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Plasma Membrane (plasma + membrane)
Kinds of Plasma Membrane Terms modified by Plasma Membrane Selected AbstractsCytokine Stimulation Promotes Increased Glucose Uptake Via Translocation at the Plasma Membrane of GLUT1 in HEK293 CellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2010Angara Zambrano PhD Abstract Interleukin-3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) are two of the best-characterized cell survival factors in hematopoietic cells; these factors induce an increase in Akt activity in multiple cell lines, a process thought to be involved in cellular survival. It is known that growth factors require sustained glucose metabolism to promote cell survival. It has been determined that IL-3 and GM-CSF signal for increased glucose uptake in hematopoietic cells. Interestingly, receptors for IL-3 and GM-CSF are present in several non-hematopoietic cell types but their roles in these cells have been poorly described. In this study, we demonstrated the expression of IL-3 and GM-CSF receptors in HEK293 cells and analyzed their effect on glucose uptake. In these cells, both IL-3 and GM-CSF, increased glucose uptake. The results indicated that this increase involves the subcellular redistribution of GLUT1, affecting glucose transporter levels at the cell surface in HEK293 cells. Also the data directly demonstrates that the PI 3-kinase/Akt pathway is an important mediator of this process. Altogether these results show a role for non-insulin growth factors in the regulation of GLUT1 trafficking that has not yet been directly determined in non-hematopoietic cells. J. Cell. Biochem. 110: 1471,1480, 2010. © 2010 Wiley-Liss, Inc. [source] A Cytotoxic Ruthenium Tris(Bipyridyl) Complex that Accumulates at Plasma MembranesCHEMBIOCHEM, Issue 11 2009Olivier Zava Dr. Shine a light: A ruthenium tris(bipyridyl) complex that was originally designed as a photosensitizer for solar cells has been found to adhere to plasma membranes. [source] Ion-Exchange Plasma Membranes for Fuel Cells on a Micrometer Scale,CHEMICAL VAPOR DEPOSITION, Issue 6-7 2007S. Roualdès Abstract Recent advances in,miniaturization technology make polymer electrolyte membrane fuel cells very attractive as power sources for portable devices. Ion-exchange membranes for microscale fuel cells are synthesized by plasma polymerization (using a precursor containing ion-exchange groups) and intensively characterized. Ion-exchange plasma membranes are thin, amorphous, and dense materials with no defects. Spectroscopic analyses reveal a polymer-type matrix containing a rather high concentration of ion-exchange groups. Under the best synthesis conditions, membranes show a satisfying ionic conduction level and a high compatibility with other active layers of fuel cells, making them suitable for insertion in such power-supply devices. [source] Interspecies differences in hepatic Ca2+ -ATPase activity and the effect of cold preservation on porcine liver Ca2+ -ATPase functionLIVER TRANSPLANTATION, Issue 2 2001Piotr K. Janicki MD The accumulation of intracellular calcium ([Ca2+]i) caused by ischemia-reperfusion during liver transplantation has been implicated as a factor leading to primary graft nonfunction. Plasma membrane (PM) and endoplasmic reticulum (ER) Ca2+ -adenosinetriphosphatases (ATPases) are the primary transporters that maintain [Ca2+]i homeostasis in the liver. We hypothesized that the porcine liver is better than the rat liver as a model for the study of human liver Ca2+ -ATPase activity. We also hypothesized that cold preservation would depress Ca2+ -ATPase activity in the porcine liver. Pig and rat livers were harvested, and human liver samples were obtained from surgical resection specimens. All were preserved with University of Wisconsin solution, and porcine livers were also preserved on ice for 2 to 18 hours. Ca2+ -ATPase activity was measured after incubation with 45Ca2+ and adenosine triphosphate in the presence of specific Ca2+ -ATPase inhibitors. Porcine PM and ER Ca2+ -ATPase activities were 0.47 ± 0.03 and 1.57 ± 0.10 nmol of Ca2+/mg of protein/min, respectively. This was not significantly different from human liver, whereas rat liver was significantly greater at 2.60 ± 0.03 and 9.2 ± 0.9 nmol of Ca2+/mg of protein/min, respectively. We conclude that the Ca2+ -ATPase activity in the pig liver is equivalent to that of human liver, and thus, the pig liver is a better model than the rat liver. Cold preservation studies showed a significant decrease in porcine hepatic PM Ca2+ -ATPase activity after 4 hours of storage and near-total inhibition after 12 hours. Porcine hepatic ER Ca2+ -ATPase activity showed a 45% decrease in activity by 12 hours and a 69% decrease by 18 hours. We conclude that cold ischemia at clinically relevant times depresses PM Ca2+ -ATPase more than ER Ca2+ -ATPase activity in pig liver homogenates. [source] Plasma membrane surface potential (,pm) as a determinant of ion bioavailability: A critical analysis of new and published toxicological studies and a simplified method for the computation of plant ,pmENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2006Thomas B. Kinraide Abstract Plasma membranes (PMs) are negatively charged, and this creates a negative PM surface electrical potential ,PM) that is also controlled by the ionic composition of the bathing medium. The ,PM controls the distribution of ions between the PM surface and the medium so that negative potentials increase the surface activity of cations and decrease the surface activity of anions. All cations reduce the negativity of ,PM, and these common ions are effective in the following order: Al3+ > H+ > Cu2+ > Ca2+ , Mg2+ > Na+ , K+. These ions, especially H+, Ca2+, and Mg2+, are known to reduce the uptake and biotic effectiveness of cations and to have the opposite effects on anions. Toxicologists commonly interpret the interactions between toxic cations (commonly metals) and ameliorative cations (commonly H+, Ca2+, and Mg2+) as competitions for binding sites at a PM surface ligand. The ,PM is rarely considered in this biotic ligand model, which incorporates the free ion activity model. The thesis of this article is that ,PM effects are likely to be more important to bioavailability than site-specific competition. Furthermore, ,PM effects could give the false appearance of competition even when it does not occur. The electrostatic approach can account for the bioavailability of anions, whereas the biotic ligand model cannot, and it can account for interactions among cations when competition does not occur. Finally, a simplified procedure is presented for the computation of ,PM for plants, and the possible use of ,PM in a general assessment of the bioavailability of ions is considered. [source] Presence of a Na+ -stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium Aphanothece halophyticaFEMS MICROBIOLOGY LETTERS, Issue 1 2007Kanjana Wiangnon Abstract Aphanothece cells could take up Na+ and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m -chlorophenylhydrazone (CCCP). Cells preloaded with Na+ exhibited Na+ extrusion ability upon energizing with glucose. Na+ was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na+ -ionophore. Orthovanadate and CCCP strongly inhibited Na+ uptake, whereas N, N, -dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na+ but not with K+, Ca2+ and Li+. The Km values for ATP and Na+ were 1.66±0.12 and 25.0±1.8 mM, respectively, whereas the Vmax value was 0.66±0.05 ,mol min,1 mg,1. Mg2+ was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N -ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na+/H+ antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na+ -stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition. [source] Effects of zotepine and olanzapine on noradrenaline transporter in cultured bovine adrenal medullary cellsHUMAN PSYCHOPHARMACOLOGY: CLINICAL AND EXPERIMENTAL, Issue 7 2005Reiji Yoshimura Abstract Background Previously, it was demonstrated that the inhibitory effects of atypical antipsychotic drugs such as clozapine and risperidone on noradrenaline transporter (NAT) might in part be associated with their clinical profile. The present study examined the effects of zotepine on NAT in the cells and compared them with those of olanzapine. Materials and Methods Adrenal medullary cells were isolated by a method of collagenase digestion of slices of fresh bovine adrenal medulla and the cells were plated at a density of 4,×,106 cells. Cells were incubated with [3H]noradrenaline (NA) in the presence or absence of zotepine or olanzapine. The amount of radioactivity taken into the cells was counted by a liquid scintillation counter. Plasma membranes of bovine adrenal medulla were prepared, and the binding of [3H]desipramine (DMI) was determined by incubating the membrane suspension in binding buffer together with zotepine or olanzapine. Specific binding of [3H] DMI was defined as that binding which was inhibited by nisoxetine. Results Both zotepine (10,1000,ng/ml) and olanzapine (10,1000,ng/ml) decreased [3H]NA uptake in a concentration-dependent manner. The IC50 values of zotepine and olanzapine on [3H]NA uptake were 10,±,4 and 14,±,8,ng/ml, respectively. Eadie-Hofstee analysis of [3H]NA uptake showed that treatment with zotepine and olanzapine decreased the Vmax of uptake without changing the Km. Both zotepine (10,1000,ng/ml) and olanzapine (30,1000,ng/ml) inhibited [3H]DMI binding in a concentration-dependent manner. The IC50 values of zotepine and olanzapine on [3H]DMI binding were 50,±,18, and 120,±,38,ng/ml, respectively. Scatchard plot analysis of [3H]DMI binding showed that zotepine and olanzapine decreased the Bmax of binding without altering the Kd. Conclusions The inhibitory effects of zotepine and olanzapine might be responsible in part for their clinical profile. Copyright © 2005 John Wiley & Sons, Ltd. [source] Protein kinase C mRNA and protein expressions in hypobaric hypoxia-induced cardiac hypertrophy in ratsACTA PHYSIOLOGICA, Issue 4 2010M. Uenoyama Abstract Aim:, Protein kinase C (PKC), cloned as a serine/threonine kinase, plays key roles in diverse intracellular signalling processes and in cardiovascular remodelling during pressure overload or volume overload. We looked for correlations between changes in PKC isoforms (levels and/or subcellular distributions) and cardiac remodelling during experimental hypobaric hypoxic environment (HHE)-induced pulmonary hypertension. Methods:, To study the PKC system in the heart during HHE, 148 male Wistar rats were housed for up to 21 days in a chamber at the equivalent of 5500 m altitude level (10% O2). Results:, At 14 or more days of exposure to HHE, pulmonary arterial pressure (PAP) was significantly increased. In the right ventricle (RV): (1) the expression of PKC-, protein in the cytosolic and membrane fractions was increased at 3,14 days and at 5,7 days of exposure respectively; (ii) the cytosolic expression of PKC-, protein was increased at 1,5, 14 and 21 days of exposure; (3) the membrane expressions of the proteins were decreased at 14,21 (PKC-,II), 14,21 (PKC-,), and 0.5,5 and 21 (PKC-,) days of exposure; (4) the expression of the active form of PKC-, protein on the plasma membrane was increased at 3 days of exposure (based on semiquantitative analysis of the immunohistochemistry). In the left ventricle, the expressions of the PKC mRNAs, and of their cytosolic and membrane proteins, were almost unchanged. The above changes in PKC-,, which were strongly evident in the RV, occurred alongside the increase in PAP. Conclusion:, PKC-, may help to modulate the right ventricular hypertrophy caused by pulmonary hypertension in HHE. [source] Local Ca2+ influx through CRAC channels activates temporally and spatially distinct cellular responsesACTA PHYSIOLOGICA, Issue 1 2009A. B. Parekh Abstract Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels controls a disparate array of key cellular responses. In this review, recent work will be described that shows local Ca2+ influx through CRAC channels has important spatial and temporal consequences on cell function. A localized Ca2+ rise below the plasma membrane activates, within tens of seconds, catabolic enzymes resulting in the generation of the intracellular messenger arachidonic acid and the paracrine pro-inflammatory molecule LTC4. In addition, local Ca2+ entry can activate gene expression, which develops over tens of minutes. Local Ca2+ influx through CRAC channels therefore has far-reaching consequences on intra- and intercellular communication. [source] When is high-Ca2+ microdomain required for mitochondrial Ca2+ uptake?,ACTA PHYSIOLOGICA, Issue 1 2009A. Spät Abstract Ca2+ release from IP3 -sensitive stores in the endoplasmic reticulum (ER) induced by Ca2+ -mobilizing agonists generates high-Ca2+ microdomains between ER vesicles and neighbouring mitochondria. Here we present a model that describes when such microdomains are required and when submicromolar [Ca2+] is sufficient for mitochondrial Ca2+ uptake. Mitochondrial Ca2+ uptake rate in angiotensin II-stimulated H295R adrenocortical cells correlates with the proximity between ER vesicles and the mitochondrion, reflecting the uptake promoting effect of high-Ca2+ peri-mitochondrial microdomains. Silencing or inhibition of p38 mitogen-activated protein kinase (MAPK) or inhibition of the novel isoforms of protein kinase C enhances mitochondrial Ca2+ uptake and abolishes the positive correlation between Ca2+ uptake and ER-mitochondrion proximity. Inhibition of protein phosphatases attenuates mitochondrial Ca2+ uptake and also abolishes its positive correlation with ER-mitochondrion proximity. We postulate that during IP3 -induced Ca2+ release, Ca2+ uptake is confined to ER-close mitochondria, because of the simultaneous activation of the protein kinases. Attenuation of Ca2+ uptake prevents Ca2+ overload of mitochondria and thus protects the cell against apoptosis. On the other hand, all the mitochondria accumulate Ca2+ at a non-inhibited rate during physiological Ca2+ influx through the plasma membrane. Membrane potential is higher in ER-distant mitochondria, providing a bigger driving force for Ca2+ uptake. Our model explains why comparable mitochondrial Ca2+ signals are formed in response to K+ and angiotensin II (equipotent in respect to global cytosolic Ca2+ signals), although only the latter generates high-Ca2+ microdomains. [source] Calcium handling in afferent arteriolesACTA PHYSIOLOGICA, Issue 4 2004M. Salomonsson Abstract The cytosolic intracellular calcium concentration ([Ca2+]i) is a major determining factor in the vascular smooth muscle tone. In the afferent arteriole it has been shown that agonists utilizing G-protein coupled receptors recruit Ca2+ via release from intracellular stores and entry via pathways in the plasma membrane. The relative importances of entry vs. mobilization seem to differ between different agonists, species and preparations. The entry pathway might include different types of voltage sensitive Ca2+ channels located in the plasmalemma such as dihydropyridine sensitive L-type channels, T-type channels and P/Q channels. A role for non-voltage sensitive entry pathways has also been suggested. The importance of voltage sensitive Ca2+ channels in the control of the tone of the afferent arteriole (and thus in the control of renal function and whole body control of extracellular fluid volume and blood pressure) sheds light on the control of the membrane potential of afferent arteriolar smooth muscle cells. Thus, K+ and Cl, channels are of importance in their role as major determinants of membrane potential. Some studies suggest a role for calcium-activated chloride (ClCa) channels in the renal vasoconstriction elicited by agonists. Other investigators have found evidence for several types of K+ channels in the regulation of the afferent arteriolar tone. The available literature in this field regarding afferent arterioles is, however, relatively sparse and not conclusive. This review is an attempt to summarize the results obtained by others and ourselves in the field of agonist induced afferent arteriolar Ca2+ recruitment, with special emphasis on the control of voltage sensitive Ca2+ entry. Outline of the Manuscript: This manuscript is structured as follows: it begins with an introduction where the general role for [Ca2+]i as a key factor in the regulation of the tone of vascular smooth muscles (VSMC) is detailed. In this section there is an emphasis is on observations that could be attributed to afferent arteriolar function. We then investigate the literature and describe our results regarding the relative roles for Ca2+ entry and intracellular release in afferent arterioles in response to vasoactive agents, with the focus on noradrenalin (NA) and angiotensin II (Ang II). Finally, we examine the role of ion channels (i.e. K+ and Cl, channels) for the membrane potential, and thus activation of voltage sensitive Ca2+ channels. [source] The roles of actin cytoskeleton and microtubules for membrane recycling of a food vacuole in Tetrahymena thermophilaCYTOSKELETON, Issue 7 2009Maki Sugita Abstract Phagocytosis is a fundamental cellular event for the uptake of nutrients from the environment in several kinds of eukaryote. Most ciliates egest waste and undigested materials in food vacuoles (FVs) through a cytoproct, which is a specific organelle for defecation. It is considered that FV egestion is initiated by fusion between the FV membrane and plasma membrane in a cytoproct and completed with retrieval of the membrane into a cytoplasmic space. In addition, electron microscopy indicated that microfilaments might be involved in the recycling process of the FV membrane in ciliates over 30 years ago; however, there is no conclusive evidence. Here we demonstrated actin organization on FV near a cytoproct in Tetrahymena thermophila by using a marker for a cytoproct. Moreover, it was revealed that cells treated with actin cytoskeletal inhibitor, Latrunculin B, might be suppressed for membrane retrieval in a cytoproct following FV egestion. On the other hand, the actin structures, likely to be the site of membrane retrieval, were frequently observed in the cells treated with cytoplasmic microtubules inhibitor, Nocodazole. We concluded that actin filaments were probably required for recycling of the FV membrane in a cytoproct although the role was not essential for FV egestion. In addition, it was possible that microtubules might be involved in transportation of recycling vesicles of FV coated with F-actin. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Centrioles to basal bodies in the spermiogenesis of Mastotermes darwiniensis (Insecta, Isoptera)CYTOSKELETON, Issue 5 2009Maria Giovanna Riparbelli Abstract In addition to their role in centrosome organization, the centrioles have another distinct function as basal bodies for the formation of cilia and flagella. Centriole duplication has been reported to require two alternate assembly pathways: template or de novo. Since spermiogenesis in the termite Mastotermes darwiniensis lead to the formation of multiflagellate sperm, this process represents a useful model system in which to follow basal body formation and flagella assembly. We present evidence of a possible de novo pathway for basal body formation in the differentiating germ cell. This cell also contains typical centrosomal proteins, such as centrosomin, pericentrin-like protein, ,-tubulin, that undergo redistribution as spermatid differentiation proceeds. The spermatid centrioles are long structures formed by nine doublet rather than triplet microtubules provided with short projections extending towards the surrounding cytoplasm and with links between doublets. The sperm basal bodies are aligned in parallel beneath the nucleus. They consist of long regions close to the nucleus showing nine doublets in a cartwheel array devoid of any projections; on the contrary, the short region close to the plasma membrane, where the sperm flagella emerge, is characterized by projections similar to those observed in the centrioles linking the basal body to the plasma membrane. It is hypothesized that this appearance is in connection with the centriole elongation and further with the flagellar axonemal organization. Microtubule doublets of sperm flagellar axonemes are provided with outer dynein arms, while inner arms are rarely visible. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Microtubule-dependent motility and orientation of the cortical endoplasmic reticulum in elongating characean internodal cellsCYTOSKELETON, Issue 3 2009Ilse Foissner Abstract Motility of the endoplasmic reticulum (ER) is predominantly microtubule- dependent in animal cells but thought to be entirely actomyosin-dependent in plant cells. Using live cell imaging and transmission electron microscopy to examine ER motility and structural organization in giant internodal cells of characean algae, we discovered that at the onset of cell elongation, the cortical ER situated near the plasma membrane formed a tight meshwork of predominantly transverse ER tubules that frequently coaligned with microtubules. Microtubule depolymerization increased mesh size and decreased the dynamics of the cortical ER. In contrast, perturbing the cortical actin array with cytochalasins did not affect the transverse orientation but decreased mesh size and increased ER dynamics. Our data suggest that myosin-dependent ER motility is confined to the ER strands in the streaming endoplasm, while the more sedate cortical ER uses microtubule-based mechanisms for organization and motility during early stages of cell elongation. We show further that the ER has an inherent, NEM-sensitive dynamics which can be altered via interaction with the cytoskeleton and that tubule formation and fusion events are cytoskeleton-independent. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Heterogeneous modes of uptake for latex beads revealed through live cell imaging of phagocytes expressing a probe for phosphatidylinositol-(3,4,5)-trisphosphate and phosphatidylinositol-(3,4)-bisphosphateCYTOSKELETON, Issue 9 2008Jennifer Giorgione Abstract Latex beads are the preferred phagocytic substrate in biochemical studies of phagosome composition and maturation. Using living Dictyostelium cells and fluorescent probes, we compared the properties of phagosomes formed to ingest latex beads or digestible prey. Significant differences were found during the initial steps of phagocytosis. During uptake of bacteria or yeast, PHcrac-GFP, a probe that binds to membranes enriched in PI(3,4,5)P3 and PI(3,4)P2, always labeled the nascent phagosome and faded shortly after it sealed. However, labeling of bead-containing phagosomes was highly variable. Beads were engulfed by phagosomes either lacking or displaying the PHcrac-GFP label, and that label, if present, often persisted for many minutes, revealing that early trafficking steps for bead-containing phagosomes are quite heterogeneous. Later stages of the endocytic pathway appeared more similar for phagosomes containing prey and latex beads. Both types of phagosomes fused with acidic endosomes while undergoing transport along microtubules, both acquired the V-ATPase and lost it prior to exocytosis, and both bound the late endosome marker vacuolin B, which was transferred to the plasma membrane upon exocytosis. We conclude that caution is needed in extrapolating results from latex bead phagosomes to phagosomes containing physiological substances, especially in early stages of the endocytic pathway. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] The Arabidopsis class VIII myosin ATM2 is involved in endocytosisCYTOSKELETON, Issue 6 2008Amirali Sattarzadeh Abstract Members of the class XI of the myosin superfamily comprising higher plant, actin-based molecular motors have been shown to be involved in peroxisome and Golgi vesicle trafficking comparable to yeast and animal class V myosins. The tasks of the second class of myosins of higher plants, class VIII, are unclear. In this study the class VIII myosin ATM2 from the model plant Arabidopsis thaliana was selected for the examination of cargo specificity in vivo. Fluorescent protein-fusion plasmid constructs with fragments of the ATM2 cDNA were generated and used for Agrobacterium tumefaciens -based transient transformation of Nicotiana benthamiana leaves. The resulting subcellular localization patterns were recorded by live imaging with confocal laser scanning microscopy (CLSM) in epidermal leaf cells. Expression of a nearly full-length construct displayed labeling of filaments and vesicles, a head + neck fragment led to decoration of filaments only. However, expression of fluorescent protein-tagged C-terminal tail domain constructs labeled vesicular structures of different appearance. Most importantly, coexpression of different RFP/YFP-ATM2 tail fusion proteins showed colocalization and, hence, binding to the same type of vesicular target. Further coexpression experiments of RFP/YFP-ATM2 tail fusion proteins with the endosomal marker FYVE and the endosomal tracer FM4-64 demonstrated colocalization with endosomes. Colocalization was also detected by expression of the CFP-tagged membrane receptor BRI1 as marker, which is constantly recycled via endosomes. Occasionally the ATM2 tail targeted to sites at the plasma membrane closely resembling the pattern obtained upon expression of the YFP-ATM1 C-terminal tail. ATM1 is known for its localization at the plasma membrane at sites of plasmodesmata. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] The chemotaxis defect of Shwachman-Diamond Syndrome leukocytesCYTOSKELETON, Issue 3 2004Vesna Stepanovic Abstract Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive, multisystem disorder presenting in childhood with intermittent neutropenia and pancreatic insufficiency. It is characterized by recurrent infections independent of neutropenia, suggesting a functional neutrophil defect. While mutations at a single gene locus (SBDS) appear to be responsible for SDS in a majority of patients, the function of that gene and a specific defect in SDS neutrophil behavior have not been elucidated. Therefore, employing 2D and 3D computer-assisted motion analysis systems, we have analyzed the basic motile behavior and chemotactic responsiveness of individual polymorphonuclear leukocytes (PMNs) of 14 clinically diagnosed SDS patients. It is demonstrated that the basic motile behavior of SDS PMNs is normal in the absence of chemoattractant, that SDS PMNs respond normally to increasing and decreasing temporal gradients of the chemoattractant fMLP, and that SDS PMNs exhibit a normal chemokinetic response to a spatial gradient of fMLP. fMLP receptors were also distributed uniformly through the plasma membrane of SDS PMNs as in control PMNs. SDS PMNs, however, were incapable of orienting in and chemotaxing up a spatial gradient of fMLP. This unique defect in orientation was manifested by the PMNs of every SDS patient tested. The PMNs of an SDS patient who had received an allogenic hematopoietic stem cell transplant, as well as PMNs from a cystic fibrosis patient, oriented normally. These results suggest that the defect in SDS PMNs is in a specific pathway emanating from the fMLP receptor that is involved exclusively in regulating orientation in response to a spatial gradient of fMLP. This pathway must function in parallel with additional pathways, intact in SDS patients, that emanate from the fMLP receptor and regulate responses to temporal rather than spatial changes in receptor occupancy. Cell Motil. Cytoskeleton 57:158,174, 2004. © 2004 Wiley-Liss, Inc. [source] Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: A statistical studyDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2000Fumie Suzuki-Toyota Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze-fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP-free area increased during capacitation. To correct the IMP-free area, arithmetically redistributed (ARD)-IMP density was used for statistical analysis. In the PM group, the mean density ± SD of large IMP was 379 ± 64 and 266 ± 58/,m2, and that of small IMP was 1450 ± 155 and 672 ± 252/,m2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One-fifth of the total small IMP density reduced by 2 h. Filipin-sterol complexes (FSC) were numerous in the PAPM, and FSC-free areas also increased during capacitation. The mechanism of IMP-free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability. [source] Functional analysis in Drosophila indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant-negative mechanismDEVELOPMENTAL DYNAMICS, Issue 4 2004Gary R. Hime Abstract Mutations in the human homolog of the patched gene are associated with the developmental (and cancer predisposition) condition Nevoid Basal Cell Carcinoma Syndrome (NBCCS), as well as with sporadic basal cell carcinomas. Most mutations that have been identified in the germline of NBCCS patients are truncating or frameshift mutations, with amino acid substitutions rarely found. We show that a missense mutation in the sterol-sensing domain G509V acts as a dominant negative when assayed in vivo in Drosophila. Ectopic expression of a Drosophila patched transgene, carrying the analogous mutation to G509V, causes ectopic activation of Hedgehog target genes and ectopic membrane stabilisation of Smoothened. The G509V transgene behaves in a manner similar, except in its subcellular distribution, to a C-terminal truncation that has been characterised previously as a dominant-negative protein. G509V exhibits vesicular localisation identical to the wild-type protein, but the C-terminal truncated Patched molecule is localised predominantly to the plasma membrane. This finding suggests that dominant-negative function can be conferred by interruption of different aspects of Patched protein behaviour. Another mutation at the same residue, G509R, did not exhibit dominant-negative activity, suggesting that simple removal of the glycine at 509 is not sufficient to impart dominant-negative function. Developmental Dynamics 229:780,790, 2004. © 2004 Wiley-Liss, Inc. [source] High-frequency stimuli preferentially release large dense-core vesicles located in the proximity of nonspecialized zones of the presynaptic membrane in sympathetic gangliaDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2008F. Cifuentes Abstract We characterized the effect of a brief high-frequency stimulus on the number, distribution, and optical density of large dense-core vesicles (LDCVs) in the nerve terminals of the rat superior cervical ganglia. From 4.21 ± 0.37 LDCVs/bouton detected in control nerve terminals, a stimulus of 40 Hz for 1 min released 41% of LDCVs, decreasing their number to 2.48 ± 0.14 LDCVs/bouton (p = 0.0009). In control ganglia, most dense vesicles were located close to the plasma membrane (at ,100 nm); in contrast, in stimulated ganglia they were broadly distributed with respect to the active zone. The mean distance of LDCVs to membrane and active zones was 95 ± 8 nm and 473 ± 15 nm, respectively. The analysis of the core density showed that both groups had a similar asymmetric distribution with the same average. Stimulation preferentially released those vesicles located ,100 nm from the plasma membrane that had no apparent relationship with the active zone. After the stimulus, the average distances of LDCVs to the plasma membrane and active zone did not change, suggesting that the stimulus also caused the relocation of inner LDCVs. Interestingly, optical density analysis showed that the released vesicles had low range densities, and suggested that LDCVs release their entire content. We conclude that LDCV exocytosis mainly involves those vesicles located ,100 nm from the plasma membrane and occurs in regions of synaptic boutons presumed to be nonspecialized. These results agree with the characteristics of the classical model that proposes full content release. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008. [source] Activation of a calcium entry pathway by sodium pyrithione in the bag cell neurons of AplysiaDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2004Ronald J. Knox Abstract The ability of sodium pyrithione (NaP), an agent that produces delayed neuropathy in some species, to alter neuronal physiology was accessed using ratiometric imaging of cytosolic free Ca2+ concentration ([Ca2+]i) in fura PE-filled cultured Aplysia bag cell neurons. Bath-application of NaP evoked a [Ca2+]i elevation in both somata and neurites with an EC50 of ,300 nM and a Hill coefficient of ,1. The response required the presence of external Ca2+, had an onset of 3,5 min, and generally reached a maximum within 30 min. 2-Methyl-sulfonylpyridine, a metabolite and close structural analog of NaP, did not elevate [Ca2+]i. Under whole-cell current-clamp recording, NaP produced a ,14 mV depolarization of resting membrane potential that was dependent on external Ca2+. These data suggested that NaP stimulates Ca2+ entry across the plasma membrane. To minimize the possibility that a change in cytosolic pH was the basis for NaP-induced Ca2+ entry, bag cell neuron intracellular pH was estimated with the dye 2,,7,-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester. Exposure of the neurons to NaP did not alter intracellular pH. The slow onset and sustained nature of the NaP response suggested that a cation exchange mechanism coupled either directly or indirectly to Ca2+ entry could underlie the phenomenon. However, neither ouabain, a Na+/K+ ATPase inhibitor, nor removal of extracellular Na+, which eliminates Na+/Ca2+ exchanger activity, altered the NaP-induced [Ca2+]i elevation. Finally, the possibility that NaP gates a Ca2+ -permeable ion channel in the plasma membrane was examined. NaP did not appear to activate two major forms of bag cell neuron Ca2+ -permeable ion channels, as Ca2+ entry was unaffected by inhibition of voltage-gated Ca2+ channels using nifedipine or by inhibition of a voltage-dependent, nonselective cation channel using a high concentration of tetrodotoxin. In contrast, two potential store-operated Ca2+ entry current inhibitors, SKF-96365 and Ni2+, attenuated NaP-induced Ca2+ entry. We conclude that NaP activates a slow, persistent Ca2+ influx in Aplysia bag cell neurons. © 2004 Wiley Periodicals, Inc. J Neurobiol 411,423, 2004 [source] Initial stages of neural regeneration in Helisoma trivolvis are dependent upon PLA2 activityDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2003Matthew S. Geddis Abstract Neuronal regeneration after damage to an axon tract requires the rapid sealing of the injured plasma membrane and the subsequent formation of growth cones that can lead regenerating processes to their appropriate target. Membrane sealing and growth cone formation are Ca2+ -dependent processes, but the signaling pathways activated by Ca2+ to bring about these effects remain poorly understood. An in vitro injury model was employed in which neurites from identified snail neurons (Helisoma trivolvis) were transected with a glass microknife, and the formation of new growth cones from the distal portions of transected neurites was recorded at defined times after transection. This study presents three main results. First, phospholipase A2 (PLA2), a calcium-activated enzyme, is necessary for membrane sealing in vitro. Second, PLA2 activity is also required for the formation of a new growth cone after the membrane has sealed successfully. Thus, PLA2 plays a dual role by affecting both growth cone formation and membrane sealing. Third, the injury-induced activation of PLA2 by Ca2+ controls growth cone formation through the production of leukotrienes, secondary metabolites of PLA2 activity. Taken together, these results suggest that the injury-induced Ca2+ influx acts via PLA2 and leukotriene production to assure growth cone formation. These findings indicate that events that cause an inhibition of PLA2 or lipoxygenases, enzymes that produce leukotrienes, could result in the inability of neurites to regenerate. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 555,565, 2003 [source] Histology and ultrastructure of the salivary glands and salivary pumps in the scorpionfly Panorpa obtusa (Mecoptera: Panorpidae)ACTA ZOOLOGICA, Issue 4 2010Shuyu Liu Abstract Liu, S. and Hua, B. 2009. Histology and ultrastructure of the salivary glands and salivary pumps in the scorpionfly Panorpa obtusa (Mecoptera: Panorpidae). ,Acta Zoologica (Stockholm) 91: 457,465. The morphology, histology and ultrastructure of the salivary glands and salivary pumps in the scorpionfly Panorpa obtusaCheng 1949 were investigated using light microscopy and scanning and transmission electron microscopy. The salivary glands display a distinct sexual dimorphism. The female has only two small sac-like glands located in the prothorax, while the male possesses six long tubular glands extending into the sixth abdominal segment. The male salivary glands can be divided into five distinct regions. The apical long, thin secretory region possesses numerous secretory cells containing large secretory vesicles; the salivary reservoir expands in diameter, accumulating and temporarily storing the saliva in addition to secreting saliva; the constricted region contains prismatic cells with complex infolded plasma membrane; the sac has an internal brush border to absorb water and ions; the common salivary duct contains longitudinal muscles in the male, but not in the female. The salivary pump possesses independent strong dorsal muscles and abundant internal palm spines near its orifice. The anatomy and ultrastructure of the salivary glands and the salivary pump of scorpionflies as well as their possible functions are briefly discussed. [source] Fine structure of spermatozoa of Chondrostoma nasus and Rutilus meidingerii (Teleostei, Cyprinidae), as revealed by scanning and transmission electron microscopyACTA ZOOLOGICA, Issue 1 2010Sonja Fürböck Abstract Fürböck, S., Patzner, R.A. and Lahnsteiner, F. 2008. Fine structure of spermatozoa of Chondrostoma nasus and Rutilus meidingerii (Teleostei, Cyprinidae), as revealed by scanning and transmission electron microscopy. , Acta Zoologica (Stockholm) 91: 88,95 The fine structure of spermatozoa of sneep or nase, Chondrostoma nasus, and lake chub, Rutilus meidingerii, was investigated by means of scanning and transmission electron microscopy. The uniflagellate spermatozoa of C. nasus lacked an acrosome. The flagellum contained the conventional nine peripheral doublets and one central pair of microtubules (9 + 2 pattern) and lacked lateral fins. The uniflagellate spermatozoa of R. meidingerii were made up of a head, also without an acrosome. For both species the sperm tail was covered by a plasma membrane. The midpiece of C. nasus contained five or six mitochondria on average, vesicles and glycogen granules, whereas the midpiece of R. meidingerii had seven mitochondria of a spherical or ovoid shape. The centriolar complex was located caudolaterally with respect to the nucleus. In C. nasus, the centrioles were orientated at an angle of 125° to each other, whereas the centrioles of R. meidingerii were at an angle of 110°. The fine structure of C. nasus and R. meidingerii spermatozoa showed species-specific differences in the position of the proximal centriole relative to the distal centriole, the position and number of mitochondria, size of the head and the length of the flagellum. (Correction added on 11 June 2009, after first online publication: The word ,axoneme' was deleted from the sentence ,The flagellum contained the conventional nine peripheral doublets and one central pair of microtubules (9 + 2 pattern) axoneme and lacked lateral fins.') [source] Walker tumor cells express larger amounts of the antiapoptotic protein Bcl-2 and presents higher resistance to toxic concentrations of Ca2+ than the tumor cells K 562DRUG DEVELOPMENT RESEARCH, Issue 4 2001Graziela Milani Abstract Ca2+ homeostasis was studied in two tumor cell lines (Walker 256 and K 562) previously shown to exhibit different mitochondrial Ca2+ accumulation capacity. When intact, both cells present cytosolic Ca2+ concentrations within the range expected for mammalian cells, as determined through fura-2 fluorescence ratios. In order to study intracellular Ca2+ distribution, digitonin was used to permeabilize the plasma membrane without affecting intracellular organelle structure, as assessed using electron microscopy. Digitonin-permeabilized Walker 256 cells incubated with Ca2+ presented uptake of the cation exclusively through mitochondrial activity. In addition, very large Ca2+ loads were necessary to promote a disruption of Walker 256 mitochondrial membrane potential. K 562 cells presented active Ca2+ uptake through both nonmitochondrial and mitochondrial compartments and suffered disruption of mitochondrial membrane potential at lower Ca2+ loads than Walker 256 mitochondria. The higher Ca2+ resistance in Walker 256 cells could be attributed to Bcl-2 overexpression, as evidenced by immunocytochemical staining. Thus, we correlate natural Bcl-2 overexpression, observed in Walker 256 cells, with higher resistance to mitochondrial Ca2+ overload, as was shown previously in mitochondria from cells transfected with the bcl-2 gene. Drug Dev. Res. 52:508,514, 2001. © 2001 Wiley-Liss, Inc. [source] Structure of adhesive organ of the mountain-stream catfish, Pseudocheneis sulcatus (Teleostei: Sisoridae)ACTA ZOOLOGICA, Issue 4 2005Debasish Das Abstract The structure and ultrastructure of the adhesive organ (AO) in the catfish, Pseudocheneis sulcatus (Sisoridae), an inhabitant of the sub-Himalayan streams of India, is described. The surface of the AO is thrown into folds, the ridges of which bear curved spines. The AO epidermis consists of 10,12 tiers of filament-rich cells, of which the outer tier cells project spines lined with a thick plasma membrane and bear bundles of tonofilaments (TF). Their cytoplasm contains TF and large mucus-like granules, but no obvious organelles. A second tier of living cells with spines is present beneath the outer tier and seems to replace the latter when its spines are damaged or shed. The outer tier cells react positively with antibody to cytokeratin. Actin labelling is clearly absent from the outer tier, indicating that keratinization of the outer tier occurs in the absence of actin filaments. In the cells of the third to fifth tiers, the cytoplasm possesses abundant small mucous granules (0.1,0.3 µm), and fewer TF compared to the cytoplasm in the spines. The cells of the innermost tiers and the basal layer possess few TF bundles, but no mucous granules. The potential of AO filament cells to produce both mucous granules and keratin filaments is noteworthy. The observations provide evidence that specific regions of fish epidermis can actually undergo a true process of keratinization. [source] Synaptic vesicle proteins under conditions of rest and activation: Analysis by 2-D difference gel electrophoresisELECTROPHORESIS, Issue 17 2006Jacqueline Burré Abstract Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl- n -hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification. [source] Delivery of bioactive, gel-isolated proteins into live cellsELECTROPHORESIS, Issue 9 2003Jennifer E. Taylor Abstract The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein. [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] Altered membrane glycoprotein targeting in cholestatic hepatocytesEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 5 2010Giuseppa Esterina Liquori Eur J Clin Invest 2010; 40 (5): 393,400 Abstract Background, Hepatocytes are polarized epithelial cells with three morphologically and functionally distinct membrane surfaces: the sinusoidal, lateral and canalicular surface domains. These domains differ from each other in the expression of integral proteins, which concur to their polarized functions. We hypothesize that the cholestasis-induced alterations led to partial loss of hepatocyte polarity. An altered expression of membrane proteins may be indicative of functional disorders. Alkaline liver phosphatase (ALP), one of the most representative plasma membrane glycoproteins in hepatocytes, is expressed at the apical (canalicular) pole of the cell. Because the release of ALP protein in the bloodstream is significantly increased in cholestasis, the enzymatic levels of plasma ALP have major relevance in the diagnosis of cholestatic diseases. Here we assess the cholestasis-induced redistribution of membrane glycoproteins to investigate the ALP release. Materials and methods, We performed enzymatic histochemistry, immunohistochemistry, lectin histochemistry, immunogold and lectin-and immunoblotting studies. Experimental cholestasis was induced in rats by ligation of common bile duct (BDL). Results, The BDL led to altered membrane sialoglycoprotein targeting as well as to ultrastructural and functional disorders. Disarrangement of the microtubular system, thickening of the microfilamentous pericanalicular ectoplasm and disturbance of the vectorial trafficking of membrane glycoprotein containing vesicles were found. Conclusions, Altogether, results indicate that the cholestasis-induced partial loss of hepatocyte cell polarity leads to mistranslocation of ALP to the sinusoidal plasma membrane from where the enzyme is then massively released into the bloodstream. [source] | ||||||||||||||||||||||||||||||||||||||||||||||