Normal Cell Membrane Research Articles

Allicin, a SUR2 opener: possible mechanism for the treatment of diabetic hypertension in rats

The garlic (Allium sativum L., Amaryllidaceae) has been popularly used in the treatment of diabetes and cardiac complications. In the present work, we have studied the possible mechanisms, sulfonylurea receptor (SUR) selectivity of allicin in diabetic hypertensive rats. Diabetic hypertension was induced by intraperitoneal injection of streptozotocin (50 mg/kg) followed by daily administration of dexamethasone (10 µg/kg, s.c.). Different parameters, blood pressure and blood glucose levels were studied in the rats weekly up to eight weeks. Allicin (8 mg/kg, p.o.) shows potent antidiabetic (*p<0.001) as well as antihypertensive effect (**p<0.001, *p<0.01). It may act as a vasodilator by hyperpolarizing the membrane of normal vascular smooth muscle cells. The hyperpolarization in vascular smooth muscle occurs due to K+ channel opener activity. Antihypertensive effect of allicin is inhibited by glibenclamide, nonselective SUR blocker while combination of allicin with nateglinide, selective SUR1 blocker exerted synergistic antihypertensive effect. The results indicates that allicin is effective in the treatment of diabetic hypertension; through a mechanism that might involve selective opening of SUR2.

Drosophilasosiefunctions with βH-Spectrin and actin organizers in cell migration, epithelial morphogenesis and cortical stability

SummaryMorphogenesis in multicellular organisms requires the careful coordination of cytoskeletal elements, dynamic regulation of cell adhesion and extensive cell migration. sosie (sie) is a novel gene required in various morphogenesis processes in Drosophila oogenesis. Lack of sie interferes with normal egg chamber packaging, maintenance of epithelial integrity and control of follicle cell migration, indicating that sie is involved in controlling epithelial integrity and cell migration. For these functions sie is required both in the germ line and in the soma. Consistent with this, Sosie localizes to plasma membranes in the germ line and in the somatic follicle cells and is predicted to present an EGF-like domain on the extracellular side. Two positively charged residues, C-terminal to the predicted transmembrane domain (on the cytoplasmic side), are required for normal plasma membrane localization of Sosie. Because sie also contributes to normal cortical localization of βH-Spectrin, it appears that cortical βH-Spectrin mediates some of the functions of sosie. sie also interacts with the genes coding for the actin organizers Filamin and Profilin and, in the absence of sie function, F-actin is less well organized and nurse cells frequently fuse.

Seg1 controls eisosome assembly and shape

Eisosomes are stable domains at the plasma membrane of the budding yeast Saccharomyces cerevisiae and have been proposed to function in endocytosis. Eisosomes are composed of two main cytoplasmic proteins, Pil1 and Lsp1, that form a scaffold around furrow-like plasma membrane invaginations. We show here that the poorly characterized eisosome protein Seg1/Ymr086w is important for eisosome biogenesis and architecture. Seg1 was required for efficient incorporation of Pil1 into eisosomes and the generation of normal plasma membrane furrows. Seg1 preceded Pil1 during eisosome formation and established a platform for the assembly of other eisosome components. This platform was further shaped and stabilized upon the arrival of Pil1 and Lsp1. Moreover, Seg1 abundance controlled the shape of eisosomes by determining their length. Similarly, the Schizosaccharomyces pombe Seg1-like protein Sle1 was necessary to generate the filamentous eisosomes present in fission yeast. The function of Seg1 in the stepwise biogenesis of eisosomes reveals striking architectural similarities between eisosomes in yeast and caveolae in mammals.

Rescue of a pathogenic mutant human glucagon receptor by pharmacological chaperones

We have previously demonstrated that a homozygous inactivating P86S mutation of the glucagon receptor (GCGR) causes a novel human disease of hyperglucagonemia, pancreatic α-cell hyperplasia, and pancreatic neuroendocrine tumors (Mahvash disease). The mechanisms for the decreased activity of the P86S mutant (P86S) are abnormal receptor localization to the endoplasmic reticulum (ER) and defective interaction with glucagon. To search for targeted therapies for Mahvash disease, we examined whether P86S can be trafficked to the plasma membrane by pharmacological chaperones and whether novel glucagon analogs restore effective receptor interaction. We used enhanced green fluorescent protein-tagged P86S stably expressed in HEK 293 cells to allow fluorescence imaging and western blotting and molecular modeling to design novel glucagon analogs in which alanine 19 was replaced with serine or asparagine. Incubation at 27 °C largely restored normal plasma membrane localization and normal processing of P86S but osmotic chaperones had no effects. The ER stressors thapsigargin and curcumin partially rescued P86S. The lipophilic GCGR antagonist L-168,049 also partially rescued P86S, so did Cpd 13 and 15 to a smaller degree. The rescued P86S led to more glucagon-stimulated cAMP production and was internalized by glucagon. Compared with the native glucagon, the novel glucagon analogs failed to stimulate more cAMP production by P86S. We conclude that the mutant GCGR is partially rescued by several pharmacological chaperones and our data provide proof-of-principle evidence that Mahvash disease can be potentially treated with pharmacological chaperones. The novel glucagon analogs, however, failed to interact with P86S more effectively.

Effects of Net Charge on Biological Activities of Alpha-helical Anticancer Peptides

Owing to the low possibility of induction of resistance,amphipathic α-helical anticancer peptides whose sole target is the biomembrane show their promising potentials in cancer treatments.Obtained in the previous study,an amphipathic α-helical anticancer peptide A12L/A20L(Peptide P) with significant anticancer activity was utilized as the framework to systematically design a series of peptide analogs with different net charges by amino acid substitutions on the polar face,and to study the effects of net charge on biological activitiesof cationic anticancer peptides.The results showed that there was an obvious net charge threshold amongpeptide analogs,that was the net charge of +7 to +8 and the number of electrostatic repulsion of 3—5.Increases or decreases of net charges beyond the threshold value resulted in a dramatic reduction in both anticancer activity and therapeutic index.The alteration on net charges of peptides exhibited weak effect on hemolytic activity(maximum 2-fold) but significant influence on anticancer activity and therapeutic index(maximum 10-fold),respectively.Compared with the neutral membrane of human normal cells,the negatively charged membrane of cancer cells was much more sensitive to net charge alteration of the anticancer peptide.The amphipathic α-helical anticancer peptides showed good specificity between cancer cells and human normal cells,which might play a crucial role in making further improvement in target selectivity and designing anticancer peptide agents as therapeutics with higher efficacy and lower toxicity.

Plasma Membrane Localization Is Required for RasA-Mediated Polarized Morphogenesis and Virulence of Aspergillus fumigatus

Ras is a highly conserved GTPase protein that is essential for proper polarized morphogenesis of filamentous fungi. Localization of Ras proteins to the plasma membrane and endomembranes through posttranslational addition of farnesyl and palmitoyl residues is an important mechanism through which cells provide specificity to Ras signal output. Although the Aspergillus fumigatus RasA protein is known to be a major regulator of growth and development, the membrane distribution of RasA during polarized morphogenesis and the role of properly localized Ras signaling in virulence of a pathogenic mold remain unknown. Here we demonstrate that Aspergillus fumigatus RasA localizes primarily to the plasma membrane of actively growing hyphae. We show that treatment with the palmitoylation inhibitor 2-bromopalmitate disrupts normal RasA plasma membrane association and decreases hyphal growth. Targeted mutations of the highly conserved RasA palmitoylation motif also mislocalized RasA from the plasma membrane and led to severe hyphal abnormalities, cell wall structural changes, and reduced virulence in murine invasive aspergillosis. Finally, we provide evidence that proper RasA localization is independent of the Ras palmitoyltransferase homolog, encoded by erfB, but requires the palmitoyltransferase complex subunit, encoded by erfD. Our results demonstrate that plasma membrane-associated RasA is critical for polarized morphogenesis, cell wall stability, and virulence in A. fumigatus.

Residue Ile89 in human plasma membrane monoamine transporter influences its organic cation transport activity and sensitivity to inhibition by dilazep

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter belonging to the equilibrative nucleoside transporter (ENT) family. Despite its distinct substrate specificity from the classic nucleoside transporters ENT1 and 2, PMAT appears to share similar protein architecture with ENT1/2 and retains low affinity binding to classic ENT inhibitors such as nitrobenzylmercaptopurine riboside (NBMPR) and the coronary vasodilators dilazep and dipyridamole. Here we investigated the role of residue Ile89, a position known to be important for ENT interaction with dilazep, dipyridamole, and nucleoside substrates, in PMAT transport function and its interaction with classic ENT inhibitors using Madin–Darby canine kidney (MDCK) cells stably expressing human PMAT. Substitution of Ile89 in PMAT with Met, the counterpart residue in ENT1, resulted in normal plasma membrane localization and protein expression. Transport kinetic analysis revealed that I89M mutant had a 2.7-fold reduction in maximal transport velocity (Vmax) with no significant change in apparent binding affinity (Km) towards the prototype PMAT substrate 1-methyl-4-phenylpyridinium (MPP+), suggesting that I89 is an important determinant for the catalytic activity of PMAT. Dose-dependent inhibition studies further showed that the I89M mutation significantly increased PMAT's sensitivity to dilazep by 2.5-fold without affecting its sensitivity to dipyridamole and NBMPR. Located at the extracellular end of transmembrane domain 1 of PMAT, I89 may occupy an important position close to the substrate permeation pathway and may be involved in direct interaction with the vasodilator dilazep.

Abstract LB-18: Expression of discoidin domain receptor tyrosine kinases in breast cancer

Abstract A key step in breast cancer progression is the transition from in situ to invasive carcinoma. This process is partly mediated by defective interactions between the epithelial cells and the extracellular matrix, leading to loss of cell-cell contacts and polarity, increased proliferation, and acquisition of invasive activity. Here we report that a unique set of receptor tyrosine kinases (RTKs) known as the discoidin domain receptors (DDRs) may play a key role during the transition of in situ to invasive breast carcinoma. DDRs are the only RTKs that recognize collagen as their ligands and thus mediate collagen-initiated signaling in both physiological and pathological conditions. The expression of DDRs at various stages of breast cancer progression is unknown. We used a high-density tissue microarray containing samples of normal breast, ductal carcinoma in situ (DCIS), and invasive carcinomas of different clinico-pathological characteristics to examine the expression of DDR1 by immunohistochemistry using an antibody to the C-terminus of DDR1. Expression of DDR1 was evaluated taking into consideration the percentage and intensity of staining, and categorized as low or high. DDR1 was highly expressed in the cytoplasm and membrane of normal epithelial cells in ducts and acini, and in DCIS. In contrast, 88 of 134 (66%) invasive carcinomas displayed low or absent DDR1 expression, while 44% exhibited high levels. DDR1 expression was not associated with histologic type, grade, hormonal receptor or HER2-neu status. Interestingly, 15 of 40 (37.5%) patients with high DDR1 expression developed tumor recurrence compared to 9 of 46 (19.6%) patients with low DDR1 (Kaplan Meier log-rank p=0.05). The expression of DDR1 was also evaluated in a mouse model of breast cancer progression using pre-neoplastic and malignant human MCF10A cell variants. DDR1 was highly expressed in the benign hyperplastic lesions generated by MCF10AT1 cells, and in the comedo DCIS tumors generated by MCF10DCIS.COM cells. In contrast, low or no DDR1 expression was observed in tumors generated by the highly malignant MCF10CA1 variant. Expression analyses of DDR1 isoforms in cultured cells by real-time PCR demonstrated that non-malignant MCF10A cells expressed higher levels of DDR1b mRNA while malignant MCF10CA1 cells showed higher levels of DDR1a suggesting that malignant progression is associated with a DDR1 isoform switch. At present the functional roles of the DDR1 isoforms in breast cancer progression remain unknown. Our analyses suggest that loss of or reduced DDR1 expression is associated with the transition from in situ to invasive carcinomas while patients with invasive carcinomas displaying high DDR1 expression have worse disease free survival. These novel studies highlight a complex role of DDR1 in breast cancer progression that deserves further exploration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-18. doi:1538-7445.AM2012-LB-18

17β-Estradiol stimulates the translocation of endogenous estrogen receptor α at the plasma membrane of normal anterior pituitary cells

In the present work we aimed at identifying ERα in the plasma membrane of normal anterior pituitary cells and investigated if 17β-estradiol was able to induce their subcellular redistribution. Our results show that about 8% of anterior pituitary cells expressed ERα in the plasma membrane, with the geometrical mean fluorescence intensity being increased after steroid hormone treatment. 17β-Estradiol and the selective ERα agonist PPT induced an increase of ERα expression in the plasma membrane and activated the PKCα/ERK 1/2 pathway in a time-course not compatible with genomic actions, thus supporting the notion of membrane-initiated effects. These findings suggest that 17β-estradiol stimulates the translocation of endogenous ERα to the plasma membrane, consequently modulating this ER pool and leading to cellular biological effects in normal anterior pituitary gland.

Loss of EBP50 stimulates EGFR activity to induce EMT phenotypic features in biliary cancer cells

Scaffold proteins form multiprotein complexes that are central to the regulation of intracellular signaling. The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is highly expressed at the plasma membrane of normal biliary epithelial cells and binds epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with oncogenic properties. This study investigated EBP50-EGFR interplay in biliary cancer. We report that in a collection of 106 cholangiocarcinomas, EBP50 was delocalized to the cytoplasm of tumor cells in 66% of the cases. Ectopic expression of EBP50 was correlated with the presence of satellite nodules and with the expression of EGFR, which was at the plasma membrane, implying a loss of interaction with EBP50 in these cases. In vitro, loss of interaction between EBP50 and EGFR was mimicked by EBP50 depletion using a small interfering RNA approach in human biliary carcinoma cells co-expressing the two proteins at their plasma membrane, and in which interaction between EBP50 and EGFR was validated. EBP50 depletion caused an increase in EGFR expression at their surface, and a sustained activation of the receptor and of its downstream effectors (extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3) in both basal and EGF-stimulated conditions. Cells lacking EBP50 showed epithelial-to-mesenchymal transition-associated features, including reduction in E-cadherin and cytokeratin-19 expression, induction of S100A4 and of the E-cadherin transcriptional repressor, Slug, and loss of cell polarity. Accordingly, depletion of EBP50 induced the disruption of adherens junctional complexes, the development of lamellipodia structures and the subsequent acquisition of motility properties. All these phenotypic changes were prevented upon inhibition of EGFR tyrosine kinase by gefitinib. These findings indicate that loss of EBP50 at the plasma membrane in tumor cells may contribute to biliary carcinogenesis through EGFR activation.

Surface plasmon resonance scattering and absorption of biofunctionalized gold nanoparticles for targeted cancer imaging and laser therapy

The localized surface plasmon resonance of gold nanoparticles enables them to be excellent light scattering sensing reagents and efficient lignt-heat convertors for potential diagnostics and therapeutics. In this work, gold nanoparticles of 15 nm in size were synthesized and conjugated with a kind of arginine-glycine-aspartic acid peptide (RGD) to target the cancer cells. Under a conventional dark field microscope, the scattering images from normal and cancer cells are very different. Only a few of gold nanoparticles bind to the membrane of normal cells due to nonspecific interaction. Yet for cancer cells, the concentration of gold nanoparticles inside cancer cells is much higher than that inside normal cells, showing a strong light scattering signal. By exposing these cells to an external laser irradiation, cancer cells were selectively destructed because of the heat conversion of gold nanoparticles inside them, but normal cells still kept alive due to the absence of gold nanoparticles in them. These results incidated that gold nanoparticles might be potential contrast reagents for targeted cancer imaging and laser therapy.

Wound healing and regeneration: time heals all wounds, but sometimes it needs a little help

Molecular Biology of the CellVol. 22, No. 6 ASCB Annual Meeting HighlightsFree AccessWound healing and regeneration: time heals all wounds, but sometimes it needs a little helpWilliam M. Bement, and Phillip A. NewmarkWilliam M. BementDepartment of Zoology, Program in Cellular and Molecular Biology, University of Wisconsin–Madison, Madison, WI 53706Search for more papers by this author, and Phillip A. NewmarkHoward Hughes Medical Institute, Department of Cell and Developmental Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801Search for more papers by this authorPublished Online:13 Oct 2017https://doi.org/10.1091/mbc.e11-01-0011AboutSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail The Minisymposium on wound healing and regeneration was broad in scope, covering wound repair in organisms ranging from yeast to mammals and across levels of biological organization, ranging from single cells to regeneration of tissues and organs.Bill Bement (University of Wisconsin–Madison) presented results suggesting that the single and multicellular wound responses are linked. Damage to a single cell within a Xenopus epithelium not only elicits calcium elevation and subsequent activation of the small GTPases Rho and Cdc42 around the original wound site, but it also triggers calcium elevation as well as Rho and Cdc42 activation at cell–cell junctions distal to the original wound. At least part of the wound response is mediated via Abr, a dual GEF and GAP for Rho and Cdc42. Abr is apparently responsible for controlling the differential distribution of active Rho and Cdc42 normally seen around single-cell wounds.Keiko Kono (Pellman Laboratory, Dana Farber) described work on development of budding yeast as a model system for single-cell wound repair. Kono has found that laser-induced damage during budding results in rapid redistribution of actin and protein kinase C from the bud site (i.e., the nascent daughter cell) to the site of damage. This actin redistribution is dependent on degradation of the formin, Bni, and represents a polarization switch such that machinery used for daughter cell formation is rapidly shifted toward the wound site.Amber Howard (McNeil Laboratory, Medical College of Georgia) reported on her work investigating the well-known deficit of wound healing caused by diabetes. Although it has long been thought that diabetes suppresses wound repair as a systemic consequence of the disease, Howard found that individual muscle cells from diabetic mice had deficient plasma membrane repair. Further, she demonstrated that simply culturing otherwise normal muscle cells in high glucose resulted in progressive loss of the normal plasma membrane repair response, demonstrating that the response to diabetic conditions is intrinsic to the muscle cells themselves.Michelle Juarez (McGinnis Laboratory, UCSD) described genetic approaches to identify genes coordinating the epidermal response to wounds in the Drosophila embryo. Using wound-dependent fluorescent reporter constructs, Juarez screened panels of deletion mutants to identify genes required for proper activation or inhibition of wound-responsive genes. Juarez identified Flotillin-2 as an inhibitor of wound-responsive genes: Flotillin-2 mutants show broad activation of wound-response genes throughout the embryo, whereas overexpression leads to inhibition of wound-responsive genes. Juarez provided evidence that Src may regulate Flotillin-2 to limit the spread of the epithelial response to wounding.Adrian Halme (University of Virginia) examined how organisms respond systemically to tissue damage. In Drosophila larvae, tissue damage leads to a developmental delay that postpones metamorphosis until the tissue can be repaired. During the course of normal metamorphosis, prothoracicotropic hormone (PTTH) is secreted from neurons that innervate the ring gland; in response to PTTH, the ring gland produces ecdysone, triggering metamorphosis. After tissue damage, PTTH expression is inhibited, resulting in delayed ecdysone production. Halme found that Jun N-terminal kinase (JNK) signaling is required for this damage-induced developmental delay. He also showed that neoplastic tumors produce a similar developmental delay, in which PTTH expression is inhibited by JNK signaling.Phillip Newmark (HHMI/University of Illinois at Urbana–Champaign) discussed intestinal regeneration and differentiation in the planarian. A combination of live imaging and BrdU pulse-chase experiments revealed that intestinal regeneration results from the coordinated differentiation of newly born intestinal cells and remodeling of preexisting intestinal tissue. Newmark described a novel method for purifying intestinal phagocytes, facilitating the identification of intestine-specific monoclonal antibodies and the characterization of intestinal gene expression. The latter results were used to inform a targeted RNAi screen that identified genes required for proper intestinal differentiation and remodeling.FOOTNOTESMolecular Biology of the Cell Volume 22 Page 719MBoC is pleased to publish this summary of the Minisymposium “Wound Healing and Regeneration” held at the American Society for Cell Biology 2010 Annual Meeting, Philadelphia, PA, December 12, 2010.FiguresReferencesRelatedDetails Vol. 22, No. 6 March 15, 2011715-905 Metrics Downloads & Citations Downloads: 64 History Information© 2011 Bement and Newmark. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).PDF download

Galectin-3 regulates MUC1 and EGFR cellular distribution and EGFR downstream pathways in pancreatic cancer cells

MUC1 is a transmembrane glycoprotein which is typically expressed at the apical membrane of normal epithelial cells. In cancer cells, the over-expression of MUC1 and its aberrant localization around the cell membrane and in the cytoplasm favours its interaction with different protein partners such as epidermal growth factor receptor (EGFR) and can promote tumour proliferation through the activation of oncogenic signalling pathways. Our aims were to study the mechanisms inducing MUC1 cytoplasmic localization in pancreatic cancer cells, and to decipher their impact on EGFR cellular localization and activation. Our results showed that galectin-3, an endogenous lectin, is co-expressed with MUC1 in human pancreatic ductal adenocarcinoma, and that it favours the endocytosis of MUC1 and EGFR. Depletion of galectin-3 by RNA interference increased the interaction between MUC1 and EGFR, EGFR and ERK-1,2 phosphorylation, and translocation of EGFR to the nucleus. On the contrary, silencing of galectin-3 led to a decrease of cyclin-D1 levels and of cell proliferation. The galectin-3-dependent regulation of MUC1/EGFR functions may represent an interesting mechanism modulating the EGFR-stimulated cell growth of pancreatic cancer cells.

Minocycline attenuates ischemia-induced ventricular arrhythmias in rats

Minocycline has been shown to protect against myocardial ischemia–reperfusion injury. This study investigated the effects of minocycline on ischemia-induced ventricular arrhythmias in rats. Anesthetized male rats were once treated with minocycline (45 mg/kg, i.p.) 1 h before ischemia in the absence and/or presence of 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002, 0.3 mg/kg, i.v., a PI3K inhibitor) and 5-hydroxydecanoic acid [5-HD, 10 mg/kg, i.v., a specific inhibitor of mitochondrial ATP-sensitive potassium (K ATP) channels] which were once injected 10 min before ischemia and then subjected to ischemia for 30 min. Ventricular arrhythmias were assessed. L-type Ca 2+ current was measured by the patch-clamp technique. During the 30-minute ischemia, minocycline significantly reduced the incidence of ventricular fibrillation (VF) (P < 0.05). The duration of VT + VF, the number of VT + VF episodes and the severity of arrhythmias were all significantly reduced by minocycline compared to those in myocardial ischemia group (P < 0.05 for all). Administration of LY294002 or 5-HD abolished the protective effects of minocycline on VF incidence, the duration of VT + VF, the number of VT + VF episodes and the severity of arrhythmias (P < 0.05 for all). In addition, minocycline inhibited L-type Ca 2+ currents of normal myocardial cell membrane in a dose-dependent manner. This study suggested that minocycline could attenuate ischemia-induced ventricular arrhythmias in rats in which PI3K/Akt signaling pathway, mitochondrial K ATP channels and L-type Ca 2+ channels may be involved .

Posttranslational negative regulation of glycosylated and non-glycosylated BCRP expression by Derlin-1

Human breast cancer resistance protein (BCRP)/MXR/ABCG2 is a well-recognized ABC half-transporter that is highly expressed at the apical membrane of many normal tissues and cancer cells. BCRP facilitates disposition of endogenous and exogenous harmful xenobiotics to protect cells/tissues from xenobiotic-induced toxicity. Despite the enormous impact of BCRP in the physiological and pathophysiological regulation of the transport of a wide variety of substrates, little is known about the factors that regulate posttranslational expression of BCRP. Here, we identified Derlin-1, a member of a family of proteins that bears homology to yeast Der1p, as a posttranslational regulator of BCRP expression. Overexpression of Derlin-1 suppressed ER to Golgi transport of wild-type (WT) BCRP that is known to be efficiently trafficked to the plasma membrane. On the other hand, protein expression of N596Q variant of BCRP, N-linked glycosylation-deficient mutant that preferentially undergoes ubiquitin-mediated ER-associated degradation (ERAD), was strongly suppressed by the overexpression of Derlin-1, whereas knockdown of Derlin-1 stabilized N596Q protein, suggesting a negative regulatory role of Derlin-1 for N596Q protein expression. Notably, knockdown of Derlin-1 also stabilized the expression of tunicamycin-induced deglycosylated WT BCRP protein, implying the importance of glycosylation state for the recognition of BCRP by Derlin-1. Thus, our data demonstrate that Derlin-1 is a negative regulator for both glycosylated and non-glycosylated BCRP expression and provide a novel posttranslational regulatory mechanism of BCRP by Derlin-1.

Enhanced recovery of Salmonella Typhimurium DT104 from exposure to stress at low temperature

Salmonella enterica serovar Typhimurium (S. Typhimurium) remains an important cause of food-borne infection in the developed world. In order to establish infection within a host, Salmonella must survive and recover from a range of environmental stresses. S. Typhimurium strain SL1344 is among the most extensively studied pathogenic Salmonella strains, while S. Typhimurium phage type DT104 is an important type that has been associated with pandemic spread and a high number of food-borne disease outbreaks over the last two decades. In this study, we have compared the abilities of these two S. Typhimurium types to recover from stress exposures commonly encountered in food production, including 685 mM NaCl, pH 3.8, low temperature (6 °C) and combinations thereof. Following removal from prolonged (8 days) stress, DT104 cultures that had been exposed to low temperature, with or without additional stress, resumed exponential growth more rapidly than SL1344 cultures exposed to the same conditions. SL1344 showed higher levels of filamentation than DT104 in response to NaCl exposure at low temperature. Further, SL1344 incurred higher levels of membrane damage in response to elevated NaCl and pH 3.8 at both temperatures compared with DT104. However, both strains recovered normal cell division and membrane integrity within 6 h when all stresses were removed. Expression of the Salmonella pathogenicity island 1 gene prgH, the first gene in the prg/org operon, was monitored using a chromosomal reporter in which gfp(+) expression was driven by the prgH promoter. Recovery of prgH expression was comparable for SL1344 and DT104 exposed to stress at 22 °C. However, DT104 cultures exposed to pH 3.8 or combined NaCl and low-pH stress at low temperature resumed prgH expression more rapidly than SL1344. Both strains recovered maximal levels of prgH expression after 6 h recovery from all stresses and, interestingly, maximal levels of prgH expression were significantly higher in SL1344, consistent with prgH expression in late-exponential, non-stressed SL1344 and DT104 cultures. Together, these data show that S. Typhimurium is capable of rapid recovery from environmental and food-related stresses, and give insight into the enhanced ability of DT104 compared with SL1344 to adapt to such stresses, which may contribute to the success of this globally disseminated pathogenic phage type.

Lipid profiles of sperm and seminal plasma from boars having normal or low sperm motility

Sperm plasma membrane lipids have an important role to play in determining membrane fluidity and sperm motility. The objective of the present study was to determine whether there are differences in the lipid and fatty acid (FA) composition of boar sperm and seminal plasma in the ejaculates of boars having different sperm motilities. Semen was collected from two groups of boars having normal (> 60%; n = 53) or low (< 60%; n = 53) motility sperm and the semen was evaluated for motility, morphology and vitality. The semen was then centrifuged to separate the sperm from the seminal plasma and both were kept at −20 °C until analyzed for lipid content and FA profile by gas chromatography. Total antioxidant status (TAS) of seminal plasma was determined using a commercial kit. There were differences (P ≤ 0.05) in sperm total lipids, cholesterol, saturated fatty acids (SFA), phospholipids, n-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and the ratio of n-6:n-3 PUFA between boars with normal and low motility sperm. Total lipids, cholesterol, phospholipids, PUFA, DHA and n-3 PUFA were positively correlated with sperm motility, viability, normal morphology and normal plasma membrane. In contrast, SFA and the ratio of n-6: n-3 PUFA were negatively correlated (P ≤ 0.05) with sperm motility, viability, normal morphology and normal plasma membranes. The TAS of seminal plasma from boars having normal motility sperm was higher (P ≤ 0.05) than that of boars having low motility sperm and TAS was positively correlated (P = 0.0001) with sperm motility, viability, normal morphology and normal plasma membranes. In summary, differences in sperm motility were related to n-3 PUFA content in the sperm plasma membrane and extracellular antioxidants in seminal plasma which protect sperm plasma membranes from lipid peroxidation during periods of oxidative stress.

Fas and Fas Ligand Expression In Children with Acute Lymphoblastic Leukemia, Benign Hematological Diseases and Solid Tumors

AbstractAbstract 4853Fas/FasL is a key pathway of cellular apoptosis. Fas receptor is expressed on membranes of both normal and neoplastic cells while, Fas ligand (FasL) mainly on activated T-lymphocytes. Fas-FasL abnormalities have been detected in malignancies and autoimmune diseases and implicated in resistance to treatment. The aim of the study was the determination of the levels of Fas, FasL and their coexpression in bone marrow cells of children with acute lymphoblastic leukaemia (ALL) at diagnosis, during the course of treatment and the comparison with relevant levels in other hematological and neoplastic diseases. Expression levels of Fas and FasL were determined with flow cytometry in children with ALL at diagnosis (ALLd, n=13), on day 15 of treatment (d15, n=6), on day33 (ALLd33, n=6), during consolidation (ALLhr, n=12) and at the end of therapy (ALLet, n=7) as well as in children with Langerhans Histiocytosis (LCH, n=4), cytopenias (Cytp, n=8) and solid tumors without bone marrow infiltration at diagnosis (STd, n=5) and on therapy (STther, n=6). Results:The lowest levels of Fas expression were detected at diagnosis of ALL and were gradually statistical significantly increased until remission of day 33 (ALLd vs ALLd33: 8.02±1.94 vs 24.04±6.11, p=0.035). At consolidation Fas levels were found to be decreased compared to day33 (16.1±4.18) and were again increased at the end of therapy (ALLd vs ALLet: 8.02±1.94 vs 24.96±7.95, p=0.024). On the contrary, FasL levels were gradually decreased and finally increased to similar to diagnosis levels at the end of treatment (ALLd: 4.59±1.41, ALLet: 5.89±1.99). In solid tumors at diagnosis Fas levels were similar to the ones while on chemotherapy (STd vs STth:16.04±2.2 vs 15.19±6.4). The highest FasL levels were detected in the group of STd with the relevant levels on treatment being lower in comparison (STd vs STther: 10.91±3.32 vs 2.92±0.79, p=0.052). In LCH both Fas and FasL levels were found to be as low as at ALL diagnosis. In cytopenias no significant difference was observed between groups for either Fas (11.05±4.49) or FasL (3.01±0.62). As for Fas+FasL+ coexpression no difference was evident between ALLd, ALLet and STd or STther [ALLd (0.73±0.38), ALLet (0.64±0.17), STd (0.68±0.095), STther (0.66±0.38)]. The lowest coexpreesion levels were observed in the group of cytopenias with statistical significant difference compared to STd (0.68±0.095 vs 0.26±0.08, p=0.031). In conclusion, at diagnosis of ALL Fas levels were expressed in lowest levels that were found to be gradually increased at remission and at the end of treatment. This finding probably correlates with the apoptotic process of the leukemia clone and possibly with response to treatment. Disclosures:No relevant conflicts of interest to declare.

Low-intensity pulsed ultrasound (LIPUS) therapy may enhance the negative effects of oxygen radicals in the acute phase of fracture

Though it is well accepted that low-intensity pulsed ultrasound (LIPUS) can accelerate the healing process of a fracture with very good results, we should still pay attention to its side effects and further improve its application in detail, such as the appropriate time and point for the application. In the early phase of a bone fracture, there are millions of oxygen radicals released by neutrophils in the injured area. This article focuses on whether the increased permeability of normal cell membranes by LIPUS makes the concentration of oxygen radicals increase to such a high degree that damage occurs to healthy tissue cells. It is proposed that it may be better not to use LIPUS in the acute phase of a fracture (i.e. within 1week after injury) but instead delay its application until after any inflammatory reaction has weakened to yield better results.

Salicylate restores transport function and anion exchanger activity of missense pendrin mutations

The SLC26A4 gene encodes the transmembrane protein pendrin, which is involved in the homeostasis of the ion concentration of the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Mutations in the SLC26A4 gene cause sensorineuronal hearing loss. However, the mechanisms responsible for such loss have remained unknown. Therefore, in this study, we focused on the function of ten missense pendrin mutations (p.P123S (Pendred syndrome), p.M147V (NSEVA), p.K369E (NSEVA), p.A372V (Pendred syndrome/NSEVA), p.N392Y (Pendred syndrome), p.C565Y (NSEVA), p.S657N (NSEVA), p.S666F (NSEVA), p.T721M (NSEVA) and p.H723R (Pendred syndrome/NSEVA)) reported in Japanese patients, and analyzed their cellular localization and anion exchanger activity using HEK293 cells transfected with each mutant gene. Immunofluorescent staining of the cellular localization of the pendrin mutants revealed that p.K369E and p.C565Y, as well as wild-type pendrin, were transported to the plasma membrane, while 8 other mutants were retained in the cytoplasm. Furthermore, we analyzed whether salicylate, as a pharmacological chaperone, restores normal plasma membrane localization of 8 pendrin mutants retained in the cytoplasm to the plasma membrane. Incubation with 10 mM of salicylate of the cells transfected with the mutants induced the transport of 4 pendrin mutants (p.P123S, p.M147V, p.S657Y and p.H723R) from the cytoplasm to the plasma membrane and restored the anion exchanger activity. These findings suggest that salicylate might contribute to development of a new method of medical treatment for sensorineuronal hearing loss caused by the mutation of the deafness-related proteins, including pendrin.