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Membrane Preparations (membrane + preparation)
Selected AbstractsMembrane orientation of laminin binding proteinFEBS JOURNAL, Issue 18 2003An extracellular matrix bridging molecule of Leishmania donovani Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m -iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end. [source] Nitric Oxide: The "Second Messenger" of InsulinIUBMB LIFE, Issue 5 2000Nighat N. Kahn Abstract Incubation of various tissues, including heart, liver, kidney, muscle, and intestine from mice and erythrocytes or their membrane fractions from humans, with physiologic concentration of insulin resulted in the activation of a membrane-bound nitric oxide synthase (NOS). Activation of NOS and synthesis of NO were stimulated by the binding of insulin to specific receptors on the cell surface. A Lineweaver-Burk plot of the enzymatic activity demonstrated that the stimulation of NOS by insulin was related to the decrease in the Km for L-arginine, the substrate for NOS, with a simultaneous increase of Vmax. Addition of NG-nitro-L-arginine methyl ester (LNAME), a competitive inhibitor of NOS, to the reaction mixture completely inhibited the hormone-stimulated NO synthesis in all tissues. Furthermore, NO had an insulin-like effect in stimulating glucose transport and glucose oxidation in muscle, a major site for insulin action. Addition of NAME to the reaction mixture completely blocked the stimulatory effect of insulin by inhibiting both NO production and glucose metabolism, without affecting the hormone-stimulated tyrosine or phosphatidylinositol 3-kinases of the membrane preparation. Injection of NO in alloxan-induced diabetic mice mimicked the effect of insulin in the control of hyperglycemia (i.e., lowered the glucose content in plasma). However, injection of NAME before the administration of insulin to diabetic-induced and nondiabetic mice inhibited not only the insulin-stimulated increase of NO in plasma but also the glucose-lowering effect of insulin. [source] Membranes of cellulose triacetate produced from sugarcane bagasse cellulose as alternative matrices for doxycycline incorporationJOURNAL OF APPLIED POLYMER SCIENCE, Issue 6 2009Guimes Rodrigues Filho Abstract Cellulose triacetate (CTA) membranes were prepared using polyethylene glycol, 600 g mol,1, (PEG) as additive and were utilized in essays of doxycycline (DOX) incorporation using two different procedures: (i) incorporation of the drug during the membrane preparation and (ii) incorporation of the drug to a previously prepared membrane. In the first, the produced membrane presented high compatibility between DOX and CTA, what was evidenced by analyzing the DSC curve for a CTA/PEG 50%/DOX system. Results showed that the drug is homogeneously distributed throughout the matrix, molecularly. In the second method, the drug was molecularly and superficially adsorbed, as seen through the DSC curve for the system CTA/PEG 10%/DOX, which nearly does not present alterations in relation to the original material, and through the isotherm of drug adsorption that follows the Langmuir model. Results showed that the membranes produced from sugarcane bagasse are adequate to produce matrices for drug-controlled release, both for enteric use (Method (i)) and topic use (Method (ii)). © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009 [source] Characterization and expression of a novel Porphyromonas gingivalis outer membrane protein, Omp28MOLECULAR ORAL MICROBIOLOGY, Issue 3 2002N. Slakeski We report the characterization of a Porphyromonas gingivalis gene, designated omp28, encoding a protein that we have previously purified and characterized as a 28-kDa outer membrane protein. The deduced amino acid sequence of the omp28 open reading frame displayed an outer membrane leader sequence and lipoprotein attachment site but did not exhibit any significant overall sequence identity with protein sequences in the databases. A small stretch of amino acids (19 residues) exhibits 50% sequence identity with a segment of a fimbrial protein from Dichelobacter nodosus involved in adhesion, suggesting that Omp28 may be a surface adhesin/receptor of P. gingivalis. Using the pET-24 vector we expressed recombinant Omp28 (rOmp28) in Escherichia coli. Western blot analyses of purified rOmp28 with rabbit antisera to a P. gingivalis outer membrane preparation, protective rat anti-whole P. gingivalis antisera and pooled human sera from chronic periodontitis patients showed that the recombinant was recognized by all antisera. Further, anti-rOmp28 antisera exhibited strong reactivity with a panel of four laboratory strains and 10 clinical isolates of P. gingivalis from the United States, Sudan, Romania and Norway. These results suggest that Omp28 is expressed by a wide distribution of P. gingivalis strains. [source] Coconut water as a potential resource for cellulose acetate membrane preparationPOLYMER INTERNATIONAL, Issue 3 2008Cynthia Radiman Abstract BACKGROUND: Cellulose acetate membranes are frequently used for pressure-driven membrane processes. The aim of this work was to prepare cellulose acetate membranes from nata-de-coco using coconut water as starting material. The use of this lignin-free material will certainly minimize the use of chemicals usually needed in the traditional pulps and substitute for the use of wood, which helps prevent global warming and preserves nature as well. RESULTS: Coconut water was fermented by Acetobacter xylinum for 6 days to produce nata-de-coco, which was then acetylated to produce cellulose diacetate with an acetyl content of 39.6%. Fourier transform infrared analysis showed characteristic peaks for the acetyl group at 1748 and 1236 cm,1. The resulting membranes made from the hydrolysis product showed a water flux of 210.5 L m,2 h,1 under an applied pressure of 2 kg cm,2 while the rejection coefficients of dextran T-500 and T-2000 solutions were 78 and 93.7%, respectively. CONCLUSION: Coconut water has a potential to be used in the fabrication of membranes by converting it to nata-de-coco and then to cellulose diacetate which gives an added value to its original nature. It is also highly competitive compared to the traditional pulps, by which acetylation decreases the degree of crystallinity of nata-de-coco resulting in higher membrane permeability. Copyright © 2007 Society of Chemical Industry [source] Glycoproteomics and glycomics investigation of membrane N -glycosylproteins from human colon carcinoma cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2008Anne-Sophie Vercoutter-Edouart Dr. Abstract Aberrant glycosylation of proteins is known to profoundly affect cellular adhesion or motility of tumoral cells. In this study, we used HT-29 human colon epithelial cancer cells as a cellular model of cancer progression, as they can either proliferate or differentiate into enterocyte phenotype. A glycoproteomic approach based on Con A lectin-affinity chromatography, SDS-PAGE and MS analysis, allowed the identification of membrane N -glycoproteins from Triton X-100-solubilized proteins from membrane preparation. Among them, 65% were membrane proteins, and 45% were known to be N -glycosylated, such as , chains integrin and dipeptidyl isomerase IV. By lectin-blot analysis, significant changes of ,-2,3- and ,-2,6-sialylation of membrane glycoproteins were observed between proliferating and differentiated HT-29 cells. From these results, nano-LC-MS/MS analysis of the tryptic digests of the corresponding bands was performed and led to the identification of several transmembrane glycoproteins, like members of the solute carrier family and adhesion proteins. Finally, we compared N -glycans profiles and monosaccharide composition of proliferating and enterocyte-like HT-29 cells using MALDI-MS and GC-MS analyses of permethylated derivatives. This glycomic approach allowed to underscore significant changes in N -glycans structure, in particular the expression of atypical N -acetylglucosamine (GlcNAc)-ended N -glycans in enterocyte-like HT-29 cells. [source] New poly ether ether ketones containing phosphorus for membrane preparationASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING, Issue 1 2010Francesco Trotta Abstract In this study is reported the synthetic procedure and the characterisation of novel phosphorus containing polyether ether ketone (PEEK-P). The new polymer was synthesized via direct polycondensation of 2,2-bis(3-diethylphosphono-4-hydroxyphenyl)-propane and 4,4,-difluorobenzophenone according to well known general procedure and was extensively characterized by using infrared spectroscopy (FT-IR), thermogravimetric analyses (TGA), viscosity measurements and ,H-NMR analysis. The obtained polymer is soluble in some organic solvents and the solution of phosphonated polymer could be used to obtain membranes by using the phase inversion technique. Copyright © 2009 Curtin University of Technology and John Wiley & Sons, Ltd. [source] Tandem Exploitation of Helix pomatia Glycosyltransferases: Facile Syntheses of H-Antigen-Bearing OligosaccharidesCHEMISTRY - A EUROPEAN JOURNAL, Issue 25 2007Hagen Bretting Prof. Abstract Snails from the family Helicidae produce in their albumen glands a highly branched galactan, which consists almost exclusively of D - and L -galactose. The D -Gal residues are glycosydically ,(1,6)- or ,(1,3)-linked, whereas the L -Gal moieties are attached ,(1,2). Up until the present time, two ,(1,6)- D -galactosyl transferases and one ,(1,2)- L -galactosyl transferase have been identified in a membrane preparation of these glands. These were used to synthesise various oligosaccharides by successive addition of the NDP-activated (NDP=nucleoside-5,-diphosphate) D -Gal or L -Fuc moieties, up to a heptasaccharide by starting from the disaccharide D -Gal-,(1,3)- D -Gal-,(1,OMe. Even larger oligosaccharides up to a tridecasaccharide were obtained by starting with the hexasaccharide D -Gal-[,(1,3)- D -Gal]4 -,(1,4)- D -Glc as an acceptor substrate. This tandem exploitation process has high potential for the easy introduction of D -Gal and L -Fuc residues into a great variety of oligosaccharides, which can be used in ligand/acceptor studies. [source] Cover Picture: Electrophoresis 21'2008ELECTROPHORESIS, Issue 21 2008Article first published online: 14 NOV 200 This issue has an emphasis on "Proteomics and Related Topics". It comprises 11 research articles including the "Fast Track" article on the topic of proteomics, glycoproteomics, proteins and peptides. The "Fast Track" article describes a CE-LIF detection-based assay for the simultaneous measurements of the electrophoretic mobility, catalytic activity and the variation of activity over time of the individual enzymes molecules of Escherichia coli beta-galactosidase. The remaining 10 research articles of the Emphasis deal with the development of sensitive fluorescent staining for proteomic analysis, depletion of high abundance proteins form human serum, lectin affinity chromatography in the identification of rat urinary glycoproteome, lab-on-chip screening strategy of mouse serum samples prior to proteomics analysis, identification of proteins from membrane preparations, capillary coating for CE of proteins, characterization of rabbit liver apothioneins by CE-ESI-MS, quantitative analysis of recombinant protein charge heterogeneity by imaging CIEF, dye staining and immunodetection of proteins on a PVDF membrane, and separation of multiphosphorylated peptide isomers by CZE. [source] An effective skeletal muscle prefractionation method to remove abundant structural proteins for optimized two-dimensional gel electrophoresisELECTROPHORESIS, Issue 11 2005Bradley Jarrold Abstract Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes. [source] Response of the charophyte Nitellopsis obtusa to heavy metals at the cellular, cell membrane, and enzyme levelsENVIRONMENTAL TOXICOLOGY, Issue 3 2002Levonas Manusad, ianas Abstract The responses of the freshwater macroalga Nitellopsis obtusa to heavy metal (HM) salts of Hg, Cd, Co, Cu, Cr, and Ni were assessed at different levels: whole-cell mortality (96-h LC50), in vivo cell membrane (45-min depolarization of resting potential, EC50), and enzyme in plasma membrane preparations (K+, Mg2+ -specific H+ -ATPase inhibition, IC50). To measure ATPase activity, a novel procedure for isolation of plasma membrane,enriched vesicles from charophyte cells was developed. The short-term ATPase inhibition assay (IC50 from 6.0 × 10,7 to 4.6 × 10,4 M) was slightly more sensitive than the cell mortality test (LC50 from 1.1 × 10,6 to 2.6 × 10,3 M), and the electrophysiological test with the end point of 45-min depolarization of resting potential was characterized by less sensitivity for HMs (EC50 from 1.1 × 10,4 to 2.2 × 10,2 M). The variability of IC50 values assessed for HMs in the ATPase assays was close to that of LC50 values in the mortality tests (CVs from 33.5 to 83.5 and from 12.4% to 57.7%, respectively), whereas the EC50 values in the electrophysiological tests were characterized by CVs generally below 30%. All three end points identified two separate HM groups according to their toxicity to N. obtusa: Co, Ni, and Cr comprised a group of less toxic metals, whereas Hg, Cu, and Cd comprised a group of more toxic metals. However, the adverse effects within each group were discriminated differently. For example, the maximum difference between the highest and lowest LC50 for the group of less toxic metals in the long-term mortality test was approximately 60% of the response range, whereas the corresponding difference in IC50 values in the ATPase assay was 30%. In contrast, the LC50 values of the more toxic metals occupied only 10% of the response range, whereas the IC50 values were spread over 70%. Further investigation should be done of the underlying mechanism or mechanisms responsible for the observed differences in the dynamic range of a particular end point of the groups of toxicants of varying strength. © 2002 Wiley Periodicals, Inc. Environ Toxicol 17: 275,283, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.10058 [source] Retina expresses a novel variant of the ryanodine receptorEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2007Varda Shoshan-Barmatz Abstract Calcium released from intracellular stores via the ryanodine receptor (RyR) mediates a variety of signalling processes. We previously showed that retina expresses the three known types of RyR, but retinal membrane preparations exhibit unique characteristics such as Ca2+ -independent [3H]ryanodine-binding and inhibition by caffeine. We have heretofore suggested that the major retinal RyR isoform is novel. The present study aimed to identify this receptor isoform and to localize RyR in mammalian retina. Immunoblotting with specific and pan-antibodies showed that the major retinal RyR has a mobility similar to that of RyR2 or RyR3. Real-time PCR revealed that the major type is RyR2, and RT-PCR followed by sequencing showed a transcript that encodes a protein with ~ 99% identity to RyR2, yet lacking two regions of seven and 12 amino acids and including an additional insertion of eight amino acids. An antibody against RyR2 localized this type to somas and primary dendrites of most retinal neurons. An antibody against RyR1 localized RyR to most somas but also revealed staining in photoreceptor outer segments, concentrated on the disk membranes at their rim. The ryanodine-binding properties and the electrophoretic mobility of RyR from the outer segments were similar to those of the whole retinal preparation. The results thus identify a novel variant of RyR2 which can contribute to regulating photoreceptor Ca2+ concentrations. The restricted localization of the outer segment RyR to the disk rim suggests that its activation mechanism involves a coupling between retinal RyR and the cGMP-gated channel. [source] Membrane-associated guidance cues direct the innervation of forebrain and midbrain by dorsal raphe-derived serotonergic axonsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2005Audrey Petit Abstract Unlike many neurons that extend an axon precisely to a single target, individual dorsal raphe 5-HT neurons project to multiple brain regions and their axon terminals often lack classical synaptic specializations. It is not known how 5-HT axon collaterals select between multiple target fields, or even if 5-HT axons require specific guidance cues to innervate their targets. Nor is it known how these axon collaterals are restrained within specific innervation target regions. To investigate this, we challenged explants of dorsal raphe with co-explants, or cell membrane preparations of ventral midbrain, striatum or cerebral cortex. We provide evidence for membrane-associated cues that promote 5-HT axon growth into each of these three target regions. The axon growth-promoting activity was heat-, protease- and phosphatidylinositol-phospholipase-C (PI-PLC)-sensitive. Interestingly, 5-HT axons specifically lost the ability to grow in heterotypic explants, or membrane carpets, following contact with ventral midbrain or striatal, but not cortical, explants or membranes. This inductive activity associated with striatal and ventral midbrain membranes was sensitive to both high salt extraction and PI-PLC treatment. By contrast, the activity that inhibited 5-HT axon growth onto heterotypic membranes was sensitive only to high salt extraction. These results provide evidence that a glycosylphosphatidylinositol (GPI)-linked membrane protein promotes 5-HT axon growth, and that short-range membrane-bound, as well as GPI-linked, molecules contribute to the guidance of 5-HT axon collaterals. These findings suggest that 5-HT axon collaterals acquire a target-induced growth-inhibitory response to alternative targets, increasing their selectivity for the newly innervated field. [source] Metabotropic glutamate receptor 5 localized in the limbic forebrain is critical for the development of morphine-induced rewarding effect in miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2004Takeshi Aoki Abstract The aim of the present study was to clarify the role of the metabotropic glutamate 5 (mGlu5) receptor subtype in the development of rewarding effect induced by a prototypical µ-opioid receptor agonist morphine in the mouse. In the conditioned place preference paradigm, intracerebroventricular (i.c.v.) administration of a selective mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), attenuated the morphine-induced rewarding effects. Using immunoblot analysis, we confirmed that the increased level of protein kinase C, (PKC,) isoform was observed in the limbic forebrain of ICR mice conditioned with morphine. Here we found for the first time that the treatment with MPEP significantly inhibited the up-regulation of PKC, isoform in the limbic forebrain of mice showing the significant place preference. Furthermore, it should be mentioned that the protein level of mGlu5 was significantly increased in membrane preparations of the limbic forebrain obtained from morphine-conditioned mice compared to those from saline-conditioned mice. As well as the result from the immunoblot analysis, we demonstrated using the receptor binding assay that the number of mGlu5 receptors in the mouse limbic forebrain was significantly increased by morphine conditioning. The present data provide direct evidence that the activation of mGlu5 receptor linked to the increased PKC, isoform in the mouse limbic forebrain is implicated in the development of rewarding effect of morphine. [source] Low-density caveolae-like membrane from Xenopus laevis oocytes is enriched in RasJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2001Susan E. SadlerArticle first published online: 10 JUL 200 Abstract Detergent-free discontinuous sucrose density gradient centrifugation was used to resolve low- and high-density membrane fractions from Xenopus laevis oocytes. Compared to high-density membrane, low-density oocyte membrane is enriched two-fold in cholesterol and highly enriched in ganglioside GM1. Protein immunoblotting of membrane fractions from whole cells with polyclonal anti-human caveolin antibody detected multiple bands, including a distinctive triad with apparent molecular weights of 21, 33, and 48 kDa. To more clearly determine which of these caveolin-like protein(s) is associated with the oocyte plasma membrane, microdissection was used to separate external membrane (cortical preparations containing plasma membrane) from intracellular membrane. Cortical membrane preparations displayed a single 21-kDa caveolin-like protein in low-density membrane. Internal oocyte membrane displayed the higher molecular weight bands of 33 and 48 kDa and a lesser amount of the 21-kDa protein in low-density membrane fractions. Monoclonal anti-human Ras antibody detected a single 23-kDa immunoblot band that is enriched an average of eight-fold in low-density membrane fractions prepared from whole cells. This is the first report of caveolin-associated, low-density membrane in amphibian oocytes, and is consistent with a role for caveolin and caveolae-like microdomains in oocyte signal transduction. © 2001 Wiley-Liss, Inc. [source] Transmembrane signaling through phospholipase C-, in the developing human prefrontal cortexJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2006Iñigo Ruiz de Azúa Abstract To investigate changes in muscarinic receptor-stimulated phospholipase C-, (PLC-,) activity during brain development, we examined the functional coupling of each of the three major protein components of the phosphoinositide system (M1, M3, and M5 muscarinic receptor subtypes; Gq/11 proteins; PLC-,1,4 isoforms) in membrane preparations from post-mortem human prefrontal cerebral cortex collected at several stages of prenatal and postnatal development. In human prenatal brain membranes, PLC was found to be present and could be activated by calcium, but the ability of guanosine-5,-o-3 thiotriphosphate (GTP,S) or carbachol (in the presence of GTP,S) to modulate prenatal PLC-, was significantly weaker than that associated with postnatal PLC-,. Western blot analysis revealed that the levels of G,q/11 did not change significantly during development. In contrast, dramatically higher levels of expression of PLC-,1,4 isoforms and of M1, M3, and M5 muscarinic receptors were detected in the child vs. the fetal brain, a finding that might underlie the observed increased activity of PLC. Thus, inositol phosphate production may be more efficiently regulated by altering the amount of effectors (PLC-,1,4) and receptors (M1,3,5 subtypes) than by altering the level of G,q/11 subunits. These results demonstrate that different PLC isoforms are expressed in the prefrontal cortex of the developing human brain in an age-specific manner, suggesting specific roles not only in synaptic transmission but also in the differentiation and maturation of neurons in the developing brain. © 2006 Wiley-Liss, Inc. [source] Small potent ligands to the insulin-regulated aminopeptidase (IRAP)/AT4 receptorJOURNAL OF PEPTIDE SCIENCE, Issue 7 2007Andreas Axén Abstract Angiotensin IV analogs encompassing aromatic scaffolds replacing parts of the backbone of angiotensin IV have been synthesized and evaluated in biological assays. Several of the ligands displayed high affinities to the insulin-regulated aminopeptidase (IRAP)/AT4 receptor. Displacement of the C -terminal of angiotensin IV with an o -substituted aryl acetic acid derivative delivered the ligand 4, which exhibited the highest binding affinity (Ki = 1.9 nM). The high affinity of this ligand provides support to the hypothesis that angiotensin IV adopts a ,-turn in the C -terminal of its bioactive conformation. Ligand (4) inhibits both human IRAP and aminopeptidase N-activity and induces proliferation of adult neural stem cells at low concentrations. Furthermore, ligand 4 is degraded considerably more slowly in membrane preparations than angiotensin IV. Hence, it might constitute a suitable research tool for biological studies of the (IRAP)/AT4 receptor. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Characterization of HMW-PBPs from the rod-shaped actinomycete Corynebacterium glutamicum: peptidoglycan synthesis in cells lacking actin-like cytoskeletal structuresMOLECULAR MICROBIOLOGY, Issue 3 2007Noelia Valbuena Summary Analysis of the complete genome sequence of Corynebacterium glutamicum indicated that, in addition to ftsI, there are eight proteins with sequence motifs that are strongly conserved in penicillin binding proteins (PBPs): four genes that code for high-molecular-weight (HMW)-PBPs (PBP1a, PBP1b, PBP2a and PBP2b), two genes encoding low-molecular-weight PBPs (PBP4 and PBP4b) and two probable ,-lactamases (PBP5 and PBP6). Here, the function of the four HMW-PBPs in C. glutamicum was investigated using a combination of genetic knockouts, enhanced green fluorescent protein 2 (EGFP2) fusions and penicillin staining of membrane preparations. The four HMW-PBPs were expressed in a growing culture of C. glutamicum, but none of four pbp genes was individually essential for the growth of the bacterium, and only the simultaneous disruption of both pbp1b and pbp2b was lethal. The fused EGFP2,PBP proteins were functional in vivo, which allowed correct determination of their cellular localization. EGFP2 fusions to PBP1a, PBP1b and PBP2b localized at the poles and at the septum, whereas EGFP2,PBP2a was predominantly found at the septum. Cefsulodin treatment specifically delocalized PBP1a and PBP1b (class A HMW-PBPs), whereas mecillinam caused the specific delocalization of PBP2b and PBP2a (class B HMW-PBPs). The results provide new insight into the mechanisms involved in the synthesis of the cell wall in this bacterial species, which lacks a known actin-like cytoskeletal structure. [source] Mechanism of association of adenylate cyclase toxin with the surface of Bordetella pertussis: a role for toxin,filamentous haemagglutinin interactionMOLECULAR MICROBIOLOGY, Issue 6 2002Franca R. Zaretzky Summary Adenylate cyclase (AC) toxin from Bordetella per-tussis is unusual in that, unlike most other members of the repeats-in-toxin family that are released into the extracellular milieu, it remains associated with the bacterial surface. In this study, we investigated the nature of the association of this toxin with the surface of B. pertussis. AC toxin was extracted from crude outer membrane preparations of B. pertussis with 8 M urea, but only partially with alkaline sodium carbonate and not at all with octylglucoside, suggesting that denaturation of the toxin is necessary for its removal from the membrane. B. pertussis mutants lacking filamentous haemagglutinin (FHA) released significantly more AC toxin into the medium, and AC toxin association with the bacterial surface was partially restored by expression of FHA from a plasmid, suggesting a role for FHA in surface retention of AC toxin. AC toxin distribution was unaffected by the absence of pertactin, or full-length lipopolysaccharide, or a defect in secretion of pertussis toxin. Using overlay and immunoprecipitation, we found that a direct physical association can occur between AC toxin and FHA. Combined, these findings suggest that FHA may play a role in AC toxin retention on the surface of B. pertussis and raise the possibility of an involvement of adherence mediated by FHA in delivery of AC toxin from the bacterium to the target cell. [source] Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera: Apidae)PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 7 2001Ralf Nauen Abstract Acute oral and contact toxicity tests of imidacloprid, an insecticide acting agonistically on nicotinic acetylcholine receptors (nAChR), to adult honeybees, Apis mellifera L var carnica, were carried out by seven different European research facilities. Results indicated that the 48-h oral LD50 of imidacloprid is between 41 and >81,ng per bee, and the contact LD50 between 49 and 102,ng per bee. The ingested amount of imidacloprid-containing sucrose solution decreased with increasing imidacloprid concentrations and may be attributed to dose-related sub-lethal intoxication symptoms or to antifeedant responses. Some previously reported imidacloprid metabolites occuring at low levels in planta after seed dressing, ie olefine-, 5-OH- and 4,5-OH-imidacloprid, showed lower oral LD50 values (>36, >49 and 159,ng per bee, respectively) compared with the concurrently tested parent molecule (41,ng per bee). The urea metabolite and 6-chloronicotinic acid (6-CNA) exhibited LD50 values of >99,500 and >121,500,ng per bee, respectively. The pharmacological profile of the [3H]imidacloprid binding site in honeybee head membrane preparations is consistent with that anticipated for a nAChR. IC50 values for the displacement of [3H]imidacloprid by several metabolites such as olefine, 5-OH-, 4,5-OH-imidacloprid, urea and 6-CNA were 0.45, 24, 6600, >100,000, and >100,000,nM, respectively. Displacement of [3H]imidacloprid by imidacloprid revealed an IC50 value of 2.9,nM, thus correlating well with the observed acute oral toxicity of the compounds in honeybees. Neurons isolated from the antennal lobe of A mellifera and subjected to whole-cell voltage clamp electrophysiology responded to the application of 100,µM acetylcholine with a fast inward current of between 30 and 1600 pA at ,70,mV clamp potential. Imidacloprid and two of the metabolites (olefine- and 5-OH-imidacloprid) acted agonistically on these neurons, whereas the others did not induce currents at test conencentrations up to 3,mM. The electrophysiological data revealed Hill coefficients of approximately 1, indicating a single binding site responsible for an activation of the receptor and no direct cooperativity or allosteric interaction with a second binding site. © 2001 Society of Chemical Industry [source] Proteomic examination of Leishmania chagasi plasma membrane proteins: Contrast between avirulent and virulent (metacyclic) parasite formsPROTEOMICS - CLINICAL APPLICATIONS, Issue 1 2010Chaoqun Yao Abstract Purpose: About two million new cases of leishmaniasis with 50 000 associated deaths occur worldwide each year. Promastigotes of the causative Leishmania spp. develop from the procyclic stage to the highly virulent metacyclic stage within the sand fly vector. We hypothesized that proteins important for promastigote virulence might be uniquely represented in the plasma membrane of metacyclic, but not procyclic, promastigotes. Experimental design: Procyclic (logarithmic) promastigotes and purified metacyclic promastigotes from stationary phase cultures of Leishmania chagasi were used to prepare membrane preparations either by surface biotinylation-streptavidin affinity separation or by octyl glucoside detergent extraction. Results: These membrane fractions were enriched over 130- and 250-fold, respectively, as estimated by Western blotting for the plasma membrane's major surface protease. Hundreds or dozens of proteins were identified by LC-MS/MS in the surface biotinylation or detergent extraction, respectively. Confocal microscopy suggested the difference between the lists was due to the fact that proteins localized both on the surface membrane and within the flagellar pocket were accessible to surface biotinylation, whereas only proteins on the membrane were obtained by detergent extraction. Using detergent extraction, we found different proteins were present in membranes of the procyclic stage compared to metacyclic stage promastigotes. Several dozen were stage specific. Conclusions and clinical relevance: These data provide a foundation for identifying virulence factors in the plasma membranes of Leishmania spp. promastigotes during metacyclogenesis. [source] Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysisRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2003Christian Ihling Fourier transform ion-cyclotron resonance (FTICR) mass spectrometry offers several advantages for the analysis of biological samples, including excellent mass resolution, ultra-high mass measurement accuracy, high sensitivity, and wide mass range. We report the application of a nano-HPLC system coupled to an FTICR mass spectrometer equipped with nanoelectrospray source (nano-HPLC/nano-ESI-FTICRMS) for proteome analysis. Protein identification in proteomics is usually conducted by accurately determining peptide masses resulting from enzymatic protein digests and comparing them with theoretically digested protein sequences from databases. A tryptic in-solution digest of bovine serum albumin was used to optimize experimental conditions and data processing. Spots from Coomassie Blue and silver-stained two-dimensional (2D) gels of human thyroid tissue were excised, in-gel digested with trypsin, and subsequently analyzed by nano-HPLC/nano-ESI-FTICRMS. Additionally, we analyzed 1D-gel bands of membrane preparations of COS-6 cells from African green monkey kidney as an example of more complex protein mixtures. Nano-HPLC was performed using 1-mm reverse-phase C-18 columns for pre-concentration of the samples and reverse-phase C-18 capillary columns for separation, applying water/acetonitrile gradient elution conditions at flow rates of 200,nL/min. Mass measurement accuracies smaller than 3,ppm were routinely obtained. Different methods for processing the raw data were compared in order to identify a maximum number of peptides with the highest possible degree of automation. Parallel identification of proteins from complex mixtures down to low-femtomole levels makes nano-HPLC/nano-ESI-FTICRMS an attractive approach for proteome analysis. Copyright © 2003 John Wiley & Sons, Ltd. [source] Diabetes-induced Alterations in Latissimus Dorsi Muscle Properties Impair Effectiveness of Dynamic Cardiomyoplasty in RatsARTIFICIAL ORGANS, Issue 4 2004Kátia De Angelis Abstract:, Short-term diabetes was induced in male Wistar rats with streptozotocin injection. The effects of diabetes on latissimus dorsi (LD) muscle contractile and biochemical properties and acute cardiomyoplasty (CDM) were assessed and compared with data from 16 control rats. Isometric force, contractile properties, and fatigue were measured in electrically stimulated muscles (0.3 ms, 1,256 Hz), and Na+K+ and Ca2+ATPase activities were quantified in muscle membrane preparations. Systolic arterial pressure and aortic blood flow were recorded at rest and during LD muscle stimulation. Compared with control muscle, diabetic muscle showed smaller maximum specific tetanic tension and lower rates of rise and fall in force. Diabetic LD muscle also showed lower muscle enzyme activities. Twitch tension and fatigue did not differ between groups. Smaller increases in aortic flow and systolic pressure after CDM were found in diabetic rats compared to controls. The marked decrease in CDM effectiveness in diabetic rats likely reflected the alterations in muscle properties associated with diabetes. [source] Pharmacological characterization of a novel investigational antimuscarinic drug, fesoterodine, in vitro and in vivoBJU INTERNATIONAL, Issue 8 2008Peter Ney OBJECTIVE To investigate the primary pharmacology of fesoterodine (a novel antimuscarinic drug developed for treating overactive bladder) and SPM 7605 (its active metabolite, considered to be the main pharmacologically active principle of fesoterodine in man) against human muscarinic receptor subtypes, and to investigate in vitro and in vivo functional activity of these agents on the rat bladder compared with existing standard agents. MATERIALS AND METHODS The displacement of radioligand binding by fesoterodine, SPM 7605 and standard agents in membrane preparations of Chinese hamster ovary (CHO) cells expressing the different human muscarinic receptors (M1,M5) was characterized. Agonistic and antagonistic activities were studied using different CHO cell lines stably expressing the human recombinant muscarinic receptor subtypes. The effects of fesoterodine and SPM 7605 on isolated bladder strips contracted by carbachol or electrical field stimulation (EFS) were investigated. In vivo the effects of fesoterodine and SPM 7605 on micturition variables were assessed using continuous cystometry in conscious female Sprague-Dawley rats, and compared to those of oxybutynin and atropine. RESULTS In vitro SPM 7605 potently inhibited radioligand binding at all five human muscarinic receptor subtypes with equal affinity across all five. Fesoterodine had a similar balanced selectivity profile but was less potent than SPM 7605. Both substances were competitive antagonists of cholinergic agonist-stimulated responses in human M1-M5 cell lines and had a similar potency and selectivity profile to the radioligand-binding studies. In rat bladder strips, fesoterodine and SPM 7605 caused a rightward shift of the concentration-response curve for carbachol with no depression of the maximum, and concentration-dependently reduced contractions induced by EFS. The potency of both drugs was similar to that of atropine and oxybutynin. In the presence of the esterase inhibitor neostigmine, the concentration-response curve of fesoterodine was shifted to the right, suggesting that part of the activity was caused by metabolism to SPM 7605 by tissue enzymes. In vivo, low doses (0.01 mg/kg) of fesoterodine and SPM 7605 reduced micturition pressure and increased intercontraction intervals and bladder capacity, but did not affect residual volume. CONCLUSIONS Fesoterodine and its active metabolite, SPM 7605, are nonsubtype selective, competitive antagonists of human muscarinic receptors, but SPM 7605 has greater potency than the parent compound. Pharmacodynamic studies in the rat bladder in vitro confirm the competitive muscarinic antagonist profile of these agents in a native tissue preparation, and in vivo studies in the rat showed effects on bladder function consistent with a muscarinic antagonist profile. [source] An endogenous regulator of inflammation, resolvin E1, modulates osteoclast differentiation and bone resorptionBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2008B S Herrera Background and purpose: The inflammation-resolving lipid mediator resolvin E1 (RvE1) effectively stops inflammation-induced bone loss in vivo in experimental periodontitis. It was of interest to determine whether RvE1 has direct actions on osteoclast (OC) development and bone resorption. Experimental approach: Primary OC cultures derived from mouse bone marrow were treated with RvE1 and analysed for OC differentiation, cell survival and bone substrate resorption. Receptor binding was measured using radiolabelled RvE1. Nuclear factor (NF)-,B activation and Akt phosphorylation were determined with western blotting. Lipid mediator production was assessed with liquid chromatography tandem mass spectrometry. Key results: OC growth and resorption pit formation were markedly decreased in the presence of RvE1. OC differentiation was inhibited by RvE1 as demonstrated by decreased number of multinuclear OC, a delay in the time course of OC development and attenuation of receptor activator of NF-,B ligand-induced nuclear translocation of the p50 subunit of NF-,B. OC survival and apoptosis were not altered by RvE1. Messenger RNA for both receptors of RvE1, ChemR23 and BLT1 is expressed in OC cultures. Leukotriene B4 (LTB4) competed with [3H]RvE1 binding on OC cell membrane preparations, and the LTB4 antagonist U75302 prevented RvE1 inhibition of OC growth, indicating that BLT1 mediates RvE1 actions on OC. Primary OC synthesized the RvE1 precursor 18R -hydroxy-eicosapentaenoic acid and LTB4. Co-incubation of OC with peripheral blood neutrophils resulted in transcellular RvE1 biosynthesis. Conclusions and implications: These results indicate that RvE1 inhibits OC growth and bone resorption by interfering with OC differentiation. The bone-sparing actions of RvE1 are in addition to inflammation resolution, a direct action in bone remodelling. British Journal of Pharmacology (2008) 155, 1214,1223; doi:10.1038/bjp.2008.367; published online 22 September 2008 [source] Separation of cannabinoid receptor affinity and efficacy in delta-8-tetrahydrocannabinol side-chain analoguesBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2001Graeme Griffin The activities of a number of side-chain analogues of delta-8-tetrahydrocannabinol (,8 -THC) in rat cerebellar membrane preparations were tested. The affinities of each compound for the CB1 receptor were compared by their respective abilities to displace [3H]-SR141716A and their efficacies compared by stimulation of [35S]-GTP,S binding. It was found that the affinities varied from 0.19±0.03 nM for 3-norpentyl-3-[6,-cyano,1,,1,-dimethyl]hexyl-,8 -THC to 395±66.3 nM for 5,-[N-(4-chlorophenyl)]-1,,1,-dimethyl-carboxamido-,8 -THC. The efficacies of these compounds varied greatly, ranging from the very low efficacy exhibited to acetylenic compounds such as 1,-heptyn-,8 -THC and 4,-octyn-,8 -THC to higher efficacy compounds such as 5,-(4-cyanophenoxy)-1,,1,-dimethyl-,8 -THC and 5,-[N-(4-aminosulphonylphenyl)]-1,,1, dimethyl-carboxamido ,8 -THC. All agonist activities were antagonized by the CB1 -selective antagonist SR141716A. It was found that a ligand's CB1 affinity and efficacy are differentially altered by modifications in the side-chain. Decreasing the flexibility of the side-chain reduced efficacy but largely did not alter affinity. Additionally, the positioning of electrostatic moieties, such as cyano groups, within the side-chain also has contrasting effects on these two properties. In summary, this report details the characterization of a number of novel ,8 -THC analogues in rat cerebellar membranes. It provides the first detailed pharmacological analysis of how the inclusion of electrostatic moieties in the side-chain and also how alteration of the side-chain's flexibility may differentially affect a CB1 cannabinoid receptor ligand's affinity and efficacy. British Journal of Pharmacology (2001) 132, 525,535; doi:10.1038/sj.bjp.0703827 [source] Characterisation of ligand binding to the parathyroid hormone/parathyroid hormone-related peptide receptor in MCF7 breast cancer cells and SaOS-2 osteosarcoma cellsCELL BIOCHEMISTRY AND FUNCTION, Issue 2 2007Majed S. Alokail Abstract Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH)/PTHrP-receptor, PTH/PTHrP-R, are frequently expressed in mammary carcinomas as well as in bone cells. In this study we compared the ligand binding characteristics of the PTH/PTHrP,R in SaOS-2 human osteosarcoma cells with those in MCF7 breast cancer cells. We used both Scatchard analysis of saturation kinetics for iodinated ligand and the level of expressed receptor protein by visualising the single radio-labelled receptor-ligand complex from isolated membrane preparations from the two cell lines. In MCF7 cells, ligand binding, (receptor number) was increased by prior exposure of the cultured cells to epidermal growth factor (EGF), estradiol (E2), or dexamethasone (DEX), and decreased following calcitriol (1,25 DHCC). In contrast in the SaOS-2 cells, PTH/PTHrP-R number was increased by exposure to E2 and 1,25DHCC and decreased by DEX while EGF had no effect. These data were confirmed when the PTH/PTHrP-R was cross linked with 125I-PTHrP-1-34Tyr, and extended by visualising the intensity of the isolated radiolabelled receptor complex by autoradiography following SDS-PAGE at several time points during the treatment. Copyright © 2005 John Wiley & Sons, Ltd. [source] | |||||||||||||||||||||||||||||||