Cell Membrane Extracts Research Articles

Fluorine NMR‐Based Screening on Cell Membrane Extracts

The possibility of measuring the action of inhibitors of specific enzymatic reactions in intact cells, cell lysates or membrane preparations represents a major advance in the lead discovery process. Despite the relevance of assaying in physiological conditions, only a small number of biophysical techniques, often requiring complex set-up, are applicable to these sample types. Here, we demonstrate the first application of n-fluorine atoms for biochemical screening (n-FABS), a homogeneous and versatile assay based on (19) F NMR spectroscopy, to the detection of high- and low-affinity inhibitors of a membrane enzyme in cell extracts and determination of their IC50 values. Our approach can allow the discovery of novel binding fragments against targets known to be difficult to purify or where membrane-association is required for activity. These results pave the way for future applications of the methodology to these relevant and complex biological systems.

The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.

The Soluble Form of LR11 Protein Is a Regulator of Hypoxia-induced, Urokinase-type Plasminogen Activator Receptor (uPAR)-mediated Adhesion of Immature Hematological Cells

A key property of hematopoietic stem and progenitor cells (HSPCs) regarding differentiation from the self-renewing quiescent to the proliferating stage is their adhesion to the bone marrow (BM) niche. An important molecule involved in proliferation and pool size of HSPCs in the BM is the hypoxia-induced urokinase-type plasminogen activator receptor (uPAR). Here, we show that the soluble form (sLR11) of LR11 (also called SorLA or SORL1) modulates the uPAR-mediated attachment of HSPCs under hypoxic conditions. Immunohistochemical and mRNA expression analyses revealed that hypoxia increased LR11 expression in hematological c-Kit(+) Lin(-) cells. In U937 cells, hypoxia induced a transient rise in LR11 transcription, production of cellular protein, and release of sLR11. Attachment to stromal cells of c-Kit(+) Lin(-) cells of lr11(-/-) mice was reduced by hypoxia much more than of lr11(+/+) animals. sLR11 induced the adhesion of U937 and c-Kit(+) Lin(-) cells to stromal cells. Cell attachment was increased by sLR11 and reduced in the presence of anti-uPAR antibodies. Furthermore, the fraction of uPAR co-immunoprecipitated with LR11 in membrane extracts of U937 cells was increased by hypoxia. CoCl2, a chemical inducer of HIF-1α, enhanced the levels of LR11 and sLR11 in U937 cells. The decrease in hypoxia-induced attachment of HIF-1α-knockdown cells was largely prevented by exogenously added sLR11. Finally, hypoxia induced HIF-1α binding to a consensus binding site in the LR11 promoter. Thus, we conclude that sLR11 regulates the hypoxia-enhanced adhesion of HSPCs via an uPAR-mediated pathway that stabilizes the hematological pool size by controlling cell attachment to the BM niche.

High entomotoxicity and mechanism of the fungal GalNAc/Gal-specific Rhizoctonia solani lectin in pest insects

Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cotton leafworm Spodoptera littoralis and the pea aphid Acyrthosiphon pisum demonstrated a high concentration-dependent entomotoxicity, suggesting that this GalNAc/Gal-specific fungal lectin might be a good control agent for different pest insects. RSA at 10mg/g in the solid diet of 2nd-instar caterpillars caused 84% weight reduction after 8days with none of the caterpillars reaching the 4th-instar stage. In sucking aphids, 50% mortality was achieved after 3days with 9μM of RSA in the liquid diet.Feeding of FITC-labeled RSA to both insect pest species revealed strong lectin binding at the apical/luminal side of the midgut epithelium with the brush border zone, suggesting the insect midgut as a primary insecticide target tissue for RSA. This was also confirmed with cell cultures in vitro, where there was high fluorescence binding at the microvillar zone with primary cultures of larval midgut columnar cells of S. littoralis, and also at the surface with the insect midgut CF-203 cell line without lectin uptake in the midgut cells.In vitro assays using insect midgut CF-203 cells, revealed that RSA was highly toxic with an EC50 of 0.3μM. Preincubation with GalNAc and saponin indicated that this action of RSA was carbohydrate-binding dependent and happened at the surface of the cells. Intoxicated CF-203 cells showed symptoms of apoptosis as nuclear condensation and DNA fragmentation, and this concurred with an increase of caspase-3/7, -8 and -9 activities. Finally, RSA affinity chromatography of membrane extracts of CF-203 cells followed by LC–MS/MS allowed the identification of 5747 unique peptides, among which four putatively glycosylated membrane proteins that are associated with apoptosis induction, namely Fas-associated factor, Apoptosis-linked gene-2, Neuroglian and CG2076, as potential binding targets for RSA. These data are discussed in relation to the physiological effects of RSA.

A label-free proteome analysis strategy for identifying quantitative changes in erythrocyte membranes induced by red cell disorders

Red blood cells have been extensively studied but many questions regarding membrane properties and pathophysiology remain unanswered. Proteome analysis of red cell membranes is complicated by a very wide dynamic range of protein concentrations as well as the presence of proteins that are very large, very hydrophobic, or heterogeneously glycosylated. This study investigated the removal of other blood cell types, red cell membrane extraction, differing degrees of fractionation using 1-D SDS gels, and label-free quantitative methods to determine optimized conditions for proteomic comparisons of clinical blood samples. The results showed that fractionation of red cell membranes on 1-D SDS gels was more efficient than low-ionic-strength extractions followed by 1-D gel fractionation. When gel lanes were sliced into 30 uniform slices, a good depth of analysis that included the identification of most well-characterized, low-abundance red cell membrane proteins including those present at 500 to 10,000 copies per cell was obtained. Furthermore, the size separation enabled detection of changes due to proteolysis or in vivo protein crosslinking. A combination of Rosetta Elucidator quantitation and subsequent statistical analysis enabled the robust detection of protein differences that could be used to address unresolved questions in red cell disorders. This article is part of a Special Issue entitled: Integrated omics.

IL-1β and TSH disturb thyroid epithelium integrity in autoimmune thyroid diseases

Pro-inflammatory cytokines such as IL-1β and TNFα are known to affect thyroid function. They stimulate IL-6 secretion and modify epithelium integrity by altering junction proteins. To study the role of cytokines on thyroid epithelia tightness in autoimmune thyroid diseases (AITD), we analyzed the expression profiles of junction proteins (ZO-1, Claudin, JAM-A) and cytokines in human thyroid slices and also investigated the effect of IL-1β on the epithelium integrity in primary cultures of human thyrocytes. Junction proteins expression (ZO-1, Claudin, JAM-A) has been analyzed by immunohistochemistry on thyroid slices and by Western blot on membrane proteins extracted from thyrocytes of patients suffering from Graves and Hashimoto diseases. The high expression of junction proteins we found on Graves’ disease thyroid slices as well as in cell membrane extracts acknowledges the tightness of thyroid follicular cells in this AITD. In contrast, the reduced expression of JAM and ZO-1 in thyroid cells from patients suffering from Hashimoto thyroiditis is in agreement with the loss of thyroid follicular cell integrity that occurs in this pathology. Concerning the effects on epithelium integrity of TSH and of the pro-inflammatory cytokine IL-1β in primary cultures of human thyroid cells, TSH appeared able to modify JAM-A localization but without any change in the expression levels of JAM-A, Claudin and ZO-1. Inversely, IL-1β provoked a decrease in the expression of- and a redistribution of both, Claudin and ZO-1 without modifying the expression and sub-cellular distribution patterns of JAM-A in thyroid cells. These results demonstrate (i) that Hashimoto's- and Graves’ diseases display different junction proteins expression patterns with a loss of epithelium integrity in the former and (ii) that IL-1β modifies thyroid epithelial tightness of human thyrocytes by altering the expression and localization of junction proteins. Therefore, IL-1β could play a role in the pathogenesis of thyroid autoimmunity.

Plasma membrane as the target site of cholic acid analogs

Although the mechanism is unknown, Calculus Bovis and its active components, cholic acid analogs (CAAs), have been used in China to treat a wide range of diseases. Based on the previous finding that the potency of CAA is strongly dependent on the intrinsic surface activity, this paper aimed to investigate the role of the plasma membrane in the pharmacological activity of CAAs. First, CAAs (0.1 mM) caused a surface activity-dependent depression on ATPase activity in the cell membrane extract, but it had no effects on other cellular extracts, suggesting an indispensable role of the membrane environment for pharmacological activity. Second, CAAs lowered the membrane fluidity of cultured Caco-2 cells with the same rank-order of potency sequence. Third, the hypothesis that any functional protein located on the membrane is influenced by changes in cellular membrane fluidity was supported by: ileal contraction that was induced by acetylcholine and mediated by the muscarinic receptor (M-receptor) or the relaxation induced by adrenaline and mediated by the β-adrenergic receptor (β-receptor) was inhibited by CAAs. They also had similar rank-order of potency and the effects on the plasma membrane. Collectively, the plasma membrane may be a target for the CAAs to exert the multiple pharmacological effects which are mediated by the alteration of the membrane mobility and the function of integral membrane proteins.

Chronic and acute inflammatory conditions determine MEK1 or MEK2 usage as regulators of IL-1β and sIL-1ra expression (117.18)

Abstract Deregulation of the production of IL-1β and its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra), plays an important role in chronic inflammatory diseases such as multiple sclerosis. Relevant to this condition direct cellular contact with stimulated T cells potently triggers cytokine production in human monocytes. In this study we investigated the implication of MEK1 and MEK2 in the control of IL-1β and sIL-1Ra production by human monocytes upon two different stimuli: (i) soluble extracts of plasma membranes from stimulated T cells (CEsHUT), mimicking cellular contact with T cells that is relevant to chronic/sterile inflammation; and (ii) LPS that is relevant to acute/infectious inflammation. By using MEK1/2 (U0126) and MEK1 (PD98059) inhibitors and siRNA specific to MEK1 and MEK2 we demonstrate that MEK1 and MEK2 are differentially involved in the induction of IL-1β production upon chronic/sterile and acute/infectious inflammatory conditions, MEK1 being dispensable to IL-1β induction in monocytes activated by LPS but required in CEsHUT-activated monocytes. Together with the fact that immunomodulatory treatments of multiple sclerosis - IFNβ and glatiramer acetate - are using MEK2 to induce sIL-1Ra production in human monocytes, MEK1 represents a potential therapeutic target which could participate in the restoration of IL-1β/sIL-1Ra balance in chronic/sterile inflammation without affecting normal response to infectious agents.

HAMLET Binding to α-Actinin Facilitates Tumor Cell Detachment

Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed.

Deletion of the glycosyltransferase bgsB of Enterococcus faecalis leads to a complete loss of glycolipids from the cell membrane and to impaired biofilm formation

BackgroundDeletion of the glycosyltransferase bgsA in Enterococcus faecalis leads to loss of diglucosyldiacylglycerol from the cell membrane and accumulation of its precursor monoglucosyldiacylglycerol, associated with impaired biofilm formation and reduced virulence in vivo. Here we analyzed the function of a putative glucosyltransferase EF2890 designated biofilm-associated glycolipid synthesis B (bgsB) immediately downstream of bgsA.ResultsA deletion mutant was constructed by targeted mutagenesis in E. faecalis strain 12030. Analysis of cell membrane extracts revealed a complete loss of glycolipids from the cell membrane. Cell walls of 12030ΔbgsB contained approximately fourfold more LTA, and 1H-nuclear magnetic resonance (NMR) spectroscopy suggested that the higher content of cellular LTA was due to increased length of the glycerol-phosphate polymer of LTA. 12030ΔbgsB was not altered in growth, cell morphology, or autolysis. However, attachment to Caco-2 cells was reduced to 50% of wild-type levels, and biofilm formation on polystyrene was highly impaired. Despite normal resistance to cationic antimicrobial peptides, complement and antibody-mediated opsonophagocytic killing in vitro, 12030ΔbgsB was cleared more rapidly from the bloodstream of mice than wild-type bacteria. Overall, the phenotype resembles the respective deletion mutant in the bgsA gene. Our findings suggest that loss of diglucosyldiacylglycerol or the altered structure of LTA in both mutants account for phenotypic changes observed.ConclusionsIn summary, BgsB is a glucosyltransferase that synthesizes monoglucosyldiacylglycerol. Its inactivation profoundly affects cell membrane composition and has secondary effects on LTA biosynthesis. Both cell-membrane amphiphiles are critical for biofilm formation and virulence of E. faecalis.

Some CLL Cells Bind Myosin-Exposed Apoptotic Cells. Exposure of Cytoplasmic Myosin Results From Transfer of Caspase-3 Dependent Cleavage Products to the Outer Cell Membrane

AbstractAbstract 3900Many monoclonal antibodies (mAbs) produced by B-cell chronic lymphocytic leukemia (CLL) cells bind a subset of apoptotic cells that expose intracellular myosin on the cell surface. CLL patients with mAbs that bind these myosin-exposed apoptotic cells (MEACs) have shorter overall survival. Thus, understanding the mechanism of formation of MEACs and how CLL cells interact with MEACs may help elucidate the pathogenesis of CLL.To test if formation of MEACs is part of general apoptotic mechanisms, apoptosis was induced in Jurkat T cells by either the intrinsic or extrinsic pathways. The intrinsic pathway was either achieved spontaneously by culturing at high cell density or induced by camptothecin (CPT) treatment. The extrinsic pathway was induced by Fas ligand (FasL) or anti-Fas mAb treatment. Apoptosis and myosin exposition were analyzed by flow cytometry. All four methods of apoptosis induction produced MEACs after prolonged incubation as detailed below. CPT, FasL or anti-Fas mAb incubation for 4 hrs induced significant apoptosis (43-58%) with a detectable fraction of MEACs (9-12%). After incubation for 16 hrs or longer, the majority of apoptotic cells were MEACs (61-89%). Similarly, spontaneous apoptosis produced more MEACs after longer incubation (20% on day 1 versus 59–69% on days 2–4). Both early apoptotic cells, which flip phosphatidylserine (PS) from the inner to outer membrane surface yet retain membrane integrity (AnnexinV+, 7-actinomycin D (7AAD)-), and late apoptotic cells, which become membrane permeable (AnnexinV+, 7AAD+), demonstrate a subpopulation of MEACs that increases with longer incubation times. In contrast, MEACs are not detectable in non-apoptotic cells (AnnexinV-, 7AAD-).Thus, both intrinsic and extrinsic apoptotic pathways lead to MEAC formation, suggesting that a common downstream mediator may be involved. Caspase-3 activation mediates apoptotic PS exposure and membrane permeability. Therefore, we tested a caspase-3 inhibitor, Z-DEVD-FMK, and found that it significantly reduced both apoptosis and MEAC formation. For example, Z-DEVD-FMK reduced FasL induced apoptosis and MEAC formation from 74 to 14% and from 57 to 10%, respectively. In contrast, caspase-1 inhibitor, Z-YVAD-FMK, had no effect. To test if intracellular myosin is transferred from the cytoplasm to the cell membrane surface during apoptosis, cytoplasmic and membrane protein extracts were prepared, isolated by ultracentrifugation, and blotted with anti-myosin antibody. Two protein bands of the size expected for caspase-3 cleaved myosin (149 and 94 kDa) appeared in membrane extracts of apoptotic cells, but not of live cells. A protein band of the size expected for full-length myosin (250 kDa) predominated in cytoplasmic extracts of live cells. Furthermore, Z-DEVD-FMK inhibited the formation of the 149 and 94 kDa myosin bands in membrane extracts as well as the formation of caspase-3 dependant PARP cleavage products; the same treatment did not alter CD3 membrane protein or GAPDH cytoplasmic protein levels.Taken together, these results suggest that both intrinsic and extrinsic apoptotic pathways produce MEACs at a later stage in apoptosis that involves the common downstream caspase-3 activation. In turn, myosin fragmentation occurs with subsequent exposure to the cell membrane, where the myosin fragments can serve as a potential neoantigen that may be recognized by some CLL mAbs. Because the mAbs we have used in these analyses were originally integral components of CLL surface membranes, we hypothesized that CLL cells could bind MEACs. Indeed, CLL cells binding to MEACs were visualized by confocal microscopy. To determine the functional consequences of such binding, analyses of the effects of MEAC binding on CLL cell survival in vitro are underway. Disclosures:No relevant conflicts of interest to declare.

Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro

The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca(2+) and 8 mM Mg(2+). Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2-3 nm in diameter and 200 nm to up to 5 microm in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.

CRISP-3 in equine seminal plasma is involved in selective uterine sperm transport

During ejaculation, a large amount of seminal plasma proteins interact with the sperm membrane, leading to a series of biochemical and structural changes implicated in sperm function and gamete interaction. However, the roles of the majority of these proteins remain unknown. This study aimed to investigate the proteome and functionality of the major equine proteins of seminal plasma and the sperm membrane. Seminal plasma and enriched-membrane proteins (150 μg) were separated by two-dimensional gel electrophoresis, and the respective maps were analyzed. Protein identification was performed by in-gel digestion and tandem mass spectrometry (GeLC-MS/MS). Samples were also submitted to in-solution digestion (complex protein mixture) and identified by shotgun analysis by LC-MS/MS; bioinformatic tools were used to investigate protein functions. Seminal plasma and sperm membrane extract maps contained 91.0 ± 8.2 spots and 245.3 ± 11.3 spots, respectively, within the 3–10 pH range. In total, the most abundant proteins identified in 2D maps and in complex protein mixtures included 24 proteins for seminal plasma and 33 for sperm membrane extract, with a high degree of confidence (P < 0.05). Of these, HSP1, CRISP3 and KLK1E2 were the most abundant in seminal plasma; HSP1 was highly abundant in sperm membrane extract, in many isoforms, which is related to membrane destabilization and may compromise sperm preservation. HSP1-polybromo-1 interactions suggested a role in DNA stabilization. Prosaposin was identified in seminal plasma and may play a role in the fertilization process. IZUMO4, a member of the IgSF family involved in the prefertilization stages, was identified in 2D gel and MS/MS analysis of sperm membrane extract. Ten proteins of seminal plasma were found to interact with the sperm membrane and were related to binding and catalytic activities (clusterin, CRISP3, epididymal sperm-binding protein 1, kallikrein1E2, seminal plasma protein A3, and HSP1). Additionally, other identified proteins were associated with DNA integrity, capacitation and recognition of pregnancy. These findings indicate that the binding of specific proteins to the plasma membrane during ejaculation may influence sperm survival after cryopreservation and may play a role in decreasing the quality in stallions with toxic seminal plasma. Elucidation of these interactions is an important step in understanding the biological processes related to equine fertility and facilitates future investigations on the selection and application of low freezability semen strategies.

An adhesion-based method for plasma membrane isolation: Evaluating cholesterol extraction from cells and their membranes

A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-β-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-β-cyclodextrin plasma membrane extraction properties.

The trafficking/interaction of eNOS and caveolin-1 induced by insulin modulates endothelial nitric oxide production.

Endothelial nitric oxide synthase (eNOS) activity is tightly regulated by posttranscriptional modification and its subcellular localization. Here we examined whether insulin modulates nitric oxide (NO) production by regulating eNOS subcellular localization. We used confocal microscopy and immunoblots to examine the time course for 1) subcellular targeting/association of eNOS and caveolin-1 (CAV-1); 2) eNOS Ser(1179) phosphorylation; and 3) NO production in cultured bovine aorta endothelial cells. Serum starvation increased eNOS/CAV-1 localization to the perinuclear region. Adding insulin provoked their prompt translocation to and association at the plasma membrane (PM). Specific monoclonal antibodies against either CAV-1 or eNOS coimmunoprecipitated the other from bovine aorta endothelial cell membrane extracts, and insulin increased this interaction. Insulin stimulated NO production transiently despite a persistent eNOS Ser(1179) phosphorylation. The decline of NO production correlated temporally to insulin-induced translocation of eNOS and CAV-1 to PM. Knockdown of CAV-1 expression with a specific small interfering RNA duplex resulted in eNOS redistributing to the perinuclear region and nearly doubled insulin-induced NO production. Inhibition of phosphatidylinositol 3-kinase activity with wortmannin not only significantly inhibited insulin-induced translocation of eNOS and CAV-1 to PM but also blocked insulin-induced interaction of CAV-1 with eNOS at PM. Insulin increased incorporation of [(3)H]palmitic acid into eNOS immunoprecipitates by approximately 140%. Insulin-induced translocation of eNOS and CAV-1 to PM was palmitoylation dependent. Inhibiting eNOS and CAV-1 palmitoylation enhanced the NO production while blocking the translocation of eNOS and CAV-1 to PM induced by insulin. These data show that insulin acutely regulates eNOS and CAV-1 trafficking to PM of vascular endothelial cells where their interaction can regulate eNOS activity.

Osteopontin in Seminal Plasma and Sperm Membrane of Dogs

The objective of this work was to describe the presence of osteopontin (OPN) in canine seminal plasma and sperm membranes. A pool of seminal plasma and sperm membrane extract from 30 dogs was used. Polyacrylamide electrophoresis gels were performed and the bands were transferred to nitrocellulose paper and Western blot was undertaken using an antibody anti-OPN. Two and 12 bands were marked in the seminal plasma (77.2 and 15.6 kDa) and sperm membrane extracts (70.6-26.6 kDa), respectively. However, from 12 marked bands in the sperm membrane extract, only three (46.4, 37.7 and 36.5 kDa) were strongly marked. We conclude that, seminal plasma and sperm membranes from dogs contain different isoforms of OPN; yet, further studies will be necessary to determine their function in this species.

Nuclear angiotensin II type 2 (AT2) receptors are functionally linked to nitric oxide production

Expression of nuclear angiotensin II type 1 (AT(1)) receptors in rat kidney provides further support for the concept of an intracellular renin-angiotensin system. Thus we examined the cellular distribution of renal ANG II receptors in sheep to determine the existence and functional roles of intracellular ANG receptors in higher order species. Receptor binding was performed using the nonselective ANG II antagonist (125)I-[Sar(1),Thr(8)]-ANG II ((125)I-sarthran) with the AT(1) antagonist losartan (LOS) or the AT(2) antagonist PD123319 (PD) in isolated nuclei (NUC) and plasma membrane (PM) fractions obtained by differential centrifugation or density gradient separation. In both fetal and adult sheep kidney, PD competed for the majority of cortical NUC (> or =70%) and PM (> or =80%) sites while LOS competition predominated in medullary NUC (> or =75%) and PM (> or =70%). Immunodetection with an AT(2) antibody revealed a single approximately 42-kDa band in both NUC and PM extracts, suggesting a mature molecular form of the NUC receptor. Autoradiography for receptor subtypes localized AT(2) in the tubulointerstitium, AT(1) in the medulla and vasa recta, and both AT(1) and AT(2) in glomeruli. Loading of NUC with the fluorescent nitric oxide (NO) detector DAF showed increased NO production with ANG II (1 nM), which was abolished by PD and N-nitro-l-arginine methyl ester, but not LOS. Our studies demonstrate ANG II receptor subtypes are differentially expressed in ovine kidney, while nuclear AT(2) receptors are functionally linked to NO production. These findings provide further evidence of a functional intracellular renin-angiotensin system within the kidney, which may represent a therapeutic target for the regulation of blood pressure.

The Microtubule-Targeting Agents, PBOX-6 [Pyrrolobenzoxazepine 7-[(dimethylcarbamoyl)oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine] and Paclitaxel, Induce Nucleocytoplasmic Redistribution of the Peptidyl-Prolyl Isomerases, Cyclophilin A and Pin1, in Malignant Hematopoietic Cells

Microtubule assembly and disassembly is required for the maintenance of cell structure, mobility, and division. However, the cellular and biochemical implications of microtubule disruption are not fully understood. Using a proteomic approach, we found that the peptidyl-prolyl isomerase, cyclophilin A, was increased in plasma membrane extracts from chronic myeloid leukemia cells after microtubule disruption. In addition, we found that two peptidyl-prolyl isomerases, cyclophilin A and pin1, are overexpressed up to 10-fold in hematological malignancies compared with normal peripheral blood mononuclear cells. Although previous reports suggest that cyclophilin A is localized to the cytosol of mammalian cells, we found that cyclophilin A and pin1 are both localized to the nucleus and nuclear domains in hematopoietic cells. Microtubule disruption of hematopoietic cells caused a dramatic subcellular redistribution of cyclophilin A and pin1 from the nucleus to the cytosol and plasma membrane. We suggest that this accounts for the increased cyclophilin A at the plasma membrane of chronic myeloid leukemia cells after microtubule disruption. The subcellular redistribution of cyclophilin A and pin1 occurred in a c-Jun NH(2)-terminal kinase- and serine protease-dependent manner. Moreover, the altered subcellular localization of the peptidyl-prolyl isomerases occurred in a dose- and time-dependent manner after microtubule disruption and was found to correlate with G(2)/M arrest and precede induced cell death. These results suggest that the function of peptidyl-prolyl isomerases may be influenced by microtubule dynamics throughout the cell cycle, and their altered localization may be an important part of the mechanism by which microtubule-disrupting agents exert their cytostatic effects.

Dual Activation of Phospholipase C-ϵ by Rho and Ras GTPases

Phospholipase C-epsilon (PLC-epsilon) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-epsilon to stimulate its phospholipase activity. Purified PLC-epsilon was activated in a guanine nucleotide- and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P(2)-containing phospholipid vesicles. Whereas mutation of two critical lysine residues within the second Ras-association domain of PLC-epsilon prevented K-Ras-dependent activation of the purified enzyme, guanine nucleotide-dependent activation by RhoA was retained. Deletion of a loop unique to PLC-epsilon eliminated its activation by RhoA but not H-Ras. In contrast, removal of the autoinhibitory X/Y-linker region of the catalytic core of PLC-epsilon markedly activates the enzyme (Hicks, S. N., Jezyk, M. R., Gershburg, S., Seifert, J. P., Harden, T. K., and Sondek, J. (2008) Mol. Cell, 31, 383-394), but PLC-epsilon lacking this regulatory region retained activation by both Rho and Ras GTPases. Additive activation of PLC-epsilon by RhoA and K- or H-Ras was observed in intact cell studies, and this additivity was recapitulated in experiments in which activation of purified PLC-epsilon was quantified with PtdIns(4,5)P(2)-containing phospholipid vesicles reconstituted with purified, isoprenylated GTPases. A maximally effective concentration of activated RhoA also increased the sensitivity of purified PLC-epsilon to activation by K-Ras. These results indicate that PLC-epsilon can be directly and concomitantly activated by both RhoA and individual Ras GTPases resulting in diverse upstream control of signaling cascades downstream of PLC-epsilon.

Valosin‐containing protein/p97 interacts with sperm‐activating and sperm‐attracting factor (SAAF) in the ascidian egg and modulates sperm‐attracting activity

Sperm chemotaxis toward an egg is observed in many animals, and the control of sperm-attracting activity is thought to play an important role in ensuring fertilization. However, the mechanism underlying the release of a sperm attractant from an egg is still obscure. In this study, we examined the systems involved in the release of sperm-activating and sperm-attracting factor (SAAF), which is the sperm attractant of the ascidian Ciona intestinalis. Here, we show that the egg acquires sperm-attracting activity after germinal vesicle breakdown. Further, since the cytoplasmic extracts of immature oocytes exhibit no sperm-attracting activity, the SAAF in oocytes may be activated after germinal vesicle breakdown. We found 13 SAAF-binding proteins in an egg plasma membrane extract and identified five proteins by proteomic analysis: valosin-containing protein (VCP)/p97, proteasome alpha 2 subunit, MGC97756 protein, proteasome subunit Y, and beta-tubulin. In particular, the interaction between VCP/p97 and SAAF was confirmed by a pull-down assay. VCP/p97 is initially localized in the germinal vesicle, and during oocyte maturation, it shifts to the endoplasmic reticulum in the cortical regions. Thus, VCP/p97 is a potential modulator of SAAF release from the egg.