Proteolipid plasmolipin: localization in polarized cells,regulated expression and lipid raft association in CNS and PNS myelin

The proteolipid plasmolipin is member of the expanding group of tetraspan (4TM) myelin proteins. Initially, plasmolipin was isolated from kidney plasma membranes, but subsequent northern blot analysis revealed highest expression in the nervous system. To gain more insight into the functional roles of plasmolipin, we have generated a plasmolipin-specific polyclonal antibody. Immunohistochemical staining confirms our previous observation of glial plasmolipin expression and proves plasmolipin localization in the compact myelin of rat peripheral nerve and myelinated tracts of the CNS. Western blot analysis indicates a strong temporal correlation of plasmolipin expression and (re-) myelination in the PNS and CNS. However, following axotomy plasmolipin expression is also recovered in non-regenerating distal nerve stumps. In addition, we detected plasmolipin expression in distinct neuronal subpopulations of the CNS. The observed asymmetric distribution of plasmolipin in compact myelin, as well as in epithelial cells of kidney and stomach, indicates a polarized cellular localization. Therefore, we purified myelin from the CNS and PNS and demonstrated an enrichement of phosphorylated plasmolipin protein in detergent-insoluble lipid raft fractions, suggesting selective targeting of plasmolipin to the myelin membranes. The present data indicate that the proteolipid plasmolipin is a structural component of apical membranes of polarized cells and provides the basis for further functional analysis.     

Intermediate filaments: structure, regulation of expression and possible function

New data are reviewed on intermediate filaments, i.e. on one of the cytoskeleton components. Structural proteins of intermediate filaments, their enzymatic modification, filament-associated proteins and the peculiarities of filament assembly are dealt with. The regularities of expression of intermediate filament proteins in normal tissues are analysed, as well as during differentiation and cultured cell growth. In the final part of the paper possible functions of intermediate filaments are discussed.     

孤啡肽（OFQ）的分布,特性及其可能作用

孤啡肽是１９９５年底新发现的一种结构与内阿片肽类似的物质,它的发现引发了国际上相关研究的热潮。近年来的研究已经基本阐明了孤啡肽在中枢及外周的分布及其生化与药理特性,并初步发现该物质参与痛觉调制、影响运动与行为、调控递质释放,还可能参与情绪反应与学习记忆、神经内分泌与免疫过程以及胃的活动等,但其确切作用仍有待于进一步阐明。     

Effects of disialoganglioside GD3 on the mitochondrial membrane potential

GD3 is an intracellular mediator of apoptotic signaling. Although GD3 is known to directly act on mitochondria, the dynamic responses of individual mitochondria to GD3 remain to be elucidated. In the current study, the membrane potential of single mitochondria is observed in the presence of GD3 or its analogues. Here, we report that (1) GD3 specifically induces gradual depolarizations of the inner membrane by a mechanism that differs from the permeability transition, and (2) the GD3-induced depolarizations are suppressed by cyclosporin A. These results suggest that GD3 depolarizes mitochondria by a mechanism distinct from but relevant to the permeability transition.     

Apoptosis of human breast carcinoma cells in the presence of disialosyl gangliosides: II. Treatment of SKBR3 cells with GD3 and GD1b gangliosides

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells has been monitored during the cells' progression to apoptosis by anti-cancer drugs and inhibitors of the cell surface glycolipids, gangliosides and SA-Le^x biosyntheses [Basu, S (1991) Glycobiology, 1, 469–475; and ibid, 427–435] in animal tissues and human carcinoma cells, respectively. Induction of apoptosis in cancer cells by cell surface glycolipids in the human breast cancer (SKBR3) cells is the aim in this study. We have employed the disialosyl gangliosides (GD3 and GD1b) to initiate apoptosis in SKBR3 cells grown in culture in the presence of ¹⁴C-L-Serine. At lower concentrations (0–20 μM) of exogenously added non-radioactive GD3, GD1b, or bovine ganglioside mixture (GM1:GD1a:GD1b:GT1a 2:4:4:2), the incorporation of radioactivity in both ¹⁴C-sphingolipid and ¹⁴C-ceramide was higher. However, at higher concentrations (20–100 μM), wherein apoptosis occurred in high frequency, the ¹⁴C-incorporation decreased in both GSLs and ceramide. Apoptosis induction was monitored by the concomitant appearance of caspase-3 activation and the binding of a fluorescent dye PSS-380 to the outer leaflet of phosphatidyl-serine. These results indicated that, in addition to many unknown cell surface glycoconjugates GD3 or GD1b (disialosyl ganglioside) could play an important role in the regulation of breast carcinoma cell death. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Saposins: structure, function, distribution, and molecular genetics.

Saposins A, B, C, and D are small heat-stable glycoproteins derived from a common precursor protein, prosaposin. These mature saposins, as well as prosaposin, activate several lysosomal hydrolases involved in the metabolism of various sphingolipids. All four saposins are structurally similar to one another including placement of six cysteines, a glycosylation site, and conserved prolines in identical positions. In spite of the structural similarities, the specificity and mode of activation of sphingolipid hydrolases differs among individual saposins. Saposins appear to be lysosomal proteins, exerting their action upon lysosomal hydrolases. Prosaposin is a 70 kDa glycoprotein containing four domains, one for each saposin, placed in tandem. Prosaposin is proteolytically processed to saposins A, B, C and D, apparently within lysosomes. However, prosaposin also exists as an integral membrane protein not destined for lysosomal entry and exists uncleaved in many biological fluids such as seminal plasma, human milk, and cerebrospinal fluid, where it appears to have a different function. The physiological significance of saposins is underlined by their accumulation in tissues of lysosomal storage disease patients and the occurrence of sphingolipidosis due to mutations in the prosaposin gene. This review presents an overview of the occurrence, structure and function of these saposin proteins.     

Roles for Laminin in Embryogenesis: Exencephaly,Syndactyly, and Placentopathy in Mice Lacking the Laminin α5 Chain

Laminins are the major noncollagenous glycoproteins of all basal laminae (BLs). They are α/β/γ heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin''s roles in embryonic development. Here, we show that the laminin α5 chain is required during embryogenesis. The α5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds, including those of the surface ectoderm and placental vasculature. BLs that lose α5 retain or acquire other α chains. Embryos lacking laminin α5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure (exencephaly), failure of digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth. These defects are all attributable to defects in BLs that are α5 positive in controls and that appear ultrastructurally abnormal in its absence. Other laminin α chains accumulate in these BLs, but this compensation is apparently functionally inadequate. Our results identify new roles for laminins and BLs in diverse developmental processes.     

β2 and γ3 laminins are critical cortical basement membrane components: Ablation of Lamb2 and Lamc3 genes disrupts cortical lamination and produces dysplasia

Cortical development is dependent on the timely production and migration of neurons from neurogenic sites to their mature positions. Mutations in several receptors for extracellular matrix (ECM) molecules and their downstream signaling cascades produce dysplasia in brain. Although mutation of a critical binding site in the gene that encodes the ECM molecule laminin γ1 (Lamc1) disrupts cortical lamination, the ECM ligand(s) for many ECM receptors have not been demonstrated directly in the cortex. Several isoforms of the heterotrimeric laminins, all containing the β2 and γ3 chain, have been isolated from the brain, suggesting they are important for CNS function. Here, we report that mice homozygous null for the laminin β2 and γ3 chains exhibit cortical laminar disorganization. Mice lacking both of these laminin chains exhibit hallmarks of human cobblestone lissencephaly (type II, nonclassical): they demonstrate severe laminar disruption; midline fusion; perturbation of Cajal‐Retzius cell distribution; altered radial glial cell morphology; and ectopic germinal zones. Surprisingly, heterozygous mice also exhibit laminar disruption of cortical neurons, albeit with lesser severity. In compound null mice, the pial basement membrane is fractured, and the distribution of a key laminin receptor, dystroglycan, is altered. These data suggest that β2 and γ3‐containing laminins play an important dose‐dependent role in development of the cortical pial basement membrane, which serves as an attachment site for Cajal‐Retzius and radial glial cells, thereby guiding neural development. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Gender-dependent cellular and biochemical effects of maternal deprivation on the hippocampus of neonatal rats: a possible role for the endocannabinoid system

Adult animals submitted to a single prolonged episode of maternal deprivation (MD) [24 h, postnatal days (PND) 9-10] show behavioral alterations that resemble specific symptoms of schizophrenia. These behavioral impairments may be related to neuronal loss in the hippocampus triggered by elevated glucocorticoids. Furthermore, our previous data suggested functional relationships between MD stress and the endocannabinoid system. In this study, we addressed the effects of MD on hippocampal glial cells and the possible relationship with changes in plasma corticosterone (CORT) levels. In addition, we investigated the putative involvement of the endocannabinoid system by evaluating (a) the effects of MD on hippocampal levels of endocannabinoids (b) The modulation of MD effects by two inhibitors of endocannabinoids inactivation, the fatty acid amide hydrolase inhibitor N-arachidonoyl-serotonin (AA-5-HT), and the endocannabinoid reuptake inhibitor, OMDM-2. Drug treatments were administered once daily from PND 7 to PND 12 at a dose of 5 mg/kg, and the animals were sacrificed at PND 13. MD induced increased CORT levels in both genders. MD males also showed an increased number of astrocytes in CA1 and CA3 areas and a significant increase in hippocampal 2-arachidonoylglycerol. The cannabinoid compounds reversed the endocrine and cellular effects of maternal deprivation. We provide direct evidence for gender-dependent cellular and biochemical effects of MD on developmental hippocampus, including changes in the endocannabinoid system.     

Oligomeric state and membrane binding behaviour of creatine kinase isoenzymes: Implications for cellular function and mitochondrial structure

The membrane binding properties of cytosolic and mitochondrial creatine kinase isoenzymes are reviewed in this article. Differences between both dimeric and octameric mitochondrial creatine kinase (Mi-CK) attached to membranes and the unbound form are elaborated with respect to possible biological function. The formation of crystalline mitochondrial inclusions under pathological conditions and its possible origin in the membrane attachment capabilities of Mi-CK are discussed. Finally, the implications of these results on mitochondrial energy transduction and structure are presented.     

Zinc inhibition of cellular energy production: implications for mitochondria and neurodegeneration

An increasing body of evidence suggests that high intracellular free zinc promotes neuronal death by inhibiting cellular energy production. A number of targets have been postulated, including complexes of the mitochondrial electron transport chain, components of the tricarboxylic acid cycle, and enzymes of glycolysis. Consequences of cellular zinc overload may include increased cellular reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and reduced cellular ATP levels. Additionally, zinc toxicity might involve zinc uptake by mitochondria and zinc induction of mitochondrial permeability transition. The present review discusses these processes with special emphasis on their potential involvement in brain injury.     

Down-regulation of GD3 ganglioside and its O-acetylated derivative by stable transfection with antisense vector against GD3-synthase gene expression in hamster melanoma cells: effects on cellular growth, melanogenesis, and dendricity

The expression of gangliosides in hamster melanoma cells is closely related to cellular growth and degree of differentiation, with slow-growing, highly differentiated melanotic melanoma cells expressing GM3 and fast-growing, undifferentiated amelanotic Ab melanoma cells having a preponderance of GD3 and O-acetyl-GD3. We recently showed that down-regulation of O-acetyl-GD3 expression in hamster melanoma cells by introducing the influenza C virus O-acetylesterase cDNA into the cells resulted in induction of dendricity, with a concomitant increased expression of GD3. To examine the effect of the increased GD3 expression in the plasma membrane on the dendricity of the AbC-1 cells, we first established the cDNA coding for hamster GD3-synthase. We then targeted the sialyltransferase gene expression by the antisense knockdown experiment, and the results showed that inhibition of the expression of gangliosides GD3 and O-acetyl-GD3 induced dendricity in the hamster melanoma AbC-1 cell line. These GD3- and O-acetyl-GD3-depleted cells also demonstrated a decreased rate of cell growth, but their melanogenic potential was not affected. These results rule out the possibility that GD3 may serve as an active molecule for dendrite outgrowth in this cell line and suggest that the enhanced expression of O-acetyl-GD3 ganglioside may stimulate cellular growth and suppress certain differentiated phenotypes such as dendrite formation, but not melanogenesis, in our system.     

Expression and sub cellular localization of the sodium hydrogen exchanger isoform-1 in rat tissues: A possible functional relevance

In an attempt to understand the mechanism underlying the tissue-dependent function, the expression of NHE-1 protein and its sub cellular localization was examined in the rat GI-tract and other tissues. Rat NHE-1 polyclonal antibodies were raised in rabbits using a NHE-1 fusion protein antigen. The antibodies recognized a 110 kD protein in rats and mice, but not in human or rabbit RBCs. Colon, stomach, brain, spleen and kidney expressed NHE-1 protein abundantly, whereas the skeletal muscle the least abundant. Ouabain-sensitive-K⁺-stimulated p-nitrophenylphosphatase (PNPPase), the partial activity of the sodium pump and alkaline phosphatase (Apase) were used as the markers of the basolateral and apical membranes. NHE-1 was detected predominantly in the PNPPase enriched membrane fractions, but was also detected in the apical membrane enriched fractions in the kidney cortex, jejunum and colon at a lower level. NHE-1 was detected in the plasma membrane enriched fractions from the skeletal muscle and ventricle. Immunofluorescence data showed a similar localization pattern of NHE-1 in the colon and kidney sections. These findings suggest that NHE-1 is localized both on the apical and basolateral membrane. In view of its similar sub cellular localization in the GI-tract and kidney, but a different level of expression, might suggest that the level of protein, but not the sub cellular distribution is important to regulate its tissue-dependent function.     

Annexin II tetramer: structure and function

The annexins are a family of proteins that bind acidic phospholipids in the presence of Ca²⁺. The interaction of these proteins with biological membranes has led to the suggestion that these proteins may play a role in membrane trafficking events such as exocytosis, endocytosis and cell-cell adhesion. One member of the annexin family, annexin II, has been shown to exist as a monomer, heterodimer or heterotetramer. The ability of annexin II tetramer to bridge secretory granules to plasma membrane has suggested that this protein may play a role in Ca²⁺-dependent exocytosis. Annexin II tetramer has also been demonstrated on the extracellular face of some metastatic cells where it mediates the binding of certain metastatic cells to normal cells. Annexin II tetramer is a major cellular substrate of protein kinase C and pp60^src. Phosphorylation of annexin II tetramer is a negative modulator of protein function.Supported by a grant from the Medical Research Council of Canada     

(3)H](2S,4R)-4-Methylglutamate: a novel ligand for the characterization of glutamate transporters

[(3)H](2S,4R)-4-Methylglutamate ([(3)H]4MG), used previously as a ligand for low-affinity kainate receptors, was employed to establish a binding assay for glutamate transporters (GluTs), as 4MG has also been shown to have affinity for the glial GluTs, GLT1 and GLAST. In rat brain membrane homogenates in the presence of Na(+) ions at 4 degrees C, specific binding of [(3)H]4MG was rapid and saturable (t(1/2) approximately 15 min), representing > 90% of total binding. Dissociation of [(3)H]4MG occurred in a biphasic manner, however, saturation studies and Scatchard analysis indicated a single site of binding (n(H) = 0.85) and a K(d) of 6.2 +/- 0.8 microM with a B(max) of 111.8 +/- 23.8 pmol/mg protein. Specific binding of [(3)H]4MG was Na(+)-dependent and inhibited by K(+) and HCO(3-). Pharmacological inhibition with compounds acting at GluTs revealed that Glu, D- and L-aspartate, L-serine-O-sulfate and Ltrans-pyrrolidine-2,4-dicarboxylate fully displaced specific binding. Drugs having preferential affinity for GLT1, kainate, dihydrokainate and Lthreo-3-methylglutamate, all inhibited approximately 40% of specific binding. The inhibition pattern of L-serine-O-sulfate in the presence of a saturating concentration of dihydrokainate was suggestive of [(3)H]4MG also labelling GLAST. 6-Cyano-7-nitroquinoxaline, a kainate receptor antagonist, and a range of Glu receptor agonists and antagonists failed to significantly inhibit [(3)H]4MG binding. The pharmacological profile of binding of [(3)H]4MG resembled that found for [(3)H]D-aspartate, a ligand specific for GluTs, reinforcing the hypothesis that [(3)H]4MG was labelling GluTs in this assay. Together, these data illustrate the development of an efficient, economic binding assay that is suitable for the characterization of different subtypes of GLuTs.     

Significant variability among bulls in the sperm membrane permeability for water and glycerol: possible implications for semen freezing protocols for individual males

The aim of this study was to test the hypothesis that bulls have significant intra-individual differences in the hydraulic conductivity (L(p)) and permeability coefficient for glycerol (P(s)) of the sperm cell membrane. The permeability parameters were determined at 22, 10, and 0 degrees C of sperm from 7 Holstein Frisian artificial insemination (AI) bulls, using four ejaculates per bull. A stopped-flow approach was applied to provide temporal resolution sufficient to measure rapid cell volume changes under anisosmotic conditions in the absence or presence of glycerol. This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute L(p) in the absence glycerol, and L(p) in the presence of glycerol (L(p)(gly)) and P(s). Averaged over all bulls, L(p) in the absence of glycerol was 0.28+/-0.01, 0.15+/-0.01 and 0.10+/-0.01 microm min(-1)atm(-1) (mean+/-SD) at 22, 10 and 0 degrees C, respectively, yielding an Arrhenius activation energy (E(a)) of 7.39 kcal/mol. The average L(p)(gly) value at 22 degrees C, was 3.8 times lower than L(p) in the absence of glycerol (P<0.05). L(p)(gly), P(s), and the reflection coefficient (sigma) at 22 degrees C were 0.073+/-0.015 microm min(-1)atm(-1), 0.80+/-0.33 x 10(-3)cm min(-1), and 0.92+/-0.10 (mean+/-SD), respectively. Subsequent experiments were performed at 10 and 0 degrees C. Activation energies for L(p)(gly) and P(s) were 10.08 and 8.77 kcal/mol, respectively. The significant differences between individual bulls in L(p) and P(s) indicate that individual males may require individual adjustments of the cooling protocol. Application of these data in a theoretical model to simulate the osmotic events during freezing resulted in predicted optimal cooling rates in the range of published empirical values.