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Sample records for acrosomal membrane integrity

  1. Osmotic tolerance limits and effects of cryoprotectants on the motility, plasma membrane integrity and acrosomal integrity of rat sperm.

    PubMed

    Si, Wei; Benson, James D; Men, Hongsheng; Critser, John K

    2006-12-01

    Osmotic stress is an important factor that can result in cell damage during cryopreservation. The objectives of this study were to determine: (1) isosmotic sperm cell volume; (2) osmotically inactive volume; (3) osmotic tolerance limits of rat sperm; and (4) the effects of addition and removal of glycerol (Gly), ethylene glycol (EG), propylene glycol (PG) or dimethyl sulfoxide (Me(2)SO) on rat sperm function. Sperm from Fischer 344 and Sprague-Dawley rats were used in this study. An electronic particle counter was used to measure the cell volume of rat sperm. Computer-assisted sperm motility analysis and flow-cytometric analysis were used to assess sperm motility, plasma membrane and acrosomal integrity. The isosmotic sperm cell volumes of the two strains were 37.0+/-0.1 and 36.2+/-0.2 microm(3), respectively. Rat sperm behaved as linear osmometers from 260 to 450 mOsm, and the osmotically inactive sperm volumes of the two strains were 79.8+/-1.5% and 81.4+/-2.2%, respectively. Rat sperm have very limited osmotic tolerances. The sperm motility and the sperm plasma membranes of both strains were sensitive to anisosmotic treatments, but the acrosomes of both strains were more sensitive to hyposmotic than hyperosmotic conditions. The one-step addition and removal of Me(2)SO showed the most deleterious effect on rat sperm motility, plasma membrane integrity, and acrosomal integrity among the four cryoprotectants. These data characterizing rat sperm osmotic behavior, osmotic and cryoprotectant tolerance will be used to design cryopreservation protocols for rat sperm.

  2. Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm.

    PubMed

    Valle, R R; Valle, C M R; Nichi, M; Muniz, J A P C; Nayudu, P L; Guimarães, M A B V

    2008-07-01

    Simple, rapid and stable sperm evaluation methods which have been optimized for common marmoset (Callithrix jacchus) are critical for studies involving collection and evaluation of sperm in the field. This is particularly important for new species groups such as Callitrichidae where the sperm have been little studied. Of this family, C. jacchus is the best known, and has been chosen as a model species for other members of the genus Callithrix. The fundamental evaluation parameters for sperm of any species are viability and acrosomal status. Semen samples were collected by penile vibratory stimulation. To evaluate sperm plasma membrane integrity, Eosin-Nigrosin was tested here for the common marmoset sperm to be used under field conditions. Further, a non-fluorescent stain for acrosome, the "Simple" stain, developed for domestic and wild cats, was tested on common marmoset sperm. This was compared with a fluorescent staining, Fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA), routinely used and validated for common marmoset at the German Primate Centre to evaluate acrosomal integrity. Results obtained with the "Simple" stain showed a marked differentiation between sperm with intact and non-intact acrosome both with and without ionophore treatment and closely correlated with results obtained with FITC-PSA. Temperature had no effect on the results with the "Simple" stain and the complete processing is simple enough to be carried out under field conditions. These findings indicated that the "Simple" stain and Eosin-Nigrosin provide rapid and accurate results for C. jacchus sperm and that those methods can be reliably used as field tools for sperm evaluation for this species.

  3. In vitro effects of l-carnitine and glutamine on motility, acrosomal abnormality, and plasma membrane integrity of rabbit sperm during liquid-storage.

    PubMed

    Sarıözkan, Serpil; Ozdamar, Saim; Türk, Gaffari; Cantürk, Fazile; Yay, Arzu

    2014-06-01

    This study was designed to evaluate the in vitro effects of l-carnitine and glutamine (Gln) on the sperm quality parameters of liquid-stored rabbit semen maintained up to 24 h at 5°C. Pooled and extended ejaculates were divided into two equal portions. l-Carnitine doses of 0.5, 1 and 2mM were added to the first portion, and glutamine was added at the same doses to the second portion. All samples were cooled to 5°C and examined at 0, 6, 12 and 24 h of liquid storage. Supplementation of the semen extender with three different doses of l-carnitine provided significant increases in the percentage of motile sperm at 12 h (P<0.01), and 24h (P<0.001) and enabled significant protection of the sperm plasma membrane (P<0.01) at 12 and 24h of cool-storage, in comparison to the control samples. Only the 2mM dose of l-carnitine significantly (P<0.01) decreased the rate of acrosomal damage when compared to the control group. Furthermore, all doses of Gln caused a significant (P<0.01) decrease in acrosomal damage at 6h, and provided significant improvement (P<0.01) in sperm motility, acrosomal and plasma membrane integrities at 12 and 24h of liquid storage, when compared to the controls. In conclusion, the supplementation of liquid-stored rabbit semen with l-carnitine and Gln provided a protection for sperm against cool storage-induced functional and structural damages.

  4. The Effect of Low-Level Laser Irradiation on Sperm Motility, and Integrity of the Plasma Membrane and Acrosome in Cryopreserved Bovine Sperm

    PubMed Central

    Fernandes, Guilherme Henrique C.; de Carvalho, Paulo de Tarso Camillo; Serra, Andrey Jorge; Crespilho, André Maciel; Peron, Jean Pierre Schatzman; Rossato, Cristiano; Leal-Junior, Ernesto Cesar Pinto; Albertini, Regiane

    2015-01-01

    Background and Objective Freezing changes sperm integrity remarkably. Cryopreservation involves cooling, freezing, and thawing and all these contribute to structural damage in sperm, resulting in reduced fertility potential. Low-level laser irradiation (LLLI) could increase energy supply to the cell and cause reactive oxygen species reduction (ROS), contributing to the restoration of oxygen consumption and adenosine triphosphate synthesis (ATP) in the mitochondria. Our goal was to analyze the effects of low-level laser irradiation on sperm motility and integrity of the plasma membrane and acrosome in cryopreserved bovine sperm. Study Design/Materials and Methods We analyzed 09 samples of bull semen (Bos taurus indicus), divided into three groups: a control group without laser irradiation, a 4J group subjected to a laser irradiation dose of 4 joules, and a 6J group subjected to dose of 6 joules. Samples were divided for the analysis of cell viability and acrosomal membrane integrity using flow cytometry; another portion was used for motion analysis. Irradiation was performed in petri dishes of 30 mm containing 3 ml of semen by an aluminum gallium indium phosphide laser diode with a wavelength of 660 nm, 30 mW power, and energy of 4 and 6 joules for 80 and 120 seconds respectively. Subsequently, the irradiated and control semen samples were subjected to cryopreservation and analyzed by flow cytometry (7AAD and FITC-PSA) using the ISAS - Integrated Semen Analysis System. Results Flow cytometry showed an increase in the percentage of live sperm cells and acrosome integrity in relation to control cells when subjected to irradiation of low-power laser in two different doses of 4 and 6 joules (p < 0.05). In the analysis of straightness, percentage of cell movement, and motility, a dose of 4 joules was more effective (p < 0.05). Conclusion We conclude that LLLI may exert beneficial effects in the preservation of live sperm. A dose of 4 joules prior to cryopreservation was

  5. Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes.

    PubMed

    Anzar, M; Kroetsch, T; Boswall, L

    2011-06-01

    In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.

  6. Remodeling of the plasma membrane in preparation for sperm-egg recognition: roles of acrosomal proteins.

    PubMed

    Tanphaichitr, Nongnuj; Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark

    2015-01-01

    The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm-ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm-ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm-ZP binding step.

  7. Calcium channel antagonists inhibit the acrosome reaction and bind to plasma membranes of sea urchin sperm.

    PubMed Central

    Kazazoglou, T; Schackmann, R W; Fosset, M; Shapiro, B M

    1985-01-01

    As a prerequisite to fertilization, sea urchin sperm undergo an acrosome reaction that is mediated in part by increased permeability to Ca2+, with an attendant rapid, massive intracellular Ca2+ accumulation. The acrosome reaction is inhibited by Ca2+ channel antagonists, including verapamil, D600, and dihydropyridines such as nitrendipine, nimodipine, and nisoldipine. To examine the interaction of Ca2+ antagonists with sperm, a plasma membrane preparation enriched for Na+,K+-ATPase was isolated from sea urchin sperm. These plasma membranes specifically bound [3H]nitrendipine and [3H]verapamil at concentrations similar to those that inhibit the acrosome reaction. The binding of verapamil was sigmoidal and half-maximal at 1 microM. There was a high specificity in the binding interaction, since by competition binding verapamil, (-)-D600, and (+)-D600 had different relative Kd values, 11, 2.5, and 0.5 microM, respectively. These data suggest that sperm mediate the Ca2+ influx required for induction of the acrosome reaction via Ca2+ channels with properties similar, but not identical, to those of other excitable tissues. Images PMID:3856274

  8. Membrane events in the acrosomal reaction of Limulus sperm. Membrane fusion, filament-membrane particle attachment, and the source and formation of new membrane surface

    PubMed Central

    1979-01-01

    The membranes of Limulus (horseshoe crab) sperm were examined before and during the acrosomal reaction by using the technique of freeze- fracturing and thin sectioning. We focused on three areas. First, we examined stages in the fusion of the acrosomal vacuole with the cell surface. Fusion takes place in a particle-free zone which is surrounded by a circlet of particles on the P face of the plasma membrane and an underlying circlet of particles on the P face of the acrosomal vauole membrane. These circlets of particles are present before induction. Up to nine focal points of fusion occur within the particle-free zone. Second, we describe a system of fine filaments, each 30 A in diameter, which lies between the acrosomal vacuole and the plasma membrane. These filaments change their orientation as the vacuole opens, a process that takes place in less than 50 ms. Membrane particles seen on the P face of the acrosomal vacuole membrane change their orientation at the same time and in the same way as do the filaments, thus indicating that the membrane particles and filaments are probably connected. Third, we examined the source and the point of fusion of new membrane needed to cover the acrosomal process. This new membrane is almost certainly derived from the outer nuclear envelope and appears to insert into the plasma membrane in a particle-free area adjacent to an area rich in particles. The latter is the region where the particles are probably connected to the cytoplasmic filaments. The relevance of these observations in relation to the process of fertilization of this fantastic sperm is discussed. PMID:582596

  9. ROLE OF THE GAMETE MEMBRANES IN FERTILIZATION IN SACCOGLOSSUS KOWALEVSKII (ENTEROPNEUSTA). I. THE ACROSOMAL REGION AND ITS CHANGES IN EARLY STAGES OF FERTILIZATION.

    PubMed

    COLWIN, A L; COLWIN, L H

    1963-12-01

    Previous electron microscope studies of sperm-egg association in the annelid Hydroides revealed novel aspects with respect to the acrosomal region. To determine whether these aspects were unique, a comparable study was made of a species belonging to a widely separated phylum, Hemichordata. Osmium tetroxide-fixed polyspermic material of the enteropneust, Saccoglossus, was used. The acrosomal region includes the membrane-bounded acrosome, with its large acrosomal granule and shallow adnuclear invagination, and the periacrosomal material which surrounds the acrosome except at the apex; here, the acrosomal membrane lies very close to the enclosing sperm plasma membrane. After reaching the egg envelope, the spermatozoon is activated and undergoes a series of changes: the apex dehisces and around the resulting orifice the acrosomal and sperm plasma membranes form a continuous mosaic membrane. The acrosomal granule disappears. Within 7 seconds the invagination becomes the acrosomal tubule, spans the egg envelopes, and meets the egg plasma membrane. The rest of the acrosomal vesicle everts. The periacrosomal mass changes profoundly: part becomes a fibrous core (possibly equivalent to a perforatorium); part remains as a peripheral ring. The basic pattern of structure and sperm-egg association in Saccoglossus is the same as in Hydroides. Previous evidence from four other phyla as interpreted here also indicates conformity to this pattern. The major role of the acrosome is apparently to deliver the sperm plasma membrane to the egg plasma membrane.

  10. Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction*

    PubMed Central

    De La Vega-Beltran, Jose Luis; Sánchez-Cárdenas, Claudia; Krapf, Darío; Hernandez-González, Enrique O.; Wertheimer, Eva; Treviño, Claudia L.; Visconti, Pablo E.; Darszon, Alberto

    2012-01-01

    Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pHi) and Ca2+ ([Ca2+]i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K+ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K+ or by addition of solubilized zona pellucida (sZP). Moreover, K+ and sZP were also able to increase [Ca2+]i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K+ concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca2+]i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction. PMID:23095755

  11. Fusion of artificial membranes with mammalian spermatozoa. Specific involvement of the equatorial segment after acrosome reaction.

    PubMed

    Arts, E G; Kuiken, J; Jager, S; Hoekstra, D

    1993-11-01

    The fusogenic properties of bovine and human spermatozoa membranes were investigated, using phospholipid bilayers (liposomes) as target membranes. Fusion was monitored by following lipid mixing, as revealed by an assay based on resonance-energy transfer. In addition, fusion was visualized by fluorescence microscopy, using fluorescent lipid vesicles. Cryopreserved bovine sperm fused with liposomes before induction of the acrosome reaction, fluorescence being located in essentially all spermatozoa membrane domains. Fresh bovine and human spermatozoa fused with liposomes only after the induction of the acrosome reaction, as triggered by calcium ionophore A23187 or zonae pellucidae (proteins), while the fluorescence distribution was mainly restricted to the equatorial segment (ES). However, with spermatozoa that had undergone a freeze/thawing cycle, domains other than ES also became labeled. Hence, the redistribution of the lipid probes over the entire membrane occurring during lipid mixing with cryopreserved bovine sperm is probably related to membrane perturbations caused by long-term cryopreservation. Fusion with liposomes was governed by spermatozoa factors and required the presence of acidic phospholipids like cardiolipin and phosphatidylserine in the liposomal bilayer. Incorporation of the zwitterionic lipid phosphatidylcholine in the vesicles inhibited the fusion reaction. Fusion was pH dependent. The results indicate that the ES is the primary domain of spermatozoa membranes that harbours the fusogenic capacity of sperm. Liposomes appear a valuable tool in further characterizing the properties of this domain, which has been claimed [Yanagimachi, R. (1988) in The physiology of reproduction (Knobil, E. & Neill, J., eds) pp. 135-185, Raven Press, New York] to represent the putative, initial fusion site for the oocyte.

  12. Fluorometric study of rabbit sperm head membrane phospholipid asymmetry during capacitation and acrosome reaction using Annexin-V FITC.

    PubMed

    Avalos-Rodríguez, A; Ortíz-Muñíz, A R; Ortega-Camarillo, C; Vergara-Onofre, M; Rosado-García, A; Rosales-Torres, A M

    2004-01-01

    This study was conducted to evaluate phosphatidylserine translocation in head plasma membrane of Percoll-gradient purified of rabbit cauda epididymal sperm during capacitation and acrosome reaction (AR) using Annexin-V. Propidium iodide was used as control to reject dead or dying cells. The presence and distribution of Annexin-V binding sites were analyzed using flow fluorocytometry and confocal microscopy. After 6 h of incubation of sperm in capacitation medium, the number of cells positively stained with Annexin-V showed a small but significant increment. The Annexin-V binding sites produced during capacitation were found mainly in the post-acrosomal region of the sperm head plasma membrane. After AR induction with progesterone, the localization of phosphatidylserine was changed and the Annexin-V binding sites were found almost only in the acrosomal region, but with higher number of binding sites in the equatorial area. On the contrary, after AR induction with A23187, phosphatidylserine translocation, although predominant over the acrosomal region, was also observed in the post-acrosomal region. Plasma membrane destabilization during capacitation and AR may be important for sperm-oocyte fusion.

  13. Increased chromatin fragmentation and reduced acrosome integrity in spermatozoa of red deer from lead polluted sites.

    PubMed

    Castellanos, Pilar; del Olmo, Enrique; Fernández-Santos, M Rocío; Rodríguez-Estival, Jaime; Garde, J Julián; Mateo, Rafael

    2015-02-01

    Vertebrates are constantly exposed to a diffuse pollution of heavy metals existing in the environment, but in some cases, the proximity to emission sources like mining activity increases the risk of developing adverse effects of these pollutants. Here we have studied lead (Pb) levels in spermatozoa and testis, and chromatin damage and levels of endogenous antioxidant activity in spermatozoa of red deer (Cervus elaphus) from a Pb mining area (n=37) and a control area (n=26). Deer from the Pb-polluted area showed higher Pb levels in testis parenchyma, epididymal cauda and spermatozoa, lower values of acrosome integrity, higher activity of glutathione peroxidase (GPx) and higher values of DNA fragmentation (X-DFI) and stainability (HDS) in sperm than in the control area. These results indicate that mining pollution can produce damage on chromatin and membrane spermatozoa in wildlife. The study of chromatin fragmentation has not been studied before in spermatozoa of wildlife species, and the sperm chromatin structure assay (SCSA) has been revealed as a successful tool for this purpose in species in which the amount of sperm that can be collected is very limited.

  14. Challenges in cryopreserving endangered mammal spermatozoa: morphology and the value of acrosomal integrity as markers of cryo-survival.

    PubMed

    Pukazhenthi, Budhan; Santymire, Rachel; Crosier, Adrienne; Howard, JoGayle; Wildt, David E

    2007-01-01

    The science of cryobiology is essential to the effective, practical use of semen for assisted breeding to help manage small populations of rare wildlife species. In this review, we describe challenges associated with cryopreserving gametes from wild fauna. Based on more than 25 years of experience across a diversity of mammals, it appears that the primary driving force dictating cryo-survival of a spermatozoon is its initial pre-freeze quality and morphology, especially having a morphologically normal, intact acrosome. This assertion is supported through extensive studies of three animal groups that routinely ejaculate semen containing (1) normal sperm/acrosomal quality (examples, Eld's deer, Cervus eldi and giant panda, Ailuropoda melanoleuca), (2) normal acrosomal quality, but from teratospermic donors (>70% pleiomorphic sperm; cheetah, Acinonyx jubatus and black-footed ferret, Mustela nigripes) and (3) abnormal acrosomal quality and general teratospermia (clouded leopard, Neofelis nebulosa). Data revealed that species producing high quality sperm with > 70% normal, intact acrosomes were best able to survive cryopreservation (-80% intact acrosomes post-thaw). Species that were teratospermic, but with high proportions of intact acrosomes (72 to 88%) in ejaculates varied significantly (4 to 55% intact acrosomes post-thaw) in sperm survival to freeze-thawing. Spermatozoa from the clouded leopard (that was both teratospermic while producing only 11% normal acrosomes in fresh semen) failed to survive cryopreservation despite using an array of conventional and unconventional freezing approaches. These observations (combined with zona penetration assays and artificial insemination results) suggest that proportions of malformed sperm and especially initial structural integrity of the acrosome are more important predictors of sperm survivability post-thaw than initial sperm motility scores.

  15. In vitro effects of nonylphenol on motility, mitochondrial, acrosomal and chromatin integrity of ram and boar spermatozoa.

    PubMed

    Uguz, C; Varisli, O; Agca, C; Evans, T; Agca, Y

    2015-10-01

    The objective of this study was to determine the effects of nonylphenol (NP) on viability of ram and boar sperm in vitro. Ram or boar spermatozoa were exposed to 1, 10, 100, 250 and 500 μg NP ml(-1) for 1, 2, 3 or 4 h. Computer-assisted sperm motility analysis (CASA) system was used to evaluate sperm motility characteristics. Flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity, while epifluorescent microscopy was used to determine sperm acrosomal status. Exposure of both species spermatozoa to 250 and 500 μg NP ml(-1) was detrimental to progressive motility (P < 0.05), and its adverse effect was significant at lower (100 μg NP ml(-1) ) concentration (P < 0.05). The percentages of ram and boar spermatozoa with high MMP declined drastically after exposures to ≥250 μg ml(-1) NP (P < 0.05). Unlike chromatin integrity, which did not appear to be altered by NP exposure, there were dose-dependent NP effects (P < 0.05) on acrosomal integrity of both species at as low as 1 μg ml(-1) NP for boar spermatozoa and 10 μg ml(-1) NP for ram spermatozoa. These data show adverse effects of NP on ram and boar spermatozoa and thus its potential harmful effects on male reproduction as NP is found in fruits, vegetables, human milk, fish and livestock products.

  16. Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding.

    PubMed

    Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark A; Tanphaichitr, Nongnuj

    2015-02-01

    The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

  17. Con A-binding protein Zn-α2-glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility.

    PubMed

    Liu, Y; Qu, F; Cao, X; Chen, G; Guo, Q; Ying, X; Guo, W; Lu, L; Ding, Z

    2012-04-01

    Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.

  18. Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa.

    PubMed

    Pontbriand, D; Howard, J G; Schiewe, M C; Stuart, L D; Wildt, D E

    1989-08-01

    A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested.

  19. Presence and Function of Dopamine Transporter (DAT) in Stallion Sperm: Dopamine Modulates Sperm Motility and Acrosomal Integrity

    PubMed Central

    Covarrubias, Alejandra A.; Rodríguez-Gil, Joan Enric; Ramírez-Reveco, Alfredo; Concha, Ilona I.

    2014-01-01

    Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP+), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility. PMID:25402186

  20. Effects of ascorbic acid on sperm motility, viability, acrosome reaction and DNA integrity in teratozoospermic samples

    PubMed Central

    Fanaei, Hamed; Khayat, Samira; Halvaei, Iman; Ramezani, Vahid; Azizi, Yaser; Kasaeian, Amir; Mardaneh, Jalal; Parvizi, Mohammad Reza; Akrami, Maryam

    2014-01-01

    Background: Oxidative stress in teratozoospermic semen samples caused poor assisted reproductive techniques (ART) outcomes. Among antioxidants, ascorbic acid is a naturally occurring free radical scavenger and as such its presence assists various other mechanisms in decreasing numerous disruptive free radical processes. Objective: The main goal of this study was to evaluate potential protective effects of ascorbic acid supplementation during in vitro culture of teratozoospermic specimens. Materials and Methods: Teratozoospermic semen samples that collected from 15 volunteers were processed, centrifuged and incubated at 37oC until sperm swimmed-up. Supernatant was divided into four groups and incubated at 37oC for one hour under different experimental conditions: Control, 10 µm A23187, 600µm ascorbic acid and 10 µm A23187+600 µm ascorbic acid. After incubation sperm motility, viability, acrosome reaction, DNA damage and malondialdehyde levels were evaluated. Results: Our results indicated that after one hour incubation, ascorbic acid significantly reduced malondialdehyde level in ascorbic acid group (1.4±0.11 nmol/ml) compared to control group (1.58±0.13 nmol/ml) (p<0.001). At the end of incubation, progressive motility and viability in ascorbic acid group (64.5±8.8% and 80.3±6.4%, respectively) were significantly (p<0.05 and p<0.001, respectively) higher than the control group (54.5±6.8% and 70.9±7.3%, respectively). A23187 significantly (p<0.0001) increased acrosome reaction in A23187 group (37.3±5.6%) compared to control group (8.5±3.2%) and this effect of A23187 attenuated by ascorbic acid in ascorbic acid+A23187 group (17.2±4.4%). DNA fragmentation in ascorbic acid group (20±4.1%) was significantly (p<0.001) lower than controls (28.9±4.6%). Conclusion: In vitro ascorbic acid supplementation during teratozoospermic semen processing for ART could protect teratozoospermic specimens against oxidative stress, and it could improve ART outcome. PMID

  1. Motility and acrosomal integrity comparisons between electro-ejaculated and epididymal ram sperm after exposure to a range of anisosmotic solutions, cryoprotective agents and low temperatures.

    PubMed

    Varisli, Omer; Uguz, Cevdet; Agca, Cansu; Agca, Yuksel

    2009-02-01

    Effective ram sperm cryopreservation protocols, which would yield acceptable lambing rates following artificial insemination (AI), are currently lacking. The objectives of the current studies were to compare the effects of various anisosmotic conditions, cryoprotective agents (CPAs) and chilling on the motility and acrosomal integrity of electro-ejaculated and epididymal ram sperm. Three experiments were conducted. In experiment 1, ejaculated and epididymal ram sperm were exposed to 75, 150, 225, 600, 900 and 1200 milliosmolal (mOsm)/kg sucrose solutions, held for 5 min and then returned to isosmotic condition. Motility characteristics of sperm during exposure to each anisosmotic solutions and after returning to isosmotic conditions were determined. In experiment 2, ejaculated and epididymal ram sperm were exposed to 1M glycerol (Gly), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) for 5 min and then returned to isosmotic conditions. Motility characteristics of sperm samples during exposure to each CPA solution and after returning to isosmotic conditions were determined. In experiment 3, effects of various temperatures on motility characteristics of ejaculated and epididymal ram sperm were determined after exposing them to three different sub-physiologic temperatures (4, 10 and 22 degrees C) for 30 min and subsequently returning them to 37 degrees C. The motility of ejaculated ram sperm was significantly more affected from anisosmotic stress than was epididymal ram sperm (P<0.05). While anisosmotic stress had no effects on acrosomal integrity of epididymal ram sperm, there was a significant reduction in acrosomal integrity for ejaculated ram sperm after the addition and removal of a 75 mOsm sucrose solution. The abrupt addition and removal of 1M Gly, DMSO, EG or PG had no effect on the motility and acrosomal integrity of epididymal ram sperm (P>0.05). However, there was a slight decrease in acrosomal integrity for ejaculated ram sperm

  2. Equatorin is not essential for acrosome biogenesis but is required for the acrosome reaction

    SciTech Connect

    Hao, Jianxiu; Chen, Min; Ji, Shaoyang; Wang, Xiaona; Wang, Yanbo; Huang, Xingxu; Yang, Lin; Wang, Yaqing; Cui, Xiuhong; Lv, Limin; Liu, Yixun; Gao, Fei

    2014-02-21

    Highlights: • Eqtn knockout mice were used for these experiments. • In vivo and in vitro fertilization analyses were performed. • Eqtn-deficient sperm were evaluated by transmission electron microscopy (TEM) and an A23187-induced acrosome reaction (AR) assay. • Co-immunoprecipitation (Co-IP) was performed to assess the interaction between Eqtn and the SNARE complex. - Abstract: The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in mammalian fertilization. However, the regulatory mechanisms controlling acrosome biogenesis and acrosome exocytosis during fertilization are largely unknown. Equatorin (Eqtn) is a membrane protein that is specifically localized to the acrosomal membrane. In the present study, the physiological functions of Eqtn were investigated using a gene knockout mouse model. We found that Eqtn{sup −/−} males were subfertile. Only approximately 50% of plugged females were pregnant after mating with Eqtn{sup −/−} males, whereas more than 90% of plugged females were pregnant after mating with control males. Sperm and acrosomes from Eqtn{sup −/−} mice presented normal motility and morphology. However, the fertilization and induced acrosome exocytosis rates of Eqtn-deficient sperm were dramatically reduced. Further studies revealed that the Eqtn protein might interact with Syntaxin1a and SNAP25, but loss of Eqtn did not affect the protein levels of these genes. Therefore, our study demonstrates that Eqtn is not essential for acrosome biogenesis but is required for the acrosome reaction. Eqtn is involved in the fusion of the outer acrosomal membrane and the sperm plasma membrane during the acrosome reaction, most likely via an interaction with the SNARE complex.

  3. Kinetics of human sperm acrosomal exocytosis.

    PubMed

    Sosa, C M; Pavarotti, M A; Zanetti, M N; Zoppino, F C M; De Blas, G A; Mayorga, L S

    2015-03-01

    The acrosome reaction is a unique event in the lifespan of sperm characterized by the exocytosis of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal membrane and the plasma membrane. This unique regulated exocytosis is mediated by essentially the same membrane fusion machinery present in neuroendocrine cells. However, whereas secretion in neuroendocrine cells occurs in less than a second, the acrosome reaction is normally assessed after several minutes of incubation with inducers. In this report, we measured the kinetics of human sperm exocytosis triggered by two stimuli (calcium ionophore and progesterone) by using electron microscopy and three different approaches based on the incorporation of fluorescent Pisum sativum agglutinin into the acrosome upon opening of fusion pores connecting the extracellular medium with the acrosomal lumen. The results with the different methods are consistent with a slow kinetics (t½ = 14 min). We also manipulated the system to measure different steps of the process. We observed that cytosolic calcium increased with a relatively fast kinetics (t½ = 0.1 min). In contrast, the swelling of the acrosomal granule that precedes exocytosis was a slow process (t½ = 13 min). When swelling was completed, the fusion pore opening was fast (t½ = 0.2 min). The results indicate that acrosomal swelling is the slowest step and it determines the kinetics of the acrosome reaction. After the swelling is completed, the efflux of calcium from intracellular stores triggers fusion pores opening and the release of hybrid vesicles in seconds.

  4. Expanded cumuli induce acrosome reaction in boar sperm.

    PubMed

    Mattioli, M; Lucidi, P; Barboni, B

    1998-12-01

    The authors investigated acrosomal changes occurring in boar sperm that interact with the expanded cumulus matrix surrounding ovulated pig oocytes. Samples of washed boar sperm obtained from six donors were incubated for 4 hr under capacitating conditions and exposed either to solubilized zonae pellucidae (ZP) or solubilized expanded pig cumuli (SEC) obtained from IVM oocytes. Alternatively, hyaluronic acid, laminin, or fibronectin, components of the extracellular matrix (ECM) were added to capacitated sperm. Acrosomal integrity was evaluated 1 hr later by using FITC-PSA staining. Solubilized cumuli induced acrosome reaction (AR) in a dose-dependent manner with a saturating effect exerted at 2.5 SEC/50 microl. Both 500 nM fibronectin and 500 nM laminin stimulated acrosomal exocytosis, the latter being more effective and inducing saturating levels of AR. By contrast, hyaluronic acid did not affect acrosomal status. Preincubation with anti-laminin antibodies completely prevented the inducing activity of SEC without affecting the activity of solubilized ZP. Consistent with these data, the integrin VLA-6, a receptor with high affinity for laminin, was detected by immunoblotting on the plasma membrane of capacitated boar spermatozoa. In addition, its immunoneutralization, obtained with the preincubation of capacitated sperm with the antibody raised against the alpha chain of VLA-6 integrin, prevented AR upon exposure to laminin or SEC (10.7+/-3.2 and 10.2+/-1.0% respectively), while the samples retained their responsiveness to ZP (29.6+/-1.2%). The results demonstrate that the interaction between laminin, entrapped in the expanded cumuli, and specific integrins present on the sperm membrane can initiate AR, thus taking part in the process of sperm-egg recognition.

  5. Effects of post-thaw incubation on motility, acrosomal integrity and in vitro fertilizing capacity of boar spermatozoa cryopreserved in 0.5 and 5ml straws.

    PubMed

    Córdova, Alejandro; Hernández-Gil, Rocío; Córdova-Jiménez, Cristian Alejandro; Cardilli, Diogo José; de Sousa Oliveira, Kellen; Pérez-Gutiérrez, José Félix

    2013-06-01

    The effect of post-thaw incubation (0 vs. 5h at 15°C) and straw size (5 vs. 0.5ml) on motility, acrosomal integrity and in vitro fertilizing (IVF) capacity of cryopreserved boar spermatozoa was studied. In samples assessed immediately after thawing, no differences were found between the two straw sizes. After 5h post-thaw incubation, all parameters, except polyspermy, decreased and, spermatozoa packaged in 5ml straws showed better functional and IVF parameters than these in 0.5ml straws.

  6. Slow cooling prevents cold-induced damage to sperm motility and acrosomal integrity in the black-footed ferret (Mustela nigripes).

    PubMed

    Santymire, R M; Marinari, P E; Kreeger, J S; Wildt, D E; Howard, J G

    2007-01-01

    The endangered black-footed ferret (Mustela nigripes) has benefited from artificial insemination; however, improved sperm cryopreservation protocols are still needed. The present study focused on identifying factors influencing gamete survival during processing before cryopreservation, including: (1) the presence or absence of seminal plasma; (2) temperature (25 degrees C v. 37 degrees C); (3) type of medium (Ham's F10 medium v. TEST yolk buffer [TYB]); (4) cooling rate (slow, rapid and ultra-rapid); and (5) the presence or absence of glycerol. Seminal plasma did not compromise (P > 0.05) sperm motility or acrosomal integrity. Sperm motility traits were maintained longer (P < 0.05) at 25 degrees C than at 37 degrees C in Ham's or TYB, but temperature did not affect (P > 0.05) acrosomal integrity. Overall, TYB maintained optimal (P < 0.05) sperm motility compared with Ham's medium, but Ham's medium maintained more (P < 0.05) intact acrosomes than TYB. Slow cooling (0.2 degrees C min(-1)) was optimal (P < 0.05) compared to rapid cooling (1 degrees C min(-1)), and ultra-rapid cooling (9 degrees C min(-1)) was found to be highly detrimental (P < 0.05). Results obtained in TYB with 0% or 4% glycerol were comparable (P > 0.05), indicating that 4% glycerol was non-toxic to ferret sperm; however, glycerol failed to ameliorate the detrimental effects of either rapid or ultra-rapid cooling. The results of the present study demonstrate that the damage observed to black-footed ferret spermatozoa is derived largely from the rate of cooling.

  7. Matrix membranes and integrability

    SciTech Connect

    Zachos, C.; Fairlie, D.; Curtright, T.

    1997-06-01

    This is a pedagogical digest of results reported in Curtright, Fairlie, {ampersand} Zachos 1997, and an explicit implementation of Euler`s construction for the solution of the Poisson Bracket dual Nahm equation. But it does not cover 9 and 10-dimensional systems, and subsequent progress on them Fairlie 1997. Cubic interactions are considered in 3 and 7 space dimensions, respectively, for bosonic membranes in Poisson Bracket form. Their symmetries and vacuum configurations are explored. Their associated first order equations are transformed to Nahm`s equations, and are hence seen to be integrable, for the 3-dimensional case, by virtue of the explicit Lax pair provided. Most constructions introduced also apply to matrix commutator or Moyal Bracket analogs.

  8. RIM, Munc13, and Rab3A Interplay in Acrosomal Exocytosis

    PubMed Central

    Bello, Oscar D.; Zanetti, M. Natalia; Mayorga, Luis S.; Michaut, Marcela A.

    2012-01-01

    Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have a central role in membrane docking. PMID:22248876

  9. Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls.

    PubMed

    Reis, L S L S; Ramos, A A; Camargos, A S; Oba, E

    2014-11-10

    The effect of dietary manganese (Mn(2+)) supplementation on the reproductive performance of Nelore bulls was evaluated by assessment of sperm membrane integrity. Sixty Nelore bulls (Bos taurus indicus) aged 18-20 mo were randomly divided into four groups (n=15) receiving dietary Mn(2+) supplementation at 540, 1300, 3800 and 6300mg/kg (treatments TC, T1300, T3800 and T6300, respectively). The diets were changed for the groups every 70d. Semen samples were obtained 15 and 56d after the diet change, which corresponded to the period of adjustment to the new diet and the time required for a complete spermatogenesis cycle, respectively. Sperm integrity was assessed by detection of: intact (IMe) or damaged (DMe) membranes, intact (IA) or damaged (DA) acrosomes, and high (HM) or low (LM) mitochondrial membrane potentials. Only bulls from the TC treatment showed a significant increase in the production of intact sperm [IMe/IA/LM] and decrease in the production of sperm with damaged acrosome [IMe/DA/LM] or completely damaged sperm [DMe/DA/LM] (P<0.05). The Mn(2+) concentrations in the semen were positively correlated with the incidence of sperm with IMe, DA, and LM and negatively correlated with number of sperm with DMe, IA, and LM. Therefore, dietary Mn(2+) supplementation for Nelore bulls must be limited to 540mg of Mn(2+)/kg given that higher doses are detrimental to the integrity of the plasma and acrosomal sperm membranes.

  10. Composite membrane with integral rim

    DOEpatents

    Routkevitch, Dmitri; Polyakov, Oleg G

    2015-01-27

    Composite membranes that are adapted for separation, purification, filtration, analysis, reaction and sensing. The composite membranes can include a porous support structure having elongate pore channels extending through the support structure. The composite membrane also includes an active layer comprising an active layer material, where the active layer material is completely disposed within the pore channels between the surfaces of the support structure. The active layer is intimately integrated within the support structure, thus enabling great robustness, reliability, resistance to mechanical stress and thermal cycling, and high selectivity. Methods for the fabrication of composite membranes are also provided.

  11. RIM, Munc13, and Rab3A interplay in acrosomal exocytosis

    SciTech Connect

    Bello, Oscar D.; Zanetti, M. Natalia; Mayorga, Luis S.; Michaut, Marcela A.

    2012-03-10

    Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking. -- Highlights: Black-Right-Pointing-Pointer RIM and Munc13 are present in human sperm and localize to the acrosomal region. Black-Right-Pointing-Pointer RIM and Munc13 are necessary for acrosomal exocytosis. Black-Right-Pointing-Pointer RIM and Munc13 participate before the acrosomal calcium efflux. Black

  12. Expression of a P-selectin ligand in zona pellucida of porcine oocytes and P-selectin on acrosomal membrane of porcine sperm cells. Potential implications for their involvement in sperm-egg interactions.

    PubMed

    Geng, J G; Raub, T J; Baker, C A; Sawada, G A; Ma, L; Elhammer, A P

    1997-05-05

    The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor-globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.

  13. Effect of divalent ions in acrosome reaction induced by glycosamineglycans in porcine spermatozoa.

    PubMed

    Delgado, N M; Carranco, A; Merchant, H; Reyes, R

    1985-01-01

    Magnesium, calcium, and zinc at the concentration of 10 microM are capable of inducing a "true" acrosome reaction in the pig spermatozoa judged by the criteria of the fusion of the acrosome and the plasmatic membrane at the anterior region or the sperm nucleus. The optimal percent of acrosome reaction reached by any of the ions tested as a whole was 50%. When glycosamineglycan sulfate (GAGs) plus 10 microM of Mg++, Ca++, or Zn++ was added, they reach to 70-80% of acrosome reaction. At the electrom microscope, thin sections taken from pig spermatozoa treated with ions, GAGs, or ion + GAGs under optimal experimental conditions revealed the same pattern of acrosomal reaction. Results suggest the important role that divalent cations play in general in the induction of the acrosome reaction and question the so-called essential role of calcium ions.

  14. Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein.

    PubMed

    Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

    2016-03-01

    Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome

  15. Identification of Bovine Sperm Acrosomal Proteins that Interact with a 32kDa Acrosomal Matrix Protein

    PubMed Central

    Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

    2016-01-01

    Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction is comprised of three major (54, 50, and 45kDa) and several minor (38–19kDa) polypeptides. The set of minor polypeptides (38–19kDa) termed “OMCrpf polypeptides” is selectively solubilized by high-pH extraction (pH 10.5) while the three major polypeptides (55, 50 and 45kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38–19kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment; whereas, IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidyl choline (LPC

  16. Membrane stiffness is modified by integral membrane proteins.

    PubMed

    Fowler, Philip W; Hélie, Jean; Duncan, Anna; Chavent, Matthieu; Koldsø, Heidi; Sansom, Mark S P

    2016-09-20

    The ease with which a cell membrane can bend and deform is important for a wide range of biological functions. Peripheral proteins that induce curvature in membranes (e.g. BAR domains) have been studied for a number of years. Little is known, however, about the effect of integral membrane proteins on the stiffness of a membrane (characterised by the bending rigidity, Kc). We demonstrate by computer simulation that adding integral membrane proteins at physiological densities alters the stiffness of the membrane. First we establish that the coarse-grained MARTINI forcefield is able to accurately reproduce the bending rigidity of a small patch of 1500 phosphatidyl choline lipids by comparing the calculated value to both experiment and an atomistic simulation of the same system. This enables us to simulate the dynamics of large (ca. 50 000 lipids) patches of membrane using the MARTINI coarse-grained description. We find that altering the lipid composition changes the bending rigidity. Adding integral membrane proteins to lipid bilayers also changes the bending rigidity, whilst adding a simple peripheral membrane protein has no effect. Our results suggest that integral membrane proteins can have different effects, and in the case of the bacterial outer membrane protein, BtuB, the greater the density of protein, the larger the reduction in stiffness.

  17. Proteomic analysis of integral plasma membrane proteins.

    PubMed

    Zhao, Yingxin; Zhang, Wei; Kho, Yoonjung; Zhao, Yingming

    2004-04-01

    Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 microg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.

  18. Effect of progesterone on acrosome reaction, hypoosmotic swelling test, and DNA stability in human spermatozoa.

    PubMed

    Contreras, H R; Roa, J; Ramirez, M A

    1999-01-01

    The association between different sperm parameters, an in vitro effect of progesterone, has not been studied satisfactorily. In this article, the effect of progesterone on acrosome reaction (AR), plasma membrane integrity, and chromatin stability has been assessed in human spermatozoa with normal morphology and motility. Semen samples were obtained by masturbation from 25 patients. Two criteria of classification were utilized in this study: high motility group and normal morphology group incubated with progesterone. The effect of progesterone on AR, plasma membrane integrity, and chromatin stability in human spermatozoa with normal morphology and motility was realized. The results suggest that only the subpopulation of spermatozoa with normal morphology is able to undergo the progesterone-induced AR. It is possible that in the reproductive female tract it takes place a high selection of sperm with chromatin stability determined and optimal plasma membrane to undergo the AR prerequisite for the fecundation.

  19. Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates.

    PubMed

    Serafini, R; Longobardi, V; Spadetta, M; Neri, D; Ariota, B; Gasparrini, B; Di Palo, R

    2014-02-01

    Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June-July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates.

  20. Integrable structure in discrete shell membrane theory

    PubMed Central

    Schief, W. K.

    2014-01-01

    We present natural discrete analogues of two integrable classes of shell membranes. By construction, these discrete shell membranes are in equilibrium with respect to suitably chosen internal stresses and external forces. The integrability of the underlying equilibrium equations is proved by relating the geometry of the discrete shell membranes to discrete O surface theory. We establish connections with generalized barycentric coordinates and nine-point centres and identify a discrete version of the classical Gauss equation of surface theory. PMID:24808755

  1. Identification and Characterization of a Bovine Sperm Acrosomal Matrix Protein and its Mechanism of Interaction with Acrosomal Hydrolases

    PubMed Central

    Nagdas, Subir K; Smith, Linda; Mcnamara, Allen; Hernandez–Encarnacion, Luisa; Medina-Ortiz, Ilza

    2015-01-01

    Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads is comprised of 54, 50, 45, and 38–19kDa polypeptides. The objective of this study is to identify and to characterize the 45kDa (OMC45) polypeptide and to define its role in binding acrosomal hydrolases and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI–TOF–TOF yielded 8 peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X–100–permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti–OMC45 and anti–TEKT3. The OMC45 polypeptide was solubilized by RIPA (radio-immunoprecipitation assay) buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti–OMC45 immunoprecipitation pellet. An identical blot stained with anti–TEKT3 exhibited the presence of TEKT3 polypeptide in the anti–OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide; that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N–linked and O–linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N–acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid

  2. Quantification of bovine sperm separation by a swim-up method. Relationship to sperm motility, integrity of acrosomes, sperm migration in polyacrylamide gel and fertility.

    PubMed

    Parrish, J J; Foote, R H

    1987-01-01

    The number of bovine spermatozoa separated in a swim-up procedure was quantified using an electronic cell counter. In an initial test of the swim-up procedure, non-frozen sperm samples with different ratios of live to dead cells were prepared and tested for the number of spermatozoa counted by the swim-up procedure. In ejaculates from six bulls, the number of spermatozoa swimming up was related to the number of live cells present (R2 = 0.97). Next, sperm quality of frozen-thawed semen immediately after thawing was measured at 37 C by swim-up sperm count, sperm motility, spermatozoa with an intact acrosome and migration in polyacrylamide gel and then compared with the fertility of the semen used for artificial insemination. Twenty-nine ejaculates of frozen-thawed semen from 11 bulls were evaluated. Correlations with fertility were highest on an ejaculate basis for motility (r = 0.41, P = 0.05) and for swim-up sperm count (r = 0.35, P = 0.06). On a bull basis, swim-up sperm count had the highest correlation with fertility (r = 0.59, P = 0.06). In a multiple regression model to predict male fertility that included all described measures of semen quality, a R2 value of 0.69 was obtained. This is the first report showing that the ability of spermatozoa to swim out of a more dense medium (whole milk-glycerol extender) into culture media is quantitatively related to in vivo fertility.

  3. In vitro capacitation and acrosome reaction in sperm of the phyllostomid bat Artibeus jamaicensis.

    PubMed

    Álvarez-Guerrero, Alma; González-Díaz, Francisco; Medrano, Alfredo; Moreno-Mendoza, Norma

    2016-04-01

    Sperm capacitation occurs during the passage of sperm through the female reproductive tract. Once the sperm binds to the pellucid zone, the acrosome reaction to enable penetration of the oocyte is completed. In this study, sperm of Artibeus jamaicensis bat was used to evaluate both capacitation status and the acrosome reaction under in vitro conditions, incubating sperm at 32 and 37°C with and without progesterone. Sperm was incubated at different times to assess sperm cells' functionality in terms of capacitation and acrosome reaction, using the chlortetracycline staining, lectin fluoresceinisocyanate conjugate-Pisum sativum agglutinin (FITC-PSA), and transmission electron microscopy. Sperm cells that presented uniform fluorescence throughout the head and mid-piece were classified as non-capacitated. Subsequently, sperm cells, which were observed with fluorescence only in the anterior portion of the head and mid-piece, were classified as capacitated. Sperm cells with no fluorescence in the head, but fluorescence in the mid-piece, were categorized as sperm cells that have carried out the acrosome reaction. During the acrosome reaction, sperm cells showed changes in their morphology, so it was not possible to distinguish the plasma and acrosomal membranes. Around the entire head, it was not possible to distinguish the fusion points between these membranes that made it possible for the acrosomal reaction to take place and thus to release the enzymes necessary to penetrate the pellucid zone. In conclusion, under appropriate in vitro conditions and by supplementing the culture medium with progesterone, A. jamaicensis bat sperm cells are able to be capacitated in a period from 6 to 8 h and to carry out the acrosome reaction.

  4. Effect of commercial long-term extenders on metabolic activity and membrane integrity of boar spermatozoa stored at 17 degrees C.

    PubMed

    Dziekońska, A; Fraser, L; Majewska, A; Lecewicz, M; Zasiadczyk, Ł; Kordan, W

    2013-01-01

    This study was aimed to analyze the metabolic activity and membrane integrity of boar spermatozoa following storage in long-term semen extenders. Boar semen was diluted with Androhep EnduraGuard (AeG), DILU-Cell (DC), SafeCell Plus (SCP) and Vitasem LD (VLD) extenders and stored for 10 days at 17 degrees C. Parameters of the analyzed sperm metabolic activity included total motility (TMOT), progressive motility (PMOT), high mitochondrial membrane potential (MMP) and ATP content, whereas those of the membrane integrity included plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome. Extender type was a significant (P < 0.05) source of variation in all the analyzed sperm parameters, except for ATP content. Furthermore, the storage time had a significant effect (P < 0.05) on the sperm metabolic activity and membrane integrity during semen storage. In all extenders the metabolic activity and membrane integrity of the stored spermatozoa decreased continuously over time. Among the four analyzed extenders, AeG and SCP showed the best performance in terms of TMOT and PMI on Days 5, 7 and 10 of storage. Marked differences in the proportions of spermatozoa with high MMP were observed between the extenders, particularly on Day 10 of storage. There were not any marked differences in sperm ATP content between the extenders, regardless of the storage time. Furthermore, the percentage of spermatozoa with NAR acrosomes decreased during prolonged storage, being markedly lower in DC-diluted semen compared with semen diluted with either AeG or SCP extender. The results of this study indicated that components of the long-term extenders have different effects on the sperm functionality and prolonged semen longevity by delaying the processes associated with sperm ageing during liquid storage.

  5. A mechanism for differential release of acrosomal enzymes during the acrosome reaction.

    PubMed Central

    Hardy, D M; Oda, M N; Friend, D S; Huang, T T

    1991-01-01

    To study the organization of fertilization enzymes in the sperm acrosome, we isolated and characterized two physicochemically distinct acrosomal fractions of guinea-pig spermatozoa. A soluble fraction contained the 25,000-Mr acrosomal autoantigen, AA1, and most of the acrosomal hyaluronidase and dipeptidyl peptidase II activity. A particulate fraction, designated acrosomal matrix (AM), consisted of membraneless crescent-shaped structures, and contained most of the acrosomal proacrosin. The AM also contained a 28,000-Mr putative proacrosin-binding protein, and a very-high-Mr component which, on reduction, was dissociated into 48,000-Mr and 67,000-Mr subunits. Autoproteolytic dissolution of the AM correlated with proteolysis by acrosin of the 28,000-Mr and 48,000-Mr AM molecules. Components of both the AM and the soluble fraction were localized by immuno-electron microscopy to the electron-dense region of the guinea-pig sperm acrosome. We conclude that acrosomal molecules are segregated into soluble and matrix compartments. This segregation is a function of disulphide bonding and non-covalent interactions among the relatively few components of the AM. Association of acrosin with the AM may be the mechanism by which this enzyme's release from the spermatozoon during the acrosome reaction is delayed relative to the release of other acrosomal molecules. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:1903927

  6. Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

    PubMed

    Dragileva, E; Rubinstein, S; Breitbart, H

    1999-11-01

    channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.

  7. Integrated Ceramic Membrane System for Hydrogen Production

    SciTech Connect

    Schwartz, Joseph; Lim, Hankwon; Drnevich, Raymond

    2010-08-05

    Phase I was a technoeconomic feasibility study that defined the process scheme for the integrated ceramic membrane system for hydrogen production and determined the plan for Phase II. The hydrogen production system is comprised of an oxygen transport membrane (OTM) and a hydrogen transport membrane (HTM). Two process options were evaluated: 1) Integrated OTM-HTM reactor – in this configuration, the HTM was a ceramic proton conductor operating at temperatures up to 900°C, and 2) Sequential OTM and HTM reactors – in this configuration, the HTM was assumed to be a Pd alloy operating at less than 600°C. The analysis suggested that there are no technical issues related to either system that cannot be managed. The process with the sequential reactors was found to be more efficient, less expensive, and more likely to be commercialized in a shorter time than the single reactor. Therefore, Phase II focused on the sequential reactor system, specifically, the second stage, or the HTM portion. Work on the OTM portion was conducted in a separate program. Phase IIA began in February 2003. Candidate substrate materials and alloys were identified and porous ceramic tubes were produced and coated with Pd. Much effort was made to develop porous substrates with reasonable pore sizes suitable for Pd alloy coating. The second generation of tubes showed some improvement in pore size control, but this was not enough to get a viable membrane. Further improvements were made to the porous ceramic tube manufacturing process. When a support tube was successfully coated, the membrane was tested to determine the hydrogen flux. The results from all these tests were used to update the technoeconomic analysis from Phase I to confirm that the sequential membrane reactor system can potentially be a low-cost hydrogen supply option when using an existing membrane on a larger scale. Phase IIB began in October 2004 and focused on demonstrating an integrated HTM/water gas shift (WGS) reactor to

  8. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  9. High Efficiency Solar Integrated Roof Membrane Product

    SciTech Connect

    Partyka, Eric; Shenoy, Anil

    2013-05-15

    This project was designed to address the Solar Energy Technology Program objective, to develop new methods to integrate photovoltaic (PV) cells or modules within a building-integrated photovoltaic (BIPV) application that will result in lower installed cost as well as higher efficiencies of the encapsulated/embedded PV module. The technology assessment and development focused on the evaluation and identification of manufacturing technologies and equipment capable of producing such low-cost, high-efficiency, flexible BIPV solar cells on single-ply roofing membranes.

  10. Membrane integrity and fertilizing potential of cryopreserved spermatozoa in European mouflon.

    PubMed

    Naitana, S; Ledda, S; Leoni, G; Bogliolo, L; Loi, P; Cappai, P

    1998-08-21

    There is a pressing need to develop and use assisted reproductive techniques in wildlife species living in small and captive groups. We evaluated the effect of freezing on membrane integrity and fertilizing capacity of European mouflon (Ovis gmelini musimon) spermatozoa collected during the breeding season. After thawing, the percentage of live spermatozoa, stained with fluorescein isothiocynate labeled Pisum Sativum agglutinin and propidium iodide, was 47% of which 19% showed intact acrosomal membrane. After culture in TCM 199 + 10% FCS, the number of live spermatozoa was significantly (P < 0.01) lower than in a medium with oviductal epithelial cells. The absence of oviductal cells decreased significantly the fertilization rates (P < 0.05), 24.0 vs. 63.1 with oviductal epithelial cells and 59.1 in vivo of in vitro matured ovine oocytes. Polyspermic fertilization rate of oocytes was lower (P < 0.05) with oviductal epithelial cells (1.6) than in absence of cells (12.8). However, the percentage of embryos that reached blastocyst stage was significantly higher in vivo than in vitro. These results provide interesting preliminary data for the development of genetic resource banks for European mouflon.

  11. Changes in subcellular elemental distributions accompanying the acrosome reaction in sea urchin sperm

    SciTech Connect

    Cantino, M.E.; Schackmann, R.W.; Johnson, D.E.

    1983-05-01

    Energy-dispersive x-ray microanalysis was used to analyze changes in the subcellular distributions of Na, Mg, P, S, Cl, K, and Ca associated with the acrosome reaction of sea urchin sperm. Within 5 sec after induction of the acrosome reaction, nuclear Na and mitochondrial Ca increased and nuclear and mitochondrial K decreased. Uptake of mitochondrial P was detected after several minutes, and increases in nuclear Mg were detected only after 5-10 min of incubation following induction of the reaction. The results suggest that sudden permeability changes in the sperm plasma membrane are associated with the acrosome reaction, but that complete breakdown of membrane and cell function does not occur for several minutes.

  12. Transitional states of acrosomal exocytosis and proteolytic processing of the acrosomal matrix in guinea pig sperm.

    PubMed

    Kim, Kye-Seong; Foster, James A; Kvasnicka, Kevin W; Gerton, George L

    2011-12-01

    In this study, we adapted a FluoSphere bead-binding assay to study the exposure and release of guinea pig sperm acrosomal components during the course of capacitation and acrosomal exocytosis. Prior to capacitation or the initiation of exocytosis, acrosomal proteins were not accessible to FluoSpheres coated with antibodies against two acrosomal matrix (AM) proteins, AM67 and AM50; during the course of capacitation and ionophore-induced acrosomal exocytosis, however, we detected the transient exposure of the solid-phase AM proteins on the surface of guinea pig sperm using the antibody-coated fluorescent beads. Several different transitional stages leading to complete acrosomal exocytosis were classified, and we propose these represent true, functional intermediates since some of the AM proteins are orthologues of mouse proteins that bind the zona pellucida (ZP) of unfertilized eggs. In addition, we present evidence that implicates acrosin in the proteolytic processing of AM50 during AM disassembly. Thus, we propose that the transitional states of acrosomal exocytosis involve early binding of AM proteins to the ZP (by what visually appear to be "acrosome-intact" sperm), maintenance of ZP binding that coincides with the progressive exposure of AM proteins, and gradual proteolytic disassembly of the AM to allow sperm movement through the ZP. We feel this "transitional states" model provides a more refined view of acrosomal function that supports a move away from the widely held, overly simplistic, and binary "acrosome-reaction" model, and embraces a more dynamic view of acrosomal exocytosis that involves intermediate stages of the secretory process in ZP binding and penetration.

  13. Effect of magnetized extender on sperm membrane integrity and development of oocytes in vitro fertilized with liquid storage boar semen.

    PubMed

    Lee, Sang-Hee; Park, Choon-Keun

    2015-03-01

    The objective of this study was to evaluate the effect of a magnetized extender on sperm membrane damage and development of oocytes in vitro fertilized with liquid storage boar semen. Before semen dilution, extender was flowed through a neodymium magnet (0, 2000, 4000 and 6000G) for 5min and collected semen was preserved for 168h at 18°C. In results, plasma membrane integrity with live sperm was significantly higher in semen treated with extenders magnetized at 4000G than sperm treated with extenders magnetized at 0G during semen preservation for 120-168h (p<0.05). In addition, acrosomal membrane damage was significantly lower in semen treated with extenders magnetized at 4000 and 6000G compared to 0 and 2000G during semen preservation for 168h (p<0.05). And mitochondrial membrane damage with all sperm was significantly lower in semen treated with extenders magnetized at 2000G than other groups during semen preservation for 168h. The ability of semen to achieve successful in vitro fertilization was also not significantly different among the groups during preservation. However, when the semen was preserved for 168h, the blastocyst formation rates were significantly higher at 6000G compared to 0 and 2000G (p<0.05). In conclusion, these results suggest that highly magnetized semen extender could protect the sperm membrane from damage, and improve the ability of rates of in vitro blastocyst development and magnetized semen diluter is beneficial for long liquid preservation of boar semen.

  14. Thermoelectric integrated membrane evaporation water recovery technology

    NASA Technical Reports Server (NTRS)

    Roebelen, G. J., Jr.; Winkler, H. E.; Dehner, G. F.

    1982-01-01

    The recently developed Thermoelectric Integrated Membrane Evaporation Subsystem (TIMES) offers a highly competitive approach to water recovery from waste fluids for future on-orbit stations such as the Space Operations Center. Low power, compactness and gravity insensitive operation are featured in this vacuum distillation subsystem that combines a hollow fiber membrane evaporator with a thermoelectric heat pump. The hollow fiber elements provide positive liquid/gas phase control with no moving parts other than pumps and an accumulator, thus solving problems inherent in other reclamation subsystem designs. In an extensive test program, over 850 hours of operation were accumulated during which time high quality product water was recovered from both urine and wash water at an average steady state production rate of 2.2 pounds per hour.

  15. Thermoelectric Integrated Membrane Evaporation Subsystem operational improvements

    NASA Technical Reports Server (NTRS)

    Dehner, G. F.; Winkler, H. E.; Reysa, R. P.

    1984-01-01

    A three-man preprototype Thermoelectric Integrated Membrane Evaporation Subsystem (TIMES) has been developed to provide high quality water recovery from waste fluids on extended duration space flights. In the most recent effort, a number of improvements have been made to simplify subsystem operation and increase performance. These modifications include changes to the hollow fiber membrane evaporator, the condensing section of the thermoelectric heat pump, and the electronic controller logic and display. This paper describes the results of the test program that was conducted to evaluate the implemented improvements. In addition, an advanced design concept is discussed that will provide lower electrical power consumption, greater water production capacity, lower weight, and a smaller package than the present subsystem configuration.

  16. Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm

    PubMed Central

    1995-01-01

    Calcium flux is required for the mammalian sperm acrosome reaction, an exocytotic event triggered by egg binding, which results in a dramatic rise in sperm intracellular calcium. Calcium-dependent membrane fusion results in the release of enzymes that facilitate sperm penetration through the zona pellucida during fertilization. We have characterized inositol 1,4,5-trisphosphate (IP3)-gated calcium channels and upstream components of the phosphoinositide signaling system in mammalian sperm. Peptide antibodies colocalized G alpha q/11 and the beta 1 isoform of phospholipase C (PLC beta 1) to the anterior acrosomal region of mouse sperm. Western blotting using a polyclonal antibody directed against purified brain IP3 receptor (IP3R) identified a specific 260 kD band in 1% Triton X-100 extracts of rat, hamster, mouse and dog sperm. In each species, IP3R immunostaining localized to the acrosome cap. Scatchard analysis of [3H]IP3 binding to rat sperm sonicates revealed a curvilinear plot with high affinity (Kd = 26 nM, Bmax = 30 pmol/mg) and low affinity (Kd = 1.6 microM, Bmax = 550 pmol/mg) binding sites, reflecting among the highest receptor densities in mammalian tissue. Immunoelectron microscopy confirmed the acrosomal localization in rat sperm. The IP3R fractionated with acrosomes by discontinuous sucrose gradient centrifugation and was enriched in the medium of acrosome- reacted sperm. ATP-dependent 45Ca2+ loading of digitonin permeabilized rat sperm was decreased by 45% in the presence of 10 microM IP3. The IP3-mediated release of calcium was blocked by heparin. Thapsigargin, a sequiterpene lactone inhibitor of the microsomal Ca(2+)-ATPase, stimulated the acrosome reaction of mouse sperm to the same extent as the Ca2+ ionophore, A23187. The failure of caffeine and ryanodine to affect calcium accumulation suggested that thapsigargin acted through an IP3-sensitive store. The presence of G alpha q/11, PLC beta 1 and a functional IP3R in the anterior acrosomal region

  17. Phosphoinositide-Dependent Pathways in Mouse Sperm are Regulated by Egg ZP3 and Drive the Acrosome Reaction

    PubMed Central

    Jungnickel, Melissa K.; Sutton, Keith A.; Wang, Yanli; Florman, Harvey M.

    2007-01-01

    Sperm of many animals must complete an exocytotic event, the acrosome reaction, in order to fuse with eggs. In mammals, acrosome reactions are triggered during sperm contact with the egg extracellular matrix, or zona pellucida, by the matrix glycoprotein ZP3. Here, we show that ZP3 stimulates production of phosphatidylinositol-(3,4,5)-triphosphate in sperm membranes. Phosphatidylinositol-3-kinase antagonists that prevent the production of this phosphoinositide blocked acrosome reactions and fertilization in vitro, while generation of this phosphoinositide in the absence of ZP3 triggered acrosome reactions. Downstream effectors of phosphatidylinositol-(3,4,5)-triphosphate in sperm include the protein kinases, Akt and PKCζ. These studies outline a signal transduction pathway that plays an essential role in the early events of mammalian fertilization. PMID:17258189

  18. Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction

    PubMed Central

    Frolikova, Michaela; Sebkova, Natasa; Ded, Lukas; Dvorakova-Hortova, Katerina

    2016-01-01

    The acrosome reaction (AR) is a process of membrane fusion and lytic enzyme release, which enables sperm to penetrate the egg surroundings. It is widely recognized that specific sperm proteins form an active network prior to fertilization, and their dynamic relocation is crucial for the sperm-egg fusion. The unique presence of the membrane cofactor protein CD46 in the sperm acrosomal membrane was shown, however, its behaviour and connection with other sperm proteins has not been explored further. Using super resolution microscopy, we demonstrated a dynamic CD46 reorganisation over the sperm head during the AR, and its interaction with transmembrane protein integrins, which was confirmed by proximity ligation assay. Furthermore, we propose their joint involvement in actin network rearrangement. Moreover, CD46 and β1 integrins with subunit α3, but not α6, are localized into the apical acrosome and are expected to be involved in signal transduction pathways directing the acrosome stability and essential protein network rearrangements prior to gamete fusion. PMID:27666019

  19. Integrated oxidation membrane filtration process - NOM rejection and membrane fouling.

    PubMed

    Winter, J; Uhl, W; Bérubé, P R

    2016-11-01

    The extent and mechanisms by which organic matter in a solution can be retained and foul a membrane largely depends on the molecular weight of the material being filtered and the molecular weight cut-off (MWCO) of the membrane. The present study investigated the effect of the MWCO of a membrane and the molecular weight distribution of natural organic matter (NOM) in a source water on the increase in resistance to the permeate flux over time. Of particular interest was the effect of oxidation, applied prior to membrane filtration, on the predominant fouling mechanism. Oxidation can change the molecular weight distribution of organic matter in raw water, and therefore the ability of a membrane to retain this organic matter. Oxidation, using both ozonation and UV/H2O2, could effectively reduce the extent of fouling for higher MWCO membranes. However, neither oxidation approaches could effectively reduce the extent of fouling for lower MWCO membranes, likely because oxidation could not effectively oxidize lower molecular weight organic matter. Althoug the data indicated that the extent of fouling is increasing with the amount of DOC retained by the membrane, no statistically significant correlation was observed between these parameters. The results suggest that oxidation did not affect the predominant fouling mechanism. However, it did affect the molecular weight distribution of the organic matter retained by the membranes, and as a result, the resistance offered by the foulant cake layer.

  20. Virus-Mimetic Fusogenic Exosomes for Direct Delivery of Integral Membrane Proteins to Target Cell Membranes.

    PubMed

    Yang, Yoosoo; Hong, Yeonsun; Nam, Gi-Hoon; Chung, Jin Hwa; Koh, Eunee; Kim, In-San

    2017-02-06

    An efficient system for direct delivery of integral membrane proteins is successfully developed using a new biocompatible exosome-based platform. Fusogenic exosomes harboring viral fusogen, vascular stomatitis virus (VSV)-G protein, can fuse with and modify plasma membranes in a process called "membrane editing." This can facilitate the transfer of biologically active membrane proteins into the target cell membranes both in vitro and in vivo.

  1. Localization and characterization of the acrosomal antigen recognized by GB24 on human spermatozoa.

    PubMed

    Fénichel, P; Dohr, G; Grivaux, C; Cervoni, F; Donzeau, M; Hsi, B L

    1990-10-01

    GB24, a mouse monoclonal antibody, recognizes a trophoblast-leukocyte cross-reactive antigen (TLX), which is likely identical to the membrane cofactor protein (MCP), a complement regulatory protein. GB24 reacts also with a human acrosomal sperm antigen (Fénichel et al.: J Reprod Fertil 87:699-706, 1989). By immunofluorescence or immunoperoxidase, testicular, epididymal, and ejaculated spermatozoa were found to be positive after fixation by acetone. Motile, suspended spermatozoa became positive only through conditions known to induce acrosome reaction (A23187, follicular fluid, contact with oocytes). Ultrastructural studies with immunogold staining localized this protein on the inner acrosome membrane and in the acrosomal content. By SDS-polyacrylamide gel electrophoresis, GB24 immunoprecipitated a unique protein of 48 kDa from capacitated and A23187-induced spermatozoa under reducing conditions. No cross-reactivity was found with mouse, boar, or ram spermatozoa. Localization of this human sperm antigen recognized by GB24 and its similarity with the TLX-MCP family antigens would suggest a possible role of this molecule during fertilization in sperm-egg binding or immune protection.

  2. Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa.

    PubMed

    Nandi, Pinki; Ghosh, Swatilekha; Jana, Kuladip; Sen, Parimal C

    2012-01-01

    Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.

  3. USP8/UBPy-regulated sorting and the development of sperm acrosome: the recruitment of MET.

    PubMed

    Berruti, Giovanna; Paiardi, Chiara

    2015-06-01

    The acrosome is a peculiar vacuole that at fertilization undergoes the acrosome reaction (AR), an event unique in the sperm life. Contents released promote sperm penetration through oocyte's investments; membranous components are involved in sperm-egg interaction/fusion. Therefore, both constituents play a role in fertilization. The biogenesis of this vacuole, however, has not been clarified yet; recently, it has been proposed as a novel lysosome-related organelle (LRO). Our research focuses on the involvement of the endosomal pathway in acrosomogenesis starting from the early phases. The trafficking sorted by USP8/UBPy, an endosomal regulator recently described as a compelling candidate for male fertility gene, was investigated in comparison to that of SP56, a marker of the biosynthetic pathway. Mouse spermatids were double/triple immunolabeled and examined by confocal microscopy. The contribution of the vesicular traffic assisted by the cortical microtubule array was also evaluated in nocodazole-treated spermatids. USP8/UBPy-sorted cargo contributes early to acrosomogenesis and its trafficking is microtubule mediated. It was identified, through co-immunoprecipitation/co-immunolocalization assays, that the membrane receptor MET, described herein for the first time in spermatids, as an USP8/UBPy-target substrate is delivered to the acrosome. MET and USP8/UBPy still colocalize in epididymal spermatozoa. Following the AR, MET and USP8/UBPy show a distinct fate. MET, in particular, translocates at the PAS, the post acrosomal segment known to harbor sperm-borne factors involved in oocyte activation. Overall, our results support the concept of the acrosome as a LRO and provide evidence for the identification of MET as a tyrosine kinase receptor that may play a role in fertilization.

  4. Differential release of guinea pig sperm acrosomal components during exocytosis.

    PubMed

    Kim, K S; Foster, J A; Gerton, G L

    2001-01-01

    The contents of the sperm acrosome are compartmentalized at the biochemical and morphological levels. Biochemically, the acrosome can be considered to be comprised of two compartments: one consisting of readily soluble proteins and one containing a particulate acrosomal matrix. To test the hypothesis that compartmentalization affects the release of acrosomal components during the course of secretion in guinea pig sperm, we examined the relationship between the presence of specific proteins and acrosomal status and monitored the recovery of acrosomal constituents in the medium surrounding sperm induced to undergo exocytosis with the ionophore A23187. Cysteine-rich secretory protein 2 (CRISP-2), a soluble component of the acrosome, was rapidly lost from the acrosome soon after ionophore treatment. However, acrosomal matrix components remained associated with the sperm for longer periods. AM67, a matrix component and the guinea pig orthologue of the mouse sperm zona pellucida-binding protein sp56, was released at a slower rate than was CRISP-2 but at a faster rate than were two other matrix proteins, AM50 and proacrosin. Coincident with their release from the sperm, AM50 and proacrosin were posttranslationally modified, probably by proteolysis. The release of proacrosin from the matrix appears associated with the conversion of this protein to the enzymatically active acrosin protease. These results provide strong support for the hypothesis that compartmentalization plays a significant role in regulating the release of proteins during the course of acrosomal exocytosis. Acrosomal matrix proteins remain associated with the sperm for prolonged periods of time following the induction of acrosomal exocytosis, suggesting that transitional acrosomal intermediates may have significant functions in the fertilization process.

  5. Purification of basolateral integral membrane proteins by cationic colloidal silica-based apical membrane subtraction.

    PubMed

    Goode, Robert J A; Simpson, Richard J

    2009-01-01

    Epithelial cell polarity mediates many essential biological functions and perturbation of the apical/basolateral divide is a hallmark of epithelial to mesenchymal transition in carcinoma. Therefore, correct targeting of proteins to the apical and basolateral surfaces is essential to proper epithelial cell function. However, proteomic characterisation of apical/basolateral sorting has been largely ignored, due to ineffectual separation techniques and contamination of plasma-membrane preparations with housekeeping proteins. Here we describe a method that strips the apical membrane from the adherent cells and releases the intracellular contents, thereby leaving the basolateral membrane available for stringent washes and collection. Analysis of the basolateral membrane of an adherent colon adenocarcinoma cell line resulted in 66% of identified proteins being integral membrane proteins, which possessed either a transmembrane domain or lipid modification, including 35 CD antigens. Based on the abundance of peptides from basolateral marker proteins, this method efficiently captures basolateral integral membrane proteins, with minimal contamination from other membranes and basic proteins.

  6. Effect of NGF on the motility and acrosome reaction of golden hamster spermatozoa in vitro.

    PubMed

    Jin, WanZhu; Tanaka, Akane; Watanabe, Gen; Matsuda, Hiroshi; Taya, Kazuyoshi

    2010-08-01

    Motility and fertilizing ability are known to be two important physiological attributes of a mature sperm, yet the mechanism by which spermatozoa mature and become motile remains largely unknown. It has been shown that nerve growth factor (NGF) is a protein essential for the development, maintenance and survival of the peripheral and central nervous systems. However, the presence of high levels of NGF protein and mRNA do not correlate with the innervations by NGF sensitive fibers in tissues such as the testis, prostate and seminal vesicles. These observations have shifted the attention of research to the role of NGF outside of the nervous system. Here, we demonstrate that NGF and its receptors TrkA and p75 are widely expressed in the testis, accessory reproductive organ, and the epididymal sperms. We also show that NGF stimulates two important aspects of sperm functions, motility and the acrosome reaction, in a time- and dose-dependent manner. NGF activated the sperm cell acrosome reaction, while addition of inhibitors specific for MAPK kinase significantly blocked the sperm acrosome reaction. Taken together, our findings suggest that NGF plays an integral role in sperm motility and the acrosome reaction through, at least in part, the MAPK signalling pathway.

  7. Dynamics of Sun5 Localization during Spermatogenesis in Wild Type and Dpy19l2 Knock-Out Mice Indicates That Sun5 Is Not Involved in Acrosome Attachment to the Nuclear Envelope

    PubMed Central

    Yassine, Sandra; Escoffier, Jessica; Nahed, Roland Abi; Pierre, Virginie; Karaouzene, Thomas; Ray, Pierre F.; Arnoult, Christophe

    2015-01-01

    The acrosome is an organelle that is central to sperm physiology and a defective acrosome biogenesis leads to globozoospermia, a severe male infertility. The identification of the actors involved in acrosome biogenesis is therefore particularly important to decipher the molecular pathogeny of globozoospermia. We recently showed that a defect in the DPY19L2 gene is present in more than 70% of globozoospermic men and demonstrated that Dpy19l2, located in the inner nuclear membrane, is the first protein involved in the attachment of the acrosome to the nuclear envelope (NE). SUN proteins serve to link the nuclear envelope to the cytoskeleton and are therefore good candidates to participate in acrosome-nucleus attachment, potentially by interacting with DPY19L2. In order to characterize new actors of acrosomal attachment, we focused on Sun5 (also called Spag4l), which is highly expressed in male germ cells, and investigated its localization during spermatogenesis. Using immunohistochemistry and Western blot experiments in mice, we showed that Sun5 transits through different cellular compartments during meiosis. In pachytene spermatocytes, it is located in a membranous compartment different to the reticulum. In round spermatids, it progresses to the Golgi and the NE before to be located to the tail/head junction in epididymal sperm. Interestingly, we demonstrate that Sun5 is not, as initially reported, facing the acrosome but is in fact excluded from this zone. Moreover, we show that in Dpy19l2 KO spermatids, upon the detachment of the acrosome, Sun5 relocalizes to the totality of the NE suggesting that the acrosome attachment excludes Sun5 from the NE facing the acrosome. Finally, Western-blot experiments demonstrate that Sun5 is glycosylated. Overall, our work, associated with other publications, strongly suggests that the attachment of the acrosome to the nucleus does not likely depend on the formation of SUN complexes. PMID:25775128

  8. Integrated system for extraction, purification, and digestion of membrane proteins.

    PubMed

    Liu, Yiying; Yan, Guoquan; Gao, Mingxia; Deng, Chunhui; Zhang, Xiangmin

    2016-05-01

    An integrated system was developed for directly processing living cells into peptides of membrane proteins. Living cells were directly injected into the system and cracked in a capillary column by ultrasonic treatment. Owing to hydrophilicity for broken pieces of the cell membrane, the obtained membranes were retained in a well-designed bi-filter. While cytoplasm proteins were eluted from the bi-filter, the membranes were dissolved and protein released by flushing 4% SDS buffer through the bi-filter. The membrane proteins were subsequently transferred into a micro-reactor and covalently bound in the reactor for purification and digestion. As the system greatly simplified the whole pretreatment processes and minimized both sample loss and contamination, it could be used to analyze the membrane proteome samples of thousand-cell-scales with acceptable reliability and stability. We totally identified 1348 proteins from 5000 HepG2 cells, 615 of which were annotated as membrane proteins. In contrast, with conventional method, only 233 membrane proteins were identified. It is adequately demonstrated that the integrated system shows promising practicability for the membrane proteome analysis of small amount of cells.

  9. Bilayer-thickness-mediated interactions between integral membrane proteins.

    PubMed

    Kahraman, Osman; Koch, Peter D; Klug, William S; Haselwandter, Christoph A

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  10. The effect of season on spermatozoa motility, plasma membrane and acrosome integrity in fresh and frozen-thawed semen from Xinong Saanen bucks.

    PubMed

    Wang, W; Luo, J; Sun, S; Xi, L; Gao, Q; Haile, A B; Shi, H; Zhang, W; Shi, H

    2015-02-01

    The aim of this study was to evaluate whether the season of ejaculate collection influences seminal quality parameters of pre- and post-freeze-thawing in Xinong Saanen bucks. Ejaculates were collected from eight bucks throughout the four seasons (spring, summer, autumn and winter) in a 12 months' time period, identified in the Northern Hemisphere. Semen samples were evaluated by the combinations of conventional and Computer-Assisted Sperm Analysis (CASA) when fresh and after frozen-thawed, respectively. The results clearly demonstrated that season of ejaculate collection influenced (p < 0.05) fresh semen quality. Highest semen quality was observed during autumn. On the contrary, undesirable indices (significantly lower, p < 0.05) were observed in winter as compared with the other remaining seasons. CASA has clearly shown the influences of seasonal variations on semen motility parameters. Furthermore, season of ejaculate collection was also found to influence sperm freezability. Semen characteristics after frozen-thawed followed a similar pattern with that of fresh ejaculate except in spring. The results revealed that sperm quality was higher (p < 0.01) in summer and autumn than in spring and winter. In conclusion, seasonal variation influences semen quality in Xinong Saanen bucks. In addition to summer and autumn, fresh ejaculates in spring can also be successfully used for AI. Sperm from ejaculates collected during summer and autumn are more suitable for cryopreservation. Hence, it is possible to increase the efficiency of goat breeding by manipulating the seasonal variations of semen quality for immediate AI and/or cryopreservation.

  11. Recovery of real dye bath wastewater using integrated membrane process: considering water recovery, membrane fouling and reuse potential of membranes.

    PubMed

    Balcik-Canbolat, Cigdem; Sengezer, Cisel; Sakar, Hacer; Karagunduz, Ahmet; Keskinler, Bulent

    2016-12-30

    It has been recognized by the whole world that textile industry which produce large amounts of wastewater with strong color and toxic organic compounds is a major problematical industry requiring effective treatment solutions. In this study, reverse osmosis (RO) membranes were tested on biologically treated real dye bath wastewater with and without pretreatment by nanofiltration (NF) membrane to recovery. Also membrane fouling and reuse potential of membranes were investigated by multiple filtrations. Obtained results showed that only NF is not suitable to produce enough quality to reuse the wastewater in a textile industry as process water while RO provide successfully enough permeate quality. The results recommend that integrated NF/RO membrane process is able to reduce membrane fouling and allow long-term operation for real dye bath wastewater.

  12. Removal of seminal plasma enhances membrane stability on fresh and cooled stallion spermatozoa.

    PubMed

    Barrier-Battut, I; Bonnet, C; Giraudo, A; Dubois, C; Caillaud, M; Vidament, M

    2013-02-01

    Fertility is reduced after semen cooling for a considerable number of stallions. The main hypotheses include alterations in plasma membrane following cooling and deleterious influence of seminal plasma. However, interindividual variability is controversial. We hypothesized that the removal of seminal plasma could enhance motility in some 'poor cooler' stallions, but could also affect, negatively or positively, membrane quality in some stallions. This study examined the effect of centrifugation, followed or not by removal of seminal plasma, on parameters indicating semen quality after 48 h at 4 °C: motility, plasma membrane integrity as evaluated by hypo-osmotic swelling test, acrosome integrity and response to a pharmacological induction of acrosome reaction using ionophore A23187. Sixty-six ejaculates from 14 stallions were used, including stallions showing high or low sperm motility after cooled storage. Centrifugation without removal of seminal plasma did not affect sperm parameters. Removal of seminal plasma did not affect motility, but significantly stabilized sperm membranes, as demonstrated by a higher response to the osmotic challenge, and a reduced reactivity of the acrosome. Moreover, for the same semen sample, the response to an induction of acrosome reaction was significantly higher when the induction was performed in the presence of seminal plasma, compared with the induction in the absence of seminal plasma. This was observed both for fresh and cooled semen. When the induction of acrosome reaction with ionophore A23187 is used to evaluate sperm quality, care must therefore be taken to standardize the proportion of seminal plasma between samples. For the 10 stallions serving at least 25 mares, the only variable significantly correlated with fertility was motility. The influence of membrane stabilization regarding fertility requires further investigations.

  13. Molecular basis of sperm capacitation acrosome reaction and interaction with eggs

    SciTech Connect

    Sheikhnejad, G.

    1985-01-01

    A phospholipase C (PLC) which can hydrolyze /sup 14/C-phosphatidylcholine was purified from bull seminal plasma. This PLC has an optimum at pH 7.2 and its PI was about 5.0. The enzyme was inhibited by EDTA, Cd/sup 2 +/, Pb/sup 2 +/, Ni/sup 2 +/, Fe/sup 2 +/, and Zn/sup 2 +/. PLC consists of two subunits one 69,000 and the other 55,000 daltons. The purified PLC was examined for induction of capacitation and acrosome reaction of guinea pig spermatozoa. Sperm were examined for the acrosome reaction 10 min after addition of 3.4 mM Ca/sup 2 +/. Fifty percent of the sperm underwent the acrosome reaction while the control had less than 5% acrosome reacted sperm. The antiserum to the inneracrosomal membrane isolated from sperm was labeled with FITC conjugated goat anti-guinea pig IgG. The conjugated antibody was used to localize sperm antigens. The antigens located on the IAM were only fluoresced when rabbit sperm were treated with methanol and/or MgCl/sub 2/. Therefore anti-IAM antibody did not bind to the sperm plasma membrane. In vivo capacitated rabbit sperm were incubated with anti-IAM antibody (intact IgG and F(ab')/sub 2/ fragments) for 30 min prior to addition of rabbit eggs. After 24 h the eggs were examined for cleavage. The control eggs were fertilized (90%) while the antibody completely inhibited the fertilization of ova in vitro. The eggs incubated with antibody prior to the addition of sperm were still fertilizable. Thus, anti-IAM did not have any noticeable effect on the eggs. It was also shown that antibody inhibited fertilization of zona-free rabbit eggs in vitro as well.

  14. Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa.

    PubMed

    Kútvölgyi, G; Stefler, J; Kovács, A

    2006-01-01

    A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2-4 h Giemsa staining at 25-40 degrees C is recommended for stallion semen.

  15. MALDI tissue profiling of integral membrane proteins from ocular tissues.

    PubMed

    Thibault, Danielle B; Gillam, Christopher J; Grey, Angus C; Han, Jun; Schey, Kevin L

    2008-06-01

    MALDI tissue profiling and imaging have become valuable tools for rapid, direct analysis of tissues to investigate spatial distributions of proteins, potentially leading to an enhanced understanding of the molecular basis of disease. Sample preparation methods developed to date for these techniques produce protein expression profiles from predominantly hydrophilic, soluble proteins. The ability to obtain information about the spatial distribution of integral membrane proteins is critical to more fully understand their role in physiological processes, including transport, adhesion, and signaling. In this article, a sample preparation method for direct tissue profiling of integral membrane proteins is presented. Spatially resolved profiles for the abundant lens membrane proteins aquaporin 0 (AQP0) and MP20, and the retinal membrane protein opsin, were obtained using this method. MALDI tissue profiling results were validated by analysis of dissected tissue prepared by traditional membrane protein processing methods. Furthermore, direct tissue profiling of lens membrane proteins revealed age related post-translational modifications, as well as a novel modification that had not been detected using conventional tissue homogenization methods.

  16. Prostasomes of canine seminal plasma - zinc-binding ability and effects on motility characteristics and plasma membrane integrity of spermatozoa.

    PubMed

    Mogielnicka-Brzozowska, M; Strzeżek, R; Wasilewska, K; Kordan, W

    2015-06-01

    Prostasomes are small lipid membrane-confined vesicles that are involved in various fertilization-related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross-bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc-affinity chromatography on a Chelating Sepharose Fast Flow - Zn(2 +) showed that from 93 to 100% of the prostasome proteins bind zinc ions (P(+) Zn). SDS-PAGE revealed that canine P(+) Zn comprised four protein bands, with low molecular weights (10.2-12 kDa). We have also shown a positive effect of prostasomes (p < 0.05), especially variant B (2% of total seminal plasma protein) on canine sperm motility parameters after 2 h storage at 5°C (TMOT%, 44.75 ± 5.18) and PMOT%, 12.42 ± 1.59) and VAP, VSL, VCL, when compared with Control (TMOT%, 7.30 ± 1.41 and PMOT%, 1.70 ± 0.42). Higher percentage of spermatozoa with intact plasma membrane (SYBR/PI dual staining) and intact acrosome (Giemsa stained), after 2 h storage at 5°C, was showed, in variant A (1.5% of total seminal plasma protein) and B, when compared with Control and variant C (2.5% of total seminal plasma protein). The prostasomes' effect on motility and plasma membrane integrity of canine cold-stored spermatozoa may be related to their ability to bind zinc ions and regulate their availability to the sperm.

  17. Nanocarbon-based membrane filtration integrated with electric field driving for effective membrane fouling mitigation.

    PubMed

    Fan, Xinfei; Zhao, Huimin; Quan, Xie; Liu, Yanming; Chen, Shuo

    2016-01-01

    Membrane filtration provides an effective solution for removing pollutants from water but is limited by serious membrane fouling. In this work, an effective approach was used to mitigate membrane fouling by integrating membrane filtration with electropolarization using an electroconductive nanocarbon-based membrane. The electropolarized membrane (EM) by alternating square-wave potentials between +1.0 V and -1.0 V with a pulse width of 60 s exhibited a permeate flux 8.1 times as high as that without electropolarization for filtering feed water containing bacteria, which confirms the ability of the EM to achieve biofouling mitigation. Moreover, the permeate flux of EM was 1.5 times as high as that without electropolarization when filtrating natural organic matter (NOM) from water, and demonstrated good performance in organic fouling mitigation with EM. Furthermore, the EM was also effective for complex fouling mitigation in filtering water containing coexisting bacteria and NOM, and presented an increased flux rate 1.9 times as high as that without electropolarization. The superior fouling mitigation performance of EM was attributed to the synergistic effects of electrostatic repulsion, electrochemical oxidation and electrokinetic behaviors. This work opens an effective avenue for membrane fouling mitigation of water-treatment membrane filtration systems.

  18. Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.

    PubMed

    Yi, Y-J; Zimmerman, S W; Manandhar, G; Odhiambo, J F; Kennedy, C; Jonáková, V; Maňásková-Postlerová, P; Sutovsky, M; Park, C-S; Sutovsky, P

    2012-04-01

    Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation

  19. Membranes for the Sulfur-Iodine Integrated Laboratory Scale Demonstration

    SciTech Connect

    Frederick F. Stewart

    2007-08-01

    INL has developed polymeric membrane-based chemical separations to enable the thermochemical production of hydrogen. Major activities included studies of sulfuric acid concentration membranes, hydriodic acid concentration membranes, SO2/O2 separation membranes, potential applications of a catalyst reactor system for the decomposition of HI, and evaluation of the chemical separation needs for alternate thermochemical cycles. Membranes for the concentration of sulfuric acid were studied using pervaporation. The goal of this task was to offer the sulfur-iodine (S-I) and the hybrid sulfur (HyS) cycles a method to concentrate the sulfuric acid containing effluent from the decomposer without boiling. In this work, sulfuric acid decomposer effluent needs to be concentrated from ~50 % acid to 80 %. This task continued FY 2006 efforts to characterize water selective membranes for use in sulfuric acid concentration. In FY 2007, experiments were conducted to provide specific information, including transmembrane fluxes, separation factors, and membrane durability, necessary for proper decision making on the potential inclusion of this process into the S-I or HyS Integrated Laboratory Scale demonstration.

  20. Sea urchin sperm antigens mediating the acrosome reaction

    SciTech Connect

    Trimmer, J.S.

    1987-01-01

    The study of sea urchin sperm antigens mediating the acrosome reactions (AR) has been undertaken. Monoclonal antibodies (mAbs) have been isolated reacting with a number of sperm surface antigens. These mAbs have been used in functional assays to attempt to infer the roles of these proteins in the induction of the AR. These mAbs have also been used to isolate protein for biochemical characterization and reconstitution studies. mAbs reacting with a 210 kD protein of the sea urchin sperm plasma membrane have been used to identify this protein as playing a role in the regulation of ion fluxes during the induction of the AR. mAbs reacting with certain extracellular regions inhibit the induction of: the AR, the long duration {sup 45}Ca{sup 2+} uptake into the mitochondrion, and H{sup +} efflux. Addition of these same mAbs, however, induces an increase in sperm (Ca{sup 2+}){sub i} to levels much higher than those induced by FSG, as monitored by the fluorescent Ca{sup 2+} indicators fura 2 and indo 1. This (Ca{sup 2+}){sub i} increase occurs without an increase in pH{sub i}, and thus allows for the first time the analysis of the effects of increasing sperm (Ca{sup 2+}){sub i} ion the absence of increased pH{sub i}.

  1. Changes in the distribution and molecular mass of boar sperm acrosome-associated 1 proteins during the acrosome reaction; their validity as indicators for occurrence of the true acrosome reaction.

    PubMed

    Ogura, Yukari; Takagishi, Yuki; Harayama, Hiroshi

    2016-09-01

    The aims of this study were to investigate changes in the distribution and molecular mass of boar sperm acrosome-associated 1 (SPACA1) proteins during the acrosome reaction and to discuss validity of SPACA1 proteins as indicators for occurrence of the true acrosome reaction. Boar ejaculated spermatozoa were used for induction of the extracellular Ca(2+)-dependent acrosome reaction (true acrosome reaction) or acrosomal damages (false acrosome reaction) and then subjected to double staining with the anti-SPACA1 protein antibody and FITC-PNA and Western blotting. Extracellular Ca(2+)-dependently acrosome-reacted spermatozoa were characterized by appearance of SPACA1 proteins in the postacrosomal region (; these spermatozoa were classified into SP-3&AR pattern of double staining). However, SPACA1 proteins were not observed in the postacrosomal region of frozen-thawed spermatozoa with severely damaged acrosomes (; these spermatozoa were classified into SP-2&AR pattern). Moreover, the spermatozoa in which acrosomes were severely damaged by incubation with cyclodextrins and without CaCl2 were classified into either SP-2&AR or SP-3&AR pattern. Although SPACA1 proteins were detected mainly as 36-42kDa proteins in the spermatozoa with intact acrosomes, small types of SPACA1 proteins (15-28kDa) increased in extracellular Ca(2+)-dependently acrosome-reacted spermatozoa as well as frozen-thawed spermatozoa with damaged acrosomes. These results show the increase of boar spermatozoa classified into SP-3&AR pattern after incubation in the medium with CaCl2 and without cyclodextrins indicates occurrence of the true acrosome reaction. Moreover, we suggest the increase of small types of SPACA1 proteins is a valid indicator for occurrence of the acrosomal disintegration arising from the true and false acrosome reactions.

  2. Direct ultrafiltration performance and membrane integrity monitoring by microbiological analysis.

    PubMed

    Ferrer, O; Casas, S; Galvañ, C; Lucena, F; Bosch, A; Galofré, B; Mesa, J; Jofre, J; Bernat, X

    2015-10-15

    The feasibility of substituting a conventional pre-treatment, consisting of dioxi-chlorination, coagulation/flocculation, settling and sand filtration, of a drinking water treatment plant (DWTP) by direct ultrafiltration (UF) has been assessed from a microbiological standpoint. Bacterial indicators, viral indicators and human viruses have been monitored in raw river, ultrafiltered and conventionally pre-treated water samples during two years. Direct UF has proven to remove bacterial indicators quite efficiently and to a greater extent than the conventional process does. Nevertheless, the removal of small viruses such as some small bacteriophages and human viruses (e.g. enteroviruses and noroviruses) is lower than the current conventional pre-treatment. Membrane integrity has been assessed during two years by means of tailored tests based on bacteriophages with different properties (MS-2, GA and PDR-1) and bacterial spores (Bacillus spores). Membrane integrity has not been compromised despite the challenging conditions faced by directly treating raw river water. Bacteriophage PDR-1 appears as a suitable microbe to test membrane integrity, as its size is slightly larger than the considered membrane pore size. However, its implementation at full scale plant is still challenging due to difficulties in obtaining enough phages for its seeding.

  3. In vitro toxicity of mercuric chloride on rabbit spermatozoa motility and cell membrane integrity.

    PubMed

    Slivkova, Jana; Massanyi, Peter; Pizzi, Flavia; Trandzik, Jozef; Roychoudhury, Shubhadeep; Lukac, Norbert; Dankova, Marianna; Almasiova, Viera

    2010-01-01

    In this in vitro study the effects of mercuric chloride on the motility and structural integrity of rabbit spermatozoa were investigated. The spermatozoa motility was evaluated using CASA method and Annexin analysis was used for detection of structural changes. The concentration of mercury in the medium varied from 5.0 to 83.3 microg HgCl(2)/mL. At Time 0 the highest motility was detected in the control group (67.09 +/- 8.72%). Motility in groups with mercury administration was lower in comparison with control. Significant differences were detected in groups with 50.0-83.3 microg HgCl(2)/mL (P < 0.001) at Time 0. After 60 and 120 minutes of incubation with mercuric chloride the motility significantly decreased almost in all experimental groups. Progressive motility had a decreasing trend in all experimental groups. At time 60 and 120 significant differences were noted in the group receiving 6.25-83.3 microg HgCl(2)/mL. Significant differences were detected in all experimental groups, except the group with the lowest mercuric chloride administration. The concentration-dependent decrease of spermatozoa progressive motility up to 50% of control was detected for groups receiving 50.0 - 83.3 microg HgCl(2)/mL at Time 0, for groups receiving 12.5-83.3 microg HgCl(2)/mL at Time 60 and 120, decreasing from 36.46 +/- 18.73% to 1.03 +/- 2.50%. Detailed evaluation of spermatozoa distance (DAP, DCL, and DSL) and velocity (VAP, VCL, and VSL) parameters as well as straightness (STR), linearity (LIN), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) of spermatozoa revealed decrease in groups with the highest mercury concentration in comparison with the control group at all time periods. Detection of spermatozoa with disordered membrane was carried out for groups with higher mercury concentrations and control, using Annexin analysis. Analysis showed higher occurrence of positive spermatozoa in the mercury exposed groups. Some Annexin

  4. Comparison of methods for assessing integrity of equine sperm membranes.

    PubMed

    Foster, M L; Love, C C; Varner, D D; Brinsko, S P; Hinrichs, K; Teague, S; Lacaze, K; Blanchard, T L

    2011-07-15

    Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50-79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = -3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = -11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes.

  5. Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function.

    PubMed

    Johnston, S D; Satake, N; Zee, Y; López-Fernández, C; Holt, W V; Gosálvez, J

    2012-06-01

    This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic

  6. Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. II. Ultrastructural changes associated with inhibition of the acrosome reaction.

    PubMed

    Chang, M C; Schuel, H

    1991-05-01

    Pretreatment of Strongylocentrotus purpuratus sperm with delta 9-tetrahydrocannabinol (THC) prevents the triggering of the acrosome reaction by egg jelly. Examination of THC-treated sperm by transmission electron microscopy reveals that the membrane fusion reaction between the sperm plasma membrane and the acrosomal membrane is completely blocked. Electron-dense deposits are present in the subacrosomal fossa and in the centriolar fossa. The nuclear envelope is fragmented in close proximity to the electron-dense deposits. The electron-dense deposits are not bound by a limiting membrane, stain positively for lipid with thymol and farnesol, and disappear from THC-treated sperm that are extracted with chloroform:methanol (2:1) after glutaraldehyde fixation. The electron-dense deposits are lipid in nature and may be a hydrolytic product of the nuclear envelope. Electron-dense deposits are seen in sperm after 1-10 min treatment with 5-100 microM THC. The electron-dense deposits disappear after removal of THC from the sperm by washing, but the fragmented nuclear envelope in the subacrosomal fossa persists. Cannabidiol (CBD) and cannabinol (CBN) also inhibit the triggering of the acrosome reaction by egg jelly and produce ultrastructural changes in the sperm identical to those elicited by THC. Enhanced phospholipase activity stimulated by THC, CBD, and CBN may be the cause of the accumulation of lipid deposits in the sperm. Metabolites derived from this modification of membrane phospholipids may prevent triggering of the acrosome reaction by egg jelly and thereby inhibit fertilization.

  7. Integral membrane protein interaction with Triton cytoskeletons of erythrocytes.

    PubMed

    Sheetz, M P

    1979-10-19

    The organization of erythrocyte membrane lipids and proteins has been studied following the release of cytoplasmic components with the non-ionic detergent Triton X-100. After detergent extraction, a detergent-resistant complex called the erythrocyte cytoskeleton is separated from detergent, solubilized lipid and protein by sucrose buoyant density sedimentation. In cytoskeletons prepared under isotonic conditions all of the major erythrocyte membrane proteins are retained except for the integral protein, glycophorin, which is quantitatively solubilized and another integral glycoprotein, band 3, which is only 60% removed. When cytoskeletons are prepared in hypertonic KCl solutions, band 3 is fully solubilized along with bands 2.1 and 4.2 and several minor components. The resulting cytoskeletons have the same morphology as those prepared in isotonic buffer but they are composed of only three major peripheral proteins, spectrin, actin and band 4.1. We have designated this peripheral protein complex the 'shell' of the erythrocyte membrane, and have shown that the attachment of band 3 to the shell satisfies the criteria for a specific interaction. Although Triton did affect erythrocyte shape, cytoskeleton lipid content and the activity of membrane proteases, there was no indication that Triton altered the attachment of band 3 to the shell. We suggest that band 3 attaches to the shell as part of a ternary complex of bands 2.1, 3 and 4.2.

  8. Antimonide-based membranes synthesis integration and strain engineering.

    PubMed

    Zamiri, Marziyeh; Anwar, Farhana; Klein, Brianna A; Rasoulof, Amin; Dawson, Noel M; Schuler-Sandy, Ted; Deneke, Christoph F; Ferreira, Sukarno O; Cavallo, Francesca; Krishna, Sanjay

    2017-01-03

    Antimonide compounds are fabricated in membrane form to enable materials combinations that cannot be obtained by direct growth and to support strain fields that are not possible in the bulk. InAs/(InAs,Ga)Sb type II superlattices (T2SLs) with different in-plane geometries are transferred from a GaSb substrate to a variety of hosts, including Si, polydimethylsiloxane, and metal-coated substrates. Electron microscopy shows structural integrity of transferred membranes with thickness of 100 nm to 2.5 [Formula: see text]m and lateral sizes from [Formula: see text]m(2) to [Formula: see text] cm(2) Electron microscopy reveals the excellent quality of the membrane interface with the new host. The crystalline structure of the T2SL is not altered by the fabrication process, and a minimal elastic relaxation occurs during the release step, as demonstrated by X-ray diffraction and mechanical modeling. A method to locally strain-engineer antimonide-based membranes is theoretically illustrated. Continuum elasticity theory shows that up to [Formula: see text]3.5% compressive strain can be induced in an InSb quantum well through external bending. Photoluminescence spectroscopy and characterization of an IR photodetector based on InAs/GaSb bonded to Si demonstrate the functionality of transferred membranes in the IR range.

  9. Antimonide-based membranes synthesis integration and strain engineering

    PubMed Central

    Anwar, Farhana; Klein, Brianna A.; Rasoulof, Amin; Dawson, Noel M.; Schuler-Sandy, Ted; Deneke, Christoph F.; Ferreira, Sukarno O.; Cavallo, Francesca; Krishna, Sanjay

    2017-01-01

    Antimonide compounds are fabricated in membrane form to enable materials combinations that cannot be obtained by direct growth and to support strain fields that are not possible in the bulk. InAs/(InAs,Ga)Sb type II superlattices (T2SLs) with different in-plane geometries are transferred from a GaSb substrate to a variety of hosts, including Si, polydimethylsiloxane, and metal-coated substrates. Electron microscopy shows structural integrity of transferred membranes with thickness of 100 nm to 2.5 μm and lateral sizes from 24×24μm2 to 1×1 cm2. Electron microscopy reveals the excellent quality of the membrane interface with the new host. The crystalline structure of the T2SL is not altered by the fabrication process, and a minimal elastic relaxation occurs during the release step, as demonstrated by X-ray diffraction and mechanical modeling. A method to locally strain-engineer antimonide-based membranes is theoretically illustrated. Continuum elasticity theory shows that up to ∼3.5% compressive strain can be induced in an InSb quantum well through external bending. Photoluminescence spectroscopy and characterization of an IR photodetector based on InAs/GaSb bonded to Si demonstrate the functionality of transferred membranes in the IR range. PMID:27986953

  10. Association of dystrophin and an integral membrane glycoprotein.

    PubMed

    Campbell, K P; Kahl, S D

    1989-03-16

    Duchenne muscular dystrophy (DMD) is caused by a defective gene found on the X-chromosome. Dystrophin is encoded by the DMD gene and represents about 0.002% of total muscle protein. Immunochemical studies have shown that dystrophin is localized to the sarcolemma in normal muscle but is absent in muscle from DMD patients. Many features of the predicted primary structure of dystrophin are shared with membrane cytoskeletal proteins, but the precise function of dystrophin in muscle is unknown. Here we report the first isolation of dystrophin from digitonin-solubilized skeletal muscle membranes using wheat germ agglutinin (WGA)-Sepharose. We find that dystrophin is not a glycoprotein but binds to WGA-Sepharose because of its tight association with a WGA-binding glycoprotein. The association of dystrophin with this glycoprotein is disrupted by agents that dissociate cytoskeletal proteins from membranes. We conclude that dystrophin is linked to an integral membrane glycoprotein in the sarcolemma. Our results indicate that the function of dystrophin could be to link this glycoprotein to the underlying cytoskeleton and thus help either to preserve membrane stability or to keep the glycoprotein non-uniformly distributed in the sarcolemma.

  11. Conjugated carbon monolayer membranes: methods for synthesis and integration.

    PubMed

    Unarunotai, Sakulsuk; Murata, Yuya; Chialvo, Cesar E; Mason, Nadya; Petrov, Ivan; Nuzzo, Ralph G; Moore, Jeffrey S; Rogers, John A

    2010-03-12

    Monolayer membranes of conjugated carbon represent a class of nanomaterial with demonstrated uses in various areas of electronics, ranging from transparent, flexible, and stretchable thin film conductors, to semiconducting materials in moderate and high-performance field-effect transistors. Although graphene represents the most prominent example, many other more structurally and chemically diverse systems are also of interest. This article provides a review of demonstrated synthetic and integration strategies, and speculates on future directions for the field.

  12. Ethanol fermentation integrated with PDMS composite membrane: An effective process.

    PubMed

    Fu, Chaohui; Cai, Di; Hu, Song; Miao, Qi; Wang, Yong; Qin, Peiyong; Wang, Zheng; Tan, Tianwei

    2016-01-01

    The polydimethylsiloxane (PDMS) membrane, prepared in water phase, was investigated in separation ethanol from model ethanol/water mixture and fermentation-pervaporation integrated process. Results showed that the PDMS membrane could effectively separate ethanol from model solution. When integrated with batch ethanol fermentation, the ethanol productivity was enhanced compared with conventional process. Fed-batch and continuous ethanol fermentation with pervaporation were also performed and studied. 396.2-663.7g/m(2)h and 332.4-548.1g/m(2)h of total flux with separation factor of 8.6-11.7 and 8-11.6, were generated in the fed-batch and continuous fermentation with pervaporation scenario, respectively. At the same time, high titre ethanol production of ∼417.2g/L and ∼446.3g/L were also achieved on the permeate side of membrane in the two scenarios, respectively. The integrated process was environmental friendly and energy saving, and has a promising perspective in long-terms operation.

  13. ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization

    PubMed Central

    1996-01-01

    The sperm acrosome reaction is a Ca(2+)-dependent secretory event required for fertilization. Adhesion to the egg's zona pellucida promotes Ca2+ influx through voltage-sensitive channels, thereby initiating secretion. We used potentiometric fluorescent probes to determine the role of sperm membrane potential in regulating Ca2+ entry. ZP3, the glycoprotein agonist of the zona pellucida, depolarizes sperm membranes by activating a pertussis toxin-insensitive mechanism with the characteristics of a poorly selective cation channel. ZP3 also activates a pertussis toxin-sensitive pathway that produces a transient rise in internal pH. The concerted effects of depolarization and alkalinization open voltage-sensitive Ca2+ channels. These observations suggest that mammalian sperm utilize membrane potential-dependent signal transduction mechanisms and that a depolarization pathway is an upstream transducing element coupling adhesion to secretion during fertilization. PMID:8707844

  14. ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation*

    PubMed Central

    Pelletán, Leonardo E.; Suhaiman, Laila; Vaquer, Cintia C.; Bustos, Matías A.; De Blas, Gerardo A.; Vitale, Nicolas; Mayorga, Luis S.; Belmonte, Silvia A.

    2015-01-01

    Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5′-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization. PMID:25713146

  15. Fine structure of acrosome biogenesis and of mature sperm in the bivalve molluscs Glycymeris sp. (Pteriomorphia) and Eurhomalea rufa (Heterodonta)

    NASA Astrophysics Data System (ADS)

    Guerra, Rosa; Sousa, Mário; Torres, Artur; Oliveira, Elsa; Baldaia, Luis

    2003-03-01

    Proacrosomal vesicles form during the pachytene stage, being synthetized by the Golgi complex in Glycymeris sp., and by both the Golgi and the rough endoplasmic reticulum in Eurhomalea rufa. During early spermiogenesis, a single acrosomal vesicle forms and its apex becomes linked to the plasma membrane while it migrates. In Glycymeris sp., the acrosomal vesicle then turns cap-shaped (1.8 μm) and acquires a complex substructure. In E. rufa, proacrosomal vesicles differentiate their contents while still at the premeiotic stage; as the acrosomal vesicle matures and its contents further differentiate, it elongates and becomes longer than the nucleus (3.2 μm), while the subacrosomal space develops a perforatorium. Before condensation, chromatin turns fibrillar in Glycymeris sp., whereas it acquires a cordonal pattern in E. rufa. Accordingly, the sperm nucleus of Glycymeris sp. is conical and elongated (8.3 μm), and that of E. rufa is short and ovoid (1.1 μm). In the midpiece (Glycymeris sp.: 1.1 μm; E. rufa: 0.8 μm), both species have four mitochondria encircling two linked orthogonal (Glycymeris sp.) or orthogonal and tilted (30-40°; E. rufa) centrioles. In comparison with other Arcoida species, sperm of Glycymeris sp. appear distinct due to the presence of an elongated nucleus, a highly differentiated acrosome, and four instead of five mitochondria. The same occurs with E. rufa regarding other Veneracea species, with the acrosome of the mature sperm strongly resembling that of the recent Mytilinae.

  16. The Zeamine Antibiotics Affect the Integrity of Bacterial Membranes

    PubMed Central

    Masschelein, Joleen; Clauwers, Charlien; Stalmans, Karen; Nuyts, Koen; De Borggraeve, Wim; Briers, Yves; Aertsen, Abram; Michiels, Chris W.

    2014-01-01

    The zeamines (zeamine, zeamine I, and zeamine II) constitute an unusual class of cationic polyamine-polyketide-nonribosomal peptide antibiotics produced by Serratia plymuthica RVH1. They exhibit potent bactericidal activity, killing a broad range of Gram-negative and Gram-positive bacteria, including multidrug-resistant pathogens. Examination of their specific mode of action and molecular target revealed that the zeamines affect the integrity of cell membranes. The zeamines provoke rapid release of carboxyfluorescein from unilamellar vesicles with different phospholipid compositions, demonstrating that they can interact directly with the lipid bilayer in the absence of a specific target. DNA, RNA, fatty acid, and protein biosynthetic processes ceased simultaneously at subinhibitory levels of the antibiotics, presumably as a direct consequence of membrane disruption. The zeamine antibiotics also facilitated the uptake of small molecules, such as 1-N-phenylnaphtylamine, indicating their ability to permeabilize the Gram-negative outer membrane (OM). The valine-linked polyketide moiety present in zeamine and zeamine I was found to increase the efficiency of this process. In contrast, translocation of the large hydrophilic fluorescent peptidoglycan binding protein PBDKZ-GFP was not facilitated, suggesting that the zeamines cause subtle perturbation of the OM rather than drastic alterations or defined pore formation. At zeamine concentrations above those required for growth inhibition, membrane lysis occurred as indicated by time-lapse microscopy. Together, these findings show that the bactericidal activity of the zeamines derives from generalized membrane permeabilization, which likely is initiated by electrostatic interactions with negatively charged membrane components. PMID:25452285

  17. Seipin deficiency increases chromocenter fragmentation and disrupts acrosome formation leading to male infertility

    PubMed Central

    El Zowalaty, A E; Baumann, C; Li, R; Chen, W; De La Fuente, R; Ye, X

    2015-01-01

    midpiece of Bscl2−/− sperm, indicating defective mitochondrial activity that most likely contributed to reduced Bscl2−/− sperm motility. These data demonstrate novel roles of seipin in spermatid chromatin integrity, acrosome formation, and mitochondrial activity. Increased spermatid apoptosis, increased chromocenter fragmentation, defective chromatin condensation, abnormal acrosome formation, and defective mitochondrial activity contributed to decreased sperm production and defective sperm that resulted in Bscl2−/− male infertility. PMID:26181198

  18. Identification of two integral membrane proteins of Plasmodium falciparum.

    PubMed Central

    Smythe, J A; Coppel, R L; Brown, G V; Ramasamy, R; Kemp, D J; Anders, R F

    1988-01-01

    We describe the isolation and cloning of two integral membrane protein antigens of Plasmodium falciparum. The antigens were isolated by Triton X-114 temperature-dependent phase separation, electrophoretically transferred to nitrocellulose, and used to affinity-purify monospecific human antibodies. These antibodies were used to isolate the corresponding cDNA clones from a phage lambda gt11-Amp3 cDNA expression library. Clone Ag512 corresponds to a Mr 55,000 merozoite rhoptry antigen, and clone Ag513 corresponds to a Mr 45,000 merozoite surface antigen. Both proteins can be biosynthetically labeled with [3H]glucosamine and [3H]myristic acid, suggesting that they may be anchored in membranes via a glycosylphosphatidylinositol moiety. Similarities in the C-terminal sequences of the Mr 45,000 merozoite surface antigen and the Trypanosoma brucei variant surface glycoproteins provides further evidence that this antigen has a glycosylphosphatidylinositol anchor. Images PMID:3293051

  19. Fluorescent multiple staining and CASA system to assess boar sperm viability and membranes integrity in short and long-term extenders

    PubMed Central

    Lange-Consiglio, A.; Meucci, A.; Cremonesi, F.

    2013-01-01

    The aim of this study was to assess the effect on boar spermatozoa quality of in vitro storage in short and long-term extenders by fluorescent multiple staining (FMS) and computer assisted semen analyzer (CASA). Fresh ejaculates from three healthy, sexually mature boars were diluted with equal volumes of six short-term or three long-term commercial extenders and stored at 19°C for 6 days (short-term) or 12 days (long-term). The integrity of spermatozoa membranes was analyzed by FMS using propidium iodide, 5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) and fluorescein isothiocyanate-conjugated peanut agglutinin (PNA). The results obtained from this staining were compared with spermatozoa motility assessed by CASA. Our study showed that the number of viable spermatozoa with non-reacted acrosomes and intact mitochondria was positively correlated with the rate of motile spermatozoa (r2>0.9) irrespective of the extender used. In all extenders the number of motile spermatozoa significantly decreased as preservation period increased (P<0.05). FMS test is a potent indicator of sperm motility because it analyses mitochondrial integrity independently from observable alterations in motility. The best performing extenders were BTS for short-term storage and TRI-x-Cell for long-term storage. PMID:26623308

  20. A Link Between Integral Membrane Protein Expression and Simulated Integration Efficiency

    PubMed Central

    Müller, Axel; Tiemann, Katrin; Saladi, Shyam M.; Galimidi, Rachel P.; Zhang, Bin; Clemons, William M.; Miller, Thomas F.

    2016-01-01

    Integral membrane proteins (IMP) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels widely vary and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design. PMID:27524616

  1. Antimonide-based membranes synthesis integration and strain engineering

    NASA Astrophysics Data System (ADS)

    Zamiri, Marziyeh; Anwar, Farhana; Klein, Brianna A.; Rasoulof, Amin; Dawson, Noel M.; Schuler-Sandy, Ted; Deneke, Christoph F.; Ferreira, Sukarno O.; Cavallo, Francesca; Krishna, Sanjay

    2017-01-01

    Antimonide compounds are fabricated in membrane form to enable materials combinations that cannot be obtained by direct growth and to support strain fields that are not possible in the bulk. InAs/(InAs,Ga)Sb type II superlattices (T2SLs) with different in-plane geometries are transferred from a GaSb substrate to a variety of hosts, including Si, polydimethylsiloxane, and metal-coated substrates. Electron microscopy shows structural integrity of transferred membranes with thickness of 100 nm to 2.5 μμm and lateral sizes from 24×24μ24×24μm2 to 1×11×1 cm2. Electron microscopy reveals the excellent quality of the membrane interface with the new host. The crystalline structure of the T2SL is not altered by the fabrication process, and a minimal elastic relaxation occurs during the release step, as demonstrated by X-ray diffraction and mechanical modeling. A method to locally strain-engineer antimonide-based membranes is theoretically illustrated. Continuum elasticity theory shows that up to ˜˜3.5% compressive strain can be induced in an InSb quantum well through external bending. Photoluminescence spectroscopy and characterization of an IR photodetector based on InAs/GaSb bonded to Si demonstrate the functionality of transferred membranes in the IR range.

  2. Effects of photooxidation on membrane integrity in Salix nigra seeds

    PubMed Central

    Roqueiro, Gonzalo; Facorro, Graciela B.; Huarte, Mónica G.; Rubín de Celis, Emilio; García, Fernando; Maldonado, Sara; Maroder, Horacio

    2010-01-01

    Background and Aims Salix nigra seeds are desiccation-tolerant, as are orthodox seeds, although in contrast to other orthodox seeds they lose viability in a few weeks at room temperature. They also differ in that the chloroplasts of the embryo tissues conserve their chlorophyll and endomembranes. The aim of this paper was to investigate the role of chlorophyll in seed deterioration. Methods Seeds were aged at different light intensities and atmospheric conditions. Mean germination time and normal and total germination were evaluated. The formation of free radicals was assessed using electronic spin resonance spectroscopy, and changes in the fatty acid composition from phospholipids, galactolipids and triglycerides using gas–liquid chromatography. Membrane integrity was studied with electronic spin resonance spin probe techniques, electrolyte leakage and transmission electron microscopy. Key Results Light and oxygen played an important role in free-radical generation, causing a decrease in normal germination and an increase in mean germination time. Both indices were associated with a decrease in polyunsaturated fatty acids derived from membrane lipids as phospholipids and galactolipids. The detection of damage in thylakoid membranes and an increase in plasmalemma permeability were consistent with the decrease in both types of lipids. Triglycerides remained unchanged. Light-induced damage began in outermost tissues and spread inwards, decreasing normal germination. Conclusions Salix nigra seeds were very susceptible to photooxidation. The thylakoid membranes appeared to be the first target of the photooxidative process since there were large decreases in galactolipids and both these lipids and the activated chlorophyll are contiguous in the structure of that membrane. Changes in normal germination and mean germination time could be explained by the deteriorative effects of oxidation. PMID:20338949

  3. Integrated approach to characterize fouling on a flat sheet membrane gravity driven submerged membrane bioreactor.

    PubMed

    Fortunato, Luca; Jeong, Sanghyun; Wang, Yiran; Behzad, Ali R; Leiknes, TorOve

    2016-12-01

    Fouling in membrane bioreactors (MBR) is acknowledged to be complex and unclear. An integrated characterization methodology was employed in this study to understand the fouling on a gravity-driven submerged MBR (GD-SMBR). It involved the use of different analytical tools, including optical coherence tomography (OCT), liquid chromatography with organic carbon detection (LC-OCD), total organic carbon (TOC), flow cytometer (FCM), adenosine triphosphate analysis (ATP) and scanning electron microscopy (SEM). The three-dimensional (3D) biomass morphology was acquired in a real-time through non-destructive and in situ OCT scanning of 75% of the total membrane surface directly in the tank. Results showed that the biomass layer was homogeneously distributed on the membrane surface. The amount of biomass was selectively linked with final destructive autopsy techniques. The LC-OCD analysis indicated the abundance of low molecular weight (LMW) organics in the fouling composition. Three different SEM techniques were applied to investigate the detailed fouling morphology on the membrane.

  4. Zn2+-stimulation of sperm capacitation and of the acrosome reaction is mediated by EGFR activation.

    PubMed

    Michailov, Yulia; Ickowicz, Debbi; Breitbart, Haim

    2014-12-15

    Extracellular zinc regulates cell proliferation via the MAP1 kinase pathway in several cell types, and has been shown to act as a signaling molecule. The testis contains a relatively high concentration of Zn(2+), required in both the early and late stages of spermatogenesis. Despite the clinical significance of this ion, its role in mature sperm cells is poorly understood. In this study, we characterized the role of Zn(2+) in sperm capacitation and in the acrosome reaction. Western blot analysis revealed the presence of ZnR of the GPR39 type in sperm cells. We previously demonstrated the presence of active epidermal growth factor receptor (EGFR) in sperm, its possible transactivation by direct activation of G-protein coupled receptor (GPCR), and its involvement in sperm capacitation and in the acrosome reaction (AR). We show here that Zn(2+) activates the EGFR during sperm capacitation, which is mediated by activation of trans-membrane adenylyl cyclase (tmAC), protein kinase A (PKA), and the tyrosine kinase, Src. Moreover, the addition of Zn(2+) to capacitated sperm caused further stimulation of EGFR and phosphatydil-inositol-3-kinase (PI3K) phosphorylation, leading to the AR. The stimulation of the AR by Zn(2+) also occurred in the absence of Ca(2+) in the incubation medium, and required the tmAC, indicating that Zn(2+) activates a GPCR. The AR stimulated by Zn(2+) is mediated by GPR39 receptor, PKA, Src and the EGFR, as well as the EGFR down-stream effectors PI3K, phospholipase C (PLC) and protein kinase C (PKC). These data support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways in sperm capacitation and the acrosome reaction.

  5. Porous Nanocomposites with Integrated Internal Domains: Application to Separation Membranes

    PubMed Central

    Li, Wenle; Walz, John Y.

    2014-01-01

    Asymmetric membranes with layered structure have made significant achievements due to their balanced properties and multi-functionalities that come from a combination of multiple layers. However, issues such as delamination and substructure resistance are generated by the intrinsic layered structure. Here, we present a strategy to integrate the traditional layered structure into an asymmetric but continuous porous network. Through infiltrations of microparticles and nanoparticles to targeted regions, active domains are created inside the porous scaffold versus having them applied externally. The fabricated internal active domains are highly adjustable in terms of its dimensions, pore size, and materials. We demonstrate that it is a general method that can be applicable to a wide variety of particles regardless of their material, dimensions, or geometry. By eliminating the external layered structure, problems such as those mentioned above can be eliminated. This integration technique can be extended to other devices required a layered structure, such as solid oxide fuel cells and lithium ion battery. PMID:24646923

  6. Expression and purification of integral membrane metallopeptidase HtpX.

    PubMed

    Arolas, Joan L; García-Castellanos, Raquel; Goulas, Theodoros; Akiyama, Yoshinori; Gomis-Rüth, F Xavier

    2014-07-01

    Little is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli. Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His8-tag, extracted from the membranes using octyl-β-d-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and size-exclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members.

  7. Hyaluronic acid as capacitation inductor: metabolic changes and membrane-associated adenylate cyclase regulation.

    PubMed

    Fernández, S; Córdoba, M

    2014-12-01

    The aim of this research was to study the effect of hyaluronic acid on bovine cryopreserved spermatozoa compared with heparin as regards the variation of capacitation induction, cellular oxidative metabolism and intracellular signal induced by membrane-associated adenylate cyclase to propose hyaluronic acid as a capacitation inductor. Heparin or hyaluronic acid and lysophosphatidylcholine were used to induce sperm capacitation and acrosome reaction, respectively. 2',5'-dideoxyadenosine was used as a membrane-associated adenylate cyclase inhibitor. The highest percentages of capacitated spermatozoa and live spermatozoa with acrosome integrity were obtained by incubating sperm for 60 min using 1000 μg/ml hyaluronic acid. In these conditions, capacitation induced by hyaluronic acid was lower compared with heparin; nonetheless both glycosaminoglycans promote intracellular changes that allow true acrosome reaction in vitro induced by lysophosphatidylcholine in bovine spermatozoa. Oxygen consumption in heparin-capacitated spermatozoa was significantly higher than in hyaluronic acid-treated spermatozoa. With all treatments, mitochondrial coupling was observed when a specific uncoupler of the respiratory chain was added. The inhibition of membrane-associated adenylate cyclase significantly blocked capacitation induction produced by hyaluronic acid, maintaining a basal sperm oxygen uptake in contrast to heparin effect in which both sperm parameters were inhibited, suggesting that the membrane-associated adenylate cyclase activation is involved in the intracellular signal mechanisms induced by both capacitation inductors, but only regulates mitochondrial oxidative phosphorylation in heparin-capacitated spermatozoa.

  8. Efficient ethanol recovery from yeast fermentation broth with integrated distillation-membrane process

    EPA Science Inventory

    A hybrid process integrating vapor stripping with vapor compression and vapor permeation membrane separation, termed Membrane Assisted Vapor Stripping (MAVS), was evaluated for recovery and dehydration of ethanol from aqueous solution as an alternative to conventional distillatio...

  9. Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability, and acrosome reaction.

    PubMed

    Critser, J K; Huse-Benda, A R; Aaker, D V; Arneson, B W; Ball, G D

    1987-04-01

    Three experiments were conducted to evaluate effects of holding semen at +5.0 degrees C for 30 minutes or -5.0 degrees C for 10 minutes and ice crystal induction (seeding) on frozen-thawed human spermatozoa. In experiment 1, spermatozoa were frozen, and postthaw motility was evaluated immediately (0 hour) and 24 hours later. At both 0 and 24 hours, nonfrozen control samples had higher motility than all other treatment groups. At 0 hour postthaw, motility was higher in samples held at -5.0 degrees C for 10 minutes with no significant effect of seeding. At 24 hours, samples held at -5.0 degrees C for 10 minutes and seeded, but not samples held at -5.0 degrees C and not seeded, had higher motility than samples held at +5.0 degrees C. In experiment 2, semen samples were frozen, and fertilizability was evaluated in a zona-free hamster egg penetration assay. Seeded samples had a higher frequency of sperm penetration than either nonfrozen or nonseeded samples. In experiment 3, nonfrozen controls and frozen treatment groups were evaluated for the frequency of survival and acrosomal integrity. Seeded samples had higher frequencies of survival and loss of acrosomal integrity than nonseeded samples. All frozen-thawed samples had a lower frequency of survival and a higher frequency of loss of acrosomal integrity than nonfrozen controls. Although altered patterns of fertilizability and acrosomal integrity are induced, collectively these data suggest that incorporating a holding temperature of -5.0 degrees C for 10 minutes and seeding may result in a superior protocol for freezing human spermatozoa.

  10. A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation

    PubMed Central

    1991-01-01

    The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)- Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like

  11. Using quantitative interference phase microscopy for sperm acrosome evaluation (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Balberg, Michal; Kalinowski, Ksawery; Levi, Mattan; Shaked, Natan T.

    2016-03-01

    We demonstrate quantitative assessment of sperm cell morphology, primarily acrosomal volume, using quantitative interference phase microscopy (IPM). Normally, the area of the acrosome is assessed using dyes that stain the acrosomal part of the cell. We have imaged fixed individual sperm cells using IPM. Following, the sample was stained and the same cells were imaged using bright field microscopy (BFM). We identified the acrosome using the stained BFM image, and used it to define a quantitative corresponding area in the IPM image and determine a quantitative threshold for evaluating the volume of the acrosome.

  12. Spacesuit Water Membrane Evaporator Integration with the ISS Extravehicular Mobility

    NASA Technical Reports Server (NTRS)

    Margiott, Victoria; Boyle, Robert

    2014-01-01

    NASA has developed a Solid Water Membrane Evaporation (SWME) to provide cooling for the next generation spacesuit. One approach to increasing the TRL of the system is to incorporate this hardware with the existing EMU. Several integration issues were addressed to support a potential demonstration of the SWME with the existing EMU. Systems analysis was performed to assess the capability of the SWME to maintain crewmember cooling and comfort as a replacement for sublimation. The materials of the SWME were reviewed to address compatibility with the EMU. Conceptual system placement and integration with the EMU via an EVA umbilical system to ensure crew mobility and Airlock egress were performed. A concept of operation for EVA use was identified that is compatible with the existing system. This concept is extensible as a means to provide cooling for the existing EMU. The cooling system of one of the EMUs on orbit has degraded, with the root cause undetermined. Should there be a common cause resident on ISS, this integration could provide a means to recover cooling capability for EMUs on orbit.

  13. Development of an integrated membrane process for water reclamation.

    PubMed

    Lew, C H; Hu, J Y; Song, L F; Lee, L Y; Ong, S L; Ng, W J; Seah, H

    2005-01-01

    An integrated membrane process (IMP) comprising a membrane bioreactor (MBR) and a reverse osmosis (RO) process was developed for water reclamation. Wastewater was treated by an MBR operated at a sludge retention time (SRT) of 20 days and a hydraulic retention time (HRT) of 5.5 h. The IMP had an overall recovery efficiency of 80%. A unique feature of the IMP was the recycling of a fraction of RO concentrate back to the MBR. Experimental results revealed that a portion of the slow- and hard-to-degrade organic constituents in the recycle stream could be degraded by an acclimated biomass leading to an improved MBR treatment efficiency. Although recycling concentrated constituents could impose an inhibitory effect on the biomass and suppress their respiratory activities, results obtained suggested that operating MBR (in the novel IMP) at an F/M ratio below 0.03 g TOC/g VSS.day could yield an effluent quality comparable to that achievable without concentrate recycling. It is noted in this study that the novel IMP could achieve an average overall TOC removal efficiency of 88.940% and it consistently produced product water usable for high value reuse applications.

  14. Integration between chemical oxidation and membrane thermophilic biological process.

    PubMed

    Bertanza, G; Collivignarelli, M C; Crotti, B M; Pedrazzani, R

    2010-01-01

    Full scale applications of activated sludge thermophilic aerobic process for treatment of liquid wastes are rare. This experimental work was carried out at a facility, where a thermophilic reactor (1,000 m(3) volume) is operated. In order to improve the global performance of the plant, it was decided to upgrade it, by means of two membrane filtration units (ultrafiltration -UF-, in place of the final sedimentation, and nanofiltration -NF-). Subsequently, the integration with chemical oxidation (O(3) and H(2)O(2)/UV processes) was taken into consideration. Studied solutions dealt with oxidation of both the NF effluents (permeate and concentrate). Based on experimental results and economic evaluation, an algorithm was proposed for defining limits of convenience of this process.

  15. Acrosome reaction in spermatozoa from the amphioxus Branchiostoma belcheri (Cephalochordata, Chordata).

    PubMed

    Morisawa, Sachiko; Mizuta, Takanobu; Kubokawa, Kaoru; Tanaka, Hiroyuki; Morisawa, Masaaki

    2004-11-01

    The formation of an acrosomal process at acrosomal exocytosis in spermatozoa of the amphioxus was described in the present report for the first time. A non-reacted acrosome was located in front of the nucleus, where a cup-shaped acrosomal vesicle covered a conical accumulation of subacrosomal material. When naturally spawned spermatozoa were treated with a calcium ionophore, ionomycin, the acrosomal vesicle opened at the apex and an acrosomal process was projected. The process exhibited a filamentous structure. The reaction followed the mode typically seen in marine invertebrates. These observations suggest that the features and function of the acrosome of amphioxus, whose position is on the border between invertebrates and vertebrates, reflect their ecological adaptation and phylogenic position.

  16. Low-pressure membrane integrity tests for drinking water treatment: A review.

    PubMed

    Guo, H; Wyart, Y; Perot, J; Nauleau, F; Moulin, P

    2010-01-01

    Low-pressure membrane systems, including microfiltration (MF) and ultrafiltration (UF) membranes, are being increasingly used in drinking water treatments due to their high level of pathogen removal. However, the pathogen will pass through the membrane and contaminate the product if the membrane integrity is compromised. Therefore, an effective on-line integrity monitoring method for MF and UF membrane systems is essential to guarantee the regulatory requirements for pathogen removal. A lot of works on low-pressure membrane integrity tests have been conducted by many researchers. This paper provides a literature review about different low-pressure membrane integrity monitoring methods for the drinking water treatment, including direct methods (pressure-based tests, acoustic sensor test, liquid porosimetry, etc.) and indirect methods (particle counting, particle monitoring, turbidity monitoring, surrogate challenge tests). Additionally, some information about the operation of membrane integrity tests is presented here. It can be realized from this review that it remains urgent to develop an alternative on-line detection technique for a quick, accurate, simple, continuous and relatively inexpensive evaluation of low-pressure membrane integrity. To better satisfy regulatory requirements for drinking water treatments, the characteristic of this ideal membrane integrity test is proposed at the end of this paper.

  17. Quantitative changes of Ricinus communis agglutinin I and Helix pomatia lectin binding sites in the acrosome of rat spermatozoa during epididymal transit.

    PubMed

    Hermo, L; Winikoff, R; Kan, F W

    1992-09-01

    During passage through the epididymis, spermatozoa undergo a number of changes which result in their acquisition of fertility and motility. Some of the changes that occur include loss of the cytoplasmic droplet and changes in sperm morphology, metabolism and properties of the nucleus and plasma membrane. Changes have also been reported in the acrosomic system of mammalian spermatozoa during their transit through the epididymis. In the present study, the quantitative changes of the glycoconjugate content in the acrosome of rat spermatozoa were examined during their passage through the epididymis using lectin-colloidal gold cytochemistry. Various regions of the epididymis (initial segment, caput, corpus and cauda epididymidis) were fixed by perfusion with 1% or 2% glutaraldehyde buffered in sodium cacodylate (0.1 M), dehydrated in ethanol and embedded without osmication in Lowicryl K4M. Lectin-colloidal gold labeling was performed on thin sections using Ricinus communis agglutinin I (RCA I) or Helix pomatia lectin (HPL) to detect D-galactose- and N-acetyl-D-galactosamine-containing glycoconjugates, respectively. The labeling density over the acrosome of the acrosomic system was evaluated as the number of gold particles per microns 2 of profile area using a Zeiss MOP-3 image analyzer. The overall mean labeling densities over the acrosome of spermatozoa for each lectin was estimated from 4 rats and over the four distinct epididymal regions. The mean labeling density of the acrosome with RCA I and HPL showed a similar pattern along the epididymis, although RCA I revealed approximately twice as many gold particles per epididymal region. In either case, there was a significant decrease in the labeling density of the acrosome of spermatozoa between the initial segment or caput epididymidis and cauda epididymidis (p less than 0.01). A similar decrease was also noted between the initial segment and corpus epididymidis (p less than 0.01). No change was found between the

  18. Phase separation in biological membranes: integration of theory and experiment.

    PubMed

    Elson, Elliot L; Fried, Eliot; Dolbow, John E; Genin, Guy M

    2010-01-01

    Lipid bilayer model membranes that contain a single lipid species can undergo transitions between ordered and disordered phases, and membranes that contain a mixture of lipid species can undergo phase separations. Studies of these transformations are of interest for what they can tell us about the interaction energies of lipid molecules of different species and conformations. Nanoscopic phases (<200 nm) can provide a model for membrane rafts, specialized membrane domains enriched in cholesterol and sphingomyelin, which are believed to have essential biological functions in cell membranes. Crucial questions are whether lipid nanodomains can exist in stable equilibrium in membranes and what is the distribution of their sizes and lifetimes in membranes of different composition. Theoretical methods have supplied much information on these questions, but better experimental methods are needed to detect and characterize nanodomains under normal membrane conditions. This review summarizes linkages between theoretical and experimental studies of phase separation in lipid bilayer model membranes.

  19. Effects of Bloom-Forming Algae on Fouling of Integrated Membrane Systems in Seawater Desalination

    ERIC Educational Resources Information Center

    Ladner, David Allen

    2009-01-01

    Combining low- and high-pressure membranes into an integrated membrane system is an effective treatment strategy for seawater desalination. Low-pressure microfiltration (MF) and ultrafiltration (UF) membranes remove particulate material, colloids, and high-molecular-weight organics leaving a relatively foulant-free salt solution for treatment by…

  20. Proteomic characterization of integral membrane proteins using thermostatted liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Moore, Sarah M; Wu, Christine C

    2012-01-01

    Due to the hydrophobicity and localization of integral membrane proteins, they are difficult to study using conventional biochemical methods that are compatible with proteomic analyses. This chapter describes the coupling of multiple crucial steps that lead to the optimized shotgun proteomic analysis of integral membrane proteins while maintaining empirical topology information. Namely, a membrane shaving method is utilized to separate protease accessible peptides from membrane embedded peptides and elevated temperatures during chromatographic separation is utilized to augment the recovery of hydrophobic peptides for in-line analysis using tandem mass spectrometry. This combination of steps facilitates increased identification of membrane proteins while also maintaining information regarding protein topology.

  1. Phase separation in biological membranes: integration of theory and experiment

    PubMed Central

    Elson, Elliot L.; Fried, Eliot; Dolbow, John E.; Genin, Guy M.

    2013-01-01

    Lipid bilayer model membranes can undergo transitions between ordered and disordered phases, and membranes that contain a mixture of lipid species can undergo phase separations. Studies of these transformations are of interest for what they can tell us about the interaction energies of lipid molecules of different species and conformations. Nanoscopic phases can provide a model for membrane rafts, which have important biological functions in cell membranes. Important questions are whether lipid nanodomains can exist in stable equilibrium in membranes and what is the distribution of their sizes in membranes of different composition. It is also important to know the lifetimes of nanodomains. Theoretical methods have supplied much important information on these questions, but better experimental methods are needed to detect and characterize nanodomains under normal membrane conditions. This review summarizes linkages between theoretical and experimental studies of phase separation in lipid bilayer model membranes. PMID:20192775

  2. Process Intensification with Integrated Water-Gas-Shift Membrane Reactor

    SciTech Connect

    2009-11-01

    This factsheet describes a research project whose objective is to develop hydrogen-selective membranes for an innovative gas-separation process based on a water-gas-shift membrane reactor (WGS-MR) for the production of hydrogen.

  3. Air separation by integrally asymmetric hollow-fiber membranes

    SciTech Connect

    Feng, X.; Ivory, J.; Rajan, V.S.V.

    1999-10-01

    Integrally asymmetric hollow-fiber membranes each with an outer skin layer and a porous substrate were studied for air separation to produce nitrogen and oxygen enriched air. The test on both bore-side feed and shell-side feed with concurrent and countercurrent flow arrangements for a wide range of stage cuts shows that the bore-side feed countercurrent flow was the most advantageous configuration in the permeator design. When operated in the bore-side feed countercurrent configuration, the permeator performance compared favorably with the commercial systems available for nitrogen production. A mathematical model was developed for this configuration. Since the concentration polarization in the substrate was a major concern for the bore-side feed configuration, especially for high stage-cut operations, a theoretical approach was pursued to formulate the concentration polarization. This allows for the diagnosis of the significance of concentration polarization in a specific permeation process, although it is difficult to predict concentration polarization accurately due to limited knowledge of the detailed membrance structure.

  4. Acrosomal Component of Rat Round Spermatids Recognized by a Novel Monoclonal Antibody.

    PubMed

    Russinova; Atanassova; Paskaleva; Kancheva

    1998-09-01

    OBJECTIVE: To characterize immunocytochemically the antigen recognized which appears at specific stages of germ cell development and acrosomal biogenesis by the novel monoclonal antibody (Mab 3C2). METHODS: The novel monoclonal antibody (Mab 3C2) raised against testicular Sertoli and germ cells. RESULTS: The immunoreactivity of this Mab in testicular sections from immature 20-day-old rats was confined to the pachytene spermatocytes. In adult testis the Mab 3C2, besides meiotic cells, recognized also acrosomal component of round spermatids. The immune reaction was observed in Golgi and cap phases of acrosomal development until the stage VIII of the cycle of the seminiferous epithelium. Immunostaining was absent in acrosome of elongating and mature spermatids and indicated that some modifications in acrosomal protein may exist in subsequent stages of acrosomal development. CONCLUSIONS: Novel Mab 3C2 shares a common antigen in pachytene spermatocytes and round spermatids. Therefore, it may be a marker of meiotic and postmeiotic germ cells.

  5. Membrane curvature in cell biology: An integration of molecular mechanisms.

    PubMed

    Jarsch, Iris K; Daste, Frederic; Gallop, Jennifer L

    2016-08-15

    Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists.

  6. Membrane curvature in cell biology: An integration of molecular mechanisms

    PubMed Central

    Daste, Frederic

    2016-01-01

    Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists. PMID:27528656

  7. An Integrated Framework Advancing Membrane Protein Modeling and Design

    PubMed Central

    Weitzner, Brian D.; Duran, Amanda M.; Tilley, Drew C.; Elazar, Assaf; Gray, Jeffrey J.

    2015-01-01

    Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design. PMID:26325167

  8. MUNI Ways and Structures Building Integrated Solar Membrane Project

    SciTech Connect

    Smith, Randall

    2014-07-03

    The initial goal of the MUNI Ways and Structures Building Integrated Solar Membrane Installation Project was for the City and County of San Francisco (CCSF) to gain experience using the integrated higher efficiency solar photovoltaic (PV) single-ply membrane product, as it differs from the conventional, low efficiency, thin-film PV products, to determine the feasibility of success of larger deployment. As several of CCSF’s municipal rooftops are constrained with respect to weight restrictions, staff of the Energy Generation Group of the San Francisco Public Utilities Commission (SFPUC) proposed to install a solar PV system using single-ply membrane The installation of the 100 kW (DC-STC) lightweight photo voltaic (PV) system at the MUNI Ways and Structures Center (700 Pennsylvania Ave., San Francisco) is a continuation of the commitment of the City and County of San Francisco (CCSF) to increase the pace of municipal solar development, and serve its municipal facilities with clean renewable energy. The fourteen (14) solar photovoltaic systems that have already been installed at CCSF municipal facilities are assisting in the reduction of fossil-fuel use, and reduction of greenhouse gases from fossil combustion. The MUNI Ways & Structures Center roof has a relatively low weight-bearing capacity (3.25 pounds per square foot) and use of traditional crystalline panels was therefore rejected. Consequently it was decided to use the best available highest efficiency Building-Integrated PV (BIPV) technology, with consideration for reliability and experience of the manufacturer which can meet the low weight-bearing capacity criteria. The original goal of the project was to provide an opportunity to monitor the results of the BIPV technology and compare these results to other City and County of San Francisco installed PV systems. The MUNI Ways and Structures Center was acquired from the Cookson Doors Company, which had run the Center for many decades. The building was

  9. Efficient ethanol recovery from fermentation broths with integrated distillation-membrane process

    EPA Science Inventory

    The energy demand of distillation-molecular sieve systems for ethanol recovery/dehydration can be significant, particularly for dilute solutions. An alternative process integrating vapor stripping (like a beer still) with vapor compression and a vapor permeation membrane separati...

  10. Binding of plasma membrane lipids recruits the yeast integral membrane protein Ist2 to the cortical ER.

    PubMed

    Fischer, Marcel André; Temmerman, Koen; Ercan, Ebru; Nickel, Walter; Seedorf, Matthias

    2009-08-01

    Recruitment of cytosolic proteins to individual membranes is governed by a combination of protein-protein and protein-membrane interactions. Many proteins recognize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] at the cytosolic surface of the plasma membrane (PM). Here, we show that a protein-lipid interaction can also serve as a dominant signal for the sorting of integral membrane proteins. Interaction with phosphatidly-inositolphosphates (PIPs) at the PM is involved in the targeting of the polytopic yeast protein Ist2 to PM-associated domains of the cortical endoplasmic reticulum (ER). Moreover, binding of PI(4,5)P(2) at the PM functions as a dominant mechanism that targets other integral membrane proteins to PM-associated domains of the cortical ER. This sorting to a subdomain of the ER abolishes proteasomal degradation and trafficking along the classical secretory (sec) pathway. In combination with the localization of IST2 mRNA to the bud tip and other redundant signals in Ist2, binding of PIPs leads to efficient accumulation of Ist2 at domains of the cortical ER from where the protein may reach the PM independently of the function of the sec-pathway.

  11. Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification.

    PubMed

    Shirzad-Wasei, Nazhat; van Oostrum, Jenny; Bovee-Geurts, Petra H M; Kusters, Lisanne J A; Bosman, Giel J C G M; DeGrip, Willem J

    2015-08-01

    Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

  12. Protective effect of black tea on integral membrane proteins in rat liver.

    PubMed

    Szachowicz-Petelska, Barbara; Skrzydlewska, Elżbieta; Figaszewski, Zbigniew

    2013-01-01

    Ethanol intoxication is accompanied by oxidative stress formation. Consequently, it leads to disturbances in cellular metabolism that can alter the structure and function of cell membrane components. Black tea displays antioxidant properties, protects membrane phospholipids and may protect integral membrane proteins. In the present study, we examined whether black tea induces changes in the liver integral membrane proteins of 12-months old rats chronically intoxicated with ethanol. To estimate qualitatively and quantitatively the levels of the liver integral membrane proteins, the proteins were selectively hydrolyzed by trypsin, the obtained peptides were resolved by HPLC and the levels of specific amino acids within the individual peptides were determined. All of the obtained peptides contained phenylalanine (Phe), cysteine (Cys) and lysine (Lys). Compared to the control group, rats in the ethanol intoxication group showed decreased liver levels of integral membrane proteins as well as fewer trypsin-hydrolyzed peptides and amino acids in the hydrolyzed peptides. Administration of black tea to ethanol-intoxicated rats partially protected proteins against the structural changes caused by ethanol. Black tea prevented decreases in the levels of cysteine (in about 90% of cases), lysine (in about 60% of cases), phenylalanine (in about 70% of cases) and examined peptides (in about 60% of cases). The liver protein level was higher (by about 18%) in rats who received black tea and ethanol than in those who received ethanol alone. In conclusion, black tea partially protects the composition and level of rat liver cell integral membrane proteins against changes caused by ethanol intoxication.

  13. Role of amino acids as additives on sperm motility, plasma membrane integrity and lipid peroxidation levels at pre-freeze and post-thawed ram semen.

    PubMed

    Sangeeta, Sharon; Arangasamy, A; Kulkarni, S; Selvaraju, S

    2015-10-01

    The possibility of including amino acids for cryopreservation of ram semen to improve the quality of frozen semen was explored in this study in sheep model. 24 samples were collected in triplicate from 8 rams of 2-3 year old Bannur cross bred rams maintained at the Institute Experimental Livestock Unit. Semen was diluted in tris-egg yolk glycerol diluent and made into 7 aliquots as follows: aliquot 1 served as control, "l-alanine" was added at 100 and 135mM in the aliquots 2 and 3, "l-glutamine" was added at 20 and 25mM in the aliquots 4 and 5 and "l-proline" was added at 25 and 50mM in the aliquots 6 and 7, respectively. Diluted semen was filled in 0.25ml French straws and frozen in LN2. Inclusion of "l-proline" and "l-glutamine" in the diluent increased the percent live sperm (P<0.001), total motility (P<0.05) and maintained higher functional membrane and acrosomal integrity (P<0.001) by decreasing lipid peroxidation (P<0.001) compared to the control group. In contrast, "l-alanine" decreased the percentage of total motility, fast progressive spermatozoa and increased (P<0.01) the percentage of immotile spermatozoa. It can be concluded that 20mM "l-glutamine" and 25mM "l-proline" can be used as semen additive to freeze ram semen as they prevented cryoinjuries to sperm and improved the pre-freeze and post-thaw semen characteristics.

  14. Integrating membrane filtration into bioelectrochemical systems as next generation energy-efficient wastewater treatment technologies for water reclamation: A review.

    PubMed

    Yuan, Heyang; He, Zhen

    2015-11-01

    Bioelectrochemical systems (BES) represent an energy-efficient approach for wastewater treatment, but the effluent still requires further treatment for direct discharge or reuse. Integrating membrane filtration in BES can achieve high-quality effluents with additional benefits. Three types of filtration membranes, dynamic membrane, ultrafiltration membrane and forward osmosis membrane that are grouped based on pore size, have been studied for integration in BES. The integration can be accomplished either in an internal or an external configuration. In an internal configuration, membranes can act as a separator between the electrodes, or be immersed in the anode/cathode chamber as a filtration component. The external configuration allows BES and membrane module to be operated independently. Given much progress and interest in the integration of membrane filtration into BES, this paper has reviewed the past studies, described various integration methods, discussed the advantages and limitations of each integration, and presented challenges for future development.

  15. Poloxamer-188 and citicoline provide neuronal membrane integrity and protect membrane stability in cortical spreading depression.

    PubMed

    Yıldırım, Timur; Eylen, Alpaslan; Lule, Sevda; Erdener, Sefik Evren; Vural, Atay; Karatas, Hulya; Ozveren, Mehmet Faik; Dalkara, Turgay; Gursoy-Ozdemir, Yasemin

    2015-01-01

    Under pathological conditions such as brain trauma, subarachnoid hemorrhage and stroke, cortical spreading depression (CSD) or peri-infarct depolarizations contribute to brain damage in animal models of neurological disorders as well as in human neurological diseases. CSD causes transient megachannel opening on the neuronal membrane, which may compromise neuronal survival under pathological conditions. Poloxamer-188 (P-188) and citicoline are neuroprotectants with membrane sealing properties. The aim of this study is to investigate the effect of P-188 and citicoline on the neuronal megachannel opening induced by CSD in the mouse brain. We have monitored megachannel opening with propidium iodide, a membrane impermeable fluorescent dye and, demonstrate that P-188 and citicoline strikingly decreased CSD-induced neuronal PI influx in cortex and hippocampal dentate gyrus. Therefore, these agents may be providing neuroprotection by blocking megachannel opening, which may be related to their membrane sealing action and warrant further investigation for treatment of traumatic brain injury and ischemic stroke.

  16. Correlation study of microalgae carbonation in membrane integrated photobioreactor

    NASA Astrophysics Data System (ADS)

    Suali, E.; Sarbatly, R.; Shaleh, S. R. M.; Lahin, F. A.; Anisuzzaman, S. M.

    2016-06-01

    Microalgae ability to utilise CO2 higher compared to terrestrial plant making it suitable for biomass production and as CO2 utiliser. This could be one of many ways to preserve a safer and healthier environment with less air pollutant. For study purposes, CO2 usually transported to microalgae culture broth with the aid of membrane technology to prevent formation of large bubble and to accelerate the carbonation of microalgal media. However, membrane susceptible to accumulation of CO2, which can cause extreme acidic to microalgal media. This prevents microalgae to assimilate CO2. Thus, this study proposes correlations to prevent the extreme acidic: which represents the relationship of: (1) CO2 inlet and accumulation, (2) CO2 inlet and CO2 at the membrane-liquid interphase and (3) CO2 inlet and CO2 solubility in the media. The correlations were successfully validated with a deviation of less than 20% compared to the theoretical value.

  17. High quality single crystal Ge nano-membranes for opto-electronic integrated circuitry

    SciTech Connect

    Shah, V. A. Gammon, P. M.; Rhead, S. D.; Halpin, J. E.; Trushkevych, O.; Wilson, N. R.; Myronov, M.; Edwards, R. S.; Patchett, D. H.; Allred, P. S.; Prest, M. J.; Whall, T. E.; Parker, E. H. C.; Leadley, D. R.; Chávez-Ángel, E.; Shchepetov, A.; Prunnila, M.; Kachkanov, V.; Dolbnya, I. P.; Reparaz, J. S.; and others

    2014-04-14

    A thin, flat, and single crystal germanium membrane would be an ideal platform on which to mount sensors or integrate photonic and electronic devices, using standard silicon processing technology. We present a fabrication technique compatible with integrated-circuit wafer scale processing to produce membranes of thickness between 60 nm and 800 nm, with large areas of up to 3.5 mm{sup 2}. We show how the optical properties change with thickness, including appearance of Fabry-Pérot type interference in thin membranes. The membranes have low Q-factors, which allow the platforms to counteract distortion during agitation and movement. Finally, we report on the physical characteristics showing sub-nm roughness and a homogenous strain profile throughout the freestanding layer, making the single crystal Ge membrane an excellent platform for further epitaxial growth or deposition of materials.

  18. integrating Solid State NMR and Computations in Membrane Protein Science

    NASA Astrophysics Data System (ADS)

    Cross, Timothy

    2015-03-01

    Helical membrane protein structures are influenced by their native environment. Therefore the characterization of their structure in an environment that models as closely as possible their native environment is critical for achieving not only structural but functional understanding of these proteins. Solid state NMR spectroscopy in liquid crystalline lipid bilayers provides an excellent tool for such characterizations. Two classes of restraints can be obtained - absolute restraints that constrain the structure to a laboratory frame of reference when using uniformly oriented samples (approximately 1° of mosaic spread) and relative restraints that restrain one part of the structure with respect to another part such as torsional and distance restraints. Here, I will discuss unique restraints derived from uniformly oriented samples and the characterization of initial structures utilizing both restraint types, followed by restrained molecular dynamics refinement in the same lipid bilayer environment as that used for the experimental restraint collection. Protein examples will be taken from Influenza virus and Mycobacterium tuberculosis. When available comparisons of structures to those obtained using different membrane mimetic environments will be shown and the causes for structural distortions explained based on an understanding of membrane biophysics and its sophisticated influence on membrane proteins.

  19. The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri.

    PubMed

    Wang, Xia; Jiang, Feng; Zheng, Jianhua; Chen, Lihong; Dong, Jie; Sun, Lilian; Zhu, Yafang; Liu, Bo; Yang, Jian; Yang, Guowei; Jin, Qi

    2016-09-01

    Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri, termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support that PldA, which is widespread across Gram-negative bacteria, is an important factor for the bacterial life cycle, particularly in human pathogens.

  20. Monitoring changes in membrane polarity, membrane integrity, and intracellular ion concentrations in Streptococcus pneumoniae using fluorescent dyes.

    PubMed

    Clementi, Emily A; Marks, Laura R; Roche-Håkansson, Hazeline; Håkansson, Anders P

    2014-02-17

    Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca(2+), K(+), and H(+), respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial

  1. Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

    PubMed Central

    Roche-Håkansson, Hazeline; Håkansson, Anders P.

    2014-01-01

    Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species

  2. Fouling and long-term durability of an integrated forward osmosis and membrane distillation system.

    PubMed

    Husnain, T; Mi, B; Riffat, R

    2015-01-01

    An integrated forward osmosis (FO) and membrane distillation (MD) system has great potential for sustainable wastewater reuse. However, the fouling and long-term durability of the system remains largely unknown. This study investigates the fouling behaviour and efficiency of cleaning procedures of FO and MD membranes used for treating domestic wastewater. Results showed that a significant decline in flux of both FO and MD membranes were observed during treatment of wastewater with organic foulants. However, shear force generated by the increased cross-flow physically removed the loosely attached foulants from the FO membrane surface and resulted in 86-88% recovery of flux by cleaning with tap water. For the MD membrane, almost no flux recovery was achieved due to adsorption of organic foulants on the hydrophobic membrane surface, thus indicating significant irreversible fouling/wetting, which may not be effectively cleaned even with chemical reagents. Long-term (10 d) tests showed consistent performance of the FO membrane by rejecting the contaminants. However, organic foulants reduced the hydrophobicity of the MD membrane, caused wetting problems and allowed contaminants to pass through. The results demonstrate that combination of the FO and MD processes can effectively reduce irreversible membrane fouling and solve the wetting problem of the MD membrane.

  3. Iron Deprivation Affects Drug Susceptibilities of Mycobacteria Targeting Membrane Integrity

    PubMed Central

    Pal, Rahul; Hameed, Saif; Fatima, Zeeshan

    2015-01-01

    Multidrug resistance (MDR) acquired by Mycobacterium tuberculosis (MTB) through continuous deployment of antitubercular drugs warrants immediate search for novel targets and mechanisms. The ability of MTB to sense and become accustomed to changes in the host is essential for survival and confers the basis of infection. A crucial condition that MTB must surmount is iron limitation, during the establishment of infection, since iron is required by both bacteria and humans. This study focuses on how iron deprivation affects drug susceptibilities of known anti-TB drugs in Mycobacterium smegmatis, a “surrogate of MTB.” We showed that iron deprivation leads to enhanced potency of most commonly used first line anti-TB drugs that could be reverted upon iron supplementation. We explored that membrane homeostasis is disrupted upon iron deprivation as revealed by enhanced membrane permeability and hypersensitivity to membrane perturbing agent leading to increased passive diffusion of drug and TEM images showing detectable differences in cell envelope thickness. Furthermore, iron seems to be indispensable to sustain genotoxic stress suggesting its possible role in DNA repair machinery. Taken together, we for the first time established a link between cellular iron and drug susceptibility of mycobacteria suggesting iron as novel determinant to combat MDR. PMID:26779346

  4. Integrating complex functions: coordination of nuclear pore complex assembly and membrane expansion of the nuclear envelope requires a family of integral membrane proteins.

    PubMed

    Schneiter, Roger; Cole, Charles N

    2010-01-01

    The nuclear envelope harbors numerous large proteinaceous channels, the nuclear pore complexes (NPCs), through which macromolecular exchange between the cytosol and the nucleoplasm occurs. This double-membrane nuclear envelope is continuous with the endoplasmic reticulum and thus functionally connected to such diverse processes as vesicular transport, protein maturation and lipid synthesis. Recent results obtained from studies in Saccharomyces cerevisiae indicate that assembly of the nuclear pore complex is functionally dependent upon maintenance of lipid homeostasis of the ER membrane. Previous work from one of our laboratories has revealed that an integral membrane protein Apq12 is important for the assembly of functional nuclear pores. Cells lacking APQ12 are viable but cannot grow at low temperatures, have aberrant NPCs and a defect in mRNA export. Remarkably, these defects in NPC assembly can be overcome by supplementing cells with a membrane fluidizing agent, benzyl alcohol, suggesting that Apq12 impacts the flexibility of the nuclear membrane, possibly by adjusting its lipid composition when cells are shifted to a reduced temperature. Our new study now expands these findings and reveals that an essential membrane protein, Brr6, shares at least partially overlapping functions with Apq12 and is also required for assembly of functional NPCs. A third nuclear envelope membrane protein, Brl1, is related to Brr6, and is also required for NPC assembly. Because maintenance of membrane homeostasis is essential for cellular survival, the fact that these three proteins are conserved in fungi that undergo closed mitoses, but are not found in metazoans or plants, may indicate that their functions are performed by proteins unrelated at the primary sequence level to Brr6, Brl1 and Apq12 in cells that disassemble their nuclear envelopes during mitosis.

  5. TM7SF1 (GPR137B): a novel lysosome integral membrane protein.

    PubMed

    Gao, Jialin; Xia, Libin; Lu, Meiqing; Zhang, Binhua; Chen, Yueping; Xu, Rang; Wang, Lizhuo

    2012-09-01

    In the previous proteomic study of human placenta, transmembrane 7 superfamily member 1 (TM7SF1) was found enriched in lysosome compartments. TM7SF1 encodes a 399-amino acid protein with a calculated molecular mass of 45 kDa. Bioinformatic analysis of its amino acid sequence showed that it is a multipass transmembrane protein containing a potential dileucine-based lysosomal targeting signal and four putative N-glycosylation sites. By percoll-gradient centrifugation and further subfraction ways, the lysosomal solute and membrane compartments were isolated respectively. Immunoblotting analysis indicated that TM7SF1 was co-fractioned with lysosome associated membrane protein 2 (LAMP2), which was only detected in lysosomal membrane compartments whereas not detected in the solute compartments. Using specific anti-TM7SF1 antibody and double-immunofluorescence with lysosome membrane protein LAMP1 and Lyso-Tracker Red, the colocalisations of endogenous TM7SF1 with lysosome and late endosome markers were demonstrated. All of this indicated that TM7SF1 is an integral lysosome membrane protein. Rat ortholog of TM7SF1 was found to be strongly expressed in heart, liver, kidney and brain while not or low detected in other tissues. In summary, TM7SF1 was a lysosomal integral membrane protein that shows tissue-specific expression. As a G-protein-coupled receptor in lysosome membrane, TM7SF1 was predicted function as signal transduction across lysosome membrane.

  6. Membrane composition influences the topology bias of bacterial integral membrane proteins.

    PubMed

    Bay, Denice C; Turner, Raymond J

    2013-02-01

    Small multidrug resistance (SMR) protein family members confer bacterial resistance to toxic antiseptics and are believed to function as dual topology oligomers. If dual topology is essential for SMR activity, then the topology bias should change as bacterial membrane lipid compositions alter to maintain a "neutral" topology bias. To test this hypothesis, a bioinformatic analysis of bacterial SMR protein sequences was performed to determine a membrane protein topology based on charged amino acid residues within loops, and termini regions according to the positive inside rule. Three bacterial lipid membrane parameters were examined, providing the proportion of polar lipid head group charges at the membrane surface (PLH), the relative hydrophobic fatty acid length (FAL), and the proportion of fatty acid unsaturation (FAU). Our analysis indicates that individual SMR pairs, and to a lesser extent SMR singleton topology biases, are significantly correlated to increasing PLH, FAL and FAU differences validating the hypothesis. Correlations between the topology biases of SMR proteins identified in Gram+ compared to Gram- species and each lipid parameter demonstrated a linear inverse relationship.

  7. Integrated Water Gas Shift Membrane Reactors Utilizing Novel, Non Precious Metal Mixed Matrix Membrane

    SciTech Connect

    Ferraris, John

    2013-09-30

    Nanoparticles of zeolitic imidazolate frameworks and other related hybrid materials were prepared by modifying published synthesis procedures by introducing bases, changing stoichiometric ratios, or adjusting reaction conditions. These materials were stable at temperatures >300 °C and were compatible with the polymer matrices used to prepare mixed- matrix membranes (MMMs). MMMs tested at 300 °C exhibited a >30 fold increase in permeability, compared to those measured at 35 °C, while maintaining H{sub 2}/CO{sub 2} selectivity. Measurements at high pressure (up to 30 atm) and high temperature (up to 300 °C) resulted in an increase in gas flux across the membrane with retention of selectivity. No variations in permeability were observed at high pressures at either 35 or 300 °C. CO{sub 2}-induced plasticization was not observed for Matrimid®, VTEC, and PBI polymers or their MMMs at 30 atm and 300 °C. Membrane surface modification by cross-linking with ethanol diamine resulted in an increase in H{sub 2}/CO{sub 2} selectivity at 35 °C. Spectrometric analysis showed that the cross-linking was effective to temperatures <150 °C. At higher temperatures, the cross-linked membranes exhibit a H{sub 2}/CO{sub 2} selectivity similar to the uncross-linked polymer. Performance of the polybenzimidazole (PBI) hollow fibers prepared at Santa Fe Science and Technology (SFST, Inc.) showed increased flux o to a flat PBI membrane. A water-gas shift reactor has been built and currently being optimized for testing under DOE conditions.

  8. A Novel and Facile Method to Prepare Integrated Electrospun Nanofibrous Membrane with Soldered Junctions.

    PubMed

    Shen, Lingdi; Chen, Jiajia; Hong, Guishan; Wang, Xuefen

    2016-01-01

    Integrated electrospun nanofibrous membrane was prepared by creating soldered junctions between nanofibers via a facile strategy. Polyacrylonitrile (PAN) mixed with poly(vinylidene fluoride) (PVDF) at different ratios of PVDF were prepared in N,N'-dimethyl formamide (DMF), then electrospun to fabricate PAN/PVDF membranes. PVDF can form microgels in DMF which slows down volatile speed of DMF and affects the solidification of PAN/PVDF nanofibers. The resulting membranes were investigated by Fourier transform infrared spectroscopy, scanning electron microscopy, dynamic water contact angle and tensile testing to confirm the morphology and mechanical properties. Soldered junctions were observed between nanofibers with the increase of PVDF content. These junctions made the membrane integrated and greatly enhanced tensile strength from 5.1 to 8.1 MPa (increased by ~60%) and tensile modulus from 49.4 to 117.9 MPa (increased by ~139%) without compromising porosity when the content of PVDF increased from 0 to 60 wt%.

  9. Phase separation of integral membrane proteins in Triton X-114 solution.

    PubMed

    Bordier, C

    1981-02-25

    A solution of the nonionic detergent Triton X-114 is homogeneous at 0 degrees C but separates in an aqueous phase and a detergent phase above 20 degrees C. The extent of this detergent phase separation increases with the temperature and is sensitive to the presence of other surfactants. The partition of proteins during phase separation in solutions of Triton X-114 is investigated. Hydrophilic proteins are found exclusively in the aqueous phase, and integral membrane proteins with an amphiphilic nature are recovered in the detergent phase. Triton X-114 is used to solubilize membranes and whole cells, and the soluble material is submitted to phase separation. Integral membrane proteins can thus be separated from hydrophilic proteins and identified as such in crude membrane or cellular detergent extracts.

  10. Effects of PEO-PPO-PEO triblock copolymers on phospholipid membrane integrity under osmotic stress.

    PubMed

    Wang, Jia-Yu; Chin, Jaemin; Marks, Jeremy D; Lee, Ka Yee C

    2010-08-03

    The effects of PEO-PPO-PEO triblock copolymers, mainly Poloxamer 188, on phospholipid membrane integrity under osmotic gradients were explored using giant unilamellar vesicles (GUVs). Fluorescence leakage assays showed two opposing effects of P188 on the structural integrity of GUVs depending on the duration of their incubation time. A two-state transition mechanism of interaction between the triblock copolymers and the phospholipid membrane is proposed: an adsorption (I) and an insertion (II) state. While the triblock copolymer in state I acts to moderately retard the leakage, their insertion in state II perturbs the lipid packing, thus increasing the membrane permeability. Our results suggest that the biomedical application of PEO-PPO-PEO triblock copolymers, either as cell membrane resealing agents or as accelerators for drug delivery, is directed by the delicate balance between these two states.

  11. Integration of nanoporous membranes for sample filtration/preconcentration in microchip electrophoresis.

    PubMed

    Long, Zhicheng; Liu, Dayu; Ye, Nannan; Qin, Jianhua; Lin, Bingcheng

    2006-12-01

    Microfluidic devices integrating membrane-based sample preparation with electrophoretic separation are demonstrated. These multilayer devices consist of 10 nm pore diameter membranes sandwiched between two layers of PDMS substrates with embedded microchannels. Because of the membrane isolation, material exchange between two fluidic layers can be precisely controlled by applied voltages. More importantly, since only small molecules can pass through the nanopores, the integrated membrane can serve as a filter or a concentrator prior to microchip electrophoresis under different design and operation modes. As a filter, they can be used for separation and selective injection of small analytes from sample matrix. This has been effectively applied in rapid determination of reduced glutathione in human plasma and red blood cells without any off-chip deproteinization procedure. Alternatively, in the concentrator mode, they can be used for online purification and preconcentration of macromolecules, which was illustrated by removing primers and preconcentrating the product DNA from a PCR product mixture.

  12. Membrane photobioreactors for integrated microalgae cultivation and nutrient remediation of membrane bioreactors effluent.

    PubMed

    Marbelia, L; Bilad, M R; Passaris, I; Discart, V; Vandamme, D; Beuckels, A; Muylaert, K; Vankelecom, Ivo F J

    2014-07-01

    The feasibility of a new concept of wastewater treatment by combining a membrane bioreactor (MBR) and a microalgae membrane photobioreactor (MPBR) is assessed in this study. In this system, the organic carbon present in wastewater is expected to be fully oxidized in the MBR, while the nutrients are removed via the subsequent MPBR treatment. The effluent of a lab-scale MBR was fed into a PBR and a MPBR which served as growing medium for Chlorella vulgaris. The MPBRs demonstrated their superiority by limiting the algae wash-out, thus increasing the allowable optimum dilution rate (Dopt). At these corresponding Dopt values, 3.5 and 2 times higher biomass concentrations and volumetric productivities respectively were achieved by the MPBR. It is also possible to run the MPBR at still higher biomass concentration, thus enabling a smaller footprint and higher nutrient removal efficiency. However, reduced nutrient removal efficiencies were found to be one possible drawback.

  13. Pervaporation behavior and integrated process for concentrating lignocellulosic ethanol through polydimethylsiloxane (PDMS) membrane.

    PubMed

    Chen, Jingwen; Zhang, Hongman; Wei, Ping; Zhang, Lin; Huang, He

    2014-02-01

    The effects of by-products from ethanol fermentation and hydrolysates of lignocelluloses on ethanol diffusion through polydimethylsiloxane (PDMS) membranes with/without silicalite-1 were investigated. A pervaporation process was integrated with lignocellulosic fermentation to concentrate bioethanol using bare PDMS membranes. Results showed that yeasts, solid particles, and salts increased ethanol flux and selectivity through the membranes (PDMS with/without silicalite-1), whereas glucose exerted negative effects on the performance. On bare PDMS membrane, the performance was not obviously affected by the existence of aliphatic acids. However, on PDMS-silicalite-1 membrane, a remarkable decrease in ethanol selectivity and a rapid growth of total flux in the presence of aliphatic acids were observed. These phenomena were due to the interaction of acids with silanol (Si-OH) groups to break the dense membrane surface. On the PDMS membranes with/without silicalite-1, degradation products of lignocellulosic hydrolysates such as furfural and hydroxyacetone slightly influenced separation performance. These results revealed that an integrated process can effectively eliminate product inhibition, improve ethanol productivity, and enhance the glucose conversion rate.

  14. Self-assembly of single integral membrane proteins into soluble nanoscale phospholipid bilayers

    PubMed Central

    Bayburt, Timothy H.; Sligar, Stephen G.

    2003-01-01

    One of the biggest challenges in pharmaceutical research is obtaining integral membrane proteins in a functional, solubilized, and monodisperse state that provides a native-like environment that maintains the spectrum of in vivo activities. Many of these integral membrane proteins are receptors, enzymes, or other macromolecular assemblies that are important drug targets. An example is the general class of proteins composed of seven-transmembrane segments (7-TM) as exemplified by the G-protein–coupled receptors. In this article, we describe a simple system for self-assembling bacteriorhodopsin, as a model protein containing 7-TM helices, with phospholipids to form a nanometer-scale soluble bilayer structure encircled by a 200 amino acid scaffold protein. The result is the single molecule incorporation of an integral membrane protein target into a soluble and monodisperse structure that allows the structural and functional tools of solution biochemistry to be applied. PMID:14573860

  15. Reduced Graphene Oxide Bipolar Membranes for Integrated Solar Water Splitting in Optimal pH.

    PubMed

    McDonald, Michael B; Bruce, Jared P; McEleney, Kevin; Freund, Michael S

    2015-08-24

    The integration of light absorbers and catalysts for the water splitting process requires a membrane capable of both ion and electron management and product separation to realize efficient solar fuels systems. Bipolar membranes can maintain a pH gradient for optimal reaction conditions by the dissociation of water. Such membranes that contain graphene in the interfacial layer are fabricated by the chemical reduction of a uniformly deposited graphene oxide layer to convert sp(3) catalyst regions to sp(2) conductive regions. The resulting electrical and water dissociation properties are optimized by adjusting the exposure conditions, and treatments of less than 5 min render an interface that exceeds the conductivity requirements for integrated solar water splitting and increases the overpotential by <0.3 V. Integration with photoelectrodes is examined by characterizing the electrical interface formed between graphene and Si microwires, and we found that efficient Ohmic junctions are possible.

  16. Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

    PubMed Central

    Alonso, Jose L.; Mascellaro, Salvatore; Moreno, Yolanda; Ferrús, María A.; Hernández, Javier

    2002-01-01

    We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells. PMID:12324366

  17. Effect of powdered activated carbon on integrated submerged membrane bioreactor-nanofiltration process for wastewater reclamation.

    PubMed

    Woo, Yun Chul; Lee, Jeong Jun; Shim, Wang-Geun; Shon, Ho Kyong; Tijing, Leonard D; Yao, Minwei; Kim, Han-Seung

    2016-06-01

    The aim of this study was to determine the effect of powdered activated carbon (PAC) on the overall performance of a submerged membrane bioreactor (SMBR) system integrated with nanofiltration (NF) for wastewater reclamation. It was found that the trans-membrane pressure of SMBR increased continuously while that of the SMBR with PAC was more stable, mainly because water could still pass through the PACs and membrane even though foulants adhered on the PAC surface. The presence of PAC was able to mitigate fouling in SMBR as well as in NF. SMBR-NF with PAC obtained a higher flux of 8.1 LMH compared to that without PAC (6.6 LMH). In addition, better permeate quality was obtained with SMBR-NF integrated process added with PAC. The present results suggest that the addition of PAC in integrated SMBR-NF process could possibly lead to satisfying water quality and can be operated for a long-term duration.

  18. Synthesis of a major integral membrane polypeptide of rat liver peroxisomes on free polysomes.

    PubMed Central

    Fujiki, Y; Rachubinski, R A; Lazarow, P B

    1984-01-01

    The manner of synthesis and assembly of the peroxisomal membrane proteins is unknown. Understanding these processes is essential to an understanding of the formation of the organelle. We have investigated the biogenesis of the previously identified major 21.7-kDa integral peroxisomal membrane polypeptide [Fujiki, Y., Fowler, S., Shio, H., Hubbard, A. L. & Lazarow, P. B. (1982) J. Cell Biol. 93, 103-110]. This protein was purified to apparent homogeneity and used to elicit a rabbit antiserum. In immunoblotting analysis, antibody bound only to the 22-kDa membrane polypeptide present exclusively in peroxisomal membranes. Total rat liver RNA was translated in a nuclease-treated rabbit reticulocyte cell-free protein-synthesizing system. The in vitro translation product, isolated by means of the antibody and Staphylococcus aureus cells, comigrated with the mature 22-kDa polypeptide in NaDodSO4/PAGE. Analysis of the translation products of RNAs from free and membrane-bound polysomes indicated that the mRNA for the 22-kDa membrane polypeptide is found predominantly in free polysomes. The results imply post-translational insertion of the membrane polypeptide into the peroxisomal membrane without proteolytic processing and suggest that peroxisomes, like mitochondria and chloroplasts, form by fission from preexisting organelles. Images PMID:6594687

  19. Membrane separation of carbon dioxide in the integrated gasification combined cycle systems

    NASA Astrophysics Data System (ADS)

    Kotowicz, Janusz; Skorek-osikowska, Anna; Janusz-szymańska, Katarzyna

    2010-09-01

    Integrated gasification combined cycle systems (IGCC) are becoming more popular because of the characteristics, by which they are characterized, including low pollutants emissions, relatively high efficiency of electricity production and the ability to integrate the installation of carbon capture and storage (CCS). Currently, the most frequently used CO2 capture technology in IGCC systems is based on the absorption process. This method causes a significant increase of the internal load and decreases the efficiency of the entire system. It is therefore necessary to look for new methods of carbon dioxide capture. The authors of the present paper propose the use of membrane separation. The paper reviews available membranes for use in IGCC systems, indicates, inter alia, possible places of their implementation in the system and the required operation parameters. Attention is drawn to the most important parameters of membranes (among other selectivity and permeability) influencing the cost and performance of the whole installation. Numerical model of a membrane was used, among others, to analyze the influence of the basic parameters of the selected membranes on the purity and recovery ratio of the obtained permeate, as well as to determine the energetic cost of the use of membranes for the CO2 separation in IGCC systems. The calculations were made within the environment of the commercial package Aspen Plus. For the calculations both, membranes selective for carbon dioxide and membranes selective for hydrogen were used. Properly selected pressure before and after membrane module allowed for minimization of energy input on CCS installation assuring high purity and recovery ratio of separated gas.

  20. Alterations of domains in the plasmatic membrane due to damages of the perinuclear theca of pig preserved spermatozoa.

    PubMed

    Orozco Benítez, María Guadalupe; Lemus Flores, Clemente; Hernández Ballesteros, Juan Antonio; Navarrete Méndez, Raúl; Juárez-Mosqueda, María de Lourdes

    2008-05-15

    Samples of semen from 12 pigs, three from Yorkshire, Landrace, Duroc and Mexican Hairless each where obtained to study cryopreservation methods. Three stages of boar semen cryopreservation were evaluated: none (fresh stage), cooling at 5 degrees C and freezing at -196 degrees C then thawing to 56 degrees C for 12 sec. Perinuclear theca damage and domain alterations were selected as indices of seminal quality, as measured by electronic and fluorescence microcopy, respectively according to two lineal models considering by separately the effect of semen preservation and breed. Integrity and absence of perinuclear theca significantly (p < 0.001) decreased and increased, respectively according to a decrease in temperature of cryopreservation, from 87.4 to 58.8% and from 0.8 to 26.2%, respectively. This same significant (p < 0.001) effect was found for acrosomal and post-acrosomal membrane distribution of domains, from 92.1 to 76.8% and from 3.1 to 13.1% in this same order. Slight but highly significant (p < 0.001) differences were observed when theca integrity was evaluated as affected by breed, with highest and lowest values for Yorkshire and Pel6n Mexicano pigs, respectively. No breed effect was encountered for presence of acrosomal domains. A strong interdependence was found between perinuclear theca damage and domain distribution. In this connection, a highly significant (p < 0.001) positive, interdependence was observed between the theca damage and acrosomal domain (r = 0.87), while this same relationship was although highly significant (p < 0.001), negative in nature for equatorial and post-acrosonal domains (r = -0.77 and -0.85, respectively). This experiment confirmed that cryopreservation methods may severely affect semen quality of pigs and that genotype may further influence these same indices. More research is needed for improving methods of preservation of pig semen quality, from the point of view of perinuclear theca and domain characteristics of

  1. Acrosome intactness and seminal hyaluronidase activity: relationship with conventional seminal parameters.

    PubMed

    Tambe, A S; Kaore, S B; Sawane, M V; Gosavi, G B

    2001-03-01

    Seminal hyaluronidase activity was estimated after liquefaction in semen samples of 100 male partners of infertile couples including 16 azoospermic (no spermatozoon) men and 48 fertility proven men by a method based on measurement of the area of digestion of substrate (hyaluronic acid) in agar plate. Semen samples were also evaluated for Acrosomal Intactness (AI) test except the azoospermics of the studied samples. Seminal hyaluronidase activity was completely absent in azoospermic specimens confirming its cellular origin. Seminal hyaluronidase activity was found to be significantly correlated, statistically, with sperm density (r = 0.708, p < 0.001), % motility (r = 0.6478, p < 0.001) and % normal sperm morphology (r = 0.5724, p < 0.001). Acrosomal Intactness (AI) test scores were also well correlated with sperm density (r = 0.6477, p < 0.001), % motility (r = 0.5965, p < 0.001) and % normal morphology (r = 0.6237, p < 0.001). Both values were higher in semen samples with normal routine parameters (proven fertility and normozoospermic infertile groups) than those compared with abnormal routine parameters (oligozoospermic). We also found very highly significant correlation (r = 0.8442) between seminal hyaluronidase activity and Acrosomal Intactness scores, statistically (p < 0.001). This could be because; normal germinal semineferous epithelium generates abundant number of sperms with normal motility and morphology that are also having intact acrosome. Intact acrosome prevents loss of acrosomal enzymatic activity (e.g. hyaluronidase) until released after liquefaction during seminal analysis and during acrosomal reaction in female genital tract prior to fertilization. Seminal hyaluronidase activity, thus determined, is primarily dependent upon the intact status of acrosome. As each sperm contributes to the seminal hyaluronidase activity, it is directly correlated with sperm density; but at the same time it exhibits goods correlation with % motility and % normal

  2. Development and application of a procedure for evaluating the long-term integrity of membranes for the anaerobic fluidized membrane bioreactor (AFMBR).

    PubMed

    Shin, Chungheon; Kim, Kihyun; McCarty, Perry L; Kim, Jeonghwan; Bae, Jaeho

    A bench-scale short-term test, developed to predict the long-term integrity of membranes with potential for use in anaerobic fluidized-bed membrane bioreactors, was used to evaluate several commercial hollow-fiber membranes. It was found that membrane performance varied widely, some membranes failing much more rapidly than others. Also found was that larger sizes of the fluidized media, in this case granular activated carbon (GAC), severely affected membrane structural integrity more than did smaller sizes, as did the method used for membrane attachment. Within the limits studied, the GAC packing ratio had only a minor impact. A decrease in membrane permeability that sometimes resulted during the testing and was caused by the deposition of fine GAC particles could be eliminated without membrane damage through simultaneous chemical cleaning and sonication. This new testing procedure should be useful for selecting membranes and reactor operating conditions to better ensure long-term operating performance of anaerobic fluidized-bed membrane bioreactors.

  3. Function of nuclear membrane proteins in shaping the nuclear envelope integrity during closed mitosis.

    PubMed

    Yang, Hui-Ju; Iwamoto, Masaaki; Hiraoka, Yasushi; Haraguchi, Tokuko

    2017-04-08

    The nuclear envelope (NE) not only protects the genome from being directly accessed by detrimental agents but also regulates genome organization. Breaches in NE integrity threaten genome stability and impede cellular function. Nonetheless, the NE constantly remodels, and NE integrity is endangered in dividing or differentiating cells. Specifically, in unicellular eukaryotes undergoing closed mitosis, the NE expands instead of breaking down during chromosome segregation. The newly assembling nuclear pore complexes (NPCs) penetrate the existing NE in interphase. A peculiar example of NE remodeling during nuclear differentiation in Tetrahymena involves formation of the redundant NE and clustered NPCs. Even under these conditions, the NE remains intact. Many recent studies on unicellular organisms have revealed that nuclear membrane proteins, such as LEM-domain proteins, play a role in maintaining NE integrity. This review summarizes and discusses how nuclear membrane proteins participate in NE integrity.

  4. Biogenesis of sperm acrosome is regulated by pre-mRNA alternative splicing of Acrbp in the mouse

    PubMed Central

    Kanemori, Yoshinori; Koga, Yoshitaka; Sudo, Mai; Kang, Woojin; Kashiwabara, Shin-ichi; Ikawa, Masahito; Hasuwa, Hidetoshi; Nagashima, Kiyoshi; Ishikawa, Yu; Ogonuki, Narumi; Ogura, Atsuo; Baba, Tadashi

    2016-01-01

    Proper biogenesis of a sperm-specific organelle, the acrosome, is essential for gamete interaction. An acrosomal matrix protein, ACRBP, is known as a proacrosin-binding protein. In mice, two forms of ACRBP, wild-type ACRBP-W and variant ACRBP-V5, are generated by pre-mRNA alternative splicing of Acrbp. Here, we demonstrate the functional roles of these two ACRBP proteins. ACRBP-null male mice lacking both proteins showed a severely reduced fertility, because of malformation of the acrosome. Notably, ACRBP-null spermatids failed to form a large acrosomal granule, leading to the fragmented structure of the acrosome. The acrosome malformation was rescued by transgenic expression of ACRBP-V5 in ACRBP-null spermatids. Moreover, exogenously expressed ACRBP-W blocked autoactivation of proacrosin in the acrosome. Thus, ACRBP-V5 functions in the formation and configuration of the acrosomal granule during early spermiogenesis. The major function of ACRBP-W is to retain the inactive status of proacrosin in the acrosome until acrosomal exocytosis. PMID:27303034

  5. A Bioluminescent Cytotoxicity Assay for Assessment of Membrane Integrity Using a Proteolytic Biomarker

    PubMed Central

    Cho, Ming-Hsuang; Niles, Andrew; Huang, Ruili; Inglese, James; Austin, Christopher P.; Riss, Terry; Xia, Menghang

    2008-01-01

    Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic “release” assays which measure the extracellular activities of lactate dehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known toxins from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity. PMID:18400464

  6. Membranous nephropathy: integrating basic science into improved clinical management.

    PubMed

    Cattran, Daniel C; Brenchley, Paul E

    2017-03-01

    Idiopathic membranous nephropathy (INM) remains a common cause of the nephrotic syndrome in adults. The autoimmune nature of IMN was clearly delineated in 2009 with the identification of the glomerular-deposited IgG to be a podocyte receptor, phospholipase A2 receptor (PLA2R) in 70% to 75% of cases. This anti-PLA2R autoantibody, predominantly the IgG4 subclass, has been quantitated in serum using an enzyme-linked immunosorbent assay and has been used to aid diagnosis and monitor response to immunosuppressive therapy. In 2014, a second autoantigen, thrombospondin type 1 domain-containing 7A (THSD7A), was identified. Immunostaining of biopsy specimens has further detected either PLA2R or THSD7A antigen in the deposited immune complexes in 5% to 10% of cases autoantibody seronegative at the time of biopsy. Therefore, the term IMN should now be superseded by the term primary or autoimmune MN (AMN) (anti-PLA2R or anti-THSD7A positive) classifying ∼80% to 90% of cases previously designated IMN. Cases of secondary MN associated with other diseases show much lower association with these autoantibodies, but their true incidence in secondary cases still needs to be defined. How knowledge of the autoimmune mechanism and the sequential measurement of these autoantibodies is likely to change the clinical management and trajectory of AMN by more precisely defining its diagnosis, prognosis, and treatment is discussed. Their application early in the disease course to new and old therapies will provide additional precision to AMN management. We also review innovative therapeutic approaches on the horizon that are expected to lead to our ultimate goal of improved patient care in A(I)MN.

  7. Integrative Signaling Networks of Membrane Guanylate Cyclases: Biochemistry and Physiology

    PubMed Central

    Sharma, Rameshwar K.; Duda, Teresa; Makino, Clint L.

    2016-01-01

    This monograph presents a historical perspective of cornerstone developments on the biochemistry and physiology of mammalian membrane guanylate cyclases (MGCs), highlighting contributions made by the authors and their collaborators. Upon resolution of early contentious studies, cyclic GMP emerged alongside cyclic AMP, as an important intracellular second messenger for hormonal signaling. However, the two signaling pathways differ in significant ways. In the cyclic AMP pathway, hormone binding to a G protein coupled receptor leads to stimulation or inhibition of an adenylate cyclase, whereas the cyclic GMP pathway dispenses with intermediaries; hormone binds to an MGC to affect its activity. Although the cyclic GMP pathway is direct, it is by no means simple. The modular design of the molecule incorporates regulation by ATP binding and phosphorylation. MGCs can form complexes with Ca2+-sensing subunits that either increase or decrease cyclic GMP synthesis, depending on subunit identity. In some systems, co-expression of two Ca2+ sensors, GCAP1 and S100B with ROS-GC1 confers bimodal signaling marked by increases in cyclic GMP synthesis when intracellular Ca2+ concentration rises or falls. Some MGCs monitor or are modulated by carbon dioxide via its conversion to bicarbonate. One MGC even functions as a thermosensor as well as a chemosensor; activity reaches a maximum with a mild drop in temperature. The complexity afforded by these multiple limbs of operation enables MGC networks to perform transductions traditionally reserved for G protein coupled receptors and Transient Receptor Potential (TRP) ion channels and to serve a diverse array of functions, including control over cardiac vasculature, smooth muscle relaxation, blood pressure regulation, cellular growth, sensory transductions, neural plasticity and memory. PMID:27695398

  8. Microbial responses to membrane cleaning using sodium hypochlorite in membrane bioreactors: Cell integrity, key enzymes and intracellular reactive oxygen species.

    PubMed

    Han, Xiaomeng; Wang, Zhiwei; Wang, Xueye; Zheng, Xiang; Ma, Jinxing; Wu, Zhichao

    2016-01-01

    Sodium hypochlorite (NaClO) is a commonly used reagent for membrane cleaning in membrane bioreactors (MBRs), while it, being a kind of disinfectant (oxidant), may impair viability of microbes or even totally inactivate them upon its diffusion into mixed liquor during membrane cleaning. In this study, we systematically examine the effects of NaClO on microorganisms in terms of microbial cell integrity, metabolism behaviours (key enzymes), and intracellular reactive oxygen species (ROS) under various NaClO concentrations. Different proportions of microbial cells in activated sludge were damaged within several minutes dependent on NaClO dosages (5-50 mg/g-SS), and correspondingly organic matters were released to bulk solution. Inhibition of key enzymes involved in organic matter biodegradation, nitrification and denitrification was observed in the presence of NaClO above 1 mg/g-SS, and thus organic matter and nitrogen removal efficiencies were decreased. It was also demonstrated that intracellular ROS production was increased with the NaClO dosage higher than 1 mg/g-SS, which likely induced further damage to microbial cells.

  9. Coating cells with cationic silica-magnetite nanocomposites for rapid purification of integral plasma membrane proteins.

    PubMed

    Zhang, Wei; Zhao, Chao; Wang, Sheng; Fang, Caiyun; Xu, Yawei; Lu, Haojie; Yang, Pengyuan

    2011-09-01

    This study developed a simple and rapid purification method for plasma membrane with high yields from adherent cells. The plasma membrane (PM) sheets could be absorbed specifically by the cationic silica-magnetite nanocomposites (CSMN) under acidic conditions, and recovered directly in cell-lysis-buffer with no need for precipitation. The binding between CSMN and PM sheets was confirmed by electron microscopy. Western blot analysis demonstrated a >10-fold relative enrichment factor. Up to 422 integral membrane proteins were identified from 10(7) Huh7 cells. Notably, we found 29 Ras family proteins by classification according to their biological functions. The whole enrichment procedure took <30 min. The CSMN-based procedure demonstrates a simple, economical and efficient enrichment of integral PM proteins in proteomic study.

  10. Development of a preprototype thermoelectric integrated membrane evaporation subsystem for water recovery

    NASA Technical Reports Server (NTRS)

    Winkler, H. E.; Roebelen, G. J., Jr.

    1980-01-01

    A three-man urine water recovery preprototype subsystem using a new concept to provide efficient potable water recovery from waste fluids on extended duration space flights has been designed, fabricated, and tested. Low power, compactness, and gravity insensitive operation are featured in this vacuum distillation subsystem that combines a hollow fiber polysulfone membrane evaporator with a thermoelectric heat pump. Application and integration of these key elements have solved problems inherent in previous reclamation subsystem designs. The hollow fiber elements provide positive liquid/gas phase control with no moving parts other than a waste liquid recirculation pump and a product water withdrawal pump. Tubular membranes provide structural integrity, improving on previous flat sheet membrane designs. A thermoelectric heat pump provides latent energy recovery.

  11. Integral membrane proteins in proteomics. How to break open the black box?

    PubMed

    Vit, O; Petrak, J

    2017-02-05

    Integral membrane proteins (IMPs) are coded by 20-30% of human genes and execute important functions - transmembrane transport, signal transduction, cell-cell communication, cell adhesion to the extracellular matrix, and many other processes. Due to their hydrophobicity, low expression and lack of trypsin cleavage sites in their transmembrane segments, IMPs have been generally under-represented in routine proteomic analyses. However, the field of membrane proteomics has changed markedly in the past decade, namely due to the introduction of filter assisted sample preparation (FASP), the establishment of cell surface capture (CSC) protocols, and the development of methods that enable analysis of the hydrophobic transmembrane segments. This review will summarize the recent developments in the field and outline the most successful strategies for the analysis of integral membrane proteins.

  12. Use of pressure-hold test for sterilizing filter membrane integrity in radiopharmaceutical manufacturing.

    PubMed

    Belanger, Anthony P; Byrne, John F; Paolino, Justin M; DeGrado, Timothy R

    2009-11-01

    The bubble point test is the de facto standard for postproduction filter membrane integrity test in the radiopharmaceutical community. However, the bubble point test depends on a subjective visual assessment of bubbling rate that can be obscured by significant diffusive gas flows below the manufacturer's prescribed bubble point. To provide a more objective means to assess filter membrane integrity, this study evaluates the pressure-hold test as an alternative to the bubble point test. In our application of the pressure-hold test, the nonsterile side of the sterilizing filter is pressurized to 85% of the predetermined bubble point with nitrogen, the filter system is closed off from the pressurizing gas and the pressure is monitored over a prescribed time interval. The drop in pressure, which has a known relationship with diffusive gas flow, is used as a quantitative measure of membrane integrity. Characterization of the gas flow vs. pressure relationship of each filter/solution combination provides an objective and quantitative means for defining a critical value of pressure drop over which the membrane is indicated to be nonintegral. The method is applied to sterilizing filter integrity testing associated with the commonly produced radiopharmaceuticals, [(18)F]FDG and [(11)C]PIB. The method is shown to be robust, practical and amenable to automation in radiopharmaceutical manufacturing environments (e.g., hot cells).

  13. Molecular Cloning of Spergen-4, Encoding a Spermatogenic Cell-Specific Protein Associated with Sperm Flagella and the Acrosome Region in Rat Spermatozoa.

    PubMed

    Howida, Ali; Salaheldeen, Elsaid; Iida, Hiroshi

    2016-04-01

    We used a differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene LOC690919 with an open reading frame of 1227-length nucleotides encoding a protein of 409 amino acids. This gene was designated as Spergen-4 (a spermatogenic cell-specific gene-4). Spergen-4 mRNA was highly expressed in testis, and its expression was detected in rat testis starting at three weeks of postnatal development and persisting up to adulthood. Mouse and human orthologs, which lack N-terminal 77 amino acid residues of rat Spegen-4, were found in the database. Immunofluorescence microscopy and immunoblot analysis demonstrated that Spergen-4 was not expressed in spermatogonia, spermatocytes, and round spermatids, but was restrictedly detected at sperm head, cytoplasm, and developing flagella of elongated spermatids in rat testis. In mature spermatozoa, Spergen-4 was detected at the acrosome region as well as the principal piece of flagella. Spergen-4 immunosignal disappeared from sperm heads on acrosome reaction induced by progesterone. These data suggest that Spergen-4 integrated into elongated spermatids during spermiogenesis serves as a constituent for acrosome region and flagella of rat spermatozoa.

  14. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis

    SciTech Connect

    Bricker, T.M.; Boyer, M.J.; Keith, J.; Watson-McKown, R.; Wise, K.S.

    1988-02-01

    Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 (/sup 35/S)methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of (/sup 3/H)palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from /sup 125/I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of (/sup 3/H)palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins.

  15. ERK1/2 mediates sperm acrosome reaction through elevation of intracellular calcium concentration.

    PubMed

    Jaldety, Yael; Breitbart, Haim

    2015-10-01

    Mammalian sperm acquire fertilization capacity after residing in the female reproductive tract for a few hours in a process called capacitation. Only capacitated sperm can bind the zona pellucida (ZP) of the egg and undergo the acrosome reaction, a process that allows penetration and fertilization. Extracellular signal regulated kinase (ERK1/2) mediates signalling in many cell types, however its role in sperm function is largely unknown. Here we show that ERK1/2 is highly phosphorylated/activated after a short incubation of mouse sperm under capacitation conditions and that this phosphorylation is reduced after longer incubation. Further phosphorylation was observed upon addition of crude extract of egg ZP or epidermal growth factor (EGF). The mitogen-activated ERK-kinase (MEK) inhibitor U0126 abolished ERK1/2 phosphorylation, in vitro fertilization rate and the acrosome reaction induced by ZP or EGF but not by the Ca2+-ionophore A23187. Moreover, inhibition of ERK1/2 along the capacitation process diminished almost completely the sperm's ability to go through the acrosome reaction, while inhibition at the end of capacitation attenuated the acrosome reaction rate by only 45%. The fact that the acrosome reaction, induced by the Ca2+ -ionophore A23187, was not inhibited by U0126 suggests that ERK1/2 mediates the acrosome reaction by activating Ca2+ transport into the cell. Direct determination of intracellular [Ca2+] revealed that Ca2+ influx induced by EGF or ZP was completely blocked by U0126. Thus, it has been established that the increase in ERK1/2 phosphorylation/activation in response to ZP or by activation of the EGF receptor (EGFR) by EGF, is a key event for intracellular Ca2+ elevation and the subsequent occurrence of the acrosome reaction.

  16. α-Synuclein Oligomers Induced by Docosahexaenoic Acid Affect Membrane Integrity

    PubMed Central

    Fecchio, Chiara; De Franceschi, Giorgia; Relini, Annalisa; Greggio, Elisa; Dalla Serra, Mauro; Bubacco, Luigi; Polverino de Laureto, Patrizia

    2013-01-01

    A key feature of Parkinson disease is the aggregation of α-synuclein and its intracellular deposition in fibrillar form. Increasing evidence suggests that the pathogenicity of α-synuclein is correlated with the activity of oligomers formed in the early stages of its aggregation process. Oligomers toxicity seems to be associated with both their ability to bind and affect the integrity of lipid membranes. Previously, we demonstrated that α-synuclein forms oligomeric species in the presence of docosahexaenoic acid and that these species are toxic to cells. Here we studied how interaction of these oligomers with membranes results in cell toxicity, using cellular membrane-mimetic and cell model systems. We found that α-synuclein oligomers are able to interact with large and small unilamellar negatively charged vesicles acquiring an increased amount of α-helical structure, which induces small molecules release. We explored the possibility that oligomers effects on membranes could be due to pore formation, to a detergent-like effect or to fibril growth on the membrane. Our biophysical and cellular findings are consistent with a model where α-synuclein oligomers are embedded into the lipid bilayer causing transient alteration of membrane permeability. PMID:24312431

  17. Recovery of Flavonoids from Orange Press Liquor by an Integrated Membrane Process

    PubMed Central

    Cassano, Alfredo; Conidi, Carmela; Ruby-Figueroa, René

    2014-01-01

    Orange press liquor is a by-product generated by the citrus processing industry containing huge amounts of natural phenolic compounds with recognized antioxidant activity. In this work, an integrated membrane process for the recovery of flavonoids from orange press liquors was investigated on a laboratory scale. The liquor was previously clarified by ultrafiltration (UF) in selected operating conditions by using hollow fiber polysulfone membranes. Then, the clarified liquor with a total soluble solids (TSS) content of 10 g·100 g−1 was pre-concentrated by nanofiltration (NF) up to 32 g TSS 100 g−1 by using a polyethersulfone spiral-wound membrane. A final concentration step, up to 47 g TSS 100 g−1, was performed by using an osmotic distillation (OD) apparatus equipped with polypropylene hollow fiber membranes. Suspended solids were completely removed in the UF step producing a clarified liquor containing most part of the flavonoids of the original press liquor due to the low rejection of the UF membrane towards these compounds. Flavanones and anthocyanins were highly rejected by the NF membrane, producing a permeate stream with a TSS content of 4.5 g·100 g−1. An increasing of both the flavanones and anthocyanins concentration was observed in the NF retentate by increasing the volume reduction factor (VRF). The final concentration of flavonoids by OD produced a concentrated solution of interest for nutraceutical and pharmaceutical applications. PMID:25116725

  18. Multichannel detection of ionic currents through two nanopores fabricated on integrated Si3N4 membranes.

    PubMed

    Yanagi, Itaru; Akahori, Rena; Aoki, Mayu; Harada, Kunio; Takeda, Ken-Ichi

    2016-08-16

    Integration of solid-state nanopores and multichannel detection of signals from each nanopore are effective measures for realizing high-throughput nanopore sensors. In the present study, we demonstrated fabrication of Si3N4 membrane arrays and the simultaneous measurement of ionic currents through two nanopores formed in two adjacent membranes. Membranes with thicknesses as low as 6.4 nm and small nanopores with diameters of less than 2 nm could be fabricated using the poly-Si sacrificial-layer process and multilevel pulse-voltage injection. Using the fabricated nanopore membranes, we successfully achieved simultaneous detection of clear ionic-current blockades when single-stranded short homopolymers (poly(dA)60) passed through two nanopores. In addition, we investigated the signal crosstalk and leakage current among separated chambers. When two nanopores were isolated on the front surface of the membrane, there was no signal crosstalk or leakage current between the chambers. However, when two nanopores were isolated on the backside of the Si substrate, signal crosstalk and leakage current were observed owing to high-capacitance coupling between the chambers and electrolysis of water on the surface of the Si substrate. The signal crosstalk and leakage current could be suppressed by oxidizing the exposed Si surface in the membrane chip. Finally, the observed ionic-current blockade when poly(dA)60 passed through the nanopore in the oxidized chip was approximately half of that observed in the non-oxidized chip.

  19. Nanowire-integrated microporous silicon membrane for continuous fluid transport in micro cooling device

    NASA Astrophysics Data System (ADS)

    So, Hongyun; Cheng, Jim C.; Pisano, Albert P.

    2013-10-01

    We report an efficient passive micro pump system combining the physical properties of nanowires and micropores. This nanowire-integrated microporous silicon membrane was created to feed coolant continuously onto the surface of the wick in a micro cooling device to ensure it remains hydrated and in case of dryout, allow for regeneration of the system. The membrane was fabricated by photoelectrochemical etching to form micropores followed by hydrothermal growth of nanowires. This study shows a promising approach to address thermal management challenges for next generation electronic devices with absence of external power.

  20. Nanowire-integrated microporous silicon membrane for continuous fluid transport in micro cooling device

    SciTech Connect

    So, Hongyun; Pisano, Albert P.; Cheng, Jim C.

    2013-10-14

    We report an efficient passive micro pump system combining the physical properties of nanowires and micropores. This nanowire-integrated microporous silicon membrane was created to feed coolant continuously onto the surface of the wick in a micro cooling device to ensure it remains hydrated and in case of dryout, allow for regeneration of the system. The membrane was fabricated by photoelectrochemical etching to form micropores followed by hydrothermal growth of nanowires. This study shows a promising approach to address thermal management challenges for next generation electronic devices with absence of external power.

  1. Influence of ethanolic extract of Tephrosia purpurea Linn. on mast cells and erythrocytes membrane integrity.

    PubMed

    Gokhale, A B; Dikshit, V J; Damre, A S; Kulkarni, K R; Saraf, M N

    2000-08-01

    The ethanolic extract of T. purpurea Linn. was studied for its in vitro effect on rat mast cell degranulation and erythrocyte membrane integrity in vitro. The extract in concentration of 25-200 microg/ml showed a dose-dependant inhibition of rat mast cell degranulation induded by compound 48/80 and egg albumin. T. purpurea extract was found to inhibit haemolysis of erythrocytes induced by hypotonic solution but accelerated haemolysis induced by heat at a concentration of 100 microg/ml. The studies reveal that the ethanolic extract of T. purpurea may inhibit degranulation of mast cells by a mechanism other than membrane stabilization.

  2. Studies on improved integrated membrane-based chromatographic process for bioseparation

    NASA Astrophysics Data System (ADS)

    Xu, Yanke

    To improve protein separation and purification directly from a fermentation broth, a novel membrane filtration-cum-chromatography device configuration having a relatively impermeable coated zone near the hollow fiber module outlet has been developed. The integrated membrane filtration-cum-chromatography unit packed with chromatographic beads on the shell side of the hollow fiber unit enjoys the advantages of both membrane filtration and chromatography; it allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane while leaving the rest of the hollow fiber membrane unaffected. Myoglobin (Mb), bovine serum albumin (BSA) and a-lactalbumin (a-LA) were used as model proteins in binary mixtures. Separation behaviors of binary protein mixtures were studied in devices using either an ultrafiltration (UF) membrane or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, the Mb and a-LA elution profiles for the four consecutive cyclic runs were almost superimposable. Due to the lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem as it is in conventional microfiltration. A mathematical model describing the hydrodynamic and protein loading behaviors of the integrated device using UF membrane with a coated zone was developed. The simulation results for the breakthrough agree well with the experimental breakthrough curves. The optimal length of the coated zone was obtained from the simulation. A theoretical analysis of the protein mass transfer was performed using a diffusion-convection model

  3. Acrosomal reaction of thyone sperm. I. Changes in the sperm head visualized by high resolution video microscopy

    PubMed Central

    1982-01-01

    Structural changes inside the head of Thyone sperm undergoing the acrosomal reaction were followed with a high-resolution, differential interference contrast (DIC) video microscope. The beating sperm, adhering by their midpiece to the cover slip of a wedge perfusion chamber, were activated by a calcium ionophore (20 microM A23187) suspended in sea water containing 50 mM excess CaCl2. Before activation of the sperm, the acrosomal region appears as a 1.1-microM diameter sphere, slightly less dense than the rest of the sperm head. Upon activation, the acrosome pops; the acrosomal region suddenly swells and its refractive index drops. After approximately 1 s, a crescent-shaped periacrosomal cup appears behind the acrosomal vacuole. In the next several seconds, the cup loses more refractive index and expands forward as the acrosomal process extends. The acrosomal vacuole becomes smaller, but without appreciable drop in refractive index. These observations, coupled with the behavior of the extending acrosomal process reported in the companion paper, and in electron microscopy (EM) and early physiological studies, suggest that the acrosomal process is extended by a combination of the explosive polymerization of actin and the osmotic swelling of the periacrosomal cup material. In this paper, we also consider the meaning of the enhanced DIC image seen in the high-resolution video microscope, and discuss the reliability of measurements on small linear dimensions made with the DIC microscope. PMID:6811599

  4. Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm

    PubMed Central

    1995-01-01

    The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin. PMID:7822422

  5. Spatial differences in an integral membrane proteome detected in laser capture microdissected samples.

    PubMed

    Wang, Zhen; Han, Jun; Schey, Kevin L

    2008-07-01

    The combination of laser capture microdissection and mass spectrometry represents a powerful technology for studying spatially resolved proteomes. Moreover, the compositions of integral membrane proteomes have rarely been studied in a spatially resolved manner. In this study, ocular lens tissue was carefully dissected by laser capture microdissection and conditions for membrane protein enrichment, trypsin digestion, and mass spectrometry analysis were optimized. Proteomic analysis allowed the identification of 170 proteins, 136 of which were identified with more than one peptide match. Spatial differences in protein expression were observed between cortical and nuclear samples. In addition, the spatial distribution of post-translational modifications to lens membrane proteins, such as the lens major intrinsic protein AQP0, were investigated and regional differences were measured for AQP0 C-terminal phosphorylation and truncation.

  6. Effect of sinus membrane perforation on dental implant integration: a retrospective study on 128 patients.

    PubMed

    Oh, Eric; Kraut, Richard A

    2011-02-01

    A common complication of sinus augmentation is perforation of the sinus membrane during augmentation and/or implant placement. This retrospective study examines the effect of sinus membrane perforation with regard to graft survival and implant integration. A total of 175 sinuses were augmented with 115 of the membranes being reported intact at the time of surgery. A total of three infections occurred in patients who sustained perforated sinuses and one infection occurred in a patient who had an intact sinus. All four infections resolved after culture sensitivity and placement of the patient on an appropriate antibiotic for 10 days. Of 438 dental implants placed in the augmented sinuses, five implants failed, four of which were associated with perforated sinuses and and which was not associated with a perforated grafted sinus.

  7. Acrosome-specific gene AEP1: identification, characterization and roles in spermatogenesis.

    PubMed

    Luk, John M; Lee, Nikki P Y; Shum, Cathy K; Lam, Brian Y; Siu, Annie F M; Che, Chi-Ming; Tam, Po-Chor; Cheung, Annie N Y; Yang, Z M; Lin, Yi-Nan; Matzuk, Martin M; Lee, Kai-Fai; Yeung, William S B

    2006-12-01

    Spermatogenesis is a tightly regulated process leading to the development of spermatozoa. To elucidate the molecular spermatogenic mechanisms, we identified an acrosome-specific gene AEP1 in spermatids, which is located in rat chromosome 17p14 with a transcript size of 3,091 bp encoding a signal peptide, zinc finger-like motif, coiled-coil region, several predicted glycosylation and phosphorylation sites. Northern blot and RT-PCR analyses revealed the restricted expression of AEP1 to the testis only. In postnatal rat testes, AEP1 mRNA became detectable from postnatal 25 dpp (round spermatids) and onwards. By using in situ hybridization (ISH) and flow cytometry-fluorescent ISH, only the haploid spermatids yielded the positive AEP1 signal. Immunohistochemistry showed that AEP1 was expressed in the acrosomal cap of late-staged germ cells in rat testis, and co-localized with the acrosomal marker, peanut agglutinin. The spatial expression of AEP1 immunoreactivity in testis was conserved among diverse mammalian species (rat, pig, monkey, human). To further study its roles in spermatogenesis, we showed AEP1 and beta-actin was associated together in complex by co-immunoprecipitation in adult germ cells and by immunofluorescence assay in isolated spermatozoon. In human testes diagnosed with hypospermatogenesis, lower expression of AEP1 was observed, whereas there was no detectable signal in undescended testes. In short, AEP1 is an evolutionary-conserved acrosome-specific gene and likely functions in acrosome-cap formation.

  8. Localization of Tektin 1 at both acrosome and flagella of mouse and bull spermatozoa.

    PubMed

    Oiki, Sayoko; Hiyama, Erina; Gotoh, Takafumi; Iida, Hiroshi

    2014-02-01

    Tektins (TEKTs) are constitutive filamentous proteins of microtubules in cilia, flagella, basal bodies, and centrioles. In mammals, five TEKTs (TEKT1, 2, 3, 4, and 5) have been identified in testis and spermatozoa. With the exception of TEKT1, these TEKTs have been reported to be present in spermatozoa with predominant localization at the peri-axoneme structures of flagella, i.e., mitochondria and outer dense fibers. In the present study, we produced an antibody against TEKT1 to examine the localization of TEKT1 in mouse, bull, and rat spermatozoa. By immunoblot analyses and immunofluorescence microscopy, we found TEKT1 to be present in sperm flagella and at the apical region of acrosome cap in spermatozoa of all these species. Acrosome-associated TEKT1 disappeared after in vitro acrosome reaction in mouse spermatozoa. These observations suggest another potential role for TEKT1 as a cytoskeletal element in the sperm head, or as a molecule involved in acrosome-related phenomena, such as acrosome reaction.

  9. Ultrastructural study on the fertilisation process in sturgeon (Acipenser), function of acrosome and prevention of polyspermy.

    PubMed

    Psenicka, Martin; Rodina, Marek; Linhart, Otomar

    2010-01-01

    Sturgeon gametes differ from other fish in that their spermatozoa possess acrosome with finger-like posterolateral projections, which undergo exocytosis and filament formation, whereas eggs possess numerous micropyles. The fertilisation process in Acipenser baerii was investigated by fluorescence and electron microscopy. A suitable activation solution containing 2.5 mM CaCl(2), 15 mM Tris, pH 10 was found for detailed description of acrosomal reaction. The acrosome reaction includes the formation of a spear-like fertilisation filament coming from three endonuclear canals and implantation fossa through the acrosomes. It can accelerate the process of polyspermy prevention. Another unique feature of the acrosome was an anchor-like opening of the posterolateral projections. Mature eggs of A. baerii possessed 2-10 micropyles in the animal pole region. The eggs consisted of three principal layers and an outermost jelly coat blocking micropyle, and a layer of cortical granules in unfertilised eggs. With the exposure to freshwater, the jelly like layer separated from the egg surface, whereas the cortical granules swelled. No change between the layers of fertilised and unfertilised eggs, apart from the generation of an increasing perivitelline space by dissolution of the cortical granules, had been observed after the fusion of spermatozoon with an egg. A fertilisation cone blocked a fusion of other spermatozoa with cytoplasmatic projection in the fertilised micropyle.

  10. The opioid peptide beta-endorphin stimulates acrosome reaction in human spermatozoa.

    PubMed

    Urizar-Arenaza, I; Estomba, H; Muñoa-Hoyos, I; Matorras, R; Esposito, A; Candenas, L; Pinto, F M; Valdivia, A; Irazusta, J; Subirán, N

    2016-01-01

    The acrosome reaction occurs in vivo following sperm capacitation and is essential for the acquisition of sperm fertilization ability. However, little is known about the molecular identity of the physiological acrosome reaction regulators. In addition to progesterone, which is produced by cumulus oophorus cells and known to regulate acrosome reaction by activating the specific calcium channel CatSper, endogenous opioid peptides such as beta-endorphin and met-enkephalin are present at high concentrations in the follicular fluid suggesting that the opioid system may be involved in the mechanisms regulating the acrosome reaction in humans. By using Reverse Transcription-PCR, western blot and immunofluorescence approaches, we described the presence and localization of the beta-endorphin precursor, pro-opiomelanocortinin the middle section and in flagellum of human spermatozoa, and inside the seminiferous tubules of human testis. Flow cytometry and intracellular calcium analyses showed that beta-endorphin causes an inversely dose-dependent increase in the percentage of acrosome-reacted sperm cells by a calcium-independent protein kinase C pathway. These findings are important for future studies of sperm physiology and provide new insight into the function of the opioid system as a target of fertility management.

  11. α-SNAP Prevents Docking of the Acrosome during Sperm Exocytosis because It Sequesters Monomeric Syntaxin

    PubMed Central

    Rodríguez, Facundo; Bustos, Matías A.; Zanetti, María N.; Ruete, María C.; Mayorga, Luis S.; Tomes, Claudia N.

    2011-01-01

    α-SNAP has an essential role in membrane fusion that consists of bridging cis SNARE complexes to NSF. α-SNAP stimulates NSF, which releases itself, α-SNAP, and individual SNAREs that subsequently re-engage in the trans arrays indispensable for fusion. α-SNAP also binds monomeric syntaxin and NSF disengages the α-SNAP/syntaxin dimer. Here, we examine why recombinant α-SNAP blocks secretion in permeabilized human sperm despite the fact that the endogenous protein is essential for membrane fusion. The only mammalian organism with a genetically modified α-SNAP is the hyh mouse strain, which bears a M105I point mutation; males are subfertile due to defective sperm exocytosis. We report here that recombinant α-SNAP-M105I has greater affinity for the cytosolic portion of immunoprecipitated syntaxin than the wild type protein and in consequence NSF is less efficient in releasing the mutant. α-SNAP-M105I is a more potent sperm exocytosis blocker than the wild type and requires higher concentrations of NSF to rescue its effect. Unlike other fusion scenarios where SNAREs are subjected to an assembly/disassembly cycle, the fusion machinery in sperm is tuned so that SNAREs progress uni-directionally from a cis configuration in resting cells to monomeric and subsequently trans arrays in cells challenged with exocytosis inducers. By means of functional and indirect immunofluorescense assays, we show that recombinant α-SNAPs — wild type and M105I — inhibit exocytosis because they bind monomeric syntaxin and prevent this SNARE from assembling with its cognates in trans. Sequestration of free syntaxin impedes docking of the acrosome to the plasma membrane assessed by transmission electron microscopy. The N-terminal deletion mutant α-SNAP-(160–295), unable to bind syntaxin, affects neither docking nor secretion. The implications of this study are twofold: our findings explain the fertility defect of hyh mice and indicate that assembly of SNAREs in trans complexes is

  12. The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis.

    PubMed

    Sánchez-Alcázar, José A; Rodríguez-Hernández, Angeles; Cordero, Mario D; Fernández-Ayala, Daniel J M; Brea-Calvo, Gloria; Garcia, Katherina; Navas, Plácido

    2007-07-01

    It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic microtubule network possesses a novel organization, whose nucleation appears independent of conventional gamma-tubulin ring complex containing structures. Our analysis suggests that microtubules are closely associated with the plasma membrane, forming a cortical ring or cellular "cocoon". Concomitantly other components of the cytoskeleton, such as actin and cytokeratins disassemble. We found that colchicine-mediated disruption of apoptotic microtubule network results in enhanced plasma membrane permeability and secondary necrosis, suggesting that the reformation of a microtubule cytoskeleton plays an important role in preserving plasma membrane integrity during apoptosis. Significantly, cells induced to enter apoptosis in the presence of the pan-caspase inhibitor z-VAD, nevertheless form microtubule-like structures suggesting that microtubule formation is not dependent on caspase activation. In contrast we found that treatment with EGTA-AM, an intracellular calcium chelator, prevents apoptotic microtubule network formation, suggesting that intracellular calcium may play an essential role in the microtubule reformation. We propose that apoptotic microtubule network is required to maintain plasma membrane integrity during the execution phase of apoptosis.

  13. Direct effects of ionizing radiation on integral membrane proteins. Noncovalent energy transfer requires specific interpeptide interactions

    SciTech Connect

    Jhun, E.; Jhun, B.H.; Jones, L.R.; Jung, C.Y. )

    1991-05-25

    The 12 transmembrane alpha helices (TMHs) of human erythrocyte glucose transporter were individually cut by pepsin digestion as membrane-bound 2.5-3.5-kDa peptide fragments. Radiation-induced chemical degradation of these fragments showed an average target size of 34 kDa. This is 10-12 x larger than the average size of an individual TMH, demonstrating that a significant energy transfer occurs among these TMHs in the absence of covalent linkage. Heating this TMH preparation at 100{degree}C for 15 min reduced the target size to 5 kDa or less, suggesting that the noncovalent energy transfer requires specific helix-helix interactions. Purified phospholamban, a small (6-kDa) integral membrane protein containing a single TMH, formed a pentameric assembly in sodium dodecyl sulfate. The chemical degradation target size of this phospholamban pentamer was 5-6 kDa, illustrating that not all integral membrane protein assemblies permit intersubunit energy transfer. These findings together with other published observations suggest strongly that significant noncovalent energy transfer can occur within the tertiary and quaternary structure of membrane proteins and that as yet undefined proper molecular interactions are required for such covalent energy transfer. Our results with pepsin-digested glucose transporter also illustrate the importance of the interhelical interaction as a predominating force in maintaining the tertiary structure of a transmembrane protein.

  14. Integral Membrane Protein Sorting to Vacuoles in Plant Cells: Evidence for Two Pathways

    PubMed Central

    Jiang, Liwen; Rogers, John C.

    1998-01-01

    Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of α-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of γ-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of γ-TIP did not alter this traffic. In contrast, the α-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and α-TIP protein colocalized in them with the α-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV. PMID:9832548

  15. Integration of micro-filtration into osmotic membrane bioreactors to prevent salinity build-up.

    PubMed

    Wang, Xinhua; Yuan, Bo; Chen, Yao; Li, Xiufen; Ren, Yueping

    2014-09-01

    The high salinity remains as one of major obstacles of the osmotic membrane bioreactor (OMBR). In this study, a new pathway was explored to prevent the salinity build-up by integrating the micro-filtration (MF) membrane to the OMBR (MF-OMBR). The results indicated that the salinity characterized by conductivity in the MF-OMBR was effectively alleviated and controlled at a lower value of about 5 mS/cm, and the stable flux of forward osmosis (FO) membrane correspondingly increased to approximately 5.5L/(m(2)h). Besides, the addition of MF membrane in the OMBR could increase the total organic carbon (TOC) and ammonium nitrogen (NH3-N) removals due to the activated sludge by improving the microbial activity. The membrane fouling especially the reversible fouling in the MF-OMBR was severer compared to that in the conventional OMBR, which resulted in a lower water flux than the expectation due to the increase of filtration resistance and external concentration polarization.

  16. Comparison of different methods for assessment of sperm concentration and membrane integrity with bull semen.

    PubMed

    Anzar, Muhammad; Kroetsch, Tom; Buhr, Mary M

    2009-01-01

    Assessing semen quality is crucially important for the exploitation of genetically superior sires in an artificial insemination (AI) program. In this study, we compare modern and conventional techniques to estimate bovine sperm concentration and membrane integrity. First, the NucleoCounter SP-100 was validated for sperm concentration and provided statistically reliable and repeatable estimates among aliquots and replicates of 25 fresh ejaculates. Sperm concentrations in 78 ejaculates were then determined with hemacytometer, flow cytometer, and NucleoCounter SP-100 and were significantly correlated (P < .001), with regression coefficients among these 3 techniques close to 1 (P < .01). However, the sperm concentration determined by hemacytometer was lower (P < .01) than by flow cytometer and NucleoCounter SP-100. Forty frozen-thawed semen samples were then assessed for sperm concentration and membrane integrity with hemacytometer, flow cytometer and NucleoCounter SP-100. Significant relationships were found for sperm concentration determined by hemacytometer and NucleoCounter SP-100 and for sperm membrane integrity determined by flow cytometer and NucleoCounter SP-100 (P < .01). Finally, the standard curves of sperm concentrations in 6 spectrophotometers, comparing optical density against counts drawn by hemacytometer and NucleoCounter SP-100 (n = 94 fresh ejaculates) showed different (P < .01) intercepts and regression coefficients (linear, quadratic, cubic). It was calculated that a breeding station can improve its production potential by 13% with the use of NucleoCounter SP-100 instead of hemacytometer for calibration of spectrophotometers. Flow cytometer and NucleoCounter SP-100 can be used with equal confidence to estimate sperm concentration and membrane integrity in domestic animals and human semen.

  17. An integrated membrane sub-harmonic Schottky diode mixers at 340GHz

    NASA Astrophysics Data System (ADS)

    Wang, Junlong; Yang, Dabao; Xing, Dong; Liang, Shixiong; Zhang, Lisen; Zhao, Xiangyang; Feng, Zhihong

    2015-11-01

    This paper presents a sub-harmonic mixer operating over the spectral band 332-348 GHz. The mixers employ integrated GaAs membrane Schottky diode technology. The simulated results show that the conversion loss of the mixer is below dB in the band from 333 GHz to 347 GHz with a local oscillator power requirement of 5mW.The minimum is 8.2dB at 344GHz.

  18. Irvalec Inserts into the Plasma Membrane Causing Rapid Loss of Integrity and Necrotic Cell Death in Tumor Cells

    PubMed Central

    Molina-Guijarro, José M.; Macías, Álvaro; García, Carolina; Muñoz, Eva; García-Fernández, Luis F.; David, Miren; Núñez, Lucía; Martínez-Leal, Juan F.; Moneo, Victoria; Cuevas, Carmen; Lillo, M. Pilar; Villalobos Jorge, Carlos; Valenzuela, Carmen; Galmarini, Carlos M.

    2011-01-01

    Irvalec is a marine-derived antitumor agent currently undergoing phase II clinical trials. In vitro, Irvalec induces a rapid loss of membrane integrity in tumor cells, accompanied of a significant Ca2+ influx, perturbations of membrane conductivity, severe swelling and the formation of giant membranous vesicles. All these effects are not observed in Irvalec-resistant cells, or are significantly delayed by pretreating the cells with Zn2+. Using fluorescent derivatives of Irvalec it was demonstrated that the compound rapidly interacts with the plasma membrane of tumor cells promoting lipid bilayer restructuration. Also, FRET experiments demonstrated that Irvalec molecules localize in the cell membrane close enough to each other as to suggest that the compound could self-organize, forming supramolecular structures that likely trigger cell death by necrosis through the disruption of membrane integrity. PMID:21556352

  19. High-fat Diet Accelerates Intestinal Tumorigenesis Through Disrupting Intestinal Cell Membrane Integrity

    PubMed Central

    Park, Mi-Young; Kim, Min Young; Seo, Young Rok; Kim, Jong-Sang; Sung, Mi-Kyung

    2016-01-01

    Background: Excess energy supply induces chronic low-grade inflammation in association with oxidative stress in various tissues including intestinal epithelium. The objective of this study was to investigate the effect of high-fat diet (HFD) on intestinal cell membrane integrity and intestinal tumorigenesis in ApcMin/+ mice. Methods: Mice were fed with either normal diet (ND) or HFD for 12 weeks. The number of intestinal tumors were counted and biomarkers of endotoxemia, oxidative stress, and inflammation were determined. Changes in intestinal integrity was measured by fluorescein isothiocyanate (FITC)-dextran penetration and membrane gap junction protein expression. Results: HFD group had significantly higher number of tumors compared to ND group (P < 0.05). Blood total antioxidant capacity was lower in HFD group, while colonic 8-hydroxy-2′-deoxyguanosine level, a marker of oxidative damage, was higher in HFD group compared to that of ND group (P < 0.05). The penetration of FITC-dextran was substantially increased in HFD group (P < 0.05) while the expressions of membrane gap junction proteins including zonula occludens-1, claudin-1, and occludin were lower in HFD group (P < 0.05) compared to those in ND group. Serum concentration of lipopolysaccharide (LPS) receptor (CD14) and colonic toll-like receptor 4 (a LPS receptor) mRNA expression were significantly higher in HFD group than in ND group (P < 0.05), suggesting that significant endotoxemia may occur in HFD group due to the increased membrane permeability. Serum interleukin-6 concentration and myeloperoxidase activity were also higher in HFD group compared to those of ND group (P < 0.05). Conclusions: HFD increases oxidative stress disrupting intestinal gap junction proteins, thereby accelerating membrane permeability endotoxemia, inflammation, and intestinal tumorigenesis. PMID:27390738

  20. Tris buffer improves fluorescence yield of ram spermatozoa when evaluating membrane integrity.

    PubMed

    Yániz, Jesús Luis; Mateos, José Angel; Santolaria, Pilar

    2012-04-01

    This study was designed to evaluate the effect of various buffers on the fluorescence signal intensity of two fluorochromes (IP and CFDA) when used to assess the membrane integrity of ram sperm. Second ejaculates (18) from nine adult males were collected using an artificial vagina and diluted in either MOPS, TRIS, TES, HEPES, citrate, or phosphate-based extenders. Semen samples were stored at 15°C and the membrane integrity was assessed within the first 24 h of storage. Mean fluorescence intensity (FI) of PI- and CDFA-labeled sperm heads and fluorescence background noise (FBN) were determined quantitatively using Image J software. Fluorescence contrast (FC) was expressed as the difference between FI and FBN. Significantly, higher FI and FC were recorded when TRIS diluent was used, rather than the other diluents, both in the propidium- and fluorescein-labeled cells. The citrate and phosphate-based extenders showed intermediate results of FC between those of TRIS and zwitterionic (MOPS, TES and HEPES) groups for the PI-labeled sperm. However, in the CFDA-labeled sperm, the lower values of FC were obtained in the citrate and phosphate groups due to increased levels of FBN. For the membrane-damaged sperm, fluorescent labeling was limited to the sperm heads when TRIS-buffer was used, whereas in the other groups, the sperm tail was also frequently observed. It was concluded that TRIS buffer solution markedly increases the fluorescence yield of IP/CFDA-labeled sperm cells in the ram and that this should be considered when evaluating their membrane integrity.

  1. The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly

    PubMed Central

    1993-01-01

    Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F- actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization. PMID:8432731

  2. A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis.

    PubMed

    McPartlin, L A; Littell, J; Mark, E; Nelson, J L; Travis, A J; Bedford-Guaus, S J

    2008-03-15

    Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating medium) yielded high rates of protein tyrosine phosphorylation. Either HCO3(-) or BSA was required to support these changes, with the combination of both providing the most intense results. When a membrane-permeable form of cAMP and a phosphodiesterase inhibitor (IBMX) were added to MW in the absence of HCO3(-) and BSA, the tyrosine phosphorylation results obtained in our capacitating conditions could not be replicated, suggesting either effects apart from cAMP were responsible for tyrosine phosphorylation, or that stallion sperm might respond differently to these reagents as compared to sperm from other mammals. Sperm incubation in capacitating conditions was also associated with high percentages (Pacrosomal exocytosis upon exposure to progesterone (44.6%) or calcium ionophore (51.6%), as compared to sperm incubated in medium devoid of BSA and NaHCO3. Our results were novel in that we report protein tyrosine phosphorylation in stallion sperm incubated in defined conditions, coupled with significant percentages of acrosome reacted sperm. The continuation of these studies might help to elucidate the conditions and pathways supporting sperm capacitation in the horse.

  3. Surface characteristics of implants influence their bone integration after simultaneous placement of implant and GBR membrane.

    PubMed

    Lima, Luiz A; Fuchs-Wehrle, Anita M; Lang, Niklaus P; Hämmerle, Christoph H F; Liberti, Edson; Pompeu, Eduardo; Todescan, José H

    2003-12-01

    The purpose of this study was to evaluate the influence of titanium surface characteristics on bone integration of implants, and to describe the pattern of peri-implant tissue healing after simultaneous implant placement and guided bone regeneration. In four healthy mongrel dogs mandibular premolars were extracted. Two weeks following full mouth prophylaxis and 4 months after extractions, simultaneous membrane and implant surgeries were performed. Efforts were made to produce bony defects with dimensions of 7 x 7 x 7 mm. Into these, 24 standard ITI implants (diameter = 4.1 mm; length = 8 mm) with either a titanium plasma-sprayed (TPS) or a machined surface (MS) were placed. Although implants were inserted 4 mm into cancellous bone, difficulties in achieving optimal primary stability were encountered. All dogs were maintained on a soft diet. Chlorhexidine rinses were performed three times a week. Full mouth prophylaxis was performed every 2 weeks. In the case of membrane exposure, the membranes were removed prematurely (4-6 or 14-15 weeks after surgery). Two dogs were sacrificed at 16 weeks and two at 24 weeks after surgery. Nondecalcified histologic sections were processed and histometric analyses were carried out. When membranes were removed after 4-6 weeks, a vertical bone growth (VB) of 45-61% of the original defect was noted. After membrane removal at 14-15 weeks, similar VB was observed. However, if membranes were left in situ for 24 weeks, VB was between 79% and 96%. In this group of sites, the VB was 66% at 16 weeks and 86% at 24 weeks. Osseointegration in the regenerated bone area ranged from 12% to 32% for the TPS and from 0.0% to 3.6% for the MS implants at 16 and 24 weeks combined. Osseointegration in the pristine host bone area ranged from 16% to 35% for the TPS and from 0.0% to 11% for the MS sites at 16 and 24 weeks. In conclusion, the fraction of implant-bone integration was much higher in the pristine bone compared to that in the regenerated bone

  4. Conformational Stability and Pathogenic Misfolding of the Integral Membrane Protein PMP22

    PubMed Central

    2016-01-01

    Despite broad biochemical relevance, our understanding of the physiochemical reactions that limit the assembly and cellular trafficking of integral membrane proteins remains superficial. In this work, we report the first experimental assessment of the relationship between the conformational stability of a eukaryotic membrane protein and the degree to which it is retained by cellular quality control in the secretory pathway. We quantitatively assessed both the conformational equilibrium and cellular trafficking of 12 variants of the α-helical membrane protein peripheral myelin protein 22 (PMP22), the intracellular misfolding of which is known to cause peripheral neuropathies associated with Charcot–Marie–Tooth disease (CMT). We show that the extent to which these mutations influence the energetics of Zn(II)-mediated PMP22 folding is proportional to the observed reduction in cellular trafficking efficiency. Strikingly, quantitative analyses also reveal that the reduction of motor nerve conduction velocities in affected patients is proportional to the extent of the mutagenic destabilization. This finding provides compelling evidence that the effects of these mutations on the energetics of PMP22 folding lie at the heart of the molecular basis of CMT. These findings highlight conformational stability as a key factor governing membrane protein biogenesis and suggest novel therapeutic strategies for CMT. PMID:26102530

  5. Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography.

    PubMed

    Nunes, Catherine; Sousa, Angela; Nunes, José C; Morão, António M; Sousa, Fani; Queiroz, João A

    2014-06-01

    The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial-scale systems aiming at plasmid DNA purification.

  6. Evaluation of hydrogen production and internal resistance in forward osmosis membrane integrated microbial electrolysis cells.

    PubMed

    Lee, Mi-Young; Kim, Kyoung-Yeol; Yang, Euntae; Kim, In S

    2015-01-01

    In order to enhance hydrogen production by facilitated proton transport through a forward osmosis (FO) membrane, the FO membrane was integrated into microbial electrolysis cells (MECs). An improved hydrogen production rate was obtained in the FO-MEC (12.5±1.84×10(-3)m(3)H2/m(3)/d) compared to that of the cation exchange membrane (CEM) - MEC (4.42±0.04×10(-3)m(3)H2/m(3)/d) during batch tests (72h). After an internal resistance analysis, it was confirmed that the enhanced hydrogen production in FO-MEC was attributed to the smaller charge transfer resistance than in the CEM-MEC (90.3Ω and 133.4Ω respectively). The calculation of partial internal resistance concluded that the transport resistance can be substantially reduced by replacing a CEM with a FO membrane; decrease of the resistance from 0.069Ωm(2) to 5.99×10(-4)Ωm(2).

  7. The role of forward osmosis and microfiltration in an integrated osmotic-microfiltration membrane bioreactor system.

    PubMed

    Luo, Wenhai; Hai, Faisal I; Kang, Jinguo; Price, William E; Nghiem, Long D; Elimelech, Menachem

    2015-10-01

    This study investigates the performance of an integrated osmotic and microfiltration membrane bioreactor (O/MF-MBR) system for wastewater treatment and reclamation. The O/MF-MBR system simultaneously used microfiltration (MF) and forward osmosis (FO) membranes to extract water from the mixed liquor of an aerobic bioreactor. The MF membrane facilitated the bleeding of dissolved inorganic salts and thus prevented the build-up of salinity in the bioreactor. As a result, sludge production and microbial activity were relatively stable over 60 days of operation. Compared to MF, the FO process produced a better permeate quality in terms of nutrients, total organic carbon, as well as hydrophilic and biologically persistent trace organic chemicals (TrOCs). The high rejection by the FO membrane also led to accumulation of hydrophilic and biologically persistent TrOCs in the bioreactor, consequently increasing their concentration in the MF permeate. On the other hand, hydrophobic and readily biodegradable TrOCs were minimally detected in both MF and FO permeates, with no clear difference in the removal efficiencies between two processes.

  8. A Two-Stage Model for Lipid Modulation of the Activity of Integral Membrane Proteins

    PubMed Central

    Dodes Traian, Martín M.; Cattoni, Diego I.; Levi, Valeria; González Flecha, F. Luis

    2012-01-01

    Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes. PMID:22723977

  9. Effect of integral membrane proteins on the lateral mobility of plastoquinone in phosphatidylcholine proteoliposomes

    PubMed Central

    Blackwell, Mary F.; Whitmarsh, John

    1990-01-01

    Pyrene fluorescence quenching by plastoquinone was used to estimate the rate of plastoquinone lateral diffusion in soybean phosphatidylcholine proteoliposomes containing the following integral membrane proteins: gramicidin D, spinach cytochrome bf complex, spinach cytochrome f, reaction centers from Rhodobacter sphaeroides, beef heart mitochondrial cytochrome bc1, and beef heart mitochondrial cytochrome oxidase. The measured plastoquinone lateral diffusion coefficient varied between 1 and 3 · 10-7 cm2 s-1 in control liposomes that lacked protein. When proteins were added, these values decreased: a 10-fold decrease was observed when 16-26% of the membrane surface area was occupied by protein for all the proteins but gramicidin. The larger protein complexes (cytochrome bf, Rhodobacter sphaeroides reaction centers, cytochrome bc1, and cytochrome oxidase), whose hydrophobic volumes were 15-20 times as large as that of cytochrome f and the gramicidin transmembrane dimer, were 15-20 times as effective in decreasing the lateral-diffusion coefficient over the range of concentrations studied. These proteins had a much stronger effect than that observed for bacteriorhodopsin in fluorescence photobleaching recovery measurements. The effect of high-protein concentrations in gramicidin proteoliposomes was in close agreement with fluorescence photobleaching measurements. The results are compared with the predictions of several theoretical models of lateral mobility as a function of integral membrane concentration. PMID:19431774

  10. A membrane-integrated advanced scheme for treatment of industrial wastewater: dynamic modeling towards scale up.

    PubMed

    Kumar, Ramesh; Pal, Parimal

    2013-08-01

    Modeling and simulation was carried out for an advanced membrane-integrated hybrid treatment process that ensures reuse of water with conversion and recovery of ammoniacal nitrogen as value-added struvite fertilizer from coke wastewater. While toxic cyanide was largely removed in a pre-chemical treatment unit using Fenton's reagents under optimized conditions, more than 95% of NH4(+)-N could be recovered as a valuable by-product called struvite through addition of appropriate doses of magnesium and phosphate salts. Water could be turned reusable through a polishing treatment by nanofiltration membranes in a largely fouling free membrane module following a biodegradation step. Mathematical modeling of such an integrated process was done with Haldane-Andrew approach for the associated microbial degradation of phenol by Pseudomonas putida. Residual NH4(+) was degraded by nitrification and denitrification following the modified Monod kinetics. The model could successfully predict the plant performance as reflected in reasonably low relative error (0.03-0.18) and high Willmott d-index (>0.98).

  11. FAD oxidizes the ERO1-PDI electron transfer chain: The role of membrane integrity

    SciTech Connect

    Papp, Eszter; Nardai, Gabor; Mandl, Jozsef; Banhegyi, Gabor; Csermely, Peter . E-mail: csermely@puskin.sote.hu

    2005-12-16

    The molecular steps of the electron transfer in the endoplasmic reticulum from the secreted proteins during their oxidation are relatively unknown. We present here that flavine adenine dinucleotide (FAD) is a powerful oxidizer of the oxidoreductase system, Ero1 and PDI, besides the proteins of rat liver microsomes and HepG2 hepatoma cells. Inhibition of FAD transport hindered the action of FAD. Microsomal membrane integrity was mandatory for all FAD-related oxidation steps downstream of Ero1. The PDI inhibitor bacitracin could inhibit FAD-mediated oxidation of microsomal proteins and PDI, but did not hinder the FAD-driven oxidation of Ero1. Our data demonstrated that Ero1 can utilize FAD as an electron acceptor and that FAD-driven protein oxidation goes through the Ero1-PDI pathway and requires the integrity of the endoplasmic reticulum membrane. Our findings prompt further studies to elucidate the membrane-dependent steps of PDI oxidation and the role of FAD in redox folding.

  12. Ethylhexylglycerin Impairs Membrane Integrity and Enhances the Lethal Effect of Phenoxyethanol

    PubMed Central

    Langsrud, Solveig; Steinhauer, Katrin; Lüthje, Sonja; Weber, Klaus; Goroncy-Bermes, Peter; Holck, Askild L.

    2016-01-01

    Preservatives are added to cosmetics to protect the consumers from infections and prevent product spoilage. The concentration of preservatives should be kept as low as possible and this can be achieved by adding potentiating agents. The aim of the study was to investigate the mechanisms behind potentiation of the bactericidal effect of a commonly used preservative, 2-phenoxyethanol (PE), by the potentiating agent ethylhexylglycerin (EHG). Sub-lethal concentrations of EHG (0.075%) and PE (0.675%) in combination led to rapid killing of E. coli (> 5 log reduction of cfu after 30 min), leakage of cellular constituents, disruption of the energy metabolism, morphological deformities of cells and condensation of DNA. Used alone, EHG disrupted the membrane integrity even at low concentrations. In conclusion, sub-lethal concentrations of EHG potentiate the effect of PE through damage of the cell membrane integrity. Thus, adding EHG to PE in a 1:9 ratio has a similar effect on membrane damage and bacterial viability as doubling the concentration of PE. This study provides insight about the mechanism of action of a strong potentiating agent, EHG, which is commonly used in cosmetics together with PE. PMID:27783695

  13. Structure determination of an integral membrane protein at room temperature from crystals in situ

    SciTech Connect

    Axford, Danny; Foadi, James; Hu, Nien-Jen; Choudhury, Hassanul Ghani; Iwata, So; Beis, Konstantinos; Evans, Gwyndaf; Alguel, Yilmaz

    2015-05-14

    The X-ray structure determination of an integral membrane protein using synchrotron diffraction data measured in situ at room temperature is demonstrated. The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.

  14. Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa.

    PubMed

    Cocchia, N; Pasolini, M P; Mancini, R; Petrazzuolo, O; Cristofaro, I; Rosapane, I; Sica, A; Tortora, G; Lorizio, R; Paraggio, G; Mancini, A

    2011-04-15

    New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H(2)O(2) and O(2) production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: native semen (control 1); native semen diluted 1:3 with Kenney semen extender (control 2); spermatozoa diluted after centrifugation in extender without (control 3) or with SOD at 25 IU/ml (experimental 1) or 50 IU/ml (experimental 2). Each sample was analyzed for motility, viability and acrosome status, immediately after semen preparation and again after storage at 5 °C for 24 h, 48 h and 7 2h. Acrosome integrity was evaluated by Chlortetracycline (CTC) and Fluorescent-labeled peanut lectin agglutinin (PNA-FITC conjugated staining). A proteomic approach of quantifying extracellular signal regulated kinase (ERK) was also evaluated as an indirect indicator of oxidative stress. In all samples sperm progressive motility and sperm acrosomal integrity showed a significant reduction between fresh and cooled spermatozoa at 24 h, 48 h and 72 h. Quality parameters of sperm were significantly higher (Progressive Motility P < 0.01; Viability P < 0.001) in aliquots supplemented with SOD. ERK phosphorylation was statistically higher (P < 0.01) in aliquots without SOD. The Authors concluded that addition of SOD to semen extenders improves the quality of chilled equine semen and reduces ERK activation.

  15. Synthesis gas production by mixed conducting membranes with integrated conversion into liquid products

    DOEpatents

    Nataraj, Shankar; Russek, Steven Lee; Dyer, Paul Nigel

    2000-01-01

    Natural gas or other methane-containing feed gas is converted to a C.sub.5 -C.sub.19 hydrocarbon liquid in an integrated system comprising an oxygenative synthesis gas generator, a non-oxygenative synthesis gas generator, and a hydrocarbon synthesis process such as the Fischer-Tropsch process. The oxygenative synthesis gas generator is a mixed conducting membrane reactor system and the non-oxygenative synthesis gas generator is preferably a heat exchange reformer wherein heat is provided by hot synthesis gas product from the mixed conducting membrane reactor system. Offgas and water from the Fischer-Tropsch process can be recycled to the synthesis gas generation system individually or in combination.

  16. Efficient Nanoporous Silicon Membranes for Integrated Microfluidic Separation and Sensing Systems

    SciTech Connect

    Ileri, N; L?tant, S E; Britten, J; Nguyen, H; Larson, C; Zaidi, S; Palazoglu, A; Faller, R; Tringe, J W; Stroeve, P

    2009-04-06

    Nanoporous devices constitute emerging platforms for selective molecule separation and sensing, with great potential for high throughput and economy in manufacturing and operation. Acting as mass transfer diodes similar to a solid-state device based on electron conduction, conical pores are shown to have superior performance characteristics compared to traditional cylindrical pores. Such phenomena, however, remain to be exploited for molecular separation. Here we present performance results from silicon membranes created by a new synthesis technique based on interferometric lithography. This method creates millimeter sized planar arrays of uniformly tapered nanopores in silicon with pore diameter 100 nm or smaller, ideally-suited for integration into a multi-scale microfluidic processing system. Molecular transport properties of these devices are compared against state-of-the-art polycarbonate track etched (PCTE) membranes. Mass transfer rates of up to fifteen-fold greater than commercial sieve technology are obtained. Complementary results from molecular dynamics simulations on molecular transport are reported.

  17. Highly efficient metal grating coupler for membrane-based integrated photonics.

    PubMed

    Dolores-Calzadilla, V; Heiss, D; Smit, M

    2014-05-01

    We present the design of a metal grating coupler compatible with membrane photonic circuit platforms, consisting of a buried metal grating and a metal mirror. A nonapodized design provides a fiber-to-chip coupling efficiency at 1.55 μm up to 73%, whereas apodized designs show theoretical efficiencies as high as 89%, with a 3 dB bandwidth of 61 and 78 nm, respectively. An important advantage is that the coupling efficiency is independent from the underlying layer stack, enabling its use in diverse applications. For example, a thin buffer layer is required to achieve optical coupling for the heterogeneous integration of III-V and silicon photonics, whereas a thick buffer is of interest for thermal isolation between photonic membranes and CMOS circuits.

  18. Structure determination of an integral membrane protein at room temperature from crystals in situ.

    PubMed

    Axford, Danny; Foadi, James; Hu, Nien Jen; Choudhury, Hassanul Ghani; Iwata, So; Beis, Konstantinos; Evans, Gwyndaf; Alguel, Yilmaz

    2015-06-01

    The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.

  19. Flow Cytometric Assessment of Membrane Integrity of Ethanol-Stressed Oenococcus oeni Cells

    PubMed Central

    Graça da Silveira, M.; Vitória San Romão, M.; Loureiro-Dias, Maria C.; Rombouts, Frans M.; Abee, Tjakko

    2002-01-01

    The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into the mechanisms involved in ethanol toxicity and tolerance in this organism. Exposure to ethanol resulted in an increase in the permeability of the cytoplasmic membrane, enhancing passive proton influx and concomitant loss of intracellular material (absorbing at 260 nm). Cells grown in the presence of 8% (vol/vol) ethanol revealed adaptation to ethanol stress, since these cells showed higher retention of compounds absorbing at 260 nm. Moreover, for concentrations higher than 10% (vol/vol), lower rates of passive proton influx were observed in these ethanol-adapted cells, especially at pH 3.5. The effect of ethanol on O. oeni cells was studied as the ability to efficiently retain carboxyfluorescein (cF) as an indicator of membrane integrity and enzyme activity and the uptake of propidium iodide (PI) to assess membrane damage. Flow cytometric analysis of both ethanol-adapted and nonadapted cells with a mixture of the two fluorescent dyes, cF and PI, revealed three main subpopulations of cells: cF-stained intact cells; cF- and PI-stained permeable cells, and PI-stained damaged cells. The subpopulation of O. oeni cells that maintained their membrane integrity, i.e., cells stained only with cF, was three times larger in the population grown in the presence of ethanol, reflecting the protective effect of ethanol adaptation. This information is of major importance in studies of microbial fermentations in order to assign bulk activities measured by classical methods to the very active cells that are effectively responsible for the observations. PMID:12450832

  20. Trehalose enhances osmotic tolerance and suppresses lysophosphatidylcholine-induced acrosome reaction in ram spermatozoon.

    PubMed

    Ahmad, E; Naseer, Z; Aksoy, M; Küçük, N; Uçan, U; Serin, I; Ceylan, A

    2015-09-01

    This study was aimed to investigate the influence of trehalose on osmotic tolerance and the ability of ram spermatozoon to undergo acrosome reaction induced by lysophosphatidylcholine (LPC). In experiment 1, the diluted ejaculates were exposed to anisosmotic fructose solutions (70, 500, 750 and 1000 mOsm l(-1) ) with or without 50 mm trehalose. The presence of trehalose in hyperosmotic conditions enhanced (P < 0.05) the percentage of live, live-intact and intact spermatozoa. Similarly, trehalose enhanced (P < 0.05) the live and live-intact spermatozoa during hypo-osmotic conditions. In experiment 2, the centrifuged ejaculates were diluted with TCG only or TCG containing either 50 or 100 mm trehalose. The acrosome reaction was induced by LPC. The percentage of acrosome-reacted spermatozoon was less (P < 0.05) in trehalose-supplemented groups compared to control. In experiment 3, the ejaculates were cryopreserved in an extender containing 0 mm (control), 50 mm or 100 mm trehalose. Supplementation of extender with trehalose, either 50 mm or 100 mm, enhanced the cryosurvival rate (P < 0.05) compared to the control. In conclusion, the presence of trehalose in anisosmotic conditions enhances the osmotic tolerance, cryosurvival rate of ram spermatozoon and suppresses their ability to undergo LPC and cryo-induced acrosome reaction.

  1. Integrated forward osmosis-membrane distillation process for human urine treatment.

    PubMed

    Liu, Qianliang; Liu, Caihong; Zhao, Lei; Ma, Weichao; Liu, Huiling; Ma, Jun

    2016-03-15

    This study demonstrated a forward osmosis-membrane distillation (FO-MD) hybrid system for real human urine treatment. A series of NaCl solutions at different concentrations were adopted for draw solutions in FO process, which were also the feed solutions of MD process. To establish a stable and continuous integrated FO-MD system, individual FO process with different NaCl concentrations and individual direct contact membrane distillation (DCMD) process with different feed temperatures were firstly investigated separately. Four stable equilibrium conditions were obtained from matching the water transfer rates of individual FO and MD processes. It was found that the integrated system is stable and sustainable when the water transfer rate of FO subsystem is equal to that of MD subsystem. The rejections to main contaminants in human urine were also investigated. Although individual FO process had relatively high rejection to Total Organic Carbon (TOC), Total Nitrogen (TN) and Ammonium Nitrogen (NH4(+)-N) in human urine, these contaminants could also accumulate in draw solution after long term performance. The MD process provided an effective rejection to contaminants in draw solution after FO process and the integrated system revealed nearly complete rejection to TOC, TN and NH4(+)-N. This work provided a potential treatment process for human urine in some fields such as water regeneration in space station and water or nutrient recovery from source-separated urine.

  2. The Crystal Structure of an Integral Membrane Fatty Acid α-Hydroxylase*

    PubMed Central

    Zhu, Guangyu; Koszelak-Rosenblum, Mary; Connelly, Sara M.; Dumont, Mark E.; Malkowski, Michael G.

    2015-01-01

    Neuronal electrical impulse propagation is facilitated by the myelin sheath, a compact membrane surrounding the axon. The myelin sheath is highly enriched in galactosylceramide (GalCer) and its sulfated derivative sulfatide. Over 50% of GalCer and sulfatide in myelin is hydroxylated by the integral membrane enzyme fatty acid 2-hydroxylase (FA2H). GalCer hydroxylation contributes to the compact nature of the myelin membrane, and mutations in FA2H result in debilitating leukodystrophies and spastic paraparesis. We report here the 2.6 Å crystal structure of sphingolipid α-hydroxylase (Scs7p), a yeast homolog of FA2H. The Scs7p core is composed of a helical catalytic cap domain that sits atop four transmembrane helices that anchor the enzyme in the endoplasmic reticulum. The structure contains two zinc atoms coordinated by the side chains of 10 highly conserved histidines within a dimetal center located near the plane of the cytosolic membrane. We used a yeast genetic approach to confirm the important role of the dimetal-binding histidines in catalysis and identified Tyr-322 and Asp-323 as critical determinants involved in the hydroxylase reaction. Examination of the Scs7p structure, coupled with molecular dynamics simulations, allowed for the generation of a model of ceramide binding to Scs7p. Comparison of the Scs7p structure and substrate-binding model to the structure of steroyl-CoA desaturase revealed significant differences in the architecture of the catalytic cap domain and location of the dimetal centers with respect to the membrane. These observations provide insight into the different mechanisms of substrate binding and recognition of substrates by the hydroxylase and desaturase enzymes. PMID:26515067

  3. Integrated atomic force microscopy techniques for analysis of biomaterials: Study of membrane proteins

    NASA Astrophysics Data System (ADS)

    Connelly, Laura S.

    Atomic Force Microscopy (AFM) is the prominent techniques for structural studies of biological materials in physiological relevant fluidic environments. AFM has been used to resolve the three-dimensional (3D) surface structure of cells, membranes, and proteins structures. Ion channels, formed by membrane proteins, are the key structures that control the activity of all living systems. This dissertation focuses on the structural evaluation of membrane proteins through atomic force microscopy. In Part I, AFM is utilized to study one of the most prominent medical issues facing our society, Alzheimer's Disease (AD). AD is a misfolded protein disease characterized by the accumulation of beta-amyloid (Abeta) peptide as senile plaques, progressive neurodegeneration, and memory loss. Recent evidence suggests that AD pathology is linked to the destabilization of cellular ionic homeostasis mediated by toxic channel structures composed of Abeta peptides. Selectively engineered sequences of Abeta were examined by AFM to elucidate the substructures and thus activity Abeta channels. Key residues were evaluated with the intent better understand the exact nature by which these pores conduct electrical and molecular signals, which could aid in identifying potential therapeutic targets for the prevention/treatment of AD. Additionally, AFM was used to analyze brain derived Abeta and newly developed pharmacological agents to study membranes and Abeta. Part II, presents a novel technology that incorporates electrophysiology into the AFM interface, enabling simultaneous imaging and complementary conductance measurements. The activity of ion channels is studied by various techniques, including patch clamp, free standing lipid bilayers, droplet interface bilayers, and supported lipid bilayers. However, direct correlation with channel structures has remained a challenge. The integrated atomic force microscopy system presented offers a solution to this challenge. The functionality of the

  4. Lipid Bilayer-Bound Conformation of an Integral Membrane Beta Barrel Protein by Multidimensional MAS NMR

    PubMed Central

    Eddy, Matthew T.; Su, Yongchao; Silvers, Robert; Andreas, Loren; Clark, Lindsay; Wagner, Gerhard; Pintacuda, Guido; Emsley, Lyndon; Griffin, Robert G.

    2015-01-01

    The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms 2-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for 13C line width and less than 0.5 ppm 15N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported

  5. Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR.

    PubMed

    Eddy, Matthew T; Su, Yongchao; Silvers, Robert; Andreas, Loren; Clark, Lindsay; Wagner, Gerhard; Pintacuda, Guido; Emsley, Lyndon; Griffin, Robert G

    2015-04-01

    The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5-0.3 ppm for (13)C line widths and <0.5 ppm (15)N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported

  6. The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex Function

    PubMed Central

    Ho, Albert K.; Raczniak, Gregory A.; Ives, Eric B.; Wente, Susan R.

    1998-01-01

    Integral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered the nup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23°C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethal nup116-C phenotype was conducted. One gene (designated SNL1 for suppressor of nup116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity of gle2–1 and nic96-G3 mutant cells. The nic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporin nup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function

  7. Insights into the structure and function of membrane-integrated processive glycosyltransferases

    SciTech Connect

    Bi, Yunchen; Hubbard, Caitlin; Purushotham, Pallinti; Zimmer, Jochen

    2015-09-02

    Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. In this paper, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin, alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).

  8. Insights into the structure and function of membrane-integrated processive glycosyltransferases

    PubMed Central

    Bi, Yunchen; Hubbard, Caitlin; Purushotham, Pallinti; Zimmer, Jochen

    2015-01-01

    Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. Here, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin, alginate, hyaluronan and poly-N-acetylglucosamine (PNAG). PMID:26342143

  9. An ELISA for screening hybridoma cultures for monoclonal antibodies against a detergent solubilized integral membrane protein.

    PubMed

    Noteboom, W D; Knurr, K E; Kim, H S; Richmond, W G; Martin, A P; Vorbeck, M L

    1984-12-14

    A method is described for the binding of a detergent solubilized integral membrane protein to polystyrene immunoassay plates. Addition of Bouin's fluid, a histochemical fixative, to wells of plates containing the detergent solubilized antigen, followed by low speed centrifugation, is sufficient to promote binding of antigen in the presence of Triton X-100 concentrations as high as 1.75%. The binding of antigen is rapid and the entire binding procedure, including removal of fixative and washing of the plates, can be accomplished in less than 15 min. Immunological specificity of the bound antigen is retained. This method has been used to effectively screen hybridoma cultures for specific antibodies.

  10. The role and importance of cofilin in human sperm capacitation and the acrosome reaction.

    PubMed

    Megnagi, Bar; Finkelstein, Maya; Shabtay, Ortal; Breitbart, Haim

    2015-12-01

    The spermatozoon is capable of fertilizing an oocyte only after undergoing several biochemical changes in the female reproductive tract, referred to as capacitation. The capacitated spermatozoon interacts with the egg zona pellucida and undergoes the acrosome reaction, which enables its penetration into the egg and fertilization. Actin dynamics play a major role throughout all these processes. Actin polymerization occurs during capacitation, whereas prior to the acrosome reaction, F-actin must undergo depolymerization. In the present study, we describe the presence of the actin-severing protein, cofilin, in human sperm. We examined the function and regulation of cofilin during human sperm capacitation and compared it to gelsolin, an actin-severing protein that was previously investigated by our group. In contrast to gelsolin, we found that cofilin is mainly phosphorylated/inhibited at the beginning of capacitation, and dephosphorylation occurs towards the end of the process. In addition, unlike gelsolin, cofilin phosphorylation is not affected by changing the cellular levels of PIP2. Despite the different regulation of the two proteins, the role of cofilin appears similar to that of gelsolin, and its activation leads to actin depolymerization, inhibition of sperm motility and induction of the acrosome reaction. Moreover, like gelsolin, cofilin translocates from the tail to the head during capacitation. In summary, gelsolin and cofilin play a similar role in F-actin depolymerization prior to the acrosome reaction but their pattern of phosphorylation/inactivation during the capacitation process is different. Thus, for the sperm to achieve high levels of F-actin along the capacitation process, both proteins must be inactivated at different times and, in order to depolymerize F-actin, both must be activated prior to the acrosome reaction.

  11. Anandamide (arachidonylethanolamide), a brain cannabinoid receptor agonist, reduces sperm fertilizing capacity in sea urchins by inhibiting the acrosome reaction.

    PubMed Central

    Schuel, H; Goldstein, E; Mechoulam, R; Zimmerman, A M; Zimmerman, S

    1994-01-01

    Anandamide (arachidonylethanolamide) is an endogenous cannabinoid receptor agonist in mammalian brain. Sea urchin sperm contain a high-affinity cannabinoid receptor similar to the cannabinoid receptor in mammalian brain. (-)-delta 9-Tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. We now report that anandamide produces effects similar to those previously obtained with THC in Strongylocentrotus purpuratus in reducing sperm fertilizing capacity and inhibiting the egg jelly-stimulated acrosome reaction. Arachidonic acid does not inhibit the acrosome reaction under similar conditions. The adverse effects of anandamide on sperm fertilizing capacity and the acrosome reaction are reversible. The receptivity of unfertilized eggs to sperm and sperm motility are not impaired by anandamide. Under conditions where anandamide completely blocks the egg jelly-stimulated acrosome reaction, it does not inhibit the acrosome reaction artificially initiated by ionomycin, which promotes Ca2+ influx, and nigericin, which activates K+ channels in sperm. These findings provide additional evidence that the cannabinoid receptor in sperm plays a role in blocking the acrosome reaction, indicate that anandamide or a related molecule may be the natural ligand for the cannabinoid receptor in sea urchin sperm, and suggest that binding of anandamide to the cannabinoid receptor modulates stimulus-secretion-coupling in sperm by affecting an event prior to ion channel opening. PMID:8052642

  12. An enzymatic immunoassay microfluidics integrated with membrane valves for microsphere retention and reagent mixing.

    PubMed

    Ren, Li; Wang, Jian-Chun; Liu, Wenming; Tu, Qin; Liu, Rui; Wang, Xueqin; Xu, Juan; Wang, Yaolei; Zhang, Yanrong; Li, Li; Wang, Jinyi

    2012-05-15

    The present study presents a new microfluidic device integrated with pneumatic microvalves and a membrane mixer for enzyme-based immunoassay of acute myocardial infarction (AMI) biomarkers, namely, myoglobin, and heart-type fatty acid binding protein (H-FABP). Superparamagnetic microspheres with carboxyl groups on their surfaces were used as antibody solid carriers. A membrane mixer consisting of four ψ-type membrane valves was assembled under the reaction chamber for on-chip performing microsphere trapping and reagent mixing. The entire immunoassay process, including microsphere capture, reagent input, mixing, and subsequent reaction, was accomplished on the device either automatically or manually. The post-reaction substrate resultant was analyzed using a microplate reader. The results show that the average absorbance value is correlated with the concentration of cardiac markers, in agreement with the results obtained using a conventional microsphere-based immunoassay; this indicated that the proposed on-chip immunoassay protocol could be used to detect both myoglobin and H-FABP. The minimum detectable concentration is 5 ng/mL for myoglobin and 1 ng/mL for H-FABP.

  13. Integral membrane protease fibroblast activation protein sensitizes fibrosarcoma to chemotherapy and alters cell death mechanisms.

    PubMed

    Baird, Sarah K; Rigopoulos, Angela; Cao, Diana; Allan, Laura; Renner, Christoph; Scott, Fiona E; Scott, Andrew M

    2015-11-01

    Fibroblast activation protein (FAP), an integral membrane serine protease, is found on fibro- and osteo-sarcoma and on myofibroblasts in epithelial carcinoma, but rarely on other adult tissue. FAP has been demonstrated to be an excellent target for tumor imaging in clinical trials, and antibodies and other FAP-targeting drugs are in development. Here we have shown that FAP overexpression increased the growth of HT1080 fibrosarcoma cells in vitro and in vivo, and found that the expression of FAP affects response to chemotherapy. When treated with doxorubicin, expression of FAP increased susceptibility to the drug. In spite of this, FAP-HT1080 cells had fewer markers of classical apoptosis than HT1080 cells and neither necrosis nor necroptosis were enhanced. However, levels of early mitochondrial and lysosomal membrane permeability markers were increased, and autophagy switched from a protective function in HT1080 cells to part of the cell death mechanism with FAP expression. Therefore, FAP may affect how the tumor responds to chemotherapeutic drugs overall, which should be considered in targeted drug development. The overexpression of FAP also alters cell signaling and responses to the environment in this cell line. This includes cell death mechanisms, changing the response of HT1080 cells to doxorubicin from classical apoptosis to an organelle membrane permeability-dependent form of cell death.

  14. Integrity of Lichen Cell Membranes in Relation to Concentration of Airborne Elements

    PubMed

    Garty; Kloog; Cohen

    1998-02-01

    This study dealt with the impact of air pollution on the integrity of cell membranes in the lichen Ramalina duriaei. The lichen was transplanted from a relatively unpolluted site in Israel to more polluted sites, adjacent to a 40-year-old town, for a period of 10 months. The concentrations of K, B, Al, Cr, Fe, Si, Ti, Zn, P, Ba, Cu, Mg, Na, Pb, Ca, Mn, Sr, and S in lichen thalli were compared with injury caused to the cell membranes. The study indicates that electric conductivity reflects adequately cellular injury to lichen thalli transplanted to sites near a motorway, an oil-fueled power plant and a rural community (a kibbutz). The concentrations of S, B, Al, Cr, Fe, Si, Ti, and Zn correlated with injury in cell membranes of R. duriaei retrieved from the biomonitoring sites, whereas the concentration of K in the thalli correlated inversely with the electric conductivity parameter. Leakage of K from lichen thalli as a result of air pollutants is suggested. This K leakage correlates positively with concentrations of S and Cr in transplants of R. duriaei to the Ashdod region.

  15. Reversible air electrodes integrated with an anion-exchange membrane for secondary air batteries

    NASA Astrophysics Data System (ADS)

    Fujiwara, Naoko; Yao, Masaru; Siroma, Zyun; Senoh, Hiroshi; Ioroi, Tsutomu; Yasuda, Kazuaki

    Reversible air electrodes integrated with a polymer electrolyte membrane have been proposed for use in rechargeable metal-air batteries or unitized regenerative fuel cells to reduce the impact of atmospheric carbon dioxide. Reversible air electrodes were prepared with an anion-exchange membrane (AEM) as a polymer electrolyte membrane and platinum-based catalysts. The AEM at the interface between the alkaline electrolyte and the air electrode layer plays major roles in AEM-type air electrodes as follows: it blocks (a) the permeation of cations in the alkaline electrolyte into the air electrode layer to prevent carbonate precipitation, (b) penetration of the alkaline solution itself, and (c) neutralization of the alkaline electrolyte by carbon dioxide, all of which prevent performance degradation of oxygen reactions. Catalysts for decreasing the overvoltage of oxygen reactions were also investigated with the AEM-type air electrode, and the overall efficiency was improved due to a remarkable decrease in the potential for the oxygen evolution reaction with Pt-Ir catalysts.

  16. Amphipathic polymers: tools to fold integral membrane proteins to their active form.

    PubMed

    Pocanschi, Cosmin L; Dahmane, Tassadite; Gohon, Yann; Rappaport, Fabrice; Apell, Hans-Jürgen; Kleinschmidt, Jörg H; Popot, Jean-Luc

    2006-11-28

    Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dimensional structure has proven to be a major challenge. We describe here the use of an amphipathic polymer, amphipol A8-35, as a novel environment that allows both beta-barrel and alpha-helical MPs to fold to their native state, in the absence of detergents or lipids. Amphipols, which are extremely mild surfactants, appear to favor the formation of native intramolecular protein-protein interactions over intermolecular or protein-surfactant ones. The feasibility of the approach is demonstrated using as models OmpA and FomA, two outer membrane proteins from the eubacteria Escherichia coli and Fusobacterium nucleatum, respectively, and bacteriorhodopsin, a light-driven proton pump from the plasma membrane of the archaebacterium Halobacterium salinarium.

  17. The effect of creosote on growth and membrane integrity of the aquatic macrophyte, Myriophyllum sp.

    SciTech Connect

    McCann, J.; Day, K.; Solomon, K.; Greenberg, B.

    1995-12-31

    Creosote is a coal-tar distillate used as a wood preservative on railway ties and dock pilings. Its use in aquatic systems indicates a possible risk to the aquatic community through leaching of creosote components into the water column or sediment. A study has been initiated at the University of Guelph (Ontario, Canada) to determine bioindicators of exposure and effects of creosote on freshwater systems. A laboratory study was conducted to determine the effects of creosote exposure on the rooted, aquatic macrophyte Myriophyllum sp. Using an aseptic culture of Myriophyllum and 3 cm buds grown from single nodes, a growth assay was conducted during which Myriophyllum were exposed for 14 days to concentrations ranging from 0.16 mg/L to 200 mg/L creosote. Growth measurements included: shoot length; number of nodes, buds and roots; total shoot and root length; and growth curves over the exposure period. From the information gathered from the growth assay, 5 creosote concentrations were chosen and used for membrane integrity studies. Myriophyllum were exposed to creosote for either 4 or 12 day periods, after which membrane fluidity was determined by fluorescence depolarization, and electrolyte and K+ leakage were determined by conductivity and atomic absorption spectrophotometry, respectively. The results of both the growth and membrane assays will be discussed.

  18. High Permeate Recovery for Concentrate Reduction by Integrated Membrane Process in Textile Effluent.

    PubMed

    Sudhakar, M; Vijayalakshmi, P; Nilavunesan, D; Thiruvengadaravi, K V; Baskaralingam, P; Sivanesan, S

      The textile dyeing industry consumes a significant amount of high-quality water for processing, which stresses water resources. In recent decades, technologies have been developed to recover water from wastewater. This study describes the high recovery (greater than 92%) of reusable water from an industrial-scale hosiery dye-water recovery facility, consisting of three stages of reverse osmosis and ultrafiltration. The effluent was pre-treated before the membrane process was performed to prevent biofouling. The process performance results in the generation of a consistent water quality that is required for dyeing operations. An average feed flux of 15 l/m(2)h was maintained in the reverse osmosis membrane by regular chemical dosing and cleaning. The integrated membrane process achieved a permeate with a pH of 6.5 and total dissolved solids (TDS) of 160 mg/l, with no other contaminants, which is of sufficient quality for reuse in the cotton hosiery dyeing process.

  19. Influence of estrogenic pesticides on membrane integrity and membrane transfer of monosaccharide into the human red cell

    SciTech Connect

    Ingermann, R.L. )

    1989-09-01

    Some natural and synthetic estrogens inhibit carrier-mediated transport of glucose into human red blood cells and membrane vesicles from the placenta. The inhibitory action of these estrogens on transport appears to be a direct effect at the membrane and does not involve receptor binding and protein synthesis. It is not clear, however, whether such inhibition is a common feature among estrogenic agents. Several chlorinated hydrocarbon pesticides have been shown to possess estrogenic activity. These pesticides could have inhibitory effects on the human sodium-independent glucose transporter. Owing to the apparent importance of this membrane transporter in human tissues, direct interaction of hormones and xenobiotics with the glucose transporter is of fundamental significance. Some pesticides have been shown to alter membrane structure directly and alter the passive permeability of membranes. Whether the estrogenic pesticides influence passive diffusion of sugars across membranes has not been established. Finally, preliminary observations have suggested that some estrogens and pesticides have lytic effects on intact cells. Consequently, this study focuses on the ability of several estrogens and estrogenic pesticides to disrupt the cell membrane, influence the monosaccharide transporter, and alter the rate of monosaccharide permeation through the membrane by simple diffusion.

  20. An integrated process: ester synthesis in an enzymatic membrane reactor and water sorption.

    PubMed

    Trusek-Holownia, Anna; Noworyta, Andrzej

    2007-05-31

    In the case of such reactions as ester synthesis, water is produced during the reaction. Because these reactions are carried out in hydrophobic solvents an additional (water) phase in the system must not be allowed, i.e. the concentration of water saturation in the organic solvent should not be exceeded. In such a case, the reaction kinetics and product equilibrium concentration undergo undesirable changes because of the partition coefficient of the components and hampered process of product separation. Hence, removal of the water produced in the reaction determines whether the process is successful or not. For this purpose, the integrated process with water sorption in the column with molecular sieves was applied. Integration of the process of synthesis and dehydration of a reaction phase, in which a biocatalyst is suspended and not dissolved as in water solutions, requires holding up of the catalyst in the reactor before directing the stream of reaction mixture to dehydration process. This hold-up and a possibility of multiple use of the catalyst may be accomplished by using a separating barrier, e.g. an ultrafiltration membrane or by permanent fixing of the catalyst to the matrix, e.g. a polymeric membrane. The efficiency and activity of a biocatalyst (lipase CAL-B) immobilized on a polymer membrane by sorption and chemical binding, were determined. A subject of study was the synthesis of geranyl acetate, one of the most known aromatic compound. A hydrophobic (polypropylene) matrix was shown to be a much better carrier in the reactions performed in an organic solvent than a hydrophilic (polyamide) membrane being tested. The reaction kinetics of geranyl acetate synthesis with the use of geraniol and acetic acid as substrates, was described by the equation defining the "Ping-Pong Bi Bi" mechanism that was related additionally to the inhibition of a substrate (acetic acid). The following constants of kinetic equation were obtained k(3)(')=0.344 mol g(-1)h(-1), K

  1. Type II integral membrane protein, TM of J paramyxovirus promotes cell-to-cell fusion.

    PubMed

    Li, Zhuo; Hung, Cher; Paterson, Reay G; Michel, Frank; Fuentes, Sandra; Place, Ryan; Lin, Yuan; Hogan, Robert J; Lamb, Robert A; He, Biao

    2015-10-06

    Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin-neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion.

  2. Vitrification of Sperm from Marine Fishes: Effect on Motility and Membrane Integrity.

    PubMed

    Cuevas-Uribe, Rafael; Chesney, Edward J; Daly, Jonathan; Tiersch, Terrence R

    2015-06-01

    Our goal was to develop a standardized approach for sperm vitrification of marine fishes that can be applied generally in aquatic species. The objectives were to: 1) estimate acute toxicity of cryoprotectants over a range of concentrations; 2) evaluate the properties of vitrification solutions (VS); 3) evaluate different thawing solutions, and 4) evaluate sperm quality after thawing by examination of motility and membrane integrity. Sperm were collected from red snapper (Lutjanus campechanus), spotted seatrout (Cynoscion nebulosus), and red drum (Sciaenops ocellatus). A total of 29 combinations of cryoprotectants were evaluated for toxicity and glass formation. Samples were loaded onto 10-µL polystyrene loops and plunged into liquid nitrogen. There was a significant difference (P < 0.05) in post-thaw motility among VS and among species when using the same VS. The sperm in VS of 15% DMSO + 15% ethylene glycol + 10% glycerol + 1% X-1000™ + 1% Z-1000™ had an average post-thaw motility of 58% and membrane integrity of 19% for spotted seatrout, 38% and 9% for red snapper, and 30% and 19% for red drum. Adaptations by marine fish to high osmotic pressures could explain the survival in the high cryoprotectant concentrations. Vitrification offers an alternative to conventional cryopreservation.

  3. Impact of saffron on rat sperm membrane integrity and spermatogenesis status

    PubMed Central

    Vaez, Ahmad; Mardani, Mohammad; Razavi, Shahnaz

    2014-01-01

    Background: Male factor has been considered as a 50% of infertility causes. One of the reasons for poor semen quality is oxidative stress. Saffron and vitamin E as antioxidant agent can be involved in free radical scavenging and improvement of semen quality. Materials and Methods: We divided 30 adult male Wistar rats into saffron (n = 10), vitamin E (n = 10) and control (n = 10) groups randomly. Saffron (100 mg/kg/day), vitamin E (100 mg/kg/day), and distilled water (0.5 ml/day) were fed by gavage to the animals for 60 consecutive days in aforementioned groups. After cervical dislocation, both testes and left epididymis of each animal were removed and the cauda epididymal spermatozoa were aspirated for analysis of sperm parameters. Sperm membrane integrity was assessed by hypo-osmotic swelling test (HOST). In different groups, seminiferous tubule histological assessments were done after Hematoxylin -Eosin staining. Results: The mean percentage of HOST positive sperm was increased in vitamin E and saffron groups as compared to control group. As we can see there was a significant difference among control and experimental groups (P < 0.001); also a significant difference was obtained between vitamin E and saffron groups (P = 0.002). The evaluation of seminiferous tubules has shown no significant differences among groups. Conclusions: The present data suggest that saffron had superior antioxidant properties which can improve sperm parameters and membrane integrity so it can lead to develop fertility potential. PMID:25161993

  4. Membrane and Integrative Nuclear Fibroblastic Growth Factor Receptor (FGFR) Regulation of FGF-23*

    PubMed Central

    Han, Xiaobin; Xiao, Zhousheng; Quarles, L. Darryl

    2015-01-01

    Fibroblastic growth factor receptor 1 (FGFR1) signaling pathways are implicated in the regulation of FGF-23 gene transcription, but the molecular pathways remain poorly defined. We used low molecular weight (LMW, 18 kDa) FGF-2 and high molecular weight (HMW) FGF-2 isoforms, which, respectively, activate cell surface FGF receptors and intranuclear FGFR1, to determine the roles of membrane FGFRs and integrative nuclear FGFR1 signaling (INFS) in the regulation of FGF-23 gene transcription in osteoblasts. We found that LMW-FGF-2 induced NFAT and Ets1 binding to conserved cis-elements in the proximal FGF-23 promoter and stimulated FGF-23 promoter activity through PLCγ/calcineurin/NFAT and MAPK pathways in SaOS-2 and MC3T3-E1 osteoblasts. In contrast, HMW-FGF-2 stimulated FGF-23 promoter activity in osteoblasts through a cAMP-dependent binding of FGFR1 and cAMP-response element-binding protein (CREB) to a conserved cAMP response element (CRE) contiguous with the NFAT binding site in the FGF-23 promoter. Mutagenesis of the NFAT and CRE binding sites, respectively, inhibited the effects of LMW-FGF-2 and HMW-FGF-23 to stimulate FGF-23 promoter activity. FGF-2 activation of both membrane FGFRs and INFS-dependent FGFR1 pathways may provide a means to integrate systemic and local regulation of FGF-23 transcription under diverse physiological and pathological conditions. PMID:25752607

  5. Integration of an interferometric IR absorber into an epoxy membrane based CO2 detector

    NASA Astrophysics Data System (ADS)

    Ashraf, S.; Mattsson, C. G.; Thungström, G.; Rödjegård, H.

    2014-05-01

    Measurements of carbon dioxide levels in the environment are commonly performed by using non-dispersive infrared technology (NDIR). Thermopile detectors are often used in NDIR systems because of their non-cooling advantages. The infrared absorber has a major influence on the detector responsivity. In this paper, the fabrication of a SU-8 epoxy membrane based Al/Bi thermopile detector and the integration of an interferometric infrared absorber structure of wavelength around 4 μm into the detector is reported. The membrane of thermopile detector has been utilized as a dielectric medium in an interferometric absorption structure. By doing so, a reduction in both thermal conductance and capacitance is achieved. In the fabrication of the thermopile, metal evaporation and lift off process had been used for the deposition of serially interconnected Al/Bi thermocouples. Serial resistance of fabricated thermopile was measured as 220 kΩ. The response of fabricated thermopile detector was measured using a visible to infrared source of radiation flux 3.23 mW mm-2. The radiation incident on the detector was limited using a band pass filter of wavelength 4.26 μm in front of the detector. A responsivity of 27.86 V mm2 W-1 at room temperature was achieved using this setup. The fabricated detector was compared to a reference detector with a broad band absorber. From the comparison it was concluded that the integrated interferometric absorber is functioning correctly.

  6. Membrane and integrative nuclear fibroblastic growth factor receptor (FGFR) regulation of FGF-23.

    PubMed

    Han, Xiaobin; Xiao, Zhousheng; Quarles, L Darryl

    2015-04-17

    Fibroblastic growth factor receptor 1 (FGFR1) signaling pathways are implicated in the regulation of FGF-23 gene transcription, but the molecular pathways remain poorly defined. We used low molecular weight (LMW, 18 kDa) FGF-2 and high molecular weight (HMW) FGF-2 isoforms, which, respectively, activate cell surface FGF receptors and intranuclear FGFR1, to determine the roles of membrane FGFRs and integrative nuclear FGFR1 signaling (INFS) in the regulation of FGF-23 gene transcription in osteoblasts. We found that LMW-FGF-2 induced NFAT and Ets1 binding to conserved cis-elements in the proximal FGF-23 promoter and stimulated FGF-23 promoter activity through PLCγ/calcineurin/NFAT and MAPK pathways in SaOS-2 and MC3T3-E1 osteoblasts. In contrast, HMW-FGF-2 stimulated FGF-23 promoter activity in osteoblasts through a cAMP-dependent binding of FGFR1 and cAMP-response element-binding protein (CREB) to a conserved cAMP response element (CRE) contiguous with the NFAT binding site in the FGF-23 promoter. Mutagenesis of the NFAT and CRE binding sites, respectively, inhibited the effects of LMW-FGF-2 and HMW-FGF-23 to stimulate FGF-23 promoter activity. FGF-2 activation of both membrane FGFRs and INFS-dependent FGFR1 pathways may provide a means to integrate systemic and local regulation of FGF-23 transcription under diverse physiological and pathological conditions.

  7. When are bacteria dead? A step towards interpreting flow cytometry profiles after chlorine disinfection and membrane integrity staining.

    PubMed

    Nocker, Andreas; Cheswick, Ryan; Dutheil de la Rochere, Pierre-Marie; Denis, Matthieu; Léziart, Tangui; Jarvis, Peter

    2017-04-01

    Flow cytometry is increasingly employed by drinking water providers. Its use with appropriate fluorescent stains allows the distinction between intact and membrane-damaged bacteria, which makes it ideally suited for assessment of disinfection efficiency. In contrast to plate counting, the technology allows the visualization of the gradual loss of membrane integrity. Although this sensitivity per se is very positive, it creates the problem of how this detailed viability information compares with binary plate counts where a colony is either formed or not. Guidelines are therefore needed to facilitate interpretation of flow cytometry results and to determine a degree of membrane damage where bacteria can be considered 'dead'. In this study we subjected Escherichia coli and environmental microorganisms in real water to increasing chlorine concentrations. Resulting flow cytometric patterns after membrane integrity staining were compared with culturability and in part with redox activity. For laboratory-grown bacteria, culturability was lost at lower disinfectant concentrations than membrane integrity making the latter a conservative viability parameter. No recovery from chlorine was observed for four days. For real water, loss of membrane integrity had to be much more substantial to completely suppress colony formation, probably due to the heterogenic composition of the natural microbial community with different members having different susceptibilities to the disinfectant.

  8. Retained functional integrity of bull spermatozoa after double freezing and thawing using PureSperm density gradient centrifugation.

    PubMed

    Maxwell, W M C; Parrilla, I; Caballero, I; Garcia, E; Roca, J; Martinez, E A; Vazquez, J M; Rath, D

    2007-10-01

    .2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.

  9. MALDI imaging mass spectrometry of integral membrane proteins from ocular lens and retinal tissue.

    PubMed

    Grey, Angus C; Chaurand, Pierre; Caprioli, Richard M; Schey, Kevin L

    2009-07-01

    A tissue preparation protocol for MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry of integral membrane proteins was developed using ocular lens and retinal tissues as model samples. Frozen bovine and human lenses were cryosectioned equatorially or axially at -20 degrees C into 20 mum-thick tissue sections. Lens sections were mounted onto gold-coated MALDI targets by methanol soft-landing to maintain tissue integrity. Tissue sections underwent extensive water washing to deplete the samples of highly abundant water-soluble proteins. Automated matrix deposition was achieved using an acoustic reagent multispotter, with sinapinic acid as matrix and high percentage acetonitrile as solvent, with a center-to-center spot spacing of 200-300 mum. Molecular images of full-length Aquaporin-0 (AQP0) and its most abundant truncation products were obtained from mass spectral data acquired across whole bovine and human lens sections. In equatorial and axial sections of bovine lenses, full-length AQP0 was detected throughout the lens. A truncation product corresponding to AQP0 (1-260) was detected in the bovine lens core at low abundance. In axial lens sections, no antero-posterior variation was detected. In 11 year-old human lens sections, full-length AQP0 was most abundant in the lens periphery, but was detected throughout the lens. The major truncation product, consisting of AQP0 residues 1-246, was absent from the lens periphery and increased in abundance in the lens core. This tissue preparation protocol was then applied to image the distribution of the G-protein coupled receptor, opsin, in the rabbit retina. This protocol has expanded the variety of target analytes which can be detected by MALDI imaging mass spectrometry to include intact integral membrane proteins.

  10. Integration of hollow fiber membranes improves nutrient supply in three-dimensional tissue constructs.

    PubMed

    Bettahalli, N M S; Vicente, J; Moroni, L; Higuera, G A; van Blitterswijk, C A; Wessling, M; Stamatialis, D F

    2011-09-01

    Sufficient nutrient and oxygen transport is a potent modulator of cell proliferation in in vitro tissue-engineered constructs. The lack of oxygen and culture medium can create a potentially lethal environment and limit cellular metabolic activity and growth. Diffusion through scaffold and multi-cellular tissue typically limits transport in vitro, leading to potential hypoxic regions and reduction in the viable tissue thickness. For the in vitro generation of clinically relevant tissue-engineered grafts, current nutrient diffusion limitations should be addressed. Major approaches to overcoming these include culture with bioreactors, scaffolds with artificial microvasculature, oxygen carriers and pre-vascularization of the engineered tissues. This study focuses on the development and utilization of a new perfusion culture system to provide adequate nutrient delivery to cells within large three-dimensional (3D) scaffolds. Perfusion of oxygenated culture medium through porous hollow fiber (HF) integrated within 3D free form fabricated (FFF) scaffolds is proposed. Mouse pre-myoblast (C2C12) cells cultured on scaffolds of poly(ethylene-oxide-terephthalate)-poly(butylene-terephthalate) block copolymer (300PEOT55PBT45) integrated with porous HF membranes of modified poly(ether-sulfone) (mPES, Gambro GmbH) is used as a model system. Various parameters such as fiber transport properties, fiber spacing within a scaffold and medium flow conditions are optimized. The results show that four HF membranes integrated with the scaffold significantly improve the cell density and cell distribution. This study provides a basis for the development of a new HF perfusion culture methodology to overcome the limitations of nutrient diffusion in the culture of large 3D tissue constructs.

  11. In-situ integration of microbial fuel cell with hollow-fiber membrane bioreactor for wastewater treatment and membrane fouling mitigation.

    PubMed

    Tian, Yu; Li, Hui; Li, Lipin; Su, Xinying; Lu, Yaobin; Zuo, Wei; Zhang, Jun

    2015-02-15

    A hollow-fiber membrane bioreactor was integrated with a microbial fuel cell to develop a novel system of MFC-MBR based on the utilization of electricity recovered by the MFC for wastewater treatment improvement and membrane fouling mitigation in the MBR. In this system, a maximum power density of 2.18 W/m(3) and an average voltage output of 0.15 V were achieved at an external resistance of 50 Ω. The removal efficiencies of COD, ammonia nitrogen ( [Formula: see text] ) and total nitrogen (TN) in the MFC-MBR were improved by 4.4%, 1.2% and 10.3%, respectively. It is worth noting that, in addition to reducing the deposition of sludge on the membrane surface by the electric field force, the MFC-MBR also alleviated the membrane fouling by sludge modification. Compared with the control MBR (C-MBR), less loosely bound extracellular polymeric substances (LB-EPS), lower SMPp/SMPc ratio, more homogenized sludge flocs and less filamentous bacteria were obtained in the MFC-MBR, which improved the dewaterability and filterability of the sludge. The cake layer on the membrane formed by the modified sludge was more porous with lower compressibility, significantly enhancing the membrane filterability. A proof of concept of an MFC-MBR was provided and shown to be effective in membrane fouling mitigation with efficient wastewater treatment and energy recovery, demonstrating the feasibility of the minute electricity generated by the MFC for membrane fouling alleviation in the MBR.

  12. Spermatogenesis of the green-lipped mussel Perna viridis with dual patterns of acrosome and tail development in spermatids

    NASA Astrophysics Data System (ADS)

    Reunov, A. A.; Au, D. W. T.; Wu, R. S. S.

    1999-08-01

    Spermatogenesis in the mussel Perna viridis was studied by electron microscopy. Results demonstrated that cytological development in spermatogonia and spermatocytes was similar to that previously described in other Mytilidae. Acrosome formation began with the arising of proacrosomal vesicles in spermatogonia. The abundance of proacrosomal vesicles increased in spermatocytes, which were flagellated. However, during spermiogenesis, dual patterns of acrosome development as well as flagellum development could be found among spermatids in a male gonad. The two lines of acrosome formation in spermatids ultimately gave rise to morphologically similar acrosomes. The two lines of flagellum development in spermatids resulted in the formation of sperm cells with either a typically posteriorly directed tail or an anteriorly directed tail.

  13. Evaluation of mitochondrial function and membrane integrity by dual fluorescent staining for assessment of sperm status in rats.

    PubMed

    Kato, Masashi; Makino, Sachiko; Kimura, Hitoshi; Ota, Takao; Furuhashi, Tadakazu; Nagamura, Yoichi

    2002-02-01

    Dual fluorescent staining (DFS) with calcein acetoxy methyl ester (CAM), which labels the cellular esterase activity that is a major component of energy metabolism in cellular mitochondria, and with ethidium homodimer-1 (EthD-1) was used to evaluate mitochondrial function and membrane integrity in rat spermatozoa. The spermatozoa stained by DFS could be classified into three different populations microscopically when excited at 490 nm after 60 min incubation. 1) Spermatozoa, which were stained with CAM alone and had maintained either mitochondrial function or membrane integrity, were identified as live during incubation. 2) Spermatozoa, which were stained with EthD-1 alone and had lost either mitochondrial function or membrane integrity, were identified as already dead at the beginning of incubation. 3) Spermatozoa, which were stained with both CAM and EthD-1 and had maintained mitochondrial function with membrane breached, were identified as having died during incubation. Two toxicological tests, an in vitro triton X-100 experiment and an in vivo nitrobenzene experiment, were done. All spermatozoa were immobilized and lost either mitochondrial function or membrane integrity by 1.0% triton X-100 treatment. Almost no motile sperm were found at 0.1% in the triton X-100 group and in the groups treated with 60 and 40 mg/kg/day of nitrobenzene, and these spermatozoa maintained their mitochondrial function but had their membrane breached. In conclusion, the DFS procedure, which uses CAM and EthD-1, can clearly and visually identify the population of viable and dead spermatozoa simultaneously by fluorescence microscopy in rats. This is a useful technique to characterize sperm status, which is determined by the mitochondrial function assessed by CAM and membrane integrity evaluated by EthD-1.

  14. Spacesuit Water Membrane Evaporator Integration with the ISS Extravehicular Mobility Unit

    NASA Technical Reports Server (NTRS)

    Margiott, Victoria; Boyle, Robert

    2014-01-01

    NASA has developed a Solid Water Membrane Evaporation (SWME) to provide cooling for the next generation spacesuit. The current spacesuit team has looked at this technology from the standpoint of using the ISS EMU to demonstrate the SWME technology while EVA, and from the standpoint of augmenting EMU cooling in the case of a fouled EMU cooling system. One approach to increasing the TRL of the system is to incorporate this hardware with the existing EMU. Several integration issues were addressed to support a potential demonstration of the SWME with the existing EMU. Systems analysis was performed to assess the capability of the SWME to maintain crewmember cooling and comfort as a replacement for sublimation. The materials of the SWME were reviewed to address compatibility with the EMU. Conceptual system placement and integration with the EMU via an EVA umbilical system to ensure crew mobility and Airlock egress were performed. A concept of operation for EVA use was identified that is compatible with the existing system. This concept is extensible as a means to provide cooling for the existing EMU. The cooling system of one of the EMUs on orbit has degraded, with the root cause undetermined. Should there be a common cause resident on ISS, this integration could provide a means to recover cooling capability for EMUs on orbit.

  15. Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking

    SciTech Connect

    Bleil, J.D.; Wassarman, P.M. )

    1990-07-01

    During the process of fertilization in mammals, sperm bind in a relatively species-specific manner to the zona pellucida (ZP) of ovulated eggs. ZP3, a glycoprotein found in the mouse egg zona pellucida, serves as receptor for sperm during gamete adhesion. We report here that a Mr 56,000 protein found on mouse sperm has properties expected for a sperm component that recognizes and binds to ZP3. This sperm protein is radiolabeled preferentially by a photoactivatable heterobifunctional crosslinker (Denny-Jaffee reagent) covalently linked to purified ZP3, binds very tightly to ZP3-affinity columns, and is localized to heads of acrosome-intact but not acrosome-reacted sperm. These and other findings suggest that this protein may be a ZP3-binding protein that, together with the sperm receptor, supports species-specific binding of mouse sperm to unfertilized eggs.

  16. Effects of acrosomal conditions of frozen-thawed spermatozoa on the results of artificial insemination in Japanese Black cattle.

    PubMed

    Kishida, Kazumi; Sakase, Mitsuhiro; Minami, Kenta; Arai, Miyuki M; Syoji, Reiko; Kohama, Namiko; Akiyama, Takayuki; Oka, Akio; Harayama, Hiroshi; Fukushima, Moriyuki

    2015-01-01

    The purposes of this study were to examine the relationship between male artificial insemination (AI) fertility and sperm acrosomal conditions assessed by new and conventional staining techniques and to identify possible reproductive dysfunctions causing low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions in Japanese Black bulls. We investigated individual differences among bulls in the results concerning (1) acrosomal conditions of frozen-thawed spermatozoa as assessed by not merely peanut agglutinin-lectin staining (a conventional staining technique) but also immunostaining of acrosomal tyrosine-phosphorylated proteins (a new staining technique), (2) routine AI using frozen-thawed spermatozoa as assessed by pregnancy diagnosis, (3) in vivo fertilization of frozen-thawed spermatozoa and early development of fertilized eggs as assessed by superovulation/AI-embryo collection tests and (4) in vitro fertilization of frozen-thawed spermatozoa with oocytes. The percentages of frozen-thawed spermatozoa with normal acrosomal conditions assessed by the abovementioned staining techniques were significantly correlated with the conception rates of routine AI, rates of transferable embryos in superovulation/AI-embryo collection tests and in vitro fertilization rates. These results are consistent with new suggestions that the distribution of acrosomal tyrosine-phosphorylated proteins as well as the acrosomal morphology of frozen-thawed spermatozoa are AI fertility-associated markers that are valid for the prediction of AI results and that low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions result from reproductive dysfunctions in the processes between sperm insemination into females and early embryo development, probably failed fertilization of frozen-thawed spermatozoa with oocytes.

  17. Plasma Membrane Integrity and Survival of Melanoma Cells After Nanosecond Laser Pulses

    PubMed Central

    Pérez-Gutiérrez, Francisco G.; Camacho-López, Santiago; Evans, Rodger; Guillén, Gabriel; Goldschmidt, Benjamin S.; Viator, John A.

    2010-01-01

    Circulating tumor cells (CTCs) photoacoustic detection systems can aid clinical decision-making in the treatment of cancer. Interaction of melanin within melanoma cells with nanosecond laser pulses generates photoacoustic waves that make its detection possible. This study aims at: (1) determining melanoma cell survival after laser pulses of 6 ns at λ = 355 and 532 nm; (2) comparing the potential enhancement in the photoacoustic signal using λ = 355 nm in contrast with λ = 532 nm; (3) determining the critical laser fluence at which melanin begins to leak out from melanoma cells; and (4) developing a time-resolved imaging (TRI) system to study the intracellular interactions and their effect on the plasma membrane integrity. Monolayers of melanoma cells were grown on tissue culture-treated clusters and irradiated with up to 1.0 J/cm2. Surviving cells were stained with trypan blue and counted using a hemacytometer. The phosphate buffered saline absorbance was measured with a nanodrop spectrophotometer to detect melanin leakage from the melanoma cells post-laser irradiation. Photoacoustic signal magnitude was studied at both wavelengths using piezoelectric sensors. TRI with 6 ns resolution was used to image plasma membrane damage. Cell survival decreased proportionally with increasing laser fluence for both wavelengths, although the decrease is more pronounced for 355 nm radiation than for 532 nm. It was found that melanin leaks from cells equally for both wavelengths. No significant difference in photoacoustic signal was found between wavelengths. TRI showed clear damage to plasma membrane due to laser-induced bubble formation. PMID:20589533

  18. The immiscible cholesterol bilayer domain exists as an integral part of phospholipid bilayer membranes.

    PubMed

    Raguz, Marija; Mainali, Laxman; Widomska, Justyna; Subczynski, Witold K

    2011-04-01

    Electron paramagnetic resonance (EPR) spin-labeling methods were used to study the organization of cholesterol and phospholipids in membranes formed from Chol/POPS (cholesterol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine) mixtures, with mixing ratios from 0 to 3. It was confirmed using the discrimination by oxygen transport and polar relaxation agent accessibility methods that the immiscible cholesterol bilayer domain (CBD) was present in all of the suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST) in the POPS membrane. The behavior of phospholipid molecules was monitored with phospholipid analogue spin labels (n-PCs), and the behavior of cholesterol was monitored with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio of ~2. Additionally, EPR spectra for n-PC, ASL, and CSL indicated that both phospholipids and cholesterol exist in these suspensions in the lipid-bilayer-like structures. EPR spectral characteristics of n-PCs (spin labels located in the phospholipid cholesterol bilayer, outside the CBD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CBD forms an integral part of the phospholipid bilayer when formed from a Chol/POPS mixture up to a mixing ratio of ~2. Interestingly, CSL in cholesterol alone (without phospholipids) when suspended in buffer does not detect formation of bilayer-like structures. A broad, single-line EPR signal is given, similar to that obtained for the dry film of cholesterol before addition of the buffer. This broad, single-line signal is also observed in suspensions formed for Chol/POPS mixtures (as a background signal) when the Chol/POPS ratio is much greater than 3. It is suggested that the EPR spin-labeling approach can discriminate and characterize the fraction of cholesterol that forms the CBD within the

  19. Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential.

    PubMed Central

    Toner, M; Cravalho, E G; Stachecki, J; Fitzgerald, T; Tompkins, R G; Yarmush, M L; Armant, D R

    1993-01-01

    A thermodynamic model was used to evaluate and optimize a rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120 degrees C/min when a 5-min holding period was inserted at -10 degrees C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1 degree C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other than IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45 degrees C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos. Images FIGURE 2 FIGURE 10 FIGURE 11 PMID:8369414

  20. Risk assessment of Giardia from a full scale MBR sewage treatment plant caused by membrane integrity failure.

    PubMed

    Zhang, Yu; Chen, Zhimin; An, Wei; Xiao, Shumin; Yuan, Hongying; Zhang, Dongqing; Yang, Min

    2015-04-01

    Membrane bioreactors (MBR) are highly efficient at intercepting particles and microbes and have become an important technology for wastewater reclamation. However, many pathogens can accumulate in activated sludge due to the long residence time usually adopted in MBR, and thus may pose health risks when membrane integrity problems occur. This study presents data from a survey on the occurrence of water-borne Giardia pathogens in reclaimed water from a full-scale wastewater treatment plant with MBR experiencing membrane integrity failure, and assessed the associated risk for green space irrigation. Due to membrane integrity failure, the MBR effluent turbidity varied between 0.23 and 1.90 NTU over a period of eight months. Though this turbidity level still met reclaimed water quality standards (≤5 NTU), Giardia were detected at concentrations of 0.3 to 95 cysts/10 L, with a close correlation between effluent turbidity and Giardia concentration. All β-giardin gene sequences of Giardia in the WWTP influents were genotyped as Assemblages A and B, both of which are known to infect humans. An exponential dose-response model was applied to assess the risk of infection by Giardia. The risk in the MBR effluent with chlorination was 9.83×10(-3), higher than the acceptable annual risk of 1.0×10(-4). This study suggested that membrane integrity is very important for keeping a low pathogen level, and multiple barriers are needed to ensure the biological safety of MBR effluent.

  1. Osmotic versus conventional membrane bioreactors integrated with reverse osmosis for water reuse: Biological stability, membrane fouling, and contaminant removal.

    PubMed

    Luo, Wenhai; Phan, Hop V; Xie, Ming; Hai, Faisal I; Price, William E; Elimelech, Menachem; Nghiem, Long D

    2017-02-01

    This study systematically compares the performance of osmotic membrane bioreactor - reverse osmosis (OMBR-RO) and conventional membrane bioreactor - reverse osmosis (MBR-RO) for advanced wastewater treatment and water reuse. Both systems achieved effective removal of bulk organic matter and nutrients, and almost complete removal of all 31 trace organic contaminants investigated. They both could produce high quality water suitable for recycling applications. During OMBR-RO operation, salinity build-up in the bioreactor reduced the water flux and negatively impacted the system biological treatment by altering biomass characteristics and microbial community structure. In addition, the elevated salinity also increased soluble microbial products and extracellular polymeric substances in the mixed liquor, which induced fouling of the forward osmosis (FO) membrane. Nevertheless, microbial analysis indicated that salinity stress resulted in the development of halotolerant bacteria, consequently sustaining biodegradation in the OMBR system. By contrast, biological performance was relatively stable throughout conventional MBR-RO operation. Compared to conventional MBR-RO, the FO process effectively prevented foulants from permeating into the draw solution, thereby significantly reducing fouling of the downstream RO membrane in OMBR-RO operation. Accumulation of organic matter, including humic- and protein-like substances, as well as inorganic salts in the MBR effluent resulted in severe RO membrane fouling in conventional MBR-RO operation.

  2. A membrane-bound NAC transcription factor as an integrator of biotic and abiotic stress signals.

    PubMed

    Seo, Pil Joon; Park, Chung-Mo

    2010-05-01

    Transcription factors are central components of gene regulatory networks that mediate virtually all aspects of growth and developmental processes in biological systems. The activity of transcription factors is regulated at multiple steps, such as gene transcription, posttranscriptional RNA processing, posttranslational modification, protein-protein interactions, and controlled protein turnover. Controlled activation of dormant, membrane-bound transcription factor (MTF) is an intriguing regulatory mechanism that ensures quick transcriptional responses to environmental fluctuations in plants, in which various stress hormones serve as signaling mediators. NTL6 is proteolytically activated upon exposure to cold and induces expression of the Pathogenesis-Related (PR) genes. The membrane-mediated cold signaling in inducing pathogen resistance is considered to be an adaptive strategy that protects plants against infection by hydrophilic pathogens frequently occurring during cold season. We found that NTL6 also mediates abscisic acid (ABA) regulation of abiotic stress responses in Arabidopsis. NTL6 is proteolytically activated by ABA. Transgenic plants overexpressing a nuclear NTL6 form (35S:6ΔC) exhibited a hypersensitive response to ABA and high salinity in seed germination. Taken together, these observations indicate that NTL6 plays an integrative role in plant responses to both biotic and abiotic stress conditions.

  3. The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation

    PubMed Central

    1995-01-01

    We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis. Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene. In either methanol or methylamine medium, conditions that normally induce the organelles, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol. The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD. Analysis of the primary sequence of Per8p revealed a 39-amino-acid cysteine-rich segment with similarity to the C3HC4 family of zinc-finger motifs. Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers. We show that Per8p is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organelles. We propose that Per8p is a component of the molecular machinery that controls the proliferation of this organelle. PMID:7844145

  4. Removal of emerging contaminants from municipal wastewater with an integrated membrane system, MBR-RO.

    PubMed

    Dolar, Davor; Gros, Meritxell; Rodriguez-Mozaz, Sara; Moreno, Jordi; Comas, Joaquim; Rodriguez-Roda, Ignasi; Barceló, Damià

    2012-11-15

    The presence of emerging contaminants in the aquatic environment and their potential effects on living organisms has become an issue of growing concern. Among emerging contaminants, pharmaceuticals may enter the aquatic environment due to their high consumption and their incomplete removal in conventional municipal wastewater treatment plants (WWTPs). The main goal of this study was the assessment of the removal efficiency of pharmaceuticals found in municipal wastewater of a coastal WWTP (Castell-Platja d'Aro, Spain) using an integrated pilot scale membrane system (MBR-RO). Twenty multiple-class pharmaceuticals (including psychiatric drugs, macrolide antibiotics, β-blockers, sulfonamide and fluoroquinolone antibiotics, histamine H2 receptor antagonists, anti-inflammatories, nitroimidazole, β-agonist and antiplatelet agent) were measured in real influent with the lowest average concentration for psychiatric drugs (0.017 μg L(-1)) to the highest for macrolide antibiotics (2.02 μg L(-1)). Although some contaminants were in relatively high concentrations (even up to 2.90 μg L(-1) in the case of ofloxacin). The combination of MBR and RO treatment showed excellent overall removal of target emerging contaminants with removal rates above 99% for all of them. For some compounds (metronidazole, hydrocodone, codein, ranitidine) MBR provided high removal efficiency (up to 95%). Additionally RO membrane showed removal rates always higher than 99%.

  5. Functional dissection of SseF, a membrane-integral effector protein of intracellular Salmonella enterica.

    PubMed

    Müller, Petra; Chikkaballi, Deepak; Hensel, Michael

    2012-01-01

    During intracellular life, the bacterial pathogen Salmonella enterica translocates a complex cocktail of effector proteins by means of the SPI2-encoded type III secretions system. The effectors jointly modify the endosomal system and vesicular transport in host cells. SseF and SseG are two effectors encoded by genes within Salmonella Pathogenicity Island 2 and both effector associate with endosomal membranes and microtubules and are involved in the formation of Salmonella-induced filaments. Our previous deletional analyses identified protein domains of SseF required for the effector function. Here we present a detailed mutational analysis that identifies a short hydrophobic motif as functionally essential. We demonstrate that SseF and SseG are still functional if translocated as a single fusion protein, but also mediate effector function if translocated in cells co-infected with sseF and sseG strains. SseF has characteristics of an integral membrane protein after translocation into host cells.

  6. Stabilized liquid membrane device (SLMD) for the passive, integrative sampling of labile metals in water

    USGS Publications Warehouse

    Brumbaugh, W.G.; Petty, J.D.; Huckins, J.N.; Manahan, S.E.

    2002-01-01

    A stabilized liquid membrane device (SLMD) is described for potential use as an in situ, passive, integrative sampler for cadmium (Cd), cobalt (Co), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn) in natural waters. The SLMD (patent pending) consists of a 2.5-cm-wide by 15-cm-long strip of low-density polyethylene (LDPE) layflat tubing containing 1 mL of an equal mixture (v/v) of oleic acid (cis-9-octadecenoic acid) and EMO-8Q (7-[4-ethyl-1-methyloctyl]-8-quinolinol). The reagent mixture continuously diffuses to the exterior surface of the LDPE membrane, and provides for sequestration of several divalent metals for up to several weeks. Depending on sampler configuration, concentration factors of several thousand can be realized for these metal ions after just a few days. In addition to in situ deployment, the SLMD may be useful for laboratory determination of labile metal species in grab samples. Methods for minimizing the effects of water flow on the sampling rate are currently under investigation.

  7. Anti-Candida activity of geraniol involves disruption of cell membrane integrity and function.

    PubMed

    Sharma, Y; Khan, L A; Manzoor, N

    2016-09-01

    Candidiasis is a major problem in immunocompromised patients. Candida, an opportunistic fungal pathogen, is a major health concern today as conventional drugs are highly toxic with undesirable side effects. Their fungistatic nature is responsible for drug resistance in continuously evolving strains. Geraniol, an acyclic monoterpene alcohol, is a component of several plant essential oils. In the present study, an attempt has been made to understand the antifungal activity of geraniol at the cell membrane level in three Candida species. With an MIC of 30-130μg/mL, this natural compound was fungicidal at concentrations 2×MIC. There was complete suppression of fungal growth at MIC values (growth curves) and encouragingly geraniol is non-toxic even at the concentrations approaching 5×MIC (hemolysis assay). Exposed cells showed altered morphology, wherein the cells appeared either broken or shrivelled up (SEM studies). Significant reduction was seen in ergosterol levels at sub-MIC and glucose-induced H(+) efflux at concentrations>MIC values. Our results suggest that geraniol disrupts cell membrane integrity by interfering with ergosterol biosynthesis and inhibiting the very crucial PM-ATPase. It may hence be used in the management and treatment of both superficial and invasive candidiasis but further studies are required to elaborate its mode of action.

  8. Consequences of lipidic nanoemulsions on membrane integrity and ultrastructural morphology of Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Singh, Neeru; Manaswita Verma, Saurabh; Singh, Sandeep Kumar; Ranjan Prasad Verma, Priya

    2014-04-01

    The present study divulges the consequences of lipidic nanoemulsions (cationized and non-cationized) on morphology and membrane integrity of Staphylococcus aureus using transmission electron microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Transmission electron microscopic (TEM) images reveal that the cationized lipidic emulsions (CLEs) remained adhered even after the hostile treatment to remove nanoemulsions by centrifugation owing to electrostatic attraction between CLE and negatively charged bacterial surface. TEM images portray the extensive cell lyses owing to the release of cytoplasmic content when treated with both CLE and Non-CLE (NCLE). The AFM analysis of the NCLE and CLE treated S. aureus cells showed the root mean square roughness of 11.3 ± 2.8 nm and 17.7 ± 3.2 nm, respectively. The complete losses of bacterial colonies after 45 min of contact with NCLE were observed. No viable bacterial colonies were noticeable after 10 min of contact when treated with CLE, indicating better rate of killing with respect to NCLE. Similar results were obtained in the zone of inhibition studies. Significant (p < 0.05) increase of cytoplasmic material was observed both in NCLE (0.192 ± 0.003) and CLE (0.308 ± 0.012) as compared to control (0.019 ± 0.002). The present finding illustrates that the NCLE and CLE had caused significant membrane disorganization leading to release of cytoplasmic content causing irreversible cell damage, which is in accordance with the TEM, SEM and AFM studies.

  9. Microfluidic Microdialysis: Spatiotemporal Control over Solution Microenvironments Using Integrated Hydrogel Membrane Microwindows

    NASA Astrophysics Data System (ADS)

    Paustian, Joel S.; Azevedo, Rodrigo Nery; Lundin, Sean-Thomas B.; Gilkey, Matthew J.; Squires, Todd M.

    2013-10-01

    We present a powerful and versatile technique that enables exquisite spatial and temporal control over local solution chemistry in microfluidic devices. Using a microscope and a UV lamp, we use projection lithography to photopolymerize thin (10-25μm) hydrogel membrane “microwindows” (HMMs) into standard microfluidic devices. These microwindows are permeable to solute and solvent diffusion and to electric fields, yet act as rigid walls from the standpoint of fluid flow. Reservoirs of solution may thus be rapidly imposed, switched, and maintained on one side of a HMM using standard microfluidic techniques, provoking changes in solution conditions on the other side without active mixing, stirring, or diluting. We highlight three paradigmatic experimental capabilities enabled by HMMs: (1) rapid dialysis and swapping of solute and/or solvent, (2) stable and convection-free localized concentration gradients, and (3) local electric permeability. The functional versatility of hydrogel microwindow membranes, coupled with the ease and speed of their fabrication and integration into simple microchannels or multilayer devices, will open a variety of novel applications and studies in a broad range of fields.

  10. Membrane integrity of Campylobacter jejuni subjected to high pressure is pH-dependent

    NASA Astrophysics Data System (ADS)

    Lerasle, M.; Guillou, S.; Simonin, H.; Laroche, M.; de Lamballerie, M.; Federighi, M.

    2012-03-01

    Our study focuses on a foodborne pathogen, Campylobacter, which is responsible for the most frequent bacterial enteritis worldwide. Membrane integrity of Campylobacter jejuni NCTC 11168 cells treated at high pressure (300 MPa, 20°C, 10 min) at pH 7.0 and pH 5.6 was measured by fluorescence spectroscopy of propidium iodide (PI) uptake. The percentage of membrane-damaged cells by high pressure, in which PI is allowed to penetrate, was determined using two calibration methods based on the PI fluorescence signal obtained with cells killed either by a heat treatment (80°C for 15 min) or by a pressure treatment (400 MPa, 20°C, 10 min). Both calibrations were shown to be statistically different (P<0.05), particularly at acidic pH, suggesting that a difference in the penetration of PI into bacterial cells might depend on the mode of cell inactivation. These results corroborate the fact that the mechanism of microbial inactivation by high pressure is pH-dependent.

  11. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

    PubMed Central

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-01-01

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance. PMID:27436903

  12. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria.

    PubMed

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-08-02

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance.

  13. Improvement of hydrophobic integral membrane protein identification by mild performic acid oxidation-assisted digestion.

    PubMed

    Cao, Rui; Liu, Yisong; Chen, Ping; Lv, Rong; Song, Qin; Sheng, Tingting; He, Quanyuan; Wang, Yin; Wang, Xianchun; Liang, Songping

    2010-12-15

    Integral membrane proteins (IMPs) are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of IMPs make them difficult to analyze. In proteomic analyses, hydrophobic peptides including transmembrane domains are often underrepresented, and this reduces the sequence coverage and reliability of the identified IMPs. Here we report a new strategy, mild performic acid oxidation treatment (mPAOT), for improvement of IMP identification. In the mPAOT strategy, the hydrophobicity of IMPs is significantly decreased by oxidizing their methionine and cysteine residues with performic acid, thereby improving the solubility and enzymolysis of these proteins. The application of the mPAOT strategy to the analysis of IMPs from human nasopharyngeal carcinoma CNE1 cell line demonstrated that many IMPs, including those with high hydrophobicity, could be reliably identified.

  14. Integrated carbon composite bipolar plate for polymer-electrolyte membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Yu, Ha Na; Hwang, In Uk; Kim, Seong Su; Lee, Dai Gil

    The electrical resistance of bipolar plates for polymer-electrolyte membrane fuel cells (PEMFCs) should be very low to conduct the electricity generated with minimum electrical loss. The resistance of a bipolar plate consists of the bulk material resistance and the interfacial contact resistance when two such plates are contacted to provide channels for fuel and air (oxygen) supplies. Since the interfacial contact resistance is much larger than the bulk resistance in an actual fuel cell stack, an integrated carbon composite bipolar plate is developed in this study to eliminate the contact resistance between contacting bipolar plates. To fabricate this plate with channels for fuel, air and coolant, many stainless-steel pipes of 1 mm diameter are uniformly embedded in the carbon fiber/epoxy composite prepreg and co-cured. The contact resistance, flexural strength and thermal degradation temperatures of the developed composite bipolar plate are then measured.

  15. Insights into the structure and function of membrane-integrated processive glycosyltransferases

    DOE PAGES

    Bi, Yunchen; Hubbard, Caitlin; Purushotham, Pallinti; ...

    2015-09-02

    Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. In this paper, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin,more » alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).« less

  16. Aspergillus carbonarius polygalacturonases purified by integrated membrane process and affinity precipitation for apple juice production.

    PubMed

    Nakkeeran, Ekambaram; Umesh-Kumar, Sukumaran; Subramanian, Rangaswamy

    2011-02-01

    Aspergillus carbonarius, when grown by submerged and solid-state fermentation, produces different molecular forms of polygalacturonase (PG; EC 3.2.1.15), among them a 42 kDa PG with a high specific activity of 7000 U/mg protein. When the enzymes were purified by integrated membrane process (IMP) and alginate affinity precipitation (AAP), the two processes concentrated different forms of the enzyme. The AAP process selectively purified and concentrated the high active PG whereas the IMP yielded different PGs and also amylase and protease. Evaluation of the AAP enzyme preparations for apple juice preparation under conditions usually employed commercially demonstrated that the high activity PG did not result in good juice clarity. With IMP processed enzymes, juice yields and clarity were similar to that obtained with commercial PG from A. niger.

  17. Hydrogen peroxide induces premature acrosome reaction in rat sperm and reduces their penetration of the zona pellucida.

    PubMed

    Hsu, P C; Hsu, C C; Guo, Y L

    1999-11-29

    Recent studies have demonstrated that mammalian sperm are capable of generating reactive oxygen species (ROS) and that this activity is significantly accelerated in subfertile subjects. The observed decrease in penetration of zona-intact oocyte might be explained by chemical-induced ROS-related early onset of capacitation and premature acrosome reaction, but the mechanism is not clear. We determine whether zona-intact oocyte penetration capability in rat epididymal sperm was affected by premature acrosome reaction in rat sperm treated with hydrogen peroxide (H2O2) and calcium ionophore A23187 or H2O2 and lysophosphatidyl choline. Chlortetracycline fluorescence assay was used to study the status of acrosome reaction on epididymal sperm. The sperm-oocyte binding and penetration assay was used to evaluate the capability for zona pellucida penetration. There was a positive linear correlation between the frequency of acrosome-reacted sperm and capability of sperm-oocyte binding and penetration in zona-free oocytes. In the zona-intact oocytes, the sperm-oocyte penetration rate was suppressed as the proportions of acrosome-reacted sperm increased. In summary, this study showed that premature acrosome reaction reduced rat sperm's capability of penetrating zona-intact oocytes. However, this reduction is not seen in zona-free oocytes. These findings may provide a basis for understanding the effects of sperm ROS generation on zona pellucida penetration in male reproductive toxicology.

  18. Sirt1 regulates acrosome biogenesis by modulating autophagic flux during spermiogenesis in mice.

    PubMed

    Liu, Chao; Song, Zhenhua; Wang, Lina; Yu, Haiyan; Liu, Weixiao; Shang, Yongliang; Xu, Zhiliang; Zhao, Haichao; Gao, Fengyi; Wen, Jiamin; Zhao, Linan; Gui, Yaoting; Jiao, Jianwei; Gao, Fei; Li, Wei

    2017-02-01

    Sirt1 is a member of the sirtuin family of proteins and has important roles in numerous biological processes. Sirt1(-/-) mice display an increased frequency of abnormal spermatozoa, but the mechanism of Sirt1 in spermiogenesis remains largely unknown. Here, we report that Sirt1 might be directly involved in spermiogenesis in germ cells but not in steroidogenic cells. Germ cell-specific Sirt1 knockout mice were almost completely infertile; the early mitotic and meiotic progression of germ cells in spermatogenesis were not obviously affected after Sirt1 depletion, but subsequent spermiogenesis was disrupted by a defect in acrosome biogenesis, which resulted in a phenotype similar to that observed in human globozoospermia. In addition, LC3 and Atg7 deacetylation was disrupted in spermatids after knocking out Sirt1, which affected the redistribution of LC3 from the nucleus to the cytoplasm and the activation of autophagy. Furthermore, Sirt1 depletion resulted in the failure of LC3 to be recruited to Golgi apparatus-derived vesicles and in the failure of GOPC and PICK1 to be recruited to nucleus-associated acrosomal vesicles. Taken together, these findings reveal that Sirt1 has a novel physiological function in acrosome biogenesis.

  19. Lectin staining and flow cytometry reveals female-induced sperm acrosome reaction and surface carbohydrate reorganization

    PubMed Central

    Kekäläinen, Jukka; Larma, Irma; Linden, Matthew; Evans, Jonathan P.

    2015-01-01

    All cells are covered by glycans, an individually unique layer of oligo- and polysaccharides that are critical moderators of self-recognition and other cellular-level interactions (e.g. fertilization). The functional similarity between these processes suggests that gamete surface glycans may also have an important, but currently overlooked, role in sexual selection. Here we develop a user-friendly methodological approach designed to facilitate future tests of this possibility. Our proposed method is based on flow cytometric quantification of female-induced sperm acrosome reaction and sperm surface glycan modifications in the Mediterranean mussel Mytilus galloprovincialis. In this species, as with many other taxa, eggs release water-soluble factors that attract conspecific sperm (chemoattraction) and promote potentially measurable changes in sperm behavior and physiology. We demonstrate that flow cytometry is able to identify sperm from other seawater particles as well as accurately measure both acrosome reaction and structural modifications in sperm glycans. This methodological approach can increase our understanding of chemically-moderated gamete-level interactions and individual-specific gamete recognition in Mytilus sp. and other taxa with similar, easily identifiable acrosome structure. Our approach is also likely to be applicable to several other species, since carbohydrate-mediated cellular-level interactions between gametes are universal among externally and internally fertilizing species. PMID:26470849

  20. ¹H-NMR study of the impact of high pressure and thermal processing on cell membrane integrity of onions.

    PubMed

    Gonzalez, Maria E; Barrett, Diane M; McCarthy, Michael J; Vergeldt, Frank J; Gerkema, Edo; Matser, Ariette M; Van As, Henk

    2010-09-01

    Proton nuclear magnetic resonance (¹H-NMR) relaxometry was used to study the effects of high pressure and thermal processing on membrane permeability and cell compartmentalization, important components of plant tissue texture. High pressure treated onions were subjected to pressure levels from 20 to 200 MPa at 5 min hold time at initial temperatures of 5 and 20 °C. Thermally treated onions were exposed for 30 min at temperatures from 40 to 90 °C. Loss of membrane integrity was clearly shown by changes in transverse relaxation time (T(2)) of water at temperatures of 60 °C and above. Destabilization effects on membranes exposed to high pressure were observed at 200 MPa as indicated by T(2) measurements and cryo-scanning electron microscopy (Cryo-SEM). T(2) relaxation successfully discriminated different degrees of membrane damage based on the T(2) shift of the vacuolar component. Analyses of the average water self-diffusion coefficient indicated less restricted diffusion after membrane rupture occurred in cases of severe thermal treatments. Milder processing treatments yielded lower average diffusion coefficients than the controls. ¹H-NMR proved to be an effective method for quantification of cell membrane damage in onions and allowed for the comparison of different food processes based on their impact on tissue integrity.

  1. Effects of glycerol and sugar mixing temperature on the morphologic and functional integrity of cryopreserved ram sperm.

    PubMed

    Pelufo, V; López Armengol, M F; Malcotti, V; Venturino, A; Aisen, E G

    2015-01-01

    Sperm deep freezing procedures for ram semen have considerable variations regarding the steps being employed for cooling, freezing, and addition of cryoprotectants. In this work, we evaluated the effects of the addition of glycerol and/or the disaccharides sucrose and trehalose to hypertonic diluents either before or after cooling from 30 °C to 5 °C in Merino Australian ram semen cryopreservation. Using optical and transmission electron microscopy techniques, we assessed that glycerol was beneficial to the cooling process independently of its addition at 30 °C or 5 °C in terms of sperm membrane integrity in different regions of the plasma membrane (acrosomal region, 14.5% higher integrity; postacrosomal region, 8.0% higher integrity [P < 0.01]; hypoosmotic swelling test [HOST], 10.8% higher integrity [P < 0.001]). Disaccharides were necessary for a better cryopreservation in liquid nitrogen, and the best procedure was their addition after cooling at 5 °C (12% higher sperm motility [P < 0.001]; 8% higher acrosome integrity, [P < 0.05]; 9.5% higher plasma membrane integrity assessed by HOST [P < 0.001]). Trehalose showed a greater preservation cryoprotectant capacity than sucrose, as indicated by sperm motility after thawing (8.1% greater [P < 0.01]) and by the integrity of the intermediate piece (20% greater [P < 0.05]). From these results, we conclude that the best procedure for ram semen cryopreservation in hypertonic disaccharide-containing diluents is the addition of glycerol and trehalose after the cooling process, at 5 °C.

  2. The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop

    PubMed Central

    1996-01-01

    No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it. PMID:8609161

  3. Integrated pyrolucite fluidized bed-membrane hybrid process for improved iron and manganese control in drinking water.

    PubMed

    Dashtban Kenari, Seyedeh Laleh; Barbeau, Benoit

    2017-04-15

    Newly developed ceramic membrane technologies offer numerous advantages over the conventional polymeric membranes. This work proposes a new configuration, an integrated pyrolucite fluidized bed (PFB)-ceramic MF/UF hybrid process, for improved iron and manganese control in drinking water. A pilot-scale study was undertaken to evaluate the performance of this process with respect to iron and manganese control as well as membrane fouling. In addition, the fouling of commercially available ceramic membranes in conventional preoxidation-MF/UF process was compared with the hybrid process configuration. In this regard, a series of experiments were conducted under different influent water quality and operating conditions. Fouling mechanisms and reversibility were analyzed using blocking law and resistance-in-series models. The results evidenced that the flux rate and the concentration of calcium and humic acids in the feed water have a substantial impact on the filtration behavior of both membranes. The model for constant flux compressible cake formation well described the rise in transmembrane pressure. The compressibility of the filter cake substantially increased in the presence of 2 mg/L humic acids. The presence of calcium ions caused significant aggregation of manganese dioxide and humic acid which severely impacted the extent of membrane fouling. The PFB pretreatment properly alleviated membrane fouling by removing more than 75% and 95% of iron and manganese, respectively.

  4. Formation of lipid bilayer membrane in a poly(dimethylsiloxane) microchip integrated with a stacked polycarbonate membrane support and an on-site nanoinjector.

    PubMed

    Teng, Wei; Ban, Changill; Hahn, Jong Hoon

    2015-03-01

    This paper describes a new and facile approach for the formation of pore-spanning bilayer lipid membranes (BLMs) within a poly(dimethylsiloxane) (PDMS) microfluidic device. Commercially, readily available polycarbonate (PC) membranes are employed for the support of BLMs. PC sheets with 5 μm, 2 μm, and 0.4 μm pore diameters, respectively, are thermally bonded into a multilayer-stack, reducing the pore density of 0.4 μm-pore PC by a factor of 200. The BLMs on this support are considerably stable (a mean lifetime: 17 h). This multilayer-stack PC (MSPC) membrane is integrated into the PDMS chip by an epoxy bonding method developed to secure durable bonding under the use of organic solvents. The microchip has a special channel for guiding a micropipette in the proximity of the MSPC support. With this on-site injection technique, tens to hundreds of nanoliters of solutions can be directly dispensed to the support. Incorporating gramicidin ion channels into BLMs on the MSPC support has confirmed the formation of single BLMs, which is based on the observation from current signals of 20 pS conductance that is typical to single channel opening. Based on the bilayer capacitance (1.4 pF), about 15% of through pores across the MSPC membrane are estimated to be covered with BLMs.

  5. Metabolic Dysfunction and Unabated Respiration Precede the Loss of Membrane Integrity during Dehydration of Germinating Radicles1

    PubMed Central

    Leprince, Olivier; Harren, Frans J.M.; Buitink, Julia; Alberda, Mark; Hoekstra, Folkert A.

    2000-01-01

    This study shows that dehydration induces imbalanced metabolism before loss of membrane integrity in desiccation-sensitive germinated radicles. Using a photoacoustic detection system, responses of CO2 emission and fermentation to drying were analyzed non-invasively in desiccation-tolerant and -intolerant radicles of cucumber (Cucumis sativa) and pea (Pisum sativum). Survival after drying and a membrane integrity assay showed that desiccation tolerance was present during early imbibition and lost in germinated radicles. However, tolerance could be re-induced in germinated cucumber radicles by incubation in polyethylene glycol before drying. Tolerant and polyethylene glycol (PEG)-induced tolerant radicles exhibited a much-reduced CO2 production before dehydration compared with desiccation-sensitive radicles. This difference was maintained during dehydration. In desiccation-sensitive tissues, dehydration induced an increase in the emission of acetaldehyde and ethanol that peaked well before the loss of membrane integrity. Acetaldehyde emission from sensitive radicles was significantly reduced when dehydration occurred in 50% O2 instead of air. Acetaldehyde/ethanol were not detected in dehydrating tolerant radicles of either species or in polyethylene glycol-induced tolerant cucumber radicles. Thus, a balance between down-regulation of metabolism during drying and O2 availability appears to be associated with desiccation tolerance. Using Fourier transform infrared spectroscopy, acetaldehyde was found to disturb the phase behavior of phospholipid vesicles, suggesting that the products resulting from imbalanced metabolism in seeds may aggravate membrane damage induced by dehydration. PMID:10677452

  6. Integrating Membrane Transport with Male Gametophyte Development and Function through Transcriptomics.

    SciTech Connect

    Bock KW; D Honys; JM. Ward; S Padmanaban; EP Nawrocki; KD Hirschi; D Twell; H Sze

    2006-01-01

    Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. About 1269 genes encoding classified transporters were collected from the Arabidopsis thaliana genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3 and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9); while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, were developmentally-regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter::GUS analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.

  7. Tunable integration of absorption-membrane-adsorption for efficiently separating low boiling gas mixtures near normal temperature

    PubMed Central

    Liu, Huang; Pan, Yong; Liu, Bei; Sun, Changyu; Guo, Ping; Gao, Xueteng; Yang, Lanying; Ma, Qinglan; Chen, Guangjin

    2016-01-01

    Separation of low boiling gas mixtures is widely concerned in process industries. Now their separations heavily rely upon energy-intensive cryogenic processes. Here, we report a pseudo-absorption process for separating low boiling gas mixtures near normal temperature. In this process, absorption-membrane-adsorption is integrated by suspending suitable porous ZIF material in suitable solvent and forming selectively permeable liquid membrane around ZIF particles. Green solvents like water and glycol were used to form ZIF-8 slurry and tune the permeability of liquid membrane surrounding ZIF-8 particles. We found glycol molecules form tighter membrane while water molecules form looser membrane because of the hydrophobicity of ZIF-8. When using mixing solvents composed of glycol and water, the permeability of liquid membrane becomes tunable. It is shown that ZIF-8/water slurry always manifests remarkable higher separation selectivity than solid ZIF-8 and it could be tuned to further enhance the capture of light hydrocarbons by adding suitable quantity of glycol to water. Because of its lower viscosity and higher sorption/desorption rate, tunable ZIF-8/water-glycol slurry could be readily used as liquid absorbent to separate different kinds of low boiling gas mixtures by applying a multistage separation process in one traditional absorption tower, especially for the capture of light hydrocarbons. PMID:26892255

  8. Membrane gas absorbers for H2S removal--design, operation and technology integration into existing odour treatment strategies.

    PubMed

    Jefferson, B; Nazareno, C; Georgaki, S; Gostelow, P; Stuetz, R M; Longhurst, P; Robinson, T

    2005-07-01

    A hollow fibre (HF) polypropylene membrane gas absorber was investigated for the removal of hydrogen sulphide (H2S) from gas streams. Gas concentrations between 25-2010 ppmV were fed into the shell side of a membrane module whilst water-NaOH solutions flowed counter-currently in the fibre lumens. The process was effective at removing the H2S (96% at G:L ratios up to 50 and pH 13) from the gas phase in a single pass through the membrane at all the concentrations of HaS investigated. Analysis of the mass transfer process revealed the rate of transfer to be controlled by the gas phase transfer coefficient with a value between 1 and 25 x 10(-4) m.s(-1). The possible integration of a membrane absorber system into existing odour treatment strategies was assessed by comparing the membrane system, based on the experimentally determined mass transfer coefficient, with existing full scale biofiltration plants. The membrane system became economically favourable at gas flow rates lower than 1630 m(3) x h(-1).

  9. Tunable integration of absorption-membrane-adsorption for efficiently separating low boiling gas mixtures near normal temperature.

    PubMed

    Liu, Huang; Pan, Yong; Liu, Bei; Sun, Changyu; Guo, Ping; Gao, Xueteng; Yang, Lanying; Ma, Qinglan; Chen, Guangjin

    2016-02-19

    Separation of low boiling gas mixtures is widely concerned in process industries. Now their separations heavily rely upon energy-intensive cryogenic processes. Here, we report a pseudo-absorption process for separating low boiling gas mixtures near normal temperature. In this process, absorption-membrane-adsorption is integrated by suspending suitable porous ZIF material in suitable solvent and forming selectively permeable liquid membrane around ZIF particles. Green solvents like water and glycol were used to form ZIF-8 slurry and tune the permeability of liquid membrane surrounding ZIF-8 particles. We found glycol molecules form tighter membrane while water molecules form looser membrane because of the hydrophobicity of ZIF-8. When using mixing solvents composed of glycol and water, the permeability of liquid membrane becomes tunable. It is shown that ZIF-8/water slurry always manifests remarkable higher separation selectivity than solid ZIF-8 and it could be tuned to further enhance the capture of light hydrocarbons by adding suitable quantity of glycol to water. Because of its lower viscosity and higher sorption/desorption rate, tunable ZIF-8/water-glycol slurry could be readily used as liquid absorbent to separate different kinds of low boiling gas mixtures by applying a multistage separation process in one traditional absorption tower, especially for the capture of light hydrocarbons.

  10. Fucose, mannose, and β-N-acetylglucosamine glycopolymers initiate the mouse sperm acrosome reaction through convergent signaling pathways.

    PubMed

    Wu, Linghui; Sampson, Nicole S

    2014-02-21

    The sperm acrosome reaction (AR), an essential exocytosis step in mammalian fertilization, is mediated by a species-specific interaction of sperm surface molecules with glycans on the egg. Previous studies indicate that a subset of terminal carbohydrates on the mouse egg zona pellucida (ZP) trigger the AR by cross-linking or aggregating receptors on the sperm membrane. However, the exact role of those carbohydrates in AR has not been identified and the mechanism underlying the AR still needs further investigation. To study this process, a series of glycopolymers was synthesized. The glycopolymers are composed of a multivalent scaffold (norbornene), a functional ligand (previously identified ZP terminal monosaccharides), and a linker connecting the ligand and the scaffold. The polymers were tested for their ability to initiate AR and through which signaling pathways AR induction occurred. Our data demonstrate that mannose, fucose, and β-N-acetylglucosamine 10-mers and 100-mers initiate AR in a dose-dependent manner, and the 100-mers are more potent on a per monomer basis than the 10-mers. Although nearly equipotent in inducing the AR at the optimal concentrations, their AR activation kinetics are not identical. Similar to mouse ZP3, all 100-mer-activated AR are sensitive to guanine-binding regulatory proteins (G-proteins), tyrosine kinase, protein kinase A, protein kinase C, and Ca(2+)-related antagonists. Thus, the chemotypes of synthetic glycopolymers imitate the physiologic AR-activation agents and provide evidence that occupation of one of at least three different receptor binding sites is sufficient to initiate the AR.

  11. Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38).

    PubMed Central

    Luzio, J P; Brake, B; Banting, G; Howell, K E; Braghetta, P; Stanley, K K

    1990-01-01

    Organelle-specific integral membrane proteins were identified by a novel strategy which gives rise to monospecific antibodies to these proteins as well as to the cDNA clones encoding them. A cDNA expression library was screened with a polyclonal antiserum raised against Triton X-114-extracted organelle proteins and clones were then grouped using antibodies affinity-purified on individual fusion proteins. The identification, molecular cloning and sequencing are described of a type 1 membrane protein (TGN38) which is located specifically in the trans-Golgi network. Images Fig. 1. Fig. 3. PMID:2204342

  12. Lysosomal integral membrane protein Sidt2 plays a vital role in insulin secretion.

    PubMed

    Gao, Jialin; Yu, Cui; Xiong, Qianyin; Zhang, Yao; Wang, Lizhuo

    2015-01-01

    Abnormal insulin secretion results in impaired glucose tolerance and is one of the causal factors in the etiology of type 2 diabetes mellitus. Sidt2, a lysosomal integral membrane protein, plays a critical role in insulin secretion. Here, we further investigate its regulation in insulin secretion. We show that Sidt2(-/-) mice exhibit weight loss, decreased postnatal survival rate with aging, increased fasting glucose and impaired glucose tolerance. After loading high levels of glucose in their diet, Sidt2(-/-) mice produce notably lower insulin levels at the first-phase secretion compared with Sidt2(+/+) mice. Consistent with the in vivo study, INS-1 cells treated with Sidt2 siRNA produced less insulin when loaded with 16.7 mM of glucose. Only 2 of the 13 genes, synap1 and synap3 which encode soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, showed significantly decreased expression in Sidt2(-/-) mice. In conclusion, Sdit2 may play a vital role in the regulation of insulin secretion via two SNARE proteins synap1 and syanp3.

  13. Structure of the integral membrane domain of the GLP1 receptor.

    PubMed

    Frimurer, T M; Bywater, R P

    1999-06-01

    A three-dimensional (3D) model of the integral membrane domain of the GLP1 receptor, a member of the secretin receptor family of the G-protein-coupled receptor superfamily is proposed. The probable arrangement of the seven helices in this receptor was deduced from a detailed analysis of all the sequences in the secretin receptor family. The analysis includes: 1) identifying the transmembrane helices, 2) charge distribution analysis to estimate to which extent the transmembrane helices are buried, 3) Fourier transform analysis of different property profiles within the transmembrane helices to determine the orientation of exposed and buried faces of the helices, 4) alignment of sequences with those of the rhodopsin-like family using the novel "cold spot" method reported herein, 5) determination of lengths of transmembrane helices and their connecting loops and the constraints these impose on packing, tilting and organization, 6) incorporation of mutagenesis and ligand specificity data. We find that there is a close similarity between the structural properties of receptors of the secretin family and those of the rhodopsin-like family as typified by the frog rhodopsin structure recently solved by electron cryomicroscopy.

  14. Three-dimensional integrated microfluidic architectures enabled through electrically switchable nanocapillary array membranes

    PubMed Central

    Gatimu, E. N.; King, T. L.; Sweedler, J. V.; Bohn, P. W.

    2007-01-01

    The extension of microfluidic devices to three dimensions requires innovative methods to interface fluidic layers. Externally controllable interconnects employing nanocapillary array membranes (NCAMs) have been exploited to produce hybrid three-dimensional fluidic architectures capable of performing linked sequential chemical manipulations of great power and utility. Because the solution Debye length, κ−1, is of the order of the channel diameter, a, in the nanopores, fluidic transfer is controlled through applied bias, polarity and density of the immobile nanopore surface charge, solution ionic strength and the impedance of the nanopore relative to the microfluidic channels. Analyte transport between vertically separated microchannels can be saturated at two stable transfer levels, corresponding to reverse and forward bias. These NCAM-mediated integrated microfluidic architectures have been used to achieve highly reproducible and tunable injections down to attoliter volumes, sample stacking for preconcentration, preparative analyte band collection from an electrophoretic separation, and an actively-tunable size-dependent transport in hybrid structures with grafted polymers displaying thermally-regulated swelling behavior. The synthetic elaboration of the nanopore interior has also been used to great effect to realize molecular separations of high efficiency. All of these manipulations depend critically on the transport properties of individual nanocapillaries, and the study of transport in single nanopores has recently attracted significant attention. Both computation and experimental studies have utilized single nanopores as test beds to understand the fundamental chemical and physical properties of chemistry and fluid flow at nanometer length scales. PMID:19693375

  15. Evolution of the integral membrane desaturase gene family in moths and flies.

    PubMed Central

    Knipple, Douglas C; Rosenfield, Claire-Lise; Nielsen, Rasmus; You, Kyung Man; Jeong, Seong Eun

    2002-01-01

    Lepidopteran insects use sex pheromones derived from fatty acids in their species-specific mate recognition system. Desaturases play a particularly prominent role in the generation of structural diversity in lepidopteran pheromone biosynthesis as a result of the diverse enzymatic properties they have evolved. These enzymes are homologous to the integral membrane desaturases, which play a primary role in cold adaptation in eukaryotic cells. In this investigation, we screened for desaturase-encoding sequences in pheromone glands of adult females of eight lepidopteran species. We found, on average, six unique desaturase-encoding sequences in moth pheromone glands, the same number as is found in the genome database of the fly, Drosophila melanogaster, vs. only one to three in other characterized eukaryotic genomes. The latter observation suggests the expansion of this gene family in insects before the divergence of lepidopteran and dipteran lineages. We present the inferred homology relationships among these sequences, analyze nonsynonymous and synonymous substitution rates for evidence of positive selection, identify sequence and structural correlates of three lineages containing characterized enzymatically distinct desaturases, and discuss the evolution of this sequence family in insects. PMID:12524345

  16. Application of integrated ozone biological aerated filters and membrane filtration in water reuse of textile effluents.

    PubMed

    He, Yaozhong; Wang, Xiaojun; Xu, Jinling; Yan, Jinli; Ge, Qilong; Gu, Xiaoyang; Jian, Lei

    2013-04-01

    A combined process including integrated ozone-BAFs (ozone biological aerated filters) and membrane filtration was first applied for recycling textile effluents in a cotton textile mill with capacity of 5000 m(3)/d. Influent COD (chemical oxygen demand) in the range of 82-120 mg/L, BOD5 (5-day biochemical oxygen demand) of 12.6-23.1 mg/L, suspended solids (SSs) of 38-52 mg/L and color of 32-64° were observed during operation. Outflows with COD≤45 mg/L, BOD5≤7.6 mg/L, SS≤15 mg/L, color≤8° were obtained after being decontaminated by ozone-BAF with ozone dosage of 20-25 mg/L. Besides, the average removal rates of PVA (polyvinyl alcohol) and UV254 were 100% and 73.4% respectively. Permeate water produced by RO (reverse osmosis) could be reused in dyeing and finishing processes, while the RO concentrates could be discharged directly under local regulations with COD≤100 mg/L, BOD5≤21 mg/L, SS≤52 mg/L, color≤32°. Results showed that the combined process could guarantee water reuse with high quality, and solve the problem of RO concentrate disposal.

  17. Integrated Microfluidic Membrane Transistor Utilizing Chemical Information for On-Chip Flow Control

    PubMed Central

    Frank, Philipp; Schreiter, Joerg; Haefner, Sebastian; Paschew, Georgi; Voigt, Andreas; Richter, Andreas

    2016-01-01

    Microfluidics is a great enabling technology for biology, biotechnology, chemistry and general life sciences. Despite many promising predictions of its progress, microfluidics has not reached its full potential yet. To unleash this potential, we propose the use of intrinsically active hydrogels, which work as sensors and actuators at the same time, in microfluidic channel networks. These materials transfer a chemical input signal such as a substance concentration into a mechanical output. This way chemical information is processed and analyzed on the spot without the need for an external control unit. Inspired by the development electronics, our approach focuses on the development of single transistor-like components, which have the potential to be used in an integrated circuit technology. Here, we present membrane isolated chemical volume phase transition transistor (MIS-CVPT). The device is characterized in terms of the flow rate from source to drain, depending on the chemical concentration in the control channel, the source-drain pressure drop and the operating temperature. PMID:27571209

  18. Chronic restraint stress induces sperm acrosome reaction and changes in testicular tyrosine phosphorylated proteins in rats

    PubMed Central

    Arun, Supatcharee; Burawat, Jaturon; Sukhorum, Wannisa; Sampannang, Apichakan; Maneenin, Chanwit; Iamsaard, Sitthichai

    2016-01-01

    Background: Stress is a cause of male infertility. Although sex hormones and sperm quality have been shown to be low in stress, sperm physiology and testicular functional proteins, such as phosphotyrosine proteins, have not been documented. Objective: To investigate the acrosome status and alterations of testicular proteins involved in spermatogenesis and testosterone synthesis in chronic stress in rats. Materials and Methods: In this experimental study, male rats were divided into 2 groups (control and chronic stress (CS), n=7). CS rats were immobilized (4 hr/day) for 42 consecutive days. The blood glucose level (BGL), corticosterone, testosterone, acrosome status, and histopathology were examined. The expressions of testicular steroidogenic acute regulatory (StAR), cytochrome P450 side chain cleavage (CYP11A1), and phosphorylated proteins were analyzed. Results: Results showed that BGL (71.25±2.22 vs. 95.60±3.36 mg/dl), corticosterone level (24.33±4.23 vs. 36.9±2.01 ng/ml), acrosome reacted sperm (3.25±1.55 vs. 17.71±5.03%), and sperm head abnormality (3.29±0.71 vs. 6.21±1.18%) were significantly higher in CS group in comparison with control. In contrast, seminal vesicle (0.41±0.05 vs. 0.24±0.07 g/100g), testosterone level (3.37±0.79 vs. 0.61±0.29 ng/ml), and sperm concentration (115.33±7.70 vs. 79.13±3.65×106 cells/ml) of CS were significantly lower (p<0.05) than controls. Some atrophic seminiferous tubules and low sperm mass were apparent in CS rats. The expression of CYP11A1 except StAR protein was markedly decreased in CS rats. In contrast, a 55 kDa phosphorylated protein was higher in CS testes. Conclusion: CS decreased the expression of CYP11A, resulting in decreased testosterone, and increased acrosome-reacted sperm, assumed to be the result of an increase of 55 kDa phosphorylated protein. PMID:27525328

  19. Role of Wasp and the small GTPases RhoA, RhoB, and Cdc42 during capacitation and acrosome reaction in spermatozoa of English guinea pigs.

    PubMed

    Delgado-Buenrostro, Norma L; Mújica, Adela; Chiquete-Felix, Natalia; Déciga-Alcaraz, Alejandro; Medina-Reyes, Estefany I; Uribe-Carvajal, Salvador; Chirino, Yolanda I

    2016-10-01

    Cytoskeleton remodeling is necessary for capacitation and the acrosome reaction in spermatozoa. F-actin is located in the acrosome and equatorial region during capacitation, but is relocated in the post-acrosomal region during the acrosome reaction in spermatozoa from bull, rat, mice, and guinea pig. Actin polymerization and relocalization are generally regulated by small GTPases that activate Wasp protein, which coordinates with Arp2/3, profilin I, and profilin II to complete cytoskeletal remodeling. This sequence of events is not completely described in spermatozoa, though. Therefore, the aim of this study was to determine if Wasp interacts with small GTPases (RhoA, RhoB, and Cdc42) and proteins (Arp2/3, profilin I, and profilin II) that co-localize with F-actin during capacitation and the acrosome reaction in English guinea pig spermatozoa obtained from the vas deferens. The spermatozoa were capacitated in calcium-free medium, incubated with an activator or an inhibitor of GTPases, and then induced to acrosome react using calcium. The distribution patterns of F-actin were compared to the patterns of Wasp and its putative interaction partners: Wasp and RhoB, but not RhoA or Cdc42, localization overlap with F-actin during capacitation and the acrosome reaction. Activation of small GTPases localized RhoB to the post-acrosomal region whereas their inhibition prevented acrosome exocytosis. Arp2/3 and profilin II appear to interact with Wasp in the post-acrosomal region and flagellum, while profilin I and Wasp could be found in the equatorial region. Thus, Wasp and F-actin distribution overlap during capacitation and acrosome reaction, and small GTPases play an important role in cytoskeleton remodeling during these processes in spermatozoa. Mol. Reprod. Dev. 83: 927-937, 2016 © 2016 Wiley Periodicals, Inc.

  20. Modelling bioprocesses and membrane fouling in membrane bioreactor (MBR): a review towards finding an integrated model framework.

    PubMed

    Zuthi, M F R; Ngo, H H; Guo, W S

    2012-10-01

    The bioprocesses taking place in activated sludge wastewater treatment system itself are characterized by great complexity and yet incomplete understanding of some of the phenomena involved. The MBR technology inherent deficiencies for its simulation due to additional intrinsic complexities resulting from the interaction between concurrently occurring and dynamic biological processes with membrane filtration and the straightforward adoption of the activated sludge models' (ASM) frameworks or their modified variations. In this backdrop, this paper compiles a brief overview of the previous developments to the current state-of-the-art mathematical modelling approaches of the MBR system. With extended discussions on particular topics such as applications of modified ASMs to MBR modelling, ASM extensions incorporating soluble microbial products (SMP)/extracellular polymeric substances (EPS) concepts, this paper also provides a guide for different end-users of mathematical models of MBR systems.

  1. Methods for Studying Interactions of Detergents and Lipids withα-Helical and β-Barrel Integral Membrane Proteins

    PubMed Central

    Hasan, S. Saif; Baniulis, Danas; Yamashita, Eiki; Zhalnina, Mariya V.; Zakharov, Stanislav D.; Stofleth, Jason T.; Cramer, William A.

    2014-01-01

    Methods for studying interactions of protein with lipids and detergents are described for representatives of two major classes of membrane proteins: (1) the α-helical heterooligomeric integral cytochrome b6f complex of oxygenic photosynthesis from cyanobacteria, and (2) the outer membrane β-barrel proteins BtuB and OmpF from Gram-negative Escherichia coli bacteria. Details are presented on the use of detergents for purification and crystallization of the b6f complex as well as a method for lipid exchange. The positions of detergent and lipid molecules, which define eight potential lipid-binding sites in the b6f complex, are described. Differences in detergent strategies for isolation and crystallization of β-barrel proteins relative to those for oligomeric helical membrane proteins are discussed, and purification and assessment of protein quality by circular dichroism (CD) is presented. PMID:24510648

  2. Membrane-integrated physico-chemical treatment of coke-oven wastewater: transport modelling and economic evaluation.

    PubMed

    Kumar, Ramesh; Chakrabortty, Sankha; Pal, Parimal

    2015-04-01

    A modelling and simulation study with economic evaluation was carried out for an advanced membrane-integrated hybrid treatment process that ensures reuse of water with recovery of ammoniacal nitrogen as struvite from coke-oven wastewater. Linearized transport model was developed based on extended Nernst-Plank and concentration polarization modulus equation. Effects of pH, transmembrane pressure and cross-flow rate of interest on membrane charge density, solute rejection and solvent flux were investigated. The membrane module was successful in yielding a pure water flux as high as 120 L m(-2) h(-1) removing more than 95 and 96% of the cyanide and phenol, respectively, while permeating more than 90% NH4 (+)-N at a transmembrane pressure of only 15 × 10(2) KPa and at a pH of 10 for a volumetric cross-flow rate of 800 L h(-1). The Fenton's reagents were used to degrade more than 99% of pollutants present in the concentrated stream. The developed model could successfully predict the plant performance as reflected in the very low relative error (0.01-0.12) and overall high correlation coefficient (R(2) > 0.96). Economic analysis indicated that such a membrane-integrated hybrid system could be quite promising in coke wastewater treatment at low cost i.e. $0.934/m(2) of wastewater.

  3. Smart polymer brush nanostructures guide the self-assembly of pore-spanning lipid bilayers with integrated membrane proteins

    NASA Astrophysics Data System (ADS)

    Wilhelmina de Groot, G.; Demarche, Sophie; Santonicola, M. Gabriella; Tiefenauer, Louis; Vancso, G. Julius

    2014-01-01

    Nanopores in arrays on silicon chips are functionalized with pH-responsive poly(methacrylic acid) (PMAA) brushes and used as supports for pore-spanning lipid bilayers with integrated membrane proteins. Robust platforms are created by the covalent grafting of polymer brushes using surface-initiated atom transfer radical polymerization (ATRP), resulting in sensor chips that can be successfully reused over several assays. His-tagged proteins are selectively and reversibly bound to the nitrilotriacetic acid (NTA) functionalization of the PMAA brush, and consequently lipid bilayer membranes are formed. The enhanced membrane resistance as determined by electrochemical impedance spectroscopy and free diffusion of dyed lipids observed as fluorescence recovery after photobleaching confirmed the presence of lipid bilayers. Immobilization of the His-tagged membrane proteins on the NTA-modified PMAA brush near the pore edges is characterized by fluorescence microscopy. This system allows us to adjust the protein density in free-standing bilayers, which are stabilized by the polymer brush underneath. The potential application of the integrated platform for ion channel protein assays is demonstrated.

  4. CaMKII prevents spontaneous acrosomal exocytosis in sperm through induction of actin polymerization.

    PubMed

    Shabtay, Ortal; Breitbart, Haim

    2016-07-01

    In order to interact with the egg and undergo acrosomal exocytosis or the acrosome reaction (AR), mammalian spermatozoa must undergo a series of biochemical changes in the female reproductive tract, collectively called capacitation. We showed that F-actin is formed during sperm capacitation and fast depolymerization occurs prior to the AR. We hypothesized that F-actin protects the sperm from undergoing spontaneous-AR (sAR) which decreases fertilization rate. We show that activation of the actin-severing protein gelsolin induces a significant increase in sAR. Moreover, inhibition of CaMKII or PLD during sperm capacitation, caused an increase in sAR and inhibition of F-actin formation. Spermine, which leads to PLD activation, was able to reverse the effects of CaMKII inhibition on sAR-increase and F-actin-decrease. Furthermore, the increase in sAR and the decrease in F-actin caused by the inactivation of the PLD-pathway, were reversed by activation of CaMKII using H2O2 or by inhibiting protein phosphatase 1 which enhance the phosphorylation and oxidation states of CaMKII. These results indicate that two distinct pathways lead to F-actin formation in the sperm capacitation process which prevents the occurrence of sAR.

  5. Participation of the sperm proteasome during in vitro fertilisation and the acrosome reaction in cattle.

    PubMed

    Sánchez, R; Deppe, M; Schulz, M; Bravo, P; Villegas, J; Morales, P; Risopatrón, J

    2011-04-01

    In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.

  6. Uptake of iodinated contrast material in ischemic myocardium as an indicator of loss of cellular membrane integrity.

    PubMed

    Abraham, J L; Higgins, C B; Newell, J D

    1980-11-01

    Differential uptake of iodine containing radiographic contrast medium (I) in myocardial infarcts compared with normal mycardium has been detected by computerized transmission tomography (CTT). In this study the histologic and cellular distribution of I in ischemically damaged canine myocardium after intravenous administration of contrast material was examined by the use of scanning electron microscopy and energy dispersive X-ray microanalysis of fresh frozen cryosections. Analysis of individual cells in 6-mu thick sections mounted on carbon substrates showed that I was detectable in the ischemically damaged but not the normal myocardial cells. A decline in the potassium-to-sodium ratio confirmed the loss of membrane integrity in the ischemically damaged cells that accumulated I. These results indicate that I enters ischemically damaged but not normal myocardial cells suggesting that CTT scans after intravenous administration of contrast material may be capable of defining the area of the myocardium in which cells have lost membrane integrity after an ischemic injury.

  7. Simple production of membrane-based LiNbO3 micro-modulators with integrated tapers.

    PubMed

    Courjal, Nadège; Caspar, Alexis; Calero, Venancio; Ulliac, Gwenn; Suarez, Miguel; Guyot, Clément; Bernal, Maria-Pilar

    2016-11-01

    We report on free-standing electro-optical LiNbO3 waveguides with integrated tapers made by optical grade dicing. Membranes with a calibrated thickness are produced simultaneously with tapers acting as spot-size converters. Thereby, thicknesses from 450 to 500 μm can simply be achieved together with integrated tapers guaranteeing low insertion losses. These developments open the way to the low-cost production of compact and low-power-consuming electro-optical components. As an example, a 200 μm-long free-standing electro-optical Fabry-Perot is demonstrated with a figure of merit of only 0.19 V·cm in a 4.5 μm-thick membrane.

  8. Sulfated polysaccharides from the egg jelly layer are species-specific inducers of acrosomal reaction in sperms of sea urchins.

    PubMed

    Alves, A P; Mulloy, B; Diniz, J A; Mourão, P A

    1997-03-14

    We have characterized the fine structure of sulfated polysaccharides from the egg jelly layer of three species of sea urchins and tested the ability of these purified polysaccharides to induce the acrosome reaction in spermatozoa. The sea urchin Echinometra lucunter contains a homopolymer of 2-sulfated, 3-linked alpha-L-galactan. The species Arbacia lixula and Lytechinus variegatus contain linear sulfated alpha-L-fucans with regular tetrasaccharide repeating units. Each of these sulfated polysaccharides induces the acrosome reaction in conspecific but not in heterospecific spermatozoa. These results demonstrate that species specificity of fertilization in sea urchins depends in part on the fine structure of egg jelly sulfated polysaccharide.

  9. Effects of two copper compounds on Microcystis aeruginosa cell density, membrane integrity, and microcystin release.

    PubMed

    Tsai, Kuo-Pei

    2015-10-01

    Microcystin release following Microcystis aeruginosa cell lysis after copper-based algaecide treatment is often cited as a concern leading to restricted use of algaecide in restoration of natural water resources. To examine this concern, bench-scale experiments were conducted to study responses of M. aeruginosa to 8-day copper exposures as copper sulfate and copper-ethanolamine (Cu-EA). M. aeruginosa UTEX 2385 was cultured in BG11 medium to cell density of 10(6)cells/mL with total and extracellular microcystin of 93 and 53μg/L, respectively. Exposures of copper concentration ranged from 40 to 1000μgCu/L. Cell membrane integrity was indicated by erythrosine B. In the end of experiment, total microcystin and cell density in untreated control (313μg/L and 10(7)cells/mL) was 3.3 and 10 times greater than pretreatment value, respectively. Minimum amount of copper required to reduce M. aeruginosa population within 8 days was 160μgCu/L as copper sulfate and 80μgCu/L as Cu-EA, where total and extracellular microcystin concentrations (47 and 44μg/L for copper sulfate; 56 and 44μg/L for Cu-EA) were degraded with degradation rate coefficient 0.1 day(-1) and were less than pretreatment values. Given a copper concentration at 80µgCu/L as Cu-EA, M. aeruginosa cells were intact and less microcystin were released compared to treatments at 160-1000µgCu/L, where lysed cells and relatively greater microcystin release were observed. Based on the laboratory results, a minimum amount of copper required for reducing M. aeruginosa population could decrease total microcystin concentration and not compromise cells and minimize microcystin release.

  10. Evaluating the mechanical integrity of bilayer lipid membranes using a high-precision pressurization system

    NASA Astrophysics Data System (ADS)

    Hopkinson, David; Leo, Donald J.

    2007-04-01

    A new methodology has been developed to measure the mechanical integrity of a bilayer lipid membrane (BLM) formed over porous substrates. A custom test fixture was fabricated in which a stepper motor linear actuator drives a piston in order to apply pressure to a BLM in very fine increments. The pressure, monitored with a pressure transducer, is observed to increase until the BLM reaches its failure pressure, and then drop. This experiment was performed on 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC) lipid bilayers formed over porous polycarbonate substrates with various pore sizes ranging from 0.05 - 10 μm in diameter. A trend of increasing failure pressure with decreasing pore size was observed. The same set of experiments was repeated for BLMs that were formed from a mixture of SOPC and cholesterol (CHOL) at a cholesterol concentration of 50 mol%. The presence of cholesterol was found to increase the failure pressure of the BLMs by 1.5 times on average. A model of the characteristic pressure curve from this experiment was developed based on an initially closed fluid system in which pressure increases as it is loaded by a moving piston, and which upon reaching a critical failure pressure allows pressure to decrease as fluid escapes through a porous medium. Since the BLM is formed over many pores, this model assumes that the failure pressure for each micro-BLM follows a normal distribution over all pores. The model is able to accurately predict the major trends in the pressurization curves by curve-fitting a few statistical parameters.

  11. Functional assessment of SLC4A11, an integral membrane protein mutated in corneal dystrophies.

    PubMed

    Loganathan, Sampath K; Schneider, Hans-Peter; Morgan, Patricio E; Deitmer, Joachim W; Casey, Joseph R

    2016-11-01

    SLC4A11, a member of the SLC4 family of bicarbonate transporters, is a widely expressed integral membrane protein, abundant in kidney and cornea. Mutations of SLC4A11 cause some cases of the blinding corneal dystrophies, congenital hereditary endothelial dystrophy, and Fuchs endothelial corneal dystrophy. These diseases are marked by fluid accumulation in the corneal stroma, secondary to defective fluid reabsorption by the corneal endothelium. The role of SLC4A11 in these corneal dystrophies is not firmly established, as SLC4A11 function remains unclear. To clarify the normal function(s) of SLC4A11, we characterized the protein following expression in the simple, low-background expression system Xenopus laevis oocytes. Since plant and fungal SLC4A11 orthologs transport borate, we measured cell swelling associated with accumulation of solute borate. The plant water/borate transporter NIP5;1 manifested borate transport, whereas human SLC4A11 did not. SLC4A11 supported osmotically driven water accumulation that was electroneutral and Na(+) independent. Studies in oocytes and HEK293 cells could not detect Na(+)-coupled HCO3(-) transport or Cl(-)/HCO3(-) exchange by SLC4A11. SLC4A11 mediated electroneutral NH3 transport in oocytes. Voltage-dependent OH(-) or H(+) movement was not measurable in SLC4A11-expressing oocytes, but SLC4A11-expressing HEK293 cells manifested low-level cytosolic acidification at baseline. In mammalian cells, but not oocytes, OH(-)/H(+) conductance may arise when SLC4A11 activates another protein or itself is activated by another protein. These data argue against a role of human SLC4A11 in bicarbonate or borate transport. This work provides additional support for water and ammonia transport by SLC4A11. When expressed in oocytes, SLC4A11 transported NH3, not NH3/H().

  12. Nitrous oxide from moving bed based integrated fixed film activated sludge membrane bioreactors.

    PubMed

    Mannina, Giorgio; Capodici, Marco; Cosenza, Alida; Di Trapani, Daniele; Laudicina, Vito Armando; Ødegaard, Hallvard

    2017-02-01

    The present paper reports the results of a nitrous oxide (N2O) production investigation in a moving bed based integrated fixed film activated sludge (IFAS) membrane bioreactor (MBR) pilot plant designed in accordance with the University of Cape Town layout for biological phosphorous removal. Gaseous and liquid samples were collected in order to measure the gaseous as well as the dissolved concentration of N2O. Furthermore, the gas flow rate from each reactor was measured and the gas flux was estimated. The results confirmed that the anoxic reactor represents the main source of nitrous oxide production. A significant production of N2O was, however, also found in the anaerobic reactor, thus indicating a probable occurrence of the denitrifying phosphate accumulating organism activity. The highest N2O fluxes were emitted from the aerated reactors (3.09 g N2ON m(-2) h(-1) and 9.87 g N2ON m(-2) h(-1), aerobic and MBR tank, respectively). The emission factor highlighted that only 1% of the total treated nitrogen was emitted from the pilot plant. Furthermore, the measured N2O concentrations in the permeate flow were comparable with other reactors. Nitrous oxide mass balances outlined a moderate production also in the MBR reactor despite the low hydraulic retention time. On the other hand, the mass balance showed that in the aerobic reactor a constant consumption of nitrous oxide (up to almost 15 mg N2O h(-1)) took place, due to the high amount of stripped gas.

  13. Design and synthesis of non-hydrolyzable homoisoprenoid α-monofluorophosphonate inhibitors of PPAPDC family integral membrane lipid phosphatases.

    PubMed

    Subramanian, Thangaiah; Ren, Hongmei; Subramanian, Karunai Leela; Sunkara, Manjula; Onono, Fredrick O; Morris, Andrew J; Spielmann, H Peter

    2014-09-15

    An efficient, diversity oriented synthesis of homoisoprenoid α-monofluorophosphonates utilizing electrophilic fluorination is presented along with their activity as inhibitors of PPAPDC2 family integral membrane lipid phosphatases. These novel phosphatase-resistant analogues of isoprenoid monophosphates are a platform for further structure-activity relationship studies and provide access to other isoprenoid family members where the phosphate ester oxygen is replaced by a α-monofluoromethylene moiety.

  14. Advanced treatment of biologically pretreated coal gasification wastewater by a novel integration of catalytic ultrasound oxidation and membrane bioreactor.

    PubMed

    Jia, Shengyong; Han, Hongjun; Zhuang, Haifeng; Xu, Peng; Hou, Baolin

    2015-01-01

    Laboratorial scale experiments were conducted to investigate a novel system integrating catalytic ultrasound oxidation (CUO) with membrane bioreactor (CUO-MBR) on advanced treatment of biologically pretreated coal gasification wastewater. Results indicated that CUO with catalyst of FeOx/SBAC (sewage sludge based activated carbon (SBAC) which loaded Fe oxides) represented high efficiencies in eliminating TOC as well as improving the biodegradability. The integrated CUO-MBR system with low energy intensity and high frequency was more effective in eliminating COD, BOD5, TOC and reducing transmembrane pressure than either conventional MBR or ultrasound oxidation integrated MBR. The enhanced hydroxyl radical oxidation, facilitation of substrate diffusion and improvement of cell enzyme secretion were the mechanisms for CUO-MBR performance. Therefore, the integrated CUO-MBR was the promising technology for advanced treatment in engineering applications.

  15. A carboxy-terminal affinity tag for the purification and mass spectrometric characterization of integral membrane proteins.

    PubMed

    Wong, Julie P; Reboul, Emmanuelle; Molday, Robert S; Kast, Juergen

    2009-05-01

    G-protein-coupled receptors (GPCRs) and other structurally and functionally related membrane proteins represent particularly attractive targets for drug discovery. Integral membrane proteins are often difficult to purify from native contexts, and lack of sufficient quantities hampers subsequent structural and functional proteomic studies. We describe here an optimized enrichment strategy involving a membrane protein-compatible 1D4 affinity tag that is derived from the carboxy-terminal nine amino residues of bovine rhodopsin, and its corresponding tag-specific, high-affinity monoclonal antibody. When two GPCRs as well as two related ATP binding cassette (ABC) transporters are expressed in their functional forms in human cell lines, we have shown that a single detergent and wash condition can be employed for the purification of all said membrane proteins. Subsequent in-gel digestion with trypsin and mass spectrometric peptide analysis resulted in high sequence coverage for the ABC transporters ABCA1-1D4 and ABCA4-1D4. In contrast, digestion by various enzymatic combinations was necessary to obtain the best sequence coverage for affinity-enriched GPCRs CXCR4-1D4 and CCR5-1D4 as compared against other entries in an annotated spectrum library. Furthermore, specific enzyme combinations were necessary to produce suitable peptides for deducing N-glycosylation sites on CXCR4. Our results demonstrate that the 1D4-tag enrichment strategy is a versatile tool for the characterization of integral membrane proteins that can be employed for functional proteomic studies.

  16. Measurement of permeation of membranes by ethanol and water with integrated optics and Raman spectroscopy

    SciTech Connect

    Scherer, J.R.; Bailey, G.F.; Malladi, D.P.

    1983-03-25

    A new method for measuring the permeability of synthetic membranes to water and ethanol has been developed. The technique involves using the membrane as a laser waveguide and making Raman scattering measurements from the guided laser beam. The measurements show that alcohol can be detected at low levels within damp cellulose acetate membranes. The results suggest that further exploration using waveguide and Raman measurements might lead to a better understanding of the mechanisms which affect the relative permeability of alcohol and water. 6 references, 4 figures, 1 table.

  17. Thermodynamic characterization of the exchange of detergents and amphipols at the surfaces of integral membrane proteins.

    PubMed

    Tribet, C; Diab, C; Dahmane, T; Zoonens, M; Popot, J-L; Winnik, F M

    2009-11-03

    The aggregation of integral membrane proteins (IMPs) in aqueous media is a significant concern for mechanistic investigations and pharmaceutical applications of this important class of proteins. Complexation of IMPs with amphiphiles, either detergents or short amphiphilic polymers known as amphipols (APols), renders IMPs water-soluble. It is common knowledge that IMP-detergent complexes are labile, while IMP-APol complexes are exceptionally stable and do not dissociate even under conditions of extreme dilution. To understand the thermodynamic origin of this difference in stability and to guide the design of new APols, we have studied by isothermal titration calorimetry (ITC) the heat exchanges during two reciprocal processes, the "trapping" of detergent-solubilized IMPs in APols and the "stripping" of IMP-APol complexes by detergents, using two IMPs (the transmembrane domain of porin OmpA from Escherichia coli and bacteriorhodopsin from Halobium salinarium), two APols [an anionic polymer derived from acrylic acid (A8-35) and a cationic phosphorylcholine-based polymer (C22-43)], and two neutral detergents [n-octyl thioglucoside (OTG) and n-octyltetraethylene glycol (C(8)E(4))]. In the presence of detergent, free APols and IMP-APol complexes form mixed particles, APol-detergent and IMP-APol-detergent, respectively, according to the regular mixing model. Diluting IMP-APol-detergent complexes below the critical micellar concentration (CMC) of the detergent triggers the dispersion of detergent molecules as monomers, a process characterized by an enthalpy of demicellization. The enthalpy of APol <--> detergent exchange on the hydrophobic surface of IMPs is negligibly small, an indication of the similarity of the molecular interactions of IMPs with the two types of amphiphiles. The enhanced stability against dilution of IMP-APol complexes, compared to IMP-detergent ones, originates from the difference in entropy gain achieved upon release in water of a few APol molecules

  18. Novel fatty acid acylation of lens integral membrane protein aquaporin-0.

    PubMed

    Schey, Kevin L; Gutierrez, Danielle B; Wang, Zhen; Wei, Junhua; Grey, Angus C

    2010-11-16

    Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0. LC/MS/MS proteomic analysis of hydrophobic tryptic peptides from lens membrane proteins revealed both N-terminal and C-terminal peptides modified by 238 and 264 Da which were subsequently assigned by accurate mass measurement as palmitoylation and oleoylation, respectively. Specific sites of modification were the N-terminal methionine residue and lysine 238 revealing, for the first time, an oleic acid modification via an amide linkage to a lysine residue. The specific fatty acids involved reflect their abundance in the lens fiber cell plasma membrane. Imaging mass spectrometry indicated abundant acylated AQP0 in the inner cortical region of both bovine and human lenses and acylated truncation products in the lens nucleus. Additional analyses revealed that the lipid-modified forms partitioned exclusively to a detergent-resistant membrane fraction, suggesting a role in membrane domain targeting.

  19. Tyrosine Hydroxylase Binding to Phospholipid Membranes Prompts Its Amyloid Aggregation and Compromises Bilayer Integrity

    PubMed Central

    Baumann, Anne; Jorge-Finnigan, Ana; Jung-KC, Kunwar; Sauter, Alexander; Horvath, Istvan; Morozova-Roche, Ludmilla A.; Martinez, Aurora

    2016-01-01

    Tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of catecholamine neurotransmitters and hormones, binds to negatively charged phospholipid membranes. Binding to both large and giant unilamellar vesicles causes membrane permeabilization, as observed by efflux and influx of fluorescence dyes. Whereas the initial protein-membrane interaction involves the N-terminal tail that constitutes an extension of the regulatory ACT-domain, prolonged membrane binding induces misfolding and self-oligomerization of TH over time as shown by circular dichroism and Thioflavin T fluorescence. The gradual amyloid-like aggregation likely occurs through cross-β interactions involving aggregation-prone motives in the catalytic domains, consistent with the formation of chain and ring-like protofilaments observed by atomic force microscopy in monolayer-bound TH. PC12 cells treated with the neurotoxin 6-hydroxydopamine displayed increased TH levels in the mitochondrial fraction, while incubation of isolated mitochondria with TH led to a decrease in the mitochondrial membrane potential. Furthermore, cell-substrate impedance and viability assays showed that supplementing the culture media with TH compromises cell viability over time. Our results revealed that the disruptive effect of TH on cell membranes may be a cytotoxic and pathogenic factor if the regulation and intracellular stability of TH is compromised. PMID:28004763

  20. Integrated distillation-membrane process for bio-ethanol and bio-butanol recovery from actual fermentation broths: Separation energy efficiency and fate of secondary fermentation products

    EPA Science Inventory

    A hybrid process integrating vapor stripping with vapor compression and vapor permeation membrane separation, termed Membrane Assisted Vapor Stripping (MAVS), was evaluated for recovery and dehydration of ethanol and/or 1-butanol from aqueous solution as an alternative to convent...

  1. Intramolecular Disulfide Bond of Tim22 Protein Maintains Integrity of the TIM22 Complex in the Mitochondrial Inner Membrane*

    PubMed Central

    Okamoto, Hiroaki; Miyagawa, Akiko; Shiota, Takuya; Tamura, Yasushi; Endo, Toshiya

    2014-01-01

    Mitochondrial proteins require protein machineries called translocators in the outer and inner membranes for import into and sorting to their destination submitochondrial compartments. Among them, the TIM22 complex mediates insertion of polytopic membrane proteins into the inner membrane, and Tim22 constitutes its central insertion channel. Here we report that the conserved Cys residues of Tim22 form an intramolecular disulfide bond. By comparison of Tim22 Cys → Ser mutants with wild-type Tim22, we show that the disulfide bond of Tim22 stabilizes Tim22 especially at elevated temperature through interactions with Tim18, which are also important for the stability of the TIM22 complex. We also show that lack of the disulfide bond in Tim22 impairs the assembly of TIM22 pathway substrate proteins into the inner membrane especially when the TIM22 complex handles excess amounts of substrate proteins. Our findings provide a new insight into the mechanism of the maintenance of the structural and functional integrity of the TIM22 complex. PMID:24385427

  2. Fully efficient chromosome dimer resolution in Escherichia coli cells lacking the integral membrane domain of FtsK

    PubMed Central

    Dubarry, Nelly; Barre, François-Xavier

    2010-01-01

    In bacteria, septum formation frequently initiates before the last steps of chromosome segregation. This is notably the case when chromosome dimers are formed by homologous recombination. Chromosome segregation then requires the activity of a double-stranded DNA transporter anchored at the septum by an integral membrane domain, FtsK. It was proposed that the transmembrane segments of proteins of the FtsK family form pores across lipid bilayers for the transport of DNA. Here, we show that truncated Escherichia coli FtsK proteins lacking all of the FtsK transmembrane segments allow for the efficient resolution of chromosome dimers if they are connected to a septal targeting peptide through a sufficiently long linker. These results indicate that FtsK does not need to transport DNA through a pore formed by its integral membrane domain. We propose therefore that FtsK transports DNA before membrane fusion, at a time when there is still an opening in the constricted septum. PMID:20033058

  3. The cytoplasmic domain is essential for transport function of the integral membrane transport protein SLC4A11.

    PubMed

    Loganathan, Sampath K; Lukowski, Chris M; Casey, Joseph R

    2016-01-15

    Large cytoplasmic domains (CD) are a common feature among integral membrane proteins. In virtually all cases, these CD have a function (e.g., binding cytoskeleton or regulatory factors) separate from that of the membrane domain (MD). Strong associations between CD and MD are rare. Here we studied SLC4A11, a membrane transport protein of corneal endothelial cells, the mutations of which cause genetic corneal blindness. SLC4A11 has a 41-kDa CD and a 57-kDa integral MD. One disease-causing mutation in the CD, R125H, manifests a catalytic defect, suggesting a role of the CD in transport function. Expressed in HEK-293 cells without the CD, MD-SLC4A11 is retained in the endoplasmic reticulum, indicating a folding defect. Replacement of CD-SLC4A11 with green fluorescent protein did not rescue MD-SLC4A11, suggesting some specific role of CD-SLC4A11. Homology modeling revealed that the structure of CD-SLC4A11 is similar to that of the Cl(-)/HCO3(-) exchange protein AE1 (SLC4A1) CD. Fusion to CD-AE1 partially rescued MD-SLC4A11 to the cell surface, suggesting that the structure of CD-AE1 is similar to that of CD-SLC4A11. The CD-AE1-MD-SLC4a11 chimera, however, had no functional activity. We conclude that CD-SLC4A11 has an indispensable role in the transport function of SLC4A11. CD-SLC4A11 forms insoluble precipitates when expressed in bacteria, suggesting that the domain cannot fold properly when expressed alone. Consistent with a strong association between CD-SLC4A11 and MD-SLC4A11, these domains specifically associate when coexpressed in HEK-293 cells. We conclude that SLC4A11 is a rare integral membrane protein in which the CD has strong associations with the integral MD, which contributes to membrane transport function.

  4. The cytoplasmic domain is essential for transport function of the integral membrane transport protein SLC4A11

    PubMed Central

    Loganathan, Sampath K.; Lukowski, Chris M.

    2015-01-01

    Large cytoplasmic domains (CD) are a common feature among integral membrane proteins. In virtually all cases, these CD have a function (e.g., binding cytoskeleton or regulatory factors) separate from that of the membrane domain (MD). Strong associations between CD and MD are rare. Here we studied SLC4A11, a membrane transport protein of corneal endothelial cells, the mutations of which cause genetic corneal blindness. SLC4A11 has a 41-kDa CD and a 57-kDa integral MD. One disease-causing mutation in the CD, R125H, manifests a catalytic defect, suggesting a role of the CD in transport function. Expressed in HEK-293 cells without the CD, MD-SLC4A11 is retained in the endoplasmic reticulum, indicating a folding defect. Replacement of CD-SLC4A11 with green fluorescent protein did not rescue MD-SLC4A11, suggesting some specific role of CD-SLC4A11. Homology modeling revealed that the structure of CD-SLC4A11 is similar to that of the Cl−/HCO3− exchange protein AE1 (SLC4A1) CD. Fusion to CD-AE1 partially rescued MD-SLC4A11 to the cell surface, suggesting that the structure of CD-AE1 is similar to that of CD-SLC4A11. The CD-AE1-MD-SLC4a11 chimera, however, had no functional activity. We conclude that CD-SLC4A11 has an indispensable role in the transport function of SLC4A11. CD-SLC4A11 forms insoluble precipitates when expressed in bacteria, suggesting that the domain cannot fold properly when expressed alone. Consistent with a strong association between CD-SLC4A11 and MD-SLC4A11, these domains specifically associate when coexpressed in HEK-293 cells. We conclude that SLC4A11 is a rare integral membrane protein in which the CD has strong associations with the integral MD, which contributes to membrane transport function. PMID:26582474

  5. Membrane-type photonic integration of InGaN/GaN multiple-quantum-well diodes and waveguide

    NASA Astrophysics Data System (ADS)

    Gao, Xumin; Bai, Dan; Cai, Wei; Xu, Yin; Yuan, Jialei; Yang, Yongchao; Zhu, Guixia; Cao, Xun; Zhu, Hongbo; Wang, Yongjin

    2017-02-01

    We report here a membrane-type integration of InGaN/GaN multiple-quantum-well diodes (MQWDs) with a waveguide to build a highly integrated photonic system to perform functionalities on a GaN-on-silicon platform. Suspended MQWDs can be used as either for light-emitting diode (LED) or photodiode. In the fabricated photonic system, part of the LED emission is coupled into a suspended waveguide, and the guided light laterally propagates along the waveguide and is finally sensed by the photodiode. The photonic system can detect the in-plane guided light and the external incident light simultaneously. Planar optical communication experimentally demonstrates that the proof-of-concept monolithic photonic integration system can achieve the in-plane visible light communication. This work paves the way towards novel active electro-optical sensing systems and planar optical communication in the visible range.

  6. Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

    SciTech Connect

    Golczak, Marcin; Kiser, Philip D.; Lodowski, David T.; Maeda, Akiko; Palczewski, Krzysztof

    2010-04-05

    Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A{sup 2} treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and

  7. Involvement of zinc in the regulation of pHi, motility, and acrosome reactions in sea urchin sperm

    PubMed Central

    1985-01-01

    When sperm of Strongylocentrotus purpuratus or Lytechinus pictus are diluted into seawater, motility is initiated; and when exposed to egg jelly, an acrosome reaction is induced. In the presence of a variety of structurally different metal chelators (0.1-1 mM EDTA, EGTA, phenanthroline, dipyridyl, cysteine, or dithiothreitol), motility initiation is delayed and the acrosome reaction is inhibited. Of the metals detected in the sperm of these two species, very low levels of Zn+2 (0.1 microM free Zn+2) uniquely prevent this chelator inhibition. L. pictus sperm concentrate 65Zn+2 from seawater, and EDTA removes 50% of the accumulated 65Zn+2 by 5 min. Since both sperm motility and acrosome reactions are in part regulated by intracellular pH (pHi), the effect of chelators on the sperm pHi was examined by using the fluorescent pH sensitive probe, 9-aminoacridine, EDTA depresses sperm pHi in both species, and 0.1 microM free Zn+2 reverses this pHi depression. When sperm are diluted into media that contain chelators, both NH4Cl and monensin (a Na+/H+ ionophore) increase the sperm pHi and reverse the chelator inhibition of sperm motility and acrosome reactions. The results of this study are consistent with the involvement of a trace metal (probably zinc) in the pHi regulation of sea urchin sperm and indicate a likely mechanism for the previously observed effects of chelators on sperm motility and acrosome reactions. PMID:3922992

  8. Manchette-acrosome disorders during spermiogenesis and low efficiency of seminiferous tubules in hypercholesterolemic rabbit model

    PubMed Central

    Simón, Layla; Funes, Abi K.; Yapur, Martín A.; Cabrillana, María E.; Monclus, María A.; Boarelli, Paola V.; Vincenti, Amanda E.

    2017-01-01

    Hypercholesterolemia is a marker for several adult chronic diseases. Recently we demonstrated that sub/infertility is also associated to Hypercholesterolemia in rabbits. Seminal alterations included: abnormal sperm morphology, decreased sperm number and declined percentage of motile sperm, among others. In this work, our objective was to evaluate the effects of hypercholesterolemia on testicular efficiency and spermiogenesis, as the latter are directly related to sperm number and morphology respectively. Tubular efficiency was determined by comparing total number of spermatogenic cells with each cell type within the proliferation/differentiation compartments. We found lower testicular efficiency related to both a decrease in spermatogonial cells and an increase in germ cell apoptosis in hypercholesterolemic rabbits. On the other hand, spermiogenesis–the last step of spermatogenesis involved in sperm shaping–was detaily analyzed, particularly the acrosome-nucleus-manchette complex. The manchette is a microtubular-based temporary structure responsible in sperm cell elongation. We analyzed the contribution of actin filaments and raft microdomains in the arrangement of the manchette. Under fluorescence microscopy, spermatocyte to sperm cell development was followed in cells isolated from V to VIII tubular stages. In cells from hypercholesterolemic rabbits, abnormal development of acrosome, nucleus and inaccurate tail implantation were associated with actin–alpha-tubulin–GM1 sphingolipid altered distribution. Morphological alterations were also observed at electron microscopy. We demonstrated for the first time that GM1-enriched microdomains together with actin filaments and microtubules are involved in allowing the correct anchoring of the manchette complex. In conclusion, cholesterol enriched diets promote male fertility alterations by affecting critical steps in sperm development: spermatogenesis and spermiogenesis. It was also demonstrated that

  9. Manchette-acrosome disorders during spermiogenesis and low efficiency of seminiferous tubules in hypercholesterolemic rabbit model.

    PubMed

    Simón, Layla; Funes, Abi K; Yapur, Martín A; Cabrillana, María E; Monclus, María A; Boarelli, Paola V; Vincenti, Amanda E; Saez Lancellotti, Tania E; Fornés, Miguel W

    2017-01-01

    Hypercholesterolemia is a marker for several adult chronic diseases. Recently we demonstrated that sub/infertility is also associated to Hypercholesterolemia in rabbits. Seminal alterations included: abnormal sperm morphology, decreased sperm number and declined percentage of motile sperm, among others. In this work, our objective was to evaluate the effects of hypercholesterolemia on testicular efficiency and spermiogenesis, as the latter are directly related to sperm number and morphology respectively. Tubular efficiency was determined by comparing total number of spermatogenic cells with each cell type within the proliferation/differentiation compartments. We found lower testicular efficiency related to both a decrease in spermatogonial cells and an increase in germ cell apoptosis in hypercholesterolemic rabbits. On the other hand, spermiogenesis-the last step of spermatogenesis involved in sperm shaping-was detaily analyzed, particularly the acrosome-nucleus-manchette complex. The manchette is a microtubular-based temporary structure responsible in sperm cell elongation. We analyzed the contribution of actin filaments and raft microdomains in the arrangement of the manchette. Under fluorescence microscopy, spermatocyte to sperm cell development was followed in cells isolated from V to VIII tubular stages. In cells from hypercholesterolemic rabbits, abnormal development of acrosome, nucleus and inaccurate tail implantation were associated with actin-alpha-tubulin-GM1 sphingolipid altered distribution. Morphological alterations were also observed at electron microscopy. We demonstrated for the first time that GM1-enriched microdomains together with actin filaments and microtubules are involved in allowing the correct anchoring of the manchette complex. In conclusion, cholesterol enriched diets promote male fertility alterations by affecting critical steps in sperm development: spermatogenesis and spermiogenesis. It was also demonstrated that hypercholesterolemic

  10. POM152 is an integral protein of the pore membrane domain of the yeast nuclear envelope

    PubMed Central

    1994-01-01

    We have identified a concanavalin A-reactive glycoprotein of 150 kD that coenriches with isolated yeast nuclear pore complexes. Molecular cloning and sequencing of this protein revealed a single canonical transmembrane segment. Epitope tagging and localization by both immunofluorescence and immunoelectron microscopy confirmed that it is a pore membrane protein. The protein was termed POM152 (for pore membrane protein of 152 kD) on the basis of its location and cDNA-deduced molecular mass. POM152 is likely to be a type II membrane protein with its NH2-terminal region (175 residues) and its COOH-terminal region (1,142 residues) positioned on the pore side and cisternal side of the pore membrane, respectively. The proposed cisternally exposed domain contains eight repetitive motifs of approximately 24 residues. Surprisingly, POM152 deletion mutants were viable and their growth rate was indistinguishable from that of wild-type cells at temperatures between 17 and 37 degrees C. However, overproduction of POM152 inhibited cell growth. When expressed in mouse 3T3 cells, POM152 was found to be localized to the pore membrane, suggesting a conserved sorting pathway between yeast and mammals. PMID:8138573

  11. Platelet-activating factor in Iberian pig spermatozoa: receptor expression and role as enhancer of the calcium-induced acrosome reaction.

    PubMed

    Bragado, M J; Gil, M C; Garcia-Marin, L J

    2011-12-01

    Platelet-activating factor (PAF) is a phospholipid involved in reproductive physiology. PAF receptor is expressed in some mammalian spermatozoa species where it plays a role in these germ-cell-specific processes. The aim of this study is to identify PAF receptor in Iberian pig spermatozoa and to evaluate PAF's effects on motility, viability and acrosome reaction. Semen samples from Iberian boars were used. PAF receptor identification was performed by Western blotting. Spermatozoa motility was analysed by computer-assisted sperm analysis system, whereas spermatozoa viability and acrosome reaction were evaluated by flow cytometry. Different PAF concentrations added to non-capacitating medium during 60 min have no effect on any spermatozoa motility parameter measured. Acrosome reaction was rapid and potently induced by 1 μm calcium ionophore A23187 showing an effect at 60 min and maximum at 240 min. PAF added to a capacitating medium is not able to induce spermatozoa acrosome reaction at any time studied. However, PAF, in the presence of A23187, significantly accelerates and enhances the calcium-induced acrosome reaction in a concentration-dependent manner in Iberian boar spermatozoa. Exogenous PAF does not affect at all spermatozoa viability, whereas slightly exacerbated the A23187-induced loss in viability. This work demonstrates that PAF receptor is expressed in Iberian pig spermatozoa and that its stimulation by PAF regulates the calcium-induced acrosome reaction. This work contributes to further elucidate the physiological regulation of the most relevant spermatozoa functions for successful fertilization: acrosome reaction.

  12. Effects of X-ray and carbon ion beam irradiation on membrane permeability and integrity in Saccharomyces cerevisiae cells.

    PubMed

    Cao, Guozhen; Zhang, Miaomiao; Miao, Jianshun; Li, Wenjian; Wang, Jufang; Lu, Dong; Xia, Jiefang

    2015-03-01

    Saccharomyces cerevisiae has served as a eukaryotic model in radiation biology studies of cellular responses to ionizing radiation (IR). Research in this field has thus far mainly been focused on DNA strand breaks, DNA base damage, or inhibition of protein activity. However, the effects of IR on S. cerevisiae cell membranes have barely been studied. Here, we investigated the changes in the permeability and integrity of S. cerevisiae cell membranes induced by high-linear energy transfer carbon ion (CI) beam or low-linear energy transfer X-ray. After CI exposure, protein elution and nucleotide diffusion were more pronounced than after X-ray treatment at the same doses, although these features were most prevalent following irradiation doses of 25-175 Gy. Flow cytometry of forward scatter light versus side scatter light and double-staining with fluorescein diacetate and propidium iodide showed that CI and X-ray irradiation significantly affected S. cerevisiae cell membrane integrity and cellular enzyme activity compared with untreated control cells. The extent of lesions in CI-irradiated cells, which exhibited markedly altered morphology and size, was greater than that in X-ray-irradiated cells. The relationships between permeabilized cells, esterase activity, and non-viable cell numbers furthermore indicated that irradiation-induced increases in cell permeabilization and decreases in esterase activity are dependent on the type of radiation and that these parameters correspond well with cell viability. These results also indicate that the patterns of cell inactivity due to X-ray or CI irradiation may be similar in terms of cell membrane damage.

  13. Highly integrated hybrid process with ceramic ultrafiltration-membrane for advanced treatment of drinking water: a pilot study.

    PubMed

    Guo, Jianning; Wang, Lingyun; Zhu, Jia; Zhang, Jianguo; Sheng, Deyang; Zhang, Xihui

    2013-01-01

    This article presents a highly integrated hybrid process for the advanced treatment of drinking water in dealing with the micro-polluted raw water. A flat sheet ceramic membrane with the pore size of 50∼60 nm for ultrafiltration (UF) is used to integrate coagulation and ozonation together. At the same time, biological activated carbon filtration (BAC) is used to remove the ammonia and organic pollutants in raw water. A pilot study in the scale of 120 m(3)/d has been conducted in Southern China. The mainly-analyzed parameters include turbidity, particle counts, ammonia, total organic carbon (TOC), UV254, biological dissolved organic carbon (BDOC), dissolved oxygen (DO) as well as trans-membrane pressure (TMP). The experiments demonstrated that ceramic UF-membrane was able to remove most of turbidity and suspended particulate matters. The final effluent turbidity reached to 0.14 NTU on average. BAC was effective in removing ammonia and organic matters. Dissolved oxygen (DO) is necessary for the biodegradation of ammonia at high concentration. The removal efficiencies reached to 90% for ammonia with the initial concentration of 3.6 mg/L and 76% for TOC with the initial concentration of 3.8 mg/L. Ozonation can alter the molecular structure of organics in terms of UV254, reduce membrane fouling, and extend the operation circle. It is believed the hybrid treatment process developed in this article can achieve high performance with less land occupation and lower cost compared with the conventional processes. It is especially suitable for the developing countries in order to obtain high-quality drinking water in a cost-effective way.

  14. Cold Stress Makes Escherichia coli Susceptible to Glycopeptide Antibiotics by Altering Outer Membrane Integrity.

    PubMed

    Stokes, Jonathan M; French, Shawn; Ovchinnikova, Olga G; Bouwman, Catrien; Whitfield, Chris; Brown, Eric D

    2016-02-18

    A poor understanding of the mechanisms by which antibiotics traverse the outer membrane remains a considerable obstacle to the development of novel Gram-negative antibiotics. Herein, we demonstrate that the Gram-negative bacterium Escherichia coli becomes susceptible to the narrow-spectrum antibiotic vancomycin during growth at low temperatures. Heterologous expression of an Enterococcus vanHBX vancomycin resistance cluster in E. coli confirmed that the mechanism of action was through inhibition of peptidoglycan biosynthesis. To understand the nature of vancomycin permeability, we screened for strains of E. coli that displayed resistance to vancomycin at low temperature. Surprisingly, we observed that mutations in outer membrane biosynthesis suppressed vancomycin activity. Subsequent chemical analysis of lipopolysaccharide from vancomycin-sensitive and -resistant strains confirmed that suppression was correlated with truncations in the core oligosaccharide of lipopolysaccharide. These unexpected observations challenge the current understanding of outer membrane permeability, and provide new chemical insights into the susceptibility of E. coli to glycopeptide antibiotics.

  15. ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function*

    PubMed Central

    Darshi, Manjula; Mendiola, Vincent L.; Mackey, Mason R.; Murphy, Anne N.; Koller, Antonius; Perkins, Guy A.; Ellisman, Mark H.; Taylor, Susan S.

    2011-01-01

    The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952–14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function. PMID:21081504

  16. AM67, a secretory component of the guinea pig sperm acrosomal matrix, is related to mouse sperm protein sp56 and the complement component 4-binding proteins.

    PubMed

    Foster, J A; Friday, B B; Maulit, M T; Blobel, C; Winfrey, V P; Olson, G E; Kim, K S; Gerton, G L

    1997-05-09

    The guinea pig sperm acrosomal matrix is the dense core of the acrosome and is likely to be important in acrosome biogenesis and fertilization. Isolated acrosomal matrices are composed of a limited number of major bands when analyzed by SDS-polyacrylamide gel electrophoresis, among which is a Mr 67,000 protein that we have termed AM67. Indirect immunofluorescence demonstrated that AM67 is localized to the apical segment of the cauda epididymal sperm acrosome. Immunoelectron microscopy further refined the localization of AM67 to the M1 (dorsal bulge) domain within the acrosome. Using a polymerase chain reaction product based upon tryptic peptide sequences from AM67, a lambdagt11 guinea pig testis cDNA library was screened to yield two cDNA clones that encode the AM67 peptides. Northern analysis revealed that AM67 is transcribed as a 1. 9-kilobase testis-specific mRNA. The complete AM67 sequence encodes a prepropolypeptide of 533 amino acids with a calculated Mr of 59, 768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have a Mr of 56,851 and seven consensus sites for asparagine-linked glycosylation. The deduced amino acid sequence of AM67 is most similar to those of the mouse sperm protein sp56 and the alpha-subunits of complement component 4-binding proteins from various mammalian species. Although mouse sp56 has been reported to be a cell-surface receptor for the murine zona pellucida glycoprotein ZP3, standard immunoelectron microscopy using the anti-sp56 monoclonal antibody 7C5 detected sp56 within the mouse sperm acrosome, but failed to detect sp56 on the surface of acrosome-intact mouse sperm. Furthermore, acrosomal labeling was detected in mouse sperm prepared for immunofluorescence using paraformaldehyde fixation, but was not observed with live unfixed sperm. Thus, the finding that sp56 is present within the acrosome provides further support that sp56 and AM67 are orthologues and suggests that sp56 may function in

  17. Encapsulation of sex sorted boar semen: sperm membrane status and oocyte penetration parameters.

    PubMed

    Spinaci, Marcella; Chlapanidas, Theodora; Bucci, Diego; Vallorani, Claudia; Perteghella, Sara; Lucconi, Giulia; Communod, Ricardo; Vigo, Daniele; Galeati, Giovanna; Faustini, Massimo; Torre, Maria Luisa

    2013-03-01

    Although sorted semen is experimentally used for artificial, intrauterine, and intratubal insemination and in vitro fertilization, its commercial application in swine species is still far from a reality. This is because of the low sort rate and the large number of sperm required for routine artificial insemination in the pig, compared with other production animals, and the greater susceptibility of porcine spermatozoa to stress induced by the different sex sorting steps and the postsorting handling protocols. The encapsulation technology could overcome this limitation in vivo, protecting and allowing the slow release of low-dose sorted semen. The aim of this work was to evaluate the impact of the encapsulation process on viability, acrosome integrity, and on the in vitro fertilizing potential of sorted boar semen. Our results indicate that the encapsulation technique does not damage boar sorted semen; in fact, during a 72-hour storage, no differences were observed between liquid-stored sorted semen and encapsulated sorted semen in terms of plasma membrane (39.98 ± 14.38% vs. 44.32 ± 11.72%, respectively) and acrosome integrity (74.32 ± 12.17% vs. 66.07 ± 10.83%, respectively). Encapsulated sorted spermatozoa presented a lower penetration potential than nonencapsulated ones (47.02% vs. 24.57%, respectively, P < 0.0001), and a significant reduction of polyspermic fertilization (60.76% vs. 36.43%, respectively, polyspermic ova/total ova; P < 0.0001). However, no difference (P > 0.05) was observed in terms of total efficiency of fertilization expressed as normospermic oocytes/total oocytes (18.45% vs. 15.43% for sorted diluted and sorted encapsulated semen, respectively). The encapsulation could be an alternative method of storing of pig sex sorted spermatozoa and is potentially a promising technique in order to optimize the use of low dose of sexed spermatozoa in vivo.

  18. Integrated nitrogen removal biofilter system with ceramic membrane for advanced post-treatment of municipal wastewater.

    PubMed

    Son, Dong-Jin; Yun, Chan-Young; Kim, Woo-Yeol; Zhang, Xing-Ya; Kim, Dae-Gun; Chang, Duk; Sunwoo, Young; Hong, Ki-Ho

    2016-12-01

    The pre-denitrification biofilm process for nitrogen removal was combined with ceramic membrane with pore sizes of 0.05-0.1 µm as a system for advanced post-treatment of municipal wastewater. The system was operated under an empty bed hydraulic retention time of 7.8 h, recirculation ratio of 3, and transmembrane pressure of 0.47 bar. The system showed average removals of organics, total nitrogen, and solids as high as 93%, 80%, and 100%, respectively. Rapid nitrification could be achieved and denitrification was performed in the anoxic filter without external carbon supplements. The residual particulate organics and nitrogen in effluent from biofilm process could be also removed successfully through membrane filtration and the removal of total coliform was noticeably improved after membrane filtration. Thus, a system composed of the pre-denitrification biofilm process with ceramic membrane would be a compact and flexible option for advanced post-treatment of municipal wastewater.

  19. A micropatterned cell array with an integrated oxygen-sensitive fluorescent membrane.

    PubMed

    Montagne, Kevin; Komori, Kikuo; Yang, Fei; Tatsuma, Tetsu; Fujii, Teruo; Sakai, Yasuyuki

    2009-11-01

    We propose a simple method for producing micropatterned cell spots by photocatalytic lithography on a Pt porphyrin-based oxygen-sensitive polystyrene membrane that enables real-time imaging of oxygen consumption of patterned cell spots with sub-millimetre resolution.

  20. Contactless microfluidic pumping using microchannel-integrated carbon black composite membranes

    PubMed Central

    Fu, Xiaotong; Gagnon, Zachary

    2015-01-01

    The ability to pump and manipulate fluid at the micron-scale is a basic requirement for microfluidic platforms. Many current manipulation methods, however, require expensive and bulky external supporting equipment, which are not typically compatible for portable applications. We have developed a contactless metal electro-osmotic micropump capable of pumping conductive buffers. The pump operates using two pairs of gallium metal electrodes, which are activated using an external voltage source and separated from a main flow channel by a thin micron-scale polydimethylsiloxane (PDMS) membrane. The thin contactless membrane allows for field penetration and electro-osmotic flow within the microchannel, but eliminates electrode damage and sample contamination commonly associated with traditional DC electro-osmotic pumps that utilize electrodes in direct contact with the working fluid. Our previous work has demonstrated the effectiveness of this method in pumping deionized water. However, due to the high resistivity of PDMS, this method proved difficult to apply towards manipulating conductive buffers. To overcome this limitation, we fabricated conductive carbon black (CB) powder directly into the contactless PDMS membranes. The increased electrical conductivity of the contactless PDMS membrane significantly increased micropump performance. Using a microfluidic T-channel device and an electro-osmotic flow model, we determined the influence that CB has on pump pressure for CB weight percents varying between 0 and 20. The results demonstrate that the CB increases pump pressure by two orders of magnitude and enables effective operations with conductive buffers. PMID:26543514

  1. Effects of two 6-quinolinyl chalcones on the integrity of plasma membrane of Paracoccidioides brasiliensis.

    PubMed

    de Sá, Nívea P; Cisalpino, Patrícia S; Tavares, Luciana C; Espíndola, Leandro; Borelli, Beatriz M; Barbeira, Paulo Js; Cardoso Perdigão, Gabriele de Matos; Souza-Fagundes, Elaine M; Rosa, Carlos A; Pizzolatti, Moacir G; Johann, Susana

    2017-03-01

    Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, yet few therapeutic options exist. Our aim was to search for new compounds with high efficacy, low toxicity, shorter treatment time and affordable cost. We studied two synthetic 6-quinolinyl chalcones, 3b and 3e, to determine their effects on VERO cells, antifungal activity, survival curve, interaction with other drugs and phenotypic effects against several isolates of Paracoccidioides spp. In this study, we verified that the compounds were not toxic, exhibited superior in vitro activity compared with that shown by trimethoprim-sulfamethoxazole, and after 5 days of treatment, decreased the fungal cell viability by approximately 70%. Additionally, no interactions were observed between the tested compounds and other drugs. We also found that these compounds induced morphological changes, such as shriveling of cells, fragmentation of the plasma membrane and cytoplasmic disorganization in vitro. The changes observed by microscopy assays corroborate the observation made with propidium iodide, where the number of cells stained with the compounds was higher than that observed after amphotericin B treatment. We observed an increase in the efflux of K(+) and a loss of intracellular contents in cells treated with 3b and 3e, confirming their effects on fungal membranes. However, damage to the membrane was not associated with a decrease in membrane ergosterol levels. The experimental evidences showed no direct indications of cellular wall damage caused by these compounds. Thus, these results confirm the antifungal potential of 3b and 3e against Paracoccidioides spp. with possible action on the membrane.

  2. The fate of Transparent Exopolymer Particles (TEP) in integrated membrane systems: removal through pre-treatment processes and deposition on reverse osmosis membranes.

    PubMed

    Villacorte, Loreen O; Kennedy, Maria D; Amy, Gary L; Schippers, Jan C

    2009-12-01

    The abundance of Transparent Exopolymer Particles (TEP) in surface waters has been unnoticed for many years until recently as a potential foulant in reverse osmosis systems. Recent studies indicate that TEP may cause organic and biological fouling and may enhance particulate/colloidal fouling in reverse osmosis membranes. The presence of TEP was measured in the raw water, the pre-treatment processes and reverse osmosis (RO) systems of 6 integrated membrane installations. A spectrophotometric method was used to measure TEP in the particulate size range (>0.40microm) and was extended to measure TEP in the colloidal size range (0.05-0.40microm). Ultrafiltration pre-treatment applied in 4 plants, totally removed particulate TEP while microfiltration systems (2 plants) and coagulation/sedimentation/rapid sand filtration systems (3 plants) partially removed this fraction. None of the pre-treatment systems investigated totally removed colloidal TEP. Biopolymer analysis using LC-OCD showed consistency between colloidal TEP and polysaccharide removal by UF pre-treatment and further verified the presence of TEP in the RO feedwater. TEP deposition in the RO system was determined after measuring total TEP concentrations in the RO feed and concentrate. The TEP deposition factors and specific deposition rates indicate that TEP accumulation had occurred in all plants investigated. This observation was verified by an autopsy of RO modules from two RO plants. Further improvement and verification of the (modified) TEP method, in particular the calibration, is necessary so that it can be employed to investigate the role of TEP in the fouling of RO systems.

  3. Thermal, High Pressure, and Electric Field Processing Effects on Plant Cell Membrane Integrity and Relevance to Fruit and Vegetable Quality

    PubMed Central

    Gonzalez, Maria E; Barrett, Diane M

    2010-01-01

    Advanced food processing methods that accomplish inactivation of microorganisms but minimize adverse thermal exposure are of great interest to the food industry. High pressure (HP) and pulsed electric field (PEF) processing are commercially applied to produce high quality fruit and vegetable products in the United States, Europe, and Japan. Both microbial and plant cell membranes are significantly altered following exposure to heat, HP, or PEF. Our research group sought to quantify the degree of damage to plant cell membranes that occurs as a result of exposure to heat, HP, or PEF, using the same analytical methods. In order to evaluate whether new advanced processing methods are superior to traditional thermal processing methods, it is necessary to compare them. In this review, we describe the existing state of knowledge related to effects of heat, HP, and PEF on both microbial and plant cells. The importance and relevance of compartmentalization in plant cells as it relates to fruit and vegetable quality is described and various methods for quantification of plant cell membrane integrity are discussed. These include electrolyte leakage, cell viability, and proton nuclear magnetic resonance (1H-NMR). PMID:20492210

  4. An Essential Tyrosine Phosphatase Homolog Regulates Cell Separation, Outer Membrane Integrity, and Morphology in Caulobacter crescentus ▿ †

    PubMed Central

    Shapland, Elaine B.; Reisinger, Sarah J.; Bajwa, Amrita K.; Ryan, Kathleen R.

    2011-01-01

    Although reversible phosphorylation on tyrosine residues regulates the activity of many eukaryotic proteins, there are few examples of this type of regulation in bacteria. We have identified the first essential tyrosine phosphatase homolog in a bacterium, Caulobacter crescentusCtpA. ctpAmutants with altered active-site residues are nonviable, and depletion of CtpA yields chains of cells with blebbed outer membranes, linked by unresolved peptidoglycan. CtpA overexpression reduces cell curvature in a manner similar to deleting the intermediate filament protein crescentin, but it does not disrupt crescentin localization or membrane attachment. Although it has no obvious signal sequence or transmembrane-spanning domains, CtpA associates with the Caulobacterinner membrane. Immunolocalization experiments suggest that CtpA accumulates at the division site during the last quarter of the cell cycle. We propose that CtpA dephosphorylates one or more proteins involved in peptidoglycan biosynthesis or remodeling, which in turn affect cell separation, cell envelope integrity, and vibrioid morphology. PMID:21705597

  5. Cell membrane integrity and internalization of ingested TiO(2) nanoparticles by digestive gland cells of a terrestrial isopod.

    PubMed

    Novak, Sara; Drobne, Damjana; Valant, Janez; Pipan-Tkalec, Živa; Pelicon, Primož; Vavpetič, Primož; Grlj, Nataša; Falnoga, Ingrid; Mazej, Darja; Remškar, Maja

    2012-05-01

    The present study was motivated by the paucity of reports on cellular internalization of ingested titanium dioxide (TiO(2)) nanoparticles (nano-TiO(2)). The model invertebrate (Porcellio scaber, Isopoda, Crustacea) was exposed to food dosed with nano-TiO(2) containing 100, 1,000, 3,000, or 5,000 µg nano-TiO(2) per gram of food. After 14 d of exposure, the amount of Ti in the entire body was analyzed by inductively coupled plasma-mass spectrometry, and elemental analyses of tissue cross sections were performed by particle induced X-ray emission. In addition, a series of toxicological markers including feeding parameters, weight change, and survival, as well as cytotoxic effects such as digestive gland cell membrane stability, were monitored. Internalization of ingested nano-TiO(2) by the isopod's digestive gland epithelial cells was shown to depend on cell membrane integrity. Cell membranes were found to be destabilized by TiO(2) particles, and at higher extracellular concentrations of nano-TiO(2), the nanoparticles were internalized.

  6. Thermal, high pressure, and electric field processing effects on plant cell membrane integrity and relevance to fruit and vegetable quality.

    PubMed

    Gonzalez, Maria E; Barrett, Diane M

    2010-09-01

    Advanced food processing methods that accomplish inactivation of microorganisms but minimize adverse thermal exposure are of great interest to the food industry. High pressure (HP) and pulsed electric field (PEF) processing are commercially applied to produce high quality fruit and vegetable products in the United States, Europe, and Japan. Both microbial and plant cell membranes are significantly altered following exposure to heat, HP, or PEF. Our research group sought to quantify the degree of damage to plant cell membranes that occurs as a result of exposure to heat, HP, or PEF, using the same analytical methods. In order to evaluate whether new advanced processing methods are superior to traditional thermal processing methods, it is necessary to compare them. In this review, we describe the existing state of knowledge related to effects of heat, HP, and PEF on both microbial and plant cells. The importance and relevance of compartmentalization in plant cells as it relates to fruit and vegetable quality is described and various methods for quantification of plant cell membrane integrity are discussed. These include electrolyte leakage, cell viability, and proton nuclear magnetic resonance (¹H-NMR).

  7. Integrated multilayer microfluidic device with a nanoporous membrane interconnect for online coupling of solid-phase extraction to microchip electrophoresis.

    PubMed

    Long, Zhicheng; Shen, Zheng; Wu, Dapeng; Qin, Jianhua; Lin, Bingcheng

    2007-12-01

    An integrated microfluidic device was developed for online coupling of solid-phase extraction to microchip electrophoresis (chip SPE-CE). With a nanoporous membrane sandwiched between two PDMS substrates, SPE preconcentration and electrophoretic separation can be carried out in upper and lower fluidic layers, separately and sequentially. During the SPE process, the thin membrane can act as a fluid isolator to prevent intermixing between two fluidic channels. However, when a pulse voltage is applied, the membrane becomes a gateable interconnect so that a small plug of concentrated analytes can be online injected into the lower channel for subsequent separations. This multilayer design provides a universal solution to online SPE-CE hyphenation. Both electroosmotic flow and hydrodynamic pumps have been adopted for SPE operation. SPE was performed on a 2.5 mm long microcolumn, with two weirs on both sides to retain the C(18)-coated silica beads. Rhodamine 123 and FITC-labelled ephedrine were used to test the operational performance of the hyphenation system. High separation efficiency and thousand-fold signal enhancement were achieved.

  8. The Usefulness of Selected Physicochemical Indices, Cell Membrane Integrity and Sperm Chromatin Structure in Assessments of Boar Semen Sensitivity

    PubMed Central

    Wysokińska, A.; Kondracki, S.; Iwanina, M.

    2015-01-01

    The present work describes experiments undertaken to evaluate the usefulness of selected physicochemical indices of semen, cell membrane integrity and sperm chromatin structure for the assessment of boar semen sensitivity to processes connected with pre-insemination procedures. The experiments were carried out on 30 boars: including 15 regarded as providers of sensitive semen and 15 regarded as providers of semen that is little sensitive to laboratory processing. The selection of boars for both groups was based on sperm morphology analyses, assuming secondary morphological change incidence in spermatozoa as the criterion. Two ejaculates were manually collected from each boar at an interval of 3 to 4 months. The following analyses were carried out for each ejaculate: sperm motility assessment, sperm pH measurement, sperm morphology assessment, sperm chromatin structure evaluation and cell membrane integrity assessment. The analyses were performed three times. Semen storage did not cause an increase in the incidence of secondary morphological changes in the group of boars considered to provide sperm of low sensitivity. On the other hand, with continued storage there was a marked increase in the incidence of spermatozoa with secondary morphological changes in the group of boars regarded as producing more sensitive semen. Ejaculates of group I boars evaluated directly after collection had an approximately 6% smaller share of spermatozoa with undamaged cell membranes than the ejaculates of boars in group II (p≤0.05). In the process of time the percentage of spermatozoa with undamaged cell membranes decreased. The sperm of group I boars was characterised with a lower sperm motility than the semen of group II boars. After 1 hour of storing diluted semen, the sperm motility of boars producing highly sensitive semen was already 4% lower (p≤0.05), and after 24 hours of storage it was 6.33% lower than that of the boars that produced semen with a low sensitivity. Factors

  9. A Procedure-Spanning Analysis of Plasma Membrane Integrity for Assessment of Cell Viability in Sperm Cryopreservation of Zebrafish Danio rerio.

    PubMed

    Yang, Huiping; Daly, Jonathan; Carmichael, Carrie; Matthews, Jen; Varga, Zoltan M; Tiersch, Terrence

    2016-04-01

    The goal of this study was to evaluate plasma membrane integrity and motility for zebrafish sperm quality assessment along the cryopreservation pathway-from sample collection through refrigerated storage, cryoprotectant equilibration, freezing, thawing, and fertilization. The objectives were to: (1) evaluate the effects of osmolality, extender, and refrigerated storage on sperm plasma membrane integrity and motility, and (2) compare cryopreservation of sperm from farm-raised and well-characterized research populations by evaluating motility and membrane integrity of fresh, post-equilibration (before freezing) and post-thaw sperm, and post-thaw fertility. Osmolality, extender, and storage time each influenced sperm motility and membrane integrity. Isotonic osmolality showed the best protection for motility and membrane integrity compared to hypotonic and hypertonic osmolalities. Of the four tested extenders, Hanks' balanced salt solution (HBSS) and Ca(2+)-free HBSS showed the best protection compared with NaCl and glucose, and sperm retained motility and membrane integrity for 24 h of refrigerated storage. Sperm cryopreservation of zebrafish from a farm population (n = 20) and an AB research line (n = 20) showed significant differences in post-thaw fertility (32% ± 18% vs. 73% ± 21%). No differences were found in post-thaw motility, although the farm-raised zebrafish possessed a larger body size, testis weight, and higher fresh motility. Correlation analysis of pooled data did not identify correlations among motility, flow cytometry analysis of membrane integrity and recognizable cells, and post-thaw sperm fertility (p ≥ 0.202). More research is needed to standardize the fertilization conditions especially sperm-to-egg ratio to avoid possible overabundance of sperm to obscure the differences.

  10. Free-standing alumina nanobottles and nanotubes pre-integrated into nanoporous alumina membranes

    NASA Astrophysics Data System (ADS)

    Fang, Jinghua; Levchenko, Igor; (Ken Ostrikov, Kostya

    2014-08-01

    A novel interfacial structure consisting of long (up to 5 μm), thin (about 300 nm), highly-ordered, free-standing, highly-reproducible aluminum oxide nanobottles and long tubular nanocapsules attached to a rigid, thin (less than 1 μm) nanoporous anodic alumina membrane is fabricated by simple, fast, catalyst-free, environmentally friendly voltage-pulse anodization. A growth mechanism is proposed based on the formation of straight channels in alumina membrane by anodization, followed by neck formation due to a sophisticated voltage control during the process. This process can be used for the fabrication of alumina nanocontainers with highly controllable geometrical size and volume, vitally important for various applications such as material and energy storage, targeted drug and diagnostic agent delivery, controlled drug and active agent release, gene and biomolecule reservoirs, micro-biologically protected platforms, nano-bioreactors, tissue engineering and hydrogen storage.

  11. The integral nuclear membrane protein nurim plays a role in the suppression of apoptosis.

    PubMed

    Chen, H; Chen, K; Chen, J; Cheng, H; Zhou, R

    2012-12-01

    As an essential component of eukaryotic cells, the nuclear envelope (NE) plays a crucial role in many physiological processes. At present, a few membrane proteins from NE have been functionally characterized. To determine whether the inner nuclear membrane (INM) protein Nurim is expressed in cancer cells with evidence of apoptosis, we identified three isoforms of this protein that are specific for human testicular seminoma and are generated by alternative splicing. We observed that Nurim is expressed in a broad range of cancer types and that its expression level is correlated with a higher tumor grade. Biochemical analysis showed that Nurim b, like a, is tightly bound to the nuclear envelope. Furthermore, knockdown using miR-Nurim resulted in an abnormal shape change of the nuclear envelope. Notably, Nurim knockdown obviously increased apoptosis induced by ultraviolet in HeLa cells. Together, these findings implicate that the INM protein Nurim plays an important role in the suppression of apoptosis.

  12. Fluorescence-Detectino Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins

    SciTech Connect

    Kawate,T.; Gouaux, E.

    2006-01-01

    Formation of well-ordered crystals of membrane proteins is a bottleneck for structure determination by X-ray crystallography. Nevertheless, one can increase the probability of successful crystallization by precrystallization screening, a process by which one analyzes the monodispersity and stability of the protein-detergent complex. Traditionally, this has required microgram to milligram quantities of purified protein and a concomitant investment of time and resources. Here, we describe a rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass. We show the application of this precrystallization screening to four membrane proteins derived from prokaryotic or eukaryotic organisms.

  13. Semicontinuous Production of Lactic Acid From Cheese Whey Using Integrated Membrane Reactor

    NASA Astrophysics Data System (ADS)

    Li, Yebo; Shahbazi, Abolghasem; Coulibaly, Sekou; Mims, Michele M.

    Semicontinuous production of lactic acid from cheese whey using free cells of Bifidobacterium longum with and without nanofiltration was studied. For the semicontinuous fermentation without membrane separation, the lactic acid productivity of the second and third runs is much lower than the first run. The semicontinuous fermentation with nanoseparation was run semicontinuously for 72 h with lactic acid to be harvested every 24 h using a nanofiltration membrane unit. The cells and unutilized lactose were kept in the reactor and mixed with newly added cheese whey in the subsequent runs. Slight increase in the lactic acid productivity was observed in the second and third runs during the semicontinuous fermentation with nanofiltration. It can be concluded that nanoseparation could improve the lactic acid productivity of the semicontinuous fermentation process.

  14. Promoting Tag Removal of a MBP-Fused Integral Membrane Protein by TEV Protease.

    PubMed

    Chen, Yanke; Li, Qichang; Yang, Jun; Xie, Hao

    2017-03-01

    Tag removal is a prerequisite issue for structural and functional analysis of affinity-purified membrane proteins. The present study took a MBP-fused membrane protein, MrpF, as a model to investigate the tag removal by TEV protease. Influences of the linking sequence between TEV cleavage site and MrpF on protein expression and predicted secondary structure were investigated. The steric accessibility of TEV protease to cleavage site of MBP-fused MrpF was explored. It was found that reducing the size of hydrophilic group of detergents and/or extending the linking sequence between cleavage site and target protein can significantly improve the accessibility of the cleavage site and promote tag removal by TEV protease.

  15. Integration of aerobic granular sludge and mesh filter membrane bioreactor for cost-effective wastewater treatment.

    PubMed

    Li, Wen-Wei; Wang, Yun-Kun; Sheng, Guo-Ping; Gui, Yong-Xin; Yu, Lei; Xie, Tong-Qing; Yu, Han-Qing

    2012-10-01

    Conventional MBR has been mostly based on floc sludge and the use of costly microfiltration membranes. Here, a novel aerobic granule (AG)-mesh filter MBR (MMBR) process was developed for cost-effective wastewater treatment. During 32-day continuous operation, a predominance of granules was maintained in the system, and good filtration performance was achieved at a low trans-membrane pressure (TMP) of below 0.025 m. The granules showed a lower fouling propensity than sludge flocs, attributed to the formation of more porous biocake layer at mesh surface. A low-flux and low-TMP filtration favored a stable system operation. In addition, the reactor had high pollutant removal efficiencies, with a 91.4% chemical oxygen demand removal, 95.7% NH(4)(+) removal, and a low effluent turbidity of 4.1 NTU at the stable stage. This AG-MMBR process offers a promising technology for low-cost and efficient treatment of wastewaters.

  16. Free-standing alumina nanobottles and nanotubes pre-integrated into nanoporous alumina membranes

    PubMed Central

    Fang, Jinghua; Levchenko, Igor; Ostrikov, Kostya (Ken)

    2014-01-01

    A novel interfacial structure consisting of long (up to 5 μm), thin (about 300 nm), highly-ordered, free-standing, highly-reproducible aluminum oxide nanobottles and long tubular nanocapsules attached to a rigid, thin (less than 1 μm) nanoporous anodic alumina membrane is fabricated by simple, fast, catalyst-free, environmentally friendly voltage-pulse anodization. A growth mechanism is proposed based on the formation of straight channels in alumina membrane by anodization, followed by neck formation due to a sophisticated voltage control during the process. This process can be used for the fabrication of alumina nanocontainers with highly controllable geometrical size and volume, vitally important for various applications such as material and energy storage, targeted drug and diagnostic agent delivery, controlled drug and active agent release, gene and biomolecule reservoirs, micro-biologically protected platforms, nano-bioreactors, tissue engineering and hydrogen storage. PMID:27877705

  17. Two-Dimensional Crystallization of Integral Membrane Proteins for Electron Crystallography

    PubMed Central

    Stokes, David L.; Rice, William J.; Hu, Minghui; Kim, Changki; Ubarretxena, Iban

    2011-01-01

    Although membrane proteins make up 30% of the proteome and are a common target for therapeutic drugs, determination of their atomic structure remains a technical challenge. Electron crystallography represents an alternative to the conventional methods of X-ray diffraction and NMR and relies on the formation of two-dimensional crystals. These crystals are produced by reconstituting purified, detergent-solubilized membrane proteins back into the native environment of a lipid bilayer. This chapter reviews methods for producing two-dimensional crystals and for screening them by negative stain electron microscopy. In addition, we show examples of the different morphologies that are commonly obtained and describe basic image analysis procedures that can be used to evaluate their promise for structure determination by cryoelectron microsopy. PMID:20665267

  18. Simultaneous topographic and amperometric membrane mapping using an AFM probe integrated biosensor.

    PubMed

    Stanca, Sarmiza Elena; Csaki, Andrea; Urban, Matthias; Nietzsche, Sandor; Biskup, Christoph; Fritzsche, Wolfgang

    2011-02-15

    The investigation of the plasma membrane with intercorrelated multiparameter techniques is a prerequisite for understanding its function. Presented here, is a simultaneous electrochemical and topographic study of the cell membrane using a miniaturized amperometric enzymatic biosensor. The fabrication of this biosensor is also reported. The biosensor combines a scanning force microscopy (AFM) gold-coated cantilever and an enzymatic transducer layer of peroxidases (PODs). When these enzymes are brought in contact with the substrate, the specific redox reaction produces an electric current. The intensity of this current is detected simultaneously with the surface imaging. For sensor characterization, hydroquinone-2-carboxylic acid (HQ) is selected as an intrinsic source of H(2)O(2). HQ has been electrochemically regenerated by the reduction of antraquinone-2-carboxylic acid (AQ). The biosensor reaches the steady state value of the current intensity in 1 ± 0.2s.

  19. Integrated hollow fiber membranes for gas delivery into optical waveguide based photobioreactors.

    PubMed

    Ahsan, Syed Saad; Gumus, Abdurrahman; Jain, Aadhar; Angenent, Largus T; Erickson, David

    2015-09-01

    Compact algal reactors are presented with: (1) closely stacked layers of waveguides to decrease light-path to enable larger optimal light-zones; (2) waveguides containing scatterers to uniformly distribute light; and (3) hollow fiber membranes to reduce energy required for gas transfer. The reactors are optimized by characterizing the aeration of different gases through hollow fiber membranes and characterizing light intensities at different culture densities. Close to 65% improvement in plateau peak productivities was achieved under low light-intensity growth experiments while maintaining 90% average/peak productivity output during 7-h light cycles. With associated mixing costs of ∼ 1 mW/L, several magnitudes smaller than closed photobioreactors, a twofold increase is realized in growth ramp rates with carbonated gas streams under high light intensities, and close to 20% output improvement across light intensities in reactors loaded with high density cultures.

  20. Elucidating in Vivo Structural Dynamics in Integral Membrane Protein by Hydroxyl Radical Footprinting*

    PubMed Central

    Zhu, Yi; Guo, Tiannan; Park, Jung Eun; Li, Xin; Meng, Wei; Datta, Arnab; Bern, Marshall; Lim, Sai Kiang; Sze, Siu Kwan

    2009-01-01

    We describe here a novel footprinting technique to probe the in vivo structural dynamics of membrane protein. This method utilized in situ generation of hydroxyl radicals to oxidize and covalently modify biomolecules on living Escherichia coli cell surface. After enriching and purifying the membrane proteome, the modified amino acid residues of the protein were identified with tandem mass spectrometry to map the solvent-accessible surface of the protein that will form the footprint of in vivo structure of the protein. Of about 100 outer membrane proteins identified, we investigated the structure details of a typical β-barrel structure, the porin OmpF. We found that six modified tryptic peptides of OmpF were reproducibly detected with 19 amino acids modified under the physiological condition. The modified amino acid residues were widely distributed in the external loop area, β-strands, and periplasmic turning area, and all of them were validated as solvent-accessible according to the crystallography data. We further extended this method to study the dynamics of the voltage gating of OmpF in vivo using mimic changes of physiological circumstance either by pH or by ionic strength. Our data showed the voltage gating of porin OmpF in vivo for the first time and supported the proposed mechanism that the local electrostatic field changes in the eyelet region may alter the porin channels to switch. Thus, this novel method can be a potentially efficient method to study the structural dynamics of the membrane proteins of a living cell. PMID:19473960

  1. Uptake of iodinated contrast material in ischemic myocardium as an indicator of loss of cellular membrane integrity.

    PubMed Central

    Abraham, J. L.; Higgins, C. B.; Newell, J. D.

    1980-01-01

    Differential uptake of iodine containing radiographic contrast medium (I) in myocardial infarcts compared with normal mycardium has been detected by computerized transmission tomography (CTT). In this study the histologic and cellular distribution of I in ischemically damaged canine myocardium after intravenous administration of contrast material was examined by the use of scanning electron microscopy and energy dispersive X-ray microanalysis of fresh frozen cryosections. Analysis of individual cells in 6-mu thick sections mounted on carbon substrates showed that I was detectable in the ischemically damaged but not the normal myocardial cells. A decline in the potassium-to-sodium ratio confirmed the loss of membrane integrity in the ischemically damaged cells that accumulated I. These results indicate that I enters ischemically damaged but not normal myocardial cells suggesting that CTT scans after intravenous administration of contrast material may be capable of defining the area of the myocardium in which cells have lost membrane integrity after an ischemic injury. Images Figure 1 p[329]-a Figure 2 PMID:7435540

  2. Activity ranking of synthetic analogs targeting vascular endothelial growth factor receptor 2 by an integrated cell membrane chromatography system.

    PubMed

    Wang, Dongyao; Lv, Diya; Chen, Xiaofei; Liu, Yue; Ding, Xuan; Jia, Dan; Chen, Langdong; Zhu, Zhenyu; Cao, Yan; Chai, Yifeng

    2015-12-01

    Evaluating the biological activities of small molecules represents an important part of the drug discovery process. Cell membrane chromatography (CMC) is a well-developed biological chromatographic technique. In this study, we have developed combined SMMC-7721/CMC and HepG2/CMC with high-performance liquid chromatography and time-of-flight mass spectrometry to establish an integrated screening platform. These systems was subsequently validated and used for evaluating the activity of quinazoline compounds, which were designed and synthesized to target vascular endothelial growth factor receptor 2. The inhibitory activities of these compounds towards this receptor were also tested using a classical caliper mobility shift assay. The results revealed a significant correlation between these two methods (R(2) = 0.9565 or 0.9420) for evaluating the activities of these compounds. Compared with traditional methods of evaluating the activities analogous compounds, this integrated cell membrane chromatography screening system took less time and was more cost effective, indicating that it could be used as a practical method in drug discovery.

  3. Effect of supplementation of butylated hydroxytoluene on post-thaw sperm viability, motility and membrane integrity of Hariana bulls

    PubMed Central

    Patel, Akhil; Saxena, Atul; Swain, Dilip Kumar; Yadav, Dushyant; Yadav, Sanjay Singh; Kumar, Abhishek; Kumar, Anuj

    2015-01-01

    Aim: This study was aimed to see the beneficial effect of butylated hydroxytoluene (BHT) as a semen additive of Hariana bull semen. Materials and Methods: The study was carried out in Hariana bulls. Twenty-four ejaculates from two bulls were used for this study. Each ejaculate was extended with standard glycerolated egg yolk tris extender and supplemented with BHT at two concentrations as 0.5 mM (T1) and 1.0 mM (T2). After dilution, equilibration and 24 h of cryopreservation, the samples were analyzed for progressive motility, sperm viability and membrane integrity. Results: Progressive motility, sperm viability and sperm membrane integrity were significantly (p<0.05) increased in the samples fortified with BHT as compared to the control during the process of cryopreservation and thawing. The BHT concentration of 1 mM revealed better results as compared to 0.5 mM. Conclusion: Addition of 1.0 mM BHT was found better in cryopreservation of Hariana bull semen compared to 0.5 mM BHT and control samples. The addition of BHT has improved the sperm quality by acting as an antioxidant thereby reducing the lipid peroxidation of the sperms. PMID:27065652

  4. The effects of various control and water treatment processes on the membrane integrity and toxin fate of cyanobacteria.

    PubMed

    Fan, Jiajia; Hobson, Peter; Ho, Lionel; Daly, Robert; Brookes, Justin

    2014-01-15

    Cyanobacterial blooms are one of the main contaminants that can degrade drinking water quality with the associated taste, odour and toxic compounds. Although a wide range of techniques have shown promise for cyanobacterial bloom control and cyanobacterial cell/metabolite removal in reservoirs and water treatment plants (WTPs), these treatments may have negative consequences through release of intracellular metabolites into the surrounding water. This study assessed the impact of copper sulphate (CuSO4), chlorine, potassium permanganate (KMnO4), hydrogen peroxide (H2O2) and ozone on Microcystis aeruginosa culture and the toxins it produced. All of these agents induced the loss of cyanobacterial membrane integrity. However, no associated increase in dissolved toxins was detected during chlorine and H2O2 treatments which may be due to faster toxin oxidation rates than release rates. KMnO4 doses of 1 and 3mgL(-1) degraded dissolved toxins while having no impact on cyanobacterial membrane integrity. In contrast, ozone induced a significant increase in extracellular toxins but it was unable to degrade these toxins to the same degree as the other oxidants which may due to the lack of residual. All chemicals, except CuSO4, were able to reduce cyanotoxins and chlorine was the most effective with a rate up to 2161M(-1)s(-1).

  5. The Synthetic Amphipathic Peptidomimetic LTX109 Is a Potent Fungicide That Disturbs Plasma Membrane Integrity in a Sphingolipid Dependent Manner

    PubMed Central

    Larsen, Camilla Eggert; Folkesson, Anders; Regenberg, Birgitte

    2013-01-01

    The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)2C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids. PMID:23874964

  6. Scalable Fabrication of Integrated Nanophotonic Circuits on Arrays of Thin Single Crystal Diamond Membrane Windows.

    PubMed

    Piracha, Afaq H; Rath, Patrik; Ganesan, Kumaravelu; Kühn, Stefan; Pernice, Wolfram H P; Prawer, Steven

    2016-05-11

    Diamond has emerged as a promising platform for nanophotonic, optical, and quantum technologies. High-quality, single crystalline substrates of acceptable size are a prerequisite to meet the demanding requirements on low-level impurities and low absorption loss when targeting large photonic circuits. Here, we describe a scalable fabrication method for single crystal diamond membrane windows that achieves three major goals with one fabrication method: providing high quality diamond, as confirmed by Raman spectroscopy; achieving homogeneously thin membranes, enabled by ion implantation; and providing compatibility with established planar fabrication via lithography and vertical etching. On such suspended diamond membranes we demonstrate a suite of photonic components as building blocks for nanophotonic circuits. Monolithic grating couplers are used to efficiently couple light between photonic circuits and optical fibers. In waveguide coupled optical ring resonators, we find loaded quality factors up to 66 000 at a wavelength of 1560 nm, corresponding to propagation loss below 7.2 dB/cm. Our approach holds promise for the scalable implementation of future diamond quantum photonic technologies and all-diamond photonic metrology tools.

  7. Preconcentration of diluted biochemical samples using microchannel with integrated nanoscale Nafion membrane.

    PubMed

    Chao, Chen-Chiao; Chiu, Ping-Hsien; Yang, Ruey-Jen

    2015-04-01

    A microfluidic preconcentration device comprising a microchannel and a surface-patterned nanoscale Nafion membrane is proposed. Given the application of an electric field across the chip, the nanopore within Nafion membrane becomes ion selective due to an overlapping of the electric double layer. The resulting difference in flux of the co- and counter-ions within the membrane nanopore prompts the formation of a concentration gradient and leads to a gradual accumulation of the co-ions at the micro-nano junction. It is shown experimentally that the rate of concentration and the preconcentration factor both increase with an increasing electrical field intensity. The preconcentration performance in a straight microchannel is compared with that in a convergent microchannel using fluorescein disodium salt dehydrate and Fluorescein isothiocyanate (FITC)-labeled bovine serum albumin samples. The results show that the reduced cross-sectional area of the convergent microchannel increases the preconcentration factor compared to that obtained in a straight microchannel and yields a significant reduction in the preconcentration time.

  8. Formaldehyde gas sensor based on TiO2 thin membrane integrated with nano silicon structure

    NASA Astrophysics Data System (ADS)

    Zheng, Xuan; Ming, An-jie; Ye, Li; Chen, Feng-hua; Sun, Xi-long; Liu, Wei-bing; Li, Chao-bo; Ou, Wen; Wang, Wei-bing; Chen, Da-peng

    2016-07-01

    An innovative formaldehyde gas sensor based on thin membrane type metal oxide of TiO2 layer was designed and fabricated. This sensor under ultraviolet (UV) light emitting diode (LED) illumination exhibits a higher response to formaldehyde than that without UV illumination at low temperature. The sensitivities of the sensor under steady working condition were calculated for different gas concentrations. The sensitivity to formaldehyde of 7.14 mg/m3 is about 15.91 under UV illumination with response time of 580 s and recovery time of 500 s. The device was fabricated through micro-electro-mechanical system (MEMS) processing technology. First, plasma immersion ion implantation (PIII) was adopted to form black polysilicon, then a nanoscale TiO2 membrane with thickness of 53 nm was deposited by DC reactive magnetron sputtering to obtain the sensing layer. By such fabrication approaches, the nanoscale polysilicon presents continuous rough surface with thickness of 50 nm, which could improve the porosity of the sensing membrane. The fabrication process can be mass-produced for the MEMS process compatibility.

  9. The importance of extracellular speciation and corrosion of copper nanoparticles on lung cell membrane integrity.

    PubMed

    Hedberg, Jonas; Karlsson, Hanna L; Hedberg, Yolanda; Blomberg, Eva; Odnevall Wallinder, Inger

    2016-05-01

    Copper nanoparticles (Cu NPs) are increasingly used in various biologically relevant applications and products, e.g., due to their antimicrobial and catalytic properties. This inevitably demands for an improved understanding on their interactions and potential toxic effects on humans. The aim of this study was to investigate the corrosion of copper nanoparticles in various biological media and to elucidate the speciation of released copper in solution. Furthermore, reactive oxygen species (ROS) generation and lung cell (A549 type II) membrane damage induced by Cu NPs in the various media were studied. The used biological media of different complexity are of relevance for nanotoxicological studies: Dulbecco's modified eagle medium (DMEM), DMEM(+) (includes fetal bovine serum), phosphate buffered saline (PBS), and PBS+histidine. The results show that both copper release and corrosion are enhanced in DMEM(+), DMEM, and PBS+histidine compared with PBS alone. Speciation results show that essentially no free copper ions are present in the released fraction of Cu NPs in neither DMEM(+), DMEM nor histidine, while labile Cu complexes form in PBS. The Cu NPs were substantially more membrane reactive in PBS compared to the other media and the NPs caused larger effects compared to the same mass of Cu ions. Similarly, the Cu NPs caused much more ROS generation compared to the released fraction only. Taken together, the results suggest that membrane damage and ROS formation are stronger induced by Cu NPs and by free or labile Cu ions/complexes compared with Cu bound to biomolecules.

  10. Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

    PubMed Central

    Cartron, Michaël L.; Olsen, John D.; Sener, Melih; Jackson, Philip J.; Brindley, Amanda A.; Qian, Pu; Dickman, Mark J.; Leggett, Graham J.; Schulten, Klaus; Hunter, C. Neil

    2014-01-01

    Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1, whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1-PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities. PMID:24530865

  11. Integrated CO2, Humidity and Thermal Control by Membrane Gas Absorption, Results of Breadboard Testing

    NASA Astrophysics Data System (ADS)

    van Driel, C.; Eckhard, F.; Feron, P. H. M.; Savage, C. J.

    2002-01-01

    Membrane gas absorption for the removal of CO2 in manned spacecrafts is subject of study by Stork and TNO for many years. The system is based on the combination of membrane separation and gas absorption. The air is fed along one side of a hydrophobic membrane and diffuses through the membrane after which the CO2 is selectively absorbed by an absorption liquid. Great advantage is that the system not only can be used to remove the carbon dioxide but also can be applied to control the relative humidity and temperature of the cabin atmosphere. Absorption of moisture and heat is achieved by cooling the absorption liquid below the dewpoint temperature of the gas stream. In the studies, the Crew Transfer Vehicle is used as a basis. Compared to the planned h/w for this vehicle, an air conditioning system, consisting of a condensing heat exchanger, LiOH cartridges to remove the carbon dioxide and a water evaporator assembly, the MGA/MGD has a large volume and a small mass advantage. The absorption liquid circulates through the spacecraft thermal control loop, replacing the coolant water. This set-up has two advantages. At first, by increasing the absorption liquid temperature the CO2 desorption rate in the desorber is favoured and secondly, should additional heat rejection aside from the basic heat rejection system be required (off nominal case), this can be established by dumping extra water via the desorption module, using the associated heat of vaporisation. Control of the water desorption rate is achieved by adjusting the permeate pressure with the throttle valve. In the nominal case the water absorption rate is equal to the desorption rate. The CO2 absorption capacity of the absorption liquid is restored in a desorption unit. This process is based on pervaporation. On one side of the membrane the absorption liquid is fed, on the other side a reduced pressure is maintained. Due to this pressure difference a driving force for water vapour and CO2 is created. The water

  12. Evidence that aggregation of mouse sperm receptors by ZP3 triggers the acrosome reaction

    PubMed Central

    1989-01-01

    In the mouse, considerable evidence indicates that initial sperm binding to the zona pellucida (ZP) is mediated by ZP3. In addition, this same glycoprotein is also responsible for inducing the acrosome reaction (AR). Whereas the O-linked oligosaccharides of ZP3 appear to mediate sperm-ZP binding, the portion of ZP3 bearing AR activity has not been defined. To try to understand the bifunctional role of ZP3 (binding and AR inducing activities), we have examined the hypothesis that ZP3 aggregates sperm receptor molecules. By analogy with findings in a variety of other extracellular signal transducing systems, including receptors for growth factors and insulin, this aggregation event could initiate the cascade resulting in the AR. To test this hypothesis, we have generated monospecific polyclonal antibodies against ZP2 and against ZP3, and examined the effects of these probes on capacitated sperm incubated in the absence or presence of various ZP protein preparations. For some experiments, we have used proteolytic fragments of ZP3, a preparation known to retain specific binding, but not AR-inducing, activity. We show here that capacitated mouse sperm, incubated with ZP glycopeptides, displayed ARs when incubated subsequently with anti-ZP3 IgG; ARs did not occur when parallel sperm samples were incubated with anti-ZP2 IgG or with anti-ZP3 Fab fragments. When capacitated sperm were treated successively, with (a) ZP3 glycopeptides, (b) anti-ZP3 Fab fragments, and (c) goat anti-rabbit IgG, ARs occurred in the majority of sperm. An alternative approach to examine this hypothesis used ZP proteins obtained from tubal eggs treated previously with bioactive phorbol diester (12-O-tetradecanoyl phorbol-13-acetate [TPA]). This preparation arrests capacitated sperm in an intermediate state of the AR. We demonstrate here that these sperm can be induced to undergo a complete AR by subsequent treatment with anti-ZP3 IgG. Together, these findings are consistent with the hypothesis

  13. Participation of protein kinases and phosphatases in the progesterone-induced acrosome reaction and calcium influx in human spermatozoa.

    PubMed

    Barón, L; Fara, K; Zapata-Carmona, H; Zuñiga, L; Kong, M; Signorelli, J; Díaz, E S; Morales, P

    2016-11-01

    In human spermatozoa, protein kinases have a role in the acrosome reaction (AR) induced by a variety of stimuli. However, there is disagreement or a lack of information regarding the role of protein kinases and phosphatases in the progesterone (P)-induced increase in intracellular calcium concentration ([Ca(2+) ]i ). In addition, there are no studies regarding the role of Ser/Thr and Tyr phosphatases and there are contradictory results regarding the role of Tyr kinases in the P-induced acrosome reaction. Here, we performed a simultaneous evaluation of the involvement of protein kinases and phosphatases in the P-induced acrosome reaction and in the P-induced calcium influx. Motile spermatozoa were capacitated for 18 h and different aliquots were allocated to treated or control groups and then evaluated for their ability to undergo the acrosome reaction and to increase [Ca(2+) ]i in response to P. The acrosome reaction was evaluated using Pisum sativum agglutinin (PSA)-FITC, and [Ca(2+) ]i was evaluated using fura 2AM. At all of the concentrations tested, PKA inhibitors significantly reduced the percentage of the P-induced acrosome reaction (p < 0.001). However, only the highest concentrations of PKA inhibitors reduced the P-induced calcium influx; lower concentrations of PKA inhibitors did not affect it. Similar results were apparent for PKC inhibitors and for tyrosine kinase inhibitors. None of the Ser/Thr phosphatase inhibitors affected the P-induced acrosome reaction or the P-induced calcium influx, except for the PP2B inhibitors that significantly reduced the P-induced acrosome reaction without affecting calcium influx. Finally, the protein tyrosine phosphatase inhibitors significantly blocked the P-induced acrosome reaction and reduced the amplitude of the P-induced calcium transient (p < 0.001) as well as the amplitude of the plateau phase (p < 0.01). The data suggest that protein kinases and possibly PP2B have a role on the acrosome reaction at some

  14. SLXL1, a Novel Acrosomal Protein, Interacts with DKKL1 and Is Involved in Fertilization in Mice

    PubMed Central

    Zhuang, Xin-jie; Hou, Xiao-jun; Liao, Shang-Ying; Wang, Xiu-Xia; Cooke, Howard J.; Zhang, Ming; Han, Chunsheng

    2011-01-01

    Background Spermatogenesis is a complex cellular developmental process which involves diverse families of genes. The Xlr (X-linked, lymphocyte regulated) family includes multiple members, only a few of which have reported functions in meiosis, post-meiotic maturation, and fertilization of germ cells. Slx-like1 (Slxl1) is a member of the Xlr family, whose expression and function in spermatogenesis need to be elucidated. Methodology/Principal Findings The mRNA and protein expression and localization of Slxl1 were investigated by RT-PCR, Western blotting and immunohistochemistry in different tissues and at different stages of spermatogenesis. The interacting partner of SLXL1 was examined by co-immunoprecipitation and co-localization. Assessment of the role of SLXL1 in capacitation, acrosome reaction, zona pellucida binding/penetration, and fertilization was carried out in vitro using blocking antisera. The results showed that Slxl1 mRNA and protein were specifically expressed in the testis. SLXL1 was exclusively located in the acrosome of post-meiotic germ cells and interacts with DKKL1 (Dickkopf-like1), which is an acrosome-associated protein and plays an important role in fertilization. The rates of zona pellucida binding/penetration and fertilization were significantly reduced by the anti-SLXL1 polyclonal antiserum. Conclusions/Significance SLXL1 is the first identified member of the XLR family that is associated with acrosome and is involved in zona pellucid binding/penetration and subsequent fertilization. These results, together with previous studies, suggest that Xlr family members participate in diverse processes from meiosis to fertilization during spermatogenesis. PMID:21698294

  15. Integrated photografted molecularly imprinted polymers with a cellulose acetate membrane for the extraction of melamine from dry milk before HPLC analysis.

    PubMed

    Akbari-Adergani, Behrouz; Sadeghian, Gholam-Hossein; Alimohammadi, Alireza; Esfandiari, Zahra

    2017-03-01

    In this study, a new separation technique based on membrane extraction is described for the determination of melamine in dry milk. The water-compatible cellulose acetate membrane, which is photografted by melamine imprinted nanospheres, was prepared by placing the membrane into the polymerization solution containing methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as cross-linker, acetonitrile as porogen, and melamine as the template molecule. The characterization of the polymeric membrane was performed by Fourier transmission infrared spectroscopy and scanning electron microscopy. This integrated composite membrane was used as a solid-phase extraction medium for the extraction of melamine from dry milk samples. Various parameters affecting the extraction efficiency of the membrane were evaluated. The results showed higher binding capacity for melamine imprinted membranes in comparison with the nonimprinted membranes. High-performance liquid chromatography analysis showed that the extraction of melamine from dry milk by the photografted cellulose acetate membrane had a linear calibration curve in the range of 0.02-11.80 μg/mL with an excellent precision of 2.73%. The limit of detection and quantification of melamine was 0.007 and 0.020 μg/mL, respectively. The recoveries of melamine were in the range of 88.7-94.8%.

  16. Lipid-protein interactions of integral membrane proteins: a comparative simulation study.

    PubMed

    Deol, Sundeep S; Bond, Peter J; Domene, Carmen; Sansom, Mark S P

    2004-12-01

    The interactions between membrane proteins and their lipid bilayer environment play important roles in the stability and function of such proteins. Extended (15-20 ns) molecular dynamics simulations have been used to explore the interactions of two membrane proteins with phosphatidylcholine bilayers. One protein (KcsA) is an alpha-helix bundle and embedded in a palmitoyl oleoyl phosphatidylcholine bilayer; the other (OmpA) is a beta-barrel outer-membrane protein and is in a dimyristoyl phosphatidylcholine bilayer. The simulations enable analysis in detail of a number of aspects of lipid-protein interactions. In particular, the interactions of aromatic amphipathic side chains (i.e., Trp, Tyr) with lipid headgroups, and "snorkeling" interactions of basic side chains (i.e., Lys, Arg) with phosphate groups are explored. Analysis of the number of contacts and of H-bonds reveal fluctuations on an approximately 1- to 5-ns timescale. There are two clear bands of interacting residues on the surface of KcsA, whereas there are three such bands on OmpA. A large number of Arg-phosphate interactions are seen for KcsA; for OmpA, the number of basic-phosphate interactions is smaller and shows more marked fluctuations with respect to time. Both classes of interaction occur in clearly defined interfacial regions of width approximately 1 nm. Analysis of lateral diffusion of lipid molecules reveals that "boundary" lipid molecules diffuse at about half the rate of bulk lipid. Overall, these simulations present a dynamic picture of lipid-protein interactions: there are a number of more specific interactions but even these fluctuate on an approximately 1- to 5-ns timescale.

  17. Intracellular calcium movements of boar spermatozoa during 'in vitro' capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular calcium-dependent model.

    PubMed

    Yeste, M; Fernández-Novell, J M; Ramió-Lluch, L; Estrada, E; Rocha, L G; Cebrián-Pérez, J A; Muiño-Blanco, T; Concha, I I; Ramírez, A; Rodríguez-Gil, J E

    2015-07-01

    This work analysed intracellular calcium stores of boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico-chemical properties, Fluo-3 and Rhod-5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo-3 was located at both the midpiece and the whole head. Rhod-5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca(2+) signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca(2+) labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca(2+) signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo-3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca(2+) . The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca(2+) -induced peak, O2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.

  18. Probing the potential of apigenin liposomes in enhancing bacterial membrane perturbation and integrity loss.

    PubMed

    Banerjee, Kacoli; Banerjee, Shubhadeep; Das, Subhayan; Mandal, Mahitosh

    2015-09-01

    Along with discovery of new antibacterial agents, it is important to develop novel drug delivery systems to effectively deliver drugs within bacterial cells for enhanced therapeutic activity. Liposomes have been extensively investigated as pharmaceutical carriers for improvement of therapeutic index of antimicrobial agents. The aim of this present study was to evaluate the antibacterial activity of free and liposomal formulation of apigenin, a plant based isoflavone and elucidate the mode of action. Distearoylphosphatidylcholine liposomes were prepared having nano-range particle size (104.3±1.8 nm), narrow particle distribution (0.204) and high encapsulation efficiency of apigenin (89.9±2.31%). Antibacterial activity of apigenin and efficacy of liposome-mediated apigenin delivery were determined from minimum inhibitory concentration values. Interaction studies using electron microscopy revealed adherence and fusion of liposomal apigenin with the bacteria causing membrane perturbation through reactive oxygen species generation which was evaluated by epi-fluorescence microscopy and fluorescence activated cell sorting. The interaction of apigenin liposomes with bacterial membrane increased intracellular drug concentration and thus, can be employed to deliver apigenin within cells to augment its antibacterial activity. Increased efficacy and hemocompatibility of this formulation paves way for future evaluation of underlying molecular mechanisms and in vivo testing for enhanced therapeutic effects.

  19. Biodegradation characteristics and size fractionation of landfill leachate for integrated membrane treatment.

    PubMed

    Insel, Güçlü; Dagdar, Mina; Dogruel, Serdar; Dizge, Nadir; Ubay Cokgor, Emine; Keskinler, Bülent

    2013-09-15

    The fate of organics and nitrogen during the biological treatment with MBR and subsequent membrane filtration processes (nano filtration, NF; reverse osmosis, RO) were investigated for a landfill leachate. The chemical oxygen demand (COD) and total Kjeldahl nitrogen (TKN) removal performances of membrane bioreactor (MBR) were obtained to be around 89% and 85%, respectively. The effluent COD of MBR was measured to be 1935 mg/L (30 kDa) which is much lower than experimentally determined soluble inert COD of 3200 mg/L using 0.45 μm filter. The readily and slowly biodegradable COD fractions were estimated to be 17% and 52% of raw influent COD, respectively. The respirometry based modeling test performed on raw leachate exhibited much slower degradation kinetics compared to municipal wastewater. A unique subset of model parameters was extracted from batch respirometry by using acclimated MBR sludge. The sequential ultrafiltration (UF) experiments (particle size distribution, PSD) revealed that most of the organics was below 2 nm filter mesh size. In addition, NF/RO post treatment after MBR system was required to increase COD and total nitrogen (TN) removal performances up to 99%. Relatively lower salt rejection rates around 94% was obtained for RO system as a post treatment of MBR system.

  20. The design of a membrane-based integrated ethanol production process

    SciTech Connect

    Groot, W.J.; Van Der Lans, R.G.J.M.; Luyben, K.C.A.M.

    1991-12-31

    Alcohol production is an important topic in biotechnology. The research in this field aims at the development of efficient production schemes for alcohol as a fuel or beverage. In the case of fuel alcohol the validation of low-cost substrates or wastes also is aimed at. The efficiency of the production process depends on several aspects: operational simplicity, productivity and product concentration (cq. substrate consumption) in the fermentation, and product recovery. In a fermentation the productivity is proportional to the biocatalyst concentration, and techniques to retain the biocatalyst in the fermentation section must ensure a high productivity. In addition, product inhibition of the biocatalyst determines the productivity and the substrate consumption. These inhibition effects can be partly reduced by alcohol removal during fermentation. Another advantage of this technology is that the recovery method is an economically feasible alternative to conventional distillation for purification of alcohol. Promising techniques for biomass retention are (auto)flocculation (200 m{sup 3} scale), immobilization (4 m{sup 3} scale), and microfiltration (0.8 m{sup 3} scale). In situ product recovery by stripping, combined with biomass retention by centrifugation, is applied in the Biostill-process (scale 150 m{sup 3}/d ethanol). A novel separation technique is pervaporation, in which an ethanol/water mixture is recovered from the broth by evaporation via a selective membrane. High-flux membranes are now available, which makes it possible to investigate pervaporation for laboratory-scale in situ product recover.

  1. CARBONIC ANHYDRASE ACTIVITY OF INTEGRAL-FUNCTIONAL COMPLEXES OF THYLAKOID MEMBRANES OF SPINACH CHLOROPLASTS.

    PubMed

    Semenihin, A V; Zolotareva, O K

    2015-01-01

    Isolated thylakoid membranes were disrupted by treatment with nonionic detergents digitonin or dodecyl maltoside. Solubilized polypeptide complexes were separated by native gel charge shift electrophoresis. The position of ATP-synthase complex and its isolated catalytic part (CF1) within gel was determined using the color reaction for ATPase activity. Due to the presence of cytochromes, the red band in unstained gels corresponded to the cytochrome b6f complex. Localization of the cytochrome b6f complex, ATP synthase and coupling CF1 in the native gel was confirmed by their subunit composition determined after SDS-electrophoretic analysis. Carbonic anhydrase (CA) activity in polypeptide zones of PS II, cytochrome b6f complex, and ATP-synthase CF1 was identified in native gels using indicator bromothymol blue. CA activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration. The water-soluble acetazolamide, an inhibitor of CA, unlike lipophilic ethoxyzolamide inhibited CA activity of CF1 Thus, it was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange. The data obtained suggest the presence of multiple forms of carbonic anhydrase in the thylakoid membranes of spinach chloroplasts and confirm their involvement in the proton transfer to the ATP synthase.

  2. Cristae remodeling causes acidification detected by integrated graphene sensor during mitochondrial outer membrane permeabilization

    PubMed Central

    Pham, Ted D.; Pham, Phi Q.; Li, Jinfeng; Letai, Anthony G.; Wallace, Douglas C.; Burke, Peter J.

    2016-01-01

    The intrinsic apoptotic pathway and the resultant mitochondrial outer membrane permeabilization (MOMP) via BAK and BAX oligomerization, cytochrome c (cytc) release, and caspase activation are well studied, but their effect on cytosolic pH is poorly understood. Using isolated mitochondria, we show that MOMP results in acidification of the surrounding medium. BAK conformational changes associated with MOMP activate the OMA1 protease to cleave OPA1 resulting in remodeling of the cristae and release of the highly concentrated protons within the cristae invaginations. This was revealed by utilizing a nanomaterial graphene as an optically clear and ultrasensitive pH sensor that can measure ionic changes induced by tethered mitochondria. With this platform, we have found that activation of mitochondrial apoptosis is accompanied by a gradual drop in extra-mitochondrial pH and a decline in membrane potential, both of which can be rescued by adding exogenous cytc. These findings have importance for potential pharmacological manipulation of apoptosis, in the treatment of cancer. PMID:27786282

  3. Efficient biosynthetic incorporation of tryptophan and indole analogs in an integral membrane protein

    PubMed Central

    Broos, Jaap; Gabellieri, Edi; Biemans-Oldehinkel, Esther; Strambini, Giovanni B.

    2003-01-01

    Biosynthetic incorporation of tryptophan (Trp) analogs such as 7-azatryptophan, 5-hydroxytryptophan, and fluorotryptophan into a protein can facilitate its structural analysis by spectroscopic techniques such as fluorescence, phosphorescence, nuclear magnetic resonance, and Fourier transform infrared. Until now, the approach has dealt primarily with soluble proteins. In this article, we demonstrate that four different Trp analogs can be very efficiently incorporated into a membrane protein as demonstrated for the mannitol transporter of Escherichia coli (EIImtl). EIImtl overexpression was under control of the λPR promoter, and the E. coli Trp auxotroph M5219 was used as host. This strain constitutively expresses the heat labile repressor protein of the λPR promoter. Together with the presence of the repressor gene on the EIImtl plasmid, this resulted in a tightly controlled promoter system, a prerequisite for high Trp analog incorporation. A new method for determining the analog incorporation efficiency is presented that is suitable for membrane proteins. The procedure involves fitting of the phosphorescence spectrum as a linear combination of the Trp and Trp analog contributions, taking into account the influence of the protein environment on the Trp analog spectrum. The data show that the analog content of EIImtl samples is very high (>95%). In addition, we report here that biosynthetic incorporation of Trp analogs can also be effected with less expensive indole analogs, which in vivo are converted to L-Trp analogs. PMID:12930998

  4. Using proximity biotinylation to detect herpesvirus entry glycoprotein interactions: Limitations for integral membrane glycoproteins.

    PubMed

    Lajko, Michelle; Haddad, Alexander F; Robinson, Carolyn A; Connolly, Sarah A

    2015-09-01

    Herpesvirus entry into cells requires coordinated interactions among several viral transmembrane glycoproteins. Viral glycoproteins bind to receptors and interact with other glycoproteins to trigger virus-cell membrane fusion. Details of these glycoprotein interactions are not well understood because they are likely transient and/or low affinity. Proximity biotinylation is a promising protein-protein interaction assay that can capture transient interactions in live cells. One protein is linked to a biotin ligase and a second protein is linked to a short specific acceptor peptide (AP). If the two proteins interact, the ligase will biotinylate the AP, without requiring a sustained interaction. To examine herpesvirus glycoprotein interactions, the ligase and AP were linked to herpes simplex virus 1 (HSV1) gD and Epstein Barr virus (EBV) gB. Interactions between monomers of these oligomeric proteins (homotypic interactions) served as positive controls to demonstrate assay sensitivity. Heterotypic combinations served as negative controls to determine assay specificity, since HSV1 gD and EBV gB do not interact functionally. Positive controls showed strong biotinylation, indicating that viral glycoprotein proximity can be detected. Unexpectedly, the negative controls also showed biotinylation. These results demonstrate the special circumstances that must be considered when examining interactions among glycosylated proteins that are constrained within a membrane.

  5. Effect of cholesterol-loaded-cyclodextrin on sperm viability and acrosome reaction in boar semen cryopreservation.

    PubMed

    Lee, Yong-Seung; Lee, Seunghyung; Lee, Sang-Hee; Yang, Boo-Keun; Park, Choon-Keun

    2015-08-01

    This study was undertaken to examine the effect of cholesterol-loaded-cyclodextrin (CLC) on boar sperm viability and spermatozoa cryosurvival during boar semen cryopreservation, and methyl-β-cyclodextrin (MBCD) was treated for comparing with CLC. Boar semen treated with CLC and MBCD before freezing process to monitor the effect on survival and capacitation status by flow cytometry with appropriate fluorescent probes. Sperm viability was higher in 1.5mg CLC-treated sperm (76.9±1.01%, P<0.05) than un-treated and MBCD-treated sperm before cryopreservation (58.7±1.31% and 60.3±0.31%, respectively). For CTC patterns, F-pattern was higher in CLC treated sperm than MBCD-treated sperm, for B-pattern was higher in CLC-treated sperm than fresh sperm (P<0.05). For AR pattern (an acrosome-reacted sperm) was lower in CLC-treated sperm than MBCD-treated sperm (P<0.05). Moreover, we examined in vitro development of porcine oocytes after in vitro fertilization using CLC-treated frozen-thawed semen, in which CLC treatment prior to freezing and thawing increased the development of oocytes to blastocyst stage in vitro. In conclusion, CLC could protect the viability of spermatozoa from cryodamage prior to cryopreservation in boar semen.

  6. A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm.

    PubMed

    Romarowski, Ana; Sánchez-Cárdenas, Claudia; Ramírez-Gómez, Héctor V; Puga Molina, Lis del C; Treviño, Claudia L; Hernández-Cruz, Arturo; Darszon, Alberto; Buffone, Mariano G

    2016-03-01

    During capacitation, sperm acquire the ability to undergo the acrosome reaction (AR), an essential step in fertilization. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AR. An increase in intracellular Ca(2+) ([Ca(2+)]i) is necessary for AR to occur. In this study, we investigated the spatiotemporal correlation between the changes in [Ca(2+)]i and AR in single mouse spermatozoa in response to progesterone. We found that progesterone stimulates an [Ca(2+)]i increase in five different patterns: gradual increase, oscillatory, late transitory, immediate transitory, and sustained. We also observed that the [Ca(2+)]i increase promoted by progesterone starts at either the flagellum or the head. We validated the use of FM4-64 as an indicator for the occurrence of the AR by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, we have simultaneously visualized the rise in [Ca(2+)]i and the process of exocytosis in response to progesterone and found that only a specific transitory increase in [Ca(2+)]i originating in the sperm head promotes the initiation of AR.

  7. Role and regulation of EGFR in actin remodeling in sperm capacitation and the acrosome reaction

    PubMed Central

    Breitbart, Haim; Etkovitz, Nir

    2011-01-01

    To bind and fertilize the egg, the spermatozoon should undergo few biochemical and motility changes in the female reproductive tract collectively called capacitation. The capacitated spermatozoon binds to the egg zona pellucida, and then undergoes the acrosome reaction (AR), which allows its penetration into the egg. The mechanisms regulating sperm capacitation and the AR are not completely understood. In the present review, we summarize some data regarding the role and regulation of the epidermal growth factor receptor (EGFR) in these processes. In the capacitation process, the EGFR is partially activated by protein kinase A (PKA), resulting in phospholipase D (PLD) activation and actin polymerization. Protein kinase C alpha (PKCα), which is already activated at the beginning of the capacitation, also participates in PLD activation. Further activation of the EGFR at the end of the capacitation enhances intracellular Ca2+ concentration leading to F-actin breakdown and allows the AR to take place. Under in vivo conditions, the EGFR can be directly activated by its known ligand epidermal growth factor (EGF), and indirectly by activating PKA or by transactivation mediated by G protein-coupled receptors (GPCRs) activation or by ouabain. Under physiological conditions, sperm PKA is activated mainly by bicarbonate, which activates the soluble adenylyl cyclase to produce cyclic adenosine monophosphate (cAMP), the activator of PKA. The GPCR activators angiotensin II or lysophosphatidic acid, as well as ouabain and EGF are physiological components present in the female reproductive tract. PMID:21200378

  8. Identification of egg-jelly substances triggering sperm acrosome reaction in the newt, Cynops pyrrhogaster.

    PubMed

    Watanabe, Akihiko; Fukutomi, Keiko; Kubo, Hideo; Ohta, Manami; Takayama-Watanabe, Eriko; Onitake, Kazuo

    2009-04-01

    Our previous studies have shown that the acrosome reaction (AR) occurs in egg-jelly of the Japanese newt, Cynops pyrrhogaster. This is analogous to the substances of echinoderms but distinct from those of many other vertebrates derived from the egg envelope or its derivative, the zona pellucida. To identify the AR-inducing substances in newt egg jelly, a monoclonal antibody (mAb) was generated against the jelly by screening the culture supernatants to find the one that best neutralized the AR-inducing activity of the jelly substance. The mAb specifically reacted to protein bands in the jelly. These proteins, with apparent molecular weights of 122 and 90 kDa, exhibited AR-inducing activity, indicating that they are definitely AR-inducing substances. Western blotting using the mAb indicated that the 122 and 90 kDa proteins are present only in the egg jelly's outermost layer, where AR-inducing activity is known to occur. Both proteins were recognized with wheat germ agglutinin (WGA), a lectin that inhibits AR-induction in egg jelly extract. Taken together, these findings indicate that the 122 and 90 kDa proteins are the AR-inducing substances in the egg jelly of C. pyrrhogaster. The WGA recognition of the proteins was lost by N-glycosidase digestion, suggesting that N-linked carbohydrate moieties in these proteins may be responsible for the AR-inducing activity.

  9. One-Step Fabrication of a Microfluidic Device with an Integrated Membrane and Embedded Reagents by Multimaterial 3D Printing.

    PubMed

    Li, Feng; Smejkal, Petr; Macdonald, Niall P; Guijt, Rosanne M; Breadmore, Michael C

    2017-04-05

    One of the largest impediments in the development of microfluidic-based smart sensing systems is the manufacturability of integrated, complex devices. Here we propose multimaterial 3D printing for the fabrication of such devices in a single step. A microfluidic device containing an integrated porous membrane and embedded liquid reagents was made by 3D printing and applied for the analysis of nitrate in soil. The manufacture of the integrated, sealed device was realized as a single print within 30 min. The body of the device was printed in transparent acrylonitrile butadiene styrene (ABS) and contained a 400 μm wide structure printed from a commercially available composite filament. The composite filament can be turned into a porous material through dissolution of a water-soluble material. Liquid reagents were integrated by briefly pausing the printing before resuming for sealing the device. The devices were evaluated by the determination of nitrate in a soil slurry containing zinc particles for the reduction of nitrate to nitrite using the Griess reagent. Using a consumer digital camera, the linear range of the detector response ranged from 0 to 60 ppm, covering the normal range of nitrate in soil. To ensure that the sealing of the reagent chamber is maintained, aqueous reagents should be avoided. When using the nonaqueous reagent, the multimaterial device containing the Griess reagent could be stored for over 4 days but increased the detection range to 100-500 ppm. Multimaterial 3D printing is a potentially new approach for the manufacture of microfluidic devices with multiple integrated functional components.

  10. Comparative study of metal induced phospholipid modifications in the heavy metal tolerant filamentous fungus Paecilomyces marquandii and implications for the fungal membrane integrity.

    PubMed

    Słaba, Mirosława; Bernat, Przemysław; Różalska, Sylwia; Nykiel, Justyna; Długoński, Jerzy

    2013-01-01

    In this work we compared the effect of five heavy metals: Zn, Pb, Cd, Ni and Cu on phospholipid composition of the ubiquitous soil fungus Paecilomyces marquandii, originating from a strongly metal polluted area and characterized by high tolerance to these elements. Cd, Ni and Cu caused an increase in phosphatidylcholine (PC). Only Pb decreased PC content, which was accompanied by a significant rise in the phosphatidic acids (PA) level, probably due to activation of phospholipase D which hydrolyzes PC to PA. This could result in membrane fluidity disturbance, and thus affect its integrity. The assessment of propidium iodide influx showed strong disturbance of membrane integrity for Cu and Pb stressed mycelia, whereas mycelia treated with Ni were impermeable to this dye. The results obtained revealed a strong Cu and Pb toxicity involving disruption of membrane integrity. Pb action was reflected by lipid composition, whereas changes in Cu treated mycelia did not completely elucidate its harmful effect on the membrane, which was most probably caused by Cu induced lipid peroxidation. Zn did not induce quantitative changes in PC and phosphatidylethanolamine (PE) but caused changes in phospholipid lipid saturation, which appears to be important for fungus adaptation to the presence of metals. The enhanced PC content balanced by higher PC saturation can help in the maintenance of proper membrane fluidity and result in alleviating the Cd and Ni induced stress. These results will allow to clarify the mechanism of Pb toxicity and help to elucidate the cellular basis of fungal membrane adaptation to heavy metals.

  11. High-fidelity simulation and reduced-order modelling of integrally-actuated membrane wings with feedback control

    NASA Astrophysics Data System (ADS)

    Buoso, Stefano; Palacios, Rafael

    2016-04-01

    This work presents a numerical framework for the simulation and design of integrally actuated membrane wings with feedback control. The performance of the aeroelastic system are evaluated using a high-fidelity model. It consists in a fluid solver based on the direct numerical integration of the unsteady Navier-Stokes equations implicitly coupled with a geometrically non-linear dynamic structural model which has been calibrated using experimental data. The rate-dependent constitutive law for the dielectric elastomer considered for the integral wing actuation is based on a non-linear formulation. The framework also includes a methodology for the model reduction of the fully-coupled system. The resulting low-order description showed to retain the main system dynamics, and can therefore be used for the design of the control scheme for the wing. Results highlights the potential to achieve on-demand aerodynamics using the actuation concept proposed. In particular, it is shown that the wing aerodynamic performance is noticeably enhanced through the actuation and the disturbances on the lift in case of gusts can be reduced up to 60%.

  12. Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family.

    PubMed

    Preston, G M; Agre, P

    1991-12-15

    CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5' untranslated nucleotide sequence, an 807-bp open reading frame, and approximately 2 kilobases of 3' untranslated sequence containing a polyadenylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DNA sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.

  13. Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins

    PubMed Central

    Gasanov, Sardar E.; Shrivastava, Indira H.; Israilov, Firuz S.; Kim, Aleksandr A.; Rylova, Kamila A.; Zhang, Boris; Dagda, Ruben K.

    2015-01-01

    Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL) to disrupt mitochondrial membranes. Collectively, 31P-NMR, electron paramagnetic resonance (EPR), proton NMR (1H-NMR), deuterium NMR (2H-NMR) spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC) liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS). Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity. PMID:26091109

  14. Solution structure of the integral human membrane protein VDAC-1 in detergent micelles**

    PubMed Central

    Hiller, Sebastian; Garces, Robert G.; Malia, Thomas J.; Orekhov, Vladislav Y.; Colombini, Marco; Wagner, Gerhard

    2008-01-01

    The voltage-dependent anion channel (VDAC) mediates trafficking of small molecules and ions across the eukaryotic outer mitochondrial membrane. VDAC also interacts with anti-apoptotic proteins from the Bcl-2 family and this interaction inhibits release of apoptogenic proteins from the mitochondrion. We present the NMR solution structure of recombinant human VDAC-1 reconstituted in detergent micelles. It forms a 19-stranded β-barrel with the first and last strand parallel. The hydrophobic outside perimeter of the barrel is covered by detergent molecules in a belt-like fashion. In the presence of cholesterol recombinant VDAC-1 can form voltage-gated channels in phospholipid bilayers similar to the native protein. NMR measurements revealed the binding sites of VDAC-1 for the Bcl-2 protein Bcl-xL, for β-NADH and for cholesterol. Bcl-xL interacts with the VDAC barrel laterally at strands 17 and 18. PMID:18755977

  15. A novel integrated thermal-/membrane-based solar energy-driven hybrid desalination system: Concept description and simulation results.

    PubMed

    Kim, Young-Deuk; Thu, Kyaw; Ng, Kim Choon; Amy, Gary L; Ghaffour, Noreddine

    2016-09-01

    In this paper, a hybrid desalination system consisting of vacuum membrane distillation (VMD) and adsorption desalination (AD) units, designated as VMD-AD cycle, is proposed. The synergetic integration of the VMD and AD is demonstrated where a useful effect of the AD cycle is channelled to boost the operation of the VMD process, namely the low vacuum environment to maintain the high pressure gradient across the microporous hydrophobic membrane. A solar-assisted multi-stage VMD-AD hybrid desalination system with temperature modulating unit is first designed, and its performance is then examined with a mathematical model of each component in the system and compared with the VMD-only system with temperature modulating and heat recovery units. The total water production and water recovery ratio of a solar-assisted 24-stage VMD-AD hybrid system are found to be about 21% and 23% higher, respectively, as compared to the VMD-only system. For the solar-assisted 24-stage VMD-AD desalination system having 150 m(2) of evacuated-tube collectors and 10 m(3) seawater storage tanks, both annual collector efficiency and solar fraction are close to 60%.

  16. Design, purification and characterization of a soluble variant of the integral membrane protein MotB for structural studies.

    PubMed

    Andrews, Daniel A; Xie, Meng; Hughes, Victoria; Wilce, Matthew C; Roujeinikova, Anna

    2013-02-01

    The bacterial flagellar motor is an intricate nanomachine powered by a transmembrane electrochemical gradient. Rotation is driven by the cumulative action of several peptidoglycan-anchored stator complexes on the rotor. In proton-motive force-driven motors, the stator complex is composed of a motility protein B (MotB) dimer surrounded by four copies of MotA, where both MotA and MotB are integral membrane proteins. The lack of full-length MotA and MotB structures hinders understanding of the mechanism of torque generation. Given the low levels of expression and low stability of detergent-solubilized MotB, a soluble chimaeric variant was engineered, where the two transmembrane helices of the MotB dimer were replaced by a leucine zipper. The biochemical and biophysical analysis of the resultant protein showed that it was properly folded, stable, behaved as a monodisperse dimer at low pH, had molecular dimensions close to those expected for native MotB and yielded reproducible crystals. The chimaeric protein is, therefore, a good candidate for structural studies. This 'solubilization by design' approach may be generally applicable to the production of soluble forms of other dimeric, trimeric and tetrameric single-span membrane proteins for functional and structural studies.

  17. Determination of the temperature-dependent cell membrane permeabilities using microfluidics with integrated flow and temperature control.

    PubMed

    Fang, Cifeng; Ji, Fujun; Shu, Zhiquan; Gao, Dayong

    2017-02-28

    We developed an integrated microfluidic platform for instantaneous flow and localized temperature control. The platform consisted of a flow-focusing region for sample delivery and a cross-junction region embedded with a microheater for cell trapping and localized temperature control by using an active feedback control system. We further used it to measure the membrane transport properties of Jurkat cells, including the osmotically inactive cell volume (Vb) and cell membrane permeabilities to water (Lp) and to cryoprotective agent (CPA) solutions (dimethyl sulfoxide (DMSO) in this study) (PS) at various temperatures (room temperature, 30 °C, and 37 °C). Such characteristics of cells are of great importance in many applications, especially in optimal cryopreservation. With the results, the corresponding activation energy for water and CPA transport was calculated. The comparison of the results from the current study with reference data indicates that the developed platform is a reliable tool for temperature-dependent cell behavior study, which provides valuable tools for general cell manipulation applications with precise temperature control.

  18. Membrane-bound trafficking regulates nuclear transport of integral epidermal growth factor receptor (EGFR) and ErbB-2.

    PubMed

    Wang, Ying-Nai; Lee, Heng-Huan; Lee, Hong-Jen; Du, Yi; Yamaguchi, Hirohito; Hung, Mien-Chie

    2012-05-11

    Nuclear localization of multiple receptor-tyrosine kinases (RTKs), such as EGF receptor (EGFR), ErbB-2, FGF receptor (FGFR), and many others, has been reported by several groups. We previously showed that cell surface EGFR is trafficked to the nucleus through a retrograde pathway from the Golgi to the endoplasmic reticulum (ER) and that EGFR is then translocated to the inner nuclear membrane (INM) through the INTERNET (integral trafficking from the ER to the nuclear envelope transport) pathway. However, the nuclear trafficking mechanisms of other membrane RTKs, apart from EGFR, remain unclear. The purpose of this study was to compare the nuclear transport of EGFR family proteins with that of FGFR-1. Interestingly, we found that digitonin permeabilization, which selectively releases soluble nuclear transporters from the cytoplasm and has been shown to inhibit nuclear transport of FGFR-1, had no effects on EGFR nuclear transport, raising the possibility that EGFR and FGFR-1 use different pathways to be translocated into the nucleus. Using the subnuclear fractionation assay, we further demonstrated that biotinylated cell surface ErbB-2, but not FGFR-1, is targeted to the INM, associating with Sec61β in the INM, similar to the nuclear trafficking of EGFR. Thus, ErbB-2, but not FGFR-1, shows a similar trafficking pathway to EGFR for translocation to the nucleus, indicating that at least two different pathways of nuclear transport exist for cell surface receptors. This finding provides a new direction for investigating the trafficking mechanisms of various nuclear RTKs.

  19. Structural basis of the signalling through a bacterial membrane receptor HasR deciphered by an integrative approach

    PubMed Central

    Wojtowicz, Halina; Prochnicka-Chalufour, Ada; deAmorim, Gisele Cardoso; Roudenko, Olga; Simenel, Catherine; Malki, Idir; Pehau-Arnaudet, Gérard; Gubellini, Francesca; Koutsioubas, Alexandros; Pérez, Javier; Delepelaire, Philippe; Delepierre, Muriel; Fronzes, Rémi; Izadi-Pruneyre, Nadia

    2016-01-01

    Bacteria use diverse signalling pathways to adapt gene expression to external stimuli. In Gram-negative bacteria, the binding of scarce nutrients to membrane transporters triggers a signalling process that up-regulates the expression of genes of various functions, from uptake of nutrient to production of virulence factors. Although proteins involved in this process have been identified, signal transduction through this family of transporters is not well understood. In the present study, using an integrative approach (EM, SAXS, X-ray crystallography and NMR), we have studied the structure of the haem transporter HasR captured in two stages of the signalling process, i.e. before and after the arrival of signalling activators (haem and its carrier protein). We show for the first time that the HasR domain responsible for signal transfer: (i) is highly flexible in two stages of signalling; (ii) extends into the periplasm at approximately 70–90 Å (1 Å=0.1 nm) from the HasR β-barrel; and (iii) exhibits local conformational changes in response to the arrival of signalling activators. These features would favour the signal transfer from HasR to its cytoplasmic membrane partners. PMID:27208170

  20. Integrated thermophilic submerged aerobic membrane bioreactor and electrochemical oxidation for pulp and paper effluent treatment--towards system closure.

    PubMed

    Qu, X; Gao, W J; Han, M N; Chen, A; Liao, B Q

    2012-07-01

    A novel integrated thermophilic submerged aerobic membrane bioreactor (TSAMBR) and electrochemical oxidation (EO) technology was developed for thermomechanical pulping pressate treatment with the aim of system closure. The TSAMBR was able to achieve a chemical oxygen demand (COD) removal efficiency of 88.6 ± 1.9-92.3 ± 0.7% under the organic loading rate of 2.76 ± 0.13-3.98 ± 0.23 kg COD/(m(3) d). An optimal hydraulic retention time (HRT) of 1.1 ± 0.1d was identified for COD removal. Cake formation was identified as the dominant mechanism of membrane fouling. The EO of the TSAMBR permeate was performed using a Ti/SnO(2)-Sb(2)O(5)-IrO(2) electrode. After 6-h EO, a complete decolourization was achieved and the COD removal efficiency was increased to 96.2 ± 1.2-98.2 ± 0.3%. The high-quality effluent produced by the TSAMBR-EO system can be reused as process water for system closure in pulp and paper mill.

  1. Partial Purification of Integral Membrane Antigenic Proteins from Trypanosoma evansi That Display Immunological Cross-Reactivity with Trypanosoma vivax

    PubMed Central

    Velásquez, Norma P.; Camargo, Rocío E.; Uzcanga, Graciela L.; Bubis, José

    2014-01-01

    Trypanosoma evansi and Trypanosoma vivax, which are the major causative agents of animal trypanosomosis in Venezuela, have shown a very high immunological cross-reactivity. Since the production of T. vivax antigens is a limiting factor as this parasite is difficult to propagate in experimental animal models, our goal has been to identify and isolate antigens from T. evansi that cross-react with T. vivax. Here, we used the Venezuelan T. evansi TEVA1 isolate to prepare the total parasite lysate and its corresponding cytosolic and membranous fractions. In order to extract the T. evansi integral membrane proteins, the particulate portion was further extracted first with Triton X-100, and then with sodium dodecyl sulfate. After discarding the cytosolic and Triton X-100 solubilized proteins, we employed sedimentation by centrifugation on linear sucrose gradients to partially purify the sodium dodecyl sulfate-solubilized proteins from the Triton X-100 resistant particulate fraction of T. evansi. We obtained enriched pools containing polypeptide bands with apparent molecular masses of 27 kDa, 31 kDa, and 53 kDa, which were recognized by anti-T. vivax antibodies from experimentally and naturally infected bovines. PMID:24757558

  2. Facile fabrication of Ag dendrite-integrated anodic aluminum oxide membrane as effective three-dimensional SERS substrate

    NASA Astrophysics Data System (ADS)

    Zhang, Cong-yun; Lu, Ya; Zhao, Bin; Hao, Yao-wu; Liu, Ya-qing

    2016-07-01

    A novel surface enhanced Raman scattering (SERS)-active substrate has been successfully developed, where Ag-dendrites are assembled on the surface and embedded in the channels of anodic aluminum oxide (AAO) membrane, via electrodeposition in AgNO3/PVP aqueous system. Reaction conditions were systematically investigated to attain the best Raman enhancement. The growth mechanism of Ag dendritic nanostructures has been proposed. The Ag dendrite-integrated AAO membrane with unique hierarchical structures exhibits high SERS activity for detecting rhodamine 6G with a detection limit as low as 1 × 10-11 M. Furthermore, the three-dimensional (3D) substrates display a good reproducibility with the average intensity variations at the major Raman peak less than 12%. Most importantly, the 3D SERS substrates without any surface modification show an outstanding SERS response for the molecules with weak affinity for noble metal surfaces. The potential application for the detection of polycyclic aromatic hydrocarbons (PAHs) was evaluated with fluoranthene as Raman target molecule and a sensitive SERS detection with a limit down to 10-8 M was reached. The 3D SERS-active substrate shows promising potential for rapid detection of trace organic pollutants even weak affinity molecules in the environment.

  3. Purification, crystallization and preliminary crystallographic studies of an integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli.

    PubMed

    Abramson, J; Larsson, G; Byrne, B; Puustinen, A; Garcia-Horsman, A; Iwata, S

    2000-08-01

    Cytochrome bo(3) ubiquinol oxidase has been successfully purified for crystallization. Single crystals of this integral membrane protein diffract X-rays to 3.5 A resolution and belong to the orthorhombic space group C222(1). From the diffraction data, the unit-cell parameters were determined to be a = 91.3, b = 370.3, c = 232.4 A. The crystals have a solvent content of 59% and contain two molecules per asymmetric unit. A search model generated from the structures of cytochrome c oxidase from Paracoccus denitrificans and the extrinsic domain of cytochrome bo(3) ubiquinol oxidase from Escherichia coli was used for molecular-replacement studies, resulting in a solution with sensible molecular packing.

  4. Phosphorylation of Golgi Peripheral Membrane Protein Grasp65 Is an Integral Step in the Formation of the Human Cytomegalovirus Cytoplasmic Assembly Compartment.

    PubMed

    Rebmann, G Michael; Grabski, Robert; Sanchez, Veronica; Britt, William J

    2016-10-04

    Human cytomegalovirus (HCMV) is the largest member of the Herpesviridae and represents a significant cause of disease. During virus replication, HCMV alters cellular functions to facilitate its replication, including significant reorganization of the secretory and endocytic pathways of the infected cell. A defining morphologic change of the infected cell is the formation of a membranous structure in the cytoplasm that is designated the virion assembly compartment (AC), which consists of virion structural proteins surrounded by cellular membranes. The loss of normal Golgi compartment morphology and its relocalization from a juxtanuclear ribbonlike structure to a series of concentric rings on the periphery of the AC represents a readily recognized reorganization of cellular membranes in the HCMV-infected cell. Although trafficking of viral proteins to this compartment is required for the assembly of infectious virions, the functional significance of the reorganization of intracellular membranes like the Golgi membranes into the AC in the assembly of infectious virus remains understudied. In this study, we determined that Golgi membrane ribbon fragmentation increased during the early cytoplasmic phase of virion assembly and that Golgi membrane fragmentation in infected cells was dependent on the phosphorylation of an integral cis-Golgi protein, Grasp65. Inhibition of Golgi membrane fragmentation and of its reorganization into the AC resulted in decreased production of infectious particles and alteration of the incorporation of an essential protein into the envelope of the mature virion. These results demonstrated the complexity of the virus-host cell interactions required for efficient assembly of this large DNA virus.

  5. Piscine reovirus encodes a cytotoxic, non-fusogenic, integral membrane protein and previously unrecognized virion outer-capsid proteins.

    PubMed

    Key, Tim; Read, Jolene; Nibert, Max L; Duncan, Roy

    2013-05-01

    Piscine reovirus (PRV) is a tentative new member of the family Reoviridae and has been linked to heart and skeletal muscle inflammation in farmed Atlantic salmon (Salmo salar L.). Recent sequence-based evidence suggests that PRV is about equally related to members of the genera Orthoreovirus and Aquareovirus. Sequence similarities have also suggested that PRV might encode a fusion-associated small transmembrane (FAST) protein, which in turn suggests that PRV might be the prototype of a new genus with syncytium-inducing potential. In previous support of this designation has been the absence of identifiable PRV-encoded homologues of either the virion outer-clamp protein of ortho- and aquareoviruses or the virion outer-fibre protein of most orthoreoviruses. In the current report, we have provided experimental evidence that the putative p13 FAST protein of PRV lacks the defining feature of the FAST protein family - the ability to induce syncytium formation. Instead, p13 is the first example of a cytosolic, integral membrane protein encoded by ortho- or aquareoviruses, and induces cytotoxicity in the absence of cell-cell fusion. Sequence analysis also identified signature motifs of the outer-clamp and outer-fibre proteins of other reoviruses in two of the predicted PRV gene products. Based on these findings, we conclude that PRV does not encode a FAST protein and is therefore unlikely to be a new fusogenic reovirus. The presence of a novel integral membrane protein and two previously unrecognized, essential outer-capsid proteins has important implications for the biology, evolution and taxonomic classification of this virus.

  6. A survey of membrane oxygenator heat-exchanger integrity testing at cardiac surgery centres in Great Britain and Ireland.

    PubMed

    Carlton, Matthew; Campbell, John

    2013-11-01

    Membrane oxygenator heat exchanger (HE) device failure is reported to be very low for both short- and long-term extracorporeal devices. All oxygenator manufacturers provide instructions for leak testing of their HE devices prior to patient use. In addition to these recommendations, since 2006 at Nottingham University Hospitals (NUH) we have also additionally pressure tested HE devices prior to use. We conducted a national survey of cardiac centers in Great Britain and Ireland to determine the methods undertaken in individual centers for validation of the integrity of HE devices. Furthermore, we also collected information on the routine maintenance techniques utilized within these centers to inhibit microbial growth in the water used in the heater-cooler units (HCUs). In total, 34 responses were collected from the 57 centers performing cardiac surgery, producing a response rate of 60%. Of the responding centers, 71% are adhering to manufacturer's recommended guidelines of circulating the water through the device for 5 minutes. Of these centers, 17% reported detecting a leak between the HE and membrane compartment of the oxygenator. In responding centers, 29% reported using the pressure test technique. In the centers utilizing pressure testing, 60% reported detecting a leak. This survey reports an association of a greater HE leak detection rate using the pressure test technique compared to using water testing in isolation (p = 0.034). We believe the pressure testing method provides the perfusionist with confidence in the integrity of the HE for short- and long-term circulatory support devices prior to use in both elective and emergency situations.

  7. Evidence for substantial maintenance of membrane integrity and cell viability in normally developing grape (Vitis vinifera L.) berries throughout development.

    PubMed

    Krasnow, Mark; Matthews, Mark; Shackel, Ken

    2008-01-01

    Fluorescein diacetate (FDA) was used as a vital stain to assay membrane integrity (cell viability) in mesocarp tissue of the developing grape (Vitis vinifera L.) berry in order to test the hypothesis that there is a substantial loss of compartmentation in these cells during ripening. This technique was also used to determine whether loss of viability was associated with symptoms of a ripening disorder known as berry shrivel. FDA fluorescence of berry cells was rapid, bright, and stable for over 1 h at room temperature. Confocal microscopy detected FDA staining through two to three intact surface cell layers (300-400 mum) of bisected berries, and showed that the fluorescence was confined to the cytoplasm, indicating the maintenance of integrity in both cytoplasmic as well as vacuolar membranes, and the presence of active cytoplasmic esterases. FDA clearly discriminated between living cells and freeze-killed cells, and exhibited little, if any, non-specific staining. Propidium iodide and DAPI, both widely used to assess cell viability, were unable to discriminate between living and freeze-killed cells, and did not specifically stain the nuclei of dead cells. For normally developing berries under field conditions there was no evidence of viability loss until about 40 d after veraison, and the majority (80%) of mesocarp cells remained viable past commercial harvest (26 degrees Brix). These results are inconsistent with current models of grape berry development which hypothesize that veraison is associated with a general loss of compartmentation in mesocarp cells. The observed viability loss was primarily in the locule area around the seeds, suggesting that a localized loss of viability and compartmentation may occur as part of normal fruit development. The cell viability of berry shrivel-affected berries was similar to that of normally developing berries until the onset of visible symptoms (i.e. shrivelling), at which time viability declined in visibly shrivelled

  8. Flow cytometry immunodetection and membrane integrity assessment of Escherichia coli O157:H7 in ready-to-eat pasta salad during refrigerated storage.

    PubMed

    Subires, Alicia; Yuste, Josep; Capellas, Marta

    2014-01-03

    Over the past years, products of non-animal origin have been increasingly linked to foodborne diseases caused by the enterohemorrhagic pathogen Escherichia coli O157:H7. Contaminated fresh produce and derived ready-to-eat meals are of major concern, since no further or only minimal processing is applied. In this study, flow cytometry was evaluated as a rapid technique to detect E. coli O157:H7 by immunofluorescence, using polyclonal antibodies conjugated to R-phycoerythrin, in refrigerated ready-to-eat pasta salad containing acetic acid and benzoic acid. Signal filtering strategies were applied during sample analysis to reduce the limit of detection of the technique to 5 log CFU/g. Simultaneously with pathogen detection, physiological state was assessed by staining with the membrane integrity indicators propidium iodide and SYBR Green I. Fine tuning of dye concentrations and ratios allowed discrimination of not only cells with intact or damaged membranes, but also of cells with partially damaged membranes, which were considered injured cells. Then, changes in membrane integrity of inoculated E. coli O157:H7 cells were monitored throughout 14-day refrigerated storage. Most cells were injured at the beginning of refrigeration, but showed an intact membrane at the end. This suggests that injured E. coli O157:H7 cells underwent a membrane repair during exposure to refrigeration and acid stresses, and survived in ready-to-eat pasta salad. This highlights the importance of the implementation of control measures to limit the presence of this pathogen in non-animal origin food products. Additionally, the proposed immunodetection and membrane integrity three-color assay in food is a good tool to monitor the effect of a number of food-related treatments on E. coli O157:H7 cell membrane.

  9. Integrated antifouling and bactericidal polymer membranes through bioinspired polydopamine/poly(N-vinyl pyrrolidone) coating

    NASA Astrophysics Data System (ADS)

    Wang, Xianghong; Yuan, Shuaishuai; Shi, Dean; Yang, Yingkui; Jiang, Tao; Yan, Shunjie; Shi, Hengchong; Luan, Shifang; Yin, Jinghua

    2016-07-01

    Polypropylene (PP) non-woven has been widely used as wound dressing; however, the hydrophobic nature of PP can initiate bacterial attachment and subsequent biofilm formation. Herein, we propose a facile approach to functionalize PP non-woven with poly(ethylene glycol) (PEG) and poly(N-vinyl pyrrolidone)-iodine complex (PVP-I). PVP and PEG were successively tethered onto PP non-woven surface via versatile bioinspired dopamine (DA) chemistry, followed by complexing iodine with PVP moieties. It was demonstrated through the field emission scanning electron microscope (SEM) and spread plate method that the as-modified PP non-woven integrated both antifouling property of PEG for suppressing bacterial adhesion, and bactericidal property of PVP-I for killing the few adherent bacteria. Meanwhile, it could greatly resist platelet and red blood cell adhesion. The integrated antifouling and bactericidal PP non-woven surfaces might have great potential in various wound dressing applications.

  10. Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach.

    PubMed

    Lydka, M; Piasecka, M; Gaczarzewicz, D; Koziorowski, M; Bilinska, B

    2012-08-01

    Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 μg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p < 0.05) in flutamide-treated boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.

  11. Dimethyl Sulfoxide Damages Mitochondrial Integrity and Membrane Potential in Cultured Astrocytes

    PubMed Central

    Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming

    2014-01-01

    Dimethyl sulfoxide (DMSO) is a polar organic solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacological and pathological effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species production, and subsequent cytochrome c release and caspase-3 activation. DMSO at concentrations of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO. PMID:25238609

  12. Integrating Activities of Laminins that Drive Basement Membrane Assembly and Function.

    PubMed

    Yurchenco, Peter D

    2015-01-01

    Studies on extracellular matrix proteins, cells, and genetically modified animals have converged to reveal mechanisms of basement membrane self-assembly as mediated by γ1 subunit-containing laminins, the focus of this chapter. The basic model is as follows: A member of the laminin family adheres to a competent cell surface and typically polymerizes followed by laminin binding to the extracellular adaptor proteins nidogen, perlecan, and agrin. Assembly is completed by the linking of nidogen and heparan sulfates to type IV collagen, allowing it to form a second stabilizing network polymer. The assembled matrix provides structural support, anchoring the extracellular matrix to the cytoskeleton, and acts as a signaling platform. Heterogeneity of function is created in part by the isoforms of laminin that vary in their ability to polymerize and to interact with integrins, dystroglycan, and other receptors. Mutations in laminin subunits, affecting expression or LN domain-specific functions, are a cause of human diseases that include those of muscle, nerve, brain, and kidney.

  13. Integral: collector solar greenhouse using solar membrane and external rock storage

    SciTech Connect

    Droll, P.W.

    1980-12-31

    The results of a three-year study to evaluate engineering aspects of converting a commercial greenhouse to more effectively used solar energy as a heating source are summarized. The solar retrofit greenhouse and nine other similar buildings are located in northern California. They are large Quonset style greenhouses glazed on all curved surfaces with a conventional corrugated fiberglass covering. The solar retrofit building was modified in 1978 by installing on the inside surface of the original corrugated fiberglass three air-separated layers of a material called Solar Membrane which reduces convective losses through the overlapping fiberglass panels and effectively prevents long wave infrared transmission out of the greenhouse. A large above-ground rock storage bin was also constructed. Two control buildings were also monitored, one for two years, and the second for only the second year. The measured values of electrical and gas consumption indicate that the modified solar building: (1) used less than 50% of the gas by the two-year control building, and (2) used 40% less gas than the one-year control building. Detailed mechanical design data are included, along with a development of the computer program used to predict actual modified and unmodified greenhouse performance. Good agreement was obtained between actual performance and the theoretical values predicted by the simulation. (LEW)

  14. Integrated structural model and membrane targeting mechanism of the human ESCRT-II complex

    PubMed Central

    Im, Young Jun; Hurley, James H.

    2008-01-01

    Summary ESCRT-II plays a pivotal role in receptor downregulation and multivesicular body biogenesis, and is conserved from yeast to humans. The crystal structures of two human ESCRT-II complex structures have been determined at 2.6 and 2.9 Å resolution, respectively. The complex has three lobes and contains one copy each of VPS22 and VPS36, and two copies of VPS25. The structure reveals a dynamic helical domain to which both the VPS22 and VPS36 subunits contribute, which connects the GLUE domain to the rest of the ESCRT-II core. Hydrodynamic analysis shows that intact ESCRT-II has a compact, closed conformation. ESCRT-II binds to the ESCRT-I VPS28 C-terminal domain subunit through a helix immediately C-terminal to the VPS36-GLUE domain. ESCRT-II is targeted to endosomal membranes by the lipid binding activities of both the Vps36 GLUE domain and the first helix of Vps22. These data provide a unifying structural and functional framework for the ESCRT-II complex. PMID:18539118

  15. Integrated structural model and membrane targeting mechanism of the human ESCRT-II complex

    SciTech Connect

    Im, Young Jun; Hurley, James H.

    2009-01-15

    ESCRT-II plays a pivotal role in receptor downregulation and multivesicular body biogenesis and is conserved from yeast to humans. The crystal structures of two human ESCRT-II complex structures have been determined at 2.6 and 2.9 {angstrom} resolution, respectively. The complex has three lobes and contains one copy each of VPS22 and VPS36 and two copies of VPS25. The structure reveals a dynamic helical domain to which both the VPS22 and VPS36 subunits contribute that connects the GLUE domain to the rest of the ESCRT-II core. Hydrodynamic analysis shows that intact ESCRT-II has a compact, closed conformation. ESCRT-II binds to the ESCRT-I VPS28 C-terminal domain subunit through a helix immediately C-terminal to the VPS36-GLUE domain. ESCRT-II is targeted to endosomal membranes by the lipid-binding activities of both the Vps36 GLUE domain and the first helix of Vps22. These data provide a unifying structural and functional framework for the ESCRT-II complex.

  16. Improvement of proton exchange membrane fuel cell overall efficiency by integrating heat-to-electricity conversion

    NASA Astrophysics Data System (ADS)

    Xie, Chungang; Wang, Shuxin; Zhang, Lianhong; Hu, S. Jack

    Proton exchange membrane fuel cells (PEMFCs) have shown to be well suited for distributed power generation due to their excellent performance. However, a PEMFC produces a considerable amount of heat in the process of electrochemical reaction. It is desirable to use thermal energy for electricity generation in addition to heating applications. Based on the operating characteristics of a PEMFC, an advanced thermal energy conversion system using "ocean thermal energy conversion" (OTEC) technology is applied to exploit the thermal energy of the PEMFC for electricity generation. Through this combination of technology, this unique PEMFC power plant not only achieves the combined heat and power efficiency, but also adequately utilizes heat to generate more valuable electricity. Exergy analysis illustrates the improvement of overall efficiency and energy flow distribution in the power plant. Analytical results show that the overall efficiency of the PEMFC is increased by 0.4-2.3% due to the thermal energy conversion (TEC) system. It is also evident that the PEMFC should operate within the optimal load range by balancing the design parameters of the PEMFC and of the TEC system.

  17. Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes.

    PubMed

    Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming

    2014-01-01

    Dimethyl sulfoxide (DMSO) is a polar organic solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacological and pathological effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species production, and subsequent cytochrome c release and caspase-3 activation. DMSO at concentrations of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO.

  18. Realization of efficient metal grating couplers for membrane-based integrated photonics.

    PubMed

    Higuera-Rodriguez, Aura; Dolores-Calzadilla, Victor; Jiao, Yuqing; Geluk, Erik J; Heiss, Dominik; Smit, Meint K

    2015-06-15

    Grating couplers are widely used to couple light between photonic integrated circuits and optical fibers. Here, we fabricate and characterize a device based on a buried metal grating. In contrast to dielectric gratings, simulations predict strongly reduced parasitic leakage of light to the substrate and are performance independent of the optical buffer thickness, while using standard fabrication processes with high yield. The gratings show a 3 dB bandwidth of 61 nm and chip-to-fiber coupling efficiency of 54%, which makes them attractive building blocks for on-wafer testing and dense optical interconnects.

  19. Human spermatozoa possess a calcium-dependent chloride channel that may participate in the acrosomal reaction

    PubMed Central

    Orta, Gerardo; Ferreira, Gonzalo; José, Omar; Treviño, Claudia L; Beltrán, Carmen; Darszon, Alberto

    2012-01-01

    Motility, maturation and the acrosome reaction (AR) are fundamental functions of mammalian spermatozoa. While travelling through the female reproductive tract, spermatozoa must mature through a process named capacitation, so that they can reach the egg and undergo the AR, an exocytotic event necessary to fertilize the egg. Though Cl− is important for sperm capacitation and for the AR, not much is known about the molecular identity of the Cl− transporters involved in these processes. We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa. Our whole cell recordings revealed the presence of a Ca2+-dependent Cl− current. The biophysical characteristics of this current and its sensitivity to niflumic acid (NFA) and 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid (DIDIS) are consistent with those displayed by the Ca2+-dependent Cl− channel from the anoctamin family (TMEM16). Whole cell patch clamp recordings in the cytoplasmic droplet of human spermatozoa corroborated the presence of these currents, which were sensitive to NFA and to a small molecule TMEM16A inhibitor (TMEM16Ainh, an aminophenylthiazole). Importantly, the human sperm AR induced by a recombinant human glycoprotein from the zona pellucida, rhZP3, displayed a similar sensitivity to NFA, DIDS and TMEM16Ainh as the sperm Ca2+-dependent Cl− currents. Our findings indicate the presence of Ca2+-dependent Cl− currents in human spermatozoa, that TMEM16A may contribute to these currents and also that sperm Ca2+-dependent Cl− currents may participate in the rhZP3-induced AR. PMID:22473777

  20. Intracellular sodium changes during the speract response and the acrosome reaction in sea urchin sperm

    PubMed Central

    Rodríguez, Esmeralda; Darszon, Alberto

    2003-01-01

    The sperm-activating peptide speract and fucose-sulphate glycoconjugate (FSG) are sea urchin egg-envelope components that modulate sperm ion permeability. They influence motility and induce acrosomal reaction (AR), respectively. A fluorescent Na+-sensitive dye (Na+-binding benzofuran isophthalate, SBFI) was used to determine how these egg envelope components influence sperm Na+ permeability. [Ca2+]i and pHi were also measured to correlate their changes in response to speract and FSG with those observed in [Na+]i. SBFI determinations indicate that the resting [Na+]i is 20 ± 8 mm in sea urchin sperm. Saturating levels of speract increased [Na+]i by ≈15 mm, while similar levels of FSG caused a further elevation of ≈30 mm. The kinetics of the [Na+]i, [Ca2+]i and pHi changes induced by saturating levels of speract were faster than those induced by FSG. Both egg ligands appeared to activate more than one Na+ transport system. Nifedipine, Ni2+ and TEA+ inhibited the ionic changes and the AR induced by FSG but, importantly, did not alter those caused by speract. Thus, there are differences in some of the ionic transport mechanisms that operate in the speract and FSG responses. ZD2788, a blocker of hyperpolarization and cyclic-nucleotide-gated (HCN) channels such as SpHCN present in sea urchin sperm, did not decrease the speract-induced [Na+]i increase, but slowed its kinetics. Therefore, SpHCN does not play a major role in the uptake of Na+ triggered by this decapeptide. KB-R7943, an inhibitor of Na+/Ca2+ exchangers, decreased the resting [Na+]i and did not change significantly the speract-induced [Ca2+]i increase, but slowed its recovery. PMID:12509481

  1. Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases

    PubMed Central

    Ickowicz, Debby; Finkelstein, Maya; Breitbart, Haim

    2012-01-01

    Mammalian sperm must undergo a series of biochemical and physiological modifications, collectively called capacitation, in the female reproductive tract prior to the acrosome reaction (AR). The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca2+ during both capacitation and the AR. In the present review, we summarize some of the signaling events that are involved in capacitation. During the capacitation process, phosphatidyl-inositol-3-kinase (PI3K) is phosphorylated/activated via a protein kinase A (PKA)-dependent cascade, and downregulated by protein kinase C α (PKCα). PKCα is active at the beginning of capacitation, resulting in PI3K inactivation. During capacitation, PKCα as well as PP1γ2 is degraded by a PKA-dependent mechanism, allowing the activation of PI3K. The activation of PKA during capacitation depends mainly on cyclic adenosine monophosphate (cAMP) produced by the bicarbonate-dependent soluble adenylyl cyclase. This activation of PKA leads to an increase in actin polymerization, an essential process for the development of hyperactivated motility, which is necessary for successful fertilization. Actin polymerization is mediated by PIP2 in two ways: first, PIP2 acts as a cofactor for phospholipase D (PLD) activation, and second, as a molecule that binds and inhibits actin-severing proteins such as gelsolin. Tyrosine phosphorylation of gelsolin during capacitation by Src family kinase (SFK) is also important for its inactivation. Prior to the AR, gelsolin is released from PIP2 and undergoes dephosphorylation/activation, resulting in fast F-actin depolymerization, leading to the AR. PMID:23001443

  2. Regulation of Sperm Capacitation and the Acrosome Reaction by PIP 2 and Actin Modulation.

    PubMed

    Breitbart, Haim; Finkelstein, Maya

    2015-01-01

    Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. Actin polymerization occurs during capacitation and prior to the acrosome reaction, fast F-actin breakdown takes place. The increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP 2 ) and its phosphorylation on tyrosine-438 by Src. Activation of gelsolin following its release from PIP 2 is known to cause F-actin breakdown and inhibition of sperm motility, which can be restored by adding PIP 2 to the cells. Reduction of PIP 2 synthesis inhibits actin polymerization and motility, while increasing PIP 2 synthesis enhances these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP 2 and F-actin. During capacitation there was an increase in PIP 2 and F-actin levels in the sperm head and a decrease in the tail. In spermatozoa with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends upon its binding to PIP 2 . Stimulation of phospholipase C, by Ca 2 + -ionophore or by activating the epidermal-growth-factor-receptor, inhibits tyrosine phosphorylation of gelsolin and enhances enzyme activity. In conclusion, these data indicate that the increase of PIP 2 and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result, the decrease of gelsolin in the tail allows the maintenance of high levels of F-actin in this structure, which is essential for the development of HA motility.

  3. Regulation of sperm capacitation and the acrosome reaction by PIP2 and actin modulation

    PubMed Central

    Breitbart, Haim; Finkelstein, Maya

    2015-01-01

    Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. Actin polymerization occurs during capacitation and prior to the acrosome reaction, fast F-actin breakdown takes place. The increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP2) and its phosphorylation on tyrosine-438 by Src. Activation of gelsolin following its release from PIP2 is known to cause F-actin breakdown and inhibition of sperm motility, which can be restored by adding PIP2 to the cells. Reduction of PIP2 synthesis inhibits actin polymerization and motility, while increasing PIP2 synthesis enhances these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP2 and F-actin. During capacitation there was an increase in PIP2 and F-actin levels in the sperm head and a decrease in the tail. In spermatozoa with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends upon its binding to PIP2. Stimulation of phospholipase C, by Ca2+-ionophore or by activating the epidermal-growth-factor-receptor, inhibits tyrosine phosphorylation of gelsolin and enhances enzyme activity. In conclusion, these data indicate that the increase of PIP2 and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result, the decrease of gelsolin in the tail allows the maintenance of high levels of F-actin in this structure, which is essential for the development of HA motility. PMID:25966627

  4. Beneficial phosphate recovery from reverse osmosis (RO) concentrate of an integrated membrane system using polymeric ligand exchanger (PLE).

    PubMed

    Kumar, Manish; Badruzzaman, Mohammad; Adham, Samer; Oppenheimer, Joan

    2007-05-01

    Phosphorus (P) discharge to surface water is a major environmental problem. Wastewater treatment is targeted towards removal of this nutrient to prevent degradation of surface water. Integrated membrane systems (IMS) are increasingly being considered for wastewater reclamation, and provide excellent removal of P compounds. However, reverse osmosis (RO), which forms an integral part of these IMSs, concentrates most dissolved substances including P-species such as phosphates in the RO waste stream. In this study, removal of phosphate from this stream using polymeric ligand exchange (PLE) resins was investigated. Further, the possibility of phosphate recovery through struvite (MgNH(4)PO(4).6H(2)O) precipitation was tested. Struvite has been promoted as a slow release fertilizer in recent years. This study demonstrates that PLEs can be successfully used to remove phosphate from RO-concentrate, and to recover more than 85% of the adsorbed phosphorus from the exhausted media and precipitated as a beneficial product (struvite). The approach, presented in this study, suggests advantages of providing economic benefit from a waste product (RO) while avoiding phosphorus discharge to the environment.

  5. Arsenic removal from contaminated groundwater by membrane-integrated hybrid plant: optimization and control using Visual Basic platform.

    PubMed

    Chakrabortty, S; Sen, M; Pal, P

    2014-03-01

    A simulation software (ARRPA) has been developed in Microsoft Visual Basic platform for optimization and control of a novel membrane-integrated arsenic separation plant in the backdrop of absence of such software. The user-friendly, menu-driven software is based on a dynamic linearized mathematical model, developed for the hybrid treatment scheme. The model captures the chemical kinetics in the pre-treating chemical reactor and the separation and transport phenomena involved in nanofiltration. The software has been validated through extensive experimental investigations. The agreement between the outputs from computer simulation program and the experimental findings are excellent and consistent under varying operating conditions reflecting high degree of accuracy and reliability of the software. High values of the overall correlation coefficient (R (2) = 0.989) and Willmott d-index (0.989) are indicators of the capability of the software in analyzing performance of the plant. The software permits pre-analysis, manipulation of input data, helps in optimization and exhibits performance of an integrated plant visually on a graphical platform. Performance analysis of the whole system as well as the individual units is possible using the tool. The software first of its kind in its domain and in the well-known Microsoft Excel environment is likely to be very useful in successful design, optimization and operation of an advanced hybrid treatment plant for removal of arsenic from contaminated groundwater.

  6. Integrated profiling of microRNA expression in membranous nephropathy using high-throughput sequencing technology.

    PubMed

    Chen, Wenbiao; Lin, Xiaocong; Huang, Jianrong; Tan, Kuibi; Chen, Yuyu; Peng, Wujian; Li, Wuxian; Dai, Yong

    2014-01-01

    The present study analyzed microRNA (miRNA) expression profiles in peripheral blood lymphocyte cells (PBLCs) from patients with membranous nephropathy (MN) and normal controls (NC), in an effort to improve the understanding of the pathogenesis of MN. High-throughput sequencing was performed on 30 MN patients and 30 healthy individuals (NC group). Known and novel miRNAs were analyzed and the results were confirmed by quantitative reverse transcription PCR (qRT-PCR). In total, 326 miRNAs showed a significant difference in expression between the MN and NC groups. This included 286 downregulated miRNAs and 40 upregulated miRNAs. In addition, there were 6 novel miRNAs that presented differential levels of expression between the MN and NC groups. The miRNAs were mapped to the genome, using a short oligonucleotide alignment program (SOAP), to analyze their expression and distribution. Twenty-five percent of the unique miRNAs in the MN group and 52.1% in the NC group were mapped to the genome. One hundred and eight mismatches were identified. Seventy-seven mismatches were detected in a higher proportion of the MN samples, compared with the NC samples. Twenty-five mismatches were detected in a higher proportion of the NC samples than the MN samples. Differential miRNA expression was also detected between 10 randomly selected pair groups, as depicted in a cluster analysis diagram. These data indicate that differential miRNA expression may be involved in the pathogenesis of MN. In addition, the discrepancies between the MN and NC groups, in the mismatched miRNAs that were mapped to the genome, strongly suggest that miRNAs play an important role in the pathogenesis of human disorders. miRNAs may provide a potential breakthrough in the research of MN and may provide a novel biomarker for the diagnosis and treatment of the disease.

  7. Neuronal activity causes rapid changes of lateral amygdala neuronal membrane properties and reduction of synaptic integration and synaptic plasticity in vivo

    PubMed Central

    Rosenkranz, J. Amiel

    2011-01-01

    Neuronal membrane properties dictate neuronal responsiveness. Plasticity of membrane properties alters neuronal function and can arise in response to robust neuronal activity. Despite the potential for great impact, there is little evidence for a rapid effect of activity-dependent changes of membrane properties on many neuronal functions in vivo in mammalian brain. In this study it was tested whether periods of neuronal firing lead to a rapid change of membrane properties in neurons of a rat brain region important for some forms of learning, the lateral nucleus of the amygdala (LAT), using in vivo intracellular recordings. Our results demonstrate that rapid plasticity of membrane properties occurs in vivo, in response to action potential firing. This plasticity of membrane properties leads to changes of synaptic integration and subsequent synaptic plasticity. These changes require Ca2+, but are NMDA independent. Furthermore, the parameters and timecourse of these changes would not have been predicted from most in vitro studies. The plasticity of membrane properties demonstrated here may represent a basic form of in vivo short-term plasticity that modifies neuronal function. PMID:21508236

  8. Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis

    PubMed Central

    Lone, Museer A.; Atkinson, Aaron E.; Hodge, Christine A.; Cottier, Stéphanie; Martínez-Montañés, Fernando; Maithel, Shelley; Mène-Saffrané, Laurent

    2015-01-01

    Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously—Brr6, which is closely related to Brl1, and Apq12—function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE. PMID:26432634

  9. A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

    PubMed

    Fernández, Silvina; Córdoba, Mariana

    2017-04-01

    Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC.

  10. Photothermal performance of an amorphous silicon photovoltaic panel integrated in a membrane structure

    NASA Astrophysics Data System (ADS)

    Zhao, Bing; Hu, Jianhui; Chen, Wujun; Qiu, Zhenyu; Zhou, Jinyu; Qu, Yegao; Ge, Binbin

    2016-10-01

    The amorphous silicon photovoltaic (a-Si PV) cells are widely used for electricity generation from solar energy. When the a-Si PV cells are integrated into building roofs, such as ETFE (ethylene-tetrafouoroethylene) cushions, the temperature characteristics are indispensible for evaluating the thermal performances of a-Si PV and its constructions. This temperature value is directly dependent on the solar irradiance, wind velocity, ambient temperature and installation form. This paper concerns the field experiments and numerical modeling on the temperature characteristics and temperature value of the a-Si PV integrated in a double-layer ETFE cushion structure. To this end, an experimental model composed of two a-Si PV cells and a double-layer ETFE cushion was developed, and the corresponding experiments were carried out under two typical weather conditions (summer sunny and summer cloudy). The theoretical thermal model was developed based on an energy balance equation taking the short wave radiation, long wave radiation, convection and generated power into account. The measured solar irradiance and air temperature were used as real weather conditions for the thermal model. The corresponding differential equation of the a-Si PV temperature varying with the solar irradiance and air temperature was solved by a newly developed program based on the numerical method. The measured results show that the influence of solar irradiance on the temperature is much more significant than the other parameters, and the maximum temperature variation under sunny conditions is greater than that under cloudy conditions. The comparative study between the experimental and numerical results shows the correct predictions of the a-Si PV temperature under the sunny and cloudy conditions. The maximum difference is 3.9 °C with the acceptable reasons of the solar irradiance fluctuation and the PV thermal response time. These findings will provide useful observations and explanations for

  11. Comprehensive Phylogenetic Analysis Sheds Light on the Diversity and Origin of the MLO Family of Integral Membrane Proteins

    PubMed Central

    Kusch, Stefan; Pesch, Lina; Panstruga, Ralph

    2016-01-01

    Mildew resistance Locus O (MLO) proteins are polytopic integral membrane proteins that have long been considered as plant-specific and being primarily involved in plant–powdery mildew interactions. However, research in the past decade has revealed that MLO proteins diverged into a family with several clades whose members are associated with different physiological processes. We provide a largely increased dataset of MLO amino acid sequences, comprising nearly all major land plant lineages. Based on this comprehensive dataset, we defined seven phylogenetic clades and reconstructed the likely evolution of the MLO family in embryophytes. We further identified several MLO peptide motifs that are either conserved in all MLO proteins or confined to one or several clades, supporting the notion that clade-specific diversification of MLO functions is associated with particular sequence motifs. In baker’s yeast, some of these motifs are functionally linked to transmembrane (TM) transport of organic molecules and ions. In addition, we attempted to define the evolutionary origin of the MLO family and found that MLO-like proteins with highly diverse membrane topologies are present in green algae, but also in the distinctly related red algae (Rhodophyta), Amoebozoa, and Chromalveolata. Finally, we discovered several instances of putative fusion events between MLO proteins and different kinds of proteins. Such Rosetta stone-type hybrid proteins might be instructive for future analysis of potential MLO functions. Our findings suggest that MLO is an ancient protein that possibly evolved in unicellular photosynthetic eukaryotes, and consolidated in land plants with a conserved topology, comprising seven TM domains and an intrinsically unstructured C-terminus. PMID:26893454

  12. Comprehensive Phylogenetic Analysis Sheds Light on the Diversity and Origin of the MLO Family of Integral Membrane Proteins.

    PubMed

    Kusch, Stefan; Pesch, Lina; Panstruga, Ralph

    2016-03-26

    Mildew resistanceLocusO(MLO) proteins are polytopic integral membrane proteins that have long been considered as plant-specific and being primarily involved in plant-powdery mildew interactions. However, research in the past decade has revealed that MLO proteins diverged into a family with several clades whose members are associated with different physiological processes. We provide a largely increased dataset of MLO amino acid sequences, comprising nearly all major land plant lineages. Based on this comprehensive dataset, we defined seven phylogenetic clades and reconstructed the likely evolution of the MLO family in embryophytes. We further identified several MLO peptide motifs that are either conserved in all MLO proteins or confined to one or several clades, supporting the notion that clade-specific diversification of MLO functions is associated with particular sequence motifs. In baker's yeast, some of these motifs are functionally linked to transmembrane (TM) transport of organic molecules and ions. In addition, we attempted to define the evolutionary origin of the MLO family and found that MLO-like proteins with highly diverse membrane topologies are present in green algae, but also in the distinctly related red algae (Rhodophyta), Amoebozoa, and Chromalveolata. Finally, we discovered several instances of putative fusion events between MLO proteins and different kinds of proteins. Such Rosetta stone-type hybrid proteins might be instructive for future analysis of potential MLO functions. Our findings suggest that MLO is an ancient protein that possibly evolved in unicellular photosynthetic eukaryotes, and consolidated in land plants with a conserved topology, comprising seven TM domains and an intrinsically unstructured C-terminus.

  13. Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status

    SciTech Connect

    Cummins, J.M.; Fleming, A.D.; Crozet, N.; Kuehl, T.J.; Kosower, N.S.; Yanagimachi, R.

    1986-03-01

    Living spermatozoa of seven mammalian species were treated with the thiol-alkylating fluorescent labelling compound, monobromobimane (MBBR). MB-labelling alone had no effect on sperm motility, nor on the time course or ability of golden hamster spermatozoa to undergo the acrosome reaction when capacitated in vitro. Exposure of MB-labelled spermatozoa to ultraviolet (UV) light and excitation of the MB fluorochrome resulted in virtually immediate immobilization of the spermatozoa without affecting acrosomal status. UV exposure of unlabelled spermatozoa for up to 30 sec had no effect upon motility. Immobilization of MB-labelled spermatozoa depended on the midpiece being irradiated, as irradiation of the head alone, or of the more distal parts of the principal piece, had little or no effect upon motility. Labelling with MB followed by immobilization of individually selected spermatozoa was most useful for detailing the course and site of occurrence of the acrosome reaction during penetration of the cumulus oophorus by golden hamster spermatozoa in vitro. In these often hyperactivated spermatozoa, precise determination of the acrosomal status could not often otherwise be made due to the difficulty in visualizing the acrosomal region of a vigorously thrashing, hyperactivated spermatozoon. This technique should prove valuable in a variety of studies on sperm motility, capacitation and fertilization, and could also be extended to other cell systems.

  14. Desmosterol, the main sterol in rabbit semen: distribution among semen subfractions and its role in the in vitro spermatozoa acrosome reaction and motility

    PubMed Central

    Mourvaki, Evangelia; Cardinali, Raffaella; Roberti, Rita; Dal Bosco, Alessandro; Castellini, Cesare

    2010-01-01

    Sterols are essential components of the cell membrane lipid bilayer that include molecules such as cholesterol and desmosterol, which are significantly found in the spermatozoa of several animal species. However, the presence of desmosterol in rabbit semen has never been investigated. The aims of this study were to characterize the sterol composition of subfractions of ejaculated rabbit semen and evaluate the in vitro effects of sterol on the spermatozoa acrosome reaction and motility. Two sterols, occurring prevalently in the free form (94.3%), were identified in whole semen collected from 10 fertile New Zealand White rabbits, specifically desmosterol (58.5% of total sterols) and cholesterol (35.9% of total sterols). Desmosterol was the predominant sterol found in all subfractions of rabbit semen, varying from 56.7% (in the prostatic secretory granules, PSGs) to 63.8% (in the seminal plasma). Spermatozoa contained an intermediate proportion of desmosterol (59.8%), which was asymmetrically distributed between the heads (52.0% of the total content of sterols) and the tails (81.8%). Results showed that both desmosterol and cholesterol can be transferred from the PSGs to the spermatozoa and are equally effective in inhibiting in vitro spermatozoa capacitation at a concentration higher than 1 mg L−1. In contrast, neither desmosterol nor cholesterol had a significant effect on spermatozoa motility. Thus, it was concluded that, the various fractions of rabbit seminal fluid differ from each other in sterol composition and quantity, probably due to their different functional properties, and these fractions may undergo significant sterol changes depending on the stage of spermatozoa capacitation. PMID:20729867

  15. Human sperm acrosome reaction-initiating activity associated with the human cumulus oophorus and mural granulosa cells.

    PubMed

    Siiteri, J E; Dandekar, P; Meizel, S

    1988-04-01

    This report describes the detection and partial characterization of preovulatory human cumulus oophorus and mural granulosa cell-associated activity capable of initiating the human sperm acrosome reaction (AR) in vitro. Fragments of preovulatory human cumulus (cells plus extracellular matrix) were washed 3 times, incubated for 24 hr and the spent media and washes assayed for their ability to initiate the human sperm acrosome reaction (AR) in vitro. AR activity was present in the first two washes but not the third wash; however, AR activity was recovered in the spent medium after 3 X-washed fragments were incubated for 24 hr under conditions which maintained the viability of the cumulus cells. The spent media of preovulatory human mural granulosa cells contained AR-initiating activity after 1-3, 3-6, and 6-9 days of culture. The properties of the AR activity present in spent media of human cumulus fragments included resistance to loss of activity during treatment with pronase; resistance to loss of activity during treatment with chondroitinase ABC or bacterial hyaluronidase; heat stability after overnight incubation; lack of extraction by chloroform-methanol; an apparent molecular weight (MW) of 50,000, as determined by Sephadex G-75 column chromatography; conversion to a lower apparent MW activity by incubation with pronase. These properties are also characteristic of a fraction derived by Sephadex G-75 chromatography of preovulatory human follicular fluid which also has been shown to stimulate the human sperm acrosome reaction in vitro. The AR activity from spent media of human mural granulosa cells is also found in a 50,000 MW Sephadex G-75 fraction. We propose that the sources of the 50,000 MW human follicular fluid AR activity are the cumulus oophorus and the mural granulosa cells.

  16. Porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Membrane modification of host subcellular compartments is critical to the replication of many RNA viruses. Enveloped viruses additionally require the ability to requisition cellular membranes during egress for the development of infectious progeny. Porcine reproductive and respiratory syndrome virus...

  17. Monolithic Flexible Supercapacitors Integrated into Single Sheets of Paper and Membrane via Vapor Printing.

    PubMed

    Liu, Andong; Kovacik, Peter; Peard, Nolan; Tian, Wenda; Goktas, Hilal; Lau, Jonathan; Dunn, Bruce; Gleason, Karen K

    2017-03-10

    A novel approach to fabricate supercapacitors (SCs) via vapor printing, specifically oxidative chemical vapor deposition (oCVD), is demonstrated. Compared to stacking multiple layers into a SC, this method enables the monolithic integration of all components into a single-sheet substrate, minimizing the inactive materials and eliminating the possibility of multilayer delamination. Electrodes comprised of pseudocapacitive material, poly(3,4-ethylenedioxythiophene) (PEDOT), are deposited into both sides of a sheet of flexible porous substrate. The film deposition and patterning are achieved in a single step. The oCVD PEDOT penetrates partially into the porous substrate from both surfaces, while leaving the interior of the substrate serving as a separator. Near the surface, the PEDOT coating conforms to the substrate's structure without blocking the pores, resembling the substrate's intrinsic morphology with high surface area. The porously structured PEDOT coating, paired with in situ ion gel electrolyte synthesis, gives enhanced electrode-electrolyte interfaces. The monolithic device demonstrates high volumetric capacitance (11.3 F cm(-3) ), energy density (2.98 mWh cm(-3) ), and power density (0.42 W cm(-3) ). These outstanding performance metrics are attributed to the large loading of active materials, minimization of inactive materials, and good electrode-electrolyte interfaces. SC arrays can be printed on a single substrate without the use of wire interconnects.

  18. Pig Spermatozoa Defect in Acrosome Formation Caused Poor Motion Parameters and Fertilization Failure through Artificial Insemination and In vitro Fertilization.

    PubMed

    Lee, Won Young; Lee, Ran; Kim, Hee Chan; Lee, Kyung Hoon; Cui, Xiang Shun; Kim, Nam Hyung; Kim, Sang Hyun; Lee, Il Joo; Uhm, Sang Jun; Yoon, Min Jung; Song, Hyuk

    2014-10-01

    The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not

  19. Fast Pyrolysis Oil Stabilization: An Integrated Catalytic and Membrane Approach for Improved Bio-oils. Final Report

    SciTech Connect

    George W. Huber; Upadhye, Aniruddha A.; Ford, David M.; Bhatia, Surita R.; Badger, Phillip C.

    2012-10-19

    This University of Massachusetts, Amherst project, "Fast Pyrolysis Oil Stabilization: An Integrated Catalytic and Membrane Approach for Improved Bio-oils" started on 1st February 2009 and finished on August 31st 2011. The project consisted following tasks: Task 1.0: Char Removal by Membrane Separation Technology The presence of char particles in the bio-oil causes problems in storage and end-use. Currently there is no well-established technology to remove char particles less than 10 micron in size. This study focused on the application of a liquid-phase microfiltration process to remove char particles from bio-oil down to slightly sub-micron levels. Tubular ceramic membranes of nominal pore sizes 0.5 and 0.8m were employed to carry out the microfiltration, which was conducted in the cross-flow mode at temperatures ranging from 38 to 45 C and at three different trans-membrane pressures varying from 1 to 3 bars. The results demonstrated the removal of the major quantity of char particles with a significant reduction in overall ash content of the bio-oil. The results clearly showed that the cake formation mechanism of fouling is predominant in this process. Task 2.0 Acid Removal by Membrane Separation Technology The feasibility of removing small organic acids from the aqueous fraction of fast pyrolysis bio-oils using nanofiltration (NF) and reverse osmosis (RO) membranes was studied. Experiments were carried out with a single solute solutions of acetic acid and glucose, binary solute solutions containing both acetic acid and glucose, and a model aqueous fraction of bio-oil (AFBO). Retention factors above 90% for glucose and below 0% for acetic acid were observed at feed pressures near 40 bar for single and binary solutions, so that their separation in the model AFBO was expected to be feasible. However, all of the membranes were irreversibly damaged when experiments were conducted with the model AFBO due to the presence of guaiacol in the feed solution. Experiments

  20. Integrating microbial fuel cells with anaerobic acidification and forward osmosis membrane for enhancing bio-electricity and water recovery from low-strength wastewater.

    PubMed

    Liu, Jinmeng; Wang, Xinhua; Wang, Zhiwei; Lu, Yuqin; Li, Xiufen; Ren, Yueping

    2017-03-01

    Microbial fuel cells (MFCs) and forward osmosis (FO) are two emerging technologies with great potential for energy-efficient wastewater treatment. In this study, anaerobic acidification and FO membrane were simultaneously integrated into an air-cathode MFC (AAFO-MFC) for enhancing bio-electricity and water recovery from low-strength wastewater. During a long-term operation of approximately 40 days, the AAFO-MFC system achieved a continuous and relatively stable power generation, and the maximum power density reached 4.38 W/m(3). The higher bio-electricity production in the AAFO-MFC system was mainly due to the accumulation of ethanol resulted from anaerobic acidification process and the rejection of FO membrane. In addition, a proper salinity environment in the system controlled by the addition of MF membrane enhanced the electricity production. Furthermore, the AAFO-MFC system produced a high quality effluent, with the removal rates of organic matters and total phosphorus of more than 97%. However, the nitrogen removal was limited for the lower rejection of FO membrane. The combined biofouling and inorganic fouling were responsible for the lower water flux of FO membrane, and the Desulfuromonas sp. utilized the ethanol for bio-electricity production was observed in the anode. These results substantially improve the prospects for simultaneous wastewater treatment and energy recovery, and further studies are needed to optimize the system integration and operating parameters.

  1. Integrated stereological and biochemical studies on hepatocytic membranes. I.V. Heterogeneous distribution of marker enzymes on endoplasmic reticulum membranes in fractions

    PubMed Central

    1980-01-01

    The purpose of the study was to consider quantitatively the relationships between the surface area of the endoplasmic reticulum (ER) and constituent marker enzyme activities, as they occur in fractions collected from rat liver homogenates. The ER surface area was estimated in five membrane-containing fractions by use of a combined cytochemical-stereological technique (5), while, at the same time, ER marker enzymes were assayed biochemically. Fraction/homogenate recoveries for the ER enzymes averaged 100%, total membrane surface area 98%, and ER surface area 96%. Relative specific activities, which compare the relative amounts of ER marker enzyme activities to the relative ER surface area in the membrane-containing fractions, indicate variable distributions for glucose-6-phosphatase and NADPH cytochrome c reductase, but not for esterase. PMID:6248565

  2. Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib.

    PubMed Central

    Fox, J E

    1985-01-01

    Experiments were performed to determine whether platelets contain a membrane skeleton. Platelets were labeled by a sodium periodate/sodium [3H]borohydride method and lysed with Triton X-100. Much of the filamentous actin could be sedimented at low g forces (15,600 g, 4 min), but some of the actin filaments required high-speed centrifugation for their sedimentation (100,000 g, 3 h). The latter filaments differed from those in the low-speed pellet in that they could not be depolymerized by Ca2+ and could not be sedimented at low g forces even from Triton X-100 lysates of platelets that had been activated with thrombin. Actin-binding protein sedimented with both types of filaments, but 3H-labeled membrane glycoproteins were recovered mainly with the high-speed filaments. The primary 3H-labeled glycoprotein recovered with this "membrane skeleton" was glycoprotein (GP) Ib. Approximately 70% of the platelet GP Ib was present in this skeleton. Several other minor glycoproteins, including greater than 50% of the GP Ia and small amounts of three unidentified glycoproteins of Mr greater than 200,000, were also recovered with the membrane skeleton. The Triton X-100 insolubility of GP Ib, GP Ia, a minor membrane glycoprotein of 250,000 Mr, and actin-binding protein resulted from their association with actin filaments as they were rendered Triton X-100-soluble when actin filaments were depolymerized with deoxyribonuclease I and co-isolated with actin filaments on sucrose gradients. When isolated platelet plasma membranes were extracted with Triton X-100, actin, actin-binding protein, and GP Ib were recovered as the Triton X-100 residue. These studies show that unstimulated platelets contain a membrane skeleton composed of actin filaments and actin-binding protein that is distinct from the rest of the cytoskeleton and is attached to GP Ib, GP Ia, and a minor glycoprotein of 250,000 Mr on the plasma membrane. Images PMID:2932470

  3. Maintenance of membrane integrity and increase of taxanes production in hazel (Corylus avellana L.) cells induced by low-intensity ultrasound.

    PubMed

    Safari, Masoumeh; Ghanati, Faezeh; Hajnoruzi, Abazar; Rezaei, Ayatollah; Abdolmaleki, Parviz; Mokhtari-Dizaji, Manigeh

    2012-06-01

    Suspension-cultured hazel cells were ultrasonicated at power densities of 4 and 455 mW for 4-40 min. Both treatments stimulated the production of major taxanes: Taxol, 10-deacetylbaccatin, and baccatin III. The highest amounts of these taxanes (0.46, 0.26, and 0.07 mg/l, respectively) were obtained at 8 and 20 min of the treatment at 455 mW. Ultrasound had no adverse effects on cell viability, growth, or membrane integrity. Increased release of taxanes by ultrasound resulted not from increased membrane permeability but more likely from stimulation of taxanes biosynthesis.

  4. Analysis of the control structures for an integrated ethanol processor for proton exchange membrane fuel cell systems

    NASA Astrophysics Data System (ADS)

    Biset, S.; Nieto Deglioumini, L.; Basualdo, M.; Garcia, V. M.; Serra, M.

    The aim of this work is to investigate which would be a good preliminary plantwide control structure for the process of Hydrogen production from bioethanol to be used in a proton exchange membrane (PEM) accounting only steady-state information. The objective is to keep the process under optimal operation point, that is doing energy integration to achieve the maximum efficiency. Ethanol, produced from renewable feedstocks, feeds a fuel processor investigated for steam reforming, followed by high- and low-temperature shift reactors and preferential oxidation, which are coupled to a polymeric fuel cell. Applying steady-state simulation techniques and using thermodynamic models the performance of the complete system with two different control structures have been evaluated for the most typical perturbations. A sensitivity analysis for the key process variables together with the rigorous operability requirements for the fuel cell are taking into account for defining acceptable plantwide control structure. This is the first work showing an alternative control structure applied to this kind of process.

  5. Effect of staining and sorting on boar sperm membrane integrity, mitochondrial activity and in vitro blastocyst development.

    PubMed

    Spinaci, M; De Ambrogi, M; Volpe, S; Galeati, G; Tamanini, C; Seren, E

    2005-07-01

    The objective of this study was to determine the effects of staining with Hoechst 33342 and of the entire sorting procedure on boar sperm membrane integrity (using Annexin-V/PI), mitochondrial activity (using JC-1/SYBR/PI) and blastocyst development in vitro; the effect of storage at 17 degrees C for 24h prior to Hoechst staining and sorting was also investigated. The Hoechst staining and the whole sorting procedure reduced the percent of live spermatozoa in both fresh (day 0) and stored (day 1) semen, as determined by both assays; nevertheless, there was no increase in live sperm cells showing signs of early damage (Annexin-V positive, propidium negative), whose percentages remained nearly zero. The majority of Annexin-V positive cells were propidium positive, therefore dead. JC-1 staining evidenced a correlation between mitochondrial activity and viability. However, a significant difference between viable sperm cells and sperm cells with active mitochondria was detected in control and stained sperm, whereas almost all viable sorted spermatozoa had active mitochondria. No significant differences in the in vitro produced blastocysts both on day 0 and 1 were observed. In conclusion, despite the damages induced by sorting procedures, semen sorted as fresh or after storage at 17 degrees C can be successfully used for in vitro production of pig embryos.

  6. Solution NMR Structure and Functional Analysis of the Integral Membrane Protein YgaP from Escherichia coli

    PubMed Central

    Eichmann, Cédric; Tzitzilonis, Christos; Bordignon, Enrica; Maslennikov, Innokentiy; Choe, Senyon; Riek, Roland

    2014-01-01

    The solution NMR structure of the α-helical integral membrane protein YgaP from Escherichia coli in mixed 1,2-diheptanoyl-sn-glycerol-3-phosphocholine/1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) micelles is presented. In these micelles, YgaP forms a homodimer with the two transmembrane helices being the dimer i