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Sample records for acrosomal membrane integrity

  1. Silymarin protects plasma membrane and acrosome integrity in sperm treated with sodium arsenite

    PubMed Central

    Eskandari, Farzaneh; Momeni, Hamid Reza

    2016-01-01

    Background: Exposure to arsenic is associated with impairment of male reproductive function by inducing oxidative stress. Silymarin with an antioxidant property scavenges free radicals. Objective: The aim of this study was to investigate if silymarin can prevent the adverse effects of sodium arsenite on ram sperm plasma membrane and acrosome integrity. Materials and Methods: Ram epidydimal spermatozoa were divided into five groups: spermatozoa at 0 hr, spermatozoa at 180 min (control), spermatozoa treated with silymarin (20 μM) + sodium arsenite (10 μM) for 180 min, spermatozoa treated with sodium arsenite (10 μM) for 180 min and spermatozoa treated with silymarin (20 μM) for 180 min. Double staining of Hoechst and propidium iodide was performed to evaluate sperm plasma membrane integrity, whereas comassie brilliant blue staining was used to assess acrosome integrity. Results: Plasma membrane (p< 0.001) and acrosome integrity (p< 0.05) of the spermatozoa were significantly reduced in sodium arsenite group compared to the control. In silymarin + sodium arsenite group, silymarin was able to significantly (p< 0.001) ameliorate the adverse effects of sodium arsenite on these sperm parameters compared to sodium arsenite group. The incubation of sperm for 180 min (control group) showed a significant (p< 0.001) decrease in acrosome integrity compared to the spermatozoa at 0 hour. The application of silymarin alone for 180 min could also significantly (p< 0.05) increase sperm acrosome integrity compared to the control. Conclusion: Silymarin as a potent antioxidant could compensate the adverse effects of sodium arsenite on the ram sperm plasma membrane and acrosome integrity. PMID:27141548

  2. 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

  3. [Inhibitors of acrosomal proteinase as antifertility agents. A problem of acrosomal membrane permeability (author's transl)].

    PubMed

    Schill, W B; Feifel, M; Fritz, H; Hammerstein, J

    1982-01-01

    In vitro studies were performed to investigate the accessibility of acrosin to various proteinase inhibitors inside the intact acrosome of testicular, ejaculated, and uterine human spermatozoa. As test system, the gelatin plate assay was used. For this assay, it was shown formerly that a correlation exists between the size of the digested lysis areas (halo formation) and acrosin activity estimated with synthetic substrates. In addition, saturation of the gelatin substrate membranes with acrosin inhibitors including highly specific ones before application of spermatozoa completely prevented halo formation indicating that the gelatinolytic activity of human spermatozoa is caused exclusively by acrosin. When human spermatozoa were incubated with various acrosin inhibitors (concentration: 1 mmol/1) prior to application to the gelatine membrane, reduction of halo formation could not be observed, however. This result indicates that most of the tested acrosin inhibitors (9 naturally occurring protein inhibitors, 2 microbial peptide inhibitors, 19 synthetic inhibitors) were unable to penetrate the acrosomal membranes of testicular, ejaculated, and uterine human spermatozoa. Only 2 inhibitors caused moderate to complete inhibition of the gelatinolytic activity of the spermatozoa if applied in concentrations between 1-10 mmol/l: the proteinase inhibitor aprotinin and the synthetic inhibitor NPGB (4-nitrophenyl 4-guanidinobenzoate). Obviously, human acrosomal membranes seem to be especially impenetrable to proteins, polypeptides, and synthetic agents. Those acrosin inhibitors penetrating the human sperm head membranes are either too toxic or the local concentration necessary for effective acrosin inhibition in vivo cannot be achieved within the male or female genital tract secretions. Therefore, acrosin inhitibors cannot be used for human contraception at present. Thus, it is mandatory to continue the search for suitable acrosin inhibitors with low toxicity easily penetrating

  4. 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.

  5. Specific strychnine binding sites on acrosome-associated membranes of golden hamster spermatozoa.

    PubMed

    Llanos, Miguel N; Ronco, Ana M; Aguirre, María C

    2003-06-27

    This study demonstrates for the first time, that membrane vesicles originated from the hamster sperm head after the occurrence of the acrosome reaction, possess specific strychnine binding sites. [3H]Strychnine binding was saturable and reversible, being displaced by unlabeled strychnine (IC(50)=26.7+/-2.3 microM). Kinetic analysis revealed one binding site with K(d)=120nM and B(max)=142fmol/10(6) spermatozoa. Glycine receptor agonists beta-alanine and taurine inhibited strychnine binding by 20-30%. Surprisingly, glycine stimulated binding by about 40-50%. Results obtained in this study strongly suggest the presence of glycine receptors-with distinctive kinetic properties on the periacrosomal plasma membrane of hamster spermatozoa. Localization of this receptor fits well with its previously proposed role in acrosomal exocytosis during mammalian fertilization.

  6. 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. PMID:14086133

  7. 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.

  8. Nanovid microscopy for assessing sperm membrane changes induced by in vitro capacitating and acrosomal reacting procedures.

    PubMed

    Degelos, S D; Wilson, M P; Chandler, J E

    1994-01-01

    This study was to verify the usefulness of Nanovid microscopy techniques for evaluating induced modifications in bovine spermatozoal membranes. Frozen thawed bovine sperm were labeled with 20-nm colloidal gold particles bound to concanavalin A (ConA) or heparin ligands. Sperm membrane changes were induced in vitro by capacitating and acrosome-reacting procedures. Capacitation was induced by incubation with 10 micrograms/ml of heparin for 4 hours at 37 degrees C, 5% CO2, and high humidity. Membrane changes associated with the acrosome reaction were induced by addition of lysophosphatidylcholine (100 micrograms/ml) and incubation for 15 minutes at 37 degrees C, 5% CO2, and high humidity. Gray intensity (black = 0; white = 255) of sperm (ONCELL) and background (OFFCELL) were evaluated with computer-enhanced videomicroscopy with either differential interference contrast (DIC) or Nanovid optics. A high gold concentration on a membrane region produced blacker video pictures with Nanovid microscopy. Gray intensity of video pictures of a region with little or no gold would have a gray intensity equal to or greater than that of the background, that is, toward white. Weighted least squares methods were used to analyze ONCELL data using OFFCELL as a covariate. In experiment 1, ONCELL intensities of cells labeled with ConA-gold complex were lower than those labeled with heparin-gold at the same treatment level. In experiment 2, ONCELL intensity decreased as the concentration of heparin-gold increased from 0 to 0.041 microgram/microliter heparin. ONCELL intensity significantly decreased after sperm were treated with the highest heparin-gold level and acrosome reacted.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. 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.

  10. Lipid Regulation of Acrosome Exocytosis.

    PubMed

    Cohen, Roy; Mukai, Chinatsu; Travis, Alexander J

    2016-01-01

    Lipids are critical regulators of mammalian sperm function, first helping prevent premature acrosome exocytosis, then enabling sperm to become competent to fertilize at the right place/time through the process of capacitation, and ultimately triggering acrosome exocytosis. Yet because they do not fit neatly into the "DNA--RNA-protein" synthetic pathway, they are understudied and poorly understood. Here, we focus on three lipids or lipid classes-cholesterol, phospholipids, and the ganglioside G(M1)--in context of the modern paradigm of acrosome exocytosis. We describe how these various- species are precisely segregated into membrane macrodomains and microdomains, simultaneously preventing premature exocytosis while acting as foci for organizing regulatory and effector molecules that will enable exocytosis. Although the mechanisms responsible for these domains are poorly defined, there is substantial evidence for their composition and functions. We present diverse ways that lipids and lipid modifications regulate capacitation and acrosome exocytosis, describing in more detail how removal of cholesterol plays a master regulatory role in enabling exocytosis through at least two complementary pathways. First, cholesterol efflux leads to proteolytic activation of phospholipase B, which cleaves both phospholipid tails. The resultant changes in membrane curvature provide a mechanism for the point fusions now known to occur far before a sperm physically interacts with the zona pellucida. Cholesterol efflux also enables G(M1) to regulate the voltage-dependent cation channel, Ca(V)2.3, triggering focal calcium transients required for acrosome exocytosis in response to subsequent whole-cell calcium rises. We close with a model integrating functions for lipids in regulating acrosome exocytosis. PMID:27194352

  11. 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.

  12. The Acrosome Reaction: A Historical Perspective.

    PubMed

    Okabe, Masaru

    2016-01-01

    Acrosome reaction is often referred to as acrosomal exocytosis, but it differs significantly from normal exocytosis. While the vesicle membrane initially holding excreting molecules remains on the cell surface during exocytosis, the outer acrosomal membrane and plasma membrane are lost by forming vesicles during acrosome reaction. In this context, the latter process resembles a release of exosome. However, recent experimental data indicate that the most important roles of acrosome reaction lie not in the release of acrosomal contents (or "vesiculated" plasma and outer acrosomal membrane complexes) but rather in changes in sperm membrane. This review describes the mechanism of fertilization vis-a-vis sperm membrane change, with a brief historical overview of the half-century study of acrosome reaction. PMID:27194347

  13. Effects of hypothermic liquid storage and cryopreservation on basal and induced plasma membrane phospholipid disorder and acrosome exocytosis in boar spermatozoa.

    PubMed

    Guthrie, H D; Welch, G R

    2005-01-01

    Flow cytometry was utilised to determine whether short-term (Day 1) or long-term hypothermic liquid storage (Day 5), or cryopreservation of boar spermatozoa (1) caused changes in plasma membrane phospholipid disorder (MPLD) and acrosome exocytosis (AE), indicative of an advanced stage of capacitation or acrosome status, and (2) facilitated or inhibited the induction of capacitation and the acrosome reaction. Merocyanine with Yo-Pro-1 and peanut agglutinin-fluorescein isothiocyanate with propidium iodide were used to identify MPLD and AE, respectively, in viable spermatozoa. The incidence of basal sperm MPLD and AE in fresh semen was very low (1.1 and 2.2%, respectively) and was increased (P < 0.05) only a small amount in Day 5 and cryopreserved semen (3-8%). Compared to no bicarbonate, incubation with bicarbonate increased MPLD, but the response was greatest (P < 0.05) in fresh sperm (52.3%) compared with Day 1 (36.6%), Day 5 (13.9%) and cryopreserved sperm (13.6%). Incubation with calcium ionophore A23187 increased AE in spermatozoa, but the response was less (P < 0.05) for fresh (34%) and cryopreserved (27%) semen than for Day 1 (45%) and Day 5 (57%) semen. In summary, hypothermic liquid storage and cryopreservation of boar spermatozoa did not advance capacitation or acrosome status in viable spermatozoa, but did alter their responses to induction of capacitation and the acrosome reaction. PMID:15899159

  14. 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

  15. The Acrosomal Matrix.

    PubMed

    Foster, James A; Gerton, George L

    2016-01-01

    The acrosome, a single exocytotic vesicle on the head of sperm, has an essential role in fertilization, but the exact mechanisms by which it facilitates sperm-egg interactions remain unresolved. The acrosome contains dozens of secretory proteins that are packaged into the forming structure during spermatogenesis; many of these proteins are localized into specific topographical areas of the acrosome, while others are more diffusely distributed. Acrosomal proteins can also be biochemically classified as components of the acrosomal matrix, a large, relatively insoluble complex, or as soluble proteins. This review focuses on recent findings using genetically modified mice (gene knockouts and transgenic "green acrosome" mice) to study the effects of eliminating acrosomal matrix-associated proteins on sperm structure and function. Some gene knockouts produce infertile phenotypes with obviously missing, specific activities that affect acrosome biogenesis during spermatogenesis or interfere with acrosome function in mature sperm. Mutations that delete some components produce fertile phenotypes with subtler effects that provide useful insights into acrosomal matrix function in fertilization. In general, these studies enable the reassessment of paradigms to explain acrosome formation and function and provide novel, objective insights into the roles of acrosomal matrix proteins in fertilization. The use of genetically engineered mouse models has yielded new mechanistic information that complements recent, important in vivo imaging studies. PMID:27194348

  16. 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.

  17. The acrosome of eutherian mammals.

    PubMed

    Fléchon, Jacques-Edmond

    2016-01-01

    The acrosome is not just a bag of enzymes, most of which, if not all, are singly non-essential for sperm-oocyte interaction. The Golgi-derived acrosomal cap reveals some extraordinary development and structure particularities. The acrosome of eutherian spermatozoa basically consists of two parts, the anterior and equatorial segments; the present review is devoted to the former, the initial actor in fertilization. Its occasional fanciful morphological changes during epididymal maturation are analyzed, together with its heterogeneous contents: enzymes, zona binding proteins, structural proteins (matrix) and yet to be chemically characterized crystalloids. The plasma and acrosomal membranes present stabilized ordered domains, whereas glycoprotein-free areas appear during capacitation and before fusion. Exocytosis, induced by the cumulus oophorus and/or the zona pellucida, may generally start proximally and progress anteriorly, resulting in the detachment of a hybrid membrane shroud, whose entity is probably maintained by the bound matrix. Immediately released soluble enzymes must be active during the first interactions of the gametes, whereas other lysins, bound to the matrix or stored as proenzymes, are only progressively released. Zona binding is probably achieved via the shroud and/or the IAM (depending on species). Penetration along an incurved slit through the stratified zona is allowed by the rigid and denuded head tip and flagellar hyperactivity, and assisted by the local proteolytic activity of proteasomes bound to the IAM, the unique essential zona lysin system.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. Acrosomal Swelling Is Triggered by cAMP Downstream of the Opening of Store-Operated Calcium Channels During Acrosomal Exocytosis in Human Sperm.

    PubMed

    Sosa, Claudia M; Zanetti, M Natalia; Pocognoni, Cristian A; Mayorga, Luis S

    2016-03-01

    Acrosomal exocytosis in mammalian sperm is a regulated secretion with unusual characteristics. One of its most striking features is the postfusion loss of the outer acrosomal membrane and the overlying plasma membrane as hybrid vesicles. We have previously reported in human sperm that, by preventing the release of calcium from the acrosome, the exocytic process can be arrested at a stage where the acrosomes are profusely swollen, with invaginations of the outer acrosomal membrane. In this report, we show by transmission electron microcopy swelling with similar characteristics without arresting the exocytic process. Acrosomal swelling was observed when secretion was promoted by pharmacological and physiological inducers of the acrosome reaction that trigger exocytosis by different mechanisms. We show that progesterone- and thapsigargin-induced swelling depended on a calcium influx from the extracellular medium through store-operated calcium channels. However, calcium was dispensable when sperm were stimulated with cAMP analogs. KH7, an inhibitor of the soluble adenylyl cyclase, blocked progesterone-induced swelling. Our results indicate that swelling is a required process for acrosomal exocytosis triggered by activation of an adenylyl cyclase downstream of the opening of store-operated calcium channels.

  3. Acrosome Reaction as a Preparation for Gamete Fusion.

    PubMed

    Cuasnicú, Patricia S; Da Ros, Vanina G; Weigel Muñoz, Mariana; Cohen, Débora J

    2016-01-01

    The acrosome reaction (AR) is a universal requisite for sperm-egg fusion. However, whereas through the animal kingdom fusion of spermatozoa with the egg plasma membrane occurs via the inner acrosomal membrane exposed after the AR, in eutherian mammals, gamete fusion takes place through a specialized region of the acrosome known as the equatorial segment (ES) which becomes fusogenic only after the AR is completed. This chapter focuses on the different molecular mechanisms involved in the acquisition of the fusogenicity of the ES after the AR. We provide an update of the knowledge about the proteins proposed to have a role in this process either by modifying cytoskeletal and/or membrane molecules or by relocalizing to the ES after the AR to subsequently participate in gamete fusion. PMID:27194355

  4. Sperm surface changes during the acrosome reaction as observed by freeze-fracture.

    PubMed

    Fléchon, J E

    1985-11-01

    The mammalian acrosome reaction is an exocytotic process that can be analyzed by the technique of freeze-fracture; only sperm cells capacitated in vitro or treated to elicit the acrosome reaction in vitro have been studied, and all pictures published are from material fixed before freezing. All the authors point out the appearance of particle-free areas in the plasma membrane of the acrosomal region during capacitation and before any fusion. This is interpreted as an increase in membrane fluidity as suggested by studies on membrane lipid composition in guinea-pig sperm. We have recently described the induced acrosome reaction in ram spermatozoa. Fusion starts at the limit of the anterior and equatorial segments and progresses forward in the anterior segment along ramified paths, resulting in a fenestration gradient of the acrosomal cap. Fusion propagation may be controlled by fluidity increase in the plasma membrane of the anterior segment, and it is probably inhibited in the equatorial segment by the ordered structure of the acrosomal membrane.

  5. Cytochemical characterization of glycoproteins in the developing acrosome of rats. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation.

    PubMed

    Martínez-Menárguez, J A; Ballesta, J; Avilés, M; Castells, M T; Madrid, J F

    1992-01-01

    The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with beta-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane. PMID:1500300

  6. Integral Membrane Proteins and Bilayer Proteomics

    PubMed Central

    Whitelegge, Julian P.

    2013-01-01

    Integral membrane proteins reside within the bilayer membranes that surround cells and organelles, playing critical roles in movement of molecules across them and the transduction of energy and signals. While their extreme amphipathicity presents technical challenges, biological mass spectrometry has been applied to all aspects of membrane protein chemistry and biology, including analysis of primary, secondary, tertiary and quaternary structure, as well as the dynamics that accompany functional cycles and catalysis. PMID:23301778

  7. 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. PMID:26744028

  8. 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.

  9. Membrane Integration of Poliovirus 2B Viroporin▿

    PubMed Central

    Martínez-Gil, Luis; Bañó-Polo, Manuel; Redondo, Natalia; Sánchez-Martínez, Silvia; Nieva, José Luis; Carrasco, Luis; Mingarro, Ismael

    2011-01-01

    Virus infections can result in a variety of cellular injuries, and these often involve the permeabilization of host membranes by viral proteins of the viroporin family. Prototypical viroporin 2B is responsible for the alterations in host cell membrane permeability that take place in enterovirus-infected cells. 2B protein can be localized at the endoplasmic reticulum (ER) and the Golgi complex, inducing membrane remodeling and the blockade of glycoprotein trafficking. These findings suggest that 2B has the potential to integrate into the ER membrane, but specific information regarding its biogenesis and mechanism of membrane insertion is lacking. Here, we report experimental results of in vitro translation-glycosylation compatible with the translocon-mediated insertion of the 2B product into the ER membrane as a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. A similar topology was found when 2B was synthesized in cultured cells. In addition, the in vitro translation of several truncated versions of the 2B protein suggests that the two hydrophobic regions cooperate to insert into the ER-derived microsomal membranes. PMID:21835803

  10. Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa.

    PubMed

    Prapaiwan, N; Tharasanit, T; Punjachaipornpol, S; Yamtang, D; Roongsitthichai, A; Moonarmart, W; Kaeoket, K; Manee-In, S

    2016-05-01

    Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa. PMID:26954170

  11. A 33 kDa molecular marker of sperm acrosome differentiation and maturation in the tammar wallaby (Macropus eugenii).

    PubMed

    Wade, M A; Lin, M

    1999-09-01

    This study was undertaken to identify potential molecular markers of acrosomal biogenesis and post-testicular maturation in marsupials, using the tammar wallaby as a model species. A two-step sperm extraction procedure yielded two protein extracts of apparent acrosomal origin and a tail extract. The extracts were analysed by SDS-PAGE under reducing conditions. Several prominent polypeptide bands (45, 38 and 33 kDa) appeared common to both acrosomal extracts. Antiserum raised against the 33 kDa polypeptide from the inner acrosomal membrane matrix (IAMM) extract showed immunoreactivity with 45, 38 and 33 kDa polypeptides in both acrosomal extracts, indicating that the 33 kDa polypeptide was related to the proteins in the 45 and 38 kDa bands. Therefore, the antiserum was used as a molecular probe. Indirect immuno-fluorescence indicated that the acrosome was the major location of the 33 kDa polypeptide. This contention was confirmed by ultrastructural study: immunogold labelling indicated that the 33 kDa polypeptide associated with acrosomal matrix components throughout acrosomal development in the testes and throughout post-testicular maturation in the epididymis. The label clearly delineated the changing morphology of the maturing marsupial acrosome. This is the first study to use immunocytochemical techniques to chart testicular and post-testicular development of any sperm organelle in a marsupial. As a result of this study, a 33 kDa molecular marker of marsupial acrosome differentiation and maturation has been identified. It may be possible to chart similar events in other marsupial species and identify opportunities for manipulating fertility. PMID:10645248

  12. 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.

  13. Fertilization in a chiton: acrosome-mediated sperm-egg fusion.

    PubMed

    Buckland-Nicks, J; Koss, R; Chia, F S

    1988-11-01

    Contrary to the widely accepted view that chiton sperm lack acrosomes and that fertilization in this group occurs via a micropyle, we demonstrate here that fertilization in Tonicella lineata occurs by acrosome-mediated sperm-egg fusion. The acrosome is a small vesicle containing two granules located at the tip of the sperm. The eggs have an elaborate hull (= chorion), which is formed into cupules that remain covered by follicle cells until maturity. When dissected ripe eggs were exposed to sperm in vitro, the sperm were attracted only to open cupules, inside which they swam through one of seven channels to the base where they penetrated the hull. The acrosome fired on contact with, or in, the hull, and during passage through it the apical granule was exhausted while the basal granule was exposed. If sperm contacted follicle cells between the cupules the acrosome did not react. The vitelline layer beneath the hull contains pores arranged in a regular pattern. Embedded in the base of each pore is an egg microvillus. Having penetrated the hull the sperm anterior filament located a pore and fused with the tip of the egg microvillus projecting into it. This created a membranous tube, through which the sperm nucleus was injected into the egg. The egg membrane appeared to be raised up into a small fertilization cone around the penetrating sperm, the vitelline layer became slightly elevated, and some cortical granules were released by exocytosis.

  14. Glycopolymer induction of mouse sperm acrosomal exocytosis shows highly cooperative self-antagonism.

    PubMed

    Rodolis, Maria T; Huang, He; Sampson, Nicole S

    2016-06-01

    Identifying inducers of sperm acrosomal exocytosis (AE) to understand sperm functionality is important for both mechanistic and clinical studies in mammalian fertilization. Epifluorescence microscopy methods, while reproducible, are laborious and incompatible for high throughput screening. Flow cytometry methods are ideal for quantitative measurements on large numbers of samples, yet typically rely on the use of lectins that can interfere with physiologic AE-inducers. Here, we present an optimized triple stain flow cytometric method that is suitable for high-throughput screening of AE activation by glycopolymers. SYTO-17 and propidium iodide (PI) were used to differentiate cells based on their membrane integrity or viability, and membrane impermeable soybean trypsin inhibitor (SBTI) was used to monitor acrosome exocytosis. The SBTI/PI/SYTO-17 combination provides a positive screen for viability and AE of live sperm cells with minimal noise or false positives. A scattering gate enables the use of samples that may be contaminated with non-cellular aggregates, e.g., cryopreservation agents. This assay format enabled detailed analysis of glycopolymer dose response curves. We found that fucose polymer has a narrow effective dose range (EC50 = 1.6 μM; IC50 = 13.5 μM); whereas mannose polymer and β-N-acetylglucosamine polymer have broader effective dose ranges (EC50 = 1.2 μM and 3.4 μM, respectively). These results highlight the importance of testing inducers over a large concentration range in small increments for accurate comparison. PMID:27150629

  15. 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

  16. Site of Mammalian Sperm Acrosome Reaction.

    PubMed

    Hirohashi, Noritaka

    2016-01-01

    Until recently, no special attention has been paid to the question of the site of mammalian sperm acrosome reaction (AR) in the female reproductive tract. Because AR is an essential process that enables the spermatozoon to fertilize, it is generally believed that it occurs at a specific step during sperm-egg interaction. It is generally thought that "the site of action coincides with the site of commitment." Thus, understanding the roles of AR and acrosomal substances is needed to gain insight into the site of the sperm commitment to undergo AR. PMID:27194354

  17. 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

  18. 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.

  19. Effects of taurine and hypotaurine supplementation and ionophore concentrations on post-thaw acrosome reaction of dog spermatozoa.

    PubMed

    Martins-Bessa, A; Rocha, A; Mayenco-Aguirre, A

    2009-01-15

    The aim of this work was to study of the effect of the amino acids (AA) taurine (T) and hypotaurine (H) and of different calcium ionophore concentrations on the ability of capacitated frozen-thawed dog sperm to undergo the acrosome reaction (AR). Fifteen ejaculates grouped into five pools were used. Sperm was frozen at a concentration of 80x10(6)sperm cells/mL in the Uppsala Equex extender (UE) supplemented with 25, 50 and 75mM of either AA. The UE extender without T or H was used as control. After thawing, sperm was capacitated with Canine Capacitation Medium for 20min. Sperm was then challenged with calcium ionophore A23178 at 0, 2.5 and 10microM concentration and evaluated for integrity of plasma and acrosome membranes after 5, 15 and 30min of incubation, utilizing PI/Fitc-PNA fluorescent staining and flow cytometry. Sperm cryopreserved in UE supplemented with 50mM T (UE 50T) had higher AR rates than sperm cryopreserved with UE 75T, UE 25H and UE 50H, but AR rates were similar to semen frozen with the control extender. Challenges with 2.5 and 10microM/L of calcium ionophore increased AR in frozen-thawed sperm incubated for 5, 15 and 30min. The combination of calcium ionophore concentration and incubation time resulting in the highest AR rate was 10microM and 15min.

  20. 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.

  1. Regulatory mechanisms of the acrosome reaction revealed by multiview microscopy of single starfish sperm

    PubMed Central

    1995-01-01

    The acrosome reaction in many animals is a coupled reaction involving an exocytotic step and a dramatic change in cell shape. It has been proposed that these morphological changes are regulated by intracellular ions such as Ca2+ and H+. We report here simultaneous visualization, under a multiview microscope, of intracellular free Ca2+ concentration ([Ca2+]i), intracellular pH (pHi), and morphological changes in a single starfish sperm (Asterina pectinifera). [Ca2+]i and pHi were monitored with the fluorescent probes indo-1 and SNARF-1, respectively. The acrosome reaction was induced with ionomycin. After the introduction of ionomycin in the medium, [Ca2+]i increased gradually and reached a plateau in approximately 30 s. The fusion of the acrosomal vacuole took place abruptly before the plateau, during the rising phase. Although the speed of the [Ca2+]i increase varied among the many sperm tested, exocytosis in all cases occurred at the same [Ca2+]i of approximately 2 microM (estimated using the dissociation constant of indo-1 for Ca2+ of 1.1 microM). This result suggests that the exocytotic mechanism in starfish sperm responds to [Ca2+]i rapidly, with a reaction time of the order of one second or less. Unlike the change in [Ca2+]i, an abrupt increase in pHi was observed immediately after exocytosis, suggesting the presence of a proton mobilizing system that is triggered by exocytosis. The rapid increase in pHi coincided with the formation of the acrosomal rod and the beginning of vigorous movement of the flagellum, both of which have been proposed to be pHi dependent. The exocytotic event itself was visualized with the fluorescent membrane probe RH292. The membrane of the acrosomal vacuole, concealed from the external medium in an unreacted sperm, was seen to fuse with the plasma membrane. PMID:7490297

  2. The Knobbed Acrosome Defect in Beef Bulls

    PubMed Central

    Barth, Albert D.

    1986-01-01

    The knobbed acrosome defect was found at levels of 25 to 100 percent of spermatozoa from 16 of 2054 beef bulls. The incidence of this defect appeared to be particularly high in the Charolais breed. Pedigree analysis of some of the affected Charolais bulls indicated there may be a genetic predisposition for this sperm defect. In eosin-nigrosin stained semen smears the most common form of the abnormality was a flattened or indented apex of the sperm head. A refractile bead at the apex of the sperm head was seen less commonly. Electron microscopy of the spermatozoa from one bull showed that the abnormality was similar to the knobbed sperm defect previously described in Friesian bulls. A breeding trial confirmed that bulls producing spermatozoa with a high incidence of knobbed acrosomes are infertile. ImagesFigure 2 and 3.Figure 4.Figure 5.Figure 6 and 7.Figure 8.Figure 9.Figure 10. PMID:17422706

  3. Acrosome reaction is subfamily specific in sea star fertilization.

    PubMed

    Nakachi, Mia; Moriyama, Hideaki; Hoshi, Motonori; Matsumoto, Midori

    2006-10-15

    In the fertilization process of sea stars, sperm is activated to go through the acrosome reaction before cell fusion. We focused on induction of the acrosome reaction as a key process in fertilization. Six species of sea stars were used in this study: Asterias amurensis, Asterias rubens, Asterias forbesi, Aphelasterias japonica, Distolasterias nipon, and Asterina pectinifera. Acrosome reaction assays indicate that the acrosome reaction can be induced across species within Asteriinae subfamily. However, cross-fertilization assays indicate that sea stars have species specificity in fertilization. Therefore, steps after the acrosome reaction are responsible for the species specificity. To explain acrosome reaction subfamily specificity at the molecular level, the sugar components of egg jelly were examined and analyzed by principal component analysis. A. amurensis and A. forbesi belong to the same induction group of the acrosome reaction. D. nipon and An. pectinifera are in a unique group. Enzyme-linked immunosorbent assays indicate that Asteriinae subfamily share a common glycan structure, the Fragment 1 of Acrosome Reaction-Inducing Substance from A. amurensis. Fragment 1 plays an important role in the subfamily specificity of acrosome reaction induction. In addition, A. amurensis sperm activating peptide was recognized by sperm from the same superorder. These results demonstrate that the specificity of acrosome reaction induction is present at the subfamily level in sea stars.

  4. 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.

  5. 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

  6. Dynamics of the induced acrosome reaction in boar sperm evaluated by flow cytometry.

    PubMed

    Birck, Anders; Labouriau, Rodrigo; Christensen, Preben

    2009-10-01

    The present study investigated the dynamics of the in vitro induced acrosome reaction (AR) in boar sperm in response to medium composition, incubation time and ionophore concentration. The AR is a prerequisite for normal sperm fertilizing capability and can be studied in vitro following induction by various agents. The ability of a sperm population to undergo the AR in vivo is expected to influence male fertilizing potential, and attempts to relate the in vitro induced AR to fertility has been reported. However, to relate the induced AR to fertility one should be aware of the dynamics of the in vitro induced AR. A detailed description of the dynamics of sperm viability and acrosomal status of boar sperm following in vitro induction of the AR has to our knowledge not previously been conducted. In the present study, a triple color flow cytometric detection technique was used, which gave simultaneous information on sperm viability and acrosomal status. The ionophore induced AR was dependent on extracellular Ca(2+), but could be easily induced in boar sperm without capacitation. Capacitation-associated plasma membrane phospholipid scrambling was assessed and a medium specific ability to induce these membrane changes was observed. Both sperm viability and the induced AR were significantly affected by sperm capacitation, incubation time and ionophore concentration. The results lead to suggestions for an optimized AR induction protocol that takes both sperm viability and the effectiveness of AR induction into consideration. PMID:19084358

  7. Valorization of artichoke wastewaters by integrated membrane process.

    PubMed

    Conidi, C; Cassano, A; Garcia-Castello, E

    2014-01-01

    In this work an integrated membrane system was developed on laboratory scale to fractionate artichoke wastewaters. In particular, a preliminary ultrafiltration (UF) step, based on the use of hollow fibre membranes, was investigated to remove suspended solids from an artichoke extract. The clarified solution was then submitted to a nanofiltration (NF) step. Two different 2.5 × 21 in. spiral-wound membranes (Desal DL and NP030) with different properties were investigated. Both membranes showed a high rejection towards the phenolic compounds analysed (chlorogenic acid, cynarin and apigenin-7-O-glucoside) and, consequently, towards the total antioxidant activity (TAA). On the other hand, the Desal DL membrane was characterized by a high rejection towards sugar compounds (glucose, fructose and sucrose) (100%) when compared with the NP030 membrane (4.02%). The performance of selected membranes in terms of permeate flux, fouling index and water permeability recovery was also evaluated. On the base of experimental results, an integrated membrane process for the fractionation of artichoke wastewaters was proposed. This conceptual process design permitted to obtain different valuable products: a retentate fraction (from the NP030 membrane) enriched in phenolic compounds suitable for nutraceutical, cosmeceutical or food application; a retentate fraction (from the Desal DL membrane), enriched in sugar compounds, of interest for food applications; a clear permeate (from the Desal DL membrane) which can be reused as process water or for membrane cleaning. PMID:24125635

  8. 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.

  9. Monoclonal antibodies which recognize equatorial segment epitopes presented de novo following the A23187-induced acrosome reaction of guinea pig sperm.

    PubMed

    Allen, C A; Green, D P

    1995-02-01

    formaldehyde-fixed sperm with fluorescein isothiocyanate- or gold-labelled second antibody, with or without permeabilization with 0.05% Triton X-100, showed dense labelling on the cytoplasmic face of the plasma membrane overlying the convex surface of the acrosome but little labelling elsewhere. Cryosections of acrosome-intact sperm labelled indirectly with immuno-gold showed labelling consistent with the same location, as well as sporadic labelling at other intracellular sites overlying the acrosome. Since there is no evidence that sperm can translocate intact membrane protein from the cytoplasmic face to the extracellular face of the plasma membrane during the acrosome reaction, the evidence suggests that there are two isolated antigen pools.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7539438

  10. 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.

  11. 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. PMID:27449406

  12. 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}.

  13. 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

  14. Large-scale identification of yeast integral membrane protein interactions

    PubMed Central

    Miller, John P.; Lo, Russell S.; Ben-Hur, Asa; Desmarais, Cynthia; Stagljar, Igor; Noble, William Stafford; Fields, Stanley

    2005-01-01

    We carried out a large-scale screen to identify interactions between integral membrane proteins of Saccharomyces cerevisiae by using a modified split-ubiquitin technique. Among 705 proteins annotated as integral membrane, we identified 1,985 putative interactions involving 536 proteins. To ascribe confidence levels to the interactions, we used a support vector machine algorithm to classify interactions based on the assay results and protein data derived from the literature. Previously identified and computationally supported interactions were used to train the support vector machine, which identified 131 interactions of highest confidence, 209 of the next highest confidence, 468 of the next highest, and the remaining 1,085 of low confidence. This study provides numerous putative interactions among a class of proteins that have been difficult to analyze on a high-throughput basis by other approaches. The results identify potential previously undescribed components of established biological processes and roles for integral membrane proteins of ascribed functions. PMID:16093310

  15. 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.

  16. Integration of lateral porous silicon membranes into planar microfluidics.

    PubMed

    Leïchlé, Thierry; Bourrier, David

    2015-02-01

    In this work, we present a novel fabrication process that enables the monolithic integration of lateral porous silicon membranes into single-layer planar microchannels. This fabrication technique relies on the patterning of local electrodes to guide pore formation horizontally within the membrane and on the use of silicon-on-insulator substrates to spatially localize porous silicon within the channel depth. The feasibility of our approach is studied by current flow analysis using the finite element method and supported by creating 10 μm long mesoporous membranes within 20 μm deep microchannels. The fabricated membranes are demonstrated to be potentially useful for dead-end microfiltration by adequately retaining 300 nm diameter beads while macromolecules such as single-stranded DNA and immunoglobulin G permeate the membrane. The experimentally determined fluidic resistance is in accordance with the theoretical value expected from the estimated pore size and porosity. The work presented here is expected to greatly simplify the integration of membranes capable of size exclusion based separation into fluidic devices and opens doors to the use of porous silicon in planar lab on a chip devices.

  17. Processing, localization and binding activity of zonadhesin suggest a function in sperm adhesion to the zona pellucida during exocytosis of the acrosome.

    PubMed Central

    Bi, Ming; Hickox, John R; Winfrey, Virginia P; Olson, Gary E; Hardy, Daniel M

    2003-01-01

    Zonadhesin is a sperm protein that binds in a species-specific manner to the extracellular matrix ZP (zona pellucida) of the mammalian oocyte. The pig zonadhesin precursor is a 267000-Da mosaic protein with a Type I membrane topology and a large extracellular region comprising meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and five tandem von Willebrand D (VWD) domains. Multiple mature forms of zonadhesin in the sperm head differ in their avidities for the ZP. To determine the potential functions of zonadhesin forms in gamete adhesion, we characterized the processing, activation and localization of protein in pig spermatozoa. The predominant polypeptides of processed zonadhesin were M(r) 300000 (p300), 105000 (p105) and 45000 (p45). p45 and p105, comprised primarily the D1, D2-D3 domains respectively, and were N-glycosylated. p300 was heavily O-glycosylated, and spanned the meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and D0 domains. Hydrolysis of the precursor polypeptide occurred in the testis, and N-terminal sequencing of p45 and p105 identified Asp806-Pro and Asp1191-Pro in the D1 and D2 domains respectively as bonds cleaved in the protein's functional maturation. Testicular zonadhesin was extractable with non-ionic detergents, and localized to the developing outer acrosomal membrane of round and elongating spermatids. As spermatozoa transited the epididymis, most of the protein became incorporated into an extraction-resistant fraction, and the proportions of active and of multimeric zonadhesins in the cells increased. Zonadhesin localized to the perimeter of the acrosome in intact ejaculated spermatozoa and to the leading edge of acrosomal matrix overlying cells with disrupted acrosomal membranes. We conclude that the zonadhesin precursor is specifically proteolysed, glycosylated and assembled into particulate structures in the distal parts of the acrosome where it may mediate specific adhesion to the ZP during the initial stages of

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. VAMP-1: a synaptic vesicle-associated integral membrane protein.

    PubMed Central

    Trimble, W S; Cowan, D M; Scheller, R H

    1988-01-01

    Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters. Images PMID:3380805

  5. Improved preimplantation development of bovine ICSI embryos generated with spermatozoa pretreated with membrane-destabilizing agents lysolecithin and Triton X-100.

    PubMed

    Zambrano, Fabiola; Aguila, Luis; Arias, María E; Sánchez, Raúl; Felmer, Ricardo

    2016-10-01

    In cattle, intracytoplasmic sperm injection (ICSI) has a low efficiency. The acrosome content may be responsible for this effect because of the large amount of hydrolytic enzymes that are released within the oocyte. With the aim of removing the acrosome and destabilize the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX) at different concentrations. We evaluated the membrane integrity, the acrosome integrity, DNA integrity, and the variation of phospholipase C zeta. The rates of development (cleavage and blastocysts) were also evaluated along with pronuclear formation and the embryo quality. Spermatozoa incubated with LL and TX (0.01%, 0.02%, 0.03%, and 0.04%) decreased (P < 0.0001) sperm viability in a dose-dependent manner. The acrosome reaction was also increased (P < 0.0001) in all tested concentrations of LL and TX achieving 100% at 0.05% concentration in both treatments. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay reported an increase (P < 0.05) in DNA fragmentation only with the highest concentration of LL (0.06%), whereas all concentrations assessed of TX reported an increased respect to the control. Phospholipase C zeta expression decreased (P < 0.05) in spermatozoa treated with LL and TX at all concentrations tested. A higher cleavage rate was observed in ICSI-TX (66%) and ICSI-LL (65%) groups compared with the untreated control group (51%) and the blastocyst formation rate significantly increased in the ICSI-LL group (29%) compared with the control (21%). No differences were observed in the pronuclear formation and quality of the embryos. In conclusion, the destabilization of the plasma membrane and the release of the acrosomal content with LL and TX before ICSI improve the rate of embryonic development, without affecting the quality of the embryos produced by this technique. PMID:27325573

  6. 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.

  7. 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

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

    PubMed

    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 (r(2)>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.

  9. Solid-state NMR structures of integral membrane proteins.

    PubMed

    Patching, Simon G

    2015-01-01

    Solid-state NMR is unique for its ability to obtain three-dimensional structures and to measure atomic-resolution structural and dynamic information for membrane proteins in native lipid bilayers. An increasing number and complexity of integral membrane protein structures have been determined by solid-state NMR using two main methods. Oriented sample solid-state NMR uses macroscopically aligned lipid bilayers to obtain orientational restraints that define secondary structure and global fold of embedded peptides and proteins and their orientation and topology in lipid bilayers. Magic angle spinning (MAS) solid-state NMR uses unoriented rapidly spinning samples to obtain distance and torsion angle restraints that define tertiary structure and helix packing arrangements. Details of all current protein structures are described, highlighting developments in experimental strategy and other technological advancements. Some structures originate from combining solid- and solution-state NMR information and some have used solid-state NMR to refine X-ray crystal structures. Solid-state NMR has also validated the structures of proteins determined in different membrane mimetics by solution-state NMR and X-ray crystallography and is therefore complementary to other structural biology techniques. By continuing efforts in identifying membrane protein targets and developing expression, isotope labelling and sample preparation strategies, probe technology, NMR experiments, calculation and modelling methods and combination with other techniques, it should be feasible to determine the structures of many more membrane proteins of biological and biomedical importance using solid-state NMR. This will provide three-dimensional structures and atomic-resolution structural information for characterising ligand and drug interactions, dynamics and molecular mechanisms of membrane proteins under physiological lipid bilayer conditions.

  10. Solid-state NMR structures of integral membrane proteins.

    PubMed

    Patching, Simon G

    2015-01-01

    Solid-state NMR is unique for its ability to obtain three-dimensional structures and to measure atomic-resolution structural and dynamic information for membrane proteins in native lipid bilayers. An increasing number and complexity of integral membrane protein structures have been determined by solid-state NMR using two main methods. Oriented sample solid-state NMR uses macroscopically aligned lipid bilayers to obtain orientational restraints that define secondary structure and global fold of embedded peptides and proteins and their orientation and topology in lipid bilayers. Magic angle spinning (MAS) solid-state NMR uses unoriented rapidly spinning samples to obtain distance and torsion angle restraints that define tertiary structure and helix packing arrangements. Details of all current protein structures are described, highlighting developments in experimental strategy and other technological advancements. Some structures originate from combining solid- and solution-state NMR information and some have used solid-state NMR to refine X-ray crystal structures. Solid-state NMR has also validated the structures of proteins determined in different membrane mimetics by solution-state NMR and X-ray crystallography and is therefore complementary to other structural biology techniques. By continuing efforts in identifying membrane protein targets and developing expression, isotope labelling and sample preparation strategies, probe technology, NMR experiments, calculation and modelling methods and combination with other techniques, it should be feasible to determine the structures of many more membrane proteins of biological and biomedical importance using solid-state NMR. This will provide three-dimensional structures and atomic-resolution structural information for characterising ligand and drug interactions, dynamics and molecular mechanisms of membrane proteins under physiological lipid bilayer conditions. PMID:26857803

  11. 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.

  12. Morphological and acrosomal changes of canine spermatozoa during epididymal transit

    PubMed Central

    2013-01-01

    Background During epididymal transit, functional and structural modifications leading to full maturation enable male gametes to reach, recognize and fertilize the oocytes. In dogs, little is known on the modifications of spermatozoa during the passage in the epididymis. The aim of this study was to describe the motility, morphology and acrosomal patterns of canine spermatozoa retrieved from the epididymis caput, corpus and cauda. Results After the dilution required for the collection of epididymal content, sperm motility was significantly higher (P <0.0001) in the cauda compared to corpus and caput. Proportions of spermatozoa with normal morphology were significantly higher in corpus (P =0.02) and cauda (P <0.0001) compared to caput. Overall morphological abnormalities of the head and neck/midpiece were similar in the three different epididymal regions. A significantly increased prevalence of tail defects, mainly represented by single bent tails, was observed in the corpus compared to caput (P <0.0001) and cauda (P =0.006). Numbers of immature sperm with cytoplasmic droplets decreased from the proximal to the distal region of the epididymis. Particularly, proximal cytoplasmic droplets were more frequently found in spermatozoa collected from the caput epididymis than in the corpus (P <0.0001) and in the cauda (P <0.0001), whereas the occurrence of distal cytoplasmic droplets was higher in the corpus than in the caput (P =0.0003) and in the cauda (P <0.05). Significantly higher proportions of spermatozoa with intact acrosomes were retrieved from the cauda epididymis than from the caput (P =0.03) and the corpus (P =0.008). This difference was mainly due to a lower proportion of spermatozoa with abnormal acrosomes (mainly swollen acrosomes) rather than with absent acrosomes. Conclusions Canine spermatozoa undergo several modifications in the epididymis. The acquisition of progressive motility, migration of the cytoplasmic droplet and acrosomal reshaping lead to mature

  13. 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

  14. A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency.

    PubMed

    Marshall, Stephen S; Niesen, Michiel J M; Müller, Axel; Tiemann, Katrin; Saladi, Shyam M; Galimidi, Rachel P; Zhang, Bin; Clemons, William M; Miller, Thomas F

    2016-08-23

    Integral membrane proteins (IMPs) 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 vary widely 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

  15. 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

  16. Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins.

    PubMed

    Duong-Thi, Minh-Dao; Bergström, Maria; Edwards, Katarina; Eriksson, Jonny; Ohlson, Sten; Ying, Janet To Yiu; Torres, Jaume; Hernández, Víctor Agmo

    2016-02-01

    Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures. PMID:26673836

  17. 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

  18. Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum

    PubMed Central

    Li, Xiaochun; Roberti, Rita; Blobel, Günter

    2014-01-01

    Sterols are essential biological molecules in the majority of life forms. Sterol reductases1 including Delta-14 sterol reductase (C14SR), 7-dehydrocholesterol reductase (DHCR7) and 24-dehydrocholesterol reductase (DHCR24) reduce specific carbon-carbon double bonds of the sterol moiety using a reducing cofactor during sterol biosynthesis. Lamin B Receptor2 (LBR), an integral inner nuclear membrane protein, also contains a functional C14SR domain. Here we report the crystal structure of a Delta-14 sterol reductase (maSR1) from the methanotrophic bacterium Methylomicrobium alcaliphilum 20Z, a homolog of human C14SR, LBR, and DHCR7, with the cofactor NADPH. The enzyme contains 10 transmembrane segments (TM). Its catalytic domain comprises the C-terminal half (containing TM6-10) and envelops two interconnected pockets, one of which faces the cytoplasm and houses NADPH, while the other one is accessible from the lipid bilayer. Comparison with a soluble steroid 5β-reductase structure3 suggests that the reducing end of NADPH meets the sterol substrate at the juncture of the two pockets. A sterol reductase activity assay proves maSR1 can reduce the double bond of a cholesterol biosynthetic intermediate demonstrating functional conservation to human C14SR. Therefore, our structure as a prototype of integral membrane sterol reductases provides molecular insight into mutations in DHCR7 and LBR for inborn human diseases. PMID:25307054

  19. 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...

  20. Assembly of the fluorescent acrosomal matrix and its fate in fertilization in the water strider, Aquarius remigis

    PubMed Central

    Miyata, Haruhiko; Noda, Naoki; Fairbairn, Daphne J.; Oldenbourg, Rudolf; Cardullo, Richard A.

    2011-01-01

    Animal sperm show remarkable diversity in both morphology and molecular composition. Here we provide the first report of intense intrinsic fluorescence in an animal sperm. The sperm from a semi-aquatic insect, the water strider, Aquarius remigis, contains an intrinsically fluorescent molecule with properties consistent with those of Flavin Adenine Dinucleotid (FAD) which appears first in the acrosomal vesicle of round spermatids and persists in the acrosome throughout spermiogenesis. Fluorescence recovery after photobleaching reveals that the fluorescent molecule exhibits unrestricted mobility in the acrosomal vesicle of round spermatids, but is completely immobile in the acrosome of mature sperm. Fluorescence polarization microscopy shows a net alignment of the fluorescent molecules in the acrosome of the mature sperm but not in the acrosomal vesicle of round spermatids. These results suggest that acrosomal molecules are rearranged in the elongating acrosome and FAD is incorporated into the acrosomal matrix during its formation. Further, we followed the fate of the acrosomal matrix in fertilization utilizing the intrinsic fluorescence. The fluorescent acrosomal matrix was observed inside the fertilized egg and remained structurally intact even after gastrulation started. This observation suggests that FAD is not released from the acrosomal matrix during the fertilization process or early development and supports an idea that FAD is involved in the formation of the acrosomal matrix. The intrinsic fluorescence of the A. remigis acrosome will be a useful marker for following spermatogenesis and fertilization. PMID:20857404

  1. 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.

  2. Acrosomal reaction of the Thyone sperm. III. The relationship between actin assembly and water influx during the extension of the acrosomal process.

    PubMed

    Tilney, L G; Inoué, S

    1985-04-01

    In an attempt to investigate the role of water influx in the extension of the acrosomal process of Thyone sperm, we induced the acrosomal reaction in sea water whose osmolarity varied from 50 to 150% of that of sea water. (a) Video sequences of the elongation of the acrosomal processes were made; plots of the length of the acrosomal process as a function of (time)1/2 produced a straight line except at the beginning of elongation and at the end in both hypotonic and hypertonic sea water (up to 1.33 times the osmolarity of sea water), although the rate of elongation was fastest in hypotonic sea water and was progressively slower as the tonicity was raised. (b) Close examination of the video sequences revealed that regardless of the tonicity of the sea water, the morphology of the acrosomal processes were similar. (c) From thin sections of fixed sperm, the amount of actin polymerization that takes place is roughly coupled to the length of the acrosomal process formed so that sperm with short processes only polymerize a portion of the actin that must be present in those sperm. From these facts we conclude that the influx of water and the release of actin monomers from their storage form in the profilactin (so that these monomers can polymerize) are coupled. The exact role of water influx, why it occurs, and whether it could contribute to the extension of the acrosomal process by a hydrostatic pressure mechanism is discussed. PMID:3920226

  3. 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.

  4. 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.

  5. 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…

  6. 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.

  7. 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

  8. 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.

  9. The acrosomal matrix from guinea pig sperm contains structural proteins, suggesting the presence of an actin skeleton.

    PubMed

    Zepeda-Bastida, Armando; Chiquete-Felix, Natalia; Uribe-Carvajal, Salvador; Mujica, Adela

    2011-01-01

    The mammalian sperm acrosome contains a large number of hydrolytic enzymes. When the acrosomal reaction and fertilization occur, these enzymes are released in an orderly fashion, suggesting that the acrosomal matrix is highly organized. It was decided to determine the identity of the structural scaffold underlying the organization of the acrosome. In permeabilized acrosomes and in the Triton X-100-extracted acrosomal matrices from guinea pig sperm, we used indirect immunofluorescence, immunogold labeling, and Western blotting to identify F-actin, spectrin, myosin, calmodulin, and gelsolin. These proteins were detected in the acrosomal matrix for the first time. In noncapacitated, intact spermatozoa the addition of the F-actin monomerizing agent cytochalasin D resulted in loss of the acrosome, suggesting that F-actin is needed to preserve an intact acrosome. Our results suggest that the acrosomal architecture is supported by a dynamic F-actin skeleton, which probably regulates the differential rate of release of the acrosomal enzymes during acrosomal reaction and fertilization.

  10. Integration of nanoporous membranes into microfluidic devices: electrokinetic bio-sample pre-concentration.

    PubMed

    Kim, Minseok; Kim, Taesung

    2013-10-21

    The integration of nanoporous membranes into microfluidic devices allows a wide range of analytical and biochemical applications such as stable concentration gradient generation, sample pre-concentration, and ion and biomolecule filtration in a controllable manner. However, further applications of nanoporous membranes in microfluidic devices require rapid and controllable fabrication methods of various nanoporous precursor materials; currently, few such methods exist. Here, we describe simple and robust methods that can be used for microfabricating four different precursor materials as leakage-tight membranes in a microfluidic channel network. The methods consist of a common integration process and individual solidification processes such as solvent evaporation, UV-curing, and temperature treatment. We demonstrate that the fabricated membranes can be used for electrokinetic, nanofluidic pre-concentration of bio-samples such as proteins, cells, and microspheres on either the anodic or cathodic side of the membranes. In addition, we not only characterize the physicochemical properties of the membranes such as conductance of membrane-integrated microchannels, relative permselectivity, and pre-concentration ability, but also compare fabrication availability, membrane robustness, surface charge density tunability and biocompatibility with buffer solutions. The methods are versatile for many nanoporous precursor materials and easy to control the location and dimension of the membranes. Hence, the methods developed and the characterized properties of the membranes tested in this work could be widely employed for further applications of nanoporous membranes in microfluidic systems.

  11. 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.

  12. 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. PMID:27528656

  13. Monoclonal antibody to an integral membrane protein, the lactose permease.

    PubMed

    Eash, J; Villarejo, M R

    1983-02-01

    A monoclonal IgG antibody directed against the lactose permease was produced from animals inoculated with membranes of a lac Y plasmid strain. The appropriate antibody was selected by a series of ELISA assays in which membranes, purified permease, or a lac Y-Z chimeric protein was the immobilized antigen. The antibody recognizes a portion of the permease exposed on the surface of membrane vesicles but does not inhibit lactose transport.

  14. 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

  15. An Integrated Framework Advancing Membrane Protein Modeling and Design.

    PubMed

    Alford, Rebecca F; Koehler Leman, Julia; Weitzner, Brian D; Duran, Amanda M; Tilley, Drew C; Elazar, Assaf; Gray, Jeffrey J

    2015-09-01

    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

  16. 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

  17. Topological Predictions for Integral Membrane Channel and Carrier Proteins

    PubMed Central

    Abhinay, Reddy; Jaehoon, Cho; Sam, Ling; Vamsee, Reddy; Maksim, Shlykov; Milton, Saier

    2014-01-01

    We evaluated topological predictions for nine different programs, HMMTOP, TMHMM, SVMTOP, DAS, SOSUI, TOPCONS, PHOBIUS, MEMSAT-SVM (hereinafter referred to as MEMSAT), and SPOCTOPUS. These programs were first evaluated using four large topologically well-defined families of secondary transporters, and the three best programs were further evaluated using topologically more diverse families of channels and carriers. In the initial studies, the order of accuracy was: SPOCTOPUS>MEMSAT>HMMTOP>TOPCONS>PHOBIUS>TMHMM>SVMTOP>DAS>S OSUI. Some families, such as the Sugar Porter family (2.A.1.1) of the Major Facilitator Superfamily (MFS; TC# 2.A.1) and the Amino acid/Polyamine/Organocation (APC) Family (TC# 2.A.3), were correctly predicted with high accuracy while others, such as the Mitochondrial Carrier (MC) (TC# 2.A.29) and the K+ transporter (Trk) families (TC# 2.A.38), were predicted with much lower accuracy. For small, topologically homogeneous families, SPOCTOPUS and MEMSAT were generally most reliable, while with large, more diverse superfamilies, HMMTOP often proved to have the greatest prediction accuracy. We next developed a novel program, TM-STATS, that tabulates HMMTOP, SPOCTOPUS or MEMSAT-based topological predictions for any subdivision (class, subclass, superfamily, family, subfamily, or any combination of these) of the Transporter Classification Database (TCDB; www.tcdb.org) and examined the following subclasses: α-type channel proteins (TC subclasses 1.A and 1.E), secreted poreforming toxins (TC subclass 1.C) and secondary carriers (subclass 2.A). Histograms 3 were generated for each of these subclasses, and the results were analyzed according to subclass, family and protein. The results provide an update of topological predictions for integral membrane transport proteins as well as guides for the development of more reliable topological prediction programs, taking family-specific characteristics into account. PMID:24992992

  18. Daptomycin exerts rapid bactericidal activity against Bacillus anthracis without disrupting membrane integrity

    PubMed Central

    Xing, Yu-hua; Wang, Wei; Dai, Su-qin; Liu, Ti-yan; Tan, Jun-jie; Qu, Guo-long; Li, Yu-xia; Ling, Yan; Liu, Gang; Fu, Xue-qi; Chen, Hui-peng

    2014-01-01

    Aim: To examine whether the novel cyclic lipopeptide antibiotic daptomycin could be used to treat anthrax and to study the mechanisms underlying its bactericidal action against Bacillus anthracis. Methods: Spore-forming B anthracis AP422 was tested. MIC values of antibiotics were determined. Cell membrane potential was measured using flow cytometric assays with membrane potential-sensitive fluorescent dyes. Cell membrane integrity was detected using To-Pro-3 iodide staining and transmission electron microscopy. K+ efflux and Na+ influx were measured using the fluorescent probes PBFI and SBFI-AM, respectively. Results: Daptomycin exhibited rapid bactericidal activity against vegetative B anthracis with a MIC value of 0.78 μg/mL, which was comparable to those of ciprofloxacin and penicillin G. Furthermore, daptomycin prevented the germinated spores from growing into vegetative bacteria. Daptomycin concentration-dependently dissipated the membrane potential of B anthracis and caused K+ efflux and Na+ influx without disrupting membrane integrity. In contrast, both ciprofloxacin and penicillin G did not change the membrane potential of vegetative bacteria or spores. Penicillin G disrupted membrane integrity of B anthracis, whereas ciprofloxacin had no such effect. Conclusion: Daptomycin exerts rapid bactericidal action against B anthracis via reducing membrane potential without disrupting membrane integrity. This antibiotic can be used as an alternate therapy for B anthracis infections. PMID:24362329

  19. Detection of cooling-induced membrane changes in the response of boar sperm to capacitating conditions.

    PubMed

    Petrunkina, Anna M; Volker, Gabriele; Weitze, Karl-Fritz; Beyerbach, Martin; Töpfer-Petersen, Edda; Waberski, Dagmar

    2005-05-01

    There is a need for methods of rapid and sensitive sperm function assessment. As spermatozoa are not able to fertilize an oocyte before having undergone a series of complex physiological changes collectively called capacitation, it is logical to assess sperm function under fertilizing conditions in vitro. In this study, the responsiveness of sperm to capacitating conditions in vitro was monitored by changes in sperm response to ionophore and by changes in the amount of intracellular calcium ions in stored boar semen. Boar semen was diluted at 32 and 20 degrees C and stored for 24 and 72 h at 16 and 10 degrees C. Ionophore-induced changes and increased intracellular calcium ion content in boar spermatozoa were recorded by flow cytometry and found to progress as a function of time during incubation under capacitating conditions. All responsiveness parameters (increases in proportions of membrane-defective spermatozoa, acrosome-reacted spermatozoa, and cells with high intracellular calcium levels) were shown to be sensitive to subtle physiological changes occurring at low storage temperatures. The initial levels of sperm with a high calcium content were higher in semen stored at 10 degrees C, but the accumulation of internal calcium was lower than in semen stored at 16 degrees C. The loss of membrane integrity and increase in the proportion of acrosome-reacted cells were higher in semen stored at 10 degrees C. Dilution at 20 degrees C had no negative effect on membrane integrity or responsiveness to capacitating conditions. There was no significant difference between semen stored for 24 and 72 h in terms of membrane integrity, acrosome reaction, and intracellular calcium after capacitation treatment. However, dynamics of cell death and acrosome reaction in response to capacitating conditions were somewhat accelerated after 72 h storage, especially in semen stored at 10 degrees C. It can be concluded that the simultaneous use of the sperm membrane responsiveness and

  20. GMAP210 AND IFT88 ARE PRESENT IN THE SPERMATID GOLGI APPARATUS AND PARTICIPATE IN THE DEVELOPMENT OF THE ACROSOME-ACROPLAXOME COMPLEX, HEAD-TAIL COUPLING APPARATUS AND TAIL

    PubMed Central

    Kierszenbaum, Abraham L.; Rivkin, Eugene; Tres, Laura L.; Yoder, Bradley K.; Haycraft, Courtney J.; Bornens, Michel; Rios, Rosa M.

    2014-01-01

    We describe the localization of the golgin GMAP210 and the intraflagellar protein IFT88 in the Golgi of spermatids and the participation of these two proteins in the development of the acrosome-acroplaxome complex, the head-tail coupling apparatus (HTCA) and the spermatid tail. Immunocytochemical experiments show that GMAP210 predominates in the cis-Golgi whereas IFT88 prevails in the trans-Golgi network. Both proteins co-localize in proacrosomal vesicles, along acrosome membranes, the HTCA and the developing tail. IFT88 persists in the acrosome-acroplaxome region of the sperm head whereas GMAP210 is not longer seen there. Spermatids of the Ift88 mouse mutant display abnormal head shaping and are tail-less. GMAP210 is visualized in the Ift88 mutant during acrosome-acroplaxome biogenesis. However, GMAP210–stained vesicles, mitochondria and outer dense fiber material build up in the manchette region and fail to reach the abortive tail stump in the mutant. In vitro disruption of the spermatid Golgi and microtubules with Brefeldin-A and nocodazole blocks the progression of GMAP210- and IFT88-stained proacrosomal vesicles to the acrosome-acroplaxome complex but F-actin distribution in the acroplaxome is not affected. We provide the first evidence that IFT88 is present in the Golgi of spermatids, that the microtubule-associated golgin GMAP210 and IFT88 participate in acrosome, HTCA and tail biogenesis, and that defective intramanchette transport of cargos disrupts spermatid tail development. PMID:21337470

  1. 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. PMID:26362050

  2. 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.

  3. An integrated membrane system for the biocatalytic production of 3'-sialyllactose from dairy by-products.

    PubMed

    Luo, Jianquan; Nordvang, Rune T; Morthensen, Sofie T; Zeuner, Birgitte; Meyer, Anne S; Mikkelsen, Jørn Dalgaard; Pinelo, Manuel

    2014-08-01

    An integrated membrane system was investigated for the production of 3'-sialyllactose by an engineered sialidase using casein glycomacropeptide (CGMP) and lactose as substrates. CGMP was purified by ultrafiltration (UF) to remove any small molecules present and then an enzymatic membrane reactor (EMR) was used to separate the product and reuse the enzyme. A PLCC regenerated cellulose membrane was found to be the most suitable for both the UF purification and EMR. Subsequently, nanofiltration (NF) was conducted to increase the purity of the 3'-sialyllactose by removing the excess lactose present. The NTR7450 membrane outperformed others in NF due to its high retention of 3'-sialyllactose (98%) and relatively low rejection of lactose (40%). The lactose in the permeate could be concentrated by the NF45 membrane and recycled into the EMR. The described integrated membrane system enables a more economic and efficient enzymatic production of 3'-sialyllactose. PMID:24880807

  4. Characterization of the major integral protein of vacuolar membrane.

    PubMed

    Maeshima, M

    1992-04-01

    The vacuolar membrane of radish (Raphanus sativus) taproot contained a large quantity of a protein of 23 kilodaltons that accounted for more than 25% of the total membrane proteins. The protein, tentatively named VM 23, was purified and characterized. VM 23 tends to aggregate at high temperature even in the presence of 1% sodium dodecyl sulfate. The apparent molecular size of VM 23 was estimated to be about 400 kilodaltons by polyacrylamide gel electrophoresis in the presence of 0.1% Triton X-100. VM 23 was partially extracted from the vacuolar membranes with chloroform:methanol, indicating its high hydrophobicity. The hydrophobic carboxyl modifier N,N'-dicyclohexylcarbodiimide bound covalently to VM 23. The results suggest that VM 23 may act as a secondary transport system coupled with the proton transport. The antibody against radish VM 23 reacted with the major proteins in the vacuolar membranes of mung bean (Vigna radiata) and castor bean (Ricinus communis) hypocotyls and pumpkin (Cucurbita moschata) epicotyl, but not with that of sugar beet (Beta vulgaris) taproot. VM 23 comigrated with vacuolar H(+)-pyrophosphatase on sucrose density gradient centrifugation after sonication of membranes, indicating that it is associated with the vacuolar membrane.

  5. 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...

  6. HAMLET Interacts with Lipid Membranes and Perturbs Their Structure and Integrity

    PubMed Central

    Baumann, Anne; Lanekoff, Ingela; Chao, Yinxia; Martinez, Aurora; Svanborg, Catharina; Karlsson, Roger

    2010-01-01

    Background Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. Methodology/Principal Findings We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLAall-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. Conclusions/Significance The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death. PMID:20186341

  7. 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.

  8. 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.

  9. 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.

  10. FAM170B, a novel acrosomal protein involved in fertilization in mice.

    PubMed

    Li, Yuchi; Lin, Shouren; Luo, Manling; Guo, Huan; Chen, Jianbo; Ma, Qian; Gu, Yanli; Jiang, Zhimao; Gui, Yaoting

    2015-10-01

    The acrosome is a specialized organelle that covers the anterior region of the sperm nucleus, and plays an essential role in mammalian fertilization. Although acrosome biogenesis is an important aspect of spermiogenesis, the molecular mechanism that regulates this event remains unknown. In the present study, we identified a novel gene, Fam170b (family with sequence similarity 170, member B), exclusively expressed in mouse testes. Fam170b expression first started at postnatal week 3, and increased in an age-dependent manner until plateauing in adulthood. Immunofluorescence staining revealed its enrichment in round spermatids, and redistribution to a perinuclear spot adjacent to the Golgi and the acrosome of elongating spermatids and spermatozoa; this localization was shared between mouse and human spermatozoa. Anti-FAM170B antibody was remarkably found to inhibit murine in vitro fertilization, specifically blocking the acrosome reaction. We further determined that FAM170B interacts with GOPC (Golgi-associated PDZ and coiled-coil motif containing protein) during acrosome formation, as verified by immunofluorescence and co-immunoprecipitation assays. Thus, we document the expression and function for the endogenous acrosomal protein FAM170B during spermiogenesis and fertilization.

  11. Expression of Flotilin-2 and Acrosome Biogenesis Are Regulated by MiR-124 during Spermatogenesis

    PubMed Central

    Zheng, Haoyu; Jiang, Min; Xia, Zhengrong; Yu, Jinjin; Chen, Ling; Huang, Xiaoyan

    2015-01-01

    MicroRNAs (miRNAs) are a class of short non-coding RNA molecules, which diversely regulate gene expression in organisms. Although the regulatory role of these small RNA molecules has been recently explored in animal spermatogenesis, the role of miR-124 in male germ cells is poorly defined. In our previous study, flotillin-2 was investigated as a novel Golgi-related protein involved in sperm acrosome biogenesis. The current study was designed to analyze the contribution of miR-124 in the regulation of flotillin-2 expression during mouse acrosome biogenesis. Luciferase assays revealed the target effects of miR-124 on flotillin-2 expression. Following intratesticular injection of miR-124 in 3-week-old male mice, quantitative real-time RT-PCR and western blot analysis were employed to confirm the function of miR-124 in regulating flotillin-2 after 48 hours. Sperm abnormalities were assessed 3 weeks later by ordinary optical microscopy, the acrosome abnormalities were also assessed by PNA staining and transmission electron microscopy. The results showed the proportion of sperm acrosome abnormalities was significantly higher than that of the control group. The expression of flotillin-2 and caveolin-1 was significantly downregulated during acrosome biogenesis. These results indicated that miR-124 could potentially play a role in caveolin-independent vesicle trafficking and modulation of flotillin-2 expression in mouse acrosome biogenesis. PMID:26313572

  12. 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.

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

    PubMed

    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

    2016-06-28

    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

  14. Peptides from sperm acrosomal protein that initiate egg development.

    PubMed

    Gould, M C; Stephano, J L

    1991-08-01

    How sperm initiate egg development is being investigated with gametes of the marine worm Urechis. Sperm acrosomal protein, previously shown to activate eggs (Gould et al., 1986, Dev. Biol. 117, 306-318; Gould and Stephano, 1987, Science 235, 1654-1656), was enzymatically cleaved into soluble peptide fragments. When this mixture was added to eggs they activated, and parthenogenetic cleavage often occurred. An active peptide (P23) was purified from the mixture and its sequence was determined to be Val-Ala-Lys-Lys-Pro-Lys. Synthetic peptide had the same biological activity. P23 induced eggs to undergo the complete sequence of changes that normally follows fertilization, including the fertilization potential, completion of meiosis, and DNA replication. When a sperm centrosome was introduced into eggs by prefertilization without activation, and the eggs were subsequently activated by P23, they developed normally to trochophore larvae (the contribution of another sperm component is not ruled out by this experiment). P23 covalently coupled to bovine serum albumin also activated eggs, showing that it acted on the external surface of the egg. The peptide did not activate sea urchin eggs, but did cause oyster eggs to undergo germinal vesicle breakdown.

  15. 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.

  16. Loss of Elongation Factor P Disrupts Bacterial Outer Membrane Integrity

    PubMed Central

    Hersch, Steven J.; Roy, Hervé; Wiggers, J. Brad; Leung, Andrea S.; Buranyi, Stephen; Xie, Jinglin Lucy; Dare, Kiley; Ibba, Michael; Navarre, William Wiley

    2012-01-01

    Elongation factor P (EF-P) is posttranslationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, and detergents and enhanced resistance to the compound S-nitrosoglutathione. The susceptibility phenotypes are largely explained by the enhanced membrane permeability of the efp mutant, which exhibits increased uptake of the hydrophobic dye 1-N-phenylnaphthylamine (NPN). Analysis of the membrane proteomes of wild-type and efp mutant Salmonella strains reveals few changes, including the prominent overexpression of a single porin, KdgM, in the efp mutant outer membrane. Removal of KdgM in the efp mutant background ameliorates the detergent, antibiotic, and osmosensitivity phenotypes and restores wild-type permeability to NPN. Our data support a role for EF-P in the translational regulation of a limited number of proteins that, when perturbed, renders the cell susceptible to stress by the adventitious overexpression of an outer membrane porin. PMID:22081389

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

    PubMed Central

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

    2016-01-01

    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. PMID:27655730

  18. Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein

    PubMed Central

    1996-01-01

    We have identified an integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD that cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) which contributes the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. We suggest that p56 is a sea urchin LBR homologue that targets membranes to chromatin and later anchors the membrane to the lamina. PMID:8991085

  19. 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. PMID:26606094

  20. 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.

  1. 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. PMID:27032685

  2. 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.

  3. Successful integration of membrane technologies in a conventional purification process of tannery wastewater streams.

    PubMed

    Stoller, Marco; Sacco, Olga; Sannino, Diana; Chianese, Angelo

    2013-07-11

    The aim of this work is to design and integrate an optimized batch membrane process in a conventional purification process used for the treatment of tannery wastewater. The integration was performed by using two spiral wound membrane modules in series, that is, nanofiltration and reverse osmosis, as substitutes to the biological reactor. The membrane process was designed in terms of sensible fouling issues reduction, which may be observed on the nanofiltration membrane if no optimization is performed. The entity of the fouling phenomena was estimated by pressure cycling measurements, determining both the critical and the threshold flux on the nanofiltration membrane. The obtained results were used to estimate the need of the overdesign of the membrane plant, as well as to define optimized operating conditions in order to handle fouling issues correctly for a long period of time. Finally, the developed membrane process was compared, from a technical and economic point of view, with the conventional biological process, widely offered as an external service near tannery production sites, and, here, proposed to be substituted by membrane technologies.

  4. Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for Proteome-Wide Analysis

    SciTech Connect

    Goshe, Michael B.; Blonder, Josip; Smith, Richard D.

    2003-03-01

    The ability to identify and quantify integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and derivatize these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified of which 40 were integral membrane proteins containing from 1 to 9 mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g. ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g. pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.

  5. 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.

  6. 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.

  7. 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.

  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%. PMID:27398532

  9. Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters.

    PubMed

    Bird, Louise E; Nettleship, Joanne E; Järvinen, Valtteri; Rada, Heather; Verma, Anil; Owens, Raymond J

    2016-01-01

    The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years. PMID:27553231

  10. A Method for Solution NMR Structural Studies of Large Integral Membrane Proteins: Reverse Micelle Encapsulation

    PubMed Central

    Kielec, Joseph M.; Valentine, Kathleen G.; Wand, A. Joshua

    2009-01-01

    The structural study of membrane proteins perhaps represents one of the greatest challenges of the post-genomic era. While membrane proteins comprise over 50% of current and potential drug targets, their structural characterization lags far behind that of soluble proteins. Nuclear magnetic resonance (NMR) offers great potential not only with respect to structural characterization of integral membrane proteins but may also provide the ability to study the details of small ligand interactions. However, the size limitations of solution NMR have restricted comprehensive structural characterization of membrane protein NMR structures to the relatively small β-barrel proteins or helical proteins of relatively simple topology. In an effort to escape the barriers presented by slow molecular reorientation of large integral membrane proteins solubilized by detergent micelles in water, we have adapted the reverse micelle encapsulation strategy originally developed for the study of large soluble proteins by solution NMR methods. Here we review a novel approach to the solubilization of large integral membrane proteins in reverse micelle surfactants dissolved in low viscosity alkane solvents. The procedure is illustrated with a 54 kDa construct of the homotetrameric KcsA potassium channel. PMID:19665988

  11. Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm.

    PubMed

    Waterhouse, K E; Hofmo, P O; Tverdal, A; Miller, R R

    2006-05-01

    The response of sperm to cryopreservation and the fertility of frozen-thawed semen varies between species. Besides species differences in sperm physiology, structure and biochemistry, factors such as sperm transport and female reproductive tract anatomy will affect fertility of frozen-thawed semen. Therefore, studying differences in sperm cryotolerance between breeds and individuals instead of between species may reveal sources of variability in sperm cryotolerance. In the present study, the effect of cooling, re-warming and freezing and thawing on plasma membrane and acrosome integrity of sperm within and between Norwegian Landrace and Duroc breeds was studied. Furthermore, the relation between post-thaw survival rate and fatty acid composition of the sperm plasma membranes was investigated. Flow cytometry assessments of plasma membrane and acrosome integrity revealed no significant differences between breeds; however there were significant male-to-male variations within breeds in post-thaw percentages of live sperm (plasma membrane intact). The most abundant fatty acids in the plasma membranes from both breeds were palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1, n-9), docosapentaenoic acid (22:5, n-6) and docosahexaenoic acid (22:6, n-3). The ratio of sigma operator 22:5, n-6 and 22:6, n-3/ sigma operator all other membrane fatty acids was significantly related to survival rate (plasma membrane integrity) of sperm for both Norwegian Landrace (correlation coefficient (r(s)) = 0.64, P < 0.05) and Duroc (r(s) = 0.67, P < 0.05) boars. In conclusion, male-to-male differences in sperm survival rate after freezing and thawing may be partly related to the amount of long-chain polyunsaturated fatty acids in the sperm plasma membranes. PMID:16672353

  12. 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.

  13. 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.

  14. A biofilter integrated with gas membrane separation unit for the treatment of fluctuating styrene loads.

    PubMed

    Li, Lin; Lian, Jing; Han, Yunping; Liu, Junxin

    2012-05-01

    Biofiltration for volatile organic compound control in waste gas streams is best operated at steady contaminant loadings. To provide long-term stable operation of a biofilter under adverse contaminant feeding conditions, an integrated bioreactor system with a gas separation membrane module installed after a biofilter was proposed for styrene treatment. Styrene was treated effectively, with average styrene effluent concentrations maintained at less than 50 mg m(-3) and a total removal efficiency of over 96% achieved when the biofiltration column faced fluctuating loads. The maximum elimination capacity of the integrated bioreactor system was 93.8 g m(-3)h(-1), which was higher than that obtained with the biofiltration column alone. The combination of these two processes (microbial and chemical) led to more efficient elimination of styrene and buffering of the fluctuating loads. The factors on gas membrane separation, microbial characteristics in the integrated bioreactor and membrane fouling were also investigated in this study.

  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. PMID:26204850

  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. Double-staining method for differentiation of morphological changes and membrane integrity of Campylobacter coli cells.

    PubMed

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

    2002-10-01

    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.

  18. 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.

  19. Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa.

    PubMed

    Parrish, J J; Susko-Parrish, J L; Handrow, R R; Ax, R L; First, N L

    1989-12-01

    The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 MW dextran sulfate fertilized more bovine oocytes than control sperm (P less than 0.05), sperm treated with 500,000 MW dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.

  20. 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

    2016-01-01

    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. PMID:27438251

  1. 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.

  2. 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

  3. 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.

  4. The Architecture of EssB, an Integral Membrane Component of the Type VII Secretion System

    PubMed Central

    Zoltner, Martin; Norman, David G.; Fyfe, Paul K.; El Mkami, Hassane; Palmer, Tracy; Hunter, William N.

    2013-01-01

    Summary The membrane-bound EssB is an integral and essential component of the bacterial type VII secretion system that can contribute to pathogenicity. The architecture of Geobacillus thermodenitrificans EssB has been investigated by combining crystallographic and EPR spectroscopic methods. The protein forms a dimer that straddles the cytoplasmic membrane. A helical fold is observed for the C-terminal segment, which is positioned on the exterior of the membrane. This segment contributes most to dimer formation. The N-terminal segment displays a structure related to the pseudokinase fold and may contribute to function by recognizing substrates or secretion system partners. The remaining part of EssB may serve as an anchor point for the secretion apparatus, which is embedded in the cytoplasmic membrane with the C-terminal domain protruding out to interact with partner proteins or components of peptidoglycan. PMID:23499020

  5. Yeast cell wall integrity sensors form specific plasma membrane microdomains important for signalling.

    PubMed

    Kock, Christian; Arlt, Henning; Ungermann, Christian; Heinisch, Jürgen J

    2016-09-01

    The cell wall integrity (CWI) pathway of the yeast Saccharomyces cerevisiae relies on the detection of cell surface stress by five sensors (Wsc1, Wsc2, Wsc3, Mid2, Mtl1). Each sensor contains a single transmembrane domain and a highly mannosylated extracellular region, and probably detects mechanical stress in the cell wall or the plasma membrane. We here studied the distribution of the five sensors at the cell surface by using fluorescently tagged variants in conjunction with marker proteins for established membrane compartments. We find that each of the sensors occupies a specific microdomain at the plasma membrane. The novel punctate 'membrane compartment occupied by Wsc1' (MCW) shows moderate overlap with other Wsc-type sensors, but not with those of the Mid-type sensors or other established plasma membrane domains. We further observed that sensor density and formation of the MCW compartment depends on the cysteine-rich head group near the N-terminus of Wsc1. Yet, signalling capacity depends more on the sensor density in the plasma membrane than on clustering within its microcompartment. We propose that the MCW microcompartment provides a quality control mechanism for retaining functional sensors at the plasma membrane to prevent them from endocytosis.

  6. Cell-free Expression and In Meso Crystallisation of an Integral Membrane Kinase for Structure Determination

    PubMed Central

    Shah, Syed Tasadaque Ali; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank; Caffrey, Martin

    2014-01-01

    Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a 3-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipidic mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28 Å resolution. The quality of cellular and cell-free expressed kinase samples have been evaluated systematically by comparing i) spectroscopic properties, ii) purity and oligomer formation, iii) lipid content and iv) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved. PMID:25012698

  7. 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.

  8. 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).

  9. Actin filaments elongate from their membrane-associated ends

    PubMed Central

    Tilney, LG; Bonder, EM; DeRosier, DJ

    1981-01-01

    In limulus sperm an actin filament bundle 55 mum in length extends from the acrosomal vacuole membrane through a canal in the nucleus and then coils in a regular fashion around the base of the nucleus. The bundle expands systematically from 15 filaments near the acrosomal vacuole to 85 filaments at the basal end. Thin sections of sperm fixed during stages in spermatid maturation reveal that the filament bundle begins to assemble on dense material attached to the acrosomal vacuole membrane. In micrographs fo these early stages in maturation, short bundles are seen extending posteriorly from the dense material. The significance is that these short, developing bundles have about 85 filaments, suggesting that the 85-filament end of the bundle is assembled first. By using filament bundles isolated and incubated in vitro with G actin from muscle, we can determine the end “preferred” for addition of actin monomers during polymerization. The end that would be associated with the acrosomal vacuole membrane, a membrane destined to be continuous with the plasma membrane, is preferred about 10 times over the other, thicker end. Decoration of the newly polymerized portions of the filament bundle with subfragment 1 of myosin reveals that the arrowheads point away from the acrosomal vacuole membrane, as is true of other actin filament bundles attached to membranes. From these observations we conclude that the bundle is nucleated from the dense material associated with the acrosomal vacuole and that monomers are added to the membrane-associated end. As monomers are added at the dense material, the thick first-made end of the filament bundle is pushed down through the nucleus where, upon reaching the base of the nucleus, it coils up. Tapering is brought about by the capping of the peripheral filaments in the bundle. PMID:7197276

  10. Lipid Modulation of Calcium Flux through CaV2.3 Regulates Acrosome Exocytosis and Fertilization

    PubMed Central

    Cohen, Roy; Buttke, Danielle E.; Asano, Atsushi; Mukai, Chinatsu; Nelson, Jacquelyn L.; Ren, Dongjun; Miller, Richard; Cohen-Kutner, Moshe; Atlas, Daphne; Travis, Alexander J.

    2014-01-01

    Summary Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca2+ influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3’s pore-forming α1E subunit showed altered Ca2+ responses, reduced AE, and a strong sub-fertility phenotype. Surprisingly, AE depended on spatio-temporal information encoded by flux through CaV2.3—not merely the presence/ amplitude of Ca2+ waves. Using both studies in sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1’s lipid and sugar components and CaV2.3’s α1E and α2δ subunits. Our results provide mechanistic understanding of membrane lipid regulation of Ca2+ flux and therefore Ca2+-dependent cellular and developmental processes such as exocytosis and fertilization. PMID:24525187

  11. 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

  12. Recovery of flavonoids from orange press liquor by an integrated membrane process.

    PubMed

    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

  13. Effect of Akti-2 on sperm motility, capacitation and acrosome reaction in a mouse model

    PubMed Central

    QUAN, YANMEI; LIU, QIANG

    2016-01-01

    The aim of the present study was to investigate the effect of the Akt inhibitor, Akti-2, on the sperm motility and acrosome reaction in mice. Mature sperms from the adult mice, aged 8 weeks, were co-incubated with Akti-2 for ~30 min at 37°C in 5% CO2, and the sperm viability was assessed by eosin-nigrosin staining. The sperm total and progressive motility were analyzed by computer-aided sperm analysis. In addition, the acrosome reaction of sperms was detected by the acid phosphatase assay, Coomassie Brilliant Blue staining and fluorescein-isothiocyanate conjugated pisum sativum lectin staining, respectively. Compared with the control (dimethyl sulfoxide), Akti-2 had no effect on sperm viability, but it suppressed the total and progressive motility significantly. Furthermore, the capacitation-associated protein tyrosine phosphorylation and the acrosome reaction induced by calcium ionophore A23187 could be suppressed by Akti-2. These experiments confirmed that Akti-2 significantly impaired the sperm functions, including motility, capacitation and acrosome reaction, and provide the proof for its potential in male reproductive toxicity. PMID:27123250

  14. Mouse sperm begin to undergo acrosomal exocytosis in the upper isthmus of the oviduct.

    PubMed

    La Spina, Florenza A; Puga Molina, Lis C; Romarowski, Ana; Vitale, Alejandra M; Falzone, Tomas L; Krapf, Dario; Hirohashi, Noritaka; Buffone, Mariano G

    2016-03-15

    Recent evidence demonstrated that most fertilizing mouse sperm undergo acrosomal exocytosis (AE) before binding to the zona pellucida of the eggs. However, the sites where fertilizing sperm could initiate AE and what stimuli trigger it remain unknown. Therefore, the aim of this study was to determine physiological sites of AE by using double transgenic mouse sperm, which carried EGFP in the acrosome and DsRed2 fluorescence in mitochondria. Using live imaging of sperm during in vitro fertilization of cumulus-oocyte complexes, it was observed that most sperm did not undergo AE. Thus, the occurrence of AE within the female reproductive tract was evaluated in the physiological context where this process occurs. Most sperm in the lower segments of the oviduct were acrosome-intact; however, a significant number of sperm that reached the upper isthmus had undergone AE. In the ampulla, only 5% of the sperm were acrosome-intact. These results support our previous observations that most of mouse sperm do not initiate AE close to or on the ZP, and further demonstrate that a significant proportion of sperm initiate AE in the upper segments of the oviductal isthmus.

  15. 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. PMID:25269572

  16. 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.

  17. 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.

  18. Primary structure analysis of an integral membrane glycoprotein of the nuclear pore

    PubMed Central

    1989-01-01

    The complete primary structure of an integral membrane glycoprotein of the nuclear pore was deduced from the cDNA sequence. The cDNA encodes a polypeptide of 204,205 D containing a 25-residue-long signal sequence, two hydrophobic segments that could function as transmembrane segments, and 13 potential N-linked oligosaccharide addition sites. Endoglycosidase H reduces the molecular mass by approximately 9 kD suggesting that not all of these 13 sites are used. We discuss possible models for the topology of this protein in the pore membrane as well as a possible role in the formation of pores and pore complexes. PMID:2738089

  19. 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.

  20. Freezing dog semen in presence of the antioxidant butylated hydroxytoluene improves postthaw sperm membrane integrity.

    PubMed

    Neagu, V R; García, B Macías; Sandoval, C Salazar; Rodríguez, A Morillo; Ferrusola, C Ortega; Fernández, L González; Tapia, J A; Peña, F J

    2010-03-15

    In an attempt to evaluate the protective effect of a lipid-soluble antioxidant (butylated hydroxytoluene; BHT), semen from four dogs (Canis familiaris) was frozen in two different extenders (Uppsala or INRA-96 plus glycerol) with or without 1mM BHT. Sperm membrane integrity using flow cytometry and motility using a computerized system were evaluated in each experimental group. The Uppsala extender was superior in all aspects of sperm function. The percentage of sperm membranes was significantly higher in semen samples frozen in presence of BHT. Our results suggest that the Uppsala extender can be improved with the addition of BHT.

  1. 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

  2. Integration of ceramic membrane and compressed air-assisted solvent extraction (CASX) for metal recovery.

    PubMed

    Li, Chi-Wang; Chiu, Chun-Hao; Lee, Yu-Cheng; Chang, Chia-Hao; Lee, Yu-Hsun; Chen, Yi-Ming

    2010-01-01

    In our previous publications, compressed air-assisted solvent extraction process (CASX) was developed and proved to be kinetically efficient process for metal removal. In the current study, CASX with a ceramic MF membrane integrated for separation of spent solvent was employed to remove and recover metal from wastewater. MF was operated either in crossflow mode or dead-end with intermittent flushing mode. Under crossflow mode, three distinct stages of flux vs. TMP (trans-membrane pressure) relationship were observed. In the first stage, flux increases with increasing TMP which is followed by the stage of stable flux with increasing TMP. After reaching a threshold TMP which is dependent of crossflow velocity, flux increases again with increasing TMP. At the last stage, solvent was pushed through membrane pores as indicated by increasing permeate COD. In dead-end with intermittent flushing mode, an intermittent flushing flow (2 min after a 10-min or a 30-min dead-end filtration) was incorporated to reduce membrane fouling by flush out MSAB accumulated on membrane surface. Effects of solvent concentration and composition were also investigated. Solvent concentrations ranging from 0.1 to 1% (w/w) have no adverse effect in terms of membrane fouling. However, solvent composition, i.e. D(2)EHPA/kerosene ratio, shows impact on membrane fouling. The type of metal extractants employed in CASX has significant impact on both membrane fouling and the quality of filtrate due to the differences in their viscosity and water solubility. Separation of MSAB was the limiting process controlling metal removal efficiency, and the removal efficiency of Cd(II) and Cr(VI) followed the same trend as that for COD.

  3. Research resource: Monitoring endoplasmic reticulum membrane integrity in β-cells at the single-cell level.

    PubMed

    Kanekura, Kohsuke; Ou, Jianhong; Hara, Takashi; Zhu, Lihua J; Urano, Fumihiko

    2015-03-01

    Endoplasmic reticulum (ER) membrane integrity is an emerging target for human chronic diseases associated with ER stress. Despite the underlying importance of compromised ER membrane integrity in disease states, the entire process leading to ER membrane permeabilization and cell death is still not clear due to technical limitations. Here we describe a novel method for monitoring ER membrane integrity at the single-cell level in real time. Using a β-cell line expressing ER-targeted redox sensitive green fluorescent protein, we could identify a β-cell population undergoing ER membrane permeabilization induced by palmitate and could monitor cell fate and ER stress of these cells at the single-cell level. Our method could be used to develop a novel therapeutic modality targeting the ER membrane for ER-associated disorders, including β-cell death in diabetes, neurodegeneration, and Wolfram syndrome.

  4. 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.

  5. Electrocoagulation-integrated hybrid membrane processes for the treatment of tannery wastewater.

    PubMed

    Keerthi; Vinduja, V; Balasubramanian, N

    2013-10-01

    Three different combinations of treatment techniques, i.e. electrocoagulation combined with microfiltration (EMR), membrane bioreactor (MBR) and electrocoagulation integrated with membrane bioreactor (hybrid MBR, (HMBR)), were analysed and compared for the treatment of tannery wastewater operated for 7 days under the constant trans-membrane pressure of 5 kPa. HMBR was found to be most suitable in performance as well as fouling reduction, with 94 % of chemical oxygen demand (COD) removal, 100 % chromium removal and 8 % improvement in percentage reduction in permeate flux compared to MBR with only 90 % COD removal and 67 % chromium removal. The effect of mixed liquor suspended solids on fouling was also investigated and was found to be insignificant. EMR was capable of elevating the flux but was not as efficient as HMBR and MBR in COD removal. Fouling reduction by HMBR was further confirmed by SEM-EDX and particle size analysis.

  6. Structural and Functional Dynamics of an Integral Membrane Protein Complex Modulated by Lipid Headgroup Charge

    PubMed Central

    Li, Ji; James, Zachary M.; Dong, Xiaoqiong; Karim, Christine B.; Thomas, David D.

    2012-01-01

    We have used membrane surface charge to modulate the structural dynamics of an integral membrane protein, phospholamban (PLB), and thereby its functional inhibition of the sarcoplasmic reticulum Ca-ATPase (SERCA). It was previously shown by EPR, in vesicles of neutral lipids, that the PLB cytoplasmic domain is in equilibrium between an ordered T state and a dynamically disordered R state, and that phosphorylation of PLB increases the R state and relieves SERCA inhibition, suggesting that R is less inhibitory. Here we sought to control the T/R equilibrium by an alternative means – varying the lipid headgroup charge, thus perturbing the electrostatic interaction of PLB’s cationic cytoplasmic domain with the membrane surface. We resolved the T and R states not only by EPR in the absence of SERCA, but also by time-resolved fluorescence resonance energy transfer (TR-FRET) from SERCA to PLB, thus probing directly the SERCA-PLB complex. Compared to neutral lipids, anionic lipids increased both the T population and SERCA inhibition, while cationic lipids had the opposite effects. In contrast to conventional models, decreased inhibition was not accompanied by decreased binding. We conclude that PLB binds to SERCA in two distinct structural states of the cytoplasmic domain, an inhibitory T state that interacts strongly with the membrane surface, and a less inhibitory R state that interacts more strongly with the anionic SERCA cytoplasmic domain. Modulating membrane surface charge provides an effective way of investigating the correlation between structural dynamics and function of integral membrane proteins. PMID:22381409

  7. Preservation of Supported Lipid Membrane Integrity from Thermal Disruption: Osmotic Effect.

    PubMed

    Zhu, Tao; Jiang, Zhongying; Ma, Yuqiang; Hu, Yong

    2016-03-01

    Preservation of structural integrity under various environmental conditions is one major concern in the development of the supported lipid membrane (SLM)-based devices. It is common for SLMs to experience temperature shifts from manufacture, processing, storage, and transport to operation. In this work, we studied the thermal adaption of the supported membranes on silica substrates. Homogenous SLMs with little defects were formed through the vesicle fusion method. The mass and fluidity of the bilayers were found to deteriorate from a heating process but not a cooling process. Fluorescence characterizations showed that the membranes initially budded as a result of heating-induced lipid lateral area expansion, followed by the possible fates including maintenance, retraction, and fission, among which the last contributes to the irreversible compromise of the SLM integrity and spontaneous release of the interlipid stress accumulated. Based on the mechanism, we developed a strategy to protect SLMs from thermal disruption by increasing the solute concentration in medium. An improved preservation of the membrane mass and fluidity against the heating process was observed, accompanied by a decrease in the retraction and fission of the buds. Theoretical analysis revealed a high osmotic energy penalty for the fission, which accounts for the depressed disruption. This osmotic-based protection strategy is facile, solute nonspecific, and long-term efficient and has little impact on the original SLM properties. The results may help broaden SLM applications and sustain the robustness of SLM-based devices under multiple thermal conditions. PMID:26886864

  8. A band 3-based macrocomplex of integral and peripheral proteins in the RBC membrane

    SciTech Connect

    Bruce, Lesley J.; Beckmann, Roland; Ribeiro, M. Leticia; Peters, Luanne L.; Chasis, Joel A.; Delaunay, Jean; Mohandas, Narla; Anstee, David J.; Tanner, Michael J.A.

    2003-06-18

    We have studied the membrane proteins of band 3 anion exchanger (AE1)-deficient mouse and human red blood cells. It has been shown previously that proteins of the band 3 complex are reduced or absent in these cells. In this study we show that proteins of the Rh complex are also greatly reduced (Rh-associated glycoprotein, Rh polypeptides, CD47, glycophorin B) or absent (LW). These observations suggest that the Rh complex is associated with the band 3 complex in healthy RBCs. Mouse band 3 RBCs differed from the human band 3-deficient RBCs in that they retained CD47. Aquaporin 1 was reduced, and its glycosylation was altered in mouse and human band 3-deficient RBCs. Proteins of the glycophorin complex, and other proteins with independent cytoskeletal interactions, were present in normal or increased amounts. To obtain direct evidence for the association of the band 3 and the Rh protein complexes in the RBC, we examined whether Rh complex proteins were coimmunoprecipitated with band 3 from membranes. RhAG and Rh were found to be efficiently coimmunoprecipitated with band 3 from deoxycholate-solubilized membranes. Results suggest that band 3 forms the core of a macrocomplex of integral and peripheral RBC membrane proteins. The presence of these proteins in a single structural Macrocomplex makes it likely that they have linked functional or regulatory roles. We speculate that this macrocomplex may function as an integrated CO2/O2 gas exchange unit (metabolon) in the erythrocyte.

  9. 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

  10. Integration of Solid-State Nanopores in Microfluidic Networks via Transfer Printing of Suspended Membranes

    PubMed Central

    Jain, Tarun; Guerrero, Ricardo Jose S.; Aguilar, Carlos A.; Karnik, Rohit

    2013-01-01

    Solid-state nanopores have emerged as versatile single-molecule sensors for applications including DNA sequencing, protein unfolding, micro-RNA detection, label-free detection of single nucleotide polymorphisms, and mapping of DNA-binding proteins involved in homologous recombination. While machining nanopores in dielectric membranes provides nanometer-scale precision, the rigid silicon support for the membrane contributes capacitive noise and limits integration with microfluidic networks for sample pre-processing. Herein, we demonstrate a technique to directly transfer solid-state nanopores machined in dielectric membranes from a silicon support into a microfluidic network. The resulting microfluidic-addressable nanopores can sense single DNA molecules at high bandwidths and with low noise, owing to significant reductions in membrane capacitance. This strategy will enable large-scale integration of solid-state nanopores with microfluidic upstream and downstream processing and permit new functions with nanopores such as complex manipulations for multidimensional analysis and parallel sensing in two and three-dimensional architectures. PMID:23347165

  11. Integration of solid-state nanopores in microfluidic networks via transfer printing of suspended membranes.

    PubMed

    Jain, Tarun; Guerrero, Ricardo Jose S; Aguilar, Carlos A; Karnik, Rohit

    2013-04-16

    Solid-state nanopores have emerged as versatile single-molecule sensors for applications including DNA sequencing, protein unfolding, micro-RNA detection, label-free detection of single nucleotide polymorphisms, and mapping of DNA-binding proteins involved in homologous recombination. While machining nanopores in dielectric membranes provides nanometer-scale precision, the rigid silicon support for the membrane contributes capacitive noise and limits integration with microfluidic networks for sample preprocessing. Herein, we demonstrate a technique to directly transfer solid-state nanopores machined in dielectric membranes from a silicon support into a microfluidic network. The resulting microfluidic-addressable nanopores can sense single DNA molecules at high bandwidths and with low noise, owing to significant reductions in membrane capacitance. This strategy will enable large-scale integration of solid-state nanopores with microfluidic upstream and downstream processing and permit new functions with nanopores such as complex manipulations for multidimensional analysis and parallel sensing in two and three-dimensional architectures. PMID:23347165

  12. Preservation of Supported Lipid Membrane Integrity from Thermal Disruption: Osmotic Effect.

    PubMed

    Zhu, Tao; Jiang, Zhongying; Ma, Yuqiang; Hu, Yong

    2016-03-01

    Preservation of structural integrity under various environmental conditions is one major concern in the development of the supported lipid membrane (SLM)-based devices. It is common for SLMs to experience temperature shifts from manufacture, processing, storage, and transport to operation. In this work, we studied the thermal adaption of the supported membranes on silica substrates. Homogenous SLMs with little defects were formed through the vesicle fusion method. The mass and fluidity of the bilayers were found to deteriorate from a heating process but not a cooling process. Fluorescence characterizations showed that the membranes initially budded as a result of heating-induced lipid lateral area expansion, followed by the possible fates including maintenance, retraction, and fission, among which the last contributes to the irreversible compromise of the SLM integrity and spontaneous release of the interlipid stress accumulated. Based on the mechanism, we developed a strategy to protect SLMs from thermal disruption by increasing the solute concentration in medium. An improved preservation of the membrane mass and fluidity against the heating process was observed, accompanied by a decrease in the retraction and fission of the buds. Theoretical analysis revealed a high osmotic energy penalty for the fission, which accounts for the depressed disruption. This osmotic-based protection strategy is facile, solute nonspecific, and long-term efficient and has little impact on the original SLM properties. The results may help broaden SLM applications and sustain the robustness of SLM-based devices under multiple thermal conditions.

  13. Identification of Two Novel Endoplasmic Reticulum Body-Specific Integral Membrane Proteins1[W][OA

    PubMed Central

    Yamada, Kenji; Nagano, Atsushi J.; Nishina, Momoko; Hara-Nishimura, Ikuko; Nishimura, Mikio

    2013-01-01

    The endoplasmic reticulum (ER) body, a large compartment specific to the Brassicales, accumulates β-glucosidase and possibly plays a role in the defense against pathogens and herbivores. Although the ER body is a subdomain of the ER, it is unclear whether any ER body-specific membrane protein exists. In this study, we identified two integral membrane proteins of the ER body in Arabidopsis (Arabidopsis thaliana) and termed them MEMBRANE PROTEIN OF ENDOPLASMIC RETICULUM BODY1 (MEB1) and MEB2. In Arabidopsis, a basic helix-loop-helix transcription factor, NAI1, and an ER body component, NAI2, regulate ER body formation. The expression profiles of MEB1 and MEB2 are similar to those of NAI1, NAI2, and ER body β-glucosidase PYK10 in Arabidopsis. The expression of MEB1 and MEB2 was reduced in the nai1 mutant, indicating that NAI1 regulates the expression of MEB1 and MEB2 genes. MEB1 and MEB2 proteins localize to the ER body membrane but not to the ER network, suggesting that these proteins are specifically recruited to the ER body membrane. MEB1 and MEB2 physically interacted with ER body component NAI2, and they were diffused throughout the ER network in the nai2 mutant, which has no ER body. Heterologous expression of MEB1 and MEB2 in yeast (Saccharomyces cerevisiae) suppresses iron and manganese toxicity, suggesting that MEB1 and MEB2 are metal transporters. These results indicate that the membrane of ER bodies has specific membrane proteins and suggest that the ER body is involved in defense against metal stress as well as pathogens and herbivores. PMID:23166355

  14. On the Efficiency of NHS Ester Cross-Linkers for Stabilizing Integral Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Chen, Fan; Gerber, Sabina; Korkhov, Volodymyr M.; Mireku, Samantha; Bucher, Monika; Locher, Kaspar P.; Zenobi, Renato

    2015-03-01

    We have previously presented a straightforward approach based on high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to study membrane proteins. In addition, the stoichiometry of integral membrane protein complexes could be determined by MALDI-MS, following chemical cross-linking via glutaraldehyde. However, glutaraldehyde polymerizes in solution and reacts nonspecifically with various functional groups of proteins, limiting its usefulness for structural studies of protein complexes. Here, we investigated the capability of N-hydroxysuccinimide (NHS) esters, which react much more specifically, to cross-link membrane protein complexes such as PglK and BtuC2D2. We present clear evidence that NHS esters are capable of stabilizing membrane protein complexes in situ, in the presence of detergents such as DDM, C12E8, and LDAO. The stabilization efficiency strongly depends on the membrane protein structure (i.e, the number of primary amine groups and the distances between primary amines). A minimum number of primary amine groups is required, and the distances between primary amines govern whether a cross-linker with a specific spacer arm length is able to bridge two amine groups.

  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. 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.

  17. Integration of isolated cell membrane patches in nanomachined apertures for single ion channel recording

    NASA Astrophysics Data System (ADS)

    Niels, Fertig; Behrends, Jan; Blick, Robert

    2001-03-01

    We apply nanostructuring techniques to machine apertures in semiconductor materials with the aim of single channel recording of ion channels in cell membranes. This approach will overcome many limitations of the classical patch-clamp technique. The integration of nanostructured devices 'on-chip' promises novel types of experiments on single ion channels. We investigate the microscopic contact of a cell/semiconductor hybrid and demonstrate single channel recording.

  18. Bat3 promotes the membrane integration of tail-anchored proteins.

    PubMed

    Leznicki, Pawel; Clancy, Anne; Schwappach, Blanche; High, Stephen

    2010-07-01

    The membrane integration of tail-anchored proteins at the endoplasmic reticulum (ER) is post-translational, with different tail-anchored proteins exploiting distinct cytosolic factors. For example, mammalian TRC40 has a well-defined role during delivery of tail-anchored proteins to the ER. Although its Saccharomyces cerevisiae equivalent, Get3, is known to function in concert with at least four other components, Get1, Get2, Get4 and Get5 (Mdy2), the role of additional mammalian proteins during tail-anchored protein biogenesis is unclear. To this end, we analysed the cytosolic binding partners of Sec61beta, a well-defined substrate of TRC40, and identified Bat3 as a previously unknown interacting partner. Depletion of Bat3 inhibits the membrane integration of Sec61beta, but not of a second, TRC40-independent, tail-anchored protein, cytochrome b5. Thus, Bat3 influences the in vitro membrane integration of tail-anchored proteins using the TRC40 pathway. When expressed in Saccharomyces cerevisiae lacking a functional GET pathway for tail-anchored protein biogenesis, Bat3 associates with the resulting cytosolic pool of non-targeted chains and diverts it to the nucleus. This Bat3-mediated mislocalisation is not dependent upon Sgt2, a recently identified component of the yeast GET pathway, and we propose that Bat3 either modulates the TRC40 pathway in higher eukaryotes or provides an alternative fate for newly synthesised tail-anchored proteins.

  19. Different Transmembrane Domains Associate with Distinct Endoplasmic Reticulum Components during Membrane Integration of a Polytopic Protein

    PubMed Central

    Meacock, Suzanna L.; Lecomte, Fabienne J.L.; Crawshaw, Samuel G.; High, Stephen

    2002-01-01

    We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the α and β subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61α and Sec61β during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the “stage” of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide. PMID:12475939

  20. 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. PMID:26879205

  1. Refinement of the localization of the gene for human intra-acrosomal protein SP-10 (ACRV1) to the junction of bands q23 [yields] q24 of chromosome 11 by nonisotopic in situ hybridization

    SciTech Connect

    Golden, W.L.; Von Kap-Herr, C.; Kurth, B.; Kurth, B.; Wright, R.M.; Flickinger, C.J.; Herr, J.C. ); Eddy, R.; Shows, T. )

    1993-11-01

    The human sperm antigen SP-10 is a testis-specific protein associated with the matrix of the acrosomal vesicle in developing spermatids and the acrosomal matrix and membranes of ejaculated sperm. A previous study, utilizing somatic cell hybrids, localized the gene for SP-10 to chromosome 11 and assigned the gene symbol ACRV1 (acrosomal vesicle protein-1). Although previous analysis of several somatic cell hybrids containing portions of chromosome 11 indicated that ACRV1 was in the p12 [yields] q13 region, the present fluorescence in situ hybridization studies using cDNA, ribo, and genomic versions of probes for SP-10 coupled to analysis of an expanded series of somatic cell hybrids demonstrated the refined localization of ACRV1 to the junction of bands q23 and q24 of chromosome 11. A comparison of the three types of probes used for the in situ study demonstrated that while the genomic probe hybridized most efficiently, the riboprobe hybridized to the same location and was superior to the cDNA probe in mapping this single-copy gene. This report emphasizes the utility of riboprobes for chromosome localization of single-copy genes. 12 refs., 2 figs.

  2. Mechanisms of integration of de novo-synthesized polypeptides into membranes: signal-recognition particle is required for integration into microsomal membranes of calcium ATPase and of lens MP26 but not of cytochrome b5.

    PubMed

    Anderson, D J; Mostov, K E; Blobel, G

    1983-12-01

    We have investigated the in vitro integration into dog pancreas microsomal membranes of three integral membrane proteins that were synthesized de novo in a wheat germ cell-free translation system: calcium ATPase of rabbit sarcoplasmic reticulum, MP26 of bovine lens fiber plasma membrane, and rat liver cytochrome b5. Biosynthetically these proteins show a common feature in that they are synthesized without a transient NH2-terminal signal sequence. Two of these proteins, ATPase and MP26, were shown to require the recently discovered signal-recognition particle (SRP) [Walter, P. & Blobel, G. (1982) Nature (London) 299, 691-698] for integration. By this criterion, therefore, they each contain at least one uncleaved signal sequence. Surprisingly, however, the uncleaved signal sequence(s) of these two proteins did not induce the characteristic SRP-mediated translation arrest that was previously shown for a cleaved signal sequence. Unlike ATPase and MP26, cytochrome b5 did not require SRP for integration into microsomal membrane. Thus, the distinction between an "insertion" sequence (specifying unassisted and opportunistic integration into any exposed membrane) and a "signal" sequence (directing integration into a specific membrane by a receptor-mediated mechanism) is a valid one. By assaying for SRP dependence, the two mechanisms of integration can now be experimentally distinguished.

  3. 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

  4. 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

  5. Enriching membrane cholesterol improves stability and cryosurvival of buffalo spermatozoa.

    PubMed

    Rajoriya, J S; Prasad, J K; Ramteke, S S; Perumal, P; Ghosh, S K; Singh, M; Pande, Megha; Srivastava, N

    2016-01-01

    Buffalo spermatozoa are comparatively more susceptible to freezing hazards than cattle spermatozoa. In recent times incubation of spermatozoa with cholesterol-loaded-cyclodextrins (CLC) has shown improvements in semen quality in several species. Therefore, this study was undertaken to evaluate the incubation level of CLC at which maximum benefit is derived for the buffalo spermatozoa. For the study, 120 million spermatozoa were incubated in 2, 3 and 4 mg/mL of CLC (Gr II, III and IV, respectively) and cholesterol and phospholipids content, their ratio, flow cytometric evaluation of plasma membrane integrity (PMI), plasma membrane fluidity and extent of cryoinjury (Chlortetracycline, CTC assay) were compared with an untreated control (Gr I). Additionally the ability of cholesterol-loaded-spermatozoa to undergo induced acrosome reaction (IAR) using ionophore calcium (A23187) was evaluated in frozen-thaw samples. Data show a significant and linear increase (CV=0.88) in cholesterol content of spermatozoa in Gr II, III and IV and a significant decrease in phospholipids content at frozen-thaw stage in Gr IV than Gr III spermatozoa. The study revealed a significant improvement in PMI and significant reduction in plasma membrane fluidity and cryoinjury of CLC treated spermatozoa at progressive stages in three groups compared to control. Nevertheless, spermatozoa of Gr II, III and IV were significantly less responsive to ionophore calcium (A23187) than Gr I. This study shows for the first time that incubation of buffalo bull spermatozoa with CLC (3mg/120×10(6)) prior to processing permits greater numbers of sperm to survive cryopreservation while allowing spermatozoa to capacitate and the acrosome to react to AR inducer ionophore calcium (A23187).

  6. Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability[W

    PubMed Central

    Schapire, Arnaldo L.; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A.

    2008-01-01

    Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca2+-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca2+-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness. PMID:19088329

  7. 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.

  8. 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

  9. 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). PMID:25841189

  10. 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).

  11. 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.

  12. 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. PMID:25966331

  13. Eugenol alters the integrity of cell membrane and acts against the nosocomial pathogen Proteus mirabilis.

    PubMed

    Devi, K Pandima; Sakthivel, R; Nisha, S Arif; Suganthy, N; Pandian, S Karutha

    2013-03-01

    Eugenol, a member of the phenylpropanoids class of chemical compounds, is a clear to pale yellow oily liquid extracted from certain essential oils especially from clove oil, nutmeg, cinnamon, and bay leaf. The antibacterial activity of eugenol and its mechanism of bactericidal action against Proteus mirabilis were evaluated. Treatment with eugenol at their minimum inhibitory concentration [0.125 % (v/v)] and minimum bactericidal concentration [0.25 % (v/v)] reduced the viability and resulted in complete inhibition of P. mirabilis. A strong bactericidal effect on P. mirabilis was also evident, as eugenol inactivated the bacterial population within 30 min exposure. Chemo-attractant property and the observance of highest antibacterial activity at alkaline pH suggest that eugenol can work more effectively when given in vivo. Eugenol inhibits the virulence factors produced by P. mirabilis as observed by swimming motility, swarming behavior and urease activity. It interacts with cellular membrane of P. mirabilis and makes it highly permeable, forming nonspecific pores on plasma membrane, which in turn directs the release of 260 nm absorbing materials and uptake of more crystal violet from the medium into the cells. SDS-polyacrylamide gel, scanning electron microscopy and Fourier transform infrared analysis further proves the disruptive action of eugenol on the plasma membrane of P. mirabilis. The findings reveal that eugenol shows an excellent bactericidal activity against P. mirabilis by altering the integrity of cell membrane. PMID:23444040

  14. 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

  15. 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.

  16. 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

  17. Nicotinic infertility: assessing DNA and plasma membrane integrity of human spermatozoa.

    PubMed

    Arabi, M

    2004-10-01

    Infertility remains a major problem in society, with recent data suggesting its presence in one of four couples. The objective of the present study was to evaluate the impact of nicotine (0.25, 0.5 and 0.75 mm), as a major component of cigarette smoke, in vitro, on sperm membrane [by spermatocrit and lipoperoxidation (LPO) tests], DNA integrity (by Comet assay), and viability of spermatozoa (by eosin staining) from normozoospermic men. Sperm samples were washed and diluted with phosphate-buffered saline. A drop in spermatocrit values and an increase in thiobarbituric acid-reactive substances/LPO rate was observed with the addition of nicotine, predominantly at a concentration of 0.75 mm, indicating a deleterious effect of nicotine on sperm membrane intactness. There was also a strong negative correlation between LPO rate and percentage viable sperm cell (r = -0.990). Data obtained from Comet assay technique revealed that nicotine could induce double-stranded DNA breaks (11% in 0.75 mm concentration) in the sperm nuclei. The value of r between LPO rate and percentage Comets was found to be +0.976. Taken together, nicotine proved to be a potential oxidant agent in the category of environmental factors to the integrity of sperm plasma membrane and DNA. PMID:15458549

  18. 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.

  19. Cell-free synthesis, functional refolding, and spectroscopic characterization of bacteriorhodopsin, an integral membrane protein.

    PubMed

    Sonar, S; Patel, N; Fischer, W; Rothschild, K J

    1993-12-21

    Bacteriorhodopsin (bR) is an integral membrane protein which functions as a light-driven proton pump in Halobacterium halobium (also known as Halobacterium salinarium). The cell-free synthesis of bR in quantities sufficient for FTIR and NMR spectroscopy and the ability to selectively isotope label bR using aminoacylated suppressor tRNAs would provide a powerful approach for studying the role of specific amino acid residues. However, no integral membrane protein has yet been expressed in a cell-free system in quantities sufficient for such biophysical studies. We report the cell-free synthesis of bacterioopsin, its purification, its refolding in polar lipids from H. halobium, and its regeneration with all-trans-retinal to yield bacteriorhodopsin in a form functionally similar to bR in purple membrane. Importantly, the yields obtained from in vitro and in vivo expression are comparable. Functionality of the cell-free expressed bR is established using static and time-resolved absorption spectroscopy and FTIR difference spectroscopy.

  20. Effects of cigarette smoking on sperm plasma membrane integrity and DNA fragmentation.

    PubMed

    Belcheva, Antoaneta; Ivanova-Kicheva, Maria; Tzvetkova, Petia; Marinov, Mihail

    2004-10-01

    Cigarette smoking is a serious health problem of our society. It is known that cigarette smoke is a cell mutagen and carcinogen, and that it may affect adversely male fertility. The possible detrimental effects on sperm cells are of great interest but the data available to support this statement are somewhat elusive. To approach this problem we examined conventional semen parameters, plasma membrane translocation of phosphatidylserine (PS) (annexin V/6-CFDA cell staining) and sperm DNA integrity (comet assay) in a group of healthy man smoking cigarettes on a regular basis. The results of the study were compared with the results of the same tests in healthy non-smoking donors. Significant difference in standard sperm parameters between the two groups was not found. Intensive expression of PS on the sperm plasma membrane surface (assayed by annexin V positive staining) was detected in the smokers group. There is a significant increase of population of apoptotic spermatozoa in ejaculates of smokers. Albeit DNA damages (high frequencies of double- and single- stranded DNA breaks) in spermatozoa of smokers are increased compared with non-smokers, but this difference is not statistically significant. Sperm DNA integrity of healthy smokers remains in the normal range, but a clear negative trend is observed, especially in respect of disturbance of plasma membrane phospholipid asymmetry.

  1. The signaling module cAMP/Epac/Rap1/PLCε/IP3 mobilizes acrosomal calcium during sperm exocytosis.

    PubMed

    Lucchesi, Ornella; Ruete, María C; Bustos, Matías A; Quevedo, María F; Tomes, Claudia N

    2016-04-01

    Exocytosis of the sperm's single secretory granule, or acrosome, is a regulated exocytosis triggered by components of the egg's investments. In addition to external calcium, sperm exocytosis (termed the acrosome reaction) requires cAMP synthesized endogenously and calcium mobilized from the acrosome through IP3-sensitive channels. The relevant cAMP target is Epac. In the first part of this paper, we present a novel tool (the TAT-cAMP sponge) to investigate cAMP-related signaling pathways in response to progesterone as acrosome reaction trigger. The TAT-cAMP sponge consists of the cAMP-binding sites of protein kinase A regulatory subunit RIβ fused to the protein transduction domain TAT of the human immunodeficiency virus-1. The sponge permeated into sperm, sequestered endogenous cAMP, and blocked exocytosis. Progesterone increased the population of sperm with Rap1-GTP, Rab3-GTP, and Rab27-GTP in the acrosomal region; pretreatment with the TAT-cAMP sponge prevented the activation of all three GTPases. In the second part of this manuscript, we show that phospholipase Cε (PLCε) is required for the acrosome reaction downstream of Rap1 and upstream of intra-acrosomal calcium mobilization. Last, we present direct evidence that cAMP, Epac, Rap1, and PLCε are necessary for calcium mobilization from sperm's secretory granule. In summary, we describe here a pathway that connects cAMP to calcium mobilization from the acrosome during sperm exocytosis. Never before had direct evidence for each step of the cascade been put together in the same study.

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

    PubMed Central

    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. PMID:26300347

  3. Capacitated acrosome-intact mouse spermatozoa bind to Sepharose beads coated with functional neoglycoproteins.

    PubMed

    Bendahmane, Malika; Tulsiani, Daulat R P

    2003-07-15

    Capacitated acrosome-intact mouse spermatozoa bind to the egg's extracellular coat, the zona pellucida (ZP), in a carbohydrate-mediated receptor-ligand manner. The tight irreversible binding of the opposite gametes triggers a signal transduction pathway resulting in the exocytosis of acrosomal contents (i.e., induction of the acrosome reaction [AR]). Previously, we demonstrated that a hexose (mannose) and two amino sugars (N-acetylglucosamine and N-acetylgalactosamine), when covalently conjugated to bovine serum albumin (BSA) (functional neoglycoproteins, ngps), mimicked mZP3 and induced the AR [Biol. Reprod. 60 (1999) 94-101]. To further elucidate the specificity of sperm-ngp interaction and the mZP3 mimicking role of the functional ngps, we have examined binding of the mouse spermatozoa to Sepharose 4B beads coated with the functional and non-functional ngps as well as BSA, ovalbumin (OVA), or asialofetuin (ASF). A significantly greater number of capacitated acrosome-intact spermatozoa bound to the beads coated with functional ngps than the beads coated with non-functional ngps, BSA, OVA, or ASF. The binding was temperature-sensitive and was highest when the sperm-bead assay was carried out at 37 degrees C. Blocking of in vitro capacitation, by including calmodulin antagonists in the incubation medium, prevented sperm from binding to the beads. Furthermore, inclusion of free sugars (mannose, N-acetylglucosamine, or N-acetylgalactosamine) in the binding assay, either individually or as a mixture, inhibited sperm-bead binding in a concentration-dependent manner. Taken together, our data provide evidence strongly suggesting that binding of capacitated spermatozoa to the ngp-coated Sepharose beads is specific. The beads that mimic zona-intact eggs provide an excellent tool for examining pharmacological effects of reagents that alter the sperm function. In addition, the immobilized ngp(s) will be useful as an affinity medium to isolate the sperm surface receptor

  4. 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

  5. Sperm Nuclear Vacuoles in relation to Acrosome Reactions and Sperm Motility

    PubMed Central

    Kawauchi, Yoko; Kato, Tomonori; Tanii, Ichiro; Fuse, Hideki

    2014-01-01

    We investigated sperm nuclear vacuolation in relation to acrosome reactions and the maintenance of sperm motility. Thirty male patients who visited our Male Infertility Clinic were enrolled. These patients underwent conventional semen analyses, Acrobeads tests, and high-magnification observation of the sperm head to evaluate the degree of nuclear vacuolation on the Acrobeads test scoring after 24 hours of incubation. The presence of acrosome reactions was evaluated using the Acrobeads test. The spermatozoa were classified into three groups: (I) those bound to MH61-beads, (II) motile spermatozoa that did not bind to MH61-beads, and (III) immotile spermatozoa that did not bind to MH61-beads. The percentage of spermatozoa with large nuclear vacuoles (%LNV) was compared between the three groups. The degree of sperm nuclear vacuolation was evaluated in 17,992 ejaculated spermatozoa. The mean %LNVs were 2.4% in group I, 5.8% in group II, and 9.8% in group III. These values were significantly different from each other (P < 0.001, paired t-test). There were no correlations between the %LNV values and the Acrobeads scores. In conclusion, the degree of sperm nuclear vacuolation was significantly lower in the acrosome-reacted spermatozoa and spermatozoa with maintained motility, and higher in the immotile spermatozoa that did not bind to MH61-beads. PMID:25097868

  6. Sperm nuclear vacuoles in relation to acrosome reactions and sperm motility.

    PubMed

    Komiya, Akira; Kawauchi, Yoko; Kato, Tomonori; Watanabe, Akihiko; Tanii, Ichiro; Fuse, Hideki

    2014-01-01

    We investigated sperm nuclear vacuolation in relation to acrosome reactions and the maintenance of sperm motility. Thirty male patients who visited our Male Infertility Clinic were enrolled. These patients underwent conventional semen analyses, Acrobeads tests, and high-magnification observation of the sperm head to evaluate the degree of nuclear vacuolation on the Acrobeads test scoring after 24 hours of incubation. The presence of acrosome reactions was evaluated using the Acrobeads test. The spermatozoa were classified into three groups: (I) those bound to MH61-beads, (II) motile spermatozoa that did not bind to MH61-beads, and (III) immotile spermatozoa that did not bind to MH61-beads. The percentage of spermatozoa with large nuclear vacuoles (%LNV) was compared between the three groups. The degree of sperm nuclear vacuolation was evaluated in 17,992 ejaculated spermatozoa. The mean %LNVs were 2.4% in group I, 5.8% in group II, and 9.8% in group III. These values were significantly different from each other (P < 0.001, paired t-test). There were no correlations between the %LNV values and the Acrobeads scores. In conclusion, the degree of sperm nuclear vacuolation was significantly lower in the acrosome-reacted spermatozoa and spermatozoa with maintained motility, and higher in the immotile spermatozoa that did not bind to MH61-beads.

  7. Beclin orthologs: integrative hubs of cell signaling, membrane trafficking, and physiology

    PubMed Central

    Levine, Beth; Liu, Rong; Dong, Xiaonan; Zhong, Qing

    2015-01-01

    The Beclin family, including yeast Atg6 (autophagy related gene 6), its orthologs in higher eukaryotic species, and the more recently characterized mammalian-specific Beclin 2, are essential molecules in autophagy and other membrane-trafficking events. Extensive studies of Beclin orthologs have provided considerable insights into the regulation of autophagy, the diverse roles of autophagy in physiology and disease, and potential new strategies to modulate autophagy in a variety of clinical diseases. In this review we discuss the functions of Beclin 1 orthologs, the regulation of such functions by diverse cellular signaling pathways, and the effects of such regulation on downstream cellular processes including tumor suppression and metabolism. These findings suggest that Beclin orthologs serve as crucial molecules that integrate diverse environmental signals with membrane trafficking events to ensure optimal responses of the cell to stressful stimuli. PMID:26071895

  8. 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.

  9. Up against the wall: is yeast cell wall integrity ensured by mechanosensing in plasma membrane microdomains?

    PubMed

    Kock, Christian; Dufrêne, Yves F; Heinisch, Jürgen J

    2015-02-01

    Yeast cell wall integrity (CWI) signaling serves as a model of the regulation of fungal cell wall synthesis and provides the basis for the development of antifungal drugs. A set of five membrane-spanning sensors (Wsc1 to Wsc3, Mid2, and Mtl1) detect cell surface stress and commence the signaling pathway upon perturbations of either the cell wall structure or the plasma membrane. We here summarize the latest advances in the structure/function relationship primarily of the Wsc1 sensor and critically review the evidence that it acts as a mechanosensor. The relevance and physiological significance of the information obtained for the function of the other CWI sensors, as well as expected future developments, are discussed.

  10. Reverse micelles in integral membrane protein structural biology by solution NMR spectroscopy

    PubMed Central

    Kielec, Joseph M.; Valentine, Kathleen G.; Babu, Charles R.; Wand, A. Joshua

    2009-01-01

    SUMMARY Integral membrane proteins remain a significant challenge to structural studies by solution NMR spectroscopy. This is due not only to spectral complexity but also because the effects of slow molecular reorientation are exacerbated by the need to solublize the protein in aqueous detergent micelles. These assemblies can be quite large and require deuteration for use of the TROSY effect. In principle, another approach is to employ reverse micelle encapsulation to solublize the protein in a low viscosity solvent where the rapid tumbling of the resulting particle allows use of standard triple resonance methods. The preparation of such samples of membrane proteins is difficult. Using a 54 kDa construct of the homotetrameric potassium channel KcsA we demonstrate a strategy that employs a hybrid surfactant to transfer the protein to the reverse micelle system. PMID:19278649

  11. 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.

  12. 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

  13. 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. PMID:26773483

  14. 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.

  15. Biodegradable, Tethered Lipid Bilayer-Microsphere Systems with Membrane-Integrated α-Helical Peptide Anchors.

    PubMed

    Fried, Eric S; Luchan, Joshua; Gilchrist, M Lane

    2016-04-12

    Supported lipid bilayers (SLBs) are ideally suited for the study of biomembrane-biomembrane interactions and for the biomimicry of cell-to-cell communication, allowing for surface ligand displays that contain laterally mobile elements. However, the SLB paradigm does not include three-dimensionality and biocompatibility. As a way to bypass these limitations, we have developed a biodegradable form of microsphere SLBs, also known as proteolipobeads (PLBs), using PLGA microspheres. Microspheres were synthesized using solvent evaporation and size selected with fluorescence activated cell sorting (FACS). Biomembranes were covalently tethered upon fusion to microsphere supports via short-chain PEG spacers connecting membrane-integrated α-helical peptides and the microsphere surface, affecting membrane diffusivity and mobility as indicated by confocal FRAP analysis. Membrane heterogeneities, which are attributed to PLGA hydrophobicity and rough surface topography, are curtailed by the addition of PEG tethers. This method allows for the presentation of tethered, laterally mobile biomembranes in three dimensions with functionally embedded attachment peptides for mobile ligand displays.

  16. 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.

  17. Biodegradable, Tethered Lipid Bilayer-Microsphere Systems with Membrane-Integrated α-Helical Peptide Anchors.

    PubMed

    Fried, Eric S; Luchan, Joshua; Gilchrist, M Lane

    2016-04-12

    Supported lipid bilayers (SLBs) are ideally suited for the study of biomembrane-biomembrane interactions and for the biomimicry of cell-to-cell communication, allowing for surface ligand displays that contain laterally mobile elements. However, the SLB paradigm does not include three-dimensionality and biocompatibility. As a way to bypass these limitations, we have developed a biodegradable form of microsphere SLBs, also known as proteolipobeads (PLBs), using PLGA microspheres. Microspheres were synthesized using solvent evaporation and size selected with fluorescence activated cell sorting (FACS). Biomembranes were covalently tethered upon fusion to microsphere supports via short-chain PEG spacers connecting membrane-integrated α-helical peptides and the microsphere surface, affecting membrane diffusivity and mobility as indicated by confocal FRAP analysis. Membrane heterogeneities, which are attributed to PLGA hydrophobicity and rough surface topography, are curtailed by the addition of PEG tethers. This method allows for the presentation of tethered, laterally mobile biomembranes in three dimensions with functionally embedded attachment peptides for mobile ligand displays. PMID:26972467

  18. 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.

  19. Membrane-integral pyrophosphatase subfamily capable of translocating both Na+ and H+.

    PubMed

    Luoto, Heidi H; Baykov, Alexander A; Lahti, Reijo; Malinen, Anssi M

    2013-01-22

    One of the strategies used by organisms to adapt to life under conditions of short energy supply is to use the by-product pyrophosphate to support cation gradients in membranes. Transport reactions are catalyzed by membrane-integral pyrophosphatases (PPases), which are classified into two homologous subfamilies: H(+)-transporting (found in prokaryotes, protists, and plants) and Na(+)-transporting (found in prokaryotes). Transport activities have been believed to require specific machinery for each ion, in accordance with the prevailing paradigm in membrane transport. However, experiments using a fluorescent pH probe and (22)Na(+) measurements in the current study revealed that five bacterial PPases expressed in Escherichia coli have the ability to simultaneously translocate H(+) and Na(+) into inverted membrane vesicles under physiological conditions. Consistent with data from phylogenetic analyses, our results support the existence of a third, dual-specificity bacterial Na(+),H(+)-PPase subfamily, which apparently evolved from Na(+)-PPases. Interestingly, genes for Na(+),H(+)-PPase have been found in the major microbes colonizing the human gastrointestinal tract. The Na(+),H(+)-PPases require Na(+) for hydrolytic and transport activities and are further activated by K(+). Based on ionophore effects, we conclude that the Na(+) and H(+) transport reactions are electrogenic and do not result from secondary antiport effects. Sequence comparisons further disclosed four Na(+),H(+)-PPase signature residues located outside the ion conductance channel identified earlier in PPases using X-ray crystallography. Our results collectively support the emerging paradigm that both Na(+) and H(+) can be transported via the same mechanism, with switching between Na(+) and H(+) specificities requiring only subtle changes in the transporter structure.

  20. 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

  1. Hemorrhagic shock impairs myocardial cell volume regulation and membrane integrity in dogs

    SciTech Connect

    Horton, J.W.

    1987-06-01

    An in vitro myocardial slice technique was used to quantitate alterations in cell volume regulation and membrane integrity after 2 h or hemorrhagic shock. After in vitro incubation in Krebs-Ringer-phosphate medium containing trace (/sup 14/C)inulin, values (ml H/sub 2/O/g dry wt) for control nonshocked myocardial slices were 4.03 /plus minus/ 0.11 (SE) for total water, 2.16 /plus minus/ 0.07 for inulin impermeable space, and 1.76 /plus minus/ 0.15 for inulin diffusible space. Shocked myocardial slices showed impaired response to cold incubation. After 2 h of in vivo shock, total tissue water, inulin diffusible space, and inulin impermeable space increased significantly for subendocardium, whereas changes in subepicardium parameters were minimal. Shock-induced cellular swelling was accompanied by an increased total tissue sodium, but no change in tissue potassium. Calcium entry blockade in vivo significantly reduced subendocardial total tissue water as compared with shock-untreated dogs. In addition, calcium entry blockade reduced shock-induced increases in inulin diffusible space. In vitro myocardial slice studies confirm alterations in subendocardial membrane integrity after 2 h of in vivo hemorrhagic shock. Shock-induced abnormalities in myocardial cell volume regulation are reduced by calcium entry blockade in vivo.

  2. 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

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

    PubMed Central

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

    2013-01-01

    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. PMID:26074721

  4. 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.

  5. 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. PMID:25218103

  6. Sperm cephalic vacuoles: new arguments for their non acrosomal origin in two cases of total globozoospermia.

    PubMed

    Gatimel, N; Léandri, R D; Foliguet, B; Bujan, L; Parinaud, J

    2013-01-01

    To add new arguments concerning the origin of the sperm-head vacuoles observed under high magnification with interference contrast microscopy, we carried out in two patients with total globozoospermia confirmed using transmission electron microscopy (TEM), a detailed sperm morphometric analysis with high magnification (×6000) under Nomarski contrast, an acrosomal status analysis (using fluorescent labelling with peanut agglutinin (PNA) lectins and anti-CD46 antibodies) and a nuclear status analysis (using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay TUNEL, sperm chromatin structure assay SCSA and aniline blue staining). Our two patients with globozoospermia had relative sperm vacuole areas of 6.3% and 5%, similar to those observed in a reference population of 12 fertile men (5.9%). TUNEL and SCSA assays gave normal results in both patients, although the percentage of immature nuclei using aniline blue staining was increased (27 and 46% for patient 1 and 2 respectively). Cytofluorescence and TEM analysis evidence differences between the two patients: although no acrosomal neither Golgi residue could be detected in patient 1, patient 2 had positive PNA lectin labelling for 9% of spermatozoa and Golgi residues were seen using electron microscopy. Unlike patient 1, a live birth could be obtained after intracytoplasmic sperm injection (ICSI) for patient 2. This descriptive study of two patients with total globozoospermia confirmed using TEM argue in favour of a deep analysis of total globozoospermia before assisted reproductive technology and provides further information on the non-acrosomal origin of the sperm-head vacuoles observed under high magnification.

  7. [The study on the characters of membrane protein interaction and its network based on integrated intelligence method].

    PubMed

    Shen, Yizhen; Ding, Yongsheng; Hao, Kuangrong

    2011-08-01

    Membrane protein and its interaction network have become a novel research direction in bioinformatics. In this paper, a novel membrane protein interaction network simulator is proposed for system biology studies by integrated intelligence method including spectrum analysis, fuzzy K-Nearest Neighbor(KNN) algorithm and so on. We consider biological system as a set of active computational components interacting with each other and with the external environment. Then we can use the network simulator to construct membrane protein interaction networks. Based on the proposed approach, we found that the membrane protein interaction network almost has some dynamic and collective characteristics, such as small-world network, scale free distributing, and hierarchical module structure. These properties are similar to those of other extensively studied protein interaction networks. The present studies on the characteristics of the membrane protein interaction network will be valuable for its relatively biological and medical studies. PMID:21936357

  8. Charged gels as orienting media for measurement of residual dipolar couplings in soluble and integral membrane proteins.

    PubMed

    Cierpicki, Tomasz; Bushweller, John H

    2004-12-15

    Measurement of residual dipolar couplings for membrane proteins will dramatically improve the quality of the structures obtainable by solution NMR spectroscopy. While there has been some success in achieving alignment of membrane-bound peptides, there has been very limited success in achieving alignment for functional membrane proteins. Herein, we demonstrate that charged polyacrylamide-based copolymers are suitable for obtaining weak alignment of membrane proteins reconstituted in detergent micelles. Varying the copolymer compositions, we prepared positively, zwitterionic, and negatively charged gels that are very stable at low concentration and can be used for obtaining weak alignment by compression in an NMR tube. Application of this method is demonstrated for the integral membrane protein OmpA in DPC micelles.

  9. Charged gels as orienting media for measurement of residual dipolar couplings in soluble and integral membrane proteins.

    PubMed

    Cierpicki, Tomasz; Bushweller, John H

    2004-12-15

    Measurement of residual dipolar couplings for membrane proteins will dramatically improve the quality of the structures obtainable by solution NMR spectroscopy. While there has been some success in achieving alignment of membrane-bound peptides, there has been very limited success in achieving alignment for functional membrane proteins. Herein, we demonstrate that charged polyacrylamide-based copolymers are suitable for obtaining weak alignment of membrane proteins reconstituted in detergent micelles. Varying the copolymer compositions, we prepared positively, zwitterionic, and negatively charged gels that are very stable at low concentration and can be used for obtaining weak alignment by compression in an NMR tube. Application of this method is demonstrated for the integral membrane protein OmpA in DPC micelles. PMID:15584763

  10. Folded state of the integral membrane colicin E1 immunity protein in solvents of mixed polarity.

    PubMed

    Taylor, R M; Zakharov, S D; Bernard Heymann, J; Girvin, M E; Cramer, W A

    2000-10-10

    The colicin E1 immunity protein (ImmE1), a 13.2-kDa hydrophobic integral membrane protein localized in the Escherichia coli cytoplasmic membrane, protects the cell from the lethal, channel-forming activity of the bacteriocin, colicin E1. Utilizing its solubility in organic solvents, ImmE1 was purified by 1-butanol extraction of isolated membranes, followed by gel filtration and ion-exchange chromatography in a chloroform/methanol/H(2)O (4:4:1) solvent system. Circular dichroism analysis indicated that the alpha-helical content of ImmE1 is approximately 80% in 1-butanol or 2,2,2-trifluoroethanol, consistent with a previous membrane-folding model with three extended hydrophobic transmembrane helical domains, H1-H3. Each of these extended hydrophobic domains contains a centrally located single Cys residue that could be used as a probe of protein structure. The presence of tertiary structure of purified ImmE1 in a solvent of mixed polarity, chloroform/methanol/H(2)O (4:4:1) was demonstrated by (i) the constraints on Tyr residues shown by the amplitude of near-UV circular dichroism spectra in the wavelength interval, 270-285 nm; (ii) the correlation between the near-UV Tyr CD spectrum of single and double Cys-to-X mutants of the Imm protein and their in vivo activity; (iii) the upfield shift of methyl groups in a 1D NMR spectrum, a 2D- HSQC NMR spectrum of ImmE1 in the mixed polarity solvent mixture, and a broadening and disappearance of the indole (1)H proton resonance from Trp94 in H3 by a spin label attached to Cys16 in the H2 hydrophobic domain; (iv) near-UV circular dichroism spectra with a prominent ellipticity band centered at 290 nm from a single Trp inserted into the extended hydrophobic domains. It was concluded that the colicin E1 immunity protein adopts a folded conformation in chloroform/methanol/H(2)O (4:4:1) that is stabilized by helix-helix interactions. Analysis of the probable membrane folding topology indicated that several Tyr residues in the bilayer

  11. 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.

  12. GABA/progesterone-induced polyphosphoinositide (PPI) breakdown and its role in the acrosome reaction of guinea pig spermatozoa in vitro.

    PubMed

    Yuan, Y; Mao, L; Shi, Q; Roldan, E R; Chen, W; Yu, S; Zhuang, Y; Xu, S

    2001-08-01

    To investigate whether GABA/progesterone (P(4)) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were preincubated in MCM-LCa(2+) for 5.5 h and then labeled with [(32)P]pi for 1 h. Samples were washed through a three-step gradient Percoll, adjusted to 5x10(7) cells/mL and exposed to 2 mmol/L Ca(2+), 5 micromol/L GABA, 10 micromol/L P(4) and other agents. Lipids were separated by t.l.c. and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microscopy. The results showed that (i) when spermatozoa were treated with GABA,(32)P-label diminished rapidly in phosphatidylinositol 4, 5-bisphosphate (PIP(2)), phosphatidylinositol 4-phosphate (PIP), and increased in phosphatidic acid (PA). The loss of label from PPI was almost completed by 10 min. The time-course of the AR was much slower than PPI when spermatozoa reached a maximal response by 15 min; (ii) the pattern of PPI hydrolysis and stimulation of AR was similar for the three agonists tested; their potency followed the order A23187>progesterone> or =GABA; (iii) GABA-induced PIP(2) hydrolysis and rise in PA and the AR were prevented by inclusion of 10 mmol/L neomycin; (iv) the loss of PIP(2) labeling and the increase in PA labeling abolished when spermatozoa were exposed to EGTA or Ca(2+) channel blocker. These results indicate that GABA or P(4)-induced PPI breakdown is an important and essential event in the series of changes to membrane fusion during the AR of guinea pig spermatozoa and this effect is mediated via calcium by activation of phosphatidylinositol-specific phospholipase C. PMID:18726415

  13. 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

  14. 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. PMID:25312817

  15. 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.

  16. Effects of surface integrity on the fatigue life of thin flexing membranes.

    PubMed

    Sinnott, M M; Hoeppner, D W; Romney, E; Dew, P A

    1989-01-01

    It has been known for some time that surface integrity has an effect on the fatigue life of metals and "brittle" polymers. In cardiovascular applications of polymeric materials, emphasis is placed on elastomers having extended flexure lifetimes (i.e., fatigue life). The effect of surface integrity on the performance properties of Biomer (Ethicon, Inc, Somerville, NJ) a segmented polyurethane used in many blood contacting devices, is being investigated using uniaxial tensile tests in air at room temperature, and biaxial fatigue tests in deionized water at body temperature. Tensile tests were done using ASTM-D-882: Standard Test Methods for Tensile Properties of Thin Plastic Sheeting. No significant differences were noted in the stress-strain curves for specimens with various surface finishes. Fatigue tests were performed using an apparatus developed to allow for the exposure of thin-sheet polymer specimens to fluid at body temperature, while being biaxially strained. Because no standard test method was available, a test protocol was developed with reference to ASTM-D-671-78: Standard Test Methods for the Flexural Fatigue of Plastics by Constant Amplitude of Force. Stress versus life cycle data for specimens with differing surface finishes are being collected. Results to date suggest fatigue life of thin flexing membranes will decrease with increasing order of surface roughness, and fatigue properties are more sensitive to effects of changes in surface integrity than tensile properties measured by monotonic loading.

  17. 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.

  18. 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

  19. 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. PMID:27554866

  20. 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%.

  1. 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.

  2. Can Ozone Alter the Terpenoid Composition and Membrane Integrity of in vitro Melissa officinalis Shoots?

    PubMed

    D'Angiolillo, Francesca; Tonelli, Mariagrazia; Pellegrini, Elisa; Nali, Cristina; Lorenzini, Giacomo; Pistelli, Luisa; Pistelli, Laura

    2015-06-01

    Ozone affects volatile organic compounds that protect plants from biotic and abiotic stress. In vitro Melissa officinalis shoots were exposed to ozone (200 ppb, 3 h) in controlled environmental conditions: leaf pigments, membrane integrity and headspace composition were assayed during fumigation and after the recovery period (3 h from the beginning of the exposure, FBE). At the end of the exposure, no injury was observed in untreated and treated shoots, although an evident increase in lipid peroxidation was reported (+38.5 and +37.2% of TBARS levels in comparison with controls, respectively after 1 and 3 h FBE). The levels of total carotenoids significantly rose as a normal response mechanism to oxidative stress. SPME-GS-MS analysis showed that, as a consequence of the fumigation, the trends in non-terpenoid compounds increased after 1 and 3 h FBE. This suggests that the concentration and the duration of the treatment were enough to cause a breakdown of cells (as evidenced by increased TBARS levels) and involves an association between volatile products of the lipoxygenase pathway (LOX products) and membrane degradation.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Antimicrobial peptide protonectin disturbs the membrane integrity and induces ROS production in yeast cells.

    PubMed

    Wang, Kairong; Dang, Wen; Xie, Junqiu; Zhu, Ranran; Sun, Mengyang; Jia, Fengjing; Zhao, Yanyan; An, Xiaoping; Qiu, Shuai; Li, Xiaoyuan; Ma, Zelin; Yan, Wenjin; Wang, Rui

    2015-10-01

    Candidiasis is often observed in immunocompromised patients and is the 4th most common cause of bloodstream infections. However, antifungals are limited, so novel antifungal agents are urgently needed. Antimicrobial peptides (AMPs) are considered as potential alternatives of conventional antibiotics. In the present study, antimicrobial peptide protonectin was chemically synthesized and its antifungal activity and mode of action were studied. Our results showed that protonectin has potent antifungal activity and fungicidal activity against the tested fungi cells. Its action mode involved the disruption of the membrane integrity and the inducing of the production of cellular ROS. Furthermore, protonectin could inhibit the formation of biofilm and kill the adherent fungi cells. In conclusion, with the increase of fungal infection, protonectin may offer a new strategy and be considered as a potential therapeutic agent against fungal disease. PMID:26209560

  8. Zipper encodes a putative integral membrane protein required for normal axon patterning during Drosophila neurogenesis.

    PubMed Central

    Zhao, D B; Côté, S; Jähnig, F; Haller, J; Jäckle, H

    1988-01-01

    During the development of the central nervous system, Drosophila embryo axons become organized in a stereo-typed fasciculation pattern. We have found that the zipper (zip) gene, initially identified on the basis of a defective larval cuticle in zip mutant embryos, is possibly involved in the establishment or maintenance of the axon pattern during the late stages of neurogenesis. The zip wild-type gene is expressed in the developing nervous system. It codes for a putative integral membrane protein. Both the molecular features of zipper and its biological effect in the nervous system of mutants suggest that zipper is an essential component for cell surface interactions involved in axon patterning, and that the cuticle phenotype of zip mutants is dependent on the primary defects observed in the nervous system. Images PMID:3402433

  9. Consequences of defective vitamin A transportation on mitochondrial membrane integrity during protein depletion.

    PubMed

    Olowookere, J O

    1986-01-01

    The relationships between the structural integrity and functionality of rat liver mitochondrial membranes, and different levels of dietary protein and vitamin A transportation during protein depletion in animals have been investigated. Although the vitamin A content of the protein-depleted diet was 1680 +/- 35 IU/kg diet, and that of the control diet was 1,650 +/- 30 IU/kg diet, the vitamin A content of the liver of depleted rats was reduced to 16.7% of controls. The hepatic mitochondria of rats fed a protein-depleted diet showed excessive passive swelling (about 3-fold of controls) in isotonic solutions. Whereas a seemingly inverse relationship existed between the vitamin A content of the liver and the osmotic behaviour of hepatic mitochondria of rats fed a protein-depleted diet, there is a direct relationship between their hepatic mitochondrial vitamin A and the respiratory control ratio. The implications of these observations are discussed.

  10. IGF1 stabilizes sperm membrane proteins to reduce cryoinjury and maintain post-thaw sperm motility in buffalo (Bubalus bubalis) spermatozoa.

    PubMed

    Selvaraju, Sellappan; Krishnan, Binsila B; Archana, Santhanahalli Siddalingappa; Ravindra, Janivara Parameshwaraiah

    2016-08-01

    Insulin like growth factor 1 (IGF1) in the seminal plasma is reported to improve sperm motility by reducing oxidative stress. The present study was conducted to assess the effect of addition of IGF1 on sperm function and protein composition during cryopreservation process. Semen samples were collected from six Murrah buffaloes (2 ejaculates from each animal) and diluted (80 million/ml) in tris egg yolk extender and divided into control, T1, T2 and T3, groups supplemented with 0, 50, 100 and 150 ng of IGF1/mL, respectively. The semen was filled in straws (250 μL) and straws from each group were divided into two batches. One batch was processed for freezing and another batch was incubated at 4 °C for 4 h. The sperm kinematic and functional parameters were studied in both the batches. A significant (P < 0.05) positive effect of IGF1 was observed on functional membrane integrity (%) during incubation at 4 °C for 4 h in T3 as compared to control group. The spermatozoa (%) positive for structural membrane integrity, mitochondrial membrane potential and the metabolic activity in post-thaw semen were significantly (P < 0.05) high in T3 than the control group. The acrosomal integrity was significantly (P < 0.05) higher in T2 group as compared to control. The proteins (kDa) of 17.3 with pI 4.2 (calmodulin), 11.3 with pI 6.5 (dermcidin) and 18.1 with pI 5.5 (sperm acrosome membrane associated protein3) were protected in IGF1 group. The study suggests that IGF1 can be added to the extender for improving cryosurvial of buffalo spermatozoa.

  11. Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors

    PubMed Central

    West, E.L.; Pearson, R.A.; Tschernutter, M.; Sowden, J.C.; MacLaren, R.E.; Ali, R.R.

    2008-01-01

    Retinal degeneration is the leading cause of untreatable blindness in the developed world. Cell transplantation strategies provide a novel therapeutic approach to repair the retina and restore sight. Previously, we have shown that photoreceptor precursor cells can integrate and form functional photoreceptors after transplantation into the subretinal space of the adult mouse. In a clinical setting, however, it is likely that far greater numbers of integrated photoreceptors would be required to restore visual function. We therefore sought to assess whether the outer limiting membrane (OLM), a natural barrier between the subretinal space and the outer nuclear layer (ONL), could be reversibly disrupted and if disruption of this barrier could lead to enhanced numbers of transplanted photoreceptors integrating into the ONL. Transient chemical disruption of the OLM was induced in adult mice using the glial toxin, dl-alpha-aminoadipic acid (AAA). Dissociated early post-natal neural retinal cells were transplanted via subretinal injection at various time-points after AAA administration. At 3 weeks post-injection, the number of integrated, differentiated photoreceptor cells was assessed and compared with those found in the PBS-treated contralateral eye. We demonstrate for the first time that the OLM can be reversibly disrupted in adult mice, using a specific dose of AAA administered by intravitreal injection. In this model, OLM disruption is maximal at 72 h, and recovers by 2 weeks. When combined with cell transplantation, disruption of the OLM leads to a significant increase in the number of photoreceptors integrated within the ONL compared with PBS-treated controls. This effect was only seen in animals in which AAA had been administered 72 h prior to transplantation, i.e. when precursor cells were delivered into the subretinal space at a time coincident with maximal OLM disruption. These findings suggest that the OLM presents a physical barrier to photoreceptor

  12. PKA activation in concert with ARIS and asterosap induces the acrosome reaction in starfish.

    PubMed

    Islam, M Sadiqul; Kawase, O; Hase, S; Hoshi, M; Matsumoto, M

    2006-11-01

    The acrosome reaction (AR) is a fundamental event for fertilization, which is induced in concert with acrosome reaction-inducing substance (ARIS) and asterosap, both of which are components of starfish egg jelly (EJ). During the AR, a spermatozoon undergoes a series of physiological changes, such as in intracellular cGMP concentration ([cGMP]i), pHi and intracellular Ca2+ concentration ([Ca2+]i). Affinity purification of cGMP-binding protein resulted in the isolation of a regulatory subunit of the cAMP-dependent protein kinase A (PKA), suggesting the involvement of a cAMP-dependent pathway in the AR. By using a cAMP enzyme immunoassay, [cAMP]i was found to increase in starfish spermatozoa when stimulated with ARIS and asterosap. ARIS could also increase the [cAMP]i in the presence of high pH seawater. Pretreatment of spermatozoa with two specific and cell-permeable PKA inhibitors, H89 and KT5720, prevented the induction of the AR in a concentration-dependent manner. These results suggest that PKA activity participates in the induction of the AR with ARIS and asterosap. To investigate this, we have cloned a gene that encodes a regulatory subunit of PKA that had been identified in starfish spermatozoa.

  13. Effect of acute tadalafil on sperm motility and acrosome reaction: in vitro and in vivo studies.

    PubMed

    Yang, Y; Ma, Y; Yang, H; Jin, Y; Hu, K; Wang, H-X; Wang, Y-X; Huang, Y-R; Chen, B

    2014-05-01

    Effects of acute tadalafil on sperm motility and acrosome reaction were investigated, both in vitro and in vivo. Twenty asthenozoospermic and 20 normozoospermic patients as control were randomly enrolled. For in vitro part, 0.5 ml tadalafil solutions with different concentrations were added (0.2, 0.1, 0.05 and 0.025 μg ml(-1) , respectively) into semen samples. In both groups, samples treated with 0.2 μg ml(-1) tadalafil had significant increase in sperm motility after 2 h incubation. For in vivo part, oral administration of tadalafil (20 mg) or sildenafil (100 mg) was given. In both groups, computer-assisted semen analysis parameters showed no significant difference. After the administration of tadalafil (2 h) and sildenafil (1 h), there was no significant difference observed in premature acrosome reaction incidence rate. Taking both in vitro and in vivo results into consideration, acute on-demand administration of tadalafil would have no adverse effect on semen parameters.

  14. [Effect of diluents for long-term storage on the acrosomes of ram spermatozoa].

    PubMed

    Fornůsek, L; Vĕtvicka, V; Petelíková, J

    1981-04-01

    Samples of ram sperm, stored for a long time and diluted in a lactose-yolk or TRIS-glucose-yolk diluent in formulation currently used in A. I. practice in the Czech Socialist Republic, were subjected to morphological evaluation under optical and electron microscope. No visible differences between the diluents were observed under the optical microscope and the amount of acrosomal defects was just slightly higher than in the control fresh sperm. However, differences and changes were observed in the electron-microscopic pictures. When the former diluent was used, the percentage of acrosomal defects in the sperm stored for a long time was 44% and in the case of the latter diluent this percentage was as high as 59%, whereas in the control it was not higher than 16%. Most of the defects are caused during diluting and equilibration; less damage is suffered during freezing. The control osmometric determinations demonstrated a high hyperosmolality (495 mOsmol.kg-1) of the TRIS-glucose-yolk diluent. Hence the lactose-yolk diluent appears to be better, although the solubility of egg yolk in it is worse.

  15. 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.

  16. 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.

  17. 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.

  18. Electron microscopic localization of a fucose-binding protein in acrosome reacted boar spermatozoa by the fucosyl-peroxidase-gold method.

    PubMed

    Friess, A E; Toepfer-Petersen, E; Nguyen, H; Schill, W B

    1987-01-01

    In this study we have examined the behaviour and the localization of the fucose-binding protein (FBP) in boar spermatozoa during ionophore induced acrosome reaction (AR) by means of normal TEM and specimen preparation in toto. During early stages of AR the FBP is first localized at the border between equatorial segment and anterior acrosome. With the propagation of the AR the FBP is dramatically expressed and visible over the entire surface of the acrosome and equatorial segment. TEM pictures of this stages show that the FBP is associated with the OAM. At later stages of AR, when acrosomal ghost formation occur, the FBP is associated with the acrosomal ghost, and equatorial segment and to a very low degree also with the IAM. It is concluded from this data that the FBP is responsible for the specific binding of the ghost-sperm unit to the zona pellucida.

  19. Effects of high ambient temperature on fish sperm plasma membrane integrity and mitochondrial activity - A flow cytometric study.

    PubMed

    Nagy, Szabolcs Tamás; Kakasi, Balázs; Pál, László; Havasi, Máté; Bercsényi, Miklós; Husvéth, Ferenc

    2016-06-01

    Local extreme climatic conditions occurring as a result of global climate change may interfere with the reproduction of animals. In the present study fish spermatozoa were incubated at different temperatures (20, 25, 30 and 40 °C) for 10 and 30 minutes, respectively and plasma membrane integrity and mitochondrial membrane potential changes were evaluated with flow cytometry using SYBR-14/PI and Mitotracker Deep Red FM fluorescent dyes. No significant differences were found in plasma membrane integrity at either incubation temperatures or time points. Mitotracker Deep Red FM histogram profiles indicating mitochondrial activity showed significant (p < 0.001) alterations in all cases of higher (25, 30 and 40 °C) temperature treatments as compared to the samples incubated at 20 °C. Our results indicate that fish spermatozoa exposed to high temperatures suffer sublethal damage that cannot be detected with conventional, vital staining techniques. PMID:27165524

  20. Hsp30, the integral plasma membrane heat shock protein of Saccharomyces cerevisiae, is a stress-inducible regulator of plasma membrane H(+)-ATPase.

    PubMed

    Piper, P W; Ortiz-Calderon, C; Holyoak, C; Coote, P; Cole, M

    1997-03-01

    Saccharomyces cerevisiae has a single integral plasma membrane heat shock protein (Hsp). This Hsp30 is induced by several stresses, including heat shock, ethanol exposure, severe osmostress, weak organic acid exposure and glucose limitation. Plasma membrane H(+)-ATPase activities of heat shocked and weak acid-adapted, hsp30 mutant and wild-type cells, revealed that Hsp30 induction leads to a downregulation of the stress-stimulation of this H(+)-ATPase. Plasma membrane H(+)-ATPase activity consumes a substantial fraction of the ATP generated by the cell, a usage that will be increased by the H(+)-ATPase stimulation occurring with several Hsp30-inducing stresses. Hsp30 might therefore provide an energy conservation role, limiting excessive ATP consumption by plasma membrane H(+)-ATPase during prolonged stress exposure or glucose limitation. Consistent with the role of Hsp30 being energy conservation, Hsp30 null cultures give lower final biomass yields. They also have lower ATP levels, consistent with higher H(+)-ATPase activity, at the glucose exhaustion stage of batch fermentations (diauxic lag), when Hsp30 is normally induced. Loss of Hsp30 does not affect several stress tolerances but it extends the time needed for cells to adapt to growth under several stressful conditions where the maintenance of homeostasis will demand an unusually high usage of energy, hsp30 is the first yeast gene identified as both weak organic acid-inducible and assisting the adaptation to growth in the presence of these acids.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26015832

  2. ERK is involved in the process of acrosome reaction in vitro of the Chinese mitten crab, Eriocheir sinensis.

    PubMed

    Sun, Wen-Juan; Zhu, Ming; Wang, Yuan-Li; Li, Qing; Yang, Hong-Dan; Duan, Ze-Lin; He, Lin; Wang, Qun

    2015-06-01

    Mitogen-activated protein kinases (MAPKs), also termed extracellular signal-regulated kinases (ERKs), are cytoplasmic and nuclear serine/threonine kinases involved in signal transduction of several extracellular effectors. In mammals, ERKs participate in the regulation of spermatogenesis, mature spermatozoa motility, hyperactivation, and the acrosome reaction. To investigate ERK functions in Eriocheir sinensis reproduction, we successfully cloned the full-length ERK from the testis of E. sinensis (ES-ERK). The 1098-nucleotide open reading frame encodes a 365-amino-acid protein with a predicted molecular weight of 42 kDa. Expressions of ES-ERK in different tissues and testis development stages were detected by the quantitative RT-PCR and Western blotting. ES-ERK is expressed relatively highly in the testis. The expression of ES-ERK protein gradually increased in the spermatid stage, reaching a peak in sperm stage. Western blotting showed a similar expression pattern for the total ES-ERK protein, but phospho-ERK (p-ERK) showed the higher expression in spermatid than sperm stage. We also used trypan blue and hematoxylin and eosin staining to identify structural changes in E. sinensis spermatozoa during the process of acrosome reaction (AR). After stimulating the process of AR, the ES-ERK has translocated from the nucleus to the acrosomal tubule. This result suggested that the ERK MAPK might be involved in the regulation of the E. sinensis acrosome reaction.

  3. 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

  4. 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.

  5. 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.

  6. 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

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

    PubMed

    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.

  8. 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

  9. 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.

  10. Applicability of in vitro methods to study patulin bioaccessibility and its effects on intestinal membrane integrity.

    PubMed

    Assunção, Ricardo; Ferreira, Mariana; Martins, Carla; Diaz, Irene; Padilla, Beatriz; Dupont, Didier; Bragança, Mauro; Alvito, Paula

    2014-01-01

    In human health risk assessment, ingestion of food is considered a major route of exposure to many contaminants, although the total amount of an ingested contaminant (external dose) does not always reflect the quantity available for the body (internal dose). In this study, two in vitro methods were applied to study bioaccessibility and intestinal membrane integrity of cells exposed to patulin, a mycotoxin with significant public health risk. Seven artificially contaminated fruit juices were assayed in the presence or absence of a standard meal, showing a significant difference for bioaccessibility values between contaminated samples alone (mean 27.65 ± 13.50%) and combinations with a standard meal (mean 7.89 ± 4.03%). Different concentrations of patulin (PAT) and cysteine (CYS) (protector agent) were assayed in Caco-2 cells monolayers. At 95 μM, PAT produced a marked decrease in transepithelial electrical resistance (TEER). This effect was significantly reduced when 400 μM and 4000 μM CYS was added to the cells. Combined use of in vitro digestion models with other techniques using intestinal cell lines, such as in vitro intestinal absorption models that use Caco-2 cells, may offer a more comprehensive model of what is occurring during digestion and absorption processes. The study of beneficial effects of protective agents would also be enhanced. PMID:25072729

  11. 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

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

    PubMed

    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

  13. Peripheral and integral membrane binding of peptides characterized by time-dependent fluorescence shifts: focus on antimicrobial peptide LAH₄.

    PubMed

    Macháň, Radek; Jurkiewicz, Piotr; Olżyńska, Agnieszka; Olšinová, Marie; Cebecauer, Marek; Marquette, Arnaud; Bechinger, Burkhard; Hof, Martin

    2014-06-01

    Positioning of peptides with respect to membranes is an important parameter for biological and biophysical studies using model systems. Our experiments using five different membrane peptides suggest that the time-dependent fluorescence shift (TDFS) of Laurdan can help when distinguishing between peripheral and integral membrane binding and can be a useful, novel tool for studying the impact of transmembrane peptides (TMP) on membrane organization under near-physiological conditions. This article focuses on LAH4, a model α-helical peptide with high antimicrobial and nucleic acid transfection efficiencies. The predominantly helical peptide has been shown to orient in supported model membranes parallel to the membrane surface at acidic and, in a transmembrane manner, at basic pH. Here we investigate its interaction with fully hydrated large unilamellar vesicles (LUVs) by TDFS and fluorescence correlation spectroscopy (FCS). TDFS shows that at acidic pH LAH4 does not influence the glycerol region while at basic pH it makes acyl groups at the glycerol level of the membrane less mobile. TDFS experiments with antimicrobial peptides alamethicin and magainin 2, which are known to assume transmembrane and peripheral orientations, respectively, prove that changes in acyl group mobility at the glycerol level correlate with the orientation of membrane-associated peptide molecules. Analogous experiments with the TMPs LW21 and LAT show similar effects on the mobility of those acyl groups as alamethicin and LAH4 at basic pH. FCS, on the same neutral lipid bilayer vesicles, shows that the peripheral binding mode of LAH4 is more efficient in bilayer permeation than the transmembrane mode. In both cases, the addition of LAH4 does not lead to vesicle disintegration. The influence of negatively charged lipids on the bilayer permeation is also addressed.

  14. 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. PMID:22023717

  15. Marine sponge cyclic peptide theonellamide A disrupts lipid bilayer integrity without forming distinct membrane pores.

    PubMed

    Espiritu, Rafael Atillo; Cornelio, Kimberly; Kinoshita, Masanao; Matsumori, Nobuaki; Murata, Michio; Nishimura, Shinichi; Kakeya, Hideaki; Yoshida, Minoru; Matsunaga, Shigeki

    2016-06-01

    Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides derived from the marine sponge Theonella sp. These peptides specifically bind to 3β-hydroxysterols, resulting in 1,3-β-D-glucan overproduction and membrane damage in yeasts. The inclusion of cholesterol or ergosterol in phosphatidylcholine membranes significantly enhanced the membrane affinity of theonellamide A (TNM-A) because of its direct interaction with 3β-hydroxyl groups of sterols. To better understand TNM-induced membrane alterations, we investigated the effects of TNM-A on liposome morphology. (31)P nuclear magnetic resonance (NMR) and dynamic light scattering (DLS) measurements revealed that the premixing of TNM-A with lipids induced smaller vesicle formation. When giant unilamellar vesicles were incubated with exogenously added TNM-A, confocal micrographs showed dynamic changes in membrane morphology, which were more frequently observed in cholesterol-containing than sterol-free liposomes. In conjunction with our previous data, these results suggest that the membrane action of TNM-A proceeds in two steps: 1) TNM-A binds to the membrane surface through direct interaction with sterols and 2) accumulated TNM-A modifies the local membrane curvature in a concentration-dependent manner, resulting in dramatic membrane morphological changes and membrane disruption.

  16. Marine sponge cyclic peptide theonellamide A disrupts lipid bilayer integrity without forming distinct membrane pores.

    PubMed

    Espiritu, Rafael Atillo; Cornelio, Kimberly; Kinoshita, Masanao; Matsumori, Nobuaki; Murata, Michio; Nishimura, Shinichi; Kakeya, Hideaki; Yoshida, Minoru; Matsunaga, Shigeki

    2016-06-01

    Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides derived from the marine sponge Theonella sp. These peptides specifically bind to 3β-hydroxysterols, resulting in 1,3-β-D-glucan overproduction and membrane damage in yeasts. The inclusion of cholesterol or ergosterol in phosphatidylcholine membranes significantly enhanced the membrane affinity of theonellamide A (TNM-A) because of its direct interaction with 3β-hydroxyl groups of sterols. To better understand TNM-induced membrane alterations, we investigated the effects of TNM-A on liposome morphology. (31)P nuclear magnetic resonance (NMR) and dynamic light scattering (DLS) measurements revealed that the premixing of TNM-A with lipids induced smaller vesicle formation. When giant unilamellar vesicles were incubated with exogenously added TNM-A, confocal micrographs showed dynamic changes in membrane morphology, which were more frequently observed in cholesterol-containing than sterol-free liposomes. In conjunction with our previous data, these results suggest that the membrane action of TNM-A proceeds in two steps: 1) TNM-A binds to the membrane surface through direct interaction with sterols and 2) accumulated TNM-A modifies the local membrane curvature in a concentration-dependent manner, resulting in dramatic membrane morphological changes and membrane disruption. PMID:27003125

  17. 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

  18. Effect of integral proteins in the phase stability of a lipid bilayer: Application to raft formation in cell membranes

    NASA Astrophysics Data System (ADS)

    Gómez, Jordi; Sagués, Francesc; Reigada, Ramon

    2010-04-01

    The existence of lipid rafts is a controversial issue. The affinity of cholesterol for saturated lipids is manifested in macroscopic phase separation in model membranes, and is believed to be the thermodynamic driving force for raft formation. However, there is no clear reason to explain the small (nanometric) size of raft domains in cell membranes. In a recent paper Yethiraj and Weisshaar [Biophys. J. 93, 3113 (2007)] proposed that the effect of neutral integral membrane proteins may prevent from the formation of large lipid domains. In this paper we extend this approach by studying the effect of the protein size, as well as the lipid-protein interaction. Depending on these factors, two different mechanisms for nanodomain stabilization are shown to be possible for static proteins. The application of these results to a biological context is discussed.

  19. A Novel Family of Soluble Minimal Scaffolds Provides Structural Insight into the Catalytic Domains of Integral Membrane Metallopeptidases*

    PubMed Central

    López-Pelegrín, Mar; Cerdà-Costa, Núria; Martínez-Jiménez, Francisco; Cintas-Pedrola, Anna; Canals, Albert; Peinado, Juan R.; Marti-Renom, Marc A.; López-Otín, Carlos; Arolas, Joan L.; Gomis-Rüth, F. Xavier

    2013-01-01

    In the search for structural models of integral-membrane metallopeptidases (MPs), we discovered three related proteins from thermophilic prokaryotes, which we grouped into a novel family called “minigluzincins.” We determined the crystal structures of the zymogens of two of these (Pyrococcus abyssi proabylysin and Methanocaldococcus jannaschii projannalysin), which are soluble and, with ∼100 residues, constitute the shortest structurally characterized MPs to date. Despite relevant sequence and structural similarity, the structures revealed two unique mechanisms of latency maintenance through the C-terminal segments previously unseen in MPs as follows: intramolecular, through an extended tail, in proabylysin, and crosswise intermolecular, through a helix swap, in projannalysin. In addition, structural and sequence comparisons revealed large similarity with MPs of the gluzincin tribe such as thermolysin, leukotriene A4 hydrolase relatives, and cowrins. Noteworthy, gluzincins mostly contain a glutamate as third characteristic zinc ligand, whereas minigluzincins have a histidine. Sequence and structural similarity further allowed us to ascertain that minigluzincins are very similar to the catalytic domains of integral membrane MPs of the MEROPS database families M48 and M56, such as FACE1, HtpX, Oma1, and BlaR1/MecR1, which are provided with trans-membrane helices flanking or inserted into a minigluzincin-like catalytic domain. In a time where structural biochemistry of integral-membrane proteins in general still faces formidable challenges, the minigluzincin soluble minimal scaffold may contribute to our understanding of the working mechanisms of these membrane MPs and to the design of novel inhibitors through structure-aided rational drug design approaches. PMID:23733187

  20. 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.

  1. 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. PMID:25380632

  2. Integrated membrane systems incorporating coagulation, activated carbon and ultrafiltration for the removal of toxic cyanobacterial metabolites from Anabaena circinalis.

    PubMed

    Dixon, M B; Richard, Y; Ho, L; Chow, C W K; O'Neill, B K; Newcombe, G

    2011-01-01

    The use of integrated membrane systems (a train of treatment processes incorporating one or more membranes) is increasing globally as the technology is very effective for the production of high quality drinking water. In this investigation a laboratory scale integrated membrane system (IMS) featuring coagulation, powdered activated carbon (PAC) and ultrafiltration (UF) was investigated for the removal of an Australian strain of the cyanobacteria Anabaena circinalis and the cyanotoxin it produced. Three coagulants were compared, aluminium chlorohydrate (ACH), aluminium sulphate (alum) and an engineered aluminium coagulant referred to as high performance aluminium chlorohydrate (HPAC). PAC (Acticarb PS1000) was tested to determine adsorption of extracellular saxitoxin. Removal of A. circinalis cells was 100% by UF alone and the removal of cells prior to the membrane by coagulation reduced fouling attributed to algogenic organic material. Alum was the least efficient coagulant for removal of cells while ACH and HPAC were similar. Saxitoxin removal reached a maximum of 80% using ACH and PAC. The UF-IMS was challenged using a natural bloom of A. circinalis that occurred in the Myponga Reservoir in South Australia. PMID:21508543

  3. Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

    PubMed Central

    Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

    2013-01-01

    Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

  4. 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.

  5. Pathogen specificity of Treponema pallidum subsp. pallidum integral membrane proteins identified by phase partitioning with Triton X-114.

    PubMed

    Radolf, J D; Norgard, M V

    1988-07-01

    The antigenically conserved proteins of Treponema pallidum subsp. pallidum and four nonpathogenic cultivatable treponemes were investigated by phase partitioning with the nonionic detergent Triton X-114 and immunoblot analysis. None of the T. pallidum integral membrane proteins identified by phase partitioning (detergent-phase proteins) appeared to be antigenically related to proteins of the nonpathogens. Protease-resistant material similar to lipopolysaccharide was identified in the detergent phase from T. phagedenis biotype Reiter but was not detected in T. pallidum.

  6. Effects of air pollution on cell membrane integrity, spectral reflectance and metal and sulfur concentrations in lichens

    SciTech Connect

    Garty, J.; Cohen, Y.; Kloog, N.; Karnieli, A.

    1997-07-01

    The fruticose lichen Ramalina duriaei is generally considered to be sensitive to air pollution. In the present study the authors sought to determine whether thalli of this lichen collected in a remote unpolluted site (the HaZorea Forest, northeast Israel) and transplanted to the Ashdod region (southwest Israel) could provide information on the quality of the air in this area. For this purpose, the concentrations of Pb, Cu, Cd, Ni, Mn, Fe, S, Ca, Mg, Na, and K were determined in in situ thalli collected in the HaZorea Forest in March 1993 and in in situ and transplanted thalli retrieved in June 1993. The concentration of these elements in R. duriaei thalli was analyzed in comparison with physiological parameters such as the integrity of cell membranes, chlorophyll content, and alterations in reflectance responses from lichen thalli. Thalli transplanted to several industrial sites in the town of Ashdod for a period of 100 d accumulated high concentrations of Pb, Cd, Ni, Fe, S, Mg, Na, Ca, and K. The concentration of S in thalli transplanted to the Ashdod region was found to correlate with damage caused to cell membranes and showed and inverse correlation with the chlorophyll content and with the reflectance response of the lichen. The electrical conductivity values corresponding to membrane integrity in the lichen thallus showed an inverse correlation with the ratio of chlorophyll a to pheophytin a, indicating the integrity of the photobiontic chlorophyll and with normalized-difference vegetation index values corresponding to the reflectance response of the thallus. The chlorophyll integrity correlated with the reflectance response. Magnesium accumulated in the lichen thalli in dusty sites and was found to correlate with damage caused to membranes.

  7. MORPHOLOGY OF GAMETE MEMBRANE FUSION AND OF SPERM ENTRY INTO OOCYTES OF THE SEA URCHIN

    PubMed Central

    Franklin, Luther E.

    1965-01-01

    Sea urchin gametes predominate in molecular studies of fertilization, yet relatively little is known of the subcellular aspects of sperm entry in this group. Accordingly, it seemed desirable to make a detailed examination of sperm entry phenomena in sea urchins with the electron microscope. Gametes of the sea urchins Arbacia punctulata and Lytechinus variegatus were used in this study. Samples of eggs containing 2 to 8 per cent oocytes were selected and fixed with osmium tetroxide in sea water at various intervals after insemination. Fixed specimens were embedded in Epon 812, sectioned, and examined with an electron microscope. An apical vesicle was observed at the anterior end of the acrosome. The presence of this structure, together with other observations, suggested that initiation of the acrosome reaction in sea urchin sperm involves dehiscence of the acrosomal region with the subsequent release of the acrosomal granule. Contact and initial fusion of gamete membranes was observed in mature eggs and oocytes and invariably involved the extended acrosomal tubule of the spermatozoon. Only one spermatozoon normally enters the mature egg. The probability of locating such a sperm in ultrathin sections is exceedingly low. Several sperm do normally enter oocytes. Consequently, observations of sperm entry were primarily restricted to the latter. The manner of sperm entry into oocytes did not resemble phagocytosis. Organelles of the spermatozoon were progressively divested of their plasma membrane as they entered the ground cytoplasm of the oocyte fertilization cone. Initiation of the acrosome reaction, contact and initial fusion of gamete membranes, and sperm entry into oocytes of sea urchins conform to the Hydroides-Saccoglossus pattern of early fertilization events as described by Colwin and Colwin (13). PMID:19866669

  8. Effects of in vitro storage time and semen-extender on membrane quality of boar sperm assessed by flow cytometry.

    PubMed

    Waterhouse, K E; De Angelis, P M; Haugan, T; Paulenz, H; Hofmo, P O; Farstad, W

    2004-12-01

    The Norwegian AI company Norsvin has used the short-term semen-extender BTS to extend and store boar semen since the late 1980s. Fertility results have been consistent when extended semen has been used for AI within 3 days after collection, however, from a production and economic point of view it is preferable that semen stored for up to 5 days can be used. The aim of this study was to compare membrane quality of sperm stored in BTS for 3 days with sperm stored in the long-term semen-extenders Androstar, Mulberry III and X-cell for 5 days. Using a split-sample design, plasma membrane- and acrosome-integrity were assessed flow cytometrically by use of Yo-Pro-1 and PNA-FITC, and fluidity and phospholipid asymmetry of the membrane were assessed by use of MC540 and Annexin V-FITC. Due to observed sperm fragmentation in Androstar after Day 1, the data for Androstar were excluded from the analyses. After 5 days of storage, the membrane quality of X-cell-stored sperm was not statistically different from that of sperm stored in BTS for 3 days, while membrane quality of sperm stored in Mulberry III was statistically better on Day 5 compared to BTS on Day 3. In conclusion, Mulberry III and X-cell preserve sperm quality, as well as that of BTS on Day 3, for up to 5 days after collection. PMID:15511551

  9. Integration of biological method and membrane technology in treating palm oil mill effluent.

    PubMed

    Zhang, Yejian; Yan, Li; Qiao, Xiangli; Chi, Lina; Niu, Xiangjun; Mei, Zhijian; Zhang, Zhenjia

    2008-01-01

    Palm oil industry is the most important agro-industry in Malaysia, but its by-product-palm oil mill effluent (POME), posed a great threat to water environment. In the past decades, several treatment and disposal methods have been proposed and investigated to solve this problem. A two-stage pilot-scale plant was designed and constructed for POME treatment. Anaerobic digestion and aerobic biodegradation constituted the first biological stage, while ultrafiltration (UF) and reverse osmosis (RO) membrane units were combined as the second membrane separation stage. In the anaerobic expanded granular sludge bed (EGSB) reactor, about 43% organic matter in POME was converted into biogas, and COD reduction efficiency reached 93% and 22% in EGSB and the following aerobic reactor, respectively. With the treatment in the first biological stage, suspended solids and oil also decreased to a low degree. All these alleviated the membrane fouling and prolonged the membrane life. In the membrane process unit, almost all the suspended solids were captured by UF membranes, while RO membrane excluded most of the dissolved solids or inorganic salts from RO permeate. After the whole treatment processes, organic matter in POME expressed by BOD and COD was removed almost thoroughly. Suspended solids and color were not detectable in RO permeate any more, and mineral elements only existed in trace amount (except for K and Na). The high-quality effluent was crystal clear and could be used as the boiler feed water.

  10. Towards integrated operation of membrane bioreactors: effects of aeration on biological and filtration performance.

    PubMed

    Dalmau, M; Monclús, H; Gabarrón, S; Rodriguez-Roda, I; Comas, J

    2014-11-01

    Two experimental studies evaluated the effect of aerobic and membrane aeration changes on sludge properties, biological nutrient removal and filtration processes in a pilot plant membrane bioreactor. The optimal operating conditions were found at an aerobic dissolved oxygen set-point (DO) of 0.5 mg O2 L(-1) and a membrane specific aeration demand (SADm) of 1 m h(-1), where membrane aeration can be used for nitrification. Under these conditions, a total flow reduction of 42% was achieved (75% energy reduction) without compromising nutrient removal efficiencies, maintaining sludge characteristics and controlled filtration. Below these optimal operating conditions, the nutrient removal efficiency was reduced, increasing 20% for soluble microbial products, 14% for capillarity suction time and reducing a 15% for filterability. Below this DO set-point, fouling increased with a transmembrane pressure 75% higher. SADm below 1 m h(-1) doubled the values of transmembrane pressure, without recovery after achieving the initial conditions.

  11. 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. PMID:23261305

  12. 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...

  13. 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

    2009-11-01

    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

  14. Decreased content of integral membrane calcium-binding protein (IMCAL) in tissues of the spontaneously hypertensive rat

    SciTech Connect

    Kowarski, S.; Cowen, L.A.; Schachter, D.

    1986-02-01

    Prior studies report that plasma membranes of the spontaneously hypertensive rat (Okamoto-Aoki strain) bind less calcium than do the corresponding preparations from Wistar Kyoto controls. The possibility that the differences result from a decrease in the content of integral membrane calcium-binding protein (IMCAL) was explored by the application of immunoassays with polyclonal antisera and a mouse monoclonal antibody. IMCAL binds calcium with relatively high affinity, and its content in many rat tissues is regulated by vitamin D and the level of dietary calcium. Radioimmunoassays of tissue IMCAL demonstrate significant reductions in content in the erythrocyte ghost, intestinal mucosa, kidney, heart, testis, and liver of the spontaneously hypertensive rat as compared to the control strains. The decreases are observed both at 4-5 weeks of age, before the onset of severe hypertension and at 8-9 weeks in the presence of severe hypertension. Moreover, the magnitude of the decrease in erythrocyte IMCAL can account for much of the decrease in the calcium-binding capacity of erythrocyte membranes reported by others. The results are especially significant because an abnormality in the membrane binding of calcium and in the regulation of cytosolic calcium ion concentration could underlie the pathogenesis of the hypertension.

  15. Reconstitution of integral membrane proteins into isotropic bicelles with improved sample stability and expanded lipid composition profile.

    PubMed

    Morrison, Emma A; Henzler-Wildman, Katherine A

    2012-03-01

    Reconstitution of integral membrane proteins into membrane mimetic environments suitable for biophysical and structural studies has long been a challenge. Isotropic bicelles promise the best of both worlds-keeping a membrane protein surrounded by a small patch of bilayer-forming lipids while remaining small enough to tumble isotropically and yield good solution NMR spectra. However, traditional methods for the reconstitution of membrane proteins into isotropic bicelles expose the proteins to potentially destabilizing environments. Reconstituting the protein into liposomes and then adding short-chain lipid to this mixture produces bicelle samples while minimizing protein exposure to unfavorable environments. The result is higher yield of protein reconstituted into bicelles and improved long-term stability, homogeneity, and sample-to-sample reproducibility. This suggests better preservation of protein structure during the reconstitution procedure and leads to decreased cost per sample, production of fewer samples, and reduction of the NMR time needed to collect a high quality spectrum. Furthermore, this approach enabled reconstitution of protein into isotropic bicelles with a wider range of lipid compositions. These results are demonstrated with the small multidrug resistance transporter EmrE, a protein known to be highly sensitive to its environment.

  16. 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. PMID:26091957

  17. 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. PMID:26819478

  18. Host Lipid and Temperature as Important Screening Variables for Crystallizing Integral Membrane Proteins in Lipidic Mesophases. Trials with Diacylglycerol Kinase

    PubMed Central

    Li, Dianfan; Shah, Syed T. A.; Caffrey, Martin

    2013-01-01

    A systematic study of the crystallization of an α-helical, integral membrane enzyme, diacylglycerol kinase, DgkA, using the lipidic cubic mesophase or in meso method is described. These trials have resulted in the production of blocky, rhombohedron-shaped crystals of diffraction quality currently in use for structure determination. Dramatic improvements in crystal quality were obtained when the identity of the lipid used to form the mesophase bilayer into which the protein was reconstituted as a prelude to crystallogenesis was varied. These monoacylglycerol lipids incorporated fatty acyl chains ranging from 14 to 18 carbon atoms long with cis olefinic bonds located toward the middle of the chain. Best crystals were obtained with a lipid that had an acyl chain 15 carbon atoms long with the double bond between carbons 7 and 8. It is speculated that the effectiveness of this lipid derives from hydrophobic mismatch between the target integral membrane protein and the bilayer of the host mesophase. Low temperature (4 °C) worked in concert with the short chain lipid to provide high quality crystals. Recommended screening strategies for crystallizing membrane proteins that include host lipid type and low temperature are made on the basis of this and related in meso crystallization trials. PMID:23956688

  19. An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases

    PubMed Central

    Liu, Changchun; Geva, Eran; Mauk, Michael; Qiu, Xianbo; Abrams, William R.; Malamud, Daniel; Curtis, Kelly; Owen, S. Michele; Bau, Haim H.

    2015-01-01

    A simple, point of care, inexpensive, disposable cassette for the detection of nucleic acids extracted from pathogens was designed, constructed, and tested. The cassette utilizes a single reaction chamber for isothermal amplification of nucleic acids. The chamber is equipped with an integrated, flow-through, Flinders Technology Associates (Whatman FTA®) membrane for the isolation, concentration, and purification of DNA and/or RNA. The nucleic acids captured by the membrane are used directly as templates for amplification without elution, thus simplifying the cassette’s flow control. The FTA membrane also serves another critical role—enabling the removal of inhibitors that dramatically reduce detection sensitivity. Thermal control is provided with a thin film heater external to the cassette. The amplification process was monitored in real time with a portable, compact fluorescent reader. The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids. The HIV RNA was reverse transcribed and subjected to loop-mediated, isothermal amplification (LAMP). A detection limit of less than 10 HIV particles was demonstrated. The cassette is particularly suitable for resource poor regions, where funds and trained personnel are in short supply. The cassette can be readily modified to detect nucleic acids associated with other pathogens borne in saliva, urine, and other body fluids as well as in water and food. PMID:21455542

  20. 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.

  1. 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

  2. High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

    PubMed Central

    Bruni, Renato

    2014-01-01

    Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

  3. 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.

  4. 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.

  5. Towards the molecular mechanism of the integration of peroxisomal membrane proteins☆

    PubMed Central

    Giannopoulou, Evdokia-Anastasia; Emmanouilidis, Leonidas; Sattler, Michael; Dodt, Gabriele; Wilmanns, Matthias

    2016-01-01

    The correct topogenesis of peroxisomal membrane proteins is a crucial step for the formation of functioning peroxisomes. Although this process has been widely studied, the exact mechanism with which it occurs has not yet been fully characterized. Nevertheless, it is generally accepted that peroxisomes employ three proteins – Pex3, Pex19 and Pex16 in mammals – for the insertion of peroxisomal membrane proteins into the peroxisomal membrane. Structural biology approaches have been utilized for the elucidation of the mechanistic questions of peroxisome biogenesis, mainly by providing information on the architecture of the proteins significant for this process. This review aims to summarize, compare and put into perspective the structural knowledge that has been generated mainly for Pex3 and Pex19 and their interaction partners in recent years. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann. PMID:26434995

  6. Ethylene Action and Loss of Membrane Integrity during Petal Senescence in Tradescantia.

    PubMed

    Suttle, J C; Kende, H

    1980-06-01

    Senescence of isolated petals of Tradescantia is accompanied by a large increase in membrane permeability, and application of ethylene hastens the onset of this increase. There is a 1- to 2.5-hour lag between ethylene application and the onset of anthocyanin efflux (an indicator of increased membrane permeability). Simultaneous application of 0.1 millimolar cordycepin or cycloheximide with ethylene abolishes the response to ethylene. Analysis of phospholipid levels in these petals during senescence has shown that the increase in membrane permeability is accompanied by a massive loss of phospholipids. Factors which enhance or retard the rate of anthocyanin efflux exert a corresponding effect on the rate of phospholipid loss. The composition of the phospholipid fraction remains unchanged during senescence. The activity of phospholipase D declines during senescence whereas that of acyl hydrolase remains essentially constant.

  7. Ethylene action and loss of membrane integrity during petal senescence in Tradescantia

    SciTech Connect

    Suttle, J.; Kende, H.

    1980-06-01

    Senescence of isolated petals of Tradescantia is accompanied by a large increase in membrane permeability, and application of ethylene hastens the onset of this increase. There is a 1- to 2.5-hour lag between ethylene application and the onset of anthocyanin efflux (an indicator of increased membrane permeability). Simultaneous application of 0.1 millimolar cordycepin or cycloheximide with ethylene abolishes the response to ethylene. Analysis of phospholipid levels in these petals during senescence has shown that the increase in membrane permeability is accompanied by a massive loss of phospholipids. Factors which enhance or retard the rate of anthocyanin efflux exert a corresponding effect on the rate of phospholipid loss. The composition of the phospholipid fraction remains unchanged during senescence. The activity of phospholipase D declines during senescence whereas that of acyl hydrolase remains essentially constant.

  8. Melatonin protects the integrity of granulosa cells by reducing oxidative stress in nuclei, mitochondria, and plasma membranes in mice

    PubMed Central

    TANABE, Manabu; TAMURA, Hiroshi; TAKETANI, Toshiaki; OKADA, Maki; LEE, Lifa; TAMURA, Isao; MAEKAWA, Ryo; ASADA, Hiromi; YAMAGATA, Yoshiaki; SUGINO, Norihiro

    2014-01-01

    Melatonin protects luteinized granulosa cells (GCs) from oxidative stress in the follicle during ovulation. However, it is unclear in which cellular components (e.g., nuclei, mitochondria, or plasma membranes) melatonin works as an antioxidant. GCs from immature (3 wks) ICR mice were incubated with hydrogen peroxide (H2O2; 0.01, 0.1, 1, 10 mM) in the presence or absence of melatonin (100 μg/ml) for 2 h. DNA damage was assessed by fluorescence-based immunocytochemistry using specific antibodies for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative guanine base damage in DNA, and for histone H2AX phosphorylation (γH2AX), a marker of double-strand breaks of DNA. Mitochondrial function was assessed by the fluorescence intensity of MitoTracker Red probes, which diffuse across the membrane and accumulate in mitochondria with active membrane potentials. Lipid peroxidation of plasma membranes was analyzed by measuring hexanoyl-lysine (HEL), a oxidative stress marker for lipid peroxidation. Apoptosis of GCs was assessed by nuclear fragmentation using DAPI staining, and apoptotic activities were evaluated by caspase-3/7 activities. H2O2 treatment significantly increased the fluorescence intensities of 8-OHdG and γH2AX, reduced the intensity of MitoTracker Red in the mitochondria, increased HEL concentrations in GCs, and enhanced the number of apoptotic cells and caspase-3/7 activities. All these changes were significantly decreased by melatonin treatment. Melatonin reduced oxidative stress-induced DNA damage, mitochondrial dysfunction, lipid peroxidation, and apoptosis in GCs, suggesting that melatonin protects GCs by reducing oxidative stress of cellular components including nuclei, mitochondria, and plasma membranes. Melatonin helps to maintain the integrity of GCs as an antioxidant in the preovulatory follicle. PMID:25366368

  9. Melatonin protects the integrity of granulosa cells by reducing oxidative stress in nuclei, mitochondria, and plasma membranes in mice.

    PubMed

    Tanabe, Manabu; Tamura, Hiroshi; Taketani, Toshiaki; Okada, Maki; Lee, Lifa; Tamura, Isao; Maekawa, Ryo; Asada, Hiromi; Yamagata, Yoshiaki; Sugino, Norihiro

    2015-01-01

    Melatonin protects luteinized granulosa cells (GCs) from oxidative stress in the follicle during ovulation. However, it is unclear in which cellular components (e.g., nuclei, mitochondria, or plasma membranes) melatonin works as an antioxidant. GCs from immature (3 wks) ICR mice were incubated with hydrogen peroxide (H2O2; 0.01, 0.1, 1, 10 mM) in the presence or absence of melatonin (100 μg/ml) for 2 h. DNA damage was assessed by fluorescence-based immunocytochemistry using specific antibodies for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative guanine base damage in DNA, and for histone H2AX phosphorylation (γH2AX), a marker of double-strand breaks of DNA. Mitochondrial function was assessed by the fluorescence intensity of MitoTracker Red probes, which diffuse across the membrane and accumulate in mitochondria with active membrane potentials. Lipid peroxidation of plasma membranes was analyzed by measuring hexanoyl-lysine (HEL), a oxidative stress marker for lipid peroxidation. Apoptosis of GCs was assessed by nuclear fragmentation using DAPI staining, and apoptotic activities were evaluated by caspase-3/7 activities. H2O2 treatment significantly increased the fluorescence intensities of 8-OHdG and γH2AX, reduced the intensity of MitoTracker Red in the mitochondria, increased HEL concentrations in GCs, and enhanced the number of apoptotic cells and caspase-3/7 activities. All these changes were significantly decreased by melatonin treatment. Melatonin reduced oxidative stress-induced DNA damage, mitochondrial dysfunction, lipid peroxidation, and apoptosis in GCs, suggesting that melatonin protects GCs by reducing oxidative stress of cellular components including nuclei, mitochondria, and plasma membranes. Melatonin helps to maintain the integrity of GCs as an antioxidant in the preovulatory follicle.

  10. Unsaturated lipids protect the integral membrane peptide gramicidin A from singlet oxygen.

    PubMed

    Rokitskaya, Tatyana I; Kotova, Elena A; Agapov, Igor I; Moisenovich, Mikhail M; Antonenko, Yuri N

    2014-05-01

    In contrast to expectations that unsaturated fatty acids contribute to oxidative stress by providing a source of lipid peroxides, we demonstrated the protective effect of double bonds in lipids on oxidative damage to membrane proteins. Photodynamic inactivation of gramicidin channels was decreased in unsaturated lipid compared to saturated lipid bilayers. By estimating photosensitizer (boronated chlorine e6 amide) binding to the membrane with the current relaxation technique, the decrease in gramicidin photoinactivation was attributed to singlet oxygen scavenging by double bonds in lipids rather than to the reduction in photosensitizer binding. Gramicidin protection by unsaturated lipids was also observed upon induction of oxidative stress with tert-butyl hydroperoxide.

  11. In vitro synthesis and integration into mitochondria of porin, a major protein of the outer mitochondrial membrane of Saccharomyces cerevisiae.

    PubMed

    Mihara, K; Blobel, G; Sato, R

    1982-12-01

    We have isolated an outer mitochondrial membrane (OMM) fraction from baker's yeast. Saccharomyces cerevisiae, that possesses porin activity and contains a major polypeptide of 29,000 daltons. By analogy to similar data for an OMM fraction from rat liver and mung bean [Zalman, L. S., Nikaido, N. & Kagawa, Y. (1980) J. Biol. Chem. 255, 1771-1774], the 29,000-dalton polypeptide of the isolated yeast OMM fraction has been tentatively identified as porin. Evidence to substantiate this identification was provided by the finding that both the porin activity and the 29,000-dalton polypeptide were entirely resistant when the OMM fraction was exposed to trypsin digestion, with the 29,000-dalton polypeptide being virtually the only polypeptide in the OMM fraction to be unaffected by trypsin digestion. There was no protection when trypsin digestion was carried out in the presence of detergent. Using monospecific antibodies, we have shown that yeast porin is apparently not synthesized as a larger precursor in a cell-free translation system. In vitro-synthesized porin could not be integrated into dog pancreas microsomal vesicles or into an isolated OMM fraction from yeast, either co- or posttranslationally. In vitro-synthesized porin, however, could be integrated posttranslationally into whole isolated mitochondria. This membrane specificity suggests that integration does not proceed by unassisted partitioning. The integration of porin into whole mitochondria occurred with fidelity by the criterion of its resistance to trypsin. Moreover, integration was not inhibited in the presence of the protonophore carbonyl cyanide m-chlorophenyl-hydrazone whereas translocation into the mitochondrial matrix of the in vitro-synthesized gamma subunit of F1-ATPase was inhibited.

  12. Membrane distributed-reflector laser integrated with SiOx-based spot-size converter on Si substrate.

    PubMed

    Nishi, Hidetaka; Fujii, Takuro; Takeda, Koji; Hasebe, Koichi; Kakitsuka, Takaaki; Tsuchizawa, Tai; Yamamoto, Tsuyoshi; Yamada, Koji; Matsuo, Shinji

    2016-08-01

    We demonstrate monolithic integration of a 50-μm-long-cavity membrane distributed-reflector laser with a spot-size converter, consisting of a tapered InP wire waveguide and an SiOx waveguide, on SiO2/Si substrate. The device exhibits 9.4-GHz/mA0.5 modulation efficiency with a 2.2-dB fiber coupling loss. We demonstrate 25.8-Gbit/s direct modulation with a bias current of 2.5 mA, resulting in a low energy cost of 132 fJ/bit.

  13. 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

  14. 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. PMID:27108849

  15. Integral membrane proteins and free electron lasers - a compatible couple indeed!

    PubMed

    Wiener, Michael C

    2015-07-01

    Several structures of membrane transport proteins in complex with mechanistically-relevant ligands, determined by serial femtosecond crystallography of microcrystals at an X-ray free-electron source source, are presented. These results, including investigation of approaches to data quality assessment and refinement from low-redundancy data, indicate the feasibility of using this approach for ligand screening.

  16. SPACA7 is a novel male germ cell-specific protein localized to the sperm acrosome that is involved in fertilization in mice.

    PubMed

    Nguyen, Edward B; Westmuckett, Andrew D; Moore, Kevin L

    2014-01-01

    Sperm acrosome associated 7 (SPACA7) is a novel protein of unknown function with no homology to any known protein. Spaca7 transcripts are detected only in testis and predict a 158-residue mature polypeptide with one potential N-glycosylation site and no cysteines. Orthologs are present in various species, including mice and humans. We developed a polyclonal antibody to mouse SPACA7 to study its expression and function. Western blotting and immunofluorescence microscopy detected SPACA7 only in testis, and it was detected in testis starting at Postnatal Day 21 and into adulthood. Immunofluorescence staining of testicular germ cells detected weak SPACA7 expression as early as zygotene spermatocytes. Higher expression was observed in round spermatids, where SPACA7 was localized to a perinuclear spot adjacent to the Golgi and to the acrosome of elongating spermatids and spermatozoa. Immunogold electron microscopy demonstrated that SPACA7 is localized within the proacrosomal granule of round spermatids and the acrosome of spermatozoa. Finally, we showed that SPACA7 was retained within the acrosome of epididymal sperm and was released upon the acrosome reaction. To assess if SPACA7 was involved in fertilization, in vitro fertilization assays in the presence of anti-SPACA7 IgG were performed. Anti-SPACA7 inhibited fertilization of cumulus-intact eggs and prominently delayed cumulus dispersal. However, anti-SPACA7 did not inhibit fertilization of cumulus-free eggs. Our findings indicate that release of SPACA7 from the acrosome accelerates cumulus dispersal and facilitates fertilization via unknown mechanisms. This study is the first to document the expression of endogenous SPACA7 and a function for this novel acrosomal protein.

  17. The acrosomal protein SP-10 (Acrv1) is an ideal marker for staging of the cycle of seminiferous epithelium in the mouse.

    PubMed

    Osuru, Hari Prasad; Monroe, Jennifer E; Chebolu, Apoorv P; Akamune, Joycelyn; Pramoonjago, Patcharin; Ranpura, Sandeep A; Reddi, Prabhakara P

    2014-10-01

    The study of spermatogenesis requires accurate identification of the stages of the cycle of the seminiferous epithelium. A stage refers to the unique association of germ cell types at a particular phase of development, as seen in a cross-sectioned seminiferous tubule. Stage-identification, however, is a daunting task. There are 12 stages represented in the mouse seminiferous epithelium. Stages are typically identified on the basis of the morphology of the developing acrosome of spermatids. Although the characteristic features of the acrosome are well-documented in ultrastructure images, a reagent that can highlight the subtle differences in acrosome shape under the light microscope is lacking. Here we demonstrate that a polyclonal antibody raised against the mouse acrosomal protein SP-10 is extremely useful for stage identification. Immunohistochemistry showed that the anti-SP-10 antibody is highly specific for the acrosome of spermatids, as no other cell type in the epithelium showed immunoreactivity. At lower magnification, the gross shape of the acrosome and the increasing intensity of immunostaining served as a guide for the identification of stages I-XII. At higher magnification, characteristic morphological features-such as whether the part of the acrosome that contacts the nuclear surface is round (stage III) or flat (stage IV) or curved (stage VI)-could be identified unambiguously. Overall, we present evidence that SP-10 is a useful marker for staging the cycle of the seminiferous epithelium. The anti-SP-10 antibody works well in different fixatives, on paraffin-embedded as well as cryosections, and has been shown to be useful for characterizing spermatogenic defects in mutant mice.

  18. Characterization of integral membrane proteins of Leishmania major by Triton X-114 fractionation and analysis of vaccination effects in mice.

    PubMed

    Murray, P J; Spithill, T W; Handman, E

    1989-07-01

    The total integral membrane proteins of promastigotes of Leishmania major were extracted by using the Triton X-114 phase separation technique and were characterized by immunoprecipitation, Western blotting (immunoblotting), and lectin chromatography. Of the 40 or more proteins which partitioned into the detergent phase, only about 10 proteins could be surface radioiodinated on live promastigotes, suggesting their surface orientation. The abundance of the gp58-63 antigen varied markedly between two strains of L. major. Sera from patients with visceral leishmaniasis caused by Leishmania donovani chagasi recognized the gp58-63 complex and an additional Mr-42,000 polypeptide shared between L. major and L. donovani chagasi. A subpopulation of six surface proteins, including the abundant gp58-63 antigen and a group of proteins of Mr 81,000 to 105,000, were glycoproteins recognized by antiserum to wheat germ agglutinin- or concanavalin A-binding proteins. The membrane proteins of the LRC-L119 isolate of L. major could successfully vaccinate genetically susceptible mice, thus opening the way for a molecularly defined subunit vaccine composed of glycolipid and membrane protein antigens.

  19. 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

  20. Characterization of integral membrane proteins of Leishmania major by Triton X-114 fractionation and analysis of vaccination effects in mice.

    PubMed Central

    Murray, P J; Spithill, T W; Handman, E

    1989-01-01

    The total integral membrane proteins of promastigotes of Leishmania major were extracted by using the Triton X-114 phase separation technique and were characterized by immunoprecipitation, Western blotting (immunoblotting), and lectin chromatography. Of the 40 or more proteins which partitioned into the detergent phase, only about 10 proteins could be surface radioiodinated on live promastigotes, suggesting their surface orientation. The abundance of the gp58-63 antigen varied markedly between two strains of L. major. Sera from patients with visceral leishmaniasis caused by Leishmania donovani chagasi recognized the gp58-63 complex and an additional Mr-42,000 polypeptide shared between L. major and L. donovani chagasi. A subpopulation of six surface proteins, including the abundant gp58-63 antigen and a group of proteins of Mr 81,000 to 105,000, were glycoproteins recognized by antiserum to wheat germ agglutinin- or concanavalin A-binding proteins. The membrane proteins of the LRC-L119 isolate of L. major could successfully vaccinate genetically susceptible mice, thus opening the way for a molecularly defined subunit vaccine composed of glycolipid and membrane protein antigens. Images PMID:2731987

  1. A kinetic model for voltage-gated ion channels in cell membranes based on the path integral method

    NASA Astrophysics Data System (ADS)

    Erdem, Rıza; Ekiz, Cesur

    2005-04-01

    A kinetic model of cell membrane ion channels is proposed based on the path integral method. From the Pauli-type master equations valid on a macroscopic time scale, we derive a first-order differential equation or the kinetic equation which governs temporal evolution of the channel system along the paths of extreme probability. Using known parameters for the batrachotoxin (BTX)-modified sodium channels in squid giant axon, the time dependence of the channel activation and the voltage dependence of the corresponding time constants ( τ) are examined numerically. It is found that the channel activation relaxes to the steady (or equilibrium)-state values for a given membrane potential and the corresponding time constant reaches a maximum at a certain potential and thereafter decreases in magnitude as the membrane potential increases. A qualitative comparison between these results and the results of Hodgkin-Huxley theory, path probability method and thermodynamic models as well as the cut-open axon technique is presented. Good agreement is achieved.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. Two-dimensional crystallization of integral membrane proteins for electron crystallography.

    PubMed

    Stokes, David L; Rice, William J; Hu, Minghui; Kim, Changki; Ubarretxena-Belandia, Iban

    2010-01-01

    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 microscopy.

  9. Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling.

    PubMed

    Zhang, T; Rawson, D M; Tosti, L; Carnevali, O

    2008-04-01

    This study investigated enzymatic activity of cathepsins and the membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling. Stage III oocytes (>0.5mm), obtained through dissection of anaesthetised female fish and desegregation of ovarian cumulus, were exposed to 2M methanol or 2M DMSO (both prepared in Hank's medium) for 30min at 22 degrees C before being loaded into 0.5ml plastic straws and placed into a programmable cooler. After controlled slow freezing, samples were plunged into liquid nitrogen (LN) and held for at least 10min, and thawed by immersing straws into a 27 degrees C water bath for 10s. Thawed oocytes were washed twice in Hank's medium. Cathepsin activity and membrane integrity of oocytes were assessed both after cryoprotectant treatment at 22 degrees C and after freezing in LN. Cathepsin B and L colorimetric analyses were performed using substrates Z-Arg-ArgNNap and Z-Phe-Arg-4MbetaNA-HCl, respectively, and 2-naphthylamine and 4-methoxy-2-naphthylamine were used as standards. Cathepsin D activity was performed by analysing the level of hydrolytic action on haemoglobin. Oocytes membrane integrity was assessed using 0.2% Trypan blue staining for 5min. Analysis of cathepsin activities showed that whilst the activity of cathepsin B and D was not affected by 2M DMSO treatment, their activity was lowered when treated with 2M methanol. Following freezing to -196 degrees C, the activity of all cathepsins (B, D and L) was significantly decreased in both 2M DMSO and 2M methanol. Trypan blue staining showed that 63.0+/-11.3% and 72.7+/-5.2% oocytes membrane stayed intact after DMSO and methanol treatment for 30min at 22 degrees C, respectively, whilst 14.9+/-2.6% and 1.4+/-0.8% stayed intact after freezing in DMSO and methanol to -196 degrees C. The results indicate that cryoprotectant treatment and freezing modified the activities of lysosomal enzymes involved in oocyte maturation and yolk

  10. Thermodynamic and kinetic investigations of the release of oxidized phospholipids from lipid membranes and its effect on vascular integrity

    PubMed Central

    Heffern, Charles T.R.; Pocivavsek, Luka; Birukova, Anna A.; Moldobaeva, Nurgul; Bochkov, Valery N.; Lee, Ka Yee C.; Birukov, Konstantin G.

    2013-01-01

    The lipid membrane not only provides a rich interface with an array of receptor signaling complexes with which a cell communicates, but it also serves as a source of lipid derived bioactive molecules. In pathologic conditions of acute lung injury (ALI) associated with activation of oxidative stress, unsaturated phosphatidyl cholines overlooking a luminal space undergo oxidation leading to generation of fragmented phospholipids such as 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPC), or 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) full length oxygenation products (oxPAPC). Using Langmuir monolayers as models of the lipid bilayer, we evaluated the propensity of these phospholipids to solubilize from the cell membrane. The results suggest that lysoPC is rapidly released as it is produced, while oxPAPC has a longer membrane bound lifetime. After being released from cell membranes, these oxidized phospholipids exhibit potent agonist-like effects on neighboring cells. Therefore, we correlate the presence of the two phospholipid groups with the onset and resolution of increased vascular leakiness associated with ALI through testing their effect on vascular endothelial barrier integrity. Our work shows that cells respond differently to these two groups of products of phosphatidyl choline oxidation. LysoPC disrupts cell–cell junctions and increases endothelial permeability while oxPAPC enhances endothelial barrier. These data suggest a model whereby rapid release of lysoPC results in onset of ALI associated vascular leak, and the release of a reserve of oxPAPC as oxidative stress subsides restores the vascular barrier properties. PMID:23911706

  11. The synthetic amphipathic peptidomimetic LTX109 is a potent fungicide that disturbs plasma membrane integrity in a sphingolipid dependent manner.

    PubMed

    Bojsen, Rasmus; Torbensen, Rasmus; 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.

  12. Proteasome-mediated degradation of integral inner nuclear membrane protein emerin in fibroblasts lacking A-type lamins

    SciTech Connect

    Muchir, Antoine; Massart, Catherine; Engelen, Baziel G. van; Lammens, Martin; Bonne, Gisele; Worman, Howard J. . E-mail: hjw14@columbia.edu

    2006-12-29

    We previously identified and characterized a homozygous LMNA nonsense mutation leading to the absence of A-type lamins in a premature neonate who died at birth. We show here that the absence of A-type lamins is due to degradation of the aberrant mRNA transcript with a premature termination codon. In cultured fibroblasts from the subject with the homozygous LMNA nonsense mutation, there was a decreased steady-state expression of the integral inner nuclear membrane proteins emerin and nesprin-1{alpha} associated with their mislocalization to the bulk endoplasmic reticulum and a hyperphosphorylation of emerin. To determine if decreased emerin expression occurred post-translationally, we treated cells with a selective proteasome inhibitor and observed an increase in expression. Our results show that mislocalization of integral inner nuclear membrane proteins to the endoplasmic reticulum in human cells lacking A-type lamins leads to their degradation and provides the first evidence that their degradation is mediated by the proteasome.

  13. Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family.

    PubMed

    Shanklin, John; Whittle, Edward

    2003-06-19

    Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site. PMID:12804773

  14. 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. PMID:27111636

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. Integrated SDS removal and protein digestion by hollow fiber membrane based device for SDS-assisted proteome analysis.

    PubMed

    Xia, Simin; Yuan, Huiming; Chen, Yuanbo; Liang, Zheng; Zhang, Lihua; Zhang, Yukui

    2015-08-15

    In this work, a novel integrated sample preparation device for SDS-assisted proteome analysis was developed, by which proteins dissolved in 4% (w/v) SDS were first diluted by 50% methanol, and then SDS was online removed by a hollow fiber membrane interface (HFMI) with 50mM ammonium bicarbonate (pH 8.0) as an exchange buffer, finally digested by an immobilized enzyme reactor (IMER). To evaluate the performance of such an integrated device, bovine serum albumin dissolved in 4% (w/v) SDS as a model sample was analyzed; it could be found that similar to that obtained by direct analysis of BSA digests without SDS (the sequence coverage of 60.3±1.0%, n=3), with HFMI as an interface for SDS removal, BSA was identified with the sequence coverage of 61.0±1.0% (n=3). However, without SDS removal by HFMI, BSA could not be digested by the IMER and none peptides could be detected. In addition, such an integrated sample preparation device was also applied for the analysis of SDS extracted proteins from rat brain, compared to those obtained by filter-aided sample preparation (FASP), not only the identified protein group and unique peptide number were increased by 12% and 39% respectively, but also the sample pretreatment time was shortened from 24h to 4h. All these results demonstrated that such an integrated sample preparation device would provide an alternative tool for SDS assisted proteome analysis.

  1. 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

  2. Direct Measurement of Cyclic Current-Voltage Responses of Integral Membrane Proteins at a Self-Assembled Lipid-Bilayer-Modified Electrode: Cytochrome f and Cytochrome c Oxidase

    NASA Astrophysics Data System (ADS)

    Salamon, Z.; Hazzard, J. T.; Tollin, G.

    1993-07-01

    Direct cyclic voltage-current responses, produced in the absence of redox mediators, for two detergent-solubilized integral membrane proteins, spinach cytochrome f and beef heart cytochrome c oxidase, have been obtained at an optically transparent indium oxide electrode modified with a self-assembled lipid-bilayer membrane. The results indicate that both proteins interact with the lipid membrane so as to support quasi-reversible electron transfer redox reactions at the semiconductor electrode. The redox potentials that were obtained from analysis of the cyclic "voltammograms," 365 mV for cytochrome f and 250 and 380 mV for cytochrome c oxidase (vs. normal hydrogen electrode), compare quite well with the values reported by using conventional titration methods. The ability to obtain direct electrochemical measurements opens up another approach to the investigation of the properties of integral membrane redox proteins.

  3. Direct measurement of cyclic current-voltage responses of integral membrane proteins at a self-assembled lipid-bilayer-modified electrode: Cytochrome f and cytochrome c oxidase

    SciTech Connect

    Salamon, Z.; Hazzard, J.T.; Tollin, G. )

    1993-07-15

    Direct cyclic voltage-current responses, produced in the absence of redox mediators, for two detergent-solubilized integral membrane proteins, spinach cytochrome f and beef heart cytochrome c oxidase, have been obtained at an optically transparent indium oxide electrode modified with a self-assembled lipid-bilayer membrane. The results indicate that both proteins interact with the lipid membrane so as to support quasi-reversible electron transfer redox reactions at the semiconductor electrode. The redox potentials that were obtained from analysis of the cyclic [open quotes]voltammograms,[close quotes] 365 mV for cytochrome f and 250 and 380 mV for cytochrome c oxidase (vs. normal hydrogen electrode), compare quite well with the values reported by using conventional titration methods. The ability to obtain direct electrochemical measurements opens up another approach to the investigation of the properties of integral membrane redox proteins. 63 refs., 2 figs., 1 tab.

  4. The Cell-Free Integration of a Polytopic Mitochondrial Membrane Protein into Liposomes Occurs Cotranslationally and in a Lipid-Dependent Manner

    PubMed Central

    Long, Ashley R.; O'Brien, Catherine C.; Alder, Nathan N.

    2012-01-01

    The ADP/ATP Carrier (AAC) is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids. PMID:23050015

  5. 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

  6. A major integral protein of the plant plasma membrane binds flavin.

    PubMed

    Lorenz, Astrid; Kaldenhoff, Ralf; Hertel, Rainer

    2003-05-01

    Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor. PMID:12768338

  7. Interfacing Zwitterionic Liposomes with Inorganic Nanomaterials: Surface Forces, Membrane Integrity, and Applications.

    PubMed

    Liu, Juewen

    2016-05-10

    Zwitterionic phosphocholine (PC) lipids are the main constituent of the mammalian cell membrane. PC bilayers are known for their antifouling properties, yet they are adsorbed by all tested inorganic nanoparticles. This feature article is focused on the developments in my laboratory in the past few years on this topic. The main experimental techniques include fluorescence-based liposome leakage assays, adsorption and desorption, and cryo-TEM. Different materials interact with PC liposomes differently. PC liposomes adsorb on SiO2, followed by membrane fusion with the surface forming supported lipid bilayers. TiO2 and other metal oxides adsorb only intact PC liposomes via lipid phosphate bonding; the steric effect from the choline group hinders subsequent liposome fusion onto the particles. Citrate-capped AuNPs are adsorbed very strongly via van der Waals forces, inducing local gelation. The result is transient liposome leakage upon AuNP adsorption or desorption and AuNP aggregation on the liposome surface. All carbon-based nanomaterials (graphene oxides, carbon nanotubes, and nanodiamond) are adsorbed mainly via hydrogen bonding. The oxidation level of graphene oxide strongly influences the outcome of the final hybrid material. In the context of inorganic nanoparticle adsorption, insights are given regarding the lack of protein adsorption by PC bilayers. These inorganic/lipid hybrid materials can be used for controlled release, drug delivery, and fundamental studies. A few examples of application are covered toward the end, and future perspectives are given. PMID:27093351

  8. 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.

  9. Ice-induced partial unfolding and aggregation of an integral membrane protein.

    PubMed

    Garber Cohen, Iona P; Castello, Pablo R; González Flecha, F Luis

    2010-11-01

    Although the deleterious effects of ice on water-soluble proteins are well established, little is known about the freeze stability of membrane proteins. Here we explore this issue through a combined kinetic and spectroscopic approach using micellar-purified plasma membrane calcium pump as a model. The ATPase activity of this protein significantly diminished after freezing using a slow-cooling procedure, with the decrease in the activity being an exponential function of the storage time at 253K, with t(½)=3.9±0.6h. On the contrary, no significant changes on enzyme activity were detected when a fast cooling procedure was performed. Regardless of the cooling rate, successive freeze-thaw cycles produced an exponential decrease in the Ca(2+)-ATPase activity, with the number of cycles at which the activity was reduced to half being 9.2±0.3 (fast cooling) and 3.7±0.2 (slow cooling). PAGE analysis showed that neither degradation nor formation of SDS-stable aggregates of the protein takes place during protein inactivation. Instead, the inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, far UV circular dichroism, and 1-anilino-naphtalene-8-sulfonate binding. This inactive protein undergoes, in a later stage, a further irreversible transformation leading to large aggregates.

  10. Zinc protection against aluminium induced altered lipid profile and membrane integrity.

    PubMed

    Singla, Neha; Dhawan, D K

    2013-05-01

    The aim of the present study was to investigate the effects of Zinc (Zn) supplementation on lipid profile and fluidity of cerebrum and cerebellum membranes of rats treated with aluminium (Al). Sprague dawley male rats were divided into four different treatment groups viz: Control, aluminium treated, zinc treated and aluminium+zinc treated. Aluminium (AlCl3) was administered orally at a dose of 100mg/kgb.wt./day (dissolved in drinking water). Zinc as zinc sulphate was supplemented to rats at a dose of 227mg/l in drinking water. A significant decrease in the levels of total lipids, glycolipids, phospholipids, cholesterol and gangliosides contents were observed in both the cerebrum and cerebellum following Al exposure, which were found to be significantly increased following Zn supplementation. On the contrary, Al treatment caused a significant increase in the formation of conjugated dienes, which were observed to be reduced on Zn co-treatment. Further, Al treatment significantly elevated the fluorescence polarization, anisotropy and order parameter, which however were normalized upon Zn co-administration. Hence, the present study depicts the potential of Zn in moderating the changes caused by Al on membrane composition and fluidity in rat brain.

  11. 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. PMID:26502699

  12. Interfacing Zwitterionic Liposomes with Inorganic Nanomaterials: Surface Forces, Membrane Integrity, and Applications.

    PubMed

    Liu, Juewen

    2016-05-10

    Zwitterionic phosphocholine (PC) lipids are the main constituent of the mammalian cell membrane. PC bilayers are known for their antifouling properties, yet they are adsorbed by all tested inorganic nanoparticles. This feature article is focused on the developments in my laboratory in the past few years on this topic. The main experimental techniques include fluorescence-based liposome leakage assays, adsorption and desorption, and cryo-TEM. Different materials interact with PC liposomes differently. PC liposomes adsorb on SiO2, followed by membrane fusion with the surface forming supported lipid bilayers. TiO2 and other metal oxides adsorb only intact PC liposomes via lipid phosphate bonding; the steric effect from the choline group hinders subsequent liposome fusion onto the particles. Citrate-capped AuNPs are adsorbed very strongly via van der Waals forces, inducing local gelation. The result is transient liposome leakage upon AuNP adsorption or desorption and AuNP aggregation on the liposome surface. All carbon-based nanomaterials (graphene oxides, carbon nanotubes, and nanodiamond) are adsorbed mainly via hydrogen bonding. The oxidation level of graphene oxide strongly influences the outcome of the final hybrid material. In the context of inorganic nanoparticle adsorption, insights are given regarding the lack of protein adsorption by PC bilayers. These inorganic/lipid hybrid materials can be used for controlled release, drug delivery, and fundamental studies. A few examples of application are covered toward the end, and future perspectives are given.

  13. 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%.

  14. Effectiveness of heat-integrated methanol steam reformer and polymer electrolyte membrane fuel cell stack systems for portable applications

    NASA Astrophysics Data System (ADS)

    Lotrič, A.; Sekavčnik, M.; Hočevar, S.

    2014-12-01

    Efficiently combining proton exchange membrane fuel cell (PEMFC) stack with methanol steam reformer (MSR) into a small portable system is still quite a topical issue. Using methanol as a fuel in PEMFC stack includes a series of chemical processes where each proceeds at a unique temperature. In a combined MSR-PEMFC-stack system with integrated auxiliary fuel processors (vaporizer, catalytic combustor, etc.) the processes are both endothermic and exothermic hence their proper thermal integration can help raising the system efficiency. A concept of such fully integrated and compact system is proposed in this study. Three separate systems are designed based on different PEMFC stacks and MSR. Low-temperature (LT) and conventional high-temperature (cHT) PEMFC stack characteristics are based on available data from suppliers. Also, a novel high-temperature (nHT) PEMFC stack is proposed because its operating temperature coincides with that of MSR. A comparative study of modelled systems is performed using a mass and energy balances zero-dimensional model, which is interdependently coupled to a physical model based on finite element method (FEM). The results indicate that a system with nHT PEMFC stack is feasible and has the potential to reach higher system efficiencies than systems with LT or cHT PEMFC stacks.

  15. Relationship of flow cytometric sperm integrity assessments with boar fertility performance under optimized field conditions.

    PubMed

    Broekhuijse, M L W J; Šoštarić, E; Feitsma, H; Gadella, B M

    2012-12-01

    The number of intact and functional spermatozoa in semen can be assessed with flow cytometry and is believed to relate to male fertility. The aim of this study was to examine whether currently used sperm integrity assessments with flow cytometry correlate with field fertility data obtained for boar semen. For this purpose, 20 boars were followed for a 20-wk period (with a total average production of 33 ejaculates per boar) and the obtained fertility results (farrowing rate and number of piglets born) of commercial artificial insemination doses made from these ejaculates were recorded. Fertility results were corrected for farm, sow, boar, and semen-related parameters. From the same semen samples, sperm cell integrity was assessed with respect to DNA and to membrane integrity, acrosome intactness and responsiveness, and mitochondrial potential using established flow cytometric assays. This was done on freshly produced semen and on semen stored for up to 15 d. Remarkably, none of the individual membrane integrity variables was significantly related to fertility results. In contrast, the amount of DNA damage as assessed at 7 to 10 d and at 14 to 15 d of semen storage related to farrowing rate (P = 0.0400) and total number of piglets born (P = 0.0310), respectively. Therefore, the degree of DNA damage in stored boar semen samples may be a useful factor to evaluate semen as an indicator for litter size and farrowing rate. PMID:23255815

  16. Bioinformatic Analyses of Integral Membrane Transport Proteins Encoded Within the Genome of the Planctomycetes species, Rhodopirellula baltica

    PubMed Central

    Paparoditis, Philipp; Vastermark, Ake; Le, Andrew J.; Fuerst, John A.; Saier, Milton H.

    2013-01-01

    Rhodopirellula baltica (R. baltica) is a Planctomycete, known to have intracellular membranes. Because of its unusual cell structure and ecological significance, we have conducted comprehensive analyses of its transmembrane transport proteins. The complete proteome of R. baltica was screened against the Transporter Classification Database (TCDB) to identify recognizable integral membrane transport proteins. 342 proteins were identified with a high degree of confidence, and these fell into several different classes. R. baltica encodes in its genome channels (12%), secondary carriers (33%), and primary active transport proteins (41%) in addition to classes represented in smaller numbers. Relative to most non-marine bacteria, R. baltica possesses a larger number of sodium-dependent symporters but fewer proton-dependent symporters, and it has dimethylsulfoxide (DMSO) and trimethyl-amine-oxide (TMAO) reductases, consistent with its Na+-rich marine environment. R. baltica also possesses a Na+-translocating NADH:quinone dehydrogenase (Na+-NDH), a Na+ efflux decarboxylase, two Na+-exporting ABC pumps, two Na+-translocating F-type ATPases, two Na+:H+ antiporters and two K+:H+ antiporters. Flagellar motility probably depends on the sodium electrochemical gradient. Surprisingly, R. baltica also has a complete set of H+-translocating electron transport complexes similar to those present in β-proteobacteria and eukaryotic mitochondria. The transport proteins identified proved to be typical of the bacterial domain with little or no indication of the presence of eukaryotic-type transporters. However, novel functionally uncharacterized multispanning membrane proteins were identified, some of which are found only in Rhodopirellula species, but others of which are widely distributed in bacteria. The analyses lead to predictions regarding the physiology, ecology and evolution of R. baltica. PMID:23969110

  17. 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

  18. Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family.

    PubMed Central

    Preston, G M; Agre, P

    1991-01-01

    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. Images PMID:1722319

  19. Bioinformatic analyses of integral membrane transport proteins encoded within the genome of the planctomycetes species, Rhodopirellula baltica.

    PubMed

    Paparoditis, Philipp; Västermark, Ake; Le, Andrew J; Fuerst, John A; Saier, Milton H

    2014-01-01

    Rhodopirellula baltica (R. baltica) is a Planctomycete, known to have intracellular membranes. Because of its unusual cell structure and ecological significance, we have conducted comprehensive analyses of its transmembrane transport proteins. The complete proteome of R. baltica was screened against the Transporter Classification Database (TCDB) to identify recognizable integral membrane transport proteins. 342 proteins were identified with a high degree of confidence, and these fell into several different classes. R. baltica encodes in its genome channels (12%), secondary carriers (33%), and primary active transport proteins (41%) in addition to classes represented in smaller numbers. Relative to most non-marine bacteria, R. baltica possesses a larger number of sodium-dependent symporters but fewer proton-dependent symporters, and it has dimethylsulfoxide (DMSO) and trimethyl-amine-oxide (TMAO) reductases, consistent with its Na(+)-rich marine environment. R. baltica also possesses a Na(+)-translocating NADH:quinone dehydrogenase (Na(+)-NDH), a Na(+) efflux decarboxylase, two Na(+)-exporting ABC pumps, two Na(+)-translocating F-type ATPases, two Na(+):H(+) antiporters and two K(+):H(+) antiporters. Flagellar motility probably depends on the sodium electrochemical gradient. Surprisingly, R. baltica also has a complete set of H(+)-translocating electron transport complexes similar to those present in α-proteobacteria and eukaryotic mitochondria. The transport proteins identified proved to be typical of the bacterial domain with little or no indication of the presence of eukaryotic-type transporters. However, novel functionally uncharacterized multispanning membrane proteins were identified, some of which are found only in Rhodopirellula species, but others of which are widely distributed in bacteria. The analyses lead to predictions regarding the physiology, ecology and evolution of R. baltica. PMID:23969110

  20. N-terminal domain of the V-ATPase a2-subunit displays integral membrane protein properties.

    PubMed

    Merkulova, Maria; McKee, Mary; Dip, Phat Vinh; Grüber, Gerhard; Marshansky, Vladimir

    2010-10-01

    V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N(1-402)) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N(1-402) recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINE®12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N(1-402) in NDSB-256 was successfully used in pull-down and BIAcore™ protein-protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N(1-402) and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called "cytosolic" tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N(1-402) should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.

  1. Charge equilibration force fields for molecular dynamics simulations of lipids, bilayers, and integral membrane protein systems.

    PubMed

    Lucas, Timothy R; Bauer, Brad A; Patel, Sandeep

    2012-02-01

    With the continuing advances in computational hardware and novel force fields constructed using quantum mechanics, the outlook for non-additive force fields is promising. Our work in the past several years has demonstrated the utility of polarizable force fields, those based on the charge equilibration formalism, for a broad range of physical and biophysical systems. We have constructed and applied polarizable force fields for lipids and lipid bilayers. In this review of our recent work, we discuss the formalism we have adopted for implementing the charge equilibration (CHEQ) method for lipid molecules. We discuss the methodology, related issues, and briefly discuss results from recent applications of such force fields. Application areas include DPPC-water monolayers, potassium ion permeation free energetics in the gramicidin A bacterial channel, and free energetics of permeation of charged amino acid analogs across the water-bilayer interface. This article is part of a Special Issue entitled: Membrane protein structure and function.

  2. 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%.

  3. Effects of Lewisite on cell membrane integrity and energy metabolism in human keratinocytes and SCL II cells.

    PubMed

    Kehe, K; Flohé, S; Krebs, G; Kreppel, H; Reichl, F X; Liebl, B; Szinicz, L

    2001-06-21

    Lewisite is a highly toxic arsenic compound which can cause skin damage. In the present study effects of Lewisite on cell membrane integrity and energy metabolism as well as antidotal effects of DL-2,3-dimercaptopropanesulfonate (DMPS), and meso-2,3-dimercaptosuccinic acid (m-DMSA) were investigated in a keratinocyte derived cell line (SCL II) and primary human keratinocytes (HK). Cells were incubated in Lewisite (60 microM) containing medium for 5 min. During the following 6 h lactate dehydrogenase (LDH) activity in the supernatant, intracellular ATP content, tetrazolium reduction, glucose consumption and lactate formation were measured. Glucose consumption and lactate production were decreased in both cell lines after Lewisite exposure. In SCL II cells an increase of LDH activity in the supernatant, a decrease of ATP content, and an impaired ability to reduce tetrazolium was found 3 h after Lewisite exposure. In HK cultures tetrazolium reduction was significantly decreased already after 2 h, whereas LDH increase in the supernatant and ATP content decrease occurred only at 6 h after Lewisite exposure. When DMPS or m-DMSA was added directly after Lewisite exposure to SCL II cells, glucose consumption and lactate formation were restored and LDH leakage was prevented. SCL II cells might be more prone to membrane damage whereas in keratinocytes mitochondrial impairment seems to be the predominant effect of Lewisite.

  4. 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.

  5. 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

  6. 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%. PMID:27176649

  7. Design, purification and characterization of a soluble variant of the integral membrane protein MotB for structural studies

    PubMed Central

    Andrews, Daniel A.; Xie, Meng; Hughes, Victoria; Wilce, Matthew C.; Roujeinikova, Anna

    2013-01-01

    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. PMID:23193105

  8. 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.

  9. Controlled Architecture of Dual-Functional Block Copolymer Brushes on Thin-Film Composite Membranes for Integrated "Defending" and "Attacking" Strategies against Biofouling.

    PubMed

    Ye, Gang; Lee, Jongho; Perreault, François; Elimelech, Menachem

    2015-10-21

    We report a new macromolecular architecture of dual functional block copolymer brushes on commercial thin-film composite (TFC) membranes for integrated "defending" and "attacking" strategies against biofouling. Mussel-inspired catechol chemistry is used for a convenient immobilization of initiator molecules to the membrane surface with the aid of polydopamine (PDA). Zwitterionic polymer brushes with strong hydration capacity and quaternary ammonium salt (QAS) polymer brushes with bactericidal ability are sequentially grafted on TFC membranes via activators regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP), an environmentally benign and controlled polymerization method. Measurement of membrane intrinsic transport properties in reverse osmosis experiments shows that the modified TFC membrane maintains the same water permeability and salt selectivity as the pristine TFC membrane. Chemical force microscopy and protein/bacterial adhesion studies are carried out for a comprehensive evaluation of the biofouling resistance and antimicrobial ability, demonstrating low biofouling propensity and excellent bacterial inactivation for the modified TFC membrane. We conclude that this polymer architecture, with complementary "defending" and "attacking" capabilities, can effectively prevent the attachment of biofoulants and formation of biofilms and thereby significantly mitigate biofouling on TFC membranes. PMID:26378606

  10. Controlled Architecture of Dual-Functional Block Copolymer Brushes on Thin-Film Composite Membranes for Integrated "Defending" and "Attacking" Strategies against Biofouling.

    PubMed

    Ye, Gang; Lee, Jongho; Perreault, François; Elimelech, Menachem

    2015-10-21

    We report a new macromolecular architecture of dual functional block copolymer brushes on commercial thin-film composite (TFC) membranes for integrated "defending" and "attacking" strategies against biofouling. Mussel-inspired catechol chemistry is used for a convenient immobilization of initiator molecules to the membrane surface with the aid of polydopamine (PDA). Zwitterionic polymer brushes with strong hydration capacity and quaternary ammonium salt (QAS) polymer brushes with bactericidal ability are sequentially grafted on TFC membranes via activators regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP), an environmentally benign and controlled polymerization method. Measurement of membrane intrinsic transport properties in reverse osmosis experiments shows that the modified TFC membrane maintains the same water permeability and salt selectivity as the pristine TFC membrane. Chemical force microscopy and protein/bacterial adhesion studies are carried out for a comprehensive evaluation of the biofouling resistance and antimicrobial ability, demonstrating low biofouling propensity and excellent bacterial inactivation for the modified TFC membrane. We conclude that this polymer architecture, with complementary "defending" and "attacking" capabilities, can effectively prevent the attachment of biofoulants and formation of biofilms and thereby significantly mitigate biofouling on TFC membranes.

  11. Essential factors of an integrated moving bed biofilm reactor-membrane bioreactor: Adhesion characteristics and microbial community of the biofilm.

    PubMed

    Tang, Bing; Yu, Chunfei; Bin, Liying; Zhao, Yiliang; Feng, Xianfeng; Huang, Shaosong; Fu, Fenglian; Ding, Jiewei; Chen, Cuiqun; Li, Ping; Chen, Qianyu

    2016-07-01

    This work aims at revealing the adhesion characteristics and microbial community of the biofilm in an integrated moving bed biofilm reactor-membrane bioreactor, and further evaluating their variations over time. With multiple methods, the adhesion characteristics and microbial community of the biofilm on the carriers were comprehensively illuminated, which showed their dynamic variation along with the operational time. Results indicated that: (1) the roughness of biofilm on the carriers increased very quickly to a maximum value at the start-up stage, then, decreased to become a flat curve, which indicated a layer of smooth biofilm formed on the surface; (2) the tightly-bound protein and polysaccharide was the most important factor influencing the stability of biofilm; (3) the development of biofilm could be divided into three stages, and Gammaproteobacteria were the most dominant microbial species in class level at the last stage, which occupied the largest ratio (51.48%) among all microbes.

  12. 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.

  13. 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

    2014-01-01

    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.

  14. 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

    2014-01-01

    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. PMID:24239975

  15. 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.

  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. 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)

  18. Regulatory design in a simple system integrating membrane potential generation and metabolic ATP consumption. Robustness and the role of energy dissipating processes.

    PubMed

    Acerenza, Luis; Cristina, Ernesto; Hernández, Julio A

    2011-12-01

    Bacterial physiological responses integrate energy-coupling processes at the membrane level with metabolic energy demand. The regulatory design behind these responses remains largely unexplored. Propionigenium modestum is an adequate organism to study these responses because it presents the simplest scheme known integrating membrane potential generation and metabolic ATP consumption. A hypothetical sodium leak is added to the scheme as the sole regulatory site. Allosteric regulation is assumed to be absent. Information of the rate equations is not available. However, relevant features of the patterns of responses may be obtained using Metabolic Control Analysis (MCA) and Metabolic Control Design (MCD). With these tools, we show that membrane potential disturbances can be compensated by adjusting the leak flux, without significant perturbations of ATP consumption. Perturbations of membrane potential by ATP demand are inevitable and also require compensatory changes in the leak. Numerical simulations were performed with a kinetic model exhibiting the responses for small changes obtained with MCA and MCD. A modest leak (10% of input) was assumed for the reference state. We found that disturbances in membrane potential and ATP consumption, produced by environmental perturbations of the cation concentration, may be reverted to the reference state adjusting the leak. Leak changes can also compensate for undesirable effects on membrane potential produced by changes in nutrient availability or ATP demand, in a wide range of values. The system is highly robust to parameter fluctuations. The regulatory role of energy dissipating processes and the trade-off between energetic efficiency and regulatory capacity are discussed.

  19. 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.

  20. 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.

  1. Effect of storage in short--and long-term commercial semen extenders on the motility, plasma membrane and chromatin integrity of boar spermatozoa.

    PubMed

    De Ambrogi, Marco; Ballester, Juan; Saravia, Fernando; Caballero, Ignacio; Johannisson, Anders; Wallgren, Margareta; Andersson, Magnus; Rodriguez-Martinez, Heriberto

    2006-10-01

    For artificial insemination (AI) in pigs, preservation of liquid boar semen at 16-20 degrees C is still common practice as sperm cryopreservation remains suboptimal in this species. To meet the different needs of the swine industry, several extenders have been developed to preserve semen in liquid form for short--and long-term storage. In the present study, three different commercial extenders devised for short-term (BTS+) or long-term preservation (MR-A and X-Cell), were used to test whether storage of semen from four mature, fertile boars at 17 degrees C for 96 h would affect sperm characteristics relevant for fertility, such as motility, membrane integrity and chromatin stability. Computer-assisted sperm analysis, and stainings with the acylated membrane dye SYBR-14/propidium iodide, and acridine orange in connection with flow cytometry were used to evaluate these variables. Percentages of total motile spermatozoa decreased slightly, but significantly, after 72-96 h. While membrane integrity values varied during the period of study, no significant changes in either membrane integrity or chromatin stability were, however, registered. This suggests a customary 96-day storage at 17 degrees C in these extenders was too short an interval to cause losses of integrity in nuclear DNA in the boar population studied. PMID:16573706

  2. 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

  3. 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

  4. 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

    2016-03-01

    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. PMID:26893454

  5. 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...

  6. Heparin-glutathione III: study with fluorescent probes as indicators of membrane status of bull sperm.

    PubMed

    Reyes, R; Martinez, J C; Delgado, N M; Merchant-Larios, H

    2002-01-01

    Sperm obtained from bull epididymes were used to validate in vitro the effect of heparin and reduced glutathione on sperm membrane status, with the use of sodium dodecyl sulfate (SDS) and Triton X-100 in the presence of propidium iodide (IP) and diacetate fluorescein (FDA). The metabolic activities of treated sperm were qualitatively monitored using an alamar Blue Redox fluorescence indicator. Heparin did not damage the sperm plasma membrane, whereas GSH and SDS at 26 h of incubation dissolved the plasma membrane and the acrosome. On the other hand, at time zero, Triton X-100 showed 75% of sperm stained with IP, indicating plasma membrane damage. Results validated by electron microscopy of thin sections of treated sperm showed complete lack of the membrane, acrosome, and postacrosomal membrane system with 0.01% Triton X-100. Extracellular 15 mM GSH completely disappeared the plasma membrane over the sperm nucleus, leaving the postacrosomal membrane system and nucleus without apparent damage. The metabolic activity was supported over 52 h of incubation in any of the incubation systems tested, including Triton X-100, which showed a spermaticide effect. The authors propose that membrane damage does not mean they are dead, no matter the vital stain employed, and also that FDA-IP staining can be used as a fluorescent marker of sperm plasmatic membrane permeabilization and nuclear swelling.

  7. 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

  8. Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice

    PubMed Central

    Escoffier, Jessica; Jemel, Ikram; Tanemoto, Akemi; Taketomi, Yoshitaka; Payre, Christine; Coatrieux, Christelle; Sato, Hiroyasu; Yamamoto, Kei; Masuda, Seiko; Pernet-Gallay, Karin; Pierre, Virginie; Hara, Shuntaro; Murakami, Makoto; De Waard, Michel; Lambeau, Gérard; Arnoult, Christophe

    2010-01-01

    Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization. PMID:20424324

  9. 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

  10. Identification and localization of integral membrane proteins of virulent Treponema pallidum subsp. pallidum by phase partitioning with the nonionic detergent triton X-114.

    PubMed

    Radolf, J D; Chamberlain, N R; Clausell, A; Norgard, M V

    1988-02-01

    Integral membrane proteins of Treponema pallidum subsp. pallidum (T. pallidum) were identified by phase partitioning with the nonionic detergent Triton X-114; antigens with apparent molecular masses of 47, 38, 36, 34, 32, 17, and 15 kilodaltons (kDa) were identified in the detergent phase. Immunoblotting with murine monoclonal antibodies directed against pathogen-specific 47- and 34-kDa T. pallidum antigens confirmed their presence in the detergent phase. Endoflagellar proteins of T. pallidum were not detected in immunoblots of detergent-phase proteins when monospecific antisera directed against endoflagella of the nonpathogenic T. phagedenis biotype Reiter were used. At detergent concentrations (0.02 and 0.1%) which appeared to solubilize selectively the outer membranes of treponemes radiolabeled with 35S in vitro, limited amounts of detergent-phase proteins were immunoprecipitated. Greater amounts of detergent-phase proteins were extracted at higher detergent concentrations (0.5 and 2.0%) which resulted in both outer membrane solubilization and ultrastructural derangements of the residual cytoplasmic bodies. Furthermore, Triton X-114 extraction of both intact treponemes and organisms without outer membranes yielded detergent phases with similar protein profiles. The results of these experiments indicate that the hydrophobic proteins identified by Triton X-114 are not located exclusively in the T. pallidum outer membrane. The results are also consistent with the hypothesis that the T. pallidum outer membrane is a protein-deficient lipid bilayer.

  11. A high resolution electro-optical approach for investigating transition of soluble proteins to integral membrane proteins probed by colicin A.

    PubMed

    Honigmann, Alf; Pulagam, Lakshmi Padmavathi; Sippach, Michael; Bartsch, Philipp; Steinhoff, Heinz-Jürgen; Wagner, Richard

    2012-10-19

    The transition from water soluble state to an integral membrane protein state is a crucial step in the formation of the active form of many pore-forming or receptor proteins. Albeit this, high resolution techniques which allow assay of protein membrane binding and concomitant development of the final active form in the membrane await further development. Here, we describe a horizontal artificial bilayers setup allowing for simultaneous electrical and optical measurements at a single molecule level. We use the membrane binding and subsequent channel formation of colicin A (ColA) a water soluble bacteriocin secreted by some strains of Escherichia coli to demonstrate the potential of the combined electro-optical technique. Our results expand the knowledge on ColA molecular details which show that active ColA is monomeric; membrane binding is pH but not membrane-potential (Δϕ) dependent. ColA is at Δϕ=0 permeable for molecules ≥1 nm. Although ColA exhibits low ion conductance it facilitates permeation of large molecules. Our electro-optical recordings reveal ColA monomeric state and the chimeric character of its pore. PMID:23000162

  12. 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.

  13. Sensitive dependence of the coefficient of variation of interspike intervals on the lower boundary of membrane potential for the leaky integrate-and-fire neuron model.

    PubMed

    Inoue, Junko; Doi, Shinji

    2007-01-01

    After the report of Softky and Koch [Softky, W.R., Koch, C., 1993. The highly irregular firing of cortical cells is inconsistent with temporal integration of random EPSPs. J. Neurosci. 13, 334-350], leaky integrate-and-fire models have been investigated to explain high coefficient of variation (CV) of interspike intervals (ISIs) at high firing rates observed in the cortex. The purpose of this paper is to study the effect of the position of a lower boundary of membrane potential on the possible value of CV of ISIs based on the diffusional leaky integrate-and-fire models with and without reversal potentials. Our result shows that the irregularity of ISIs for the diffusional leaky integrate-and-fire neuron significantly changes by imposing a lower boundary of membrane potential, which suggests the importance of the position of the lower boundary as well as that of the firing threshold when we study the statistical properties of leaky integrate-and-fire neuron models. It is worth pointing out that the mean-CV plot of ISIs for the diffusional leaky integrate-and-fire neuron with reversal potentials shows a close similarity to the experimental result obtained in Softky and Koch [Softky, W.R., Koch, C., 1993. The highly irregular firing of cortical cells is inconsistent with temporal integration of random EPSPs. J. Neurosci. 13, 334-350].

  14. Comparison of Mycoplasma gallisepticum strains and identification of immunogenic integral membrane proteins with Triton X-114 by immunoblotting.

    PubMed

    Avakian, A P; Kleven, S H; Ley, D H

    1991-11-01

    Pooled chicken antisera from 33 and 77 days post Mycoplasma gallisepticum strain R contact-exposure reacted with cell proteins of 19 M. gallisepticum strains. These pooled antisera reacted with more proteins and with greater intensity to reference strains (R, PG31, S6, and A5969) and nine field strains than they did with six other field strains including three (703, 503, and 730) that have been described as serological variants. Following extraction with Triton X-114 the majority of immunogenic M. gallisepticum proteins partitioned exclusively or primarily into the detergent phase indicating that they are integral membrane proteins. This included three immunogenic species-specific proteins (p64, p56 and p26). M. gallisepticum p56 was detected, by immunoblotting, in 18 of 19 strains suggesting that it could serve as an antigen for serological tests. P26 was evident in 13 of 19 strains. Hyperimmune antiserum to p64 reacted with a 64 kDa protein in 19 M. gallisepticum strains, but did not react with seven other avian Mycoplasma spp. There was no evidence found supporting the view that p64 is the hemagglutinin of M. gallisepticum.

  15. 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. PMID:15935852

  16. 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.

  17. 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 interface, whereas the N-terminal cytoplasmic domain includes a rhodanese-fold in accordance to its sequence homology to the rhodanese family of sulfurtransferases. The enzymatic sulfur transfer activity of full-length YgaP as well as of the N-terminal rhodanese domain only was investigated performing a series of titrations with sodium thiosulfate and potassium cyanide monitored by NMR and EPR. The data indicate the thiosulfate concentration-dependent addition of several sulfur atoms to the catalytic Cys-63, which process can be reversed by the addition of potassium cyanide. The catalytic reaction induces thereby conformational changes within the rhodanese domain, as well as on the transmembrane α-helices of YgaP. These results provide insights into a potential mechanism of YgaP during the catalytic thiosulfate activity in vivo. PMID:24958726

  18. Quantum dots for single-pair fluorescence resonance energy transfer in membrane- integrated EFoF1.

    PubMed

    Galvez, Eva; Düser, Monika; Börsch, Michael; Wrachtrup, Jörg; Gräber, Peter

    2008-10-01

    spFRET (single-pair fluorescence resonance energy transfer) with organic fluorophores has been used to demonstrate rotation of the subunits gamma and epsilon in membrane-integrated FoF1 during proton transport-coupled ATP synthesis. Owing to the high light intensities used in single-molecule spectroscopy, organic fluorophores show a high probability for photobleaching. Luminescent CdSe/ZnS nanocrystals with a hydrophilic shell have been covalently bound to FoF1 either to the stator subunit b or to the rotor subunit c. TIRFM (total internal reflection microscopy) shows that covalent binding of the QD (quantum dot) via cysteine to FoF1 leads to a significant decrease in the blinking probability in the microsecond-to-second time range. This effect allows the observation of subunit movements in an extended time range. If the QD is bound to the rotor subunit c, the fluorescence anisotropy shows fluctuations in the presence of ATP, in contrast with the constant anisotropy observed in the absence of ATP.

  19. Simultaneous effective carbon and nitrogen removals and phosphorus recovery in an intermittently aerated membrane bioreactor integrated system

    PubMed Central

    Wang, Yun-Kun; Pan, Xin-Rong; Geng, Yi-Kun; Sheng, Guo-Ping

    2015-01-01

    Recovering nutrients, especially phosphate resource, from wastewater have attracted increasing interest recently. Herein, an intermittently aerated membrane bioreactor (MBR) with a mesh filter was developed for simultaneous chemical oxygen demand (COD), total nitrogen (TN) and phosphorous removal, followed by phosphorus recovery from the phosphorus-rich sludge. This integrated system showed enhanced performances in nitrification and denitrification and phosphorous removal without excess sludge discharged. The removal of COD, TN and total phosphorus (TP) in a modified MBR were averaged at 94.4 ± 2.5%, 94.2 ± 5.7% and 53.3 ± 29.7%, respectively. The removed TP was stored in biomass, and 68.7% of the stored phosphorous in the sludge could be recovered as concentrated phosphate solution with a concentration of phosphate above 350 mg/L. The sludge after phosphorus release could be returned back to the MBR for phosphorus uptake, and 83.8% of its capacity could be recovered. PMID:26541793

  20. Internal limiting membrane layer visualization and vitreoretinal surgery guidance using a common-path OCT integrated microsurgical tool

    NASA Astrophysics Data System (ADS)

    Liu, Xuan; Meisne, Eric; Han, Jae-Ho; Zhang, Kang; Gehlbach, Peter; Taylor, Russell; Kang, Jin U.

    2010-02-01

    Contemporary retinal microsurgery is performed by skilled surgeons through operating microscopes, utilizing free hand techniques and manually operated micro-instruments. One technically challenging procedure is the incising and peeling of the internal limiting membrane (ILM) while minimizing damage to the underlying retina. One strategy for minimizing damage is to improve visualization of the ILM layer. Here we present a preliminary evaluation of a prototype tool that integrates an ultra high resolution Fourier domain common path Optical Coherence Tomography (OCT) with an intelligent microsurgical instrument. The tool provides OCT guided visualization of the ILM layer at the point of tissue contact by the surgical tool. We have evaluated the imaging properties of the common path OCT system. The common path OCT system used in this study has a maximum imaging depth of 1.3mm and a sensitivity of 91dB. We have achieved an experimental axial resolution of 3μm in air and this appears to be sufficient to both identify the ILM and to perform surgical maneuvers. We scanned the single mode fiber probe using an intelligent microsurgical instrument to form B-Mode images. We imaged a porcine eye with both anterior eye segment and the vitreous removed. The image obtained show distinct functional layers of retina.

  1. Probing the roles of Ca(2+) and Mg(2+) in humic acids-induced ultrafiltration membrane fouling using an integrated approach.

    PubMed

    Wang, Long-Fei; He, Dong-Qin; Chen, Wei; Yu, Han-Qing

    2015-09-15

    Membrane fouling induced by natural organic matter (NOM) negatively affects the performance of ultrafiltration (UF) technology in producing drinking water. Divalent cation is found to be an important factor that affects the NOM-induced membrane fouling process. In this work, attenuated total reflection-Fourier transformation infrared spectroscopy (ATR-FTIR) coupled with quartz crystal microbalance (QCM), assisted by isothermal titration calorimetry (ITC), is used to explore the contribution of Mg(2+) and Ca(2+), the two abundant divalent cations in natural water, to the UF membrane fouling caused by humic acid (HA) at a molecular level. The results show that Ca(2+) exhibited superior performance in accelerating fouling compared to Mg(2+). The hydrophobic polyethersulfone (PES) membrane exhibited greater complexation with HA in the presence of Mg(2+) and Ca(2+), compared to the hydrophilic cellulose membrane, as evidenced by the more intense polysaccharide C-O, aromatic C=C and carboxylic C=O bands in the FTIR spectra. The QCM and ITC measurements provide quantitative evidence to support that Ca(2+) was more effective than Mg(2+) in binding with HA and accumulating foulants on the membrane surfaces. The higher charge neutralization capacity and more favorable binding ability of Ca(2+) were found to be responsible for its greater contribution to the NOM-induced membrane fouling than Mg(2+). This work offers a new insight into the mechanism of cation-mediated NOM-induced membrane fouling process, and demonstrates that such an integrated ATR-FTIR/QCM/ITC approach could be a useful tool to explore other complicated interaction processes in natural and engineered environments. PMID:26094086

  2. Phosphorylation of Golgi Peripheral Membrane Protein Grasp65 Is an Integral Step in the Formation of the Human Cytomegalovirus Cytoplasmic Assembly Compartment

    PubMed Central

    Rebmann, G. Michael; Grabski, Robert; Sanchez, Veronica

    2016-01-01

    ABSTRACT 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. PMID:27703074

  3. A transmission electron microscopy investigation: the membrane complex in spermatogenesis of Fenneropenaeus chinensis

    PubMed Central

    Ge, Shaoqin; Guo, Mingshen; Liu, Guirong; Mu, Shumei

    2008-01-01

    The transforming characteristics of the membrane complex in spermatogenesis of Fenneropenaeus chinensis have been studied by using transmission electron microscopy. Two types of membrane complex have been investigated based on their sources: one originating from nucleus and the other from cytoplasm. The first one, consisted of annular structures, monolayer membrane blebs, and double or multi-lamellar membrane vesicles, emerges in the primary spermatocyte, then diffuses with the nuclear membrane and finally enters the cytoplasm. This type of membrane complex seems to play an important role in the materials transfusion from nucleus to cytoplasm, and it mainly exists inside the primary spermatocyte with some inside the secondary spermatocyte. The latter, originated from cytoplasm, is formed during the anaphase of spermiogenesis. It also exists in mature sperm, locating at both sides of the nucleus under the acrosomal cap. This type of membrane complex mainly comprises rings of convoluted membrane pouches, together with mitochondria, annular lamina bodies, fragments of endoplasmic reticulum, nuclear membrane and some nuclear particles. It releases vesicles and particles into the acrosomal area during the formation of the perforatorium, suggesting a combined function of the endoplasmic reticulum, mitochondria and Golgi’s mechanism. PMID:19002849

  4. Effects of high hydrostatic pressure on Escherichia coli ultrastructure, membrane integrity and molecular composition as assessed by FTIR spectroscopy and microscopic imaging techniques.

    PubMed

    Prieto-Calvo, María; Prieto, Miguel; López, Mercedes; Alvarez-Ordóñez, Avelino

    2014-12-18

    High hydrostatic pressure (HHP) is a novel food processing technology that is considered as an attractive alternative to conventional heat treatments for the preservation of foods, due to its lethal effects on pathogenic and spoilage microorganisms, while causing minor effects on food quality and sensorial attributes. This study is aimed at investigating how HHP treatments at varying intensities in the range 50-900 MPa affect the viability, membrane integrity, ultrastructure and molecular composition of Escherichia coli. Results of membrane integrity tests (measurement of cellular leakage and monitoring of propidium iodide uptake through fluorescence microscopy) and ultrastructural observations by transmission electron microscopy demonstrated that HHP gave rise to cellular enlargement, membrane damage or detachment, DNA and protein denaturation and loss of intracellular contents. Fourier-transform infrared (FTIR) spectroscopy analyses evidenced minor changes in molecular composition in response to high pressures, which were mostly observed on the spectral region w4 (1200-900 cm-1), mainly informative of carbohydrates and polysaccharides of the cell wall. These findings suggest that exposure of E. coli cells to HHP causes alterations in their physical integrity while producing minor modifications in biochemical cellular composition. The current study increases the knowledge on the mechanisms of E. coli inactivation by HHP and provides valuable information for the design of more effective food preservation regimes based on the integration of mild HHP in combination with other food preservation strategies into a multi-target hurdle technology approach.

  5. Demonstration by phase-partitioning in Triton X-114 solutions that phosphoprotein B-50 (F-1) from rat brain is an integral membrane protein.

    PubMed

    Dosemeci, A; Rodnight, R

    1987-03-01

    The Triton X-114 phase separation technique was employed to fractionate phosphoproteins present in membrane fragments from rat brain. Membranes were labelled with [gamma-32P]ATP in media containing Ca2+, Ca2+ plus calmodulin or cyclic AMP, and then treated with Triton X-114. Phosphoproteins recovered in the detergent-insoluble fraction, aqueous and detergent phases were detected by SDS-polyacrylamide gel electrophoresis and autoradiography. Of the proteins solubilised by the detergent, a known substrate of protein kinase C, the B-50 phosphoprotein (45 kD; also known as F-1), partitioned quantitatively into the detergent-rich phase, making it very probable that this phosphoprotein is an integral membrane protein. The detergent-rich phase also contained an 80 kD phosphoprotein, which probably corresponds to the widespread acidic 87 kD substrate of protein kinase C.

  6. Presence of the peroxisomal 22-kDa integral membrane protein in the liver of a person lacking recognizable peroxisomes (Zellweger syndrome).

    PubMed Central

    Lazarow, P B; Fujiki, Y; Small, G M; Watkins, P; Moser, H

    1986-01-01

    Peroxisomes have not been detected in liver and kidney of patients with Zellweger syndrome. Some peroxisome proteins are missing; others are present in normal amounts but are located in the cytosol. We have prepared an antiserum against the 22-kDa integral membrane protein characteristic of rat liver peroxisomes. The antiserum crossreacts with the human liver counterpart, which likewise has a mass of 22 kDa. By immunoblot analysis, we demonstrate that the 22-kDa protein is present in normal amount in Zellweger liver and is integral to a membrane. The result suggests that peroxisome membranes are assembled in Zellweger syndrome but may be defective for the import of matrix proteins. As a result, newly synthesized proteins are left in the cytosol, where some persist and others are degraded. Lacking their usual content, such aberrant peroxisomal membranes would be unrecognizable morphologically. Immunoblot analyses also showed that the peroxisomal hydratase-dehydrogenase is deficient in Zellweger kidney as well as liver, but catalase is present in both organs. Images PMID:3538019

  7. Integral membrane proteins Brr6 and Apq12 link assembly of the nuclear pore complex to lipid homeostasis in the endoplasmic reticulum

    PubMed Central

    Hodg, Christine A.; Choudhary, Vineet; Wolyniak, Michael J.; Scarcelli, John J.; Schneiter, Roger; Col, Charles N.

    2010-01-01

    Summary Cells of Saccharomyces cerevisiae lacking Apq12, a nuclear envelope (NE)-endoplasmic reticulum (ER) integral membrane protein, are defective in assembly of nuclear pore complexes (NPCs), possibly because of defects in regulating membrane fluidity. We identified BRR6, which encodes an essential integral membrane protein of the NE-ER, as a dosage suppressor of apq12 Δ. Cells carrying the temperature-sensitive brr6-1 allele have been shown to have defects in nucleoporin localization, mRNA metabolism and nuclear transport. Electron microscopy revealed that brr6-1 cells have gross NE abnormalities and proliferation of the ER. brr6-1 cells were hypersensitive to compounds that affect membrane biophysical properties and to inhibitors of lipid biosynthetic pathways, and displayed strong genetic interactions with genes encoding non-essential lipid biosynthetic enzymes. Strikingly, brr6-1 cells accumulated, in or near the NE, elevated levels of the two classes of neutral lipids, steryl esters and triacylglycerols, and over-accumulated sterols when they were provided exogenously. Although neutral lipid synthesis is dispensable in wild-type cells, viability of brr6-1 cells was fully dependent on neutral lipid production. These data indicate that Brr6 has an essential function in regulating lipid homeostasis in the NE-ER, thereby impacting NPC formation and nucleocytoplasmic transport. PMID:20016074

  8. Characterization of the complex formed by β-glucocerebrosidase and the lysosomal integral membrane protein type-2

    PubMed Central

    Zunke, Friederike; Andresen, Lisa; Wesseler, Sophia; Groth, Johann; Arnold, Philipp; Rothaug, Michelle; Mazzulli, Joseph R.; Krainc, Dimitri; Blanz, Judith; Saftig, Paul; Schwake, Michael

    2016-01-01

    The lysosomal integral membrane protein type-2 (LIMP-2) plays a pivotal role in the delivery of β-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced α-synuclein levels, suggesting that LIMP-2–derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies. PMID:27001828

  9. Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish

    PubMed Central

    Takeuchi, Miki; Yamaguchi, Shingo; Yonemura, Shigenobu; Kakiguchi, Kisa; Sato, Yoshikatsu; Higashiyama, Tetsuya; Shimizu, Takashi; Hibi, Masahiko

    2015-01-01

    Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets. PMID:26451951

  10. Reliability of nine programs of topological predictions and their application to integral membrane channel and carrier proteins.

    PubMed

    Reddy, Abhinay; Cho, Jaehoon; Ling, Sam; Reddy, Vamsee; Shlykov, Maksim; Saier, Milton H

    2014-01-01

    We evaluated topological predictions for nine different programs, HMMTOP, TMHMM, SVMTOP, DAS, SOSUI, TOPCONS, PHOBIUS, MEMSAT-SVM (hereinafter referred to as MEMSAT), and SPOCTOPUS. These programs were first evaluated using four large topologically well-defined families of secondary transporters, and the three best programs were further evaluated using topologically more diverse families of channels and carriers. In the initial studies, the order of accuracy was: SPOCTOPUS > MEMSAT > HMMTOP > TOPCONS > PHOBIUS > TMHMM > SVMTOP > DAS > SOSUI. Some families, such as the Sugar Porter Family (2.A.1.1) of the Major Facilitator Superfamily (MFS; TC #2.A.1) and the Amino Acid/Polyamine/Organocation (APC) Family (TC #2.A.3), were correctly predicted with high accuracy while others, such as the Mitochondrial Carrier (MC) (TC #2.A.29) and the K(+) transporter (Trk) families (TC #2.A.38), were predicted with much lower accuracy. For small, topologically homogeneous families, SPOCTOPUS and MEMSAT were generally most reliable, while with large, more diverse superfamilies, HMMTOP often proved to have the greatest prediction accuracy. We next developed a novel program, TM-STATS, that tabulates HMMTOP, SPOCTOPUS or MEMSAT-based topological predictions for any subdivision (class, subclass, superfamily, family, subfamily, or any combination of these) of the Transporter Classification Database (TCDB; www.tcdb.org) and examined the following subclasses: α-type channel proteins (TC subclasses 1.A and 1.E), secreted pore-forming toxins (TC subclass 1.C) and secondary carriers (subclass 2.A). Histograms were generated for each of these subclasses, and the results were analyzed according to subclass, family and protein. The results provide an update of topological predictions for integral membrane transport proteins as well as guides for the development of more reliable topological prediction programs, taking family-specific characteristics into account.

  11. The SH integral membrane protein of the paramyxovirus simian virus 5 is required to block apoptosis in MDBK cells.

    PubMed

    He, B; Lin, G Y; Durbin, J E; Durbin, R K; Lamb, R A

    2001-05-01

    In some cell types the paramyxovirus simian virus 5 (SV5) causes little cytopathic effect (CPE) and infection continues productively for long periods of time; e.g., SV5 can be produced from MDBK cells for up to 40 days with little CPE. SV5 differs from most paramyxoviruses in that it encodes a small (44-amino-acid) hydrophobic integral membrane protein (SH). When MDBK cells were infected with a recombinant SV5 containing a deletion of the SH gene (rSV5DeltaSH), the MDBK cells exhibited an increase in CPE compared to cells infected with wild-type SV5 (recovered from cDNA; rSV5). The increased CPE correlated with an increase in apoptosis in rSV5DeltaSH-infected cells over mock-infected and rSV5-infected cells when assayed for annexin V binding, DNA content (propidium iodide staining), and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). In rSV5DeltaSH-infected MDBK cells an increase in caspase-2 and caspase-3 activities was observed. By using peptide inhibitors of individual caspases it was found that caspase-2 and caspase-3 were activated separately in rSV5DeltaSH-infected cells. Expression of caspase-2 and -3 in rSV5DeltaSH-infected MDBK cells appeared not to require STAT1 protein, as STAT1 protein could not be detected in SV5-infected MDBK cells. When mutant mice homologous for a targeted disruption of STAT1 were used as a model animal system and infected with the viruses it was found that rSV5DeltaSH caused less mortality than wild-type rSV5, consistent with the notion of clearance of apoptotic cells in a host species.

  12. Effect of equilibration time on the motility and functional integrity of canine spermatozoa frozen in three different extenders.

    PubMed

    Belala, R; Briand-Amirat, L; Vinciguerra, L; Tainturier, D; Kaidi, R; Thorin, C; Michaud, S; Anton, M; Bencharif, D

    2016-06-01

    The present work aimed to assess the effect of equilibration time on post-thaw motility parameters of canine sperm frozen in three extenders: 6% low-density lipoproteins (LDL), 6% liposomes (LIPO), and 40% egg yolk plasma (EYP). A second experiment is aimed at evaluating the functional integrity of canine spermatozoa frozen in the three extenders at the best equilibration time found in the experiment one. In the first experiment, 20 ejaculates harvested from 7 dogs, were frozen in three extenders (LDL, LIPO, and EYP) after four equilibration times (30min, 1h, 3h, and 6h). The semen was evaluated after thawing using an image analyser (HT-IVOS 14.0). The 6h equilibration time gave better results of motility and progressive motility in the three studied extenders. (LDL: 58.9% vs. 42.7%; LIPO: 54.4% vs. 31.9%; EYP: 55.4% vs 40.5% for motility 6 vs. 1h). In the second experiment, 10 ejaculates taken from 6 dogs were frozen under the same conditions as the previous experiment, after 6h equilibration time. The integrity parameters of the spermatozoal membrane (hypo-osmotic swelling test, and SYBR14/propidium Iodide staining), acrosome (FITC-Pisium sativum Aglutinin staining), and DNA (acridine orange staining) were evaluated at three different stages: post-dilution (T0), post-equilibration, and post-thawing. Post-thaw results were as follows: membrane integrity (HOSt: 62;6% vs 58% vs 64.4%; SYBR14/IP: 63.6% vs 57.9% vs 64.8%); acrosome integrity (FITC-PSA: 79.4% vs 74% vs 76.2%) and DNA integrity (Acridine-orange: 98.9% vs 98.5% vs 98.7%) respectively for LDL vs. LIPO vs. EYP. No significant difference existed between the extenders tested; thus 6%LIPO and 40%EYP could be good candidates for replacement of 6%LDL in the protection of canine sperm during the freeze-thaw process without altering motility and integrity parameters. PMID:27234538

  13. Integration of Nine Steps into One Membrane Reactor To Produce Synthesis Gases for Ammonia and Liquid Fuel.

    PubMed

    Li, Wenping; Zhu, Xuefeng; Chen, Shuguang; Yang, Weishen

    2016-07-18

    The synthesis of ammonia and liquid fuel are two important chemical processes in which most of the energy is consumed in the production of H2 /N2 and H2 /CO synthesis gases from natural gas (methane). Here, we report a membrane reactor with a mixed ionic-electronic conducting membrane, in which the nine steps for the production of the two types of synthesis gases are shortened to one step by using water, air, and methane as feeds. In the membrane reactor, there is no direct CO2 emission and no CO or H2 S present in the ammonia synthesis gas. The energy consumption for the production of the two synthesis gases can be reduced by 63 % by using this membrane reactor. This promising membrane reactor process has been successfully demonstrated by experiment.

  14. Integration of Nine Steps into One Membrane Reactor To Produce Synthesis Gases for Ammonia and Liquid Fuel.

    PubMed

    Li, Wenping; Zhu, Xuefeng; Chen, Shuguang; Yang, Weishen

    2016-07-18

    The synthesis of ammonia and liquid fuel are two important chemical processes in which most of the energy is consumed in the production of H2 /N2 and H2 /CO synthesis gases from natural gas (methane). Here, we report a membrane reactor with a mixed ionic-electronic conducting membrane, in which the nine steps for the production of the two types of synthesis gases are shortened to one step by using water, air, and methane as feeds. In the membrane reactor, there is no direct CO2 emission and no CO or H2 S present in the ammonia synthesis gas. The energy consumption for the production of the two synthesis gases can be reduced by 63 % by using this membrane reactor. This promising membrane reactor process has been successfully demonstrated by experiment. PMID:27264787

  15. Putrescine-Induced Wounding and Its Effects on Membrane Integrity and Ion Transport Processes in Roots of Intact Corn Seedlings

    PubMed Central

    DiTomaso, Joseph M.; Shaff, Jon E.; Kochian, Leon V.

    1989-01-01

    Interactions between putrescine and membrane function were examined with the use of a recently developed microelectrode system that enables us simultaneously to quantify membrane potentials and net K+ fluxes associated with individual cells at the root surface of an intact corn (Zea mays L.) seedling. In contrast to the results of others, our analyses indicate that exogenous putrescine (0.5 millimolar), in the absence of calcium, does not maintain membrane stability. In addition, putrescine caused a wound response characterized by a gradual depolarization of the membrane potential and a considerable net efflux of K+ from the root. In the presence of calcium, both short term (20 minutes) and long term (24 hours) exposure to a high concentration of exogenous putrescine (5 millimolar) also caused a reduction in the resting membrane potential and a significant K+ efflux. However, preincubating corn roots in a solution containing the antioxidant ascorbate ameliorated the wounding effects of putrescine and slightly increased potassium uptake. A similar preincubation in the absence of calcium did not protect membranes against putrescine-induced damage. The ameliorating effect of ascorbate on putrescine-induced membrane damage suggests that the wounding response of high putrescine levels in corn roots involves the catabolism of the polyamine by a cell wall diamine oxidase, with the concomitant production of hydrogen peroxide and free radicals resulting in peroxidative damage of the plasmalemma. PMID:16666910

  16. Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus.

    PubMed

    Yeste, M; Estrada, E; Rocha, L G; Marín, H; Rodríguez-Gil, J E; Miró, J

    2015-03-01

    Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) and poor (poor freezability ejaculates: PFE) freezability stallion ejaculates in other sperm parameters, including peroxide and superoxide levels, potential of mitochondrial membrane and nuclear integrity. With this purpose, a total of 24 stallion ejaculates were cryopreserved and classified into two groups (GFE vs. PFE), depending on their sperm membrane integrity and motility after freeze-thawing. From the total of 24 ejaculates, 13 were classified as GFE and the other 11 were classified as PFE. Apart from differences in sperm membrane permeability and lipid disorder after freeze-thawing, GFE presented significantly (p < 0.05) higher percentages of viable spermatozoa with high content of peroxides and of superoxides than PFE. In contrast, and despite cryopreservation of stallion spermatozoa increasing DNA fragmentation and disrupting disulphide bonds in sperm head proteins, no significant differences between GFE and PFE were seen. We can thus conclude that good and poor freezability stallion ejaculates differ in their reactive oxygen species levels after cryopreservation, but not in the damage extent on sperm nucleus.

  17. Fully integrated PDMS/SU-8/quartz microfluidic chip with a novel macroporous poly dimethylsiloxane (PDMS) membrane for isoelectric focusing of proteins using whole-channel imaging detection.

    PubMed

    Shameli, Seyed Mostafa; Elbuken, Caglar; Ou, Junjie; Ren, Carolyn L; Pawliszyn, Janusz

    2011-02-01

    A fully integrated polydimethylsiloxane (PDMS)/modified PDMS membrane/SU-8/quartz hybrid chip was developed for protein separation using isoelectric focusing (IEF) mechanism coupled with whole-channel imaging detection (WCID) method. This microfluidic chip integrates three components into one single chip: (i) modified PDMS membranes for separating electrolytes in the reservoirs from the sample in the microchannel and thus reducing pressure disturbance, (ii) SU-8 optical slit to block UV light (below 300 nm) outside the channel aiming to increase detection sensitivity, and (iii) injection and discharge capillaries for continuous operation. Integration of all these components on a single chip is challenging because it requires fabrication techniques for perfect bonding between different materials and is prone to leakage and blockage. This study has addressed all the challenges and presented a fully integrated chip, which is more robust with higher sensitivity than the previously developed IEF chips. This chip was tested by performing protein and pI marker separation. The separation results obtained in this chip were compared with that obtained in commercial cartridges. Side-by-side comparison validated the developed chip and fabrication techniques.

  18. Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin.

    PubMed

    Collin, Séverine; Guilvout, Ingrid; Nickerson, Nicholas N; Pugsley, Anthony P

    2011-05-01

    The lipoprotein PulS is a dedicated chaperone that is required to target the secretin PulD to the outer membrane in Klebsiella or Escherichia coli, and to protect it from proteolysis. Here, we present indirect evidence that PulD protomers do not assemble into the secretin dodecamer before they reach the outer membrane, and that PulS reaches the outer membrane in a soluble heterodimer with the general lipoprotein chaperone LolA. However, we could not find any direct evidence for PulD protomer association with the PulS-LolA heterodimer. Instead, in cells producing PulD and a permanently locked PulS-LolA dimer (in which LolA carries an R43L substitution that prevents lipoprotein transfer to LolB in the outer membrane), LolAR43L was found in the inner membrane, probably still associated with PulS bound to PulD that had been incorrectly targeted because of the LolAR43L substitution. It is speculated that PulD protomers normally cross the periplasm together with PulS bound to LolA but when the latter cannot be separated (due to the mutation in lolA), the PulD protomers form dodecamers that insert into the inner membrane. PMID:21338419

  19. Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin.

    PubMed

    Collin, Séverine; Guilvout, Ingrid; Nickerson, Nicholas N; Pugsley, Anthony P

    2011-05-01

    The lipoprotein PulS is a dedicated chaperone that is required to target the secretin PulD to the outer membrane in Klebsiella or Escherichia coli, and to protect it from proteolysis. Here, we present indirect evidence that PulD protomers do not assemble into the secretin dodecamer before they reach the outer membrane, and that PulS reaches the outer membrane in a soluble heterodimer with the general lipoprotein chaperone LolA. However, we could not find any direct evidence for PulD protomer association with the PulS-LolA heterodimer. Instead, in cells producing PulD and a permanently locked PulS-LolA dimer (in which LolA carries an R43L substitution that prevents lipoprotein transfer to LolB in the outer membrane), LolAR43L was found in the inner membrane, probably still associated with PulS bound to PulD that had been incorrectly targeted because of the LolAR43L substitution. It is speculated that PulD protomers normally cross the periplasm together with PulS bound to LolA but when the latter cannot be separated (due to the mutation in lolA), the PulD protomers form dodecamers that insert into the inner membrane.

  20. Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Sánchez-Mir, Laura; Franco, Alejandro; Martín-García, Rebeca; Madrid, Marisa; Vicente-Soler, Jero; Soto, Teresa; Gacto, Mariano; Pérez, Pilar

    2014-01-01

    The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade. PMID:24820419

  1. Mechanochemistry of protein 4.1's spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening

    PubMed Central

    1995-01-01

    Mechanical strength of the red cell membrane is dependent on ternary interactions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a constitutive exon encoding 59 aa. A series of truncated SAB peptides were engineered to define the sequences involved in spectrin-actin interactions, and also membrane strength. Analysis of in vitro supramolecular assemblies showed that gelation activity of SAB peptides correlates with their ability to recruit a critical amount of spectrin into the complex to cross-link actin filaments. Also, several SAB peptides appeared to exhibit a weak, cooperative actin-binding activity which mapped to the first 26 residues of the constitutive 59 aa. Fluorescence-imaged microdeformation was used to show SAB peptide integration into the elastic skeletal network of spectrin, actin, and protein 4.1. In situ membrane-binding and membrane-strengthening abilities of the SAB peptides correlated with their in vitro gelation activity. The findings imply that sites for strong spectrin binding include both the alternative 21-aa cassette and a conserved region near the middle of the 59 aa. However, it is shown that only weak SAB affinity is necessary for physiologically relevant action. Alternatively spliced exons can thus translate into strong modulation of specific protein interactions, economizing protein function in the cell without, in and of themselves, imparting unique function. PMID:7642705

  2. Increase of seed germination, growth and membrane integrity of wheat seedlings by exposure to static and a 10-KHz electromagnetic field.

    PubMed

    Payez, Atefeh; Ghanati, Faezeh; Behmanesh, Mehrdad; Abdolmaleki, Parviz; Hajnorouzi, Abazar; Rajabbeigi, Elham

    2013-12-01

    There is a large body of experimental data demonstrating various effects of magnetic field (MF) on plants growth and development. Although the mechanism(s) of perception of MF by plants is not yet elucidated, there is a possibility that like other stimuli, MF exerts its effects on plants by changing membrane integrity and conductance of its water channels, thereby influencing growth characteristics. In this study, the seeds of wheat (Triticum aestivum L. cv. Kavir) were imbibed in water overnight and then treated with or without a 30-mT static magnetic field (SMF) and a 10-kHz electromagnetic field (EMF) for 4 days, each 5 h. Water uptake of seeds reduced 5 h of the treatment with EMF but did not show changes in SMF treatment. Exposure to both magnetic fields did not affect germination percent of the seeds but increased the speed of germination, compared to the control group. Treatment with EMF significantly reduced seedling length and subsequently vigor index I, while SMF had no effects on these parameters. Both treatments significantly increased vigor index II, compared to the control group. These treatments also remarkably increased catalase activity and proline contents of seedlings but reduced the activity of peroxidase, the rate of lipid peroxidation and electrolyte leakages of membranes. The results suggest promotional effects of EMFs on membrane integrity and growth characteristics of wheat seedlings.

  3. Fermentative production of poly (γ-glutamic acid) from renewable carbon source and downstream purification through a continuous membrane-integrated hybrid process.

    PubMed

    Kumar, Ramesh; Pal, Parimal

    2015-02-01

    Experimental investigations were carried out on continuous and direct production of poly-(γ-glutamic acid) in a hybrid reactor system that integrated conventional fermentative production step with membrane-based downstream separation and purification. Novelty of the integrated system lies in high degree of purity, conversion, yield and productivity of poly-(γ-glutamic acid) through elimination of substrate-product inhibitions of traditional batch production system. This new system is compact, flexible, eco-friendly and largely fouling-free ensuring steady and continuous production of poly-(γ-glutamic acid) directly from a renewable carbon source at the rate of 0.91 g/L/h. Cross-flow microfiltration membrane modules ensured almost complete separation and recycle of cells without much fouling problem. Well-screened ultrafiltration membrane module helped to concentrate poly-(γ-glutamic acid) while ensuring recovery and recycle of 96% unconverted carbon source resulting in yield of 0.6g/g along with high product purity.

  4. Bioelectricity generation in an integrated system combining microbial fuel cell and tubular membrane reactor: effects of operation parameters performing a microbial fuel cell-based biosensor for tubular membrane bioreactor.

    PubMed

    Wang, Jie; Zheng, Yawen; Jia, Hui; Zhang, Hongwei

    2014-10-01

    A bio-cathode microbial fuel cell (MFC) with tubular membrane was integrated to construct a microbial fuel cell-tubular membrane bioreactor (MFC-TMBR) system, in which the bio-cathode MFC was developed as a biosensor for COD real-time monitoring in TMBR and the performance was analyzed in terms of its current variation caused by operation parameters. With a constant anode potential, the effect of HRT demonstrated that higher rate of mass transport increased the response of the system. The system was further explored an inverse relationship between TMP and current peak by using EPS concentration under the different MLSS concentration. The sensor output had a linear relationship with COD up to 1000mg/L (regression coefficient, R(2)=0.97) and MLSS (regression coefficient, R(2)=0.94). The simple and compact bio-cathode MFC biosensor for TMBR using MFC-TMBR integrated system showed promising potential for direct and economical COD online monitoring, and provided an opportunity to widen the application of MFC-based biosensor.

  5. Biodiesel production from waste chicken fat with low free fatty acids by an integrated catalytic process of composite membrane and sodium methoxide.

    PubMed

    Shi, Wenying; Li, Jianxin; He, Benqiao; Yan, Feng; Cui, Zhenyu; Wu, Kaiwei; Lin, Ligang; Qian, Xiaomin; Cheng, Yu

    2013-07-01

    An integrated process of catalytic composite membranes (CCMs) and sodium methoxide was developed to produce biodiesel from waste chicken fat. The free fatty acids (FFAs) in the chicken oil were converted to methyl esters by esterification with methanol using a novel sulfonated polyethersulfone (SPES)/PES/non-woven fabric (NWF) CCMs in a flow-through catalytic membrane reactor. The CCM is that the NWF fibers were fully embedded in SPES/PES with a homogeneous and microporous structure. The oil obtained after esterification was carried out by transesterification of sodium methoxide. The results showed that the FFAs conversion obtained by CCMs with the acid capacity of 25.28 mmol (H(+)) was 92.8% at the residence time 258s. The CCMs present a good stability during the continuous running of 500 h. The conversion of transesterification was 98.1% under the optimum conditions. The quality of the biodiesel met the international standards. PMID:23665693

  6. Biodiesel production from waste chicken fat with low free fatty acids by an integrated catalytic process of composite membrane and sodium methoxide.

    PubMed

    Shi, Wenying; Li, Jianxin; He, Benqiao; Yan, Feng; Cui, Zhenyu; Wu, Kaiwei; Lin, Ligang; Qian, Xiaomin; Cheng, Yu

    2013-07-01

    An integrated process of catalytic composite membranes (CCMs) and sodium methoxide was developed to produce biodiesel from waste chicken fat. The free fatty acids (FFAs) in the chicken oil were converted to methyl esters by esterification with methanol using a novel sulfonated polyethersulfone (SPES)/PES/non-woven fabric (NWF) CCMs in a flow-through catalytic membrane reactor. The CCM is that the NWF fibers were fully embedded in SPES/PES with a homogeneous and microporous structure. The oil obtained after esterification was carried out by transesterification of sodium methoxide. The results showed that the FFAs conversion obtained by CCMs with the acid capacity of 25.28 mmol (H(+)) was 92.8% at the residence time 258s. The CCMs present a good stability during the continuous running of 500 h. The conversion of transesterification was 98.1% under the optimum conditions. The quality of the biodiesel met the international standards.

  7. Integrative Structure–Function Mapping of the Nucleoporin Nup133 Suggests a Conserved Mechanism for Membrane Anchoring of the Nuclear Pore Complex*

    PubMed Central

    Kim, Seung Joong; Fernandez-Martinez, Javier; Sampathkumar, Parthasarathy; Martel, Anne; Matsui, Tsutomu; Tsuruta, Hiro; Weiss, Thomas M.; Shi, Yi; Markina-Inarrairaegui, Ane; Bonanno, Jeffery B.; Sauder, J. Michael; Burley, Stephen K.; Chait, Brian T.; Almo, Steven C.; Rout, Michael P.; Sali, Andrej

    2014-01-01

    The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup13355–502) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup1332–1157. Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes. PMID:25139911

  8. Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex.

    PubMed

    Kim, Seung Joong; Fernandez-Martinez, Javier; Sampathkumar, Parthasarathy; Martel, Anne; Matsui, Tsutomu; Tsuruta, Hiro; Weiss, Thomas M; Shi, Yi; Markina-Inarrairaegui, Ane; Bonanno, Jeffery B; Sauder, J Michael; Burley, Stephen K; Chait, Brian T; Almo, Steven C; Rout, Michael P; Sali, Andrej

    2014-11-01

    The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup133(55-502)) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup133(2-1157). Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes. PMID:25139911

  9. Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex.

    PubMed

    Kim, Seung Joong; Fernandez-Martinez, Javier; Sampathkumar, Parthasarathy; Martel, Anne; Matsui, Tsutomu; Tsuruta, Hiro; Weiss, Thomas M; Shi, Yi; Markina-Inarrairaegui, Ane; Bonanno, Jeffery B; Sauder, J Michael; Burley, Stephen K; Chait, Brian T; Almo, Steven C; Rout, Michael P; Sali, Andrej

    2014-11-01

    The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup133(55-502)) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup133(2-1157). Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes.

  10. Restricted diffusion of integral membrane proteins and polyphosphoinositides leads to their depletion in microvesicles released from human erythrocytes.

    PubMed Central

    Hagelberg, C; Allan, D

    1990-01-01

    The protein and phospholipid composition of microvesicles released from normal human erythrocytes after ATP depletion, on aging or by treatment with merocyanine 540, dimyristoyl phosphatidylcholine or Ca2+/ionophore A23187 has been compared with the composition of the original cell membrane. It has been shown that these microvesicles are depleted of band 3, glycophorin and phosphatidylinositol 4,5-bisphosphate relative to phospholipid by 40% or more. These data are interpreted to mean that less than half of these membrane components are free to diffuse laterally in the lipid bilayer. Acetylcholinesterase was found to be enriched 2-3-fold in microvesicles, possibly because the removal of non-diffusing proteins from the vesiculating region of the lipid bilayer allows more space for freely diffusing proteins like acetylcholinesterase to enter the microvesicle membrane. PMID:2173910

  11. Correlation analysis of the progesterone-induced sperm acrosome reaction rate and the fertilisation rate in vitro.

    PubMed

    Jiang, T; Qin, Y; Ye, T; Wang, Y; Pan, J; Zhu, Y; Duan, L; Li, K; Teng, X

    2015-10-01

    In this study, we aimed to investigate whether progesterone-induced acrosome reaction (AR) rate could be an indicator for fertilisation rate in vitro. Twenty-six couples with unexplained infertility and undergoing in vitro fertilisation (IVF) treatment were involved. On the oocytes retrieval day after routine IVF, residual sperm samples were collected to receive progesterone induction (progesterone group) or not (control group). AR rate was calculated and fertilisation rate was recorded. The correlation between progesterone-induced AR and fertilisation rate and between sperm normal morphology and 3PN (tripronuclear) were analysed using the Spearman correlation analysis. The AR rate of progesterone group was statistically higher than that of the control group (15.6 ± 5.88% versus 9.66 ± 5.771%, P < 0.05), but not significantly correlated with fertilisation rate (r = -0.053, P > 0.01) or rate of high-quality embryo development (r = -0.055, P > 0.01). Normal sperm morphology also showed no significant correlation with the amount of 3PN zygotes (r = 0.029, P > 0.01), rate of 3PN zygotes production (r = 0.20, P > 0.01), rate of 3PN embryo development (r = -0.406, P > 0.01), fertilisation rate (r = -0.148, P > 0.01) or progesterone-induced AR rate (r = 0.214, P > 0.01). Progesterone can induce AR in vitro significantly; however, the progesterone-induced AR may not be used to indicate fertilisation rate.

  12. Structural requirements for species-specific induction of the sperm acrosome reaction by sea urchin egg sulfated fucan.

    PubMed

    Hirohashi, Noritaka; Vilela-Silva, Ana-Cristina E S; Mourão, Paulo A S; Vacquier, Victor D

    2002-11-01

    The sulfated fucan (SF) of egg jelly induces the acrosome reaction (AR) of sea urchin sperm. Strongylocentrotus franciscanus (Sf) SF is sulfated only at the 2-position. Strongylocentrotus purpuratus (Sp) has two SF isotypes, each one being female specific. One is rich in sulfate at both the 2- and 4-positionS (SF-1), and the other is rich in sulfate at the 4-position, but not the 2-position (SF-2). Sf SF is poor at inducing the AR of Sp sperm, presumably due to lack of 4-sulfation. Sp SF-1 is better at inducing the AR of Sf sperm than Sp SF-2, hypothetically due to increased 2-sulfation. Chemical oversulfation of Sf SF increases the percentage of AR of Sp sperm, showing that 4-sulfation is important for recognition of SF by Sp sperm. Chemically oversulfated Sp SF-2 is better at inducing the Sf sperm AR, presumably because of increased 2-sulfation. The species, Strongylocentrotus drobachiensis (Sd), has an SF-2 that is exclusively 2-sulfated (like Sf), except the glycosidic linkage in Sd is alpha(1-->4), whereas in Sf it is alpha(1-->3). Sd SF-2 does not induce the AR of Sf sperm, showing the strict requirement for the alpha(1-->3) linkage in recognition between Sf sperm and SF. Egg jelly from Echinometra lucunter (El) contains sulfated galactan (SG) which differs from Sf SF only in that the monosaccharide is L-galactose, not L-fucose. This SG and Sf SF are equally potent in inducing the AR of Sf sperm, showing that modification at C6 of L-fucose is not important for proper recognition between SF and Sf sperm receptors. This system permits study of the structural basis for recognition between sulfated polysaccharide and receptors controlling signal transduction pathways in animal cells.

  13. A Recurrent Deletion of DPY19L2 Causes Infertility in Man by Blocking Sperm Head Elongation and Acrosome Formation

    PubMed Central

    Harbuz, Radu; Zouari, Raoudha; Pierre, Virginie; Ben Khelifa, Mariem; Kharouf, Mahmoud; Coutton, Charles; Merdassi, Ghaya; Abada, Farid; Escoffier, Jessica; Nikas, Yorgos; Vialard, François; Koscinski, Isabelle; Triki, Chema; Sermondade, Nathalie; Schweitzer, Thérèse; Zhioua, Amel; Zhioua, Fethi; Latrous, Habib; Halouani, Lazhar; Ouafi, Marrakchi; Makni, Mounir; Jouk, Pierre-Simon; Sèle, Bernard; Hennebicq, Sylviane; Satre, Véronique; Viville, Stéphane; Arnoult, Christophe; Lunardi, Joël; Ray, Pierre F.

    2011-01-01

    An increasing number of couples require medical assistance to achieve a pregnancy, and more than 2% of the births in Western countries now result from assisted reproductive technologies. To identify genetic variants responsible for male infertility, we performed a whole-genome SNP scan on patients presenting with total globozoospermia, a primary infertility phenotype characterized by the presence of 100% round acrosomeless spermatozoa in the ejaculate. This strategy allowed us to identify in most patients (15/20) a 200 kb homozygous deletion encompassing only DPY19L2, which is highly expressed in the testis. Although there was no known function for DPY19L2 in humans, previous work indicated that its ortholog in C. elegans is involved in cell polarity. In man, the DPY19L2 region has been described as a copy-number variant (CNV) found to be duplicated and heterozygously deleted in healthy individuals. We show here that the breakpoints of the deletions are located on a highly homologous 28 kb low copy repeat (LCR) sequence present on each side of DPY19L2, indicating that the identified deletions were probably produced by nonallelic homologous recombination (NAHR) between these two regions. We demonstrate that patients with globozoospermia have a homozygous deletion of DPY19L2, thus indicating that DPY19L2 is necessary in men for sperm head elongation and acrosome formation. A molecular diagnosis can now be proposed to affected men; the presence of the deletion confirms the diagnosis of globozoospermia and assigns a poor prognosis for the success of in vitro fertilization. PMID:21397064

  14. Water-quality data from semipermeable-membrane devices and polar organic chemical integrative samplers deployed in the McKenzie River basin, Oregon

    USGS Publications Warehouse

    McCarthy, Kathleen A.; Alvarez, David A.

    2012-01-01

    Two types of passive samplers—the semipermeable membrane device (SPMD) and the polar organic chemical integrative sampler (POCIS)—are being used to collect data from the McKenzie River, Oregon. The McKenzie River is the source of drinking water for the City of Eugene, Oregon, and passive-sampler data are part of an ongoing monitoring effort designed to help understand and protect the drinking water source. Data from the passive samplers are reported here. This data report is dynamic and will be appended with additional data as they become available.

  15. Integrating solid-state NMR and computational modeling to investigate the structure and dynamics of membrane-associated ghrelin.

    PubMed

    Vortmeier, Gerrit; DeLuca, Stephanie H; Els-Heindl, Sylvia; Chollet, Constance; Scheidt, Holger A; Beck-Sickinger, Annette G; Meiler, Jens; Huster, Daniel

    2015-01-01

    The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide's secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide's positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8-17 form an α-helix, while residues 21-23 and 26-27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane. PMID:25803439

  16. Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

    PubMed Central

    Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A. Pedro; Starr, Trevor L.

    2015-01-01

    The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion. PMID:25595444

  17. Structure of an E. coli integral membrane sulfurtransferase and its structural transition upon SCN- binding defined by EPR-based hybrid method

    NASA Astrophysics Data System (ADS)

    Ling, Shenglong; Wang, Wei; Yu, Lu; Peng, Junhui; Cai, Xiaoying; Xiong, Ying; Hayati, Zahra; Zhang, Longhua; Zhang, Zhiyong; Song, Likai; Tian, Changlin

    2016-01-01

    Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN-, which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane.

  18. Purge gas recovery of ammonia synthesis plant by integrated configuration of catalytic hydrogen-permselective membrane reactor and solid oxide fuel cell as a novel technology

    NASA Astrophysics Data System (ADS)

    Siavashi, Fakhteh; Saidi, Majid; Rahimpour, Mohammad Reza

    2014-12-01

    The purge gas emission of ammonia synthesis plant which contains hazardous components is one of the major sources of environmental pollution. Using integrated configuration of catalytic hydrogen-permselective membrane reactor and solid oxide fuel cell (SOFC) system is a new approach which has a great impact to reduce the pollutant emission. By application of this method, not only emission of ammonia and methane in the atmosphere is prevented, hydrogen is produced through the methane steam reforming and ammonia decomposition reactions that take place simultaneously in a catalytic membrane reactor. The pure generated hydrogen by recovery of the purge gas in the Pd-Ag membrane reactor is used as a feed of SOFC. Since water is the only byproduct of the electrochemical reaction in the SOFC, it is recycled to the reactor for providing the required water of the reforming reaction. Performance investigation of the reactor represents that the rate of hydrogen permeation increases with enhancing the reactor temperature and pressure. Also modeling results indicate that the SOFC performance improves with increasing the temperature and fuel utilization ratio. The generated power by recovery of the purging gas stream of ammonia synthesis plant in the Razi petrochemical complex is about 8 MW.

  19. The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1.

    PubMed Central

    Waterham, H R; de Vries, Y; Russel, K A; Xie, W; Veenhuis, M; Cregg, J M

    1996-01-01

    We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1. PMID:8628321

  20. Ophiobolin A from Bipolaris oryzae perturbs motility and membrane integrities of porcine sperm and induces cell death on mammalian somatic cell lines.

    PubMed

    Bencsik, Ottó; Papp, Tamás; Berta, Máté; Zana, Annamária; Forgó, Péter; Dombi, György; Andersson, Maria A; Salkinoja-Salonen, Mirja; Vágvölgyi, Csaba; Szekeres, András

    2014-09-01

    Bipolaris oryzae is a phytopathogenic fungus causing a brown spot disease in rice, and produces substance that strongly perturbs motility and membrane integrities of boar spermatozoa. The substance was isolated from the liquid culture of the fungal strain using extraction and a multi-step semi-preparative HPLC procedures. Based on the results of mass spectrometric and 2D NMR techniques, the bioactive molecule was identified as ophiobolin A, a previously described sesterterpene-type compound. The purified ophiobolin A exhibited strong motility inhibition and viability reduction on boar spermatozoa. Furthermore, it damaged the sperm mitochondria significantly at sublethal concentration by the dissipation of transmembrane potential in the mitochondrial inner membrane, while the plasma membrane permeability barrier remained intact. The study demonstrated that the cytotoxicity of ophiobolin A toward somatic cell lines is higher by 1-2 orders of magnitude compared to other mitochondriotoxic mycotoxins, and towards sperm cells unique by replacing the progressive motility by shivering tail beating at low exposure concentration. PMID:25251540

  1. Structure of an E. coli integral membrane sulfurtransferase and its structural transition upon SCN‑ binding defined by EPR-based hybrid method

    NASA Astrophysics Data System (ADS)

    Ling, Shenglong; Wang, Wei; Yu, Lu; Peng, Junhui; Cai, Xiaoying; Xiong, Ying; Hayati, Zahra; Zhang, Longhua; Zhang, Zhiyong; Song, Likai; Tian, Changlin

    2016-01-01

    Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN‑, which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane.

  2. Structure of an E. coli integral membrane sulfurtransferase and its structural transition upon SCN− binding defined by EPR-based hybrid method

    PubMed Central

    Ling, Shenglong; Wang, Wei; Yu, Lu; Peng, Junhui; Cai, Xiaoying; Xiong, Ying; Hayati, Zahra; Zhang, Longhua; Zhang, Zhiyong; Song, Likai; Tian, Changlin

    2016-01-01

    Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN−, which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane. PMID:26817826

  3. An Integrated 520-600 GHz Sub-Harmonic Mixer and Tripler Combination Based on GaAs MMIC Membrane Planar Schottky Diodes

    NASA Technical Reports Server (NTRS)

    Thomas, B.; Gill, J.; Maestrini, A.; Lee, C.; Lin, R.; Sin, S.; Peralta, A.; Mehdi, I.

    2011-01-01

    We present here the design, development and test of an integrated sub-millimeter front-end featuring a 520-600 GHz sub-harmonic mixer and a 260-300 GHz frequency tripler in a single cavity. Both devices used GaAs MMIC membrane planar Schottky diode technology. The sub-harmonic mixer/tripler circuit has been tested using conventional machined as well as silicon micro-machined blocks. Measurement results on the metal block give best DSB mixer noise temperature of 2360 K and conversion losses of 7.7 dB at 520 GHz. Preliminary results on the silicon micro-machined blocks give a DSB mixer noise temperature of 4860 K and conversion losses of 12.16 dB at 540 GHz. The LO input power required to pump the integrated tripler/sub-harmonic mixer for both packages is between 30 and 50 mW

  4. An Integrated 520-600 GHz Sub-Harmonic Mixer and Tripler Combination Based on GaAs MMIC Membrane Planar Schottky Diodes

    NASA Technical Reports Server (NTRS)

    Thomas, B.; Gill, J.; Maestrini, A.; Lee, C.; Lin, R.; Sin, S.; Peralta, A.; Mehdi, I.

    2010-01-01

    We present here the design, development and test of an integrated sub-millimeter front-end featuring a 520-600 GHz sub-harmonic mixer and a 260-300 GHz frequency tripler in a single cavity. Both devices used GaAs MMIC membrane planar Schottky diode technology. The sub-harmonic mixer/tripler circuit has been tested using conventional machined as well as silicon micro-machined blocks. Measurement results on the metal block give best DSB mixer noise temperature of 2360 K and conversion losses of 7.7 dB at 520 GHz. Preliminary results on the silicon micro-machined blocks give a DSB mixer noise temperature of 4860 K and conversion losses of 12.16 dB at 540 GHz. The LO input power required to pump the integrated tripler/sub-harmonic mixer for both packages is between 30 and 50 mW.

  5. Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding.

    PubMed

    Chiu, Philip C N; Chung, Man-Kin; Koistinen, Riitta; Koistinen, Hannu; Seppala, Markku; Ho, Pak-Chung; Ng, Ernest H Y; Lee, Kai-Fai; Yeung, William S B

    2007-01-01

    Fertilization depends on successful binding of the spermatozoa to the zona pellucida of the oocyte. Glycodelin-A inhibits spermatozoa-zona pellucida binding. Previous data showed that glycodelin-A receptor(s) and zona pellucida protein receptor(s) on human spermatozoa are closely related. Using a chemical cross-linking approach, the glycodelin-A-sperm receptor complex was isolated. The receptor was identified to be fucosyltransferase-5 (FUT5) by mass spectrometry and confirmed with the use of anti-FUT5 antibodies. Sperm FUT5 was an externally oriented integral membrane protein in the acrosomal region of human spermatozoa. Biologically active FUT5 was purified from spermatozoa. Co-immunoprecipitation confirmed the interaction between glycodelin-A and sperm FUT5. Solubilized zona pellucida reduced the binding of glycodelin-A to sperm FUT5. An anti-FUT5 antibody and FUT5 acceptor blocked the binding of glycodelin-A to spermatozoa and the zona binding inhibitory activity of glycodelin-A. Sperm FUT5 bound strongly to intact and solubilized human zona pellucida. The equilibrium dissociation constant of sperm FUT5 binding to solubilized zona pellucida was 42.82 pmol/ml. These observations suggest that human sperm FUT5 is a receptor of glycodelin-A and zona pellucida proteins, and that glycodelin-A inhibits spermatozoa-zona binding by blocking the binding of sperm FUT5 to the zona pellucida.

  6. Inhibitors of serine proteases decrease sperm penetration during porcine fertilization in vitro by inhibiting sperm binding to the zona pellucida and acrosome reaction.

    PubMed

    Beek, J; Nauwynck, H; Appeltant, R; Maes, D; Van Soom, A

    2015-11-01

    Serine proteases are involved in mammalian fertilization. Inhibitors of serine proteases can be applied to investigate at which point these enzymes exert their action. We selected two serine protease inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, 100 μM) and soybean trypsin inhibitor (STI, 5 μM) from Glycine max, via previous dose-response IVF experiments and sperm toxicity tests. In the present study, we evaluated how these inhibitors affect porcine fertilization in vitro as calculated on total fertilization rate, polyspermy rate, and the sperm number per fertilized oocyte of cumulus-intact, cumulus-free, and zona-free oocytes. In the control group (no inhibitor), these parameters were 86%, 49%, and 2.2 for cumulus-intact oocytes and 77%, 43%, and 2.2 for cumulus-free oocytes (6-hour gamete incubation period, 1.25 × 10(5) spermatozoa/mL). 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride and STI significantly reduced total fertilization and polyspermy rate in cumulus-intact and cumulus-free oocytes (P < 0.05). Total fertilization rates were respectively 65% and 53% (AEBSF) and 36% and 17% (STI). Inhibition rates were higher in cumulus-free oocytes than in cumulus-intact oocytes, indicating that inhibitors exerted their action after sperm passage through the cumulus. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride but not STI reduced sperm binding to the ZP. The acrosome reaction was significantly inhibited by both inhibitors. Only 40.4% (AEBSF) and 11.4% (STI) of spermatozoa completed a calcium-induced acrosome reaction compared to 86.7% of spermatozoa in the control group. There was no effect on sperm binding or fertilization parameters in zona-free oocytes. In conclusion, sperm-zona binding and acrosome reaction were inhibited by serine protease inhibitors during porcine IVF.

  7. Expression of the male reproduction-related gene in spermatic ducts of the blue swimming crab, Portunus pelagicus, and transfer of modified protein to the sperm acrosome.

    PubMed

    Sroyraya, Morakot; Hanna, Peter J; Changklungmoa, Narin; Senarai, Thanyaporn; Siangcham, Tanapan; Tinikul, Yotsawan; Sobhon, Prasert

    2013-01-01

    Expression of a sex-specific gene in Macrobrachium rosenbergii (Mr-Mrr), encoding a male reproduction-related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp-Mrr) and Scylla serrata (Ss-Mrr). We then investigated expression of Pp-Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp-Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp-Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti-Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp-Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs.

  8. A change in twist of actin provides the force for the extension of the acrosomal process in Limulus sperm: the false-discharge reaction.

    PubMed

    DeRosier, D J; Tilney, L G; Bonder, E M; Frankl, P

    1982-05-01

    One of the most spectacular motions is the generation of the acrosomal process in the limulus sperm. On contact with the egg, the sperm generates a 60-mum-long process that literally drills its way through the jelly surrounding the egg. This irresversible reaction takes only a few seconds. We suggested earlier that this motion is driven by a change in twist of the actin filaments comprising the acrosomal process. In this paper we analyze the so-called false discharge, a reversible reaction, in which the acrosomal filament bundle extends laterally from the base of the sperm and not anteriorly from the apex. Unlike the true discharge, which is straight, the false discharge is helical. Before extension, the filament bundle is coiled about the base of the sperm. In the coil, the bundle is not smoothly bent but consists of arms (straight segments) and elbows (corners) so that the coil looks like a 14-sided polygon. The extension of the false discharge works as follows: starting at the base of the bundle, the filaments change their twist which concomitantly changes the orientations of the elbows relative to each other; that is, in the coil, the elbows all like in a common plane, but after the change in twist, the plane of each elbow is rotated to be perpendicular to that of its neighbors. This change transforms the bundle from a compact coil into an extended left- handed helix. Because the basal end of the bundle is unconstrained, the extension is lateral. The true discharge works the same way but starts at the apical end of the bundle. The apical end, however, is constrained by its passage through the nuclear canal, which directs the extention anteriorly. Unlike the false discharge, during the true discharge the elbows are melted out, making the reaction irreversible. This study shows that rapid movement can be regenerated by actin without myosin and gives us insight into the molecular mechanism.

  9. Nanoengineered membrane electrode assembly interface

    DOEpatents

    Song, Yujiang; Shelnutt, John A

    2013-08-06

    A membrane electrode structure suitable for use in a membrane electrode assembly (MEA) that comprises membrane-affixed metal nanoparticles whose formation is controlled by a photochemical process that controls deposition of the metal nanoparticles using a photocatalyst integrated with a polymer electrolyte membrane, such as an ionomer membrane. Impregnation of the polymer membrane with the photocatalyst prior to metal deposition greatly reduces the required amount of metal precursor in the deposition reaction solution by restricting metal reduction substantially to the formation of metal nanoparticles affixed on or near the surface of the polymer membrane with minimal formation of metallic particles not directly associated with the membrane.

  10. Roles of Na(+)/K(+)-dependent ATPase, Na(+)/H(+) antiporter and GLUT hexose transporters in the cryosurvival of dog spermatozoa: effects on viability, acrosome state and motile sperm subpopulation structure.

    PubMed

    Mogas, M T; Alamo, M Rivera Del; Rodríguez-Gil, J E

    2011-06-01

    To assess the roles of Na(+)/K(+)-dependent ATPase, Na(+)/H(+) antiporter and GLUT hexose transporters in the cryosurvival of dog sperm, semen samples were frozen in a standard freezing medium supplemented with the specific inhibitors of these factors ouabain, amiloride or phloretin, respectively. The presence of ouabain did not counteract the effects of freeze-thawing on the percentages of motile sperm and altered acrosomes, although a small recovery effect was observed in motility parameter means. Amiloride had a similar effect, although motility was more intensely recovered. Phloretin significantly (P < 0.05) impaired viability when added at a maximal concentration of 10(-)4 M (57.3 ± 5.1% vs 76.5 ± 5.7% in cells frozen without inhibitors), although partial recovery of motility parameters was also observed. These effects were accompanied with specific changes in both motility parameters and the percentages of motile sperm in each of the 4 subpopulations comprising the motile sperm population of the ejaculate. Our findings indicate a role for Na(+)/K(+)-dependent ATPase and Na(+)/H(+) antiporter in the mechanisms involved in determining specific sperm motility patterns in response to freeze-thawing, although neither pump seems to be important for the resistance of cell membrane structures to freezing-thawing. In addition, a role for GLUTs in regulating water exchange in dog sperm during freeze-thawing seems unlikely. In contrast, the precise structure of dog sperm in terms of its motile subpopulations was found to condition both cryosurvival and sperm cell sensitivity to the inhibitors used. PMID:21334054

  11. Solubilization of native integral membrane proteins in aqueous buffer by non-covalent chelation with monomethoxy polyethylene glycol (mPEG) polymers

    PubMed Central

    Janaratne, Thamara K.; Okach, Linda; Brock, Ansgar; Lesley, Scott A.

    2011-01-01

    Highly hydrophobic integral membrane proteins (IMPs) are typically purified in excess detergent media, often resulting in rapid inactivation and denaturation of the protein. One promising approach to solve this problem is to couple hydrophilic polymers, such as monomethoxypolyethylene glycol (mPEG) to IMPs under mild conditions in place of detergents. However, the broad application of this approach is hampered by poor reaction efficiencies, low tolerance of detergent stabilized membrane proteins to reaction conditions and a lack of proper site-specific reversible approaches. Here we have developed a straightforward, efficient and mild approach to site-specific non-covalent binding of long-chain polymers to recombinant IMPs. This method uses the hexa-histidine tag (His-Tag) often used for purification of recombinant proteins as an attachment site for mPEGs. Solubility studies performed using five different IMPs confirmed that all tested mPEG-bound IMPs were completely soluble and stable in detergent free aqueous buffer compared to their precipitated native proteins under the identical circumstances. Activity assays and circular dichroism (CD) spectroscopy confirmed the structural integrity of modified IMPs. PMID:21740061

  12. Effect of recombinant bovine somatotropin on plasma concentrations of insulin-like growth factor I, insulin and membrane integrity of bull spermatozoa.

    PubMed

    Vieira, M B; Bianchi, I; Madeira, E M; Roll, V F B; Oliveira, C A; Viau, P; Pivato, I; Severo, N C; Del Pino, F A B; Schneider, A; Corrêa, M N

    2010-12-01

    This study aimed to evaluate the effect of the exogenous recombinant bovine somatotropin (rbST) on plasma concentrations of insulin-like growth factor I (IGF-I), insulin and semen quality of bulls. Twenty bulls (Aberdeen Angus and Brangus) were divided by breed into two groups. Placebo group was injected with NaCl 0.9% (s.c.) and treatment group with rbST (s.c., 500 mg) at days 0 and 14 of the experiment. Immediately after semen collection, blood samples were taken on days 0, 14, 28, 42 and 56 of the experiment. Semen was also collected on day 70 of the experiment. Evaluation of sperm motility was performed at pre-freezing and post-thawing stage, whereas assessment of sperm membrane integrity was performed after freezing and thawing. Analysis of data revealed that the effect of treatment and treatment-by-collection day on plasma concentrations of IGF-I and insulin was not significant. However, mean plasma concentrations of IGF-I and insulin were affected (p < 0.0001) by days of blood sampling. Effect of treatment and treatment-by-collection day on motility of spermatozoa was similar (p > 0.05) at pre-freezing and post-thawing stage. Intactness of plasmalemma and tail membrane of spermatozoa at post-thawing stage was higher (p < 0.05) in rbST-treated group than in control. In conclusion, rbST did not affect plasma concentrations of IGF-I and insulin, however, it did improve post-thaw sperm membrane integrity. PMID:19663813

  13. The division abnormally delayed (dally) gene: a putative integral membrane proteoglycan required for cell division patterning during postembryonic development of the nervous system in Drosophila.

    PubMed

    Nakato, H; Futch, T A; Selleck, S B

    1995-11-01

    We have devised a genetic screen to obtain mutants affecting cell division patterning in the developing central nervous system of Drosophila. The division abnormally delayed (dally) locus was identified using a combination of "enhancer trap" and behavioral screening methods. The ordered cell cycle progression of lamina precursor cells, which generate synaptic target neurons for photoreceptors, is disrupted in dally mutants. The first of two lamina precursor cell divisions shows a delayed entry into mitosis. The second division, one that is triggered by an intercellular signal from photoreceptor axons, fails to take place. Similar to lamina precursors, cells that generate the ommatidia of the adult eye show two synchronized divisions found along the morphogenetic furrow in the eye disc and the first division cycle in dally mutants displays a delayed progression into M phase like that found in the first lamina precursor cell division. dally mutations also affect viability and produce morphological defects in several adult tissues, including the eye, antenna, wing and genitalia. Sequencing of a dally cDNA reveals a potential open reading frame of 626 amino acids with homology to a family of Glypican-related integral membrane proteoglycans. These heparan sulfate-containing proteins are attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol linkage. Heparan sulfate proteoglycans may serve as co-receptors for a variety of secreted proteins including fibroblast growth factor, vascular endothelial growth factor, hepatocyte growth factor and members of the Wnt, TGF-beta and Hedgehog families. The cell division defects found in dally mutants implicate the Glypican group of integral membrane proteoglycans in the control of cell division during development.

  14. Methyl glycol, methanol and DMSO effects on post-thaw motility, velocities, membrane integrity and mitochondrial function of Brycon orbignyanus and Prochilodus lineatus (Characiformes) sperm.

    PubMed

    Viveiros, Ana T M; Nascimento, Ariane F; Leal, Marcelo C; Gonçalves, Antônio C S; Orfão, Laura H; Cosson, Jacky

    2015-02-01

    The aim of this study was to use more accurate techniques to investigate the effects of cryoprotectants (CPAs) and extenders on post-thaw sperm quality of Brycon orbignyanus and Prochilodus lineatus. Six freezing media comprising the combination of three CPAs (DMSO, methanol and methyl glycol) and two extenders (BTS and glucose) were used. Sperm was diluted in each medium, loaded into 0.5-mL straws, frozen in a nitrogen vapor vessel (dry-shipper), and stored in liquid nitrogen at -196 °C. Post-thaw sperm motility rate and velocities (curvilinear = VCL; straight line = VSL; average path = VAP) were evaluated using a computer-assisted sperm analyzer. Membrane integrity and mitochondrial function were determined using fluorochromes. Post-thaw quality was considered high when samples presented the following minimum values: 60 % motile sperm, 140 µm/s of VCL, 50 % intact sperm membrane and 50 % mitochondrial function integrity. High post-thaw quality was observed in B. orbignyanus sperm frozen in BTS-methyl glycol and in P. lineatus sperm frozen in BTS-methyl glycol, glucose-methyl glycol and glucose-methanol. All samples frozen in DMSO yielded low quality. The presence of ions in the BTS extender affected post-thaw sperm quality positively in B. orbignyanus and negatively in P. lineatus. Methyl glycol was the most suitable CPA for both fish species, leading to a good protection of cell membrane, mitochondrial function and motility apparatus during the cryopreservation process. For an improved protection, B. orbignyanus sperm should be frozen in an ionic freezing medium. PMID:25433690

  15. p-Aminobenzamidine-sensitive acrosomal protease(s) other than acrosin serve the sperm penetration of the egg zona pellucida in mouse.

    PubMed

    Yamagata, K; Murayama, K; Kohno, N; Kashiwabara, S; Baba, T

    1998-11-01

    It has been reported that a significant delay in protein dispersal from the acrosomal matrix is observed in wild-type sperm by adding p-aminobenzamidine, a trypsin/acrosin inhibitor, to the incubation medium. The pattern of this delayed release was similar to that of the acrosin-deficient mutant mouse sperm (Yamagata et al., J. Biol. Chem., 273, 10470-4, 1998). In the present study, no further delay in protein dispersal was found when the acrosin-deficient sperm were treated with p-aminobenzamidine, indicating that among the p-aminobenzamidine-sensitive protease(s) only acrosin may function to accelerate this process. Although the acrosin-deficient sperm penetrated the zona pellucida (Baba et al., J. Biol. Chem., 269, 31845-9, 1994), the addition of p-aminobenzamidine to the fertilisation medium caused a significant inhibition of fertilisation in vitro. This indicates that there is a p-aminobenzamidine-sensitive protease(s) other than acrosin participating in the zona penetration step. Indeed, we demonstrated that a non-acrosin protease with a size of 42 kDa was present in the supernatant of the acrosome-reacted sperm suspension. The enzyme was inhibited by p-aminobenzamidine, diisopropyl fluorophosphate and N alpha-tosyl-L-lysine chloromethyl ketone, and was apparently activated by acrosin. PMID:9921641

  16. Ocimum sanctum essential oil and its active principles exert their antifungal activity by disrupting ergosterol biosynthesis and membrane integrity.

    PubMed

    Khan, Amber; Ahmad, Aijaz; Akhtar, Feroz; Yousuf, Snowber; Xess, Immaculata; Khan, Luqman A; Manzoor, Nikhat

    2010-12-01

    The increasing incidence of drug-resistant pathogens and host toxicity of existing antifungals attracts attention toward the efficacy of natural products as antifungals in mucocutaneous infections and combinational therapies. The composition and antifungal activity of the essential oil obtained from Ocimum sanctum (OSEO) was studied. On GC-MS analysis, OSEO showed a high content of methyl chavicol (44.63%) and linalool (21.84%). Antifungal activity of OSEO and its two main constituents was determined against sixty clinical and five standard laboratory isolates of Candida. OSEO, methyl chavicol and linalool showed inhibitory activity toward all tested strains. The mechanism of their fungicidal action was assessed by studying their effect on the plasma membrane using flow cytometry, confocal imaging and determination of the levels of ergosterol, a fungal-specific sterol. Propidium iodide rapidly penetrated a majority of yeast cells when they were treated with OSEO concentrations just above MIC, implying that fungicidal activity resulted from extensive lesions of the plasma membrane. OSEO and its components also caused a considerable reduction in the amount of ergosterol. The present study indicates that OSEO, methyl chavicol and linalool have significant antifungal activity against Candida, including azole-resistant strains, advocating further investigation for clinical applications in the treatment of fungal infections. PMID:20868749

  17. Alleviation of Waterlogging Damage in Winter Rape by Uniconazole Application: Effects on Enzyme Activity, Lipid Peroxidation, and Membrane Integrity.

    PubMed

    Leul; Zhou

    1999-08-01

    Oilseed rape (Brassica napus L.) seedlings treated with uniconazole [(E)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-l-yl)-l-penten-3-ol] were transplanted at the five-leaf stage into specially designed experimental containers and then exposed to waterlogging for 3 weeks. After waterlogging stress, uniconazole-treated seedlings had significantly higher activities of superoxide dismutase, catalase, and peroxidase enzymes and endogenous free proline content at both the seedling and flowering stages. Uniconazole plus waterlogging-treated plants had a significantly higher content of unsaturated fatty acids than the waterlogged plants. There was a parallel increase in the lipid peroxidation level and electrolyte leakage rate from the leaves of waterlogged plants. Leaves from uniconazole plus waterlogging-treated plants had a significantly lower lipid peroxidation level and electrolyte leakage rate compared with waterlogged plants at both the seedling and flowering stages. Pretreatment of seedlings with uniconazole could effectively delay stress-induced degradation of chlorophyll and reduction of root oxidizability. Uniconazole did not alter the soluble sugar content of leaves and stems, after waterlogging of seedlings. Uniconazole improved waterlogged plant performance and increased seed yield, possibly because of improved antioxidation defense mechanisms, and it retarded lipid peroxidation and membrane deterioration of plants.Key Words. Waterlogging-Uniconazole-Brassica napus L.-Enzymes-Lipid peroxidation-Membrane integrityhttp://link.springer-ny.com/link/service/journals/00344/bibs/18n1p9.html PMID:10467014

  18. Deconvoluting the Effect of the Hydrophobic and Hydrophilic Domains of an Amphiphilic Integral Membrane Protein in Lipid Bicontinuous Cubic Mesophases.

    PubMed

    van 't Hag, Leonie; Shen, Hsin-Hui; Lu, Jingxiong; Hawley, Adrian M; Gras, Sally L; Drummond, Calum J; Conn, Charlotte E

    2015-11-10

    Lipidic bicontinuous cubic mesophases with encapsulated amphiphilic proteins are widely used in a range of biological and biomedical applications, including in meso crystallization, as drug delivery vehicles for therapeutic proteins, and as biosensors and biofuel cells. However, the effect of amphiphilic protein encapsulation on the cubic phase nanostructure is not well-understood. In this study, we illustrate the effect of incorporating the bacterial amphiphilic membrane protein Ag43, and its individual hydrophobic β(43) and hydrophilic α(43) domains, in bicontinuous cubic mesophases. For the monoolein, monoalmitolein, and phytantriol cubic phases with and without 8% w/w cholesterol, the effect of the full length amphiphilic protein Ag43 on the cubic phase nanostructure was more significant than the sum of the individual hydrophobic β(43) and hydrophilic α(43) domains. Several factors were found to potentially influence the impact of the hydrophobic β(43) domain on the cubic phase internal nanostructure. These include the size of the hydrophobic β(43) domain relative to the thickness of the lipid bilayer, as well as its charge and diameter. The size of the hydrophilic α(43) domain relative to the water channel radius of the cubic mesophase was also found to be important. The secondary structure of the Ag43 proteins was affected by the hydrophobic thickness and physicochemical properties of the lipid bilayer and the water channel diameter of the cubic phase. Such structural changes may be small but could potentially affect membrane protein function.

  19. Subthreshold membrane currents confer distinct tuning properties that enable neurons to encode the integral or derivative of their input

    PubMed Central

    Ratté, Stéphanie; Lankarany, Milad; Rho, Young-Ah; Patterson, Adam; Prescott, Steven A.

    2015-01-01

    Neurons rely on action potentials, or spikes, to encode information. But spikes can encode different stimulus features in different neurons. We show here through simulations and experiments how neurons encode the integral or derivative of their input based on the distinct tuning properties conferred upon them by subthreshold currents. Slow-activating subthreshold inward (depolarizing) current mediates positive feedback control of subthreshold voltage, sustaining depolarization and allowing the neuron to spike on the basis of its integrated stimulus waveform. Slow-activating subthreshold outward (hyperpolarizing) current mediates negative feedback control of subthreshold voltage, truncating depolarization and forcing the neuron to spike on the basis of its differentiated stimulus waveform. Depending on its direction, slow-activating subthreshold current cooperates or competes with fast-activating inward current during spike initiation. This explanation predicts that sensitivity to the rate of change of stimulus intensity differs qualitatively between integrators and differentiators. This was confirmed experimentally in spinal sensory neurons that naturally behave as specialized integrators or differentiators. Predicted sensitivity to different stimulus features was confirmed by covariance analysis. Integration and differentiation, which are themselves inverse operations, are thus shown to be implemented by the slow feedback mediated by oppositely directed subthreshold currents expressed in different neurons. PMID:25620913

  20. Evaluation of agonist selectivity for the NMDA receptor ion channel in bilayer lipid membranes based on integrated single-channel currents.

    PubMed

    Hirano, A; Sugawara, M; Umezawa, Y; Uchino, S; Nakajima-Iijima, S

    2000-06-01

    A new method for evaluating chemical selectivity of agonists to activate the N-methyl-D-aspartate (NMDA) receptor was presented by using typical agonists NMDA, L-glutamate and (2S, 3R, 4S)-2-(carboxycyclopropyl)glycine (L-CCG-IV) and the mouse epsilon1/zeta1 NMDA receptor incorporated in bilayer lipid membranes (BLMs) as an illustrative example. The method was based on the magnitude of an agonist-induced integrated single-channel current corresponding to the number of total ions passed through the open channel. The very magnitudes of the integrated single-channel currents were compared with the different BLMs as a new measure of agonist selectivity. The epsilon1/zeta1 NMDA receptor was partially purified from Chinese hamster ovary (CHO) cells expressing the epsilon1/zeta1 NMDA receptor and incorporated in BLMs formed by the tip-dip method. The agonist-induced integrated single-channel currents were obtained at 50 microM agonist concentration, where the integrated current for NMDA was shown to reach its saturated value. The obtained integrated currents were found to be (4.5 +/- 0.55) x 10(-13) C/s for NMDA, (5.8 +/- 0.72) x 10(-13) C/s for L-glutamate and (6.6 +/- 0.61) x 10(-13) C/s for L-CCG-IV, respectively. These results suggest that the agonist selectivity in terms of the total ion flux through the single epsilon1/zeta1 NMDA receptor is in the order of L-CCG-IV approximately = L-glutamate > NMDA.

  1. Analysis of saturation transfer electron paramagnetic resonance spectra of a spin-labeled integral membrane protein, band 3, in terms of the uniaxial rotational diffusion model.

    PubMed Central

    Hustedt, E J; Beth, A H

    1995-01-01

    Algorithms have been developed for the calculation of saturation transfer electron paramagnetic resonance (ST-EPR) spectra of a nitroxide spin-label assuming uniaxial rotational diffusion, a model that is frequently used to describe the global rotational dynamics of large integral membrane proteins. One algorithm explicitly includes terms describing Zeeman overmodulation effects, whereas the second more rapid algorithm treats these effects approximately using modified electron spin-lattice and spin-spin relaxation times. Simulations are presented to demonstrate the sensitivity of X-band ST-EPR spectra to the rate of uniaxial rotational diffusion and the orientation of the nitroxide probe with respect to the diffusion axis. Results obtained by using the algorithms presented, which are based on the transition-rate formalism, are in close agreement with those obtained by using an eigenfunction expansion approach. The effects of various approximations used in the simulation algorithms are considered in detail. Optimizing the transition-rate formalism to model uniaxial rotational diffusion results in over an order of magnitude reduction in computation time while allowing treatment of nonaxial A- and g-tensors. The algorithms presented here are used to perform nonlinear least-squares analyses of ST-EPR spectra of the anion exchange protein of the human erythrocyte membrane, band 3, which has been affinity spin-labeled with a recently developed dihydrostilbene disulfonate derivative, [15N,2H13]-SL-H2DADS-MAL. These results suggest that all copies of band 3 present in intact erythrocytes undergo rotational diffusion about the membrane normal axis at a rate consistent with a band 3 dimer. PMID:8534811

  2. Transfer of a lipophilic drug (temoporfin) between small unilamellar liposomes and human plasma proteins: influence of membrane composition on vesicle integrity and release characteristics.

    PubMed

    Decker, Christiane; Steiniger, Frank; Fahr, Alfred

    2013-06-01

    The introduction of PEG lipid conjugates into lipid bilayers leads to long circulating liposomes with improved pharmacokinetics and pharmacodynamics characteristics. The concentration range of PEG-lipids is limited by their micelle forming properties. We investigated two phosphatidyl oligoglycerols as potential alternatives to PEG-lipid conjugates and compared their micelle forming properties after incorporation of increasing amounts of oligoglycerols into gel-phase liposomes via cryo-transmission electron microscopy. The incorporation of highly hydrophobic drugs into liposomes makes water soluble formulations possible and improves the therapeutic properties of the drug. We incorporated the hydrophobic photosensitizer temoporfin into liposomes varying in membrane fluidity and nature of surface modifying agents. The main purpose of this study was the investigation of liposome integrity and temoporfin incorporation stability in the presence of plasma. After incubation of temoporfin-loaded liposomes with human plasma for different time intervals, liposomes and the single lipoprotein fractions were separated via size-exclusion chromatography. Liposome stability and temoporfin distribution profile over the lipoprotein fractions were determined with the help of a non-exchangeable ³H-lipid label and ¹⁴C-labeled temoporfin. The results demonstrate that both oligoglycerols are suitable alternatives to PEG-lipid conjugates because of the lack of micelle forming properties, comparable liposome stability, and a reduced temoporfin transfer rate compared to PEG-lipids. Furthermore, the incorporation stability of temoporfin is--at least to some extent--influenced by membrane fluidity, indicating that fluid membranes may be better suited for retention of lipophilic drugs.

  3. Alternative oxidase of potato is an integral membrane protein synthesized de novo during aging of tuber slices. [Solanum tuberosum

    SciTech Connect

    Hiser, C.; McIntosh, L. )

    1990-05-01

    The rise in alternative respiratory capacity upon aging of potato (Solanum tuberosum) tuber slices is correlated with changes in mitochondrial membrane protein composition and a requirement for cytoplasmic protein synthesis. However, the lack of an antibody specific to the alternative oxidase has, until recently, prevented examination of the alternative oxidase protein(s) itself. We have employed a monoclonal antibody raised against the Sauromatum guttatum alternative oxidase to investigate developmental changes in the alternative pathway of aging potato slice mitochondria and to characterize the potato alternative oxidase by one- and two-dimensional gel electrophoresis. The relative levels of a 36 kilodalton protein parallel the rise in alternative path capacity. A plausible interpretation is that this alternative oxidase protein is synthesized de novo during aging of potato slices.

  4. Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase*

    PubMed Central

    Fritsch, Johannes; Siebert, Elisabeth; Priebe, Jacqueline; Zebger, Ingo; Lendzian, Friedhelm; Teutloff, Christian; Friedrich, Bärbel; Lenz, Oliver

    2014-01-01

    The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys6[4Fe-3S] cluster. PMID:24448806

  5. Immunostimulatory cellular responses of cured Leishmania-infected patients and hamsters against the integral membrane proteins and non-membranous soluble proteins of a recent clinical isolate of Leishmania donovani

    PubMed Central

    Garg, R; Gupta, S K; Tripathi, P; Naik, S; Sundar, S; Dube, A

    2005-01-01

    Development of an effective immunoprophylactic agent for visceral leishmaniasis (VL) has become imperative due to the increasing number of cases of drug resistance and relapse. Live and killed whole parasites as well as fractionated and recombinant preparations have been evaluated for vaccine potential. However, a successful vaccine against the disease has been elusive. Because protective immunity in human and experimental leishmaniasis is predominantly of the Th1 type, immunogens with Th1 stimulatory potential would make good vaccine candidates. In the present study, the integral membrane proteins (IMPs) and non-membranous soluble proteins (NSPs), purified from promastigotes of a recent field isolate, Leishmania donovani stain 2001, were evaluated for their ability to induce cellular responses in cured patients (n = 9), endemic controls (n = 5) of visceral leishmaniasis (VL) and treated hamsters (n = 10). IMPs and NSPs induced significant proliferative responses (SI 6·3 ± 4·1 and 5·6 ± 2·3, respectively; P < 0·01) and IFN-γ production (356·3 ± 213·4 and 294·29 ± 107·6 pg/ml, respectively) in lymphocytes isolated from cured VL patients. Significant lymphoproliferative responses against IMPs and NSPs were also noticed in cured Leishmania animals (SI 7·2 ± 4·7 & 6·4 ± 4·1, respectively; P < 0·01). In addition, significant NO production in response both IMPs and NSPs was also noticed in macrophages of hamsters and different cell lines (J774A-1 and THP1). These results suggest that protective, immunostimulatory molecules are present in the IMP and NSP fractions, which may be exploited for development of a subunit vaccine for VL. PMID:15762886

  6. Myocardial metabolism of fluorodeoxyglucose compared to cell membrane integrity for the potassium analogue rubidium-82 for assessing infarct size in man by PET

    SciTech Connect

    Gould, K.L.; Yoshida, K.; Hess, M.J.; Haynie, M.; Mullani, N.; Smalling, R.W. )

    1991-01-01

    Potassium loss from damaged myocardial cells is linearly related to CPK enzyme loss reflecting extent of necrosis. The potassium analog, rubidium-82 (82Rb), is extracted after i.v. injection and retained in viable myocardium but is not trapped or washed out of necrotic regions. To compare myocardial cell metabolism with membrane dysfunction as indicators of necrosis/viability, 43 patients with evolving myocardial infarction and coronary arteriography had positron emission tomography using fluorodeoxyglucose (FDG) and the potassium analog 82Rb. Percent of heart showing FDG defects and 82Rb washout on sequential images indicating failure to retain the potassium analogue were visually assessed and quantified by automated software. Infarct size based on rubidium kinetics correlated closely with size and location on FDG images (visual r = 0.93, automated r = 0.82), suggesting that loss of cell membrane integrity for trapping the potassium analog 82Rb parallels loss of intracellular glucose metabolism, both comparable quantitative markers of myocardial necrosis/viability.

  7. Lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) is a substrate of cathepsin-F, a cysteine protease mutated in type-B-Kufs-disease.

    PubMed

    Peters, Judith; Rittger, Andrea; Weisner, Rebecca; Knabbe, Johannes; Zunke, Friederike; Rothaug, Michelle; Damme, Markus; Berkovic, Samuel F; Blanz, Judith; Saftig, Paul; Schwake, Michael

    2015-02-13

    The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor for enterovirus 71 uptake and mannose-6-phosphate-independent lysosomal trafficking of the acid hydrolase β-glucocerebrosidase. Here we show that LIMP-2 undergoes proteolytic cleavage mediated by lysosomal cysteine proteases. Heterologous expression and in vitro studies suggest that cathepsin-F is mainly responsible for the lysosomal processing of wild-type LIMP-2. Furthermore, examination of purified lysosomes revealed that LIMP-2 undergoes proteolysis in vivo. Mutations in the gene encoding cathepsin-F (CTSF) have recently been associated with type-B-Kufs-disease, an adult form of neuronal ceroid-lipofuscinosis. In this study we show that disease-causing cathepsin-F mutants fail to cleave LIMP-2. Our findings provide evidence that LIMP-2 represents an in vivo substrate of cathepsin-F with relevance for understanding the pathophysiology of type-B-Kufs-disease.

  8. Development of a Next-Generation Membrane-Integrated Adsorption Processor for CO2 Removal and Compression for Closed-Loop Air Revitalization Systems

    NASA Technical Reports Server (NTRS)

    Mulloth, Lila; LeVan, Douglas

    2002-01-01

    The current CO2 removal technology of NASA is very energy intensive and contains many non-optimized subsystems. This paper discusses the concept of a next-generation, membrane integrated, adsorption processor for CO2 removal nd compression in closed-loop air revitalization systems. This processor will use many times less power than NASA's current CO2 removal technology and will be capable of maintaining a lower CO2 concentration in the cabin than that can be achieved by the existing CO2 removal systems. The compact, consolidated, configuration of gas dryer, CO2 separator, and CO2 compressor will allow continuous recycling of humid air in the cabin and supply of compressed CO2 to the reduction unit for oxygen recovery. The device has potential application to the International Space Station and future, long duration, transit, and planetary missions.

  9. Molecular cloning of a highly conserved mouse and human integral membrane protein (Itm1) and genetic mapping to mouse chromosome 9

    SciTech Connect

    Hong, Guizhu; Tylzanowski, P.; Deleersnijder, W.

    1996-02-01

    We have isolated and characterized a novel cDNA coding for a highly hydrophobic protein (B5) from a fetal mouse mandibular condyle cDNA library. The full-length mouse B5 cDNA is 3095 nucleotides long and contains a potential open reading frame coding for a protein of 705 amino acids with a calculated molecular weight of 80.5 kDa. The B5 mRNA is differentially polyadenylated, with the most abundant transcript having a length of 2.7 kb. The human homolog of B5 was isolated from a cDNA testis library. The predicted amino acid sequence of the human B5 is 98.5% identical to that of mouse. The most striking feature of the B5 protein is the presence of numerous (10-14) potential transmembrane domains, characteristic of an integral membrane protein. Similarity searches in public databanks reveal that B5 is 58% similar to the T12A2.2 gene of Caenorhabditis elegans and 60% similar to the