evaluation of 18F-labeled choline as an oncologic tracer for positron emission tomography: initial findings in prostate cancer.

The up-regulation of rates of choline uptake and phosphorylation in certain malignancies has motivated the development of positron-labeled choline analogues for noninvasive detection of cancer using positron emission tomography (PET). The choline analogue, no-carrier-added [18F]fluoromethyl-dimethyl-2-hydroxyethyl-ammonium (FCH), was synthesized through the intermediate [18F]fluorobromomethane. FCH was evaluated in relationship to 2-[18F]fluoro-2-deoxyglucose (FDG) as an oncological probe in cultured PC-3 human prostate cancer cells, a murine PC-3 human prostate cancer xenograft model, and in PET imaging studies of patients with prostate cancer. FCH was synthesized in 20-40% radiochemical yield and >98% radiochemical purity. Accumulation of FCH and FDG were comparable in cultured prostate cancer cells, whereas only FCH was inhibited (90%) by hemicholinium-3, a specific inhibitor of choline transport and phosphorylation. FCH showed similar biodistribution to [14C]choline in the tumor-bearing mouse, with prominent renal and hepatic uptake. Tumor uptake of FCH was similar to choline and FDG in the mouse model, although tumor:blood ratios were moderately higher for FCH. Initial PET imaging studies in prostate cancer patients showed high uptake of FCH in advanced prostate carcinoma and detection of osseous and soft tissue metastases. FCH uptake by tumors was markedly reduced in patients rescanned during androgen deprivation therapy. It is concluded that FCH closely mimics choline uptake by normal tissues and prostate cancer neoplasms. FCH is potentially useful as a PET tracer for detection and localization of prostate cancer and monitoring effects of therapy.

The monosaccharide transport system of the human erythrocyte. Orientation upon reconstitution.

Treatment of intact human erythrocytes with trypsin had no effect upon either the rate of hexose transport or the binding of cytochalasin B to the transport system. In contrast, proteolysis of inside-out vesicles prepared from human erythrocyte membranes inactivated both hexose transport and cytochalasin B binding. When purified hexose transporter, reconstituted into phospholipid vesicles of undetermined size, was treated with trypsin, approx. 50% of the cytochalasin B binding activity was lost. This loss correlated with a decrease in the amount of the transporter polypeptide, as assayed by gel electrophoresis. These results show that the orientation of the transporter can be established through trypsin treatment in conjunction with cytochalasin B binding. Small unilamellar vesicles containing transporter were prepared by sonication of larger species and by a cycle of cholate solubilization and removal of the detergent. In the former case, the transporter orients almost randomly, whereas in the latter approx. 75% of the transporters have the cytoplasmic domain external.

Purification of the cytochalasin B binding component of the human erythrocyte monosaccharide transport system.

The cytochalasin B binding component of the human erythrocyte monosaccharide transport system has been purified. The preparation appears to contain one major protein with an apparent polypeptide chain molecular weight of 55,000 and about 0.4 binding sites per chain. Cytochalasin B binds to the reconstituted preparation with a dissociation constant of 1.3.10(-7) M, a value which is similar to that reported for the transport system in the intact erythrocyte.

Characterization of vesicles containing insulin-responsive intracellular glucose transporters isolated from 3T3-L1 adipocytes by an improved procedure.

Our previously described immunoadsorption method for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters from 3T3-L1 adipocytes has been improved in two ways. First, the minimal number of g minutes required to sediment the plasma membranes from the cell homogenate has been determined and, as a result, the supernatant used for immunoadsorption in the new procedure contained twice as much of the intracellular transporters. Second, the immunoadsorption has been performed with affinity-purified antibodies directed against the carboxy terminal peptide of the transporter, rather than against the entire protein. 10(7) cells (10 mg protein) yielded about 12 micrograms of vesicular protein and 11 micrograms of vesicular phospholipid. The transporter constituted 3% of the protein in the vesicles; this amount equates to approx. eight copies of the transporter per 50 nm vesicle. The polypeptide composition of the vesicles was determined by gel electrophoresis and protein staining. Major components, other than the glucose transporter, are polypeptides of Mr 270,000, 245,000, 165,000 and 115,000. The vesicles contained several phosphoproteins; the major ones have a Mr of 245,000, 190,000, 115,000 and 25,000. Insulin treatment of adipocytes did not significantly change the phosphoprotein composition of the vesicles. The vesicles were not enriched in the Golgi marker enzyme, galactosyltransferase. The cellular content of the marker for the trans-Golgi reticulum, sialyltransferase, was too low to detect.