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Sample records for acrosomal membrane integrity

  1. Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry.

    PubMed

    Kanno, Chihiro; Kang, Sung-Sik; Kitade, Yasuyuki; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

    2016-08-01

    The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen-thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE-PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE-PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE-PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE-PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously. PMID:26369275

  2. 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

  3. 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

  4. Evolution and development of the outer acrosomal membrane (OAM) and evidence that acrosin-inhibitors are proteins of the OAM.

    PubMed

    Flörke-Gerloff, S; Töpfer-Petersen, E; Schill, W B; Engel, W

    1987-01-01

    An antiserum to the purified porcine outer acrosomal membrane (OAM) was raised in rabbits and the IgG fraction isolated by ammonium sulphate precipitation and ion exchange chromatography. The antibodies reacted exclusively with the acrosomal cap of the sperm head as revealed by indirect immunofluorescence. In addition they cross-reacted not only with the acrosomal part of the spermatozoa of all mammalian species tested (bull, horse, rabbit, rat, mouse, hamster, mole, antelope, monkey, man) but also with the spermatozoa of the cock (Class: birds) and the rainbow trout (Class: fish). All the species exhibited similar development of the acrosomal cap during spermatogenesis, with the appearance of the immunofluorescent stain in early round spermatids. In the mole the localization of the acrosome in elongated testicular spermatids differed from that in all other species: Instead of prominent fluorescence over the apical part of the sperm an equatorial belt was formed. The cross-reactivity of the anti-boar OAM antibody with the acrosomes of different vertebrate species at the morphological level was supported by the results of Western blotting experiments with purified boar OAM proteins and the SDS-extractable proteins of bull and human spermatozoa, respectively. Using anti-OAM antibodies and antibodies against the acrosin inhibitors I and II described recently by Tschesche et al. (1982), in absorption and Western blotting experiments, it was demonstrated that the acrosin inhibitor proteins are integrated in the outer acrosomal membrane. PMID:2446533

  5. 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

  6. 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. PMID:12804573

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

    PubMed Central

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

    2012-01-01

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

  8. The osmotic properties of the acrosome of guinea-pig sperm.

    PubMed

    Green, D P

    1978-08-01

    The osmotic behaviour of the acrosome in intact guinea-pig sperm has been examined by light and electron microscopy. Because the acrosome is retained within the cell, it can only experience the changes in tonicity of the cytoplasm and these have first to be related to the changes in tonicity in the external medium. Nevertheless, a qualitative description of the osmotic properties of the acrosome can be obtained. The evidence suggests that the acrosomal contents are normally close to their limit of compression. It has been proposed that a hydrostatic pressure is exerted inwards on the acrosomal membrane and that this is responsible for the maintenance of acrosomal shape. Swelling of the acrosome when sperm are suspended in hypotonic medium suggests that the acrosomal membrane does not have an abnormally low water permeability. Two-thirds of the acrosomal volume are occupied by a single matrix which undergoes cavitation in the course of the acrosome reaction. It also undergoes cavitation when guinea-pig sperm are suspended in calcium-free medium containing the non-ionic detergent Triton X-100. This suggests that cavitation is caused by the loss of integrity of the plasma and acrosomal membranes. A mechanism for cavitation is proposed in which a colloid osmotic pressure within the acrosomal matrix is allowed expression when the crystalloids of the external medium pass into it, this internal colloid osmotic pressure forcing the matrix apart to produce a cavity. PMID:701392

  9. 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

  10. Analysis of the complexity of the sperm acrosomal membrane by super-resolution stimulated emission depletion microscopy compared with transmission electron microscopy.

    PubMed

    Ito, Chizuru; Yamatoya, Kenji; Toshimori, Kiyotaka

    2015-08-01

    The acrosome is a Golgi-derived sperm cell organelle enclosed by a continuous acrosomal membrane. The acrosomal membrane complexes with surrounding matrices containing molecules necessary for fertilization; however, the complex of acrosomal membrane and associating matrices (CAMAM) has not been visualized in detail under living conditions. Here, we analyzed the CAMAM at the nanometer level using super-resolution stimulated emission depletion (STED) fluorescence microscopy and equatorin-enhanced green fluorescent protein transgenic mice. The STED images were compared with the corresponding images taken by immunoelectron microscopy. Consequently, the substructure of CAMAM could be differentiated at nanometer-scale resolution by STED microscopy without the need for sectioning. The information obtained in this study will be beneficial not only for understanding the molecular mechanism of fertilization but also for cell imaging under living conditions. PMID:25430742

  11. 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

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

    PubMed

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

    1989-08-01

    A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested. PMID:2766782

  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. Acrosome integrity assessment of boar spermatozoa images using an early fusion of texture and contour descriptors.

    PubMed

    García-Olalla, Oscar; Alegre, Enrique; Fernández-Robles, Laura; Malm, Patrik; Bengtsson, Ewert

    2015-06-01

    The assessment of the state of the acrosome is a priority in artificial insemination centres since it is one of the main causes of function loss. In this work, boar spermatozoa present in gray scale images acquired with a phase-contrast microscope have been classified as acrosome-intact or acrosome-damaged, after using fluorescent images for creating the ground truth. Based on shape prior criteria combined with Otsu's thresholding, regional minima and watershed transform, the spermatozoa heads were segmented and registered. One of the main novelties of this proposal is that, unlike what previous works stated, the obtained results show that the contour information of the spermatozoon head is important for improving description and classification. Other of this work novelties is that it confirms that combining different texture descriptors and contour descriptors yield the best classification rates for this problem up to date. The classification was performed with a Support Vector Machine backed by a Least Squares training algorithm and a linear kernel. Using the biggest acrosome intact-damaged dataset ever created, the early fusion approach followed provides a 0.9913 F-Score, outperforming all previous related works. PMID:25887848

  15. 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

  16. The effect of glycerol-related osmotic changes on post-thaw motility and acrosomal integrity of ram spermatozoa.

    PubMed

    Fiser, P S; Fairfull, R W

    1989-02-01

    Ram semen, collected by artificial vagina, was diluted and processed for long-term storage as described by P. S. Fiser, L. Ainsworth, and R. W. Fairfull (Canad. J. Anim. Sci. 62, 425-428, 1982). The concentration of the cryoprotectant, glycerol, was adjusted to 4% in the diluted semen prior to freezing by a one-step addition at 30 degrees C (Method 1), by cooling the semen to 5 degrees C and addition of the glycerol gradually over 30 min (Method 2), by one-step addition of glycerol prior to equilibration for 2 hr (Method 3), or by cooling to 5 degrees C, followed by a holding period of 2 hr at 5 degrees C, and the one-step addition of glycerol just prior to freezing (Method 4). After thawing, the glycerol concentration of the semen was reduced by stepwise dilution from 4 to 0.4% over 15 or 30 min or by a one-step ten-fold dilution. The average post-thaw percentage of motile spermatozoa was significantly lower after addition of glycerol by Method 1 (39.9%) than when the glycerol was added by the other three methods (range, 44.0-46.4% averaged over the glycerol dilution). The average post-thaw percentage of intact acrosomes (61.2%), highest in semen in which the glycerol was added by Method 2, was not significantly different from those in which glycerol was added to semen by Methods 3 and 4, but it was significantly higher than that found in semen in which the glycerol was added by Method 1 (54.4%). However, when averaged over the method of glycerolation, the post-thaw percentage of motile spermatozoa (range, 43.7-44.2%) and the percentage of intact acrosomes (range, 56.8-59.5%) did not differ significantly in semen subjected to gradual decrease in glycerol concentration and diluent osmolality (over 15 and 30 min) or by a one-step, 10-fold dilution. These data indicate that post-thaw survival of spermatozoa can be influenced by the way in which glycerol is added prior to freezing. However, post-thaw spermatozoa motility and acrosomal integrity can be maintained even

  17. 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.

  18. 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

  19. Acrosomal ultrastructure of stallion spermatozoa cryopreserved with ethylene glycol using two packaging systems.

    PubMed

    Alvarenga, M A; Landim-Alvarenga, F C; Moreira, R M; Cesarino, M M

    2000-11-01

    The present experiments aimed to examine the substitution of glycerol (G) by ethylene glycol (E) as a cryoprotective agent for stallion spermatozoa. Two different ethylene glycol concentrations (5% and 10%) and also the association of glycerol (2%) and ethylene glycol (3%) (E/G) were studied (Experiment 1). In Experiment 2, two packing systems (0.5 x 4.0 ml) were evaluated using both cryoprotectors. In both experiments, the sperm membrane integrity after freezing was evaluated using transmission electron microscopy. The mean post-thaw motility was 34.25, 36.5, 29.25 and 34.75% for G5%, E5%, E10% and E/G, respectively. It was observed that the percentage of motile spermatozoa was significantly smaller (P<0.05) when semen was processed with E10%. A decrease in the acrosome integrity was observed in frozen thawed spermatozoa from all treated groups. It was observed that 28.0, 22.5, 25.5 and 22.5% of the sperm cells had a normal acrosome following freezing with G5%, E5%, E10% and E/G, respectively. Undulation of the outer acrosomal membrane, acrosomal swelling and loss of acrosomal content density and homogeneity were the most evident ultrastructural alterations observed. In Experiment 2, the post-thaw motility was higher (P<0.05) for sperm frozen in 0.5 ml straws than in 4.0 ml straws, regardless of the cryoprotector used. The ultrastructural evaluation showed 26.7 and 16.0% of intact acrosomes for sperm frozen in 0.5 ml and 4.0 ml straws, respectively. We concluded that ethylene glycol has similar cryoprotective properties to glycerol and that utilisation of 0.5 ml straws improved the ability of horse sperm cells to withstand damage after the cryopreservation process. PMID:11093629

  20. Sperm Capacitation and Acrosome Reaction in Mammalian Sperm.

    PubMed

    Stival, Cintia; Puga Molina, Lis Del C; Paudel, Bidur; Buffone, Mariano G; Visconti, Pablo E; Krapf, Dario

    2016-01-01

    Physiological changes that endow mammalian sperm with fertilizing capacity are known as sperm capacitation. As part of capacitation, sperm develop an asymmetrical flagellar beating known as hyperactivation and acquire the ability to undergo the acrosome reaction. Together, these processes promote fertilizing competence in sperm. At the molecular level, capacitation involves a series of signal transduction events which include activation of cAMP-dependent phosphorylation pathways, removal of cholesterol, hyperpolarization of the sperm plasma membrane, and changes in ion permeability. In recent years, new technologies have aided in the study of sperm signaling molecules with better resolution, at both spatial and temporal levels, unraveling how different cascades integrate and cooperate to render a fertilizing sperm. Despite this new information, the molecular mechanisms connecting capacitation with acrosomal exocytosis and hyperactivation are not well understood. This review brings together results obtained in mammalian species in the field of sperm capacitation with special focus on those pathways involved in the preparation to undergo the acrosomal reaction. PMID:27194351

  1. Cholesterol-loaded cyclodextrin enhances osmotic tolerance and inhibits the acrosome reaction in rabbit spermatozoa.

    PubMed

    Aksoy, Melih; Akman, Orhan; Lehimcioğlu, Necdet Cankat; Erdem, Hüseyin

    2010-07-01

    The effects of cholesterol-loaded cyclodextrin (CLC) treatment on the osmotic tolerance and ability to undergo the acrosome reaction of rabbit spermatozoa, with an unusually high cholesterol/phospholipid ratio in plasma membranes, were examined in two successive experiments. In the first experiment, CLC-pretreated and untreated sperm cells were exposed for 15min to one of five fructose solutions, adjusted to 20, 80, 290, 500 or 1500mOsm/L. After the anisoosmotic challenge, the integrity of sperm membranes in the CLC-supplemented (at a dose level of 3mg/120x10(6)spermatozoa) and control groups was estimated by a modified hypoosmotic swelling test (HOST) associated with a supravital eosin staining test (HE-test). In the second part of the study, the influence of cholesterol supplementation on the acrosome reaction of sperm cells stimulated by either calcium ionophore A23187 (CI) or lysophosphatidylcholine (LPC) was evaluated. CLC pretreatment increased viable and live-HOST-responsive sperm rates (P<0.01) after incubation in anisoosmotic solutions varying from 80 to 1500mOsm/L. However, CLC supplementation did not influence the percentage of HOST-responsive sperm cells (P>0.05). A significant interaction was determined between CLC pretreatment and the level of osmotic pressure in maintaining the functional and physical integrities of sperm membranes undergoing osmotic challenges. Both CI and LPC successfully induced the acrosome reaction in rabbit spermatozoa (P<0.001). Compared with CI, LPC was more effective (P<0.0001). CLC pretreatment resulted in a significant reduction (P<0.01) in the percentage of acrosome reacted sperm cells irrespective of the inducing agent, either CI or LPC. In conclusion, CLC treatment enhanced the anisoosmotic tolerance of rabbit spermatozoa and reduced their ability to undergo the acrosome reaction after stimulation by CI or LPC. PMID:20304567

  2. 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.

  3. The hypo-osmotic swelling test for evaluation of sperm membrane integrity.

    PubMed

    Ramu, Sivakumar; Jeyendran, Rajasingam S

    2013-01-01

    A functional membrane is requisite for the fertilizing ability of spermatozoa, as it plays an integral role in sperm capacitation, acrosome reaction, and binding of the spermatozoon to the egg surface. The hypo-osmotic swelling (HOS) test evaluates the functional integrity of the sperm's plasma membrane and also serves as a useful indicator of fertility potential of sperm. The HOS test predicts membrane integrity by determining the ability of the sperm membrane to maintain equilibrium between the sperm cell and its environment. Influx of the fluid due to hypo-osmotic stress causes the sperm tail to coil and balloon or "swell." A higher percentage of swollen sperm indicates the presence of sperm having a functional and intact plasma membrane. Here, we present the detailed protocol for performing the HOS test and explain the results for interpretation. PMID:22992900

  4. Capacitation and acrosome reaction induction on thawed Dama dama deer spermatozoa: glycine effect as cryopreservation diluent supplement.

    PubMed

    Fernández, Silvina; Sestelo, Adrián; Rivolta, Miguel; Córdoba, Mariana

    2013-12-01

    The aim of this research was to evaluate two different diluents for sperm cryopreservation and to study functional parameters in relation to the response to heparin, lysophosphatidylcholine and progesterone, in frozen-thawed semen of fallow deer (Dama dama) during the reproductive season (brama). In this way, fallow deer can be used as a biological model of endangered cervids. Semen was obtained by electroejaculation. Heparin, progesterone and lysophosphatidylcholine were used as capacitation and acrosome reaction inducers, respectively. Capacitation and acrosome reaction were evaluated by chlorotetracycline epifluorescence technique (CTC), membrane integrity by Hypo-osmotic swelling test (HOS) and viability and acrosome integrity by trypan blue stain/DIC. Data was analyzed by ANOVA and Tukey Test (P < 0.05). Semen was cryopreserved in different diluents and Fructose-Tris-Glycine extender was selected. Capacitation with heparin at different incubation times determined that the highest capacitation percentage was obtained at 45 minutes incubation. Progesterone (1 'M) and lysophosphatidylcholine in heparin capacitated sperm induced acrosome reaction (P < 0.05). This study contributes to improve cryopreservation methods and to increase the knowledge about capacitation and acrosome reaction in vitro in deer spermatozoa, allowing an advance in the development of reproductive biotechnologies. PMID:24320190

  5. 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

  6. Morphological change of the acrosome on motile bovine spermatozoa due to storage at 4 degrees C.

    PubMed

    Aalseth, E P; Saacke, R G

    1985-07-01

    Swelling of the apical ridge and anterior acrosome of motile bovine spermatozoa was observed during in-vitro storage using differential interference-contrast optics. This morphological alteration is different from that described as the false acrosome reaction on immotile spermatozoa, apparent in ageing semen samples and which has been associated with cell death. In this study, transmission electron microscopy revealed that the apical ridge acrosomal matrix was extended into complex folds and/or projections. Acrosomal and plasma membrane integrity was retained. Storing spermatozoa (1500 X 10(6)/ml) in seminal plasma at 4 degrees C for 1 day was most conducive to the swelling of the apical ridge. Replacing seminal plasma with egg yolk-citrate inhibited swelling. However, incubating semen at 37 degrees C in egg yolk-Tris-fructose extender (25 X 10(6) spermatozoa/ml) after storage in egg yolk-citrate at 4 degrees C for greater than or equal to 3 days restored the swelling characteristic. PMID:3900381

  7. The effect of different extenders on post-thaw sperm survival, acrosomal integrity and longevity in cryopreserved semen of Formosan Sika deer and Formosan Sambar deer.

    PubMed

    Cheng, Feng-Pang; Wu, Jui-Te; Chan, Jacky Peng-Wen; Wang, Jiunn-Shiow; Fung, Hang-Poung; Colenbrander, Ben; Tung, Kwong-Chung

    2004-06-01

    This study investigates the efficacy of five extenders in contributing to the outcome of semen cryopreservation in Formosan Sika and Sambar deer. Pooled semen (n=4) of six males of each breed was used. In Sika deer, semen collection rate was 96% (23/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 0.5+/-0.4 ml, 77+/-6% and 1471.3+/-940.0 x 10(6) ml(-1), respectively. Post-thaw motility in respective extender was A: 66+/-16%; B: 71+/-2%; C: 73+/-6%; D: 9+/-4% and E: 26+/-12% (mean+/-S.D.). In extender C (74+/-14%) more viable spermatozoa were preserved than in the others (A: 64+/-10%; B: 48+/-11%; D: 41+/-16%; E: 47+/-6%; P<0.05). Acrosomal integrity was not influenced by extender composition. Post-thaw motility did not decrease during a 4-h incubation period, irrespective of the extender used (P>0.05). In Sambar deer, semen collection rate was 88% (21/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 1.3+/-0.5 ml, 82+/-4% and 379.1+/-252.2 x 10(6) ml(-1), respectively. Post-thaw motility was in respective extenders A: 69+/-2%; B: 74+/-6%; C: 73+/-2%; D: 13+/-6% and E: 31+/-20%. Extenders B and C were superior (P>0.05) with respect to sperm motility. Similarly, post-thaw viability in extenders A (70+/-7%), B (76+/-7%) and C (79+/-2%) was higher than that D (25+/-19%) and E (29+/-17%) (P<0.01). Sperm acrosomal integrity was better preserved in extenders B (86+/-4%) and C (83+/-4%) than in extenders A (54+/-13%), D (39+/-22%) and E (46+/-22%) (P<0.05). Post-thaw sperm longevity in extender A reduced from 69 to 16% during incubation (P<0.05) whereas only a slight decrease was observed in the other extenders after 4 h. In conclusion these data show that egg-yolk-Tris-Tes-glycerol based extender C containing Equex STM paste is optimal for freezing semen of Formosan Sika deer while egg-yolk-Tris-citric acid-glycerol based extender B containing Equex and

  8. Composite membrane with integral rim

    SciTech Connect

    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.

  9. Functional Amyloids in the Mouse Sperm Acrosome

    PubMed Central

    Guyonnet, Benoit; Egge, Nathan

    2014-01-01

    The acrosomal matrix (AM) is an insoluble structure within the sperm acrosome that serves as a scaffold controlling the release of AM-associated proteins during the sperm acrosome reaction. The AM also interacts with the zona pellucida (ZP) that surrounds the oocyte, suggesting a remarkable stability that allows its survival despite being surrounded by proteolytic and hydrolytic enzymes released during the acrosome reaction. To date, the mechanism responsible for the stability of the AM is not known. Our studies demonstrate that amyloids are present within the sperm AM and contribute to the formation of an SDS- and formic-acid-resistant core. The AM core contained several known amyloidogenic proteins, as well as many proteins predicted to form amyloid, including several ZP binding proteins, suggesting a functional role for the amyloid core in sperm-ZP interactions. While stable at pH 3, at pH 7, the sperm AM rapidly destabilized. The pH-dependent dispersion of the AM correlated with a change in amyloid structure leading to a loss of mature forms and a gain of immature forms, suggesting that the reversal of amyloid is integral to AM dispersion. PMID:24797071

  10. 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. PMID:26792943

  11. 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

  12. Role of Ion Channels in the Sperm Acrosome Reaction.

    PubMed

    Beltrán, Carmen; Treviño, Claudia L; Mata-Martínez, Esperanza; Chávez, Julio C; Sánchez-Cárdenas, Claudia; Baker, Mark; Darszon, Alberto

    2016-01-01

    The acrosome reaction (AR) is a unique exocytotic process where the acrosome, a single membrane-delimited specialized organelle, overlying the nucleus in the sperm head of many species, fuses with the overlying plasma membrane. This reaction, triggered by physiological inducers from the female gamete, its vicinity, or other stimuli, discharges the acrosomal content modifying the plasma membrane, incorporating the inner acrosomal membrane, and exposing it to the extracellular medium. The AR is essential for sperm-egg coat penetration, fusion with the eggs' plasma membrane, and fertilization. As in most exocytotic processes Ca(2+) is crucial for the AR, as well as intracellular pH and membrane potential changes. Thus, among the required processes needed for this reaction, ion permeability changes involving channels are pivotal. In spite of the key role ion channels play in the AR, their identity and regulation is not fully understood. Though molecular and pharmacological evidence indicates that various ionic channels participate during the AR, such as store-operated Ca(2+) channels and voltage-dependent Ca(2+) channels, whole cell patch clamp recordings have failed to detect some of them until now. Since sperm display a very high resistance and a minute cytoplasmic volume, very few channels are needed to achieve large membrane potential and concentration changes. Functional detection of few channels in the morphologically complex and tiny sperm poses technical problems, especially when their conductance is very small, as in the case of SOCs. Single channel recordings and novel fluorescence microscopy strategies will help to define the participation of ionic channels in the intertwined signaling network that orchestrates the AR. PMID:27194349

  13. The human sperm acrosome reaction: physiology and regulatory mechanisms. An update.

    PubMed

    Brucker, C; Lipford, G B

    1995-01-01

    The acrosome reaction is a crucial step during gamete interaction in all species, including man. It allows spermatozoa to penetrate the zona pellucida and fuse with the oocyte membrane. Spermatozoa unable to undergo the acrosome reaction will not fertilize intact oocytes. This article concentrates on the characteristics and regulatory mechanisms of the acrosome reaction in human spermatozoa. During recent years, various entities found in the vicinity of the ovulated oocyte have been identified as stimulators of the acrosome reaction, of which zona protein is considered the prime physiological inducer in vivo. The steroid hormone progesterone has been shown to evoke critical responses in sperm cells leading to the acrosome reaction. Calcium has also been shown to play a central role during the acrosome reaction. Calcium flux is induced specifically by progesterone in capacitated and uncapacitated sperm cells, whereas only capacitated spermatozoa are able to subsequently complete the acrosome reaction. Progesterone as well as zona protein has been shown to evoke crucial responses within human spermatozoa, shedding light on the cascade of intracellular signalling events leading to the completion of the acrosome reaction. Furthermore, chemical agents which bring about the reaction in vitro, such as the ionophores ionomycin or A23187, have been used to shed light on its regulatory mechanisms. A number of molecules have been postulated to regulate the acrosome reaction in mammals, for example a galactosyl-transferase and a sperm protein tyrosine kinase. In addition, a novel protein, termed SAA-1, that was first detected on human spermatozoa is discussed with respect to its potential role as a regulatory protein closely involved in the initiation of the acrosome reaction. PMID:9080206

  14. Lead chloride affects sperm motility and acrosome reaction in mice: lead affects mice sperm motility and acrosome reaction.

    PubMed

    Oliveira, Helena; Spanò, Marcello; Santos, Conceição; Pereira, Maria de Lourdes

    2009-08-01

    Lead is highly toxic and persistent in the environment and, thus, a major concern for public health. In this study, the effects of lead chloride (PbCl2) on mouse epididymal sperm were evaluated. Male mice were subcutaneously injected with 74 and 100 mg PbCl2/kg body weight for four consecutive days. Sperm was collected from the epididymis and several parameters of sperm function, such as sperm density, motility, viability, mitochondrial function, acrosome integrity and morphology, were evaluated. Furthermore, DNA fragmentation was assessed by the terminal deoxylnucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay and chromatin integrity was evaluated by sperm chromatin structure assay (SCSA). In order to assess direct effects on existing sperm population, we sacrificed one group for each condition at day 5. The effects of lead upon one entire spermatogenic cycle were evaluated on day 35. Both lead concentrations used in this work affected sperm motility, although no significant differences were observed in sperm viability, mitochondrial function and DNA/chromatin integrity. However, a decrease in the percentage of intact acrosomes was also observed, mirroring a lead-induced premature acrosome reaction. Thus, the results obtained indicate that, together with impaired motility, the effect of lead toxicity on acrosome integrity, leading to premature reaction, may compromise the ability of sperm to fertilize the oocyte. PMID:18594995

  15. Loss of Zona Pellucida Binding Proteins in the Acrosomal Matrix Disrupts Acrosome Biogenesis and Sperm Morphogenesis▿ †

    PubMed Central

    Lin, Yi-Nan; Roy, Angshumoy; Yan, Wei; Burns, Kathleen H.; Matzuk, Martin M.

    2007-01-01

    Zona pellucida binding protein 1 (ZPBP1), a spermatid and spermatozoon protein that localizes to the acrosome, was originally identified in pigs and named for its binding to the oocyte zona pellucida. In an in silico search for germ cell-specific genes, Zpbp1 and its novel paralog, Zpbp2, were discovered and confirmed to be expressed only in the testes in both mice and humans. To study the in vivo functions of both ZPBP proteins, we disrupted Zpbp1 and Zpbp2 in mice. Males lacking ZPBP1 were sterile, with abnormal round-headed sperm morphology and no forward sperm motility. Ultrastructural studies demonstrated that absence of ZPBP1 prevents proper acrosome compaction, resulting in acrosome fragmentation and disruption of the Sertoli-spermatid junctions. Males null for ZPBP2 were subfertile, demonstrated aberrant acrosomal membrane invaginations, and produced dysmorphic sperm with reduced ability to penetrate zona pellucida. Molecular phylogenetic analysis of ZPBPs from amphibians, birds, and mammals suggests that these paralogous genes coevolved to play cooperative roles during spermiogenesis. Whereas ZPBP1 was discovered for an in vitro role in sperm-egg interactions, we have shown that both ZPBP proteins play an earlier structural role during spermiogenesis. PMID:17664285

  16. 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

  17. 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

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

    PubMed Central

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

    2016-01-01

    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

  19. Ultrastructural localization of acrosome reaction-inducing substance (ARIS) on sperm of the starfish Asterias amurensis.

    PubMed

    Longo, F J; Ushiyama, A; Chiba, K; Hoshi, M

    1995-05-01

    Using colloidal gold tagged ligands we have identified the ultrastructural site of ARIS binding to intact and acrosome-reacted starfish sperm. In intact sperm, colloidal gold conjugated ARIS was specifically localized to a single domain (0.1-0.3 micron in diameter) on the plasma membrane. This site was located on the anterior-lateral aspect of the sperm head, that is, just peripheral to the region occupied by the acrosomal vesicle and periacrosomal components. When sperm were labeled with colloidal gold conjugated ARIS, washed to remove unbound label, and then induced to undergo the acrosome reaction, the labeled patch remained associated with the plasma membrane and was positioned just lateral to the acrosomal process. However, when sperm were suspended in labeled ARIS and induced to undergo the acrosome reaction, label was observed along the entire anterior aspect of the sperm head with the exception of the acrosomal process. Labeling along the entire anterior aspect of the sperm head in this case was deemed to be nonspecific and due to binding of colloidal gold tagged molecules to components formerly located within the acrosomal vesicle, as the same pattern was obtained using colloidal gold tagged bovine serum albumin. Quantitative and qualitative aspects of ARIS binding observed here by electron microscopy are in agreement with measured binding characteristics previously reported (Ushiyama et al., 1993a: Zygote 1:121-127; Ushiyama et al., 1993b: J Reprod Dev 39:53-54), and indicate that the site of labeled ARIS binding represents a specific plasma membrane domain occupied by ARIS receptors. PMID:7619512

  20. Sperm-engulfing response of sea urchin egg surfaces inseminated with acrosome-reacted starfish sperm.

    PubMed

    Kyozuka, K; Osanai, K

    1988-10-01

    Sperm-egg interaction was examined in two interclass combinations of sea urchin (Strongylocentrotus nudus and Hemicentrotus pulcherrimus) eggs and starfish (Asterina pectinifera and Asterias amurensis) sperm. Cross-fertilization was unsuccessful between these combinations. When the vitelline coat-free sea urchin eggs were mixed with acrosome-reacted starfish sperm, the elongated microvilli on the egg surface wrapped the sperm head. This sperm-engulfing response observed on the denuded egg surface was induced only in sperm immediately after initiation of the acrosome reaction. Further fertilization events, such as gamete membrane fusion or discharge of cortical granules, did not proceed. These observations suggest that acrosome-reacted sperm can induce a local response on the heterologous egg surface, that is independent of gamete membrane fusion and egg activation. PMID:3229729

  1. 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.

  2. Modulation of bovine sperm signalling pathways: correlation between intracellular parameters and sperm capacitation and acrosome exocytosis.

    PubMed

    Pons-Rejraji, Hanae; Bailey, Janice L; Leclerc, Pierre

    2009-01-01

    In the present study, the viability, intracellular pH (pHi), cAMP ([cAMP]i), calcium concentration and protein phosphotyrosine content were evaluated in relation to the acrosomal and capacitation status of freshly ejaculated bull spermatozoa. These parameters were evaluated before and after incubation with the capacitation inducer heparin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), the phosphotyrosyl-protein phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate, and hydrogen peroxide. The results obtained were integrated to address the physiological interactions between the different signalling events affecting sperm capacitation and acrosome reaction. As expected, heparin promoted the expression of the 'B' pattern of chlortetracycline binding, increased pHi, [cAMP]i and the phosphotyrosine content of sperm proteins. The effects of heparin were enhanced by IBMX. Both PAO and sodium orthovanadate stimulated protein phosphotyrosine content and acrosomal exocytosis, although only PAO affected pH, Ca2+ and cAMP levels. Intracellular pH was increased while both Ca2+ and [cAMP]i were decreased. Physiological concentrations of H2O2 increased [cAMP]i and promoted acrosomal exocytosis. A significant positive correlation was found between sperm capacitation, protein phosphotyrosine content and stored Ca2+ concentration, whereas the acrosome reaction was correlated with pHi and Ca2+ concentration. This study presents the first global analysis of the major elements individually described during sperm capacitation and acrosome reaction signalling pathways, supported by statistical correlations. PMID:19383258

  3. 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

  4. 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

  5. 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.

  6. Assessing equine sperm-membrane integrity.

    PubMed

    Lagares, M A; Petzoldt, R; Sieme, H; Klug, E

    2000-05-01

    The swelling of cells in a hypo-osmotic medium has been described as an important criterion for assessing the functional integrity of the sperm plasma membrane. The resistance of equine spermatozoa to osmolarity changes was studied by extending 98 semen samples collected from nine stallions in media at five osmolarities (300, 200, 150, 100, and 50 mOsmol l(-1)). The response of the cells was measured by the spermatocrit technique and eosin staining. Spermatocrit determines the increase on spermatozoal volume under hypo-osmotic conditions, a sign of functional integrity of sperm plasma membrane, whereas the eosin staining evaluates the viability of spermatozoa. A significant positive correlation (P<0.01) was observed between spermatocrit values and percentage of eosin-unstained cells. Spermatocrit measurements and eosin staining proved to be useful methods to evaluate the integrity of sperm plasma membrane under hypo-osmotic conditions and could be used as an additional criterion to predict semen preservation ability. PMID:10863971

  7. 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

  8. Thermoelectric Integrated Membrane Evaporation Subsystem operational improvements

    NASA Technical Reports Server (NTRS)

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

    1984-01-01

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

  9. 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.

  10. Quality control of integral membrane proteins by assembly-dependent membrane integration.

    PubMed

    Feige, Matthias J; Hendershot, Linda M

    2013-08-01

    Cell-surface multiprotein complexes are synthesized in the endoplasmic reticulum (ER), where they undergo cotranslational membrane integration and assembly. The quality control mechanisms that oversee these processes remain poorly understood. We show that less hydrophobic transmembrane (TM) regions derived from several single-pass TM proteins can enter the ER lumen completely. Once mislocalized, they are recognized by the Hsp70 chaperone BiP. In a detailed analysis for one of these proteins, the αβT cell receptor (αβTCR), we show that unassembled ER-lumenal subunits are rapidly degraded, whereas specific subunit interactions en route to the native receptor promote membrane integration of the less hydrophobic TM segments, thereby stabilizing the protein. For the TCR α chain, both complete ER import and subunit assembly depend on the same pivotal residue in its TM region. Thus, membrane integration linked to protein assembly allows cellular quality control of membrane proteins and connects the lumenal ER chaperone machinery to membrane protein biogenesis. PMID:23932713

  11. 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. PMID:16934796

  12. 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

  13. Purification and properties of rabbit spermatozoal acrosomal neuraminidase.

    PubMed Central

    Srivastava, P N; Abou-Issa, H

    1977-01-01

    Treatment of rabbit spermatozoa with 50mM-MgCl2 removes the plasma and the outer acrosomal membranes. Subsequent treatment with the detergents Hyamine 2389 and Triton X-100 solubilizes spermatozoal neuraminidase bound to the inner acrosomal membrane. The enzyme was further purified by DEAE-cellulose, Sephadex G-150 and Bio-Gel P-300 column chromato. The enzyme showed a single major band, with the possibility of some minor contaminants, on disc-gel electrophoresis. It had a specific activity of 0.37 micronmal of sialic acid released/min per mg with purified boar Cowper's-gland mucin as the substrate. The enzyme had marked specificity for 2 leads to 6'-linked sialic acid in glycoproteins. The Km of spermatozoal neuraminidase was 1.72 X 10(-6)M with Cowper's-gland mucin, 1.17 X 10(-5)M with fetuin and 8.8 X 10(-4)M with sialyl-lactose as a substrates. The Vmax. was 0.112 micronmol/min per mg with the Cowper's-gland mucin, 0.071 micronmol/min per mg with fetuin and 0.033 micronmol/min per mg with sialyl-lactose as substrate. The enzyme hydrolysed sheep submaxillary-gland mucin as readily as the Cowper's-gland mucin. The optimum of enzyme activity was at pH 5.0 on the Cowper's-gland mucin and at pH4.3 on sialyl-lactose. The enzyme activity was unaffected by 20mM-Na+ and-K+, but was inhibited by 20mM-Ca2+,-Mn2+,-Co2+ and -Cu2+. The enzyme was unstable in dilute solutions, but could be stored indefinitely freeze-dried at --20 degrees C. Images PLATE 1 PMID:66917

  14. 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

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

    PubMed

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

    2016-05-01

    An integrated system was developed for directly processing living cells into peptides of membrane proteins. Living cells were directly injected into the system and cracked in a capillary column by ultrasonic treatment. Owing to hydrophilicity for broken pieces of the cell membrane, the obtained membranes were retained in a well-designed bi-filter. While cytoplasm proteins were eluted from the bi-filter, the membranes were dissolved and protein released by flushing 4 % SDS buffer through the bi-filter. The membrane proteins were subsequently transferred into a micro-reactor and covalently bound in the reactor for purification and digestion. As the system greatly simplified the whole pretreatment processes and minimized both sample loss and contamination, it could be used to analyze the membrane proteome samples of thousand-cell-scales with acceptable reliability and stability. We totally identified 1348 proteins from 5000 HepG2 cells, 615 of which were annotated as membrane proteins. In contrast, with conventional method, only 233 membrane proteins were identified. It is adequately demonstrated that the integrated system shows promising practicability for the membrane proteome analysis of small amount of cells. Graphical Abstract The legend of online abstract figure is (a) schematic illustration of membrane proteins extraction, purification and digestion from living cells; (b) diagrammatic sketch of the automatic integrated membrane proteome analysis system. PMID:26922343

  16. 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

  17. Effects of extender and equilibration time on post-thaw motility and membrane integrity of cryopreserved Gyr bull semen evaluated by CASA and flow cytometry.

    PubMed

    Leite, Ticiano Guimarães; do Vale Filho, Vicente Ribeiro; de Arruda, Rubens Paes; de Andrade, André Furugen Cesar; Emerick, Lucas Luz; Zaffalon, Fabiane Gilli; Martins, Jorge André Matias; de Andrade, Venício José

    2010-07-01

    The objectives of the present study were to investigate the effects of three equilibration times (0, 2, and 4h) and two extenders (TRIS or Bioxcell) for cryopreservation of bull semen. Semen from 12 Gyr bulls was cryopreserved using an automated freezing machine. There were significant interactions between equilibration times and extenders for sperm motility and membrane integrity. The control treatment (0h equilibration) had the lowest values (P<0.05) for total (MOT) and progressive motilities (PROG), and percentage of sperm with intact plasma and acrosomal membranes (IPIA), with no significant differences between extenders. Extender TRIS had greater cryoprotective action than Bioxcell, with greater MOT, PROG, IPIA at 2 and 4h, as well as the lowest proportion of damaged plasma membrane (DPM, 72.2% vs. 85.8%) for all times. Equilibration for 4h yielded the most desirable (P<0.05) for MOT, PROG, and IPIA, and the least DPM percentage (86.5, 78.0, and 72.6% for 0, 2, and 4h, respectively). Overall, the combination of TRIS and 4h of equilibration was the most desirable semen cryopreservation method, with greatest MOT, PROG, and IPIA (TRIS-T4=26.8%; BIO-T4=18.3%) and the least DPM. In conclusion, based on objective analyses, equilibration during cryopreservation was essential for maintaining motility and integrity of sperm membranes; equilibration for 4h yielded the greatest sperm survival, independent of the extender used. PMID:20434857

  18. 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

  19. 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.

  20. Virus removal and integrity in aged RO membranes.

    PubMed

    Pype, Marie-Laure; Donose, Bogdan C; Martí, Llucia; Patureau, Dominique; Wery, Nathalie; Gernjak, Wolfgang

    2016-03-01

    Membrane ageing reduces the quality of the filtered water. Therefore, in order to warrant public health, monitoring membrane performances are of utmost importance. Reverse osmosis (RO) membranes are generally used to remove viruses and salt. However, there is no detailed study demonstrating the impact of aged membrane on the rejection of viruses and of membrane integrity indicators. In this paper, the impact of hypochlorite induced RO ageing on the rejection of a virus surrogate (MS2 phage) and four membrane integrity indicators (salt, dissolved organic matter, rhodamine WT and sulphate) was evaluated. Hypochlorite exposure was either active (under filtration) or passive (soaking), and the changes of the membrane surface chemistry were characterised using several autopsy techniques. Under this accelerated ageing condition, the introduction of chlorine in the membrane chemistry and the breakage of amide bonds caused an increase of the water permeability and a decrease of the virus surrogate's and indicators' rejection. Ageing resulted in a more negatively charged membrane and also in a higher hydrophobicity, which lead to the adsorption of MS2 phage. Despite severe physical membrane damage leading to a reduction of salt rejection to 1.2 log (94%), the minimum rejection of MS2 phage stayed on or above 4 log. PMID:26724450

  1. 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

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

    NASA Astrophysics Data System (ADS)

    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

  3. 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

  4. 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}.

  5. Egg signals for triggering the acrosome reaction in starfish spermatozoa.

    PubMed

    Hoshi, M; Amano, T; Okita, Y; Okinaga, T; Matsui, T

    1990-01-01

    Upon encountering the jelly coat of an egg, starfish spermatozoa undergo the acrosome reaction. To induce the acrosome reaction, 3 jelly components act in concert on the spermatozoa: a sulphated glycoprotein named acrosome reaction-inducing substance (ARIS), a group of steroidal saponins named Co-ARIS, and an oligopeptide presumably having an ability to increase the intracellular pH of the spermatozoon. All three are required to mimic the full ability of jelly coat to induce the acrosome reaction instantaneously. A combination of ARIS and Co-ARIS is enough for induction in normal sea water, although its action is almost 2 min slower than the jelly. ARIS can induce the acrosome reaction by itself in high Ca2+ or high pH sea water. When spermatozoa meet the jelly coat, the acrosome reaction is eventually induced because ARIS and Co-ARIS co-operatively increase the intracellular Ca2+ by stimulating verapamil- and maitotoxin-sensitive Ca2+ channels and the oligopeptide increases the intracellular pH by stimulating Na+/H+ exchange systems. PMID:2077126

  6. MALDI Tissue Profiling of Integral Membrane Proteins from Ocular Tissues

    PubMed Central

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

    2008-01-01

    MALDI tissue profiling and imaging have become valuable tools for rapid, direct analysis of tissues to investigate spatial distributions of proteins, potentially leading to an enhanced understanding of the molecular basis of disease. Sample preparation methods developed to date for these techniques produce protein expression profiles from predominantly hydrophilic, soluble proteins. The ability to obtain information about the spatial distribution of integral membrane proteins is critical to more fully understand their role in physiological processes, including transport, adhesion, and signaling. In this communication, a sample preparation method for direct tissue profiling of integral membrane proteins is presented. Spatially resolved profiles for the abundant lens membrane proteins aquaporin 0 (AQP0) and MP20, and the retinal membrane protein opsin, were obtained using this method. MALDI tissue profiling results were validated by analysis of dissected tissue prepared by traditional membrane protein processing methods. Furthermore, direct tissue profiling of lens membrane proteins revealed aged related post-translational modifications, as well as a novel modification that had not been detected using conventional tissue homogenization methods. PMID:18396059

  7. 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.

  8. Photonic integration in indium-phosphide membranes on silicon (IMOS)

    NASA Astrophysics Data System (ADS)

    van der Tol, Jos; Pello, Josselin; Bhat, Shrivatsa; Jiao, Yuqing; Heiss, Dominik; Roelkens, Gunther; Ambrosius, Huub; Smit, Meint

    2014-03-01

    A new photonic integration technique is presented, based on the use of an indium phosphide membrane on top of a silicon chip. This can provide electronic chips (CMOS) with an added optical layer (IMOS) for resolving the communication bottleneck. A major advantage of InP is the possibility to integrate passive and active components (SOAs, lasers) in a single membrane. In this paper we describe progress achieved in both the passive and active components. For the passive part of the circuit we succeeded to bring the propagation loss of our circuits close to the values obtained with silicon; we achieved propagation loss as low as 3.3 dB/cm through optimization of the lithography and the introduction of C60 (fullerene) in an electro resist. Further we report the smallest polarisation converter reported for membrane waveguides ( <10 μm) with low-loss (< 1 dB from 1520- 1550 nm), > 95% polarisation conversion efficiency over the whole C-band and tolerant fabrication. We also demonstrate an InP-membrane wavelength demultiplexer with a loss of 2.8 dB, a crosstalk level of better than 18 dB and a uniformity over the 8 channels of better than 1.2 dB. For the integration of active components we are testing a twin guide integration scheme. We present our design based on optical and electrical simulations and the fabrication techniques.

  9. 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

  10. 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

  11. 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

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

    PubMed

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

    2011-07-15

    Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50-79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = -3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = -11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes. PMID:21496902

  13. 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

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

    PubMed

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

    1988-06-01

    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. PMID:3380805

  15. A new integrated membrane filtration and chromatographic device.

    PubMed

    Xu, Yanke; Sirkar, Kamalesh K; Dai, Xiao-Ping; Luo, Robert G

    2005-01-01

    To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also 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; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration. PMID:15801803

  16. Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor

    PubMed Central

    Bono, Petri; Rubin, Kristofer; Higgins, Jonathan M. G.; Hynes, Richard O.

    2001-01-01

    The actin cytoskeleton plays a significant role in changes of cell shape and motility, and interactions between the actin filaments and the cell membrane are crucial for a variety of cellular processes. Several adaptor proteins, including talin, maintain the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Layilin, a recently characterized transmembrane protein with homology to C-type lectins, is a membrane-binding site for talin in peripheral ruffles of spreading cells. To facilitate studies of layilin's function, we have generated a layilin-Fc fusion protein comprising the extracellular part of layilin joined to human immunoglobulin G heavy chain and used this chimera to identify layilin ligands. Here, we demonstrate that layilin-Fc fusion protein binds to hyaluronan immobilized to Sepharose. Microtiter plate-binding assays, coprecipitation experiments, and staining of sections predigested with different glycosaminoglycan-degrading enzymes and cell adhesion assays all revealed that layilin binds specifically to hyaluronan but not to other tested glycosaminoglycans. Layilin's ability to bind hyaluronan, a ubiquitous extracellular matrix component, reveals an interesting parallel between layilin and CD44, because both can bind to cytoskeleton-membrane linker proteins through their cytoplasmic domains and to hyaluronan through their extracellular domains. This parallelism suggests a role for layilin in cell adhesion and motility. PMID:11294894

  17. 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

  18. Protein involvement in the fusion between the equatorial segment of acrosome-reacted human spermatozoa and liposomes.

    PubMed Central

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

    1997-01-01

    Artificial membranes (liposomes) can interact with the equatorial segment (ES) of human spermatozoa, provided that the acrosome reaction (AR) has occurred [Arts, Kuiken, Jager and Hoekstra (1993) Eur. J. Biochem. 217, 1001-1009]. Using fluorescently labelled liposomes, this interaction can be seen as either punctate fluorescence in the ES (lip-ESp), reflecting only bound liposomes, or as diffuse fluorescence in this region (lip-ESd), indicating that the liposomes have fused with the ES membrane. Only equatorial segments that still contain constituents of the acrosomal matrix have the capacity to bind liposomes and eventually to fuse with them. Since the exposure of such intact equatorial segments is the exclusive result of induction of the AR under physiological conditions, these results imply that liposomes can be used for the rapid detection of acrosome-reacted spermatozoa. The lip-ESp and lip-ESd patterns were shown to be reflections of two distinct properties of the ES. Proteolytic treatment after AR completely inhibited the formation of a lip-ESd pattern, whereas formation of the lip-ESp pattern was only marginally inhibited by the proteolytic treatment. The same results were obtained using anti-sperm antibodies, which did not react with acrosome-intact spermatozoa. Proteolytic treatment of spermatozoa before AR induction had no effect on the fusion capacity of the ES after subsequent AR, which implies that the putative fusion protein is not accessible before AR. Thus fusion of liposomes with the ES of human spermatozoa is mediated by a sperm protein(s), whereas the lip-ESp pattern is not likely to represent the liposome-binding stage that precedes the fusion step. PMID:9224646

  19. Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins.

    PubMed

    Brandt, M E; Riley, B S; Radolf, J D; Norgard, M V

    1990-04-01

    The pathogenic spirochete Borrelia burgdorferi contains a set of integral membrane proteins which were selectively extracted into the detergent phase upon solubilization of intact B. burgdorferi with the nonionic detergent Triton X-114. Virtually all of these hydrophobic proteins were recognized by antibodies in pooled sera from patients with chronic Lyme arthritis, demonstrating that proteins partitioning into the detergent phase of Triton X-114 encompass the major B. burgdorferi immunogens. Furthermore, most of these immunogenic proteins, including the previously characterized OspA and OspB membrane antigens, could be biosynthetically labeled when B. burgdorferi was incubated in vitro with [3H]palmitate. The OspA and OspB antigens were radioimmunoprecipitated from [3H]palmitate-labeled detergent-phase proteins with monoclonal antibodies, and [3H]palmitate was recovered unaltered from these proteins after sequential alkaline and acid hydrolyses. The combined results provide formal confirmation that the major B. burgdorferi immunogens extracted by Triton X-114 are lipoproteins. The demonstration that B. burgdorferi integral membrane antigens are lipoproteins may explain the basis of their immunogenicity and may help to improve our understanding of the surface topology of B. burgdorferi. PMID:2318538

  20. 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.

  1. Solution NMR Structure of Membrane-Integral Diacylglycerol Kinase

    PubMed Central

    Van Horn, Wade D.; Kim, Hak-Jun; Ellis, Charles D.; Hadziselimovic, Arina; Sulistijo, Endah S.; Karra, Murthy D.; Tian, Changlin; Sönnichsen, Frank D.; Sanders, Charles R.

    2009-01-01

    Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer using solution NMR. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK’s three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK’s lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis. PMID:19556511

  2. Determining the Topology of Integral Membrane Peptides Using EPR Spectroscopy

    PubMed Central

    Inbaraj, Johnson J.; Cardon, Thomas B.; Laryukhin, Mikhail; Grosser, Stuart M.

    2008-01-01

    This paper reports on the development of a new structural biology technique for determining the membrane topology of an integral membrane protein inserted into magnetically aligned phospholipid bilayers (bicelles) using EPR spectroscopy. The nitroxide spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was attached to the pore-lining transmembrane domain (M2δ) of the nicotinic acetylcholine receptor (AChR) and incorporated into a bicelle. The corresponding EPR spectra revealed hyperfine splittings that were highly dependent on the macroscopic orientation of the bicelles with respect to the static magnetic field. The helical tilt of the peptide can be easily calculated using the hyperfine splittings gleaned from the orientational dependent EPR spectra. A helical tilt of 14° was calculated for the M2δ peptide with respect to the bilayer normal of the membrane, which agrees well with previous 15N solid-state NMR studies. The helical tilt of the peptide was verified by simulating the corresponding EPR spectra using the standardized MOMD approach. This new method is advantageous because: (1) bicelle samples are easy to prepare, (2) the helical tilt can be directly calculated from the orientational-dependent hyperfine splitting in the EPR spectra, and (3) EPR spectroscopy is approximately 1000 fold more sensitive than 15N solid-state NMR spectroscopy; thus, the helical tilt of an integral membrane peptide can be determined with only 100 μg of peptide. The helical tilt can be determined more accurately by placing TOAC spin labels at several positions with this technique. PMID:16848493

  3. 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

  4. 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

  5. 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

  6. Myosin Va Participates in Acrosomal Formation and Nuclear Morphogenesis during Spermatogenesis of Chinese Mitten Crab Eriocheir sinensis

    PubMed Central

    Sun, Xiao; He, Ying; Hou, Lin; Yang, Wan-Xi

    2010-01-01

    Background The Chinese mitten crab Eriocheir sinensis belongs to the Class Crustacea, Decapoda, Brachyura. The spermatozoon of this species is of aflagellated type, it has a spherical acrosome surrounded by the cup-shaped nucleus, which are unique to brachyurans. For the past several decades, studies on the spermatogenesis of the mitten crab mainly focus on the morphology. Compared with the extensive study of molecular mechanism of spermatogenesis in mammals, relatively less information is available in crustacean species. Myosin Va, a member of Class V myosin, has been implicated in acrosome biogenesis and vesicle transport during spermatogenesis in mammals. In the present study we demonstrate the expression and cellular localization of myosin Va during spermatogenesis in E. sinensis. Methodology/Principal Findings Western blot demonstrated that myosin Va is expressed during spermatogenesis. Immunocytochemical and ultrastructural analyses showed that myosin Va mainly localizes in the cytoplasm in spermatocytes. At the early stage of spermiogenesis, myosin Va binds to the endoplasmic reticulum vesicle (EV) and proacrosomal granule (PG). Subsequently, myosin Va localizes within the proacrosomal vesicle (PV) formed by PG and EV fusion and locates in the membrane complex (MC) at the mid spermatid stage. At the late spermatid stage, myosin Va is associated with the shaping nucleus and mitochondria. In mature spermatozoon, myosin Va predominates in acrosomal tubule (AT) and nucleus. Conclusions/Significance Our study demonstrates that myosin Va may be involved in acrosome biogenesis and nuclear morphogenesis during spermatogenesis in E. sinensis. Considering the distribution and molecular characteristics of myosin Va, we also propose a hypothesis of AT formation in this species. It is the first time to uncover the role of myosin Va in crustacean spermatogenesis. PMID:20856877

  7. Expression and purification of integral membrane metallopeptidase HtpX.

    PubMed

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

    2014-07-01

    Little is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli. Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His8-tag, extracted from the membranes using octyl-β-d-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and size-exclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members. PMID:24769134

  8. 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

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

    NASA Astrophysics Data System (ADS)

    Li, Wenle; Walz, John Y.

    2014-03-01

    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.

  10. 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

  11. Comparative genomic analysis of integral membrane transport proteins in ciliates.

    PubMed

    Kumar, Ujjwal; Saier, Milton H

    2015-01-01

    Integral membrane transport proteins homologous to those found in the Transporter Classification Database (TCDB; www.tcdb.org) were identified and bioinformatically characterized by transporter class, family, and substrate specificity in three ciliates, Paramecium tetraurelia (Para), Tetrahymena thermophila (Tetra), and Ichthyophthirius multifiliis (Ich). In these three organisms, 1,326 of 39,600 proteins (3.4%), 1,017 of 24,800 proteins (4.2%), and 504 out of 8,100 proteins (6.2%) integral membrane transport proteins were identified, respectively. Thus, an inverse relationship was observed between the % transporters identified and the number of total proteins per genome reported. This surprising observation provides insight into the evolutionary process, giving rise to genome reduction following whole genome duplication (as in the case of Para) or during pathogenic association with a host organism (Ich). Of these transport proteins in Para and Tetra, about 41% were channels (more than any other type of organism studied), 31% were secondary carriers (fewer than most eukaryotes) and 26% were primary active transporters, mostly ATP-hydrolysis driven (more than most other eukaryotes). In Ich, the number of channels was selectively reduced by 66%, relative to Para and Tetra. Para has four times more inorganic anion transporters than Tetra, and Ich has nonselectively lost most of these. Tetra and Ich preferentially transport sugars and monocarboxylates while Para prefers di- and tricarboxylates. These observations serve to characterize the transport proteins of these related ciliates, providing insight into their nutrition and metabolism. PMID:25099884

  12. Evidence for multiple mechanisms for membrane binding and integration via carboxyl-terminal insertion sequences.

    PubMed

    Kim, P K; Janiak-Spens, F; Trimble, W S; Leber, B; Andrews, D W

    1997-07-22

    Subcellular localization of proteins with carboxyl-terminal insertion sequences requires the molecule be both targeted to and integrated into the correct membrane. The mechanism of membrane integration of cytochrome b5 has been shown to be promiscuous, spontaneous, nonsaturable, and independent of membrane proteins. Thus endoplasmic reticulum localization for cytochrome b5 depends primarily on accurate targeting to the appropriate membrane. Here direct comparison of this mechanism with that of three other proteins integrated into membranes via carboxyl-terminal insertion sequences [vesicle-associated membrane protein 1(Vamp1), polyomavirus middle-T antigen, and Bcl-2] revealed that, unlike cytochrome b5, membrane selectivity for these molecules is conferred at least in part by the mechanisms of membrane integration. Bcl-2 membrane integration was similar to that of cytochrome b5 except that insertion into lipid vesicles was inefficient. Unlike cytochrome b5 and Bcl-2, Vamp1 binding to canine pancreatic microsomes was saturable, ATP-dependent, and abolished by mild trypsin treatment of microsomes. Surprisingly, although the insertion sequence of polyomavirus middle-T antigen was sufficient to mediate electrostatic binding to membranes, binding did not lead to integration into the bilayer. Together these results demonstrate that there are at least two different mechanisms for correct membrane integration of proteins with insertion sequences, one mediated primarily by targeting and one relying on factors in the target membrane to mediate selective integration. Our results also demonstrate that, contrary to expectation, hydrophobicity is not sufficient for insertion sequence-mediated membrane integration. We suggest that the structure of the insertion sequence determines whether or not specific membrane-bound receptor proteins are required for membrane integration. PMID:9220974

  13. 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...

  14. Characterization of the sperm receptor for acrosome reaction-inducing substance of the starfish, Asterias amurensis.

    PubMed

    Kawamura, Mayu; Matsumoto, Midori; Hoshi, Motonori

    2002-04-01

    Acrosome reaction-inducing substance (ARIS) in the jelly coat of starfish eggs is a highly sulfated proteoglycan-like molecule of an apparent molecular size over 10(4) kDa and plays a pivotal role in the induction of acrosome reaction in homologous spermatozoa. It is known in Asterias amurensis that ARIS binds to a restricted area of the anterior portion of sperm head, and that a glycan fragment of ARIS, named Fragment 1, consisting of 10 repeats or so of a pentasaccharide unit retains the biological activity of ARIS to an appreciable extent. In this report, we have shown the binding of Fragment 1, a relatively small pure glycan fragment of ARIS, to the putative ARIS receptor on the sperm surface by three independent methods. First, the specific binding of P-ARIS to isolated sperm membranes was monitored in real-time by using a surface plasmon resonance detector, namely a Biacore sensor system. The specific and quantitative binding of Fragment 1 to the intact sperm and to isolated sperm membranes was similarly monitored. Secondly, the binding of 125I-labeled Fragment 1 to the intact sperm was stoichiometrically measured, for which we had developed a unique procedure for radioiodination of saccharide chains. It is found that Fragment 1 competes with P-ARIS for the binding to ARIS-receptor, suggesting that Fragment 1 is a useful ligand in the search for ARIS receptor protein(s). Thirdly, the putative receptor molecules were specifically labeled by using Fragment 1 as a ligand for photoaffinity crosslink technique. Taking these results into account, we conclude that starfish sperm have the ARIS receptor, which consists most probably of 50 to 60 kDa proteins, of reasonably high affinity (for Fragment 1, Kd = 15 microM, Bmax = 8.4 x 10(4) per cell). PMID:12130821

  15. 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.

  16. Identification of two integral membrane proteins of Plasmodium falciparum

    SciTech Connect

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

    1988-07-01

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

  17. Plant aquaporins: membrane channels with multiple integrated functions.

    PubMed

    Maurel, Christophe; Verdoucq, Lionel; Luu, Doan-Trung; Santoni, Véronique

    2008-01-01

    Aquaporins are channel proteins present in the plasma and intracellular membranes of plant cells, where they facilitate the transport of water and/or small neutral solutes (urea, boric acid, silicic acid) or gases (ammonia, carbon dioxide). Recent progress was made in understanding the molecular bases of aquaporin transport selectivity and gating. The present review examines how a wide range of selectivity profiles and regulation properties allows aquaporins to be integrated in numerous functions, throughout plant development, and during adaptations to variable living conditions. Although they play a central role in water relations of roots, leaves, seeds, and flowers, aquaporins have also been linked to plant mineral nutrition and carbon and nitrogen fixation. PMID:18444909

  18. Acrosome damage and enzyme leakage of goat spermatozoa during dilution, cooling and freezing.

    PubMed

    Chauhan, M S; Kapila, R; Gandhi, K K; Anand, S R

    1994-01-01

    Semen of Jamunapari goat bucks was frozen in three diluents egg yolk-tris, egg yolk/citrate/glucose, and skim milk/egg yolk. In fresh ejaculated semen over 90% of the spermatozoa had normal head and acrosome morphology. Quantification of goat sperm structure with Giemsa stain revealed significant (P < 0.01) damage to acrosome on freezing which varied between 38 to 43% in three diluents. Scanning electron microscopy defined and revealed greater damage during freezing with 50% sperm heads having normal acrosome structure in three diluents. The ultrastructural changes detected in frozen goat sperm was protrusion at the anterior cap, broken tail, swelling of acrosome, and loss of acrosomal contents. The leakage of five enzymes GOT, GPT, hyaluronoglucosaminidase, acid phosphatase, and alkaline phosphatase measured simultaneously revealed a positive correlation between enzyme release and acrosomal damage. PMID:8185056

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

    ERIC Educational Resources Information Center

    Ladner, David Allen

    2009-01-01

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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. Acrosome reaction-related steroidal saponin, Co-ARIS, from the starfish induces structural changes in microdomains.

    PubMed

    Naruse, Masahiro; Suetomo, Hiroyuki; Matsubara, Teruhiko; Sato, Toshinori; Yanagawa, Hiroshi; Hoshi, Motonori; Matsumoto, Midori

    2010-11-01

    Cofactor for acrosome reaction-inducing substance (Co-ARIS) is a steroidal saponin from the starfish Asterias amurensis. Saponins exist in many plants and few animals as self-defensive chemicals, but Co-ARIS has been identified as a cofactor for inducing the acrosome reaction (AR). In A. amurensis, the AR is induced by the cooperative action of egg coat components (ARIS, Co-ARIS, and asterosap); however, the mechanism of action of Co-ARIS is obscure. In this study we elucidated the membrane dynamics involved in the action of Co-ARIS. We found that cholesterol specifically inhibited the Co-ARIS activity for AR induction and detected the binding of labeled compounds with sperm using radioisotope-labeled Co-ARIS. Co-ARIS treatment did not reduce the content of sperm sterols, however, the condition was changed and localization of GM1 ganglioside on the periacrosomal region disappeared. We then developed a caveola-breaking assay, a novel method to detect the effect of chemicals on microdomains of culture cell, and confirmed the disturbance of somatic cell caveolae in the presence of Co-ARIS. Finally, by atomic force microscopy observations and surface plasmon resonance measurements using an artificial membrane, we revealed that Co-ARIS colocalized with GM1 clusters on the microdomains. Through this study, we revealed a capacitation-like event for AR in starfish sperm. PMID:20816679

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. On the mode of integration of the thylakoid membrane protein cytochrome b(6) into cytoplasmic membrane of Escherichia coli.

    PubMed

    Króliczewski, Jaroslaw; Gubernator, Beata; Rögner, Matthias; Szczepaniak, Andrzej

    2011-01-01

    In the stroma compartment, several pathways are used for integration/translocation of chloroplast proteins into or across the thylakoid membrane. In this study we investigated the mode of incorporation of the chloroplast-encoded cytochrome b(6) into the bacterial membrane. Cytochrome b(6) naturally comprises of four transmembrane helices (A,B,C,D) and contains two b-type hemes. In the present study, mature cytochrome b(6) or constructed deletion mutants of cytochrome were expressed in E. coli cells. The membrane insertion of cytochrome b(6) in this bacterial model system requires an artificially added presequence that directs the protein to use an E. coli membrane-insertion pathway. This could be accomplished by fusion to maltose-binding protein (MBP) or to the bacterial Sec-dependent signal peptide (SSpelB). The integration of mature cytochrome b(6) into the bacterial cytoplasmic membrane by the Sec pathway has been reported previously by our group (Kroliczewski et al., 2005, Biochemistry, 44: 7570). The results presented here show that cytochrome b(6) devoid of the first helix A can be inserted into the membrane, as can the entire ABCD. On the other hand, the construct devoid of helices A and B is translocated through the membrane into the periplasm without any effective insertion. This suggests the importance of the membrane-anchoring sequences that are likely to be present in only the A and B part, and it is consistent with the results of computational prediction which did not identify any membrane-anchoring sequences for the C or D helices. We also show that the incorporation of hemes into the truncated form of cytochrome b(6) is possible, as long as the B and D helices bearing axial ligands to heme are present. PMID:21725502

  11. A new integrated approach for dye removal from wastewater by polyoxometalates functionalized membranes.

    PubMed

    Yao, Lei; Zhang, Lizhi; Wang, Rong; Chou, Shuren; Dong, ZhiLi

    2016-01-15

    Membrane technique is a promising way for the removal of dyes from wastewater. A unique approach combining both the adsorptive and the catalytic membrane processes was proposed on the basis of a new functionalized membrane. The membrane integrating both the adsorptive and catalytic activities was developed by introducing polyoxometalates (POMs) as an ideal candidate for the membrane functionalization via a novel sol-gel method. A two-step protocol, adsorptive separation and catalytic degradation, was designed for dye removal, realizing an excellent dye rejection with easy and economic membrane regeneration through simply soaking the membrane in a limited volume of dilute oxidant solution. This approach is feasible and versatile owing to the flexible selection of distinct POMs and design of catalytic degradation routes as required. As a result, the current research provides insight into a new methodology of the membrane technique in dye removal applications. PMID:26410275

  12. 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

  13. Fragmentation of Integral Membrane Proteins in the Gas Phase

    PubMed Central

    2015-01-01

    Integral membrane proteins (IMPs) are of great biophysical and clinical interest because of the key role they play in many cellular processes. Here, a comprehensive top down study of 152 IMPs and 277 soluble proteins from human H1299 cells including 11 087 fragments obtained from collisionally activated dissociation (CAD), 6452 from higher-energy collisional dissociation (HCD), and 2981 from electron transfer dissociation (ETD) shows their great utility and complementarity for the identification and characterization of IMPs. A central finding is that ETD is ∼2-fold more likely to cleave in soluble regions than threshold fragmentation methods, whereas the reverse is observed in transmembrane domains with an observed ∼4-fold bias toward CAD and HCD. The location of charges just prior to dissociation is consistent with this directed fragmentation: protons remain localized on basic residues during ETD but easily mobilize along the backbone during collisional activation. The fragmentation driven by these protons, which is most often observed in transmembrane domains, both is of higher yield and occurs over a greater number of backbone cleavage sites. Further, while threshold dissociation events in transmembrane domains are on average 10.1 (CAD) and 9.2 (HCD) residues distant from the nearest charge site (R, K, H, N-terminus), fragmentation is strongly influenced by the N- or C-terminal position relative to that site: the ratio of observed b- to y-fragments is ∼1:3 if the cleavage occurs >7 residues N-terminal and ∼3:1 if it occurs >7 residues C-terminal to the nearest basic site. Threshold dissociation products driven by a mobilized proton appear to be strongly dependent on not only relative position of a charge site but also N- or C-terminal directionality of proton movement. PMID:24689519

  14. 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

  15. Enrichment of Integral Membrane Proteins for Proteomic Analysis Using Liquid Chromatography-Tandem Mass Spectrometry

    SciTech Connect

    Blonder, Josip; Goshe, Michael B.; Moore, Ronald J.; Pasa-Tolic, Liljiana; Masselon, Christophe D.; Lipton, Mary S.; Smith, Richard D.

    2002-04-01

    Currently, most proteomic studies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents, chaotropes, or reducing reagents that often interfere with electrospray ionization (ESI). To increase the identification of integral membrane proteins by LC-ESI-MS/MS, a sample preparation method combining carbonate extraction and surfactant-free organics solvent-assisted solubilization and proteolysis was developed and used to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic based on their positive grand average of hydropathicity values that covers 15% of the theoretical hydrophobic proteome. Using the PSORT algorithm, 268 identified proteins were recognized as integral membrane proteins covering 21% and 43% of the predicted integral cytoplasmic and outer membrane proteins, respectively. Of the integral cytoplasmic membrane proteins containing four or more predicted transmembrane domains (TMDs), 65% were identified by detecting at least one peptide spanning a TMD using LC-MS/MS. The extensive identification of highly hydrophobic proteins containing multiple TMDs confirms the efficacy of the described sample preparation protocol to isolate and solubilize integral membrane proteins and validates the method for large-scale analysis of bacterial membrane subproteomes using LC-ESI-MS/MS.

  16. Mechanisms of integral membrane protein insertion and folding

    PubMed Central

    2014-01-01

    The biogenesis, folding, and structure of α-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, and the transport of solutes and signals across membranes. Nearly all MPs require translocons—often referred to as protein-conducting channels—for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of membrane protein insertion and the folding of polytopic membrane proteins in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure, it is the driving force for the evolution of the ribosome-translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion. PMID:25277655

  17. Perspectives of solution NMR spectroscopy for structural and functional studies of integral membrane proteins

    NASA Astrophysics Data System (ADS)

    Reckel, Sina; Hiller, Sebastian

    2013-04-01

    This article discusses future perspectives of solution NMR spectroscopy to study structures and functions of integral membrane proteins at atomic resolution, based on a review of recent progress in this area. Several selected examples of structure determinations, as well as functional studies of integral membrane proteins are highlighted. We expect NMR spectroscopy to make future key scientific contributions to understanding membrane protein function, in particular for large membrane protein systems with known three-dimensional structure. Such situations can benefit from the fact that functional NMR studies have substantially less limitations by molecular size than a full de novo structure determination. Therefore, the general potential for NMR spectroscopy to solve biologic key questions associated with integral membrane proteins is very promising.

  18. 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

  19. Sequential focal and global elevations of sperm intracellular Ca2+ are initiated by the zona pellucida during acrosomal exocytosis.

    PubMed

    Florman, H M

    1994-09-01

    The acrosome reaction is a Cai(2+)-dependent secretory event that is initiated in mammalian sperm by ZP3, the stimulatory agonist in the egg's zona pellucida. Video image processing-enhanced fluorescence microscopy was used to monitor sperm Cai2+ responses to agonist during exocytosis. Two types of agonist-dependent Cai2+ transport pathways were defined. The first mediates small, transient Cai2+ elevations that are restricted to the sperm head. Dye emission and quenching studies indicate that this focal channel is not voltage-regulated, conducts several divalent metal cations (Co2+, Mn2+, and Ni2+) in addition to Ca2+, and has the properties of a poorly selective cation channel. The second transporter mediates sustained Cai2+ elevations throughout the cell and is pharmacologically identical to the L-type of voltage-sensitive Ca2+ channel. These channels are distinguished by inhibitor sensitivity and by their regulation during sperm maturation. A model is proposed in which ZP3 initiates a G protein-independent opening of the cation channel, producing depolarization of sperm membrane potential and consequent opening of L channels. The coordinate regulation of sperm Ca2+ channels by ZP3 during gamete adhesion promotes acrosomal exocytosis. PMID:8088433

  20. Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability.

    PubMed

    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

    2008-12-01

    Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca(2+)-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca(2+)-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

  1. Alteration of membrane integrity by delta1-tetrahydrocannabinol.

    PubMed

    Laurent, B; Roy, P E

    1975-07-01

    Delta1-tetrahydrocannabinol was found to be a potent inhibitor of some membrane-bound enzymes, such as Mg-ATPase, Na-K-ATPase and acetylcholinesterase. At a given concentration, the degree of inhibition varied for each enzyme; the inhibition was more pronounced for the enzymes that are parts of the membranes. As the kinetic parameters of these enzymes are functions of the membrane composition and organization, these parameters were studied in vitro in the presence of THC. Although the Mg-ATPase was inhibited by THC, there was no change in the allosteric behaviour of the enzyme, indicating that the alterations caused by THC did not affect the enzymatic structure. The Na-K-ATPase and acetylcholinesterase had a different allosteric behaviour as compared to controls; these modifications were like the alterations caused by the decrease in membrane fluidity. These results suggest the fact that THC is incorporated in the membranes and causes alterations in the physical organization of the membranes. PMID:126214

  2. 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...

  3. 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. PMID:26026232

  4. Spin chain from membrane and the Neumann-Rosochatius integrable system

    SciTech Connect

    Bozhilov, P.

    2007-11-15

    We find membrane configurations in AdS{sub 4}xS{sup 7}, which correspond to the continuous limit of the SU(2) integrable spin chain, considered as a limit of the SU(3) spin chain, arising in N=4 SYM in four dimensions, dual to strings in AdS{sub 5}xS{sup 5}. We also discuss the relationship with the Neumann-Rosochatius integrable system at the level of Lagrangians, comparing the string and membrane cases.

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

    PubMed

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

    2015-01-01

    Under pathological conditions such as brain trauma, subarachnoid hemorrhage and stroke, cortical spreading depression (CSD) or peri-infarct depolarizations contribute to brain damage in animal models of neurological disorders as well as in human neurological diseases. CSD causes transient megachannel opening on the neuronal membrane, which may compromise neuronal survival under pathological conditions. Poloxamer-188 (P-188) and citicoline are neuroprotectants with membrane sealing properties. The aim of this study is to investigate the effect of P-188 and citicoline on the neuronal megachannel opening induced by CSD in the mouse brain. We have monitored megachannel opening with propidium iodide, a membrane impermeable fluorescent dye and, demonstrate that P-188 and citicoline strikingly decreased CSD-induced neuronal PI influx in cortex and hippocampal dentate gyrus. Therefore, these agents may be providing neuroprotection by blocking megachannel opening, which may be related to their membrane sealing action and warrant further investigation for treatment of traumatic brain injury and ischemic stroke. PMID:25340256

  6. Integrated membrane systems for gas separation in biotechnology: potential and prospects.

    PubMed

    Teplyakov, V; Sostina, E; Beckman, I; Netrusov, A

    1996-09-01

    Integrated non-porous membrane systems were applied for microbial combustible gas separation processes. Methane/CO2 mixtures of various concentrations from methane fermentation processes (biogas) were separated using a membrane-separation complex of permabsorber type into individual components of technical grade (more than 95% purity). In experiments with three-component mixtures, using a selective membrane valve with various liquid carriers, all the gases of interest (H2, CH4 and CO2) were obtained at greater than 90% purity in one separation step. The perspectives for the further application of non-porous membrane separating devices for various gaseous mixtures from different microbial processes are discussed. PMID:24415378

  7. 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.

  8. 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.

  9. A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase.

    PubMed

    Hatzixanthis, Kostas; Palmer, Tracy; Sargent, Frank

    2003-09-01

    A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK 'twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or 'C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins. PMID:12940994

  10. Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile.

    PubMed

    van den Brink-van der Laan, Els; Killian, J Antoinette; de Kruijff, Ben

    2004-11-01

    Nonbilayer lipids can be defined as cone-shaped lipids with a preference for nonbilayer structures with a negative curvature, such as the hexagonal phase. All membranes contain these lipids in large amounts. Yet, the lipids in biological membranes are organized in a bilayer. This leads to the question: what is the physiological role of nonbilayer lipids? Different models are discussed in this review, with a focus on the lateral pressure profile within the membrane. Based on this lateral pressure model, predictions can be made for the effect of nonbilayer lipids on peripheral and integral membrane proteins. Recent data on the catalytic domain of Leader Peptidase and the potassium channel KcsA are discussed in relation to these predictions and in relation to the different models on the function of nonbilayer lipids. The data suggest a general mechanism for the interaction between nonbilayer lipids and membrane proteins via the membrane lateral pressure. PMID:15519321

  11. 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.

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

    PubMed Central

    Roche-Håkansson, Hazeline; Håkansson, Anders P.

    2014-01-01

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

  13. 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

  14. 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

  15. Successful Integration of Membrane Technologies in a Conventional Purification Process of Tannery Wastewater Streams

    PubMed Central

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

    2013-01-01

    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. PMID:24956941

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

    PubMed

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

    2012-09-01

    In the previous proteomic study of human placenta, transmembrane 7 superfamily member 1 (TM7SF1) was found enriched in lysosome compartments. TM7SF1 encodes a 399-amino acid protein with a calculated molecular mass of 45 kDa. Bioinformatic analysis of its amino acid sequence showed that it is a multipass transmembrane protein containing a potential dileucine-based lysosomal targeting signal and four putative N-glycosylation sites. By percoll-gradient centrifugation and further subfraction ways, the lysosomal solute and membrane compartments were isolated respectively. Immunoblotting analysis indicated that TM7SF1 was co-fractioned with lysosome associated membrane protein 2 (LAMP2), which was only detected in lysosomal membrane compartments whereas not detected in the solute compartments. Using specific anti-TM7SF1 antibody and double-immunofluorescence with lysosome membrane protein LAMP1 and Lyso-Tracker Red, the colocalisations of endogenous TM7SF1 with lysosome and late endosome markers were demonstrated. All of this indicated that TM7SF1 is an integral lysosome membrane protein. Rat ortholog of TM7SF1 was found to be strongly expressed in heart, liver, kidney and brain while not or low detected in other tissues. In summary, TM7SF1 was a lysosomal integral membrane protein that shows tissue-specific expression. As a G-protein-coupled receptor in lysosome membrane, TM7SF1 was predicted function as signal transduction across lysosome membrane. PMID:22729905

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

    PubMed Central

    Pal, Rahul; Hameed, Saif; Fatima, Zeeshan

    2015-01-01

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

  18. Beyond membrane integrity: Assessing the functionality of human umbilical vein endothelial cells after cryopreservation.

    PubMed

    Marquez-Curtis, Leah A; Sultani, A Billal; McGann, Locksley E; Elliott, Janet A W

    2016-06-01

    Assessment of cell membrane integrity is one of the most widely used methods to measure post-cryopreservation viability of cells such as human umbilical vein endothelial cells (HUVECs). However, an evaluation of cell function provides a better measure of cell quality following cryopreservation. The tube formation assay mimics angiogenesis in vitro and can be used to quantitate the ability of endothelial cells to form capillary-like tubular structures when cultured on reconstituted basement membrane (Matrigel). We compared the membrane integrity (measured by flow cytometry) and tube forming ability of HUVEC suspensions exposed to 10% dimethyl sulfoxide (Me2SO), cooled at 1 °C/min to various sub-zero temperatures, plunged directly into liquid nitrogen, stored for an hour, and thawed rapidly. We found that as membrane integrity increased so did the various parameters associated with the extent of in vitro angiogenesis; however, in comparison to fresh cells with a similar percentage of membrane-intact cells, the extent of tube formation, expressed as total tube length, is significantly lower in previously frozen cells for the lower range of post-thaw membrane integrities. Our findings underscore the value of an assay that quantifies a specific function that a cell is known to perform in vivo to measure the success of cryopreservation protocols. PMID:27182035

  19. 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.

  20. 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

  1. Atg7 is required for acrosome biogenesis during spermatogenesis in mice

    PubMed Central

    Wang, Hongna; Wan, Haifeng; Li, Xixia; Liu, Weixiao; Chen, Qi; Wang, Yaqing; Yang, Lin; Tang, Hongmei; Zhang, Xiujun; Duan, Enkui; Zhao, Xiaoyang; Gao, Fei; Li, Wei

    2014-01-01

    The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in the process of fertilization. The molecular mechanism underlying the biogenesis of this lysosome-related organelle (LRO) is still largely unknown. Here, we show that germ cell-specific Atg7-knockout mice were infertile due to a defect in acrosome biogenesis and displayed a phenotype similar to human globozoospermia; this reproductive defect was successfully rescued by intracytoplasmic sperm injections. Furthermore, the depletion of Atg7 in germ cells did not affect the early stages of development of germ cells, but at later stages of spermatogenesis, the proacrosomal vesicles failed to fuse into a single acrosomal vesicle during the Golgi phase, which finally resulted in irregular or nearly round-headed spermatozoa. Autophagic flux was disrupted in Atg7-depleted germ cells, finally leading to the failure of LC3 conjugation to Golgi apparatus-derived vesicles. In addition, Atg7 partially regulated another globozoospermia-related protein, Golgi-associated PDZ- and coiled-coil motif-containing protein (GOPC), during acrosome biogenesis. Finally, the injection of either autophagy or lysosome inhibitors into testis resulted in a similar phenotype to that of germ cell-specific Atg7-knockout mice. Altogether, our results uncover a new role for Atg7 in the biogenesis of the acrosome, and we provide evidence to support the autolysosome origination hypothesis for the acrosome. PMID:24853953

  2. 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.

  3. Selecting Optimum Eukaryotic Integral Membrane Proteins for Structure Determination by Rapid Expression and Solubilization Screening

    PubMed Central

    Li, Min; Hays, Franklin A.; Roe-Zurz, Zygy; Vuong, Linda; Kelly, Libusha; Ho, Chi-Min; Robbins, Renée M.; Pieper, Ursula; O’Connell, Joseph D.; Miercke, Larry J. W.; Giacomini, Kathleen M.; Sali, Andrej; Stroud, Robert M.

    2009-01-01

    A medium throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from the budding yeast Saccharomyces cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism’s membrane proteome. 272 targets were expressed and of these 234 solubilized in the detergent n-dodecyl-β-D-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of ten human integral membrane proteins from the solute carrier superfamily (SLC). This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others. PMID:19061901

  4. 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

  5. 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

  6. 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

  7. 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

  8. Seminal plasma arising from the whole boar sperm-rich fraction increases the stability of sperm membrane after thawing.

    PubMed

    Torres, M A; Ravagnani, G M; Leal, D F; Martins, S M M K; Muro, B B D; Meirelles, F V; Papa, F O; Dell'aqua, J A; Alvarenga, M A; Moretti, A S; De Andrade, A F C

    2016-05-01

    Boar spermatozoa arising from the sperm-rich ejaculate fraction are reported to have a more stable plasma membrane and are more resistant to cold shock and premature acrosome reaction than spermatozoa from the whole ejaculate. Furthermore, seminal plasma (SP) can increase the cryotolerance of boar spermatozoa, and in other domestic species, it has the ability to reverse cryopreservation damage. This study aimed to evaluate the effects of boar SP arising from the whole sperm-rich ejaculate fraction (SP-SRF) on the integrity, stability, and peroxidation of sperm membranes after thawing. Each ejaculate ( = 24) was divided among 4 treatments: control (CT), centrifuged and suspended in autologous SP-SRF (CS), centrifuged with withdrawn SP-SRF (CW), and post-thawed SP arising from the whole sperm-rich fraction addition to CW (CWSP). After thawing, all treatments were incubated for 5, 60, and 120 min and were analyzed for membrane integrity, fluidity, and peroxidation by flow cytometer. The absence of SP-SRF increased the lipid disorder ( < 0.05) but had no effect on lipid peroxidation ( > 0.05) or membrane integrity ( > 0.05). However, the increase in lipid disorder by withdrawal of SP-SRF was reversed by SP-SRF addition ( < 0.05) to the post-thawing medium, whereas plasma and acrosomal membrane integrity ( > 0.05) and lipid peroxidation ( > 0.05) were unchanged. In conclusion, despite the centrifugation effects, the addition of SP arising from the whole sperm-rich fraction to post-thawed boar semen decreased sperm lipid disorder without an influence of the sperm membrane integrity and peroxidation. PMID:27285688

  9. Cell Membrane Integrity in Myotonic Dystrophy Type 1: Implications for Therapy

    PubMed Central

    González-Barriga, Anchel; Kranzen, Julia; Croes, Huib J. E.; Bijl, Suzanne; van den Broek, Walther J. A. A.; van Kessel, Ingeborg D. G.; van Engelen, Baziel G. M.; van Deutekom, Judith C. T.; Wieringa, Bé; Mulders, Susan A. M.; Wansink, Derick G.

    2015-01-01

    Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy. PMID:25799359

  10. 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

  11. Model lipid bilayer with facile diffusion of lipids and integral membrane proteins.

    PubMed

    Wang, Tingting; Ingram, Colin; Weisshaar, James C

    2010-07-01

    A model membrane system is formed by the rupture of giant unilamellar vesicles (GUVs) onto a passivating layer comprising a PEG polymer cushion anchored in a lipid bilayer supported on glass. The novel use of pH-dependent electrostatic interactions between NeutrAvidin in the passivating layer and anionic lipids in the GUV drives vesicle rupture. The resulting "GUV pancakes" are single, planar lipid bilayer patches whose diameters vary from approximately 20 to 50 microm. The pancakes have several potential advantages for the in vitro study of protein-lipid interactions and integral membrane protein function. All components are commercially available. The pancakes resist nonspecific binding of vesicles containing protein. Both lipids and integral membrane proteins exhibit good lateral mobility in the GUV pancakes, as evidenced by single-particle tracking (SPT) of the DiD double-tailed fluorescent probe and of the integral membrane protein syntaxin-1A, labeled with AlexaFluor 633 (AF633-Syx). At least 80% of both probes exhibit free, homogeneous diffusion with a diffusion coefficient of approximately 5.5 microm(2) s(-1), which is more than 10 times faster than diffusion in a GUV pancake supported on bare glass. Atomic force microscopy (AFM) suggests that the polymer cushion has a height of approximately 4 nm. The mobility of a large fraction of the AF633-Syx probe suggests that even integral membrane proteins with large domains on both sides of the lipid bilayer should exhibit free diffusion within a GUV pancake. PMID:20459075

  12. Methodological factors affecting the results of staining frozen-thawed fertile and subfertile Japanese Black bull spermatozoa for acrosomal status.

    PubMed

    Almadaly, Essam; El-Kon, Ismail; Heleil, Bassiouni; Fattouh, El-Sayed; Mukoujima, Koushi; Ueda, Takuya; Hoshino, Youichirou; Takasu, Masaki; Murase, Tetsuma

    2012-12-01

    In the present study, some methodological factors affecting the acrosomal staining of frozen-thawed Japanese Black bull spermatozoa were investigated by examining; the effect of fixation/permeabilization procedure on intact acrosome percentage after fluorescein isothiocyanate peanut agglutinin (FITC-PNA) staining, the acrosomal staining patterns by using two types of fluorescent probes FITC-PSA (Pisum Sativum Agglutinin) and FITC-PNA and the effect of staining methods, either smear or vial, on intact acrosome percentage. Then intact acrosome percentage was compared between the samples stained by thus established method and those simply fixed with glutaraldehyde (glutaraldehyde fixation method). A possibility that FITC-PNA staining or the glutaraldehyde fixation methods could detect any difference in intact acrosome percentage or acrosomal staining patterns between fertile and subfertile bulls was also examined. The results showed that (1) 4% paraformaldehyde fixation plus 1% Triton X-100 permeabilization was better than absolute ethanol alone, (2) FITC-PNA acrosomal labeling was more specific than FITC-PSA, (3) sperm suspensions should be smeared and gently processed before acrosomal staining rather than spotted onto glass slides after staining in vial in order to avoid excessive mechanical damage of the sperm acrosome, and (4) staining spermatozoa with FITC-PNA had no major advantages over examination of simply glutaraldehyde fixed sperm samples and both failed to detect any significant difference in intact acrosome percentage between the fertile and the subfertile bulls used here. The present study demonstrates important methodological considerations which need to be taken into account in order to design a reliable and reproducible protocol for the study of the acrosome. PMID:23182469

  13. Imbalanced lipid homeostasis in the conditional Dicer1 knockout mouse epididymis causes instability of the sperm membrane.

    PubMed

    Björkgren, Ida; Gylling, Helena; Turunen, Heikki; Huhtaniemi, Ilpo; Strauss, Leena; Poutanen, Matti; Sipilä, Petra

    2015-02-01

    During epididymal sperm maturation, the lipid content of the sperm membrane is modified, which facilitates sperm motility and fertility. However, little is known about the mechanisms regulating the maturation process. By generating a conditional knockout (cKO) of Dicer1 in the proximal part of the mouse epididymis, we studied the role of RNA interference in epididymal functions. The Dicer1 cKO epididymis displayed an altered lipid homeostasis associated with a 0.6-fold reduction in the expression of the gene elongation of very long chain fatty acids-like 2, an enzyme needed for production of long-chain polyunsaturated fatty acids (PUFAs). Furthermore, the expression of several factors involved in cholesterol synthesis was up-regulated. Accordingly, the Dicer1 cKO sperm membrane showed a 0.7-fold decrease in long-chain PUFAs, whereas the amount of cholesterol in acrosome-reacted sperm displayed a 1.7-fold increase. The increased cholesterol:PUFA ratio of the sperm membrane caused breakage of the neck and acrosome region and immotility of sperm. Dicer1 cKO mice sperm also displayed reduced ability to bind to and fertilize the oocyte in vitro. This study thus shows that Dicer1 is critical for lipid synthesis in the epididymis, which directly affects sperm membrane integrity and male fertility. PMID:25366345

  14. Egg-jelly signal molecules for triggering the acrosome reaction in starfish spermatozoa.

    PubMed

    Hoshi, M; Nishigaki, T; Ushiyama, A; Okinaga, T; Chiba, K; Matsumoto, M

    1994-06-01

    It was in the early 1950s that J.C. Dan discovered the acrosome reaction in sea urchins, starfishes and several other marine invertebrates at Misaki Marine Biological Station on the Pacific coast of Japan. We now know that in many animals including mammals the acrosome reaction is an essential, and probably the most central, change in spermatozoa for fertilization. Starfish spermatozoa undergo the acrosome reaction upon encountering the jelly coat consisting of glycoproteins, steroid saponins, oligopeptides and inorganic components. To induce the acrosome reaction, three egg jelly components act in concert on the spermatozoa: a highly sulfated glycoprotein named acrosome reaction-inducing substance (ARIS), a group of sulfated steroidal saponins named Co-ARIS, and a group of glutamine-rich tetratriacontapeptides named sperm activating peptide (SAP). The action of ARIS is quite species-specific due to the specificity of ARIS-receptors in a restricted domain of the sperm surface and depends very much on sulfated saccharide chains. Co-ARIS is not much species-specific and its action depends on the sulfate group and steroid side chain. SAPs have a ring of 25 residues and increase the intracellular pH of spermatozoa. None of them can induce the acrosome reaction by itself in normal sea water, but ARIS does induce it in high Ca2+ or high pH sea water. Although a combination of ARIS and either Co-ARIS or SAP induces the acrosome reaction in normal sea water, all three are required to mimic the full activity of dissolved jelly coat. PMID:7981026

  15. 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

  16. Integral fill yarn insertion and beatup method using inflatable membrane

    NASA Technical Reports Server (NTRS)

    Farley, Gary L. (Inventor)

    1993-01-01

    An apparatus and method for integral fill yarn insertion and beatup are disclosed. A modified rapier contains a channel for holding fill yarn. The channel is covered with a flexible and inflatable boot, and an inflating apparatus for this boot is also attached. Fill yarn is inserted into the channel, and the rapier is extended into a shed formed by warp yarn. Next, the rapier is pushed into the fell of the fabric, and the flexible and inflatable cover inflated, which both pushes the yarn into the fell of the fabric and performs beatup. The rapier is withdrawn and the shed closed to complete one step of the weaving process.

  17. 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. PMID:26512807

  18. Guanylyl cyclase and cGMP-specific phosphodiesterase participate in the acrosome reaction of starfish sperm.

    PubMed

    Kawase, Osamu; Ueno, Seiichi; Minakata, Hiroyuki; Hoshi, Motonori; Matsumoto, Midori

    2004-11-01

    In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, i.e. ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for inducing the acrosome reaction. Experimentally, ARIS and asterosap are sufficient for the induction. However, when sperm are treated only with asterosap, they become unresponsive to the egg jelly to undergo the reaction. In this study, we analysed the mechanism of the acrosome reaction, using sperm inactivation by asterosap as a clue. Asterosap causes a rapid and transient increase in intracellular cGMP through the activation of the asterosap receptor, a guanylyl cyclase, and causes an increase in intracellular Ca(2+). When sperm were pretreated with asterosap, the guanylyl cyclase seemed to be inactivated irreversibly by dephosphorylation. They were still responsive to ARIS but no longer to asterosap. However, in the presence of IBMX or zaprinast, inhibitors against phosphodiesterases (PDEs), they retained their capacity to undergo the acrosome reaction in response to the egg jelly or ARIS alone. IBMX and zaprinast suppressed the intracellular catabolism of cGMP, but not of cAMP. These results suggest that guanylyl cyclase and cGMP-specific, IBMX- and zaprinast-susceptible PDEs are involved in the regulation of the acrosome reaction. PMID:15751545

  19. Cryopreservation affects bovine sperm intracellular parameters associated with capacitation and acrosome exocytosis.

    PubMed

    Pons-Rejraji, Hanae; Bailey, Janice L; Leclerc, Pierre

    2009-01-01

    Although semen cryopreservation is widely and commonly used in the bovine breeding industry, half the spermatozoa do not survive and most of those that do survive undergo numerous physiological changes that affect their fertilising ability. The aim of the present study was to determine how cryopreservation affects the intracellular events involved in sperm capacitation and acrosome reaction. Immediately after thawing and washing, almost 50% of spermatozoa were capacitated and more than 20% had lost their acrosome. The sperm cAMP concentration was lower than that in freshly ejaculated spermatozoa, but the cytosolic pH (pHcyt) was in the expected range. The free cytosolic Ca2+ concentration ([Ca2+]cyt) was higher than in fresh spermatozoa and cryopreserved spermatozoa had internally stored Ca2+. Phenylarsine oxide increased pHcyt and both cytosolic and stored Ca2+ concentrations, whereas orthovanadate enhanced acrosome loss and protein tyrosine phosphorylation (P-Tyr). Heparin increased the percentage of spermatozoa expressing the B (capacitated) chlortetracycline binding pattern, pHcyt, P-Tyr and Ca2+ storage. Moreover, positive correlations exist between capacitation, cAMP, P-Tyr and stored Ca2+, whereas the acrosome reaction is positively correlated with pHcyt and [Ca2+]cyt. These results demonstrate that sperm regulatory mechanisms may be affected by the cryopreservation procedure, but frozen-thawed sperm can still regulate their capacitation and acrosome reaction signalling pathways. PMID:19383259

  20. Prediction of Multi-Type Membrane Proteins in Human by an Integrated Approach

    PubMed Central

    Chen, Lei; Zhang, Ning; Huang, Tao; Cai, Yu-Dong

    2014-01-01

    Membrane proteins were found to be involved in various cellular processes performing various important functions, which are mainly associated to their types. However, it is very time-consuming and expensive for traditional biophysical methods to identify membrane protein types. Although some computational tools predicting membrane protein types have been developed, most of them can only recognize one kind of type. Therefore, they are not as effective as one membrane protein can have several types at the same time. To our knowledge, few methods handling multiple types of membrane proteins were reported. In this study, we proposed an integrated approach to predict multiple types of membrane proteins by employing sequence homology and protein-protein interaction network. As a result, the prediction accuracies reached 87.65%, 81.39% and 70.79%, respectively, by the leave-one-out test on three datasets. It outperformed the nearest neighbor algorithm adopting pseudo amino acid composition. The method is anticipated to be an alternative tool for identifying membrane protein types. New metrics for evaluating performances of methods dealing with multi-label problems were also presented. The program of the method is available upon request. PMID:24676214

  1. 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. PMID:27337501

  2. 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

  3. Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination.

    PubMed

    Boland, Coilín; Li, Dianfan; Shah, Syed Tasadaque Ali; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank; Caffrey, Martin

    2014-12-01

    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 three-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 lipid 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 has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) 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

  4. 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.

  5. An Integrated Balanced Superconductor-Insulator-Superconductor Heterodyne Mixer on a Silicon Membrane

    NASA Astrophysics Data System (ADS)

    Westig, M. P.; Jacobs, K.; Schultz, M.; Justen, M.; Stutzki, J.; Honingh, C. E.

    2011-03-01

    We have designed and fabricated a 380-520 GHz integrated balanced NbAlAlOxNb superconductor-insulator-superconductor (SIS) heterodyne waveguide mixer for submillimeter astrophysics. The response of the mixer measured with a Fourier transform spectrometer shows excellent agreement with the design. The novelty of our device is that we deposit the complete superconducting mixer circuit (tapered slotline antennas, hybrid coupler, MIM capacitors, SIS junctions, tuning circuits and blocking filters) on top of a 9 μ m silicon membrane. The membrane is held suspended in a waveguide by 2.5 μ m thick gold plated beamleads. We will show that silicon membrane technology and a thorough device design render the integration of SIS devices with larger circuits feasible. This is an important step towards large arrays of mixers. When using an appropriate superconductor technology, these devices are scalable to higher frequencies. We will present the design, fabrication results and first results of heterodyne measurements.

  6. Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity.

    PubMed

    Royce, Liam A; Yoon, Jong Moon; Chen, Yingxi; Rickenbach, Emily; Shanks, Jacqueline V; Jarboe, Laura R

    2015-05-01

    Carboxylic acids are an attractive biorenewable chemical, but as with many biorenewables, their toxicity to microbial biocatalysts limits their fermentative production. While it is generally accepted that membrane damage is the main mechanism of fatty acid toxicity, previous metabolic engineering efforts that increased membrane integrity did not enable increased carboxylic acid production. Here we used an evolutionary approach to improve tolerance to exogenous octanoic acid, with the goal of learning design strategies from this evolved strain. This evolution of an Escherichia coli MG1655 derivative at neutral pH in minimal media produced a strain with increased tolerance not only to octanoic acid, but also to hexanoic acid, decanoic acid, n-butanol and isobutanol. This evolved strain also produced carboxylic acids at a 5-fold higher titer than its parent strain when expressing the Anaerococcus tetradius thioesterase. While it has been previously suggested that intracellular acidification may contribute to carboxylic acid toxicity, we saw no evidence that the evolved strain has increased resistance to this acidification. Characterization of the evolved strain membrane showed that it had significantly altered membrane polarization (fluidity), integrity (leakage) and composition relative to its parent. The changes in membrane composition included a significant increase in average lipid length in a variety of growth conditions, including 30°C, 42°C, carboxylic acid challenge and ethanol challenge. The evolved strain has a more dynamic membrane composition, showing both a larger number of significant changes and larger fold changes in the relative abundance of membrane lipids. These results highlight the importance of the cell membrane in increasing microbial tolerance and production of biorenewable fuels and chemicals. PMID:25839166

  7. INTEGRATION OF FILTRATION AND ADVANCED OXIDATION: DEVELOPMENT OF A MEMBRANE LIQUID-PHASE PLASMA REACTOR

    EPA Science Inventory

    A tiered approach will be undertaken to achieve the overall project goal of demonstrating the integrated membrane/plasma process as an innovative, affordable, sustainable and effective treatment technology for small treatment systems. The team will first use a regimented ap...

  8. Proenkephalin products are stored in the sperm acrosome and may function in fertilization.

    PubMed Central

    Kew, D; Muffly, K E; Kilpatrick, D L

    1990-01-01

    Previous studies have shown that spermatogenic cells are a major source of testicular RNA encoding the opioid peptide precursor proenkephalin, suggesting that proenkephalin-derived peptides may function as intratesticular paracrine factors produced by male germ cells. However, direct evidence for the production of proenkephalin by spermatogenic cells has been lacking. In this report, we have used polysome profile analysis, peptide quantitation, and immunocytochemistry to show that proenkephalin products are synthesized during spermatogenesis and are retained within spermatozoa of humans, hamsters, rats, and sheep. We further show that these peptides are stored in the sperm acrosome and are depleted from sperm following the acrosome reaction, an exocytotic event required for fertilization. Proenkephalin products thus may serve a dual function as sperm acrosomal factors released during the fertilization process as well as intratesticular regulators secreted by spermatogenic cells. Images PMID:1701253

  9. NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

    PubMed Central

    Arndt, Lilli; Castonguay, Jan; Arlt, Elisabeth; Meyer, Dorke; Hassan, Sami; Borth, Heike; Zierler, Susanna; Wennemuth, Gunther; Breit, Andreas; Biel, Martin; Wahl-Schott, Christian; Gudermann, Thomas; Klugbauer, Norbert; Boekhoff, Ingrid

    2014-01-01

    The functional relationship between the formation of hundreds of fusion pores during the acrosome reaction in spermatozoa and the mobilization of calcium from the acrosome has been determined only partially. Hence, the second messenger NAADP, promoting efflux of calcium from lysosome-like compartments and one of its potential molecular targets, the two-pore channel 1 (TPC1), were analyzed for its involvement in triggering the acrosome reaction using a TPCN1 gene–deficient mouse strain. The present study documents that TPC1 and NAADP-binding sites showed a colocalization at the acrosomal region and that treatment of spermatozoa with NAADP resulted in a loss of the acrosomal vesicle that showed typical properties described for TPCs: Registered responses were not detectable for its chemical analogue NADP and were blocked by the NAADP antagonist trans-Ned-19. In addition, two narrow bell-shaped dose-response curves were identified with maxima in either the nanomolar or low micromolar NAADP concentration range, where TPC1 was found to be responsible for activating the low affinity pathway. Our finding that two convergent NAADP-dependent pathways are operative in driving acrosomal exocytosis supports the concept that both NAADP-gated cascades match local NAADP concentrations with the efflux of acrosomal calcium, thereby ensuring complete fusion of the large acrosomal vesicle. PMID:24451262

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

    SciTech Connect

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

    1988-02-01

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

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

    PubMed

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

    2016-08-16

    Integration of solid-state nanopores and multichannel detection of signals from each nanopore are effective measures for realizing high-throughput nanopore sensors. In the present study, we demonstrated fabrication of Si3N4 membrane arrays and the simultaneous measurement of ionic currents through two nanopores formed in two adjacent membranes. Membranes with thicknesses as low as 6.4 nm and small nanopores with diameters of less than 2 nm could be fabricated using the poly-Si sacrificial-layer process and multilevel pulse-voltage injection. Using the fabricated nanopore membranes, we successfully achieved simultaneous detection of clear ionic-current blockades when single-stranded short homopolymers (poly(dA)60) passed through two nanopores. In addition, we investigated the signal crosstalk and leakage current among separated chambers. When two nanopores were isolated on the front surface of the membrane, there was no signal crosstalk or leakage current between the chambers. However, when two nanopores were isolated on the backside of the Si substrate, signal crosstalk and leakage current were observed owing to high-capacitance coupling between the chambers and electrolysis of water on the surface of the Si substrate. The signal crosstalk and leakage current could be suppressed by oxidizing the exposed Si surface in the membrane chip. Finally, the observed ionic-current blockade when poly(dA)60 passed through the nanopore in the oxidized chip was approximately half of that observed in the non-oxidized chip. PMID:27440476

  13. Egg jelly components responsible for histone degradation and acrosome reaction in the starfish, Asterina pectinifera.

    PubMed

    Amano, T; Okita, Y; Okinaga, T; Matsui, T; Nishiyama, I; Hoshi, M

    1992-08-31

    In the starfish, Asterina pectinifera, egg jelly induces the degradation of sperm histones as well as the acrosome reaction. We have isolated histone degradation-inducing components from the egg jelly. The histone degradation and the acrosome reaction are induced by a co-operative action of ARIS, which is an extremely large, sulfated glycoprotein with diffusible substance(s) in the jelly. Co-ARIS I, a steroidal saponin of the jelly, is effective to induce both reactions in the presence of ARIS. PMID:1520308

  14. 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.

  15. 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

  16. Research Resource: Monitoring Endoplasmic Reticulum Membrane Integrity in β-Cells at the Single-Cell Level

    PubMed Central

    Kanekura, Kohsuke; Ou, Jianhong; Hara, Takashi; Zhu, Lihua J.

    2015-01-01

    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. PMID:25584413

  17. Investigating the role of viral integral membrane proteins in promoting the assembly of nepovirus and comovirus replication factories

    PubMed Central

    Sanfaçon, Hélène

    2013-01-01

    Formation of plant virus membrane-associated replication factories requires the association of viral replication proteins and viral RNA with intracellular membranes, the recruitment of host factors and the modification of membranes to form novel structures that house the replication complex. Many viruses encode integral membrane proteins that act as anchors for the replication complex. These hydrophobic proteins contain transmembrane domains and/or amphipathic helices that associate with the membrane and modify its structure. The comovirus Co-Pro and NTP-binding (NTB, putative helicase) proteins and the cognate nepovirus X2 and NTB proteins are among the best characterized plant virus integral membrane replication proteins and are functionally related to the picornavirus 2B, 2C, and 3A membrane proteins. The identification of membrane association domains and analysis of the membrane topology of these proteins is discussed. The evidence suggesting that these proteins have the ability to induce membrane proliferation, alter the structure and integrity of intracellular membranes, and modulate the induction of symptoms in infected plants is also reviewed. Finally, areas of research that need further investigation are highlighted. PMID:23439982

  18. Unexplained in-vitro fertilization failure: implication of acrosomes with a small reacting region, as revealed by a monoclonal antibody.

    PubMed

    Albert, M; Gallo, J M; Escalier, D; Parseghian, N; Jouannet, P; Schrevel, J; David, G

    1992-10-01

    To determine the acrosomal characteristics related to in-vitro fertilization (IVF) outcome, spermatozoa from 50 men whose wives had resorted to IVF have been studied by indirect immunofluorescence microscopy with anti-human pro-acrosin monoclonal antibody 4D4 (mAb 4D4), prior to and after incubation in a capacitating medium. The antibody labelled only the acrosomal principal region (APR), revealing its shape (i.e. normal, small or amorphous) and its status (i.e. unreacted, partially or totally reacted). The IVF outcome distinguished: (i) spermatozoa which were able to fertilize at least one oocyte in vitro (group I; n = 25) and (ii) spermatozoa which failed to fertilize any oocyte in vitro (group II; n = 25). The semen characteristics of the two sperm groups, including the acrosome morphology, were similar according to conventional analysis. The mAb 4D4 detected in both the whole and the swim-up sperm cell fractions a lower percentage of normal APR in group II (< 50% for 10 patients in group II versus one patient from group I), which was related to a higher percentage of small APR. Moreover, after 21 h incubation, group II had a lower acrosomal loss index. The spermatozoa of five patients of this infertile group II did not undergo acrosomal modification whereas spermatozoa of all group I patients underwent the acrosomal reaction. The data showed that the relationship between acrosomal anomalies and IVF failure is mainly due to an increased incidence of acrosomes with a reduced size of the region involved in the acrosome reaction. Immunodiagnosis of this acrosomal region by means of mAb 4D4 is informative for IVF outcome. PMID:1479007

  19. 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

  20. 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

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

    SciTech Connect

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

    1991-05-25

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

  2. 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.

  3. 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

  4. 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

  5. Implications of modifying membrane fatty acid composition on membrane oxidation, integrity, and storage viability of freeze-dried probiotic, Lactobacillus acidophilus La-5.

    PubMed

    Hansen, Marie-Louise R W; Petersen, Mikael A; Risbo, Jens; Hümmer, Magdalena; Clausen, Anders

    2015-01-01

    The aim of this study was to investigate the effect of altering the fatty acid profile of the lipid membrane on storage survival of freeze-dried probiotic, Lactobacillus acidophilus La-5, as well as study the membrane integrity and lipid oxidation. The fatty acid composition of the lipid membrane of L. acidophilus La-5 was significantly different upon growth in MRS (containing Tween 80, an oleic acid source), or in MRS with Tween 20 (containing C12:0 and C14:0), linoleic, or linolenic acid supplemented. Bacteria grown in MRS showed the highest storage survival rates. No indications of loss of membrane integrity could be found, and membrane integrity could therefore not be connected with loss of viability. Survival of bacteria grown with linoleic or linolenic acid was more negatively affected by the presence of oxygen, than bacteria grown in MRS or with Tween 20 supplemented. A small, but significant, loss of linolenic acid during storage could be identified, and an increase of volatile secondary oxidation products during storage was found for bacteria grown in MRS, or with linoleic, or linolenic acid supplemented, but not for bacteria grown with Tween 20. Overall, the results indicate that lipid oxidation and loss of membrane integrity are not the only or most important detrimental reactions which can occur during storage. By altering the fatty acid composition, it was also found that properties of oleic acid gave rise to more robust bacteria than more saturated or unsaturated fatty acids did. PMID:25823709

  6. Accurate quantification of diffusion and binding kinetics of non-integral membrane proteins by FRAP.

    PubMed

    Berkovich, Ronen; Wolfenson, Haguy; Eisenberg, Sharon; Ehrlich, Marcelo; Weiss, Matthias; Klafter, Joseph; Henis, Yoav I; Urbakh, Michael

    2011-11-01

    Non-integral membrane proteins frequently act as transduction hubs in vital signaling pathways initiated at the plasma membrane (PM). Their biological activity depends on dynamic interactions with the PM, which are governed by their lateral and cytoplasmic diffusion and membrane binding/unbinding kinetics. Accurate quantification of the multiple kinetic parameters characterizing their membrane interaction dynamics has been challenging. Despite a fair number of approximate fitting functions for analyzing fluorescence recovery after photobleaching (FRAP) data, no approach was able to cope with the full diffusion-exchange problem. Here, we present an exact solution and matlab fitting programs for FRAP with a stationary Gaussian laser beam, allowing simultaneous determination of the membrane (un)binding rates and the diffusion coefficients. To reduce the number of fitting parameters, the cytoplasmic diffusion coefficient is determined separately. Notably, our equations include the dependence of the exchange kinetics on the distribution of the measured protein between the PM and the cytoplasm, enabling the derivation of both k(on) and k(off) without prior assumptions. After validating the fitting function by computer simulations, we confirm the applicability of our approach to live-cell data by monitoring the dynamics of GFP-N-Ras mutants under conditions with different contributions of lateral diffusion and exchange to the FRAP kinetics. PMID:21810156

  7. 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.

  8. Morphological alterations of all stages of spermatogenesis and acrosome reaction in Chinese mitten crab Eriocheir sinensis.

    PubMed

    Wang, Yuan-Li; Sun, Wen-Juan; He, Lin; Li, Qing; Wang, Qun

    2015-05-01

    The Chinese mitten crab (Eriocheir sinensis) is a seasonally breeding species and its reproductive system comprises paired symmetrical structures: testes, vasa deferentia, seminal vesicles, accessory glands and ejaculatory ducts. Histological examination of the testis of mature males reveals regression of the gonads and inhibition of the process of spermatogenesis during December to April of the following year, the regeneration of the gonads during June to July and the occurrence of the highest level of spermatogenesis during August to October. Microscopic assessments and hematoxylin and eosin (H&E) staining were used to describe all spermatogenic stages (spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoids). To observe the morphological changes during spermiogenesis, we successfully initiated primary cell culture using testis tissue of E. sinensis, which will lay a solid foundation for further work on the immortalization of crab cells. During the interaction between the sperm and oocyte, the fertilizing spermatozoon must undergo a series of terminal morphological changes, called the acrosome reaction (AR). This study also provides a detailed description of the structural alterations of the acrosome reaction of E. sinensis. The acrosome complex and cup-shaped nucleus are located at the anterior and posterior of the spermatozoon, respectively. Male germ cell development involves a tightly controlled sequence of differentiation switches. The purpose of this study is to increase our knowledge of the morphological alterations during spermatogenesis and the acrosome reaction, whose changes are a fundamental requirement for fertilization of E. sinensis. PMID:25648553

  9. Specific binding of acrosome-reaction-inducing substance to the head of starfish spermatozoa.

    PubMed

    Ushiyama, A; Araki, T; Chiba, K; Hoshi, M

    1993-05-01

    In the starfish, spermatozoa undergo the acrosome reaction upon encountering the jelly coat of eggs. A highly sulphated glycoprotein in the jelly coat is called acrosome-reaction-inducing substance (ARIS) because it is the key signal molecule to trigger the acrosome reaction. The activity of ARIS is mainly attributed to its sulphate and saccharide residues. The extremely large molecular size and species-specific action of ARIS suggest the presence of a specific ARIS receptor on the sperm surface, but no experimental evidence for the receptor has been presented. We therefore measured specific binding of ARIS and its pronase digest (P-ARIS), which retains the full activity of ARIS, to homologous spermatozoa by using fluorescein-isothiocyanate-labelled ARIS and 125I-labelled P-ARIS, respectively. The spermatozoa had the ability to bind ARIS, as well as P-ARIS, specifically. The binding was species-specific and mostly localised to the head region of spermatozoa. Scatchard plot analysis indicated the presence of one class of ARIS receptor on the surface of acrosome-intact spermatozoa. Furthermore, the specific binding of P-ARIS to the anterior region of sperm heads was microscopically confirmed by using P-ARIS conjugated to polystyrene latex beads with intense fluorescence. It is concluded that starfish spermatozoa have a specific receptor for ARIS on the surface of the anterior region of heads. PMID:8081808

  10. 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

  11. 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

  12. 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.

  13. Fabrication and characterization of an integrated ionic device from suspended polypyrrole and alamethicin-reconstituted lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

    Northcutt, Robert; Sundaresan, Vishnu-Baba

    2012-09-01

    Conducting polymers are electroactive materials that undergo conformal relaxation of the polymer backbone in the presence of an electrical field through ion exchange with solid or aqueous electrolytes. This conformal relaxation and the associated morphological changes make conducting polymers highly suitable for actuation and sensing applications. Among smart materials, bioderived active materials also use ion transport for sensing and actuation functions via selective ion transport. The transporter proteins extracted from biological cell membranes and reconstituted into a bilayer lipid membrane in bioderived active materials regulate ion transport for engineering functions. The protein transporter reconstituted in the bilayer lipid membrane is referred to as the bioderived membrane and serves as the active component in bioderived active materials. Inspired by the similarities in the physics of transduction in conducting polymers and bioderived active materials, an integrated ionic device is formed from the bioderived membrane and the conducting polymer membrane. This ionic device is fabricated into a laminated thin-film membrane and a common ion that can be processed by the bioderived and the conducting polymer membranes couple the ionic function of these two membranes. An integrated ionic device, fabricated from polypyrrole (PPy) doped with sodium dodecylbenzenesulfonate (NaDBS) and an alamethicin-reconstituted DPhPC bilayer lipid membrane, is presented in this paper. A voltage-gated sodium current regulates the electrochemical response in the PPy(DBS) layer. The integrated device is fabricated on silicon-based substrates through microfabrication, electropolymerization, and vesicle fusion, and ionic activity is characterized through electrochemical measurements.

  14. 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

  15. 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

  16. 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). PMID:26619942

  17. The Transition from Closed to Open Conformation of Treponema pallidum Outer Membrane-associated Lipoprotein TP0453 Involves Membrane Sensing and Integration by Two Amphipathic Helices*

    PubMed Central

    Luthra, Amit; Zhu, Guangyu; Desrosiers, Daniel C.; Eggers, Christian H.; Mulay, Vishwaroop; Anand, Arvind; McArthur, Fiona A.; Romano, Fabian B.; Caimano, Melissa J.; Heuck, Alejandro P.; Malkowski, Michael G.; Radolf, Justin D.

    2011-01-01

    The molecular architecture and composition of the outer membrane (OM) of Treponema pallidum (Tp), the noncultivable agent of venereal syphilis, differ considerably from those of typical Gram-negative bacteria. Several years ago we described TP0453, the only lipoprotein associated with the inner leaflet of the Tp OM. Whereas polypeptides of other treponemal lipoproteins are hydrophilic, non-lipidated TP0453 can integrate into membranes, a property attributed to its multiple amphipathic helices (AHs). Furthermore, membrane integration of the TP0453 polypeptide was found to increase membrane permeability, suggesting the molecule functions in a porin-like manner. To better understand the mechanism of membrane integration of TP0453 and its physiological role in Tp OM biogenesis, we solved its crystal structure and used mutagenesis to identify membrane insertion elements. The crystal structure of TP0453 consists of an α/β/α-fold and includes five stably folded AHs. In high concentrations of detergent, TP0453 transitions from a closed to open conformation by lateral movement of two groups of AHs, exposing a large hydrophobic cavity. Triton X-114 phase partitioning, liposome floatation assay, and bis-1-anilino-8-naphthalenesulfonate binding revealed that two adjacent AHs are critical for membrane sensing/integration. Using terbium-dipicolinic acid complex-loaded large unilamellar vesicles, we found that TP0453 increased efflux of fluorophore only at acidic pH. Gel filtration and cross-linking experiments demonstrated that one AH critical for membrane sensing/insertion also forms a dimeric interface. Based on structural dynamics and comparison with Mycobacterium tuberculosis lipoproteins LprG and LppX, we propose that TP0453 functions as a carrier of lipids, glycolipids, and/or derivatives during OM biogenesis. PMID:21965687

  18. 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

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

    PubMed

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

    2012-04-01

    This study was designed to evaluate the effect of various buffers on the fluorescence signal intensity of two fluorochromes (IP and CFDA) when used to assess the membrane integrity of ram sperm. Second ejaculates (18) from nine adult males were collected using an artificial vagina and diluted in either MOPS, TRIS, TES, HEPES, citrate, or phosphate-based extenders. Semen samples were stored at 15°C and the membrane integrity was assessed within the first 24 h of storage. Mean fluorescence intensity (FI) of PI- and CDFA-labeled sperm heads and fluorescence background noise (FBN) were determined quantitatively using Image J software. Fluorescence contrast (FC) was expressed as the difference between FI and FBN. Significantly, higher FI and FC were recorded when TRIS diluent was used, rather than the other diluents, both in the propidium- and fluorescein-labeled cells. The citrate and phosphate-based extenders showed intermediate results of FC between those of TRIS and zwitterionic (MOPS, TES and HEPES) groups for the PI-labeled sperm. However, in the CFDA-labeled sperm, the lower values of FC were obtained in the citrate and phosphate groups due to increased levels of FBN. For the membrane-damaged sperm, fluorescent labeling was limited to the sperm heads when TRIS-buffer was used, whereas in the other groups, the sperm tail was also frequently observed. It was concluded that TRIS buffer solution markedly increases the fluorescence yield of IP/CFDA-labeled sperm cells in the ram and that this should be considered when evaluating their membrane integrity. PMID:22553829

  20. 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

  1. 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

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

    PubMed

    Schlebach, Jonathan P; Narayan, Malathi; Alford, Catherine; Mittendorf, Kathleen F; Carter, Bruce D; Li, Jun; Sanders, Charles R

    2015-07-15

    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

  3. 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

  4. 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

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

    PubMed

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

    2007-10-01

    .2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing. PMID:17845604

  6. 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.

  7. Effect of antifungal agents on lipid biosynthesis and membrane integrity in Candida albicans.

    PubMed Central

    Georgopapadakou, N H; Dix, B A; Smith, S A; Freudenberger, J; Funke, P T

    1987-01-01

    Eight antifungal agents were examined for effects on lipid biosynthesis and membrane integrity in Candida albicans. Lipids were labeled in vivo or in vitro with [14C]acetate and analyzed by thin-layer and gas chromatography. Membrane integrity was measured by a recently developed [14C]aminoisobutyric acid radiolabel release assay. The imidazole antifungal agents miconazole, econazole, clotrimazole, and ketoconazole, at concentrations inhibiting ergosterol biosynthesis (0.1 microM), decreased the ratio of unsaturated to saturated fatty acids in vivo but not in vitro. Similarly, naftifine, tolnaftate, and the azasterol A25822B, at concentrations inhibiting ergosterol biosynthesis (10, 100, and 1 microM, respectively), decreased the ratio of unsaturated to saturated fatty acids in vivo only. This suggests that the effect on fatty acids observed with ergosterol biosynthesis inhibitors may be secondary to the effect on ergosterol. With imidazoles, oleic acid antagonized inhibition of cell growth but not inhibition of ergosterol. This suggests that, with the C-14 demethylase inhibitors, decreased unsaturated fatty acids, rather than decreased ergosterol, are responsible for growth inhibition. Cerulenin, previously reported to be a potent inhibitor of both fatty acid and ergosterol biosynthesis, was found in the present study to inhibit the former (at 5 microM) but not the latter (up to 100 microM). Of the antifungal agents tested, econazole and miconazole (at 100 microM) produced complete release of [14C]aminoisobutyric acid, which is consistent with membrane damage. PMID:3551826

  8. The ssrA-Tag Facilitated Degradation of an Integral Membrane Protein.

    PubMed

    Chai, Qian; Wang, Zhaoshuai; Webb, Stacy R; Dutch, Rebecca E; Wei, Yinan

    2016-04-26

    ATP-dependent degradation plays a critical role in the quality control and recycling of proteins in cells. However, complete degradation of membrane proteins by ATP-dependent proteases in bacteria is not well-studied. We discovered that the degradation of a multidomain and multispan integral membrane protein AcrB could be facilitated by the introduction of a ssrA-tag at the C-terminus of the protein sequence and demonstrated that the cytoplasmic unfoldase-protease complex ClpXP was involved in the degradation. This is the first report to our knowledge to reveal that the ClpXP complex is capable of degrading integral membrane proteins. The chaperone SspB also played a role in the degradation. Using purified proteins, we demonstrated that the addition of the ssrA-tag did not drastically affect the structure of AcrB, and the degradation of detergent solubilized AcrB by purified ClpXP could be observed in vitro. PMID:27078234

  9. 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. PMID:8268152

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

    PubMed

    Kumar, Ramesh; Pal, Parimal

    2013-08-01

    Modeling and simulation was carried out for an advanced membrane-integrated hybrid treatment process that ensures reuse of water with conversion and recovery of ammoniacal nitrogen as value-added struvite fertilizer from coke wastewater. While toxic cyanide was largely removed in a pre-chemical treatment unit using Fenton's reagents under optimized conditions, more than 95% of NH4(+)-N could be recovered as a valuable by-product called struvite through addition of appropriate doses of magnesium and phosphate salts. Water could be turned reusable through a polishing treatment by nanofiltration membranes in a largely fouling free membrane module following a biodegradation step. Mathematical modeling of such an integrated process was done with Haldane-Andrew approach for the associated microbial degradation of phenol by Pseudomonas putida. Residual NH4(+) was degraded by nitrification and denitrification following the modified Monod kinetics. The model could successfully predict the plant performance as reflected in reasonably low relative error (0.03-0.18) and high Willmott d-index (>0.98). PMID:23735488

  11. 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.

  12. 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

  13. Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy

    PubMed Central

    Mizrachi, Dario; Chen, Yujie; Liu, Jiayan; Peng, Hwei-Ming; Ke, Ailong; Pollack, Lois; Turner, Raymond J.; Auchus, Richard J.; DeLisa, Matthew P.

    2015-01-01

    Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic ‘shield' that sequesters the IMP from water and promotes its solubilization. PMID:25851941

  14. 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.

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

    DOEpatents

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

    2000-01-01

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

  16. Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase.

    PubMed

    Van Horn, Wade D; Kim, Hak-Jun; Ellis, Charles D; Hadziselimovic, Arina; Sulistijo, Endah S; Karra, Murthy D; Tian, Changlin; Sönnichsen, Frank D; Sanders, Charles R

    2009-06-26

    Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer with the use of solution nuclear magnetic resonance. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK's three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK's lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis. PMID:19556511

  17. Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy.

    PubMed

    Mizrachi, Dario; Chen, Yujie; Liu, Jiayan; Peng, Hwei-Ming; Ke, Ailong; Pollack, Lois; Turner, Raymond J; Auchus, Richard J; DeLisa, Matthew P

    2015-01-01

    Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic 'shield' that sequesters the IMP from water and promotes its solubilization. PMID:25851941

  18. 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

  19. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-04-01

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

  2. 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

  3. 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. PMID:26704387

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

    PubMed Central

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

    2015-01-01

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

  5. 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.

  6. Correlation of Post Swim-Up Acrosome Index with in-vitro Fertilization Outcomes

    PubMed Central

    Evirgen, Oya; Oral, Baha

    2009-01-01

    Purpose A prospective study was planned to determine the relationship between post swim-up acrosome index (AI) evaluation and fertilization outcomes in an in vitro fertilization (IVF) program. Materials and Methods Infertile couples who have applied to IVF were admitted into this study when the male partner's sperm concentration was > 20×106/mL and motility > 30%. Pre- and post swim-up semen quality parameters including concentration, motility, sperm morphology and AI were evaluated in a prospective, randomized and blinded fashion. The couples were divided prospectively into 2 groups. In group I (25 couples) 50 000 sperm per oocyte were used for insemination considering post swim-up acrosome index, and in group II (25 couples) 50 000 sperm per oocyte were used for insemination without considering post swim-up acrosome index. Results Pre- and post swim-up AI were 30.8 ± 3.4 and 17.8 ± 4.5 in group I, and 31.4 ± 3.6 and 16.3 ± 4.7 in group II (p > 0.05) respectively. The significant improvement in morphology and motility after double wash swim-up procedure has been observed. However, double wash swim-up procedure could not eliminate head and especially acrosomal defects which would directly effect fertilization capacity in conventional IVF program. In group I, 85.3% of oocytes were fertilized, with a 48% pregnancy rate; in group II, 71.0% of oocytes were fertilized, with a pregnancy rate of 20%. Fertilization and pregnancy rates were significantly different (p < 0.05) between the two groups. Conclusion We have concluded that it could be useful to consider post swim-up AI of sperm inseminated in conventional IVF cycles, which correlates with high fertilization and pregnancy rates. PMID:19568596

  7. Evaluating the role of silver nanoparticles on acrosomal reaction and spermatogenic cells in rat

    PubMed Central

    Miresmaeili, Sayyed Mohsen; Halvaei, Iman; Fesahat, Farzaneh; Fallah, Asghar; Nikonahad, Narges; Taherinejad, Mohaddeseh

    2013-01-01

    Background: Nanoparticles have wide range of application while there are some reports regarding their probable effects on male reproductive system and spermatozoa. Objective: The aim of this study was to evaluate the effect of different doses of silver nanoparticles (AgNPs) (70nm) on acrosome of rat spermatozoa and number of spermatogenic cells. Materials and Methods: In this experimental study, in experimental group, 32 male wistar rats (8 rats/group) received oral feeding AgNPs every 12 hr in one spermatogenesis period (48 days) by means of gavages in 25, 50 , 100 and 200 mg/kg concentration (experimental groups 1-4, respectively). The control group (8 rats) was treated on schedule with distilled water. Spermatozoa were stained by triple staining protocol for acrosome reaction. Histological evaluation on testis sections was performed using tissue processing and hematoxylin-eosin (H&E) staining. Results: There was significant difference between the control group and the experimental group 1 for acrosome reaction (11.00±0.00 and 24.25±3.68, respectively, p=0.01). There was only significant reduction in spermatogonia cells in experimental group 4. Experimental groups 2, 3 and 4 showed a significant reduction in the number of primary spermatocytes and spermatids as well as spermatozoa. But there were no significant differences between different groups for Sertoli cell number and seminiferous tubule diameter. Conclusion: It seems that Ag NPs have acute and significant effects on spermatogenesis and number of spermatogenic cells and also on acrosome reaction in sperm cells. More experimental investigations are necessary to elucidate better conclusion regarding the safety of nanoparticles on male reproduction system. PMID:24639775

  8. 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.

  9. 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

  10. 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

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

    PubMed

    Trusek-Holownia, Anna; Noworyta, Andrzej

    2007-05-31

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

  12. Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum.

    PubMed

    Öjemalm, Karin; Higuchi, Takashi; Jiang, Yang; Langel, Ülo; Nilsson, IngMarie; White, Stephen H; Suga, Hiroaki; von Heijne, Gunnar

    2011-08-01

    Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) Nature 427:36-44; Zimmer J, et al. (2008) Nature 455:936-943; Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107:17182-17187]. To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid's contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal/(mol · Å(2)), the solvation parameter for transfer from translocon to bilayer is 6-10 cal/(mol · Å(2)), pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a thermodynamic partitioning model and insights into the physical properties of the translocon. PMID:21606334

  13. 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. PMID:23297210

  14. Effect of reactive oxygen species on lysosomal membrane integrity. A study on a lysosomal fraction.

    PubMed

    Zdolsek, J M; Svensson, I

    1993-01-01

    Using a lysosome-enriched "light mitochondrial" fraction of a rat liver homogenate, the effects of the reactive oxygen species hydrogen peroxide, superoxide- and hydroxyl radicals were determined. Alterations in the intralysosomal pH and the release of a lysosomal marker enzyme, N-acetyl-glucosaminidase, were used as indicators of changes in the lysosomal membrane integrity. Lipid peroxidation of the fraction was assayed by TBARS measurement. Neither superoxide radicals, generated by hypoxanthine/xanthine oxidase, nor a bolus dose of hydrogen peroxide (0.5-1.5 mM) induced any lysosomal damage. If, however, Fe(III)ADP was included in the superoxide radical-generating system, lysosomal membrane damage was detected, both as an increase in lysosomal pH and as a release of N-acetyl-glucosaminidase, but only after a lag phase of about 7 min. Lipid peroxidation, on the other hand, proceeded gradually. Lysosomes treated with hydrogen peroxide displayed similar dose-dependent alterations, albeit only if both Fe(III)ADP and the reducing amino acid cysteine were added. In the latter system, however, alterations of the lysosomal membrane stability occurred more rapidly, showing a lag phase of only 2 min. Lipid peroxidation, which proceeded faster and displayed no lag phase, levelled out within 10 min. The results indicate that neither superoxide radicals nor hydrogen peroxide are by themselves damaging to lysosomes. Available catalytically active iron in Fe(II) form, however, allows reactions yielding powerful oxidative species--probably hydroxyl radicals formed via Fenton reactions--to take place inducing peroxidation of the lysosomal membranes resulting in dissipation of the proton-gradient and leakage of their enzyme contents. PMID:8148962

  15. TMEM115 is an integral membrane protein of the Golgi complex involved in retrograde transport

    PubMed Central

    Ong, Yan Shan; Tran, Ton Hoai Thi; Gounko, Natalia V.; Hong, Wanjin

    2014-01-01

    ABSTRACT Searching and evaluating the Human Protein Atlas for transmembrane proteins enabled us to identify an integral membrane protein, TMEM115, that is enriched in the Golgi complex. Biochemical and cell biological analysis suggested that TMEM115 has four candidate transmembrane domains located in the N-terminal region. Both the N- and C-terminal domains are oriented towards the cytoplasm. Immunofluorescence analysis supports that TMEM115 is enriched in the Golgi cisternae. Functionally, TMEM115 knockdown or overexpression delays Brefeldin-A-induced Golgi-to-ER retrograde transport, phenocopying cells with mutations or silencing of the conserved oligomeric Golgi (COG) complex. Co-immunoprecipitation and in vitro binding experiments reveals that TMEM115 interacts with the COG complex, and might self-interact to form dimers or oligomers. A short region (residues 206–229) immediately to the C-terminal side of the fourth transmembrane domain is both necessary and sufficient for Golgi targeting. Knockdown of TMEM115 also reduces the binding of the lectins peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA), suggesting an altered O-linked glycosylation profile. These results establish that TMEM115 is an integral membrane protein of the Golgi stack regulating Golgi-to-ER retrograde transport and is likely to be part of the machinery of the COG complex. PMID:24806965

  16. Type IV Pilus Proteins Form an Integrated Structure Extending from the Cytoplasm to the Outer Membrane

    PubMed Central

    Li, Chengyun; Wallace, Regina A.; Black, Wesley P.; Li, Yue-zhong; Yang, Zhaomin

    2013-01-01

    The bacterial type IV pilus (T4P) is the strongest biological motor known to date as its retraction can generate forces well over 100 pN. Myxococcus xanthus, a δ-proteobacterium, provides a good model for T4P investigations because its social (S) gliding motility is powered by T4P. In this study, the interactions among M. xanthus T4P proteins were investigated using genetics and the yeast two-hybrid (Y2H) system. Our genetic analysis suggests that there is an integrated T4P structure that crosses the inner membrane (IM), periplasm and the outer membrane (OM). Moreover, this structure exists in the absence of the pilus filament. A systematic Y2H survey provided evidence for direct interactions among IM and OM proteins exposed to the periplasm. For example, the IM lipoprotein PilP interacted with its cognate OM protein PilQ. In addition, interactions among T4P proteins from the thermophile Thermus thermophilus were investigated by Y2H. The results indicated similar protein-protein interactions in the T4P system of this non-proteobacterium despite significant sequence divergence between T4P proteins in T. thermophilus and M. xanthus. The observations here support the model of an integrated T4P structure in the absence of a pilus in diverse bacterial species. PMID:23922942

  17. 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

  18. 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

  19. A miniaturized extracorporeal membrane oxygenator with integrated rotary blood pump: preclinical in vivo testing.

    PubMed

    Kopp, Ruedger; Bensberg, Ralf; Arens, Jutta; Steinseifer, Ulrich; Schmitz-Rode, Thomas; Rossaint, Rolf; Henzler, Dietrich

    2011-01-01

    Extracorporeal membrane oxygenation can achieve sufficient gas exchange in severe acute respiratory distress syndrome. A highly integrated extracorporeal membrane oxygenator (HEXMO) was developed to reduce filling volume and simplify management. Six female pigs were connected to venovenous HEXMO with a total priming volume of 125 ml for 4 hours during hypoxemia induced by a hypoxic inspired gas mixture. Animals were anticoagulated with intravenous heparin. Gas exchange, hemodynamics, hemolysis, and coagulation activation were examined. One device failed at the magnetic motor coupling of the integrated diagonal pump. In the remaining five experiments, the oxygenation increased significantly (arterial oxygen saturation [SaO2] from 79 ± 5% before HEXMO to 92% ± 11% after 4 hours) facilitated by a mean oxygen transfer of 66 ± 29 ml/dl through the oxygenator. The CO2 elimination by the HEXMO reduced arterial PaCO2 only marginal. Extracorporeal blood flow was maintained at 32% ± 6% of cardiac output. Hemodynamic instability or hemolysis was not observed. The plasmatic coagulation was only mildly activated without significant platelet consumption. The HEXMO prototype provided sufficient gas exchange to prevent hypoxemia. This proof of concept study supports further development and design modifications to increase performance and to reduce coagulation activation for potential long-term application. PMID:21317635

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

    PubMed Central

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

    2015-01-01

    Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin–neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion. PMID:26392524

  1. 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

  2. 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

  3. [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

  4. 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

  5. Cryopreservation of human spermatozoa with pentoxifylline improves the post-thaw agonist-induced acrosome reaction rate.

    PubMed

    Esteves, S C; Sharma, R K; Thomas, A J; Agarwal, A

    1998-12-01

    Cryopreservation causes extensive damage to spermatozoa, thereby impairing their fertilizing ability. The purpose of this study was to determine if the direct addition of pentoxifylline to the seminal plasma before cryopreservation improved sperm motility and acrosome reaction. Semen specimens from 15 healthy volunteers were divided into two aliquots. One aliquot was treated by adding 5 mM pentoxifylline directly to the seminal plasma (treatment group) and the other aliquot received no treatment (control group). Both aliquots were then cryopreserved by using the liquid nitrogen freezing method. The percentage of motile spermatozoa and various motion characteristics were then evaluated by performing computer-assisted semen analysis. The sperm viability was determined with a supra-vital dye, Hoechst-33258, and the acrosome reaction (spontaneous and calcium ionophore-induced) was monitored using fluorescein isothiocyanate-conjugated peanut lectin (FITC-PNA) binding assays. Pentoxifylline treatment significantly increased the sperm motility, the amplitude of lateral head displacement, the hyperactivation status, and the frequency of spontaneous acrosome reactions before freezing (P < 0.05). After post-thaw, no difference in motion characteristics (except percentage motility) between treated and control groups were observed. Acrosome loss due to the freeze-thaw process was less in the pentoxifylline-treated group (P = 0.0003). In addition, the percentage of cryopreserved acrosome-intact spermatozoa that underwent further acrosome reactions in response to calcium-ionophore challenge was significantly higher in the treated group (P = 0.03). Pentoxifylline treatment before freezing improved the acrosome reaction to ionophore challenge in cryopreserved spermatozoa. Treatment with pentoxifylline appears to minimize sperm damage during the freeze-thaw process and may improve fertilization rates with assisted reproductive procedures such as intrauterine insemination or in

  6. 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. PMID:27256665

  7. Porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes.

    PubMed

    Kappes, Matthew A; Miller, Cathy L; Faaberg, Kay S

    2015-07-01

    The membrane insertion and topology of nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) strain VR-2332 was assessed using a cell free translation system in the presence or absence of artificial membranes. Expression of PRRSV nsp2 in the absence of all other viral factors resulted in the genesis of both full-length nsp2 as well as a select number of C-terminal nsp2 isoforms. Addition of membranes to the translation stabilized the translation reaction, resulting in predominantly full-length nsp2 as assessed by immunoprecipitation. Analysis further showed full-length nsp2 strongly associates with membranes, along with two additional large nsp2 isoforms. Membrane integration of full-length nsp2 was confirmed through high-speed density fractionation, protection from protease digestion, and immunoprecipitation. The results demonstrated that nsp2 integrated into the membranes with an unexpected topology, where the amino (N)-terminal (cytoplasmic) and C-terminal (luminal) domains were orientated on opposite sides of the membrane surface. PMID:25768891

  8. 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.

  9. 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

  10. The theory of semipermeable vesicles and membranes: An integral-equation approach. II. Donnan equilibrium

    NASA Astrophysics Data System (ADS)

    Zhou, Yaoqi; Stell, George

    1988-12-01

    Integral equations that yield the charge and density profiles are derived for a Donnan system, in which an ionic solution is separated into two regions by a semipermeable membrane (SPM) or a spherical semipermeable vesicle (SPV). These equations are obtained from the Ornstein-Zernike (OZ) equation. We show how quantitative results can be obtained from either the mean spherical approximation (MSA) closure or the hypernetted-chain (HNC) closure for profiles. Use is made of bulk-correlation input obtained by means of the Debye-Hückel approximation, the MSA approximation, or the HNC approximation. The resulting approximations will be referred as MSA/DH, HNC/DH, MSA/MSA, etc. The system on which we focus contains three charged hard-sphere species: cation, anion, and a large ion (a protein or polymer ion) separated by a plane SPM, through which the large ion cannot pass, and to one side of which all large ions are confined, or a spherical SPV, outside of which the large ions are confined. Analytical expressions for the bulk density ratio between the two sides of a plane membrane as well as the membrane potential in various approximations are obtained. Results obtained from these expresssions are compared with the results obtained by equating electrochemical potentials. A new contact-value theorem is provided for the plane SPM system. Analytical solutions for the charge profile and the potential profile in the MSA/DH approximation are obtained. It turns out that results obtained in the HNC/DH approximation are exactly the same as those obtained by using 1D nonlinear Poisson-Boltzmann equations if the repulsive cores of the macroions are neglected.

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

    PubMed

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

    2015-04-01

    Membrane bioreactors (MBR) are highly efficient at intercepting particles and microbes and have become an important technology for wastewater reclamation. However, many pathogens can accumulate in activated sludge due to the long residence time usually adopted in MBR, and thus may pose health risks when membrane integrity problems occur. This study presents data from a survey on the occurrence of water-borne Giardia pathogens in reclaimed water from a full-scale wastewater treatment plant with MBR experiencing membrane integrity failure, and assessed the associated risk for green space irrigation. Due to membrane integrity failure, the MBR effluent turbidity varied between 0.23 and 1.90 NTU over a period of eight months. Though this turbidity level still met reclaimed water quality standards (≤5 NTU), Giardia were detected at concentrations of 0.3 to 95 cysts/10 L, with a close correlation between effluent turbidity and Giardia concentration. All β-giardin gene sequences of Giardia in the WWTP influents were genotyped as Assemblages A and B, both of which are known to infect humans. An exponential dose-response model was applied to assess the risk of infection by Giardia. The risk in the MBR effluent with chlorination was 9.83×10(-3), higher than the acceptable annual risk of 1.0×10(-4). This study suggested that membrane integrity is very important for keeping a low pathogen level, and multiple barriers are needed to ensure the biological safety of MBR effluent. PMID:25872734

  12. 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.

  13. Monte Carlo simulation studies of lipid order parameter profiles near integral membrane proteins.

    PubMed Central

    Sperotto, M M; Mouritsen, O G

    1991-01-01

    Monte Carlo simulation techniques have been applied to a statistical mechanical lattice model in order to study the coherence length for the spatial fluctuations of the lipid order parameter profiles around integral membrane proteins in dipalmitoyl phosphatidylcholine bilayers. The model, which provides a detailed description of the pure lipid bilayer main transition, incorporates hydrophobic matching between the lipid and protein hydrophobic thicknesses as a major contribution to the lipid-protein interactions in lipid membranes. The model is studied at low protein-to-lipid ratios. The temperature dependence of the coherence length is found to have a dramatic peak at the phase transition temperature. The dependence on protein circumference as well as hydrophobic length is determined and it is concluded that in some cases the coherence length is much longer than previously anticipated. The long coherence length provides a mechanism for indirect lipid-mediated protein-protein long-range attraction and hence plays an important role in regulating protein segregation. Images FIGURE 5 FIGURE 6 PMID:2009352

  14. 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.

  15. 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

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

    PubMed

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

    2016-08-01

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

  17. 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.

  18. Effects of quasiactive membrane on multiply periodic traveling waves in integrate-and-fire systems

    NASA Astrophysics Data System (ADS)

    James, M. P.; Coombes, S.; Bressloff, P. C.

    2003-05-01

    We consider the dynamics of a one-dimensional continuum of synaptically interacting integrate-and-fire neurons with realistic forms of axodendritic interaction. The speed and stability of traveling waves are investigated as a function of discrete communication delays, distributed synaptic delays, and axodendritic delays arising from the spatially extended nature of the model neuron. In particular, dispersion curves for periodic traveling waves are constructed. Nonlinear ionic channels in the dendrite responsible for a so-called quasiactive bandpass response are shown to significantly influence the shape of dispersion curves. Moreover, a kinematic theory of spike train propagation suggests that period-doubling bifurcations of a singly periodic wave can occur in dendritic systems with a quasiactive membrane. The explicit construction of period-doubled solutions is used to confirm this prediction.

  19. 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. PMID:3717896

  20. 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

  1. Event monitoring of herbicides with naked and membrane-covered Empore disk integrative passive sampling devices.

    PubMed

    Stephens, B Scott; Kapernick, Anita P; Eaglesham, Geoff; Mueller, Jochen F

    2009-08-01

    Subsequent to an initial wet season flood event in the Brisbane River, Australia, both fast (naked disk) and slow (membrane-covered) variants of SDB-RPS Empore disk passive sampling devices were deployed with an automated grab sampling program. A trend increase in the aquatic dissolved concentrations of diuron and simazine was observed over a 10-day period. Kinetic and equilibrium parameters for each sampler were calculated based on the dynamic concentration. Absolute percent difference for duplicate passive samples was <10% in the fast and <25% in the slow samplers. For kinetic sampling, significantly shortened integrative periods are available with the fast compared with the slow variant, with higher sampling rates offering improved detection limits. The study demonstrates a method for determining kinetic parameters of passive samplers in a variable concentration field deployment, and illustrates the differences in quality between active and passive data, in terms of capturing changes in concentration associated with rainfall events. PMID:19520390

  2. Genome-Wide Association Study Implicates Testis-Sperm Specific FKBP6 as a Susceptibility Locus for Impaired Acrosome Reaction in Stallions

    PubMed Central

    Raudsepp, Terje; Dobson, Lauren; Vishnoi, Monika; Fritz, Krista L.; Schaefer, Robert; Rendahl, Aaron K.; Derr, James N.; Love, Charles C.; Varner, Dickson D.; Chowdhary, Bhanu P.

    2012-01-01

    Impaired acrosomal reaction (IAR) of sperm causes male subfertility in humans and animals. Despite compelling evidence about the genetic control over acrosome biogenesis and function, the genomics of IAR is as yet poorly understood, providing no molecular tools for diagnostics. Here we conducted Equine SNP50 Beadchip genotyping and GWAS using 7 IAR–affected and 37 control Thoroughbred stallions. A significant (P<6.75E-08) genotype–phenotype association was found in horse chromosome 13 in FK506 binding protein 6 (FKBP6). The gene belongs to the immunophilins FKBP family known to be involved in meiosis, calcium homeostasis, clathrin-coated vesicles, and membrane fusions. Direct sequencing of FKBP6 exons in cases and controls identified SNPs g.11040315G>A and g.11040379C>A (p.166H>N) in exon 4 that were significantly associated with the IAR phenotype both in the GWAS cohort (n = 44) and in a large multi-breed cohort of 265 horses. All IAR stallions were homozygous for the A-alleles, while this genotype was found only in 2% of controls. The equine FKBP6 was exclusively expressed in testis and sperm and had 5 different transcripts, of which 4 were novel. The expression of this gene in AC/AG heterozygous controls was monoallelic, and we observed a tendency for FKBP6 up-regulation in IAR stallions compared to controls. Because exon 4 SNPs had no effect on the protein structure, it is likely that FKBP6 relates to the IAR phenotype via regulatory or modifying functions. In conclusion, FKBP6 was considered a susceptibility gene of incomplete penetrance for IAR in stallions and a candidate gene for male subfertility in mammals. FKBP6 genotyping is recommended for the detection of IAR–susceptible individuals among potential breeding stallions. Successful use of sperm as a source of DNA and RNA propagates non-invasive sample procurement for fertility genomics in animals and humans. PMID:23284302

  3. Evaluation of chondrocyte survival in situ using WST-1 and membrane integrity stains.

    PubMed

    Jomha, Nadr M; Elliott, Janet A W; Law, Garson K; McGann, Locksley E

    2007-01-01

    Evaluating chondrocytes in situ to document the effectiveness of cartilage preservation techniques has proven exceedingly difficult. This study was conducted to determine the effectiveness of WST-1 on porcine chondrocytes in situ after cooling to -10 degrees C (without ice formation) compared to membrane integrity stains (MIS). Osteochondral dowels (10 mm in diameter) were harvested from sexually mature pigs within 24 h of sacrifice and randomized into three groups: (1) untreated control, (2) one day storage at -10 degrees C (in cryoprotectant solution to prevent ice formation), and (3) seven day storage at -10 degrees C (in cryoprotectant solution). Fluorescent MISs (Syto 13 and ethidium bromide) were used on 70 microm slices. Representative images were digitized and green and red pixel numbers determined the percent recovery of intact cells. Mitochondrial activity (WST-1) was determined using 20 slices of 70 microm thickness per sample to obtain reliable readings using a spectrophotometer at 450 nm. All samples underwent repeated measures of membrane integrity and metabolic activity obtained after 0, 3, 24, 48, 72, and 144 h incubation in growth media. WST-1 consistently overestimated cell recovery with results greater than fresh controls. After hypothermic storage for 7 days, the WST-1 measurement demonstrated decreased mitochondrial activity that recovered by 48 h. MIS was most accurate when "absolute" cell recovery was compared to original controls, taking into account cell density. In conclusion, WST-1 can track metabolic activity of chondrocytes in situ over time but "absolute" cell recovery determined by MISs after 48 h incubation may be the most accurate determination of the number of live chondrocytes in situ. PMID:17094019

  4. 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

  5. Effects of water- and lipid-soluble antioxidants on turkey sperm viability, membrane integrity, and motility during liquid storage.

    PubMed

    Donoghue, A M; Donoghue, D J

    1997-10-01

    Aerobic conditions are required to maintain the viability of turkey sperm in vitro. In mammalian sperm, excess oxygen during in vitro storage results in lipid peroxidation, causing membrane damage and reduced sperm motility and subsequent fertility. The effect of adding antioxidants to turkey sperm during liquid storage was studied. Semen was collected and pooled from 20 toms and antioxidants were tested at a minimum of six concentrations, n = 6 observations per concentration. Semen was diluted into Beltsville Poultry Semen Extender. Extended semen served as a control; treatments were extended semen supplemented with tocopherol (vitamin E, 1 to 80 micrograms/mL); butylated hydroxytoluene (BHT, 0.02 to 1.25 mM); Tempo (0.039 to 1.25 microM), or vitamin C (1 to 400 micrograms/mL) and stored at 5 C for 48 h. Sperm viability in extended semen was evaluated after 0, 24, or 48 h storage. Membrane integrity and motility were also measured. Flow cytometric analysis was done using the live/dead stain combination (SYBR-14/propidium iodide) for sperm viability, and membrane integrity was assessed using a hypo-osmotic stress test. Sperm motility was evaluated subjectively. Control sperm viability was reduced almost 50% between 0 and 48 h. However, supplementation with vitamin E, Tempo, and BHT maintained populations of viable sperm similar to the 0 h levels at 48 h. Hypo-osmotic membrane integrity in the control sperm was reduced to approximately 22% (at 24 h, P < or = 0.05) and 5% (at 48 h, P < or = 0.05) of the total sperm population. Similar to controls after 24 h in vitro storage, sperm treated with the antioxidants vitamin E, Tempo, and BHT had reduced hypo-osmotic membrane integrity compared to 0 h samples. However, many of these treated samples maintained hypo-osmotic membrane integrity observed from 24 through 48 h (range, 21.5 to 44.6%), whereas hypo-osmotic membrane integrity fell to 4.6% at 48 h for the control (P < or = 0.05). Vitamin C treatments were similar to

  6. Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore

    PubMed Central

    Ohba, Tomoyuki; Schirmer, Eric C.; Nishimoto, Takeharu; Gerace, Larry

    2004-01-01

    Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex. PMID:15611332

  7. 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

  8. Bacterial binding protein-dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins.

    PubMed

    Saurin, W; Köster, W; Dassa, E

    1994-06-01

    Bacterial binding protein-dependent transport systems belong to the superfamily of ABC transporters, which is widely distributed among living organisms. Their hydrophobic membrane proteins are the least characterized components. The primary structures of 61 integral membrane proteins from 35 uptake systems were compared in order to characterize a short conserved hydrophilic segment, with a consensus EAA---G---------I-LP, located approximately 100 residues from the C-terminus. Secondary structure predictions indicated that this conserved region might be formed by two amphipathic alpha-helices connected by a loop containing the invariant G residue. We classified the conserved motifs and found that membrane proteins from systems transporting structurally related substrates specifically display a greater number of identical residues in the conserved region. We determined a consensus for each class of membrane protein and showed that these can be considered as signatures. PMID:7934906

  9. 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

  10. Genome-wide association study for sperm membrane integrity in frozen-thawed semen of Holstein-Friesian bulls.

    PubMed

    Kamiński, Stanisław; Hering, Dorota M; Oleński, Kamil; Lecewicz, Marek; Kordan, Władysław

    2016-07-01

    The aim of the study was to screen the entire bull genome to identify SNP markers and propose candidate genes potentially involved in the variation of sperm membrane integrity in Holstein-Friesian bulls. Two hundred eighty eight bulls kept in one AI center were included in the study. Each bull was genotyped for 54.001 Single Nucleotide Polymorpisms (SNP) by the Illumina BovineSNP50 BeadChip. Commercial straws of frozen-thawed semen were used for the evaluation of sperm plasma membrane integrity (SYBR-14/PI staining) and sperm mitochondrial function (JC1/PI staining). An additive model for Linear Regression Analysis was applied to estimate the effect of SNP marker for sperm membrane integrity (by the use of GoldenHelix SVS7 software). Five significant markers (encompassing 2,2 MB region located on chromosome 6) for SYBR-14/PI were found. Among them one marker-rs41570391 passed Bonferroni correction test. Within approximately 3 Mb genomic region including significant markers three candidate genes: SGMS2 (Sphingomyelin Synthase 2), TET2 (Methylcytosine dioxygenase 2) and GSTCD genes (Gluthatione S-transferase C terminal domain) were proposed as potentially involved in sperm membrane integrity in frozen-thawed semen of Holstein-Friesian bulls. PMID:27236378

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

    PubMed

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

    2015-03-01

    This paper describes a new and facile approach for the formation of pore-spanning bilayer lipid membranes (BLMs) within a poly(dimethylsiloxane) (PDMS) microfluidic device. Commercially, readily available polycarbonate (PC) membranes are employed for the support of BLMs. PC sheets with 5 μm, 2 μm, and 0.4 μm pore diameters, respectively, are thermally bonded into a multilayer-stack, reducing the pore density of 0.4 μm-pore PC by a factor of 200. The BLMs on this support are considerably stable (a mean lifetime: 17 h). This multilayer-stack PC (MSPC) membrane is integrated into the PDMS chip by an epoxy bonding method developed to secure durable bonding under the use of organic solvents. The microchip has a special channel for guiding a micropipette in the proximity of the MSPC support. With this on-site injection technique, tens to hundreds of nanoliters of solutions can be directly dispensed to the support. Incorporating gramicidin ion channels into BLMs on the MSPC support has confirmed the formation of single BLMs, which is based on the observation from current signals of 20 pS conductance that is typical to single channel opening. Based on the bilayer capacitance (1.4 pF), about 15% of through pores across the MSPC membrane are estimated to be covered with BLMs. PMID:26015832

  12. 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

  13. FoxC1 is essential for vascular basement membrane integrity and hyaloid vessel morphogenesis

    PubMed Central

    Skarie, Jonathan M.; Link, Brian A.

    2013-01-01

    Purpose Alterations in FOXC1 dosage lead to a spectrum of highly penetrant, ocular anterior segment dysgenesis phenotypes. The most serious outcome is development of glaucoma, but this only occurs in 50–75% of patients. Therefore, the need to identify specific pathways and genes that interact with FOXC1 to promote glaucoma is great. In this study, we investigated loss of foxC1 in the zebrafish to characterize phenotypes and gene interactions that may impact glaucoma pathogenesis. Methods Morpholino knockdown in zebrafish, RNA and protein marker analyses, transgenic reporter lines, and angiography, along with histology and transmission electron microscopy were used to study foxC1 function and gene interactions. Results Zebrafish foxC1 genes were expressed dynamically in the developing vasculature and periocular mesenchyme during development. Multiple ocular and vascular defects were found after knockdown of foxC1. Defects in the hyaloid vasculature, arterial-venous malformations, and coarctation of the aorta were observed with maximal depletion of foxC1. Partial loss of foxC1 resulted in CNS and ocular hemorrhages, defects in intersegmental vessel patterning, and increased vascular permeability. To investigate the basis for these disruptions, ultrastructure of foxC1-depleted hyaloid vascular cells was studied. These experiments, along with Laminin-111 immunoreactivity, revealed disruptions in basement membrane integrity. Finally, co-depletion of laminin alpha-1 and foxC1 uncovered a genetic interaction between these genes during development. Conclusions Genetic interactions between FOXC1 and basement membrane components influence vascular stability and may impact glaucoma development and increase stroke risk in FOXC1 patients. PMID:19458328

  14. Low doses of arginine butyrate derivatives improve dystrophic phenotype and restore membrane integrity in DMD models.

    PubMed

    Vianello, Sara; Consolaro, Francesca; Bich, Claudia; Cancela, José-Manuel; Roulot, Morgane; Lanchec, Erwan; Touboul, David; Brunelle, Alain; Israël, Maurice; Benoit, Evelyne; de la Porte, Sabine

    2014-06-01

    A new approach to treating Duchenne muscular dystrophy was investigated by using the ester or amide covalent association of arginine [nitric oxide (NO) pathway] and butyrate [histone deacetylase (HDAC) inhibition] in mdx mice and patient myotubes. Two prodrugs were synthesized, and the beneficial effects on dystrophic phenotype were studied. Nerve excitability abnormalities detected in saline-treated mice were almost totally rescued in animals treated at low doses (50-100 mg/kg/d). Force and fatigue resistance were improved ≈60% and 3.5-fold, respectively, and the percentage of necrosis in heart sections was reduced ≈90% in the treated mice. A decrease of >50% in serum creatine kinase indicated an overall improvement in the muscles. Restoration of membrane integrity was studied directly by measuring the reduction (≈74%) of Evans blue incorporation in the limb muscles of the treated animals, the increase in utrophin level, and the normalization of lipid composition of the heart. In cultures of human myotubes (primary cells and cell line), both prodrugs and HDAC inhibitors increased by 2- to 4-fold the utrophin level, which was correctly localized at the membrane. β-Dystroglycan and embryonic myosin protein levels were also increased. Finally, a 50% reduction in the number of spontaneous Ca(2+) spikes was observed after treatment with NO synthase substrate and HDAC inhibitors. Overall, the beneficial effects were obtained with doses 10 (in vivo) and 5 (in vitro) times lower than those of the salt formulation. Altogether, these data constitute proof of principle of the beneficial effects of low doses of arginine butyrate derivatives on muscular dystrophy, enhancing the NO pathway and inhibiting HDAC. PMID:24604079

  15. The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator.

    PubMed

    Baturin, Simon; Galka, Jamie J; Piyadasa, Hadeesha; Gajjeraman, S; O'Neil, Joe D

    2014-12-01

    Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric α-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis. TMAO increases the heat stability of the GF tetramer an average of 10 °C in the 4 detergents and also protects the protein from denaturation by SDS. However, it did not promote re-association to the tetramer when added to SDS-dissociated protein. TMAO also promotes the formation of rod-like detergent micelles, and DLS was found to be useful for monitoring the structure of the protein and the redistribution of detergent during thermal dissociation of the protein. The protein is more thermally stable in detergents with the phosphatidylcholine head group (LLPC and LMPC) than in the maltoside detergents. The implications of the results for osmolyte mechanism, membrane protein stability, and protein-protein interactions are discussed. PMID:25387032

  16. Structure of the main saccharide chain in the acrosome reaction-inducing substance of the starfish, Asterias amurensis.

    PubMed

    Koyota, S; Wimalasiri, K M; Hoshi, M

    1997-04-18

    The structure of the main saccharide chain of the acrosome reaction-inducing substance in the egg jelly coat of the starfish, Asterias amurensis, is composed of the following pentasaccharide repeating units (Structure I). A polymer consisting of 10-11 repeating units has been observed to induce the acrosome reaction in starfish sperm at high calcium concentrations. [STRUCTURE I:see text] The identities and linkage positions of constituent sugars were established using sugar, methylation, and sulfate analyses together with one- and two-dimensional nmr spectroscopy. The structure was supported by the data obtained for desulfation products and the Smith degradation of the polysaccharide. PMID:9099675

  17. 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. PMID:17266791

  18. Novel conserved structural domains of acrosome reaction-inducing substance are widespread in invertebrates.

    PubMed

    Naruse, Masahiro; Ishikawa, Riho; Sakaya, Hiroshi; Moriyama, Hideaki; Hoshi, Motonori; Matsumoto, Midori

    2011-01-01

    In the starfish Asterias amurensis, acrosome reaction inducing substance (ARIS) is the main factor responsible for allowing sperm to recognize the egg jelly and begin the acrosome reaction (AR). ARIS is a large proteoglycan-like molecule, and its pentasaccharide repeat, Fragment 1 (Fr. 1), is responsible for inducing AR. Here, we investigated the primary structure of ARIS for the first time in order to improve our understanding of its functionality. Electrophoretic analysis revealed that ARIS is a complex of three proteins, all of which are modified by the Fr. 1 sugar chain. Sequencing indicated that there are two novel, conserved domains in all three ARIS proteins: ARIS N-terminus (AR-N) and ARIS C-terminus (AR-C) domains. We also found that other echinoderms possess ARIS proteins that are capable of inducing the AR for homologous sperm, indicating that ARIS proteins may be a ubiquitous component for echinoderm fertilization. Moreover, we identified ARIS-like genes from Ctenophora to Protochordata. PMID:21268183

  19. CaMKII prevents spontaneous acrosomal exocytosis in sperm through induction of actin polymerization.

    PubMed

    Shabtay, Ortal; Breitbart, Haim

    2016-07-01

    In order to interact with the egg and undergo acrosomal exocytosis or the acrosome reaction (AR), mammalian spermatozoa must undergo a series of biochemical changes in the female reproductive tract, collectively called capacitation. We showed that F-actin is formed during sperm capacitation and fast depolymerization occurs prior to the AR. We hypothesized that F-actin protects the sperm from undergoing spontaneous-AR (sAR) which decreases fertilization rate. We show that activation of the actin-severing protein gelsolin induces a significant increase in sAR. Moreover, inhibition of CaMKII or PLD during sperm capacitation, caused an increase in sAR and inhibition of F-actin formation. Spermine, which leads to PLD activation, was able to reverse the effects of CaMKII inhibition on sAR-increase and F-actin-decrease. Furthermore, the increase in sAR and the decrease in F-actin caused by the inactivation of the PLD-pathway, were reversed by activation of CaMKII using H2O2 or by inhibiting protein phosphatase 1 which enhance the phosphorylation and oxidation states of CaMKII. These results indicate that two distinct pathways lead to F-actin formation in the sperm capacitation process which prevents the occurrence of sAR. PMID:27178669

  20. Asterosap-induced elevation in intracellular pH is indispensable for ARIS-induced sustained increase in intracellular Ca2+ and following acrosome reaction in starfish spermatozoa.

    PubMed

    Kawase, Osamu; Minakata, Hiroyuki; Hoshi, Motonori; Matsumoto, Midori

    2005-02-01

    In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, namely ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for the induction of acrosome reaction. For the induction, ARIS alone is enough in high-Ca2+ or high-pH seawater, but, besides ARIS, the addition of either Co-ARIS or asterosap is required in normal seawater. Asterosap transiently increased both the intracellular pH (pHi) and Ca2+ ([Ca2+]i), while ARIS slightly elevated the basal level of [Ca2+]i. However, a sustained elevation of [Ca2+]i and acrosome reaction occurred if sperm were simultaneously treated with ARIS and asterosap. EGTA inhibited the sustained [Ca2+]i elevation and acrosome reaction. The sustained [Ca2+]i elevation and acrosome reaction were highly susceptible to SKF96365 and Ni2+, specific blockers of the store-operated Ca2+ channel (SOC). These results suggest that sustained [Ca2+]i elevation, mediated by the SOC-like channel, is a prerequisite for the acrosome reaction. In high-pH seawater, ARIS alone induced a prominent [Ca2+]i increase and acrosome reaction, which were similarly sensitive to SKF96365. The acrosome reaction was effectively induced by ARIS alone when pHi was artificially increased to more than 7.7. Asterosap increased pHi from 7.6 +/- 0.1 to 7.7 +/- 0.1. Furthermore, the sustained [Ca2+]i elevation and acrosome reaction, induced by a combination of ARIS and asterosap, were drastically inhibited by a slight reduction in pHi. Taking these results into account, we suggest that an asterosap-induced pHi elevation is required for triggering the ARIS-induced sustained [Ca2+]i elevation and consequent acrosome reaction. PMID:15984164

  1. 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

  2. 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

    2016-01-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

    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.

  4. Integrating Membrane Transport with Male Gametophyte Development and Function through Transcriptomics1[W

    PubMed Central

    Bock, Kevin W.; Honys, David; Ward, John M.; Padmanaban, Senthilkumar; Nawrocki, Eric P.; Hirschi, Kendal D.; Twell, David; Sze, Heven

    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. Approximately 1,269 genes encoding classified transporters were collected from the Arabidopsis (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, was developmentally regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter∷β-glucuronidase 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. PMID:16607029

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

    PubMed Central

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

    2002-01-01

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

  7. Lysosomal Integral Membrane Protein-2: A New Player in Lysosome-Related Pathology

    PubMed Central

    Gonzalez, Ashley; Valeiras, Mark; Sidransky, Ellen; Tayebi, Nahid

    2014-01-01

    Lysosomes require the presence of many specialized proteins to facilitate their roles in cellular maintenance. One such protein that has proven to be an important player in the lysosomal field is lysosomal integral membrane protein-2 (LIMP-2), encoded by the gene SCARB2. LIMP-2 is required for the normal biogenesis and maintenance of lysosomes and endosomes and has been identified as the specific receptor for glucocerebrosidase, the enzyme deficient in Gaucher disease. Research into LIMP-2 and the SCARB2 gene indicate that it may be a factor contributing to the clinical heterogeneity seen among patients with Gaucher disease. Mutations in SCARB2 have also been identified as the cause of action myoclonus renal failure (AMRF), and in some cases progressive myoclonic epilepsy. A total of 14 disease-causing SCARB2 mutations have been identified to date. The role of LIMP-2 in human pathology has expanded with its identification as a component of the intercalated disc in cardiac muscle and as a receptor for specific enteroviruses, two unanticipated findings that reaffirm the myriad roles of lysosomal proteins. Studies into the full impact of LIMP-2 deficiency and the LIMP2/glucocerebrosidase molecular pathway will lead to a better understanding of disease pathogenesis in Gaucher disease and AMRF, and to new insights into lysosomal processing, trafficking and function. PMID:24389070

  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. 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

  10. 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

  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. 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. PMID:23422307

  13. 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

  14. Separation of the sperm agglutinin and the acrosome reaction-inducing substance in egg jelly of starfish.

    PubMed

    Uno, Y; Hoshi, M

    1978-04-01

    The egg jelly of the starfish Asterias amurensis was separated into the fractions J1, J2, and J3 on a Sephadex G-100 column. The Jl fraction induced the acrosome reaction and J2 induced sperm agglutination. Chemical analysis and chromatography revealed that sperm agglutinin is similar to asterosaponin A. PMID:17847329

  15. 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. PMID:25663286

  16. 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

  17. 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

  18. Smart polymer brush nanostructures guide the self-assembly of pore-spanning lipid bilayers with integrated membrane proteins

    NASA Astrophysics Data System (ADS)

    Wilhelmina de Groot, G.; Demarche, Sophie; Santonicola, M. Gabriella; Tiefenauer, Louis; Vancso, G. Julius

    2014-01-01

    Nanopores in arrays on silicon chips are functionalized with pH-responsive poly(methacrylic acid) (PMAA) brushes and used as supports for pore-spanning lipid bilayers with integrated membrane proteins. Robust platforms are created by the covalent grafting of polymer brushes using surface-initiated atom transfer radical polymerization (ATRP), resulting in sensor chips that can be successfully reused over several assays. His-tagged proteins are selectively and reversibly bound to the nitrilotriacetic acid (NTA) functionalization of the PMAA brush, and consequently lipid bilayer membranes are formed. The enhanced membrane resistance as determined by electrochemical impedance spectroscopy and free diffusion of dyed lipids observed as fluorescence recovery after photobleaching confirmed the presence of lipid bilayers. Immobilization of the His-tagged membrane proteins on the NTA-modified PMAA brush near the pore edges is characterized by fluorescence microscopy. This system allows us to adjust the protein density in free-standing bilayers, which are stabilized by the polymer brush underneath. The potential application of the integrated platform for ion channel protein assays is demonstrated.

  19. 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

  20. 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

  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. THE POLYMERIZATION OF ACTIN: ITS ROLE IN THE GENERATION OF THE ACROSOMAL PROCESS OF CERTAIN ECHINODERM SPERM

    PubMed Central

    Tilney, Lewis G.; Hatano, Sadashi; Ishikawa, Harunori; Mooseker, Mark S.

    1973-01-01

    When Asterias or Thyone sperm come in contact with egg jelly, a long process which in Thyone measures up to 90 µm in length is formed from the acrosomal region. This process can be generated in less than 30 s. Within this process is a bundle of microfilaments. Water extracts prepared from acetone powders of Asterias sperm contain a protein which binds rabbit skeletal muscle myosin forming a complex whose viscosity is reduced by ATP. Within this extract is a protein with the same molecular weight as muscle actin. It can be purified either by collecting the pellet produced after the addition of Mg++ or by reextracting an acetone powder of actomyosin prepared by the addition of highly purified muscle myosin to the extract. The sperm actin can be polymerized and by electron microscopy the polymer is indistinguishable from muscle F-actin. The sperm actin was shown to be localized in the microfilaments in the acrosomal processes by: (a) heavy meromyosin binding in situ, (b) sodium dodecyl sulfate (SDS) gel electrophoresis of the isolated acrosomal processes and a comparison to gels of flagella which contain no band corresponding to the molecular weight of actin, and (c) SDS gel electrophoresis of the extract from isolated acrosomal caps. Since the precursor for the microfilaments in the unreacted sperm appears amorphous, we suspected that the force for the generation of the acrosomal process is brought about by the polymerization of the sperm actin. This supposition was confirmed, for when unreacted sperm were lysed with the detergent Triton X-100 and the state of the actin in the sperm extract was analyzed by centrifugation, we determined that at least 80% of the actin in the unreacted sperm was in the monomeric state. PMID:4356568

  3. 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

  4. 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

  5. An Integrated Field-Effect Microdevice for Monitoring Membrane Transport in Xenopus laevis Oocytes via Lateral Proton Diffusion

    PubMed Central

    Schaffhauser, Daniel Felix; Patti, Monica; Goda, Tatsuro; Miyahara, Yuji; Forster, Ian Cameron; Dittrich, Petra Stephanie

    2012-01-01

    An integrated microdevice for measuring proton-dependent membrane activity at the surface of Xenopus laevis oocytes is presented. By establishing a stable contact between the oocyte vitelline membrane and an ion-sensitive field-effect (ISFET) sensor inside a microperfusion channel, changes in surface pH that are hypothesized to result from facilitated proton lateral diffusion along the membrane were detected. The solute diffusion barrier created between the sensor and the active membrane area allowed detection of surface proton concentration free from interference of solutes in bulk solution. The proposed sensor mechanism was verified by heterologously expressing membrane transport proteins and recording changes in surface pH during application of the specific substrates. Experiments conducted on two families of phosphate-sodium cotransporters (SLC20 & SLC34) demonstrated that it is possible to detect phosphate transport for both electrogenic and electroneutral isoforms and distinguish between transport of different phosphate species. Furthermore, the transport activity of the proton/amino acid cotransporter PAT1 assayed using conventional whole cell electrophysiology correlated well with changes in surface pH, confirming the ability of the system to detect activity proportional to expression level. PMID:22792166

  6. Study of Acrosome Formation, Interspecific and Intraspecific, in the Testicular Lobes of Some Pentatomid Species

    PubMed Central

    Souza, Hederson V.; Itoyama, Mary M.

    2010-01-01

    The objective was to compare the formation of the acrosome in the testicular lobes of the species Antiteuchus tripterus F. and Platycarenus umbractulatus F., belonging to the subfamily Discocephalinae, and Euschistus heros F., Mormidea quinqueluteum L., Oebalus sp. and Thyanta perditor F., belonging to the Pentatominae (Heteroptera: Pentatomidae). It was found that, in general, the behavior of periodic acid Schiff-positive granules for all of the species analyzed is similar for these species. In the beginning of spermiogenesis, there is a central granule that migrates to one of the extremities of the spermatid, and later, it becomes elongated and cannot be distinguished in the spermatozoa. Some species such as A. tripterus, E. heros and P. umbractulatus showed significant differences in the behavior of the PAS-positive granule in certain lobes, suggesting the formation of spermatozoa with non-fertile functions. PMID:20883131

  7. 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.

  8. 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. PMID:26141781

  9. The integral membrane enzyme PagP alternates between two dynamically distinct states.

    PubMed

    Hwang, Peter M; Bishop, Russell E; Kay, Lewis E

    2004-06-29

    PhoPQ-activated gene P (PagP) is an integral membrane enzyme that transfers the sn-1 palmitate chain from phospholipid to lipopolysaccharide in Gram-negative bacteria. A recent x-ray crystallographic study established that the sn-1 palmitate binds within a long cavity at the center of the PagP beta barrel. The high mobility required to permit substrate entry into the central core of the barrel contrasts with the need to assemble a well defined structure in the peripheral loops, where many key catalytic residues are located. To gain insight into how dynamics relate to the function of PagP, the enzyme was reconstituted into CYFOS-7, a detergent that supports enzymatic activity. Under these conditions, PagP exists in equilibrium between two states, relaxed (R) and tense (T). The kinetics and thermodynamics of the interchange have been investigated by (1)H-(15)N NMR spectroscopy, with Delta H = -10.7 kcal/mol and Delta S = -37.5 cal/mol.K for the R--> T transition. A comparison of chemical shifts between the two states indicates that major structural changes occur in the large extracellular L1 loop and adjacent regions of the beta barrel. In addition to the R,T interconversion, other conformational exchange processes are observed in the R state, showing it to be quite flexible. Thus a picture emerges in which substrate entry is facilitated by the mobility of the R state, whereas the relatively rigid T state adopts a radically different conformation in a region of the protein known to be essential for catalysis. The ability to switch between dynamically distinct states may be a key feature of the catalytic cycle of PagP. PMID:15210985

  10. Advanced treatment of biologically pretreated coal gasification wastewater by a novel integration of catalytic ultrasound oxidation and membrane bioreactor.

    PubMed

    Jia, Shengyong; Han, Hongjun; Zhuang, Haifeng; Xu, Peng; Hou, Baolin

    2015-01-01

    Laboratorial scale experiments were conducted to investigate a novel system integrating catalytic ultrasound oxidation (CUO) with membrane bioreactor (CUO-MBR) on advanced treatment of biologically pretreated coal gasification wastewater. Results indicated that CUO with catalyst of FeOx/SBAC (sewage sludge based activated carbon (SBAC) which loaded Fe oxides) represented high efficiencies in eliminating TOC as well as improving the biodegradability. The integrated CUO-MBR system with low energy intensity and high frequency was more effective in eliminating COD, BOD5, TOC and reducing transmembrane pressure than either conventional MBR or ultrasound oxidation integrated MBR. The enhanced hydroxyl radical oxidation, facilitation of substrate diffusion and improvement of cell enzyme secretion were the mechanisms for CUO-MBR performance. Therefore, the integrated CUO-MBR was the promising technology for advanced treatment in engineering applications. PMID:25936898

  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. 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. PMID:18575108

  13. 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...

  14. Treatment of domestic wastewater by an integrated anaerobic fluidized-bed membrane bioreactor under moderate to low temperature conditions.

    PubMed

    Gao, Da-Wen; Hu, Qi; Yao, Chen; Ren, Nan-Qi

    2014-05-01

    The performance of a novel integrated anaerobic fluidized-bed membrane bioreactor (IAFMBR) for treating practical domestic wastewater was investigated at a step dropped temperature from 35, 25, to 15°C. The COD removal was 74.0 ± 3.7%, 67.1 ± 2.9% and 51.1 ± 2.6% at 35, 25 and 15°C, respectively. The COD removal depended both on influent strength and operational temperature. The accumulation of VFAs (Volatile Fatty Acids) was affected by temperature, and acetic acid was the most sensitive one to the decrease of temperature. The methanogenic activity of the sludge decreased eventually and the methane yield was dropped from 0.17 ± 0.03, 0.15 ± 0.02 to 0.10 ± 0.01 L/Ld. And as compared with a mesophilic temperature, a low temperature can accelerate membrane biofouling. Proteins were the dominant matters causing membrane fouling at low temperature and membrane fouling can be mitigated by granular active carbon (GAC) through protein absorption. PMID:24650533

  15. Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein--localization of a targeting domain.

    PubMed

    Söderqvist, H; Imreh, G; Kihlmark, M; Linnman, C; Ringertz, N; Hallberg, E

    1997-12-15

    The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear pores of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticulum/nuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121. PMID:9461306

  16. 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

  17. 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. PMID:26582474

  18. Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity*

    PubMed Central

    Golczak, Marcin; Kiser, Philip D.; Lodowski, David T.; Maeda, Akiko; Palczewski, Krzysztof

    2010-01-01

    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 A2 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 phospholipid

  19. A yeast toxic mutant of HET-s amyloid disrupts membrane integrity.

    PubMed

    Ta, Ha Phuong; Berthelot, Karine; Coulary-Salin, Bénédicte; Castano, Sabine; Desbat, Bernard; Bonnafous, Pierre; Lambert, Olivier; Alves, Isabel; Cullin, Christophe; Lecomte, Sophie

    2012-09-01

    Many studies have pointed out the interaction between amyloids and membranes, and their potential involvement in amyloid toxicity. Previously, we generated a yeast toxic amyloid mutant (M8) from the harmless amyloid protein by changing a few residues of the Prion Forming Domain of HET-s (PFD HET-s(218-289)) and clearly demonstrated the complete different behaviors of the non-toxic Wild Type (WT) and toxic amyloid (called M8) in terms of fiber morphology, aggregation kinetics and secondary structure. In this study, we compared the interaction of both proteins (WT and M8) with membrane models, as liposomes or supported bilayers. We first demonstrated that the toxic protein (M8) induces a significant leakage of liposomes formed with negatively charged lipids and promotes the formation of microdomains inside the lipid bilayer (as potential "amyloid raft"), whereas the non-toxic amyloid (WT) only binds to the membrane without further perturbations. The secondary structure of both amyloids interacting with membrane is preserved, but the anti-symmetric PO(2)(-) vibration is strongly shifted in the presence of M8. Secondly, we established that the presence of membrane models catalyzes the amyloidogenesis of both proteins. Cryo-TEM (cryo-transmission electron microscopy) images show the formation of long HET-s fibers attached to liposomes, whereas a large aggregation of the toxic M8 seems to promote a membrane disruption. This study allows us to conclude that the toxicity of the M8 mutant could be due to its high propensity to interact and disrupt lipid membranes. PMID:22562024

  20. 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

  1. 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

  2. Evidence in support of a role for Ca(2+)-activated K+ channels in the hamster sperm acrosome reaction.

    PubMed

    Llanos, M N

    1994-08-01

    The sperm acrosome reaction (AR) is a crucial step for mammalian fertilization. This work describes experiments to test the effect of the cesium ion (Cs+) and charybdotoxin (ChTX) on the Ca2+ or Na+/K+ ionophores stimulated hamster sperm AR in vitro. Cs+ and ChTX, a polypeptide toxin from the venom of the scorpion Leirus quinquestriatus, are considered blockers of Ca(2+)-activated K+ channels in several somatic cells. Both agents inhibited the AR by 55-66%. The inhibition was completely reversed by the Na+/K+ ionophore nigericin, but not by the Ca2+ ionophore A-23187. Results give evidence in support of a role for Ca(2+)-activated K+ channels in K+ influx required for the occurrence of the hamster sperm acrosome reaction. PMID:7520055

  3. 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

  4. Quality control of mitochondrial protein synthesis is required for membrane integrity and cell fitness

    PubMed Central

    Richter, Uwe; Lahtinen, Taina; Marttinen, Paula; Suomi, Fumi

    2015-01-01

    Mitochondrial ribosomes synthesize a subset of hydrophobic proteins required for assembly of the oxidative phosphorylation complexes. This process requires temporal and spatial coordination and regulation, so quality control of mitochondrial protein synthesis is paramount to maintain proteostasis. We show how impaired turnover of de novo mitochondrial proteins leads to aberrant protein accumulation in the mitochondrial inner membrane. This creates a stress in the inner membrane that progressively dissipates the mitochondrial membrane potential, which in turn stalls mitochondrial protein synthesis and fragments the mitochondrial network. The mitochondrial m-AAA protease subunit AFG3L2 is critical to this surveillance mechanism that we propose acts as a sensor to couple the synthesis of mitochondrial proteins with organelle fitness, thus ensuring coordinated assembly of the oxidative phosphorylation complexes from two sets of ribosomes. PMID:26504172

  5. Ethylene Action and Loss of Membrane Integrity during Petal Senescence in Tradescantia1

    PubMed Central

    Suttle, Jeffrey C.; Kende, Hans

    1980-01-01

    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. PMID:16661332

  6. Cold Stress Makes Escherichia coli Susceptible to Glycopeptide Antibiotics by Altering Outer Membrane Integrity.

    PubMed

    Stokes, Jonathan M; French, Shawn; Ovchinnikova, Olga G; Bouwman, Catrien; Whitfield, Chris; Brown, Eric D

    2016-02-18

    A poor understanding of the mechanisms by which antibiotics traverse the outer membrane remains a considerable obstacle to the development of novel Gram-negative antibiotics. Herein, we demonstrate that the Gram-negative bacterium Escherichia coli becomes susceptible to the narrow-spectrum antibiotic vancomycin during growth at low temperatures. Heterologous expression of an Enterococcus vanHBX vancomycin resistance cluster in E. coli confirmed that the mechanism of action was through inhibition of peptidoglycan biosynthesis. To understand the nature of vancomycin permeability, we screened for strains of E. coli that displayed resistance to vancomycin at low temperature. Surprisingly, we observed that mutations in outer membrane biosynthesis suppressed vancomycin activity. Subsequent chemical analysis of lipopolysaccharide from vancomycin-sensitive and -resistant strains confirmed that suppression was correlated with truncations in the core oligosaccharide of lipopolysaccharide. These unexpected observations challenge the current understanding of outer membrane permeability, and provide new chemical insights into the susceptibility of E. coli to glycopeptide antibiotics. PMID:26853624

  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. Evaluation of Time-Temperature Integrators (TTIs) with Microorganism-Entrapped Microbeads Produced Using Homogenization and SPG Membrane Emulsification Techniques.

    PubMed

    Rahman, A T M Mijanur; Lee, Seung Ju; Jung, Seung Won

    2015-12-28

    A comparative study was conducted to evaluate precision and accuracy in controlling the temperature dependence of encapsulated microbial time-temperature integrators (TTIs) developed using two different emulsification techniques. Weissela cibaria CIFP 009 cells, immobilized within 2% Na-alginate gel microbeads using homogenization (5,000, 7,000, and 10,000 rpm) and Shirasu porous glass (SPG) membrane technologies (10 μm), were applied to microbial TTIs. The prepared micobeads were characterized with respect to their size, size distribution, shape and morphology, entrapment efficiency, and bead production yield. Additionally, fermentation process parameters including growth rate were investigated. The TTI responses (changes in pH and titratable acidity (TA)) were evaluated as a function of temperature (20°C, 25°C, and 30°C). In comparison with conventional methods, SPG membrane technology was able not only to produce highly uniform, small-sized beads with the narrowest size distribution, but also the bead production yield was found to be nearly 3.0 to 4.5 times higher. However, among the TTIs produced using the homogenization technique, poor linearity (R(2)) in terms of TA was observed for the 5,000 and 7,000 rpm treatments. Consequently, microbeads produced by the SPG membrane and by homogenization at 10,000 rpm were selected for adjusting the temperature dependence. The Ea values of TTIs containing 0.5, 1.0, and 1.5 g microbeads, prepared by SPG membrane and conventional methods, were estimated to be 86.0, 83.5, and 76.6 kJ/mol, and 85.5, 73.5, and 62.2 kJ/mol, respectively. Therefore, microbial TTIs developed using SPG membrane technology are much more efficient in controlling temperature dependence. PMID:26370796

  10. Effect of bile salts on the DNA and membrane integrity of enteric bacteria.

    PubMed

    Merritt, Megan E; Donaldson, Janet R

    2009-12-01

    Enteric bacteria are able to resist the high concentrations of bile encountered throughout the gastrointestinal tract. Here we review the current mechanisms identified in the enteric bacteria Salmonella, Escherichia coli, Bacillus cereus and Listeria monocytogenes to resist the dangerous effects of bile. We describe the role of membrane transport systems, and their connection with DNA repair pathways, in conferring bile resistance to these enterics. We discuss the findings from recent investigations that indicate bile tolerance is dependent upon being able to resist the detergent properties of bile at both the membrane and DNA level. PMID:19762477

  11. 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. PMID:24613917

  12. Acrosome reaction of sperm in the mud crab Scylla serrata as a sensitive toxicity test for metal exposures.

    PubMed

    Zhang, Ziping; Cheng, Hua; Wang, Yilei; Wang, Shuhong; Xie, Fangjing; Li, Shaojin

    2010-01-01

    In order to test the sensitivity of the sperm cell of the mud crab Scylla serrata to heavy metals, the toxic effects of Ag+, Cd2+, Cu2+, and Zn2+ on the acrosome reaction (AR) were studied by artificially inducing the AR of sperm exposed to heavy metals, counting the AR rates by light microscopy, and observing structural changes in sperm by transmission electron microscopy. The AR in S. serrata occurs at two stages. The first stage (ARI) is the eversion of the subacrosomal material. The second stage (ARII) is the ejection of the acrosomal filament. The results showed the EC50 values of the AR based on (ARI + ARII)% for Ag+, Cd2+, Cu2+, and Zn2+ were 10.02, 2.14, 13.69, and 2.21 microg/L, and the EC50 values based on ARII % of Ag+, Cd2+, Cu2+, and Zn2+ were 1.96, 0.20, 1.46, and 0.34 microg/L. The order of toxicity is Cd2+ > Zn2+ > Cu2+ > Ag+ based on the percentage of reacted sperm at the second stage. Sperm cells exposed to heavy metals showed an increased rate of swelling, shape irregularities, and the acrosomal filament of some sperm cells was, crooked, ruptured, and even dissolved. The AR of the sperm cell from S. serrata is more sensitive to the tested heavy metals compared to sea urchin sperm cell toxicity tests. PMID:19399549

  13. Contactless microfluidic pumping using microchannel-integrated carbon black composite membranes.

    PubMed

    Fu, Xiaotong; Gagnon, Zachary

    2015-09-01

    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. Valproic Acid Affects Membrane Trafficking and Cell-Wall Integrity in Fission Yeast

    PubMed Central

    Miyatake, Makoto; Kuno, Takayoshi; Kita, Ayako; Katsura, Kosaku; Takegawa, Kaoru; Uno, Satoshi; Nabata, Toshiya; Sugiura, Reiko

    2007-01-01

    Valproic acid (VPA) is widely used to treat epilepsy and manic-depressive illness. Although VPA has been reported to exert a variety of biochemical effects, the exact mechanisms underlying its therapeutic effects remain elusive. To gain further insights into the molecular mechanisms of VPA action, a genetic screen for fission yeast mutants that show hypersensitivity to VPA was performed. One of the genes that we identified was vps45+, which encodes a member of the Sec1/Munc18 family that is implicated in membrane trafficking. Notably, several mutations affecting membrane trafficking also resulted in hypersensitivity to VPA. These include ypt3+ and ryh1+, both encoding a Rab family protein, and apm1+, encoding the μ1 subunit of the adaptor protein complex AP-1. More importantly, VPA caused vacuolar fragmentation and inhibited the glycosylation and the secretion of acid phosphatase in wild-type cells, suggesting that VPA affects membrane trafficking. Interestingly, the cell-wall-damaging agents such as micafungin or the inhibition of calcineurin dramatically enhanced the sensitivity of wild-type cells to VPA. Consistently, VPA treatment of wild-type cells enhanced their sensitivity to the cell-wall-digesting enzymes. Altogether, our results suggest that VPA affects membrane trafficking, which leads to the enhanced sensitivity to cell-wall damage in fission yeast. PMID:17287531

  15. A tag at the carboxy terminus prevents membrane integration of VDAC1 in mammalian mitochondria.

    PubMed

    Kozjak-Pavlovic, Vera; Ross, Katharina; Götz, Monika; Goosmann, Christian; Rudel, Thomas

    2010-03-19

    beta-Barrel proteins are found in the outer membranes of bacteria, chloroplasts and mitochondria. The evolutionary conserved sorting and assembly machinery (SAM complex) assembles mitochondrial beta-barrel proteins, such as voltage-dependent anion-selective channel 1 (VDAC1), into complexes in the outer membrane by recognizing a sorting beta-signal in the carboxy-terminal part of the protein. Here we show that in mammalian mitochondria, masking of the C-terminus of beta-barrel proteins by a tag leads to accumulation of soluble misassembled protein in the intermembrane space, which causes mitochondrial fragmentation and loss of membrane potential. A similar phenotype is observed if the beta-signal is shortened, removed or when the conserved hydrophobic residues in the beta-signal are mutated. The length of the tag at the C-terminus is critical for the assembly of VDAC1, as well as the amino acid residues at positions 130, 222, 225 and 251 of the protein. We propose that if the recognition of the beta-signal or the folding of the beta-barrel proteins is inhibited, the nonassembled protein will accumulate in the intermembrane space, aggregate and damage mitochondria. This effect offers easy tools for studying the requirements for the membrane assembly of beta-barrel proteins, but also advises caution when interpreting the outcome of the beta-barrel protein overexpression experiments. PMID:20117113

  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. Tudor Domain Containing Protein TDRD12 Expresses at the Acrosome of Spermatids in Mouse Testis.

    PubMed

    Kim, Min; Ki, Byeong Seong; Hong, Kwonho; Park, Se-Pill; Ko, Jung-Jae; Choi, Youngsok

    2016-07-01

    Tdrd12 is one of tudor domain containing (Tdrd) family members. However, the expression pattern of Tdrd12 has not been well studied. To compare the expression levels of Tdrd12 in various tissues, real time-polymerase chain reaction was performed using total RNAs from liver, small intestine, heart, brain, kidney, lung, spleen, stomach, uterus, ovary, and testis. Tdrd12 mRNA was highly expressed in testis. Antibody against mouse TDRD12 were generated using amino acid residues SQRPNEKPLRLTEKKDC of TDRD12 to investigate TDRD12 localization in testis. Immunostaining assay shows that TDRD12 is mainly localized at the spermatid in the seminiferous tubules of adult testes. During postnatal development, TDRD12 is differentially expressed. TDRD12 was detected in early spermatocytes at 2 weeks and TDRD12 was localized at acrosome of the round spermatids. TDRD12 expression was not co-localized with TDRD1 which is an important component of piRNA pathway in germ cells. Our results indicate that TDRD12 may play an important role in spermatids and function as a regulator of spermatogenesis in dependent of TDRD1. PMID:26954166

  18. 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

  19. 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

  20. 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

  1. An Essential Tyrosine Phosphatase Homolog Regulates Cell Separation, Outer Membrane Integrity, and Morphology in Caulobacter crescentus ▿ †

    PubMed Central

    Shapland, Elaine B.; Reisinger, Sarah J.; Bajwa, Amrita K.; Ryan, Kathleen R.

    2011-01-01

    Although reversible phosphorylation on tyrosine residues regulates the activity of many eukaryotic proteins, there are few examples of this type of regulation in bacteria. We have identified the first essential tyrosine phosphatase homolog in a bacterium, Caulobacter crescentusCtpA. ctpAmutants with altered active-site residues are nonviable, and depletion of CtpA yields chains of cells with blebbed outer membranes, linked by unresolved peptidoglycan. CtpA overexpression reduces cell curvature in a manner similar to deleting the intermediate filament protein crescentin, but it does not disrupt crescentin localization or membrane attachment. Although it has no obvious signal sequence or transmembrane-spanning domains, CtpA associates with the Caulobacterinner membrane. Immunolocalization experiments suggest that CtpA accumulates at the division site during the last quarter of the cell cycle. We propose that CtpA dephosphorylates one or more proteins involved in peptidoglycan biosynthesis or remodeling, which in turn affect cell separation, cell envelope integrity, and vibrioid morphology. PMID:21705597

  2. Influence of thermal extraction of extracellular polymeric substances on cell integrity in activated sludge and membrane bioreactor samples.

    PubMed

    Antonelli, M; Bialek, K; Teli, A; Citterio, S; Malpei, F

    2011-02-01

    The influence of the soluble microbial products (SMP) and extracellular polymeric substances (EPS) heating extraction method on cell viability was evaluated for each phase of the protocol using epifluorescence microscopy. In addition, the effect of different centrifugation conditions (2700 g at 24 degrees C; 12,000 g at 4 degrees C) was also tested. Sludge samples were collected from a conventional wastewater treatment and a membrane bioreactor (MBR) pilot plant fed in parallel. Results show that different centrifugation parameters do not induce cell membrane damaging. Heating significantly influences membrane integrity; for instance, 75 to 90% of initial viable cells are damaged during this phase, possibly leading to the predominance of protein compared to carbohydrate content. The protein content in EPS is 60 to 88 mg bovine serum albumin/ g volatile suspended solids (VSS); higher values observed in MBR sludge samples are probably attributable to the different characteristics of microbial flocs and process operating parameters. Carbohydrate concentrations are not significantly different regardless of applied procedure and sludge type, and are between 10.4 to 11.6 mg glucose/g VSS. PMID:21449471

  3. The theory of semipermeable vesicles and membranes: An integral-equation approach. III. Vesicles with internal nonpermeating ions

    NASA Astrophysics Data System (ADS)

    Zhou, Yaoqi; Stell, George

    1989-09-01

    The system of a fluid in the presence of a spherical semipermeable vesicle (SPV) with the freely mobile nonpermeating species inside the vesicle is investigated via an integral-equation approach. This system can be used to model certain feature of a biological cell, permeable to simple ions, in which solute proteins inside the cell are unable to permeate its walls. As an illustrative example of the use of our integral equations, the analytical solution for density profiles in the mean-spherical approximation/Debye-Hückel approximation (MSA/DH) is obtained, where the MSA is used to obtain the density profiles near a membrane and the DH approximation to obtain the bulk pair correlation functions. A method which applies to nonmobile protein fixed inside a cell is also considered.

  4. 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

  5. A Procedure-Spanning Analysis of Plasma Membrane Integrity for Assessment of Cell Viability in Sperm Cryopreservation of Zebrafish Danio rerio.

    PubMed

    Yang, Huiping; Daly, Jonathan; Carmichael, Carrie; Matthews, Jen; Varga, Zoltan M; Tiersch, Terrence

    2016-04-01

    The goal of this study was to evaluate plasma membrane integrity and motility for zebrafish sperm quality assessment along the cryopreservation pathway-from sample collection through refrigerated storage, cryoprotectant equilibration, freezing, thawing, and fertilization. The objectives were to: (1) evaluate the effects of osmolality, extender, and refrigerated storage on sperm plasma membrane integrity and motility, and (2) compare cryopreservation of sperm from farm-raised and well-characterized research populations by evaluating motility and membrane integrity of fresh, post-equilibration (before freezing) and post-thaw sperm, and post-thaw fertility. Osmolality, extender, and storage time each influenced sperm motility and membrane integrity. Isotonic osmolality showed the best protection for motility and membrane integrity compared to hypotonic and hypertonic osmolalities. Of the four tested extenders, Hanks' balanced salt solution (HBSS) and Ca(2+)-free HBSS showed the best protection compared with NaCl and glucose, and sperm retained motility and membrane integrity for 24 h of refrigerated storage. Sperm cryopreservation of zebrafish from a farm population (n = 20) and an AB research line (n = 20) showed significant differences in post-thaw fertility (32% ± 18% vs. 73% ± 21%). No differences were found in post-thaw motility, although the farm-raised zebrafish possessed a larger body size, testis weight, and higher fresh motility. Correlation analysis of pooled data did not identify correlations among motility, flow cytometry analysis of membrane integrity and recognizable cells, and post-thaw sperm fertility (p ≥ 0.202). More research is needed to standardize the fertilization conditions especially sperm-to-egg ratio to avoid possible overabundance of sperm to obscure the differences. PMID:26859531

  6. 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

  7. 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.

  8. Integrated hollow fiber membranes for gas delivery into optical waveguide based photobioreactors.

    PubMed

    Ahsan, Syed Saad; Gumus, Abdurrahman; Jain, Aadhar; Angenent, Largus T; Erickson, David

    2015-09-01

    Compact algal reactors are presented with: (1) closely stacked layers of waveguides to decrease light-path to enable larger optimal light-zones; (2) waveguides containing scatterers to uniformly distribute light; and (3) hollow fiber membranes to reduce energy required for gas transfer. The reactors are optimized by characterizing the aeration of different gases through hollow fiber membranes and characterizing light intensities at different culture densities. Close to 65% improvement in plateau peak productivities was achieved under low light-intensity growth experiments while maintaining 90% average/peak productivity output during 7-h light cycles. With associated mixing costs of ∼ 1 mW/L, several magnitudes smaller than closed photobioreactors, a twofold increase is realized in growth ramp rates with carbonated gas streams under high light intensities, and close to 20% output improvement across light intensities in reactors loaded with high density cultures. PMID:26116445

  9. 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

  10. Free-standing alumina nanobottles and nanotubes pre-integrated into nanoporous alumina membranes

    NASA Astrophysics Data System (ADS)

    Fang, Jinghua; Levchenko, Igor; (Ken Ostrikov, Kostya

    2014-08-01

    A novel interfacial structure consisting of long (up to 5 μm), thin (about 300 nm), highly-ordered, free-standing, highly-reproducible aluminum oxide nanobottles and long tubular nanocapsules attached to a rigid, thin (less than 1 μm) nanoporous anodic alumina membrane is fabricated by simple, fast, catalyst-free, environmentally friendly voltage-pulse anodization. A growth mechanism is proposed based on the formation of straight channels in alumina membrane by anodization, followed by neck formation due to a sophisticated voltage control during the process. This process can be used for the fabrication of alumina nanocontainers with highly controllable geometrical size and volume, vitally important for various applications such as material and energy storage, targeted drug and diagnostic agent delivery, controlled drug and active agent release, gene and biomolecule reservoirs, micro-biologically protected platforms, nano-bioreactors, tissue engineering and hydrogen storage.

  11. Influence of the interfacial phase on the structural integrity and oxygen permeability of a dual-phase membrane.

    PubMed

    Sun, Ming; Chen, Xinwei; Hong, Liang

    2013-09-25

    Compositing fluorite Ce0.8Gd0.2O2-δ (CGO) oxide with perovskite La0.4Ba0.6Fe0.8Zn0.2O3-δ (LBFZ) oxide leads to the formation of a minor interfacial BaCeO3 phase upon sintering at 1400 °C. This interfacial composition assures a gastight ceramic membrane with fine grain-boundary structure, in which the LBFZ phase exhibits an improved oxygen permeability over the pristine LBFZ membrane on the same volumetric basis. The presence of the BaCeO3 phase effectively preserves the structural integrity of the composition by limiting the interfacial diffusion of barium ions between LBFZ and CGO. In comparison, replacing CGO with Y0.08Zr0.92O2-δ in the system results in a substantially low oxygen flux due to an overwhelming interfacial diffusion and, consequently, a heavy degradation of LBFZ. Besides structural reinforcement, the high interface between LBFZ and CGO benefits oxygen transport, as is proven through variation of the oxygen partial pressure on the feed side of the membrane and operation temperature. Furthermore, the trade-off between LBFZ loading and interfacial diffusion yields an optimal CGO loading at 40 wt %, which exhibits an oxygen flux of 0.84 cm(3)/cm(2)·min at 950 °C. In summary, the minor interfacial binding between CGO and LBFZ grains is constructive in easing oxygen crossover in the phase boundary with the exception of maintaining membrane structural stability under oxygen permeation conditions. PMID:23977996

  12. 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

  13. A novel approach to analyze membrane proteins by laser mass spectrometry: from protein subunits to the integral complex.

    PubMed

    Morgner, Nina; Kleinschroth, Thomas; Barth, Hans-Dieter; Ludwig, Bernd; Brutschy, Bernhard

    2007-08-01

    A novel laser-based mass spectrometry method termed LILBID (laser-induced liquid bead ion desorption) is applied to analyze large integral membrane protein complexes and their subunits. In this method the ions are IR-laser desorbed from aqueous microdroplets containing the hydrophobic protein complexes solubilized by detergent. The method is highly sensitive, very efficient in sample handling, relatively tolerant to various buffers, and detects the ions in narrow, mainly low-charge state distributions. The crucial experimental parameter determining whether the integral complex or its subunits are observed is the laser intensity: At very low intensity level corresponding to an ultrasoft desorption, the intact complexes, together with few detergent molecules, are transferred into vacuum. Under these conditions the oligomerization state of the complex (i.e., its quaternary structure) may be analyzed. At higher laser intensity, complexes are thermolyzed into subunits, with any residual detergent being stripped off to yield the true mass of the polypeptides. The model complexes studied are derived from the respiratory chain of the soil bacterium Paracoccus denitrificans and include complexes III (cytochrome bc(1) complex) and IV (cytochrome c oxidase). These are well characterized multi-subunit membrane proteins, with the individual hydrophobic subunits being composed of up to 12 transmembrane helices. PMID:17544294

  14. Effect of supplementation of butylated hydroxytoluene on post-thaw sperm viability, motility and membrane integrity of Hariana bulls

    PubMed Central

    Patel, Akhil; Saxena, Atul; Swain, Dilip Kumar; Yadav, Dushyant; Yadav, Sanjay Singh; Kumar, Abhishek; Kumar, Anuj

    2015-01-01

    Aim: This study was aimed to see the beneficial effect of butylated hydroxytoluene (BHT) as a semen additive of Hariana bull semen. Materials and Methods: The study was carried out in Hariana bulls. Twenty-four ejaculates from two bulls were used for this study. Each ejaculate was extended with standard glycerolated egg yolk tris extender and supplemented with BHT at two concentrations as 0.5 mM (T1) and 1.0 mM (T2). After dilution, equilibration and 24 h of cryopreservation, the samples were analyzed for progressive motility, sperm viability and membrane integrity. Results: Progressive motility, sperm viability and sperm membrane integrity were significantly (p<0.05) increased in the samples fortified with BHT as compared to the control during the process of cryopreservation and thawing. The BHT concentration of 1 mM revealed better results as compared to 0.5 mM. Conclusion: Addition of 1.0 mM BHT was found better in cryopreservation of Hariana bull semen compared to 0.5 mM BHT and control samples. The addition of BHT has improved the sperm quality by acting as an antioxidant thereby reducing the lipid peroxidation of the sperms. PMID:27065652

  15. 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.

  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. PMID:26859121

  17. Investigating the Structural Properties of Integral Membrane Proteins with Pulsed EPR Spectroscopy

    NASA Astrophysics Data System (ADS)

    Lorigan, Gary

    2013-03-01

    Very limited structural and dynamic information on proteins embedded inside a membrane currently exist, because they are difficulty to crystalize. New biophysical/structural biology methods are needed to probe these systems in a lipid bilayer. The Lorigan lab is applying unique hybrid NMR and spin-label EPR spectroscopic techniques to study membrane proteins. Magnetic resonance spectroscopic data of 15N-, 2H-labeled and/or spin-labeled membrane proteins incorporated into vesicles and bicelles will be presented. State-of-the-art pulsed EPR techniques such as Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy, and Double Electron-Electron Resonance (DEER) spectroscopy will be used. The ESEEM technique can determine short to medium range distances (out to about 8 Å) between a site-specific nitroxide spin label and a nearby NMR-active isotopic labeled residue for a variety of different peptides and proteins which ultimately can be used to determine the difference between an α-helical and β-sheet secondary structure. DEER can be used to measure distances between 2 spin labels out to about 70 Å. We have shown a huge improvement is sensitivity with DEER measurements at Q-band when compared to X-band.

  18. Integrated carboxylic carbon nanotube pathways with membranes for voltage-activated humidity detection and microclimate regulation.

    PubMed

    Pingitore, V; Miriello, D; Drioli, E; Gugliuzza, A

    2015-06-14

    This work describes some single walled carboxylic carbon nanotubes with outstanding transport properties when assembled in a 3D microarray working like a humidity membrane-sensor and an adjustable moisture regulator. Combined nano-assembly approaches are used to build up a better quality pathway through which assisted-charge and mass transport synchronically takes place. The structure-electrical response relationship is found, while controllable and tunable donor-acceptor interactions established at material interfaces are regarded as key factors for the accomplishment of charge transportation, enhanced electrical responses and adjustable moisture exchange. Raman and infrared spectroscopy provides indications about the fine structural and chemical features of the hybrid-composite membranes, resulting in perfect agreement with related morphology and electrical properties. Enhanced and modular electrical response to changes in the surrounding atmosphere is concerned with doping events, while assisted moisture regulation is discussed in relation to swelling and hopping actions. The electro-activated hybrid-composite membrane proposed in this work can be regarded as an attractive 'sense-to-act' precursor for smart long-distance monitoring systems with capability to adapt itself and provide local comfortable microenvironments. PMID:25939404

  19. Micro-chip initiator realized by integrating Al/CuO multilayer nanothermite on polymeric membrane

    NASA Astrophysics Data System (ADS)

    Taton, G.; Lagrange, D.; Conedera, V.; Renaud, L.; Rossi, C.

    2013-10-01

    We have developed a new nanothermite based polymeric electro-thermal initiator for non-contact ignition of a propellant. A reactive Al/CuO multilayer nanothermite resides on a 100 µm thick SU-8/PET (polyethyleneterephtalate) membrane to insulate the reactive layer from the silicon bulk substrate. When current is supplied to the initiator, the chemical reaction Al+CuO occurs and sparkles are spread to a distance of several millimeters. A micro-manufacturing process for fabricating the initiator is presented and the electrical behaviors of the ignition elements are also investigated. The characteristics of the initiator made on a 100 µm thick SU-8/PET membrane were compared to two bulk electro-thermal initiators: one on a silicon and one on a Pyrex substrate. The PET devices give 100% of Al/CuO ignition success for an electrical current >250 mA. Glass based reactive initiators give 100% of Al/CuO ignition success for an electrical current >500 mA. Reactive initiators directly on silicon cannot initiate even with a 4 A current. At low currents (<1 A), the initiation time is two orders of magnitude longer for Pyrex initiator compared to those obtained for PET initiator technology. We also observed that, the Al/CuO thermite film on PET membrane reacts within 1 ms (sparkles duration) whereas it reacts within 4 ms on Pyrex. The thermite reaction is 40 times greater in intensity using the PET substrate in comparison to Pyrex.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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. PMID:25158006

  5. Membrane potential-dependent integration of synaptic inputs in entorhinal stellate neurons.

    PubMed

    Economo, Michael N; Martínez, Joan José; White, John A

    2014-12-01

    Stellate cells (SCs) of the medial entorhinal cortex exhibit robust spontaneous membrane-potential oscillations (MPOs) in the theta (4-12 Hz) frequency band as well as theta-frequency resonance in their membrane impedance spectra. Past experimental and modeling work suggests that these features may contribute to the phase-locking of SCs to the entorhinal theta rhythm and may be important for forming the hexagonally tiled grid cell place fields exhibited by these neurons in vivo. Among the major biophysical mechanisms contributing to MPOs is a population of persistent (non-inactivating or slowly inactivating) sodium channels. The resulting persistent sodium conductance (GNaP ) gives rise to an apparent increase in input resistance as the cell approaches threshold. In this study, we used dynamic clamp to test the hypothesis that this increased input resistance gives rise to voltage-dependent, and thus MPO phase-dependent, changes in the amplitude of excitatory and inhibitory post-synaptic potential (PSP) amplitudes. We find that PSP amplitude depends on membrane potential, exhibiting a 5-10% increase in amplitude per mV depolarization. The effect is larger than-and sums quasi-linearly with-the effect of the synaptic driving force, V - Esyn . Given that input-driven MPOs 10 mV in amplitude are commonly observed in MEC stellate cells in vivo, this voltage- and phase-dependent synaptic gain is large enough to modulate PSP amplitude by over 50% during theta-frequency MPOs. Phase-dependent synaptic gain may therefore impact the phase locking and phase precession of grid cells in vivo to ongoing network oscillations. © 2014 Wiley Periodicals, Inc. PMID:25044927

  6. 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

  7. 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

  8. Probing the potential of apigenin liposomes in enhancing bacterial membrane perturbation and integrity loss.

    PubMed

    Banerjee, Kacoli; Banerjee, Shubhadeep; Das, Subhayan; Mandal, Mahitosh

    2015-09-01

    Along with discovery of new antibacterial agents, it is important to develop novel drug delivery systems to effectively deliver drugs within bacterial cells for enhanced therapeutic activity. Liposomes have been extensively investigated as pharmaceutical carriers for improvement of therapeutic index of antimicrobial agents. The aim of this present study was to evaluate the antibacterial activity of free and liposomal formulation of apigenin, a plant based isoflavone and elucidate the mode of action. Distearoylphosphatidylcholine liposomes were prepared having nano-range particle size (104.3±1.8 nm), narrow particle distribution (0.204) and high encapsulation efficiency of apigenin (89.9±2.31%). Antibacterial activity of apigenin and efficacy of liposome-mediated apigenin delivery were determined from minimum inhibitory concentration values. Interaction studies using electron microscopy revealed adherence and fusion of liposomal apigenin with the bacteria causing membrane perturbation through reactive oxygen species generation which was evaluated by epi-fluorescence microscopy and fluorescence activated cell sorting. The interaction of apigenin liposomes with bacterial membrane increased intracellular drug concentration and thus, can be employed to deliver apigenin within cells to augment its antibacterial activity. Increased efficacy and hemocompatibility of this formulation paves way for future evaluation of underlying molecular mechanisms and in vivo testing for enhanced therapeutic effects. PMID:25965432

  9. Biodegradation characteristics and size fractionation of landfill leachate for integrated membrane treatment.

    PubMed

    Insel, Güçlü; Dagdar, Mina; Dogruel, Serdar; Dizge, Nadir; Ubay Cokgor, Emine; Keskinler, Bülent

    2013-09-15

    The fate of organics and nitrogen during the biological treatment with MBR and subsequent membrane filtration processes (nano filtration, NF; reverse osmosis, RO) were investigated for a landfill leachate. The chemical oxygen demand (COD) and total Kjeldahl nitrogen (TKN) removal performances of membrane bioreactor (MBR) were obtained to be around 89% and 85%, respectively. The effluent COD of MBR was measured to be 1935 mg/L (30 kDa) which is much lower than experimentally determined soluble inert COD of 3200 mg/L using 0.45 μm filter. The readily and slowly biodegradable COD fractions were estimated to be 17% and 52% of raw influent COD, respectively. The respirometry based modeling test performed on raw leachate exhibited much slower degradation kinetics compared to municipal wastewater. A unique subset of model parameters was extracted from batch respirometry by using acclimated MBR sludge. The sequential ultrafiltration (UF) experiments (particle size distribution, PSD) revealed that most of the organics was below 2 nm filter mesh size. In addition, NF/RO post treatment after MBR system was required to increase COD and total nitrogen (TN) removal performances up to 99%. Relatively lower salt rejection rates around 94% was obtained for RO system as a post treatment of MBR system. PMID:23856313

  10. 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

  11. 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

  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. PMID:27093351

  13. Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status

    SciTech Connect

    Cummins, J.M.; Fleming, A.D.; Crozet, N.; Kuehl, T.J.; Kosower, N.S.; Yanagimachi, R.

    1986-03-01

    Living spermatozoa of seven mammalian species were treated with the thiol-alkylating fluorescent labelling compound, monobromobimane (MBBR). MB-labelling alone had no effect on sperm motility, nor on the time course or ability of golden hamster spermatozoa to undergo the acrosome reaction when capacitated in vitro. Exposure of MB-labelled spermatozoa to ultraviolet (UV) light and excitation of the MB fluorochrome resulted in virtually immediate immobilization of the spermatozoa without affecting acrosomal status. UV exposure of unlabelled spermatozoa for up to 30 sec had no effect upon motility. Immobilization of MB-labelled spermatozoa depended on the midpiece being irradiated, as irradiation of the head alone, or of the more distal parts of the principal piece, had little or no effect upon motility. Labelling with MB followed by immobilization of individually selected spermatozoa was most useful for detailing the course and site of occurrence of the acrosome reaction during penetration of the cumulus oophorus by golden hamster spermatozoa in vitro. In these often hyperactivated spermatozoa, precise determination of the acrosomal status could not often otherwise be made due to the difficulty in visualizing the acrosomal region of a vigorously thrashing, hyperactivated spermatozoon. This technique should prove valuable in a variety of studies on sperm motility, capacitation and fertilization, and could also be extended to other cell systems.

  14. 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. 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%.

  16. Modeling of an integrated fermentation/membrane extraction process for the production of 2-phenylethanol and 2-phenylethylacetate.

    PubMed

    Adler, Philipp; Hugen, Thorsten; Wiewiora, Marzena; Kunz, Benno

    2011-03-01

    An unstructured model for an integrated fermentation/membrane extraction process for the production of the aroma compounds 2-phenylethanol and 2-phenylethylacetate by Kluyveromyces marxianus CBS 600 was developed. The extent to which this model, based only on data from the conventional fermentation and separation processes, provided an estimation of the integrated process was evaluated. The effect of product inhibition on specific growth rate and on biomass yield by both aroma compounds was approximated by multivariate regression. Simulations of the respective submodels for fermentation and the separation process matched well with experimental results. With respect to the in situ product removal (ISPR) process, the effect of reduced product inhibition due to product removal on specific growth rate and biomass yield was predicted adequately by the model simulations. Overall product yields were increased considerably in this process (4.0 g/L 2-PE+2-PEA vs. 1.4 g/L in conventional fermentation) and were even higher than predicted by the model. To describe the effect of product concentration on product formation itself, the model was extended using results from the conventional and the ISPR process, thus agreement between model and experimental data improved notably. Therefore, this model can be a useful tool for the development and optimization of an efficient integrated bioprocess. PMID:22112913

  17. 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

  18. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms

    PubMed Central

    Widdows, Kate L.; Panitchob, Nuttanont; Crocker, Ian P.; Please, Colin P.; Hanson, Mark A.; Sibley, Colin P.; Johnstone, Edward D.; Sengers, Bram G.; Lewis, Rohan M.; Glazier, Jocelyn D.

    2015-01-01

    Uptake of system L amino acid substrates into isolated placental plasma membrane vesicles in the absence of opposing side amino acid (zero-trans uptake) is incompatible with the concept of obligatory exchange, where influx of amino acid is coupled to efflux. We therefore hypothesized that system L amino acid exchange transporters are not fully obligatory and/or that amino acids are initially present inside the vesicles. To address this, we combined computational modeling with vesicle transport assays and transporter localization studies to investigate the mechanisms mediating [14C]l-serine (a system L substrate) transport into human placental microvillous plasma membrane (MVM) vesicles. The carrier model provided a quantitative framework to test the 2 hypotheses that l-serine transport occurs by either obligate exchange or nonobligate exchange coupled with facilitated transport (mixed transport model). The computational model could only account for experimental [14C]l-serine uptake data when the transporter was not exclusively in exchange mode, best described by the mixed transport model. MVM vesicle isolates contained endogenous amino acids allowing for potential contribution to zero-trans uptake. Both L-type amino acid transporter (LAT)1 and LAT2 subtypes of system L were distributed to MVM, with l-serine transport attributed to LAT2. These findings suggest that exchange transporters do not function exclusively as obligate exchangers.—Widdows, K. L., Panitchob, N., Crocker, I. P., Please, C. P., Hanson, M. A., Sibley, C. P., Johnstone, E. D., Sengers, B. G., Lewis, R. M., Glazier, J. D. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms. PMID:25761365

  19. 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

  20. Kinetic folding mechanism of an integral membrane protein examined by pulsed oxidative labeling and mass spectrometry.

    PubMed

    Pan, Yan; Brown, Leonid; Konermann, Lars

    2011-07-01

    We report the application of pulsed oxidative labeling for deciphering the folding mechanism of a membrane protein. SDS-denatured bacteriorhodopsin (BR) was refolded by mixing with bicelles in the presence of free retinal. At various time points (20 ms to 1 day), the protein was exposed to a microsecond ·OH pulse that induces oxidative modifications at solvent-accessible methionine side chains. The extent of labeling was determined by mass spectrometry. These measurements were complemented by stopped-flow spectroscopy. Major time-dependent changes in solvent accessibility were detected for M20 (helix A) and M118 (helix D). Our kinetic data indicate a sequential folding mechanism, consistent with models previously suggested by others on the basis of optical data. Yet, ·OH labeling provides additional structural insights. An initial folding intermediate I(1) gets populated within 20 ms, concomitantly with formation of helix A. Subsequent structural consolidation leads to a transient species I(2). Noncovalent retinal binding to I(2) induces folding of helix D, thereby generating an intermediate I(R). In the absence of retinal, the latter transition does not take place. Hence, formation of helix D depends on retinal binding, whereas this is not the case for helix A. As the cofactor settles deeper into its binding pocket, a final transient species I(R) is generated. This intermediate converts into native BR within minutes by formation of the retinal-K216 Schiff base linkage. The combination of pulsed covalent labeling and optical spectroscopy employed here should also be suitable for exploring the folding mechanisms of other membrane proteins. PMID:21570983

  1. Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation.

    PubMed

    Behringer, Erik J; Segal, Steven S

    2015-10-15

    In resistance arteries, coupling a rise of intracellular calcium concentration ([Ca(2+)]i) to endothelial cell hyperpolarization underlies smooth muscle cell relaxation and vasodilatation, thereby increasing tissue blood flow and oxygen delivery. A controversy persists as to whether changes in membrane potential (V(m)) alter endothelial cell [Ca(2+)]i. We tested the hypothesis that V(m) governs [Ca(2+)]i in endothelium of resistance arteries by performing Fura-2 photometry while recording and controlling V(m) of intact endothelial tubes freshly isolated from superior epigastric arteries of C57BL/6 mice. Under resting conditions, [Ca(2+)]i did not change when V(m) shifted from baseline (∼-40 mV) via exposure to 10 μM NS309 (hyperpolarization to ∼-80 mV), via equilibration with 145 mm [K(+)]o (depolarization to ∼-5 mV), or during intracellular current injection (±0.5 to 5 nA, 20 s pulses) while V(m) changed linearly between ∼-80 mV and +10 mV. In contrast, during the plateau (i.e. Ca(2+) influx) phase of the [Ca(2+)]i response to approximately half-maximal stimulation with 100 nm ACh (∼EC50), [Ca(2+)]i increased as V(m) hyperpolarized below -40 mV and decreased as V(m) depolarized above -40 mV. The magnitude of [Ca(2+)]i reduction during depolarizing current injections correlated with the amplitude of the plateau [Ca(2+)]i response to ACh. The effect of hyperpolarization on [Ca(2+)]i was abolished following removal of extracellular Ca(2+), was enhanced subtly by raising extracellular [Ca(2+)] from 2 mm to 10 mm and was reduced by half in endothelium of TRPV4(-/-) mice. Thus, during submaximal activation of muscarinic receptors, V(m) can modulate Ca(2+) entry through the plasma membrane in accord with the electrochemical driving force. PMID:26260126

  2. Poor lysosomal membrane integrity in proximal tubule cells of haptoglobin 2-2 genotype mice with diabetes mellitus

    PubMed Central

    Asleh, Rabea; Nakhoul, Farid M.; Miller-Lotan, Rachel; Awad, Hoda; Farbstein, Dan; Levy, Nina S.; Nakhoul, Nakhoul; Iancu, Theodore C.; Manov, Irena; Laue, Michael; Traber, Maret G.; Lebold, Katie M.; Levy, Andrew P.

    2013-01-01

    The haptoglobin (Hp) genotype is a major determinant of progression of nephropathy in individuals with diabetes mellitus (DM). The major function of the Hp protein is to bind and modulate the fate of extracorpuscular hemoglobin and its iron cargo. We have previously demonstrated an interaction between the Hp genotype and the DM on the accumulation of iron in renal proximal tubule cells. The primary objective of this study was to determine the intracellular localization of this iron in the proximal tubule cell and to assess its potential toxicity. Transmission electron microscopy demonstrated a marked accumulation of electron-dense deposits in the lysosomes of proximal tubules cells in Hp 2-2 DM mice. Energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to perform elemental analysis of these deposits and demonstrated that these deposits were iron rich. These deposits were associated with lysosomal membrane lipid peroxidation and loss of lysosomal membrane integrity. Vitamin E administration to Hp 2-2 DM mice resulted in a significant decrease in both intralysosomal iron-induced oxidation and lysosomal destabilization. Iron-induced renal tubular injury may play a major role in the development of diabetic nephropathy and may be a target for slowing the progression of renal disease. PMID:22749805

  3. 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

  4. Structural basis of the signalling through a bacterial membrane receptor HasR deciphered by an integrative approach.

    PubMed

    Wojtowicz, Halina; Prochnicka-Chalufour, Ada; de Amorim, 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

    2016-07-15

    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

  5. 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.

  6. An Unexpected Outcome of Surface Engineering An Integral Membrane Protein: Improved Crystallization of Cytochrome Ba(3) From Thermus Thermophilus

    SciTech Connect

    Liu, B.; Luna, V.M.; Chen, Y.; Stout, C.D.; Fee, J.A.

    2009-06-01

    Past work has shown that it is feasible to mutate surface residues of soluble proteins and to a lesser extent membrane proteins in order to improve their crystallization behavior. Described here is a successful application of this approach to the integral membrane protein Thermus thermophilus cytochrome ba(3) oxidase. Two mutant forms of this enzyme (I-K258R and I-K258R/II-E4Q) were created in which symmetrical crystal contacts within crystals of wild-type enzyme were modified. These mutant proteins had greatly shortened crystallization times, decreasing from approximately 30 d for the wild type to 1-3 d for the mutants, and crystallization was highly reproducible. Native-like proteins crystallize in space group P4(3)2(1)2, whereas the mutant proteins crystallize in space group P4(1)2(1)2 with a different packing arrangement. Crystals of the P4(3)2(1)2 form occasionally diffracted to 2.4-2.3 A resolution following controlled dehydration, while those of the P4(1)2(1)2 form routinely diffracted to between 3.0 and 2.6 A for crystals that had been cryoprotected but not dehydrated.

  7. 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

  8. Partial Purification of Integral Membrane Antigenic Proteins from Trypanosoma evansi That Display Immunological Cross-Reactivity with Trypanosoma vivax

    PubMed Central

    Velásquez, Norma P.; Camargo, Rocío E.; Uzcanga, Graciela L.; Bubis, José

    2014-01-01

    Trypanosoma evansi and Trypanosoma vivax, which are the major causative agents of animal trypanosomosis in Venezuela, have shown a very high immunological cross-reactivity. Since the production of T. vivax antigens is a limiting factor as this parasite is difficult to propagate in experimental animal models, our goal has been to identify and isolate antigens from T. evansi that cross-react with T. vivax. Here, we used the Venezuelan T. evansi TEVA1 isolate to prepare the total parasite lysate and its corresponding cytosolic and membranous fractions. In order to extract the T. evansi integral membrane proteins, the particulate portion was further extracted first with Triton X-100, and then with sodium dodecyl sulfate. After discarding the cytosolic and Triton X-100 solubilized proteins, we employed sedimentation by centrifugation on linear sucrose gradients to partially purify the sodium dodecyl sulfate-solubilized proteins from the Triton X-100 resistant particulate fraction of T. evansi. We obtained enriched pools containing polypeptide bands with apparent molecular masses of 27 kDa, 31 kDa, and 53 kDa, which were recognized by anti-T. vivax antibodies from experimentally and naturally infected bovines. PMID:24757558

  9. The Escherichia coli Cpx Envelope Stress Response Regulates Genes of Diverse Function That Impact Antibiotic Resistance and Membrane Integrity

    PubMed Central

    Leblanc, Shannon K. D.; Price, Nancy L.

    2013-01-01

    The Cpx envelope stress response mediates adaptation to stresses that cause envelope protein misfolding. Adaptation is partly conferred through increased expression of protein folding and degradation factors. The Cpx response also plays a conserved role in the regulation of virulence determinant expression and impacts antibiotic resistance. We sought to identify adaptive mechanisms that may be involved in these important functions by characterizing changes in the transcriptome of two different Escherichia coli strains when the Cpx response is induced. We show that, while there is considerable strain- and condition-specific variability in the Cpx response, the regulon is enriched for proteins and functions that are inner membrane associated under all conditions. Genes that were changed by Cpx pathway induction under all conditions were involved in a number of cellular functions and included several intergenic regions, suggesting that posttranscriptional regulation is important during Cpx-mediated adaptation. Some Cpx-regulated genes are centrally involved in energetics and play a role in antibiotic resistance. We show that a number of small, uncharacterized envelope proteins are Cpx regulated and at least two of these affect phenotypes associated with membrane integrity. Altogether, our work suggests new mechanisms of Cpx-mediated envelope stress adaptation and antibiotic resistance. PMID:23564175

  10. 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. PMID:27038266

  11. 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.

  12. 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. PMID:25966408

  13. Astaxanthin Protecting Membrane Integrity against Photosensitized Oxidation through Synergism with Other Carotenoids.

    PubMed

    Du, Hui-Hui; Liang, Ran; Han, Rui-Min; Zhang, Jian-Ping; Skibsted, Leif H

    2015-10-21

    Incorporation of astaxanthin or zeaxanthin in giant unilamellar vesicles (GUVs) of phosphatidylcholine resulted in a longer lag phase than incorporation of β-carotene or lycopene for the onset of budding induced by chlorophyll a photosensitization and quantified by a dimensionless entropy parameter using optical microscopy and digital image heterogeneity analysis. The lowest initial rate of GUV budding after the lag phase was seen for GUVs with astaxanthin as the least reducing carotenoid, while the lowest final level of entropy appeared for those with lycopene or β-carotene as a more reducing carotenoid. The combination of astaxanthin and lycopene gave optimal protection against budding with respect to both a longer lag phase and lower final level of entropy by combining good electron acceptance and good electron donation. Quenching of singlet oxygen by carotenoids close to chlorophyll a in the membrane interior in parallel with scavenging of superoxide radicals by astaxanthin anchored in the surface may explain the synergism between carotenoids involving both type I and type II photosensitization by chlorophyll a. PMID:26429551

  14. 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)

  15. F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes

    PubMed Central

    1987-01-01

    F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N- ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin- membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge. PMID:3312238

  16. Seasonal changes in semen characteristics, composition of seminal plasma and frequency of acrosome reaction induced by calcium and calcium ionophore A23187 in Large White boars.

    PubMed

    Murase, Tetsuma; Imaeda, Noriaki; Yamada, Hiroto; Miyazawa, Kiyoshi

    2007-08-01

    This study attempted to explain the mechanisms regulating boar fertility by examining seasonal changes in semen characteristics, the composition of seminal plasma and responsiveness of sperm acrosomes to Ca(2+) and the Ca(2+) ionophore A23187 (Ca(2+)/A23187). Sperm-rich and sperm-poor fractions were separately collected from 3 mature fertile Large White boars once a month over a one-year period. During the period of study, ambient temperature and relative humidity were recorded for within the stall in which the boars were kept and the semen characteristics, composition of the seminal plasma of sperm-rich fractions, and occurrence of the acrosome reaction in response to Ca(2+) (3 mM)/A23187 (0.3 microM) were examined. The highest mean maximum and minimum ambient temperatures were recorded in August-September, whereas the lowest mean maximum and minimum ambient temperatures were recorded in December and January, respectively. There was a moderate peak in relative humidity from July to October. The lowest percentages of motile spermatozoa and of spermatozoa with intact acrosomes and highest percentage of spermatozoa with abnormal morphology and strongest agglutination were seen in August-September. The total protein and albumin concentrations were lowest in August-September. Testosterone levels increased gradually as day length decreased after the summer solstice (June) and peaked in October-November. The percentage of acrosome reactions in response to Ca(2+)/A23187 was highest with the quickest response in August-September, as shown by the shortest time required for 50% of relative acrosome reactions. The farrowing rates were lowest in these same 2 months. These results suggest that seasonal infertility in Large White boars may be due, at least in part, to a combination of low motility, abnormal morphology including acrosomal abnormality, and early occurrence of the acrosome reaction in response to stimulus, possibly resulting from a decrease in acrosomal stabilizing

  17. 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. PMID:17379269

  18. Anti-ganglioside antibody-induced tumor cell death by loss of membrane integrity.

    PubMed

    Roque-Navarro, Lourdes; Chakrabandhu, Krittalak; de León, Joel; Rodríguez, Sandra; Toledo, Carlos; Carr, Adriana; de Acosta, Cristina Mateo; Hueber, Anne-Odile; Pérez, Rolando

    2008-07-01

    Gangliosides have been involved in multiple cellular processes such as growth, differentiation and adhesion, and more recently as regulators of cell death signaling pathways. Some of these molecules can be considered as tumor-associated antigens, in particular, N-glycolyl sialic acid-containing gangliosides, which are promising candidates for cancer-targeted therapy because of their low expression in normal human tissues. In this study, we provided the molecular and cellular characterization of a novel cell death mechanism induced by the anti-NGcGM3 14F7 monoclonal antibody (mAb) in L1210 murine tumor cell line but not in mouse normal cells (B and CD4(+) T lymphocytes) that expressed the antigen. Impairment of ganglioside synthesis in tumor cells abrogated the 14F7 mAb cytotoxic effect; however, exogenous reincorporation of the ganglioside did not restore tumor cell sensitivity to 14F7 mAb-induced cytotoxicity. 14F7 F(ab')(2) but not Fab fragments retained the cytotoxic capacity of the whole mAb. By contrary, other mAb, which recognizes N-glycolylated gangliosides, did not show any cytotoxic effect. These mAbs showed quite different capacities to bind NGcGM3-positive cell lines measured by binding inhibition experiments. Interestingly, this complement-independent cell death mechanism did not resemble apoptosis, because no DNA fragmentation, caspase activation, or Fas mediation were observed. However, NGcGM3 ganglioside-mediated 14F7 mAb-induced cell death was accompanied by cellular swelling, membrane lesion formation, and cytoskeleton activation, suggesting an oncosis-like phenomenon. This novel mechanism of cell death lets us to support further therapeutic approaches using NGcGM3 as a molecular target for antibody-based cancer immunotherapy. PMID:18645013

  19. Regulation of the starfish sperm acrosome reaction by cGMP, pH, cAMP and Ca2+.

    PubMed

    Matsumoto, Midori; Kawase, Osamu; Islam, M Sadiqul; Naruse, Masahiro; Watanabe, Shin-Nosuke; Ishikawa, Riho; Hoshi, Motonori

    2008-01-01

    In the starfish, Asterias amurensis, three components in the jelly coat of eggs, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to induce the acrosome reaction (AR). Molecular recognition between the sperm surface molecules and the egg jelly molecules must underlie signal transduction events triggering the AR. Asterosap is a sperm-activating molecule, which stimulates rapid synthesis of intracellular cGMP, pH and Ca2+. This transient elevation of Ca2+ level is caused by a K+-dependent Na+/Ca2+ exchanger, and the increase of intracellular pH is sufficient for ARIS to induce the AR. The concerted action of ARIS and asterosap could induce elevate intracellular cAMP levels in starfish sperm and the sustained increase in [Ca2+], which is essential for the AR. The signaling pathway induced by these factors seems to be synergistically regulated to trigger the AR in starfish sperm. PMID:18649265

  20. P38 participates in spermatogenesis and acrosome reaction prior to fertilization in Chinese mitten crab Eriocheir sinensis.

    PubMed

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

    2015-04-01

    P38 mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine protein kinases that play important roles in cellular responses to inflammatory cytokines and environmental stresses. These kinases are involved in controlling cell division, differentiation and death in mammalian testes and therefore are critical to spermatogenesis. To explore their functions in male reproduction of Chinese mitten crabs, Eriocheir sinensis p38 (Es-p38) protein expression was determined in different tissues including testes at different developmental stages by Western blot. Es-p38 was expressed in various tissues, with higher levels in the heart, stomach, gills and testes. Total Es-p38 protein levels increased gradually during spermatogenesis, but phosphorylated Es-p38 was much higher in the spermatid (August-October) than the spermatocyte (July-August) and sperm (October-January) stages. Trypan blue staining and hematoxylin/eosin staining were both used to detect sperm motility and changes in sperm morphology during the acrosome reaction (AR) induced by pre-incubation with A23187 in vitro, activated Es-p38 proteins detected by fluorescent microscopy were translocated gradually to nuclear and apical cap regions, accumulating at the anterior of the acrosomal tubule. The results suggest the involvement of p38 MAPK in spermatogenesis and the AR in E. sinensis. PMID:25433091

  1. Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis.

    PubMed

    Lone, Museer A; Atkinson, Aaron E; Hodge, Christine A; Cottier, Stéphanie; Martínez-Montañés, Fernando; Maithel, Shelley; Mène-Saffrané, Laurent; Cole, Charles N; Schneiter, Roger

    2015-12-01

    Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously-Brr6, which is closely related to Brl1, and Apq12-function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE. PMID:26432634

  2. Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis

    PubMed Central

    Lone, Museer A.; Atkinson, Aaron E.; Hodge, Christine A.; Cottier, Stéphanie; Martínez-Montañés, Fernando; Maithel, Shelley; Mène-Saffrané, Laurent

    2015-01-01

    Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously—Brr6, which is closely related to Brl1, and Apq12—function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE. PMID:26432634

  3. Cardiac muscle cell cytoskeletal protein 4.1: analysis of transcripts and subcellular location--relevance to membrane integrity, microstructure, and possible role in heart failure.

    PubMed

    Taylor-Harris, Pamela M; Keating, Lisa A; Maggs, Alison M; Phillips, Gareth W; Birks, Emma J; Franklin, Rodney C G; Yacoub, Magdi H; Baines, Anthony J; Pinder, Jennifer C

    2005-03-01

    The spectrin-based cytoskeleton assembly has emerged as a major player in heart functioning; however, cardiac protein 4.1, a key constituent, is uncharacterized. Protein 4.1 evolved to protect cell membranes against mechanical stresses and to organize membrane microstructure. 4.1 Proteins are multifunctional and, among other activities, link integral/signaling proteins on the plasma and internal membranes with the spectrin-based cytoskeleton. Four genes, EPB41, EPB41L1, EPB41L2, and EPB41L3 encode proteins 4.1R, 4.1N, 4.1G, and 4.1B, respectively. All are extensively spliced. Different isoforms are expressed according to tissue and developmental state, individual function being controlled through inclusion/exclusion of interactive domains. We have defined mouse and human cardiac 4.1 transcripts; other than 4. 1B in humans, all genes show activity. Cardiac transcripts constitutively include conserved FERM and C-terminal domains; both interact with membrane-bound signaling/transport/cell adhesion molecules. Variable splicing within and adjacent to the central spectrin/actin-binding domain enables regulation of cytoskeleton-binding activity. A novel heart-specific exon occurs in human 4.1G, but not in mouse. Immunofluorescence reveals 4.1 staining within mouse cardiomyocytes; thus, both at the plasma membrane and, interdigitated with sarcomeric myosin, across myofibrils in regions close to the sarcoplasmic reticulum. These are all regions to which spectrin locates. 4.1R in human heart shows similar distribution; however, there is limited plasma membrane staining. We conclude that cardiac 4.1s are highly regulated in their ability to crosslink plasma/integral cell membranes with the spectrin-actin cytoskeleton. We speculate that over the repetitive cycles of heart muscle contraction and relaxation, 4.1s are likely to locate, support, and coordinate functioning of key membrane-bound macromolecular assemblies. PMID:15834631

  4. 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

  5. Design, integration and control of proton exchange membrane electrolyzer for wind based renewable energy applications

    NASA Astrophysics Data System (ADS)

    Harrison, Kevin W.

    This research endeavor began with the design and construction of a new hydrogen test facility at the National Renewable Energy Laboratory (NREL). To improve the electrical link of wind-based electrolysis the characterization of a proton exchange membrane (PEM) electrolyzer under varying input power was performed at NRELs new test facility. The commercially available electrolyzer from Proton Energy Systems (PES) was characterized using constant direct current (DC), sinusoidally varying DC, photovoltaics and variable magnitude and frequency energy from a 10 kW wind turbine. At rated stack current and ˜ 40°C the system efficiency of the commercial electrolyzer was measured to be 55%. At lower stack current it was shown that commercial electrolyzer system efficiency falls because of the continuous hydrogen purge (˜0.1 Nm3 hr-1) used to maintain the hydrogen desiccant drying system. A novel thermoelectric-based dew point controller is designed and modeled to reduce the penalty to renewable sources because they do not always operate at 100% of rated stack current. It is predicted that the thermoelectric design when operated 100% of the time at full current to the thermoelectric modules would consume 3.1 kWh kg -1 of hydrogen. Using the higher heating value of hydrogen and a stack efficiency of 60% to produce the hydrogen that is continuously vented, the desiccant system consumes about 5.7 kWh kg-1. Design of the UND electrolyzer sub-systems responsible for all aspects of water, power to the stack, and hydrogen conditioning enables more flexible and precise experimental data to be obtained than from an off-the-shelf system. Current-voltage (IV) characteristic curves were obtained on the UND system at temperatures between 7--70°C. The anode and cathode exchange current densities are fitted to 2.0 E-06 e0.043T and 0.12 e 0.026T A cm-2 respectively. Stack conductivity was fitted to 0.001T + 0.03 S cm-1. The three coefficients represent physical stack parameters and are

  6. 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

  7. 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

  8. 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...

  9. Monitoring Protein-Protein Interactions between the Mammalian Integral Membrane Transporters and PDZ-interacting Partners Using a Modified Split-ubiquitin Membrane Yeast Two-hybrid System*S⃞

    PubMed Central

    Gisler, Serge M.; Kittanakom, Saranya; Fuster, Daniel; Wong, Victoria; Bertic, Mia; Radanovic, Tamara; Hall, Randy A.; Murer, Heini; Biber, Jürg; Markovich, Daniel; Moe, Orson W.; Stagljar, Igor

    2008-01-01

    PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this “MYTH 2.0” system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes. PMID:18407958

  10. 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

  11. Determination of sperm concentration using flow cytometry with simultaneous analysis of sperm plasma membrane integrity in zebrafish Danio rerio.

    PubMed

    Yang, Huiping; Daly, Jonathan; Tiersch, Terrence R

    2016-04-01

    Control of sperm concentration is required to ensure consistent and reproducible results for cryopreservation and in vitro fertilization protocols. Determination of sperm concentration is traditionally performed with a counting chamber (e.g., hemocytometer), or more recently with a spectrophotometer. For small-sized biomedical model fishes, the availability of sperm sample is limited to microliters, so it is desirable to develop fast and accurate approaches for concentration determination that also minimize sample use. In this study, a new approach was developed for sperm concentration determination using a flow cytometer (Accuri C6, BD Biosciences, San Jose, CA) with simultaneous measurement of sperm membrane integrity after fluorescent staining with SYBR(®) -14 and propidium iodide (PI) in sperm from Zebrafish Danio rerio. The goal was to develop a protocol for simultaneous determination of sperm quality and quantity by flow cytometry. The objectives were to (1) determine the effects of sample volume (250 and 500 µl) and analysis volume (10 and 50 µl) on the accuracy of particle counting using standard volumetric validation beads; (2) identify the effective range of sperm concentrations that flow cytometry can measure; (3) test the precision and reproducibility of the sperm concentration measurements; and (4) verify the flow cytometry approach by comparison with measurement with a hemocytometer and a microspectrophotometer. Sample volumes of 250 and 500 µl and analysis volumes of 10 and 50 µl did not affect bead count with the factory-set flow rates of "medium" or "fast," and the precision and accuracy was retained across a concentration range of 1 × 10(3) -1 × 10(7) cells/ml. The approach developed in this study was comparable to traditional methodologies such as hemocytometer or microspectrophotometer. This study provides an efficient, accurate, and rapid method for determination of sperm concentration using flow cytometry while providing

  12. The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins

    PubMed Central

    Love, James; Mancia, Filippo; Shapiro, Lawrence; Punta, Marco; Rost, Burkhard; Girvin, Mark; Wang, Da-Neng; Zhou, Ming; Hunt, John F.; Szyperski, Thomas; Gouaux, Eric; MacKinnon, Roderick; McDermott, Ann; Honig, Barry; Inouye, Masayori; Montelione, Gaetano

    2011-01-01

    The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NY-COMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform high-level protein production and structure determination by x-ray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline. PMID:20690043

  13. Effects of antibacterial mineral leachates on the cellular ultrastructure, morphology, and membrane integrity of Escherichia coli and methicillin-resistant Staphylococcus aureus

    PubMed Central

    2010-01-01

    Background We have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli after exposure to CB07 and BY07 aqueous leachates. Methods We used scanning and transmission electron microscopy to evaluate E. coli and MRSA ultrastructure and morphology following exposure to antibacterial leachates. Additionally, we employed Baclight LIVE/DEAD staining and flow cytometry to investigate the cellular membrane as a possible target for antibacterial activity. Results Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging of E. coli and MRSA revealed intact cells following exposure to antibacterial mineral leachates. TEM images of MRSA showed disruption of the cytoplasmic contents, distorted cell shape, irregular membranes, and distorted septa of dividing cells. TEM images of E. coli exposed to leachates exhibited different patterns of cytoplasmic condensation with respect to the controls and no apparent change in cell envelope structure. Although bactericidal activity of the leachates occurs more rapidly in E. coli than in MRSA, LIVE/DEAD staining demonstrated that the membrane of E. coli remains intact, while the MRSA membrane is permeabilized following exposure to the leachates. Conclusions These data suggest that the leachate antibacterial mechanism of action differs for Gram-positive and Gram-negative organisms. Upon antibacterial mineral leachate exposure, structural integrity is retained, however, compromised membrane integrity accounts for bactericidal activity in Gram-positive, but not in Gram-negative cells. PMID:20846374

  14. 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

  15. Caenorhabditis elegans Teneurin, ten-1, Is Required for Gonadal and Pharyngeal Basement Membrane Integrity and Acts Redundantly with Integrin ina-1 and Dystroglycan dgn-1

    PubMed Central

    Trzebiatowska, Agnieszka; Topf, Ulrike; Sauder, Ursula; Drabikowski, Krzysztof

    2008-01-01

    The Caenorhabditis elegans teneurin ortholog, ten-1, plays an important role in gonad and pharynx development. We found that lack of TEN-1 does not affect germline proliferation but leads to local basement membrane deficiency and early gonad disruption. Teneurin is expressed in the somatic precursor cells of the gonad that appear to be crucial for gonad epithelialization and basement membrane integrity. Ten-1 null mutants also arrest as L1 larvae with malformed pharynges and disorganized pharyngeal basement membranes. The pleiotropic phenotype of ten-1 mutant worms is similar to defects found in basement membrane receptor mutants ina-1 and dgn-1 as well as in the mutants of the extracellular matrix component laminin, epi-1. We show that the ten-1 mutation is synthetic lethal with mutations of genes encoding basement membrane components and receptors due to pharyngeal or hypodermal defects. This indicates that TEN-1 could act redundantly with integrin INA-1, dystroglycan DGN-1, and laminin EPI-1 in C. elegans development. Moreover, ten-1 deletion sensitizes worms to loss of nidogen nid-1 causing a pharynx unattached phenotype in ten-1;nid-1 double mutants. We conclude that TEN-1 is important for basement membrane maintenance and/or adhesion in particular organs and affects the function of somatic gonad precursor cells. PMID:18632986

  16. 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

  17. 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

  18. P13, an Integral Membrane Protein of Borrelia burgdorferi, Is C-Terminally Processed and Contains Surface-Exposed Domains

    PubMed Central

    Noppa, Laila; Östberg, Yngve; Lavrinovicha, Marija; Bergström, Sven

    2001-01-01

    To elucidate antigens present on the bacterial surface of Borrelia burgdorferi sensu lato that may be involved in pathogenesis, we characterized a protein, P13, with an apparent molecular mass of 13 kDa. The protein was immunogenic and was expressed in large amounts during in vitro cultivation compared to other known antigens. An immunofluorescence assay, immunoelectron microscopy, and protease sensitivity assays indicated that P13 is surface exposed. The deduced sequence of the P13 peptide revealed a possible signal peptidase type I cleavage site, and computer analysis predicted that P13 is an integral membrane protein with three transmembrane-spanning domains. Mass spectrometry, in vitro translation, and N- and C-terminal amino acid sequencing analyses indicated that P13 was posttranslationally processed at both ends and modified by an unknown mechanism. Furthermore, p13 belongs to a gene family with five additional members in B. burgdorferi sensu stricto. The p13 gene is located on the linear chromosome of the bacterium, in contrast to five paralogous genes, which are located on extrachromosomal plasmids. The size of the p13 transcript was consistent with a monocistronic transcript. This new gene family may be involved in functions that are specific for this spirochete and its pathogenesis. PMID:11292755

  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. 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

  1. 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

    2014-01-01

    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. PMID:25529018

  2. Scorpion toxins that block T-type Ca2+ channels in spermatogenic cells inhibit the sperm acrosome reaction.

    PubMed

    López-González, Ignacio; Olamendi-Portugal, Timoteo; De la Vega-Beltrán, José L; Van der Walt, Jurg; Dyason, Karin; Possani, Lourival D; Felix, Ricardo; Darszon, Alberto

    2003-01-10

    The acrosome reaction (AR) is a Ca(2+)-dependent event required for sperm to fertilize the egg. The activation of T-type voltage-gated Ca(2+) channels plays a key role in the induction of this process. This report describes the actions of two toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively) on sperm Ca(2+) channels. Both toxins decrease T-type Ca(2+) channel activity in mouse spermatogenic cells and inhibit the AR in mature sperm. Saturating concentrations of the toxins inhibited at most approximately 70% of the whole-cell Ca(2+) current, suggesting the presence of a toxin-resistant component. In addition, both toxins inhibited approximately 60% of the AR, which is consistent with the participation of T-type Ca(2+) channels in the sperm AR. PMID:12504099

  3. Pretreatment effects of jelly components on the sperm acrosome reaction and histone degradation in the starfish, Asterina pectinifera.

    PubMed

    Amano, T; Okita, Y; Matsui, T; Hoshi, M

    1992-08-31

    Acrosome reaction (AR) and histone degradation (HD) of Asterina pectinifera sperm are induced by co-operation of ARIS and a diffusible fraction (M8) of egg jelly. Once sperm are treated with ARIS or M8 separately for several minutes, they do not undergo the AR in response to the egg jelly. Preincubation of sperm with M8 at 0 degrees C is not effective to block the jelly-induced AR whereas inhibitory effects of ARIS remain at 0 degrees C. Jelly-induced HD is inhibited by pretreatment of sperm with ARIS but is not affected by the incubation with M8. The blockage of the jelly-induced reactions, both AR and HD, by ARIS- or M8-pretreatment can be bypassed by ionophores, A23187 and monensin. PMID:1520307

  4. The contribution of a covalently bound cofactor to the folding and thermodynamic stability of an integral membrane protein.

    PubMed

    Curnow, Paul; Booth, Paula J

    2010-11-01

    The factors controlling the stability, folding, and dynamics of integral membrane proteins are not fully understood. The high stability of the membrane protein bacteriorhodopsin (bR), an archetypal member of the rhodopsin photoreceptor family, has been ascribed to its covalently bound retinal cofactor. We investigate here the role of this cofactor in the thermodynamic stability and folding kinetics of bR. Multiple spectroscopic probes were used to determine the kinetics and energetics of protein folding in mixed lipid/detergent micelles in the presence and absence of retinal. The presence of retinal increases extrapolated values for the overall unfolding free energy from 6.3 ± 0.4 kcal mol(-1) to 23.4 ± 1.5 kcal mol(-1) at zero denaturant, suggesting that the cofactor contributes 17.1 kcal mol(-1) towards the overall stability of bR. In addition, the cooperativity of equilibrium unfolding curves is markedly reduced in the absence of retinal with overall m-values decreasing from 31.0 ± 2.0 kcal mol(-1) to 10.9 ± 1.0 kcal mol(-1), indicating that the folded state of the apoprotein is less compact than the equivalent for the holoprotein. This change in the denaturant response means that the difference in the unfolding free energy at a denaturant concentration midway between the two unfolding curves is only ca 3-6 kcal mol(-1). Kinetic data show that the decrease in stability upon removal of retinal is associated with an increase in the apparent intrinsic rate constant of unfolding, k(u)(H2O), from ~1 × 10(-16) s(-1) to ~1 × 10(-4) s(-1) at 25 °C. This correlates with a decrease in the unfolding activation energy by 16.3 kcal mol(-1) in the apoprotein, extrapolated to zero SDS. These results suggest that changes in bR stability induced by retinal binding are mediated solely by changes in the activation barrier for unfolding. The results are consistent with a model in which bR is kinetically stabilized via a very slow rate of unfolding arising from protein

  5. A cell surface integral membrane glycoprotein of 85,000 mol wt (gp85) associated with triton X-100-insoluble cell skeleton.

    PubMed

    Tarone, G; Ferracini, R; Galetto, G; Comoglio, P

    1984-08-01

    The Triton X-100-insoluble skeleton of baby hamster kidney BHK cells consists of the nucleus, intermediate-size filaments, and actin fibers. By transmission electron microscopy, membrane fragments were found to be associated with these insoluble structures. When radioiodinated or [3H]glucosamine-labeled cells were extracted with 0.5% Triton, most plasma membrane glycoproteins were solubilized except for a glycoprotein with a molecular weight of 85,000 (gp85) that remained associated with the insoluble skeletons. Immunoprecipitation with a specific antiserum indicated that the gp85 is not a proteolytic degradation product of fibronectin, an extracellular matrix glycoprotein insoluble in detergent. A monoclonal antibody of BHK cells specific for gp85 was produced. Immunofluorescence analysis with this monoclonal antibody indicated that gp85 is not associated with the extracellular matrix, but is confined to the cell membrane. Both in fixed and unfixed intact cells, fluorescence was concentrated in dots preferentially aligned in streaks on the cell surface. Gp85 was found to behave as an integral membrane protein interacting with the hydrophobic core of the lipid bilayer since it was extracted from membrane preparations by ionic detergents such as SDS, but not by 0.1 N NaOH (pH 12) in the absence of detergents, a condition known to release peripheral molecules. Association of gp85 with the cell skeleton was unaffected by increasing the Triton concentration up to 5%, but it was affected when actin filaments were dissociated or when a protein-denaturing agent (6 M urea) was used in the presence of Triton, suggesting that protein-protein interactions are involved in the association of gp85 with the cell skeleton. We conclude that gp85 is an integral plasma membrane glycoprotein that might have a role in cell surface-cytoskeleton interaction. PMID:6378925

  6. 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

  7. Effect of cis-9,trans-11 and trans-10,cis-12 isomers of conjugated linoleic acid on the integrity and functionality of cryopreserved bovine spermatozoa.

    PubMed

    Soares, Marcio Pereira; Brandelli, Adriano; Celeghini, Eneiva Carla Carvalho; de Arruda, Rubens Paes; Rodriguez, Shirley Andrea Florez

    2013-08-01

    Plasma membranes of sperm subjected to low temperatures undergo changes in their structure and permeability. The addition of fatty acids in semen cryopreservation media may influence the sperm motility after thawing, possibly by maintaining the membrane fluidity due to their incorporation in lipid bilayers. In this work, different concentrations of the isomers cis-9,trans-11 and trans-10,cis-12 of conjugated linoleic acid (CLA) were added in the cryopreservation medium of bovine sperm. Four Jersey bulls were used, and the ejaculates were processed as a pool. The Tris-based extender (Dilutris®) was supplemented with 20% egg yolk (MB). The treatments with CLA (Luta-CLA®), which had oily presentation, were prepared from MB with addition of 1% sodium lauryl sulfate, and denominated MBL. The concentrations of CLA tested were 50, 100, and 150 μM. The motility characteristics of the post-thaw semen were analyzed by computerized analysis system (CASA), and plasma membrane integrity and acrosomal and mitochondrial function assessed by the association of the fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), JC-1 and Hoechst 33342. No significant differences were observed among treatments, excepting for a decreased mitochondrial potential of cells treated with 150 μM CLA. The addition of CLA, at the concentrations used, showed no advantages on the integrity and functionality of bovine sperm submitted to cryopreservation. PMID:23721967

  8. Association of a Protective Monoclonal IgA with the O Antigen of Salmonella enterica Serovar Typhimurium Impacts Type 3 Secretion and Outer Membrane Integrity

    PubMed Central

    Forbes, Stephen J.; Martinelli, Daniel; Hsieh, Chyongere; Ault, Jeffrey G.; Marko, Michael; Mannella, Carmen A.

    2012-01-01

    Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium is an energetically demanding process, involving the transfer of effector proteins from invading bacteria into host cells via a specialized organelle known as the Salmonella pathogenicity island 1 (SPI-1) type 3 secretion system (T3SS). By a mechanism that remains poorly understood, entry of S. Typhimurium into epithelial cells is inhibited by Sal4, a monoclonal, polymeric IgA antibody that binds an immunodominant epitope within the O-antigen (O-Ag) component of lipopolysaccharide. In this study, we investigated how the binding of Sal4 to the surface of S. Typhimurium influences T3SS activity, bacterial energetics, and outer membrane integrity. We found that Sal4 treatment impaired T3SS-mediated translocon formation and attenuated the delivery of tagged effector proteins into epithelial cells. Sal4 treatment coincided with a partial reduction in membrane energetics and intracellular ATP levels, possibly explaining the impairment in T3SS activity. Sal4's effects on bacterial secretion and energetics occurred concurrently with an increase in O-Ag levels in culture supernatants, alterations in outer membrane permeability, and changes in surface ultrastructure, as revealed by transmission electron microscopy and cryo-electron microscopy. We propose that Sal4, by virtue of its ability to bind and cross-link the O-Ag, induces a form of outer membrane stress that compromises the integrity of the S. Typhimurium cell envelope and temporarily renders the bacterium avirulent. PMID:22473607

  9. Multi-layered, membrane-integrated microfluidics based on replica molding of a thiol-ene epoxy thermoset for organ-on-a-chip applications.

    PubMed

    Sticker, Drago; Rothbauer, Mario; Lechner, Sarah; Hehenberger, Marie-Therese; Ertl, Peter

    2015-12-21

    In this study we have investigated a photosensitive thermoset (OSTEMER 322-40) as a complementary material to readily fabricate complex multi-layered microdevices for applications in life science. Simple, versatile and robust fabrication of multifunctional microfluidics is becoming increasingly important for the development of customized tissue-, organ- and body-on-a-chip systems capable of mimicking tissue interfaces and biological barriers. In the present work key material properties including optical properties, vapor permeability, hydrophilicity and biocompatibility are evaluated for cell-based assays using fibroblasts, endothelial cells and mesenchymal stem cells. The excellent bonding strength of the OSTEMER thermoset to flexible fluoropolymer (FEP) sheets and poly(dimethylsiloxane) (PDMS) membranes further allows for the fabrication of integrated microfluidic components such as membrane-based microdegassers, microvalves and micropumps. We demonstrate the application of multi-layered, membrane-integrated microdevices that consist of up to seven layers and three membranes that specially confine and separate vascular cells from the epithelial barrier and 3D tissue structures. PMID:26524977

  10. F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes.

    PubMed

    Wuestehube, L J; Luna, E J

    1987-10-01

    F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin-membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge. PMID:3312238

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

    PubMed

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

    2015-10-01

    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

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

    PubMed

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

    2016-04-01

    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

  13. 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

  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. Integrated light-scattering spectroscopy, a sensitive probe for peptide-vesicle binding: application to the membrane-bound colicin E1 channel peptide.

    PubMed Central

    Strawbridge, K. B.; Palmer, L. R.; Merrill, A. R.; Hallett, F. R.

    1995-01-01

    Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius. These results indicate that ILS spectroscopy is a sensitive sizing technique that is capable of detecting relatively small changes in membrane vesicles and may have a wide application in the determination of peptide binding to membrane vesicles. Images FIGURE 2 PMID:7711234

  17. 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

  18. Estimation by radiation inactivation of the minimum functional size of acrosome-reaction-inducing substance (ARIS) in the starfish, Asterias amurensis.

    PubMed

    Ushiyama, A; Chiba, K; Shima, A; Hoshi, M

    1995-11-01

    In the starfish Asterias amurensis, the jelly coat of the eggs contains a glycoprotein essential for the induction of the acrosome reaction in homologous spermatozoa that is termed the acrosome-reaction-inducing substance (ARIS). ARIS is a highly sulphated and fucose-rich glycoprotein of extremely high molecular mass (> 10(4) kDa). ARIS was irradiated with high-energy electrons in order to estimate the minimum size required for its biological activity. The minimum functional unit or target size of ARIS was estimated to be c. 14 kDa by target size analysis. ARIS was significantly disintegrated by the irradiation, yet the total sugar content was not apparently reduced. The binding of 125I-labelled ARIS to spermatozoa competed with that of irradiated ARIS, although the affinity of ARIS was much reduced after irradiation. PMID:8730900

  19. Sensitivity analysis of the pressure-based direct integrity test for membranes used in drinking water treatment.

    PubMed

    Minnery, John G; Jacangelo, Joseph G; Boden, Leslie I; Vorhees, Donna J; Heiger-Bernays, Wendy

    2009-12-15

    We conducted a sensitivity analysis of the commonly employed pressure-based direct integrity test (DIT), the most sensitive test for defects in low-pressure hollow fiber (LPHF) microfiltration and ultrafiltration systems used in drinking water treatment. Incorporating uncertainty to assess the practice of DIT, we find the resolution in some tests may be insufficient to verify the presence of a barrier to oocysts of Cryptosporidium. Applying distributions and boundaries derived from literature and practice, we solved for the defect size resolution (DSR) using Monte Carlo and Probability Bounds Analysis for five commercial membrane designs. Surface tension was modeled using annual temperature profiles from three rivers. Contact angle measurement error and variability were derived from literature, respectively, as a standard deviation of 5.7 degrees and +/- 9.6 degrees median change due to natural organic matter (NOM) fouling. These measures of contact angle uncertainty and variability were combined in a normal distribution with the discrete values currently applied. Additionally we considered model uncertainty, applying the maximum bubble pressure method, an established method of surface tension measurement in liquids in which the maximum air pressure in a submerged capillary is developed after the contact angle becomes zero prior to bubble formation. Where the DSR exceeds 3 microm the test design is not compliant with applicable drinking water regulations. Implications include uncertain and variable log-removal values (LRV) as determined by DIT due to the possible emergence of defects large enough to allow oocysts to pass without detection by the DIT. Specifically, we found the DSR may exceed 3 microm and may be as large as 8 microm. With the variable contact angle model, all lower bound possibilities are compliant, whereas the upper bound is over 80% noncompliant for three of five commercial designs. Using the Maximum Bubble Pressure Method, the lower bounds in

  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. 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. PMID:23343429

  2. Identification of an anti-sperm auto-monoclonal antibody (Ts4)-recognized molecule in the mouse sperm acrosomal region and its inhibitory effect on fertilization in vitro.

    PubMed

    Yoshitake, Hiroshi; Oda, Risako; Yanagida, Mitsuaki; Kawasaki, Yu; Sakuraba, Mayumi; Takamori, Kenji; Hasegawa, Akiko; Fujiwara, Hiroshi; Araki, Yoshihiko

    2016-06-01

    We previously established an anti-mouse sperm auto-monoclonal antibody, Ts4, which shows immunoreactivity against several kinds of glycoproteins in the acrosomal region of epididymal spermatozoa, testicular germ cells, and early embryo, via binding to an epitope containing a common N-linked oligosaccharide (OS) chain on the molecules. In mice, we have already demonstrated that the OS chain in the epitope for Ts4 is a fucosylated agalacto-complex-type biantennary glycan carrying bisecting N-acetylglucosamine. In the testis, one of the specific OS chain-conjugated molecules is TEX101, a germ cell-marker glycoprotein, which is expressed in spermatocytes, spermatids, and testicular spermatozoa, but not in epididymal spermatozoa. In this study, we identified a Ts4-reactive glycoprotein in mouse cauda epididymal sperm. An immunoprecipitation method together with liquid chromatography-tandem mass spectrometry showed that alpha-N-acetylglucosaminidase (Naglu; a degradation enzyme of heparan sulfate) is one of the glycoproteins recognized by Ts4 in the epididymal spermatozoa. Western blot and immunohistochemical analyses revealed that mouse Naglu exists in two forms (82 and 77kDa) and is expressed in the acrosomal region and the flagellum of cauda epididymal sperm. Of the two Naglu-forms expressed in sperm, Ts4 immunoreacted against only the 82-kDa form located on the acrosomal region. The Ts4 mAb and anti-Naglu pAb negatively affected mouse fertilization in vitro. In addition, Ts4 inhibited sperm acrosome reaction induced by heparan sulfate. The Ts4-recognized fucosylated agalactobiantennary complex-type glycan with bisecting N-acetylglucosamine and Naglu on cauda epididymal spermatozoa may play a role in the process of fertilization. PMID:27064211

  3. Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C.

    PubMed

    Chakravarty, Sanchita; Suraj, K; Gupta, Satish Kumar

    2005-05-01

    To facilitate our understanding of the role of zona pellucida glycoproteins during fertilization in humans, recombinant human zona pellucida glycoprotein-A (hZPA), -B (hZPB) and -C (hZPC) were obtained by using Escherichia coli and baculovirus expression systems. Analysis by SDS-PAGE and Western blot of the Ni-NTA affinity purified recombinant proteins revealed that the baculovirus-expressed hZPA, hZPB and hZPC have an apparent molecular weight of approximately 110, approximately 70-75 and approximately 65 kDa, respectively, as compared to approximately 80, approximately 65 and approximately 50 kDa of the respective E. coli-expressed proteins. Lectin binding studies revealed that the baculovirus-expressed recombinant zona proteins were glycosylated. Major oligosaccharides were represented by strong reactivity with Concanavalin A (mannose alpha 1-3 or mannose alpha 1-6 residues) and Jacalin (alpha-O glycosides of Gal or GalNAc moieties). A significant increase in acrosomal exocytosis was observed when capacitated human sperm were incubated in vitro with baculovirus-expressed hZPB (P=0.0005) and hZPC (P=0.0005) The E. coli-expressed hZPB, hZPC and baculovirus-expressed hZPA failed to induce any significant increase (P>0.05) in acrosome reaction. In contrast to hZPC, the acrosome reaction induced by recombinant hZPB was not inhibited by pertussis toxin. These studies, for the first time, have demonstrated that in humans, ZPB also induces acrosomal exocytosis through a Gi independent pathway. PMID:15805145

  4. Methanotroph outer membrane preparation.

    PubMed

    Karlsen, Odd A; Berven, Frode S; Jensen, Harald B; Fjellbirkeland, Anne

    2011-01-01

    All presently known methanotrophs are gram-negative bacteria suggesting that they are surrounded by a two-layered membrane: an inner or cytoplasmic membrane and an outer membrane. In the methanotroph Methylococcus capsulatus (Bath), separation of the two membranes has allowed studies on protein and lipid composition of the outer membrane. Its outer membrane can be isolated from purified cell envelopes by selective solubilization of the inner membranes with the detergent Triton X-100. The proteins associated with the outer membrane can further be fractionated into integral and tightly associated proteins and peripheral loosely associated proteins. We present here protocols for this fractionation and show how the proteins associated with the outer leaflet of the outer membrane can be isolated and identified by whole-cell biotin surface labeling. PMID:21419921

  5. The theory of semipermeable vesicles and membranes: An integral-equation approach. I. General formalism and application to a hard-sphere mixture

    NASA Astrophysics Data System (ADS)

    Zhou, Yaoqi; Stell, George

    1988-12-01

    An exact integral-equation formalism for a system of a binary hard-sphere mixture interacting with a spherical semipermeable vesicle (SPV) and plane semipermeable membrane (SPM) is derived by using the Ornstein-Zernike (OZ) equation with appropriate closures. The Percus-Yevick (PY) closure or the hypernetted chain (HNC) closure, in which the bulk correlation is obtained by the PY approximation, are considered as examples. We refer to these as the PY/PY and HNC/PY approximations, respectively. The mixture contains solvent particles, which are permeable to the membrane, and solute particles (``protein'' or ``polymer'' particles), which can not pass through the membrane. We develop an exact general formalism for this problem and as an illustration of its use give quantative results for solvent and solute particles modeled as hard spheres of different diameters. An analytical expression for the density ratio in the PY/PY and HNC/PY approximations between two sides of a plane SPM is obtained. Results obtained from these expressions agree very well with results obtained by equating chemical potentials in the region of interest. It turns out that the protein-membrane direct correlation function can be given by a simple analytic expression for the limit of a point solvent in the PY/PY approximation. The osmotic pressure and density profiles for the system containing an ideal spherical SPV or plane SPM in the PY/PY approximation are evaluated. Extension to the nonlocal density-functional closures previously introduced by Blum and Stell is discussed. Finally, we note that certain impenetrable-wall problems previously considered elsewhere can be regarded as semipermeable membrane problems treated via McMillan-Mayer formalism in the continuum-solvent approximation.

  6. 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.

  7. Integrating Solid-State NMR and Computational Modeling to Investigate the Structure and Dynamics of Membrane-Associated Ghrelin

    PubMed Central

    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

  8. Investigation of the Biophysical and Cell Biological Properties of Ferroportin, a Multi-Pass Integral Membrane Protein Iron Exporter

    PubMed Central

    Rice, Adrian E.; Mendez, Michael J.; Hokanson, Craig A.; Rees, Douglas C.; Björkman, Pamela J.

    2009-01-01

    Ferroportin is a multi-pass membrane protein that serves as an iron exporter in many vertebrate cell types. Ferroportin-mediated iron export is controlled by the hormone hepcidin, which binds ferroportin, causing its internalization and degradation. Mutations in ferroportin cause a form of the iron overload disease hereditary hemochromatosis. Relatively little is known about ferroportin’s properties or the mechanism by which mutations cause disease. Here we expressed and purified human ferroportin to characterize its biochemical/biophysical properties in solution and conducted cell biological studies in mammalian cells. We show that purified, detergent-solubilized ferroportin was a well-folded monomer that bound hepcidin. In cell membranes, the N- and C-termini were both cytosolic, implying an even number of transmembrane regions, and ferroportin was mainly localized to the plasma membrane. Hepcidin addition resulted in a redistribution of ferroportin to intracellular compartments that labeled with early endosomal and lysosomal, but not Golgi, markers and that trafficked along microtubules. An analysis of 16 disease-related ferroportin mutants revealed that all formed well-folded monomers that localized to the plasma membrane, but some were resistant to hepcidin-induced internalization. The characterizations reported here form a basis upon which models for ferroportin’s role in regulating iron homeostasis in health and disease can be interpreted. PMID:19150361

  9. 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

  10. Identification and characterization of LFD-2, a predicted fringe protein required for membrane integrity during cell fusion in neurospora crassa.

    PubMed

    Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A Pedro; Starr, Trevor L; Glass, N Louise

    2015-03-01

    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

  11. 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.

  12. 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

  13. Plunder of Human Blood Leukocytes Containing Ingested Material, by Other Leukocytes: Where Is the Fusagen That Allows Preservation of Membrane Integrity and Motile Function?

    PubMed Central

    Malawista, Stephen E.; Chevance de Boisfleury, Anne

    2013-01-01

    In studying phagocytosis of zymosan particles by human blood monocytes in phase-contrast videomicroscopy, we found that monocytes loaded with zymosan particles became chemotactic for polymorphonuclear leukocytes (PMN) which closed on them and purloined their particle content. This despoliation usually occurred in monocytes that had begun to swell—prefiguring their death. The violent seizure of their contents by the aggressing PMN often tore the monocytes apart. However, some apparently healthy monocyte survived the removal of zymosan content by PMN or, more commonly, its removal by another monocyte. PMN—a much hardier cell in slide preparations—that were similarly loaded with zymosan particles, also attracted PMN. The latter could remove zymosan from the target cell without killing it. Thus, leukocytes were sacrificing significant portions of themselves without losing residual membrane integrity and motile function. Their behavior with respect to other particles (e.g., bacteria) will be of interest. We suggest that the membrane fusagen resides in the inner membrane leaflets when they are brought together in an extreme hourglass configuration. This event may be similar to the fragmentation of erythrocytes into intact pieces, the formation of cytokineplasts, the rear extrusion of content by migrating cells on surfaces, and the phagocytic process itself. PMID:23840370

  14. Ophiobolin A from Bipolaris oryzae Perturbs Motility and Membrane Integrities of Porcine Sperm and Induces Cell Death on Mammalian Somatic Cell Lines

    PubMed Central

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

    2014-01-01

    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

  15. 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

  16. 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

  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. 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.

  19. 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

  20. Bioconjugated Gold Nanoparticles Penetrate Into Spermatozoa Depending on Plasma Membrane Status.

    PubMed

    Barchanski, Annette; Taylor, Ulrike; Sajti, Csaba L; Gamrad, Lisa; Kues, Wilfried A; Rath, Detlef; Barcikowski, Stephan

    2015-09-01

    Spermatozoa are not only essential for animal reproduction they also represent important tools for the manipulation of animal genetics. For instance, the genetic labeling and analysis of spermatozoa could provide a prospective complementation of pre-fertilization diagnosis and could help to prevent the inheritance of defective alleles during artificial insemination or to select beneficial traits in livestock. Spermatozoa feature extremely specialized membrane organization and restricted transport mechanisms making the labeling of genetically interesting DNA-sequences, e.g., with gold nanoparticles, a particular challenge. Here, we present a systematic study on the size-related internalization of ligand-free, monovalent and bivalent polydisperse gold nanoparticles, depending on spermatozoa membrane status. While monovalent conjugates were coupled solely to either negatively-charged oligonucleotides or positively-charged cell-penetrating peptides, bivalent conjugates were functionalized with both molecules simultaneously. The results clearly indicate that the cell membrane of acrosome-intact, bovine spermatozoa was neither permeable to ligand-free or oligonucleotide-conjugated nanoparticles, nor responsive to the mechanisms of cell-penetrating peptides. Interestingly, after acrosome reaction, which comprises major changes in sperm membrane composition, fluidity and charge, high numbers of monovalent and bivalent nanoparticles were found in the postequatorial segment, depicting a close and complex correlation between particle internalization and membrane organization. Additionally, depending on the applied peptide and for nanoparticle sizes < 10 nm even a successive nuclear penetration was observed, making the bivalent conjugates promising for future genetic delivery and sorting issues. PMID:26485929