Plasma Membrane Lipid Organization Research Articles

Impaired mammalian sperm function and lower phosphorylation signaling caused by the herbicide Roundup® Ultra Plus are due to its surfactant component

The use of worldwide glyphosate-based herbicide Roundup® is growing and to date its effects on mammalian spermatozoa are controversial. This study aims to investigate the functional impact of in vitro exposure of pig spermatozoa to low concentrations of Roundup® Ultra Plus (RUP), similar to those present as environment contaminants, to its active ingredient glyphosate, and to the non-active component, surfactant polyoxyethyleneamine (POEA). Pig spermatozoa were incubated in Tyrode's basal medium (TBM) or Tyrode's complete medium (TCM) (1 h at 38.5 °C) with several RUP dilutions or equivalent concentrations of glyphosate or POEA. RUP treatment causes a significant dilution-dependent decrease in sperm motility, a significant increase in plasma membrane disorganization and reduction in GSK3β phosphorylation (TBM) and in two PKA substrates (TBM and TCM), whereas does not affect sperm viability or mitochondrial membrane potential (MMP). Equivalent glyphosate concentrations do not affect any functional sperm parameters. However, POEA concentrations equivalent to RUP dilutions mimic all RUP sperm effects: decrease sperm motility in a concentration-dependent manner, increase sperm plasma membrane lipid disorder and significantly inhibit GSK3β phosphorylation (TBM) and two PKA substrates without affecting sperm viability or MMP. In summary, low concentrations RUP herbicide cause sperm motility impairment without affecting sperm viability. This adverse effect could be likely due to a detrimental effect in the plasma membrane lipid organization and to inhibition of phosphorylation of both, GSK3β and specific PKA substrates. Importantly, our results indicate that negative effects of low RUP concentrations in pig spermatozoa function are likely caused by the surfactant included in its formulation and no by its active ingredient glyphosate.

Lysophospholipid acylation modulates plasma membrane lipid organization and insulin sensitivity in skeletal muscle.

Aberrant lipid metabolism promotes the development of skeletal muscle insulin resistance, but the exact identity of lipid-mediated mechanisms relevant to human obesity remains unclear. A comprehensive lipidomic analysis of primary myocytes from individuals who were insulin-sensitive and lean (LN) or insulin-resistant with obesity (OB) revealed several species of lysophospholipids (lyso-PLs) that were differentially abundant. These changes coincided with greater expression of lysophosphatidylcholine acyltransferase 3 (LPCAT3), an enzyme involved in phospholipid transacylation (Lands cycle). Strikingly, mice with skeletal muscle-specific knockout of LPCAT3 (LPCAT3-MKO) exhibited greater muscle lysophosphatidylcholine/phosphatidylcholine, concomitant with improved skeletal muscle insulin sensitivity. Conversely, skeletal muscle-specific overexpression of LPCAT3 (LPCAT3-MKI) promoted glucose intolerance. The absence of LPCAT3 reduced phospholipid packing of cellular membranes and increased plasma membrane lipid clustering, suggesting that LPCAT3 affects insulin receptor phosphorylation by modulating plasma membrane lipid organization. In conclusion, obesity accelerates the skeletal muscle Lands cycle, whose consequence might induce the disruption of plasma membrane organization that suppresses muscle insulin action.

Modulation of Insulin Receptor Activity Upon Plasma Membrane Sterol and Phospholipid Substitutions to Alter Lipid Domain Formation

Plasma membrane lipid organization into ordered (raft) and disordered domains has been thought to help cellular signal transduction processes by clustering membrane receptors in rafts to facilitate robust downstream signaling. Ordered (Lo) domains are enriched in sphingomyelin (SM) and cholesterol while disordered (Ld) domains are enriched in unsaturated lipids such as phosphatidyl choline (PC). Insulin receptor (IR) is a receptor tyrosine kinase that has reduced activity after cellular cholesterol depletion, and increased distribution in detergent resistant membranes, which can reflect localization in rafts, after activation.

The ABC transporter Pdr18 is required for yeast thermotolerance due to its role in ergosterol transport and plasma membrane properties.

SummaryAmong the mechanisms by which yeast overcomes multiple stresses is the expression of genes encoding ATP‐binding cassette (ABC) transporters required for resistance to a wide range of toxic compounds. These substrates may include weak acids, alcohols, agricultural pesticides, polyamines, metal cations, as in the case of Pdr18. This pleotropic drug resistance transporter was previously proposed to transport ergosterol at the plasma membrane (PM) level contributing to the maintenance of PM lipid organization and reduced diffusional permeation induced by lipophilic compounds. The present work reports a novel phenotype associated with the putative drug/xenobiotic‐efflux‐pump transporter Pdr18: the resistance to heat shock and to long‐term growth at supra‐optimal temperatures. Cultivation at 40°C was demonstrated to lead to higher PM permeabilization of a pdr18Δ cell population with the PDR18 gene deleted compared with the parental strain population, as indicated by flow cytometry analysis of propidium iodide stained cells. Cells of pdr18Δ grown at 40°C also exhibited increased transcription levels from genes of the ergosterol biosynthetic pathway, compared with parental cells. However, this adaptive response at 40°C was not enough to maintain PM physiological ergosterol levels in the population lacking the Pdr18 transporter and free ergosterol precursors accumulate in the deletion mutant cells.

Boar spermatozoa proteomic profile varies in sperm collected during the summer and winter

Boar sperm quality is less during the summer as a result of the different photoperiod or ambient temperatures as compared with the winter. The present study was conducted to elucidate possible variations in proteomic profiles of boar spermatozoa collected during the summer and winter. Effects of season on sperm viability, total motility, progressive motility, acrosome status, mitochondrial membrane potential and plasma membrane lipid organization were also analyzed. Only sperm viability and mitochondrial membrane potential were less during the summer (P < 0.05). Spermatozoa were processed and evaluated using the nano LC–MS/MS QTof procedures. A total of 1028 characterized proteins were identified in sperm collected during both seasons of the year (False Discovery Rate < 0.01) and, among the total, 85 proteins differed in sperm collected in the winter and summer, with there being a lesser abundance of these proteins when there were ejaculate collections during the summer (q-value ≤ 0.05). The results from enrichment assessments for these protein networks utilizing UniProtKB procedures for determining reproductive processes indicates there were 23 proteins that were less abundant in the summer than winter. These proteins have essential functions in spermatogenesis, sperm motility, acrosome reaction and fertilization. These results are the first where there was ascertaining of proteomic differences in boar spermatozoa collected in the summer and winter. These results might help to explain the decreased sperm quality and prolificity when semen of boars is used for artificial insemination that is collected during the season of the year when ambient temperatures are relatively greater.

Effect of boar semen supplementation with recombinant heat shock proteins during summer

Artificial insemination (AI) in pigs is mainly performed with refrigerated boar semen. There is a marked negative seasonal effect on the quality of boar sperm, mainly due to relatively greater ambient temperatures; to counteract this thermal stress, sperm cells possess natural defensive mechanisms such as Heat Shock Proteins (HSPs) that prevent protein denaturation. Thus, the objective of this research was to improve the quality of commercial boar semen collected during the summer when ambient temperatures are greater using recombinant HSPs. For this purpose, different concentrations (0.1, 0.5 and 1 μg/ml) of recombinant heat shock proteins (HSPD1, HSPA8 or HSP86) were added to commercial boar semen and there was cooling for 48 h at 17 °C. After this storage period, sperm quality was assessed by analyzing sperm viability, mitochondrial membrane potential and plasma membrane lipid organization using flow cytometry; additionally, sperm motility was examined using a CASA system. Also, in vitro fertilization (IVF) using HSP-supplemented boar semen was performed and the quality of the embryos produced was evaluated using quantitative real-time polymerase chain reaction (qPCR) analyzing the relative abundance of mRNA transcripts for genes encoding for embryo quality-related proteins (BAX, TFAM, POLG and POG2). Sperm quality variables, blastocyst rates and the abundance of mRNA transcripts for the selected genes were not affected by the presence of recombinant HSPs at any concentration. These results indicate that the supplementation of commercial seminal doses with recombinant HSPs does not improve boar sperm quality or fertility during the summer months when ambient temperatures are greater.

Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation.

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

HSP90 maintains boar spermatozoa motility and mitochondrial membrane potential during heat stress

Heat Shock Proteins (HSP) is a family of proteins that protects cells from high temperatures. The present work aimed to elucidate the role that HSP90 exerts on boar sperm incubated under heat stress conditions on viability, total motility (TM), progressive motility (PM), acrosome status, mitochondrial membrane potential and plasma membrane lipid organization. Sperm were incubated in non-capacitating conditions (Tyrode’s basal medium or TBM) for 3, 8 and 24 h or in capacitating conditions (Tyrode’s complete medium or TCM) for 4 h at 38.5 °C or 40 °C (Heat stress) in the presence or absence of 5 or 20 μM of 17-AAG, a specific HSP90 inhibitor. Sperm viability was not affected by the presence of 17-AAG in any condition tested compared with its own control (at the same temperature and incubation time). In non-capacitating conditions TM (22.7 ± 4.1 vs. 1.9 ± 1.1; % mean ± SEM), PM (3.1 ± 0.9 vs. 0) and high mitochondrial membrane potential (19.5 ± 2.2 vs. 11.8 ± 0.8) decreased significantly in sperm incubated at 40 °C for 24 h in the presence of 20 μM 17-AAG (control vs. 20 μM 17-AAG, respectively; p < 0.05). In sperm incubated at 38.5 °C only a mild decrease in TM was observed (48.7 ± 3.1 vs. 32.1 ± 4.8; control vs. 20 μM 17-AAG, respectively; p < 0.05). However, under capacitating conditions none of the sperm parameters studied were affected by 17-AAG after 4 h of incubation. These results demonstrate for the first time the role of HSP90 in the maintenance of boar sperm motility and mitochondrial membrane potential during prolonged heat stress in non-capacitating conditions.

Protein kinase C activity in boar sperm.

Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis

BackgroundMembrane protrusions play important roles in biological processes such as cell adhesion, wound healing, migration, and sensing of the external environment. Cell protrusions are a subtype of membrane microdomains composed of cholesterol and sphingolipids, and can be disrupted by cholesterol depletion. Prominins are pentaspan membrane proteins that bind cholesterol and localize to plasma membrane (PM) protrusions. Prominin-1 is of great interest as a marker for stem and cancer cells, while Prominin-2 (Prom2) is reportedly restricted to epithelial cells. AimTo characterize the effects of Prom-2 expression on PM microdomain organization. MethodsProm2-fluorescent protein was transfected in human skin fibroblasts (HSF) and Chinese hamster ovary (CHO) cells for PM raft and endocytic studies. Caveolae at PM were visualized using transmission electron microscopy. Cdc42 activation was measured and caveolin-1 knockdown was performed using siRNAs. ResultsProm2 expression in HSF and CHO cells caused extensive Prom2-positive protrusions that co-localized with lipid raft markers. Prom2 expression significantly decreased caveolae at the PM, reduced caveolar endocytosis and increased caveolin-1 phosphorylation. Prom2 expression also inhibited Cdc42-dependent fluid phase endocytosis via decreased Cdc42 activation. Effects on endocytosis were reversed by addition of cholesterol. Knockdown of caveolin-1 by siRNA restored Cdc42 dependent fluid phase endocytosis in Prom2-expressing cells. ConclusionsProm2 protrusions primarily localize to lipid rafts and recruit cholesterol into protrusions and away from caveolae, leading to increased phosphorylation of caveolin-1, which inhibits Cdc42-dependent endocytosis. This study provides a new insight for the role for prominins in the regulation of PM lipid organization.

Structural organization of plasma membrane lipids isolated from cells cultured as a monolayer and in tissue-like conditions

Complementary biophysical approaches were used to study the structural organization of plasma membrane lipids obtained from fibroblasts cultured as two-dimensional (2D) monolayer and in tissue-like three-dimensional (3D) conditions. Fluorescence microscopy experiments demonstrated different domain patterns for 2D and 3D plasma membrane lipid extracts. ESR demonstrated that 3D lipid extract is characterized with lower order parameter than 2D in the deep hydrophobic core of the lipid bilayer. Higher cholesterol and sphingomyelin content in 3D extract, known to increase the order in the glycerophospholipid matrix, was not able to compensate higher fatty acid polyunsaturation of the phospholipids. The interfacial region of the bilayer was probed by the fluorescent probe Laurdan. A higher general polarization value for 3D extract was measured. It is assigned to the increased content of sphingomyelin, cholesterol, phosphatidylethanolamine and phosphatidylserine in the 3D membranes. These results demonstrate that cells cultured under different conditions exhibit compositional heterogeneity of the constituent lipids which determine different structural organization of the membranes.

Phthiocerol Dimycocerosates of M. tuberculosis Participate in Macrophage Invasion by Inducing Changes in the Organization of Plasma Membrane Lipids

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche.

Inhibition of sphingomyelin synthase (SMS) affects intracellular sphingomyelin accumulation and plasma membrane lipid organization

Sphingomyelin plays a very important role both in cell membrane formation that may well have an impact on the development of diseases like atherosclerosis and diabetes. However, the molecular mechanism that governs intracellular and plasma membrane SM levels is largely unknown. Recently, two isoforms of sphingomyelin synthase (SMS1 and SMS2), the last enzyme for SM de novo synthesis, have been cloned. We have hypothesized that SMS1 and SMS2 are the two most likely candidates responsible for the SM levels in the cells and on the plasma membrane. To test this hypothesis, cultured cells were treated with tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of SMS, or with SMS1 and SMS2 siRNAs. Cells were then pulsed with [ 14C]- l-serine (a precursor of all sphingolipids). SMS activity and [ 14C]-SM in the cells were monitored. We found that SMS activity was significantly decreased in cells after D609 or SMS siRNA treatment, compared with controls. SMS inhibition by D609 or SMS siRNAs significantly decreased intracellular [ 14C]-SM levels. We measured cellular lipid levels, including SM, ceramide, phosphatidylcholine, and diacylglycerol and found that SMS1 and SMS2 siRNA treatment caused a significant decrease of SM levels (20% and 11%, respectively), compared to control siRNA treatment; SMS1 but not SMS2 siRNA treatment caused a significant increase of ceramide levels (10%). There was a decreasing tendency for diacylglycerol levels after both SMS1 and SMS2 siRNA treatment, however, it was not statistical significant. As shown by lipid rafts isolation and lipid determination, SMS1 and SMS2 siRNA treatment led to a decrease of SM content in detergent-resistant lipid rafts on the cell membrane. Furthermore, SMS1 and SMS2 siRNA-treated cells had a stronger resistance than did control siRNA-treated cells to lysenin (a protein that causes cell lysis due to its affinity for plasma membrane SM). These results indicate that both SMS1 and SMS2 contribute to SM de novo synthesis and control SM levels in the cells and on the cell membrane including plasma membrane, implying an important relationship between SMS activity and cell functions.

Shedding of Collagen XVII Ectodomain Depends on Plasma Membrane Microenvironment

Collagen XVII, a hemidesmosomal component, mediates the adhesion of epidermal keratinocytes to the underlying basement membrane. It exists as a full-length transmembrane protein and a soluble ectodomain that is proteolytically released from the cell surface by sheddases of a disintegrin and metalloproteinase (ADAM) family; TACE, the tumor necrosis factor-alpha-converting enzyme, is the major physiological proteinase. Because both collagen XVII and the ADAMs are transmembrane proteins, their plasma membrane microenvironment can influence shedding. Lipid rafts, assemblies of sphingolipids and cholesterol within the plasma membrane, are responsible for the separation of membrane proteins and are thought to regulate shedding of cell surface proteins. In this study we analyzed the influence of the cholesterol-depleting agent methyl-beta-cyclodextrin (MbetaCD), which disintegrates lipid rafts, on the shedding of collagen XVII in HaCaT keratinocytes and in transfected COS-7 cells. Increasing concentrations of MbetaCD led to a dose-dependent decrease of membrane cholesterol levels and to stimulation of collagen XVII shedding. The stimulation was completely inhibited by sheddase inhibitors, and experiments with COS-7 cells co-transfected with TACE and collagen XVII demonstrated that TACE mediated the low cholesterol-dependent shedding. Co-patching analysis by double immunofluorescence staining revealed co-localization of collagen XVII with the raft resident phosphatidylinositol-linked placental alkaline phosphatase and segregation from the non-raft protein human transferrin receptor, indicating that a majority of collagen XVII molecules was incorporated into lipid rafts. These data deliver the first evidence for the role of plasma membrane lipid organization in the regulation of collagen XVII shedding and, therefore, in the regulation of keratinocyte migration and differentiation.

Bicarbonate and its role in mammalian sperm function

This paper deals with the effects of bicarbonate induced signaling pathways on plasma membrane lipid organization and downstream protein signaling, and their role in sperm–egg interactions. It also provides an overview of results that indicate that bicarbonate responses are not related to cell death or apoptosis. The information presented shows that only those sperm cells that have functionally completed maturation in the epididymis are sensitive to bicarbonate whereas immature sperm fail to respond to this physiological challenge. Therefore, it is important to selectively analyze the responsive sperm subpopulations when studying sperm capacitation. Moreover, bicarbonate induced signaling responses differ within the diverse sperm structures (e.g. the tail versus the head). Consequently, dissecting sperm structures and signaling areas from each other deserves more attention in sperm capacitation research. The information discussed was obtained from a variety of mammalian species but the basic bicarbonate-mediated sperm responses are similar in most Eutherian species despite some species to species variations (most notably in kinetics rather than the sequence of events). The objective of the paper is to provide a comparative experimental overview of bicarbonate mediated sperm capacitation in the hope that this information will lead to a better understanding of the complex biochemical nature of the involvement of bicarbonate in mammalian sperm capacitation.

Bicarbonate and its role in mammalian sperm function

This paper deals with the effects of bicarbonate induced signaling pathways on plasma membrane lipid organization and downstream protein signaling, and their role in sperm–egg interactions. It also provides an overview of results that indicate that bicarbonate responses are not related to cell death or apoptosis. The information presented shows that only those sperm cells that have functionally completed maturation in the epididymis are sensitive to bicarbonate whereas immature sperm fail to respond to this physiological challenge. Therefore, it is important to selectively analyze the responsive sperm subpopulations when studying sperm capacitation. Moreover, bicarbonate induced signaling responses differ within the diverse sperm structures (e.g. the tail versus the head). Consequently, dissecting sperm structures and signaling areas from each other deserves more attention in sperm capacitation research. The information discussed was obtained from a variety of mammalian species but the basic bicarbonate-mediated sperm responses are similar in most Eutherian species despite some species to species variations (most notably in kinetics rather than the sequence of events). The objective of the paper is to provide a comparative experimental overview of bicarbonate mediated sperm capacitation in the hope that this information will lead to a better understanding of the complex biochemical nature of the involvement of bicarbonate in mammalian sperm capacitation.

Effects of Selenium on Apoptosis Induced by Methyl Mercury Chloride in RAW 264.7 Cells

Objective: This study was performed to evaluate the protective effects of selenium against the methyl mercury chloride (MeHgCl) induced cell apoptosis. M e t h o d s: The effect of selenium on the MeHgCl induced cell apoptosis was observed in mouse macrophage-derived RAW 264.7 cells, in vitro. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM). Results: MeHgCl exerted a dose dependent cytotoxicity, as demonstrated by the MTT assay, an assay dependent, in part, on mitochondrial function. Concurrent exposure to selenium pro vided complete protective effects against the cytotoxicity induced by MeHgCl. Pretreatment with selenium increased the protective effects of subsquent administrations of selenium in con junction with MeHgCl, but pretreatment of selenium alone did not provide protection against MeHgCl when given alone. Selenium administered after exposure to MeHgCl did not repair the existing MeHgCl induced cytotoxicity. Furthermore, the apoptosis induced by MeHgCl was revealed by the DNA fragmentation, using the terminal deoxynucleotidyl transferase Biotin-dUTP nick end labeling (TUNEL) assay, alterations to the nuclear morphology, by nuclei staining, and the plasma membrane lipid orga nization, as shown by cell flow cytometry. The apoptosis induced by MeHgCl was prevented by the concurrent exposure to selenium, or pretreatment with selenium, prior to the administration of selenium in conjunction with MeHgCl. However, no inhibittion of the MeHgCl induced apoptosis was observed with selenium pretreatment prior to exposure to MeHgCl alone, or with the administration of selenium after exposure to MeHgCl. Conclusions: These results suggest that the coexistence of selenium and MeHgCl are essen tial for the protective effects of selenium against the MeHgCl-induced apoptosis, and the cytotoxicity, in RAW 264.7 cells, and may involve selenium-MeHgCl binding.

Modification of plasma membrane lipid order and H+-ATPase activity as part of the response of Saccharomyces cerevisiae to cultivation under mild and high copper stress.

Plasma membrane lipid disorganization takes place in cells of Saccharomyces cerevisiae grown under copper stress, as shown by fluorescence anisotropy measurements with the lipid reporter probe 1,6-diphenyl-1,3,5-hexatriene. The extent of plasma membrane disorganization, presumably due to copper-induced lipid peroxidation, was discontinuous when measured in cells grown in media supplemented with different concentrations of CuSO4. Results suggested the existence of adaptive mechanisms that cells employ to protect themselves against the deleterious effects of copper. The adaptive mechanisms examined in this study included the coordinate increase in the activities of Cu,Zn-superoxide dismutase (up to five-fold), glutathione reductase (up to 1.7-fold), and plasma membrane H+-ATPase (up to three-fold). These enzyme activities showed maximal levels in cells grown with copper supplied at intermediate concentrations, within the range that allowed growth. Significantly, at these concentrations, plasma membrane disorganization did not increase when increasing amounts of CuSO4 were supplied. However, at copper concentrations close to the maximal that allowed growth, the capacity of the yeast cell response to cope with the deleterious effects of copper was exceeded; plasma membrane lipid organization and plasma-membrane-bound H+-ATPase activity drastically declined in response to the increased levels of copper stress and the consequences on growth kinetics were even more severe. Our results clearly suggest that modification of plasma membrane H+-ATPase activity is either part of or the result of the global response of yeast to mild or high copper stress.

Inhibition of Ca2+ uptake into A7r5 vascular smooth muscle cells by farnesol

Previous studies have shown that farnesol, a 15-carbon nonsterol derivative of mevalonic acid, inhibits vasoconstriction. Because of its lipophilic properties, we hypothesized that farnesol increased membrane dynamics, thus reducing uptake of Ca2+ and contraction. To characterize the effect of farnesol on cell membrane fluidity. The study was conducted using A7r5 cells, a rat aortic vascular smooth muscle cell line. Inhibition of Ca2+ uptake by farnesol was first established in these cells. Then, the effect of farnesol on membrane dynamics was determined. Finally, to ascertain that activation of Ca2+ extrusion and reuptake processes by farnesol did not occur, Ca2+-ATPase activity was examined. Membrane fluidity in cell homogenates was estimated using two fluorescent dyes (1,6-diphenyl-1,3,5-hexatriene) and (1-[-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene). Ca2+ uptake was determined by monitoring the changes in cytosolic Ca2+ concentration ([Ca2+]i) in fura-2-loaded cells after addition of KCI. Ca2+-ATPase activity was measured in 100000 x g cell fractions. Farnesol reduced KCI-induced (Ca2+]i transients significantly (P < 0.001), but did not modify membrane dynamic properties [0.214+/-0.007 versus 0.218+/-0.007 (n = 10) and 0.142+/-0.002 versus 0.146+/-0.003 (n = 5) for 1 -[-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene and 1,6-diphenyl-1,3,5-hexatriene anisotropies, respectively; NS]. Administration of up to 30 micromol/l farnesol did not affect Ca2+-ATPase activity. Farnesol inhibits KCI-dependent rise of [Ca2+]i in A7r5 cells. This effect of farnesol is not related to a global change in plasma membrane lipid organization or to activation of Ca2+ pumps. Other mechanisms such as direct inhibition of voltage-dependent Ca2+ channels could therefore explain the biologic action of farnesol in the vascular tissue.

Plasma membrane lipid organization and the adherence of differentiating lymphocytes to macrophages.

Changes in the packing of phospholipids in the plasma membrane of lymphocytes occur during differentiation within primary and secondary lymphoid organs. As they differentiate, lymphocytes interact with a variety of reticuloendothelial cells, including macrophages. To investigate a possible relation between these two phenomena, the strength of the interactions between lymphocytes and macrophages was measured in vitro as a function of the tightness of packing of phospholipids on the lymphocyte surface. Strength of adherence was measured by the ability of lymphocytes to remain adherent to macrophages when subjected to increasing centrifugal forces. Phospholipid packing was assessed using the fluorescent lipophilic probe merocyanine 540 (MC540), which preferentially binds to bilayers in which the lipids are more loosely packed. Three subpopulations of murine thymocytes were resolved with respect to strength of adherence to peritoneal or thymic macrophages. To determine whether these subpopulations corresponded with the three classes of cells distinguishable by MC540 fluorescence, populations enriched for staining or non-staining cells, and cells sorted on the basis of MC540 fluorescence intensity, were examined. The least fluorescent cells were the least strongly adherent; the most fluorescent cells were the most strongly adherent; and cells of intermediate fluorescence had intermediate adherence. When splenic lymphocytes were examined with respect to adherence to peritoneal or splenic macrophages, similar patterns of fluorescence and adherence were seen. These results suggest that the organization of the plasma membrane lipid bilayer of lymphocytes may be involved in their interactions with macrophages during primary and secondary differentiation. The adherence signal for lymphocytes thus may be similar to that proposed for other blood cells.