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Sample records for human amnion membrane

  1. Effect of Thrombin on Human Amnion Mesenchymal Cells, Mouse Fetal Membranes, and Preterm Birth*

    Science.gov (United States)

    Mogami, Haruta; Keller, Patrick W.; Shi, Haolin; Word, R. Ann

    2014-01-01

    Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE2 in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE2 content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion. PMID:24652285

  2. Dehydrated Human Amnion/Chorion Membrane as Adjunctive Therapy in the Multidisciplinary Treatment of Pyoderma Gangrenosum: A Case Report.

    Science.gov (United States)

    Snyder, Robert J; Ead, Joey; Glick, Brad; Cuffy, Cherlson

    2015-09-01

    Pyoderma gangrenosum (PG) is an uncommon chronic and progressive skin disorder that can lead to severe tissue necrosis, pathergy, horrendous pain, and disfigurement if not properly and promptly diagnosed and treated. Systemic treatment traditionally consists of long-term immunosuppression. Topical care of the painful wound often represents a clinical challenge. A 77-year-old woman with multiple comorbidities including venous insufficiency and diabetes mellitus was diagnosed through exclusion with refractory, painful PG. She was managed for 3 months by a multidisciplinary team comprised of an internist, 2 dermatologists, and a podiatric wound care specialist using immunosuppressive therapy, several local wound care modalities, and supportive bandages. During that time, severe wound pain continued unabated and the affected area changed from 3 separate wounds measuring 1.4 cm x 1.0 cm x .01 cm, 1.2 cm x 0.5 cm x 0.1 cm, and 0.6 cm x 0.5 cm x 0.1 cm to 1 wound measuring 8.0 cm x 10.3 cm x 0.1 cm. At that time, dehydrated human amnion/chorion membrane (dHACM) allograft, previously reported to facilitate healing venous leg and diabetic foot ulcers, was incorporated into the treatment plan. The patient reported wound pain decreased from 10 out of 10 to 5 out of 10 within hours following application. At the 4 day follow-up visit, she reported no pain; after 1 week, the wound decreased 6.4 cm x 9.4 cm x 0.1 cm in size and after 2 months (3 applications) the wound had reduced in area from 103 cm2 to 57.96 cm2 (reduced by more than half [56%]). In this patient, following the application of dHACM as an adjunct to immunosuppressive therapy, pain receded and wound healing commenced. Additional controlled studies are needed to ascertain the generalizability of this observation.

  3. Function and failure of the fetal membrane: Modelling the mechanics of the chorion and amnion.

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    Stefaan W Verbruggen

    Full Text Available The fetal membrane surrounds the fetus during pregnancy and is a thin tissue composed of two layers, the chorion and the amnion. While rupture of this membrane normally occurs at term, preterm rupture can result in increased risk of fetal mortality and morbidity, as well as danger of infection in the mother. Although structural changes have been observed in the membrane in such cases, the mechanical behaviour of the human fetal membrane in vivo remains poorly understood and is challenging to investigate experimentally. Therefore, the objective of this study was to develop simplified finite element models to investigate the mechanical behaviour and rupture of the fetal membrane, particularly its constituent layers, under various physiological conditions. It was found that modelling the chorion and amnion as a single layer predicts remarkably different behaviour compared with a more anatomically-accurate bilayer, significantly underestimating stress in the amnion and under-predicting the risk of membrane rupture. Additionally, reductions in chorion-amnion interface lubrication and chorion thickness (reported in cases of preterm rupture both resulted in increased membrane stress. Interestingly, the inclusion of a weak zone in the fetal membrane that has been observed to develop overlying the cervix would likely cause it to fail at term, during labour. Finally, these findings support the theory that the amnion is the dominant structural component of the fetal membrane and is required to maintain its integrity. The results provide a novel insight into the mechanical effect of structural changes in the chorion and amnion, in cases of both normal and preterm rupture.

  4. The Use of Micronized Dehydrated Human Amnion/Chorion Membrane Allograft for the Treatment of Diabetic Foot Ulcers: A Case Series.

    Science.gov (United States)

    Hawkins, Brandon

    2016-05-01

    Diabetic foot ulcers (DFUs) are a common problem in patients with diabetes and are associated with significant morbidity and mortality. Dehydrated human amnion/chorion membrane (dHACM) allografts have been shown to be effective in the treatment of DFUs. A micronization process produces a dHACM powder that can be sprinkled onto irregular wound surfaces or reconstituted with normal saline for injection into tunneling wounds or wound margins. The author presents a case review of 3 patients with chronic plantar surface DFUs treated with micronized dHACM over a 1-month period. Wound duration was at least 8 months, and 2 out of 3 wounds had failed to heal with cryopreserved human fibroblast-derived dermal substitute before treatment with dHACM. Micronized dHACM (40 mg) in powder form was sprinkled onto the plantar ulcers weekly after sharp debridement, followed by standard topical dressings. Weekly dressing change and wound assessment was conducted to determine the rate of closure. Off-loading shoes were provided. Within 4 weeks of the first dHACM application, all 3 wounds had healed: the first after 2 applications, the second after 3 applications, and the last after 4 applications. No adverse events were observed, and the wounds remained healed after 6 months. In the author's practice, the micronized dHACM allograft was easily applied, clinically effective, and well tolerated as a treatment for plantar ulcers in patients with diabetes.

  5. Effect of amnion membrane transplantation on corneal neovascularization in 10 patients with alkali burn

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    Yin, Lan; Pi, Yu-Li

    2011-01-01

    By observing clinical cases, we studied the curative effect of amnion membrane transplantation on decreasing corneal neovascularization (CNV). It was a non-randomized retrospective case-control study. Among 17 cases (21 eyes) of third-degree alkali burns from 2007 to 2010, 10 cases (12 eyes) were performed with amnion membrane transplantation operation, and others were not. Amnion membrane transplantation was performed at the 3rd day after burn in the treatment group. Areas of CNV in double g...

  6. Investigating the Potential of Amnion-Based Scaffolds as a Barrier Membrane for Guided Bone Regeneration.

    Science.gov (United States)

    Li, Wuwei; Ma, Guowu; Brazile, Bryn; Li, Nan; Dai, Wei; Butler, J Ryan; Claude, Andrew A; Wertheim, Jason A; Liao, Jun; Wang, Bo

    2015-08-11

    Guided bone regeneration is a new concept of large bone defect therapy, which employs a barrier membrane to afford a protected room for osteogenesis and prevent the invasion of fibroblasts. In this study, we developed a novel barrier membrane made from lyophilized multilayered acellular human amnion membranes (AHAM). After decellularization, the AHAM preserved the structural and biomechanical integrity of the amnion extracellular matrix (ECM). The AHAM also showed minimal toxic effects when cocultured with mesenchymal stem cells (MSCs), as evidenced by high cell density, good cell viability, and efficient osteogenic differentiation after 21-day culturing. The effectiveness of the multilayered AHAM in guiding bone regeneration was evaluated using an in vivo rat tibia defect model. After 6 weeks of surgery, the multilayered AHAM showed great efficiency in acting as a shield to avoid the invasion of the fibrous tissues, stabilizing the bone grafts and inducing the massive bone growth. We hence concluded that the advantages of the lyophilized multilayered AHAM barrier membrane are as follows: preservation of the structural and mechanical properties of the amnion ECM, easiness for preparation and handling, flexibility in adjusting the thickness and mechanical properties to suit the application, and efficiency in inducing bone growth and avoiding fibrous tissues invasion.

  7. The Use of Dehydrated Human Amnion/Chorion Membrane Allograft Injection for the Treatment of Tendinopathy or Arthritis: A Case Series Involving 40 Patients.

    Science.gov (United States)

    Gellhorn, Alfred C; Han, Alex

    2017-12-01

    Degenerative joint and tendon injuries remain difficult to treat, with few effective conservative treatment options available. Regenerative approaches aim to promote the inherent healing capacity of injured tissues. Micronized dehydrated human amnion/chorion membrane (dHACM) injection is an emerging regenerative option with promising preclinical results. To test the clinical effectiveness of dHACM injection in patients with chronic tendinopathy and arthropathy. Case series. Academic medical center outpatient sports medicine clinic. A total of 40 patients with chronic tendinosis or arthropathy who received dHACM over a period of 9 months. A structured interview was administered to patients by telephone to supplement the clinical information available in the medical chart. All patients received an ultrasound-guided injection of dHACM. The primary outcome was change in pain level, and the secondary outcome was change in activities of daily living (ADLs) and sports/recreation function. More than 30% improvement in average pain and function was considered a successful outcome. Patient pain and function were measured at 1, 2, and 3 months after the procedure. Patient-reported average pain scores decreased from a baseline value of 6.4 (95% confidence interval [CI] = 5.7-7.0) to 2.7 (95% CI = 2.1-3.3; P success, defined as 30% or greater improvement in pain levels, was 68% at 1 month, 82% at 2 months, and 91% at 3 months. Patient-reported functional impairment in ADLs decreased from 6.8 (95% CI = 6.0-7.5) to 2.0 (95% CI = 1.4-2.7) (P sports/recreation decreased from 8.5 (95% CI = 7.9-9.1) to 3.2 (95% CI = 2.6-3.9) (P < .001). Frequency of pain medication use decreased from 29 of 40 patients (72.5%) before the procedure to 9 of 40 patients (22.5%) at final follow up (P < .001). Localized pain at the injection site was common, but no other adverse events or side effects were reported. In the setting of tendinosis or arthropathy, dHACM injection was clinically effective in

  8. Syndecan expressions in the human amnion and chorionic plate

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    T. Lorenzi

    2010-10-01

    Full Text Available The syndecan family consists of four distinct membrane glycoproteins in mammals. Syndecans control cell proliferation, differentiation, adhesion and migration through participation in cell-cell interactions, anchorage of cells to the extracellular environment, and modulation of multiple growth factors. Therefore, syndecans may play a pivotal role in the regulation of cell behaviour depending on the cellular microenvironment. Here, we demonstrate that syndecan-1, syndecan-2 and syndecan-4 are expressed in fetal membrane tissue with different immunolocalizations. Syndecan-1 is expressed in the amniotic epithelium, localizing at basolateral cell surfaces. Syndecan-2 and syndecan-4, in contrast, are mostly localized in intracellular compartments, in the extravillous cytotrophoblastic cells and in some fibroblasts of the chorionic plate as well as in the amniotic epithelial cells. In the latter, syndecan-4 is mainly localized in the apical part of the cells. Our results strongly suggest a key role of syndecan-1, syndecan-2 and syndecan-4 in the determination of structural and functional characteristics of human amnion and chorionic plate. Since the solute exchanges between fetus and mother take place in fetal membranes, our data suggest that syndecans are important players in the placenta for the establishment of the fetal-maternal inter-communication.

  9. Genome-Wide Analysis of DNA Methylation in Human Amnion

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    Jinsil Kim

    2013-01-01

    Full Text Available The amnion is a specialized tissue in contact with the amniotic fluid, which is in a constantly changing state. To investigate the importance of epigenetic events in this tissue in the physiology and pathophysiology of pregnancy, we performed genome-wide DNA methylation profiling of human amnion from term (with and without labor and preterm deliveries. Using the Illumina Infinium HumanMethylation27 BeadChip, we identified genes exhibiting differential methylation associated with normal labor and preterm birth. Functional analysis of the differentially methylated genes revealed biologically relevant enriched gene sets. Bisulfite sequencing analysis of the promoter region of the oxytocin receptor (OXTR gene detected two CpG dinucleotides showing significant methylation differences among the three groups of samples. Hypermethylation of the CpG island of the solute carrier family 30 member 3 (SLC30A3 gene in preterm amnion was confirmed by methylation-specific PCR. This work provides preliminary evidence that DNA methylation changes in the amnion may be at least partially involved in the physiological process of labor and the etiology of preterm birth and suggests that DNA methylation profiles, in combination with other biological data, may provide valuable insight into the mechanisms underlying normal and pathological pregnancies.

  10. Amnion membrane for coverage of gingival recession: A novel application

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    Rucha Shah

    2014-01-01

    Full Text Available Introduction: Amnion allograft has been used in the field of medicine for its exceptional wound-modulating properties. However, in the field of dentistry, only a limited number of reports have explored its potential in healing of oral wounds. Materials and Methods: Amnion allograft in conjunction with coronally advanced flap has been used in the management of gingival recession. Results: A complete coverage along with excellent esthetics and an improvement in gingival biotype was observed at 6 months postoperatively. Discussion: Because of its inherent wound-modulating properties, amnion allograft may be used to enhance periodontal wound healing and enable tissue regeneration such as that in the coverage of gingival recession. Conclusion: Amnion allograft may provide an alternative to other conventional methods of treating gingival recession.

  11. Penggunaan Tetes Telinga Serum Autologous dengan Amnion untuk Penutupan Perforasi Membran Timpani

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    Hidayatul Fitria

    2012-07-01

    Full Text Available Abstrak Latar Belakang: Gangguan pendengaran atau ketulian mempunyai dampak yang merugikan bagi penderita , keluarga, masyarakat maupun negara. Salah satu penyebab ketulian yang sering dijumpai adalah radang telinga tengah, terutama yang disertai perforasi membran timpani yang menetap. Penutupan perforasi membran timpani dapat dilakukan dengan operatif dan konservatif. Secara konservatif sudah banyak cara yang dilakukan. Salah satunya dengan mengkaustik tepi perforasi dengan menggunakan silver nitrat untuk membuat luka baru, kemudian digunakan amnion sebagai jembatan (bridge dan faktor regulasi yang terdapat pada tetes telinga serum autologous. Tujuan: Untuk menjelaskan gambaran penggunaan amnion sebagai jembatan dan tetes telinga serum autologous sebagai faktor regulasi. Tinjauan pustaka: Penutupan perforasi membran timpani dapat dilakukan secara konservatif salah satunya dengan menggunakan tetes telinga serum autologous sebagai faktor regulator, amnion sebagai jembatan dan penggunaan silver nitrat pada tepi perforasi untuk membuat luka baru. Serum autologous memiliki asselerator pertumbuhan yaitu epidermal growth factor (EGF , transforming growth factor β1 (TGF- β1 dan fibronektin. Asselerator pertumbuhan ini dapat kita temukan pada penyembuhan membran timpani normal. Sedangkan membran amnion adalah jaringan semi transparan tipis yang membentuk lapisan terdalam membran fetus dengan susunan membran basalis yang tebal dan jaringan stroma avaskuler. Membran amnion mempercepat pembentukan epitel normal dengan menekan pembentukan jaringan fibrosis. Sel epitel amnion memproduksi faktor pertumbuhan seperti fibroblast growth factor dan transforming growth factor beta. Faktor pertumbuhan akan membantu komunikasi antara epitel dan sel fibroblast stroma untuk menekan proliferasi dan diferensiasi jaringan fibrosis. Kesimpulan: Diperlukan tiga elemen pada penutupan perforasi membran timpani yaitu faktor regulasi, jembatan (bridge dan membuat luka baru

  12. The human amnion is a site of MHC class lb expression: Evidence for the expression of HLA-E and HLA-G

    Energy Technology Data Exchange (ETDEWEB)

    Houlihan, J.M.; Harper, H.M.; Jenkinson, H.J. [Univ. of Bristol (United Kingdom)] [and others

    1995-06-01

    The expression of HLA class I Ag by term human amnion epithelial cells was investigated. In immunostaining and FACS analysis, mAb to monomorphic class I Ag reacted extensively with amnion cells, whereas polymorphic mAb reactivity was more limited and variable. Further studies were conducted on amnion cell preparations containing negligible contaminants. Northern analysis with use of locus-specific probes demonstrated that amnion expresses two class lb genes, HLA-E and HLA-G. Radio-immunoprecipitation with use of monomorphic mAb identified two fully glycosylated cell surface class I H chains of 44 and 41 kDa; polymorphic mAbs failed to immunoprecipitate the 41-kDa product, although 44-kDa products, typical of class la Ag, were identified in some preparations. Class I H chains were isolated from amnion by affinity chromatography. Microsequencing revealed that the first nine residues of the N-terminus of the 41-kDa product aligned perfectly only with HLA-E. Overall, amnion at term appears to express class lb Ag with limited class la Ag. HLA-G is therefore expressed in two extrafetal epithelia: amnion and trophoblast. Identification of the class lb protein HLA-E-E in amnion epithelium may have implications for preterm labor that can be associated with infection of the placental membranes. 44 refs., 5 figs., 2 tabs.

  13. Timing of Histologic Progression from Chorio-Deciduitis to Chorio-Deciduo-Amnionitis in the Setting of Preterm Labor and Preterm Premature Rupture of Membranes with Sterile Amniotic Fluid.

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    Park, Chan-Wook; Park, Joong Shin; Norwitz, Errol R; Moon, Kyung Chul; Jun, Jong Kwan; Yoon, Bo Hyun

    2015-01-01

    Histologic chorio-deciduitis and chorio-deciduo-amnionitis (amnionitis) in extra-placental membranes are known to represent the early and advanced stages of ascending intra-uterine infection. However, there are no data in humans about the time required for chorio-deciduitis to develop and for chorio-deciduitis without amnionitis to progress to chorio-deciduitis with amnionitis, and the effect of prolongation of pregnancy on the development of chorio-deciduitis and amnionitis in patients with preterm labor and intact membranes (PTL) and preterm premature rupture of membranes (preterm-PROM). We examined these issues in this study. The study population consisted of 289 women who delivered preterm (133 cases with PTL, and 156 cases with preterm-PROM) and who had sterile amniotic fluid (AF) defined as a negative AF culture and the absence of inflammation as evidenced by a matrix metalloproteinase-8 (MMP-8) level membranes (i.e., inflammation-free extra-placental membranes, choroi-deciduitis only, and chorio-deciduitis with amnionitis) in patients with PTL and preterm-PROM. Amniocentesis-to-delivery interval was longer in cases of chorio-deciduitis with amnionitis than in cases of chorio-deciduitis only in both PTL (median [interquartile-range (IQR)]; 645.4 [319.5] vs. 113.9 [526.9] hours; P = 0.005) and preterm-PROM (131.3 [135.4] vs. 95.2 [140.5] hours; Pmembranes. Moreover, prolongation of pregnancy is an independent predictor of the development of both chorio-deciduitis and amnionitis in cases of PTL with sterile AF.

  14. The Effect of Progestins on Tumor Necrosis Factor α-Induced Matrix Metalloproteinase-9 Activity and Gene Expression in Human Primary Amnion and Chorion Cells In Vitro.

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    Allen, Terrence K; Feng, Liping; Nazzal, Matthew; Grotegut, Chad A; Buhimschi, Irina A; Murtha, Amy P

    2015-05-01

    Current treatment modalities for preventing preterm premature rupture of membranes are limited, but progestins may play a role. Tumor necrosis factor α (TNFα) enhances matrix metalloproteinase-9 (MMP-9) gene expression and activity in fetal membranes, contributing to membrane weakening and rupture. We previously demonstrated that progestins attenuate TNFα-induced MMP-9 activity in a cytotrophoblast cell line. However, whether they have a similar effect in primary amnion and chorion cells of fetal membranes is unknown. In this study, we evaluated the effect of progestins on basal and TNFα-induced MMP-9 activity and gene expression in primary chorion and amnion cells harvested from the fetal membranes of term nonlaboring patients. Primary amnion and chorion cells were isolated from fetal membranes obtained from term uncomplicated nonlaboring patients following elective cesarean delivery (n = 11). Confluent primary amnion and chorion cell cultures were both pretreated with vehicle (control), progesterone (P4), 17α-hydroxyprogesterone caproate (17P), or medroxyprogesterone acetate (MPA) at 10 M concentration for 6 hours followed by stimulation with TNFα at 10 ng/mL for an additional 24 hours. Cell cultures pretreated with the vehicle only served as the unstimulated control and the vehicle stimulated with TNFα served as the stimulated control. Both controls were assigned a value of 100 units. Cell culture medium was harvested for MMP-9 enzymatic activity quantification using gelatin zymography. Total RNA was extracted for quantifying MMP-9 gene expression using real-time quantitative PCR. Basal MMP-9 activity and gene expression data were normalized to the unstimulated control. TNFα-stimulated MMP-9 activity and gene expression were normalized to the stimulated control. The primary outcome was the effect of progestins on TNFα-induced MMP-9 enzymatic activity in term human primary amnion and chorion cells in vitro. Secondary outcomes included the effect of

  15. Timing of Histologic Progression from Chorio-Deciduitis to Chorio-Deciduo-Amnionitis in the Setting of Preterm Labor and Preterm Premature Rupture of Membranes with Sterile Amniotic Fluid.

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    Chan-Wook Park

    Full Text Available Histologic chorio-deciduitis and chorio-deciduo-amnionitis (amnionitis in extra-placental membranes are known to represent the early and advanced stages of ascending intra-uterine infection. However, there are no data in humans about the time required for chorio-deciduitis to develop and for chorio-deciduitis without amnionitis to progress to chorio-deciduitis with amnionitis, and the effect of prolongation of pregnancy on the development of chorio-deciduitis and amnionitis in patients with preterm labor and intact membranes (PTL and preterm premature rupture of membranes (preterm-PROM. We examined these issues in this study.The study population consisted of 289 women who delivered preterm (133 cases with PTL, and 156 cases with preterm-PROM and who had sterile amniotic fluid (AF defined as a negative AF culture and the absence of inflammation as evidenced by a matrix metalloproteinase-8 (MMP-8 level <23 ng/ml. We examined the association between amniocentesis-to-delivery interval and inflammatory status in the extra-placental membranes (i.e., inflammation-free extra-placental membranes, choroi-deciduitis only, and chorio-deciduitis with amnionitis in patients with PTL and preterm-PROM.Amniocentesis-to-delivery interval was longer in cases of chorio-deciduitis with amnionitis than in cases of chorio-deciduitis only in both PTL (median [interquartile-range (IQR]; 645.4 [319.5] vs. 113.9 [526.9] hours; P = 0.005 and preterm-PROM (131.3 [135.4] vs. 95.2 [140.5] hours; P<0.05. Amniocentesis-to-delivery interval was an independent predictor of the development of both chorio-deciduitis and amnionitis after correction for confounding variables such as gestational age at delivery in the setting of PTL, but not preterm-PROM.These data confirm for the first time that, in cases of both PTL and preterm-PROM with sterile AF, more time is required to develop chorio-deciduitis with amnionitis than chorio-deciduitis alone in extra-placental membranes. Moreover

  16. Monovalent cations transfer through isolated human amnion: a new pharmacological model

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    Bara, M.; Guiet-Bara, A.; Durlach, J.

    1985-04-01

    Transfer of monovalent cations through the isolated human amnion consists of different factors: paracellular, coupling, ATPase dependent cellular transfer, leak cellular transfer. Understanding this transfer permits testing of the action of various substances. Physiological substances (Mg, taurine) increase ionic transfer and there is a vicarious effect between Mg and taurine. The tocolytic agents MgSO/sub 4/ and ethanol do not exhibit a good effect on the transfer: decrease with ethanol; equality between entry and exit fluxes with MgSO/sub 4/. On the other hand, amphotericin B increases mother-to-fetus transfer. Polluting metals (Pb, Cd, Hg, As) dramatically reduce exchanges and almost completely inhibit amnion permeability. Ingestion of ethanol also exhibits a dramatic effect on the exchange between mother and fetus through the amnion. Study of ionic transfer in vitro can be considered a pharmacological model to investigate the modifications of mother-fetus exchanges by various substances.

  17. Inhibition of lysyl oxidase by prostaglandin E2 via EP2/EP4 receptors in human amnion fibroblasts: Implications for parturition.

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    Liu, Chao; Zhu, Ping; Wang, Wangsheng; Li, Wenjiao; Shu, Qun; Chen, Zi-Jiang; Myatt, Leslie; Sun, Kang

    2016-03-15

    The underlying mechanism leading to rupture of the membranes at parturition is not fully understood. Lysyl oxidase (LOX) cross-links collagen fibrils thereby increasing the tensile strength of the membranes. Thus, understanding the regulation of LOX expression may be of crucial importance for elucidation of the process of rupture of the fetal membranes. Prostaglandin E2 (PGE2), mainly produced in the amnion, plays crucial roles during human parturition. However it is not known whether PGE2 regulates LOX expression in the fetal membranes. Using primary human amnion fibroblasts, we showed that addition of PGE2 decreased LOX mRNA and protein levels, which were blocked by inhibition of EP2/EP4 receptors and the receptor-coupled cAMP/PKA pathway. EP2/EP4 receptor agonists and stimulators of the cAMP/PKA pathway consistently decreased LOX expression. Furthermore, PGE2 induced cyclo-oxygenase-2 (COX-2) expression, a key enzyme in PGE2 production, via an EP2 and EP4 receptor-coupled cAMP/PKA pathway. Small interfering RNA-mediated knock-down of COX-2 expression significantly increased the basal expression of LOX. In addition, an increase in COX-2 and a reciprocal decrease in LOX abundance occurred in amnion tissue following labor at term. In conclusion, we have revealed a feed-forward loop of induction of COX-2 and reduction in LOX expression by PGE2 acting via an EP2/EP4 receptor-coupled cAMP/PKA pathway in human amnion fibroblasts toward the end of gestation, which may play a significant role in the rupture of fetal membranes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Inhibition by human recombinant tissue inhibitor of metalloproteinases of human amnion invasion and lung colonization by murine B16-F10 melanoma cells.

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    Schultz, R M; Silberman, S; Persky, B; Bajkowski, A S; Carmichael, D F

    1988-10-01

    The human tissue inhibitor of metalloproteinases (TIMP) is a glycoprotein with a molecular weight of 28,000. It appears to be ubiquitous in human mesoderm tissues and has previously been shown to be identical to the collagenase inhibitor isolated from human skin fibroblasts. TIMP inhibits type I- and IV-specific collagenases and other neutral metalloendoproteinases that may be responsible for the degradation of extracellular matrix in tumor cell metastasis. In this work we have utilized recombinant human TIMP (rTIMP) obtained by expression of its cDNA gene (Carmichael et al., Proc. Natl. Acad. Sci. USA, 83:2407, 1986). The rTIMP is shown to have similar inhibition properties as natural TIMP against human skin fibroblast collagenase. In an in vitro amnion invasion assay system, rTIMP inhibited the invasion of B16-F10 murine melanoma cells through the human amniotic membrane at an identical concentration to that reported previously for natural TIMP. The mechanism by which rTIMP inhibits amniotic membrane invasion was compared to the mechanism by which the fibronectin receptor binding peptide RGDS and the aminin receptor binding peptide YIGSR inhibit amnion invasion. RGDS and YIGSR inhibited strong binding of the tumor cells to the amniotic membrane. In contrast rTIMP did not inhibit the cell adhesion step in amnion invasion, but actually increased the number of tumor cells that were tightly bound to the amnion. Thus rTIMP appears to inhibit a later step in the amnion invasion process, following B16-F10 cell adhesion. C57BL/6 mice treated with i.p. injections of rTIMP every 12 h for 6.5 days showed a significant inhibition of metastatic lung colonization by B16-F10 murine melanoma cells. While the rTIMP inhibited the number of metastatic lung tumors formed, it had no significant effect on the size of the lung tumors. Furthermore, tumors grown s.c. in mice receiving 12-h i.p. injections of rTIMP for 6.5 days, as in the in vivo colonization assay, showed no difference

  19. DREAM Is Involved in the Genesis of Inflammation-Induced Prolabour Mediators in Human Myometrial and Amnion Cells

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    Priyanka Goradia

    2018-01-01

    Full Text Available Preterm birth is the primary cause of perinatal morbidity and mortality worldwide. Inflammation induces a cascade of events leading to preterm birth by activating nuclear factor-κB (NF-κB. In nongestational tissues, downstream regulatory element antagonist modulator (DREAM regulates NF-κB activity. Our aims were to analyse DREAM expression in myometrium and fetal membranes obtained at term and preterm and to determine the effect of DREAM inhibition on prolabour mediators in primary myometrial and amnion cells. DREAM mRNA expression was significantly higher in fetal membranes obtained after spontaneous labour compared to nonlabour and in amnion from women with histological preterm chorioamnionitis when compared to amnion from women without chorioamnionitis. In primary myometrial and amnion cells, the effect of DREAM silencing by siRNA was a significant decrease in the expression of proinflammatory cytokine IL-6, the chemokines IL-8 and MCP-1, the adhesion molecule ICAM-1, MMP-9 mRNA expression and activity, and NF-κB transcriptional activity when stimulated with the proinflammatory cytokine IL-1β, the bacterial products fsl-1 or flagellin, or the viral dsRNA analogue poly(I:C. These data suggest that, in states of heightened inflammation, DREAM mRNA expression is increased and that, in myometrial and amnion cells, DREAM regulates proinflammatory and prolabour mediators which may be mediated via NF-κB.

  20. The Human Amnion Epithelial Cell Secretome Decreases Hepatic Fibrosis in Mice with Chronic Liver Fibrosis

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    Majid Alhomrani

    2017-10-01

    Full Text Available Background: Hepatic stellate cells (HSCs are the primary collagen-secreting cells in the liver. While HSCs are the major cell type involved in the pathogenesis of liver fibrosis, hepatic macrophages also play an important role in mediating fibrogenesis and fibrosis resolution. Previously, we observed a reduction in HSC activation, proliferation, and collagen synthesis following exposure to human amnion epithelial cells (hAEC and hAEC-conditioned media (hAEC-CM. This suggested that specific factors secreted by hAEC might be effective in ameliorating liver fibrosis. hAEC-derived extracellular vesicles (hAEC-EVs, which are nanosized (40–100 nm membrane bound vesicles, may act as novel cell–cell communicators. Accordingly, we evaluated the efficacy of hAEC-EV in modulating liver fibrosis in a mouse model of chronic liver fibrosis and in human HSC.Methods: The hAEC-EVs were isolated and characterized. C57BL/6 mice with CCl4-induced liver fibrosis were administered hAEC-EV, hAEC-CM, or hAEC-EV depleted medium (hAEC-EVDM. LX2 cells, a human HSC line, and bone marrow-derived mouse macrophages were exposed to hAEC-EV, hAEC-CM, and hAEC-EVDM. Mass spectrometry was used to examine the proteome profile of each preparation.Results: The extent of liver fibrosis and number of activated HSCs were reduced significantly in CCl4-treated mice given hAEC-EVs, hAEC-CM, and hAEC EVDM compared to untreated controls. Hepatic macrophages were significantly decreased in all treatment groups, where a predominant M2 phenotype was observed. Human HSCs cultured with hAEC-EV and hAEC-CM displayed a significant reduction in collagen synthesis and hAEC-EV, hAEC-CM, and hAEC-EVDM altered macrophage polarization in bone marrow-derived mouse macrophages. Proteome analysis showed that 164 proteins were unique to hAEC-EV in comparison to hAEC-CM and hAEC-EVDM, and 51 proteins were co-identified components with the hAEC-EV fraction.Conclusion: This study provides novel data

  1. Efektivitas Membran Amnion Liofilisasi (Handmade Tubular sebagai Nerve Conduit pada Perbaikan Cedera Saraf Perifer Tikus dengan Gap 5 mm

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    R. Dadan G. Gandadikusumah

    2013-09-01

    Full Text Available Peripheral nerve injury with 5–30 mm gap which is caused by direct injury cases (87% or iatrogenic (12% become a special concern because it could cause a serious disability in the future. Therefore, we need many kinds of nerve repair methods without adding morbidity to the patient. One of the methods is entubulation method, by using natural or synthetic material. This was an animal experimental research by using simple random design in Departement Pharmachology Laboratory, Faculty of Medicine, Universitas Padjadjaran Bandung in May 2012. The samples were used 14 Wistar rats, divided into 2 groups. After creating gap on sciatic nerve, nerve conduit was installed on case group by using handmade tubular lyophilized amnion membrane. Nerve conduit was not installed in control group. After 21 days observation, conduction test and histopathology examination were done. Data were analyzed by using non-parametric statistical analysis sign test. All animals survived without any serious surgical complication. Result showed a significant difference between groups; the conduction test=0.016 (p<0.05, nerve growth to distal gap=0.063 (p<0.05, no radier direction of nerve growth=0.031 (p<0.05. Reaction of inflammation was minimum and there was no difference between two groups. In conclusion, handmade tubular lyophilized amnion membrane is effective as nerve conduit in repair of peripheral nerve injury with 5 mm gap.

  2. Osteogenic Potential Differentiation of Human Amnion Mesenchymal Stem Cell with Chitosan-Carbonate Apatite Scaffold (In Vitro Study

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    Michael J.K. Kamadjaja

    2016-09-01

    Full Text Available Background: Tissue engineering based approaches have received much attention. Incorporation of chitosan and carbonate apatite (CA improve its capability. Human mesenchymal stem cells (hMSCs is viable for xenogenic transplantation. The purpose of this study was to fabricate and evaluate the osteogenic potential diferentiation of human amnion mesenchymal stem cell with carbonate apatite–chitosan scaffolds (CA-ChSs for tissue engineering. Method: Human amniotic membrane was procured from using cesarean section. Soncini’s protocol was employed for the isolation procedure. The cells cultured on collagen-coated dishes using Dulbecco's minimal essential medium (DMEM/F12 (1:1. A chitosan powder of medium molecular weight deacetylated chitin, poly(D(glucosamine was used and mixed with CA. Immunocytochemistry and flowcytometry used for phenotypic characterization of hAMSC. Result: Amniotic membrane obtained using cesarean section under aseptic condition did not exhibit any growth of cell cultures which were not contaminated. Immunocytochemistry testing revealed that the target cells expressed strong mesenchymal stem cell marker CD 105. Characterization at passage 10 showed that CD44 was the most significant and abundant surface receptors. The number of viable cells in chitosan-carbonate apatite was 66.59%. Scanning electron microscope (SEM observation revealed that CA-ChSs had three-dimensional structure with many pores and hAMSc could attached and proliferation among the porosity of the scaffold. The formation of calcium in the cell as an indicator of osteoblast cells was detected using Alizarin Red solution. Conclusion: hAMSc harvested from human amniotic membrane seeding in CA-ChSs had the capability for in vitro osteogenesis makes them be the one of the potential options for bone tissue engineering.

  3. [Effects of culture supernatant of human amnion mesenchymal stem cells on biological characteristics of human fibroblasts].

    Science.gov (United States)

    Wu, Qi'er; Lyu, Lu; Xin, Haiming; Luo, Liang; Tong, Yalin; Mo, Yongliang; Yue, Yigang

    2016-06-01

    To investigate the effects of culture supernatant of human amnion mesenchymal stem cells (hAMSCs-CS) on biological characteristics of human fibroblasts. (1) hAMSCs were isolated from deprecated human fresh amnion tissue of placenta and then sub-cultured. The morphology of hAMSCs on culture day 3 and hAMSCs of the third passage were observed with inverted phase contrast microscope. (2) Two batches of hAMSCs of the third passage were obtained, then the expression of vimentin of cells was observed with immunofluorescence method, and the expression of cell surface marker CD90, CD73, CD105, and CD45 was detected by flow cytometer. (3) hAMSCs-CS of the third passage at culture hour 72 were collected, and the content of insulin-like growth factor Ⅰ (IGF-Ⅰ), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay. (4) Human fibroblasts were isolated from deprecated human fresh prepuce tissue of circumcision and then sub-cultured. Human fibroblasts of the third passage were used in the following experiments. Cells were divided into blank control group and 10%, 30%, 50%, and 70% hAMSCs-CS groups according to the random number table (the same grouping method below), with 48 wells in each group. Cells in blank control group were cultured with DMEM/F12 medium containing 2% fetal bovine serum (FBS), while cells in the latter 4 groups were cultured with DMEM/F12 medium containing corresponding volume fraction of hAMSCs-CS and 2% FBS. The proliferation activity of cells was detected by cell counting kit 8 and microplate reader at culture hour 12, 24, 48, and 72, respectively, and corresponding volume fraction of hAMSCs-CS which causing the best proliferation activity of human fibroblasts was used in the following experiments. (5) Human fibroblasts were divided into blank control group and 50% hAMSCs-CS group and treated as in (4), with 4 wells in each group, at post

  4. A roentgenographic assessment of regenerative efficacy of bioactive Gengigel® in conjunction with amnion membrane in grade II furcation defect

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    S Harveen Kalra

    2015-01-01

    Full Text Available Background: Nowadays, techniques are being developed to guide and instruct the specialized cellular components of the periodontium to participate in the regenerative process. This approach of reconstruction makes use of understanding of the development of the periodontium and the cellular processes that are involved. Hyaluronic acid is a naturally occurring non-sulfated high molecular weight glycosaminoglycan that forms a critical component of the extracellular matrix and contributes significantly to tissue hydrodynamics, cell migration, and proliferation. Hence, its administration to periodontal wound sites could achieve comparable beneficial effects in periodontal tissue regeneration. Hence, the purpose of the present case report was to assess roentgenographically, the regenerative capacity of Gengigel® in conjunction with bioactive amnion guided tissue regeneration (GTR membrane in a patient with Grade II furcation defect. Case Presentation: A patient complained of bleeding gums from the lower back tooth region, reportedly found Grade II furcation in the lower right mandibular first molar. After Phase, I therapy, Gengigel® along with bioactive amnion membrane was placed in the furcation area during the surgical phase. Roentgenographic assessment was done at 4 months and 6 months postoperatively. It resulted in complete defect-fill and loss of radiolucency at 6 months. Conclusion: Surgical placement of Gengigel® along with amnion membrane in the furcation defect can significantly improve the periodontal defect morphology.

  5. Epigenetic Regulation of Cytokine Production in Human Amnion and Villous Placenta

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    Murray D. Mitchell

    2012-01-01

    Full Text Available The mechanisms of human preterm labour appear inextricably linked to cytokine biosynthesis by gestational tissues. In turn, cytokine production by gestational tissues has been shown to be regulated by epigenetic mechanisms. In this paper, we demonstrate that cytokine production in gestational tissues is regulated epigenetically in a tissue-specific manner. Furthermore, we show that treatment with a histone deacetylation inhibitor can partially abrogate LPS-stimulated TNFα production in villous placenta but not amnion. LPS treatment significantly (100-fold and IL10 (~6–10-fold after 24 h of treatment in villous explants, as expected. There were no significant LPS effects on IL1Ra production. AZA treatment did not have any significant effect on any cytokines' production tested either alone or in combination with LPS. Interestingly, however, the stimulatory effects of LPS on TNFα production were partially mitigated (<0.05 by TSA treatment in villous explants. We suggest caution in the consideration of histone deacetylation inhibitors in pregnancy due to the different responses in gestational tissues.

  6. Effect of Human Amnion Epithelial Cells on the Acute Inflammatory Response in Fetal Sheep

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    Alana Westover

    2017-11-01

    Full Text Available Intra-amniotic (IA lipopolysaccharide (LPS injection in sheep induces inflammation in the fetus. Human amnion epithelial cells (hAECs moderate the effect of IA LPS on fetal development, but their influence on the acute inflammatory response to IA LPS is unknown. We aimed to determine the effects of hAECs on the acute fetal inflammatory response to IA LPS. After surgical instrumentation at 116 days' gestation (d ewes were randomized to 1 of 4 groups at 123 d: IA LPS (10 mg and intravenous (IV saline (n = 8, IA LPS and IV hAECs (n = 6, IA saline and IV saline (n = 5 or IA saline and IV hAECs (n = 5. IV injections were administered immediately after IA injections. Serial fetal blood samples were collected. At 125 d, placental, fetal lung and liver samples were collected. IA LPS increased inflammatory cell recruitment in the placenta and lungs, increased IL-1β and IL-8 mRNA levels in the lungs and increased serum amyloid A3 (SAA3 and C-reactive protein (CRP mRNA levels in the liver. IV hAECs reduced fetal lung inflammatory cell recruitment but did not otherwise alter indices of placental, fetal lung or liver inflammation. The acute fetal inflammatory response to IA LPS is not substantially altered by IV hAEC treatment.

  7. A Pilot Study Evaluating the Safety of Intravenously Administered Human Amnion Epithelial Cells for the Treatment of Hepatic Fibrosis

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    Rebecca Lim

    2017-08-01

    Full Text Available Liver cirrhosis is the 6th leading cause of death in adults aged 15–59 years in high-income countries. For many who progress to cirrhosis, the only prospect for survival is liver transplantation. While there is some indication that mesenchymal stem cells may be useful in reversing established liver fibrosis, there are limitations to their widespread use – namely their rarity, the need for extensive serial passaging and the associated potential for genomic instability and cellular senescence. To this end, we propose the use of allogeneic amnion epithelial cells. This clinical trial will assess the safety of intravenously delivered allogeneic human amnion epithelial cells (hAECs in patients with compensated liver cirrhosis. This will also provide clinical data that will inform phases 2 and 3 clinical trials with the ultimate goal of developing hAECs as a therapeutic option for patients with cirrhosis who are at significant risk of disease progression. We will recruit 12 patients with compensated cirrhosis, based on their hepatic venous pressure gradient, for a dose escalation study. Patients will be closely monitored in the first 24 h post-infusion, then via daily telephone interviews until clinical assessment on day 5. Long term follow up will include standard liver tests, transient elastography and hepatic ultrasound. Ethics approval was obtained from Monash Health for this trial 16052A, “A Pilot Study Evaluating the Safety of Intravenously Administered Human Amnion Epithelial Cells for the Treatment of Liver Fibrosis, A First in Adult Human Study.” The trial will be conducted in accordance to Monash Health Human Ethics guidelines. Outcomes from this study will be disseminated in the form of conference presentations and submission to a peer reviewed journal. This trial has been registered on the Australian and New Zealand Clinical Trials Registry ACTRN12616000437460.

  8. Human amnion cells reverse acute and chronic pulmonary damage in experimental neonatal lung injury

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    Dandan Zhu

    2017-11-01

    Full Text Available Abstract Background Despite advances in neonatal care, bronchopulmonary dysplasia (BPD remains a significant contributor to infant mortality and morbidity. While human amnion epithelial cells (hAECs have shown promise in small and large animal models of BPD, there is scarce information on long-term benefit and clinically relevant questions surrounding administration strategy remain unanswered. In assessing the therapeutic potential of hAECs, we investigated the impact of cell dosage, administration routes and timing of treatment in a pre-clinical model of BPD. Methods Lipopolysaccharide was introduced intra-amniotically at day 16 of pregnancy prior to exposure to 65% oxygen (hyperoxia at birth. hAECs were administered either 12 hours (early or 4 days (late after hyperoxia commenced. Collective lung tissues were subjected to histological analysis, multikine ELISA for inflammatory cytokines, FACS for immune cell populations and 3D lung stem cell culture at neonatal stage (postnatal day 7 and 14. Invasive lung function test and echocardiography were applied at 6 and 10 weeks of age. Results hAECs improved the tissue-to-airspace ratio and septal crest density in a dose-dependent manner, regardless of administration route. Early administration of hAECs, coinciding with the commencement of postnatal hyperoxia, was associated with reduced macrophages, dendritic cells and natural killer cells. This was not the case if hAECs were administered when lung injury was established. Fittingly, early hAEC treatment was more efficacious in reducing interleukin-1β, tumour necrosis factor alpha and monocyte chemoattractant protein-1 levels. Early hAEC treatment was also associated with reduced airway hyper-responsiveness and normalisation of pressure–volume loops. Pulmonary hypertension and right ventricle hypertrophy were also prevented in the early hAEC treatment group, and this persisted until 10 weeks of age. Conclusions Early hAEC treatment appears to

  9. Using human epithelial amnion cells in human de-epidermized dermis for skin regeneration.

    Science.gov (United States)

    Jiang, Lei-Wei; Chen, Hongduo; Lu, Hongguang

    2016-01-01

    Human amniotic epithelial cells (hAECs) is a desirable reserve of stem cells. Human de-epidermized dermis (DED) retains basic tissue structure and parts of the basement membrane (BM) components at the acelluIar dermal surface, and provides a potential tool for skin regeneration. To evaluate the potential role of hAECs in skin regeneration, we used DED to perform organotypic culture of hAECs to develop organotypic skin. HAECs were isolated and cultured. Biological characteristics of hAECs were determined by immunocytochemistry and flow cytometry. To prepare DED, the epidermis was removed and then repeated freeze-thaw cycles. HAECs and fibroblast were seeded onto DED to perform the submerged culture for 3 days and then to be maintained at the air-liquid interface for 14 days to form organotypic culture. To identify whether the obtained DED retain the BM structure and components, the histological characteristics of DED and the BM were detected by immunohistochemistry. To evaluate whether the organotypic skin has similar histological characteristics with normal human skin, the marks of epidermal proliferation and differentiation and basement membrane component were detected by immunohistochemistry. Moreover, cell ultrastructure, cell-cell contact and ultrastructure of BM were examined under the transmission electron microscopy. HAECs has stem-cell characteristics with strong pluripotent Oct-4 and embryonic marker SSEA-4 expression. DED has effectively cleansed the cell components and continuous distributions of laminin and collagen IV. The histological appearance of tissue-engineered skin in vitro has 4 to 9 continuous layers of stratified epithelium and is similar to normal human skin in morphology. Immunohistochemical studies revealed that proliferation and differentiation markers such as Ki67, CK19, CK14, CK10, filaggrin but not CK18 expressed similar pattern characteristics to normal human epidermis. In addition, Periodic acid-Schiff stain showed that a uniform red

  10. Nuclear factor kappa B activation occurs in the amnion prior to labour onset and modulates the expression of numerous labour associated genes.

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    Sheri Lim

    Full Text Available BACKGROUND: Prior to the onset of human labour there is an increase in the synthesis of prostaglandins, cytokines and chemokines in the fetal membranes, particular the amnion. This is associated with activation of the transcription factor nuclear factor kappa B (NFκB. In this study we characterised the level of NFκB activity in amnion epithelial cells as a measure of amnion activation in samples collected from women undergoing caesarean section at 39 weeks gestation prior to the onset of labour. METHODOLOGY/PRINCIPAL FINDINGS: We found that a proportion of women exhibit low or moderate NFκB activity while other women exhibit high levels of NFκB activity (n = 12. This activation process does not appear to involve classical pathways of NFκB activation but rather is correlated with an increase in nuclear p65-Rel-B dimers. To identify the full range of genes upregulated in association with amnion activation, microarray analysis was performed on carefully characterised non-activated amnion (n = 3 samples and compared to activated samples (n = 3. A total of 919 genes were upregulated in response to amnion activation including numerous inflammatory genes such cyclooxygenase-2 (COX-2, 44-fold, interleukin 8 (IL-8, 6-fold, IL-1 receptor accessory protein (IL-1RAP, 4.5-fold, thrombospondin 1 (TSP-1, 3-fold and, unexpectedly, oxytocin receptor (OTR, 24-fold. Ingenuity Pathway Analysis of the microarray data reveal the two main gene networks activated concurrently with amnion activation are i cell death, cancer and morphology and ii cell cycle, embryonic development and tissue development. CONCLUSIONS/SIGNIFICANCE: Our results indicate that assessment of amnion NFκB activation is critical for accurate sample classification and subsequent interpretation of data. Collectively, our data suggest amnion activation is largely an inflammatory event that occurs in the amnion epithelial layer as a prelude to the onset of labour.

  11. Amnion in the treatment of pediatric partial-thickness facial burns.

    Science.gov (United States)

    Branski, Ludwik K; Herndon, David N; Celis, Mario M; Norbury, William B; Masters, Oscar E; Jeschke, Marc G

    2008-05-01

    Wound coverage for second-degree burns remains a clinical challenge. Human amniotic membranes have been used for many years in the treatment of burns; however, no large prospective clinical trials have been published. In this article, we present a novel and standardized procurement and processing method for amnion and investigate, whether the use of this biological dressing is safe and may represent a new therapeutic option for children with partial-thickness facial burns compared to standard topical treatment. Patients with partial-thickness burns of the face, neck and head admitted between 2003 and 2005 were included in this study. They were divided into two groups to receive either amnion (n=53) or topical antimicrobials (n=49). Demographics (age, gender, ethnicity, TBSA, burn areas), length of hospital stay (LOS), rate of infections (RI), time to total healing, and frequency of dressing changes were compared between the two groups. The long-term outcome was assessed in nine patients in the amnion group and eight patients in the topical group, who returned for up to 12-month follow-up visits. Patients in the amnion group had significantly less dressing changes then in the control group (pburns compared to the standard topical ointments. Further studies with the use of amniotic membranes on the trunk and the extremities, as well as for coverage of grafted third-degree burns, have yet to be performed.

  12. Increased oxidative stress in human fetal membranes overlying the cervix from term non-labouring and post labour deliveries.

    Science.gov (United States)

    Chai, M; Barker, G; Menon, R; Lappas, M

    2012-08-01

    Enzymatic breakdown of the collagen-rich extracellular matrix (ECM) that connects the amnion and chorion layers of the fetal membranes is one of the key events leading to rupture of membranes. Oxidant stress caused by increased formation of reactive oxygen species and/or reduced antioxidant capacity may predispose to membrane rupture, a major cause of preterm birth. The aim of this study was to determine the effect of human labour and supracervical (SC) apposition on antioxidant enzymes and 8-isoprostane (a marker of lipid peroxidation). To determine the effect of human labour on oxidative stress status, fetal membranes from the SC site (SCS) were collected from women at term Caesarean section (no labour), and from the site of membrane rupture (SOR) after spontaneous labour onset and delivery (post labour). To determine the effect of SC apposition on oxidative stress status, amnion was collected from the SCS and a distal site (DS) in women at term Caesarean section in the absence of labour. The release of 8-isoprostane was significantly higher in amnion from the SCS compared to DS, and in fetal membranes from the SOR compared to the SCS. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity were lower in amnion from the SC compared to DS. SOD gene expression and enzyme activity were lower in fetal membranes after labour. There was no difference in expression or activity in catalase, GPx and glutathione reductase (GSR) between no labour and post labour fetal membranes. In primary amnion cells, SOD supplementation significantly augmented IL-1β induced MMP-9 expression and activity. In summary, non-labouring SC fetal membranes are characterised by reduced antioxidant enzyme activity when compared to distal membranes, and, as such, may be more susceptible to oxidative damage and thus membrane rupture. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Synthesis of oxytocin in amnion, chorion, and decidua may influence the timing of human parturition

    National Research Council Canada - National Science Library

    Chibbar, R; Miller, F D; Mitchell, B F

    1993-01-01

    Despite the widespread clinical use of oxytocin (OT) as a potent and specific stimulant of labor, previous research data have not supported a role for OT in the physiology of normal human parturition...

  14. Human amnion epithelial cells rescue cell death via immunomodulation of microglia in a mouse model of perinatal brain injury.

    Science.gov (United States)

    Leaw, Bryan; Zhu, Dandan; Tan, Jean; Muljadi, Ruth; Saad, Mohamed I; Mockler, Joanne C; Wallace, Euan M; Lim, Rebecca; Tolcos, Mary

    2017-02-28

    Human amnion epithelial cells (hAECs) are clonogenic and have been proposed to reduce inflammatory-induced tissue injury. Perturbation of the immune response is implicated in the pathogenesis of perinatal brain injury; modulating this response could thus be a novel therapy for treating or preventing such injury. The immunomodulatory properties of hAECs have been shown in other animal models, but a detailed investigation of the effects on brain immune cells following injury has not been undertaken. Here, we investigate the effects of hAECs on microglia, the first immune responders to injury within the brain. We generated a mouse model combining neonatal inflammation and perinatal hyperoxia, both of which are risk factors associated with perinatal brain injury. On embryonic day 16 we administered lipopolysaccharide (LPS), or saline (control), intra-amniotically to C57Bl/6 J mouse pups. On postnatal day (P)0, LPS pups were placed in hyperoxia (65% oxygen) and control pups in normoxia for 14 days. Pups were given either hAECs or saline intravenously on P4. At P14, relative to controls, LPS and hyperoxia pups had reduced body weight, increased density of apoptotic cells (TUNEL) in the cortex, striatum and white matter, astrocytes (GFAP) in the white matter and activated microglia (CD68) in the cortex and striatum, but no change in total microglia density (Iba1). hAEC administration rescued the decreased body weight and reduced apoptosis and astrocyte areal coverage in the white matter, but increased the density of total and activated microglia. We then stimulated primary microglia (CD45lowCD11b+) with LPS for 24 h, followed by co-culture with hAEC conditioned medium for 48 h. hAEC conditioned medium increased microglial phagocytic activity, decreased microglia apoptosis and decreased M1 activation markers (CD86). Stimulating hAECs for 24 h with LPS did not alter release of cytokines known to modulate microglia activity. These data demonstrate that hAECs can

  15. Structural changes of skin and amnion grafts for transplantation purposes following different doses of irradiation.

    Science.gov (United States)

    Mrázová, H; Koller, J; Fujeríková, G; Babál, P

    2014-09-01

    An important part of the preparation of biological material for transplantation is sterilization. The aim of our study was to assess the impact of ionizing radiation on three types of biological tissues and the impact of different doses on cells and extracellular matrix. Three types of frozen tissues (porcine skin xenografts, human skin allografts and human amnion) were divided into five groups, control and groups according to the dose of radiation to which these samples were exposed (12.5, 25, 35 and 50 kGy). The tissue samples were fixed by formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid schiff reaction and silver impregnation. The staining with hematoxylin and eosin showed hydropic degeneration of the cells of epidermis in xenografts by the dose of 12.5 kGy, in human skin it was observed by the dose of 35 kGy. The staining for elastic fibers revealed damage of fine elastic fibers in the xenografts dermis by the dose of 12.5 kGy, in the allografts by 35 kGy. Another change was the disintegration of basement membrane of epithelium, especially in the human amnion at the dose of 50 kGy. The silver impregnation visualized nuclear chromatin condensation mainly in human amnion at the dose of 12.5 kGy. Our results have shown that the porcine xenografts and human amnion were more sensitive to irradiation than the human skin. In the next phase of the project we will focus at more detailed changes in the tissues using immunohistochemical techniques.

  16. Discovery and Characterization of Human Amniochorionic Membrane Microfractures.

    Science.gov (United States)

    Richardson, Lauren S; Vargas, Gracie; Brown, Tyra; Ochoa, Lorenzo; Sheller-Miller, Samantha; Saade, George R; Taylor, Robert N; Menon, Ramkumar

    2017-12-01

    This study obtained visual evidence of novel cellular and extracellular matrix-level structural alterations in term and preterm human fetal amniochorionic membranes. Amniochorions were collected from term cesarean (not in labor) or vaginal (labor) deliveries, preterm premature rupture of membranes, and spontaneous preterm birth. To determine the effect of oxidative stress on membranes at term or preterm labor, term not in labor samples in an organ explant culture in vitro were exposed to cigarette smoke extract. Tissues were imaged using multiphoton autofluorescence and second harmonic generation microscopy. Images were analyzed using ImageJ and IMARIS software. Three-dimensional microscopic analysis of membranes revealed microfractures that were characterized by amnion cell puckering, basement membrane degradation, and tunnels that extended into the collagen matrix with migrating cells. Numbers of microfractures were similar at term regardless of labor status; however, morphometric measures (width and depth) were higher in term labor membranes. Oxidative stress induced higher numbers of microfractures in term not in labor membranes, with morphometry resembling that seen in term labor membranes. Preterm premature rupture of the membranes had the highest number of microfractures compared to membranes from term and other preterm births. Microfractures are structural alterations indicative of areas of tissue remodeling during gestation. Their increase at preterm and in response to oxidative stress may indicate failure to reseal, predisposing membranes to rupture. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  17. Role of human amnion-derived mesenchymal stem cells in promoting osteogenic differentiation by influencing p38 MAPK signaling in lipopolysaccharide -induced human bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yuli; Wu, Hongxia; Shen, Ming; Ding, Siyang; Miao, Jing; Chen, Ning, E-mail: 2927410849@qq.com

    2017-01-01

    Periodontitis is a chronic inflammatory disease induced by bacterial pathogens, which not only affect connective tissue attachments but also cause alveolar bone loss. In this study, we investigated the anti-inflammatory effects of Human amnion-derived mesenchymal stem cells (HAMSCs) on human bone marrow mesenchymal stem cells (HBMSCs) under lipopolysaccharide (LPS)-induced inflammatory conditions. Proliferation levels were measured by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of HBMSCs osteogenic marker expression. Oxidative stress induced by LPS was investigated by assaying reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity. Here, we demonstrated that HAMSCs increased the proliferation, osteoblastic differentiation, and SOD activity of LPS-induced HBMSCs, and down-regulated the ROS level. Moreover, our results suggested that the activation of p38 MAPK signal transduction pathway is essential for reversing the LPS-induced bone-destructive processes. SB203580, a selective inhibitor of p38 MAPK signaling, significantly suppressed the anti-inflammatory effects in HAMSCs. In conclusion, HAMSCs show a strong potential in treating inflammation-induced bone loss by influencing p38 MAPK signaling. - Highlights: • LPS inhibites osteogenic differentiation in HBMSCs via suppression of p38 MAPK signaling pathway. • HAMSCs promote LPS-induced HBMSCs osteogenic differentiation through p38 MAPK signaling pathway. • HAMSCs reverse LPS-induced oxidative stress in LPS-induced HBMSCs through p38 MAPK signaling pathway.

  18. Periostin as a Biomarker of the Amniotic Membrane

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    Mariya P. Dobreva

    2012-01-01

    Full Text Available Tracing the precise developmental origin of amnion and amnion-derived stem cells is still challenging and depends chiefly on analyzing powerful genetic model amniotes like mouse. Profound understanding of the fundamental differences in amnion development in both the disc-shaped primate and human embryo and the cup-shaped mouse embryo is pivotal in particular when sampling amniotic membrane from nonprimate species for isolating candidate amniotic stem cells. The availability of molecular marker genes that are specifically expressed in the amniotic membrane and not in other extraembryonic membranes would be instrumental to validate unequivocally the starting material under investigation. So far such amniotic markers have not been reported. We postulated that bone morphogenetic protein (BMP target genes are putative amniotic membrane markers mainly because deficiency in one of several components of the BMP signaling cascade in mice has been documented to result in defective development of the early amnion. Comparative gene expression analysis of acknowledged target genes for BMP in different extraembryonic tissues, combined with in situ hybridization, identified Periostin (Postn mRNA enrichment in amnion throughout gestation. In addition, we identify and propose a combination of markers as transcriptional signature for the different extraembryonic tissues in mouse.

  19. The Involvement of the Proamnion in the Development of the Anterior Amnion Fold in the Chicken

    Science.gov (United States)

    de Melo Bernardo, Ana; Chuva de Sousa Lopes, Susana M.

    2014-01-01

    The amnion was one of the most important evolutionary novelties in the animal kingdom, allowing independence of water for reproduction and subsequent exploration of terrestrial habitats, and is therefore an important structure to understand evolution. We have studied chicken amniogenesis using ex ovo culture systems and 3D-reconstructions of serially sectioned chicken embryos. We provide evidence for a transient depression of the head in the proamnion, forming a pouch, that positions the extraembryonic membranes dorsal to the head and that is fundamental for the correct formation of the amnion and chorion membranes. When this “sinking” process in the proamnion was blocked, the amnion/chorion did not form, even though the growth of the embryo per se seemed unaffected. Here, we give insight in the role of the proamnion in amniogenesis. PMID:24647352

  20. Combination of hydroxyapatite, platelet rich fibrin and amnion membrane as a novel therapeutic option in regenerative periapical endodontic surgery: Case series

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    Uday Kiran Uppada

    2017-01-01

    Conclusion: The results of this case seriessubstantiatesthe credibility of using a combination ofamnion membrane with a bone graft and PRF to enhance radiographic healing outcome with decreased post-operative discomfort and present a viable regenerative treatment modality in periapical surgery.

  1. Amnion s and radio-sterilized porcine skin use as potential matrices for the development of human skin substitutes; Uso de amnios y piel porcina radioesterilizados como matrices potenciales para el desarrollo de sustitutos de piel humana

    Energy Technology Data Exchange (ETDEWEB)

    Martinez P, M. E.; Reyes F, M. L.; Reboyo B, D. [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico); Velasquillo M, M. C.; Sanchez S, R.; Brena M, A. M.; Ibarra P, J. C., E-mail: esther.martinez@inin.gob.mx [Instituto Nacional de Rehabilitacion, Calz. Mexico-Xochimilco No. 289, Col. Arenal de Guadalupe, 14389 Mexico D. F. (Mexico)

    2014-10-15

    The injuries by burns constitute a primordial problem of public health; they cause a high mortality index, severe physical and psychological disability, etc. The autologous skin transplant is the replacement therapy recommended for its treatment, but in patients that present a high percentage of burnt skin; this is not possible to carry out. Another strategy is the transplant of donated skin; however, due to the little donation that exists in our country is not very feasible to apply this treatment. A challenge of the tissues engineering is to develop biological skin substitutes, based on cells and amnion s, favoring the cutaneous regeneration and quick repair of injuries, diminishing this way the hospitalization expenses. At present skin substitutes that can equal to the same skin do not exist. On the other hand, the mesenchymal stromal cells (Msc) represent an alternative to achieve this objective; since has been demonstrated that the Msc participate in the tissue repair by means of inhibition of pro-inflammatory cytokines and differentiation to dermal fibroblasts and keratinocytes. To apply the Msc in cutaneous injuries a support material is required that to allow transplanting these cells to a lesion or burn. The radio-sterilized human amnion and the radio-sterilized porcine skin, processed by the Radio-Sterilized Tissues Bank of the Instituto Nacional de Investigaciones Nucleares (ININ), are biomaterials that are used as temporary cutaneous coverings. We suppose that these two matrices will be appropriate for the growth and maintenance in cultivation of the Msc, to generate two biological skin substitutes, in collaboration with the Biotechnology Laboratory of the Instituto Nacional de Rehabilitacion. (Author)

  2. AMNION LIOFILISASI EFEKTIF MENYEMBUHKAN REAKSI KULIT AKIBAT RADIOTERAPI PADA PASIEN KANKER

    Directory of Open Access Journals (Sweden)

    Arifianto Arifianto

    2016-08-01

    Full Text Available Reaksi kulit jaringan sekitar sering terjadi akibat efek samping radioterapi pada tumor. Membran amnion dapat dipakai sebagai pengobatan lokal karena kemampuannya mempercepat penyembuhan luka. Penelitian ini bertujuan untuk membandingkan efektifitas amnion liofilisasi dengan salep gentamisin terhadap penyembuhan reaksi kulit akibat radioterapi. Jenis penelitian ini adalah experimental, pada 16 pasien kanker yang mengalami reaksi kulit akibat radioterapi di unit Radioterapi RSUP Dr. M. Djamil Padang. Pasien dikelompokan menjadi 2 kelompok yaitu kelompok diberi terapi amnion liofilisasi dan kelompok yang diberi salep gentamisin. Alat ukur yang digunakan adalah tabel skala RISRAS yang dikembangkan oleh Noble-Adams. Pengukuran dilakukan tiga kali yaitu sebelum perlakuan, setelah 1 minggu dan setelah 2 minggu perlakuan. Analisis statistik menggunakan T-Test dengan nilai p ˂ 0,05.  Hasil penelitian ini didapatkan perbedaan yang bermakna penurunan Skala RISRAS pada kelompok yang diberi amnion dibandingkan dengan yang diberi salep gentamisin pada penilaian setelah 1 minggu perlakuan (p=0,007. Penelitian ini dapat disimpulkan bahwa amnion liofilisasi memberikan penyembuhan luka yang lebih cepat dibandingkan salep gentamisin.

  3. Monoclonal antibody GB3, a new probe for the study of human basement membranes and hemidesmosomes

    Energy Technology Data Exchange (ETDEWEB)

    Verrando, P.; Pisani, A.; Serieys, N.; Ortonne, J.P. (UER Medecine, Nice (France)); Hsi, Baeli; Yeh, Changjing (INSERM U210, Nice (France))

    1987-05-01

    A monoclonal antibody, GB3, has been raised against human amnion. Not only does GB3 bind to amniotic basement membrane, but it also recognizes an antigenic structure expressed by epidermal as well as by some other human basement membranes. This antigen is synthesized (and excreted) by cultured normal human epidermal keratinocytes. It is expressed to a lesser extent by the A431 epidermoid carcinoma cell line, but is not expressed by the SV40 virus-transformed SVK14 keratinocyte cell line. In ultrastructural studies, this antigen was located in the epidermal basement membrane, both in the lamina densa and in the lamina lucida, associated with hemidesmosomes. It was identified as a protein by in vitro proteolytic cleavage studies. The radio-immunoprecipitates from cultured human keratinocytes, analyzed by SDS-PAGE, showed that GB3 recognized five polypeptides of 93.5, 125, 130, 146 and 150 kD under reducing conditions. The tissue distribution of the antigen and the molecular weights (MWs) of its constitutive polypeptides suggest that it is different from other known components of basement membranes. It may provide a biochemical marker for hemidesmosomes. Furthermore, GB3 represents an interesting and original clinical probe, since the antigenic structure recognized by GB3 is lacking in Junctional Epidermolysis Bullosa, a lethal genodermatosis in which a dermo-epidermal splitting occurs at the level of lamina lucida.

  4. Gamma radiation sterilized amnion: use in ophthalmology

    Energy Technology Data Exchange (ETDEWEB)

    Martinez P, M. E. [ININ, Carretera Mexico-Toluca s/n, Ocoyoacac 52750, Estado de Mexico (Mexico); Leon T, Y. [Hospital General Regional 220, IMSS, Paseo Tollocan No. 620, Col. Vertice, Toluca 50150, Estado de Mexico (Mexico); Vazquez M, L., E-mail: esther.martinez@inin.gob.m [Hospital General de Mexico, Dr. Balmis 148, Col. Doctores, 06720 Mexico D. F. (Mexico)

    2010-10-15

    Amnion processed at the Radio sterilized Tissue Bank at the National Institute of Nuclear Research, sterilized with {sup 60}Co gamma radiation, have been used in Mexico since 2005 either as a graft to replace the damaged ocular surface, or as a patch to prevent unwanted inflammatory reactions. Patients from the Hospital General de Mexico (HGM) and Instituto Mexicano del Seguro Social (IMSS), suffering diverse pathologies such as keratoconjunctivitis; recurrent pterygium associated with symblepharon; corneal neuro trophic ulcers, chemical and thermal burns, and corneal thinning s, had been successfully treated with irradiated amnion. In the HGM, a clinical prospective study on lesions of the ocular surface of 17 eyes from 15 patients, affected with the above mentioned pathologies, was successful in 88.2%. The results have proven to be excellent as much for cosmetic purposes as for functional ones. Without the treatment, the patients could have suffered a healing after-effect or loss of sight. At IMSS, a controlled clinical randomized trial with 108 eyes from 100 patients, affected with primary nasal pterygium, was performed in 2009. These eyes were treated with radio sterilized amnion and intraoperative mitomycin C to prevent recurrence after excision of the primary pterygium. The preliminary results do not shown adverse reaction, inflammation and pain were significantly reduced radio sterilized amnion also offer security because they do no express antigens HLA-A, B or Dr and the sterile irradiated tissue do not provoke rejection or transmit an infective disease. (Author)

  5. Secretion of bioactive interleukin-6 and tumor necrosis factor-alpha proteins from primary cultured human fetal membrane chorion cells infected with influenza virus.

    Science.gov (United States)

    Uchide, N; Suzuki, A; Ohyama, K; Bessho, T; Toyoda, H

    2006-01-01

    Influenza virus infection during pregnancy is implicated in one of the causes of premature delivery, abortion and stillbirth. Pro-inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha produced by fetal membranes, are postulated to facilitate premature delivery. We investigated the secretion of IL-6 and TNF-alpha from primary cultured human fetal membrane chorion and amnion cells infected with influenza virus at protein and bioactivity levels in order to understand the pathology of premature delivery during influenza virus infection. Concentrations of IL-6 and TNF-alpha proteins were significantly increased in culture supernatants of chorion cells by influenza virus infection. Culture supernatants of the virus-infected chorion cells stimulated the proliferation of IL-6-sensitive 7-TD-1 cells and induced the cytolysis of TNF-alpha-sensitive L929 cells, both activities of which were inhibited by the addition of respective antibody, whereas no such phenomena were observed in amnion cells. The results demonstrated that only chorion cells secreted significant amounts of bioactive IL-6 and TNF-alpha proteins responding to influenza virus infection. The present study suggests a possibility that the secretion of bioactive IL-6 and TNF-alpha proteins from fetal membrane chorion cells is implicated in the pathogenesis of premature delivery during influenza virus infection.

  6. The expression of aquaporin 8 and aquaporin 9 in fetal membranes and placenta in term pregnancies complicated by idiopathic polyhydramnios.

    Science.gov (United States)

    Zhu, Xueqiong; Jiang, Shanshan; Hu, Yingchun; Zheng, Xiaoqun; Zou, Shuangwei; Wang, Yuhuan; Zhu, Xuejie

    2010-10-01

    Aquaporins are a family of membrane-bound water channel proteins that regulate the flow of water across a variety of biological membranes. The expression of aquaporin 8 and aquaporin 9 has been demonstrated in human chorioamniotic membrane and placenta. But their roles in the pathophysiology of polyhydramnios are unclear. To study the expression of aquaporin 8 and aquaporin 9 in fetal membranes and placenta in term pregnancies complicated by idiopathic polyhydramnios and to explore the association between aquaporin expressions and polyhydramnios. The placentas were collected from 51 patients who underwent elective Cesarean sections at term, of which 21 cases had idiopathic polyhydramnios and the other 30 had normal amniotic fluid volume. Real-time polymerase chain reaction and immunohistochemistry techniques were used to determine the expression and localization of aquaporin 8 and aquaporin 9 in the amnion, chorion and placenta. Expression of aquaporin 8 and aquaporin 9 was detected in the amnion, chorion and placenta and located in amnion epithelia, chorion cytotrophoblasts and placental trophoblast. Compared to normal amniotic fluid volume group, the expression of aquaporin 8 in amnion, and aquaporin 9 in amnion and chorion, were significantly increased in idiopathic polyhydramnios group; however, their expression in the placenta was significantly decreased. When polyhydramnios occurs, expression of aquaporin 8 and aquaporin 9 in fetal membranes and placenta is an adaptive change, which may be involved in the regulation of amniotic fluid volume. However, the modulation factors of the aquaporin 8 and aquaporin 9 expressions need further study. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  7. HMPAS: Human Membrane Protein Analysis System.

    Science.gov (United States)

    Kim, Min-Sung; Yi, Gwan-Su

    2013-11-07

    Membrane proteins perform essential roles in diverse cellular functions and are regarded as major pharmaceutical targets. The significance of membrane proteins has led to the developing dozens of resources related with membrane proteins. However, most of these resources are built for specific well-known membrane protein groups, making it difficult to find common and specific features of various membrane protein groups. We collected human membrane proteins from the dispersed resources and predicted novel membrane protein candidates by using ortholog information and our membrane protein classifiers. The membrane proteins were classified according to the type of interaction with the membrane, subcellular localization, and molecular function. We also made new feature dataset to characterize the membrane proteins in various aspects including membrane protein topology, domain, biological process, disease, and drug. Moreover, protein structure and ICD-10-CM based integrated disease and drug information was newly included. To analyze the comprehensive information of membrane proteins, we implemented analysis tools to identify novel sequence and functional features of the classified membrane protein groups and to extract features from protein sequences. We constructed HMPAS with 28,509 collected known membrane proteins and 8,076 newly predicted candidates. This system provides integrated information of human membrane proteins individually and in groups organized by 45 subcellular locations and 1,401 molecular functions. As a case study, we identified associations between the membrane proteins and diseases and present that membrane proteins are promising targets for diseases related with nervous system and circulatory system. A web-based interface of this system was constructed to facilitate researchers not only to retrieve organized information of individual proteins but also to use the tools to analyze the membrane proteins. HMPAS provides comprehensive information about

  8. [Expression of aquaporin 8 in human fetal membrane and placenta of idiopathic polyhydramnios].

    Science.gov (United States)

    Huang, Jin; Qi, Hong-bo

    2009-01-01

    To determine the expression of Aquaporin 8(AQP8) in the fetal membrane and placenta of idiopathic polyhydramnios. The amnion, chorion and placenta were collected from 12 term pregnancies with idiopathic polyhydramnios( polyhydramnios group) and 12 term pregnancies who were normal (control group). The expression of AQP8 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of AQP8 protein was detected by immunohistochemistry. The expression of AQP8 mRNA in amnion, chorion and placenta of polyhydramnios group was (0.78 +/- 0.13), (0.58 +/- 0.10), and (0.86 +/- 0.15) respectively, and that of control group was (0. 39 0.07), (0.45 +/- 0.09), and (0.34 +/- 0.09) respectively. The expression of AQP8 protein in amnion, chorion and placenta of polyhydramnios group was (0.195 +/- 0.024), (0.170 +/- 0.028), and (0.193 +/- 0.024) respectively, and that of control group was (0.151 +/- 0.018), (0.156 +/- 0.024), and (0.152 +/- 0.023) respectively. In all 3 types of tissues the expression of AQP8 mRNA of polyhydramnios group was higher than that of control group (P polyhydramnios group was also increased compared to that of control group (P 0.05). The expression of AQP8 mRNA and protein is significantly increased in the amnion and placenta of polyhydramnios, suggesting that AQP8 may play an important role in the regulation of amniotic fluid volume.

  9. Expression of the prostaglandin F synthase AKR1B1 and the prostaglandin transporter SLCO2A1 in human fetal membranes in relation to spontaneous term and preterm labour

    Directory of Open Access Journals (Sweden)

    Hana A Alzamil

    2014-07-01

    Full Text Available Background: Human labour is a complex series of cellular and molecular events that occur at the materno-fetal and uterine levels. Many hypotheses have been proposed for the initiation of human labour, one hypothesis suggests that maturation of the fetus releases a signal in the amniotic fluid that will be transmitted to myometrium via the fetal membranes and initiate uterine contractions. There is strong evidence that prostaglandins (PGs play a central role in initiation and progression of human labour. Objectives: In this study we intended to investigate the expression of prostaglandin F synthase and the prostaglandin transporter in the human fetal membranes and to explore the relationship between cytokines and PGs in the mechanism of human labour. Methods: We used fetal membranes obtained before labour at term and after spontaneous labour at term or preterm to identify the changes in prostaglandin F synthase (AKR1B1 and human prostaglandin transporter (SLCO2A1 proteins in relation to parturition. Using fetal membranes explants we tested the effect of cytokines (interleukin-1 and tumour necrosis factor alpha on PG production and the concomitant changes in cyclooxygenase-2 (PTGS2, AKR1B1 and SLCO2A1 expression. Results: Expression of PTGS2 and AKR1B1 was upregulated in the fetal membranes in association with term labour while SLCO2A1 was downregulated with advancing gestation and during term labour. Before labour, IL-1 increased the expression of PTGS2, however during labour TNF upregulated PTGS2 and AKR1B1 proteins. Conclusions: The prostaglandin F synthase AKR1B1 is upregulated while prostaglandin transporter is downregulated during term labour. The amnion is more responsive than choriodecidua to stimulation with pro-inflammatory cytokines. The mechanisms of term and preterm labour are different.

  10. Comprehensive two-dimensional gel protein databases offer a global approach to the analysis of human cells: the transformed amnion cells (AMA) master database and its link to genome DNA sequence data

    DEFF Research Database (Denmark)

    Celis, J E; Gesser, B; Rasmussen, H H

    1990-01-01

    qualitative and quantitative annotations has been established. The protein numbers in this database differ from those reported in an earlier version (Celis et al. Leukemia 1988, 2,561-602) as a result of changes in the scanning hardware. The reported information includes: percentage of total radioactivity...... shock proteins, annexins and phosphorylated proteins. The results presented should be considered as the initial phase of a joint effort between our laboratories to undertake a general and systematic analysis of human proteins. Using this integrated approach it will be possible to identify phenotype...

  11. Comparative analysis of the basement membrane composition of the human limbus epithelium and amniotic membrane epithelium.

    Science.gov (United States)

    Dietrich-Ntoukas, Tina; Hofmann-Rummelt, Carmen; Kruse, Friedrich E; Schlötzer-Schrehardt, Ursula

    2012-05-01

    Human amniotic membrane has been widely used as substrate for ex vivo expansion and transplantation of limbal epithelial cells. To further clarify its suitability as a surrogate niche for limbal stem cells and progenitor cells, we analyzed the composition of the amniotic epithelial basement membrane, with special focus on the expression of limbus-specific matrix components. Cryosections of corneoscleral specimens obtained from 10 human donor eyes and of 6 amniotic membrane specimens obtained at cesarean section were stained by indirect immunofluorescence using a broad panel of antibodies against basement membrane components. Both amniotic and limbal epithelial basement membranes showed positive immunoreactivity for collagen type IV α1, α2, α5, and α6 chains; collagens type VII, XV, XVI, XVII, and XVIII; laminin α3, β1, β2, β3, γ1, and γ2 chains; laminin-111 and laminin-332; nidogen-1 and nidogen-2; fibronectin; fibulin-2; fibrillin-2; perlecan; and agrin. Both types of basement membrane were negative for collagen type IV α3 and α4 chains, collagen type V, and laminin α4 chain. Limbal basement membrane components, which were not detected in amniotic membrane, included laminin α1, α2, α5, and γ3 chains; BM40/SPARC; tenascin-C; matrilin-2; endostatin; and collagen type XVIII. Despite extensive similarities in basement membrane composition between amniotic and corneolimbal epithelia, the lack of limbus-specific environmental factors argues against the potential of denuded amniotic membrane as a surrogate niche for limbal stem cells but supports its suitability as a substrate to promote the formation of a well-differentiated stratified corneal epithelial equivalent for tissue engineering strategies.

  12. An elevated maternal serum C-reactive protein in the context of intra-amniotic inflammation is an indicator that the development of amnionitis, an intense fetal and AF inflammatory response are likely in patients with preterm labor: clinical implications.

    Science.gov (United States)

    Park, Chan-Wook; Yoon, Bo Hyun; Park, Joong Shin; Jun, Jong Kwan

    2013-06-01

    We propose that an elevated maternal serum C-reactive protein (CRP) concentration in the context of intra-amniotic inflammation (IAI) is a predictor for amnionitis development, known to be the most advanced stage of maternal inflammatory response during the progression of acute histologic chorioamnionitis in preterm gestations. Study population consisted of 53 singleton gestations with IAI, who underwent amniocentesis due to preterm labor and intact membranes (PTL) and delivered preterm-neonates ( 0.05), and higher median AF MMP-8 and umbilical cord plasma CRP concentration at birth than patients (26.4%,14/53) without that (AF MMP-8 (ng/mL): 373.1 versus 138.6: p = 0.05; umbilical cord plasma CRP (ng/mL): 363.4 versus 15.5: p development of amnionitis, an intense fetal and AF inflammatory response are likely in patients with PTL.

  13. Long term storage stabilizes human erythrocyte membrane in ...

    African Journals Online (AJOL)

    Osmotic fragility (OF) test was conducted in Nigerian human black male erythrocytes stored for Oh, 12h, 24h and 48h. Storage of these human erythrocytes for up to 24h failed to alter significantly their membrane characteristics. A leftward shift in osmotic fragiligrams was noted suggestive of storage-time (age) dependent ...

  14. High membrane protein oxidation in the human cerebral cortex.

    Science.gov (United States)

    Granold, Matthias; Moosmann, Bernd; Staib-Lasarzik, Irina; Arendt, Thomas; Del Rey, Adriana; Engelhard, Kristin; Behl, Christian; Hajieva, Parvana

    2015-01-01

    Oxidative stress is thought to be one of the main mediators of neuronal damage in human neurodegenerative disease. Still, the dissection of causal relationships has turned out to be remarkably difficult. Here, we have analyzed global protein oxidation in terms of carbonylation of membrane proteins and cytoplasmic proteins in three different mammalian species: aged human cortex and cerebellum from patients with or without Alzheimer's disease, mouse cortex and cerebellum from young and old animals, and adult rat hippocampus and cortex subjected or not subjected to cerebral ischemia. Most tissues showed relatively similar levels of protein oxidation. However, human cortex was affected by severe membrane protein oxidation, while exhibiting lower than average cytoplasmic protein oxidation. In contrast, ex vivo autooxidation of murine cortical tissue primarily induced aqueous protein oxidation, while in vivo biological aging or cerebral ischemia had no major effect on brain protein oxidation. The unusually high levels of membrane protein oxidation in the human cortex were also not predicted by lipid peroxidation, as the levels of isoprostane immunoreactivity in human samples were considerably lower than in rodent tissues. Our results indicate that the aged human cortex is under steady pressure from specific and potentially detrimental membrane protein oxidation. The pronounced difference between humans, mice and rats regarding the primary site of cortical oxidation might have contributed to the unresolved difficulties in translating into therapies the wealth of data describing successful antioxidant neuroprotection in rodents. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  15. High membrane protein oxidation in the human cerebral cortex

    Directory of Open Access Journals (Sweden)

    Matthias Granold

    2015-04-01

    Full Text Available Oxidative stress is thought to be one of the main mediators of neuronal damage in human neurodegenerative disease. Still, the dissection of causal relationships has turned out to be remarkably difficult. Here, we have analyzed global protein oxidation in terms of carbonylation of membrane proteins and cytoplasmic proteins in three different mammalian species: aged human cortex and cerebellum from patients with or without Alzheimer's disease, mouse cortex and cerebellum from young and old animals, and adult rat hippocampus and cortex subjected or not subjected to cerebral ischemia. Most tissues showed relatively similar levels of protein oxidation. However, human cortex was affected by severe membrane protein oxidation, while exhibiting lower than average cytoplasmic protein oxidation. In contrast, ex vivo autooxidation of murine cortical tissue primarily induced aqueous protein oxidation, while in vivo biological aging or cerebral ischemia had no major effect on brain protein oxidation. The unusually high levels of membrane protein oxidation in the human cortex were also not predicted by lipid peroxidation, as the levels of isoprostane immunoreactivity in human samples were considerably lower than in rodent tissues. Our results indicate that the aged human cortex is under steady pressure from specific and potentially detrimental membrane protein oxidation. The pronounced difference between humans, mice and rats regarding the primary site of cortical oxidation might have contributed to the unresolved difficulties in translating into therapies the wealth of data describing successful antioxidant neuroprotection in rodents.

  16. A distinct mechanism of senescence activation in amnion epithelial cells by infection, inflammation, and oxidative stress.

    Science.gov (United States)

    Dixon, Christopher Luke; Richardson, Lauren; Sheller-Miller, Samantha; Saade, George; Menon, Ramkumar

    2017-11-30

    We investigated p38MAPK activation-induced fetal membrane cell senescence in response to inflammation (tumour necrosis factor-alpha [TNF-α]) and infection (lipopolysaccharide [LPS]), factors associated with spontaneous preterm birth. Primary amnion epithelial cells (AECs) were exposed to TNF-α, 50 ng/mL and LPS, 100 ng/mL. Cigarette smoke extract (CSE), a known OS inducer, was used as positive control. AECs were cotreated with the antioxidant N-acetyl cysteine (NAC) and p38MAPK inhibitor SB203580 to determine the effect of OS and p38MAPK. Western blot analysis was performed for active (Phospho-p38MAPK) and total p38MAPK. Senescence was determined by flow cytometry, and culture supernatants were tested for IL-6 using ELISA. TNF-α, but not LPS, increased p38MAPK activation compared to untreated cells (P = .01). The number of senescent cells and senescence-associated IL-6 was higher in both TNF-α and LPS-treated cells compared to control (P = .001, P = .01, respectively). Antioxidant NAC inhibited p38MAPK activation by TNF-α. p38MAPK inhibitor SB203580 reduced the development of senescence and IL-6 by TNF-α and LPS. CSE treatment validated our current data. TNF-α caused OS-mediated p38MAPK induction, senescence, and IL-6 increase from AECs. LPS also induced senescence and IL-6 increase. Inflammatory and infectious factors may cause premature fetal cell senescence contributing to preterm birth pathophysiology. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Human Lipoproteins at Model Cell Membranes

    DEFF Research Database (Denmark)

    Browning, K L; Lind, T K; Maric, S

    2017-01-01

    High and low density lipoproteins (HDL and LDL) are thought to play vital roles in the onset and development of atherosclerosis; the biggest killer in the western world. Key issues of initial lipoprotein (LP) interactions at cellular membranes need to be addressed including LP deposition and lipid...... exchange and lipid removal can be distinguished thanks to the combined use of hydrogenated and tail-deuterated lipids. Both HDL and LDL remove lipids from the bilayer and deposit hydrogenated material into the lipid bilayer, however, the extent of removal and exchange depends on LP type. These results...... support the notion of HDL acting as the 'good' cholesterol, removing lipid material from lipid-loaded cells, whereas LDL acts as the 'bad' cholesterol, depositing lipid material into the vascular wall....

  18. Membrane ectopeptidases targeted by human coronaviruses

    NARCIS (Netherlands)

    Bosch, Berend Jan; Smits, Saskia L.; Haagmans, Bart L.

    2014-01-01

    Six coronaviruses, including the recently identified Middle East respiratory syndrome coronavirus, are known to target the human respiratory tract causing mild to severe disease. Their interaction with receptors expressed on cells located in the respiratory tract is an essential first step in the

  19. Membrane ectopeptidases targeted by human coronaviruses

    NARCIS (Netherlands)

    B.J. Bosch (Berend Jan); S.L. Smits (Saskia); B.L. Haagmans (Bart)

    2014-01-01

    textabstractSix coronaviruses, including the recently identified Middle East respiratory syndrome coronavirus, are known to target the human respiratory tract causing mild to severe disease. Their interaction with receptors expressed on cells located in the respiratory tract is an essential first

  20. The erythrocyte membrane in human muscular dystrophy

    NARCIS (Netherlands)

    W. Ruitenbeek (Willem)

    1979-01-01

    textabstractMore than 250 different forms of human neuromuscular diseases are known. They differ in age of onset, severity of weakness, rate of progression, type of inheritance, groups of muscles affected, frequency of incidence. Sometimes the clinical symptoms are not restricted to nervous and/or

  1. Limitations and options using resorbable versus nonresorbable membranes for successful guided bone regeneration.

    Science.gov (United States)

    Soldatos, Nikolaos K; Stylianou, Popi; Koidou, Vasiliki P; Angelov, Nikola; Yukna, Raymond; Romanos, Georgios E

    2017-01-01

    Deficient bony ridges often complicate the implant treatment plan. Several treatment modalities are used to regenerate bone, including guided bone regeneration (GBR). The purpose of this study was to summarize the knowledge on different types of membranes available and currently used in GBR procedures in a staged approach or with simultaneous implant placement. The primary role of the membranes is to exclude epithelial and connective tissue cells from the wound area to be regenerated, and to create and maintain the space into which pluripotential and osteogenic cells are free to migrate. A literature search was performed for articles that were published in English on the topic. A selected number of studies were chosen in order to provide a review of the main characteristics, applications, and outcomes of the different types of membranes. Resorbable membranes are made of natural or synthetic polymers like collagen and aliphatic polyesters. Collagens are the most common type used. They have similar collagen composition to the periodontal connective tissue. Other materials available include human, porcine, and bovine pericardium membranes, human amnion and chorion tissue, and human acellular freeze-dried dermal matrix. Nonresorbable membranes used in GBR include dense-polytetrafluoroethylene (d-PTFE), expanded-polytetrafluoroethylene (e-PTFE), titanium mesh, and titanium-reinforced polytetrafluoroethylene. The most common complication of nonresorbable membranes is exposure, which has detrimental effect on the final outcome with both types of membranes. For vertical bone augmentation procedures, the most appropriate membranes are the nonresorbable. For combination defects, both types result in a successful outcome.

  2. Optical properties of the human round window membrane

    Science.gov (United States)

    Höhl, Martin; DeTemple, Daphne; Lyutenski, Stefan; Leuteritz, Georg; Varkentin, Arthur; Schmitt, Heike Andrea; Lenarz, Thomas; Roth, Bernhard; Meinhardt-Wollweber, Merve; Morgner, Uwe

    2017-10-01

    Optical techniques are effective tools for diagnostic applications in medicine and are particularly attractive for the noninvasive analysis of biological tissues and fluids in vivo. Noninvasive examinations of substances via a fiber optic probe need to consider the optical properties of biological tissues obstructing the optical path. This applies to the analysis of the human perilymph, which is located behind the round window membrane. The composition of this inner ear liquid is directly correlated to inner ear hearing loss. In this work, experimental methods for studying the optical properties of the human round window membrane ex vivo are presented. For the first time, a comprehensive investigation of this tissue is performed, including optical transmission, forward scattering, and Raman scattering. The results obtained suggest the application of visible wavelengths (>400 nm) for investigating the perilymph behind the round window membrane in future.

  3. Cations Content And Membrane Properties Of Human Sickle Blood ...

    African Journals Online (AJOL)

    The effect of hydroxyurea, sodium thiocyanate and potassium tellurite on cations content and membrane properties of sickle erythrocyte was investigated in this study. Human sickle blood was incubated with the drugs in vitro at their optimum sickling inhibitory concentration. Mean corpuscular volume (MCV), mean ...

  4. High membrane protein oxidation in the human cerebral cortex

    OpenAIRE

    Granold, Matthias; Moosmann, Bernd; Staib-Lasarzik, Irina; Arendt, Thomas; del Rey, Adriana; Engelhard, Kristin; Behl, Christian; Hajieva, Parvana

    2014-01-01

    Oxidative stress is thought to be one of the main mediators of neuronal damage in human neurodegenerative disease. Still, the dissection of causal relationships has turned out to be remarkably difficult. Here, we have analyzed global protein oxidation in terms of carbonylation of membrane proteins and cytoplasmic proteins in three different mammalian species: aged human cortex and cerebellum from patients with or without Alzheimer's disease, mouse cortex and cerebellum from young and old anim...

  5. Membrane transport of anandamide through resealed human red blood cell membranes

    DEFF Research Database (Denmark)

    Bojesen, I.N.; Hansen, Harald S.

    2005-01-01

    at 0°C and pH 7.3 with albumin-free and albumin-filled human red blood cell ghosts. The efflux kinetics is biexponential and is analyzed in terms of compartment models. The distribution of anandamide on the membrane inner to outer leaflet pools is determined to be 0.275 ± 0.023, and the rate constant......The use of resealed red blood cell membranes (ghosts) allows the study of the transport of a compound in a nonmetabolizing system with a biological membrane. Transmembrane movements of anandamide (N-arachidonoylethanolamine, arachidonoylethanolamide) have been studied by exchange efflux experiments...... of unidirectional flux from inside to outside is 0.361 ± 0.023 s. The rate constant of unidirectional flux from the membrane to BSA in the medium ([BSA]) increases with the square root of [BSA] in accordance with the theory of an unstirred layer around ghosts. Anandamide passed through the red blood cell membrane...

  6. Membrane alterations induced by nonstructural proteins of human norovirus.

    Directory of Open Access Journals (Sweden)

    Sylvie Y Doerflinger

    2017-10-01

    Full Text Available Human noroviruses (huNoV are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4 variants. The viral nonstructural (NS proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV. Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER which included single membrane vesicles (SMVs, double membrane vesicles (DMVs and multi membrane vesicles (MMVs. In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and

  7. The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterization of the carbonate-washed membrane standard

    Science.gov (United States)

    Peng, Lifeng; Kapp, Eugene A.; Fenyö, David; Kwon, Min-Seok; Jiang, Pu; Wu, Songfeng; Jiang, Ying; Aguilar, Marie-Isabel; Ahmed, Nikhat; Baker, Mark S.; Cai, Zongwei; Chen, Yu-Ju; Van Chi, Phan; Chung, Maxey C. M.; He, Fuchu; Len, Alice C. L.; Liao, Pao-Chi; Nakamura, Kazuyuki; Ngai, Sai Ming; Paik, Young-Ki; Pan, Tai-Long; Poon, Terence C. W.; Salekdeh, Ghasem Hosseini; Simpson, Richard J.; Sirdeshmukh, Ravi; Srisomsap, Chantragan; Svasti, Jisnuson; Tyan, Yu-Chang; Dreyer, Florian S.; McLauchlan, Danyl; Rawson, Pisana; Jordan, T. William

    2013-01-01

    The Asia Oceania Human Proteome Organisation has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multi-laboratory project is based on analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. Here we present the strategy used for preparation and initial characterisation of the membrane sample, including validation that the carbonate-washing step enriches for integral and lipid-anchored membrane proteins. Analysis of seventeen independent datasets from five types of proteomic workflows is in progress. PMID:20486120

  8. Amnion-Epithelial-Cell-Derived Exosomes Demonstrate Physiologic State of Cell under Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Samantha Sheller

    Full Text Available At term, the signals of fetal maturity and feto-placental tissue aging prompt uterine readiness for delivery by transitioning quiescent myometrium to an active stage. It is still unclear how the signals reach the distant myometrium. Exosomes are a specific type of extracellular vesicle (EVs that transport molecular signals between cells, and are released from a wide range of cells, including the maternal and fetal cells. In this study, we hypothesize that i exosomes act as carriers of signals in utero-placental compartments and ii exosomes reflect the physiologic status of the origin cells. The primary aims of this study were to determine exosomal contents in exosomes derived from primary amnion epithelial cells (AEC. We also determined the effect of oxidative stress on AEC derived exosomal cargo contents. AEC were isolated from amniotic membrane obtained from normal, term, not in labor placentae at delivery, and culture under standard conditions. Oxidative stress was induced using cigarette smoke extract for 48 hours. AEC-conditioned media were collected and exosomes isolated by differential centrifugations. Both growth conditions (normal and oxidative stress induced produced cup shaped exosomes of around 50 nm, expressed exosomes enriched markers, such as CD9, CD63, CD81 and HSC70, embryonic stem cell marker Nanog, and contained similar amounts of cell free AEC DNA. Using confocal microscopy, the colocalization of histone (H 3, heat shock protein (HSP 70 and activated form of pro-senescence and term parturition associated marker p38 mitogen activated protein kinase (MAPK (P-p38 MAPK co-localized with exosome enrich marker CD9. HSP70 and P-p38 MAPK were significantly higher in exosomes from AEC grown under oxidative stress conditions than standard conditions (p<0.05. Finally, mass spectrometry and bioinformatics analysis identified 221 different proteins involved in immunomodulatory response and cell-to-cell communication. This study determined

  9. Effects of radiation on the permeability of human basement membranes

    Science.gov (United States)

    Fan, B.-T.; Achour, S.; Simmonet, F.; Guerin, D.

    1999-02-01

    The influence of radiation on the permeability properties of human basement membrane was investigated by measuring the diffusion rate of several organic compounds (glycine, proline, glucose, urea and insulin) through human anterior lens capsules. The basement membranes borne an γ-irradiation treatment change significantly their permeability vis-a-vis studied organic substances. This modification in physico-chemical properties is probably due to the radiation, which alters or degrades the complex structure (or architecture) of basement membranes. Moreover the change in permeability is dependent upon the diffusing compounds. An increase in diffusion has been observed for glucose, glycine and urea. However for insulin and proline, a decrease in diffusion rate was observed. L'influence de radiation sur la perméabilité de la membrane basale a été étudiée par la mesure de la vitesse de diffusion de plusieurs composés organiques d'intérêt biologique (glycine, proline, glucose, urée et insuline) à travers la lame basale antérieure du cristallin de l'oil humain. Les membranes basales qui sont traitées avec l'irradiation γ changent significativement leur perméabilité vis-à-vis des substances organiques. Ce changement de propriétés physico-chimiques est probablement dû à l'altération ou la dégradation de la structure (ou de l'architecture) de la membrane basale entraînée par l'irradiation. De plus, la modification de la perméabilité de la membrane basale est dépendante des composés diffusants. Une augmentation de la vitesse de diffusion a été observée pour le glucose, le glycine et l'urée. Par contre, dans les cas de l'insuline et de la proline, on a observé une diminution de la vitesse de diffusion.

  10. Challenges in validating the sterilisation dose for processed human amniotic membranes

    Energy Technology Data Exchange (ETDEWEB)

    Yusof, Norimah [Malaysian Nuclear Agency, Bangi, 43000 Kajang, Selangor (Malaysia)], E-mail: norimah@mint.gov.my; Hassan, Asnah [Malaysian Nuclear Agency, Bangi, 43000 Kajang, Selangor (Malaysia); Firdaus Abd Rahman, M.N.; Hamid, Suzina A. [National Tissue Bank, Hospital Universiti Sains Malaysia, Kubang Kerian, 16130 Kelantan (Malaysia)

    2007-11-15

    Most of the tissue banks in the Asia Pacific region have been using ionising radiation at 25 kGy to sterilise human tissues for save clinical usage. Under tissue banking quality system, any dose employed for sterilisation has to be validated and the validation exercise has to be a part of quality document. Tissue grafts, unlike medical items, are not produced in large number per each processing batch and tissues relatively have a different microbial population. A Code of Practice established by the International Atomic Energy Agency (IAEA) in 2004 offers several validation methods using smaller number of samples compared to ISO 11137 (1995), which is meant for medical products. The methods emphasise on bioburden determination, followed by sterility test on samples after they were exposed to verification dose for attaining of sterility assurance level (SAL) of 10{sup -1}. This paper describes our experience in using the IAEA Code of Practice in conducting the validation exercise for substantiating 25 kGy as sterilisation dose for both air-dried amnion and those preserved in 99% glycerol.

  11. Bronchial stump closure with amniotic membrane in animal model

    Directory of Open Access Journals (Sweden)

    Gholamreza Mohajeri

    2014-01-01

    Full Text Available Background: Coverage of the bronchial stumps (BSs with adjacent tissues can improve healing and reduce bronchial complications in complex thoracic surgery. There is no evidence for the application of human amnion allograft for prevention of air leak from the BS. The comparison of the amniotic membrane (AM and pleural patch for BS healing after lobectomy in dogs was our aim in this study. Materials and Methods: A total of eight males and females 12-24-month-old dogs between 17 and 22 kg body-weight were used in this study in 2010, Isfahan University of Medical Sciences. Animals were separated into two groups: group A (n = 4; amniotic membrane and group P (n = 4; pleural patch according to the BS closure technique performed. After lobectomy of the right middle lobe, the BS was closed, while a small bronchopleural fistula (BPF was created by inserting a catheter via edges of closed stump. Then, it was covered with a piece of AM3 × 3 cm in group A and with a pedicle graft of pleura in group P. Rethoracotomy was performed after 15 days of observation, and the BS was removed for histological examination. Histological healing was classified as complete or incomplete healing. Neoangiogenesis was measured by Von Willebrand expression using immunohistochemistry (IHC. Data were analyzed by SPSS version 15 using Fisher′s exact test, Mann-Whitney test, and T tests. Results: BPF complications were not seen during observation period. There was no significant difference in histological healing between two groups. Similarly, no significant difference was observed between the groups in terms of neoangiogenesis based on IHC examination (P value = 0.69. Conclusion: Human amnion allograft could be as effective as pleural patch for BS wrapping following pulmonary resections.

  12. BMP-dependent serosa and amnion specification in the scuttle fly Megaselia abdita.

    Science.gov (United States)

    Rafiqi, Ab Matteen; Park, Chee-Hyurng; Kwan, Chun Wai; Lemke, Steffen; Schmidt-Ott, Urs

    2012-09-01

    Bone morphogenetic protein (BMP) signaling is an essential factor in dorsoventral patterning of animal embryos but how BMP signaling evolved with fundamental changes in dorsoventral tissue differentiation is unclear. Flies experienced an evolutionary reduction of extra-embryonic tissue types from two (amniotic and serosal tissue) to one (amnionserosal tissue). BMP-dependent amnioserosa specification has been studied in Drosophila melanogaster. However, the mechanisms of serosal and amniotic tissue specification in less diverged flies remain unknown. To better understand potential evolutionary links between BMP signaling and extra-embryonic tissue specification, we examined the activity profile and function of BMP signaling in serosa and amnion patterning of the scuttle fly Megaselia abdita (Phoridae) and compared the BMP activity profiles between M. abdita and D. melanogaster. In blastoderm embryos of both species, BMP activity peaked at the dorsal midline. However, at the beginning of gastrulation, peak BMP activity in M. abdita shifted towards prospective amnion tissue. This transition correlated with the first signs of amnion differentiation laterally adjacent to the serosa anlage. Marker-assisted analysis of six BMP signaling components (dpp, gbb, scw, tkv, sax, sog) by RNA interference revealed that both serosa and amnion specification of M. abdita are dependent on BMP activity. Conversely, BMP gain-of-function experiments caused sharpened expression boundaries of extra-embryonic target genes indicative of positive feedback. We propose that changes in the BMP activity profile at the beginning of gastrulation might have contributed to the reduction of extra-embryonic tissue types during the radiation of cyclorrhaphan flies.

  13. Clinical observation of bio-amnion implantation used in combined trabeculectomy for refractory glaucoma

    Directory of Open Access Journals (Sweden)

    Xin-Huai Xue

    2013-05-01

    Full Text Available AIM: To observe the clinical effect of bio-amnion implantation used in combined trabeculectomy for refractory glaucoma METHODS: Totally 86 eyes of 80 glaucoma patients were randomly divided into 2 groups. In experimental group, 43 eyes underwent trabeculectomy combined with bio-amnion implantation. In control group, 43 eyes only underwent trabeculectomy combined. The intraocular pressure(IOP, filtrative bleb and complications were observed. RESULTS: Following-up for 12 months, IOP: there was significant difference between the average IOP(15.5±1.1mmHgin experimental group and the average IOP(19.7±2.5mmHgin control group(P<0.05. Filtrative bleb: the incidence of the functional filtering bleb(86%in experimental group was more than the one in control group(67%, there was significant different between the two groups(P<0.05. The incidence of complications of post-operation(shallow anterior chamber, choroidal detachment and bleb leakingwas lower than the one of the control group obviously. CONCLUSION: Combined trabeculectomy with bio-amnion implantation can increase the rate of success and reduce the incidence of complications.

  14. Human hepatocyte functions in a crossed hollow fiber membrane bioreactor.

    Science.gov (United States)

    De Bartolo, Loredana; Salerno, Simona; Curcio, Efrem; Piscioneri, Antonella; Rende, Maria; Morelli, Sabrina; Tasselli, Franco; Bader, Augustinus; Drioli, Enrico

    2009-05-01

    An important challenge in liver tissue engineering is the development of bioartificial systems that are able to favour the liver reconstruction and to modulate liver cell behaviour. A crossed hollow fiber membrane bioreactor was developed to support the long-term maintenance and differentiation of human hepatocytes. The bioreactor consists of two types of hollow fiber (HF) membranes with different molecular weight cut-off (MWCO) and physico-chemical properties cross-assembled in alternating manner: modified polyetheretherketone (PEEK-WC) and polyethersulfone (PES), used for the medium inflow and outflow, respectively. The combination of these two fiber set produces an extracapillary network for the adhesion of cells and a high mass exchange through the cross-flow of culture medium. The transport of liver specific products such as albumin and urea together with the transport of drug such as diazepam was modelled and compared with the experimental metabolic data. The theoretical metabolite concentration differed 7.5% for albumin and 5% for urea with respect to experimental data. The optimised perfusion conditions of the bioreactor allowed the maintenance of liver functions in terms of urea synthesis, albumin secretion and diazepam biotransformation up to 18 days of culture. In particular the good performance of the bioreactor was confirmed by the high rate of urea synthesis (28.7 microg/h 10(6) cells) and diazepam biotransformation. In the bioreactor human hepatocytes expressed at high levels the individual cytochrome P450 isoenzymes involved in the diazepam metabolism. The results demonstrated that crossed HF membrane bioreactor is able to support the maintenance of primary human hepatocytes preserving their liver specific functions for all investigated period. This device may be a potential tool in the liver tissue engineering for drug metabolism/toxicity testing and study of disease pathogenesis alternatively to animal experimentation.

  15. Effects of Alcohol on Membrane Fluidity of Human Erythrocyte

    OpenAIRE

    Mori, Ichiya; Hiramatsu, Midori; Toda, Naomi; Koide, Yayoi; Miyagawa, Fumio

    1994-01-01

    Membrane fluidity in human erythrocytes was measured by a spin label method using an electron spin resonance spectrometer in healthy volunteers after ingestion of alcohol (1.5 ml of whisky/kg body weight). Fluidity in the lipid bilayer closer to the hydrophilic face decreased at 30 min and 90 min, and fluidity in the hydrophobic core decreased at 90 min after ingestion of alcohol. In the same experiment, the level of thiobarbituric acid reactive substances in the serum decreased 30 min after ...

  16. Hydrophobic photolabeling in membranes: The human erythrocyte glucose transporter

    Energy Technology Data Exchange (ETDEWEB)

    Lala, A.K.; Bhat, S. (Indian Institute of Technology Bombay, Powai (India))

    1990-10-01

    Human erythrocyte membranes were labeled with a hydrophobic photoactivable reagent, 2-({sup 3}H)Diazofluorene. Electrophoretic analysis of the protein fraction showed that several membrane spanning proteins like Band 3 (the anion transporter), Band 4.5 (the glucose transporter), and the sialoglycoproteins PAS 1, 2, and 3 have been labeled. To isolate the diazofluorene-labeled glucose transporter, the membrane preparation was solubilized with Triton X-100 and passed through a DEAE-cellulose column. The flow-through fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive analysis of the gel indicated that besides the Band 4.5, two more proteins corresponding to the Band 3 and Band 6 regions also coelute with the glucose transporter in the flow-through fraction. On the other hand, use of n-octyl glucoside gave a relatively better preparation. The 2-({sup 3}H)DAF-labeled glucose transporter isolated by the latter method on tryptic digestion indicated that the Mr 18,000 fragment corresponding to the C-terminal transmembrane fragment is labeled.

  17. Release of LHRH-activity from human fetal membranes upon exposure to PGE/sub 2/, oxytocin and isoproterenol

    Energy Technology Data Exchange (ETDEWEB)

    Poisner, A.M.; Poisner, R.; Becca, C.R.; Conn, P.M.

    1986-03-01

    The authors have previously reported that superfused chorion laeve (fetal membranes) release LHRH-like immunoreactivity upon exposure to angiotensin II. They have now studied the effects of other agonists on the release of LHRH-activity and something of its chemical nature. Fetal membranes were obtained from placentas delivered by cesarean section, the amnion stripped from the chorion, and the chorion superfused in an Amicon thin-channel device with the maternal surface facing up. The whole device was submerged in a 37 C water bath and perfused with a modified Locke's solution at 0.4 - 1.0 ml/min. LHRH-activity was measured by radioimmunoassay using three different antisera against LHRH. The release of LHRH-activity was stimulated by 6-10 min exposure to PGE/sub 2/, oxytocin, and isoproterenol. Extracts of chorion were studied using gel filtration on Sephacryl S-200 and ultrafiltration with Amicon PM-10 filters. The bulk of the LHRH-activity appeared as a higher molecular weight form (about 70,000 daltons). Since oxytocin has been reported to release PGE/sub 2/ from chorion, it may release LHRH-activity by virtue of liberating endogenous PGE/sub 2/. The chemical nature of the LHRH-activity is presently under investigation.

  18. Histopathological evaluation of bone regeneration using human resorbable demineralized membrane

    Directory of Open Access Journals (Sweden)

    Tatić Zoran

    2010-01-01

    Full Text Available Background/Aim. Filling a bone defect with bone substitution materials is a therapy of choice, but the infiltration of connective tissue from the mucoperiostal flap may compromise a healing of bone substitutions with bony wall defects. Application of membrane as a barrier is indicated as a solution to this problem. The aim of this study was to show a pathohistological view of bone regeneration and the significance of human resorbable demineralized membrane (HRDM, 200 μ thick in bone regeneration regarding mandibular defects in an experiment on dogs. Methods. The experiment was performed on six dogs. Bone defects were created in all six dogs on the right side of the mandible after the elevation of the mucoperiostal flap. One defect was filled with human deproteinised bone (HDB, and in between HDB and soft tissue RHDM of 200 μ thick was placed. In the second defect, used as a control one, only HDB without RHDM was placed. Two dogs were sacrificed two months after the surgery, another two dogs four months after the surgery and the last two dogs six months after the surgery. After that, samples of bone tissue were taken for histopathological analysis. Results. In all the six dogs with defects treated with HDB and RHDM the level of bone regeneration was much higher in comparison with the control defects without RHDM. Conclusion. Membrane, as a cover of bony defect, is useful and benefits bone regeneration. Bony defects covered with RHDM show better bony healing despite the fact that bone regeneration was not fully complete for as long as six months after the RHDM implantation.

  19. Dietary Flavonoids as Therapeutics for Preterm Birth: Luteolin and Kaempferol Suppress Inflammation in Human Gestational Tissues In Vitro

    Directory of Open Access Journals (Sweden)

    Courtney Wall

    2013-01-01

    Full Text Available Infection/inflammation is commonly associated with preterm birth (PTB, initiating uterine contractions and rupture of fetal membranes. Proinflammatory cytokines induce matrix metalloproteinases (MMPs that degrade the extracellular matrix (ECM and prostaglandins which initiate uterine contractions. Nuclear factor-κB (NF-κB and activator-protein- (AP-1 have key roles in the formation of these prolabour mediators. In nongestational tissues, dietary flavonoids such as luteolin and kaempferol inhibit NF-κB, AP-1, and their downstream targets. The aim of this study was to determine if luteolin and kaempferol reduce infection-induced prolabour mediators in human gestational tissues. Fetal membranes were incubated with LPS, and primary amnion cells and myometrial cells were incubated with IL-1β in the absence or presence of luteolin or kaempferol. Luteolin and kaempferol significantly reduced LPS-induced secretion of proinflammatory cytokines (IL-6 and IL-8 and prostaglandins (PGE2 and PGF2α in fetal membranes, IL-1β-induced COX-2 gene expression and prostaglandin production in myometrium, and IL-1β-induced MMP-9 activity in amnion and myometrial cells. Luteolin and kaempferol decreased IL-1β-induced NF-κB p65 DNA binding activity and nuclear c-Jun expression. In conclusion, luteolin and kaempferol inhibit prolabour mediators in human gestational tissues. Given the central role of inflammation in provoking preterm labour, phytophenols may be a therapeutic approach to reduce the incidence of PTB.

  20. The Molecular Structure of Human Red Blood Cell Membranes from Highly Oriented, Solid Supported Multi-Lamellar Membranes

    Science.gov (United States)

    Himbert, Sebastian; Alsop, Richard J.; Rose, Markus; Hertz, Laura; Dhaliwal, Alexander; Moran-Mirabal, Jose M.; Verschoor, Chris P.; Bowdish, Dawn M. E.; Kaestner, Lars; Wagner, Christian; Rheinstädter, Maikel C.

    2017-01-01

    We prepared highly oriented, multi-lamellar stacks of human red blood cell (RBC) membranes applied on silicon wafers. RBC ghosts were prepared by hemolysis and applied onto functionalized silicon chips and annealed into multi-lamellar RBC membranes. High resolution X-ray diffraction was used to determine the molecular structure of the stacked membranes. We present direct experimental evidence that these RBC membranes consist of nanometer sized domains of integral coiled-coil peptides, as well as liquid ordered (lo) and liquid disordered (ld) lipids. Lamellar spacings, membrane and hydration water layer thicknesses, areas per lipid tail and domain sizes were determined. The common drug aspirin was added to the RBC membranes and found to interact with RBC membranes and preferably partition in the head group region of the lo domain leading to a fluidification of the membranes, i.e., a thinning of the bilayers and an increase in lipid tail spacing. Our results further support current models of RBC membranes as patchy structures and provide unprecedented structural details of the molecular organization in the different domains.

  1. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    Science.gov (United States)

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  3. The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterisation of the carbonate-washed membrane standard.

    Science.gov (United States)

    Peng, Lifeng; Kapp, Eugene A; Fenyö, David; Kwon, Min-Seok; Jiang, Pu; Wu, Songfeng; Jiang, Ying; Aguilar, Marie-Isabel; Ahmed, Nikhat; Baker, Mark S; Cai, Zongwei; Chen, Yu-Ju; Van Chi, Phan; Chung, Maxey C M; He, Fuchu; Len, Alice C L; Liao, Pao-Chi; Nakamura, Kazuyuki; Ngai, Sai Ming; Paik, Young-Ki; Pan, Tai-Long; Poon, Terence C W; Salekdeh, Ghasem Hosseini; Simpson, Richard J; Sirdeshmukh, Ravi; Srisomsap, Chantragan; Svasti, Jisnuson; Tyan, Yu-Chang; Dreyer, Florian S; McLauchlan, Danyl; Rawson, Pisana; Jordan, T William

    2010-11-01

    The Asia Oceania Human Proteome Organisation (AOHUPO) has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multilaboratory project is based on the analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. In this study, we present the strategy used for the preparation and initial characterization of the membrane sample, including validation that the carbonate-washing step enriches for integral and lipid-anchored membrane proteins. Analysis of 17 independent data sets from five types of proteomic workflows is in progress. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Membrane properties in small cutaneous nerve fibers in humans.

    Science.gov (United States)

    Hennings, Kristian; Frahm, Ken Steffen; Petrini, Laura; Andersen, Ole K; Arendt-Nielsen, Lars; Mørch, Carsten D

    2017-02-01

    Assessment of membrane properties is important for understanding the mechanisms of painful peripheral neuropathy, developing new diagnostic techniques, and screening/profiling of analgesics that target ion channels. Small cutaneous nerves were activated electrically by small diameter (0.2 mm) cathodes, and large nerves were activated by ordinary patch electrodes. This new perception threshold tracking method combines perception threshold assessment and stimulation paradigms from conventional threshold tracking. The strength-duration time-constant of large fibers (580 µs ± 160 µs) was lower than the time constant of small fibers (1060 µs ± 690 µs; P fibers showed less threshold reduction than large fibers (repeated-measures analysis of variance, Bonferroni, P = 0.006). This is a reliable method to investigate the membrane properties of small cutaneous nerve fibers in humans and may be used in clinical settings as a diagnostic or profiling tool. Muscle Nerve 55: 195-201, 2017. © 2016 Wiley Periodicals, Inc.

  5. Repairing Fetal Membranes with a Self-adhesive Ultrathin Polymeric Film: Evaluation in Mid-gestational Rabbit Model.

    Science.gov (United States)

    Pensabene, Virginia; Patel, Premal P; Williams, Phillip; Cooper, Trisha L; Kirkbride, Kellye C; Giorgio, Todd D; Tulipan, Noel B

    2015-08-01

    Preterm premature rupture of membranes causes 40% of all preterm births, affecting 150000 women each year in the United States. Prenatal diagnostic procedures and surgical interventions increase incidence of adverse events, leading to iatrogenic membrane rupture after a fetoscopic procedure in 45% of cases. We propose an ultrathin, self-adherent, poly-L-lactic acid patch ("nanofilm") as a reparative wound closure after endoscopic/fetoscopic procedures. These nanofilms are compatible with application in wet conditions and with minimally invasive instrumentation. Ex vivo studies to evaluate the nanofilm were conducted using human chorion-amnion (CA) membranes. A custom-built inflation device was used for mechanical characterization of CA membranes and for assessment of nanofilm adhesion and sealing of membrane defects up to 3 mm in size. These ex vivo tests demonstrated the ability of the nanofilm to seal human CA defects ranging in size from 1 to 3 mm in diameter. In vivo survival studies were conducted in 25 mid-gestational rabbits, defects were created by perforating the uterus and the CA membranes and subsequently using the nanofilm to seal these wounds. These in vivo studies confirmed the successful sealing of defects smaller than 3 mm observed ex vivo. Histological analysis of whole harvested uteri 7 days after surgery showed intact uterine walls in 59% of the nanofilm repaired fetuses, along with increased uterine size and intrauterine development in 63% of the cases. In summary, we have developed an ultrathin, self-adhesive nanofilm for repair of uterine membrane defects.

  6. Red wine activates plasma membrane redox system in human erythrocytes.

    Science.gov (United States)

    Tedesco, Idolo; Moccia, Stefania; Volpe, Silvestro; Alfieri, Giovanna; Strollo, Daniela; Bilotto, Stefania; Spagnuolo, Carmela; Di Renzo, Massimo; Aquino, Rita P; Russo, Gian Luigi

    2016-01-01

    In the present study, we report that polyphenols present in red wine obtained by a controlled microvinification process are able to protect human erythrocytes from oxidative stress and to activate Plasma Membrane Redox System (PMRS). Human plasma obtained from healthy subjects was incubated in the presence of whole red wine at a concentration corresponding to 9.13-73 μg/ml gallic acid equivalents to verify the capacity to protect against hypochlorous acid (HOCl)-induced plasma oxidation and to minimize chloramine formation. Red wine reduced hemolysis and chloramine formation induced by HOCl of 40 and 35%, respectively. PMRS present on human erythrocytes transfers electrons from intracellular molecules to extracellular electron acceptors. We demonstrated that whole red wine activated PMRS activity in human erythrocytes isolated from donors in a dose-dependent manner with a maximum at about 70-100 μg/ml gallic acid equivalents. We also showed that red wine increased glutathione (GSH) levels and erythrocytic antioxidant capacity, measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching assay. Furthermore, we reported that GSH played a crucial role in regulating PMRS activity in erythrocytes. In fact, the effect of iodoacetamide, an alkylating agent that induces depletion of intracellular GSH, was completely counteracted by red wine. Bioactive compounds present in red wine, such as gallic acid, resveratrol, catechin, and quercetin were unable to activate PMRS when tested at the concentrations normally present in aged red wines. On the contrary, the increase of PMRS activity was associated with the anthocyanin fraction, suggesting the capacity of this class of compounds to positively modulate PMRS enzymatic activity.

  7. Incorporation of Human Recombinant Tropoelastin into Silk Fibroin Membranes with the View to Repairing Bruch’s Membrane

    Directory of Open Access Journals (Sweden)

    Audra M. A. Shadforth

    2015-09-01

    Full Text Available Bombyx mori silk fibroin membranes provide a potential delivery vehicle for both cells and extracellular matrix (ECM components into diseased or injured tissues. We have previously demonstrated the feasibility of growing retinal pigment epithelial cells (RPE on fibroin membranes with the view to repairing the retina of patients afflicted with age-related macular degeneration (AMD. The goal of the present study was to investigate the feasibility of incorporating the ECM component elastin, in the form of human recombinant tropoelastin, into these same membranes. Two basic strategies were explored: (1 membranes prepared from blended solutions of fibroin and tropoelastin; and (2 layered constructs prepared from sequentially cast solutions of fibroin, tropoelastin, and fibroin. Optimal conditions for RPE attachment were achieved using a tropoelastin-fibroin blend ratio of 10 to 90 parts by weight. Retention of tropoelastin within the blend and layered constructs was confirmed by immunolabelling and Fourier-transform infrared spectroscopy (FTIR. In the layered constructs, the bulk of tropoelastin was apparently absorbed into the initially cast fibroin layer. Blend membranes displayed higher elastic modulus, percentage elongation, and tensile strength (p < 0.01 when compared to the layered constructs. RPE cell response to fibroin membranes was not affected by the presence of tropoelastin. These findings support the potential use of fibroin membranes for the co-delivery of RPE cells and tropoelastin.

  8. A cell culture technique for human epiretinal membranes to describe cell behavior and membrane contraction in vitro.

    Science.gov (United States)

    Wertheimer, Christian; Eibl-Lindner, Kirsten H; Compera, Denise; Kueres, Alexander; Wolf, Armin; Docheva, Denitsa; Priglinger, Siegfried G; Priglinger, Claudia; Schumann, Ricarda G

    2017-11-01

    To introduce a human cell culture technique for investigating in-vitro behavior of primary epiretinal cells and membrane contraction of fibrocellular tissue surgically removed from eyes with idiopathic macular pucker. Human epiretinal membranes were harvested from ten eyes with idiopathic macular pucker during standard vitrectomy. Specimens were fixed on cell culture plastic using small entomological pins to apply horizontal stress to the tissue, and then transferred to standard cell culture conditions. Cell behavior of 400 epiretinal cells from 10 epiretinal membranes was observed in time-lapse microscopy and analyzed in terms of cell migration, cell velocity, and membrane contraction. Immunocytochemistry was performed for cell type-specific antigens. Cell specific differences in migration behavior were observed comprising two phenotypes: (PT1) epiretinal cells moving fast, less directly, with small round phenotype and (PT2) epiretinal cells moving slowly, directly, with elongated large phenotype. No mitosis, no outgrowth and no migration onto the plastic were seen. Horizontal contraction measurements showed variation between specimens. Masses of epiretinal cells with a myofibroblast-like phenotype expressed cytoplasmatic α-SMA stress fibers and correlated with cell behavior characteristics (PT2). Fast moving epiretinal cells (PT1) were identified as microglia by immunostaining. This in-vitro technique using traction application allows for culturing surgically removed epiretinal membranes from eyes with idiopathic macular pucker, demonstrating cell behavior and membrane contraction of primary human epiretinal cells. Our findings emphasize the abundance of myofibroblasts, the presence of microglia and specific differences of cell behavior in these membranes. This technique has the potential to improve the understanding of pathologies at the vitreomacular interface and might be helpful in establishing anti-fibrotic treatment strategies.

  9. Modelling the influence of amnionicity on the severity of twin-twin transfusion syndrome in monochorionic twin pregnancies

    NARCIS (Netherlands)

    van den Wijngaard, Jeroen P. H. M.; Umur, Asli; Ross, Michael G.; van Gemert, Martin J. C.

    2004-01-01

    Clinical treatment for diamniotic-monochorionic twin-twin transfusion syndrome (TTTS) may include conversion of diamniotic pregnancies to a monoamniotic-monochorionic state by disrupting the amnion septum. We sought to test the underlying hypothesis, i.e. that a monoamniotic state reduces the

  10. Capacitation-associated changes in membrane fluidity in asthenozoospermic human spermatozoa.

    Science.gov (United States)

    Buffone, Mariano G; Doncel, Gustavo F; Calamera, Juan C; Verstraeten, Sandra V

    2009-08-01

    The fertilizing potential of human spermatozoa relies on their ability to capacitate as they travel through the female reproductive tract. During this process, cholesterol is released from the plasma membrane, altering its architecture and dynamics. Using ISolate gradients, we obtained high (L90)- and low (L45)-quality spermatozoa from asthenozoospermic human semen samples. We tested the hypothesis that the lower fertilizing ability of asthenozoospermic L90 cells could be related to a lower ability to increase their membrane fluidity during capacitation. We assessed two sets of fluorescent probes: (i) DPH, TMA-DPH and PA-DPH which senses the hydrophobic core, cytosolic and exofacial leaflets of the bilayer, respectively and (ii) Laurdan, sensitive to the amount of water molecules intercalated between lipid moieties of the membrane (membrane hydration). Before capacitation, membrane fluidity of asthenozoospermic sperm populations was similar to the corresponding fractions of normozoospermic cells when evaluated with DPH, TMA-DPH or PA-DPH. Asthenozoospermic whole samples displayed lower plasma membrane hydration than normozoospermic cells as evidenced with Laurdan. After capacitation, asthenozoospermic L45 and L90 cells failed to increase their membrane fluidity in opposition to normozoospermic cells. Interestingly, membrane hydration significantly correlated with the main sperm motion parameters analysed, being a low membrane hydration associated with poor sperm movement. These results show that low-motility spermatozoa are unable to respond to capacitation with the necessary changes in membrane fluidity. This defect in sperm plasma membrane rheology may be responsible for their poor functional quality and low fertilizing ability.

  11. Theoretical analysis of human muscle membrane behaviour in hypokalemic periodic paralysis

    NARCIS (Netherlands)

    Alberink, M.J.; Wallinga, W.; Wolters, H.; Wolters, Henk; Ypey, D.L.; Ypey, Dirk L.; Links, Thera P.; Boom, H.B.K.

    1995-01-01

    Computer simulations were performed to investigate the behavior of human muscle membrane with membrane defects supposed to be present in the muscular disease Hypokalemic Periodic Paralysis (HOPP). The model used for simulation was a Hodgkin-Huxley model. The T-tubular system was also incorporated.

  12. The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini.

    Directory of Open Access Journals (Sweden)

    Aljona Gaiko-Shcherbak

    Full Text Available The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

  13. Primary Adult Human Retinal Pigment Epithelial Cell Cultures on Human Amniotic Membranes

    Directory of Open Access Journals (Sweden)

    Singhal Shweta

    2005-01-01

    Full Text Available Purpose: Retinal pigment epithelial (RPE cells grow well on surfaces that provide an extracellular matrix. Our aim was to establish primary adult human RPE cell cultures that retain their epithelial morphology in vitro using human amniotic membrane (hAM as substrate. Materials and Methods: Human cadaver eyeballs (16 were obtained from the eye bank after corneal trephination. RPE cells were harvested by a mechanical dissection of the inner choroid surface (10, group 1 or by b enzymatic digestion using 0.25% Trypsin/0.02% EDTA (6, group 2. The cells were explanted onto de-epithelialized hAM, nourished using DMEM/HAMS F-12 media and monitored for growth under the phase contrast microscope. Cell cultures were characterised by whole mount studies and paraffin sections. Growth data in the two groups were compared using the students′ ′t′ test. Results: Eleven samples (68.75% showed positive cultures with small, hexagonal cells arising from around the explant which formed a confluent and progressively pigmented monolayer. Whole mounts showed closely placed polygonal cells with heavily pigmented cytoplasm and indistinct nuclei. The histologic sections showed monolayers of cuboidal epithelium with variable pigmentation within the cytoplasm. Growth was seen by day 6-23 (average 11.5 days in the mechanical group, significantly earlier ( P Conclusions: Primary adult human RPE cell cultures retain epithelial morphology in vitro when cultured on human amniotic membranes . Mechanical dissection of the inner choroid surface appears to be an effective method of isolating RPE cells and yields earlier growth in cultures as compared to isolation by enzymatic digestion

  14. The Role of Progesterone and a Novel Progesterone Receptor, Progesterone Receptor Membrane Component 1, in the Inflammatory Response of Fetal Membranes to Ureaplasma parvum Infection.

    Directory of Open Access Journals (Sweden)

    Liping Feng

    Full Text Available Ureaplasma parvum (U. parvum is gaining recognition as an important pathogen for chorioamnionitis and preterm premature rupture of membranes. We aimed to investigate the roles of progesterone (P4 and a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1, in the response of fetal membranes to U. parvum. Fetal membrane cells (amnion, chorion and decidua were isolated and confirmed to be free of Mycoplasmataceae. Cells were treated with U. parvum (5x106 CFU, and adherence was quantified by qPCR. Amnion and chorion cells were transfected with scrambled siRNA or validated PGRMC1 siRNA for 72h. Cells were then treated with U. parvum for 4h with or without pretreatment with P4 (10-7 M or ethanol for 1h. Interleukin-8 (IL-8, matrix metalloproteinase 9 (MMP9 and cyclooxygenase (COX-2 mRNA expression were quantified by qRT-PCR. Culture medium was harvested and analyzed for IL-8 and prostaglandin (PGE2 secretion by ELISA and MMP9 activity by zymography. U. parvum had a mean adherence of 15.0±0.6%, 16.9± 3.7% and 4.7±0.3% in cultured amnion, chorion and decidua cells, respectively. Exposure to U. parvum elicited significant inflammatory responses including induction of IL-8, COX-2, PGE2 and MMP9. A possible role of PGRMC1 was identified in the inhibition of U. parvum-stimulated COX-2 and MMP9 mRNA expression in chorion cells and MMP9 activity in amnion cells. On the other hand, it might enhance the U. parvum-stimulated IL-8 protein secretion in amnion cells. P4, mediated through PGRMC1, significantly inhibited U. Parvum-induced MMP9 mRNA and COX-2 mRNA expression in chorion cells. P4 appeared to attenuate U. parvum induced IL-8 mRNA expression in chorion cells, but this P4 effect might not mediated through PGRMC1. In summary, U. parvum preferentially adheres to and induces inflammatory responses in chorion and amnion cells. P4 and PGRMC1 appear to differentially modulate the inflammatory responses induced by U. parvum among

  15. Interactions of the antiviral and antiparkinson agent amantadine with lipid membranes and human erythrocytes.

    Science.gov (United States)

    Suwalsky, Mario; Jemiola-Rzeminska, Malgorzata; Altamirano, Mariella; Villena, Fernando; Dukes, Nathan; Strzalka, Kazimierz

    2015-07-01

    Aimed to better understand the molecular mechanisms of its interactions with cell membranes, human erythrocyte and molecular models of the red cell membrane were utilized. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of amantadine to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction, fluorescence spectroscopy and differential scanning calorimetry (DSC). In an attempt to further elucidate its effects on cell membranes, the present work also examined amantadine influence on the morphology of intact human erythrocytes by means of scanning electron microscopy (SEM). Results indicated that amantadine induced morphological changes to human erythrocytes and interacted in a concentration-dependent manner with DMPC bilayers in contrast to DMPE that was hardly affected by the presence of the drug. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

    Science.gov (United States)

    Orsini, Francesco; Santacroce, Massimo; Cremona, Andrea; Gosvami, Nitya N; Lascialfari, Alessandro; Hoogenboom, Bart W

    2014-11-01

    Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.

  17. Effects of phenylpropanolamine (PPA) on in vitro human erythrocyte membranes and molecular models

    Energy Technology Data Exchange (ETDEWEB)

    Suwalsky, Mario, E-mail: msuwalsk@udec.cl [Faculty of Chemical Sciences, University of Concepcion, Concepcion (Chile); Zambrano, Pablo; Mennickent, Sigrid [Faculty of Pharmacy, University of Concepcion, Concepcion (Chile); Villena, Fernando [Faculty of Biological Sciences, University of Concepcion, Concepcion (Chile); Sotomayor, Carlos P.; Aguilar, Luis F. [Instituto de Quimica, Pontificia Universidad Catolica de Valparaiso, Valparaiso (Chile); Bolognin, Silvia [CNR-Institute for Biomedical Technologies, University of Padova, Padova (Italy)

    2011-03-18

    Research highlights: {yields} PPA is a common ingredient in cough-cold medication and appetite suppressants. {yields} Reports on its effects on human erythrocytes are very scarce. {yields} We found that PPA induced in vitro morphological changes to human erythrocytes. {yields} PPA interacted with isolated unsealed human erythrocyte membranes. {yields} PPA interacted with class of lipid present in the erythrocyte membrane outer monolayer. -- Abstract: Norephedrine, also called phenylpropanolamine (PPA), is a synthetic form of the ephedrine alkaloid. After reports of the occurrence of intracranial hemorrhage and other adverse effects, including several deaths, PPA is no longer sold in USA and Canada. Despite the extensive information about PPA toxicity, reports on its effects on cell membranes are scarce. With the aim to better understand the molecular mechanisms of the interaction of PPA with cell membranes, ranges of concentrations were incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of cell membranes. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, respectively. The capacity of PPA to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). This study presents evidence that PPA affects human red cell membranes as follows: (a) in SEM studies on human erythrocytes it was observed that 0.5 mM PPA induced shape changes; (b) in IUM PPA induced a sharp decrease in the fluorescence anisotropy in the lipid bilayer acyl chains in a concentration range lower than 100 {mu}M; (c) X-ray diffraction studies showed that PPA in the 0.1-0.5 m

  18. Membranes

    OpenAIRE

    Junbo Hou; Min Yang

    2012-01-01

    Lithium ion batteries have proven themselves the main choice of power sources for portable electronics. Besides consumer electronics, lithium ion batteries are also growing in popularity for military, electric vehicle, and aerospace applications. The present review attempts to summarize the knowledge about some selected membranes in lithium ion batteries. Based on the type of electrolyte used, literature concerning ceramic-glass and polymer solid ion conductors, microporous filter type separa...

  19. A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.

    2008-01-01

    The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological

  20. Comparison of human amniotic membrane and hyaluronate/carboxymethylcellulose membrane for prevention of adhesion formation in rats.

    Science.gov (United States)

    Kelekci, Sefa; Uygur, Dilek; Yilmaz, Bulent; Sut, Necdet; Yesildaglar, Narter

    2007-10-01

    To investigate the effectiveness of human amniotic membrane (HAM) in the prevention of postoperative adhesion formation and to compare it with the efficacy of hyaluronate/carboxymethylcellulose (HA/CMC) membrane in a rat model. Following pilot studies and computer-generated randomization, 23 female Wistar albino rats were operated on in the full study. One of the uterine horns with standard lesions was treated with either HAM (n = 13) or HA/CMC (n = 10) and the other uterine horn served as the control. Second look laparotomies were performed 2 weeks after the operations. Main outcome measures were extent, severity, degree, total adhesion scores and histopathologic characteristics of adhesions. Uterine horns treated with HAM had significantly lower total adhesion scores than the controls (5.15 +/- 2.67 vs. 7.92 +/- 1.50, P CMC membrane were significantly lower than those of the controls (4.30 +/- 1.95 vs. 7.50 +/- 1.84, P CMC groups regarding any adhesion scores. HAM and HA/CMC membrane are both effective for prevention of adhesion formation in a rat uterine horn model; however, one does not seem to be more effective than the other.

  1. Detergent-resistant membranes in human erythrocytes and their ...

    Indian Academy of Sciences (India)

    In cell membranes, local inhomogeneity in the lateral distribution of lipids and proteins is thought to exist in vivo in the form of lipid 'rafts', microdomains enriched in cholesterol and sphingolipids, and in specific classes of proteins, that appear to play specialized roles for signal transduction, cell-cell recognition, parasite or ...

  2. Detergent-resistant membranes in human erythrocytes and their ...

    Indian Academy of Sciences (India)

    Unknown

    Introduction. Lipid microdomains enriched in cholesterol and sphin- golipids are thought to exist in vivo in the membranes of living cells (Edidin 1997, 2001; Brown and London. 1998; Pike 2004). These structures are sites of ...... induced in erythrocytes infected by malaria parasites; Cell. Microbiol. 4 383–395. Heerklotz H ...

  3. Membrane fractions from Strongyloides venezuelensis in the immunodiagnosis of human strongyloidiasis.

    Science.gov (United States)

    Corral, Marcelo Andreetta; Paula, Fabiana Martins; Gottardi, Maiara; Meisel, Dirce Mary Correia Lima; Chieffi, Pedro Paulo; Gryschek, Ronaldo César Borges

    2015-01-01

    Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.

  4. [Expression of aquaporin 3 and aquaporin 9 in placenta and fetal membrane with idiopathic polyhydramnios.].

    Science.gov (United States)

    Zhu, Xue-Qiong; Jiang, Shan-Shan; Zou, Shuang-Wei; Hu, Ying-Chun; Wang, Yu-Huan

    2009-12-01

    To investigate the pathogenesis role of aquaporin 3 and aquaporin 9 in idiopathic polyhydramnios by detecting their expression and distribution in fetal membranes and placenta. Twenty-one of term pregnancy women with idiopathic polyhydramnios were enrolled as patient group matched with 30 women with normal term pregnancy as control group. The expression and localization of aquaporin 3 and aquaporin 9 in fetal membranes and placenta were detected by real-time polymerase chain reaction and streptavidin peroxidase immunohistochemiscal staining. (1) The mRNA expressions of aquaporin 3 and aquaporin 9 were detected in amnion, chorion and placental tissue in both patient group and control group. Both aquaporin 3 and aquaporin 9 were demonstrated positive staining in the amnion epithelia, chorion cytotrophoblasts and placental trophoblast. (2) The ratio of aquaporin 3 and aquaporin 9 mRNA expressions in amnion in patient group comparing to those in control group were 5.00 and 3.25, while in chorion they were 2.03 and 2.08. When compared with those in amnion and chorion of control group, there was a significant difference (P polyhydramnios. Further investigation should be needed to clarify the regulatory mechanism of aquaporin 3 and aquaporin 9 expressions.

  5. Comparison of amnion allograft with connective tissue graft for root coverage procedures: a double-blind, randomized, controlled clinical trial.

    Science.gov (United States)

    Ghahroudi, Amir Alireza Rasouli; Khorsand, Afshin; Rokn, Amir Reza; Sabounchi, Sepideh Seyedzadeh; Shayesteh, Yadollah Soleimani; Soolari, Ahmad

    2013-10-01

    The aim of the present double-blind, randomized, controlled study was to evaluate and compare the efficacy of amnion allograft and connective tissue graft in covering denuded root surfaces. Seventy-one teeth in 22 patients with gingival recession were treated randomly with coronally displaced flap plus connective tissue graft (control group, n = 29 recessions in 10 patients) or coronally displaced flap plus amnion allograft (test group, n = 42 recessions in 12 patients). The amount of root coverage and clinical parameters (probing depth, recession depth, clinical attachment level, recession width, gingival width, and papilla dimensions) were measured at baseline and at 3 and 6 months postoperatively. Average root coverage percentages after 6 months in the test and control groups were 67% (2.3 +/- 0.289 mm) and 54% (2.24 +/- 0.519 mm), respectively, with no statistically significant differences (p = 0.054). The changes in depth and width of recessions and in gingival width were significant 3 and 6 months after surgery compared to baseline (p = 0.000). Variations in the level of attachment and probing depths after 6 months were statistically significant in the test group compared to the control group (p = 0.002). Papilla dimensions were significantly correlated with root coverage (p = 0.00). Amnion allograft might be a suitable alternative to connective tissue graft in procedures to cover denuded root surfaces and can reduce recession depth.

  6. Basement membrane abnormalities in human eyes with diabetic retinopathy

    DEFF Research Database (Denmark)

    Ljubimov, A V; Burgeson, R E; Butkowski, R J

    1996-01-01

    Vascular and parenchymal basement membranes (BMs) are thickened in diabetes, but alterations in individual BM components in diabetic eyes, especially in diabetic retinopathy (DR), are obscure. To identify abnormalities in the distribution of specific constituents, we analyzed cryostat sections...... VI, VIII, XII, and XIV collagen were newly identified. Diabetic BM thickening appears to involve qualitative alterations of specific BM markers at an advanced disease stage, with the appearance of DR....

  7. The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump

    NARCIS (Netherlands)

    Zaman, G. J.; Flens, M. J.; van Leusden, M. R.; de Haas, M.; Mülder, H. S.; Lankelma, J.; Pinedo, H. M.; Scheper, R. J.; Baas, F.; Broxterman, H. J.

    1994-01-01

    The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an

  8. Preferential incorporation of fatty acids at the inside of human erythrocyte membranes

    NARCIS (Netherlands)

    Renooij, W.; Golde, L.M.G. van; Zwaal, R.F.A.; Roelofsen, B.; Deenen, L.L.M. van

    1974-01-01

    1. 1.Phospholipase A2 isolated from Naja naja venom was used as a tool to discriminate between the outer and inner lipid monolayer of the membranes of human erythrocytes. 2. 2.Incubation of human erythorcytes with radioactive fatty acids resulted in the formation of labelled lecithin in the

  9. Training-induced changes in membrane transport proteins of human skeletal muscle

    DEFF Research Database (Denmark)

    Juel, C.

    2006-01-01

    that the same type of training affects many transport proteins, suggesting that all transport proteins increase with training, and that both sprint and endurance training in humans increase the density of most membrane transport proteins. There seems to be an upper limit for these changes: intense training......Training improves human physical performance by inducing structural and cardiovascular changes, metabolic changes, and changes in the density of membrane transport proteins. This review focuses on the training-induced changes in proteins involved in sarcolemmal membrane transport. It is concluded...... for 6-8 weeks substantially increases the density of membrane proteins, whereas years of training (as performed by athletes) have no further effect. Studies suggest that training-induced changes at the protein level are important functionally. The underlying factors responsible for these changes...

  10. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane

    DEFF Research Database (Denmark)

    Rørvig, Sara; Østergaard, Ole; Heegaard, Niels Henrik Helweg

    2013-01-01

    granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in...... subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes....

  11. Manipulation of the host cell membrane by human γ-herpesviruses EBV and KSHV for pathogenesis.

    Science.gov (United States)

    Wei, Fang; Zhu, Qing; Ding, Ling; Liang, Qing; Cai, Qiliang

    2016-10-01

    The cell membrane regulates many physiological processes including cellular communication, homing and metabolism. It is therefore not surprising that the composition of the host cell membrane is manipulated by intracellular pathogens. Among these, the human oncogenic herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) exploit the host cell membrane to avoid immune surveillance and promote viral replication. Accumulating evidence has shown that both EBV and KSHV directly encode several similar membrane-associated proteins, including receptors and receptor-specific ligands (cytokines and chemokines), to increase virus fitness in spite of host antiviral immune responses. These proteins are expressed individually at different phases of the EBV/KSHV life cycle and employ various mechanisms to manipulate the host cell membrane. In recent decades, much effort has been made to address how these membrane-based signals contribute to viral tumorigenesis. In this review, we summarize and highlight the recent understanding of how EBV and KSHV similarly manipulate host cell membrane signals, particularly how remodeling of the cell membrane allows EBV and KSHV to avoid host antiviral immune responses and favors their latent and lytic infection.

  12. Human T cell crosstalk is induced by tumor membrane transfer.

    Directory of Open Access Journals (Sweden)

    Ronny Uzana

    Full Text Available Trogocytosis is a contact-dependent unidirectional transfer of membrane fragments between immune effector cells and their targets, initially detected in T cells following interaction with professional antigen presenting cells (APC. Previously, we have demonstrated that trogocytosis also takes place between melanoma-specific cytotoxic T lymphocytes (CTLs and their cognate tumors. In the present study, we took this finding a step further, focusing on the ability of melanoma membrane-imprinted CD8+ T cells to act as APCs (CD8+ T-APCs. We demonstrate that, following trogocytosis, CD8+ T-APCs directly present a variety of melanoma derived peptides to fraternal T cells with the same TCR specificity or to T cells with different TCRs. The resulting T cell-T cell immune synapse leads to (1 Activation of effector CTLs, as determined by proliferation, cytokine secretion and degranulation; (2 Fratricide (killing of CD8+ T-APCs by the activated CTLs. Thus, trogocytosis enables cross-reactivity among CD8+ T cells with interchanging roles of effectors and APCs. This dual function of tumor-reactive CTLs may hint at their ability to amplify or restrict reactivity against the tumor and participate in modulation of the anti-cancer immune response.

  13. Creep and stress relaxation of human red cell membrane.

    Science.gov (United States)

    Fischer, Thomas M

    2017-02-01

    In contrast to most mechanical properties of the red cell, experimental information on stress relaxation (SR) of the membrane skeleton is scarce. On the other hand, many postulates or assumptions as to the value of the characteristic time of SR [Formula: see text] can be found in the literature. Here, an experiment is presented that allows measurement of [Formula: see text] up to values of about 10 h. The membrane skeleton was deformed passively by changing the spontaneous curvature of the bilayer thus transforming the natively biconcave red cells into echinocytes. This shape and the concomitant deformation of the skeleton were kept up to 4 h by incubation at 37 ℃. During this period, no plastic deformation (creep) was observed. After the incubation, the spontaneous curvature was returned to normal. The resulting shape was smooth showing no remnants of the echinocytic shape. Both observations indicate [Formula: see text] 10 h. This result is in gross disagreement to postulates or assumptions existing in the literature.

  14. Effect of fibrin glue on the biomechanical properties of human Descemet's membrane.

    Science.gov (United States)

    Chaurasia, Shyam S; Champakalakshmi, Ravi; Li, Ang; Poh, Rebekah; Tan, Xiao Wei; Lakshminarayanan, Rajamani; Lim, Chwee T; Tan, Donald T; Mehta, Jodhbir S

    2012-01-01

    Corneal transplantation has rapidly evolved from full-thickness penetrating keratoplasty (PK) to selective tissue corneal transplantation, where only the diseased portions of the patient's corneal tissue are replaced with healthy donor tissue. Descemet's membrane endothelial keratoplasty (DMEK) performed in patients with corneal endothelial dysfunction is one such example where only a single layer of endothelial cells with its basement membrane (10-15 µm in thickness), Descemet's membrane (DM) is replaced. It is challenging to replace this membrane due to its intrinsic property to roll in an aqueous environment. The main objective of this study was to determine the effects of fibrin glue (FG) on the biomechanical properties of DM using atomic force microscopy (AFM) and relates these properties to membrane folding propensity. Fibrin glue was sprayed using the EasySpray applicator system, and the biomechanical properties of human DM were determined by AFM. We studied the changes in the "rolling up" tendency of DM by examining the changes in the elasticity and flexural rigidity after the application of FG. Surface topography was assessed using scanning electron microscopy (SEM) and AFM imaging. Treatment with FG not only stabilized and stiffened DM but also led to a significant increase in hysteresis of the glue-treated membrane. In addition, flexural or bending rigidity values also increased in FG-treated membranes. Our results suggest that fibrin glue provides rigidity to the DM/endothelial cell complex that may aid in subsequent manipulation by maintaining tissue integrity.

  15. Preparative purification of human monoclonal antibody isoforms in a multi-compartment electrolyser with immobiline membranes.

    Science.gov (United States)

    Righetti, P G; Wenisch, E; Jungbauer, A; Katinger, H; Faupel, M

    1990-02-02

    The performance of a multi-compartment electrolyser with isoelectric Immobiline membranes for large-scale protein purification is evaluated. Owing to the presence of isoelectric membranes possessing a high buffering capacity and ionic strength, isoelectric protein precipitation inside the membranes, one of the major drawbacks of present membrane uses, is fully avoided. In addition, owing to this novel membrane technology, pH gradient decay, typical of isoelectric focusing in carrier ampholytes, is fully eliminated and pH and conductivity constancy is guaranteed in all flow chambers for running periods of more than 11 days (160,000 V h). The membranes described possess a unique selectivity, in that they act by modulating the surface charge (i.e., the mobility) of macroions crossing or tangential to them. The concept of isoelectric Immobiline membranes acting like a pH-stat unit is introduced. Protein homogeneity in each chamber of the electrolyser can be achieved even when purifying human monoclonal antibodies against HIV-1, which possess high pI values (9.0-9.6), are large molecules (Mr 150,000) and are fractionated in the presence of large micelles of neutral detergents.

  16. Directing membrane chromatography to manufacture α1-antitrypsin from human plasma fraction IV.

    Science.gov (United States)

    Fan, Jinxin; Luo, Jianquan; Song, Weijie; Chen, Xiangrong; Wan, Yinhua

    2015-12-04

    The surging demand for plasma proteins, mainly driven by the growing market and the development of new therapeutic indications, is promoting manufacturers to improve the throughput of plasma proteins. Due to the inherent convective mass transfer, membrane chromatography has been proved to be an efficient approach for extracting a small amount of target proteins from large-volume feed. In this study, α1-antitrypsin (AAT) was extracted from human plasma fraction IV by a two-step membrane chromatography. An anion-exchange membrane chromatography (AEMC) was used to capture the plasma proteins in bind/elute mode, and the obtained effluent was further polished by a hydrophobic interaction membrane chromatography (HIMC) in flow-through mode. Under optimal conditions, the recovery and purity of AAT achieved 87.0% and 0.58 AAT/protein (g/g) by AEMC, respectively. After the precise polishing by HIMC, the purity of AAT was 1.22 AAT/protein (g/g). The comparison results showed that membrane chromatography outperformed column chromatography in both steps because of its high throughput. This two-step membrane chromatography could obtain an AAT recovery of 83.3% and an activity recovery of 91.4%. The outcome of this work not only offers an alternative process for protein purification from plasma, but also provides guidelines for manufacturing product from a large-volume feed with multi-components by membrane chromatography. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Binding of (/sup 3/H)imipramine to human platelet membranes with compensation for saturable binding to filters and its implication for binding studies with brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, O.M.; Wood, K.M.; Williams, D.C.

    1984-08-01

    Apparent specific binding of (/sup 3/H)imipramine to human platelet membranes at high concentrations of imipramine showed deviation from that expected of a single binding site, a result consistent with a low-affinity binding site. The deviation was due to displaceable, saturable binding to the glass fibre filters used in the assays. Imipramine, chloripramine, desipramine, and fluoxetine inhibited binding to filters whereas 5-hydroxytryptamine and ethanol were ineffective. Experimental conditions were developed that eliminated filter binding, allowing assay of high- and low-affinity binding to membranes. Failure to correct for filter binding may lead to overestimation of binding parameters, Bmax and KD for high-affinity binding to membranes, and may also be misinterpreted as indicating a low-affinity binding component in both platelet and brain membranes. Low-affinity binding (KD less than 2 microM) of imipramine to human platelet membranes was demonstrated and its significance discussed.

  18. Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans

    Science.gov (United States)

    Douglas, Lois M.; Konopka, James. B.

    2017-01-01

    Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans. PMID:26920878

  19. Effects of phenylpropanolamine (PPA) on in vitro human erythrocyte membranes and molecular models.

    Science.gov (United States)

    Suwalsky, Mario; Zambrano, Pablo; Mennickent, Sigrid; Villena, Fernando; Sotomayor, Carlos P; Aguilar, Luis F; Bolognin, Silvia

    2011-03-18

    Norephedrine, also called phenylpropanolamine (PPA), is a synthetic form of the ephedrine alkaloid. After reports of the occurrence of intracranial hemorrhage and other adverse effects, including several deaths, PPA is no longer sold in USA and Canada. Despite the extensive information about PPA toxicity, reports on its effects on cell membranes are scarce. With the aim to better understand the molecular mechanisms of the interaction of PPA with cell membranes, ranges of concentrations were incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of cell membranes. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, respectively. The capacity of PPA to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). This study presents evidence that PPA affects human red cell membranes as follows: (a) in SEM studies on human erythrocytes it was observed that 0.5 mM PPA induced shape changes; (b) in IUM PPA induced a sharp decrease in the fluorescence anisotropy in the lipid bilayer acyl chains in a concentration range lower than 100 μM; (c) X-ray diffraction studies showed that PPA in the 0.1-0.5 mM range induced increasing structural perturbation to DMPC, but no effects on DMPE multibilayers were detected. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Self-organized amniogenesis by human pluripotent stem cells in a biomimetic implantation-like niche

    Science.gov (United States)

    Shao, Yue; Taniguchi, Kenichiro; Gurdziel, Katherine; Townshend, Ryan F.; Xue, Xufeng; Yong, Koh Meng Aw; Sang, Jianming; Spence, Jason R.; Gumucio, Deborah L.; Fu, Jianping

    2017-04-01

    Amniogenesis--the development of amnion--is a critical developmental milestone for early human embryogenesis and successful pregnancy. However, human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP-SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development, thereby helping advance human embryology and reproductive medicine.

  1. Group B Streptococcus Activates Transcriptomic Pathways Related to Premature Birth in Human Extraplacental Membranes In Vitro.

    Science.gov (United States)

    Park, Hae-Ryung; Harris, Sean M; Boldenow, Erica; McEachin, Richard C; Sartor, Maureen; Chames, Mark; Loch-Caruso, Rita

    2017-11-16

    Streptococcus agalactiae (group B streptococcus, GBS) infection in pregnant women is the leading cause of infectious neonatal morbidity and mortality in the United States. Although inflammation during infection has been associated with preterm birth, the contribution of GBS to preterm birth is less certain. Moreover, the early mechanisms by which GBS interacts with the gestational tissue to affect adverse pregnancy outcomes are poorly understood. We hypothesized that short term GBS inoculation activates pathways related to inflammation and premature birth in human extraplacental membranes. We tested this hypothesis using GBS-inoculated human extraplacental membranes in vitro. In agreement with our hypothesis, a microarray-based transcriptomics analysis of gene expression changes in GBS-inoculated membranes revealed that GBS activated pathways related to inflammation and preterm birth with significant gene expression changes occurring as early as 4 hours post inoculation. In addition, pathways related to DNA replication and repair were down-regulated with GBS treatment. Conclusions based on our transcriptomics data were further supported by responses of prostaglandin E2 (PGE2) and matrix metalloproteinases 1 (MMP1) and 3 (MMP3), all of which are known to be involved in parturition and premature rupture of membranes. These results support our initial hypothesis and provide new information on molecular targets of GBS infection in human extraplacental membranes. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Progesterone Inhibits Human Myometrial Contractions by Action on Membrane Receptors

    Directory of Open Access Journals (Sweden)

    Remzi Gokdeniz

    2013-02-01

    Full Text Available Background: The mechanisms for myometrial inhibition are still being investigated Aim: To examine mechanisms of progesterone (P4 inhibition of uterine contractility. Methods: Prospective study Tertiary care center at St. Joseph’s Hospital and at Maricopa Hospital, Phoenix, AZ and research center in Arizona, USA. During 2010-2011, 24 women given birth by cesarean section. Uterine tissues from women (n=24 at term were suspended in organ chambers and exposed to various agents. Contractility was registered and compared before and after addition of agents. Tissues were treated with P4 alone, a progestin (R5020 with low affinity to the progesterone membrane receptor (mPR, or a non-sex steroid (cholesterol. Other tissues were pretreated with inhibitors of adenylate cyclase (SQ 22536, phosphodiesterase (rolipram, nitric oxide (NO synthases (L-NAME or a nuclear P4 receptor antagonist (mifepristone, MIF, followed by P4. Data were analyzed by ANOVA. Results: P4 (P0.05 inhibitory effects. P4 inhibition is not blocked by MIF, SQ, ODQ, rolipram or L-NAME (P>0.05. Conclusions: P4 rapidly inhibits myometrial contractility by nongenomic mechanisms through action on mPR but not via cAMP, cGMP, or NO [Cukurova Med J 2013; 38(1.000: 92-102

  3. Membrane Estrogen and HER-2 Receptors in Human Breast Cancer

    Science.gov (United States)

    2002-07-01

    1986). Expression of the epider- mal growth factor receptors on human cervical , ovarian and vulvar carcinomas. Cancer Res.,46: 285-293. 9.) Coussens...neurone signaling; immune and inflammatory reactions; apoptosis Aldosterone Promotion of reabsorption of sodium and excretion of potassium in kidney

  4. Membrane-associated signaling in human B-lymphoma lines

    Energy Technology Data Exchange (ETDEWEB)

    Tauzin, Sebastien; Ding, Heidrun; Burdevet, Dimitri [Department of Pathology and Immunology, Centre medical universitaire, 1, rue Michel-Servet, 1211 Geneva 11 (Switzerland); Borisch, Bettina [Department of Social and Preventive Medicine, Centre medical universitaire, 1, rue Michel-Servet, 1211 Geneva 11 (Switzerland); Hoessli, Daniel C., E-mail: danielhoessli@gmail.com [Department of Pathology and Immunology, Centre medical universitaire, 1, rue Michel-Servet, 1211 Geneva 11 (Switzerland)

    2011-01-15

    In B-non-Hodgkin lymphomas, Lyn and Cbp/PAG constitute the core of an oncogenic signalosome that captures the Phosphatidylinositol-3-kinase, the Spleen tyrosine kinase and the Signal transducer and activator of transcription-3 to generate pro-survival and proliferative signals. Lymphoma lines corresponding to follicular, mantle-cell and Burkitt-derived lymphomas display type-specific signalosome organizations that differentially activate PI3K, Syk and STAT3. In the follicular lymphoma line, PI3K, Syk and STAT3 were optimally activated upon association with the Lyn-Cbp/PAG signalosome, while in the Burkitt lymphoma-derived line, the association with Cbp/PAG and activation of PI3K were interfered with by the latent membrane proteins encoded by the Epstein-Barr virus. In the Jeko-1 mantle-cell line, a weak association of Syk with the Lyn-Cbp/PAG signalosome resulted in poor activation of Syk, but in those cells, as in the follicular and Burkitt-derived lines, efficient apoptosis induction by the Syk inhibitor R406 indicated that Syk is nonetheless an important prosurvival element and therefore a valuable therapeutic target. In all configurations described herein is the Lyn-Cbp/PAG signalosome independent of external signals and provides efficient means of activation for its associated lipid and protein kinases. In follicular and Burkitt-derived lines, Syk appears to be activated following binding to Cbp/PAG and no longer requires B-cell receptor-associated activation motifs for activation. Assessment of the different modalities of Lyn-Cbp/PAG signalosome organization could help in selecting the appropriate combination of kinase inhibitors to eliminate a particular type of lymphoma cells.

  5. Biophysical characterization of genistein-membrane interaction and its correlation with biological effect on cells - The case of EYPC liposomes and human erythrocyte membranes.

    Science.gov (United States)

    Pawlikowska-Pawlęga, Bożena; Misiak, Lucjan E; Jarosz-Wilkołazka, Anna; Zarzyka, Barbara; Paduch, Roman; Gawron, Antoni; Gruszecki, Wieslaw I

    2014-08-01

    With application of EPR and (1)H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H2O2 by lowering radicals' level. In conclusion, the capacity of genistein to incorporate, to affect membrane organization and to change its biophysical properties is correlated with the changes inside the cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. A comparative biomechanical analysis of term fetal membranes in human and domestic species

    Science.gov (United States)

    The purpose of this study was to biomechanically characterize and compare human, porcine, equine, and ovine fetal membranes. Noncontact metrology was used for topographic analyses. Uniaxial tensile testing was performed to resolve specific biomechanical values. Puncture force and radial stresses we...

  7. Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma

    DEFF Research Database (Denmark)

    Ask, Kristine Skoglund; Bardakci, Turgay; Parmer, Marthe Petrine

    2016-01-01

    Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic...

  8. Apoptosis in the human periodontal membrane evaluated in primary and permanent teeth

    DEFF Research Database (Denmark)

    Bille, Marie-Louise Bastholm; Thomsen, Bjarke; Kjær, Inger

    2011-01-01

    Abstract Objective. Recent studies revealed a highly innervated layer in close proximity to the root surface in the periodontal membrane of human teeth. Persistence of the epithelial cells of Malassez along root surfaces without resorption has also been demonstrated. It is hypothesized that resor...

  9. Membrane repair of human skeletal muscle cells requires Annexin-A5.

    Science.gov (United States)

    Carmeille, Romain; Bouvet, Flora; Tan, Sisareuth; Croissant, Coralie; Gounou, Céline; Mamchaoui, Kamel; Mouly, Vincent; Brisson, Alain R; Bouter, Anthony

    2016-09-01

    Defect in membrane repair contributes to the development of limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. In healthy skeletal muscle, unraveling membrane repair mechanisms requires to establish an exhaustive list of the components of the resealing machinery. Here we show that human myotubes rendered deficient for Annexin-A5 (AnxA5) suffer from a severe defect in membrane resealing. This defect is rescued by the addition of recombinant AnxA5 while an AnxA5 mutant, which is unable to form 2D protein arrays, has no effect. Using correlative light and electron microscopy, we show that AnxA5 binds to the edges of the torn membrane, as early as a few seconds after sarcolemma injury, where it probably self-assembles into 2D arrays. In addition, we observed that membrane resealing is associated with the presence of a cluster of lipid vesicles at the wounded site. AnxA5 is present at the surface of these vesicles and may thus participate in plugging the cell membrane disruption. Finally, we show that AnxA5 behaves similarly in myotubes from a muscle cell line established from a patient suffering from LGMD2B, a myopathy due to dysferlin mutations, which indicates that trafficking of AnxA5 during sarcolemma damage is independent of the presence of dysferlin. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Immunohistochemical study of the expression of human milk fat globule membrane glycoprotein 70.

    Science.gov (United States)

    Imam, A; Taylor, C R; Tökés, Z A

    1984-05-01

    Human milk fat globule membrane, which is said to derive from apical plasma membrane of secretory epithelial cells in breast, was analyzed by sodium dodecyl sulfate-two:dimensional gel electrophoresis. More than 35 components were detected in the gels. One of the major glycoproteins with an apparent molecular weight of 70,000, human milk fat globule membrane glycoprotein, was purified to homogeneity. The pattern of distribution of this glycoprotein in tissues was studied using polyclonal rabbit antibodies to the purified component. The localization of the antigen was accomplished by an indirect immunoperoxidase staining method. Normal mammary epithelial cells display this antigen mostly on the apical plasma membrane, whereas poorly differentiated breast carcinoma cells retained it predominantly in the cytoplasm. These observations suggest that the proper insertion of this glycoprotein into an apical membrane domain may be impaired in malignant tumor cells. In addition, a small population of tumor cells in each case examined failed to express detectable amounts of this component, indicating the presence of antigenic heterogeneity among the tumor cell population.

  11. Altered Decorin and Smad Expression in Human Fetal Membranes in PPROM1

    Science.gov (United States)

    Horgan, Casie E.; Roumimper, Hailey; Tucker, Richard; Lechner, Beatrice E.

    2014-01-01

    ABSTRACT Humans with Ehlers-Danlos syndrome, a subtype of which is caused by abnormal decorin expression, are at increased risk of preterm birth due to preterm premature rupture of fetal membranes (PPROM). In the mouse model, the absence of decorin leads to fetal membrane abnormalities, preterm birth, and dysregulation of decorin's downstream pathway components, including the transcription factor p-Smad-2. However, the role of decorin and p-Smad-2 in idiopathic human PPROM is unknown. Fetal membranes from 20–25 pregnancies per group were obtained as a cross-sectional sample of births at one institution between January 2010 and December 2012. The groups were term, preterm without PPROM, and preterm with PPROM. Immunohistochemical analysis of fetal membranes was performed for decorin and p-Smad-2 using localization and quantification assessment. Decorin expression is developmentally regulated in fetal membranes and is decreased in preterm birth with PPROM compared to preterm birth without PPROM. In preterm with PPROM samples, the presence of infection is associated with significant decorin downregulation compared to preterm with PPROM samples without infection. The preterm with PPROM group exhibited decreased p-Smad-2 staining compared to both the term controls and the preterm-without-PPROM group. Our findings suggest that dysregulation of decorin and its downstream pathway component p-Smad-2 occurs in fetal membranes during the second trimester in pathological pregnancies, thus supporting a role for decorin and p-Smad-2 in the pathophysiology of fetal membranes and adverse pregnancy outcomes. These findings may lead to the discovery of new targets for the diagnosis and treatment of PPROM. PMID:25232019

  12. Membrane Drug Transporters and Chemoresistance in Human Pancreatic Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Hagmann, Wolfgang, E-mail: w.hagmann@dkfz.de; Faissner, Ralf [Clinical Cooperation Unit of Molecular Gastroenterology, DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); Schnolzer, Martina [Functional Proteome Analysis, DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); Lohr, Matthias [Clinical Cooperation Unit of Molecular Gastroenterology, DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); Department of Surgical Gastroenterology, CLINTEC, K53, Karolinska Institute, SE-14186 Stockholm (Sweden); Jesnowski, Ralf [Clinical Cooperation Unit of Molecular Gastroenterology, DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); Department of Medicine II, Medical Faculty of Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim (Germany)

    2010-12-30

    Pancreatic cancer ranks among the tumors most resistant to chemotherapy. Such chemoresistance of tumors can be mediated by various cellular mechanisms including dysregulated apoptosis or ineffective drug concentration at the intracellular target sites. In this review, we highlight recent advances in experimental chemotherapy underlining the role of cellular transporters in drug resistance. Such contribution to the chemoresistant phenotype of tumor cells or tissues can be conferred both by uptake and export transporters, as demonstrated by in vivo and in vitro data. Our studies used human pancreatic carcinoma cells, cells stably transfected with human transporter cDNAs, or cells in which a specific transporter was knocked down by RNA interference. We have previously shown that 5-fluorouracil treatment affects the expression profile of relevant cellular transporters including multidrug resistance proteins (MRPs), and that MRP5 (ABCC5) influences chemoresistance of these tumor cells. Similarly, cell treatment with the nucleoside drug gemcitabine or a combination of chemotherapeutic drugs can variably influence the expression pattern and relative amount of uptake and export transporters in pancreatic carcinoma cells or select for pre-existing subpopulations. In addition, cytotoxicity studies with MRP5-overexpressing or MRP5-silenced cells demonstrate a contribution of MRP5 also to gemcitabine resistance. These data may lead to improved strategies of future chemotherapy regimens using gemcitabine and/or 5-fluorouracil.

  13. Structure analysis of the membrane-bound dermcidin-derived peptide SSL-25 from human sweat.

    Science.gov (United States)

    Mühlhäuser, Philipp; Wadhwani, Parvesh; Strandberg, Erik; Bürck, Jochen; Ulrich, Anne S

    2017-12-01

    SSL-25 (SSLLEKGLDGAKKAVGGLGKLGKDA) is one of the shortest peptides present in human sweat and is produced after the proteolytic processing of the parent peptide dermcidin. Both peptides are reported to have antimicrobial function. To determine the structure of SSL-25 in lipid bilayers, a series of 19 F-labeled SSL-25 analogs were synthesized. Circular dichroism (CD) analysis showed that SSL-25 and all of its analogs formed α-helices in the presence of lipid vesicles, thus allowing a detailed analysis via oriented CD and solid-state NMR. The results suggest that SSL-25 resides on the membrane surface with a slight helix tilt angle. A detailed 19 F NMR analysis revealed that SSL-25 does not form a continuous helix. The α-helical structure of the N-terminal part of the peptide was preserved in membranes of different lipid compositions and at various peptide-to-lipid molar ratios, but the C-terminus was disordered and did not fold into a well-defined α-helical conformation. Furthermore, the NMR results showed that SSL-25 resides on the membrane surface and does not re-orient into the membrane in response to changes in either peptide concentration or membrane composition. SSL-25 does not aggregate and remains fully mobile within the membrane bilayer, as shown by 19 F NMR. SSL-25 has a high binding affinity toward bilayers mimicking bacterial lipid compositions, but does not bind to mammalian model membranes containing cholesterol. These observations may explain the selectivity of this peptide for bacterial membranes, and they are also in line with basic biophysical considerations on spontaneous lipid curvature and the general effect of cholesterol on peptide/lipid interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Deoxygenation Affects Composition of Membrane-Bound Proteins in Human Erythrocytes

    Directory of Open Access Journals (Sweden)

    Oksana G. Luneva

    2016-06-01

    Full Text Available Background/Aims: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. Methods: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. Results: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ∼1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ∼2-fold elevation of the content of membrane-bound protein with Mr of ∼60 kDa. Conclusion: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions.

  15. Effect of the Human Amniotic Membrane on Liver Regeneration in Rats

    Science.gov (United States)

    Sipahi, Mesut; Şahin, Sevinç; Arslan, Ergin; Börekci, Hasan; Metin, Bayram; Cantürk, Nuh Zafer

    2015-01-01

    Introduction. Operations are performed for broader liver surgery indications for a better understanding of hepatic anatomy/physiology and developments in operation technology. Surgery can cure some patients with liver metastasis of some tumors. Nevertheless, postoperative liver failure is the most feared complication causing mortality in patients who have undergone excision of a large liver mass. The human amniotic membrane has regenerative effects. Thus, we investigated the effects of the human amniotic membrane on regeneration of the resected liver. Methods. Twenty female Wistar albino rats were divided into control and experimental groups and underwent a 70% hepatectomy. The human amniotic membrane was placed over the residual liver in the experimental group. Relative liver weight, histopathological features, and biochemical parameters were assessed on postoperative day 3. Results. Total protein and albumin levels were significantly lower in the experimental group than in the control group. No difference in relative liver weight was observed between the groups. Hepatocyte mitotic count was significantly higher in the experimental group than in the control group. Hepatic steatosis was detected in the experimental group. Conclusion. Applying the amniotic membrane to residual liver adversely affected liver regeneration. However, mesenchymal stem cell research has the potential to accelerate liver regeneration investigations. PMID:26457000

  16. Effect of the Human Amniotic Membrane on Liver Regeneration in Rats

    Directory of Open Access Journals (Sweden)

    Mesut Sipahi

    2015-01-01

    Full Text Available Introduction. Operations are performed for broader liver surgery indications for a better understanding of hepatic anatomy/physiology and developments in operation technology. Surgery can cure some patients with liver metastasis of some tumors. Nevertheless, postoperative liver failure is the most feared complication causing mortality in patients who have undergone excision of a large liver mass. The human amniotic membrane has regenerative effects. Thus, we investigated the effects of the human amniotic membrane on regeneration of the resected liver. Methods. Twenty female Wistar albino rats were divided into control and experimental groups and underwent a 70% hepatectomy. The human amniotic membrane was placed over the residual liver in the experimental group. Relative liver weight, histopathological features, and biochemical parameters were assessed on postoperative day 3. Results. Total protein and albumin levels were significantly lower in the experimental group than in the control group. No difference in relative liver weight was observed between the groups. Hepatocyte mitotic count was significantly higher in the experimental group than in the control group. Hepatic steatosis was detected in the experimental group. Conclusion. Applying the amniotic membrane to residual liver adversely affected liver regeneration. However, mesenchymal stem cell research has the potential to accelerate liver regeneration investigations.

  17. Identification of calcium-binding proteins associated with the human sperm plasma membrane

    Directory of Open Access Journals (Sweden)

    Westbrook Anne

    2010-01-01

    Full Text Available Abstract Background The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane. Methods Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface. Results Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1, HSPA5 (Bip, HYOU1 (ORP150, serum amyloid P-component (SAP and protein kinase C substrate 80K-H (80K-H were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm. Conclusion The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.

  18. Membrane composition influences the activity of in vitro refolded human vitamin K epoxide reductase.

    Science.gov (United States)

    Jaenecke, Frank; Friedrich-Epler, Beatrice; Parthier, Christoph; Stubbs, Milton T

    2015-10-27

    Human vitamin K epoxide reductase (hVKOR) is an integral membrane protein responsible for the maintenance of reduced vitamin K pools, a prerequisite for the action of γ-glutamyl carboxylase and hence for hemostasis. Here we describe the recombinant expression of hVKOR as an insoluble fusion protein in Escherichia coli, followed by purification and chemical cleavage under denaturing conditions. In vitro renaturation and reconstitution of purified solubilized hVKOR in phospholipids could be established to yield active protein. Crucially, the renatured enzyme is inhibited by the powerful coumarin anticoagulant warfarin, and we demonstrate that enzyme activity depends on lipid composition. The completely synthetic system for protein production allows a rational investigation of the multiple variables in membrane protein folding and paves the way for the provision of pure, active membrane protein for structural studies.

  19. Parallel artificial liquid membrane extraction of acidic drugs from human plasma

    DEFF Research Database (Denmark)

    Roldan-Pijuan, Mercedes; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

    2015-01-01

    The new sample preparation concept “Parallel artificial liquid membrane extraction (PALME)” was evaluated for extraction of the acidic drugs ketoprofen, fenoprofen, diclofenac, flurbiprofen, ibuprofen, and gemfibrozil from human plasma samples. Plasma samples (250 μL) were loaded into individual...... wells in a 96-well donor plate and diluted with HCl to protonate the acidic drugs. The acidic drugs were extracted as protonated species from the individual plasma samples, through corresponding artificial liquid membranes each comprising 2 μL of dihexyl ether, and into corresponding acceptor solutions......-performance liquid chromatography-ultraviolet detection of the individual acceptor solutions. Important PALME parameters including the chemical composition of the liquid membrane, extraction time, and sample pH were optimized, and the extraction performance was evaluated. Except for flurbiprofen, exhaustive...

  20. Effect of fibrin glue on the biomechanical properties of human Descemet's membrane.

    Directory of Open Access Journals (Sweden)

    Shyam S Chaurasia

    Full Text Available BACKGROUND: Corneal transplantation has rapidly evolved from full-thickness penetrating keratoplasty (PK to selective tissue corneal transplantation, where only the diseased portions of the patient's corneal tissue are replaced with healthy donor tissue. Descemet's membrane endothelial keratoplasty (DMEK performed in patients with corneal endothelial dysfunction is one such example where only a single layer of endothelial cells with its basement membrane (10-15 µm in thickness, Descemet's membrane (DM is replaced. It is challenging to replace this membrane due to its intrinsic property to roll in an aqueous environment. The main objective of this study was to determine the effects of fibrin glue (FG on the biomechanical properties of DM using atomic force microscopy (AFM and relates these properties to membrane folding propensity. METHODOLOGY/PRINCIPAL FINDINGS: Fibrin glue was sprayed using the EasySpray applicator system, and the biomechanical properties of human DM were determined by AFM. We studied the changes in the "rolling up" tendency of DM by examining the changes in the elasticity and flexural rigidity after the application of FG. Surface topography was assessed using scanning electron microscopy (SEM and AFM imaging. Treatment with FG not only stabilized and stiffened DM but also led to a significant increase in hysteresis of the glue-treated membrane. In addition, flexural or bending rigidity values also increased in FG-treated membranes. CONCLUSIONS/SIGNIFICANCE: Our results suggest that fibrin glue provides rigidity to the DM/endothelial cell complex that may aid in subsequent manipulation by maintaining tissue integrity.

  1. The Human Antimicrobial Peptides Dermcidin and LL-37 Show Novel Distinct Pathways in Membrane Interactions

    Directory of Open Access Journals (Sweden)

    Kornelius Zeth

    2017-11-01

    Full Text Available Mammals protect themselves from inflammation triggered by microorganisms through secretion of antimicrobial peptides (AMPs. One mechanism by which AMPs kill bacterial cells is perforating their membranes. Membrane interactions and pore formation were investigated for α-helical AMPs leading to the formulation of three basic mechanistic models: the barrel stave, toroidal, and carpet model. One major drawback of these models is their simplicity. They do not reflect the real in vitro and in vivo conditions. To challenge and refine these models using a structure-based approach we set out to investigate how human cathelicidin (LL-37 and dermcidin (DCD interact with membranes. Both peptides are α-helical and their structures have been solved at atomic resolution. DCD assembles in solution into a hexameric pre-channel complex before the actual membrane targeting and integration step can occur, and the complex follows a deviation of the barrel stave model. LL-37 interacts with lipids and shows the formation of oligomers generating fibril-like supramolecular structures on membranes. LL-37 further assembles into transmembrane pores with yet unknown structure expressing a deviation of the toroidal pore model. Both of their specific targeting mechanisms will be discussed in the context of the “old” models propagated in the literature.

  2. The Effect of Reduced Bacterial Dilution on Human Amniotic Membrane Antibacterial Activity, in Vitro

    Directory of Open Access Journals (Sweden)

    Mohammad Mehdi Soltan-Dallal

    2013-05-01

    Full Text Available Background: The human amniotic membrane is the inner most layer of placenta and has antimicrobial effect, due to the presence of human beta-defensins and elafins. The purpose of this study is to investigate the effect of dilution reduction of 0.5 McFarland prepared from standard bacterial strains of Salmonella enterica BAA-708, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, Klebsiella pneumoniae ATCC7881, and Enterococcus faecalis ATCC29212 on antibacterial effect of human amniotic membranes in vitro.Materials and Methods: The amniotic membranes were obtained from the bank of organ transplantation in Imam Khomeini hospital, of women with elective cesarean section whose HIV, HBV, HCV and VDRL serological tests were negative. They were cut to 1.5×1.5 cm pieces. Then 0.5 McFarland suspensions of 1.5×108, 0.5×107 and 1.5×106 dilutions were prepared from bacteria which then were spread on Mueller Hinton medium agar and a piece of membrane was put in the center of each plate. After 24 hours incubation at 37ᵒC, the results were observed.Results: In 0.5 McFarland standard dilution an inhibition zone was created in three standard strains of Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica unlike the other two strains. There was no change in the above results with two other dilutions and inhibition zone of sensitive strains was not created.Conclusion: Dilution reduction of microbial strains does not affect the antibacterial impact of amniotic membrane and dilution reduction does not yield to a false positive response and the conversion of resistant to sensitive strains.

  3. A Preliminary Study of Human Amniotic Membrane as a Potential Chondrocyte Carrier

    Directory of Open Access Journals (Sweden)

    L Boo

    2009-11-01

    Full Text Available PURPOSE: To investigate the feasibility of using processed human amniotic membrane (HAM to support the attachment and proliferation of chondrocytes in vitro which in turn can be utilised as a cell delivery vehicle in tissue engineering applications. METHODS: Fresh HAM obtained from patients undergoing routine elective caesarean sections was harvested, processed and dried using either freeze drying (FD or air drying (AD methods prior to sterilisation by gamma irradiation. Isolated, processed and characterised rabbit autologous chondrocytes were seeded on processed HAM and cultured for up to three weeks. Cell attachment and proliferation were examined qualitatively using inverted brightfield microscopy. RESULTS: Processed HAM appeared to allow cell attachment when implanted with chondrocytes. Although cells seeded on AD and FD HAM did not appear to attach as strongly as those seeded on glycerol preserved intact human amniotic membrane, these cells to be proliferated in cell culture conditions. CONCLUSION: Preliminary results show that processed HAM promotes chondrocyte attachment and proliferation.

  4. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces Cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Helix Nielsen, Claus; Scharff-Poulsen, Peter

    2014-01-01

    of solutes. Aquaporins constitute a family of physiologically very important integral membrane proteins that are found in all three kingdoms, eubacteria, archaea and eukaryotes. As protein channels, they facilitate passive transport of water across cell membranes. In the present study the yeast Saccharomyces...... cerevisiae was exploited as a host for heterologous expression of human aquaporins. Aquaporin cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human aquaporin was C-terminally tagged with yeast-enhanced GFP to quantify functional expression...... transcription factor and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30oC was due to in vivo mal-folding. Reduction of the expression temperature to 15oC almost completely...

  5. Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

    2008-05-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.

  6. Thermodynamic study of 5-(/sup 3/H)hydroxytryptamine binding to human cortex membranes

    Energy Technology Data Exchange (ETDEWEB)

    Todd, R.D.; Babinski, J.

    1987-11-01

    Kinetic and equilibrium measurements of (/sup 3/H)-serotonin (5-hydroxytryptamine) binding to human frontal cortex membranes have been made between 4 and 30 degrees C. The effects of spiperone and ascorbate on binding have also been determined. Under the conditions used, binding was saturable and reversible. Affinity constants derived from kinetic and equilibrium data were comparable. Serotonin binding to several sites had substantial enthalpic as well as entropic components.

  7. Activation of Human Dendritic Cells Is Modulated by Components of the Outer Membranes of Neisseria meningitidis

    OpenAIRE

    Al-Bader, Tamara; Christodoulides, Myron; Heckels, John E.; Holloway, Judith; Semper, Amanda E.; Friedmann, Peter S.

    2003-01-01

    Neisseria meningitidis serogroup B is a major cause of life-threatening meningitis and septicemia worldwide, and no effective vaccine is available. Initiation of innate and acquired immune responses to N. meningitidis is likely to be dependent on cellular responses of dendritic cells (DC) to antigens present in the outer membrane (OM) of the meningococcus. In this study, the responses of human monocyte-derived DC (mo-DC) to OM isolated from parent (lipopolysaccharide [LPS]-replete) meningococ...

  8. The relevance of research on red cell membranes to the understanding of complex human disease: a personal perspective.

    Science.gov (United States)

    Marchesi, Vincent T

    2008-01-01

    Molecular analysis in the service of research on human disease has finally come of age, as the chapters within this volume testify. Many technical advances, among them the development of recombinant DNA and its many applications, opened the way to study cells and processes that were unapproachable in the 1960s, when I first began my research career. The state of molecular biological studies at that time limited studies of human cell membrane proteins to experimental material most available and accessible, making the human erythrocyte membrane the favored target. I describe here how studies of red blood cell membrane proteins evolved and how results from those studies still inform present-day research.

  9. Amniotic membrane as part of a skin substitute for full-thickness wounds: an experimental evaluation in a porcine model.

    Science.gov (United States)

    Loeffelbein, Denys J; Baumann, Claudia; Stoeckelhuber, Mechthild; Hasler, Rafael; Mücke, Thomas; Steinsträßer, Lars; Drecoll, Enken; Wolff, Klaus-Dietrich; Kesting, Marco R

    2012-07-01

    We evaluated the use of human amniotic membrane (HAM) as a graft material for the treatment of iatrogenic full-thickness (FT) skin wounds in a porcine model with a view to reducing donor site morbidity in free flap transfer. Forty experimental FT-wounds were covered with an autologous split-thickness skin graft (STSG) alone or in combination with a mono- or multilayer HAM or Integra(®). Untreated wounds served as controls. Clinical evaluation and biopsy-sampling for histological and immunohistochemical staining with von-Willebrand-factor (vWF) antibody, laminin antibody, Ki-67 antibody, and smooth muscle actin (αSMA) antibody were performed on days 5, 7, 10, 20, 40, and 60 after surgical intervention. Considerable disparities in the estimated criteria were observed between the various treatment groups of the FT-wounds. The use of HAM was found to have an accelerating impact on re-epithelialization. The multilayered amnion membrane showed better results than the Integra(®) and monolayer technique in terms of contraction rate, inflammation, and scarring and seemed useful as a dermal substitute in FT-wounds giving comparable results to STSG coverage alone. This study demonstrates the successful application of HAM as part of a skin substitute in FT-wounds in minipigs. The results offer promise as a simple and effective technique for the application of multilayer HAM in iatrogenic human skin defects and the acceleration of wound healing. Copyright © 2012 Wiley Periodicals, Inc.

  10. Electron Pathways through Erythrocyte Plasma Membrane in Human Physiology and Pathology: Potential Redox Biomarker?

    Directory of Open Access Journals (Sweden)

    Elena Matteucci

    2007-01-01

    Full Text Available Erythrocytes are involved in the transport of oxygen and carbon dioxide in the body. Since pH is the influential factor in the Bohr-Haldane effect, pHi is actively maintained via secondary active transports Na+/H+ exchange and HC3 -/Cl- anion exchanger. Because of the redox properties of the iron, hemoglobin generates reactive oxygen species and thus, the human erythrocyte is constantly exposed to oxidative damage. Although the adult erythrocyte lacks protein synthesis and cannot restore damaged proteins, it is equipped with high activity of protective enzymes. Redox changes in the cell initiate various signalling pathways. Plasma membrane oxido-reductases (PMORs are transmembrane electron transport systems that have been found in the membranes of all cells and have been extensively characterized in the human erythrocyte. Erythrocyte PMORs transfer reducing equivalents from intracellular reductants to extracellular oxidants, thus their most important role seems to be to enable the cell respond to changes in intra- and extra-cellular redox environments.So far the activity of erythrocyte PMORs in disease states has not been systematically investigated. This review summarizes present knowledge on erythrocyte electron transfer activity in humans (health, type 1 diabetes, diabetic nephropathy, and chronic uremia and hypothesizes an integrated model of the functional organization of erythrocyte plasma membrane where electron pathways work in parallel with transport metabolons to maintain redox homeostasis.

  11. Human amniotic membrane as an intestinal patch for neomucosal growth in the rabbit model.

    Science.gov (United States)

    Barlas, M; Gökçora, H; Erekul, S; Dindar, H; Yücesan, S

    1992-05-01

    This experiment was carried out as a preliminary study, an attempt to grow new intestinal mucosa on human amniotic membrane in the terminal ileum in 37 rabbits. After ketamin sulfate anesthesia at laparatomy, 5-cm ileal defects were patched with human amniotic membrane (5 x 2 cm). These patched intestines were investigated on the first postoperative day and the 2nd, 5th, 10th, and 20th weeks corresponding to 4, 5, 5, 10, and 10 rabbits, respectively. Only three rabbits died in the early postoperative period. There was no evidence of intestinal obstruction or dilatation with barium meal. Microscopically, the neomucosa consisted of a thin layer of columnar epithelial cells at 2 weeks with more maturity of the villi and less irregularity and branching by 20 weeks. All patches were covered with neomucosa commencing at 2 weeks and covering the whole patch area by 20 weeks. This technique's advantages are the large size and the ease of the availability of the human amniotic membrane for neonates at risk without jeopardizing the neonates tissues. It is hoped that this method might be considered when neonatal material is scarce.

  12. Fractal analysis of AFM images of the surface of Bowman's membrane of the human cornea.

    Science.gov (United States)

    Ţălu, Ştefan; Stach, Sebastian; Sueiras, Vivian; Ziebarth, Noël Marysa

    2015-04-01

    The objective of this study is to further investigate the ultrastructural details of the surface of Bowman's membrane of the human cornea, using atomic force microscopy (AFM) images. One representative image acquired of Bowman's membrane of a human cornea was investigated. The three-dimensional (3-D) surface of the sample was imaged using AFM in contact mode, while the sample was completely submerged in optisol solution. Height and deflection images were acquired at multiple scan lengths using the MFP-3D AFM system software (Asylum Research, Santa Barbara, CA), based in IGOR Pro (WaveMetrics, Lake Oswego, OR). A novel approach, based on computational algorithms for fractal analysis of surfaces applied for AFM data, was utilized to analyze the surface structure. The surfaces revealed a fractal structure at the nanometer scale. The fractal dimension, D, provided quantitative values that characterize the scale properties of surface geometry. Detailed characterization of the surface topography was obtained using statistical parameters, in accordance with ISO 25178-2: 2012. Results obtained by fractal analysis confirm the relationship between the value of the fractal dimension and the statistical surface roughness parameters. The surface structure of Bowman's membrane of the human cornea is complex. The analyzed AFM images confirm a fractal nature of the surface, which is not taken into account by classical surface statistical parameters. Surface fractal dimension could be useful in ophthalmology to quantify corneal architectural changes associated with different disease states to further our understanding of disease evolution.

  13. Effects of hypophysectomy and GH administration on bovine and human GH binding to rat liver membranes.

    Science.gov (United States)

    Messina, J L; Edén, S; Kostyo, J L

    1985-07-01

    Experiments were conducted to investigate the specific binding of highly purified bovine and human growth hormones (bGH and hGH) to purified liver plasma membranes of male rats at various times after hypophysectomy and after the acute intravenous administration of bGH. Liver membranes prepared from hypophysectomized male rats showed a two- to threefold increase in the specific binding of either [125I]iodo-bGH or [125I]iodo-hGH, when compared with membranes prepared from the livers of age-matched normal male rats. The increase in GH binding was apparent within 3 days after hypophysectomy and persisted for a number of weeks after the operation. The increase in GH binding produced by hypophysectomy appeared to be due to an increase in the number of binding sites present on the membranes. The intravenous injection of 200 micrograms of bGH into hypophysectomized male rats 5-60 min before they were killed markedly reduced the ability of liver membranes prepared from these animals to bind [125I]iodo-bGH specifically. This decrease in GH binding seen after the injection of bGH may have been due to the development of a slowly dissociating hormone-binding site complex, which thereby reduced the number of available binding sites. This conclusion is supported by the finding that bGH, which is bound in vitro to isolated liver membranes, dissociates slowly and incompletely in the presence of an excess of unlabeled hormone. Moreover, the degree to which the bound hormone can dissociate appears to depend on the length of time that association is allowed to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Enhanced chondrogenic differentiation potential of human gingival fibroblasts by spheroid formation on chitosan membranes.

    Science.gov (United States)

    Hsu, Shan-hui; Huang, Guo-Shiang; Lin, Susan Yun Fan; Feng, Fuh; Ho, Tung-Tso; Liao, Yuan-Ching

    2012-01-01

    Human gingival fibroblasts (HGF) were recently found to be a source of mesenchymal stem cells. Their behavior on a biomaterial has not been reported so far. The effect of culturing HGF on chitosan membranes on their chondrogenic differentiation was investigated in this study. HGF were first cultured on chitosan membranes and spheroid formation of HGF was observed. Next, HGF on chitosan were induced with chondrogenesis induction medium and their chondrogenic differentiation potential was expressed by assessing the expression of chondrogenesis related genes at both mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining, respectively. We discovered that the chondrogenic differentiation potential of HGF could be enhanced simply by culturing HGF on chitosan membranes. Expression of neural crest and stemness genes were also analyzed by RT-PCR to evaluate the stemness and self-renewal of HGF spheroids. We found that spheroid formation helped to increase and maintain the expression of stemness genes in HGF. To understand the aspects of the chitosan membranes that induced spheroid formation of HGF, mechanical and physical properties of the chitosan membranes were examined. The migration of HGF on chitosan membranes was also monitored to speculate the process of spheroid formation. In addition, the roles of the Rho/Rho-associated kinase (ROCK) pathway and connexin 43 (Cx43) in spheroid formation were explored. Treatment of HGF cultured on chitosan with the ROCK-activity inhibitor Y27632 clearly inhibited spheroid formation, suggesting that the Rho/ROCK pathway was involved in spheroid formation. The increased Cx43 activity of HGF spheroids on chitosan indicated that the gap junction intercellular communication was regulated by spheroid formation. It was concluded that culturing HGF on chitosan may activate the Rho/ROCK pathway, which led to spheroid formation and gap junction regulation. These changes may contribute to the

  15. Cholesterol masks membrane glycosphingolipid tumor-associated antigens to reduce their immunodetection in human cancer biopsies.

    Science.gov (United States)

    Novak, Anton; Binnington, Beth; Ngan, Bo; Chadwick, Karen; Fleshner, Neil; Lingwood, Clifford A

    2013-11-01

    Glycosphingolipids (GSLs) are neoplastic and normal/cancer stem cell markers and GSL/cholesterol-containing membrane rafts are increased in cancer cell plasma membranes. We define a novel means by which cancer cells can restrict tumor-associated GSL immunoreactivity. The GSL-cholesterol complex reorients GSL carbohydrate to a membrane parallel, rather than perpendicular conformation, largely unavailable for antibody recognition. Methyl-β-cyclodextrin cholesterol extraction of all primary human tumor frozen sections tested (ovarian, testicular, neuroblastoma, prostate, breast, colon, pheochromocytoma and ganglioneuroma), unmasked previously "invisible" membrane GSLs for immunodetection. In ovarian carcinoma, globotriaosyl ceramide (Gb3), the GSL receptor for the antineoplastic Escherichia coli-derived verotoxin, was increased throughout the tumor. In colon carcinoma, Gb3 detection was vastly increased within the neovasculature and perivascular stroma. In tumors considered Gb3 negative (neuroblastoma, Leydig testicular tumor and pheochromocytoma), neovascular Gb3 was unmasked. Tumor-associated GSL stage-specific embryonic antigen (SSEA)-1, SSEA-3, SSEA-4 and globoH were unmasked according to tumor: SSEA-1 in prostate/colon; SSEA-3 in prostate; SSEA-4 in pheochromocytoma/some colon tumors; globoH in prostate/some colon tumors. In colon, anti-SSEA-1 was tumor cell specific. Within the GSL-cholesterol complex, filipin-cholesterol binding was also reduced. These results may relate to the ill-defined benefit of statins on cancer prognosis, for example, prostate carcinoma. We found novel anti-tumor GSL antibodies circulating in 3/5 statin-treated, but not untreated, prostate cancer patients. Lowering tumor membrane cholesterol may permit immune recognition of otherwise unavailable tumor-associated GSL carbohydrate, for more effective immunosurveillance and active/passive immunotherapy. Our results show standard immunodetection of tumor GSLs significantly under assesses

  16. Distinct human and mouse membrane trafficking systems for sweet taste receptors T1r2 and T1r3.

    Science.gov (United States)

    Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko

    2014-01-01

    The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.

  17. Preterm premature rupture of membranes in pregnancies complicated by human immunodeficiency virus infection: a single center's five-year experience.

    Science.gov (United States)

    Alvarez, Jesus R; Bardeguez, Arlene; Iffy, Leslie; Apuzzio, Joseph J

    2007-12-01

    The objective of this study was to describe one center's five-year experience of the management of human immunodeficiency virus (HIV) positive gravidas with preterm premature rupture of the membranes (PPROM) not in labor at review of all HIV positive gravidas with PPROM at rupture of membranes prior to delivery.

  18. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    Science.gov (United States)

    2011-01-01

    Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL) 11 regulates human endometrial epithelial cells (hEEC) adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE) electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2) and flotillin-1 (FLOT1), were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa) and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle). Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary hEEC and ECC-1 whilst

  19. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    Directory of Open Access Journals (Sweden)

    Stanton Peter G

    2011-05-01

    Full Text Available Abstract Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL 11 regulates human endometrial epithelial cells (hEEC adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2 and flotillin-1 (FLOT1, were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle. Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary h

  20. The Role of Two Different Collagen Membranes for Dehiscence Defect Around Implants in Humans.

    Science.gov (United States)

    Lee, Dong-Woon; Kim, Kyeong-Taek; Joo, Yon-Soo; Yoo, Mi-Kyung; Yu, Jeoung-A; Ryu, Jae-Jun

    2015-08-01

    The aim of this study was to elucidate the role of 2 types of collagen membranes (cross-linked vs noncross-linked) used in conjunction with autogenous or allogenic bone followed by xenogeneic bone particles for dehiscence defect around implants in humans. Experimental groups were divided into 2 groups: Group CL (cross-linked, Ossix Plus, n = 24 implants, 16 patients) and Group NCL (noncross-linked, Bio-Gide, n = 25 implants, 18 patients). At the time of implant insertion and uncovery surgery, measurements of the dehiscence bony height, width, and surface area were made. Before applying the membrane to defects, guided bone regeneration was performed. Because it is difficult to measure the degree of exposure, early exposed cases were excluded from the result analysis. The mean percentage gain of the dehiscence defect and the mean marginal bone reduction value of follow-up radiograph did not show statistically significant differences between the 2 groups. Both membranes exhibited satisfactory results on dehiscence defects. As a result, our authors concluded the success of guided bone regeneration was performed simultaneously for dehiscence defects around the implant, regardless whether collagen membranes were cross-linked or noncross-linked.

  1. Factors influencing the flow rate through a surgical defect in human fetal membranes.

    Science.gov (United States)

    Devlieger, R; Gratacos, E; Ardon, H; Vanstraelen, S; Deprest, J

    2002-03-01

    In order to determine factors influencing the flow rate trough a created defect in human fetal membranes, an ex vivo set-up was used with fetal membranes collected from patients undergoing Caesarean section at term. The membranes were secured at the bottom of a plastic tube and traumatised with needles ranging from 14-26 Gauges (Ga), under a hydrostatic pressure of 10 to 20 cm H(2)O and an angle of 45 degrees or 90 degrees. The column was filled with amniotic fluid or Hartmann's solution. The duration of the puncture was 1 s or the time it takes to aspirate 10 ml through the needle. The flow rate through the defect in the fetal membranes and size of the defect were measured. The flow rate and defect size increased with increasing diameter of the needle. Increasing the pressure in the column resulted in a significant linear increase in the flow rate. Replacing the saline solution with amniotic fluid did not result in significant changes in the measured flow rates, except for the small needle size (24 Ga). Increasing the duration of the puncture did not result in increased flow rates, except for small needle size (24 Ga). These experiments suggest that needle diameter, angle of needle insertion, duration of the procedure, amniotic fluid pressure and composition could influence the incidence of amniotic fluid leakage following amniocentesis. Copyright 2002 John Wiley & Sons, Ltd.

  2. [Vibrations of the human tympanic membrane measured with Laser Doppler Vibrometer].

    Science.gov (United States)

    Szymański, Marcin; Rusinek, Rafał; Zadrozniak, Marek; Warmiński, Jerzy; Morshed, Kamal

    2009-01-01

    The knowledge of the physiology of the normal ear is important to understand the function of the ear. It is especially crucial in the reconstruction of the destroyed ear to apply the knowledge of the normal ear. We present results of tympanic membrane vibrations measurements using Laser Doppler Vibrometer in human temporal bone specimens. Six temporal bone specimens were harvested within 48 hours of death and stored cooled until preparation. The preparation included mastoidectomy with posterior tympanotomy and partial resection of the facial nerve to visualize the stapes with its footplate. We measured velocity and displacement of each quadrant of the tympanic membrane and the umbo with the laser Vibrometer equipped with velocity and displacement decoders. The sensor head OFV-534 produced and read the reflected laser beam directed at a measured point with a dedicated micromanipulator attached to an operating microscope. A retro-reflective tape was used to enhance the reflection of the laser beam. Vibrations were induced by a acoustic stimulation at the tympanic membrane. The results of the measurements were corrected to a sound pressure in the external ear canal. Laser Doppler Vibrometer system allows an undisturbed measurement of vibrations in the middle ear. Posterior quadrants of the tympanic membrane have greater velocity and displacement than anterior quadrants in lower frequencies up to 2 kHz.

  3. Macromolecular diffusion characteristics of ageing human Bruch's membrane: implications for age-related macular degeneration (AMD).

    Science.gov (United States)

    Hussain, A A; Starita, C; Hodgetts, A; Marshall, J

    2010-06-01

    Macromolecular species such as retinal binding protein, transferrin, ceruloplasmin, etc., released by the fenestrated choroidal capillaries must diffuse across Bruch's membrane for interaction with the basal membranes of the retinal pigment epithelium (RPE) for delivery of essential metabolites to the neural retina. The patency of this pathway through ageing Bruch's was examined by quantifying the diffusional flux of a 21.2 kDa fluorescein-isothiocyanate labelled dextran. Dextran flux measurements across Bruch's membrane from the macular region of the human fundus showed a highly significant decrease (p < 0.001) with ageing of donor such that diffusional transport in the ninth decade was about 6.5% of that in the first decade of life. Peripheral regions also showed a highly significant decline (p < 0.001) but ageing changes were considerably slowed in comparison to the macula with diffusional rates in the ninth decade being about 44% of that in the first decade. Peripheral samples from AMD donors displayed diffusional rates that were lower than the control population. The age-related decline in macromolecular diffusion across Bruch's membrane suggests that in the elderly, the patency of the conducting pathways may be compromised and in the more advanced ageing of Bruch's associated with AMD, the metabolic trafficking of carrier proteins may be severely impaired. Copyright 2010 Elsevier Ltd. All rights reserved.

  4. Biomechanical assays amniotic membrane preserved in glycerol correlating with optical coherence tomography (OCT) and thermal gravimetric analysis (TG)

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Fernando Augusto N.; Santin, Stefany P.; Martino Junior, Antonio C.; Machado, Luci Diva B.; Freitas, Anderson Z.; Mathor, Monica B., E-mail: fernandonevessoares@yahoo.com.br [Instituto de Pesquisas Energetias Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2013-07-01

    Amnion or amniotic membranes (AM) are interchangeable terms used in the literature being internal part of the fetal membranes, non-vascular and multicellular tissue. The amnion has been widely used as a graft ophthalmic surgical as well as carrier substrate stem cell tissue equivalent for ocular surface reconstruction. The AM reduces scar formation and inflammation on the ocular surface, promotes epithelization also been used as a biological bandage covering the wound or burns, reducing dehydration and allowing regeneration of these areas. The amnion has usually 0.02 to 0.5 mm thick and consists of five subsequent layers: epithelium, basement membrane, compact layer, fibroblast layer and spongy layer. The mechanical strength from the membrane structure as well as the elasticity are factors attractive to the use of amnion as a surgical graft. Higher levels of rigidity and strength may improve the graft resistance necessary to resist the stress induced during growth of the new tissue formed. The amniotic membrane is obtained at elective caesarean section and subsequently, under sterile conditions, sectioned and separated from chorion and placenta, and free blood clots. The serological tests are done at the time of collection of tissue and 6 months after delivery to confirm the results. There are different methods for storing MA in tissue banks as fresh, high concentrations of glycerol, among others. The use of fresh membrane has some limitations due to the need to rapid use and high risk of contamination, however the amniotic membrane in glycerol has antiviral and antibacterial property which is dependent on the concentration, time and temperature. The AM used in transplants must be sterile to prevent the transmission of any disease. Although sterilization by radiation is an effective procedure, it can interfere on the membrane structure. Thus, verification of potential changes caused by ionizing radiation in amnion was made using the tensile test by calculating the

  5. Human Rab small GTPase- and class V myosin-mediated membrane tethering in a chemically defined reconstitution system.

    Science.gov (United States)

    Inoshita, Motoki; Mima, Joji

    2017-11-10

    Membrane tethering is a fundamental process essential for the compartmental specificity of intracellular membrane trafficking in eukaryotic cells. Rab-family small GTPases and specific sets of Rab-interacting effector proteins, including coiled-coil tethering proteins and multisubunit tethering complexes, are reported to be responsible for membrane tethering. However, whether and how these key components directly and specifically tether subcellular membranes remains enigmatic. Using chemically defined proteoliposomal systems reconstituted with purified human Rab proteins and synthetic liposomal membranes to study the molecular basis of membrane tethering, we established here that Rab-family GTPases have a highly conserved function to directly mediate membrane tethering, even in the absence of any types of Rab effectors such as the so-called tethering proteins. Moreover, we demonstrate that membrane tethering mediated by endosomal Rab11a is drastically and selectively stimulated by its cognate Rab effectors, class V myosins (Myo5A and Myo5B), in a GTP-dependent manner. Of note, Myo5A and Myo5B exclusively recognized and cooperated with the membrane-anchored form of their cognate Rab11a to support membrane tethering mediated by trans-Rab assemblies on opposing membranes. Our findings support the novel concept that Rab-family proteins provide a bona fide membrane tether to physically and specifically link two distinct lipid bilayers of subcellular membranes. They further indicate that Rab-interacting effector proteins, including class V myosins, can regulate these Rab-mediated membrane-tethering reactions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Plasma membrane block to polyspermy in human oocytes and preimplantation embryos.

    Science.gov (United States)

    Sengoku, K; Tamate, K; Horikawa, M; Takaoka, Y; Ishikawa, M; Dukelow, W R

    1995-09-01

    The existence and time course of the human plasma membrane block to polyspermy were investigated by an in vitro fertilization assay using zona pellucida-free unfertilized oocytes, pronuclear oocytes and embryos. In the time course study using a high sperm concentration (10(5) spermatozoa ml-1), the number of penetrating spermatozoa at 30 and 60 min after insemination were 1.3 +/- 0.3 and 2.9 +/- 0.4, respectively. By 2 h after insemination, spermatozoa penetration reached a maximum. A lower maximum number of penetrating spermatozoa was observed at a low sperm concentration (10(4) spermatozoa ml-1), but the number of penetrating spermatozoa still reached a maximum by 2 h after insemination. A reinsemination experiment demonstrated that the number of penetrating spermatozoa was not significantly different between control and reinseminated oocytes, while sperm penetration was not observed in the oocyte beyond the two-cell stage. Furthermore, the number of binding spermatozoa decreased after fertilization and most of the four-cell stage embryos displayed no sperm binding. These results suggest that the plasma membrane block plays an important role in the prevention of polyspermy in the human oocyte, and that the plasma membrane block may involve permanent changes in the binding or fusion ability of spermatozoa in the oolemma after fertilization.

  7. Possible evidence that dehydroepiandrosterone sulfate (DHA-S) stimulates cervical ripening by a membrane-mediated process: Specific binding-sites in plasma membrane from human uterine cervix

    Energy Technology Data Exchange (ETDEWEB)

    Ohno, T.; Imai, A.; Tamaya, T. (Department of Obstetrics and Gynecology, Gifu University School of Medicine (Japan))

    1991-04-01

    Fetal adrenal steroid, dehydroepiandrosterone sulfate (DHA-S) is well known to promote cervical ripening in late pregnancy. The presence of sites specifically binding the DHA-S in plasma membrane was studied in human cervical fibroblasts prepared from pregnant uterus. The fibroblasts were incubated with {sup 3}H DHA-S and then fractionated into plasma membranes, cytosol, nuclei, and other organella debris. The specific activity of 3H-count in the plasma membrane fraction was enriched {approximately} 7-fold compared with the whole homogenate. When the isolated plasma membrane preparations from the fibroblasts were exposed to {sup 3}H DHA-S, the binding showed saturation kinetics; an apparent equilibrium dissociation constant (Kd) of 12 nM, and the binding capacity (Bmax) of 1.25 pmol/mg protein. The present results suggest that DHA is bound to and recognized by components in plasma membrane, and may exert its action on cervical ripening through the membrane-mediated processes.

  8. Evaluation of Human Semenogelin Membrane Strip Test for Species Cross-reactivity in Dogs.

    Science.gov (United States)

    Stern, A W; Lanka, S

    2016-09-01

    Semenogelins are proteins originating in the seminal vesicle and are useful markers for the presumptive identification of human semen. Detection of semenogelin can be done with a commercially available membrane test. In this study, a commercially available membrane test for human semenogelin proteins was used to assess for cross-reactivity in dog bodily fluids to allow for the potential utilization for detection of human semen in dog bodily fluids. The authors analyzed canine semen and other bodily fluids, including urine, saliva, vaginal secretions, fecal material, and blood. They also examined the distribution of human semenogelin I transcripts in the canine testis, prostate, and several bodily fluids by reverse transcription polymerase chain reaction. No cross-reactivity was observed in the canine bodily fluids tested except for a single rectal swab, which was negative on a second test. Further testing should be done to validate the use of this kit for screening samples from dogs suspected to have been victims of sexual abuse. © The Author(s) 2015.

  9. Quantitative membrane proteomics reveals a role for tetraspanin enriched microdomains during entry of human cytomegalovirus.

    Directory of Open Access Journals (Sweden)

    Kasinath Viswanathan

    Full Text Available Human cytomegalovirus (HCMV depends on and modulates multiple host cell membrane proteins during each stage of the viral life cycle. To gain a global view of the impact of HCMV-infection on membrane proteins, we analyzed HCMV-induced changes in the abundance of membrane proteins in fibroblasts using stable isotope labeling with amino acids (SILAC, membrane fractionation and protein identification by two-dimensional liquid chromatography and tandem mass spectrometry. This systematic approach revealed that CD81, CD44, CD98, caveolin-1 and catenin delta-1 were down-regulated during infection whereas GRP-78 was up-regulated. Since CD81 downregulation was also observed during infection with UV-inactivated virus we hypothesized that this tetraspanin is part of the viral entry process. Interestingly, additional members of the tetraspanin family, CD9 and CD151, were also downregulated during HCMV-entry. Since tetraspanin-enriched microdomains (TEM cluster host cell membrane proteins including known CMV receptors such as integrins, we studied whether TEMs are required for viral entry. When TEMs were disrupted with the cholesterol chelator methyl-β-cylcodextrin, viral entry was inhibited and this inhibition correlated with reduced surface levels of CD81, CD9 and CD151, whereas integrin levels remained unchanged. Furthermore, simultaneous siRNA-mediated knockdown of multiple tetraspanins inhibited viral entry whereas individual knockdown had little effect suggesting essential, but redundant roles for individual tetraspanins during entry. Taken together, our data suggest that TEM act as platforms for receptors utilized by HCMV for entry into cells.

  10. Staphylococcus aureus Infection of Human Gestational Membranes Induces Bacterial Biofilm Formation and Host Production of Cytokines.

    Science.gov (United States)

    Doster, Ryan S; Kirk, Leslie A; Tetz, Lauren M; Rogers, Lisa M; Aronoff, David M; Gaddy, Jennifer A

    2017-02-15

    Staphylococcus aureus, a metabolically flexible gram-positive pathogen, causes infections in a variety of tissues. Recent evidence implicates S. aureus as an emerging cause of chorioamnionitis and premature rupture of membranes, which are associated with preterm birth and neonatal disease. We demonstrate here that S. aureus infects and forms biofilms on the choriodecidual surface of explanted human gestational membranes. Concomitantly, S. aureus elicits the production of proinflammatory cytokines, which could ultimately perturb maternal-fetal tolerance during pregnancy. Therefore, targeting the immunological response to S. aureus infection during pregnancy could attenuate disease among infected individuals, especially in the context of antibiotic resistance. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  11. Direct visualization of lipid domains in human skin stratum corneum's lipid membranes

    DEFF Research Database (Denmark)

    Plasencia, I; Norlen, Lars; Bagatolli, Luis

    2007-01-01

    ; and iii), whether pH has a direct effect on the lipid matrix phase behavior. In this work the lateral structure of membranes composed of lipids extracted from human skin stratum corneum was studied in a broad temperature range (10 degrees C-90 degrees C) using different techniques such as differential...... resolution limit 300 nm) to a single gel phase at pH 7, coexistence of different gel phases between pH 5 and 6, and no fluid phase at any pH. This observation suggests that the local pH in the stratum corneum may control the physical properties of the extracellular lipid matrix by regulating membrane lateral......-dimensional morphology of the stratum corneum extracellular space. These structures can be directly visualized using the aforementioned fluorescence microscopy techniques. At skin physiological temperatures (28 degrees C-32 degrees C), the phase state of these hydrated bilayers correspond microscopically (radial...

  12. Regenerative potential of human schneiderian membrane: progenitor cells and epithelial-mesenchymal transition.

    Science.gov (United States)

    Derjac-Aramă, A I; Sarafoleanu, C; Manea, C M; Nicolescu, M I; Vrapciu, A D; Rusu, M C

    2015-12-01

    An innate osteogenic potential of the Schneiderian membrane (SM) is progressively assessed in studies ranging from non-human species to human subjects. It has relevance for endosteal placement and osseointegration. Nestin-expressing osteogenic progenitor cells are allegedly involved in bone formation and remodelling. Nestin phenotype was not assessed previously in human SM. We therefore aimed to fill that particular gap in the literature. Bioptic samples of human adult SM were obtained during surgery from eight adult patients, operated for non-malignant pathologies. Immunohistochemistry on paraffin-embedded tissue samples used primary antibodies against nestin, CD45, CD146, cytokeratin 7 (CK7), and alpha-smooth muscle actin (α-SMA). Nestin expression was consistently found in endothelial cells, and was scarcely encountered in pericytes, putative stromal stem/progenitor cells, as well as in glandular epithelial cells. Moreover, woven bone formation in the periosteal layer of the SM can also be regarded as evidence of the osteogenic potential of this membrane. Nestin and CD45 expression in cells of the primary bone supports the osteogenic potential of SM nestin-expressing cells and a possible involvement of hematopoietic stem cells in maxillary sinus floor remodeling. CD146, a known inducer of epithelial-mesenchymal transition (EMT), was expressed in epithelia, as was CK7. Isolated stromal cells were found expressing CD146, CK7 and α-SMA, suggesting that regenerative processes happening in the SM may also involve processes of EMT which generate stem/progenitor cells. This study provides additional evidence for the regenerative potential of the Schneiderian membrane and identifies potential roles for cells of its stem niche in osteogenesis. © 2015 Wiley Periodicals, Inc.

  13. Peroxynitrite-mediated nitrosative stress decreases motility and mitochondrial membrane potential in human spermatozoa.

    Science.gov (United States)

    Uribe, P; Boguen, R; Treulen, F; Sánchez, R; Villegas, J V

    2015-03-01

    Nitrosative stress is produced by high levels of reactive nitrogen species (RNS). The RNS include peroxynitrite, a highly reactive free radical produced from a diffusion-controlled reaction between nitric oxide and superoxide anion. Peroxynitrite causes nitration and oxidation of lipids, proteins and DNA, and is thus considered an important pathogenic mechanism in various diseases. Although high levels of peroxynitrite are associated with astenozoospermia, few reports exist regarding the in vitro effect of high levels of this RNS on human sperm. The aim of this study was to evaluate the in vitro effect of nitrosative stress caused by peroxynitrite on the viability, motility and mitochondrial membrane potential of human spermatozoa. To do this, human spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a molecule that generates peroxynitrite. Incubations were done at 37°C for up to 4 h with SIN-1 concentrations between 0.2 and 1.0 mmol/l. Generation of peroxynitrite was confirmed using dihydrorhodamine 123 (DHR) by spectrophotometry and flow cytometry. Sperm viability was assessed by propidium iodide staining; sperm motility was analyzed by CASA, and the state of mitochondrial membrane potential (ΔΨm) by JC-1 staining. Viability and ΔΨm were measured by flow cytometry. The results showed an increase in DHR oxidation, demonstrating the generation of peroxynitrite through SIN-1. Peroxynitrite decreased progressive and total motility, as well as some sperm kinetic parameters. Mitochondrial membrane potential also decreased. These alterations occurred with no decrease in sperm viability. In conclusion, peroxynitrite-induced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Characterization of an atypical lipoprotein-binding protein in human aortic media membranes by ligand blotting.

    Science.gov (United States)

    Kuzmenko, Y S; Bochkov, V N; Philippova, M P; Tkachuk, V A; Resink, T J

    1994-01-01

    By use of ligand-blotting techniques, this study investigated lipoprotein-binding proteins in human aortic smooth muscle. PAGE was performed under non-reducing conditions, and, using low-density lipoprotein (LDL) as ligand, with rabbit anti-apolipoprotein (apo) B and 125I-labelled goat anti-rabbit IgG as primary and secondary antibodies respectively, we demonstrate that membranes from human aortic media (and cultured human smooth-muscle cells) contain a major lipoprotein-binding protein with an apparent molecular mass of 105 kDa. Anionized preparations (carbamoyl- and acetyl-) of LDL, which did not displace 125I-LDL bound to the apo B,E receptor of cultured fibroblasts, were also recognized as ligands for the 105 kDa protein in aortic media membranes. LDL binding to 105 kDa protein was decreased in the presence of high density lipoprotein (HDL), although more than 100-fold molar excess of HDL was required to achieve 50% displacement of bound LDL. The LDL-binding activity of 105 kDa protein was inhibited by EDTA, and was also significantly decreased when samples were reduced by beta-mercaptoethanol before electrophoresis. Monoclonal antibodies against apo B,E receptor reacted with partially purified bovine adrenal apo B,E receptor, but not with 105 kDa protein of human aortic media membranes. The spectrum of properties of this vascular smooth-muscle lipoprotein-binding protein binding are clearly distinct from those of other previously characterized lipoprotein-binding molecules. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 Figure 8 PMID:7945254

  15. In vitro evaluation of the human gingival fibroblast/gingival mesenchymal stem cell dynamics through perforated guided tissue membranes: cell migration, proliferation and membrane stiffness assay.

    Science.gov (United States)

    Gamal, A Y; Al-Berry, N N; Hassan, A A; Rashed, L A; Iacono, V J

    2017-06-01

    Migration of gingival fibroblasts/gingival mesenchymal stem cells through macro-perforated barrier membranes may allow them to participate positively in periodontal regeneration. The optimal guided tissue membrane perforation diameter that could favor maximum cell migration into the defect area and at the same time act as an occlusive barrier for gingival epithelium and its associated gingival extracellular matrix component is not yet identified. Cultured human gingival fibroblasts/gingival mesenchymal stem cells were placed in the upper chambers of 12-well collagen-coated polytetrafluoroethylene transwells, which were manually perforated with 0.2, 0.4 and 0.7 mm sized pores. The lower chambers of the transwells received blood clot as an attraction medium. The number of cells that have migrated to the lower chambers was calculated. Proliferation of these cells was evaluated using MTT assay. Scanning electron microscopy images were obtained for the lower surfaces of the transwell membranes. Perforated bovine collagen membranes (Tutopatch(®) ) were subjected to mechanical testing to determine the tensile strength and modulus of elasticity. Group 3 (0.7 mm) showed significantly higher values for cell migration and proliferation. All groups showed a small degree of extracellular matrix migration through membrane perforations. Scanning electron microscopy evaluation revealed variable numbers of cells in fibrin matrices located mainly around the pore edges. There were non-significant differences between groups regarding mechanical properties. The present study demonstrated that macro-membrane perforations of 0.2, 0.4 and 0.7 mm are suitable pore diameters that could maintain membrane stiffness and allow for cellular migration. However, these membrane perforation diameters did not allow for total gingival connective tissue isolation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Modelling the influence of amnionicity on the severity of twin-twin transfusion syndrome in monochorionic twin pregnancies

    Energy Technology Data Exchange (ETDEWEB)

    Wijngaard, Jeroen P H M van den [Laser Center and Department of Obstetrics and Gynecology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Umur, Asli [Laser Center and Department of Obstetrics and Gynecology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Ross, Michael G [Department of Obstetrics and Gynecology, Harbor University of California-Los Angeles Medical Center, Torrance, CA 90502 (United States); Gemert, Martin J C van [Laser Center and Department of Obstetrics and Gynecology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands)

    2004-03-21

    Clinical treatment for diamniotic-monochorionic twin-twin transfusion syndrome (TTTS) may include conversion of diamniotic pregnancies to a monoamniotic-monochorionic state by disrupting the amnion septum. We sought to test the underlying hypothesis, i.e. that a monoamniotic state reduces the severity of TTTS. With use of our previously developed mathematical model of two equal fetoplacental circulatory units connected by various sizes and types of placental anastomoses, we compared the haemodynamic and amniotic fluid dynamics of monoamniotic and diamniotic twins that develop TTTS. We used three anastomotic patterns that produce severe, moderate or mild forms of TTTS, respectively, in our diamniotic-monochorionic twin model. Monoamnionicity was modelled by adding the two amniotic fluid volumes and using the volume-averaged amniotic fluid osmolality. The results were as follows: for severe TTTS, small differences develop between diamniotic and monoamniotic donor twins in fetal urine production, swallowed volume, blood volume, blood pressures, net fetofetal transfusion, and blood and amniotic fluid osmolality. However, the circulatory imbalance between the monoamniotic twins deteriorates similar to that of diamniotic twins. The pathophysiological differences tend to disappear for milder TTTS. In conclusion, our model suggests that the uncommon finding of TTTS in monoamniotic twins is not due to the presence of a single amniotic sac. Rather, clinically significant differences in anastomotic patterns and the delayed or lack of identification of manifestations in monoamniotic twins account for the reduced rate of TTTS diagnosis. Based on these results we expect the clinical disruption of the amnion septum in diamniotic-monochorionic TTTS pregnancies to have only minimal benefits. (note)

  17. The outer membrane protein OprQ and adherence of Pseudomonas aeruginosa to human fibronectin.

    Science.gov (United States)

    Arhin, Abraham; Boucher, Cliff

    2010-05-01

    Outer membrane proteins of the Gram-negative organism Pseudomonas aeruginosa play a significant role in membrane permeability, antibiotic resistance, nutrient uptake, and virulence in the infection site. In this study, we show that the P. aeruginosa outer membrane protein OprQ, a member of the OprD superfamily, is involved in the binding of human fibronectin (Fn). Some members of the OprD subfamily have been reported to be important in the uptake of nutrients from the environment. Comparison of wild-type and mutant strains of P. aeruginosa revealed that inactivation of the oprQ gene does not reduce the growth rate. Although it does not appear to be involved in nutrient uptake, an increased doubling time was reproducibly observed with the loss of OprQ in P. aeruginosa. Utilizing an oprQ-xylE transcriptional fusion, we determined that the PA2760 gene, encoding OprQ, was upregulated under conditions of decreased iron and magnesium. This upregulation appears to occur in early exponential phase. Insertional inactivation of PA2760 in the P. aeruginosa wild-type background did not produce a significant increase in resistance to any antibiotic tested, a phenotype that is typical of OprD family members. Interestingly, the in trans expression of OprQ in the DeltaoprQ PAO1 mutant resulted in increased sensitivity to certain antibiotics. These findings suggest that OprQ is under dual regulation with other P. aeruginosa genes. Intact P. aeruginosa cells are capable of binding human Fn. We found that loss of OprQ resulted in a reduction of binding to plasmatic Fn in vitro. Finally, we present a discussion of the possible role of the P. aeruginosa outer membrane protein OprQ in adhesion to epithelial cells, thereby increasing colonization and subsequently enhancing lung destruction by P. aeruginosa.

  18. Effects of Gamma Irradiation on Bacterial Microflora Associated with Human Amniotic Membrane

    Directory of Open Access Journals (Sweden)

    Fahmida Binte Atique

    2013-01-01

    Full Text Available Human amniotic membrane is considered a promising allograft material for the treatment of ocular surface reconstruction, burns, and other skin defects. In order to avoid the transmission of any diseases, grafts should be perfectly sterile. Twenty-five amniotic sacs were collected to determine the microbiological quality of human amniotic membrane, to analyze the radiation sensitivity pattern of the microorganism, and to detect the radiation decimal reduction dose (D10 values. All the samples were found to be contaminated, and the bioburden was ranged from 3.4×102 to 1.2×105 cfu/g. Initially, a total fifty bacterial isolates were characterized according to their cultural, morphological, and biochemical characteristics and then tested for the radiation sensitivity in an incremental series of radiation doses from 1 to 10 KGy. The results depict gradual decline in bioburden with incline of radiation doses. Staphylococcus spp. were the most frequently isolated bacterial contaminant in tissue samples (44%. The D10 values of the bacterial isolates were ranged from 0.6 to 1.27 KGy. Streptococcus spp. were found to be the highest radioresistant strain with the radiation sterilization dose (RSD of 11.4 KGy for a bioburden level of 1000. To compare the differences, D10 values were also calculated by graphical evaluations of the data with two of the representative isolates of each bacterial species which showed no significant variations. Findings of this study indicate that lower radiation dose is quite satisfactory for the sterilization of amniotic membrane grafts. Therefore, these findings would be helpful to predict the efficacy of radiation doses for the processing of amniotic membrane for various purposes.

  19. Effects of gamma irradiation on bacterial microflora associated with human amniotic membrane.

    Science.gov (United States)

    Binte Atique, Fahmida; Ahmed, Kazi Tahsin; Asaduzzaman, S M; Hasan, Kazi Nadim

    2013-01-01

    Human amniotic membrane is considered a promising allograft material for the treatment of ocular surface reconstruction, burns, and other skin defects. In order to avoid the transmission of any diseases, grafts should be perfectly sterile. Twenty-five amniotic sacs were collected to determine the microbiological quality of human amniotic membrane, to analyze the radiation sensitivity pattern of the microorganism, and to detect the radiation decimal reduction dose (D₁₀) values. All the samples were found to be contaminated, and the bioburden was ranged from 3.4 × 10² to 1.2 × 10⁵ cfu/g. Initially, a total fifty bacterial isolates were characterized according to their cultural, morphological, and biochemical characteristics and then tested for the radiation sensitivity in an incremental series of radiation doses from 1 to 10 KGy. The results depict gradual decline in bioburden with incline of radiation doses. Staphylococcus spp. were the most frequently isolated bacterial contaminant in tissue samples (44%). The D₁₀ values of the bacterial isolates were ranged from 0.6 to 1.27 KGy. Streptococcus spp. were found to be the highest radioresistant strain with the radiation sterilization dose (RSD) of 11.4 KGy for a bioburden level of 1000. To compare the differences, D₁₀ values were also calculated by graphical evaluations of the data with two of the representative isolates of each bacterial species which showed no significant variations. Findings of this study indicate that lower radiation dose is quite satisfactory for the sterilization of amniotic membrane grafts. Therefore, these findings would be helpful to predict the efficacy of radiation doses for the processing of amniotic membrane for various purposes.

  20. Progesterone receptor membrane component 1 (PGRMC1) expression in fetal membranes among women with preterm premature rupture of the membranes (PPROM).

    Science.gov (United States)

    Feng, L; Antczak, B C; Lan, L; Grotegut, C A; Thompson, J L; Allen, T K; Murtha, A P

    2014-05-01

    PGRMC1 function is implicated in maintaining fetal membrane (FM) integrity. PGRMC1 was detectable primarily in the cytoplasm of FM cells and was actively regulated in FMs and relevant for PGRMC1-mediated progesterone action. By cell type, PGRMC1 expression was higher in amnion and chorion compared with decidua. By clinical phenotype, PGRMC1 expression was higher among preterm-no-labor and term-no-labor subjects compared to PPROM. PGRMC1 expression appears to be diminished in PPROM subjects. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Clinical application of amniotic membranes on a patient with epidermolysis bullosa.

    Science.gov (United States)

    Martínez Pardo, M E; Reyes Frías, M L; Ramos Durón, L E; Gutiérrez Salgado, E; Gómez, J C; Marín, M A; Luna Zaragoza, D

    1999-01-01

    The case of a patient with dystrophic epidermolysis bullosa treated with radiosterilised amniotic membranes is presented. The disorder is a congenital disease characterised by a poor desmosomal junction in the keratinocyte membrane. After proper donor screening, amnios were collected at Hospital Central Sur de Alta Especialidad (HCSAE), PEMEX and microbiological analysis was performed at Universidad Nacional Autónoma de México, FQUNAM, (Biology Dept. of the Chemistry Faculty, National Autonomous University of Mexico), before and after radiation sterilisation. Processing, packaging and sterilisation were performed at Instituto Nacional de Investigaciones Nucleares, ININ, (National Nuclear Research Institute). The patient, a ten-year-old boy with severe malnutrition, extensive loss of skin and pseudomonad infection in the whole body, was treated with gentle debridement in a Hubbard bath. Later amnion application was performed with sterilised amnios by using two different processes, in one of which the amnion was sterilised with paracetic acid, preserved in glycerol, kindly donated by the German Institute for Tissue and Cell Replacement and applied by Dr. Johannes C. Bruck, IAEA visiting expert, and the other amnion was processed at ININ: air dried and sterilised by gamma radiation at dose of 30 kGy. After spontaneous epithelisation was successfully promoted for seven days, the pain was alleviated and mobility was improved in a few hours and the patient's general condition was so improved that in a month he was discharged. Unfortunately, because this disease is revertive and has malignant degeneration, the prognosis is not good.

  2. Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Julie Bomholt

    Full Text Available In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tagged with yeast enhanced GFP for quantification of functional expression, determination of sub-cellular localization, estimation of in vivo folding efficiency and establishment of a purification protocol. Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction of the expression temperature to 15°C almost completely prevented Aquaporin-1 mal-folding. Bioimaging of live yeast cells revealed that recombinant Aquaporin-1 accumulated in the yeast plasma membrane. A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes.

  3. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    Energy Technology Data Exchange (ETDEWEB)

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P. (F. Hoffmann-La Roche Ltd., Basel (Switzerland))

    1991-02-15

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A){sup +} RNA.

  4. Histologic features of transplanted amniotic membrane: implications for corneal wound healing.

    Science.gov (United States)

    Said, Dalia G; Nubile, Mario; Alomar, Thaer; Hopkinson, Andy; Gray, Trevor; Lowe, James; Dua, Harminder S

    2009-07-01

    To evaluate the histologic changes occurring in the transplanted amniotic membrane in human eyes. Observational consecutive case series. Seven consecutive patients who underwent amniotic membrane transplantation (AMT) for bullous keratopathy and subsequently had a penetrating keratoplasty (PK). Corneal buttons obtained at PK were examined by light and electron microscopy and by immunohistology with antibodies against CD34 (keratocytes), alpha smooth muscle actin and vimentin (myofibroblasts and fibroblasts respectively). Time from AMT to PK ranged from 2 to 32 months. Immunophenotypic characteristics of cells populating transplanted amniotic stroma. Amniotic tissue was covered with stratified corneal epithelium with well-defined desmosomes and hemidesmosomes. Transformed corneal stroma-derived cells (CSDCs) could be seen migrating from the anterior stroma, through breaks in the Bowman's zone, into connective tissue of the amniotic membrane. Immunohistology showed that the cells populating amniotic stroma were CD34 negative but positive for vimentin and alpha smooth muscle actin. In 2 samples in which corneal transplants were performed approximately 1 year or more after AMT, some cells in the amniotic stroma showed CD34+ staining. Features of increased metabolic activity and formation of new collagen were seen on electron microscopy. In 2 cases, epithelial cell nests were seen in the amniotic stroma. The amniotic basement membrane facilitates epithelial cell migration and adhesion. The amniotic stroma supports CSDCs and epithelial cells. Repopulation of the amniotic stroma by CSDCs migrating through breaks in Bowman's zone integrates the amnion with corneal tissue and allows for rebuilding of corneal stroma. Over time, some CSDCs may revert to the resting keratocyte immunophenotype.

  5. Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane.

    Science.gov (United States)

    Berndt, M C; Gregory, C; Kabral, A; Zola, H; Fournier, D; Castaldi, P A

    1985-09-16

    Human platelet glycoprotein Ib (GP Ib) is a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VIII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that GP Ib is normally complexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GP Ib complex (GP Ib plus GP IX) was purified to homogeneity in approximately 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose. GP Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170 000 under nonreducing conditions and 135 000 (alpha subunit) and 25 000 (beta subunit) under reducing conditions. GP IX had an apparent Mr of 22 000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. GP Ib alpha had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ib beta and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ib alpha and GP IX were blocked; GP Ib beta had the N-terminal sequence, Ile-Pro-Ala-Pro-. On

  6. Basement Membrane-Rich Organoids with Functional Human Blood Vessels Are Permissive Niches for Human Breast Cancer Metastasis

    Science.gov (United States)

    Fernández-Periáñez, Rodrigo; Molina-Privado, Irene; Rojo, Federico; Guijarro-Muñoz, Irene; Alonso-Camino, Vanesa; Zazo, Sandra; Compte, Marta; Álvarez-Cienfuegos, Ana; Cuesta, Ángel M.; Sánchez-Martín, David; Álvarez-Méndez, Ana M.; Sanz, Laura; Álvarez-Vallina, Luis

    2013-01-01

    Metastasic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention. PMID

  7. Basement membrane-rich organoids with functional human blood vessels are permissive niches for human breast cancer metastasis.

    Directory of Open Access Journals (Sweden)

    Rodrigo Fernández-Periáñez

    Full Text Available Metastatic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic

  8. Investigation of interaction of human platelet membrane components with anticoagulant drugs Abciximab and Eptifibatide.

    Directory of Open Access Journals (Sweden)

    Anna Sankiewicz

    2011-04-01

    Full Text Available Abciximab (Abci and eptifibatide (Epti are antiaggregate drugs which may reduce thrombotic complications in acute coronary syndromes. The aim of this work was the investigation of the interaction between the phospholipid-GPIIb/IIIa glycoprotein complex and Abci or Epti, and the influence of these drugs on the phospholipid ratio in the platelet membrane. The interaction between the phospholipid-GPIIb/IIIa glycoprotein complex and antiaggregate drugs were investigated using the Surface Plasmon Resonance Imaging technique (SPRI. Phospholipids phosphatidylinositol (PI, phosphatidylserine (PS, phosphatidylethanolamine (PE, phosphatidylcholine (PC and sphingomyelin (SM were first immobilized onto the gold chip surface. The phospholipid ratio in the platelet membrane was determined by the HPLC. Only PI, PS, PE and PC were determined. Human platelets treated 'in vitro' with Abci or Epti exhibit changes in the phospholipid ratio in the platelet membrane. The ratio of PS decreases and PC rises. The SPRI distinctly shows interactions between phospholipids and glycoprotein GPIIb/IIIa, and between the phospholipid-glycoprotein GPIIb/IIIa complex and Abci or Epti. The interaction between phospholipids and glycoprotein GPIIb/IIIa is growing in the sequence: PI

  9. Human red blood cell membrane stability testing for the estimation of anti-inflammatory activity of methanolic extract of millettia pachycarpa benth leaves

    National Research Council Canada - National Science Library

    Chowdhury, Amin; Mamun, Abdullah Al; Rahman, Shadman; Azam, Shofiul; Shams, Kishower; Jainul, Abdullah

    2013-01-01

    .... This investigation is made following the most simple, reliable and less time consuming method. As the human red blood corpuscular membrane is similar to lysosomal membranes that influence inflammatory process...

  10. In an in-vitro model using human fetal membranes, 17-α hydroxyprogesterone caproate is not an optimal progestogen for inhibition of fetal membrane weakening.

    Science.gov (United States)

    Kumar, Deepak; Moore, Robert M; Mercer, Brian M; Mansour, Joseph M; Mesiano, Sam; Schatz, Frederick; Lockwood, Charles J; Moore, John J

    2017-12-01

    The progestogen 17-α hydroxyprogesterone caproate (17-OHPC) is 1 of only 2 agents recommended for clinical use in the prevention of spontaneous preterm delivery, and studies of its efficacy have been conflicting. We have developed an in-vitro model to study the fetal membrane weakening process that leads to rupture in preterm premature rupture of the fetal membranes (pPROM). Inflammation/infection associated with tumor necrosis factor-α (TNF-α) induction and decidual bleeding/abruption associated thrombin release are leading causes of preterm premature rupture of the fetal membranes. Both agents (TNF-α and thrombin) cause fetal membrane weakening in the model system. Furthermore, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical intermediate for both TNF-α and thrombin-induced fetal membrane weakening. In a previous report, we demonstrated that 3 progestogens, progesterone, 17-alpha hydroxyprogesterone (17-OHP), and medroxyprogesterone acetate (MPA), each inhibit both TNF-α- and thrombin-induced fetal membrane weakening at 2 distinct points of the fetal membrane weakening pathway. Each block both the production of and the downstream action of the critical intermediate granulocyte-macrophage colony-stimulating factor. The objective of the study was to characterize the inhibitory effects of 17-OHPC on TNF-α- and thrombin-induced fetal membrane weakening in vitro. Full-thickness human fetal membrane fragments from uncomplicated term repeat cesarean deliveries were mounted in 2.5 cm Transwell inserts and cultured with/without 17-alpha hydroxyprogesterone caproate (10-9 to 10-7 M). After 24 hours, medium (supernatant) was removed and replaced with/without the addition of tumor necrosis factor-alpha (20 ng/mL) or thrombin (10 U/mL) or granulocyte-macrophage colony-stimulating factor (200 ng/mL). After 48 hours of culture, medium from the maternal side compartment of the model was assayed for granulocyte-macrophage colony-stimulating factor

  11. A porous membrane-mediated isolation of mesenchymal stem cells from human embryonic stem cells.

    Science.gov (United States)

    Hong, Ki-Sung; Bae, Daekyeong; Choi, Youngsok; Kang, Sun-Woong; Moon, Sung-Hwan; Lee, Hoon Taek; Chung, Hyung-Min

    2015-03-01

    Pluripotent human embryonic stem cells (hESCs) acquire mesenchymal characteristics during the epithelial-mesenchymal transition (EMT) process. Here, we report a simple and an efficient isolation method for mesenchymal stem cells (MSCs) from hESCs undergoing EMT using a commercialized porous membrane transwell culture insert. Suspension culture of hESC colonies results in the formation of embryoid bodies, which adhered on the upper compartment of 8 μm porous membrane in the presence of EMG2-MV media. The population migrating through the permeable membrane to the lower compartment not only exhibited EMT markers but also expressed high levels of a panel of typical MSC surface antigen markers, and demonstrated multipotent differentiation capability. In addition, they have a prolonged proliferation capacity without characteristics and chromosomal changes. Furthermore, the isolated MSCs significantly enhanced cardiac functions in a rat model of myocardial infarction (MI) as measured by the left ventricle wall thickness (MI control, 32.9%±3.2% vs. hESCs-MSCs, 38.7%±2.4%), scar length (MI control, 46.1%±2.5% vs. hESCs-MSCs, 41.8%±1.3%), fibrosis area (MI control, 34.3%±1.6% vs. hESCs-MSCs, 28.9%±3.5%), and capillary density. Our findings demonstrate an ease with which hESCs-MSCs can be effectively isolated using the porous membrane, which overcomes the lack of availability of MSCs for therapeutic applications in various diseased animal models.

  12. Dapsone hydroxylamine induces premature removal of human erythrocytes by membrane reorganization and antibody binding

    Science.gov (United States)

    Bordin, Luciana; Fiore, Cristina; Zen, Francesco; Coleman, Michael D; Ragazzi, Eugenio; Clari, Giulio

    2010-01-01

    BACKGROUND AND PURPOSE N-hydroxylation of dapsone leads to the formation of the toxic hydroxylamines responsible for the clinical methaemoglobinaemia associated with dapsone therapy. Dapsone has been associated with decreased lifespan of erythrocytes, with consequences such as anaemia and morbidity in patients treated with dapsone for malaria. Here, we investigated how dapsone and/or its hydroxylamine derivative (DDS-NHOH) induced erythrocyte membrane alterations that could lead to premature cell removal. EXPERIMENTAL APPROACH Erythrocytes from healthy donors were subjected to incubation with dapsone and DDS-NHOH for varying times and the band 3 protein tyrosine-phosphorylation process, band 3 aggregation, membrane alteration and IgG binding were all examined and compared with erythrocytes from two patients receiving dapsone therapy. KEY RESULTS The hydroxylamine derivative, but not dapsone (the parent sulphone) altered membrane protein interactions, leading both to aggregation of band 3 protein and to circulating autologous antibody binding, shown in erythrocytes from patients receiving dapsone therapy. The band 3 tyrosine-phosphorylation process can be used as a diagnostic system to monitor membrane alterations both in vitro, assessing concentration and time-dependent effects of DDS-NHOH treatment, and in vivo, evaluating erythrocytes from dapsone-treated patients, in resting or oxidatively stimulated conditions. CONCLUSIONS AND IMPLICATIONS DDS-NHOH-induced alterations of human erythrocytes can be directly monitored in vitro by tyrosine-phosphorylation level and formation of band 3 protein aggregates. The latter, together with antibody-mediated labelling of erythrocytes, also observed after clinical use of dapsone, may lead to shortening of erythrocyte lifespan. PMID:20662842

  13. RESEARCH ON REDUCING PREMATURITY RUPTURE OF MEMBRANE

    Directory of Open Access Journals (Sweden)

    Maria URSACHI (BOLOTA

    2016-12-01

    Full Text Available The membranes surrounding the amniotic cavity are composed from amnion and chorion, tightly adherent layers which are composed of several cell types, including epithelial cells, trophoblasts cells and mesenchyme cells, embedded in a collagenous matrix. They retain amniotic fluid, secret substances into the amniotic fluid, as well as to the uterus and protect the fetus against upward infections from urogenital tract. Normally, the membranes it breaks during labor. Premature rupture of the amniotic sac (PRAS is defined as rupture of membranes before the onset of labor. Premature rupture of the fetal membrane, which occurs before 37 weeks of gestation, usually, refers to preterm premature rupture of membranes. Despite advances in the care period, premature rupture of membranes and premature rupture of membranes preterm continue to be regarded as serious obstetric complications. On the term 8% - 10% of pregnant women have premature rupture of membranes; these women are at increased risk of intrauterine infections, where the interval between membrane rupture and expulsion is rolled-over. Premature rupture of membranes preterm occurs in approximately 1% of all pregnancies and is associated with 30% -40% of preterm births. Thus, it is important to identify the cause of pre-term birth (after less than 37 completed weeks of "gestation" and its complications, including respiratory distress syndrome, neonatal infection and intraventricular hemorrhage. Objectives: the development of the protocol of the clinical trial on patients with impending preterm birth, study clinical and statistical on the socio-demographic characteristics of patients with imminent preterm birth; clinical condition of patients and selection of cases that could benefit from the application of interventional therapy; preclinical investigation (biological and imaging of patients with imminent preterm birth; the modality therapy; clinical investigation of the effectiveness of short

  14. Requirement of sperm-oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes.

    Science.gov (United States)

    Sengoku, K; Tamate, K; Takaoka, Y; Horikawa, M; Goishi, K; Okada, R; Tsuchiya, K; Ishikawa, M

    1999-02-01

    We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida-free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5+/-0.6 and 4.3+/-0.6 for ICSI; 3.0+/-0.3 and 3.8+/-0.4 for activated and 0.2+/-0.1 and 0.6+/-0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore-puromycin-activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes.

  15. Membrane permeability of the human granulocyte to water, dimethyl sulfoxide, glycerol, propylene glycol and ethylene glycol.

    Science.gov (United States)

    Vian, Alex M; Higgins, Adam Z

    2014-02-01

    Granulocytes are currently transfused as soon as possible after collection because they rapidly deteriorate after being removed from the body. This short shelf life complicates the logistics of granulocyte collection, banking, and safety testing. Cryopreservation has the potential to significantly increase shelf life; however, cryopreservation of granulocytes has proven to be difficult. In this study, we investigate the membrane permeability properties of human granulocytes, with the ultimate goal of using membrane transport modeling to facilitate development of improved cryopreservation methods. We first measured the equilibrium volume of human granulocytes in a range of hypo- and hypertonic solutions and fit the resulting data using a Boyle-van't Hoff model. This yielded an isotonic cell volume of 378 μm(3) and an osmotically inactive volume of 165 μm(3). To determine the permeability of the granulocyte membrane to water and cryoprotectant (CPA), cells were injected into well-mixed CPA solution while collecting volume measurements using a Coulter Counter. These experiments were performed at temperatures ranging from 4 to 37°C for exposure to dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol. The best-fit water permeability was similar in the presence of all of the CPAs, with an average value at 21°C of 0.18 μmatm(-1)min(-1). The activation energy for water transport ranged from 41 to 61 kJ/mol. The CPA permeability at 21°C was 6.4, 1.0, 8.4, and 4.0 μm/min for dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol, respectively, and the activation energy for CPA transport ranged between 59 and 68 kJ/mol. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. alpha isoforms of soluble and membrane-linked folate-binding protein in human blood

    DEFF Research Database (Denmark)

    Hoier-Madsen, M.; Holm, J.; Hansen, S.I.

    2008-01-01

    supported the hypothesis that serum FBP (29 kDa) mainly originates from neutrophils. The presence of FBP/FR alpha isoforms were established for the first time in human blood using antibodies specifically directed against human milk FBP alpha. The alpha isoforms identified on erythrocyte membranes......, and in granulocytes and serum, only constituted an almost undetectable fraction of the functional FBP The FBP alpha in neutrophil granulocytes was identified as a cytoplasmic component by indirect immunofluorescence. Gel filtration of serum revealed a peak of FBP alpha (>120 kDa), which could represent receptor...... fragments from decomposed erythrocytes and granulocytes. The soluble FBPs may exert bacteriostatic effects and protect folates in plasma from biological degradation, whereas FRs on the surface of blood cells could be involved in intracellular folate uptake or serve as signal proteins. The latter receptors...

  17. Characterization of acoustically induced deformations of human tympanic membranes by digital holography and shearography

    Science.gov (United States)

    Flores-Moreno, J. M.; Furlong, Cosme; Cheng, Jeffrey T.; Rosowski, John J.; Merchant, S. N.

    2011-08-01

    Recently, we introduced a Digital Optoelectronic Holographic System (DOEHS) for measurement of acoustically induced deformations of the human tympanic membrane (TM) in order to study and diagnose pathologic conditions of the middle-ear. The DOEHS consists of laser-delivery illumination (IS), optical head (OH), image-processing computer (IP), and positioning arm (PS) subsystems. Holographic information is recorded by a CCD and numerically reconstructed by Fresnel approximation. Our holographic otoscope system is currently deployed in a clinic and is packaged in a custom design. Since digital holography is a high sensitivity measurement technique and the interfering light waves travel along different paths, it makes measurements acquired by DOEHS susceptible to external vibrations. In order to avoid this susceptibility, we are testing a shearography setup as OH. Shearography presents same advantages as holographic interferometry, but it is less susceptible to vibration and external noise, which is a characteristic needed for the use of our techniques in a clinical environment. In this paper we present work in progress in our development of a shearography technique based on a Mach-Zehnder configuration as OH and demonstrate its application by quantifying vibrations modes in thin membranes, including human TM. Results are compared with those obtained with DOEHS.

  18. Immunocytochemical localization of the translocase of the outer mitochondrial membrane (Tom20) in the human cochlea.

    Science.gov (United States)

    Balaker, Ashley E; Ishiyama, Paul; Lopez, Ivan A; Ishiyama, Gail; Ishiyama, Akira

    2013-02-01

    Mitochondrial degeneration in the inner ear is likely a contributing factor in age-related hearing loss and other otopathologies such as Meniere's disease. Most mitochondrial proteins are synthesized in the cytosol and imported through the mitochondrial membranes by translocators. The translocase of the outer membrane (Tom) is the universal entry gate for all proteins that are imported into mitochondria. Altered function of the translocator could alter protein transport into the mitochondria, and disrupt function. In this study, we determined the immunolocalization of Tom20, a major mitochondrial protein import receptor, in microdissected human cochlea frozen sections obtained from postmortem autopsy and celloidin-embedded archival specimens. We used affinity purified rabbit polyclonal antibodies against Tom20. We also determined the Tom20 immunolocalization in the mouse inner ear. In the human and mouse cochlea, Tom20 was ubiquitously distributed in the organ of Corti, allowing well-delineated visualization of inner and outer hair cells. Tom20 immunoreactivity localized in the cytoplasm of spiral ganglia neurons. In the inner ear of aged subjects with Meniere's disease, there was decreased expression of Tom20. These results suggest that Tom20 can be used in the inner ear as a marker for mitochondrial protein import. Copyright © 2012 Wiley Periodicals, Inc.

  19. Management of temporomandibular joint degenerative disorders with human amniotic membrane: Hypothesis of action.

    Science.gov (United States)

    Guarda-Nardini, Luca; Trojan, Diletta; Paolin, Adolfo; Manfredini, Daniele

    2017-07-01

    Approaches providing the positioning of human amniotic membrane (HAM) within the intra-articular space of arthritic TMJs have never been investigated. This contrasts with the increasing amount of evidence suggesting the potential positive effects of HAM on a number of surgical conditions, even included the interpositional arthroplasty for TMJ ankylosis. Thus, the possible usefulness of HAM to restore joint functions in severely damaged TMJs could be hypothesized. Based on these premises, the clinical research question "Is human amniotic membrane positioning effective to reduce symptoms and restore jaw function in patients with severe inflammatory-degenerative disorders of the temporomandibular joint?" has been addressed by performing a systematic review of the literature. Out of potential 11988 and 8883 citations in the PubMed and Scopus databases, respectively, only five were of possible interest for inclusion in the review, but none of them addressed specifically the clinical research question. Thus, the hypothetical background for usefulness was discussed. The benefits of HAM positioning in TMJs with severe inflammatory-degenerative disorders could be related with its anti-inflammatory and anti-microbial and analgesic properties as well as its low immunogenicity. Studies in which HAM is positioned within the joint space of patients with severe TMJ degeneration, either as a disc-replacing film during major surgeries for discectomy and arthroplasty or as an injectable solution that can be needle-inserted after an arthrocentesis procedure, should be designed to test the hypothesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Morphological analysis of amnion stored in glycerol sterilized with different doses of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Fernando Augusto N.; Santin, Stefany P.; Martino Junior, Antonio C.; Machado, Luci Diva B.; Mathor, Monica B., E-mail: fernandonevessoares@yahoo.com.br [Instituto de Pesquisas Energeticas e Nucleares (CTR/IPEN/CNEN-SP), Sao Paulo, RJ (Brazil). Centro de Tecnologia das Radiacoes

    2013-07-01

    The amniotic membrane (AM) is the innermost layer of the fetal membranes (placenta), widely used in transplantation being a tissue that combines anti-inflammatory, antimicrobial and antifibrotic, and limited immunogenicity. The tissue can be used a bandage biological for treatment of burns and skin wounds, chronic ulcers, reconstructions from different body areas, including ophthalmic repairs. In the last decades the amniotic membrane has been used widely also as a carrier substrate to transfer tissues cultured 'in vitro'. The use of fresh membrane has some limitations, the main ones are being necessary your quick use and the inability to obtain full security for certain infections. Other types of preservation require a processing thereof. The radiosterilization is an alternative for ensuring quality and safety of tissues used in transplants, and other clinical applications in order to minimize the risk of contamination of the receptor tissue. Therefore, the objective of this study was to test various doses of radiation using two sources of ionizing radiation: the cobalt-60 irradiator Gamma and Electron Beam Accelerator (E.B.). A tissue analysis was done by visual and tactile qualitative analysis, semi-quantitative (solid colorimetry) and light microscopy to observe morphological and physic-chemical changes after the irradiation of AM preserved in glycerol, comparing the results obtained with the sample not irradiated. It was noted that at higher doses, for both radiation sources, irradiated membranes suffered greater color change, becoming yellowish and thereby reducing their initial malleability. (author)

  1. Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.

    Directory of Open Access Journals (Sweden)

    Anusha Panjwani

    2014-08-01

    Full Text Available Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

  2. Clinical analysis of amniotic membrane patches and grafts for acute ocular surface burn

    Directory of Open Access Journals (Sweden)

    Lin Li

    2015-01-01

    Full Text Available AIM: To investigate the effect and value of amniotic membrane patches and grafts for acute ocular surface burn at different degrees.METHODS: A retrospective analysis of 28 cases(28 eyesaffected by ocular chemical or thermal burn with different degree were included in our hospital from March 2007 to March 2012. Amniotic membrane patched was undergone in 13 eyes with fresh amnion that the patients corneal burns degree Ⅱ or Ⅲ with partial limbal buns at degree Ⅳ. Amniotic membrane grafts was performed in 15 eyes with fresh amnion that the patients all corneal burns at degree Ⅲ with the whole limbal necrosis without severe eyelid defect. The follow-up time ranged 6~24mo. The postoperative visual acuity, the condition of amniotic membrane transplant, renovation of cornea and complications were observed. RESULTS: Postoperative corrected visual acuity was improved in 20 eyes(71%, it was not changed in 5 eyes(18%, the visual acuity declined in 3 eyes(11%. The amniotic membrane survived in 23 eyes and the survival rate was up to 82%. The cornea of 4 eyes recovered to transparent, nebula emceed in 8 eyes eventually, corneal macula emerged in 10 eyes, 4 eyes ended up with leukoma, 2 eyes developed corneal melting after therapy, then received lamellar keratoplasty. Corneal surface become epithelization after amnion patches or grafts, but any of them have recurrent epithelial erosion, and become stable epithalization after repeat operation.CONCLUSION: Amniotic membrane patches and grafts is an effective method to deal with acute ocular surface burn.

  3. Interaction of a peptide derived from C-terminus of human TRPA1 channel with model membranes mimicking the inner leaflet of the plasma membrane.

    Science.gov (United States)

    Witschas, Katja; Jobin, Marie-Lise; Korkut, Dursun Nizam; Vladan, Maria Magdalena; Salgado, Gilmar; Lecomte, Sophie; Vlachova, Viktorie; Alves, Isabel D

    2015-05-01

    The transient receptor potential ankyrin 1 channel (TRPA1) belongs to the TRP cation channel superfamily that responds to a panoply of stimuli such as changes in temperature, calcium levels, reactive oxygen and nitrogen species and lipid mediators among others. The TRP superfamily has been implicated in diverse pathological states including neurodegenerative disorders, kidney diseases, inflammation, pain and cancer. The intracellular C-terminus is an important regulator of TRP channel activity. Studies with this and other TRP superfamily members have shown that the C-terminus association with lipid bilayer alters channel sensitivity and activation, especially interactions occurring through basic residues. Nevertheless, it is not yet clear how this process takes place and which regions in the C-terminus would be responsible for such membrane recognition. With that in mind, herein the first putative membrane interacting region of the C-terminus of human TRPA1, (corresponding to a 29 residue peptide, IAEVQKHASLKRIAMQVELHTSLEKKLPL) named H1 due to its potential helical character was chosen for studies of membrane interaction. The affinity of H1 to lipid membranes, H1 structural changes occurring upon this interaction as well as effects of this interaction in lipid organization and integrity were investigated using a biophysical approach. Lipid models systems composed of zwitterionic and anionic lipids, namely those present in the lipid membrane inner leaflet, where H1 is prone to interact, where used. The study reveals a strong interaction and affinity of H1 as well as peptide structuration especially with membranes containing anionic lipids. Moreover, the interactions and peptide structure adoption are headgroup specific. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Antimicrobial and Antibiofilm Effects of Human Amniotic/Chorionic Membrane Extract on Streptococcus pneumoniae

    Directory of Open Access Journals (Sweden)

    Mukesh K. Yadav

    2017-10-01

    Full Text Available Background:Streptococcus pneumoniae colonize the human nasopharynx in the form of biofilms. The biofilms act as bacterial reservoirs and planktonic bacteria from these biofilms can migrate to other sterile anatomical sites to cause pneumonia, otitis media (OM, bacteremia and meningitis. Human amniotic membrane contains numerous growth factors and antimicrobial activity; however, these have not been studied in detail. In this study, we prepared amniotic membrane extract and chorionic membrane extract (AME/CME and evaluated their antibacterial and antibiofilm activities against S. pneumoniae using an in vitro biofilm model and in vivo OM rat model.Materials and Methods: The AME/CME were prepared and protein was quantified using DCTM (detergent compatible method. The minimum inhibitory concentrations were determined using broth dilution method, and the synergistic effect of AME/CME with Penicillin-streptomycin was detected checkerboard. The in vitro biofilm and in vivo colonization of S. pneumoniae were studied using microtiter plate assay and OM rat model, respectively. The AME/CME-treated biofilms were examined using scanning electron microscope and confocal microscopy. To examine the constituents of AME/CME, we determined the proteins and peptides of AME/CME using tandem mass tag-based quantitative mass spectrometry.Results: AME/CME treatment significantly (p < 0.05 inhibited S. pneumoniae growth in planktonic form and in biofilms. Combined application of AME/CME and Penicillin-streptomycin solution had a synergistic effect against S. pneumoniae. Biofilms grown with AME/CME were thin, scattered, and unorganized. AME/CME effectively eradicated pre-established pneumococci biofilms and has a bactericidal effect. AME treatment significantly (p < 0.05 reduced bacterial colonization in the rat middle ear. The proteomics analysis revealed that the AME/CME contains hydrolase, ribonuclease, protease, and other antimicrobial proteins and peptides

  5. Hemocompatible control of sulfobetaine-grafted polypropylene fibrous membranes in human whole blood via plasma-induced surface zwitterionization.

    Science.gov (United States)

    Chen, Sheng-Han; Chang, Yung; Lee, Kueir-Rarn; Wei, Ta-Chin; Higuchi, Akon; Ho, Feng-Ming; Tsou, Chia-Chun; Ho, Hsin-Tsung; Lai, Juin-Yih

    2012-12-21

    In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood.

  6. Controlled release of human growth hormone fused with a human hybrid Fc fragment through a nanoporous polymer membrane

    Science.gov (United States)

    Kim, Eung-Sam; Jang, Do Soo; Yang, Seung Yun; Lee, Mi Nam; Jin, Kyeong Sik; Cha, Hyung Jin; Kim, Jin Kon; Sung, Young Chul; Choi, Kwan Yong

    2013-05-01

    Nanotechnology has been applied to the development of more effective and compatible drug delivery systems for therapeutic proteins. Human growth hormone (hGH) was fused with a hybrid Fc fragment containing partial Fc domains of human IgD and IgG4 to produce a long-acting fusion protein. The fusion protein, hGH-hyFc, resulted in the increase of the hydrodynamic diameter (ca. 11 nm) compared with the diameter (ca. 5 nm) of the recombinant hGH. A diblock copolymer membrane with nanopores (average diameter of 14.3 nm) exhibited a constant release rate of hGH-hyFc. The hGH-hyFc protein released in a controlled manner for one month was found to trigger the phosphorylation of Janus kinase 2 (JAK2) in human B lymphocyte and to exhibit an almost identical circular dichroism spectrum to that of the original hGH-hyFc, suggesting that the released fusion protein should maintain the functional and structural integrity of hGH. Thus, the nanoporous release device could be a potential delivery system for the long-term controlled release of therapeutic proteins fused with the hybrid Fc fragment.Nanotechnology has been applied to the development of more effective and compatible drug delivery systems for therapeutic proteins. Human growth hormone (hGH) was fused with a hybrid Fc fragment containing partial Fc domains of human IgD and IgG4 to produce a long-acting fusion protein. The fusion protein, hGH-hyFc, resulted in the increase of the hydrodynamic diameter (ca. 11 nm) compared with the diameter (ca. 5 nm) of the recombinant hGH. A diblock copolymer membrane with nanopores (average diameter of 14.3 nm) exhibited a constant release rate of hGH-hyFc. The hGH-hyFc protein released in a controlled manner for one month was found to trigger the phosphorylation of Janus kinase 2 (JAK2) in human B lymphocyte and to exhibit an almost identical circular dichroism spectrum to that of the original hGH-hyFc, suggesting that the released fusion protein should maintain the functional and

  7. Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma.

    Science.gov (United States)

    Ask, Kristine Skoglund; Bardakci, Turgay; Parmer, Marthe Petrine; Halvorsen, Trine Grønhaug; Øiestad, Elisabeth Leere; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

    2016-09-10

    Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation of phosphatidylcholines (PC), sphingomyelins (SM), and lysophosphatidylcholines (Lyso-PC). In both generic methods, no phospholipids were detected in the acceptor solutions. Thus, PALME appeared to be highly efficient for phospholipid removal. To further support this, qualitative (post-column infusion) and quantitative matrix effects were investigated with fluoxetine, fluvoxamine, and quetiapine as model analytes. No signs of matrix effects were observed. Finally, PALME was evaluated for the aforementioned drug substances, and data were in accordance with European Medicines Agency (EMA) guidelines. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Investigation of interaction of human platelet membrane components with anticoagulant drugs Abciximab and Eptifibatide

    Directory of Open Access Journals (Sweden)

    Ewa Gorodkiewicz

    2010-04-01

    Full Text Available Abciximab (Abci and eptifibatide (Epti are antiaggregate drugs which may reduce thrombotic complications inacute coronary syndromes. The aim of this work was the investigation of the interaction between the phospholipid-GPIIb/IIIa glycoprotein complex and Abci or Epti, and the influence of these drugs on the phospholipid ratio in the plateletmembrane. The interaction between the phospholipid-GPIIb/IIIa glycoprotein complex and antiaggregate drugs were investigatedusing the Surface Plasmon Resonance Imaging technique (SPRI. Phospholipids phosphatidylinositol (PI, phosphatidylserine(PS, phosphatidylethanolamine (PE, phosphatidylcholine (PC and sphingomyelin (SM were first immobilizedonto the gold chip surface. The phospholipid ratio in the platelet membrane was determined by the HPLC. Only PI,PS, PE and PC were determined. Human platelets treated 'in vitro' with Abci or Epti exhibit changes in the phospholipidratio in the platelet membrane. The ratio of PS decreases and PC rises. The SPRI distinctly shows interactions between phospholipidsand glycoprotein GPIIb/IIIa, and between the phospholipid-glycoprotein GPIIb/IIIa complex and Abci or Epti.The interaction between phospholipids and glycoprotein GPIIb/IIIa is growing in the sequence: PI<

  9. Investigation of glycosylation processes in mitochondria and microsomal membranes from human skeletal muscle.

    Science.gov (United States)

    Gasnier, F; Lerme, F; Rousson, R; Roussouly, P; Vaganay, E; Louisot, P; Gateau-Roesch, O

    1991-05-31

    Glycoconjugates are directly involved in major skeletal muscle functions. As little is known about glycosylation processes in muscle, we investigated glycoconjugate synthesis in subcellular fractions from human skeletal muscle tissue. Mitochondria and microsomal membranes were prepared from muscle biopsies by thorough mechanical disruption and differential centrifugations. This procedure resulted in the isolation of intact mitochondria (1 mg protein/g muscle) and of a microsomal fraction (1.5 mg protein/g muscle). Glycosyltransferases were studied in both subcellular fractions using either dolichylmonophosphate as a polyprenic acceptor or chemically modified fetuin as a glycoprotein substrate. Our results provide evidence for high rates of glycosylation in muscle. The highest activities were obtained with GDP-mannose: dilichylmonophosphate mannosyltransferase, a key enzyme in glycosylation process (220 pmol/mg per h in mitochondria and 1,550 pmol/mg per h in microsomal membranes). Substantial individual variations were observed for dolichol pathway glycosyltransferases but low individual variations were found for glycosyltransferases involved in maturation of glycoproteins. The role which glycosylation defects may play in muscle dysfunction has yet to be defined.

  10. Production, crystallization and neutron diffraction of fully deuterated human myelin peripheral membrane protein P2.

    Science.gov (United States)

    Laulumaa, Saara; Blakeley, Matthew P; Raasakka, Arne; Moulin, Martine; Härtlein, Michael; Kursula, Petri

    2015-11-01

    The molecular details of the formation of the myelin sheath, a multilayered membrane in the nervous system, are to a large extent unknown. P2 is a peripheral membrane protein from peripheral nervous system myelin, which is believed to play a role in this process. X-ray crystallographic studies and complementary experiments have provided information on the structure-function relationships in P2. In this study, a fully deuterated sample of human P2 was produced. Crystals that were large enough for neutron diffraction were grown by a ten-month procedure of feeding, and neutron diffraction data were collected to a resolution of 2.4 Å from a crystal of 0.09 mm(3) in volume. The neutron crystal structure will allow the positions of H atoms in P2 and its fatty-acid ligand to be visualized, as well as shedding light on the fine details of the hydrogen-bonding networks within the P2 ligand-binding cavity.

  11. Redox-active nanoceria depolarize mitochondrial membrane of human colon cancer cells

    Science.gov (United States)

    Jana, Saikat Kumar; Banerjee, Priyanka; Das, Soumen; Seal, Sudipta; Chaudhury, Koel

    2014-06-01

    Nanotherapeutics is emerging as a promising option to the various limitations and side effects associated with conventional chemotherapy. The present study investigates the cytotoxic effect of redox-active cerium oxide nanoparticles (nanoceria) on human colorectal adenocarcinoma-derived cell line (HCT 15). Exposure of these cells to nanoceria for 24 h with concentration ranging between 10 and 100 μM resulted in a significant reduction of cell viability in a dose-dependent manner. Further, at a concentration of 10 µM, nanoceria exhibited time-dependent cytotoxic effect when exposed to the cells for 24, 48, and 72 h. Upon treatment of the cells with nanoceria, reactive oxygen species (ROS) and lipid peroxidation which are indicators of oxidative stress and cytotoxicity increased significantly, in a dose-dependent manner. Nanoceria was also found to depolarize the mitochondrial membrane, thereby collapsing the membrane potential and leading to initiation of apoptosis. Scanning electron microscopic study of nanoceria-treated HCT 15 cells showed morphological changes and loss of filopodia and lamellipodia, indicating arrest of metastatic spread. Summarizing, when cultured HCT 15 cells are exposed to nanoceria, a dose-dependent cytotoxic effect mediated by ROS generation is observed.

  12. CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes

    DEFF Research Database (Denmark)

    Nielsen, C H; Marquart, H V; Prodinger, W M

    2001-01-01

    Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane-bound C3 convertase. We have investigated whether this might lead to the generation of a C5...... convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4.4 mM MgCl2/20 mM EGTA, which abrogates the classical pathway of complement without affecting...

  13. Endoscopic observation of different repair patterns in human traumatic tympanic membrane perforations.

    Science.gov (United States)

    Huang, Peng; Zhang, Shujun; Gong, Xinhong; Wang, Xuesong; Lou, Zi-Han

    2017-08-03

    In the last decade, there has been an increasing use of biomaterial patches in the regeneration of traumatic tympanic membrane perforations. The major advantages of biomaterial patches are to provisionally restore the physiological function of the middle ear, thereby immediately improving ear symptoms, and act as a scaffold for epithelium migration. However, whether there are additional biological effects on eardrum regeneration is unclear for biological material patching in the clinic. This study evaluated the healing response for different repair patterns in human traumatic tympanic membrane perforations by endoscopic observation. In total, 114 patients with traumatic tympanic membrane perforations were allocated sequentially to two groups: the spontaneous healing group (n=57) and Gelfoam patch-treated group (n=57). The closure rate, closure time, and rate of otorrhea were compared between the groups at 3 months. Ultimately, 107 patients were analyzed in the two groups (52 patients in the spontaneous healing group vs. 55 patients in the Gelfoam patch-treated group). The overall closure rate at the end of the 3 month follow-up period was 90.4% in the spontaneous healing group and 94.5% in the Gelfoam patch-treated group; the difference was not statistically significant (p>0.05). However, the total average closure time was significantly different between the two groups (26.8±9.1 days in the spontaneous healing group vs. 14.7±9.1 days in the Gelfoam patch-treated group, p<0.01). In addition, the closure rate was not significantly different between the spontaneous healing group and Gelfoam patch-treated group regardless of the perforation size. The closure time in the Gelfoam patch-treated group was significantly shorter than that in the spontaneous healing group regardless of the perforation size (small perforations: 7.1±1.6 days vs. 12.6±3.9, medium-sized perforations: 13.3±2.2 days vs. 21.8±4.2 days, and large perforations: 21.2±4.7 days vs. 38.4±5.7 days

  14. Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells

    DEFF Research Database (Denmark)

    Høgh, Mette; Tannetta, D; Sargent, I

    2006-01-01

    Objective Syncytiotrophoblast membrane fragments (STBM) exist in the peripheral circulation in pregnant women and it has been shown that the level of circulating STBM is significantly increased with pre-eclampsia compared with uncomplicated pregnancies. STBM could be one of the factors which...... directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. Design Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from...... results. Results Overall, the results do not show any great changes in gene expression in endothelial cells after STBM treatment (28 genes changed two-fold or more out of approximately 10 000 genes examined by microarray). In general, the changes observed are consistent with inhibition of proliferation...

  15. Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells

    DEFF Research Database (Denmark)

    Hoegh, A M; Tannetta, D; Sargent, I

    2006-01-01

    OBJECTIVE: Syncytiotrophoblast membrane fragments (STBM) exist in the peripheral circulation in pregnant women and it has been shown that the level of circulating STBM is significantly increased with pre-eclampsia compared with uncomplicated pregnancies. STBM could be one of the factors which...... directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. DESIGN: Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from...... results. RESULTS: Overall, the results do not show any great changes in gene expression in endothelial cells after STBM treatment (28 genes changed two-fold or more out of approximately 10,000 genes examined by microarray). In general, the changes observed are consistent with inhibition of proliferation...

  16. Online determination of biophysical parameters of mucous membranes of a human body

    Science.gov (United States)

    Lisenko, S. A.; Kugeiko, M. M.

    2013-07-01

    We have developed a method for online determination of biophysical parameters of mucous membranes (MMs) of a human body (transport scattering coefficient, scattering anisotropy factor, haemoglobin concentration, degrees of blood oxygenation, average diameter of capillaries with blood) from measurements of spectral and spatial characteristics of diffuse reflection. The method is based on regression relationships between linearly independent components of the measured light signals and the unknown parameters of MMs, obtained by simulation of the radiation transfer in the MM under conditions of its general variability. We have proposed and justified the calibration-free fibre-optic method for determining the concentration of haemoglobin in MMs by measuring the light signals diffusely reflected by the tissue in four spectral regions at two different distances from the illumination spot. We have selected the optimal wavelengths of optical probing for the implementation of the method.

  17. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    Science.gov (United States)

    Laulumaa, Saara; Kursula, Petri; Natali, Francesca

    2015-01-01

    Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  18. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    Directory of Open Access Journals (Sweden)

    Laulumaa Saara

    2015-01-01

    Full Text Available Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  19. The Effect of Covalently-Attached ATRP-Synthesized Polymers on Membrane Stability and Cytoprotection in Human Erythrocytes.

    Science.gov (United States)

    Clafshenkel, William P; Murata, Hironobu; Andersen, Jill; Creeger, Yehuda; Koepsel, Richard R; Russell, Alan J

    2016-01-01

    Erythrocytes have been described as advantageous drug delivery vehicles. In order to ensure an adequate circulation half-life, erythrocytes may benefit from protective enhancements that maintain membrane integrity and neutralize oxidative damage of membrane proteins that otherwise facilitate their premature clearance from circulation. Surface modification of erythrocytes using rationally designed polymers, synthesized via atom-transfer radical polymerization (ATRP), may further expand the field of membrane-engineered red blood cells. This study describes the fate of ATRP-synthesized polymers that were covalently attached to human erythrocytes as well as the effect of membrane engineering on cell stability under physiological and oxidative conditions in vitro. The biocompatible, membrane-reactive polymers were homogenously retained on the periphery of modified erythrocytes for at least 24 hours. Membrane engineering stabilized the erythrocyte membrane and effectively neutralized oxidative species, even in the absence of free-radical scavenger-containing polymers. The targeted functionalization of Band 3 protein by NHS-pDMAA-Cy3 polymers stabilized its monomeric form preventing aggregation in the presence of the crosslinking reagent, bis(sulfosuccinimidyl)suberate (BS3). A free radical scavenging polymer, NHS-pDMAA-TEMPO˙, provided additional protection of surface modified erythrocytes in an in vitro model of oxidative stress. Preserving or augmenting cytoprotective mechanisms that extend circulation half-life is an important consideration for the use of red blood cells for drug delivery in various pathologies, as they are likely to encounter areas of imbalanced oxidative stress as they circuit the vascular system.

  20. The Effect of Covalently-Attached ATRP-Synthesized Polymers on Membrane Stability and Cytoprotection in Human Erythrocytes

    Science.gov (United States)

    Clafshenkel, William P.; Murata, Hironobu; Andersen, Jill; Creeger, Yehuda; Russell, Alan J.

    2016-01-01

    Erythrocytes have been described as advantageous drug delivery vehicles. In order to ensure an adequate circulation half-life, erythrocytes may benefit from protective enhancements that maintain membrane integrity and neutralize oxidative damage of membrane proteins that otherwise facilitate their premature clearance from circulation. Surface modification of erythrocytes using rationally designed polymers, synthesized via atom-transfer radical polymerization (ATRP), may further expand the field of membrane-engineered red blood cells. This study describes the fate of ATRP-synthesized polymers that were covalently attached to human erythrocytes as well as the effect of membrane engineering on cell stability under physiological and oxidative conditions in vitro. The biocompatible, membrane-reactive polymers were homogenously retained on the periphery of modified erythrocytes for at least 24 hours. Membrane engineering stabilized the erythrocyte membrane and effectively neutralized oxidative species, even in the absence of free-radical scavenger-containing polymers. The targeted functionalization of Band 3 protein by NHS-pDMAA-Cy3 polymers stabilized its monomeric form preventing aggregation in the presence of the crosslinking reagent, bis(sulfosuccinimidyl)suberate (BS3). A free radical scavenging polymer, NHS-pDMAA-TEMPO˙, provided additional protection of surface modified erythrocytes in an in vitro model of oxidative stress. Preserving or augmenting cytoprotective mechanisms that extend circulation half-life is an important consideration for the use of red blood cells for drug delivery in various pathologies, as they are likely to encounter areas of imbalanced oxidative stress as they circuit the vascular system. PMID:27331401

  1. Fibronectin-synthesizing activity of free and membrane-bound polyribosomes from human embryonic fibroblasts and chick embryos

    Energy Technology Data Exchange (ETDEWEB)

    Belkin, V.M.; Volodarskaya, S.M.

    1986-06-20

    The fibronectin-synthesizing activity of membrane-bound and free polyribosomes in a cell-free system was studied using immunochemical methods. It was found that fibronectin biosynthesis on membrane-bound polyribosomes from human embryonic fibroblasts accounts for 4.9% and those from 10-day-old chick embryos for 1.1% of the total amount of newly synthesized proteins, whereas on free polyribosomes it is 1.0 and 0.3%, respectively. Fibronectin monomers with a molecular weight of 220,000 were found only in the material of the cell-free system containing heavy fractions of membrane-bound polyribosomes newly synthesized in the presence of spermidine. Thus, it was shown that fibronectin is synthesized primarily on membrane-bound polyribosomes.

  2. Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation

    DEFF Research Database (Denmark)

    Foster, Leonard J; Zeemann, Patricia A; Li, Chen

    2005-01-01

    useful for analyzing complex protein expression patterns and, when applied quantitatively, can be used to resolve subtle differences between samples. Thus, we used MS to characterize changes in expression of membrane protein markers before and after short-term induction of osteoblast (OB) differentiation...... in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane...... or membrane-anchored proteins and 159 membrane-associated proteins. Twenty-nine integrins and cell adhesion molecules, 20 receptors, and 18 Ras-related small GTPases were also identified. Upon OB differentiation, the expression levels of 83 proteins increased by at least twofold whereas the levels of another...

  3. Corneal haze induced by excimer laser photoablation in rabbits is reduced by preserved human amniotic membrane graft

    Science.gov (United States)

    Wang, Ming X.; Gray, Trevor; Prabhasawat, Pinnita; Ma, Xiong; Culbertson, William; Forster, Richard; Hanna, Khalil; Tseng, Scheffer C. G.

    1998-06-01

    We conducted a study to determine if preserved human amniotic membrane can reduce corneal haze induced by excimer laser photoablation. Excimer photoablation was performed bilaterally on 40 New Zealand white rabbits with a 6 mm ablation zone and 120 micrometer depth (PTK) using the VISX Star. One eye was randomly covered with a preserved human amniotic membrane and secured using four interrupted 10 - 0 nylon sutures; the other eye served as control. The amniotic membranes were removed at one week, and the corneal haze was graded with a slit-lamp biomicroscopy by three masked corneal specialists (WC, KH and RF) biweekly for the ensuing 12 weeks. Histology and in situ TUNEL staining (for fragmented DNA as an index for apoptosis) was performed at days 1, 3 and 7 and at 12 weeks. One week after excimer photoablation, the amniotic membrane-covered corneas showed more anterior stromal edema, which resolved at the second week. A consistent grading of organized reticular corneal haze was noted among the three masked observers. Such corneal haze peaked at the seventh week in both groups. The amniotic membrane-covered group showed statistically significant less corneal haze (0.50 plus or minus 0.15) than the control groups (1.25 plus or minus 0.35) (p less than 0.001). The amniotic membrane-covered corneas had less inflammatory response at days 1 and 3, showing nearly nil DNA fragmentation on keratocytes on the ablated anterior stromal and less stromal fibroblast activation. There is less altered epithelial cell morphology and less epithelial hyperplasia at 1 week in these amniotic membrane-treated eyes. We concluded from this study that amniotic membrane matrix is effective in reducing corneal haze induced by excimer photoablation in rabbits and may have clinical applications.

  4. Evaluation of resorbable membrane in treatment of human gingival isolated buccal recession

    Directory of Open Access Journals (Sweden)

    Sumit Narang

    2011-01-01

    Conclusion: Resorbable membrane is a versatile treatment modality for coverage of isolated buccal gingival recession. Although membrane exposure occurred in four patients, it did not interfere with post operative healing.

  5. Human Fetal Membranes at Term: Dead Tissue or Signalers of Parturition?

    OpenAIRE

    Menon, Ramkumar

    2016-01-01

    Various endocrine, immune, and mechanical factors produced by feto-maternal compartments at term increase intrauterine inflammatory loads to induce labor. The role of fetal (placental) membranes (amniochorion) as providers of parturition signals has not been well investigated. Fetal membranes line the intrauterine cavity and grow with and protect the fetus. Fetal membranes exist as an entity between the mother and fetus and perform unique functions during pregnancy. Membranes undergo a telome...

  6. Ultrastructural study of the neovagina following the utilization of human amniotic membrane for treatment of congenital absence of the vagina

    Directory of Open Access Journals (Sweden)

    L.F. Bleggi-Torres

    1997-07-01

    Full Text Available We present an ultrastructural study of the utilization of human amniotic membrane in the treatment of congenital absence of the vagina in 10 patients. All patients were surgically treated with application of an amniotic membrane graft using the modified McIndoe and Bannister technique. Sixty days after surgery, samples of the vaginal neo-epithelium were collected for transmission electron microscopy analysis. The ultrastructural findings consisted of a lining of mature squamous epithelium indicating the occurrence of metaplasia of the amniotic epithelium into the vaginal epithelium. The cells were arranged in layers as in the normal vaginal epithelium, i.e., superficial, intermediate and deep layers. There were desmosomes and cytoplasmic intermediate cytokeratin filaments, as well as some remnant features of the previous amniotic epithelium. These findings suggest that human amniotic membrane is able to complete metaplasia into squamous cells but the mechanism of this cellular transformation is unknown

  7. Structural effects of the Solanum steroids solasodine, diosgenin and solanine on human erythrocytes and molecular models of eukaryotic membranes.

    Science.gov (United States)

    Manrique-Moreno, Marcela; Londoño-Londoño, Julián; Jemioła-Rzemińska, Małgorzata; Strzałka, Kazimierz; Villena, Fernando; Avello, Marcia; Suwalsky, Mario

    2014-01-01

    This report presents evidence that the following Solanum steroids: solasodine, diosgenin and solanine interact with human erythrocytes and molecular models of their membranes as follows: a) X-ray diffraction studies showed that the compounds at low molar ratios (0.1-10.0mol%) induced increasing structural perturbation to dimyristoylphosphatidylcholine bilayers and to a considerable lower extent to those of dimyristoylphosphatidylethanolamine; b) differential scanning calorimetry data showed that the compounds were able to alter the cooperativity of dimyristoylphosphatidylcholine, dimyristoylphosphatidylethanolamine and dimyristoylphosphatidylserine phase transitions in a concentration-dependent manner; c) in the presence of steroids, the fluorescence of Merocyanine 540 incorporated to the membranes decreased suggesting a fluidization of the lipid system; d) scanning electron microscopy observations showed that all steroids altered the normal shape of human erythrocytes inducing mainly echinocytosis, characterized by the formation of blebs in their surfaces, an indication that their molecules are located into the outer monolayer of the erythrocyte membrane. © 2013.

  8. Experimental and modeling study of human tympanic membrane motion in the presence of middle ear liquid.

    Science.gov (United States)

    Zhang, Xiangming; Guan, Xiying; Nakmali, Don; Palan, Vikrant; Pineda, Mario; Gan, Rong Z

    2014-12-01

    Vibration of the tympanic membrane (TM) has been measured at the umbo using laser Doppler vibrometry and analyzed with finite element (FE) models of the human ear. Recently, full-field TM surface motion has been reported using scanning laser Doppler vibrometry, holographic interferometry, and optical coherence tomography. Technologies for imaging human TM motion have the potential to lead to using a dedicated clinical diagnosis tool for identification of middle ear diseases. However, the effect of middle ear fluid (liquid) on TM surface motion is still not clear. In this study, a scanning laser Doppler vibrometer was used to measure the full-field surface motion of the TM from four human temporal bones. TM displacements were measured under normal and disease-mimicking conditions with different middle ear liquid levels over frequencies ranging from 0.2 to 8 kHz. An FE model of the human ear, including the ear canal, middle ear, and spiral cochlea was used to simulate the motion of the TM in normal and disease-mimicking conditions. The results from both experiments and FE model show that a simple deflection shape with one or two major displacement peak regions of the TM in normal ear was observed at low frequencies (1 kHz and below) while complicated ring-like pattern of the deflection shapes appeared at higher frequencies (4 kHz and above). The liquid in middle ear mainly affected TM deflection shapes at the frequencies higher than 1 kHz.

  9. Programmed Fetal Membrane Senescence and Exosome-Mediated Signaling: A Mechanism Associated With Timing of Human Parturition

    Directory of Open Access Journals (Sweden)

    Ramkumar Menon

    2017-08-01

    Full Text Available Human parturition is an inflammatory process that involves both fetal and maternal compartments. The precise immune cell interactions have not been well delineated in human uterine tissues during parturition, but insights into human labor initiation have been informed by studies in animal models. Unfortunately, the timing of parturition relative to fetal maturation varies among viviparous species—indicative of different phylogenetic clocks and alarms—but what is clear is that important common pathways must converge to control the birth process. Herein, we hypothesize a novel signaling mechanism initiated by human fetal membrane aging and senescence-associated inflammation. Programmed events of fetal membrane aging coincide with fetal growth and organ maturation. Mechanistically, senescence involves in telomere shortening and activation of p38 mitogen-activated signaling kinase resulting in aging-associated phenotypic transition. Senescent tissues release inflammatory signals that are propagated via exosomes to cause functional changes in maternal uterine tissues. In vitro, oxidative stress causes increased release of inflammatory mediators (senescence-associated secretory phenotype and damage-associated molecular pattern markers that can be packaged inside the exosomes. These exosomes traverse through tissues layers, reach maternal tissues to increase overall inflammatory load transitioning them from a quiescent to active state. Animal model studies have shown that fetal exosomes can travel from fetal to the maternal side. Thus, aging fetal membranes and membrane-derived exosomes cargo fetal signals to the uterus and cervix and may trigger parturition. This review highlights a novel hypothesis in human parturition research based on data from ongoing research using human fetal membrane model system.

  10. Effect of isoliquiritigenin on viability and differentiated functions of human hepatocytes maintained on PEEK-WC-polyurethane membranes.

    Science.gov (United States)

    De Bartolo, Loredana; Morelli, Sabrina; Gallo, Maria Carmela; Campana, Carla; Statti, Giancarlo; Rende, Maria; Salerno, Simona; Drioli, Enrico

    2005-11-01

    In this study, we tested the ability of microporous membranes synthesised from a polymeric blend of modified polyetheretherketone (PEEK-WC) and polyurethane (PU) to support long-term maintenance and differentiation of human liver cells. The effect of isoliquiritigenin (ISL), which is a component of liquorice extract, exhibiting growth stimulatory and antiproliferative dose-dependent effect was investigated by comparing cultures treated with ISL with those untreated. To this purpose, flat-sheet membranes were prepared by a blend of PEEK-WC and PU polymers by phase inverse technique. The morphological and physico-chemical properties were characterised, respectively, by scanning electron microscopy and water contact angle measurements. Human hepatocytes cultured on PEEK-WC-PU membranes were constant up to 1 month albumin production and urea synthesis as well as the synthesis of total proteins. The liver-specific functions were expressed at high levels when cells were cultured on membranes with respect to collagen. Also the biotransformation functions were maintained for all culture periods: the ISL elimination rate increased during the culture time and high values were measured up to 22 days. Thereafter, a decrease was observed. ISL stimulated the proliferation of hepatocytes cultured on both substrata but did not affect their liver-specific functions. Hepatocytes cultured on PEEK-WC-PU membranes responded very well to ISL and expressed high levels of P450 cytochrome. These results demonstrated that long-term maintenance of human liver differentiation can be achieved on PEEK-WC-PU membranes. The incubation with ISL at the investigated concentration could stimulate the proliferation of human hepatocytes in biohybrid systems.

  11. Functional characterisation of the human alpha1 glycine receptor in a fluorescence-based membrane potential assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Kristiansen, Uffe

    2004-01-01

    In the present study, we have created a stable HEK293 cell line expressing the human homomeric alpha1 glycine receptor (GlyR) and characterised its functional pharmacology in a conventional patch-clamp assay and in the FLIPR Membrane Potential (FMP) assay, a fluorescence-based high throughput scr...

  12. CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes

    DEFF Research Database (Denmark)

    Nielsen, C H; Marquart, H V; Prodinger, W M

    2001-01-01

    convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4.4 mM MgCl2/20 mM EGTA, which abrogates the classical pathway of complement without affecting...

  13. Low-pH-induced membrane fusion mediated by human metapneumovirus F protein is a rare, strain-dependent phenomenon

    NARCIS (Netherlands)

    S. Herfst (Sander); V. Mas (Vicente); L.S. Ver (Lorena); R.J. Wierda (Rutger); A.D.M.E. Osterhaus (Albert); R.A.M. Fouchier (Ron); J.A. Melero (José)

    2008-01-01

    textabstractMembrane fusion promoted by human metapneumovirus (HMPV) fusion (F) protein was suggested to require low pH (R. M. Schowalter, S. E. Smith, and R. E. Dutch, J. Virol. 80:10931-10941, 2006). Using prototype F proteins representing the four HMPV genetic lineages, we detected

  14. Analysis of Vibrant Soundbridge placement against the round window membrane in a human cadaveric temporal bone model.

    NARCIS (Netherlands)

    Pennings, R.J.E.; Ho, A.; Brown, J.; Wijhe, R.G. van; Bance, M.

    2010-01-01

    OBJECTIVE: To evaluate optimal placement of the Floating Mass Transducer of the Vibrant Soundbridge (Med-El, Innsbruck, Austria) against the round window membrane, particularly the impact of interposed coupling fascia and of covering materials. METHOD: : Six fresh human cadaveric temporal bones were

  15. Biological Activity Alterations of Human Amniotic Membrane Pre and Post Irradiation Tissue Banking.

    Science.gov (United States)

    Nemr, Waleed; Bashandy, A S; Araby, Eman; Khamiss, O

    Innate immunity of Human Amniotic Membrane (HAM) and its highly active secretome that rich with various types of growth factors and anti-inflammatory substances proposed it as a promising material for many medical studies and applications. This study evaluate the biological activity of cultivated HAM pre and post tissue banking process in which freeze-dried HAM was sterilized by 25 KGray (kGy) dose of γ radiation. The HAM's antimicrobial activity, viability, growth of isolated human amniotic epithelial cells (HAECs), hematopoietic stimulation of co-cultivated murine bone marrow cells (mammalian model), scaffold efficiency for fish brain building up (non-mammalian model) and self re-epithelialization after trypsin denuding treatment were examined as supposed biological activity features. Native HAM revealed viability indications and was active to kill all tested microorganisms; 6 bacterial species (3 Gram-positive and 3 Gram-negative) and Candida albicans as a pathogenic fungus. Also, HAM activity promoted colony formation of murine hematopoietic cells, Tilapia nilotica brain fragment building-up and self re-epithelialization after trypsin treatment. In contrary, radiation-based tissue banking of HAM caused HAM cellular death and consequently lacked almost all of examined biological activity features. Viable HAM was featured with biological activity than fixed HAM prepared by irradiation tissue banking.

  16. Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane

    Energy Technology Data Exchange (ETDEWEB)

    Rodríguez-Fuentes, Nayeli; Rodríguez-Hernández, Ana G. [Depto. Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Mexico City 04510 (Mexico); Enríquez-Jiménez, Juana [Depto. Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), México City 14000 (Mexico); Alcántara-Quintana, Luz E. [Subd. de Investigación, Centro Nacional de la Transfusión Sanguínea, Secretaria de Salud, Mexico City 07370 (Mexico); Fuentes-Mera, Lizeth [Depto. Biología Molecular e Histocompatibilidad, Hospital General “Dr. Manuel Gea González”, México City 4800 (Mexico); Piña-Barba, María C. [Depto. Materiales Metálicos y Cerámicos, Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México (UNAM), México City 04510 (Mexico); Zepeda-Rodríguez, Armando [Depto. Biología Celular y Tisular, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), México City 04510 (Mexico); and others

    2013-05-10

    Highlights: •Nukbone showed to be a good scaffold for adhesion, proliferation and differentiation of stem cells. •Nukbone induced osteoblastic differentiation of human mesenchymal stem cells. •Results showed that Nukbone offer an excellent option for bone tissue regeneration due to properties. -- Abstract: Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.

  17. The Plasma Membrane Calcium ATPases and Their Role as Major New Players in Human Disease.

    Science.gov (United States)

    Stafford, Nicholas; Wilson, Claire; Oceandy, Delvac; Neyses, Ludwig; Cartwright, Elizabeth J

    2017-07-01

    The Ca2+ extrusion function of the four mammalian isoforms of the plasma membrane calcium ATPases (PMCAs) is well established. There is also ever-increasing detail known of their roles in global and local Ca2+ homeostasis and intracellular Ca2+ signaling in a wide variety of cell types and tissues. It is becoming clear that the spatiotemporal patterns of expression of the PMCAs and the fact that their abundances and relative expression levels vary from cell type to cell type both reflect and impact on their specific functions in these cells. Over recent years it has become increasingly apparent that these genes have potentially significant roles in human health and disease, with PMCAs1-4 being associated with cardiovascular diseases, deafness, autism, ataxia, adenoma, and malarial resistance. This review will bring together evidence of the variety of tissue-specific functions of PMCAs and will highlight the roles these genes play in regulating normal physiological functions and the considerable impact the genes have on human disease. Copyright © 2017 the American Physiological Society.

  18. Effect of Amniotic Membrane Suspension (AMS) and Amniotic Membrane Homogenate (AMH) on Human Corneal Epithelial Cell Viability, Migration and Proliferation In Vitro.

    Science.gov (United States)

    Wu, Ming-Feng; Stachon, Tanja; Langenbucher, Achim; Seitz, Berthold; Szentmáry, Nóra

    2017-03-01

    To analyze the effects of different concentrations of amniotic membrane suspension (AMS) or amniotic membrane homogenate (AMH) on human corneal epithelial cell (HCEC) viability, migration and proliferation. Amniotic membranes (AMs) of 13 placentas were prepared and stored at -80°C. For AMS preparation, following de-freezing, AM pieces were inserted in six-well plates and 5 ml Dulbecco's Modified Eagle's Medium (DMEM)/F12 (with 5% fetal bovine serum, FBS) per gram tissue was added for 96 h. After removal of the AM, the remaining supernatant was collected for experiments. For AMH preparation, following de-freezing, AMs were homogenized in liquid nitrogen and 5 ml DMEM/F12 (with 5% FBS) per gram tissue was added. Following centrifugation, the supernatant was collected for experiments. HCECs were expanded and incubated in DMEM/F12, 5% FBS supplemented by 15%, 30% or 100% AMS or 15% or 30% AMH. Viability was analyzed using Cell Proliferation Kit XTT, migration using wound healing assay and proliferation by the cell proliferation ELISA BrdU kit. HCEC viability remained unchanged using 15% or 30% AMS (p = 1.0 for both); however, it decreased significantly using 100% AMS (p migration increased significantly (p migration remained unchanged and 100% AMS inhibited HCEC migration (p migration, 15% and 30% AMS application seems to be the most appropriate method to support epithelial healing.

  19. The random co-polymer glatiramer acetate rapidly kills primary human leukocytes through sialic-acid-dependent cell membrane damage

    DEFF Research Database (Denmark)

    Christiansen, Stig Hill; Zhang, Xianwei; Juul-Madsen, Kristian

    2017-01-01

    in innate immunity. It shares the positive charge and amphipathic character of GA, and, as shown here, also the ability to kill human leukocyte. The cytotoxicity of both compounds depends on sialic acid in the cell membrane. The killing was associated with the generation of CD45 + debris, derived from cell...... of certain oligomeric and chemical properties to support cytotoxic effects of cationic polymers targeting human leukocytes....

  20. Place of human amniotic membrane immunoblotting in the diagnosis of autoimmune bullous dermatoses.

    Science.gov (United States)

    Grootenboer-Mignot, S; Descamps, V; Picard-Dahan, C; Nicaise-Roland, P; Prost-Squarcioni, C; Leroux-Villet, C; Champagnat, C; Delaval, A; Aucouturier, F; Crickx, B; Chollet-Martin, S

    2010-04-01

    Fine analysis of antiskin autoantibodies can contribute to the differential diagnosis of autoimmune bullous dermatoses. To develop a high-performance immunoblotting method using human amniotic membrane as the antigen source, and to compare it with current laboratory methods. Sera from 113 patients were tested by immunoblotting (IB), rat and monkey oesophagus and salt-split skin indirect immunofluorescence (IIF), and enzyme-linked immunosorbent assay (ELISA) quantification of anti-BP180-NC16a and anti-BP230, or antidesmoglein (Dsg) 1 and 3 antibodies. There were 56 cases of bullous pemphigoid (BP), 22 cases of mucous membrane pemphigoid (MMP), eight cases of epidermolysis bullosa acquisita (EBA), two cases of bullous systemic lupus erythematosus (BSLE), 17 cases of pemphigus vulgaris (PV), and four cases each of pemphigus foliaceus (PF) and paraneoplastic pemphigus (PNP). In BP, the three methods had similar sensitivity (84-89%) for both anti-BP180-NC16a and anti-BP230 antibody detection. In MMP, autoantibodies (mainly directed against BP180 or laminin 332 subunits) were detected in 77% of patients by IB, compared with only 9% by IIF on rat and monkey oesophagus and 36% on salt-split skin, and 14% by anti-BP180-NC16a and anti-BP230 ELISA. In patients with pemphigus, ELISA had 92% sensitivity for anti-Dsg1 and 3, but IB and rat bladder IIF were necessary to confirm PNP by revealing specific and rare patterns (antidesmoplakin I/II, antienvoplakin and antiperiplakin antibodies). IB also revealed anticollagen VII antibodies in 60% of patients with EBA and BSLE, and antibodies to BP180, BP230 and Dsg3 in a few patients who were negative using the other two techniques. Amniotic membrane immunoblotting is an interesting diagnostic tool for bullous diseases, as the entire panel of autoantibodies can be detected with a single extract. This method improves the identification of complex and heterogeneous autoimmune processes in conjunction with IIF and ELISA, and is

  1. The impact of thickness of resorbable membrane of human origin on the ossification of bone defects: A pathohistologic study

    Directory of Open Access Journals (Sweden)

    Bubalo Marija

    2012-01-01

    Full Text Available Background/Aim. A wide range of resorbable and nonresorbable membranes have been investigated over the last two decades. The barrier membrane protects the defect from ingrowth of soft tissue cells and allows bone progenitor cells to develop bone within a blood clot that is formed beneath the barrier membrane. The membranes are applied to reconstruct small bony defect prior to implantation, to cover dehiscences and fenestrations around dental implants. The aim of this study was to evaluate the influence of human resorbable demineralized membrane (RHDM thickness on bone regeneration. Methods. The experiment, approved by Ethical Committee, was performed on 6 dogs and conducted into three phases. Bone defects were created in all the 6 dogs on the left side of the mandible, 8 weeks after extraction of second, third and fourth premolars. One defect was covered with RHDM 100 μ thick, one with RHDM 200 μ thick, and the third defect left empty (control defect. The histopathological analysis was done 2, 4 and 6 months after the surgery. In the third phase samples of bone tissue were taken and subjected to histopathological analysis. Results. In all the 6 dogs the defects treated with RHDM 200 μ thick showed higher level of bone regeneration in comparison with the defect treated with RHDM 100 μ thick and especially with empty defect. Conclusion. Our results demonstrated that the thicker membrane showed the least soft tissue ingrowths and promoted better bone formation at 6 months compared with a thinner one.

  2. Characterization of the Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative Standard using SDS-PAGE shotgun proteomics.

    Science.gov (United States)

    Peng, Lifeng; Kapp, Eugene A; McLauchlan, Danyl; Jordan, T William

    2011-11-01

    Although there are now multiple methods for the analysis of membrane proteomes, there is relatively little systematic characterization of proteomic workflows for membrane proteins. The Asia Oceania Human Proteome Organisation (AOHUPO) has therefore embarked on a Membrane Proteomics Initiative (MPI) using a large range of workflows. Here, we describe the characterization of the MPI mouse liver microsomal membrane Standard using SDS-PAGE prior to in-gel tryptic digestion and LC-ESI-MS/MS. The Na(2) CO(3) wash followed by SDS-PAGE prior to in-gel tryptic digestion and LC-MS/MS strategy was effective for the detection of membrane proteins with 47.1% of the identified proteins being transmembrane proteins. Gene Ontology term enrichment analysis showed that biological processes involving transport, lipid metabolism, cell communication, cell adhesion, and cellular component organization were significantly enriched. Comparison of the present data with the previously published reports on mouse liver proteomes confirmed that the MPI Standard provides an excellent resource for the analysis of membrane proteins in the AOHUPO MPI. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools.

    Science.gov (United States)

    Nováková, Zora; Foss, Catherine A; Copeland, Benjamin T; Morath, Volker; Baranová, Petra; Havlínová, Barbora; Skerra, Arne; Pomper, Martin G; Barinka, Cyril

    2017-05-01

    Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  4. High-Mr glycoprotein profiles in human milk serum and fat-globule membrane.

    Science.gov (United States)

    Shimizu, M; Yamauchi, K; Miyauchi, Y; Sakurai, T; Tokugawa, K; McIlhinney, R A

    1986-01-01

    Gradient-polyacrylamide-gel electrophoresis of human milk serum separated three high-Mr glycoprotein bands. The properties of the components corresponding to the three bands (tentatively termed 'Components C, B and A' in their order of migration) were compared by staining with four monoclonal antibodies and lectins. Components B and C both reacted with the four antibodies, but Component A did not. Components B and C were stained with peanut (Arachis hypogaea) agglutinin (PNA) and wheat (Triticum)-germ agglutinin (WGA), Component A being stained with soya-bean (Glycine max) agglutinin as well as PNA and WGA. These results suggest that Components B and C were related molecules, whereas Component A was markedly different from them. The reactivities of Components B and C were the same as those of PAS-0, a high-Mr periodate/Schiff (PAS)-positive glycoprotein previously isolated from human milk fat-globule membrane (MFGM). Component C, whose electrophoretic mobility was the same as PAS-0, could have been a soluble form of PAS-0. A high-Mr glycoprotein having the same properties as Component A was also observed in MFGM. The amino acid composition of the isolated Component A was significantly different from that of Component C and PAS-0, high threonine and serine contents being characteristic of Component A. The carbohydrate content of Component A was 65-80%, and was much higher than that of Component C and PAS-0. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid were each detected as constituent sugars of Component A. Component A represents, therefore, a new high-Mr glycoprotein species in human milk serum and MFGM. Since these glycoproteins were high-Mr mucin-like glycoproteins, the names 'HM glycoprotein-A' and 'HM glycoprotein-C' were proposed for Component A and Component C (PAS-O) respectively. Images Fig. 2. Fig. 3. Fig. 5. Fig. 6. PMID:3707520

  5. Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaojie [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Taizhou Polytechnic College, Taizhou (China); Zhang, Ling; Shao, Yueting; Liang, Zuowen; Shao, Chen; Wang, Bo; Guo, Baofeng; Li, Na; Zhao, Xuejian [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Li, Yang, E-mail: lyang@jlu.edu.cn [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Xu, Deqi [Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Neu3 is as one of the sialidases and regulates cell surface functions. Black-Right-Pointing-Pointer A Neu3-specific siRNA inhibited prostrate cancer cell invasion and migration. Black-Right-Pointing-Pointer The Neu3-specific siRNA inhibited prostate cancer metastasis in mice. Black-Right-Pointing-Pointer Targeting Neu3 may have utility for gene-based therapy of human cancer metastasis. -- Abstract: Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed

  6. The random co-polymer glatiramer acetate rapidly kills primary human leukocytes through sialic-acid-dependent cell membrane damage

    DEFF Research Database (Denmark)

    Christiansen, Stig Hill; Zhang, Xianwei; Juul-Madsen, Kristian

    2017-01-01

    in innate immunity. It shares the positive charge and amphipathic character of GA, and, as shown here, also the ability to kill human leukocyte. The cytotoxicity of both compounds depends on sialic acid in the cell membrane. The killing was associated with the generation of CD45 + debris, derived from cell...... membrane deformation. Nanoparticle tracking analysis confirmed the formation of such debris, even at low GA concentrations. Electric cell-substrate impedance sensing measurements also recorded stable alterations in T lymphocytes following such treatment. LL-37 forms oligomers through weak hydrophobic...

  7. GLUT4 expression at the plasma membrane is related to fibre volume in human skeletal muscle fibres

    DEFF Research Database (Denmark)

    Gaster, M; Vach, W; Beck-Nielsen, H

    2002-01-01

    In this study we examined the relationship between GLUT4 expression at the plasma membrane and muscle fibre size in fibre-typed human muscle fibres by immunocytochemistry and morphometry in order to gain further insight into the regulation of GLUT4 expression. At the site of the plasma membrane......, GLUT4 was more abundantly expressed in slow as compared to fast fibres at the same fibre diameter (p GLUT4 expression increased with increasing fibre radius independently of fibre type (p GLUT4 density at the surface of slow fibres of both diabetic and obese was reduced...... compared to control subjects at the same diameter (p GLUT4 expression (p

  8. Histological evaluation of human amniotic membrane graft on oral keratinizing mucosa in the rabbit model (A pilot study

    Directory of Open Access Journals (Sweden)

    Samandari Najafabadi MH

    2007-06-01

    Full Text Available Background and Aim: One of the complications following major oral surgeries is mucosal defects and delayed healing process. Up to now, various mucocutaneous grafts have been used in this field and recently, amniotic membrane has been proposed as a biological dressing in dermatologic, ophthalmologic and otolaryngologic practices. The purpose of this pilot study was to evaluate the healing process following human amniotic membrane graft on oral keratinized mucosa of rabbit.Materials and Methods: In this experimental animal study, two surgical mucosal defects with the same size were made in palatal mucosa of 10 rabbits with the same weight, gender and race and a graft of human amniotic membrane was used on one of the defects. On the 7th, 14th and 28th postoperative days, surgical biopsies were randomly obtained from grafted and ungrafted regions of 3, 4 and 3 rabbits, respectively and submitted for microscopic study. Results: According to the results, grafted regions showed more surface epithelialization and thicker newly formed epithelium. Also inflammatory cells infiltration was less in these areas. In all cases, there was a remarkable cartilage formation in the connective tissue of the recipient sites.Conclusion: The results of this study suggest that the use of amniotic membrane graft in oral surgery could be effective in healing process. Additional studies should be done using animal and human models with more samples. Furthermore, the formation of cartilage in the grafted sites and its possible potential in reconstruction of bone defects, needs to be studied.

  9. The Effect of Highly Hydroxylated Fullerenol C60(OH36 on Human Erythrocyte Membrane Organization

    Directory of Open Access Journals (Sweden)

    Jacek Grebowski

    2015-01-01

    Full Text Available The mechanism of the interaction of highly hydroxylated fullerenol C60(OH36 with erythrocyte membranes was studied by electron spin resonance spectroscopy (ESR of stearic acid derivatives labeled with a nitroxyl radical at C-12 or C-16 and with a nitroxyl derivative of maleimide covalently attached to sulfhydryl groups of membrane proteins. A significant increase in membrane fluidity in the hydrophobic region of the lipid bilayer was observed for 12-doxylstearic acid at fullerenol concentrations of 100 mg/L or 150 mg/L, while for 16-doxylstearic acid significant increase in fluidity was only observed at 150 mg/L. Fullerenol at 100 mg/L or 150 mg/L caused conformational changes in membrane proteins, expressed as an increase in the hw/hs parameter, when fullerenol was added before the maleimide spin label (MSL to the membrane suspension. The increase of the hw/hs parameter may be caused by changes in lipid-protein or protein-protein interactions which increase the mobility of the MSL label and as a result increase the membrane fluidity. Incubation of the membranes with the MSL before the addition of fullerenol blocked the available membrane protein –SH groups and minimized the interaction of fullerenol with them. This confirms that fullerenol interacts with erythrocyte membrane proteins via available protein –SH groups.

  10. The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    DEFF Research Database (Denmark)

    Hendus-Altenburger, Ruth; Pedraz Cuesta, Elena; Olesen, Christina Wilkens

    2016-01-01

    BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify...... the human Na(+)/H(+) exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D...... and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2....

  11. Tissue integrity is essential for ectopic implantation of human endometrium in the chicken chorioallantoic membrane.

    Science.gov (United States)

    Nap, Annemiek W; Groothuis, Patrick G; Demir, Ayse Y; Maas, Jacques W M; Dunselman, Gerard A J; de Goeij, Anton F P M; Evers, Johannes L H

    2003-01-01

    Not all women with patent tubes develop clinically manifest endometriosis. Quality and quantity of endometrium in retrograde menstruation may be the determining factor in the development of the disease. We hypothesize that retrograde shedding of endometrial fragments with preserved integrity facilitates implantation of endometrium in ectopic locations, resulting in endometriotic lesion development. We evaluate the impact of tissue integrity on the success of endometriosis-like lesion development in the chicken embryo chorioallantoic membrane (CAM) model. Menstrual and non-menstrual (cyclic) endometrium were collected by biopsy, and either minced or enzymatically dispersed. Spontaneously shed menstrual effluent was collected by a menstrual cup, and cells and tissue were isolated. We evaluated whether infiltration or lesion formation in the CAM occurred after transplantation of endometrium onto the CAM. Transplantation of biopsied menstrual and cyclic endometrium fragments, and of endometrium fragments >1 mm(3) isolated from menstrual effluent, resulted in lesion formation. Transplantation of endometrial cells isolated from menstrual effluent did not lead to lesion formation. After transplantation of digested biopsied cyclic endometrium, infiltration in the CAM but no lesions were observed. In the CAM assay, integrity of tissue architecture determines success of implantation of human endometrium in ectopic locations.

  12. A normative study of tympanic membrane motion in humans using a laser Doppler vibrometer (LDV).

    Science.gov (United States)

    Whittemore, Kenneth R; Merchant, Saumil N; Poon, Becky B; Rosowski, John J

    2004-01-01

    Laser Doppler vibrometry was used to measure the sound-induced tympanic membrane (TM) velocity, assessed near the umbo, in 56 normal hearing human subjects at nine sound frequencies. A second series of measurements was made in 47 subjects with sensorineural hearing loss (SNHL). Each set of measurements has features in common with previously published results. The measured velocity magnitude (normalized by the stimulus sound pressure) at any one frequency ranged among subjects by factors of 3-0.3 (+/-10 dB) from the mean and the phase angle of the normalized velocity ranged from +/-15 degrees around the mean at low frequencies to more than +/-200 degrees around the mean at 6 kHz. Measurements repeated after intervals of minutes to months were generally within 40% in magnitude (+/-3 dB) and 20 degrees in phase. Sources of variability included the effect of small differences in the location of the measurement on the TM and small static middle-ear pressures. No effects of stimulus level, ear sidedness (right or left), gender, age or the presence or absence of SNHL were found. These results provide a baseline normal response for studies of TM velocity with conductive hearing losses of different etiologies.

  13. High-risk human papillomavirus infection is associated with premature rupture of membranes.

    Science.gov (United States)

    Cho, GeumJoon; Min, Kyung-Jin; Hong, Hye-Ri; Kim, SuhngWook; Hong, Jin-Hwa; Lee, Jae-Kwan; Oh, Min-Jeong; Kim, HaiJoong

    2013-09-06

    Human papillomavirus (HPV) is known to be more prevalent in spontaneous abortions than in elective terminations of pregnancy. More recently, placental infection with HPV was shown to be associated with spontaneous preterm delivery. However, no study has evaluated the prevalence of HPV infection in pregnant Korean females and its association with adverse pregnancy outcomes. We conducted a cross-sectional study of 311 females who gave birth at Korea University Medical Center. Our sample included 45 preterm deliveries, 50 cases of premature rupture of the membranes (PROM), 21 preeclampsia cases, and 8 gestational diabetes mellitus (GDM) patients. We used the Hybrid Capture II system to detect high-risk (HR)-HPV infection at six weeks postpartum. The prevalence of HR-HPV infection was 14.1%. Women with HR-HPV infection had a higher incidence of PROM than those without HR-HPV. HR-HPV infection was associated with an increased risk of PROM (OR, 2.380; 95% CI, 1.103-5.134). The prevalence of preterm delivery, preeclampsia, or GDM was not different between the two groups. We observed a high prevalence of HR-HPV infection in pregnant women. Moreover, HR-HPV infection was associated with a risk of PROM at term. Further studies are needed to evaluate mechanisms by which HR-HPV infection induces PROM.

  14. Development of the follicular basement membrane during human gametogenesis and early folliculogenesis.

    Science.gov (United States)

    Heeren, A Marijne; van Iperen, Liesbeth; Klootwijk, Daniëlle B; de Melo Bernardo, Ana; Roost, Matthias S; Gomes Fernandes, Maria M; Louwe, Leonie A; Hilders, Carina G; Helmerhorst, Frans M; van der Westerlaken, Lucette A J; Chuva de Sousa Lopes, Susana M

    2015-01-21

    In society, there is a clear need to improve the success rate of techniques to restore fertility. Therefore a deeper knowledge of the dynamics of the complex molecular environment that regulates human gametogenesis and (early) folliculogenesis in vivo is necessary. Here, we have studied these processes focusing on the formation of the follicular basement membrane (BM) in vivo. The distribution of the main components of the extracellular matrix (ECM) collagen IV, laminin and fibronectin by week 10 of gestation (W10) in the ovarian cortex revealed the existence of ovarian cords and of a distinct mesenchymal compartment, resembling the organization in the male gonads. By W17, the first primordial follicles were assembled individually in that (cortical) mesenchymal compartment and were already encapsulated by a BM of collagen IV and laminin, but not fibronectin. In adults, in the primary and secondary follicles, collagen IV, laminin and to a lesser extent fibronectin were prominent in the follicular BM. The ECM-molecular niche compartimentalizes the female gonads from the time of germ cell colonization until adulthood. This knowledge may contribute to improve methods to recreate the environment needed for successful folliculogenesis in vitro and that would benefit a large number of infertility patients.

  15. Functional and Molecular Characterization of Ex Vivo Cultured Epiretinal Membrane Cells from Human Proliferative Diabetic Retinopathy

    Directory of Open Access Journals (Sweden)

    Zoltán Veréb

    2013-01-01

    Full Text Available Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs from proliferative diabetic retinopathy (PDR can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNFα activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2, partially positive for hematopoietic- (CD34, CD47 and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFRβ, and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1 were detected upon TNFα activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo.

  16. Synchronism in mitochondrial ROS flashes, membrane depolarization and calcium sparks in human carcinoma cells.

    Science.gov (United States)

    Kuznetsov, Andrey V; Javadov, Sabzali; Saks, Valdur; Margreiter, Raimund; Grimm, Michael

    2017-06-01

    Mitochondria are major producers of reactive oxygen species (ROS) in many cells including cancer cells. However, complex interrelationships between mitochondrial ROS (mitoROS), mitochondrial membrane potential (ΔΨm) and Ca2+ are not completely understood. Using human carcinoma cells, we further highlight biphasic ROS dynamics: - gradual mitoROS increase followed by mitoROS flash. Also, we demonstrate heterogeneity in rates of mitoROS generation and flash initiation time. Comparing mitochondrial and near-extra-mitochondrial signals, we show that mechanisms of mitoROS flashes in single mitochondria, linked to mitochondrial permeability transition pore opening (ΔΨm collapse) and calcium sparks, may involve flash triggering by certain levels of external ROS released from the same mitochondria. In addition, mitochondria-mitochondria interactions can produce wave propagations of mitoROS flashes and ΔΨm collapses in cancer cells similar to phenomena of ROS-induced ROS release (RIRR). Our data suggest that in cancer cells RIRR, activation of mitoROS flashes and mitochondrial depolarization may involve participation of extramitochondrial-ROS produced either by individual mitochondria and/or by neighboring mitochondria. This could represent general mechanisms in ROS-ROS signaling with suggested role in both mitochondrial and cellular physiology and signaling. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Oestrogen inhibits human colonic motility by a non-genomic cell membrane receptor-dependent mechanism.

    LENUS (Irish Health Repository)

    Hogan, A M

    2012-02-01

    BACKGROUND: Classical effects of oestrogen involve activation of target genes after binding nuclear receptors. Oestrogenic effects too rapid for DNA transcription (non-genomic) are known to occur. The effect of oestrogen on colonic motility is unknown despite the prevalence of gastrointestinal symptoms in pregnant and premenopausal women. METHODS: Histologically normal colon was obtained from proximal resection margins of colorectal carcinoma specimens. Circular smooth muscle strips were microdissected and suspended in organ baths under 1 g of tension. After equilibration, they were exposed to 17beta-oestradiol (n = 8) or bovine serum albumin (BSA)-conjugated 17beta-oestradiol (n = 8). Fulvestrant, an oestrogen receptor antagonist, was added to some baths (n = 8). Other strips were exposed to calphostin C or cycloheximide. Carbachol was added in increasing concentrations and contractile activity was recorded isometrically. RESULTS: Oestrogen inhibited colonic contractility (mean difference 19.7 per cent; n = 8, P < 0.001). In keeping with non-genomic, rapid-onset steroid action, the effect was apparent within minutes and reversible. It was observed with both 17beta-oestradiol and BSA-conjugated oestrogen, and was not altered by cycloheximide. Effects were inhibited by fulvestrant, suggesting receptor mediation. CONCLUSION: Oestrogen decreases contractility in human colonic smooth muscle by a non-genomic mechanism involving cell membrane coupling.

  18. Technique of cultivating limbal derived corneal epithelium on human amniotic membrane for clinical transplantation

    Directory of Open Access Journals (Sweden)

    Fatima A

    2006-01-01

    Full Text Available Background : The technique of transplantation of cultivated limbal epithelium rather than direct limbal tissue isa novel method of "cell therapy" involved in reconstructing the ocular surface in severe limbal stem celldeficiency [LSCD], caused by chemical burns. Aim : To describe a simple feeder-cell free technique of cultivating limbal epithelium on human amniotic membrane[HAM]. Materials and Methods : The limbal tissues (2 mm were harvested from patients with LSCD. These tissueswere proliferated in vitro on HAM supplemented by human corneal epithelial cell medium and autologousserum. Cultures covering more ?50% area of 2.5x5 cm HAM were considered adequate for clinical use. Thecultured epithelium was characterized by histopathology and immunophenotyping.Results: A total of 542 cultures out of 250 limbal tissues were cultivated in the laboratory from January 2001through July 2005. The culture explants showed that clusters of cells emerging from the edge of the explantsin one-three days formed a complete monolayer within 10-14 days. In 86% of cultures (464 of 542, thegrowth was observed within one-two days. Successful explant cultures were observed in 98.5% (534 of 542cultures with 91% explant cultures showing an area of ?6.25 cm2 (6.25 - 12.5 cm2 range. The cultivatedepithelium was terminated between 10-14 days for clinical transplantation. The problems encountered wereinadequate growth (2 of 542 and contamination (2 of 542. Conclusions : We demonstrate a simple technique of generating a sheet of corneal epithelium from a limbalbiopsy. This new technique could pave the way for a novel form of cell therapy.

  19. Rapid recognition and functional analysis of membrane proteins on human cancer cells using atomic force microscopy.

    Science.gov (United States)

    Li, Mi; Xiao, Xiubin; Liu, Lianqing; Xi, Ning; Wang, Yuechao

    2016-09-01

    Understanding the physicochemical properties of cell surface signalling molecules is important for us to uncover the underlying mechanisms that guide the cellular behaviors. Atomic force microscopy (AFM) has become a powerful tool for detecting the molecular interactions on individual cells with nanometer resolution. In this paper, AFM peak force tapping (PFT) imaging mode was applied to rapidly locate and visually map the CD20 molecules on human lymphoma cells using biochemically sensitive tips. First, avidin-biotin system was used to test the effectiveness of using PFT imaging mode to probe the specific molecular interactions. The adhesion images obtained on avidin-coated mica using biotin-tethered tips obviously showed the recognition spots which corresponded to the avidins in the simultaneously obtained topography images. The experiments confirmed the specificity and reproducibility of the recognition results. Then, the established procedure was applied to visualize the nanoscale organization of CD20s on the surface of human lymphoma Raji cells using rituximab (a monoclonal anti-CD20 antibody)-tethered tips. The experiments showed that the recognition spots in the adhesion images corresponded to the specific CD20-rituximab interactions. The cluster sizes of CD20s on lymphoma Raji cells were quantitatively analyzed from the recognition images. Finally, under the guidance of fluorescence recognition, the established procedure was applied to cancer cells from a clinical lymphoma patient. The results showed that there were significant differences between the adhesion images obtained on cancer cells and on normal cells (red blood cell). The CD20 distributions on ten cancer cells from the patient were quantified according to the adhesion images. The experimental results demonstrate the capability of applying PFT imaging to rapidly investigate the nanoscale biophysical properties of native membrane proteins on the cell surface, which is of potential significance in

  20. Evaluation of visual acuity and color vision in normal human eyes with a sutureless temporary amniotic membrane patch.

    Science.gov (United States)

    Ijiri, Shigeyuki; Kobayashi, Akira; Sugiyama, Kazuhisa; Tseng, Scheffer C G

    2007-12-01

    To evaluate how sutureless amniotic membrane patches may affect visual functions in normal human eyes. Prospective intervention study. Ten sets of sutureless amniotic membrane patch manufactured as PROKERA were inserted in one eye of six normal patients. Four sets (one each) were inserted in four patients, while six sets (three each) were inserted in two patients. Uncorrected distant and near visual acuities, color vision, amniotic membrane thickness measured by pachymetry, and total symptom scores were compared before and after insertion. Within 30 minutes after insertion, mean distant visual acuities decreased from -0.22 +/- 0.06 to 0.92 +/- 0.45 logarithmic minimum angle of resolution (logMAR). Among 10 sets of PROKERA inserted, the largest optotype (1.0 logMAR) of the near vision chart could not be recognized in five, but color vision evaluated by Panel D-15 was still preserved in all. Total symptom scores increased to 47.8 +/- 9.1 points (maximum, 100 points). Among symptoms, total scores for foreign body sensation (17.8 +/- 3.6) and blurred vision (17.8 +/- 4.4) were high. Loss of distant visual acuity and increases of symptom scores were not correlated with amniotic membrane thickness, of which the mean was 67.6 +/- 25.2 mum. However, amniotic membrane that was less opaque tended to provide relatively good visual acuities. Because of the relative non-transparency of sutureless amniotic membrane patches in PROKERA, distant and near visual acuities decreased in normal human eyes. The foreign body sensation noted after insertion is primarily derived from the rigid supporting skirt.

  1. Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

    Science.gov (United States)

    Sthanam, Lakshmi Kavitha; Srivastava, Rohit; Basu, Bhakti; Dutta, Suryendu; Sen, Shamik; Modi, Deepak

    2016-01-01

    Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death. PMID:27583406

  2. Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth.

    Directory of Open Access Journals (Sweden)

    Manalee Vishnu Surve

    2016-09-01

    Full Text Available Infection of the genitourinary tract with Group B Streptococcus (GBS, an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.

  3. Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis.

    Science.gov (United States)

    Ago, Hideo; Kanaoka, Yoshihide; Irikura, Daisuke; Lam, Bing K; Shimamura, Tatsuro; Austen, K Frank; Miyano, Masashi

    2007-08-02

    The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis, are lipid mediators of smooth muscle constriction and inflammation, particularly implicated in bronchial asthma. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein that belongs to a superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes 5-lipoxygenase-activating protein, microsomal glutathione S-transferases (MGSTs), and microsomal prostaglandin E synthase 1 (ref. 13). LTC4S conjugates glutathione to LTA4, the endogenous substrate derived from arachidonic acid through the 5-lipoxygenase pathway. In contrast with MGST2 and MGST3 (refs 15, 16), LTC4S does not conjugate glutathione to xenobiotics. Here we show the atomic structure of human LTC4S in a complex with glutathione at 3.3 A resolution by X-ray crystallography and provide insights into the high substrate specificity for glutathione and LTA4 that distinguishes LTC4S from other MGSTs. The LTC4S monomer has four transmembrane alpha-helices and forms a threefold symmetric trimer as a unit with functional domains across each interface. Glutathione resides in a U-shaped conformation within an interface between adjacent monomers, and this binding is stabilized by a loop structure at the top of the interface. LTA4 would fit into the interface so that Arg 104 of one monomer activates glutathione to provide the thiolate anion that attacks C6 of LTA4 to form a thioether bond, and Arg 31 in the neighbouring monomer donates a proton to form a hydroxyl group at C5, resulting in 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4). These findings provide a structural basis for the development of LTC4S inhibitors for a proinflammatory pathway mediated by three cysteinyl leukotriene ligands whose stability and potency are different

  4. Protein expression profiling of nuclear membrane protein reveals potential biomarker of human hepatocellular carcinoma

    OpenAIRE

    Khan, Rizma; Zahid, Saadia; Wan, Yu-Jui; Forster, Jameson; Karim, A-Bashar; Nawabi, Atta M; Azhar, Abid; Rahman, M; Ahmed, Nikhat

    2013-01-01

    Abstract Background Complex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes h...

  5. Effect of carbon black nanomaterial on biological membranes revealed by shape of human erythrocytes, platelets and phospholipid vesicles.

    Science.gov (United States)

    Pajnič, Manca; Drašler, Barbara; Šuštar, Vid; Krek, Judita Lea; Štukelj, Roman; Šimundić, Metka; Kononenko, Veno; Makovec, Darko; Hägerstrand, Henry; Drobne, Damjana; Kralj-Iglič, Veronika

    2015-03-28

    We studied the effect of carbon black (CB) agglomerated nanomaterial on biological membranes as revealed by shapes of human erythrocytes, platelets and giant phospholipid vesicles. Diluted human blood was incubated with CB nanomaterial and observed by different microscopic techniques. Giant unilamellar phospholipid vesicles (GUVs) created by electroformation were incubated with CB nanomaterial and observed by optical microscopy. Populations of erythrocytes and GUVs were analyzed: the effect of CB nanomaterial was assessed by the average number and distribution of erythrocyte shape types (discocytes, echinocytes, stomatocytes) and of vesicles in test suspensions, with respect to control suspensions. Ensembles of representative images were created and analyzed using computer aided image processing and statistical methods. In a population study, blood of 14 healthy human donors was incubated with CB nanomaterial. Blood cell parameters (concentration of different cell types, their volumes and distributions) were assessed. We found that CB nanomaterial formed micrometer-sized agglomerates in citrated and phosphate buffered saline, in diluted blood and in blood plasma. These agglomerates interacted with erythrocyte membranes but did not affect erythrocyte shape locally or globally. CB nanomaterial agglomerates were found to mediate attractive interaction between blood cells and to present seeds for formation of agglomerate - blood cells complexes. Distortion of disc shape of resting platelets due to incubation with CB nanomaterial was not observed. CB nanomaterial induced bursting of GUVs while the shape of the remaining vesicles was on the average more elongated than in control suspension, indicating indirect osmotic effects of CB nanomaterial. CB nanomaterial interacts with membranes of blood cells but does not have a direct effect on local or global membrane shape in physiological in vitro conditions. Blood cells and GUVs are convenient and ethically acceptable

  6. A human in vivo study of the extent to which 31-phosphorus neurospectroscopy phosphomonoesters index cerebral cell membrane phospholipid anabolism.

    Science.gov (United States)

    Puri, B K; Treasaden, I H

    2009-01-01

    The phosphomonoester narrow resonance of human in vivo 31-phosphorus neurospectroscopy studies is believed to index the anabolism of cell membrane phospholipids and has therefore been used to study phospholipid anabolism in the brain non-invasively. However, it is an indirect measure of phospholipid metabolism and although it does contain major contributions from phosphocholine, phosphoethanolamine and L-phosphoserine, which are important precursors of membrane phospholipids, many other metabolites, including sugar phosphates, can contribute to this region of the spectrum, and separation of these different peaks is not achieved with the present in vivo methodology. Recently, it has become possible to analyze signal directly from the cell membrane motion-restricted phospholipids by analysis of a broad resonance signal. We therefore hypothesized that there should be a positive correlation between the phosphomonoester narrow resonance and the broad resonance signal if the former does indeed index cell membrane phospholipid anabolism. Cerebral 31-phosphorus magnetic resonance spectroscopy was carried out in 54 human subjects, including normal volunteers and patients with schizophrenia in order to widen the range of phosphomonoester and broad resonance values. Spectra were obtained from 70x70x70mm(3) voxels using an image-selected in vivo spectroscopy pulse sequence. There was a highly significant positive correlation between the phosphomonoester resonances and the broad resonance signals (r=0.404, Panabolism in brain studies.

  7. Comparison of FRPE and Human Embryonic Stem Cell–Derived RPE Behavior on Aged Human Bruch's Membrane

    Science.gov (United States)

    Sugino, Ilene K.; Sun, Qian; Wang, Jianqiu; Nunes, Celia F.; Cheewatrakoolpong, Noounanong; Rapista, Aprille; Johnson, Adam C.; Malcuit, Christopher; Klimanskaya, Irina; Lanza, Robert

    2011-01-01

    Purpose. To compare RPE derived from human embryonic stem cells (hES-RPE) and fetal RPE (fRPE) behavior on human Bruch's membrane (BM) from aged and AMD donors. Methods. hES-RPE of 3 degrees of pigmentation and fRPE were cultured on BM explants. Explants were assessed by light, confocal, and scanning electron microscopy. Integrin mRNA levels were determined by real-time polymerase chain reaction studies. Secreted proteins in media were analyzed by multiplex protein analysis after 48-hour exposure at culture day 21. Results. hES-RPE showed impaired initial attachment compared to fRPE; pigmented hES-RPE showed nuclear densities similar to fRPE at day 21. At days 3 and 7, hES-RPE resurfaced BM to a limited degree, showed little proliferation (Ki-67), and partial retention of RPE markers (MITF, cytokeratin, and CRALBP). TUNEL-positive nuclei were abundant at day 3. fRPE exhibited substantial BM resurfacing at day 3 with decreased resurfacing at later times. Most fRPE retained RPE markers. Ki-67–positive nuclei decreased with time in culture. TUNEL staining was variable. Increased integrin mRNA expression did not appear to affect cell survival at day 21. hES-RPE and fRPE protein secretion was similar on equatorial BM except for higher levels of nerve growth factor and thrombospondin-2 (TSP2) by hES-RPE. On submacular BM, fRPE secreted more vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor, and platelet-derived growth factor; hES-RPE secreted more TSP2. Conclusions. Although pigmented hES-RPE and fRPE resurfaced aged and AMD BM to a similar, limited degree at day 21, cell behavior at earlier times was markedly dissimilar. Differences in protein secretion may indicate that hES-RPE may not function identically to native RPE after seeding on aged or AMD BM. PMID:21460262

  8. Evaluation of a PCR/DNA Probe Colorimetric Membrane Assay for Identification of Campylobacter spp. in Human Stool Specimens

    OpenAIRE

    Collins, Evelyn; Glennon, Maura; Hanley, Shirley; Murray, Anne-Marie; Cormican, Martin; Smith, Terry; Maher, Majella

    2001-01-01

    DNA was extracted from 50 human stool specimens using the QIAamp DNA stool minikit. PCR amplification was followed by post-PCR hybridization to DNA probes specific for the Campylobacter genus, Campylobacter jejuni, and Campylobacter coli in a colorimetric membrane assay. Thirty-two of 38 culture-positive specimens were PCR/DNA probe positive for C. jejuni. The assay is rapid and simple and can be applied to stool specimens for the detection of Campylobacter.

  9. Effects of darbepoetin injections on erythrocyte membrane transport protein expressions in humans

    DEFF Research Database (Denmark)

    Rentsch, R.; Damsgaard, Rasmus; Lundby, C.

    2006-01-01

    .05) during the injection period than in the preinjection period. Age separation experiments using self-creating Percoll gradients demonstrated a higher density of membrane transport proteins in young red blood cells. These data suggest that the NESP-induced increase in membrane transport proteins is caused...... in hematocrit, red cell volume, and reticulocyte fraction. The density of aquaporin 1 protein was higher (maximal increase +59%) (P ...The present study investigated the effects of injected darbepoetin [novel erythropoietin stimulating protein (NESP)] on the density of three erythrocyte membrane transport proteins: the lactate-H+ cotransporter (monocarboxylate transporter 1), the chloride/bicarbonate exchanger 1 (anion exchanger 1...

  10. 2DE maps in the discovery of human autoimmune kidney diseases: the case of membranous glomerulonephritis.

    Science.gov (United States)

    Bruschi, Maurizio; Santucci, Laura; Ghiggeri, Gian Marco; Candiano, Giovanni

    2015-01-01

    The identification of antigens in the autoimmune diseases is a primary point to elucidate the pathogenesis of disease. Here, we propose an "in vivo" proteomics approach to identify the antigens of auto-antibodies in membranous glomerulonephritis. In this approach, podocyte proteins resolved by two-dimensional electrophoresis were semidry blotted to nitrocellulose membrane. Then the antibodies eluted from microdissected glomeruli and serum samples were used as a probe for the detection of podocyte antigens and characterized by means of mass spectrometry. These combined methods allowed us to identify six new antigens in membranous glomerulonephritis.

  11. Activation of human dendritic cells is modulated by components of the outer membranes of Neisseria meningitidis.

    Science.gov (United States)

    Al-Bader, Tamara; Christodoulides, Myron; Heckels, John E; Holloway, Judith; Semper, Amanda E; Friedmann, Peter S

    2003-10-01

    Neisseria meningitidis serogroup B is a major cause of life-threatening meningitis and septicemia worldwide, and no effective vaccine is available. Initiation of innate and acquired immune responses to N. meningitidis is likely to be dependent on cellular responses of dendritic cells (DC) to antigens present in the outer membrane (OM) of the meningococcus. In this study, the responses of human monocyte-derived DC (mo-DC) to OM isolated from parent (lipopolysaccharide [LPS]-replete) meningococci and from a mutant deficient in LPS were investigated. Parent OM selectively up-regulated Toll-like receptor 4 (TLR4) mRNA expression and induced mo-DC maturation, as reflected by increased production of chemokines, proinflammatory cytokines, and CD83, CD80, CD86, CD40, and major histocompatibility complex (MHC) class II molecules. In contrast, LPS-deficient OM selectively up-regulated TLR2 mRNA expression and induced moderate increases in both cytokine production and expression of CD86 and MHC class II molecules. Preexposure to OM, with or without LPS, augmented the allostimulatory properties of mo-DC, which induced proliferation of naive CD4+ CD45RA+ T cells. In addition, LPS-replete OM induced a greater gamma interferon/interleukin-13 ratio in naive T cells, whereas LPS-deficient OM induced the reverse profile. These data demonstrate that components of the OM, other than LPS, are also likely to be involved in determining the levels of DC activation and the nature of the T-helper immune response.

  12. Cryopreserved human amniotic membrane injection for plantar fasciitis: a randomized, controlled, double-blind pilot study.

    Science.gov (United States)

    Hanselman, Andrew E; Tidwell, John E; Santrock, Robert D

    2015-02-01

    Treatment options for plantar fasciitis have resulted in varied patient outcomes. The aim of this study was to compare a novel treatment, cryopreserved human amniotic membrane (c-hAM), to a traditional treatment, corticosteroid. Our hypothesis was that c-hAM would be safe and comparable to corticosteroids for plantar fasciitis in regard to patient outcomes. A randomized, controlled, double-blind, single-center pilot study was completed. Patients were randomized into one of 2 treatment groups: c-hAM or corticosteroid. Patients received an injection at their initial baseline visit with an option for a second injection at their first 6-week follow-up. Total follow-up was obtained for 12 weeks after the most recent injection. The primary outcome measurement was the Foot Health Status Questionnaire (FHSQ). The secondary outcome measurements were the Visual Analog Scale (VAS) and verbally reported percentage improvement. Data were analyzed between groups for the 2 different cohorts (1 injection versus 2 injections). Twenty-three patients had complete follow-up. Fourteen were randomized to receive corticosteroid and 9 were randomized to receive c-hAM. Three patients in each group received second injections. With the numbers available, the majority of outcome measurements showed no statistical difference between groups. The corticosteroid did, however, have greater FHSQ shoe fit improvement (P = .0244) at 6 weeks, FHSQ general health improvement (P = .0132) at 6 weeks, and verbally reported improvement (P = .041) at 12 weeks in the one-injection cohort. Cryopreserved hAM had greater FHSQ foot pain improvement (P = .0113) at 18 weeks in the 2-injection cohort. Cryopreserved hAM injection may be safe and comparable to corticosteroid injection for treatment of plantar fasciitis. This is a pilot study and requires further investigation. Level I, prospective randomized trial. © The Author(s) 2014.

  13. Accommodative Movements of the Vitreous Membrane, Choroid, and Sclera in Young and Presbyopic Human and Nonhuman Primate Eyes

    Science.gov (United States)

    Croft, Mary Ann; Nork, T. Michael; McDonald, Jared P.; Katz, Alexander; Lütjen-Drecoll, Elke; Kaufman, Paul L.

    2013-01-01

    Purpose. We report, for the first time to our knowledge, dynamic movements of the vitreous membrane and peripheral choroid during accommodation, and age-related changes in the anterior sclera. Methods. We studied 11 rhesus monkeys (ages 6–27 years) and 12 human subjects (ages 19–65 years). Accommodation was induced pharmacologically in human subjects and by central electrical stimulation in the monkeys. Ultrasound biomicroscopy, endoscopy, and contrast agents were used to image various intraocular structures. Results. In the monkey, the anterior hyaloid membrane bows backward during accommodation in proportion to accommodative amplitude and lens thickening. A cleft exists between the pars plicata region and the anterior hyaloid membrane, and the cleft width increases during accommodation from 0.79 ± 0.01 mm to 1.01 ± 0.02 mm in young eyes (n = 2, P vitreous zonule, and the neighboring vitreous interconnected with the vitreous zonule. Among the humans, in the older eyes the scleral contour bowed inward in the region of the limbus, compared to the young eyes. Conclusions. The monkey anterior hyaloid bends posteriorly during accommodation in proportion to accommodative amplitude and the sclera bows inward with increasing age in both species. Future descriptions of the accommodative mechanism, and approaches to presbyopia therapy, may need to incorporate these findings. PMID:23745005

  14. /sup 125/I-human epidermal growth factor specific binding to placentas and fetal membranes from varoius pregnancy states

    Energy Technology Data Exchange (ETDEWEB)

    Hofmann, G.E.; Siddiqi, T.A.; Rao, Ch. V.; Carman, F.R.

    1988-01-01

    Specific binding of /sup 125/I-human epidermal growth factor (hEGF) to homogenates of term human placentas and fetal membranes from normal and appropriate for gestational age (N = 20), intrauterine growth retarded (N = 9), twin (N = 11), White class AB diabetic (N = 12), and large for gestational age (N = 13) pregnancies was measured. In all pregnancy states, placentas bound approximately four times more /sup 125/I-hEGF than did fetal membranes (P<0.0001). There was no significant differnce in /sup 125/I-hEGF binding to fetal membranes from the various pregnancy states (P<0.05). /sup 125/I-hEGF specific binding to placentas from intrauterine growth retarded or twin pregnancies was significantly greater compared with placentas from normal and appropriate for gestational age pregnancies (P<0.05). The binding to placentas from pregnancies complicated by White class AB diabetes or large for gestational age infants, on the other hand, was not significantly different from that to placentas from normal and appropriate for gestational age pregnancies. /sup 125/I-hEGF specific binding did not differ between placentas from intrauterine growth retarded or twin pregnancies (P<0.05). Placental and fetal membrane /sup 125/I-hEGF binding did not vary with fetal sex, maternal race, placental weight, or gestational age between 37 to 42 weeks (P<0.05). Placental but not fetal membrane /sup 125/I-hEGF binding increased with increasing infant weight when appropriate for gestational age and large for gestational age infants were included (P<0.05, r = 0.38, N = 32) but not for intrauterine growth retarded, appropriate for gestational age, or large for gestational age infants alone.

  15. Characterization of Membrane Integrity and Morphological Stability of Human Salivary Exosomes

    National Research Council Canada - National Science Library

    Kumeda, Nahoko; Ogawa, Yuko; Akimoto, Yoshihiro; Kawakami, Hayato; Tsujimoto, Masafumi; Yanoshita, Ryohei

    2017-01-01

    .... Moreover, intact exosomes could be isolated from whole saliva that had been stored at 4°C. Membrane disruption with detergents such as Triton X-100 and Nonidet P-40 caused partial solubilization of DPP IV and release...

  16. Localized ridge defect augmentation using human pericardium membrane and demineralized bone matrix

    Directory of Open Access Journals (Sweden)

    Arun Kumar Vidyadharan

    2014-01-01

    Conclusion and Clinical Implications: The results suggested that HP Allograft membrane may be a suitable component for augmentation of localized alveolar ridge defects in conjunction with DBM with bone chips.

  17. Exercise-induced increase in glucose transport, GLUT-4, and VAMP-2 in plasma membrane from human muscle

    DEFF Research Database (Denmark)

    Kristiansen, S; Hargreaves, Mark; Richter, Erik

    1996-01-01

    contractions may induce trafficking of GLUT-4-containing vesicles via a mechanism similar to neurotransmitter release. Our results demonstrate for the first time exercise-induced translocation of GLUT-4 and VAMP-2 to the plasma membrane of human muscle and increased sarcolemmal glucose transport.......A major effect of muscle contractions is an increase in sarcolemmal glucose transport. We have used a recently developed technique to produce sarcolemmal giant vesicles from human muscle biopsy samples obtained before and after exercise. Six men exercised for 10 min at 50% maximal O2 uptake (Vo2max...

  18. Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 in a fluorescence-based membrane potential assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Bräuner-Osborne, Hans

    2004-01-01

    We have expressed the human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 stably in HEK293 cells and characterized the transporters pharmacologically in a conventional [(3) H]-d-aspartate uptake assay and in a fluorescence-based membrane potential assay, the FLIPR Membrane Potential (...

  19. The human selenoprotein VCP-interacting membrane protein (VIMP) is non-globular and harbors a reductase function in an intrinsically disordered region

    DEFF Research Database (Denmark)

    Christensen, Lea Cecilie; Jensen, Njal Winther; Lages Lino Vala, Andrea

    2012-01-01

    The human selenoprotein VIMP (VCP-interacting membrane protein)/SelS (selenoprotein S) localizes to the endoplasmic reticulum (ER) membrane and is involved in the process of ER-associated degradation (ERAD). To date, little is known about the presumed redox activity of VIMP, its structure and how...

  20. Analysis of retinal pigment epithelium integrin expression and adhesion to aged submacular human Bruch's membrane.

    OpenAIRE

    Zarbin, Marco A.

    2003-01-01

    PURPOSE: Uncultured aged retinal pigment epithelium (RPE) does not resurface aged Bruch's membrane after 24 hours in organ culture. These experiments assess whether culturing alters RPE integrin expression and resurfacing of Bruch's membrane. METHODS: RNA was isolated from uncultured and cultured RPE of aged adult donor and fetal eyes. Integrin subunit messenger RNA (mRNA) expression was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and semiquantitative analysis of the a...

  1. Erythrocyte membrane fluidity and indices of plasmatic oxidative damage after acute physical exercise in humans.

    Science.gov (United States)

    Berzosa, C; Gómez-Trullén, E M; Piedrafita, E; Cebrián, I; Martínez-Ballarín, E; Miana-Mena, F J; Fuentes-Broto, L; García, J J

    2011-06-01

    Optimal levels of membrane fluidity are essential for numerous cell functions including cell growth, solute transport and signal transduction. Since exercise enhances free radical production, our aim was to evaluate in healthy male subjects the effects of an acute bout of maximal and submaximal exercise on the erythrocyte membrane fluidity and its possible relation to the oxidative damage overproduction due to exercise. Subjects (n = 34) performed three cycloergometric tests: a continuous progressive exercise, a strenuous exercise until exhaustion and an acute bout of exercise at an intensity corresponding to 70% of maximal work capacity for 30 min. Venous blood samples were collected before and immediately after these exercises. Erythrocyte membrane fluidity was assessed by fluorescence spectroscopy. Plasma malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations and carbonyl content of plasmatic proteins were used as an index of lipid and protein oxidation, respectively. Exercise produced a dramatic drop in the erythrocyte membrane fluidity as compared to resting time, but this was not accompanied by significant changes in the plasmatic MDA and 4-HDA concentrations. The highest erythrocyte membrane rigidity was detected immediately after strenuous exercise until exhaustion was performed. Protein carbonyl levels were higher after exhaustive exercises than at rest. Continuous progressive and strenuous exercises until exhaustion, but not submaximal workload, resulted in a significant enhanced accumulation of carbonylated proteins in the plasma. These findings are consistent with the idea that exercise exaggerates oxidative damage, which may contribute, at least partially, to explain the rigidity in the membrane of the erythrocytes due to acute exercise.

  2. Protective Effect of Sundakai (Solanum torvum) Seed Protein (SP) Against Oxidative Membrane Damage in Human Erythrocytes.

    Science.gov (United States)

    Sivapriya, M; Gowda, S S Thammanna; Srinivas, Leela

    2015-12-01

    Lipid peroxidation by ROS at the membrane level disturbs the inherit integrity of components activating subsequent alterations in the function. In this study, the protective effect of purified Sundakai (Solanum torvum) seed protein (SP) was tested against oxidative membrane damage in erythrocyte membrane. SP prevented oxidative RBC lysis induced by pro-oxidants; Fe:As (2:20 μmol), periodate (0.4 mM), and t-BOOH (1 mM) up to 86, 81, and 86 %, respectively. Further, SP prevented the Fe:As-induced K(+) leakage up to the tune of 95 %. The inhibition offered by SP on K(+) leakage was comparable to inhibition offered by quinine sulfate, a known K(+) channel blocker. SP dose dependently restored Na(+)K(+) ATPase and Ca(2+)Mg(2+) ATPase activities in erythrocyte membrane. The restoration of ATPase activity by SP was two times more than standard antioxidants BHA and α-tocopherol. Besides, SP at 1.6 μmol restored the membrane proteins over Fe:As induction when analyzed by SDS-PAGE, which was comparable to protection offered by BHA. In conclusion, SP is an effective antioxidant in preventing oxidative membrane damage and associated functions mediated by ROS. As SP is non-toxic, it can be used as an effective bioprotective antioxidant agent to cellular components.

  3. [Procedural guidelines. Good practice procedures for acquisition and preparation of cryopreserved human amniotic membranes from donor placentas].

    Science.gov (United States)

    Hahn, A; Thanos, M; Reinhard, T; Seitz, B; Steuhl, K-P; Meller, D

    2010-11-01

    A cornea/tissue bank must have an organizational structure in which responsibility and authority to issue directives are clearly defined. It must also use a documented quality management system on the basis of good practice procedures which is maintained to the current standards. The personnel of a cornea/tissue bank must be present in sufficient numbers and be suitably qualified. A cornea/tissue bank must be in possession of appropriate facilities which are suitable for the main purpose of preparation of cryopreserved human amniotic membranes from donor placentas. All equipment must be designed and maintained corresponding to the intended purpose. Deviations from the stipulated quality and safety standards must give rise to documented investigations which include decisions on options for correctional and preventive measures. Acquisition of donors and tissue sampling must be strictly controlled and documented. This also applies to entry of donor tissue in the cornea/tissue bank. Cryopreserved human amniotic membranes can only be preserved from donors undergoing caesarean section and who did not present any known infection of the abdominal cavity or any systemic blood borne infection. Contamination of media used for cryopreservation of donor placenta must be ruled out at least once. Measures must be taken to keep the risk of contamination as low as possible. Cryopreserved human amniotic membranes from donor placentas can only be released if defined criteria are fulfilled. Any suspicion of severe undesired reactions and events for the recipient of an amniotic membrane transplant must be registered with the authorities. The activities of a cornea/tissue bank must maintain and adapt to the state-of-the-art with respect to scientific progress.

  4. Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

    Directory of Open Access Journals (Sweden)

    Campbell Kevin P

    2007-08-01

    Full Text Available Abstract Background Polymorphonuclear neutrophils (PMN constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. Results To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP and dysferlin were further validated by immunoblot analysis. Conclusion Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.

  5. Transforming growth factor-beta isoforms regulate the surface expression of membrane cofactor protein (CD46) and CD59 on human keratinocytes [corrected

    NARCIS (Netherlands)

    Pasch, M. C.; Bos, J. D.; Daha, M. R.; Asghar, S. S.

    1999-01-01

    We studied the regulation of the expression of complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF) and CD59, on human keratinocytes by supernatant of activated mononuclear cells and by some individual cytokines present therein. Cultured keratinocytes

  6. A clinical evaluation of a bioresorbable membrane and porous hydroxyapatite in the treatment of human molar class II furcations

    Directory of Open Access Journals (Sweden)

    K Gita Malathi

    2013-01-01

    Full Text Available Background: The ultimate goal of periodontal therapy is predictable regeneration of a functional attachment apparatus destroyed as a result of periodontitis. Reconstructive procedures have been used with varying success during the past decades to accomplish this goal. Aim: To evaluate whether the use of porous hydroxyapatite alone or a bioresorbable membrane alone would enhance the clinical results in the treatment of class II furcation defects in human lower molars. Materials and Methods: Fifteen patients with chronic periodontitis, aged between 39 and 49 years, with a pair of similar bilateral class II furcation defects (classification of Hamp et al. in mandibular first molars were selected. A split-mouth design was incorporated and the selected 30 furcation defects were assigned to one of the two treatment groups, i.e., Group I treated with a bioresorbable membrane from bovine-derived collagen guided tissue regeneration membrane and Group II treated using porous hydroxyapatite bone graft material on the contralateral sides. Evaluation of clinical parameters, probing depths and attachment levels, and radiographs was done preoperatively and 6 months postoperatively. Results: Both the groups showed statistically significant mean reduction in probing depths and gain in clinical attachment levels and linear bone fill. Comparison between Group I and Group II showed insignificant difference. Conclusion: Within the limits of this study, both the treatment modalities are beneficial for the treatment of human mandibular class II furcation defects.

  7. Thermal dielectroscopy - A new method for studying the membrane skeleton of human erythrocytes

    Science.gov (United States)

    Paarvanova, Boyana; Tacheva, Bilyana; Karabaliev, Miroslav; Ivanov, Ivan T.

    2017-11-01

    The structure and mechanical properties of erythrocyte plasma membrane are strongly affected by both the dephosphorylation and thermal denaturation (49.5°C) of erythrocyte under-membrane spectrin skeleton. Here, the dielectric loss (DL) of suspensions, containing native erythrocytes or erythrocyte ghost membranes (EGMs), was determined applying a mathematical method to remove the conductive loss from the imaginary capacitance, Cim, of the suspensions. The DL frequency profile of spectrin skeleton was obtained subtracting the DL data collected prior to, and after the denaturation of spectrin at 49.5°C. Spectrin skeleton exhibited narrow bell-shaped DL frequency curve, centered at 1.5 MHz, presumably reflecting the segmental mobility of spectrin. The area of this curve was reduced by 30 % after mild dephosphorylation (starvation of erythrocytes at 37°C for 5 h) and reduced to zero at EGMs resealed with alkaline phosphatase (full dephosphorylation). These results, combined with others, indicate the relevance of dielectric analysis for the study of dynamics and separation of membrane skeleton from the lipid membrane of erythrocytes.

  8. Outer Membrane Proteome of Veillonella parvula: A Diderm Firmicute of the Human Microbiome

    Directory of Open Access Journals (Sweden)

    Daniel I. Poppleton

    2017-06-01

    Full Text Available Veillonella parvula is a biofilm-forming commensal found in the lungs, vagina, mouth, and gastro-intestinal tract of humans, yet it may develop into an opportunistic pathogen. Furthermore, the presence of Veillonella has been associated with the development of a healthy immune system in infants. Veillonella belongs to the Negativicutes, a diverse clade of bacteria that represent an evolutionary enigma: they phylogenetically belong to Gram-positive (monoderm Firmicutes yet maintain an outer membrane (OM with lipopolysaccharide similar to classic Gram-negative (diderm bacteria. The OMs of Negativicutes have unique characteristics including the replacement of Braun's lipoprotein by OmpM for tethering the OM to the peptidoglycan. Through phylogenomic analysis, we have recently provided bioinformatic annotation of the Negativicutes diderm cell envelope. We showed that it is a unique type of envelope that was present in the ancestor of present-day Firmicutes and lost multiple times independently in this phylum, giving rise to the monoderm architecture; however, little experimental data is presently available for any Negativicutes cell envelope. Here, we performed the first experimental proteomic characterization of the cell envelope of a diderm Firmicute, producing an OM proteome of V. parvula. We initially conducted a thorough bioinformatics analysis of all 1,844 predicted proteins from V. parvula DSM 2008's genome using 12 different localization prediction programs. These results were complemented by protein extraction with surface exposed (SE protein tags and by subcellular fractionation, both of which were analyzed by liquid chromatography tandem mass spectrometry. The merging of proteomics and bioinformatics results allowed identification of 78 OM proteins. These include a number of receptors for TonB-dependent transport, the main component of the BAM system for OM protein biogenesis (BamA, the Lpt system component LptD, which is responsible for

  9. Progesterone receptor membrane component 1 deficiency attenuates growth while promoting chemosensitivity of human endometrial xenograft tumors.

    Science.gov (United States)

    Friel, Anne M; Zhang, Ling; Pru, Cindy A; Clark, Nicole C; McCallum, Melissa L; Blok, Leen J; Shioda, Toshi; Peluso, John J; Rueda, Bo R; Pru, James K

    2015-01-28

    Endometrial cancer is the leading gynecologic cancer in women in the United States with 52,630 women predicted to be diagnosed with the disease in 2014. The objective of this study was to determine if progesterone (P4) receptor membrane component 1 (PGRMC1) influenced endometrial cancer cell viability in response to chemotherapy in vitro and in vivo. A lentiviral-based shRNA knockdown approach was used to generate stable PGRMC1-intact and PGRMC1-deplete Ishikawa endometrial cancer cell lines that also lacked expression of the classical progesterone receptor (PGR). Progesterone treatment inhibited mitosis of PGRMC1-intact, but not PGRMC1-deplete cells, suggesting that PGRMC1 mediates the anti-mitotic actions of P4. To test the hypothesis that PGRMC1 attenuates chemotherapy-induced apoptosis, PGRMC1-intact and PGRMC1-deplete cells were treated in vitro with vehicle, P4 (1 µM), doxorubicin (Dox, 2 µg/ml), or P4 + Dox for 48 h. Doxorubicin treatment of PGRMC1-intact cells resulted in a significant increase in cell death; however, co-treatment with P4 significantly attenuated Dox-induced cell death. This response to P4 was lost in PGRMC1-deplete cells. To extend these observations in vivo, a xenograft model was employed where PGRMC1-intact and PGRMC1-deplete endometrial tumors were generated following subcutaneous and intraperitoneal inoculation of immunocompromised NOD/SCID and nude mice, respectively. Tumors derived from PGRMC1-deplete cells grew slower than tumors from PGRMC1-intact cells. Mice harboring endometrial tumors were then given three treatments of vehicle (1:1 cremophor EL: ethanol + 0.9% saline) or chemotherapy [Paclitaxel (15 mg/kg, i.p.) followed after an interval of 30 minutes by CARBOplatin (50 mg/kg)] at five day intervals. In response to chemotherapy, tumor volume decreased approximately four-fold more in PGRMC1-deplete tumors when compared with PGRMC1-intact control tumors, suggesting that PGRMC1 promotes tumor cell viability

  10. Hyperpolarization of the Membrane Potential Caused by Somatostatin in Dissociated Human Pituitary Adenoma Cells that Secrete Growth Hormone

    Science.gov (United States)

    Yamashita, Naohide; Shibuya, Naohiko; Ogata, Etsuro

    1986-08-01

    Membrane electrical properties and the response to somatostatin were examined in dissociated human pituitary adenoma cells that secrete growth hormone (GH). Under current clamp condition with a patch electrode, the resting potential was -52.4 ± 8.0 mV, and spontaneous action potentials were observed in 58% of the cells. Under voltage clamp condition an outward K+ current, a tetrodotoxin-sensitive Na+ current, and a Ca2+ current were observed. Cobalt ions suppressed the Ca2+ current. The threshold of Ca2+ current activation was about -60 mV. Somatostatin elicited a membrane hyperpolarization associated with increased membrane permeability in these cells. The reversal potential of somatostatin-induced hyperpolarization was -78.4 ± 4.3 mV in 6 mM K+ medium and -97.2 ± 6.4 mV in 3 mM K+ medium. These reversal potential values and a shift with the external K+ concentration indicated that membrane hyperpolarization was caused by increased permeability to K+. The hyperpolarized membrane potential induced by somatostatin was -63.6 ± 5.9 mV in the standard medium. This level was subthreshold for Ca2+ and Na+ currents and was sufficient to inhibit spontaneous action potentials. Hormone secretion was significantly suppressed by somatostatin and cobalt ions. Therefore, we suggest that Ca2+ entering the cell through voltage-dependent channels are playing an important role for GH secretion and that somatostatin suppresses GH secretion by blocking Ca2+ currents. Finally, we discuss other possibilities for the inhibitory effect of somatostatin on GH secretion.

  11. Human skin basement membrane-associated heparan sulphate proteoglycan: distinctive differences in ultrastructural localization as a function of developmental age

    DEFF Research Database (Denmark)

    Horiguchi, Y; Fine, J D; Couchman, J R

    1991-01-01

    at different developmental ages using two monoclonal antibodies to a well-characterized basement membrane-associated heparan sulphate proteoglycan. A series of foetal skin specimens (range, 54-142 gestational days) were examined using an immunoperoxidase immunoelectron microscopic technique. In specimens...... representing very early developmental ages, very diffuse immunoreaction products were detected. However, by approximately 76 gestational days, some accentuation of heparan sulphate proteoglycan was noted along the lamina densa, and by 142 gestational days, the distribution of heparan sulphate proteoglycan...... was identical to that observed in neonatal and adult human skin. These findings demonstrate that active remodelling of the dermo-epidermal junction occurs during at least the first two trimesters, and affects not only basement membrane-associated structures but also specific antigens....

  12. Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells

    DEFF Research Database (Denmark)

    Melo-Braga, Marcella Nunes; Schulz, Melanie; Liu, Qiuyue

    2014-01-01

    Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important...... in cellular signaling and recognition, and their function and activity are frequently regulated by post-translational modifications such as phosphorylation and glycosylation. To obtain information about membrane-associated proteins and their modified amino acids potentially involved in changes of h...... in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development...

  13. The human gonadotropin releasing hormone type I receptor is a functional intracellular GPCR expressed on the nuclear membrane.

    Directory of Open Access Journals (Sweden)

    Michelle Re

    Full Text Available The mammalian type I gonadotropin releasing hormone receptor (GnRH-R is a structurally unique G protein-coupled receptor (GPCR that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME. Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.

  14. Acetylsalicylic acid (aspirin) and salicylic acid interaction with the human erythrocyte membrane bilayer induce in vitro changes in the morphology of erythrocytes.

    Science.gov (United States)

    Suwalsky, Mario; Belmar, Jessica; Villena, Fernando; Gallardo, María José; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

    2013-11-01

    Despite the well-documented information, there are insufficient reports concerning the effects of salicylate compounds on the structure and functions of cell membranes, particularly those of human erythrocytes. With the aim to better understand the molecular mechanisms of the interaction of acetylsalicylic acid (ASA) and salicylic acid (SA) with cell membranes, human erythrocyte membranes and molecular models were utilized. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of ASA and SA to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction while DMPC unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. Moreover, we took advantage of the capability of differential scanning calorimetry (DSC) to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from ASA and SA interaction with PC and PE molecules. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy, while isolated unsealed human erythrocyte membranes (IUM) were studied by fluorescence spectroscopy. Results indicated that both salicylates interact with human erythrocytes and their molecular models in a concentration-dependent manner perturbing their bilayer structures. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Clusterin facilitates exchange of glycosyl phosphatidylinositol-linked SPAM1 between reproductive luminal fluids and mouse and human sperm membranes.

    Science.gov (United States)

    Griffiths, Genevieve S; Galileo, Deni S; Aravindan, Rolands G; Martin-DeLeon, Patricia A

    2009-09-01

    Glycosyl phosphatidylinositol (GPI)-linked proteins, which are involved in post-testicular maturation of sperm and have a role in fertilization, are acquired on the sperm surface from both vesicular and membrane-free soluble fractions of epididymal luminal fluid (LF) and uterine LF. Herein, we investigate the mechanism of uptake of these proteins from the soluble fraction of LFs using sperm adhesion molecule 1 (SPAM1) as a model. Ultracentrifugation and native Western blot analysis of the soluble fraction revealed that SPAM1 is present in low-molecular-weight (monomeric) and high-molecular-weight (oligomeric) complexes. The latter are incapable of transferring SPAM1 and may serve to produce monomers. Monomers are stabilized by hydrophobic interactions with clusterin (CLU), a lipid carrier that is abundantly expressed in LFs. We show that CLU is involved in the transfer of SPAM1 monomers, whose delivery was decreased by anti-CLU antibody under normal and apolipoprotein-enhanced conditions. Coimmunoprecipitation revealed an intimate association of CLU with SPAM1. Both plasma and recombinant CLU had a dose-related effect on transfer efficiency: high concentrations reduced and low concentrations enhanced delivery of SPAM1 to human and mouse sperm membranes, reflecting physiological states in the epididymal tract. We propose a lipid exchange model (akin to the lipid-poor model for cholesterol efflux) for the delivery of GPI-linked proteins to sperm membranes via CLU. Our investigation defines specific conditions for membrane-free GPI-linked protein transfer in vitro and could lead to technology for improving fertility or treating sperm pathology by the addition of relevant GPI-linked proteins critical for successful fertilization in humans and domestic animals.

  16. Effects of prolonged recombinant human erythropoietin administration on muscle membrane transport systems and metabolic marker enzymes

    DEFF Research Database (Denmark)

    Juel, C; Thomsen, J J; Rentsch, R L

    2007-01-01

    performance by approximately 54%. Membrane transport systems and carbonic anhydrases involved in pH regulation remained unchanged. Of the Na(+), K(+)-pump isoforms only the density of the alpha2 subunit was decreased (by 22%) after treatment. The marker enzymes cytochrom c and hexokinase remained unchanged...

  17. Expression of Plasmodium falciparum erythrocyte membrane protein 1 in experimentally infected humans

    DEFF Research Database (Denmark)

    Lavstsen, Thomas; Magistrado, Pamela; Hermsen, Cornelus C

    2005-01-01

    -encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which is expressed on the surface of infected erythrocytes where it mediates binding to endothelial receptors. Thus, severe malaria may be caused by parasites expressing PfEMP1 variants that afford parasites optimal sequestration...

  18. Molecular cloning, expression, and chromosomal localization of the gene encoding a human myeloid membrane antigen (gp150).

    Science.gov (United States)

    Look, A T; Peiper, S C; Rebentisch, M B; Ashmun, R A; Roussel, M F; Lemons, R S; Le Beau, M M; Rubin, C M; Sherr, C J

    1986-10-01

    DNA from a tertiary mouse cell transformant containing amplified human sequences encoding a human myeloid membrane glycoprotein, gp150, was used to construct a bacteriophage lambda library. A single recombinant phage containing 12 kilobases (kb) of human DNA was isolated, and molecular subclones were then used to isolate the complete gp150 gene from a human placental genomic DNA library. The intact gp150 gene, assembled from three recombinant phages, proved to be biologically active when transfected into NIH 3T3 cells. Molecular probes from the gp150 locus annealed with a 4.0-kb polyadenylated RNA transcript derived from human myeloid cell lines and from tertiary mouse cell transformants. The gp150 gene was assigned to human chromosome 15, and was subchromosomally localized to bands q25-26 by in situ hybridization. The chromosomal location of the gp150 gene coincides cytogenetically with the region assigned to the c-fes proto-oncogene, another human gene specifically expressed by myeloid cells.

  19. Recombinant human melatonin receptor MT1 isolated in mixed detergents shows pharmacology similar to that in mammalian cell membranes.

    Directory of Open Access Journals (Sweden)

    Christel Logez

    Full Text Available The human melatonin MT1 receptor-belonging to the large family of G protein-coupled receptors (GPCRs-plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.

  20. Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64.

    Directory of Open Access Journals (Sweden)

    Yi-Chang Liu

    Full Text Available Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen.

  1. Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64.

    Science.gov (United States)

    Liu, Yi-Chang; Lin, I-Hsuan; Chung, Wei-Jen; Hu, Wensi S; Ng, Wailap Victor; Lu, Chi-Yu; Huang, Tsung-Yen; Shu, Hung-Wei; Hsiao, Kwang-Jen; Tsai, Shih-Feng; Chang, Chuan-Hsiung; Lin, Chao-Hsiung

    2012-01-01

    Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen.

  2. Indications and Outcomes of Amniotic Membrane Transplantation

    Directory of Open Access Journals (Sweden)

    Alime Güneş

    2014-03-01

    Full Text Available Objectives: To investigate the indications and outcomes of amniotic membrane transplantation (AMT. Materials and Methods: A total of 87 eyes who underwent amnion membrane transplantation in our clinic between February 2010 and April 2013 were included in this study. Mean follow-up period was 7.49±7.84 months (range 1-29 months. Amnion membrane defrosted at room temperature and washed three times with saline covered all over the cornea independent of the position and size of the lesion and was sutured to the peripheral cornea circumferentially by 10/0 monofilament suture. At the end of the operation, therapeutic contact lens was placed. All cases were evaluated with respect to duration of surface epithelial healing, visual acuity, presence of recurrence, and infection. Results: Forty-six of the 87 patients (52.8% were men and 41 (47.1% were women. The mean age was 57.02±19.74 years (range 31-89 years in men and 66.69±16.56 years (range 8-88 years in women. The indications were: ulcers in 27 eyes, keratitis in 24 eyes, topical anesthetic abuse in 10 eyes, bullous keratopathy in 7 eyes, conjunctival mass in 7 eyes, symblepharon in 3 eyes, chemical burns in 3 eyes, pterygium in 2 eyes, endophthalmitis in 2 eyes, dellen in 1 eye, and conjunctival laceration in 1 eye. After AMT, 12 patients required second AMT, and 2 cases required third AMT. 2 eyes were eviscerated, 1 eye was exenterated. At the final follow-up visit, except for patients who underwent evisceration and exenteration, improved visual acuity was observed in 45 of the 84 eyes (53.5%. The average of healing time was between 4 and 6 weeks. No infectious, inflammatory, immunologic, or toxic/allergic reactions related to AMT was observed. Conclusion: Amniotic membrane transplantation is a safe and effective technique in ocular surface diseases. (Turk J Ophthalmol 2014; 44: 123-6

  3. Aberrant expression of novel and previously described cell membrane markers in human breast cancer cell lines and tumors.

    Science.gov (United States)

    Huang, Huayi; Groth, Jeff; Sossey-Alaoui, Khalid; Hawthorn, Lesleyann; Beall, Stephanie; Geradts, Joseph

    2005-06-15

    In a previous gene expression array study, we identified some 300 genes that were differentially expressed in human epidermal growth factor receptor tyrosine kinase 2 (HER2)-positive versus HER2-negative breast cancer cells. We have now done validation experiments on a group of three cell membrane components that had previously not been implicated in breast cancer. We also studied the expression of three other cell membrane proteins known to play a role in mammary neoplasia. By immunohistochemistry, we examined up to 130 archival breast carcinomas for Celsr2, E-cadherin, Kai1, and CD9 expression. The expression levels of NET-6 and TROP-2 were determined by quantitative reverse transcription-PCR in a subset of frozen tumors. We also studied fresh pellets and paraffin-embedded cell buttons of nine human breast cell lines. The relationship between the expression of all six membrane proteins and a variety of pathologic and biological variables, including estrogen receptor, HER2, and epidermal growth factor receptor status, was also examined. The NET-6 gene was transfected into a low-expressing cell line, and the effect on cellular morphology, growth, and invasion in vitro was recorded. Celsr2 was down-regulated in one cell line and in 7% of breast cancers. E-cadherin, Kai1, and CD9 were down-regulated in 35%, 76%, and 79% of tumors, respectively, confirming the important role of these markers in human mammary neoplasia. In breast cancer cell lines and tissues, TROP-2 was generally expressed at low levels, although a few specimens showed relative overexpression. NET-6 levels were lower in HER2-negative breast carcinoma cells. In addition, NET-6 was markedly down-regulated in estrogen receptor-negative breast cancers, and expression was lowest in "basal-like" tumors. Ectopic expression of NET-6 in low-expressing MDA-MB-231 cells altered cellular morphology, inhibited growth in vitro, and decreased invasion in a Boyden chamber assay. We have confirmed the expression of

  4. Effects of Δ(9)-tetrahydrocannabinol (THC) on human amniotic epithelial cell proliferation and migration.

    Science.gov (United States)

    Yao, J L; He, Q Z; Liu, M; Chang, X W; Wu, J T; Duan, T; Wang, K

    2018-02-01

    The deleterious effects of cannabis consumption for fertility and pregnancy outcome are recognized for years. The main psychoactive molecule of cannabis, Δ(9)-tetrahydrocannabinol (THC) is able to cross the placenta barrier and cause alterations in fetal growth, low birth weight and preterm labor. However, the effects of THC on the human placenta amnion are still unknown. The distributions of CB1R and CB2R in human amnion tissues were observed by immunohistochemistry (IHC). Human amniotic epithelial cell proliferation and migration in response to THC treatment were measured by MTS and transwell assays, respectively. The PCR array was performed to study the key regulators involved in the cell migration. The protein levels of CB1R, CB2R in amnion tissues and MMP2, MMP9 in cells were detected by western blotting. Small interfering RNAs (siRNAs) were used to knockdown MMP2 and MMP9 in WISH cells. Our results indicated that both CB1R and CB2R primarily identified in the epithelial layer of human placental amnion tissue. The CB1R expression in the amnion tissue was higher in the preterm group than normal control. High-dose of THC (30uM, but not 20 and 10uM) significantly inhibited (p<0.01) human amniotic epithelial cell lines (WISH) proliferation. Meanwhile, THC at both 10uM and 20uM (p<0.05) significantly suppressed cells migration in both WISH and primary human amniotic epithelial cells. The PCR array data and siRNA experiments demonstrated that MMP2/9 were tightly involved in the regulation of THC-inhibited cell migration in WISH cells. These results suggested that THC inhibited the migration of human amniotic epithelial cell through the regulation of MMP2 and MMP9, which in turn altered the development of the amnion during the gestation and partially resulted in preterm labor and other adverse pregnancy outcomes. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Comprehensive Characterization of Mesenchymal Stem Cells from Human Placenta and Fetal Membrane and Their Response to Osteoactivin Stimulation

    Directory of Open Access Journals (Sweden)

    C. M. Raynaud

    2012-01-01

    Full Text Available Mesenchymal stem cells (MSCs are the most promising seed cells for cell therapy and can be isolated from various sources of human adult tissues such as bone marrow (BM-MSC and adipose tissue. However, cells from these tissues must be obtained through invasive procedures. We, therefore, characterized MSCs isolated from fresh placenta (Pl-MSC and fetal membrane (Mb-MSC through morphological and fluorescent-activated cell sorting (FACS. MSC frequency is higher in membrane than placenta (2.14%  ± 0.65 versus 15.67%  ± 0.29%. Pl/Mb-MSCs in vitro expansion potential was significantly higher than BM-MSCs. We demonstrated that one of the MSC-specific marker is sufficient for MSC isolation and that culture in specific media is the optimal way for selecting very homogenous MSC population. These MSCs could be differentiated into mesodermal cells expressing cell markers and cytologic staining consistent with mature osteoblasts and adipocytes. Transcriptomic analysis and cytokine arrays demonstrated broad similarity between placenta- and membrane-derived MSCs and only discrete differences with BM-MSCs with enrichment of networks involved in bone differentiation. Pl/Mb-MSCs displayed higher osteogenic differentiation potential than BM-MSC when their response to osteoactivin was evaluated. Fetal-tissue-derived mesenchymal cells may, therefore, be considered as a major source of MSCs to reach clinical scale banking in particular for bone regeneration.

  6. Activity increase after extraction of alkaline phosphatase from human osteoblastic membranes by nonionic detergents: influence of age and sex.

    Science.gov (United States)

    Bourrat, C; Radisson, J; Chavassieux, P; Azzar, G; Roux, B; Meunier, P J

    2000-01-01

    The solubilization of alkaline phosphatase (AP) from osteoblastic cell membranes obtained from human primary bone cell cultures was studied according to the age and sex of the donors (17 females, 11 males; age range: 2-77 years). Cell membranes were treated by non-ionic (n-octyl beta-D-glucopyranoside, OG), ionic or zwitterionic detergents, then centrifuged. When OG was used almost all the AP was solubilized. AP activity in supernatant of solubilization was compared to the activity of the suspension before centrifugation. The activity ratio (AR) increased in function of age for subjects between 65 and 74. Neither total nor specific AP activities were influenced by age or sex. Electrophoresis studies showed that the AP released was a GPI (glycosyl phosphatidylinositol)-anchored protein, amphipathic form, with 140 kDa as apparent molecular mass. The activity change of AP in the presence of OG may result from age-related modifications either in the AP structure or in the constituents of the plasma membranes (proteins or phospholipids).

  7. New low-flux mixed matrix membranes that offer superior removal of protein-bound toxins from human plasma

    Science.gov (United States)

    Pavlenko, Denys; van Geffen, Esmée; van Steenbergen, Mies J.; Glorieux, Griet; Vanholder, Raymond; Gerritsen, Karin G. F.; Stamatialis, Dimitrios

    2016-10-01

    Hemodialysis is a widely available and well-established treatment for patients with End Stage Renal Disease (ESRD). However, although life-sustaining, patient mortality rates are very high. Several recent studies corroborated the link between dialysis patients’ outcomes and elevated levels of protein-bound uremic toxins (PBUT) that are poorly removed by conventional hemodialysis. Therefore, new treatments are needed to improve their removal. Recently, our group showed that the combination of dialysis and adsorption on one membrane, the mixed matrix membrane (MMM), can effectively remove those toxins from human plasma. However, these first MMMs were rather large in diameter and their mass transport characteristics needed improvement before application in the clinical setting. Therefore, in this study we developed a new generation of MMMs that have a smaller diameter and optimized characteristics offering superior ability in removing the PBUT indoxyl sulfate (IS) and p-cresyl sulfate (pCS) in comparison to first generation MMMs (30 and 125% respectively), as well as, a commercial dialysis membrane (more than 100% better removal).

  8. Optimisation of over-expression in E. coli and biophysical characterisation of human membrane protein synaptogyrin 1.

    Directory of Open Access Journals (Sweden)

    Christian Löw

    Full Text Available Progress in functional and structural studies of integral membrane proteins (IMPs is lacking behind their soluble counterparts due to the great challenge in producing stable and homogeneous IMPs. Low natural abundance, toxicity when over-expressed and potential lipid requirements of IMPs are only a few reasons for the limited progress. Here, we describe an optimised workflow for the recombinant over-expression of the human tetraspan vesicle protein (TVP synaptogyrin in Escherichia coli and its biophysical characterisation. TVPs are ubiquitous and abundant components of vesicles. They are believed to be involved in various aspects of the synaptic vesicle cycle, including vesicle biogenesis, exocytosis and endocytotic recycling. Even though TVPs are found in most cell types, high-resolution structural information for this class of membrane proteins is still missing. The optimisation of the N-terminal sequence of the gene together with the usage of the recently developed Lemo21(DE3 strain which allows the balancing of the translation with the membrane insertion rate led to a 50-fold increased expression rate compared to the classical BL21(DE3 strain. The protein was soluble and stable in a variety of mild detergents and multiple biophysical methods confirmed the folded state of the protein. Crosslinking experiments suggest an oligomeric architecture of at least four subunits. The protein stability is significantly improved in the presence of cholesteryl hemisuccinate as judged by differential light scattering. The approach described here can easily be adapted to other eukaryotic IMPs.

  9. Human corneal basement membrane heterogeneity: topographical differences in the expression of type IV collagen and laminin isoforms.

    Science.gov (United States)

    Ljubimov, A V; Burgeson, R E; Butkowski, R J; Michael, A F; Sun, T T; Kenney, M C

    1995-04-01

    The corneal epithelium converges at the peripheral zone (limbus) with the conjunctival epithelium, forming a continuous sheet with phenotypically distinct regions--central, limbal, and conjunctival. The epithelial basement membrane (EBM) is important for corneal functions and cell adhesion, but its regional composition is poorly understood. Current literature is controversial as to the occurrence of type IV collagen in the cornea. The aim of this study was to investigate in detail corneal basement membrane (BM) composition and correlate it with the differentiation state of contributing cells. Adult human corneas (N = 8) were cryosectioned and analyzed by immunofluorescence with antibodies to 15 BM components and to keratin 3, a marker of corneal epithelial differentiation. A novel type of spatial heterogeneity ("horizontal") in the EBM composition was found between the central cornea, limbus, and conjunctiva. Central EBM had type IV collagen alpha 3-alpha 5 chains, whereas limbal and conjunctival EBM contained alpha 1-alpha 2 chains and also laminin alpha 2 and beta 2 chains. Limbal EBM in addition had alpha 5(IV) chain. Laminin-1 (alpha 1 beta 1 gamma 1), laminin-5 (alpha 3 beta 3 gamma 2), perlecan, fibronectin, entactin/nidogen, and type VII collagen were seen in the entire EBM. Another novel type of BM heterogeneity ("vertical") was typical for the corneal Descemet's membrane: its stromal face had alpha 1(IV) and alpha 2(IV) chains and fibronectin, whereas alpha 3(IV)-alpha 5(IV) chains, entactin/nidogen, laminin-1, and perlecan were present on the endothelial face. Type IV collagen controversy is the result of the shifts of isoforms in the limbus and conjunctiva. These shifts and the appearance of additional laminins in the limbus may be related to the differentiation state of corneal cells contributing to the EBM formation. Novel types of BM heterogeneity in the human cornea are described: regional (horizontal) in the EBM and vertical in the Descemet

  10. The chorioallantoic membrane (CAM) assay for the study of human bone regeneration: a refinement animal model for tissue engineering

    Science.gov (United States)

    Moreno-Jiménez, Inés; Hulsart-Billstrom, Gry; Lanham, Stuart A.; Janeczek, Agnieszka A.; Kontouli, Nasia; Kanczler, Janos M.; Evans, Nicholas D.; Oreffo, Richard Oc

    2016-08-01

    Biomaterial development for tissue engineering applications is rapidly increasing but necessitates efficacy and safety testing prior to clinical application. Current in vitro and in vivo models hold a number of limitations, including expense, lack of correlation between animal models and human outcomes and the need to perform invasive procedures on animals; hence requiring new predictive screening methods. In the present study we tested the hypothesis that the chick embryo chorioallantoic membrane (CAM) can be used as a bioreactor to culture and study the regeneration of human living bone. We extracted bone cylinders from human femoral heads, simulated an injury using a drill-hole defect, and implanted the bone on CAM or in vitro control-culture. Micro-computed tomography (μCT) was used to quantify the magnitude and location of bone volume changes followed by histological analyses to assess bone repair. CAM blood vessels were observed to infiltrate the human bone cylinder and maintain human cell viability. Histological evaluation revealed extensive extracellular matrix deposition in proximity to endochondral condensations (Sox9+) on the CAM-implanted bone cylinders, correlating with a significant increase in bone volume by μCT analysis (p animal research and a step towards a humanized in vivo model for tissue engineering.

  11. Increased proliferation and decreased membrane permeability as defense mechanisms of Fusobacterium nucleatum against human neutrophilic peptide-1.

    Science.gov (United States)

    Keskin, Mutlu; Könönen, Eija; Söderling, Eva; Isik, Gülden; Firatli, Erhan; Uitto, Veli-Jukka; Gürsoy, Ulvi Kahraman

    2014-12-01

    Human neutrophilic peptides (HNPs) constitute a class of host defense molecules, which contribute to the non-oxidative killing of bacteria and other microorganisms. Since the adaptability is crucial to bacterial survival in changing environments, it is of interest to know how Fusobacterium nucleatum, the major bridge organism connecting early and late colonizers in dental biofilms, defends itself against HNPs. This study aimed to examine the planktonic growth, membrane permeability, and biofilm formation characteristics as defense mechanisms of F. nucleatum against HNP-1. In all experiments, the type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. nucleatum AHN 9508 and ssp. polymorphum AHN 9910) were used. Planktonic growth (measured in colony forming units), capsular polysaccharide production (visualized by Ziehl-Neelsen stain), membrane permeability (demonstrated as N-phenyl-1-naphthylamine uptake), biofilm formation, and established biofilm development (measured as total mass and polysaccharide levels) were analyzed in the presence of 0 μg/ml (control), 1 μg/ml, 5 μg/ml, and 10 μg/ml of HNP-1. Planktonic growth of the strains AHN 9508 and ATCC 25586 were significantly (p<0.05) increased in the presence of HNP-1, while their membrane permeability decreased (p<0.005) in the planktonic form. HNP-1 decreased the biofilm formation of the strains ATCC 25586 and AHN 9910, whereas it increased the growth of the strain AHN 9508 in established biofilms. Capsule formation and polysaccharide production were not observed in any strain. We conclude that the inhibition of the membrane permeability and the increase in planktonic and established biofilm growth could act as bacterial defense mechanisms against neutrophilic defensins. In addition, this strain-dependent survival ability against HNP-1 may explain the variation in the virulence of different F. nucleatum strains. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

    Directory of Open Access Journals (Sweden)

    Geyer Matthias

    2007-10-01

    Full Text Available Abstract Background The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. Results Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. Conclusion Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo.

  13. Plasma membrane proteomics of human breast cancer cell lines identifies potential targets for breast cancer diagnosis and treatment.

    Directory of Open Access Journals (Sweden)

    Yvonne S Ziegler

    Full Text Available The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease.

  14. Histological evidence of oxidative stress and premature senescence in preterm premature rupture of the human fetal membranes recapitulated in vitro

    National Research Council Canada - National Science Library

    Menon, Ramkumar; Boldogh, Istvan; Hawkins, Hal K; Woodson, Michael; Polettini, Jossimara; Syed, Tariq Ali; Fortunato, Stephen J; Saade, George R; Papaconstantinou, John; Taylor, Robert N

    2014-01-01

    Preterm prelabor rupture of the membranes (pPROM) may lead to preterm births (PTBs). We investigated premature senescence of fetal membranes in women with pPROM and spontaneous PTB with intact membranes...

  15. Improved preservation of human corneal basement membrane following freezing of donor tissue for epikeratophakia.

    OpenAIRE

    Young, R.D.(ARC Centre of Excellence for Particle Physics at the Terascale, CSSM, School of Chemistry and Physics, University of Adelaide, Adelaide, SA, 5005, Australia); Armitage, W J; Bowerman, P.; Cook, S D; Easty, D. L.

    1994-01-01

    Current methods for the production of lenticules for epikeratophakia involve rapid freezing, cryolathing, and slow warming of the donor cornea. We have found that this procedure causes structural damage to the epithelial basement membrane in the donor cornea which may subsequently contribute to poor postoperative re-epithelialisation of the implant, leading to graft failure. Endeavouring to overcome these problems, the effects of cryoprotection of donor cornea were investigated, using dimethy...

  16. Targeting the Human Complement Membrane Attack Complex to Selectively Kill Prostate Cancer Cells

    Science.gov (United States)

    2013-10-01

    reproductive tract of the fe- male. In this regard, seminal plasma is devoid of complement ac- tivity and actually has a strong anti-complement activity (8...article: EA, Ab-sensitized sheep erythrocyte; ES, sheep erythrocyte; PSA, prostate-specific Ag; PVDF, polyvinylidene difluoride. Copyright 2013 by The...addition of sample loading buffer. Proteins were separated by SDS-PAGE and transferred to PVDF membrane as described above. C3b/iC3b deposition assay Sheep

  17. Carbohydrate composition of peripheral, cultured and leukaemic human lymphocyte plasma membranes.

    Science.gov (United States)

    Newman, R A; Glöckner, W M; Uhlenbruck, G

    1978-03-01

    Plasma membranes isolated from peripheral blood lymphocytes of normal donors, lymphocytes from patients with chronic lymphatic leukaemia (CLL), a T cell and B cell line (MOLT-3 and RPMI-1788) were analysed and compared for total carbohydrate contents. T cells and peripheral blood lymphocytes contained the highest relative amounts of sialic acid and fucose, whereas chronic lymphatic leukaemic cells possessed the highest amounts of N-acetylgalactosamine and also more total cell surface carbohydrate. The Thomsen-Friedenreich antigen (TF) was detected serologically on membrane fractions by the use of anti-TF containing sera and specific lectins from Arachis hypogaea, Agaricus bisporus and Vicia graminea. The disaccharide beta-D-galactosyl(1-3)-N-acetyl-D-galactosamine is the immunogdominant carbohydrate group of the FT antigen and was detected as its reduced form, by gas chromatography, in all cells, thus correlating serological and analytical evidence. The haemagglutinating activity of the lectins and sera used was only inhibited by plasma membranes after the removal of sialic acid showong that the native form of this antigen is normally masked by sialic acid in CLL cells as well as normal lymphocytes.

  18. Osteonectin/SPARC/BM-40 in human decidua and carcinoma, tissues characterized by de novo formation of basement membrane

    DEFF Research Database (Denmark)

    Wewer, U M; Albrechtsen, R; Fisher, L W

    1988-01-01

    decidua tissue in organ culture demonstrated significant osteonectin biosynthesis. Further, Northern analysis and in situ hybridization showed that the osteonectin mRNA level in human decidua is very high. Immunohistochemically, the large mature decidual cells exhibited faint cytoplasmic immunoreactivity...... immunoreactivity was found in poorly differentiated carcinomas. Stromal cells in the peritumoral tissue and some vessels were immunoreactive. Using in situ hybridization, it appeared that both the tumor cells and the stromal cells contained osteonectin transcripts. In normal steady state human nonosseus tissues...... with antibodies to osteonectin and a distinct immunoreactivity of the newly deposited basement membranes encircling these cells. The intermediate-sized decidual cells exhibited a strong cytoplasmic immunostaining with antibodies to osteonectin. The small elongated stromal cells were devoid of osteonectin...

  19. Arf and RhoA regulate both the cytosolic and the membrane-bound phospholipase D from human placenta

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hansen, Harald S.; Provost, J.J.

    1997-01-01

    In this paper we demonstrate for the first time that human placenta contains a cytosolic phospholipase D (PLD) activity. This activity had a pH optimum of 7.0 and was stimulated by PIP and inhibited by oleate. Furthermore, cytosolic PLD was stimulated by 30 µM GTP¿S (6-14-fold) and by the small G...... proteins 1 µM mArf3 (2-fold) and 0.37 nM RhoA (2-fold). This is the first report to show RhoA activation of a cytosolic PLD. The activation by mArf3 was maintained after partial purification on DEAE Sepharose of the enzyme. We have previously reported the existence of a membrane-bound PLD from human...... placenta, which is stimulated by PIP, but not by oleate. Here we show that oleic acid and a-linolenic acid both dose-dependently inhibited solubilized membrane PLD (65% inhibition at 4 mM), whereas stearic acid (4 mM) had no effect. Thus, the presence of double bonds in the fatty acid is important...

  20. Structure of a membrane-attack complex/perforin (MACPF) family protein from the human gut symbiont Bacteroides thetaiotaomicron.

    Science.gov (United States)

    Xu, Qingping; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu Ju; Clayton, Thomas; Das, Debanu; Deller, Marc C; Duan, Lian; Ellrott, Kyle; Farr, Carol L; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Krishna, S Sri; Kumar, Abhinav; Lam, Winnie W; Marciano, David; Miller, Mitchell D; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Yeh, Andrew; Zhou, Jiadong; Hodgson, Keith O; Wooley, John; Elsliger, Marc André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

    2010-10-01

    Membrane-attack complex/perforin (MACPF) proteins are transmembrane pore-forming proteins that are important in both human immunity and the virulence of pathogens. Bacterial MACPFs are found in diverse bacterial species, including most human gut-associated Bacteroides species. The crystal structure of a bacterial MACPF-domain-containing protein BT_3439 (Bth-MACPF) from B. thetaiotaomicron, a predominant member of the mammalian intestinal microbiota, has been determined. Bth-MACPF contains a membrane-attack complex/perforin (MACPF) domain and two novel C-terminal domains that resemble ribonuclease H and interleukin 8, respectively. The entire protein adopts a flat crescent shape, characteristic of other MACPF proteins, that may be important for oligomerization. This Bth-MACPF structure provides new features and insights not observed in two previous MACPF structures. Genomic context analysis infers that Bth-MACPF may be involved in a novel protein-transport or nutrient-uptake system, suggesting an important role for these MACPF proteins, which were likely to have been inherited from eukaryotes via horizontal gene transfer, in the adaptation of commensal bacteria to the host environment.

  1. The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles.

    Science.gov (United States)

    Biagini, Massimiliano; Garibaldi, Manuela; Aprea, Susanna; Pezzicoli, Alfredo; Doro, Francesco; Becherelli, Marco; Taddei, Anna Rita; Tani, Chiara; Tavarini, Simona; Mora, Marirosa; Teti, Giuseppe; D'Oro, Ugo; Nuti, Sandra; Soriani, Marco; Margarit, Immaculada; Rappuoli, Rino; Grandi, Guido; Norais, Nathalie

    2015-08-01

    Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Association of lipidome remodeling in the adipocyte membrane with acquired obesity in humans.

    Directory of Open Access Journals (Sweden)

    Kirsi H Pietiläinen

    2011-06-01

    Full Text Available Identification of early mechanisms that may lead from obesity towards complications such as metabolic syndrome is of great interest. Here we performed lipidomic analyses of adipose tissue in twin pairs discordant for obesity but still metabolically compensated. In parallel we studied more evolved states of obesity by investigating a separated set of individuals considered to be morbidly obese. Despite lower dietary polyunsaturated fatty acid intake, the obese twin individuals had increased proportions of palmitoleic and arachidonic acids in their adipose tissue, including increased levels of ethanolamine plasmalogens containing arachidonic acid. Information gathered from these experimental groups was used for molecular dynamics simulations of lipid bilayers combined with dependency network analysis of combined clinical, lipidomics, and gene expression data. The simulations suggested that the observed lipid remodeling maintains the biophysical properties of lipid membranes, at the price, however, of increasing their vulnerability to inflammation. Conversely, in morbidly obese subjects, the proportion of plasmalogens containing arachidonic acid in the adipose tissue was markedly decreased. We also show by in vitro Elovl6 knockdown that the lipid network regulating the observed remodeling may be amenable to genetic modulation. Together, our novel approach suggests a physiological mechanism by which adaptation of adipocyte membranes to adipose tissue expansion associates with positive energy balance, potentially leading to higher vulnerability to inflammation in acquired obesity. Further studies will be needed to determine the cause of this effect.

  3. Advanced Glycation-Modified Human Serum Albumin Evokes Alterations in Membrane and Eryptosis in Erythrocytes.

    Science.gov (United States)

    Awasthi, Saurabh; Gayathiri, S K; Ramya, R; Duraichelvan, R; Dhason, A; Saraswathi, N T

    2015-11-01

    Increased burden of advanced glycation end-products (AGEs) in case of hyperglycemic conditions leads to the development of retinopathy, nephropathy, and cardiovascular and neurological disorders such as Alzheimer's disease. AGEs are considered as pro-oxidants, and their accumulation increases the oxidative stress. The prolonged exposure to these AGEs is the fundamental cause of chronic oxidative stress. Abnormal morphology of red blood cells (RBCs) and excessive eryptosis has been observed in diabetes, glomerulonephritis, dyslipidemia, and obesity, but yet the contribution of extracellular AGEs remains undefined. In this study, we investigated the effect of AGEs on erythrocytes to determine their impact on the occurrence of different pathological forms of these blood cells. Specifically, carboxymethyllysine (CML), carboxyethyllysine (CEL), and Arg-pyrimidine (Arg-P) which have been reported to be the most pre-dominant AGEs formed under in vivo conditions were used in this study. Results suggested the eryptotic properties of CML, CEL, and Arg-P for RBCs, which were evident from the highly damaged cell membrane and occurrence of abnormal morphologies. Methylglyoxal-modified albumin showed more severe effects, which can be attributed to the high reactivity and pro-oxidant nature of glycation end products. These findings suggest the possible role of AGE-modified albumin towards the morphological changes in erythrocyte's membrane associated with diabetic conditions.

  4. Association of Lipidome Remodeling in the Adipocyte Membrane with Acquired Obesity in Humans

    Science.gov (United States)

    Gopalacharyulu, Peddinti; Tang, Jing; Rodriguez-Cuenca, Sergio; Maciejewski, Arkadiusz; Naukkarinen, Jussi; Ruskeepää, Anna-Liisa; Niemelä, Perttu S.; Yetukuri, Laxman; Tan, Chong Yew; Velagapudi, Vidya; Castillo, Sandra; Nygren, Heli; Hyötyläinen, Tuulia; Rissanen, Aila; Kaprio, Jaakko; Yki-Järvinen, Hannele; Vattulainen, Ilpo; Vidal-Puig, Antonio; Orešič, Matej

    2011-01-01

    Identification of early mechanisms that may lead from obesity towards complications such as metabolic syndrome is of great interest. Here we performed lipidomic analyses of adipose tissue in twin pairs discordant for obesity but still metabolically compensated. In parallel we studied more evolved states of obesity by investigating a separated set of individuals considered to be morbidly obese. Despite lower dietary polyunsaturated fatty acid intake, the obese twin individuals had increased proportions of palmitoleic and arachidonic acids in their adipose tissue, including increased levels of ethanolamine plasmalogens containing arachidonic acid. Information gathered from these experimental groups was used for molecular dynamics simulations of lipid bilayers combined with dependency network analysis of combined clinical, lipidomics, and gene expression data. The simulations suggested that the observed lipid remodeling maintains the biophysical properties of lipid membranes, at the price, however, of increasing their vulnerability to inflammation. Conversely, in morbidly obese subjects, the proportion of plasmalogens containing arachidonic acid in the adipose tissue was markedly decreased. We also show by in vitro Elovl6 knockdown that the lipid network regulating the observed remodeling may be amenable to genetic modulation. Together, our novel approach suggests a physiological mechanism by which adaptation of adipocyte membranes to adipose tissue expansion associates with positive energy balance, potentially leading to higher vulnerability to inflammation in acquired obesity. Further studies will be needed to determine the cause of this effect. PMID:21666801

  5. A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

    Directory of Open Access Journals (Sweden)

    Shuhua Xu

    Full Text Available The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

  6. A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

    Science.gov (United States)

    Xu, Shuhua; Soroka, Carol J; Sun, An-Qiang; Backos, Donald S; Mennone, Albert; Suchy, Frederick J; Boyer, James L

    2016-01-01

    The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

  7. The membrane targeted apoptosis modulators erucylphosphocholine and erucylphosphohomocholine increase the radiation response of human glioblastoma cell lines in vitro

    Directory of Open Access Journals (Sweden)

    Budach Wilfried

    2006-03-01

    Full Text Available Abstract Background Alkylphosphocholines constitute a novel class of antineoplastic synthetic phospholipid derivatives that induce apoptosis of human tumor cell lines by targeting cellular membranes. We could recently show that the first intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC is a potent inducer of apoptosis in highly resistant human astrocytoma/glioblastoma cell lines in vitro. ErPC was shown to cross the blood brain barrier upon repeated intravenous injections in rats and thus constitutes a promising candidate for glioblastoma therapy. Aim of the present study was to analyze putative beneficial effects of ErPC and its clinically more advanced derivative erucylphosphohomocholine (erucyl-N, N, N-trimethylpropanolaminphosphate, ErPC3, Erufosine™ on radiation-induced apoptosis and eradication of clonogenic tumor cells in human astrocytoma/glioblastoma cell lines in vitro. Results While all cell lines showed high intrinsic resistance against radiation-induced apoptosis as determined by fluorescence microscopy, treatment with ErPC and ErPC3 strongly increased sensitivity of the cells to radiation-induced cell death (apoptosis and necrosis. T98G cells were most responsive to the combined treatment revealing highly synergistic effects while A172 showed mostly additive to synergistic effects, and U87MG cells sub-additive, additive or synergistic effects, depending on the respective radiation-dose, drug-concentration and treatment time. Combined treatment enhanced therapy-induced damage of the mitochondria and caspase-activation. Importantly, combined treatment also increased radiation-induced eradication of clonogenic T98G cells as determined by standard colony formation assays. Conclusion Our observations make the combined treatment with ionizing radiation and the membrane targeted apoptosis modulators ErPC and ErPC3 a promising approach for the treatment of patients suffering from malignant glioma. The use of this

  8. Type XV collagen exhibits a widespread distribution in human tissues but a distinct localization in basement membrane zones.

    Science.gov (United States)

    Myers, J C; Dion, A S; Abraham, V; Amenta, P S

    1996-12-01

    The collagen family of proteins consists of 19 types encoded by 33 genes. One of the more recently discovered collagens is the alpha1 chain of type XV. Type XV collagen is comprised of a 577-amino-acid, highly interrupted, triple-helical region that is flanked by amino and carboxy noncollagenous domains of 555 and 256 residues, respectively. To address questions of where this collagen is localized and what its function may entail, we produced a bacteria-expressed recombinant protein representing the first half of the type XV collagen carboxy-terminal domain in order to generate highly specific polyclonal antisera. Immunoscreening of an expression library with the affinity-purified antibody revealed three clones coding for part of the type XV triple-helical region and the entire noncollagenous carboxy-terminus. Western blot analysis of human tissue homogenates identified a 116-kDa collagenase-sensitive protein and a 27-kDa collagenase-resistant fragment, whose electrophoretic mobilities were unchanged in the presence and absence of reductant. Northern blot hybridization to human tissue RNAs indicated that type XV has a prevalent and widespread distribution. To determine the precise localization of type XV collagen, immunohistochemical analyses at the light- and electron-microscopic levels were performed. Type XV exhibited a surprisingly restricted and uniform presence in many human tissues as evidenced by a strong association with vascular, neuronal, mesenchymal, and some epithelial basement membrane zones. These data suggest that type XV collagen may function in some manner to adhere basement membrane to the underlying connective tissue stroma.

  9. Quantified colocalization reveals heterotypic histocompatibility class I antigen associations on trophoblast cell membranes: relevance for human pregnancy.

    Science.gov (United States)

    Jabeen, Asma; Miranda-Sayago, José Maria; Obara, Boguslaw; Spencer, Patrick Simon; Dealtry, Gill Barbara; Hayrabedyan, Soren; Shaikly, Valerie; Laissue, Pierre Philippe; Fernández, Nelson

    2013-10-01

    Human placental syncytiotrophoblasts lack expression of most types of human leukocyte antigen (HLA) class I and class II molecules; this is thought to contribute to a successful pregnancy. However, the HLA class Ib antigens HLA-G, -E, and -F and the HLA class Ia antigen HLA-C are selectively expressed on extravillous trophoblast cells, and they are thought to play a major role in controlling feto-maternal tolerance. We have hypothesized that selective expression, coupled with the preferential physical association of pairs of HLA molecules, contribute to the function of HLA at the feto-maternal interface and the maternal recognition of the fetus. We have developed a unique analytical model that allows detection and quantification of the heterotypic physical associations of HLA class I molecules expressed on the membrane of human trophoblast choriocarcinoma cells, ACH-3P and JEG-3. Automated image analysis was used to estimate the degree of overlap of HLA molecules labeled with different fluorochromes. This approach yields an accurate measurement of the degree of colocalization. In both JEG-3 and ACH-3P cells, HLA-C, -E, and -G were detected on the cell membrane, while the expression of HLA-F was restricted to the cytoplasm. Progesterone treatment alone induced a significant increase in the expression level of the HLA-G/HLA-E association, suggesting that this heterotypic association is modulated by this hormone. Our data shows that the cell-surface HLA class I molecules HLA-G, -E, and -C colocalize with each other and have the potential to form preferential heterotypic associations.

  10. The inhibitory effects of cephalosporin and dipeptide on ceftibuten uptake by human and rat intestinal brush-border membrane vesicles.

    Science.gov (United States)

    Sugawara, M; Toda, T; Kobayashi, M; Iseki, K; Miyazaki, K; Shiroto, H; Uchino, J; Kondo, Y

    1994-08-01

    The types of inhibitory effects caused by compound V (an analogue of ceftibuten) and alanylproline (dipeptide) on the uptake of ceftibuten by brush-border membrane vesicles (BBMV) prepared from human and rat small intestine were analysed. In the presence of an inward H(+)-gradient, the initial uptake rate of ceftibuten by both human and rat intestinal BBMV was concentration-dependent with apparent Km and Vmax values of 0.35 mM and 2.052 nmol (mg protein)-1 min-1 for human BBMV, and 0.50 mM and 3.056 nmol (mg protein)-1 min-1 for rat BBMV, respectively. For both human and rat BBMV, kinetic analysis by Dixon and Lineweaver-Burk plots demonstrated that the uptake of ceftibuten was competitively inhibited by compound V, whereas inhibition by alanylproline was noncompetitive or partially competitive. These results suggest that there is a stereospecific transport system which is common to ceftibuten and compound V, and that this system is not identical to the carrier system for the dipeptide, alanylproline.

  11. Targeting antibody responses to the membrane proximal external region of the envelope glycoprotein of human immunodeficiency virus.

    Directory of Open Access Journals (Sweden)

    Donatien Kamdem Toukam

    Full Text Available Although human immunodeficiency type 1 (HIV-1 infection induces strong antibody responses to the viral envelope glycoprotein (Env only a few of these antibodies possess the capacity to neutralize a broad range of strains. The induction of such antibodies represents an important goal in the development of a preventive vaccine against the infection. Among the broadly neutralizing monoclonal antibodies discovered so far, three (2F5, Z13 and 4E10 target the short and hidden membrane proximal external region (MPER of the gp41 transmembrane protein. Antibody responses to MPER are rarely observed in HIV-infected individuals or after immunization with Env immunogens. To initiate antibody responses to MPER in its membrane-embedded native conformation, we generated expression plasmids encoding the membrane-anchored ectodomain of gp41 with N-terminal deletions of various sizes. Following transfection of these plasmids, the MPER domains are displayed on the cell surface and incorporated into HIV virus like particles (VLP. Transfected cells displaying MPER mutants bound as efficiently to both 2F5 and 4E10 as cells transfected with a plasmid encoding full-length Env. Mice immunized with VLPs containing the MPER mutants produced MPER-specific antibodies, the levels of which could be increased by the trimerization of the displayed proteins as well as by a DNA prime-VLP boost immunization strategy. Although 2F5 competed for binding to MPER with antibodies in sera of some of the immunized mice, neutralizing activity could not be detected. Whether this is due to inefficient binding of the induced antibodies to MPER in the context of wild type Env or whether the overall MPER-specific antibody response induced by the MPER display mutants is too low to reveal neutralizing activity, remains to be determined.

  12. Unbound fraction of fluconazole and linezolid in human plasma as determined by ultrafiltration: Impact of membrane type.

    Science.gov (United States)

    Kratzer, Alexander; Kees, Frieder; Dorn, Christoph

    2016-12-15

    Ultrafiltration is a rapid and convenient method to determine the free concentrations of drugs in plasma. Several ultrafiltration devices based on Eppendorf cups are commercially available, but are not validated for such use by the manufacturer. Plasma pH, temperature and relative centrifugal force as well as membrane type can influence the results. In the present work, we developed an ultrafiltration method in order to determine the free concentrations of linezolid or fluconazole, both neutral and moderately lipophilic antiinfective drugs for parenteral as well as oral administration, in plasma of patients. Whereas both substances behaved relatively insensitive in human plasma regarding variations in pH (7.0-8.5), temperature (5-37°C) or relative centrifugal force (1000-10.000xg), losses of linezolid were observed with the Nanosep Omega device due to adsorption onto the polyethersulfone membrane (unbound fraction 75% at 100mg/L and 45% at 0.1mg/L, respectively). No losses were observed with Vivacon which is equipped with a membrane of regenerated cellulose. With fluconazole no differences between Nanosep and Vivacon were observed. Applying standard conditions (pH 7.4/37°C/1000xg/20min), the mean unbound fraction of linezolid in pooled plasma from healthy volunteers was 81.5±2.8% using Vivacon, that of fluconazole was 87.9±3.5% using Nanosep or 89.4±3.3% using Vivacon. The unbound fraction of linezolid was 85.4±3.7% in plasma samples from surgical patients and 92.1±6.2% in ICU patients, respectively. The unbound fraction of fluconazole was 93.9±3.3% in plasma samples from ICU patients. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Human Amniotic Membrane-Derived Products in Sports Medicine: Basic Science, Early Results, and Potential Clinical Applications.

    Science.gov (United States)

    Riboh, Jonathan C; Saltzman, Bryan M; Yanke, Adam B; Cole, Brian J

    2016-09-01

    Amniotic membrane (AM)-derived products have been successfully used in ophthalmology, plastic surgery, and wound care, but little is known about their potential applications in orthopaedic sports medicine. To provide an updated review of the basic science and preclinical and clinical data supporting the use of AM-derived products and to review their current applications in sports medicine. Systematic review. A systematic search of the literature was conducted using the Medline, EMBASE, and Cochrane databases. The search term amniotic membrane was used alone and in conjunction with stem cell, orthopaedic, tissue engineering, scaffold, and sports medicine. The search identified 6870 articles, 80 of which, after screening of the titles and abstracts, were considered relevant to this study. Fifty-five articles described the anatomy, basic science, and nonorthopaedic applications of AM-derived products. Twenty-five articles described preclinical and clinical trials of AM-derived products for orthopaedic sports medicine. Because the level of evidence obtained from this search was not adequate for systematic review or meta-analysis, a current concepts review on the anatomy, physiology, and clinical uses of AM-derived products is presented. Amniotic membranes have many promising applications in sports medicine. They are a source of pluripotent cells, highly organized collagen, antifibrotic and anti-inflammatory cytokines, immunomodulators, and matrix proteins. These properties may make it beneficial when applied as tissue engineering scaffolds, improving tissue organization in healing, and treatment of the arthritic joint. The current body of evidence in sports medicine is heavily biased toward in vitro and animal studies, with little to no human clinical data. Nonetheless, 14 companies or distributors offer commercial AM products. The preparation and formulation of these products alter their biological and mechanical properties, and a thorough understanding of these

  14. [Role of viscosity and permeability of erythrocyte plasmatic membrane in controlling the oxygen transport effectiveness by human hemoglobin on completion of space flight].

    Science.gov (United States)

    Ivanova, S M; Maksimov, G V; Morukov, B V; Iarlykova, Iu V; Labetskaia, O I; Luneva, O G; Maksimova, N V; Brazhe, N A; Bryzgalova, N Iu; Parshina, E Iu

    2007-01-01

    Plasmatic membrane viscosity and permeability and hemoporphyrine conformation in human hemoglobin were studied on completion of long-duration space flight (LSF). Reversible increases in viscosity and selective permeability (Na+/H+ -turnover) of erythrocyte plasmatic membrane were observed immediately after and in the period of recovery from LSF. Viscosity of lipids in both external and internal locations of plasmatic membrane in human erythrocytes was changed after LSF. The reversible rise of the Na+/H+ -turnover is likely to alter intracellular pH and oxygen binding with hemoglobin. The former is confirmed by the concurrent reversible decline in the deoxyhemoglobin ability to bind oxygen and the oxyhemoglobin ability to retain oxygen. In LSF and during readaptation to the normal gravity blood levels of hemoglobin and free iron are known to be reduced and may be answerable for the hypoxic state of human organism.

  15. Quantitative Membrane Proteomics in a Human Mesenchymal Stem Cell Line Undergoing Osteogenic Differentiation

    DEFF Research Database (Denmark)

    Christiansen, Helle

    devoid of cell senescence as encountered in primary cultures. The cell line used in this study has been shown capable of forming bone in vivo and osteogenic differentiation in vitro. We quantified a total number of 972 proteins of which 80 % fall within the categories of: transmembrane, membrane......  Mesenchymal stem cells (MSCs) (or marrow stromal cells) represent a population of cells located in the bone marrow that are capable of differentiation into several mesodermal cell types including osteoblasts. MSCs cannot presently be isolated based on protein markers, as no specific markers exist....... Mesenchymal stem cells are generally isolated based on physical-chemical characteristics such as adherence to plastic, isolating the monocyte fraction. The resultant cultures are often heterogeneous and can contain other cell types, providing a currently poorly defined basis for future clinical use...

  16. Association of Lipidome Remodeling in the Adipocyte Membrane with Acquired Obesity in Humans

    DEFF Research Database (Denmark)

    Pietilainen, K. H.; Rog, T.; Seppanen-Laakso, T.

    2011-01-01

    Identification of early mechanisms that may lead from obesity towards complications such as metabolic syndrome is of great interest. Here we performed lipidomic analyses of adipose tissue in twin pairs discordant for obesity but still metabolically compensated. In parallel we studied more evolved...... states of obesity by investigating a separated set of individuals considered to be morbidly obese. Despite lower dietary polyunsaturated fatty acid intake, the obese twin individuals had increased proportions of palmitoleic and arachidonic acids in their adipose tissue, including increased levels...... that the observed lipid remodeling maintains the biophysical properties of lipid membranes, at the price, however, of increasing their vulnerability to inflammation. Conversely, in morbidly obese subjects, the proportion of plasmalogens containing arachidonic acid in the adipose tissue was markedly decreased. We...

  17. Trace elements determination by energy dispersive X-ray fluorescence (EDXRF) in human placenta and membrane: a comparative study

    Energy Technology Data Exchange (ETDEWEB)

    Custodio, P.J.; Carvalho, M.L. [Centro Fisica Atomica, Universidade de Lisboa, Av. Prof. Gama Pinto, 2, 1649-003, Lisboa (Portugal); Nunes, F. [Hospital Garcia de Orta, Almada (Portugal)

    2003-04-01

    This work is an application of energy dispersive X-ray fluorescence (EDXRF) as an analytical technique for trace elemental determination in human membrane and placenta and elemental concentrations correlations in both tissues. Whole samples were collected during the delivery from healthy mothers and full-term pregnancies. The age of the mother was between 25 and 40 years old, and the weight of the infants ranged from 2.56 to 4.05 kg. Samples were lyophilised and analysed without any chemical treatment. No significant differences in elemental content of placenta and membrane samples were observed except for Ca. Very low levels of Se, As and Pb were observed in all the analysed samples. Zn, considered as one of the key elements in newborn health, was not significantly different in the analysed samples, all of which originated from healthy mothers and healthy babies. The obtained values agree with the literature except for Ca, which is much higher in the studied samples. (orig.)

  18. Differential inhibition of human erythrocyte acetylcholinesterase by polyphenols epigallocatechin-3-gallate and resveratrol. Relevance of the membrane-bound form.

    Science.gov (United States)

    Salazar, Paula B; de Athayde Moncorvo Collado, Alejandro; Canal-Martínez, Verónica; Minahk, Carlos J

    2017-01-02

    The activity of acetylcholinesterase (AChE) from human erythrocytes was tested in the presence of the phenolic compounds resveratrol and epigallocatechin-3-gallate (EGCG). Even though the stilbene barely changed this enzymatic activity, EGCG did inhibit AChE. Importantly, it preferentially acted on the membrane-bound enzyme rather than on its soluble form. Actually, it was shown that this flavonoid may bind to the red blood cell membrane surface, which may improve the interaction between EGCG and AChE. Therefore, caution should be taken when screening AChE inhibitors. In fact, testing compounds with the soluble form of the enzyme may underestimate the activity of some of these potential inhibitors, hence it would be advisable not to use them as a sole model system for screening. Moreover, erythrocyte AChE is proposed as a good model for these enzymatic assays. © 2016 BioFactors, 43(1):73-81, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

  19. Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR.

    Science.gov (United States)

    Chen, Yi-Yung; Rosario, Fredrick J; Shehab, Majida Abu; Powell, Theresa L; Gupta, Madhulika B; Jansson, Thomas

    2015-12-01

    Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, Pcauses down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR. © 2015 Authors; published by Portland Press Limited.

  20. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Hélix-Nielsen, Claus; Scharff-Poulsen, Peter

    2013-01-01

    In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tag......In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C...... at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction...

  1. Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Lund, Rikke; Hansen, Helle V

    2009-01-01

    clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice, but while one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane......'-nucleotidase (ecto-5'-NT, CD73), Ndrg1, integrin beta1, CD44, CD74 and MHC class II proteins. The altered expression levels of proteins identified by LC-MS/MS were validated using flow cytometry, Western blotting, immunocyto- and immunohisto-chemistry. Analysis of clinical breast cancer biopsies demonstrated...... by the two cell lines. The study demonstrates a quantitative and comparative proteomic strategy to identify clinically-relevant key molecules in the early events of metastasis, some of which may prove to be potential targets for cancer therapy....

  2. External root resorption: Different etiologies explained from the composition of the human root-close periodontal membrane

    Directory of Open Access Journals (Sweden)

    Inger Kjaer

    2013-01-01

    Full Text Available Introduction: This paper summarizes different conditions, which have a well-known influence on the resorption of tooth roots. It also highlights factors important for individual susceptibility to root resorption. Furthermore, the paper focuses on idiopathic root resorption where the provoking factor is not known. The Hypothesis: The several different disturbances causing root resorption can be either orthodontically provoked or acquired by trauma, virus or congenital diseases. It is presumed that all these conditions lead to inflammatory processes in the three main tissue layers, comprising the peri-root sheet. Evaluation of the Hypothesis: This paper explains how different etiologies behind root resorption and how different phenotypic traits in root resorption can be understood from immunohistochemical studies of the human periodontal membrane close to the root and thus, gain a new understanding of the phenomenon of root resorption.

  3. Cloning and sequence analysis of gene oipA encoding an outer membrane protein of human Helicobacter pylori.

    Science.gov (United States)

    Chen, Dao-Rong; Huang, Ai-Long; Tao, Xiao-Hong; Wang, Pi-Long; Jiang, Zheng

    2004-11-01

    To construct a recombinant E. coli strain that would highly express the proinflammatory outer membrane protein of human Helicobacter pylori (H pylori). The oipA DNA was amplified by PCR, inserted into pET-32a, and transformed into Top10 E. coli strain. This recombinant plasmid of Top10 was sent out for nucleotide sequence analysis. Finally this sequence AF479754 was compared with HP0638 and JHP0581. The sequence of the aim gene was obtained. It had 924 base pairs. The identity was 95.32% against HP0638, 95.02% against JHP0581, which was higher than the identity between HP0638 and JHP0581. Although the aim gene was obtained, but it was different from the published sequence of GenBank. It is not clear what makes this difference. Maybe it is because different strain was used or because there were some variations. So more researches are required to prove it.

  4. Transfer and expression of the gene encoding a human myeloid membrane antigen (gp150).

    Science.gov (United States)

    Look, A T; Peiper, S C; Rebentisch, M B; Ashmun, R A; Roussel, M F; Rettenmier, C W; Sherr, C J

    1985-02-01

    DNA from the human myeloid cell line HL-60 was cotransfected with the cloned thymidine kinase (tk) gene of herpes simplex virus into tk-deficient mouse L cells. tk-positive recipients expressing antigens detected on HL-60 cells were isolated with a fluorescence-activated cell sorter by use of a panel of monoclonal antibodies that detect epitopes on both normal and malignant myeloid cells. Independently sorted populations of transformed mouse cells showed concordant reactivities with four of the monoclonal antibodies in the panel (DU-HL60-4, MY7, MCS.2, and SJ-D1), which suggested that these antibodies reacted to products of a single human gene. A second round of DNA transfection and cell sorting was performed with donor DNA from primary transformants. Two different dominant selection systems were used to isolate secondary mouse L cell and NIH/3T3 cell transformants that coexpressed the same epitopes. Analysis of cellular DNA from secondary mouse cell subclones with a probe specific for human repetitive DNA sequences revealed a minimal human DNA complement containing a characteristic set of restriction fragments common to independently derived subclones. Two glycoproteins, of 130,000 (gp130) and 150,000 (gp150) mol wt, were specifically immunoprecipitated from metabolically labeled lysates of mouse cell transformants and were shown to contain [35S]methionine-labeled tryptic peptides identical to those of analogous glycoproteins expressed in the donor human myeloid cell line. Kinetic and biochemical analyses established that gp130 is a precursor that differs in its carbohydrate moiety from gp150, the mature form of the glycoprotein detected on the cell surface. The isolation of human gene sequences encoding gp150 in a mouse cell genetic background provides the possibility of molecularly cloning the gene and represents a general strategy for isolating human genes encoding differentiation-specific cell surface antigens.

  5. Analysis of in vitro release through reconstructed human epidermis and synthetic membranes of multi-vitamins from cosmetic formulations.

    Science.gov (United States)

    Gabbanini, Simone; Matera, Riccardo; Beltramini, Claudia; Minghetti, Andrea; Valgimigli, Luca

    2010-08-01

    A convenient method for in vitro investigation of the release of lipid- and water-soluble vitamins from cosmetic formulations was developed. The permeation of (d)-alpha-tocopherol (vitamin E), retinyl acetate (pro-vitamin A), ascorbic acid (vitamin C) and pyridoxine (vitamin B6) through SkinEthic reconstructed human epidermis (RHE), and synthetic polyethersulfone and polycarbonate membranes was studied in vitro using a Franz-type diffusion apparatus, coupled either to a spectrophotometer for continuous reading (dynamic setting) or to HPLC-DAD analysis of the receptor medium (static setting). O/W and W/O emulsions were compared with simple aqueous solutions for their kinetic of vitamins release, to evaluate the influence of the cosmetic formulation on the bioavailability of active ingredients. Results indicate that synthetic membranes offer a limited barrier to the diffusion of vitamins, but may provide information on the release ability of the formulation. Penetration was more effective when water was the external phase of the formulation, i.e. W/O emulsions were less effective in the release of vitamins than O/W emulsion or aqueous solutions. RHE (17 days old) offered a significantly higher barrier to penetration of vitamins, as expected for native human epidermis. The relative ranking in coefficient of permeability (Ps (cm/h)) was: ascorbic acid>pyridoxine>retinyl acetate>alpha-tocopherol approximately 0, the absolute values depending on the formulation. The method herein described showed to be a practical and convenient tool for the quality-control and efficacy evaluation of cosmetic formulations. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  6. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    Directory of Open Access Journals (Sweden)

    Manetta J

    2014-08-01

    Full Text Available Joseph Manetta, Holly Bina, Paul Ryan, Niles Fox, Derrick R Witcher, Kristine Kikly Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: B-cell activating factor (BAFF is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. Keywords: autoimmunity, B-cell malignancies, B-cell survival factor, BAFF

  7. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Helix Nielsen, Claus; Scharff-Poulsen, Peter

    2013-01-01

    In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C...... at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction...... and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes....

  8. Mechanical stress downregulates MHC class I expression on human cancer cell membrane.

    Directory of Open Access Journals (Sweden)

    Rosanna La Rocca

    Full Text Available In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal, depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700-1800 cm(-1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK cells cytotoxic recognition.

  9. Mechanical Stress Downregulates MHC Class I Expression on Human Cancer Cell Membrane

    KAUST Repository

    La Rocca, Rosanna

    2014-12-26

    In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700–1800 cm−1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition.

  10. Immunohistochemical and ultrastructural analysis of dysplastic epithelium of human ocular surface: basement membrane and intermediate filament.

    Science.gov (United States)

    Saika, S; Kawashima, Y; Okada, Y; Ohkawa, K; Yamanaka, O; Katoh, T; Ohnishi, Y; Ooshima, A; Kao, W W

    1999-05-01

    The dysplastic corneal epithelium is characterized by the abnormal proliferation of epithelial cells. The phenotypes of these cells have not been elucidated. We investigated whether such epithelium expresses the phenotypes of corneal or conjunctival epithelial cells. The corneas and conjunctivae from four normal subjects and from one patient with epithelial dysplasia of the central cornea were immunostained for IV and VII collagens and for cytokeratins. Monoclonal antibodies against collagen IV reacted to the [alpha1(IV)]2alpha2(IV) or alpha5(IV) molecule. Anti-cytokeratin antibodies were used to define epithelial cell types. The ultrastructure of the basement membrane (BM) of each specimen also was examined. Type VII collagen immunoreactivity was detected in all the specimens of epithelial BM. The anti-collagen IV [alpha1(IV)]2alpha2(IV) antibody labeled the conjunctival BMs, not the BMs of the corneal epithelia, of each subject. The normal corneal epithelial BM, not the BM of the conjunctival or dysplastic corneal epithelium, was immunolabeled with anti-alpha5(IV) antibody. The pattern of cytokeratin expression in the corneal epithelial dysplasia resembled that seen in the normal conjunctivae. Small breaks in the BM of dysplastic corneal epithelium were ultrastructurally revealed. The number of hemidesmosomes in the dysplastic corneal epithelium was decreased as compared with that in the normal BM. The composition of collagen types within the BM and the cellular phenotype of the dysplastic epithelium in the cornea resembled those of conjunctival epithelium, not of the cornea.

  11. Reflection coefficient and permeability of urea and ethylene glycol in the human red cell membrane

    Energy Technology Data Exchange (ETDEWEB)

    Levitt, D.G.; Mlekoday, H.J.

    1983-02-01

    The reflection coefficient (sigma) and permeability (P) of urea and ethylene glycol were determined by fitting the equations of Kedem and Katchalsky (1958) to the change in light scattering produced by adding a permeable solute to a red cell suspension. The measurements incorporated three important modifications: (a) the injection artifact was eliminated by using echinocyte cells; (b) the use of an additional adjustable parameter (Km), the effective dissociation constant at the inner side of the membrane; (c) the light scattering is not directly proportional to cell volume (as is usually assumed) because refractive index and scattering properties of the cell depend on the intracellular permeable solute concentration. This necessitates calibrating for known changes in refractive index (by the addition of dextran) and cell volume (by varying the NaCl concentration). The best fit was for sigma . 0.95, Po . 8.3 X 10(-4) cm/s, and Km . 100 mM for urea and sigma . 1.0, Po . 3.9 X 10(-4) cm/s, and Km . 30 mM for ethylene glycol. The effects of the inhibitors copper, phloretin, p-chloromercuriphenylsulfonate, and 5,5'-dithiobis (2-nitro) benzoic acid on the urea, ethylene glycol, and water permeability were determined. The results suggest that there are three separate, independent transport systems: one for water, one for urea and related compounds, and one for ethylene glycol and glycerol.

  12. Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers

    NARCIS (Netherlands)

    Demel, R.A.; Geurts van Kessel, W.S.M.; Zwaal, R.F.A.; Roelofsen, B.; Deenen, L.L.M. van

    1975-01-01

    The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes. The phospholipases which cannot hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A2 from

  13. Structural basis of cargo membrane protein discrimination by the human COPII coat machinery

    Energy Technology Data Exchange (ETDEWEB)

    Mancias, Joseph D.; Goldberg, Jonathan (MSKCC)

    2008-11-18

    Genomic analysis shows that the increased complexity of trafficking pathways in mammalian cells involves an expansion of the number of SNARE, Rab and COP proteins. Thus, the human genome encodes four forms of Sec24, the cargo selection subunit of the COPII vesicular coat, and this is proposed to increase the range of cargo accommodated by human COPII-coated vesicles. In this study, we combined X-ray crystallographic and biochemical analysis with functional assays of cargo packaging into COPII vesicles to establish molecular mechanisms for cargo discrimination by human Sec24 subunits. A conserved IxM packaging signal binds in a surface groove of Sec24c and Sec24d, but the groove is occluded in the Sec24a and Sec24b subunits. Conversely, LxxLE class transport signals and the DxE signal of VSV glycoprotein are selectively bound by Sec24a and Sec24b subunits. A comparative analysis of crystal structures of the four human Sec24 isoforms establishes the structural determinants for discrimination among these transport signals, and provides a framework to understand how an expansion of coat subunits extends the range of cargo proteins packaged into COPII-coated vesicles.

  14. Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids.

    Science.gov (United States)

    Raza Shaikh, Saame; Brown, David A

    2013-01-01

    Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. The Use of Fibrous, Supramolecular Membranes and Human Tubular Cells for Renal Epithelial Tissue Engineering : Towards a Suitable Membrane for a Bioartificial Kidney

    NARCIS (Netherlands)

    Dankers, Patricia Y. W.; Boomker, Jasper M.; Huizinga-van der Vlag, Ali; Smedts, Frank M. M.; Harmsen, Martin C.; van Luyn, Marja J. A.

    2010-01-01

    A bioartificial kidney, which is composed of a membrane cartridge with renal epithelial cells, can substitute important kidney functions in patients with renal failure. A particular challenge is the maintenance of monolayer integrity and specialized renal epithelial cell functions ex vivo. We

  16. Enhanced attachment of human osteoblasts on NH3-treated poly(L-lactic acid) membranes for guided bone regeneration.

    Science.gov (United States)

    Byeon, Ju-Hee; Kim, Su-Gwan; Son, Jun-Sik; Jin, Seung-Chan; Piao, Zheng-Gang; Lee, Sook-Young; Jang, Eun-Sook; Kim, Jae-Sung; Jeong, Mi-Ae; Ahn, Hoon; Park, Jong-Phil

    2013-03-01

    Barrier membranes for guided bone regeneration (GBR) were prepared by a solvent casting method using solutions of poly(L-lactic acid) (PLLA) and chitosan. PLLA and PLLA/chitosan membranes were treated with ammonia gas plasma. PLLA/chitosan membranes were successfully fabricated, and the surface of the PLLA/chitosan membrane was clearly modified by NH3 plasma treatment according to attenuated total reflectance (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) analyses. Additionally, water contact angle testing indicated that the hydrophilicity of these membranes was significantly increased. MG-63 cells were cultured on each type of membrane, and cell viability was examined using an MTT assay. After one week of culturing, MG-63 cells were more abundant on PLLA/chitosan membranes than on PLLA membranes. The cell viability of PLLA/chitosan membranes with plasma treatment was significantly higher than that of PLLA membranes. These results suggest that this plasma-treated membrane is suitable for GBR and is a promising source of bioactive membrane material for bone regeneration.

  17. Hyperdry amniotic membrane as a suitable biological dressing material for raw wounds in the oral cavity

    Directory of Open Access Journals (Sweden)

    Makoto Noguchi

    2016-06-01

    Full Text Available Raw wounds in the oral cavity are prone not only to infection but also contraction by scaring and often need a proper dressing to prevent these complications. Autografts using free mucosal and split-skin grafts, which seem biologically ideal, have been used to cover raw wounds in the oral cavity. Those grafts, however, require a separate surgical procedure at donor sites and often cause morbidity associated with delayed healing of the donor site. The amnion has been considered a suitable tissue for allografts, based on its low immunogenicity. It also possesses anti-inflammatory, would –protecting, and scar-reducing properties. Preserved amnions have been used for decades in various clinical fields.  However, there have been some problems in the storage and sterilization of the material. To resolve these problems, we developed hyperdry amniotic membrane (HAM, which can be stored at room temperature for a long period. In my lecture, I will share our clinical experiences of applying HAM into oral surgery, including results of experimental studies on would healing of the oral cavity.

  18. Expression of Plasmodium falciparum erythrocyte membrane protein 1 in experimentally infected humans

    Directory of Open Access Journals (Sweden)

    Theander Thor G

    2005-04-01

    Full Text Available Abstract Background Parasites causing severe malaria in non-immune patients express a restricted subset of variant surface antigens (VSA, which are better recognized by immune sera than VSA expressed during non-severe disease in semi-immune individuals. The most prominent VSA are the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1 family, which is expressed on the surface of infected erythrocytes where it mediates binding to endothelial receptors. Thus, severe malaria may be caused by parasites expressing PfEMP1 variants that afford parasites optimal sequestration in immunologically naïve individuals and high effective multiplication rates. Methods var gene transcription was analysed using real time PCR and PfEMP1 expression by western blots as well as immune plasma recognition of parasite cultures established from non-immune volunteers shortly after infection with NF54 sporozoites. Results In cultures representing the first generation of parasites after hepatic release, all var genes were transcribed, but GroupA var genes were transcribed at the lowest levels. In cultures established from second or third generation blood stage parasites of volunteers with high in vivo parasite multiplication rates, the var gene transcription pattern differed markedly from the transcription pattern of the cultures representing first generation parasites. This indicated that parasites expressing specific var genes, mainly belonging to group A and B, had expanded more effectively in vivo compared to parasites expressing other var genes. The differential expression of PfEMP1 was confirmed at the protein level by immunoblot analysis. In addition, serological typing showed that immune sera more often recognized second and third generation parasites than first generation parasites. Conclusion In conclusion, the results presented here support the hypothesis that parasites causing severe malaria express a subset of PfEMP1, which bestows

  19. Expression of the membrane complement regulatory proteins (CD55 and CD59 in human thymus.

    Directory of Open Access Journals (Sweden)

    Agnieszka Kinsner

    2007-01-01

    Full Text Available CD59 is one of the key molecules involved in cell protection against autologus complement. The fact that complement regulatory proteins are able to prevent hyperacute rejection of organs in pig to primate model, raises the question of possible complement regulatory protein (CRP involvement in the maturation of immunological system. We report here that in foetal and postnatal human thymus, CD59 and CD55 are primarily located on Hassall's corpuscles and medullary epithelial cells. This localization highly correlates with the expression of CD30L, which is the member of the tumour necrosis factor superfamily. Additionally, TUNEL technique was used to visualize distribution of apoptotic cells in the thymus, which revealed the presence of apoptotic cells closely associated with the Hassall's corpuscles. The observed co-localization of CD59, CD55 and CD30L might suggest an involvement of the complement system in thymic selection in humans.

  20. A Novel Function for the nm23-Hl Gene: Overexpression in Human Breast Carcinoma Cells Leads to the Formation of Basement Membrane and Growth Arrest

    Energy Technology Data Exchange (ETDEWEB)

    Howlett, Anthony R; Petersen, Ole W; Steeg, Patricia S; Bissell, Mina J

    1994-01-01

    We have developed a culture system using reconstituted basement membrane components in which normal human mammary epithelial cells exhibit several aspects of the development and differentiation process, including formation of acinar-like structures, production and basal deposition of basement membrane components, and production and apical secretion of sialomucins. Cell lines and cultures from human breast carcinomas failed to recapitulate this process. The data indicate the importance of cellular interactions with the basement membrane in the regulation of normal breast differentiation and, potentially, its loss in neoplasia. Our purpose was to use this assay to investigate the role of the putative metastasis suppressor gene nm23-H1 in mammary development and differentiation. The metastatic human breast carcinoma cell line MDA-MB-435, clones transfected with a control pCMVBamneo vector, and clones transfected with pCMVBamneo vector containing nm23-H1 complementary DNA (the latter of which exhibited a substantial reduction in spontaneous metastatic potential in vivo) were cultured within a reconstituted basement membrane. Clones were examined for formation of acinus-like spheres, deposition of basement membrane components, production of sialomucin, polarization, and growth arrest. In contrast to the parental cell line and control transfectants, MDA-MB-435 breast carcinoma cells overexpressing Nm23-H1 protein regained several aspects of the normal phenotype within reconstituted basement membrane. Nm23-H1 protein-positive cells formed organized acinus-like spheres, deposited the basement membrane components type IV collagen and, to some extent, laminin to the outside of the spheres, expressed sialomucin, and growth arrested. Growth arrest of Nm23-H1 protein-positive cells was preceded by and correlated with formation of a basement membrane, suggesting a causal relationship. The data indicate a previously unidentified cause-and-effect relationship between nm23-H1 gene

  1. The inter-relation between epithelial cells of Malassez and vessels studied immunohistochemically in the periodontal membrane of human primary and permanent teeth

    DEFF Research Database (Denmark)

    Bille, Marie-Louise Bastholm; Thomsen, Bjarke; Kjær, Inger

    2011-01-01

    Abstract Background. Only few immunohistochemical studies have focused on the periodontal membrane in human primary teeth. Recently, studies on epithelial cells of Malassez and innervation have been published. Studies on the inter-relation between vessels and the epithelial cells of Malassez are ...

  2. Fluorometric assay of oleate-activated phospholipase D isoenzyme in membranes of rat nervous tissue and human platelets.

    Science.gov (United States)

    Krzystanek, Marek; Trzeciak, Henryk I; Krzystanek, Ewa; Małecki, Andrzej

    2010-01-01

    Phospholipase D plays a key role in the biosynthesis of phosphatidic acid, a second messenger involved in essential cellular processes. Oleate-activated phospholipase D was the first mammalian phospholipase D isoform to be discovered but is the least known. The study was aimed to test a fluorometric method of assessment of oleate-activated phospholipase D activity in different biological materials. The brain cortex of male Wistar rats, cultured rat brain astrocytes, and human platelets were processed to yield plasmatic membranes for experiments. To assess phospholipase D activity the modified fluorometric method was used. Previously, the method was used only to determine H₂O₂. In this enzyme-coupled assay phospholipase D activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine. First, phospholipase D cleaves exogenous phosphatidylcholine to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, in the presence of horseradish peroxidase, H₂O₂ reacts with 10-acetyl-3,7-dihydroxyphenoxazine to generate the highly fluorescent product, resorufin. The concentration of resorufin was measured using excitation and emission at 560 nm and 590 nm, respectively. The proposed optimal parameters of the tested assay are 25 µg of rat brain cortex protein, 50 µg of rat brain astrocyte protein, and 50 µg of human platelet protein in a reaction volume of 200 µL, and 2 min enzymatic reaction at 37°C. The fluorometric method may be applied to assay phospholipase D in different biological materials.

  3. Biocompatibility of quantum dots (CdSe/ZnS ) in human amniotic membrane-derived mesenchymal stem cells in vitro.

    Science.gov (United States)

    Wang, Gongping; Zeng, Guangwei; Wang, Caie; Wang, Huasheng; Yang, Bo; Guan, Fangxia; Li, Dongpeng; Feng, Xiaoshan

    2015-06-01

    Amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) are a potential source of mesenchymal stem cells which could be used to repair skin damage. The use of mesenchymal stem cells to repair skin damage requires safe, effective and biocompatible agents to evaluate the effectiveness of the result. Quantum dots (QDs) composed of CdSe/ZnS are semiconductor nanocrystals with broad excitation and narrow emission spectra, which have been considered as a new chemical and fluorescent substance for non-invasively labeling different cells in vitro and in vivo. This study investigated the cytotoxic effects of QDs on hAM-dMSCs at different times following labeling. Using 0.75, 1.5 and 3.0 μL between quantum dots, labeled human amniotic mesenchymal stem cells were collected on days 1, 2 and 4 and observed morphological changes, performed an MTT cell growth assay and flow cytometry for mesenchymal stem cells molecular markers. Quantum dot concentration 0.75 μg/mL labeled under a fluorescence microscope, cell morphology was observed, The MTT assay showed cells in the proliferative phase. Flow cytometry expression CD29, CD31, CD34, CD44, CD90, CD105 and CD106. Within a certain range of concentrations between quantum dots labeled human amniotic mesenchymal stem cells has good biocompatibility.

  4. Cardenolide-Induced Lysosomal Membrane Permeabilization Demonstrates Therapeutic Benefits in Experimental Human Non-Small Cell Lung Cancers

    Directory of Open Access Journals (Sweden)

    Tatjana Mijatovic

    2006-05-01

    Full Text Available Non-small cell lung cancers (NSCLCs are the leading cause of cancer deaths in most developed countries. Targeting heat shock protein 70 (Hsp70 expression and function, together with the induction of lysosomal membrane permeabilization (LMP, could overcome the multiple anti-cell death mechanisms evidenced in NSCLCs that are responsible for the failure of currently used chemotherapeutic drugs. Because cardenolides bind to the sodium pump, they affect multiple signaling pathways and thus have a number of marked effects on tumor cell behavior. The aim of the present study was to characterize in vitro and in vivo the antitumor effects of a new cardenolide (UNBS1450 on experimental human NSCLCs. UNBS1450 is a potent source of in vivo antitumor activity in the case of paclitaxeland oxaliplatin-resistant subcutaneous human NCIH727 and orthotopic A549 xenografts in nude mice. In vitro UNBS1450-mediated antitumor activity results from the induction of nonapoptotic cell death. UNBS1450 mediates the decrease of Hsp70 at both mRNA and protein levels, and this is at least partly due to UNBS1450-induced downregulation of NFAT5/ TonEBP (a factor responsible for the transcriptional control of Hsp70. These effects were paralleled by the induction of LMP, as evidenced by acridine orange staining and immunofluorescence analysis for cathepsin B accumulation.

  5. Major Outer Membrane Protein Omp25 of Brucella suis Is Involved in Inhibition of Tumor Necrosis Factor Alpha Production during Infection of Human Macrophages

    OpenAIRE

    Jubier-Maurin, Véronique; Boigegrain, Rose-Anne; Cloeckaert, Axel; Gross, Antoine; Alvarez-Martinez, Maria-Teresa; Terraza, Annie; Liautard, Janny; Köhler, Stephan; Rouot, Bruno; Dornand, Jacques; Liautard, Jean Pierre

    2001-01-01

    Brucella spp. can establish themselves and cause disease in humans and animals. The mechanisms by which Brucella spp. evade the antibacterial defenses of their host, however, remain largely unknown. We have previously reported that live brucellae failed to induce tumor necrosis factor alpha (TNF-α) production upon human macrophage infection. This inhibition is associated with a nonidentified protein that is released into culture medium. Outer membrane proteins (OMPs) of gram-negative bacteria...

  6. Use of hybrid chitosan membranes and human mesenchymal stem cells from the Wharton jelly of umbilical cord for promoting nerve regeneration in an axonotmesis rat model★

    OpenAIRE

    Gärtner, Andrea; Pereira, Tiago; Simões, Maria João; Armada-da-Silva, Paulo AS; França, Miguel L; Sousa, Rosa; Bompasso, Simone; Raimondo, Stefania; Shirosaki, Yuki; Nakamura, Yuri; Hayakawa, Satoshi; Osakah, Akiyoshi; Porto, Beatriz; Luís, Ana Lúcia; Varejão, Artur SP

    2012-01-01

    Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to assess the effect on nerve regeneration, associating a hybrid chitosan membrane with non-differentiated human mesenchymal stem cells isolated from Wharton's jelly of umbilical cord, in peripheral nerve reconstruction after crush injury. Chromosome analysis on human mesenchymal stem cell line from Wharton's jelly was carried out and no structural alter...

  7. The identification of specific Rhesus-polypeptide-blood-group-ABH-active-glycoprotein complexes in the human red-cell membrane.

    Science.gov (United States)

    Moore, S; Green, C

    1987-06-15

    1. RhD,c and E immune complexes isolated from 3H- and 125I-surface-radiolabelled and unlabelled intact human red cells were analysed by SDS/polyacrylamide-gel electrophoresis. 2. Apparent Mr values of 31,900 for RhD polypeptide and 33,100 for Rhc,E polypeptide were obtained under both reducing and non-reducing conditions. Glycosylation of RhD,c and E polypeptides was not detected. 3. RhD,c and E immune complexes also contain a glycoprotein component. RhD glycoprotein (apparent Mr 45,000-100,000) is distinct from Rhc,E glycoprotein(s) (apparent Mr 35,000-65,000). Rh (Rhesus) glycoprotein carbohydrate moieties are susceptible to endo-beta-galactosidase digestion and carry blood-group-ABH determinants. This suggests the presence of polylactosaminoglycan-type structures. 4. Rh glycoproteins are not present in Rh immune complexes as a result of non-specific adsorption of membrane glycoproteins during the membrane-solubilization phase of immune-complex isolation because RhD immune complexes isolated from a 1:1 (v/v) mixture of Acde/cde and OcDE/cDE red cells do not contain blood-group-A-active glycoprotein. 5. Blood-group-A immune complexes isolated from group-A red cells of the appropriate Rh phenotypes contain the 31,900- and 33,100-apparent-Mr Rh polypeptides. 6. It was concluded from the above evidence that non-covalent Rh-glycoprotein-Rh-polypeptide complexes exist in the native red-cell membrane. 7. The 31,900- and 33,100-apparent-Mr Rh polypeptides are absent from blood-group-A immune complexes isolated from regulator type Rhnull cells (donor A.L.), but are replaced by a 33,800-apparent-Mr Rhnull-specific polypeptide (Rhnull polypeptide). It is suggested that Rhnull polypeptide is an aberrant product of the Rh gene complex.

  8. Discovery of novel DENN proteins: implications for the evolution of eukaryotic intracellular membrane structures and human disease

    Directory of Open Access Journals (Sweden)

    Dapeng eZhang

    2012-12-01

    Full Text Available The tripartite DENN module, comprised of a N-terminal longin domain, followed by DENN and d-DENN domains, is a GDP-GTP exchange factor (GEFs for Rab GTPases, which are regulators of practically all membrane trafficking events in eukaryotes. Using sequence and structure analysis we identify multiple novel homologs of the DENN module, many of which can be traced back to the ancestral eukaryote. These findings provide unexpected leads regarding key cellular processes such as autophagy, vesicle-vacuole interactions, chromosome segregation and human disease. Of these, SMCR8, the folliculin interacting protein-1 and 2 (FNIP1 and FNIP2, nitrogen permease regulator 2 (NPR2 and NPR3 are proposed to function in recruiting Rab GTPases during different steps of autophagy, fusion of autophagosomes with the vacuole and regulation of cellular metabolism. Another novel DENN protein identified in this study is C9ORF72; expansions of the hexanucleotide GGGGCC in its first intron have been recently implicated in amyotrophic lateral sclerosis (ALS and fronto-temporal dementia (FTD. While this mutation is proposed to cause a RNA-level defect, the identification of C9ORF72 as a potential DENN-type GEF raises the possibility that at least part of the pathology might relate to a specific Rab-dependent vesicular trafficking process, as has been observed in the case of some other neurological conditions with similar phenotypes. We present evidence that the longin domain, such as those found in the DENN module, are likely to have been ultimately derived from the related domains found in prokaryotic GTPase-activating proteins of MglA-like GTPases. Thus, the origin of the longin domains from this ancient GTPase-interacting domain, concomitant with the radiation of GTPases, especially of the Rab clade, played an important role in the dynamics of eukaryotic intracellular membrane systems.

  9. Contribution of ankyrin-band 3 complexes to the organization and mechanical properties of the membrane skeleton of human erythrocyte

    Energy Technology Data Exchange (ETDEWEB)

    Shen, B.W. [Argonne National Lab., IL (United States). Biological and Medical Research Div.

    1995-02-01

    To understand the role of ankyrin-band 3 complexes in the organization of the spectrin-based membrane skeleton and its contribution to the mechanical properties of human erythrocytes, intact skeletons and single-layered skeleton leaflets were prepared from intact and physically sheared membrane ghosts, expanded in low salt buffer, and examined by transmission electron microscopy. While the structures of intact skeletons and single-layered skeleton leaflets shared many common features, including rigid junctional complexes of spectrin, actin, and band 4.1; short stretches ({approximately}50 {angstrom}) of flexible spectrin filaments; and globular masses of ankyrin-band 3 complexes situated close to the middle of the spectrin filaments, the definition of structural units in the intact skeleton is obscured by the superposition of the two layers. However, the spatial disposition of structural elements can be clearly defined in the images of the single-layered skeleton leaflets. Partially expanded skeletal leaflets contain conglomerates of ankyrin-band 3 complexes arranged in a circular or clove-leaf configuration that straddles multiple strands of thick spectrin cables, presumably reflecting the association of ankyrin-band 3 complexes on neighboring spectrin tetramers as well as the lateral association of the spectrin filaments. Hyperexpansion of the skeleton leaflets led to dissociation of the conglomerates of ankyrin-band 3 complexes, full-extension of the spectrin tetramers, and separation of the individual strands of spectrin tetramers. Clearly defined stands of spectrin tetramers in the hyperexpanded single-layered skeletal leaflets often contained two sets of globular protein masses that divided the spectrin tetramers into three segments of approximately equal length.

  10. Susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to membrane-active peptides and enrofloxacin in human tissue cell cultures.

    Science.gov (United States)

    Nir-Paz, Ran; Prévost, Marie-Christine; Nicolas, Pierre; Blanchard, Alain; Wróblewski, Henri

    2002-05-01

    Mycoplasmas, which are bacteria that are devoid of a cell wall and which belong to the class Mollicutes, are pathogenic for humans and animals and are frequent contaminants of tissue cell cultures. Although contamination of cultures with mycoplasma can easily be monitored with fluorescent dyes that stain DNA and/or with molecular probes, protection and decontamination of cultures remain serious challenges. In the present work, we investigated the susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to the membrane-active peptides alamethicin, dermaseptin B2, gramicidin S, and surfactin by growth inhibition and lethality assays. In the absence of serum, the four peptides killed mycoplasmas at minimal bactericidal concentrations that ranged from 12.5 to 100 microM, but in all cases the activities were decreased by the presence of serum. As a result, under standard culture conditions (10% serum) only alamethicin and gramicidin S were able to inhibit mycoplasma growth (MICs, 50 microM), while dermaseptin B2 and surfactin were ineffective. Furthermore, 8 days of treatment of HeLa cell cultures experimentally contaminated with either mycoplasma species with 70 microM enrofloxacin cured the cultures of infection, whereas treatment with alamethicin and gramicidin S alone was not reliable because the concentrations and treatment times required were toxic to the cells. However, combination of alamethicin or gramicidin S with 70 microM enrofloxacin allowed mycoplasma eradication after 30 min or 24 h of treatment, depending on the mycoplasma and peptide considered. HeLa cell cultures experimentally infected with mycoplasmas should prove to be a useful model for study of the antimycoplasma activities of antibiotics and membrane-active peptides under conditions close to those found in vivo.

  11. Discovery of Novel DENN Proteins: Implications for the Evolution of Eukaryotic Intracellular Membrane Structures and Human Disease.

    Science.gov (United States)

    Zhang, Dapeng; Iyer, Lakshminarayan M; He, Fang; Aravind, L

    2012-01-01

    The tripartite DENN module, comprised of a N-terminal longin domain, followed by DENN, and d-DENN domains, is a GDP-GTP exchange factor (GEFs) for Rab GTPases, which are regulators of practically all membrane trafficking events in eukaryotes. Using sequence and structure analysis we identify multiple novel homologs of the DENN module, many of which can be traced back to the ancestral eukaryote. These findings provide unexpected leads regarding key cellular processes such as autophagy, vesicle-vacuole interactions, chromosome segregation, and human disease. Of these, SMCR8, the folliculin interacting protein-1 and 2 (FNIP1 and FNIP2), nitrogen permease regulator 2 (NPR2), and NPR3 are proposed to function in recruiting Rab GTPases during different steps of autophagy, fusion of autophagosomes with the vacuole and regulation of cellular metabolism. Another novel DENN protein identified in this study is C9ORF72; expansions of the hexanucleotide GGGGCC in its first intron have been recently implicated in amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). While this mutation is proposed to cause a RNA-level defect, the identification of C9ORF72 as a potential DENN-type GEF raises the possibility that at least part of the pathology might relate to a specific Rab-dependent vesicular trafficking process, as has been observed in the case of some other neurological conditions with similar phenotypes. We present evidence that the longin domain, such as those found in the DENN module, are likely to have been ultimately derived from the related domains found in prokaryotic GTPase-activating proteins of MglA-like GTPases. Thus, the origin of the longin domains from this ancient GTPase-interacting domain, concomitant with the radiation of GTPases, especially of the Rab clade, played an important role in the dynamics of eukaryotic intracellular membrane systems.

  12. Insights into the membrane interactions of the saposin-like proteins Na-SLP-1 and Ac-SLP-1 from human and dog hookworm.

    Directory of Open Access Journals (Sweden)

    Charlene Willis

    Full Text Available Saposin-like proteins (SAPLIPs from soil-transmitted helminths play pivotal roles in host-pathogen interactions and have a high potential as targets for vaccination against parasitic diseases. We have identified two non-orthologous SAPLIPs from human and dog hookworm, Na-SLP-1 and Ac-SLP-1, and solved their three-dimensional crystal structures. Both proteins share the property of membrane binding as monitored by liposome co-pelleting assays and monolayer adsorption. Neither SAPLIP displayed any significant haemolytic or bactericidal activity. Based on the structural information, as well as the results from monolayer adsorption, we propose models of membrane interactions for both SAPLIPs. Initial membrane contact of the monomeric Na-SLP-1 is most likely by electrostatic interactions between the membrane surface and a prominent basic surface patch. In case of the dimeric Ac-SLP-1, membrane interactions are most likely initiated by a unique tryptophan residue that has previously been implicated in membrane interactions in other SAPLIPs.

  13. Attachment and proliferation of human osteoblast-like cells on guided bone regeneration (GBR) membranes in the absence or presence of nicotine: an in vitro study.

    Science.gov (United States)

    Papaioannou, Konstantinos A; Markopoulou, Cleopatra E; Gioni, Vassiliki; Mamalis, Anastasios A; Vayouraki, Helen N; Kletsas, Dimitris; Vrotsos, Ioannis A

    2011-01-01

    To compare in vitro the attachment and proliferation of human osteoblast-like cells (MG63) on tissue culture plates and guided bone regeneration (GBR) membranes in the absence or presence of nicotine. Membrane samples were fixed to wells and the cell number (CN) was counted after 24 hours (attachment assay) or 5 days (proliferation assay). The ratio of cell count (RCC) (CN at 5 days/CN at 24 hours) was calculated. The study had three parts: First, five different resorbable GBR membranes were compared (Resolut Adapt LT [RALT], Biocollagen [BC], Bio-Gide [BG], OsseoGuard [OG], and Demokritos Human Tissue Bank [DEM]). Next, cells were cultured on tissue culture plates with five different concentrations of nicotine. Finally, cells were cultured on the membrane that had demonstrated the highest RCC and CN in part 1 with four different concentrations of nicotine. At 24 hours, BG showed the highest CN and OG showed the lowest CN. At 5 days, BG showed the highest CN. The order of RCC was BG > OG > DEM > RALT ~ BC. At 24 hours, lower nicotine concentrations (0.3 and 3 μg/mL) showed higher CNs versus the control, whereas CNs for high nicotine concentrations (30 and 300 μg/mL) were lower than for the control. CN at 5 days and RCC were lowest with 300 μg/mL nicotine. At 24 hours and 5 days, all differences among wells with membrane were statistically insignificant. Nevertheless, CN at 5 days and RCC were highest with the lowest nicotine concentration (3 μg/mL) and lowest with high concentrations. Membrane materials influence attachment and proliferation of bone cells and, therefore, could affect the outcomes of GBR. On both tissue culture plates and membranes, there is a tendency toward a biphasic effect of nicotine, with stimulatory effects at low concentrations and inhibitory effects at high concentrations.

  14. C{sub 18}-attached membrane funnel-based spray ionization mass spectrometry for quantification of anti-diabetic drug from human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wan [Department of Chemistry, The Chinese University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong); Chen, Xiangfeng, E-mail: xiangfchensdas@163.com [Department of Chemistry, The Chinese University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong); Shandong Analysis and Test Centre, Shandong Academy of Sciences, Jinan, Shandong (China); Wong, Y.-L. Elaine; Hung, Y.-L. Winnie; Wang, Ze; Deng, Liulin [Department of Chemistry, The Chinese University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong); Dominic Chan, T.-W., E-mail: twdchan@cuhk.edu.hk [Department of Chemistry, The Chinese University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong)

    2016-08-24

    In this work, sorbent-attached membrane funnel-based spray ionization mass spectrometry was explored for quantitative analysis of anti-diabetic drugs spiked in human plasma. C{sub 18}-attached membrane funnel was fabricated for in situ extraction and clean-up to alleviate matrix suppression effect in the ionization process. Repaglinide was used as a target analyte of anti-diabetic drugs. Under optimal working conditions, good linearity (R{sup 2} > 0.99) was obtained in the concentration range of 1–100 ng mL{sup −1}. The method detection limit of target drugs spiked in the human plasma was around 0.30 ng mL{sup −1}. Through the application of an isotope-labeled internal standard, the signal fluctuation caused by residual background matrices was largely alleviated and the precision of measurement (RSD) was below 15%. The recovery of repaglinide for 5, 25, and 100 ng mL{sup −1} of spiked human plasma matrixes ranged from 87% to 112%. The developed method was successfully applied to determine repaglinide in plasma volunteers who orally received a dose of drug association. Our results demonstrated that membrane funnel-based spray is a simple and sensitive method for rapid screening analysis of complex biological samples. - Highlights: • Sorbent attached membrane funnel based spray platform was used for drug determination in human plasma. • The matrix suppression effect of human plasma was largely eliminated. • The method was applied to determine repaglinide in plasma volunteers. • Membrane funnel-based spray is promising for analysis of biological samples.

  15. Cationic membrane-active peptides - anticancer and antifungal activity as well as penetration into human skin.

    Science.gov (United States)

    Do, Nhung; Weindl, Günther; Grohmann, Lisa; Salwiczek, Mario; Koksch, Beate; Korting, Hans Christian; Schäfer-Korting, Monika

    2014-05-01

    Cationic antimicrobial peptides are ancient natural broad-spectrum antibiotics, and several compounds also exhibit anticancer activity. However, most applications pertain to bacterial infections, and treatment for skin cancer is less frequently considered. The cytotoxicity of melittin, cecropin A, protegrin-1 and histatin 5 against squamous skin cancer cell lines and normal human keratinocytes was evaluated and compared to established drugs. The results show that melittin clearly outperforms 5-fluorouracil regarding antitumor activity. Importantly, combined melittin and 5-fluorouracil enhanced cytotoxic effects on cancer cells and reduced toxicity on normal keratinocytes. Additionally, minimum inhibitory concentrations indicate that melittin also shows superior activity against clinical and laboratory strains of Candida albicans compared to amphotericin B. To evaluate its potential for topical applications, human skin penetration of melittin was investigated ex vivo and compared to two non-toxic cell-penetrating peptides (CPPs), low molecular weight protamine (LMWP) and penetratin. The stratum corneum prevents penetration into viable epidermis over 6 h; however, the peptides gain access to the viable skin after 24 h. Inhibition of digestive enzymes during skin penetration significantly enhances the availability of intact peptide. In conclusion, melittin may represent an innovative agent for non-melanoma skin cancer and infectious skin diseases. In order to develop a drug candidate, skin absorption and proteolytic digestion by skin enzymes need to be addressed. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Lessons learned with molecular methods targeting the BCSP-31 membrane protein for diagnosis of human brucellosis.

    Science.gov (United States)

    Sanjuan-Jimenez, Rocio; Colmenero, Juan D; Morata, Pilar

    2017-06-01

    Brucellosis remains an emerging and re-emerging zoonosis worldwide causing high human morbidity. It usually affects persons who are permanently exposed to fastidious microorganisms of the Brucella genus and has a nonspecific clinical picture. Thus, diagnosis of brucellosis can sometimes be difficult. Molecular techniques have recently been found very useful in the diagnosis of brucellosis together with its common and very diverse focal complications. We herein review all the lessons learned by our group concerning the molecular diagnosis of human brucellosis over the last twenty years. The results, initially using one-step conventional PCR, later PCR-ELISA and more recently real-time PCR, using both fluorescent intercalating reagents (SYBR-Green I) and specific probes (Taqman), have shown that these techniques are all much more sensitive than bacteriological methods and more specific than the usual serological techniques for the diagnosis of primary infection, the post-treatment control of the disease, early detection of relapse and the diagnosis of focal complications. Optimization of the technique and improvements introduced over the years show that molecular methods, currently accessible for most clinical laboratories, enable easy rapid diagnosis of brucellosis at the same time as they avoid any risk to laboratory personnel while handling live Brucella spp. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. In vivo areal modulus of elasticity estimation of the human tympanic membrane system: modelling of middle ear mechanical function in normal young and aged ears.

    Science.gov (United States)

    Gaihede, Michael; Liao, Donghua; Gregersen, Hans

    2007-02-07

    The quasi-static elastic properties of the tympanic membrane system can be described by the areal modulus of elasticity determined by a middle ear model. The response of the tympanic membrane to quasi-static pressure changes is determined by its elastic properties. Several clinical problems are related to these, but studies are few and mostly not comparable. The elastic properties of membranes can be described by the areal modulus, and these may also be susceptible to age-related changes reflected by changes in the areal modulus. The areal modulus is determined by the relationship between membrane tension and change of the surface area relative to the undeformed surface area. A middle ear model determined the tension-strain relationship in vivo based on data from experimental pressure-volume deformations of the human tympanic membrane system. The areal modulus was determined in both a younger (n = 10) and an older (n = 10) group of normal subjects. The areal modulus for lateral and medial displacement of the tympanic membrane system was smaller in the older group (mean = 0.686 and 0.828 kN m(-1), respectively) compared to the younger group (mean = 1.066 and 1.206 kN m(-1), respectively), though not significantly (2p = 0.10 and 0.11, respectively). Based on the model the areal modulus was established describing the summated elastic properties of the tympanic membrane system. Future model improvements include exact determination of the tympanic membrane area accounting for its shape via 3D finite element analyses. In vivo estimates of Young's modulus in this study were a factor 2-3 smaller than previously found in vitro. No significant age-related differences were found in the elastic properties as expressed by the areal modulus.

  18. Surface-Enhanced Raman Spectroscopy (SERS Tracking of Chelerythrine, a Na+/K+ Pump Inhibitor, into Cytosol and Plasma Membrane Fractions of Human Lens Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Kevin M. Dorney

    2013-12-01

    Full Text Available Background/Aims: The quaternary benzo-phenanthridine alkaloid (QBA chelerythrine (CET is a pro-apoptotic drug and Na+/K+ pump (NKP inhibitor in human lens epithelial cells (HLECs. In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS. Methods: Silver nanoparticles (AgNPs prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm-1 marker band as a function of CET concentration. Results: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Conclusion: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET+ accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.

  19. Surface-enhanced Raman spectroscopy (SERS) tracking of chelerythrine, a Na(+)/K(+) pump inhibitor, into cytosol and plasma membrane fractions of human lens epithelial cell cultures.

    Science.gov (United States)

    Dorney, Kevin M; Sizemore, Ioana E P; Alqahtani, Tariq; Adragna, Norma C; Lauf, Peter K

    2013-01-01

    The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na(+)/K(+) pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm(-1) marker band as a function of CET concentration. SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET(+)) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect. © 2014 S. Karger AG, Basel.

  20. Membrane dynamics

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...... for the lateral organization of membranes as wells as for physical properties like bending, permeability and elasticity...

  1. The Alteration of the Epidermal Basement Membrane Complex of Human Nevus Tissue and Keratinocyte Attachment after High Hydrostatic Pressurization

    Directory of Open Access Journals (Sweden)

    Naoki Morimoto

    2016-01-01

    Full Text Available We previously reported that human nevus tissue was inactivated after high hydrostatic pressure (HHP higher than 200 MPa and that human cultured epidermis (hCE engrafted on the pressurized nevus at 200 MPa but not at 1000 MPa. In this study, we explore the changes to the epidermal basement membrane in detail and elucidate the cause of the difference in hCE engraftment. Nevus specimens of 8 mm in diameter were divided into five groups (control and 100, 200, 500, and 1000 MPa. Immediately after HHP, immunohistochemical staining was performed to detect the presence of laminin-332 and type VII collagen, and the specimens were observed by transmission electron microscopy (TEM. hCE was placed on the pressurized nevus specimens in the 200, 500, and 1000 MPa groups and implanted into the subcutis of nude mice; the specimens were harvested at 14 days after implantation. Then, human keratinocytes were seeded on the pressurized nevus and the attachment was evaluated. The immunohistochemical staining results revealed that the control and 100 MPa, 200 MPa, and 500 MPa groups were positive for type VII collagen and laminin-332 immediately after HHP. TEM showed that, in all of the groups, the lamina densa existed; however, anchoring fibrils were not clearly observed in the 500 or 1000 MPa groups. Although the hCE took in the 200 and 500 MPa groups, keratinocyte attachment was only confirmed in the 200 MPa group. This result indicates that HHP at 200 MPa is preferable for inactivating nevus tissue to allow its reuse for skin reconstruction in the clinical setting.

  2. In vitro studies of human monocyte migration across endothelium in response to leukotriene B4 and f-Met-Leu-Phe.

    OpenAIRE

    Migliorisi, G.; Folkes, E.; Pawlowski, N.; Cramer, E. B.

    1987-01-01

    Relatively little is known about monocyte emigration from the vasculature or about the factors that regulate this process. In this study, a human in vitro model of a blood vessel wall was used for examination of monocyte transendothelial migration. Umbilical vein endothelial cells were grown to confluency on amnion connective tissue, and human monocytes were stimulated to cross the monolayer in response to the chemoattractants leukotriene B4 or f-Met-Leu-Phe. The pattern and time course of mo...

  3. Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

    Science.gov (United States)

    Vu, Anh T; Wang, Xinying; Wickramasinghe, S Ranil; Yu, Bing; Yuan, Hua; Cong, Hailin; Luo, Yongli; Tang, Jianguo

    2015-08-01

    Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Use of hybrid chitosan membranes and human mesenchymal stem cells from the Wharton jelly of umbilical cord for promoting nerve regeneration in an axonotmesis rat model★

    Science.gov (United States)

    Gärtner, Andrea; Pereira, Tiago; Simões, Maria João; Armada-da-Silva, Paulo AS; França, Miguel L; Sousa, Rosa; Bompasso, Simone; Raimondo, Stefania; Shirosaki, Yuki; Nakamura, Yuri; Hayakawa, Satoshi; Osakah, Akiyoshi; Porto, Beatriz; Luís, Ana Lúcia; Varejão, Artur SP; Maurício, Ana Colette

    2012-01-01

    Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to assess the effect on nerve regeneration, associating a hybrid chitosan membrane with non-differentiated human mesenchymal stem cells isolated from Wharton's jelly of umbilical cord, in peripheral nerve reconstruction after crush injury. Chromosome analysis on human mesenchymal stem cell line from Wharton's jelly was carried out and no structural alterations were found in metaphase. Chitosan membranes were previously tested in vitro, to assess their ability in supporting human mesenchymal stem cell survival, expansion, and differentiation. For the in vivo testing, Sasco Sprague adult rats were divided in 4 groups of 6 or 7 animals each: Group 1, sciatic axonotmesis injury without any other intervention (Group 1-Crush); Group 2, the axonotmesis lesion of 3 mm was infiltrated with a suspension of 1 250–1 500 human mesenchymal stem cells (total volume of 50 μL) (Group 2-CrushCell); Group 3, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane covered with a monolayer of non-differentiated human mesenchymal stem cells (Group 3-CrushChitIIICell) and Group 4, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane (Group 4-CrushChitIII). Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index, static sciatic index, extensor postural thrust, and withdrawal reflex latency. Stereological analysis was carried out on regenerated nerve fibers. Results showed that infiltration of human mesenchymal stem cells, or the combination of chitosan membrane enwrapment and human mesenchymal stem cell enrichment after nerve crush injury provide a slight advantage to post-traumatic nerve regeneration. Results obtained with chitosan type III membrane alone confirmed that they significantly improve post-traumatic axonal regrowth and may

  5. Use of hybrid chitosan membranes and human mesenchymal stem cells from the Wharton jelly of umbilical cord for promoting nerve regeneration in an axonotmesis rat model.

    Science.gov (United States)

    Gärtner, Andrea; Pereira, Tiago; Simões, Maria João; Armada-da-Silva, Paulo As; França, Miguel L; Sousa, Rosa; Bompasso, Simone; Raimondo, Stefania; Shirosaki, Yuki; Nakamura, Yuri; Hayakawa, Satoshi; Osakah, Akiyoshi; Porto, Beatriz; Luís, Ana Lúcia; Varejão, Artur Sp; Maurício, Ana Colette

    2012-10-15

    Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to assess the effect on nerve regeneration, associating a hybrid chitosan membrane with non-differentiated human mesenchymal stem cells isolated from Wharton's jelly of umbilical cord, in peripheral nerve reconstruction after crush injury. Chromosome analysis on human mesenchymal stem cell line from Wharton's jelly was carried out and no structural alterations were found in metaphase. Chitosan membranes were previously tested in vitro, to assess their ability in supporting human mesenchymal stem cell survival, expansion, and differentiation. For the in vivo testing, Sasco Sprague adult rats were divided in 4 groups of 6 or 7 animals each: Group 1, sciatic axonotmesis injury without any other intervention (Group 1-Crush); Group 2, the axonotmesis lesion of 3 mm was infiltrated with a suspension of 1 250-1 500 human mesenchymal stem cells (total volume of 50 μL) (Group 2-CrushCell); Group 3, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane covered with a monolayer of non-differentiated human mesenchymal stem cells (Group 3-CrushChitIIICell) and Group 4, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane (Group 4-CrushChitIII). Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index, static sciatic index, extensor postural thrust, and withdrawal reflex latency. Stereological analysis was carried out on regenerated nerve fibers. Results showed that infiltration of human mesenchymal stem cells, or the combination of chitosan membrane enwrapment and human mesenchymal stem cell enrichment after nerve crush injury provide a slight advantage to post-traumatic nerve regeneration. Results obtained with chitosan type III membrane alone confirmed that they significantly improve post-traumatic axonal regrowth and may

  6. Histological evidence of oxidative stress and premature senescence in preterm premature rupture of the human fetal membranes recapitulated in vitro.

    Science.gov (United States)

    Menon, Ramkumar; Boldogh, Istvan; Hawkins, Hal K; Woodson, Michael; Polettini, Jossimara; Syed, Tariq Ali; Fortunato, Stephen J; Saade, George R; Papaconstantinou, John; Taylor, Robert N

    2014-06-01

    Preterm prelabor rupture of the membranes (pPROM) may lead to preterm births (PTBs). We investigated premature senescence of fetal membranes in women with pPROM and spontaneous PTB with intact membranes (membrane senescence phenotype by oxidative stress in vitro. IHC was performed for p53, p21, and phospho (p)-p38 mitogen-activated protein kinase (MAPK) as markers of senescence phenotype in pPROM, PTBs, and term births. Term fetal membranes were exposed to cigarette smoke extract to induce oxidative stress. Western blots documented p-p53 and p-p38 MAPK. Transmission electron microscopy assessed cellular morphologic features in clinical and cigarette smoke extract-treated membranes. A total of 80% of pPROM cells and >60% of term cells were positive for all three senescence phenotype markers, and concentrations were higher than in PTBs (P membranes from PTB and term birth pregnancies, whereas only membranes in vivo and after cigarette smoke extract treatment in vitro but was less apparent in PTBs. Histologic and biochemical resemblance of pPROM and term membranes suggests premature senescence of the membranes is a mechanistic feature in pPROM, and this can be phenocopied in an in vitro model. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  7. Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue.

    Science.gov (United States)

    Isachenko, Vladimir; Mallmann, Peter; Petrunkina, Anna M; Rahimi, Gohar; Nawroth, Frank; Hancke, Katharina; Felberbaum, Ricardo; Genze, Felicitas; Damjanoski, Ilija; Isachenko, Evgenia

    2012-01-01

    At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5-2.0×1.0-1.2×0.8-1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla

  8. Receptor-based differences in human aortic smooth muscle cell membrane stiffness

    Science.gov (United States)

    Huang, H.; Kamm, R. D.; So, P. T.; Lee, R. T.

    2001-01-01

    Cells respond to mechanical stimuli with diverse molecular responses. The nature of the sensory mechanism involved in mechanotransduction is not known, but integrins may play an important role. The integrins are linked to both the cytoskeleton and extracellular matrix, suggesting that probing cells via integrins should yield different mechanical properties than probing cells via non-cytoskeleton-associated receptors. To test the hypothesis that the mechanical properties of a cell are dependent on the receptor on which the stress is applied, human aortic smooth muscle cells were plated, and magnetic beads, targeted either to the integrins via fibronectin or to the transferrin receptor by use of an IgG antibody, were attached to the cell surface. The resistance of the cell to deformation ("stiffness") was estimated by oscillating the magnetic beads at 1 Hz by use of single-pole magnetic tweezers at 2 different magnitudes. The ratio of bead displacements at different magnitudes was used to explore the mechanical properties of the cells. Cells stressed via the integrins required approximately 10-fold more force to obtain the same bead displacements as the cells stressed via the transferrin receptors. Cells stressed via integrins showed stiffening behavior as the force was increased, whereas this stiffening was significantly less for cells stressed via the transferrin receptor (Pcells depend on the receptor by which the stress is applied, with integrin-based linkages demonstrating cell-stiffening behavior.

  9. RHBDL2 Is a Critical Membrane Protease for Anoikis Resistance in Human Malignant Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Tsung-Lin Cheng

    2014-01-01

    Full Text Available Anoikis resistance allows metastatic tumor cells to survive in a homeless environment. Activation of epithelial growth factor receptor (EGFR signaling is one of the key mechanisms for metastatic tumor cells to resist anoikis, yet the regulation mechanisms of homeless-triggered EGFR activation in metastatic tumor cells remain unclear. Rhomboid-like-2 (RHBDL2, an evolutionally conserved intramembrane serine protease, can cleave the EGF ligand and thus trigger EGFR activation. Herein, we demonstrated that RHBDL2 overexpression in human epithelial cells resulted in promotion of cell proliferation, reduction of cell adhesion, and suppression of anoikis. During long-term suspension cultures, increased RHBDL2 was only detected in aggressive tumor cell lines. Treatment with the rhomboid protease inhibitor or RHBDL2 shRNA increased cleaved caspase 3, a marker of apoptosis. Finally, inhibition of EGFR activation increased the cleaved caspase 3 and attenuated the detachment-induced focal adhesion kinase phosphorylation. Taken together, these findings provide evidence for the first time that RHBDL2 is a critical molecule in anoikis resistance of malignant epithelial cells, possibly through the EGFR-mediated signaling. Our study demonstrates RHBDL2 as a new therapeutic target for cancer metastasis.

  10. Receptor-based differences in human aortic smooth muscle cell membrane stiffness

    Science.gov (United States)

    Huang, H.; Kamm, R. D.; So, P. T.; Lee, R. T.

    2001-01-01

    Cells respond to mechanical stimuli with diverse molecular responses. The nature of the sensory mechanism involved in mechanotransduction is not known, but integrins may play an important role. The integrins are linked to both the cytoskeleton and extracellular matrix, suggesting that probing cells via integrins should yield different mechanical properties than probing cells via non-cytoskeleton-associated receptors. To test the hypothesis that the mechanical properties of a cell are dependent on the receptor on which the stress is applied, human aortic smooth muscle cells were plated, and magnetic beads, targeted either to the integrins via fibronectin or to the transferrin receptor by use of an IgG antibody, were attached to the cell surface. The resistance of the cell to deformation ("stiffness") was estimated by oscillating the magnetic beads at 1 Hz by use of single-pole magnetic tweezers at 2 different magnitudes. The ratio of bead displacements at different magnitudes was used to explore the mechanical properties of the cells. Cells stressed via the integrins required approximately 10-fold more force to obtain the same bead displacements as the cells stressed via the transferrin receptors. Cells stressed via integrins showed stiffening behavior as the force was increased, whereas this stiffening was significantly less for cells stressed via the transferrin receptor (Pmuscle cells depend on the receptor by which the stress is applied, with integrin-based linkages demonstrating cell-stiffening behavior.

  11. Cell Membrane Proteomic Analysis Identifies Proteins Differentially Expressed in Osteotropic Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Philippe Kischel

    2008-09-01

    Full Text Available Metastatic breast cancer cells are characterized by their high propensity to colonize the skeleton and form bone metastases, causing major morbidity and mortality. Identifying key proteins involved in the osteotropic phenotype would represent a major step toward the development of both new prognostic markers and new effective therapies. Cell surface proteins differentially expressed in cancer cells are preferred potential targets for antibody-based targeted therapies. In this study, using cell surface biotinylation and a mass spectrometric approach, we have compared the profile of accessible cell surface proteins between the human breast cancer cell line MDA-MB-231 and its highly osteotropic B02 subclone. This strategy allowed the identification of several proteins either up- or downregulated in the osteotropic cell line, and differential protein expressions were validated using antibody-based techniques. Class I HLAs were down-regulated in the bone metastatic variant, whereas αvβ3 integrins, among others, were consistently up-regulated in this latter cell line. These results show that comprehensive profiling of the cell surface proteome of mother cancerous cell lines and derived organ-specific metastatic cell lines provides an effective approach for the identification of potential accessible marker proteins for both prognosis and antibodybased targeted therapies.

  12. Human Papillomavirus in Oral Leukoplakia, Verrucous Carcinoma, Squamous Cell Carcinoma, and Normal Mucous Membrane.

    Science.gov (United States)

    Saghravanian, Nasrollah; Ghazi, Narges; Meshkat, Zahra; Mohtasham, Nooshin

    2015-11-01

    Squamous cell carcinoma (SCC) is the most common oral malignancy, and verrucous carcinoma (VC) is a less invasive type of SCC. Leukoplakia (LP) is the most frequent premalignant lesion in the oral cavity. The human papillomavirus (HPV) has been recognized as one of the etiologic factors of these conditions. The association of anogenital and cervical cancers with HPV particularly its high-risk subtypes (HPV HR) has been demonstrated. The purpose of our study was to investigate the hypothetical association between HPV and the mentioned oral cavity lesions. One hundred and seventy-three samples (114 SCCs, 21 VCs, 20 LPs) and 18 normal mucosa samples (as a control group) were retrieved from the Department of Oral and Maxillofacial Pathology of Mashhad Dental School, Iran. The association of HPV genotypes in LP, VC, and SCC was compared to normal oral mucosa using the polymerase chain reaction. The results showed the absence of HPV in normal mucosa and LP lesions. In three samples of VC (14.3%), we observed the presence of HPV HR (types 16 and 18). All VCs were present in the mandibular ridge of females aged over 65 years old. No statistically significant correlation between HPV and VC was observed (p=0.230). Additionally, 15 (13.1%) SCCs showed HPV positivity, but this was not significant (p=0.830). The prevalence of SCC was higher on the tongue with the dominant presence of less carcinogenic species of HPV (types 6 and 11). A statistically significant association was not observed between HPV and SCC or VC in the oral cavity. More studies are necessary to better understand the relationship between HPV and malignant/premalignant oral cavity lesions.

  13. Human Papillomavirus in Oral Leukoplakia, Verrucous Carcinoma, Squamous Cell Carcinoma, and Normal Mucous Membrane

    Directory of Open Access Journals (Sweden)

    Nasrollah Saghravanian

    2015-11-01

    Full Text Available Objectives: Squamous cell carcinoma (SCC is the most common oral malignancy, and verrucous carcinoma (VC is a less invasive type of SCC. Leukoplakia (LP is the most frequent premalignant lesion in the oral cavity. The human papillomavirus (HPV has been recognized as one of the etiologic factors of these conditions. The association of anogenital and cervical cancers with HPV particularly its high-risk subtypes (HPV HR has been demonstrated. The purpose of our study was to investigate the hypothetical association between HPV and the mentioned oral cavity lesions.  Methods: One hundred and seventy-three samples (114 SCCs, 21 VCs, 20 LPs and 18 normal mucosa samples (as a control group were retrieved from the Department of Oral and Maxillofacial Pathology of Mashhad Dental School, Iran. The association of HPV genotypes in LP, VC, and SCC was compared to normal oral mucosa using the polymerase chain reaction.  Results: The results showed the absence of HPV in normal mucosa and LP lesions. In three samples of VC (14.3%, we observed the presence of HPV HR (types 16 and 18. All VCs were present in the mandibular ridge of females aged over 65 years old. No statistically significant correlation between HPV and VC was observed (p=0.230. Additionally, 15 (13.1% SCCs showed HPV positivity, but this was not significant (p=0.830. The prevalence of SCC was higher on the tongue with the dominant presence of less carcinogenic species of HPV (types 6 and 11. A statistically significant association was not observed between HPV and SCC or VC in the oral cavity.  Conclusions: More studies are necessary to better understand the relationship between HPV and malignant/premalignant oral cavity lesions.

  14. Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation.

    Science.gov (United States)

    Pulido, David; Arranz-Trullén, Javier; Prats-Ejarque, Guillem; Velázquez, Diego; Torrent, Marc; Moussaoui, Mohammed; Boix, Ester

    2016-04-13

    Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1-45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance.

  15. Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549 cells

    Directory of Open Access Journals (Sweden)

    Wang Jianwei

    2011-08-01

    Full Text Available Abstract Background Influenza A virus mutates rapidly, rendering antiviral therapies and vaccines directed against virus-encoded targets ineffective. Knowledge of the host factors and molecular pathways exploited by influenza virus will provide further targets for novel antiviral strategies. However, the critical host factors involved in influenza virus infection have not been fully defined. Results We demonstrated that LAMP3, a member of lysosome-associated membrane glycoprotein (LAMP family, was significantly induced in human lung epithelial (A549 cells upon influenza A virus infection. Knockdown of LAMP3 expression by RNA interference attenuated production of viral nucleoprotein (NP as well as virus titers. Confocal microscopy results demonstrated that viral NP is colocalized within LAMP3 positive vesicles at early stages of virus infection. Furthermore, knockdown of LAMP3 expression led to a reduction in nuclear accumulation of viral NP and impeded virus replication. Conclusions LAMP3 is an influenza A virus inducible gene, and plays an important role in viral post-entry steps. Our observations may provide insights into the mechanism of influenza virus replication and potential targets for novel anti-influenza therapeutics.

  16. Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

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    Jing Zhang

    2017-12-01

    Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

  17. Insights into the oligomerization process of the C-terminal domain of human plasma membrane Ca²+-ATPase.

    Science.gov (United States)

    Benetti, Federico; Mičetić, Ivan; Carsughi, Flavio; Spinozzi, Francesco; Bubacco, Luigi; Beltramini, Mariano

    2011-02-15

    Plasma membrane calcium pumps (PMCAs) sustain a primary transport system for the specific removal of cytosolic calcium ions from eukaryotic cells. PMCAs are characterized by the presence of a C-terminal domain referred to as a regulatory domain. This domain is target of several regulatory mechanisms: activation by Ca²+-calmodulin complex and acidic phospholipids, phosphorylation by kinase A and C, proteolysis by calpain and oligomerization. As far as oligomerization is concerned, the C-terminal domain seems to be crucial for this process. We have cloned the C-terminal domain of the human PMCA isoform 1b, and characterized its properties in solution. The expressed protein maintains its tendency to oligomerize in aqueous solutions, but it is dissociated by amphipathic molecules such as diacylglycerol and sodium dodecyl sulphate. The presence of sodium dodecyl sulphate stabilizes the domain as a compact structure in monomeric form retaining the secondary structure elements, as shown by small angle neutron scattering and circular dichroism measurements. The importance of oligomerization for the regulation of PMCA activity and intracellular calcium concentration is discussed. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. A Pole-Zero Filter Cascade Provides Good Fits to Human Masking Data and to Basilar Membrane and Neural Data

    Science.gov (United States)

    Lyon, Richard F.

    2011-11-01

    A cascade of two-pole-two-zero filters with level-dependent pole and zero dampings, with few parameters, can provide a good match to human psychophysical and physiological data. The model has been fitted to data on detection threshold for tones in notched-noise masking, including bandwidth and filter shape changes over a wide range of levels, and has been shown to provide better fits with fewer parameters compared to other auditory filter models such as gammachirps. Originally motivated as an efficient machine implementation of auditory filtering related to the WKB analysis method of cochlear wave propagation, such filter cascades also provide good fits to mechanical basilar membrane data, and to auditory nerve data, including linear low-frequency tail response, level-dependent peak gain, sharp tuning curves, nonlinear compression curves, level-independent zero-crossing times in the impulse response, realistic instantaneous frequency glides, and appropriate level-dependent group delay even with minimum-phase response. As part of exploring different level-dependent parameterizations of such filter cascades, we have identified a simple sufficient condition for stable zero-crossing times, based on the shifting property of the Laplace transform: simply move all the s-domain poles and zeros by equal amounts in the real-s direction. Such pole-zero filter cascades are efficient front ends for machine hearing applications, such as music information retrieval, content identification, speech recognition, and sound indexing.

  19. Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation

    Directory of Open Access Journals (Sweden)

    David Pulido

    2016-04-01

    Full Text Available Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1–45 derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13 that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance.

  20. No midterm advantages in the middle term using small intestinal submucosa and human amniotic membrane in Achilles tendon transverse tenotomy.

    Science.gov (United States)

    Liu, Yushu; Peng, Yinbo; Fang, Yong; Yao, Min; Redmond, Robert W; Ni, Tao

    2016-11-24

    The study was aimed to compare the effects of small intestinal submucosa (SIS) and human amniotic membrane (HAM) on Achilles tendon healing. A total of 48 New Zealand white rabbits were divided into two groups. A full-thickness transverse tenotomy was made at the right leg of the rabbits. Then, the laceration site was wrapped with HAM (P/A group) or SIS (P/S group). The ultimate stress (US) and Young's modulus (E) of the tendons were detected for biomechanical analysis. Histological evaluation was performed using hematoxylin and eosin, immunohistochemical, and immunofluorescent stain. Expression of collagen I was detected by western blot analysis, and levels of inflammatory cytokines IL-1β, IL-6, and TNF-α were measured. Finally, adhesion formation was evaluated. There were no significant differences in filamentous adhesion, cross-sectional areas of the laceration sites, levels of inflammatory response, and collagen type I expression between the P/A and P/S groups (p > 0.05). Compared with the P/A group, the US and E values were significantly higher in the P/S group at day 7 (p Achilles tendon injury in the early stage of healing.

  1. In-vitro permeability of the human nail and of a keratin membrane from bovine hooves: penetration of chloramphenicol from lipophilic vehicles and a nail lacquer.

    Science.gov (United States)

    Mertin, D; Lippold, B C

    1997-03-01

    Lipophilic vehicles and especially nail lacquers are more appropriate for topical application on the nail than aqueous systems because of their better adhesion. This work has, therefore, studied the penetration through the human nail plate of the model compound chloramphenicol from the lipophilic vehicles medium chain triglycerides and n-octanol and from a lacquer based on quaternary poly(methyl methacrylates) (Eudragit RL). The results were compared with data obtained with a keratin membrane from bovine hooves. If the swelling of the nail plate or the hoof membrane is not altered by use of lipophilic vehicles, the maximum flux of the drug is independent of its solubility in the vehicle and is the same as that from a saturated aqueous solution. These vehicles are not able to enter the hydrophilic keratin membrane because of their non-polar character and so cannot change the solubility of the penetrating substance in the barrier. If the concentration of the drug in the nail lacquer is sufficiently high, the maximum flux through both barriers equals that from aqueous vehicles or even exceeds it because of the formation of a supersaturated system. Penetration through the nail plate follows first order kinetics after a lag-time of 400 h. The course of penetration through the hoof membrane is initially membrane-controlled and later becomes a matrix-controlled process because of the membrane's greater permeability. Chloramphenicol is dissolved in the lacquer up to a concentration of 31%. The relative release rates from these solution matrices are independent of the drug concentration but they decrease on changing to a suspension matrix. These results show that drug flux is independent of the character of the vehicle and that penetration of the drug is initially membrane-controlled and changes to being matrix-controlled as the drug content of the lacquer decreases.

  2. [Urinary bladder substitution using combined membrane based on secretions of human mesenchymal stem cells and type I collagen].

    Science.gov (United States)

    Kirpatovckii, V I; Kamalov, D M; Efimenko, A Yu; Makarevich, P I; Sagaradze, G D; Makarevich, O A; Nimiritskii, P P; Osidak, E O; Domogatskii, S P; Karpov, V K; Akopyan, Z H A; Tkachuk, V A; Kamalov, A A

    2016-12-01

    Despite the widespread use of intestinal cystoplasty, urinary bladder substitution remains a challenging problem due to the complexity of operations and the potentially high risk of complications. A promising alternative may be bio-engineered collagen-based matrices containing stem cells or their secretions. To evaluate the effectiveness of this bladder substitution modality, an experiment was conducted on 14 male rabbits. The animals underwent resection of urinary bladder, and the formed defect was substituted with a membrane of type I collagen (series 1, 5 rabbits) or a membrane of the same composition containing a conditioned medium with secretion of mesenchymal stem/stromal cells derived from human adipose tissue (series 2, 5 rabbits). In the comparison group (4 rabbits) resection of the bladder and the closure of the defect was carried out without bladder substitution (series 3). At 1 month after surgery, there was a complete epithelization of the inner surface of the implant, and body tissues replaced the collagen matrix. In series 1, the collagen implant was replaced mainly by connective tissue ingrown with occasional solitary smooth muscle cells. In series 2, the newly formed bladder wall contained numerous smooth muscle cells, growing into the collagen matrix and forming the muscular coat. In series 3, the muscular layer regeneration at the scar site was also noted, but it was less intense, which was confirmed by morphometry. In series 2, more active vascularization of the collagen implant occurred due to neo-angiogenesis, which was more intense than that in series 3, and especially in series 1. Functional studies revealed a reduced bladder functional capacity in series 1 and 3, while in series 2 it was close to normal. During filling cystometry, changes in intra-vesical pressure profile in series 2 were close to normal, while in series 1 and 3 infusion of a small volume of saline resulted in a marked increase in intra-vesical pressure, showing a reduced

  3. SEMP1, a senescence-associated cDNA isolated from human mammary epithelial cells, is a member of an epithelial membrane protein superfamily.

    Science.gov (United States)

    Swisshelm, K; Machl, A; Planitzer, S; Robertson, R; Kubbies, M; Hosier, S

    1999-01-21

    We have cloned a human cDNA, SEMP1 (senescence-associated epithelial membrane protein 1), using differential display (DD) of mRNA. We compared mRNA expression profiles between cultured normal senescent human mammary epithelial cells (HMECs) and proliferating, early passage HMECs. From the amino acid sequence of the open reading frame (ORF) of the cDNA, we infer that the protein belongs to a family of membrane-associated, epithelial cell-specific proteins. The translation product has 91% identity to a mouse protein, claudin-1, a tight junction (TJ)-associated protein. SEMP1 mRNA is expressed in human tissues, including adult and fetal liver, pancreas, placenta, adrenals, prostate and ovary but at low or undetectable levels in a number of human breast cancer cell lines. SEMP1 is a member of a superfamily of epithelial membrane proteins (EMPs), which may have multiple potential functions, including maintenance and regulation of cell polarity and permeability, perhaps through mechanisms involving tight junctions.

  4. A surface membrane protein of Entamoeba histolytica functions as a receptor for human chemokine IL-8: its role in the attraction of trophozoites to inflammation sites.

    Science.gov (United States)

    Diaz-Valencia, J Daniel; Pérez-Yépez, Eloy Andrés; Ayala-Sumuano, Jorge Tonatiuh; Franco, Elizabeth; Meza, Isaura

    2015-12-01

    Entamoeba histolytica trophozoites respond to the presence of IL-8, moving by chemotaxis towards the source of the chemokine. IL-8 binds to the trophozoite membrane and triggers a response that activates signaling pathways that in turn regulate actin/myosin cytoskeleton organisation to initiate migration towards the chemokine, suggesting the presence of a receptor for IL-8 in the parasite. Antibodies directed to the human IL-8 receptor (CXCR1) specifically recognised a 29 kDa protein in trophozoite membrane fractions. The same protein was immunoprecipitated by this antibody from total amebic extracts. Peptide analysis of the immunoprecipitated protein revealed a sequence with high homology to a previously identified amebic outer membrane peroxiredoxin and a motif within the third loop of human CXCR1, which is an important site for IL-8 binding and activation of signaling processes. Immunodetection assays demonstrated that the anti-human CXCR1 antibody binds to the 29 kDa protein in a different but close site to where IL-8 binds to the trophozoite surface membrane, suggesting that human and amebic receptors for this chemokine share common epitopes. In the context of the human intestinal environment, a receptor for IL-8 could be a great advantage for E. histolytica trophozoite survival, as they could reach an inflammatory milieu containing abundant nutrients. In addition, it has been suggested that the high content of accessible thiol groups of the protein and its peroxidase activity could provide protection in the oxygen rich milieu of colonic lesions, allowing trophozoite invasion of other tissues and escape from the host immune response. Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  5. Collagen-Coated Polytetrafluoroethane Membrane Inserts Enhances Chondrogenic Differentiation of Human Cord Blood Multi-Lineage Progenitor Cells

    DEFF Research Database (Denmark)

    Munir, Samir; Søballe, Kjeld; Ulrich-Vinther, Michael

    in standard micromass pellet system, layered on calcium polyphosphate (CPP), and on semi-permeable polytetrafluoroethane membranes with and without collagen type I, II or IV pre-coating. Findings / Results: The MPLC cell line used in this study possessed poor chondrogenic potency overall, but membrane...

  6. Optimization of micro-fabricated porous membranes for intestinal epithelial cell culture and in vitro modeling of the human intestinal barrier

    Science.gov (United States)

    Nair Gourikutty Sajay, Bhuvanendran; Yin, Chiam Su; Ramadan, Qasem

    2017-12-01

    In vitro modeling of organs could provide a controlled platform for studying physiological events and has great potential in the field of pharmaceutical development. Here, we describe the characterization of in vitro modeling of the human intestinal barrier mimicked using silicon porous membranes as a substrate. To mimic an intestinal in vivo setup as closely as possible, a porous substrate is required in a dynamic environment for the cells to grow rather than a static setup with an impermeable surface such as a petri dish. In this study, we focus on the detailed characterization of Caco-2 cells cultured on a silicon membrane with different pore sizes as well as the effect of dynamic fluid flow on the model. The porous silicon membrane together with continuous perfusion of liquid applying shear stress on the cells enhances the differentiation of polarized cells by providing access to the both their basal and apical surfaces. Membranes with pore sizes of 0.5–3 µm were used and a shear stress of ~0.03 dyne cm‑2 was created by applying a low flow rate of 20 nl s‑1. By providing these optimized conditions, cells were able to differentiate with columnar morphology, which developed microvilli structures on their apical side and tight junctions between adjacent cells like those in a healthy human intestinal barrier. In this setup, it is possible to study the important cellular functions of the intestine such as transport, absorption and secretion, and thus this model has great potential in drug screening.

  7. Localization of a membrane glycoprotein in benign fibrocystic disease and infiltrating duct carcinomas of the human breast with the use of a monoclonal antibody to guinea pig milk fat globule membrane.

    Science.gov (United States)

    Greenwalt, D. E.; Johnson, V. G.; Kuhajda, F. P.; Eggleston, J. C.; Mather, I. H.

    1985-01-01

    With monoclonal antibody D-274, raised against guinea pig milk fat globule membrane, the distribution of mucinlike glycoproteins of Mrs greater than or equal to 400,000 was determined in benign fibrocystic disease and infiltrating duct carcinoma of the human breast. These glycoproteins, called collectively PAS-I, were detected in 19 out of 20 cases of benign fibrocystic disease and in at least 26 out of 47 cases of infiltrating duct carcinoma. PAS-I was concentrated on luminal surfaces of ducts and alveoli in morphologically differentiated regions of the tumors. In areas where the glandular nature of the tissue was less evident in infiltrating duct carcinoma, the PAS-I determinant recognized by antibody D-274 was present on irregular luminal surfaces and in the cytoplasm. There was a negative correlation between the short-term recurrence (less than 2 years) of infiltrating duct carcinoma and the detection of strong positive staining with antibody D-274. The results are discussed with reference to recent studies on PAS-I in human breast tissue using monoclonal antibodies raised against human milk fat globule membrane. Images Figure 1 Figure 2 Figure 3 PMID:2579563

  8. Assessment of the efficacy of membrane filtration processes to remove human enteric viruses and the suitability of bacteriophages and a plant virus as surrogates for those viruses.

    Science.gov (United States)

    Shirasaki, N; Matsushita, T; Matsui, Y; Murai, K

    2017-05-15

    Here, we evaluated the efficacy of direct microfiltration (MF) and ultrafiltration (UF) to remove three representative human enteric viruses (i.e., adenovirus [AdV] type 40, coxsackievirus [CV] B5, and hepatitis A virus [HAV] IB), and one surrogate of human caliciviruses (i.e., murine norovirus [MNV] type 1). Eight different MF membranes and three different UF membranes were used. We also examined the ability of coagulation pretreatment with high-basicity polyaluminum chloride (PACl) to enhance virus removal by MF. The removal ratios of two bacteriophages (MS2 and φX174) and a plant virus (pepper mild mottle virus; PMMoV) were compared with the removal ratios of the human enteric viruses to assess the suitability of these viruses to be used as surrogates for human enteric viruses. The virus removal ratios obtained with direct MF with membranes with nominal pore sizes of 0.1-0.22 μm differed, depending on the membrane used; adsorptive interactions, particularly hydrophobic interactions between virus particles and the membrane surface, were dominant factors for virus removal. In contrast, direct UF with membranes with nominal molecular weight cutoffs of 1-100 kDa effectively removed viruses through size exclusion, and >4-log10 removal was achieved when a membrane with a nominal molecular weight cutoff of 1 kDa was used. At pH 7 and 8, in-line coagulation-MF with nonsulfated high-basicity PACls containing Al30 species had generally a better virus removal (i.e., >4-log10 virus removal) than the other aluminum-based coagulants, except for φX174. For all of the filtration processes, the removal ratios of AdV, CV, HAV, and MNV were comparable and strongly correlated with each other. The removal ratios of MS2 and PMMoV were comparable or smaller than those of the three human enteric viruses and MNV, and were strongly correlated with those of the three human enteric viruses and MNV. The removal ratios obtained with coagulation-MF for φX174 were markedly smaller than

  9. Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins.

    Science.gov (United States)

    Krautz-Peterson, Greice; Debatis, Michelle; Tremblay, Jacqueline M; Oliveira, Sergio C; Da'dara, Akram A; Skelly, Patrick J; Shoemaker, Charles B

    2017-01-01

    Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous Schistosoma mansoni worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by S. mansoni infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human S. mansoni infection sera antibodies. We conclude that, Schistosoma mansoni infection of both permissive (mouse) and non-permissive (rat) rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development.

  10. Mitochondrial Membrane Potential and Nuclear and Gene Expression Changes During Human Disc Cell Apoptosis: In Vitro and In Vivo Annulus Findings.

    Science.gov (United States)

    Gruber, Helen E; Hoelscher, Gretchen L; Bethea, Synthia; Hanley, Edward N

    2015-06-15

    A study using cultured human annulus cells and human annular tissue. To further explore and define mitochondrial mechanisms related to disc cell apoptosis in vitro and in vivo. Mitochondrial-dependent intrinsic signaling pathways are a well-recognized component of apoptosis (programmed cell death). Disc cell apoptosis is important because it is a major mechanism by which cell numbers decrease during disc degeneration. Our objective was to further explore and define mitochondrial mechanisms related to disc cell apoptosis. High-content screening techniques were used to study nuclear morphology and mitochondrial membrane potentials in cultured annulus cells. Gene expression in annulus tissue was studied with microarray analysis. Cultured cells showed significantly increased nuclear size (an indicator of apoptosis) with increasing Thompson grade (P potential (which results from the difference in electrical potential generated by the electrochemical gradient across the inner membrane of the mitochondrion) versus Thompson grade was identified in cultured human annulus cells in control conditions (r = 0.356, P potential was identified versus nontreated cells. Gene expression patterns in more degenerated Thompson grade III, IV, and V discs versus healthier grade I and II discs showed significant upregulation of a number of genes with well-recognized apoptosis roles in mitochondrial potential decline (ITM2B, beta-2-microglobulin, and cathepsin B, DAP, GAS1, and PDCD5) and TNF-α associations (cathepsin B, RAC1, and PPT1). Data presented here show the in vivo expression of apoptosis-related genes associated with the loss of mitochondrial membrane integrity and decreased mitochondrial membrane potential with increasing Thompson scores. These data, which mimic our novel, direct cell-based in vitro findings, stress the importance of mitochondrial changes related to apoptosis and TNF-α during human disc degeneration. N/A.

  11. Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins.

    Directory of Open Access Journals (Sweden)

    Greice Krautz-Peterson

    2017-01-01

    Full Text Available Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous Schistosoma mansoni worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by S. mansoni infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human S. mansoni infection sera antibodies. We conclude that, Schistosoma mansoni infection of both permissive (mouse and non-permissive (rat rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development.

  12. Immunoblot detection of class-specific humoral immune response to outer membrane proteins isolated from Salmonella typhi in humans with typhoid fever.

    OpenAIRE

    ORTIZ,V; Isibasi, A.; García-Ortigoza, E; Kumate, J.

    1989-01-01

    The studies reported here were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce a humoral immune response in humans with typhoid fever. OMPs were isolated with the nonionic detergent Triton X-100 and were found to be contaminated with approximately 4% lipopolysaccharide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns showed protein bands with molecular size ranges from 17 to 70 kilodaltons; the major groups of proteins we...

  13. Distribution of individual components of basement membrane in human colon polyps and adenocarcinomas as revealed by monoclonal antibodies

    DEFF Research Database (Denmark)

    Ljubimov, A V; Bartek, J; Couchman, J R

    1992-01-01

    by the presence of fibrillar deposits of basement-membrane components, mainly of collagen type IV and/or heparan sulfate proteoglycan. This reaction was never observed in polyps and may be derived from myofibroblasts reported to accumulate in colon cancer stroma. The combined use of antibodies to basement......-membrane components (laminin, entactin/nidogen, collagen type IV and large heparan sulfate proteoglycan), as well as to keratin 8. In all adenocarcinomas, including mucinous, basement membranes were altered more at the invasive front than in the parenchyma. The degree of this alteration was inversely correlated...

  14. Membrane transporters for the special amino acid glutamine: Structure/function relationships and relevance to human health.

    Science.gov (United States)

    Pochini, Lorena; Scalise, Mariafrancesca; Galluccio, Michele; Indiveri, Cesare

    2014-08-01

    Glutamine together with glucose is essential for body’s homeostasis. It is the most abundant amino acid and is involved in many biosynthetic, regulatory and energy production processes. Several membrane transporters which differ in transport modes, ensure glutamine homeostasis by coordinating its absorption, reabsorption and delivery to tissues. These transporters belong to different protein families, are redundant and ubiquitous. Their classification, originally based on functional properties, has recently been associated with the SLC nomenclature. Function of glutamine transporters is studied in cells over-expressing the transporters or, more recently in proteoliposomes harboring the proteins extracted from animal tissues or over-expressed in microorganisms. The role of the glutamine transporters is linked to their transport modes and coupling with Na+ and H+. Most transporters share specificity for other neutral or cationic amino acids. Na+-dependent co-transporters efficiently accumulate glutamine while antiporters regulate the pools of glutamine and other amino acids. The most acknowledged glutamine transporters belong to the SLC1, 6, 7 and 38 families. The members involved in the homeostasis are the co-transporters B0AT1 and the SNAT members 1, 2, 3, 5 and 7; the antiporters ASCT2, LAT1 and 2. The last two are associated to the ancillary CD98 protein. Some information on regulation of the glutamine transporters exist, which, however, need to be deepened. No information at all is available on structures, besides some homology models obtained using similar bacterial transporters as templates. Some models of rat and human glutamine transporters highlight very similar structures between the orthologues. Moreover the presence of glycosylation and/or phosphorylation sites located at the extracellular or intracellular faces has been predicted. ASCT2 and LAT1 are over-expressed in several cancers, thus representing potential targets for pharmacological intervention.

  15. Membrane transporters for the special amino acid glutamine: Structure/function relationships and relevance to human health.

    Directory of Open Access Journals (Sweden)

    Lorena ePochini

    2014-08-01

    Full Text Available Glutamine together with glucose is essential for body’s homeostasis. It is the most abundant amino acid and is involved in many biosynthetic, regulatory and energy production processes. Several membrane transporters which differ in transport modes, ensure glutamine homeostasis by coordinating its absorption, reabsorption and delivery to tissues. These transporters belong to different protein families, are redundant and ubiquitous. Their classification, originally based on functional properties, has recently been associated with the SLC nomenclature. Function of glutamine transporters is studied in cells over-expressing the transporters or, more recently in proteoliposomes harboring the proteins extracted from animal tissues or over-expressed in microorganisms. The role of the glutamine transporters is linked to their transport modes and coupling with Na+ and H+. Most transporters share specificity for other neutral or cationic amino acids. Na+-dependent co-transporters efficiently accumulate glutamine while antiporters regulate the pools of glutamine and other amino acids. The most acknowledged glutamine transporters belong to the SLC1, 6, 7 and 38 families. The members involved in the homeostasis are the co-transporters B0AT1 and the SNAT members 1, 2, 3, 5 and 7; the antiporters ASCT2, LAT1 and 2. The last two are associated to the ancillary CD98 protein. Some information on regulation of the glutamine transporters exist, which, however, need to be deepened. No information at all is available on structures, besides some homology models obtained using similar bacterial transporters as templates. Some models of rat and human glutamine transporters highlight very similar structures between the orthologues. Moreover the presence of glycosylation and/or phosphorylation sites located at the extracellular or intracellular faces has been predicted. ASCT2 and LAT1 are over-expressed in several cancers, thus representing potential targets for

  16. Insulin stimulates translocation of human GLUT4 to the membrane in fat bodies of transgenic Drosophila melanogaster.

    Science.gov (United States)

    Crivat, Georgeta; Lizunov, Vladimir A; Li, Caroline R; Stenkula, Karin G; Zimmerberg, Joshua; Cushman, Samuel W; Pick, Leslie

    2013-01-01

    The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in 'diabetic' flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM). We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to monitor insulin

  17. Insulin stimulates translocation of human GLUT4 to the membrane in fat bodies of transgenic Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Georgeta Crivat

    Full Text Available The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in 'diabetic' flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4, the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM. We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to

  18. Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

    Science.gov (United States)

    Burdick, Monica M.; Reynolds, Nathan M.; Martin, Eric W.; Hawes, Jacquelyn V.; Carlson, Grady E.; Cuckler, Chaz M.; Bates, Michael C.; Barthel, Steven R.; Dimitroff, Charles J.

    2014-01-01

    Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization

  19. The effect of salivary gland extract of Lucilia sericata maggots on human dermal fibroblast proliferation within collagen/hyaluronan membrane in vitro: transmission electron microscopy study.

    Science.gov (United States)

    Polakovičova, Simona; Polák, Štefan; Kuniaková, Marcela; Čambal, Marek; Čaplovičová, Mária; Kozánek, Milan; Danišovič, L'uboš; Kopáni, Martin

    2015-05-01

    Lucilia sericata maggots are applied to chronic wounds to aid healing when conventional treatments have failed. After their application into a necrotic wound, they potentially influence wound healing with a combination of specific proteinases that are involved in the remodeling of extracellular matrix. These proteases cause changes in fibroblast adhesion and spread upon extracellular matrix protein surfaces, affecting integrity of the protein surfaces-especially fibronectin-while maintaining cell viability. This study focused on in vitro monitoring of the effect of homogenate substances prepared from maggot salivary gland of L sericata on the ultrastructure of human neonatal fibroblasts. Collagen/hyaluronan membrane was used as the synthetic substitute of extracellular matrix. The cultured human neonatal fibroblasts B-HNF-1 were seeded on the surface of the collagen/hyaluronan membrane and cultured with maggot salivary gland extract (SGE) at a concentration of 2.4 glands/1 mL. The authors observed increased cell metabolism and protein production (euchromatic nucleus, voluminous nuclear membrane, large reticular nuclei, distended and filled cisterns of rough endoplasmic reticulum, Golgi apparatus with saccules, and vesicles packed with fine fibrillar material) after incubating the cells in culture medium with SGE. The authors believe that increased cell metabolism and protein production corresponded with formation of microfibrillar net used for migration of fibroblasts in culture, but mainly for proper production of extracellular matrix. The authors suggest that their results may help explain the effect of SGE on wound healing and support implementation of maggot therapy into human medicine.

  20. Glucose adsorption to chitosan membranes increases proliferation of human chondrocyte via mammalian target of rapamycin complex 1 and sterol regulatory element-binding protein-1 signaling.

    Science.gov (United States)

    Chang, Shun-Fu; Huang, Kuo-Chin; Cheng, Chin-Chang; Su, Yu-Ping; Lee, Ko-Chao; Chen, Cheng-Nan; Chang, Hsin-I

    2017-10-01

    Osteoarthritis (OA) is currently still an irreversible degenerative disease of the articular cartilage. Recent, dextrose (d-glucose) intraarticular injection prolotherapy for OA patients has been reported to benefit the chondrogenic stimulation of damaged cartilage. However, the detailed mechanism of glucose's effect on cartilage repair remains unclear. Chitosan, a naturally derived polysaccharide, has recently been investigated as a surgical or dental dressing to control breeding. Therefore, in this study, glucose was adsorbed to chitosan membranes (CTS-Glc), and the study aimed to investigate whether CTS-Glc complex membranes could regulate the proliferation of human OA chondrocytes and to explore the underlying mechanism. Human OA and SW1353 chondrocytes were used in this study. The experiments involving the transfection of cells used SW1353 chondrocytes. A specific inhibitor and siRNAs were used to investigate the mechanism underlying the CTS-Glc-regulated proliferation of human chondrocytes. We found that CTS-Glc significantly increased the proliferation of both human OA and SW1353 chondrocytes comparable to glucose- or chitosan-only stimulation. The role of mammalian target of rapamycin complex 1 (mTORC1) signaling, including mTOR, raptor, and S6k proteins, has been demonstrated in the regulation of CTS-Glc-increased human chondrocyte proliferation. mTORC1 signaling increased the expression levels of maturated SREBP-1 and FASN and then induced the expressions of cell cycle regulators, that is, cyclin D, cyclin-dependent kinase-4 and -6 in human chondrocytes. This study elucidates the detailed mechanism behind the effect of CTS-Glc complex membranes in promoting chondrocyte proliferation and proposes a possible clinical application of the CTS-Glc complex in the dextrose intraarticular injection of OA prolotherapy in the future to attenuate the pain and discomfort of OA patients. © 2017 Wiley Periodicals, Inc.

  1. Transplanted human amniotic membrane-derived mesenchymal stem cells ameliorate carbon tetrachloride-induced liver cirrhosis in mouse.

    Directory of Open Access Journals (Sweden)

    DingGuo Zhang

    Full Text Available BACKGROUND: Human amniotic membrane-derived mesenchymal stem cells (hAMCs have the potential to reduce heart and lung fibrosis, but whether could reduce liver fibrosis remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Hepatic cirrhosis model was established by infusion of CCl₄ (1 ml/kg body weight twice a week for 8 weeks in immunocompetent C57Bl/6J mice. hAMCs, isolated from term delivered placenta, were infused into the spleen at 4 weeks after mice were challenged with CCl₄. Control mice received only saline infusion. Animals were sacrificed at 4 weeks post-transplantation. Blood analysis was performed to evaluate alanine aminotransferase (ALT and aspartate aminotransferase (AST. Histological analysis of the livers for fibrosis, hepatic stellate cells activation, hepatocyte apoptosis, proliferation and senescence were performed. The donor cell engraftment was assessed using immunofluorescence and polymerase chain reaction. The areas of hepatic fibrosis were reduced (6.2%±2.1 vs. control 9.6%±1.7, p<0.05 and liver function parameters (ALT 539.6±545.1 U/dl, AST 589.7±342.8 U/dl,vs. control ALT 139.1±138.3 U/dl, p<0.05 and AST 212.3±110.7 U/dl, p<0.01 were markedly ameliorated in the hAMCs group compared to control group. The transplantation of hAMCs into liver-fibrotic mice suppressed activation of hepatic stellate cells, decreased hepatocyte apoptosis and promoted liver regeneration. More interesting, hepatocyte senescence was depressed significantly in hAMCs group compared to control group. Immunofluorescence and polymerase chain reaction revealed that hAMCs engraftment into host livers and expressed the hepatocyte-specific markers, human albumin and α-fetoproteinran. CONCLUSIONS/SIGNIFICANCE: The transplantation of hAMCs significantly decreased the fibrosis formation and progression of CCl₄-induced cirrhosis, providing a new approach for the treatment of fibrotic liver disease.

  2. Local membrane deformations activate Ca2+-dependent K+ and anionic currents in intact human red blood cells

    DEFF Research Database (Denmark)

    Dyrda, Agnieszka; Cytlak, Urszula; Ciuraszkiewicz, Anna

    2010-01-01

    by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents......-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca(2+)-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca(2+) content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.......BACKGROUND: The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced...

  3. The Effects of BMP-2, miR-31, miR-106a, and miR-148a on Osteogenic Differentiation of MSCs Derived from Amnion in Comparison with MSCs Derived from the Bone Marrow

    Directory of Open Access Journals (Sweden)

    Sirikul Manochantr

    2017-01-01

    Full Text Available Mesenchymal stromal cells (MSCs offering valuable anticipations for the treatment of degenerative diseases. They can be found in many tissues including amnion. MSCs from amnion (AM-MSCs can differentiate into osteoblast similar to that of bone marrow-derived MSCs (BM-MSCs. However, the ability is not much efficient compared to BM-MSCs. This study aimed to examine the effects of BMP-2 and miRNAs on osteogenic differentiation of AM-MSCs compared to those of BM-MSCs. The osteogenic differentiation capacity after miRNA treatment was assessed by ALP expression, ALP activity, and osteogenic marker gene expression. The results showed that the osteogenic differentiation capacity increased after BMP-2 treatment both in AM-MSCs and BM-MSCs. MiR-31, miR-106a, and miR-148a were downregulated during the osteogenic differentiation. After transfection with anti-miRNAs, ALP activity and osteogenic genes were increased over the time of differentiation. The data lead to the potential for using AM-MSCs as an alternative source for bone regeneration. Moreover, the information of miRNA expression and function during osteogenic differentiation may be useful for the development of new therapeutics or enhanced an in vitro culture technique required for stem cell-based therapies in the bone regeneration.

  4. F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

    Science.gov (United States)

    Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

    2015-05-09

    Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting

  5. The Effect of Covalently-Attached ATRP-Synthesized Polymers on Membrane Stability and Cytoprotection in Human Erythrocytes

    OpenAIRE

    William P Clafshenkel; Hironobu Murata; Jill Andersen; Yehuda Creeger; Koepsel, Richard R; Russell, Alan J

    2016-01-01

    Erythrocytes have been described as advantageous drug delivery vehicles. In order to ensure an adequate circulation half-life, erythrocytes may benefit from protective enhancements that maintain membrane integrity and neutralize oxidative damage of membrane proteins that otherwise facilitate their premature clearance from circulation. Surface modification of erythrocytes using rationally designed polymers, synthesized via atom-transfer radical polymerization (ATRP), may further expand the fie...

  6. Membrane tension and membrane fusion

    OpenAIRE

    Kozlov, Michael M.; Chernomordik, Leonid V.

    2015-01-01

    Diverse cell biological processes that involve shaping and remodeling of cell membranes are regulated by membrane lateral tension. Here we focus on the role of tension in driving membrane fusion. We discuss the physics of membrane tension, forces that can generate the tension in plasma membrane of a cell, and the hypothesis that tension powers expansion of membrane fusion pores in late stages of cell-to-cell and exocytotic fusion. We propose that fusion pore expansion can require unusually la...

  7. Response of exfoliated human buccal epithelium cells to combined gamma radiation, microwaves, and magnetic field exposure estimated by changes in chromatin condensation and cell membrane permeability

    Directory of Open Access Journals (Sweden)

    K. А. Kuznetsov

    2016-11-01

    Full Text Available Modulation of the biological effects produced by ionizing radiation (IR using microwave and magnetic fields has important theoretical and practical applications. Response of human buccal epithelium cells to different physical agents (single and combined exposure to 0.5–5 Gy γ-radiation (60Co; microwaves with the frequency of 36.64 GHz and power densities of 0.1 and 1 W/m2, and static magnetic field with the intensity of 25 mT has been investigated. The stress response of the cells was evaluated by counting heterochromatin granules quantity (HGQ in the cell nuclei stained with orcein. Membrane permeability was assessed by the percentage of cells stained with indigocarmine (cells with damaged membrane. The increase of heterochromatin granules quantity (HGQ, i.e. chromatin condensation was detected at the doses of 2 Gy and higher. Changes in the cell membrane permeability to indigocarmine expressed the threshold effect. Membrane permeability reached the threshold at the doses of 2–3 Gy for the cells of different donors and did not change with the increase of the dose of γ-radiation. Cells obtained from different donors revealed some individual peculiarities in their reaction to γ-radiation. The static magnetic field and microwaves applied before or after γ-radiation decreased its impact, as revealed by means of HGQ assessment.

  8. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  9. Membrane topology of human presenilin-1 in SK-N-SH cells determined by fluorescence correlation spectroscopy and fluorescent energy transfer.

    Science.gov (United States)

    Midde, Krishna; Rich, Ryan; Saxena, Ashwini; Gryczynski, Ignacy; Borejdo, Julian; Das, Hriday K

    2014-11-01

    Presenilin-1 (PS1) protein acts as passive ER Ca(2+) leak channels that facilitate passive Ca(2+) leak across ER membrane. Mutations in the gene encoding PS1 protein cause neurodegeneration in the brains of patients with familial Alzheimer's disease (FAD). FADPS1 mutations abrogate the function of ER Ca(2+) leak channel activity in human neuroblastoma SK-N-SH cells in vitro (Das et al., J Neurochem 122(3):487-500, 2012) and in mouse embryonic fibroblasts. Consequently, genetic deletion or mutations of the PS1 gene cause calcium (Ca(2+)) signaling abnormalities leading to neurodegeneration in FAD patients. By analogy with other known ion channels it has been proposed that the functional PS1 channels in ER may be multimers of several PS1 subunits. To test this hypothesis, we conjugated the human PS1 protein with an NH2-terminal YFP-tag and a COOH-terminal CFP-tag. As expected YFP-PS1, and PS1-CFP were found to be expressed on the plasma membranes by TIRF microscopy, and both these fusion proteins increased ER Ca(2+) leak channel activity similar to PS1 (WT) in SK-N-SH cells, as determined by functional calcium imaging. PS1-CFP was either expressed alone or together with YFP-PS1 into SK-N-SH cell line and the interaction between YFP-PS1 and PS1-CFP was determined by Förster resonance energy transfer analysis. Our results suggest interaction between YFP-PS1 and PS1-CFP confirming the presence of a dimeric or multimeric form of PS1 in SK-N-SH cells. Lateral diffusion of PS1-CFP and YFP-PS1 in the plasma membrane of SK-N-SH cells was measured in the absence or in the presence of glycerol by fluorescence correlation spectroscopy to show that both COOH-terminal and NH2-terminal of human PS1 are located on the cytoplasmic side of the plasma membrane. Therefore, we conclude that both COOH-terminal and NH2-terminal of human PS1 may also be oriented on the cytosolic side of ER membrane.

  10. 7-ketocholesterol inhibits Na,K-ATPase activity by decreasing expression of its α1-subunit and membrane fluidity in human endothelial cells.

    Science.gov (United States)

    Duran, M J; Pierre, S V; Lesnik, P; Pieroni, G; Bourdeaux, M; Dignat-Georges, F; Sampol, J; Maixent, J M

    2010-11-09

    As cholesterol, oxysterols, can insert the cell membrane and thereby modify the functions of membrane-bound proteins. The Na,K-ATPase is very sensitive to its lipid environment, seems to be involved in important endothelial functions as the regulation of nitric oxide (NO) release. The effects of 7-ketocholesterol , an oxysterol present in oxidized LDL, was investigated on Na,K-ATPase in isolated human endothelial cells. Cells were incubated 24h with lecithin-, cholesterol- or 7-ketocholesterol liposomes (6 μg/ml). K+-stimulated paranitrophenyl phosphatase activity, reflecting Na,K-ATPase activity, was evaluated as well as cell viability and lipoperoxidation. The expression of Na,K-ATPase subunits mRNAs and membrane fluidity were also investigated. As Na,K-ATPase and nitric oxide seem to be related, we determined the production of NO and the expression of endothelial NO synthase mRNAs. Na,K-ATPase activity was strongly decreased by 7-ketocholesterol. This decrease, not related to lipoperoxidation, was correlated with a decreased expression of the Na,K-ATPase α1-subunit messengers and with rigidity of plasma membranes. Cholesterol induced similar effects but was less potent than 7-ketocholesterol. Basal NO production and expression of endothelial NO synthase mRNAs were not modified by 7-ketocholesterol. Our new findings demonstrate that 7-ketocholesterol, used at non toxic doses, was very potent to disrupt the transport of ions by Na,K-ATPase and perturb membrane structure. These data demonstrate that 7-ketocholesterol induces endothelial dysfunction without cell death that may contribute to early events in atherosclerosis.

  11. Spontaneous complement activation on human B cells results in localized membrane depolarization and the clustering of complement receptor type 2 and C3 fragments

    DEFF Research Database (Denmark)

    Løbner, Morten; Leslie, Robert G Q; Prodinger, Wolfgang M

    2009-01-01

    While our previous studies have demonstrated that complement activation induced by complement receptors type 2 (CR2/CD21) and 1 (CR1/CD35) results in C3-fragment deposition and membrane attack complex (MAC) formation in human B cells, the consequences of these events for B-cell functions remain...... requires activation of complement via the alternative pathway, as indicated by total inhibition upon neutralization of factor D, and is abrogated by combined blockade of CR1 and CR2, but not of either receptor alone. The membrane depolarization is not associated with the apoptosis of B cells, as examined...... by co-staining with APO-2.7 or by the TdT-mediated biotin-dUTP nick-end labelling (TUNEL) assay. Confocal microscopy revealed that depolarization and C3 deposition, unlike MAC deposition, are limited to restricted areas on the B-cell surface. Double staining revealed a close association between the C3...

  12. Assignment of the human membrane-type matrix metalloproteinase (MMP14) gene to 14q11-q12 by in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Mignon, C.; Mattei, M.G. [Faculte de la Medecine de la Timone, Marseille (France); Okada, A.; Basset, P. [Institut de Genetique et de Biologie, Strasbourg (France)

    1995-07-20

    Matrix metalloproteinases (MMP) are enzymes implicated in normal and pathological tissue remodeling processes. The MMP family currently comprises 11 members, among which membrane-type matrix metalloproteinase (MMP14) has most recently been described. MMP14 is believed to correspond to the membrane-associated activator of pro-gelatinase A (MMP2), and it has been proposed that such activation occurs on the cancer cell surface in invasive carcinomas. However, our observation that MMP14 transcripts are specifically expressed in fibroblastic cells during both wound healing and human cancer progression further supports the possibility that pro-gelatinase A activation is also elicited from the fibroblast cell surface. 12 refs., 1 fig., 1 tab.

  13. Human milk fat globules: polar lipid composition and in situ structural investigations revealing the heterogeneous distribution of proteins and the lateral segregation of sphingomyelin in the biological membrane.

    Science.gov (United States)

    Lopez, Christelle; Ménard, Olivia

    2011-03-01

    Although human milk fat globules (MFG) are of primary importance since they are the exclusive lipid delivery carriers in the gastrointestinal tract of breast-fed infants, they remain the poorly understood aspect of milk. The objectives of this study were to investigate these unique colloidal assemblies and their interfacial properties, i.e. composition and structure of their biological membrane. In mature breast milk, MFG have a mean diameter of 4-5 microm, a surface area of about 2m(2)/g fat and an apparent zeta potential ζ=-6.7 ± 0.5 mV at 37°C. Human MFG contain 3-4mg polar lipids/g fat as quantified by HPLC/ELSD. The main polar lipids are sphingomyelin (SM; 36-45%, w/w), phosphatidylcholine (19-23%, w/w) and phosphatidylethanolamine (10-15%, w/w). In situ structural investigations of human MFG have been performed using light and confocal microscopy with adapted fluorescent probes, i.e. Nile Red, the extrinsic phospholipid Rh-DOPE, Fast Green and the lectin WGA-488. This study revealed a spatial heterogeneity in the human milk fat globule membrane (MFGM), with the lateral segregation of SM in liquid-ordered phase domains of various shapes and sizes surrounded by a liquid-disordered phase composed of the glycerophospholipids in which the proteins are dispersed. The glycocalyx formed by glycoproteins and cytoplasmic remnents have also been characterised around human MFG. A new model for the structure of the human MFGM is proposed and discussed. The unique composition and lateral organisation of the human MFGM components could be of metabolic significance and have health impact for the infants that need to be further explored. 2010 Elsevier B.V. All rights reserved.

  14. Genotoxicity assessment of membrane concentrates of landfill leachate treated with Fenton reagent and UV-Fenton reagent using human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Guifang [Department of Chemistry, Jinan University, Guangzhou 510632 (China); Lu, Gang [Key Laboratory of Water/Soil Toxic Pollutants Control and Bioremediation of Guangdong Higher Education Institutes, Department of Environmental Engineering, Jinan University, Guangzhou 510632 (China); Yin, Pinghe, E-mail: tyinph@jnu.edu.cn [Research Center of Analysis and Test, Jinan University, Guangzhou 510632 (China); Zhao, Ling, E-mail: zhaoling@jnu.edu.cn [Key Laboratory of Water/Soil Toxic Pollutants Control and Bioremediation of Guangdong Higher Education Institutes, Department of Environmental Engineering, Jinan University, Guangzhou 510632 (China); Jimmy Yu, Qiming [Griffith School of Engineering, Griffith University, Nathan Campus, Brisbane, Queensland 4111 (Australia)

    2016-04-15

    Highlights: • Membrane concentrates have a threat to human health and environment. • Untreated membrane concentrates induces cytotoxic and genotoxic to HepG2 cells. • Both methods were effective method for degradation of BPA and NP in concentrates. • Both methods were efficient in reducing genotoxic effects of concentrates. • UV-Fenton reagent had higher removal efficiency and provides toxicological safety. - Abstract: Membrane concentrates of landfill leachates contain organic and inorganic contaminants that could be highly toxic and carcinogenic. In this paper, the genotoxicity of membrane concentrates before and after Fenton and UV-Fenton reagent was assessed. The cytotoxicity and genotoxicity was determined by using the methods of methyltetrazolium (MTT), cytokinesis-block micronucleus (CBMN) and comet assay in human hepatoma cells. MTT assay showed a cytotoxicity of 75% after 24 h of exposure to the highest tested concentration of untreated concentrates, and no cytotoxocity for UV-Fenton and Fenton treated concentrates. Both CBMN and comet assays showed increased levels of genotoxicity in cells exposed to untreated concentrates, compared to those occurred in cells exposed to UV-Fenton and Fenton reagent treated concentrates. There was no significant difference between negative control and UV-Fenton treated concentrates for micronucleus and comet assay parameters. UV-Fenton and Fenton treatment, especially the former, were effective methods for degradation of bisphenol A and nonylphenol in concentrates. These findings showed UV-Fenton and Fenton reaction were effective methods for treatment of such complex concentrates, UV-Fenton reagent provided toxicological safety of the treated effluent, and the genotoxicity assays were found to be feasible tools for assessment of toxicity risks of complex concentrates.

  15. Local membrane deformations activate Ca2+-dependent K+ and anionic currents in intact human red blood cells.

    Directory of Open Access Journals (Sweden)

    Agnieszka Dyrda

    Full Text Available BACKGROUND: The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. METHODOLOGY/PRINCIPAL FINDINGS: The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K(+ and Cl(- currents were strictly dependent on the presence of Ca(2+. The Ca(2+-dependent currents were transient, with typical decay half-times of about 5-10 min, suggesting the spontaneous inactivation of a stretch-activated Ca(2+ permeability (PCa. These results indicate that local membrane deformations can transiently activate a Ca(2+ permeability pathway leading to increased [Ca(2+](i, secondary activation of Ca(2+-sensitive K(+ channels (Gardos channel, IK1, KCa3.1, and hyperpolarization-induced anion currents. CONCLUSIONS/SIGNIFICANCE: The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca(2+-mediated effects observed during the normal aging process of red blood cells, and

  16. Intercellular deposits of basement membrane material in active human pituitary adenomas detected by immunostaining for laminin and electron microscopy

    DEFF Research Database (Denmark)

    Holck, S; Wewer, U M; Albrechtsen, R

    1986-01-01

    and one patient with Cushing's syndrome). Concurrently, at the ultrastructural level, bunches of basement membrane-like material intermingled between the adenoma cells were demonstrated in seven of these ten active adenomas. Furthermore, secretory granules were entrapped occasionally in this intercellular...

  17. Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases.

    NARCIS (Netherlands)

    Ritzerfeld, J.; Remmele, S.; Wang, T.; Temmerman, K.; Brugger, B.; Wegehingel, S.; Tournaviti, S.; Strating, J.R.P.M.; Wieland, F.T.; Neumann, B.; Ellenberg, J.; Lawerenz, C.; Hesser, J.; Erfle, H.; Pepperkok, R.; Nickel, W.

    2011-01-01

    SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary

  18. Carrier-mediated ¿-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger

    2012-01-01

    and the anticancer prodrug d-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least...

  19. Primary structure analysis and lamin B and DNA binding of human LBR, an integral protein of the nuclear envelope inner membrane.

    Science.gov (United States)

    Ye, Q; Worman, H J

    1994-04-15

    We have determined the primary structure of human LBR, an integral protein of the nuclear envelope inner membrane, and examined its interactions with lamin B and DNA. Human LBR is 68% identical to the chicken lamin B receptor and has a basic nucleoplasmic amino-terminal domain of 208 amino acids followed by a hydrophobic domain with eight putative transmembrane segments. The amino-terminal domain contains a Ser-Arg-rich stretch and consensus sites for phosphorylation by protein kinase A and p34cdc2 protein kinase. A fusion protein containing the amino-terminal domain of human LBR is recognized by autoantibodies from patients with primary biliary cirrhosis, and these serum antibodies label the nuclear envelope when examined by immunofluorescence microscopy. The LBR amino-terminal domain precipitates lamin B from nuclear extracts and retards the migration of double-stranded DNA subjected to agarose gel electrophoresis. When immobilized on nitrocellulose, the amino-terminal domain of LBR also associates with DNA, and the stretch between amino acids 71 and 100, which contains the Ser-Arg-rich stretch, is necessary for DNA binding. These results demonstrate that LBR is conserved among vertebrate species and that its nucleoplasmic domain can potentially mediate the interaction of both the nuclear lamina and the chromatin with the inner nuclear membrane.

  20. The classical and alternative pathways of complement activation play distinct roles in spontaneous C3 fragment deposition and membrane attack complex (MAC) formation on human B lymphocytes

    DEFF Research Database (Denmark)

    Leslie, Robert Graham Quinton; Nielsen, Claus Henrik

    2004-01-01

    The contributions of the classical (CP) and alternative (AP) pathways of complement activation to the spontaneous deposition of C3 fragments and the formation of membrane attack complexes (MAC) on human B lymphocytes, were assessed by incubating peripheral blood mononuclear cells with autologous......, however, in the nature of the fragments deposited as a result of CP and AP activation: C3b fragments deposited via the CP were extensively ( approximately 90%) converted to the terminal degradation product, C3dg, whereas about 50% of those deposited by the AP persisted as C3b/iC3b fragments. The extent...

  1. Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells

    DEFF Research Database (Denmark)

    Prokhorova, Tatyana A; Rigbolt, Kristoffer T G; Johansen, Pia T

    2009-01-01

    -labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human......: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell...

  2. Application of Human Amniotic Membrane in Canine Penile Tunica Albuginea Defect: First Step toward an Innovating New Method for Treatment of Peyronie?s Disease

    Directory of Open Access Journals (Sweden)

    M. Salehipour

    2014-06-01

    Full Text Available Purposes To evaluate the efficacy of human amniotic membrane (AM grafting in the canine penile tunica albuginea defect; we developed an animal model as the first step toward an innovating new method for the treatment of Peyronie’s disease, penile cancers, and congenital deformities of the penis. Material and Methods From August to September 2011, ten healthy male dogs were selected. A rhomboid incision about 3x2cm over the tunica albuginea and its overlying squamous epithelium was made and then excised. The amniotic membrane was folded twice on itself and grafted on the defect. After 8 weeks, artificial erection was made for 5 dogs and for the other 5 dogs after 12 weeks. After artificial erection, partial penectomy was done and histopathological evaluation was performed on the grafts. Results Artificial erection performed successfully in all of the dogs. No infection or any other complication was seen. Histopathological examination showed complete re-epithelialization with squamous epithelium and collagen fiber deposition. Also, no dysplasia was seen. Conclusions The amniotic membrane can be used as a suitable substitution for tunica albuginea. It is safe, inexpensive, biodegradable, and available and may be used for the treatment of Peyronie’s disease, penile cancers, congenital penile deformities, and penile reconstructive surgery.

  3. The conserved N-terminus of human rhinovirus capsid protein VP4 contains membrane pore-forming activity and is a target for neutralizing antibodies.

    Science.gov (United States)

    Panjwani, Anusha; Asfor, Amin S; Tuthill, Tobias J

    2016-12-01

    Human rhinovirus is the causative agent of the common cold and belongs to the non-enveloped picornavirus family. A trigger such as receptor binding or low pH initiates conformational changes in the capsid that allow the virus to attach to membranes and form a pore for the translocation of viral RNA into the cytoplasm. We previously showed that recombinant capsid protein VP4 was able to form membrane pores. In this study, we show the N-terminus but not C-terminus of VP4 formed pores with properties similar to full-length VP4 and consistent with the size required for transfer of RNA. Sera against the N-terminus but not C-terminus of VP4 were shown to neutralize virus infectivity. Together, this suggests that the N-terminus of VP4 is responsible for membrane activity. This study contributes to an improved understanding of the mechanisms for involvement of VP4 in entry and its potential as an antiviral target.

  4. Effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine on membrane lipid profile and cryotolerance of human sperm.

    Science.gov (United States)

    Vireque, Alessandra A; Tata, Alessandra; Silva, Oswaldo F L L O; LoTurco, Edson G; Azzolini, Augusto; Ferreira, Christina R; Dantas, Marilda H Y; Ferriani, Rui A; Reis, Rosana M

    2016-08-01

    To study the effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine (soy-PC) on sperm cryotolerance with regard to sperm membrane lipid profile, membrane surface integrity, and routine semen parameters. Experimental study. University-affiliated tertiary hospital. A total of 20 normospermic fertile men. Semen samples examined for differences in semen parameters, sperm membrane lipid profile, and plasma membrane surface both before and after cryopreservation using basic freezing medium with N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) and tris-(hydroxymethyl)-aminomethane (TRIS) supplemented with purified soy-PC (TEST-PC) or egg yolk (TEST-Y), both alone or in association (TEST-Y-PC). Conventional semen parameters and membrane lipid profile by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). Postthaw sperm cell motility, vitality, and morphology parameters were similar for soy-PC (TEST-PC) and egg yolk (TEST-Y) cryoprotectants. However, sperm exposed to TEST-Y-PC presented better kinetic parameters, which were similar to the original quality of the fresh semen. Human sperm MALDI-MS lipid profiles revealed that the relative abundance of glycerophospholipids of m/z 760.44 [PC (34:1)+H]+, 781.55 [SM (20:0) +Na]+, 784.55 [PC (36:3) +H]+, 806.64 [PC (38:6) +H]+, 807.64 [SM (22:1) +Na]+, and 809.64 [SM (22:0) +Na]+ increased in soy-PC samples (TEST-PC). Nonetheless, only one lipid (m/z 781.55, [SM (20:0) +Na]+) statistically significantly changed when sperm was cryopreserved in TEST-Y-PC. Sphingomyelin was defined as a prospective biomarker of soy-PC treatment, and it could be related to the positive cryoprotective effects of soy-PC in human sperm, opening new perspectives to design of a more efficient synthetic cryoprotectant medium containing purified egg yolk biomolecules combined with soy-PC. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  5. Interaction of purified bovine brain A1-adenosine receptors with guanine nucleotide-binding proteins of human platelet membranes following reconstitution.

    Science.gov (United States)

    Munshi, R; Linden, J

    1990-08-01

    A1-adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) have been co-purified from bovine cerebral cortex by agonist affinity chromatography [J. Biol. Chem. 264:14853-14859 (1989)]. In this study we have reconstituted purified bovine brain A1 receptors into human platelet membranes that contain A2- but no detectable A1-adenosine receptors. The recovery of reconstituted receptors was assessed from the binding of the antagonist radioligand [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentyl-xanthine and ranged from 32 to 84%. Coupling of reconstituted A1 receptors to platelet G proteins was evaluated by measurement of the high affinity binding of an agonist radioligand, 125I-aminobenzyladenosine, to receptor-G protein complexes and by stereospecific photoaffinity labeling of a 35,000-Da receptor polypeptide with the agonist photoaffinity label 125I-azidobenzyladenosine. Fifty percent of receptors reconstituted into platelet membranes bound agonists with high affinity, indicative of coupling to platelet G proteins. Reconstituted A1 receptors bound various ligands with affinities characteristic of A1 receptors of bovine brain. Although platelets contain both pertussis toxin-sensitive and -insensitive G proteins, reconstituted high affinity agonist binding was almost completely abolished by treatment of platelet membranes with guanosine 5'-3-O-(thio)triphosphate, pertussis toxin, N-ethylmaleimide, or heparin. Following reconstitution, A1 receptors could be resolubilized in complexes with platelet G proteins. The data suggest that marked species differences in the binding affinity of ligands to adenosine receptors result from differences in the receptors rather than membrane structure or G proteins and, further, that A1 receptors couple selectively and tightly to pertussis toxin-sensitive G proteins.

  6. Parallel artificial liquid membrane extraction

    DEFF Research Database (Denmark)

    Gjelstad, Astrid; Rasmussen, Knut Einar; Parmer, Marthe Petrine

    2013-01-01

    This paper reports development of a new approach towards analytical liquid-liquid-liquid membrane extraction termed parallel artificial liquid membrane extraction. A donor plate and acceptor plate create a sandwich, in which each sample (human plasma) and acceptor solution is separated by an arti......This paper reports development of a new approach towards analytical liquid-liquid-liquid membrane extraction termed parallel artificial liquid membrane extraction. A donor plate and acceptor plate create a sandwich, in which each sample (human plasma) and acceptor solution is separated...... by an artificial liquid membrane. Parallel artificial liquid membrane extraction is a modification of hollow-fiber liquid-phase microextraction, where the hollow fibers are replaced by flat membranes in a 96-well plate format....

  7. Comprehensive quantitative comparison of the membrane proteome and PTM-ome of human embryonic stem cells and neural stem cells

    DEFF Research Database (Denmark)

    Braga, Marcella Nunes de Melo; Schulz, Melanie; Jakobsen, Lene

    pathways such as cdk5 and neuregulin signalling were found highly upregulated in hNSCs. Moreover, we identified known hNSC markers such as nestin, but also novel potential hNSC markers such as crumbs homolog 2 and protein wntless homolog. These data can be useful to gain a better understanding...... source for treatment of neurological diseases such as Parkinson´s disease. Membrane proteins are very important in cellular signaling and they are regulated by post-translational modifications such as phosphorylation and glycosylation. In order to obtain more information about important membrane proteins......-phosphopeptides from formerly SA glycopeptides prior to LC-MSMS. Phosphopeptides, former SA glycopeptides and non-modified peptides were identified and quantified; and regulated proteins were further analyzed using bioinformatics and pathway software. Preliminary data: Using our strategy, we were able to identify...

  8. Human Acyl-Coenzyme A:Cholesterol Acyltransferase Expressed in Chinese Hamster Ovary Cells: Membrane Topology and Active Site Location

    OpenAIRE

    Lin, Song; Lu, Xiaohui; Chang, Catherine C.Y.; Chang, Ta-Yuan

    2003-01-01

    Acyl-CoA:cholesterol acyltransferase (ACAT) is a membrane-bound enzyme that produces cholesteryl esters intracellularly. Two ACAT genes (ACAT1 and ACAT2) have been identified. The expression of ACAT1 is ubiquitous, whereas that of ACAT2 is tissue restricted. Previous research indicates that ACAT1 may contain seven transmembrane domains (TMDs). To study ACAT2 topology, we inserted two different antigenic tags (hemagglutinin, monoclonal antibody Mab1) at various hydrophi...

  9. Membrane Microdomains and Cytoskeleton Organization Shape and Regulate the IL-7 Receptor Signalosome in Human CD4 T-cells*

    Science.gov (United States)

    Tamarit, Blanche; Bugault, Florence; Pillet, Anne-Hélène; Lavergne, Vincent; Bochet, Pascal; Garin, Nathalie; Schwarz, Ulf; Thèze, Jacques; Rose, Thierry

    2013-01-01

    Interleukin (IL)-7 is the main homeostatic regulator of CD4 T-lymphocytes (helper) at both central and peripheral levels. Upon activation by IL-7, several signaling pathways, mainly JAK/STAT, PI3K/Akt and MAPK, induce the expression of genes involved in T-cell differentiation, activation, and proliferation. We have analyzed the early events of CD4 T-cell activation by IL-7. We have shown that IL-7 in the first few min induces the formation of cholesterol-enriched membrane microdomains that compartmentalize its activated receptor and initiate its anchoring to the cytoskeleton, supporting the formation of the signaling complex, the signalosome, on the IL-7 receptor cytoplasmic domains. Here we describe by stimulated emission depletion microscopy the key roles played by membrane microdomains and cytoskeleton transient organization in the IL-7-regulated JAK/STAT signaling pathway. We image phospho-STAT5 and cytoskeleton components along IL-7 activation kinetics using appropriate inhibitors. We show that lipid raft inhibitors delay and reduce IL-7-induced JAK1 and JAK3 phosphorylation. Drug-induced disassembly of the cytoskeleton inhibits phospho-STAT5 formation, transport, and translocation into the nucleus that controls the transcription of genes involved in T-cell activation and proliferation. We fit together the results of these quantitative analyses and propose the following mechanism. Activated IL-7 receptors embedded in membrane microdomains induce actin-microfilament meshwork formation, anchoring microtubules that grow radially from rafted receptors to the nuclear membrane. STAT5 phosphorylated by signalosomes are loaded on kinesins and glide along the microtubules across the cytoplasm to reach the nucleus 2 min after IL-7 stimulation. Radial microtubules disappear 15 min later, while transversal microtubules, independent of phospho-STAT5 transport, begin to bud from the microtubule organization center. PMID:23329834