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Surface Membrane (surface + membrane)

Distribution by Scientific Domains
Life Sciences41%
Medical Sciences33%

Selected Abstracts

Ca2+ - and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: From microtubules to the plasma membrane

GLIA, Issue 12 2009
J. W. Ou
Abstract Large-conductance, voltage- and Ca2+ -activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming ,-subunits (MaxiK,) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiK, is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiK, was visualized by three independent labeling approaches: (1) MaxiK,-specific antibodies, (2) expressed EGFP-labeled MaxiK,, and (3) fluorophore-conjugated iberiotoxin, a specific MaxiK pore-blocker. This MaxiK, association with microtubules was further confirmed by in vitro His-tag pulldown, co-immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiK, labeling at the plasma membrane. More notably, U46619, an analog of thromboxane A2 (TXA2), which triggers Ca2+ -release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiK, surface labeling. Whole-cell patch clamp recordings of U46619-stimulated cells develop a ,3-fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiK,. The results of this study provide the novel insight that suggests that Ca2+ released from intracellular stores may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiK, stores to the plasma membrane of developing murine astrocytes. © 2009 Wiley-Liss, Inc. [source]

The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2010
Jason Mulvenna
Abstract Infection with the human liver fluke, Opisthorchis viverrini, is a serious public health problem in Thailand, Laos and nearby locations in Southeast Asia. Both experimental and epidemiological evidence strongly implicate liver fluke infection in the etiology of one of the liver cancer subtypes, cholangiocarcinoma (CCA). To identify parasite proteins critical for liver fluke survival and the etiology of CCA, OFFGEL electrophoresis and multiple reaction monitoring were employed to characterize 300 parasite proteins from the O. viverrini excretory/secretory products and, utilizing selective labeling and sequential solubilization, from the host-exposed tegument. The excretory/secretory included a complex mixture of proteins that have been associated with cancers, including proteases of different mechanistic classes and orthologues of mammalian growth factors and anti-apoptotic proteins. Also identified was a cysteine protease inhibitor which, in other helminth pathogens, induces nitric oxide production by macrophages, and, hence may contribute to malignant transformation of inflamed cells. More than 160 tegumental proteins were identified using sequential solubilization of isolated teguments, and a subset of these was localized to the surface membrane of the tegument by labeling living flukes with biotin and confirming surface localization with fluorescence microscopy. These included annexins, which are potential immuno-modulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin-2. Novel roles in pathogenesis were suggested for the tegument,host interface since more than ten surface proteins had no homologues in the public databases. The O. viverrini proteins identified here provide an extensive catalogue of novel leads for research on the pathogenesis of opisthorchiasis and the development of novel interventions for this disease and CCA, as well as providing a scaffold for sequencing the genome of this fluke. [source]

Proteomic examination of Leishmania chagasi plasma membrane proteins: Contrast between avirulent and virulent (metacyclic) parasite forms

PROTEOMICS - CLINICAL APPLICATIONS, Issue 1 2010
Chaoqun 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]

Depletion of membrane cholesterol eliminates the Ca2+ -activated component of outward potassium current and decreases membrane capacitance in rat uterine myocytes

THE JOURNAL OF PHYSIOLOGY, Issue 2 2007
A. Shmygol
Changes in membrane cholesterol content have potent effects on cell signalling and contractility in rat myometrium and other smooth muscles. We have previously shown that depletion of cholesterol with methyl-,-cyclodextrin (MCD) disrupts caveolar microdomains. The aim of this work was to determine the mechanism underlying the increase in Ca2+ signalling and contractility occurring in the myometrium with MCD. Patch clamp data obtained on freshly isolated myocytes from the uterus of day 19,21 rats showed that outward K+ current was significantly reduced by MCD. Membrane capacitance was also reduced. Cholesterol-saturated MCD had no effect on the amplitude of outward current suggesting that the reduction in the outward current was due to cholesterol depletion induced by MCD rather than a direct inhibitory action of MCD on the K+ channels. Confocal visualization of the membrane bound indicator Calcium Green C18, revealed internalization of the surface membrane with MCD treatment. Large conductance, Ca2+ -sensitive K+ channel proteins have been shown to localize to caveolae. When these channels were blocked by iberiotoxin outward current was significantly reduced in the uterine myocytes; MCD treatment reduced the density of outward current. Following reduction of outward current by MCD pretreatment, iberiotoxin was unable to produce any additional decrease in the current, suggesting a common target. MCD treatment also increased the amplitude and frequency of spontaneous rises in cytosolic Ca2+ level ([Ca2+]i transients) in isolated myocytes. In intact rat myometrium, MCD treatment increased Ca2+ signalling and contractility, consistent with previous findings, and this effect was also found to be reduced by BK channel inhibition. These data suggest that (1) disruption of cholesterol-rich microdomains and caveolae by MCD leads to a decrease in the BK channel current thus increasing cell excitability, and (2) the changes in membrane excitability produced by MCD underlie the changes found in Ca2+ signalling and uterine contractility. [source]

Non-SCN5A Related Brugada Syndromes: Verification of Normal Splicing and Trafficking of SCN5A Without Exonic Mutations

ANNALS OF HUMAN GENETICS, Issue 1 2007
Yukiko Nakano
Summary Recently, it has been reported that under 20% of Brugada syndrome cases are linked to SCN5A mutations. The purpose of this study was to clarify whether abnormalities other than exonic mutations, such as splicing disorders, decreased mRNA expression levels, or membrane transport abnormalities of SCN5A, play a role in the pathogenesis of Brugada syndrome. We analyzed all SCN5A exons and splice sites using genomic DNA from 23 Brugada syndrome patients. We also analyzed the mRNA obtained from RV cardiomyocytes using real time PCR and sequencing, to study the expression levels and splicing patterns of SCN5A. The localization of SCN5A was examined by immunofluorescence analysis. A de novo heterozygous G to A transversion in a 5, splice junction of the intron between exons 21 and 22 was detected in 1 patient. In the mRNA analysis of Brugada syndrome patients without a mutation of SCN5A no splicing abnormalities were detected, and the SCN5A mRNA levels were similar to those of normal controls. Immunofluorescence analyses revealed that SCN5A is located on the surface membrane not only in the RV cardiomyocytes of normal controls but also in those with Brugada syndrome. We can confirm that some Brugada syndrome patients without exonic mutations in SCN5A had no other SCN5A abnormalities, including any involving the location of the SCN5A protein. These results suggest the involvement of other proteins in the pathogenesis in Brugada syndrome. [source]

Sub-lethal heat shock induces plasma membrane translocation of 70-kDa heat shock protein in viable, but not in apoptotic, U-937 leukaemia cells

APMIS, Issue 3 2010
ELENA B. LASUNSKAIA
Lasunskaia EB, Fridlianskaia I, Arnholdt AV, Kanashiro M, Guzhova I, Margulis B. Sub-lethal heat shock induces plasma membrane translocation of 70-kDa heat shock protein in viable, but not in apoptotic, U-937 leukaemia cells. APMIS 2010; 118: 179,87. Heat shock protein 70 kDa, Hsp70, is an important intracellular factor that protects cells from stress. Unusual plasma membrane expression of Hsp70, observed in some cancer cells, contributes to the cell's recognition and elimination by the immune system. Induction of apoptosis in cancer cells was demonstrated to increase Hsp70 translocation to the surface membrane, enhancing immunogenic effects through the stimulation of dendritic cells. As hyperthermia is proposed as a method of choice for anti-cancer therapy, we examined whether apoptosis induction by heat shock enhances Hsp70 membrane translocation in U-937 leukaemia cells. Cells were exposed to sub-lethal heat shock, and intracellular and membrane-bound Hsp70 expression was evaluated in apoptotic and viable cell sub-populations, employing flow cytometry and immunofluorescence. Heat shock induced Hsp70 membrane translocation in the viable cells that were able to enhance Hsp70 production upon heating, but not in the cells undergoing apoptosis that continued to express low basal levels of the intracellular protein. Data suggest that the protein translocation was associated with the increasing Hsp70 content rather than the apoptotic process. Apoptosis does not contribute to externalization of Hsp70, at least in the cells with low levels of this protein. [source]

Ro 60 functions as a receptor for ,2 -glycoprotein I on apoptotic cells

ARTHRITIS & RHEUMATISM, Issue 3 2009
Joanne H. Reed
Objective The autoantigens 60-kd Ro/SSA (Ro 60) and ,2 -glycoprotein I (,2GPI) are both displayed on the surface membrane of apoptotic cells. Epitope-spreading experiments have suggested that these autoantigens may be present as a complex on the apoptotic cell surface. This study was undertaken to investigate whether ,2GPI interacts with Ro 60 on apoptotic cells and alters the binding of anti,Ro 60 IgG. Methods The interaction between soluble recombinant Ro 60 fragments and ,2GPI was investigated in vitro by direct and saturation binding assays using native human ,2GPI and recombinant domain deletion mutants. Binding of ,2GPI to early and late apoptotic cells was assessed by multiparameter flow cytometry, and specificity of binding was determined by competitive inhibition with soluble recombinant Ro 60 and anti,Ro 60 IgG. Results The Ro 60 fragment expressing a surface-exposed epitope (apotope) bound with high affinity (Kd = ,15 nM) to domain V of ,2GPI in vitro. Beta2 -glycoprotein I bound to the surface of apoptotic cells in a dose-dependent manner and was blocked by the Ro 60 apotope fragment. In reciprocal competitive inhibition studies, ,2GPI blocked the binding of anti,Ro 60 autoantibodies to apoptotic cells in a dose-dependent manner, and anti,Ro 60 IgG inhibited the binding of ,2GPI. Moreover, ,2GPI showed a 2-fold increase in binding to apoptotic cells that overexpress Ro 60 on the surface. Conclusion These results demonstrate that Ro 60 functions as a novel receptor for ,2GPI on the surface of apoptotic cells. The formation of Ro 60,,2GPI complexes may protect against anti,Ro 60 autoantibody,mediated tissue injury. [source]

Intracellular function in rehydrated lyophilized platelets

BRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2000
Thomas H. Fischer
This study aimed to evaluate the effect of cross-linking and lyophilization on intracellular signalling processes in rehydrated, lyophilized (RL) platelets, which are under development as a platelet substitute for transfusion. Exposure of RL platelets to thrombin resulted in enhanced phosphorylation of several proteins, including 18 kDa and 42 kDa kinase substrates that were shown to be the substrates of myosin light chain and protein kinase C respectively. Cross-linking and lyophilization depleted the platelets of free cytoplasmic ADP and ATP, but had less effect on protein-bound nucleotides. The surface membrane of RL platelets was found to be permeable to poly dT probes less than approximately 3 kDa in size; larger nucleotide probes and proteins did not penetrate the surface membrane. Taken together, our results indicate that RL platelets retain some of the haemostatic stimulus-response functions of fresh platelets and are capable of feedback amplification in coagulation. [source]

EXCITATION,CONTRACTION COUPLING FROM THE 1950s INTO THE NEW MILLENNIUM

CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 9 2006
AF Dulhunty
SUMMARY 1Excitation,contraction coupling is broadly defined as the process linking the action potential to contraction in striated muscle or, more narrowly, as the process coupling surface membrane depolarization to Ca2+ release from the sarcoplasmic reticulum. 2We now know that excitation,contraction coupling depends on a macromolecular protein complex or ,calcium release unit'. The complex extends the extracellular space within the transverse tubule invaginations of the surface membrane, across the transverse tubule membrane into the cytoplasm and then across the sarcoplasmic reticulum membrane and into the lumen of the sarcoplasmic reticulum. 3The central element of the macromolecular complex is the ryanodine receptor calcium release channel in the sarcoplasmic reticulum membrane. The ryanodine receptor has recruited a surface membrane L-type calcium channel as a ,voltage sensor' to detect the action potential and the calcium-binding protein calsequestrin to detect in the environment within the sarcoplasmic reticulum. Consequently, the calcium release channel is able to respond to surface depolarization in a manner that depends on the Ca2+ load within the calcium store. 4The molecular components of the ,calcium release unit' are the same in skeletal and cardiac muscle. However, the mechanism of excitation,contraction coupling is different. The signal from the voltage sensor to ryanodine receptor is chemical in the heart, depending on an influx of external Ca2+ through the surface calcium channel. In contrast, conformational coupling links the voltage sensor and the ryanodine receptor in skeletal muscle. 5Our current understanding of this amazingly efficient molecular signal transduction machine has evolved over the past 50 years. None of the proteins had been identified in the 1950s; indeed, there was debate about whether the molecules involved were, in fact, protein. Nevertheless, a multitude of questions about the molecular interactions and structures of the proteins and their interaction sites remain to be answered and provide a challenge for the next 50 years. [source]

Computational form-finding of tension membrane structures,Non-finite element approaches: Part 1.

INTERNATIONAL JOURNAL FOR NUMERICAL METHODS IN ENGINEERING, Issue 5 2003
Use of cubic splines in finding minimal surface membranes
Abstract This paper, presented in three parts, discusses a computational methodology for form-finding of tension membrane structures (TMS), or fabric structures, used as roofing forms. The term ,form-finding' describes a process of finding the shape of a TMS under its initial tension. Such a shape is neither known a priori, nor can it be described by a simple mathematical function. The work is motivated by the need to provide an efficient numerical tool, which will allow a better integration of the design/analysis/manufacture of TMS. A particular category of structural forms is considered, known as minimal surface membranes (such as can be reproduced by soap films). The numerical method adopted throughout is dynamic relaxation (DR) with kinetic damping. Part 1 describes a new form-finding approach, based on the Laplace,Young equation and cubic spline fitting to give a full, piecewise, analytical description of a minimal surface. The advantages arising from the approach, particularly with regard to manufacture of cutting patterns for a membrane, are highlighted. Part 2 describes an alternative and novel form-finding approach, based on a constant tension field and faceted (triangular mesh) representation of the minimal surface. It presents techniques for controlling mesh distortion and discusses effects of mesh control on the accuracy and computational efficiency of the solution, as well as on the subsequent stages in design. Part 3 gives a comparison of the performance of the initial method (Part 1) and the faceted approximations (Part 2). Functional relations, which encapsulate the numerical efficiency of each method, are presented. Copyright © 2002 John Wiley & Sons, Ltd. [source]

Computational form-finding of tension membrane structures,Non-finite element approaches: Part 2.

INTERNATIONAL JOURNAL FOR NUMERICAL METHODS IN ENGINEERING, Issue 5 2003
Triangular mesh discretization, control of mesh distortion in modelling minimal surface membranes
Abstract This paper, presented in three parts, discusses a computational methodology for form-finding of tension membrane structures (TMS), or fabric structures, used as roofing forms. The term ,form-finding' describes a process of finding the shape of a TMS under its initial tension. Such a shape is neither known a priori, nor can it be described by a simple mathematical function. The work is motivated by the need to provide an efficient numerical tool, which will allow a better integration of the design/analysis/manufacture of TMS. A particular category of structural forms is considered, known as minimal surface membranes (such as can be reproduced by soap films). The numerical method adopted throughout is dynamic relaxation (DR) with kinetic damping. Part 1 gave a background to the problem of TMS design, described the DR method, and presented a new form-finding methodology, based on the Laplace,Young equation and cubic spline fitting to give a full, piecewise, analytical description of the surface. Part 2 describes an alternative and novel form-finding method, based on a constant tension field and faceted (triangular mesh) representation of the minimal surface. Techniques for controlling mesh distortion are presented, and their effects on the accuracy and computational efficiency of the solution, as well as on the subsequent stages in design, are examined. Part 3 gives a comparison of the performance of the initial method (Part 1) and the faceted approximations (Part 2). Functional relations, which encapsulate the numerical efficiency of each method, are presented. Copyright © 2002 John Wiley & Sons, Ltd. [source]

Nmp4/CIZ contributes to fluid shear stress induced MMP-13 gene induction in osteoblasts

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2007
Kanokwan Charoonpatrapong-Panyayong
Abstract The expression of matrix metalloproteinase-13 (MMP-13), involved in bone turnover, is elevated in stretched MC3T3-E1 osteoblast-like cells. Strain-mediated forces impact bone remodeling due in large part to the movement of fluid through the canalicular-lacunar network. The resulting fluid shear stress (FSS) over the surface membranes of bone cells initiates bone remodeling. Although the nuclear events mediating putative FSS-induced changes in osteoblast MMP-13 transcription are unknown, previous studies with bone cells suggest an overlap between osteoblast FSS- and PTH-induced signal response pathways. MMP-13 PTH response is regulated by a 110 bp 5, regulatory region, conserved across the mouse, rat, and human genes, that supports the binding of numerous transcription factors including Runx2, c-fos/c-jun, Ets-1, and nuclear matrix protein 4/cas interacting zinc finger protein (Nmp4/CIZ) a nucleocytoplasmic shuttling trans-acting protein that attenuates PTH-driven transcription. Nmp4/CIZ also binds p130cas, an adaptor protein implicated in mechanotransduction. Here we sought to determine whether Nmp4/CIZ contributes to FSS-induced changes in MMP-13 transcription. FSS (12 dynes/cm2, 3,5 h) increased MMP-13 promoter-reporter activity approximately two-fold in MC3T3-E1 osteoblast-like cells attended by a comparable increase in mRNA expression. This was accompanied by a decrease in Nmp4/CIZ binding to its cis-element within the PTH response region, the mutation of which abrogated the MMP-13 response to FSS. Interestingly, FSS enhanced Nmp4/CIZ promoter activity and induced p130cas nuclear translocation. We conclude that the PTH regulatory region of MMP-13 also contributes to FSS response and that Nmp4/CIZ plays similar but distinct roles in mediating hormone- and FSS-driven induction of MMP-13 in bone cells. J. Cell. Biochem. 102: 1202,1213, 2007. © 2007 Wiley-Liss, Inc. [source]