SCREENING OF PHOTOSYNTHETIC O2‐EVOLVING PROKARYOTES FOR AN INSULIN‐LIKE ANTIGEN1

Diabetes mellitus (DM), a metabolic disorder, is becoming a major health problem worldwide. Insulin is the single hope for management of type 1 diabetes, but it is not always available or suitable. For finding additional bioresources, the present study was performed. ELISA‐based preliminary screening of cyanobacterial biomass using antihuman insulin antibody have detected an insulin‐like antigen in Spirulina platensis S‐5, Spirulina NCCU‐482, and Spirulina NCCU‐483. Their similarity with insulin‐like antigen was further confirmed by electrophoretic mobility using bovine insulin as marker.     

cAMP inhibits bile acid-induced apoptosis by blocking caspase activation and cytochrome c release

We have previously shown that cAMP protects against bile acid-induced apoptosis in cultured rat hepatocytes in a phosphoinositide 3-kinase (PI3K)-dependent manner. In the present studies, we investigated the mechanisms involved in this anti-apoptotic effect. Hepatocyte apoptosis induced by glycodeoxycholate (GCDC) was associated with mitochondrial depolarization, activation of caspases, the release of cytochrome c from the mitochondria, and translocation of BAX from the cytosol to the mitochondria. cAMP inhibited GCDC-induced apoptosis, caspase 3 and caspase 9 activation, and cytochrome c release in a PI3K-dependent manner. cAMP activated PI3K in p85 immunoprecipitates and resulted in PI3K-dependent activation of the survival kinase Akt. Chemical inhibition of Akt phosphorylation with SB-203580 partially blocked the protective effect of cAMP. cAMP resulted in wortmannin-independent phosphorylation of BAD and was associated with translocation of BAD from the mitochondria to the cytosol. These results suggest that GCDC-induced apoptosis in cultured rat hepatocytes proceeds through a caspase-dependent intracellular stress pathway and that the survival effect of cAMP is mediated in part by PI3K-dependent Akt activation at the level of the mitochondria.     

Cross-talk between protein kinases Czeta and B in cyclic AMP-mediated sodium taurocholate co-transporting polypeptide translocation in hepatocytes

Cyclic AMP stimulates taurocholate (TC) uptake and sodium taurocholate co-transporting polypeptide (Ntcp) translocation in hepatocytes via the phosphoinositide-3 kinase (PI3K) signaling pathway. The aim of the present study was to determine whether protein kinase (PK) Czeta, one of the downstream mediators of the PI3K signaling pathway, is involved in cAMP-mediated stimulation of TC uptake. Studies were conducted in isolated rat hepatocytes and in HuH-7 cells stably transfected with rat liver Ntcp (HuH-Ntcp cells). Studies in hepatocytes showed that cAMP activates PKCzeta in a PI3K-dependent manner without inducing translocation of PKCzeta to the plasma membrane. Inhibition of cAMP-induced PKCzeta activity by myristoylated PKC (zeta/lambda) pseudosubstrate, a specific inhibitor of PKCzeta, and G? 6850, a PKC inhibitor, resulted in inhibition of cAMP-induced increases in TC uptake and Ntcp translocation. Studies in HuH-Ntcp cells showed that inhibition of cAMP-induced PKCzeta activation by dominant-negative (DN) PKCzeta resulted in inhibition of cAMP-induced increases in TC uptake and Ntcp translocation. DN PKCzeta also inhibited wild-type PKCzeta-induced increases in PKCzeta activity, TC uptake, and Ntcp translocation. Myristoylated PKC (zeta/lambda) pseudosubstrate and DN PKCzeta also inhibited cAMP-induced activation of PKB in hepatocytes and HuH-Ntcp cells, respectively. Neither DN PKB nor constitutively active PKB affected cAMP-induced activation of PKCzeta, and wild-type PKCzeta did not activate PKB. Taken together, these results suggest that cAMP-induced activation of PKB is dependent on cAMP-induced stimulation of PKCzeta. It is proposed that cAMP-induced Ntcp translocation involves the activation of the PI3K/PKCzeta signaling pathway followed by the activation of the PI3K/PKB signaling pathway.     

Dephosphorylation of Ser-226 facilitates plasma membrane retention of Ntcp

Ntcp is a phosphoprotein, and its translocation by cAMP to the plasma membrane is associated with dephosphorylation. However, the phosphorylation site(s) of Ntcp is not known. Thus, the aim of the present study was to determine the potential Ntcp phosphorylation sites and whether any of these phosphorylation sites is involved in Ntcp translocation. To determine the potential phosphorylation sites, metabolically labeled [32P]Ntcp isolated from hepatocytes was digested with clostripain and then subjected to SDS-PAGE followed by autoradiography. Clostripain digestion resulted in two phosphorylated peptides, and cAMP decreased phosphorylation of one of the peptides (7.8 K(d)), which contains the putative third cytoplasmic loop with three serine (Ser-213, Ser-226, and Ser-227) and two threonine (Thr-219 and Thr-225) residues. To determine whether any one of these serine/threonine residues is phosphorylated and/or is involved in Ntcp translocation, each of these serine/threonine residues were mutated to alanine. HuH-7 cells were transiently transfected with the wild-type and the mutated Ntcps followed by determination of taurocholate uptake and Ntcp expression, translocation and phosphorylation. Mutation of only Ser-226 resulted in 30% decrease in Ntcp phosphorylation and in 2.5 and 3.2-fold increases in taurocholate uptake and Ntcp retention in the plasma membrane, respectively. Cyclic AMP failed to further decrease phosphorylation and increase translocation of S226A-Ntcp. Taken together, these results suggest that the Ser-226 in the third cytoplasmic loop of Ntcp is phosphorylated and cAMP may increase Ntcp translocation to the plasma membrane by dephosphorylating Ntcp at this site.     

Effect of bile acids on calcium efflux from isolated rat hepatocytes and perfused rat livers

The changes in intracellular Ca2+ concentration [( Ca2+]i) of hepatocytes induced by certain bile acids are biphasic: an initial increase is followed by a more gradual decrease. This latter decline in [Ca2+]i may be due to an efflux of Ca2+ across the plasma membrane. This hypothesis was tested by studying the effect of different bile acids on the efflux of 45Ca from preloaded rat hepatocytes and isolated perfused rat livers. The following bile acids were studied: cholic (C), ursodeoxycholic (UDC), chenodeoxycholic (CDC), and deoxycholic (DC) acids; their taurine (T) conjugates (TC, TUDC, TCDC, and TDC); and the taurine, sulfate (S), and glucuronide (Glu) derivatives of lithocholic acid (TLC, LS, TLS, and LGlu, respectively). At 0.3 mM, all bile acids except C, TC, TCDC, UDC, and TUDC significantly increased 45Ca efflux from preloaded hepatocytes without affecting cell viability. Dose-response studies revealed that the minimum effective concentration needed to induce 45Ca efflux was 0.06 mM for LS, 0.8 mM for TCDC, and 10 mM for TC. Efflux of 86Rb from preloaded hepatocytes was not significantly altered by 0.1 mM LS, indicating relative specificity for calcium. TDC and DC, but not TC, increased 45Ca efflux from preloaded perfused rat livers. These results showed that bile acids known to increase [Ca2+]i (CDC, DC, TDC, and TLC) also increased 45Ca efflux from hepatocytes and perfused livers and that efflux was also stimulated by LS, TLS, and LGlu. The extent of this efflux was related to the hydrophobicity of the steroid nucleus of the bile acid. It is speculated that bile acid-induced increases in [Ca2+]i activate the plasma membrane Ca2+ pump resulting in increased Ca2+ efflux.     

Essential role of sodium and chloride for theophylline-induced choleresis in the isolated perfused rat liver

Active secretion of electrolytes by hepatocytes is believed to be responsible for bile acid-independent canalicular bile flow (BAICF). Theophylline, which enhances BAICF, has been shown to enhance electrogenic Cl- secretion in a number of other epithelia. Such transport is dependent on Na+ and Cl-. Thus, the mechanism of theophylline choleresis may also involve stimulation of electrogenic Cl- secretion of the liver. This hypothesis was tested by studying the effect of ion substitution on theophylline choleresis in isolated perfused rat livers. Addition of theophylline (0.1 mmol) and dibutyryl cAMP (0.05 mmol) to 100 ml perfusate, in a single dose, increased bile flow and biliary secretion of Na+ and Cl- reversibly. These effects of theophylline were virtually abolished when perfusate Na+ (146 mM) was replaced by Li+ (146 mM) or choline+ (120 mM), and when Cl- (127 mM) was replaced by 120 mM NO-3, acetate- or isethionate-. Since even the permeable ions like Li+ and NO-3 could not substitute for Na+ and Cl-, these results show that the effect of theophylline on BAICF is specifically dependent on the presence of Na+ and Cl- in the perfusate. We propose, by analogy to other epithelia, that an electrogenic Cl- secretion mechanism is present in the liver. Theophylline, acting via cAMP, stimulates this transport process, thereby enhancing BAICF.     

EMBalance - validation of a decision support system in the early diagnostic evaluation and management plan formulation of balance disorders in primary care: study protocol of a feasibility randomised controlled trial

BackgroundBalance problems are caused by multiple factors and often lead to falls and related fractures, bringing large socio-economic costs. The complexity of balance control mechanisms, the lack of medical expertise, and the absence of specialised equipment contribute to the delayed or incorrect diagnosis and management ofthese patients. Advances in computer science have allowed the development of computer systems that support clinical diagnosis and treatment decisions based on individualised patient data. The aim of the EMBalance decision support system (DSS) is to support doctors facing this clinical challenge, to make a definitive diagnosis and implement an effective management plan. The EMBalance study will determine the accuracy of this supportive tool when used by non-specialist doctors. This study is funded by the European Union’s Seventh Framework Programme.Methods/designEMBalance is a proof-of-concept study designed as a non-commercial, international, multi-centre, single-blind, parallel-group randomised controlled trial to be carried out at four clinical sites in the United Kingdom, Germany, Greece and Belgium. The study is comprised of three stages: internal pilot, phase I (diagnosis) and stage II (management). For this purpose, 200 patients presenting with persistent dizziness (>3 months’ duration) to primary care services will be randomised to either the intervention group (diagnostic assessment with the DSS) or a control group (diagnostic assessment without the DSS). Patients allocated to the intervention group will be assessed by a doctor with the support of the EMBalance DSS, while patients allocated to the control group will receive a visit as per standard practice. Ultimately, all patients’ diagnoses and management plans will be certified by a consultant in neuro-otology.DiscussionEMBalance is the first trial to test the accuracy of a DSS in both the diagnosis of and the management plan for vestibular disorders across the healthcare systems of four different countries. The EMBalance study is the result of a combined effort of engineers and physicians to develop an accurate tool to support non-specialist doctors, with no risk for the patient. This trial will provide reliable information about the benefits of implementing DSSs in primary care while supporting the feasibility of testing the EMBalance algorithms in further research.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02704819","term_id":"NCT02704819"}}NCT02704819. Registered 29 February 2016.

Electronic supplementary material

The online version of this article (doi:10.1186/s13063-016-1568-x) contains supplementary material, which is available to authorized users.     

Nucleocapsid protein-mediated maturation of dimer initiation complex of full-length SL1 stemloop of HIV-1: sequence effects and mechanism of RNA refolding

Specific binding of HIV-1 viral protein NCp7 to a unique 35-base RNA stem-loop SL1 is critical for formation and packaging of the genomic RNA dimer found within HIV-1 virions. NCp7 binding stimulates refolding of SL1 from a metastable kissing dimer (KD) into thermodynamically stable linear dimer (LD). Using UV melting, gel electrophoresis and heteronuclear NMR, we investigated effects of various site-specific mutations within the full-length SL1 on temperature- or NCp7-induced refolding in vitro. Refolding involved intramolecular melting of SL1 stems but not dissociation of the intermolecular KD interface. Refolding required only two NCp7 molecules per KD but was limited by the amount of NCp7 present, implying that the protein does not catalytically promote refolding. Efficient refolding depended strictly on the presence and, to a lesser degree, on sequence of a highly conserved G-rich internal loop that normally limits thermal stability of the SL1 stem. Adding two base pairs to the lower stem created a hyperstable SL1 mutant that failed to refold, even when bound by NCp7at high stoichiometries. NMR analysis of these kinetically trapped mutant RNA–protein complexes indicated that NCp7 initiates refolding by dissociating base pairs in the upper stem of SL1. This study illuminates structural transitions critical for HIV-1 assembly and replication.     

Optimization of solid-phase synthesis of difficult peptide sequences via comparison between different improved approaches

Summary. Different approaches are applied to avoid the strong aggregation of the difficult peptide sequences, which is considered as the main reason for incomplete acylation and deprotection reactions hindering the synthesis of these sequences. Temporary protection of amide nitrogen of peptide bond using 2-hydroxy-4-methoxybenzyl (Hmb) and 2,4,6-timethoxybenzyl (Tmob) amino acid derivatives, introduction of D-Ala or Pro residues in the peptide chain sequences and utilization of microwave energy are proved to be useful methods in the enhancement of solubility and in the hindrance of the aggregation during the solid-phase synthesis of oligoalanine. Oligoalanine is chosen to demonstrate the difficult sequences and to compare the efficiencies of these methods.     

Screening of potential cytotoxic activities of some medicinal plants of Saudi Arabia

Phytochemicals from plant extracts belong to an important source of natural products which have demonstrated excellent cytotoxic activities. However, plants of different origins exhibit diverse chemical composition and bioactivities. Therefore, discovery of plants based new anticancer agents from different parts of the world is always challenging. In this study, methanolic extracts of different parts of 11 plants from Saudi Arabia have been tested in vitro for their anticancer potential on human liver cancer cell line (HepG2). Particularly, for this study, plants from Asteraceae, Resedaceae and Polygonaceae families were chosen on the basis of locally available ethnobotanical data and their medicinal properties. Among 12 tested extract samples, three samples obtained from Artemisia monosperma stem, Ochradenus baccatus aerial parts and Pulicaria glutinosa stem have demonstrated interesting cytotoxic activities with a cell viability of 29.3%, 28.4% and 24.2%, respectively. Whereas, four plant extracts including Calendula arvensis aerial parts, Scorzonera musilii whole plant, A. monosperma leaves show moderate anticancer properties bearing a cell viability ranging from 11.9 to 16.7%. The remaining extracts have shown poor cytotoxic activities. Subsequently, GC-MS analysis of methanolic extracts of four most active plants extracts such as C. comosum, O. baccatus, P. glutinosa and A. monosperma detected the presence of 41 phytomolecules. Among which 3-(4-hydroxyphenyl) propionitrile (1), 8,11-octadecadiynoic acid methyl ester (2), 6,7-dimethoxycoumarin (3) and 1-(2-hydroxyphenyl) ethenone (4) were found to be the lead compounds of C. comosum, O. baccatus P. glutinosa and A. monosperma, respectively.