Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes.

Science.gov (United States)

Schröter, W; Neuvians, M

1970-12-01

Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.

Deorphanizing the human transmembrane genome: A landscape of uncharacterized membrane proteins.

Science.gov (United States)

Babcock, Joseph J; Li, Min

2014-01-01

The sequencing of the human genome has fueled the last decade of work to functionally characterize genome content. An important subset of genes encodes membrane proteins, which are the targets of many drugs. They reside in lipid bilayers, restricting their endogenous activity to a relatively specialized biochemical environment. Without a reference phenotype, the application of systematic screens to profile candidate membrane proteins is not immediately possible. Bioinformatics has begun to show its effectiveness in focusing the functional characterization of orphan proteins of a particular functional class, such as channels or receptors. Here we discuss integration of experimental and bioinformatics approaches for characterizing the orphan membrane proteome. By analyzing the human genome, a landscape reference for the human transmembrane genome is provided.

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

Etiopathogenetic, clinical and histopathological aspects regarding the involvement of dental focal infection in premature births with fetal hypotrophy.

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Rîcă, Gabriel Radu; Badi, Claudia Paula; Rîcă, Ana Maria; Sîrbu, Carmen Mirela; Rîcă, Nicolae

2014-01-01

The study conducted on a total of 1344 preterm births, of which 403 hypotrophic fetuses births (between 2010-2012 within the Maternal Clinics of Craiova, Romania), studied the involvement of dental inflammatory infections in the chorioamnionitis onset. The possibility of transferring germs, toxins and degraded materials into the blood flow, and them entering the chorioamniotic structures is quite a common issue. Subclinically often evolving chorioamniotic membrane and its existence is clearly established after birth by histopathological and bacteriological examinations, being partially responsible for the growth delay of the conception product. Our study revealed this fact, by using clinical examinations, ultrasound exams, bacteriological determinations of the amniotic fluid and the placenta, alongside the histopathological examinations. The chorioamnionitis inflammatory process is responsible for premature birth, through a high synthesis of interleukins (IL) and prostaglandins, causing uterine contractions. Our IL-6 dosage determinations show its growth that can be considered a prediction marker for preterm birth.

Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods

Science.gov (United States)

Lin, Hong Reng; Heish, Chao-Wen; Liu, Cheng-Hui; Muduli, Saradaprasan; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Hsu, Shih-Tien; Chen, Da-Chung; Benelli, Giovanni; Murugan, Kadarkarai; Cheng, Nai-Chen; Wang, Han-Chow; Wu, Gwo-Jang

2017-01-01

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 μm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not. PMID:28071738

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

DEFF Research Database (Denmark)

Juel, C.

2006-01-01

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...

Characterization of receptors for recombinant human tumor necrosis factor-alpha from human placental membranes

International Nuclear Information System (INIS)

Aiyer, R.A.; Aggarwal, B.B.

1990-01-01

High affinity receptors for recombinant human tumor necrosis factor-alpha (rhTNF-alpha) were identified on membranes prepared from full term human placenta. Highly purified rhTNF-alpha iodinated by the iodogen method was found to bind placental membranes in a displaceable manner with an approximate dissociation constant (KD) of 1.9 nM. The membrane bound TNF-alpha receptor could be solubilized by several detergents with optimum extraction being obtained with 1% Triton X-100. The binding of 125I-rhTNF-alpha to the solubilized receptor was found to be time and temperature dependent, yielding maximum binding within 1 h, 24 h and 48 h at 37 degrees C, 24 degrees C and 4 degrees C, respectively. However, the maximum binding obtainable at 4 degrees C was only 40% of that at 37 degrees C. The binding 125I-rhTNF-alpha to solubilized placental membrane extracts was displaceable by unlabeled rhTNF-alpha, but not by a related protein recombinant human tumor necrosis factor-beta (rhTNF-beta; previously called lymphotoxin). This is similar to the behavior of TNF-alpha receptors derived from detergent-solubilized cell extracts, although on intact cells, both rhTNF-alpha and rhTNF-beta bind with equal affinity to TNF receptors. The Scatchard analysis of the binding data of the solubilized receptor revealed high affinity binding sites with a KD of approximately 0.5 nM and a receptor concentration of about 1 pmole/mg protein. Gel filtration of the solubilized receptor-ligand complexes on Sephacryl S-300 revealed two different peaks of radioactivity at approximate molecular masses of 50,000 Da and 400,000 Da. The 400,000 dalton peak corresponded to the receptor-ligand complex. Overall, our results suggest that high affinity receptors for TNF-alpha are present on human placental membranes and provide evidence that these receptors may be different from that of rhTNF-beta

Assembly of the membrane domain of ATP synthase in human mitochondria.

Science.gov (United States)

He, Jiuya; Ford, Holly C; Carroll, Joe; Douglas, Corsten; Gonzales, Evvia; Ding, Shujing; Fearnley, Ian M; Walker, John E

2018-03-20

The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F 1 -c 8 complex inhibited by the ATPase inhibitor protein IF 1 and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast F o membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae.

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

that resorption is connected to apoptosis of the epithelial cells of Malassez. The purpose of this study is to localize cells undergoing apoptosis in the periodontal membrane of human primary and permanent teeth. Materials and methods. Human primary and permanent teeth were examined immunohistochemically...... for apoptosis and epithelial cells of Malassez in the periodontal membrane. All teeth examined were extracted in connection with treatment. Results. Apoptosis was seen in close proximity to the root surface and within the epithelial cells of Malassez. This pattern of apoptotis is similar in the periodontal...... membrane in primary and permanent teeth. Conclusions. The inter-relationship between apoptotis and root resorption cannot be concluded from the present study. Apoptosis seen in close proximity to the root surface presumably corresponds to the highly innervated layer of the periodontal membrane...

Effect of the Human Amniotic Membrane on Liver Regeneration in Rats

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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.

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.

Solubilization of human erythrocyte membranes by ASB detergents

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C.C. Domingues

2008-09-01

Full Text Available Understanding the membrane solubilization process and finding effective solubilizing agents are crucial challenges in biochemical research. Here we report results on the interaction of the novel linear alkylamido propyl dimethyl amino propanosulfonate detergents, ASB-14 and ASB-16, with human erythrocyte membranes. An estimation of the critical micelle concentration of these zwitterionic detergents (ASB-14 = 100 µM and ASB-16 = 10 µM was obtained using electron paramagnetic resonance. The amount of proteins and cholesterol solubilized from erythrocytes by these detergents was then determined. The hemolytic activities of the ASB detergents were assayed and the detergent/lipid molar ratios for the onset of hemolysis (Re sat and total lysis (Re sol were calculated, allowing the determination of the membrane binding constants (Kb. ASB-14 presented lower membrane affinity (Kb = 7050 M-1 than ASB-16 (Kb = 15610 M-1. The amount of proteins and cholesterol solubilized by both ASB detergents was higher while Re sat values (0.22 and 0.08 detergent/lipid for ASB-14 and ASB-16, respectively were smaller than those observed with the classic detergents CHAPS and Triton X-100. These results reveal that, besides their well-known use as membrane protein solubilizers to enhance the resolution of two dimensional electrophoresis/mass spectrometry, ASB-14 and ASB-16 are strong hemolytic agents. We propose that the physicochemical properties of ASB detergents determine their membrane disruption efficiency and can help to explain the improvement in the solubilization of membrane proteins, as reported in the literature.

Galactose oxidase labeling of membrane proteins from human brain white matter

International Nuclear Information System (INIS)

Hukkanen, V.; Frey, H.; Salmi, A.

1981-01-01

Membrane proteins of human autopsy brain white matter were subjected to a galactose oxidase/NaB 3 H 4 labeling procedure and the membranes labeled by this method or by [ 3 H]acetic anhydride techniques were studied by lectin affinity chromatography using Lens culinaris phytohemagglutinin (lentil lectin) attached to Sepharose 4B beads. (Auth.)

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

Ionic channels and membrane hyperpolarization in human macrophages

NARCIS (Netherlands)

Ince, C.; van Duijn, B.; Ypey, D. L.; van Bavel, E.; Weidema, F.; Leijh, P. C.

1987-01-01

Microelectrode impalement of human macrophages evokes a transient hyperpolarizing response (HR) of the membrane potential. This HR was found to be dependent on the extracellular concentration of K+ but not on that of Na+ or Cl-. It was not influenced by low temperature (12 degrees C) or by 0.2 mM

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

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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.

A comparative in vitro study of the viability of human keratinocytes grown on irradiated human amnion membrane and fibrin glue scaffolds

International Nuclear Information System (INIS)

Dorai, A.A.; Lim, C.K.; Azman, W.S.; Halim, A.S.

2008-01-01

Full text: The dried irradiated human amnion membrane has been used as a biological dressing for various clinical conditions. Being another biological membrane its potential as a scaffold to grow human keratinocytes is not known yet. To compare the growth patterns and cell viability of keratinocytes using fibrin glue and air dried amnion membrane as a scaffold. Keratinocytes were obtained from skin samples of six patients undergoing elective surgery. Fibrin glue (Tisseel, Baxter ) was diluted and used to coat the wells. Human dried amnion membrane was obtained and placed into the 24 well plates. Keratinocytes were seeded into the fibrin and amnion scaffold. Cell viability assay (MTT) was performed after 24, 48 and 72 hours. Finally the measurements were done by the Enzyme-Linked Immunosorbent Assay (ELISA) reader at 570 nm. Six patients consented for the study. The cells growing on the amnion scaffold showed a decreasing trend (20.67%, 17.94% and 16.78% respectively for 24, 48 and 72 hours). The cells growing on the fibrin scaffold showed a steady increase in number at 24, 48 and 72 hours (73.03%, 74.12% and 79.66%). The percentage of growth of normal human keratinocytes were significantly greater in the fibrin scaffold group (Mann - Whitney p = 0.002) for 24, 48 and 72 hours. The air dried irradiated human amnion membrane can be used as a scaffold to grow keratinocytes but however the growth pattern does not sustain with time. Fibrin glue supports the growth of human keratinocytes and shows an increasing pattern of growth with time. (Author)

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

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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.

Membrane alterations induced by nonstructural proteins of human norovirus.

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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

Partitioning the proteome: phase separation for targeted analysis of membrane proteins in human post-mortem brain.

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Jane A English

Full Text Available Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain.

Grape extract protects against γ-radiation-induced membrane damage strains of human erythrocytes

International Nuclear Information System (INIS)

Das, Subir Kumar

2017-01-01

The membrane integrity of circulating red blood cells (RBCs) is compromised by the deleterious actions of γ-radiation in humans. Grapes are the richest source of antioxidants due to presence of potentially bioactive phytochemicals. The objective of the present study was to assess the radioprotective actions of grape extracts against the γ-radiation-induced membrane permeability of human erythrocytes. The scavenging activities in seeds of grape in DPPH, hydrogen peroxide and hydroxyl radicals, were higher than skin or pulp of different cultivars. Grape extracts also showed appreciable extent of total antioxidant capacity and effective antihemolytic action. Grape extracts significantly ameliorated the γ-radiation-induced increase of the levels of thiobarbituric acid-reactive substances (TBARS, an index of lipid peroxidation) in the RBC membrane ghosts. Stored blood showed higher levels of K + ion as compared to the normal blood which was elevated by γ-radiation. Membrane ATPase was inhibited by the exposure to γ-radiation.Treatment of RBCs with the grape extracts prior to the exposure of γ-radiation significantly mitigated these changes in the erythrocyte membranes caused by the lower dose of radiation (4 Gy). (author)

The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells.

Science.gov (United States)

Alvarez-Guerra, Miriam; Hernandez, Maria Rosa; Escolar, Ginés; Chiavaroli, Carlo; Garay, Ricardo P; Hannaert, Patrick

2002-09-15

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4-5%, p ethamsylate tested (1 microM), with maximal enhancement of P-selectin expression (75-90%) at 10 microM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance. Copyright 2002 Elsevier Science Ltd.

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.

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

Transport of acidic amino acids by human jejunal brush-border membrane vesicles

International Nuclear Information System (INIS)

Rajendran, V.M.; Harig, J.M.; Adams, M.B.; Ramaswamy, K.

1987-01-01

This study characterizes the transport of radiolabeled acidic amino acids into brush-border membrane vesicles prepared from human jejunum. The uptakes of L-glutamic, L-aspartic, and D-aspartic acids were stimulated by a Na + gradient. Concentrative uptake (resulting in an overshoot phenomenon) of these dicarboxylic amino acids occurred when there was an outward K + gradient. In addition, increasing K + gradients resulted in enhanced uptake of L-glutamic acid. This K + requirement is somewhat specific as Rb + and Cs + could enhance uptake to a limited extent, whereas Li + and choline + showed no enhancement. The presence of a K + gradient did not affect the affinity of the carrier system for L-glutamic acid but it did increase the V/sub max/. The presence of extravesicular anions having differing membrane permeabilities did not altar L-glutamic acid uptake indicating an absence of an effect of membrane potential on the transport process. Finally, the human transport system for L-glutamic acid appears to be specific for acidic amino acids as demonstrated by inhibition studies. The studies demonstrate a transport system in human jejunum specific for acidic amino acids that is energized by an inward Na + gradient and an outward K + gradient

Effect of some radiosensitising drugs on human erythrocyte membrane - - spin label study

Energy Technology Data Exchange (ETDEWEB)

Mishra, K P [Bhabha Atomic Research Centre, Bombay (India). Biology and Agriculture Div.

1982-02-01

Electron spin resonance and spin label techniques have been employed to study the effects of local anaesthetic drugs, procaine and tetracaine, on human erythrocyte membrane. Both the drugs altered the protein and lipid arrangements in the membrane and these changes were reversible. Procaine had greater effect on the labels attached to proteins while tetracaine fluidized interior of lipid bilayer to a greater extent. The differential effects of these drugs on the protein and lipid labels have been interpreted in terms of their relative penetrability in the membrane. Present results have explained that radiation induced enhanced killing of cells in the presence of these drugs might be due to the alterations in membrane, particularly proteins both structural and enzymatic. In addition, these results indicate a possible relationship between drug-induced structural changes in membrane and their anaesthetic potency.

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......-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...

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.

Alternative Sources of Adult Stem Cells: Human Amniotic Membrane

Science.gov (United States)

Wolbank, Susanne; van Griensven, Martijn; Grillari-Voglauer, Regina; Peterbauer-Scherb, Anja

Human amniotic membrane is a highly promising cell source for tissue engineering. The cells thereof, human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC), may be immunoprivileged, they represent an early developmental status, and their application is ethically uncontroversial. Cell banking strategies may use freshly isolated cells or involve in vitro expansion to increase cell numbers. Therefore, we have thoroughly characterized the effect of in vitro cultivation on both phenotype and differentiation potential of hAEC. Moreover, we present different strategies to improve expansion including replacement of animal-derived supplements by human platelet products or the introduction of the catalytic subunit of human telomerase to extend the in vitro lifespan of amniotic cells. Characterization of the resulting cultures includes phenotype, growth characteristics, and differentiation potential, as well as immunogenic and immunomodulatory properties.

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

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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.

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.

Evaluation of a Silicone Membrane as an Alternative to Human Skin for Determining Skin Permeation Parameters of Chemical Compounds.

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Uchida, Takashi; Yakumaru, Masafumi; Nishioka, Keisuke; Higashi, Yoshihiro; Sano, Tomohiko; Todo, Hiroaki; Sugibayashi, Kenji

2016-01-01

We evaluated the effectiveness of a silicone membrane as an alternative to human skin using the skin permeation parameters of chemical compounds. An in vitro permeation study using 15 model compounds was conducted, and permeation parameters comprising permeability coefficient (P), diffusion parameter (DL(-2)), and partition parameter (KL) were calculated from each permeation profile. Significant correlations were obtained in log P, log DL(-2), and log KL values between the silicone membrane and human skin. DL(-2) values of model compounds, except flurbiprofen, in the silicone membrane were independent of the lipophilicity of the model compounds and were 100-fold higher than those in human skin. For antipyrine and caffeine, which are hydrophilic, KL values in the silicone membrane were 100-fold lower than those in human skin, and P values, calculated as the product of a DL(-2) and KL, were similar. For lipophilic compounds, such as n-butyl paraben and flurbiprofen, KL values for silicone were similar to or 10-fold higher than those in human skin, and P values for silicone were 100-fold higher than those in human skin. Furthermore, for amphiphilic compounds with log Ko/w values from 0.5 to 3.5, KL values in the silicone membrane were 10-fold lower than those in human skin, and P values for silicone were 10-fold higher than those in human skin. The silicone membrane was useful as a human skin alternative in an in vitro skin permeation study. However, depending on the lipophilicity of the model compounds, some parameters may be over- or underestimated.

Recruitment of human aquaporin 3 to internal membranes in the Plasmodium falciparum infected erythrocyte.

Science.gov (United States)

Bietz, Sven; Montilla, Irine; Külzer, Simone; Przyborski, Jude M; Lingelbach, Klaus

2009-09-01

The molecular mechanisms underlying the formation of the parasitophorous vacuolar membrane in Plasmodium falciparum infected erythrocytes are incompletely understood, and the protein composition of this membrane is still enigmatic. Although the differentiated mammalian erythrocyte lacks the machinery required for endocytosis, some reports have described a localisation of host cell membrane proteins at the parasitophorous vacuolar membrane. Aquaporin 3 is an abundant plasma membrane protein of various cells, including mammalian erythrocytes where it is found in distinct oligomeric states. Here we show that human aquaporin 3 is internalized into infected erythrocytes, presumably during or soon after invasion. It is integrated into the PVM where it is organized in novel oligomeric states which are not found in non-infected cells.

IL-27 induces a pro-inflammatory response in human fetal membranes mediating preterm birth.

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Yin, Nanlin; Wang, Hanbing; Zhang, Hua; Ge, Huisheng; Tan, Bing; Yuan, Yu; Luo, Xiaofang; Olson, David M; Baker, Philip N; Qi, Hongbo

2017-09-01

Inflammation at the maternal-fetal interface has been shown to be involved in the pathogenesis of preterm birth. Interleukin 27 (IL-27), a heterodimeric cytokine, is known to mediate an inflammatory response in some pregnancy complications. In this study, we aimed to determine whether IL-27 could induce an inflammatory reaction at the maternal-fetal interface that would mediate the onset of preterm birth. We found elevated expression of IL-27 in human peripheral serum and elevated expression of its specific receptor (wsx-1) on fetal membranes in cases of preterm birth. Moreover, the release of inflammatory markers (CXCL10, IFN-γ, MCP-1, IL-6, IL-1β and TNF-α), especially CXCL10, was markedly augmented upon stimulation of IL-27 in the fetal membranes. Additionally, IL-27 and IFN-γ cooperated to amplify the expression of CXCL10 in the fetal membranes. Moreover, the production of CXCL10 was increased in IL-27-treated fetal membrane through JNK, PI3K or Erk signaling pathways. Finally, MMP2 and MMP9 were activated by IL-27 in human fetal membranes, which may be related to the onset of preterm premature rupture of membranes (pPROM). In conclusion, for the first time, we reported that the aberrant expression of IL-27 could mediate an excessive inflammatory response in fetal membranes through the JNK, PI3K or Erk signaling pathways, which contributes to preterm birth. Copyright © 2017 Elsevier B.V. All rights reserved.

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....

Membrane properties for permeability testing: Skin versus synthetic membranes.

Science.gov (United States)

Haq, Anika; Dorrani, Mania; Goodyear, Benjamin; Joshi, Vivek; Michniak-Kohn, Bozena

2018-03-25

Synthetic membranes that are utilized in diffusion studies for topical and transdermal formulations are usually porous thin polymeric sheets for example cellulose acetate (CA) and polysulfones. In this study, the permeability of human skin was compared using two synthetic membranes: cellulose acetate and Strat-M® membrane and lipophilic and hydrophilic compounds either as saturated or formulated solutions as well as marketed dosage forms. Our data suggests that hydrophilic compounds have higher permeation in Strat-M membranes compared with lipophilic ones. High variation in permeability values, a typical property of biological membranes, was not observed with Strat-M. In addition, the permeability of Strat-M was closer to that of human skin than that of cellulose acetate (CA > Strat-M > Human skin). Our results suggest that Strat-M with little or no lot to lot variability can be applied in pilot studies of diffusion tests instead of human skin and is a better substitute than a cellulose acetate. Copyright © 2018 Elsevier B.V. All rights reserved.

Surface and protein analyses of normal human cell attachment on PIII-modified chitosan membranes

International Nuclear Information System (INIS)

Saranwong, N.; Inthanon, K.; Wongkham, W.; Wanichapichart, P.; Suwannakachorn, D.; Yu, L.D.

2012-01-01

Surface of chitosan membrane was modified with argon (Ar) and nitrogen (N) plasma immersion ion implantation (PIII) for human skin fibroblasts F1544 cell attachment. The modified surfaces were characterized by Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). Cell attachment patterns were evaluated by scanning electron microscopy (SEM). The enzyme-linked immunosorbent assay (ELISA) was used to quantify levels of focal adhesion kinase (FAK). The results showed that Ar PIII had an enhancement effect on the cell attachment while N-PIII had an inhibition effect. Filopodial analysis revealed more microfilament cytoplasmic spreading on the edge of cells attached on the Ar-treated membranes than N-treated membranes. Higher level FAK was found in Ar-treated membranes than that in N-treated membranes.

Effects of flaxseed oil on anti-oxidative system and membrane deformation of human peripheral blood erythrocytes in high glucose level.

Science.gov (United States)

Yang, Wei; Fu, Juan; Yu, Miao; Huang, Qingde; Wang, Di; Xu, Jiqu; Deng, Qianchun; Yao, Ping; Huang, Fenghong; Liu, Liegang

2012-07-08

The erythrocyte membrane lesion is a serious diabetic complication. A number of studies suggested that n-3 fatty acid could reduce lipid peroxidation and elevate α- or γ-tocopherol contents in membrane of erythrocytes. However, evidence regarding the protective effects of flaxseed oil, a natural product rich in n-3 fatty acid, on lipid peroxidation, antioxidative capacity and membrane deformation of erythrocytes exposed to high glucose is limited. Human peripheral blood erythrocytes were isolated and treated with 50 mM glucose to mimic hyperglycemia in the absence or presence of three different doses of flaxseed oil (50, 100 or 200 μM) in the culture medium for 24 h. The malondialdehyde (MDA) and L-glutathione (GSH) were measured by HPLC and LC/MS respectively. The phospholipids symmetry and membrane fatty acid composition of human erythrocytes were detected by flow cytometry and gas chromatograph (GC). The morphology of human erythrocyte was illuminated by ultra scanning electron microscopy. Flaxseed oil attenuated hyperglycemia-induced increase of MDA and decrease of GSH in human erythrocytes. Human erythrocytes treated with flaxseed oil contained higher C22:5 and C22:6 than those in the 50 mM glucose control group, indicating that flaxseed oil could reduce lipid asymmetric distribution and membrane perturbation. The ultra scanning electron microscopy and flow cytometer have also indicated that flaxseed oil could protect the membrane of human erythrocytes from deformation at high glucose level. The flaxseed oil supplementation may prevent lipid peroxidation and membrane dysfunction of human erythrocytes in hyperglycemia.

Development of a living membrane comprising a functional human renal proximal tubule cell monolayer on polyethersulfone polymeric membrane.

Science.gov (United States)

Schophuizen, Carolien M S; De Napoli, Ilaria E; Jansen, Jitske; Teixeira, Sandra; Wilmer, Martijn J; Hoenderop, Joost G J; Van den Heuvel, Lambert P W; Masereeuw, Rosalinde; Stamatialis, Dimitrios

2015-03-01

The need for improved renal replacement therapies has stimulated innovative research for the development of a cell-based renal assist device. A key requirement for such a device is the formation of a "living membrane", consisting of a tight kidney cell monolayer with preserved functional organic ion transporters on a suitable artificial membrane surface. In this work, we applied a unique conditionally immortalized proximal tubule epithelial cell (ciPTEC) line with an optimized coating strategy on polyethersulfone (PES) membranes to develop a living membrane with a functional proximal tubule epithelial cell layer. PES membranes were coated with combinations of 3,4-dihydroxy-l-phenylalanine and human collagen IV (Coll IV). The optimal coating time and concentrations were determined to achieve retention of vital blood components while preserving high water transport and optimal ciPTEC adhesion. The ciPTEC monolayers obtained were examined through immunocytochemistry to detect zona occludens 1 tight junction proteins. Reproducible monolayers were formed when using a combination of 2 mg ml(-1) 3,4-dihydroxy-l-phenylalanine (4 min coating, 1h dissolution) and 25 μg ml(-1) Coll IV (4 min coating). The successful transport of (14)C-creatinine through the developed living membrane system was used as an indication for organic cation transporter functionality. The addition of metformin or cimetidine significantly reduced the creatinine transepithelial flux, indicating active creatinine uptake in ciPTECs, most likely mediated by the organic cation transporter, OCT2 (SLC22A2). In conclusion, this study shows the successful development of a living membrane consisting of a reproducible ciPTEC monolayer on PES membranes, an important step towards the development of a bioartificial kidney. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

2018-03-01

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 h postinoculation. In addition, pathways related to DNA replication and repair were downregulated 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.

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

Science.gov (United States)

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

2016-03-15

This study demonstrated a forward osmosis-membrane distillation (FO-MD) hybrid system for real human urine treatment. A series of NaCl solutions at different concentrations were adopted for draw solutions in FO process, which were also the feed solutions of MD process. To establish a stable and continuous integrated FO-MD system, individual FO process with different NaCl concentrations and individual direct contact membrane distillation (DCMD) process with different feed temperatures were firstly investigated separately. Four stable equilibrium conditions were obtained from matching the water transfer rates of individual FO and MD processes. It was found that the integrated system is stable and sustainable when the water transfer rate of FO subsystem is equal to that of MD subsystem. The rejections to main contaminants in human urine were also investigated. Although individual FO process had relatively high rejection to Total Organic Carbon (TOC), Total Nitrogen (TN) and Ammonium Nitrogen (NH4(+)-N) in human urine, these contaminants could also accumulate in draw solution after long term performance. The MD process provided an effective rejection to contaminants in draw solution after FO process and the integrated system revealed nearly complete rejection to TOC, TN and NH4(+)-N. This work provided a potential treatment process for human urine in some fields such as water regeneration in space station and water or nutrient recovery from source-separated urine. Copyright © 2016 Elsevier Ltd. All rights reserved.

Radiosensitivity of angiogenic and mitogenic factors in human amniotic membrane

International Nuclear Information System (INIS)

Deocaris, Custer C.; De Guzman, Zenaida M.; Deocaris, Chester C.; Jacinto, Sonia D.

2003-01-01

Amniotic membrane as a temporary biological dressing remains as a beneficial and cost-effective means of treating burns in developing countries. This medical application is attributed mainly to placental structural and biochemical features that are important for maintaining proper embryonic development. Since fresh amnions are nevertheless for straightforward clinical use and for preservation, radiation-sterilization is been performed to improve the safety of this placental material. However, like any other sterilization method, gamma-radiation may induce physical and chemical changes that may influence the biological property of the material. Thus, the aim of this study is to compare the effects of various levels of radiation-sterilization protocols for human amnions on angiogenic (neovascularization) and epithelial-mitogenic activities, both of which are physiological processes fundamental to wound healing. Water-soluble extract of non-irradiated amnions demonstrates a strong stimulatory effect on both cell proliferation and angiogenesis. No change in biological activity is seen in amnions irradiated at 25 kGy, the sterilization dose used by the Philippine Nuclear Research Institute (PNRI) for the production of radiation-sterilized human amniotic membranes (RSHAM). However, it appears that amniotic angiogenic factors are more radiosensitive than its mitogenic components, evident from the depressed vascularization of the chorioallantoic membrane (CAM) exposed to 35 kGy-irradiated amnions. The dose of 35 kGy is at present the medical sterilization dose used at the Central Tissue Bank in Warsaw (Poland) for the preparation of their amnion allografts. (Authors)

Zymosterol is located in the plasma membrane of cultured human fibroblasts

International Nuclear Information System (INIS)

Echevarria, F.; Norton, R.A.; Nes, W.D.; Lange, Y.

1990-01-01

Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. (1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. (2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. (3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool

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

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.......Recent studies have demonstrated that skin basement membrane components are expressed within the dermo-epidermal junction in an orderly sequence during human foetal development. We have investigated the ultrastructural localization of basement membrane-related antigens in human foetal skin...... 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...

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.

Glycine uptake by microvillous and basal plasma membrane vesicles from term human placentae.

Science.gov (United States)

Dicke, J M; Verges, D; Kelley, L K; Smith, C H

1993-01-01

Like most amino acids, glycine is present in higher concentrations in the fetus than in the mother. Unlike most amino acids, animal studies suggest fetal concentrations of glycine are minimally in excess of those required for protein synthesis. Abnormal glycine utilization has also been demonstrated in small-for-gestational age human fetuses. The mechanism(s) of glycine uptake in the human placenta are unknown. In other mammalian cells glycine is a substrate for the A, ASC and Gly amino acid transport systems. In this study human placental glycine uptake was characterized using microvillous and basal plasma membrane vesicles each prepared from the same placenta. In both membranes glycine uptake was mediated predominantly by the sodium-dependent A system. Competitive inhibition studies suggest that in microvillous vesicles the small percentage of sodium-dependent glycine uptake not inhibited by methylaminoisobutyric acid (MeAIB) shares a transport system with glycine methyl ester and sarcosine, substrates of the Gly system in other tissues. In addition there are mediated sodium-independent and non-selective transport mechanisms in both plasma membranes. If fetal glycine availability is primarily contingent upon the common and highly regulated A system, glycine must compete with many other substrates potentially resulting in marginal fetal reserves, abnormal utilization and impaired growth.

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...... of human eyes obtained at autopsy (seven normal, five diabetic without DR, and 13 diabetic with DR) by immunofluorescence with antibodies to 30 BM and extracellular matrix components. In non-DR eyes, no qualitative changes of ocular BM components were seen. In some DR corneas, epithelial BM was stained...... discontinuously for laminin-1, entactin/nidogen, and alpha3-alpha4 Type IV collagen, in contrast to non-DR corneas. Major BM alterations were found in DR retinas compared to normals and non-DR diabetics. The inner limiting membrane (retinal BM) of DR eyes had accumulations of fibronectin (including cellular...

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

Science.gov (United States)

Douglas, Lois M; Konopka, James B

2016-03-01

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.

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

Viscoelastic properties of the human tympanic membrane studied with stroboscopic holography and finite element modeling.

Science.gov (United States)

De Greef, Daniel; Aernouts, Jef; Aerts, Johan; Cheng, Jeffrey Tao; Horwitz, Rachelle; Rosowski, John J; Dirckx, Joris J J

2014-06-01

A new anatomically-accurate Finite Element (FE) model of the tympanic membrane (TM) and malleus was combined with measurements of the sound-induced motion of the TM surface and the bony manubrium, in an isolated TM-malleus preparation. Using the results, we were able to address two issues related to how sound is coupled to the ossicular chain: (i) Estimate the viscous damping within the tympanic membrane itself, the presence of which may help smooth the broadband response of a potentially highly resonant TM, and (ii) Investigate the function of a peculiar feature of human middle-ear anatomy, the thin mucosal epithelial fold that couples the mid part of the human manubrium to the TM. Sound induced motions of the surface of ex vivo human eardrums and mallei were measured with stroboscopic holography, which yields maps of the amplitude and phase of the displacement of the entire membrane surface at selected frequencies. The results of these measurements were similar, but not identical to measurements made in intact ears. The holography measurements were complemented by laser-Doppler vibrometer measurements of sound-induced umbo velocity, which were made with fine-frequency resolution. Comparisons of these measurements to predictions from a new anatomically accurate FE model with varied membrane characteristics suggest the TM contains viscous elements, which provide relatively low damping, and that the epithelial fold that connects the central section of the human manubrium to the TM only loosely couples the TM to the manubrium. The laser-Doppler measurements in two preparations also suggested the presence of significant variation in the complex modulus of the TM between specimens. Some animations illustrating the model results are available at our website (www.uantwerp.be/en/rg/bimef/downloads/tympanic-membrane-motion). Copyright © 2014 Elsevier B.V. All rights reserved.

Membrane damage induced in cultured human skin fibroblasts by UVA irradiation

International Nuclear Information System (INIS)

Gaboriau, F.; Morliere, P.; Marquis, I.; Moysan, A.; Geze, M.; Dubertret, L.

1993-01-01

Irradiation of cultured human skin fibroblasts with ultraviolet light from 320 to 400 nm (UVA) leads to a decrease in the membrane fluidity exemplified by an enhanced fluorescence anisotropy of the lipophilic fluorescent probe 1-[4-trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene. This UVA-induced decrease in fluidity is associated with lactate dehydrogenase leakage in the supernatant. Vitamin E, an inhibitor of lipid peroxidation, exerts a protective effect on both phenomena. Therefore, this UVA-induced damage in membrane properties may be related to lipid peroxidation processes. Moreover, exponentially growing cells are more sensitive to these UVA-induced alterations than confluent cells. (Author)

Membrane-Wrapping Contributions to Malaria Parasite Invasion of the Human Erythrocyte

Science.gov (United States)

Dasgupta, Sabyasachi; Auth, Thorsten; Gov, Nir S.; Satchwell, Timothy J.; Hanssen, Eric; Zuccala, Elizabeth S.; Riglar, David T.; Toye, Ashley M.; Betz, Timo; Baum, Jake; Gompper, Gerhard

2014-01-01

The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells. PMID:24988340

Transport and Biodistribution of Dendrimers Across Human Fetal Membranes: Implications for Intravaginal Administration of Dendrimers

Science.gov (United States)

Menjoge, Anupa R.; Navath, Raghavendra S.; Asad, Abbas; Kannan, Sujatha; Kim, Chong Jai; Romero, Roberto; Kannan, Rangaramanujam M.

2010-01-01

Dendrimers are emerging as promising topical antimicrobial agents, and as targeted nanoscale drug delivery vehicles. Topical intravaginal antimicrobial agents are prescribed to treat the ascending genital infections in pregnant women. The fetal membranes separate the extra-amniotic space and fetus. The purpose of the study is to determine if the dendrimers can be selectively used for local intravaginal application to pregnant women without crossing the membranes into the fetus. In the present study, the transport and permeability of PAMAM (poly(amidoamine)) dendrimers, across human fetal membrane (using a side-by-side diffusion chamber), and its biodistribution (using immunofluorescence) are evaluated ex-vivo. Transport across human fetal membranes (from the maternal side) was evaluated using Fluorescein (FITC), an established transplacental marker (positive control, size~ 400 Da) and fluorophore-tagged G4-PAMAM dendrimers (~ 16 kDa). The fluorophore-tagged G4-PAMAM dendrimers were synthesized and characterized using 1H NMR, MALDI TOF-MS and HPLC analysis. Transfer was measured across the intact fetal membrane (chorioamnion), and the separated chorion and amnion layers. Over a five hour period, the dendrimer transport across all the three membranes was less than transport of FITC was relatively fast with as much as 49% transport across the amnion. The permeability of FITC (7.9 × 10-7 cm2/s) through the chorioamnion was 7-fold higher than that of the dendrimer (5.8 × 10-8 cm2/s). The biodistribution showed that the dendrimers were largely present in interstitial spaces in the decidual stromal cells and the chorionic trophoblast cells (in 2.5 to 4 h) and surprisingly, to a smaller extent internalized in nuclei of trophoblast cells and nuclei and cytoplasm of stromal cells. Passive diffusion and paracellular transport appear to be the major route for dendrimer transport. The overall findings further suggest that entry of drugs conjugated to dendrimers would be

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

Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra

International Nuclear Information System (INIS)

Emami, Sanaz; Fan Ying; Munro, Rachel; Ladizhansky, Vladimir; Brown, Leonid S.

2013-01-01

One of the biggest challenges in solid-state NMR studies of membrane proteins is to obtain a homogeneous natively folded sample giving high spectral resolution sufficient for structural studies. Eukaryotic membrane proteins are especially difficult and expensive targets in this respect. Methylotrophic yeast Pichia pastoris is a reliable producer of eukaryotic membrane proteins for crystallography and a promising economical source of isotopically labeled proteins for NMR. We show that eukaryotic membrane protein human aquaporin 1 can be doubly ( 13 C/ 15 N) isotopically labeled in this system and functionally reconstituted into phospholipids, giving excellent resolution of solid-state magic angle spinning NMR spectra.

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.

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

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. PMID:22662156

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

International Nuclear Information System (INIS)

Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P.

1991-01-01

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) + RNA

Presence of fucosyl residues on the oligosaccharide antennae of membrane glycopeptides of human neuroblastoma cells

International Nuclear Information System (INIS)

Santer, U.V.; Glick, M.C.

1983-01-01

Fucosyl residues linked alpha 1 leads to 3 or 4 to N-acetylglucosamine were found in large amounts on glycopeptides from the membranes of human tumor cells of neurectodermal origin but not on membrane glycopeptides from human fibroblasts. The fucosyl residues were detected by release of radioactive fucose from the glycopeptides with an almond alpha-L-fucosidase specific for fucosyl alpha 1 leads to 3(4)-N-acetylglucosamine. In other studies, the linkage was shown to be alpha 1 leads to 3 by nuclear magnetic resonance analysis. Glycopeptides containing these fucosyl residues from four human neuroblastoma cell lines were defined by binding to immobilized lectins. In addition, the glycopeptides from one human neuroblastoma cell line, CHP-134, were further characterized by enzyme degradation and columns calibrated for size and charge. The antennary position of fucosyl alpha 1 leads to 3-N-acetylglucosamine on the glycopeptides was demonstrated by the use of exoglycosidases and endoglycosidase D, since complete degradation to yield fucosyl-N-acetylglucosaminylasparagine was obtained only after treatment with almond alpha-L-fucosidase prior to the sequential degradation. Fucosyl alpha 1 leads to 3-N-acetylglucosamine was present on most size and charge classes of membrane glycopeptides and therefore was not limited to a few glycoproteins. Since the almond alpha-L-fucosidase cleaves fucosyl residues from glycoproteins, the physiological effects of the increased specific fucosylation on human tumors of neurectodermal origin can be examined

MHC Class II and CD9 in Human Eosinophils Localize to Detergent-Resistant Membrane Microdomains

Science.gov (United States)

Akuthota, Praveen; Melo, Rossana C. N.; Spencer, Lisa A.

2012-01-01

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor–stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR–containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4+ T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils. PMID:21885678

MHC Class II and CD9 in human eosinophils localize to detergent-resistant membrane microdomains.

Science.gov (United States)

Akuthota, Praveen; Melo, Rossana C N; Spencer, Lisa A; Weller, Peter F

2012-02-01

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor-stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR-containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4(+) T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils.

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.

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

International Nuclear Information System (INIS)

Ohno, T.; Imai, A.; Tamaya, T.

1991-01-01

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 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 ∼ 7-fold compared with the whole homogenate. When the isolated plasma membrane preparations from the fibroblasts were exposed to 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

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.

Transport and biodistribution of dendrimers across human fetal membranes: implications for intravaginal administration of dendrimer-drug conjugates.

Science.gov (United States)

Menjoge, Anupa R; Navath, Raghavendra S; Asad, Abbas; Kannan, Sujatha; Kim, Chong J; Romero, Roberto; Kannan, Rangaramanujam M

2010-06-01

Dendrimers are emerging as promising topical antimicrobial agents, and as targeted nanoscale drug delivery vehicles. Topical intravaginal antimicrobial agents are prescribed to treat the ascending genital infections in pregnant women. The fetal membranes separate the extra-amniotic space and fetus. The purpose of the study is to determine if the dendrimers can be selectively used for local intravaginal application to pregnant women without crossing the membranes into the fetus. In the present study, the transport and permeability of PAMAM (poly (amidoamine)) dendrimers, across human fetal membrane (using a side by side diffusion chamber), and its biodistribution (using immunofluorescence) are evaluated ex-vivo. Transport across human fetal membranes (from the maternal side) was evaluated using Fluorescein (FITC), an established transplacental marker (positive control, size approximately 400 Da) and fluorophore-tagged G(4)-PAMAM dendrimers (approximately 16 kDa). The fluorophore-tagged G(4)-PAMAM dendrimers were synthesized and characterized using (1)H NMR, MALDI TOF MS and HPLC analysis. Transfer was measured across the intact fetal membrane (chorioamnion), and the separated chorion and amnion layers. Over a 5 h period, the dendrimer transport across all the three membranes was less than dendrimer (5.8 x 10(-8) cm(2)/s). The biodistribution showed that the dendrimers were largely present in interstitial spaces in the decidual stromal cells and the chorionic trophoblast cells (in 2.5-4 h) and surprisingly, to a smaller extent internalized in nuclei of trophoblast cells and nuclei and cytoplasm of stromal cells. Passive diffusion and paracellular transport appear to be the major route for dendrimer transport. The overall findings further suggest that entry of drugs conjugated to dendrimers would be restricted across the human fetal membranes when administered topically by intravaginal route, suggesting new ways of selectively delivering therapeutics to the mother

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

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...

Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.

Science.gov (United States)

Kang, N-H; Hwang, K-A; Kim, S U; Kim, Y-B; Hyun, S-H; Jeung, E-B; Choi, K-C

2012-08-01

As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.

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....

Characterization of membrane lipid fluidity in human embryo cells malignantly transfer med post 238Pu α irradiation

International Nuclear Information System (INIS)

Qi Zirong; Sun Ling; Liu Guolian; Shen Zhiyuan

1992-01-01

The membrane lipid fluidity of malignantly transformed human embryo cells following 238 Pu α particlce irradiation in vitro has been studied. The results indicate that the ontogenesis depends on irradiation dose (Gy) and the membrane lipid fluidity in malignantly transformed cells is higher than that in normal embryo cells. With the microviscosity (η) of cells plotted against the cell counts, the correlation coefficient (γ) is calculated to be between 0.9936 and 0.9999. Since the malignant transformation of irradiated embryo cells is manifested early on cell membrane lipid, the fluidity of membrane lipid can be used as an oncologic marker

Evidence for the existence of multiple heparan sulfate proteoglycans in the human glomerular basement membrane and mesangial matrix

NARCIS (Netherlands)

Groffen, Alexander J A; Hop, Frank W H; Tryggvason, Karl; Dijkman, Henri; Assmann, Karel J M; Veerkamp, Jacques H.; Monnens, Leo A H; Van Den Heuvel, Lambert P W J

1997-01-01

Heparan sulfate proteoglycans (HSPGs) are essential components of the glomerular basement membrane (GBM) carrying a strong anionic charge. A well- characterized extracellular HSPG is perlecan, ubiquitously expressed in basement membranes. A cDNA construct encoding domains I and II of human perlecan

In vitro effects of the anti-Alzheimer drug memantine on the human erythrocyte membrane and molecular models

International Nuclear Information System (INIS)

Zambrano, Pablo; Suwalsky, Mario; Villena, Fernando; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

2017-01-01

Memantine is a NMDA antagonist receptor clinically used for treating Alzheimer's disease. NMDA receptors are present in the human neurons and erythrocyte membranes. The aim of the present study was to investigate the effects of memantine on human erythrocytes. With this purpose, the drug was developed to in vitro interact with human red cells and bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). The latter represent lipids respectively present in both outer and inner monolayers of the red cell membrane. Results obtained by scanning electron microscopy (SEM) showed that memantine changed the normal biconcave shape of red cells to cup-shaped stomatocytes. According to the bilayer-couple hypothesis the drug intercalated into the inner monolayer of the erythrocyte membrane. Experimental results obtained by X-ray diffraction on multibilayers of DMPC and DMPE, and by differential scanning calorimetry on multilamellar vesicles indicated that memantine preferentially interacted with DMPC in a concentration-dependent manner. Thus, it can be concluded that in the low therapeutic plasma concentration of circa 1 μM memantine is located in NMDA receptor channel without affecting the erythrocyte shape. However, at higher concentrations, once the receptors became saturated excess of memantine molecules (20 μM) would interact with phosphoinositide lipids present in the inner monolayer of the erythrocyte membrane inducing the formation of stomatocytes. However, 40–50 μM memantine was required to interact with isolated phosphatidylcholine bilayers. - Highlights: • The interaction of memantine with human erythrocytes and lipid bilayers were assessed. • Memantine induced morphological changes to human erythrocytes. • Memantine interacted with classes of phospholipids present in the erythrocyte membrane. • Results support the hypothesis that memantine interacts with NMDA receptors.

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

International Nuclear Information System (INIS)

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

1986-01-01

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

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.

Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane

NARCIS (Netherlands)

Groffen, Alexander J.; Ruegg, Markus A.; Dijkman, Henri; Van De Velden, Thea J.; Buskens, Carin A.; Van Den Born, Jacob; Assmann, Karel J.; Monnens, Leo A.; Veerkamp, Jacques H.; Van Den Heuvel, Lambert P.

Agrin is a heparan sulfate proteoglycan (HSPG) that is highly concentrated in the synaptic basal lamina at the neuromuscular junction (NMJ). Agrin-like immunoreactivity is also detected outside the NMJ. Here we show that agrin is a major HSPG component of the human glomerular basement membrane

Intrauterine Linear Echogenicities in the Gravid Uterus: What Radiologists Should Know.

Science.gov (United States)

Jensen, Kyle K; Oh, Karen Y; Kennedy, Anne M; Sohaey, Roya

2018-01-01

Intrauterine linear echogenicity (ILE) is a common ultrasonographic finding in the gravid uterus and has variable causes and variable maternal and fetal outcomes. Correctly categorizing ILE during pregnancy is crucial for guiding surveillance and advanced imaging strategies. Common causes of ILE include membranes in multiple gestations, uterine synechiae with amniotic sheets, and uterine duplication anomalies. Less common causes include circumvallate placenta, chorioamniotic separation, and hemorrhage between membranes. Amniotic band syndrome is a rare but important diagnosis to consider, as it causes severe fetal defects. Imaging findings enable body stalk anomaly, a lethal defect, to be distinguished from amniotic bands, which although destructive are not necessarily lethal. This review describes the key imaging findings used to differentiate the various types of ILE in pregnancy, thus enabling accurate diagnosis and appropriate patient counseling. Online supplemental material is available for this article. © RSNA, 2018.

Equilibrium thermodynamics of the partitioning of non-steroidal anti-inflammatory drugs into human erythrocyte ghost membranes

International Nuclear Information System (INIS)

Omran, Ahmed A.

2013-01-01

Graphical abstract: Bar diagram representing thermodynamic parameters obtained for the partitioning of NSAIDs into human erythrocyte ghost membranes at physiological pH; 7.4. Highlights: • Partition coefficients of NSAIDs into HEG membranes were determined. • Thermodynamic parameters were evaluated and successfully analyzed. • Partitioning of NSAIDs into HEG membranes was exothermic. • Partitioning of NSAIDs into HEG is spontaneous with negative free energy values. • Identical partitioning enthalpy–entropy driven compensation mechanism was shown. -- Abstract: In this work,second derivative spectrophotometry was applied for determining the partition coefficients (K p s) of four non-steroidal anti-inflammatory drugs (NSAIDs; flufenamic, meclofenamic, mefenamic and niflumic acids) into human erythrocyte ghost (HEG) membranes over a temperature range from (283.2 to 313.2) K. The proposed method allowed the evaluation and direct analyses of thermodynamic parameters; enthalpy (ΔH W→M ), Gibbs energy (ΔG W→M ) and entropy (ΔS W→M ) changes of the partitioning of NSAIDs into HEG membranes. The partitioning of NSAIDs between polar aqueous phase and non-polar lipid bilayer HEG membrane phase was exothermic with negative (ΔH W→M ) which compensated for the changes in (ΔS W→M ). The negative values of (ΔG W→M ) revealed that the partitioning of NSAIDs into HEG, owing to their transfer from polar aqueous phase and non-polar HEG phase is spontaneous. The enthalpy–entropy correlation analysis resulted in a good linearity that suggests an identical partitioning enthalpy–entropy driven compensation mechanism for the studied NSAIDs

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.

Atomic resolution view into the structure–function relationships of the human myelin peripheral membrane protein P2

Energy Technology Data Exchange (ETDEWEB)

Ruskamo, Salla [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Yadav, Ravi P. [Banaras Hindu University, Varanasi (India); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); Sharma, Satyan; Lehtimäki, Mari [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Laulumaa, Saara [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); Aggarwal, Shweta; Simons, Mikael [Max Planck Institute for Experimental Medicine, Göttingen (Germany); Bürck, Jochen; Ulrich, Anne S. [Karlsruhe Institute for Technology (KIT), Karlsruhe (Germany); Juffer, André H. [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Kursula, Inari [University of Oulu, Oulu (Finland); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); Kursula, Petri, E-mail: petri.kursula@oulu.fi [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); University of Hamburg, Hamburg (Germany)

2014-01-01

The structure of the human myelin peripheral membrane protein P2 has been refined at 0.93 Å resolution. In combination with functional experiments in vitro, in vivo and in silico, the fine details of the structure–function relationships in P2 are emerging. P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Å allows detailed structural analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.

Association of canalicular membrane enzymes with bile acid micelles and lipid aggregates in human and rat bile.

Science.gov (United States)

Accatino, L; Pizarro, M; Solís, N; Koenig, C S

1995-01-18

This study was undertaken to gain insights into the characteristics of the polymolecular association between canalicular membrane enzymes, bile acids, cholesterol and phospholipids in bile and into the celular mechanisms whereby the enzymes are secreted into bile. With this purpose, we studied the distribution of bile acids, cholesterol, phospholipids, proteins and representative canalicular membrane enzymes (alkaline phosphatase, 5'-nucleotidase and gamma-glutamyl transpeptidase), which can be considered specific marker constituents, in bile fractions enriched in phospholipid-cholesterol lamellar structures (multilamellar and unilamellar vesicles) and bile acid-mixed micelles. These fractions were isolated by ultracentrifugation from human hepatic bile, normal rat bile and bile of rats treated with diosgenin, a steroid that induces a marked increase in biliary cholesterol secretion, and were characterized by density, lipid composition and transmission electron microscopy. These studies demonstrate that alkaline phosphatase, 5'-nucleotidase and gamma-glutamyl transpeptidase are secreted into both human and rat bile where they are preferentially associated with bile acid-mixed micelles, suggesting a role for bile acids in both release of these enzymes and lipids from the canalicular membrane and solubilization in bile. In addition, heterogeneous association of these enzymes with nonmicellar, lamellar structures in human and rat bile is consistent with the hypothesis that processes independent of the detergent effects of bile acids might also result in the release of specific intrinsic membrane proteins into bile.

Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes

Directory of Open Access Journals (Sweden)

Brzezinska Agnieszka A

2006-12-01

Full Text Available Abstract Background Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection. Results Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX domain of p47phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47phox, is translocated to the membrane of mature phagosomes. Conclusion We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.

C_1_8-attached membrane funnel-based spray ionization mass spectrometry for quantification of anti-diabetic drug from human plasma

International Nuclear Information System (INIS)

Li, Wan; Chen, Xiangfeng; Wong, Y.-L. Elaine; Hung, Y.-L. Winnie; Wang, Ze; Deng, Liulin; Dominic Chan, T.-W.

2016-01-01

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_1_8-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"2 > 0.99) was obtained in the concentration range of 1–100 ng mL"−"1. The method detection limit of target drugs spiked in the human plasma was around 0.30 ng mL"−"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"−"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.

Ultraviolet radiation induces changes in membrane metabolism of human keratinocytes in culture

International Nuclear Information System (INIS)

De Leo, V.A.; Horlick, H.; Hanson, D.; Eisinger, M.; Harber, L.C.

1984-01-01

Human keratinocytes in culture were prelabeled with [ 3 H]arachidonic acid (AA) and then exposed to ultraviolet B radiation. Irradiated cells released labeled AA metabolites into media in a dose-dependent manner when compared to sham-irradiated cells. The response began immediately and continued for 24 h. Extracts from media were examined by high-performance liquid chromatography for identification of specific AA metabolites. Irradiated cells were stimulated to produce prostaglandin-like material (PGE2 and PGF2 alpha). These findings support the concept that the cell membrane of keratinocytes participates directly or indirectly in initiating the sunburn response. It is also felt that the metabolites formed following injury to the membrane are an integral component in the mediation of that response

An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

OpenAIRE

Leis, J F; Kaplan, N O

1982-01-01

The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid pho...

An in vitro study on the antioxidant capacity of usnic acid on human erythrocytes and molecular models of its membrane.

Science.gov (United States)

Suwalsky, M; Jemiola-Rzeminska, M; Astudillo, C; Gallardo, M J; Staforelli, J P; Villena, F; Strzalka, K

2015-11-01

Usnic acid (UA) has been associated with chronic diseases through its antioxidant action. Its main target is the cell membrane; however, its effect on that of human erythrocytes has been scarcely investigated. To gain insight into the molecular mechanisms of the interaction between UA and cell membranes human erythrocytes and molecular models of its membrane have been utilized. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were chosen as representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. Results by X-ray diffraction showed that UA produced structural perturbations on DMPC and DMPE bilayers. DSC studies have indicated that thermotropic behavior of DMPE was most strongly distorted by UA than DMPC, whereas the latter is mainly affected on the pretransition. Scanning electron (SEM) and defocusing microscopy (DM) showed that UA induced alterations to erythrocytes from the normal discoid shape to echinocytes. These results imply that UA molecules were located in the outer monolayer of the erythrocyte membrane. Results of its antioxidant properties showed that UA neutralized the oxidative capacity of HClO on DMPC and DMPE bilayers; SEM, DM and hemolysis assays demonstrated the protective effect of UA against the deleterious oxidant effects of HClO upon human erythrocytes. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

[Molecular organization of glutamate-sensitive chemoexcitatory membranes of nerve cells. Comparative analysis of glutamate-binding membrane proteins from the cerebral cortex of rats and humans].

Science.gov (United States)

Dambinova, S A; Gorodinskiĭ, A I; Lekomtseva, T M; Koreshonkov, O N

1987-10-01

The kinetics of 3H-L-glutamate binding to human brain synaptic membranes revealed the existence of one type of binding sites with Kd and Vmax comparable with those for freshly isolated rat brain membranes. The fraction of glutamate-binding proteins (GBP) was shown to contain three components with Mr of 14, 60 and 280 kD whose stoichiometry is specific for human and rat brain. All fractions were found to bind the radiolabeled neurotransmitter and to dissociate into subunits with Mr of 14 kD after treatment with-potent detergents (with the exception of the 56-60 kD component). Study of association-dissociation of GBP protein subunits by high performance liquid chromatography confirmed the hypothesis on the oligomeric structure of glutamate receptors which are made up of low molecular weight glycoprotein-lipid subunits and which form ionic channels by way of repeated association. Despite the similarity of antigen determinants in the active center of glutamate receptors from human and rat brain, it was assumed that the stoichiometry of structural organization of receptor subunits isolated from different sources is different. The functional role of structural complexity of human brain glutamate receptors is discussed.

Immunochemical identification of human trophoblast membrane antigens using monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Brown, P J; Molloy, C M; Johnson, P M [Liverpool Univ. (UK). Dept. of Immunology

1983-11-01

Human trophoblast membrane antigens recognised by monoclonal antibodies (H310, H315, H316 and H317) have been identified using combinations of radioimmunoprecipitation, SDS-PAGE, electroblotting, chromatographic and ELISA-type techniques. H317 is known to identify heat-stable placental-type alkaline phosphatase and accordingly was shown to react with a protein of subunit Msub(r) of 68000. H310 and H316 both recognise an antigen with a subunit Msub(r) of 34000 under reducing conditions. In non-reducing conditions, the H310/316 antigen gave oligomers of a component of Msub(r) 62000. It is unknown whether this 62000 dalton component is a dimer of the 34000 dalton protein with either itself or a second protein chain of presumed Msub(r) around 28000. H315 recognises an antigen with subunit Msub(r) of 36000; in non-reducing conditions this component readily associates to oligomeric structures. The epitope recognised by H315 may be sensitive to SDS. The two proteins recognised by H310/316 and H315 have been termed the p34 and p36 trophoblast membrane proteins, respectively.

An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

Science.gov (United States)

Leis, J F; Kaplan, N O

1982-11-01

The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

Premature delivery due to intrauterine Candida infection that caused neonatal congenital cutaneous candidiasis: a case report.

Science.gov (United States)

Ito, Fumitake; Okubo, Tomoharu; Yasuo, Tadahiro; Mori, Taisuke; Iwasa, Koichi; Iwasaku, Kazuhiro; Kitawaki, Jo

2013-01-01

Congenital cutaneous candidiasis is a very rare disease with less than 100 cases published in the medical literature. Neonates having this disease present with systemic skin lesions caused by intrauterine Candida infections. We present a case of threatened premature delivery due to Candida chorioamnionitis, which caused both maternal postpartum endometritis and neonatal congenital cutaneous candidiasis. A 34-year-old woman who was admitted for fetal membrane bulging at 20 weeks of gestation underwent McDonald cervical cerclage. We diagnosed threatened premature delivery due to intrauterine infection; therefore, we terminated the gestation by cesarean section at 24 weeks of gestation. Fungi-like yeast was detected in infantile gastric juice. Histopathological findings of the placenta revealed that Candida albicans mycelium invaded the placenta, chorioamniotic membrane and umbilical cord. © 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.

Dermal-epidermal membrane systems by using human keratinocytes and mesenchymal stem cells isolated from dermis

Energy Technology Data Exchange (ETDEWEB)

Salerno, Simona, E-mail: s.salerno@itm.cnr.it [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy); Messina, Antonietta [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy); Giordano, Francesca [Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, I-87036 Rende, (CS) (Italy); Bader, Augustinus [Biomedical-Biotechnological Center, BBZ, University of Leipzig, D-04103 Leipzig (Germany); Drioli, Enrico [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy); WCU Energy Engineering Department, Hanyang University, Seoul (Korea, Republic of); De Bartolo, Loredana, E-mail: l.debartolo@itm.cnr.it [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy)

2017-02-01

Dermal-epidermal membrane systems were developed by co-culturing human keratinocytes with Skin derived Stem Cells (SSCs), which are Mesenchymal Stem Cells (MSCs) isolated from dermis, on biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT and PCL. The membranes display physico-chemical, morphological, mechanical and biodegradation properties that could satisfy and fulfil specific requirements in skin tissue engineering. CHT membrane exhibits an optimal biodegradation rate for acute wounds; CHT-PCL for the chronic ones. On the other hand, PCL membrane in spite of its very slow biodegradation rate exhibits mechanical properties similar to in vivo dermis, a lower hydrophilic character, and a surface roughness, all properties that make it able to sustain cell adhesion and proliferation for in vitro skin models. Both CHT–PCL and PCL membranes guided epidermal and dermal differentiation of SSCs as pointed out by the expression of cytokeratins and the deposition of the ECM protein fibronectin, respectively. In the dermal-epidermal membrane systems, a more suitable microenvironment for the SSCs differentiation was promoted by the interactions and the mutual interplay with keratinocytes. Being skin tissue-biased stem cells committed to their specific final dermal and/or epidermal cell differentiation, SSCs are more suitable for skin tissue engineering than other adult MSCs with different origin. For this reason, they represent a useful autologous cell source for engineering skin substitutes for both in vivo and in vitro applications.

Specific binding of prostaglandin E2 to membrane preparations from human skin: receptor modulation by UVB-irradiation and chemical agents

International Nuclear Information System (INIS)

Lord, J.T.; Ziboh, V.A.

1979-01-01

Human skin membranes bind prostaglandin E2 (PGE2) with high affinity and specificity. This binding is inhibited by trypsin or heat treatment suggesting that PGE2 receptors have protein components. Exposure of the membranes to ultraviolet irradiation (UVB) resulted in the loss of the membrane binding capacity for PGE2. This UVB-inhibitory effect could be prevented by a known protein sulfhydryl-oxidizing agent and a known lipid anti-oxidant

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......ESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well...... 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...

Activation of AMPK in human fetal membranes alleviates infection-induced expression of pro-inflammatory and pro-labour mediators.

Science.gov (United States)

Lim, R; Barker, G; Lappas, M

2015-04-01

In non-gestational tissues, the activation of adenosine monophosphate (AMP)-activated kinase (AMPK) is associated with potent anti-inflammatory actions. Infection and/or inflammation, by stimulating pro-inflammatory cytokines and matrix metalloproteinase (MMP)-9, play a central role in the rupture of fetal membranes. However, no studies have examined the role of AMPK in human labour. Fetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and after preterm pre-labour rupture of membranes (PPROM). AMPK activity was assessed by Western blotting of phosphorylated AMPK expression. To determine the effect of AMPK activators on pro-inflammatory cytokines, fetal membranes were pre-treated with AMPK activators then stimulated with bacterial products LPS and flagellin or viral dsDNA analogue poly(I:C). Primary amnion cells were used to determine the effect of AMPK activators on IL-1β-stimulated MMP-9 expression. AMPK activity was decreased with term labour. There was no effect of preterm labour. AMPK activity was also decreased in preterm fetal membranes, in the absence of labour, with PROM compared to intact membranes. AMPK activators AICAR, phenformin and A769662 significantly decreased IL-6 and IL-8 stimulated by LPS, flagellin and poly(I:C). Primary amnion cells treated with AMPK activators significantly decreased IL-1β-induced MMP-9 expression. The decrease in AMPK activity in fetal membranes after spontaneous term labour and PPROM indicates an anti-inflammatory role for AMPK in human labour and delivery. The use of AMPK activators as possible therapeutics for threatened preterm labour would be an exciting future avenue of research. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane between normal and preeclampsia pregnancies with high-resolution mass spectrometry.

Directory of Open Access Journals (Sweden)

Fuqiang Wang

Full Text Available Preeclampsia is a serious complication of pregnancy, which affects 2-8% of all pregnancies and is one of the leading causes of maternal and perinatal mortality and morbidity worldwide. To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, we used high-resolution LC-MS/MS technologies to construct a comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane in normal and preeclamptic pregnancies. A total of 1027 N-glyco- and 2094 phospho- sites were detected in human placental plasma membrane, and 5 N-glyco- and 38 phospho- proteins, respectively, with differentially expression were definitively identified between control and preeclamptic placental plasma membrane. Further bioinformatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes occurring during pathological changes of preeclamptic placental plasma membrane.

Inhibition of PIM1 kinase attenuates inflammation-induced pro-labour mediators in human foetal membranes in vitro.

Science.gov (United States)

Lim, Ratana; Barker, Gillian; Lappas, Martha

2017-06-01

Does proviral integration site for Moloney murine leukaemic virus (PIM)1 kinase play a role in regulating the inflammatory processes of human labour and delivery? PIM1 kinase plays a critical role in foetal membranes in regulating pro-inflammatory and pro-labour mediators. Infection and inflammation have strong causal links to preterm delivery by stimulating pro-inflammatory cytokines and collagen degrading enzymes, which can lead to rupture of membranes. PIM1 has been shown to have a role in immune regulation and inflammation in non-gestational tissues; however, its role has not been explored in the field of human labour. PIM1 expression was analysed in myometrium and/or foetal membranes obtained at term and preterm (n = 8-9 patients per group). Foetal membranes, freshly isolated amnion cells and primary myometrial cells were used to investigate the effect of PIM1 inhibition on pro-labour mediators (n = 5 patients per treatment group). Foetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and from preterm pre-labour rupture of membranes (PPROM) (n = 9 per group). Amnion was collected from women with and without preterm chorioamnionitis (n = 8 per group). Expression of PIM1 kinase was determined by qRT-PCR and western blotting. To determine the effect of PIM1 kinase inhibition on the expression of pro-inflammatory and pro-labour mediators induced by bacterial products lipopolysaccharide (LPS) (10 μg/ml) and flagellin (1 μg/ml) and pro-inflammatory cytokine tumour necrosis factor (TNF) (10 ng/ml), chemical inhibitors SMI-4a (20 μM) and AZD1208 (50 μM) were used in foetal membrane explants and siRNA against PIM1 was used in primary amnion cells. Statistical significance was set at P membranes after spontaneous term labour compared to no labour at term and in amnion with preterm chorioamnionitis compared to preterm with no chorioamnionitis. There was no change in PIM1 expression with preterm labour or PPROM

Structural remodeling and oligomerization of human cathelicidin on membranes suggest fibril-like structures as active species

DEFF Research Database (Denmark)

Sancho-Vaello, Enea; François, Patrice; Bonetti, Eve-Julie

2017-01-01

Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens, often through irreversible damage of their cellular membranes. To explore the mechanism by which the important cathelicidin peptide LL-37 of the human innate immune system interacts w...... that these supramolecular structures represent the LL-37-membrane active state. Collectively, our study provides new insights into the fascinating plasticity of LL-37 demonstrated at atomic resolution and opens the venue for LL-37-based molecules as novel antibiotics....

Membrane Disordering is not Sufficient for Membrane Permeabilization by Islet Amyloidogenic Polypeptide: Studies of IAPP(20-29) Fragments

Science.gov (United States)

Brender, Jeffrey R.; Heyl, Deborah L.; Samisetti, Shyamprasad; Kotler, Samuel A.; Osborne, Joshua M.; Pesaru, Ranadheer R.; Ramamoorthy, Ayyalusamy

2013-01-01

A key factor in the development of type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide protein (human-IAPP) is believed to play a crucial role in this process by forming small aggregates that exhibit toxicity by disrupting the cell membrane. The actual mechanism of membrane disruption is complex and appears to involve an early component before fiber formation and later component associated with fiber formation on the membrane. By comparing the peptide-lipid interactions derived from solid-state NMR experiments of two IAPP fragments that bind the membrane and cause membrane disordering to IAPP derived peptides known to cause significant early membrane permeabilization, we show here that membrane disordering is not likely to be sufficient by itself to cause the early membrane permeabilization observed by IAPP, and may play a lesser role in IAPP membrane disruption than expected. PMID:23493863

Retinoid inhibition of in vitro invasion of human amnion basement membrane by human tumor cells

International Nuclear Information System (INIS)

Fazely, F.; Ledinko, N.; Smith, D.J.

1986-01-01

The biological activity of retinoids was assayed in an in vitro quantitative assay of human tumor cell invasion using human amnion basement membrane (BM). The effects measured were the inhibition of tumor cell migration through the BM and tumor cell degradative enzyme activity on 14 C-proline labeled collagenous and noncollagenous components of the BM. The human lung carcinoma A549 or the human Ewing's sarcoma TC-106 cell lines treated with retinoids for two days were incubated on the BM in the absence of retinoids. A dose-dependent inhibition of cell invasion was produced by retinoids. Among the retinoids tested, the most powerful was retinol acetate which inhibited invasion by 50% of A549 cells at a concentration of 0.009 μg/mL, and of TC-106 cells at 0.07 μg/mL. Retinol acetate inhibited A549 and TC-106 cell growth by approximately 50% at levels over 100-fold higher than those needed for antiinvasive activity. Retinol acetate was about 20 times more potent than retinoic acid and 30 times more potent than retinol palmitate. The model system will be useful for investigating antiinvasive activity of other retinoids as well as other compounds

Graphene oxide improves the biocompatibility of collagen membranes in an in vitro model of human primary gingival fibroblasts.

Science.gov (United States)

De Marco, Patrizia; Zara, Susi; De Colli, Marianna; Radunovic, Milena; Lazović, Vladimir; Ettorre, Valeria; Di Crescenzo, Antonello; Piattelli, Adriano; Cataldi, Amelia; Fontana, Antonella

2017-09-13

Commercial collagen membranes are used in oral surgical procedures as scaffolds for bone deposition in guided bone regeneration. Here, we have enriched them with graphene oxide (GO) via a simple non-covalent functionalization, exploiting the capacity of oxygenated carbon functional moieties of GO to interact through hydrogen bonding with collagen. In the present paper, the GO-coated membranes have been characterized in terms of stability, nano-roughness, biocompatibility and induction of inflammatory response in human primary gingival fibroblast cells. The obtained coated membranes are demonstrated not to leak GO in the bulk solution, and to change some features of the membrane, such as stiffness and adhesion between the membrane and the atomic force microscopy (AFM) tip. Moreover, the presence of GO increases the roughness and the total surface exposed to the cells, as demonstrated by AFM analyses. The obtained material is biocompatible, and does not induce inflammation in the tested cells.

Effects of cytokine-suppressive anti-inflammatory drugs on inflammatory activation in ex vivo human and ovine fetal membranes.

Science.gov (United States)

Stinson, Lisa F; Ireland, Demelza J; Kemp, Matthew W; Payne, Matthew S; Stock, Sarah J; Newnham, John P; Keelan, Jeffrey A

2014-03-01

Intrauterine infection and inflammation are responsible for the majority of early (PTBs). Anti-inflammatory agents, delivered intra-amniotically together with antibiotics, may be an effective strategy for preventing PTB. In this study, the effects of four cytokine-suppressive anti-inflammatory drugs (CSAIDs: N-acetyl cysteine (NAC), SB239063, TPCA-1 and NEMO binding domain inhibitor (NBDI)) were assessed on human and ovine gestational membrane inflammation. Full-thickness membranes were collected from healthy, term, human placentas delivered by Caesarean section (n=5). Using a Transwell model, they were stimulated ex vivo with γ-irradiation-killed Escherichia coli applied to the amniotic face. Membranes from near-term, ovine placentas were stimulated in utero with lipopolysaccharide, Ureaplasma parvum or saline control and subjected to explant culture. The effects of treatment with CSAIDs or vehicle (1% DMSO) on accumulation of PGE2 and cytokines (human interleukin 6 (IL6), IL10 and TNFα; ovine IL8 (oIL8)) were assessed in conditioned media at various time points (3-20  h). In human membranes, the IKKβ inhibitor TPCA-1 (7  μM) and p38 MAPK inhibitor SB239063 (20  μM) administered to the amniotic compartment were the most effective in inhibiting accumulation of cytokines and PGE2 in the fetal compartment. NAC (10  mM) inhibited accumulation of PGE2 and IL10 only; NBDI (10  μM) had no significant effect. In addition to the fetal compartment, SB239063 also exerted consistent and significant inhibitory effects in the maternal compartment. TPCA-1 and SB239063 suppressed oIL8 production, while all CSAIDs tested suppressed ovine PGE2 production. These results support the further investigation of intra-amniotically delivered CSAIDs for the prevention of inflammation-mediated PTB.

Influence of membrane fluidity on human immunodeficiency virus type 1 entry

International Nuclear Information System (INIS)

Harada, Shinji; Yusa, Keisuke; Monde, Kazuaki; Akaike, Takaaki; Maeda, Yosuke

2005-01-01

For penetration of human immunodeficiency virus type 1 (HIV-1), formation of fusion-pores might be required for accumulating critical numbers of fusion-activated gp41, followed by multiple-site binding of gp120 with receptors, with the help of fluidization of the plasma membrane and viral envelope. Correlation between HIV-1 infectivity and fluidity was observed by treatment of fluidity-modulators, indicating that infectivity was dependent on fluidity. A 5% decrease in fluidity suppressed the HIV-1 infectivity by 56%. Contrarily, a 5% increase in fluidity augmented the infectivity by 2.4-fold. An increased temperature of 40 deg C or treatment of 0.2% xylocaine after viral adsorption at room temperature enhanced the infectivity by 2.6- and 1.5-fold, respectively. These were inhibited by anti-CXCR4 peptide, implying that multiple-site binding was accelerated at 40 deg C or by xylocaine. Thus, fluidity of both the plasma membrane and viral envelope was required to form the fusion-pore and to complete the entry of HIV-1

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

on the expression of muscle membrane transport proteins. Likewise, improvements in performance may involve upregulation of metabolic enzymes. Since Epo is known to augment performance we tested the effect of rHuEpo on some marker enzymes that are related to aerobic capacity. For these purposes eight subjects...... 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......Adaptations to chronic hypoxia involve changes in membrane transport proteins. The underlying mechanism of this response may be related to concomitant occurring changes in erythropoietin (Epo) levels. We therefore tested the direct effects of recombinant human erythropoietin (rHuEpo) treatment...

Maculoplasty for age-related macular degeneration: reengineering Bruch's membrane and the human macula.

Science.gov (United States)

Del Priore, Lucian V; Tezel, Tongalp H; Kaplan, Henry J

2006-11-01

Age-related macular degeneration (AMD) is the leading cause of blindness in the western world. Over the last decade, there have been significant advances in the management of exudative AMD with the introduction of anti-VEGF drugs; however, many patients with exudative AMD continue to lose vision and there are no effective treatments for advanced exudative AMD or geographic atrophy. Initial attempts at macular reconstruction using cellular transplantation have not been effective in reversing vision loss. Herein we discuss the current status of surgical attempts to reconstruct damaged subretinal anatomy in advanced AMD. We reinforce the concept of maculoplasty for advanced AMD, which is defined as reconstruction of macular anatomy in patients with advanced vision loss. Successful maculoplasty is a three-step process that includes replacing or repairing damaged cells (using transplantation, translocation or stimulation of autologous cell proliferation); immune suppression (if allografts are used to replace damaged cells); and reconstruction or replacement of Bruch's membrane (to restore the integrity of the substrate for proper cell attachment). In the current article we will review the rationale for maculoplasty in advanced AMD, and discuss the results of initial clinical attempts at macular reconstruction. We will then discuss the role of Bruch's membrane damage in limiting transplant survival and visual recovery, and discuss the effects of age-related changes within human Bruch's membrane on the initial attachment and subsequent proliferation of transplanted cells. We will discuss attempts to repair Bruch's membrane by coating with extracellular matrix ligands, anatomic reconstitution of the inner collagen layer, and the effects of Bruch's membrane reconstruction of ultrastuctural anatomy and subsequent cell behavior. Lastly, we will emphasize the importance of continued efforts required for successful maculoplasty.

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.

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

Directory of Open Access Journals (Sweden)

William P Clafshenkel

Full Text Available 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(sulfosuccinimidylsuberate (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.

Comparative proteomic analysis of plasma membrane proteins between human osteosarcoma and normal osteoblastic cell lines

International Nuclear Information System (INIS)

Zhang, Zhiyu; Ma, Fang; Cai, Zhengdong; Zhang, Lijun; Hua, Yingqi; Jia, Xiaofang; Li, Jian; Hu, Shuo; Peng, Xia; Yang, Pengyuan; Sun, Mengxiong

2010-01-01

Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. However, the knowledge in diagnostic modalities has progressed less. To identify new biomarkers for the early diagnosis of OS as well as for potential novel therapeutic candidates, we performed a sub-cellular comparative proteomic research. An osteosarcoma cell line (MG-63) and human osteoblastic cells (hFOB1.19) were used as our comparative model. Plasma membrane (PM) was obtained by aqueous two-phase partition. Proteins were analyzed through iTRAQ-based quantitative differential LC/MS/MS. The location and function of differential proteins were analyzed through GO database. Protein-protein interaction was examined through String software. One of differentially expressed proteins was verified by immunohistochemistry. 342 non-redundant proteins were identified, 68 of which were differentially expressed with 1.5-fold difference, with 25 up-regulated and 43 down-regulated. Among those differential proteins, 69% ware plasma membrane, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and interaction with each other. One protein--CD151 located in net nodes was verified to be over-expressed in osteosarcoma tissue by immunohistochemistry. It is the first time to use plasma membrane proteomics for studying the OS membrane proteins according to our knowledge. We generated preliminary but comprehensive data about membrane protein of osteosarcoma. Among these, CD151 was further validated in patient samples, and this small molecule membrane might be a new target for OS research. The plasma membrane proteins identified in this study may provide new insight into osteosarcoma biology and potential diagnostic and therapeutic biomarkers

Human cytotoxic T-lymphocyte membrane-camouflaged nanoparticles combined with low-dose irradiation: a new approach to enhance drug targeting in gastric cancer

Directory of Open Access Journals (Sweden)

Zhang L

2017-03-01

Full Text Available Lianru Zhang, Rutian Li, Hong Chen, Jia Wei, Hanqing Qian, Shu Su, Jie Shao, Lifeng Wang, Xiaoping Qian, Baorui Liu The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University and Clinical Cancer Institute of Nanjing University, Nanjing, People’s Republic of China Abstract: Cell membrane-derived nanoparticles are becoming more attractive because of their ability to mimic many features of their source cells. This study reports on a biomimetic delivery platform based on human cytotoxic T-lymphocyte membranes. In this system, the surface of poly-lactic-co-glycolic acid nanoparticles was camouflaged using T-lymphocyte membranes, and local low-dose irradiation (LDI was used as a chemoattractant for nanoparticle targeting. The T-lymphocyte membrane coating was verified using dynamic light scattering, transmission electron microscopy, and confocal laser scanning microscopy. This new platform reduced nanoparticle phagocytosis by macrophages to 23.99% (P=0.002. Systemic administration of paclitaxel-loaded T-lymphocyte membrane-coated nanoparticles inhibited the growth of human gastric cancer by 56.68% in Balb/c nude mice. Application of LDI at the tumor site significantly increased the tumor growth inhibition rate to 88.50%, and two mice achieved complete remission. Furthermore, LDI could upregulate the expression of adhesion molecules in tumor vessels, which is important in the process of leukocyte adhesion and might contribute to the localization of T-lymphocyte membrane-encapsulated nanoparticles in tumors. Therefore, this new drug-delivery platform retained both the long circulation time and tumor site accumulation ability of human cytotoxic T lymphocytes, while local LDI could significantly enhance tumor localization. Keywords: cell membrane, drug delivery system, gastric cancer, low-dose irradiation, nanoparticles

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.

Cell membrane softening in human breast and cervical cancer cells

Science.gov (United States)

Händel, Chris; Schmidt, B. U. Sebastian; Schiller, Jürgen; Dietrich, Undine; Möhn, Till; Kießling, Tobias R.; Pawlizak, Steve; Fritsch, Anatol W.; Horn, Lars-Christian; Briest, Susanne; Höckel, Michael; Zink, Mareike; Käs, Josef A.

2015-08-01

Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.

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. Here we present a protocol for monitoring the in situ kinetics of lipoprotein deposition and lipid exchange/removal at model cellular membranes using the non-invasive, surface sensitive methods of neutron reflection and quartz crystal microbalance with dissipation. For neutron reflection, lipid...... 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....

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...

Degradation of endothelial basement membrane by human breast cancer cell lines

International Nuclear Information System (INIS)

Yee, C.; Shiu, R.P.

1986-01-01

During metastasis, it is believed that tumor cells destroy the basement membrane (BM) of blood vessels in order to disseminate through the circulatory system. By radioactively labeling the extracellular matrix produced by primary endothelial cells in vitro, the ability of human breast cancer cells to degrade BM components was studied. We found that T-47D, a human breast cancer line, was able to degrade significant amounts of [35S]methionine-labeled and [3H]proline-labeled BM, but not 35SO4-labeled BM. Six other tumor cell lines of human breast origin were assayed in the same manner and were found to degrade BM to varying degrees. Several non-tumor cell lines tested showed relatively little degrading activity. The use of serum-free medium greatly enhanced degradation of the BM by tumor cells, suggesting a role for naturally occurring enzyme inhibitors in the serum. Direct cell contact with the BM was required for BM degradation, suggesting that the active enzymes are cell associated. The addition of hormones implicated in the etiology of breast cancer did not significantly alter the ability of T-47D cells to degrade the BM. The use of this assay affords future studies on the mechanism of invasion and metastasis of human breast cancer

Amniotic membrane for burn trauma

International Nuclear Information System (INIS)

Jamaluddin Zainol; Hasim Mohammad

1999-01-01

Amniotic membranes are derived from human placentae at birth. They have two layers mainly the amniotic and the chorionic surfaces which are separated by a thin layer of connective tissues. The two layers are separated during procurement, the placenta and the chorionic side are discarded and the amnion membranes are then further processed. Amnion membranes are normally procured from placentae which are normally free of infections, i.e; the mothers are antenatally screened for sexually transmitted diseases or AlDs related diseases. Intrapartum the mother should not be having chorioamnionitis or jaundice. Sometimes the amniotic membranes are acquired from fresh elective caeserian sections. After processing, the amniotic membranes are packed in two layers of polypropylene and radiated with cobalt 60 at a dose of about 25 kGy. The amniotic membranes are clinically used to cover burn surfaces especially effective for superficial or partial thickness burns. The thin membranes adhered well to the trauma areas and peeled off automatically by the second week. No change of dressing were necessary during these times because of the close adherence, there were less chance of external contamination or infections of these wounds. Due to their flexibility they are very useful to cover difference contours of the human body for example the face, body, elbows or knees. However our experience revealed that amniotic membranes are not useful for third degree bums because the membranes dissolves by the enzymes present in the wounds

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

The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multi-step process. The cancer cell proteins, and plasma membrane proteins in particular, involved in this process are poorly defined and a study of the very early events of the metastatic process using...... 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...... purification and comparative quantitative LC-MS/MS proteomic analysis identified 13 membrane proteins that were expressed at higher levels and 3 that were under-expressed in the metastatic compared to the non-metastatic cell line from a total of 1919 identified protein entries. Among the proteins were ecto-5...

Local anesthetics: interaction with human erythrocyte membranes as studied by 1H and 31P nuclear magnetic resonance

International Nuclear Information System (INIS)

Fraceto, Leonardo Fernandes; Paula, Eneida de

2004-01-01

The literature carries many theories about the mechanism of action of local anesthetics (LA). We can highlight those focusing the direct effect of LA on the sodium channel protein and the ones that consider the interaction of anesthetic molecules with the lipid membrane phase. The interaction between local anesthetics and human erythrocyte membranes has been studied by 1 H and 31 P nuclear magnetic resonance spectroscopy. It was found that lidocaine (LDC) and benzocaine (BZC) bind to the membranes, increase the mobility of the protons of the phospholipids acyl chains, and decrease the mobility and/or change the structure of the polar head groups. The results indicate that lidocaine molecules are inserted across the polar and liquid interface of the membrane, establishing both electrostatic (charged form) and hydrophobic (neutral form) interactions. Benzocaine locates itself a little deeper in the bilayer, between the interfacial glycerol region and the hydrophobic core. These changes in mobility or conformation of membrane lipids could affect the Na + -channel protein insertion in the bilayer, stabilizing it in the inactivated state, thus causing anesthesia. (author)

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

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

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

International Nuclear Information System (INIS)

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

1988-01-01

Specific binding of 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 A/B diabetic (N = 12), and large for gestational age (N = 13) pregnancies was measured. In all pregnancy states, placentas bound approximately four times more 125 I-hEGF than did fetal membranes (P 125 I-hEGF binding to fetal membranes from the various pregnancy states (P 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 125 I-hEGF specific binding did not differ between placentas from intrauterine growth retarded or twin pregnancies (P 125 I-hEGF binding did not vary with fetal sex, maternal race, placental weight, or gestational age between 37 to 42 weeks (P 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. (author)

Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

Science.gov (United States)

Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

2013-10-01

Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters. Copyright © 2013 John Wiley & Sons, Ltd.

/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.

C5a binding to human polymorphonuclear leukocyte plasma membrane (PMNLM) receptors

International Nuclear Information System (INIS)

Conway, R.G.; Mollison, K.W.; Carter, G.W.; Lane, B.

1986-01-01

Previous investigations of the C5a receptor have been performed using intact human PMNL. To circumvent some of the potential problems with such whole cell assays (e.g. internalization or metabolism of radioligand) the authors have developed a PMNLM binding assay. Human PMNLM were prepared by nitrogen cavitation and Percoll gradient centrifugation. Specific binding of [ 125 I]C5a to PMNLM was: high affinity, K/sub D/ = 0.6 nM; saturable, B/sub max/ = 8.7 pmol/mg protein; and reversible. Kinetic measurements agree with the K/sub D/ value obtained by Scatchard analysis. Furthermore, the binding activity of C5a correlates with biological activity as measured by myeloperoxidase release from human PMNL. Human serum C5a and recombinant C5a bind with similar affinities when measured by competition or direct binding and label the same number of sites in human PMNLM. The nonhydrolyzable GTP analog, GppNHp, induces a low affinity state of the C5a receptor (4-6 fold shift in K/sub D/) with little effect on B/sub max/. In summary, the criteria have been satisfied for identification of a biologically relevant C5a binding site in human PMNLM. Regulation of the C5a receptor and its membrane transduction mechanism(s) appears to involve guanyl nucleotides, as has been found for other chemoattractant receptors

Cell Membrane Transport Mechanisms: Ion Channels and Electrical Properties of Cell Membranes.

Science.gov (United States)

Kulbacka, Julita; Choromańska, Anna; Rossowska, Joanna; Weżgowiec, Joanna; Saczko, Jolanta; Rols, Marie-Pierre

2017-01-01

Cellular life strongly depends on the membrane ability to precisely control exchange of solutes between the internal and external (environmental) compartments. This barrier regulates which types of solutes can enter and leave the cell. Transmembrane transport involves complex mechanisms responsible for passive and active carriage of ions and small- and medium-size molecules. Transport mechanisms existing in the biological membranes highly determine proper cellular functions and contribute to drug transport. The present chapter deals with features and electrical properties of the cell membrane and addresses the questions how the cell membrane accomplishes transport functions and how transmembrane transport can be affected. Since dysfunctions of plasma membrane transporters very often are the cause of human diseases, we also report how specific transport mechanisms can be modulated or inhibited in order to enhance the therapeutic effect.

Phosphorylation of chloroform soluble compounds in plasma membranes of human epidermoid carcinoma A431 cells

International Nuclear Information System (INIS)

Brautigan, D.L.; Randazzo, P.; Shriner, C.; Fain, J.N.

1985-01-01

This study investigated a possible role for the epidermal growth factor (EGF) receptor protein tyrosine kinase in phosphoinositide metabolism with plasma membrane vesicles from human epidermoid carcinoma (A431) cells. The authors found a novel chloroform-soluble product radiolabeled with [gamma- 32 P]ATP that did not migrate from the origin in the thin layer system designed to separate the phosphoinositides, appeared as a single band of Mr = 3500 on polyacrylamide gels in the presence of dodecyl sulfate, had an ultraviolet absorbance spectrum with a maximum at 275 nm and stained with Coomassie dye. Based on these properties this phosphorylation product is referred to as a proteolipid. The 32 P label was not detected in phosphotyrosine [Tyr(P)], phosphoserine [Ser(P)] or phosphothreonine [Thr(P)] and was lost during acid or base hydrolysis. Phosphorylation of proteolipid was increased significantly by EGF, whereas phosphorylation of phosphatidic acid was decreased and labeling of phosphoinositides was unaffected. Thus, it appears that in A431 membranes the EGF receptor/kinase does not utilize phosphatidylinositol as a substrate, but does phosphorylate a membrane proteolipid

Influence of high energy electron irradiation and gamma irradiation on the osmotic resistance of human erythrocyte membranes

International Nuclear Information System (INIS)

Catana, D.; Hategan, Alina; Moraru, Rodica; Popescu, Alina; Morariu, V. V.

1998-01-01

The effects of 5 MeV electrons and of gamma irradiation at 0 deg. C on the osmotic fragility of human erythrocyte membranes are presented. Both electron and gamma radiation in the range 0-400 Gy induced no hemolysis indicating that the membrane modifications due to radiation interaction do not reach a critical point as to cause swelling of the cells and subsequent lysis. The osmotic stress experiments performed after irradiation showed that the gamma irradiated erythrocytes exhibited an almost similar sigmoidal behavior for all irradiation doses, whereas the electron irradiated samples showed a much larger increase in hemolysis degree and, in the case of a given electron dose (100 Gy), the hemolysis was found much smaller than for the control sample (a similar behavior of the erythrocytes was found in the case of microwave irradiation at temperatures under 0 deg. C). Our experimental data suggest that electron radiation and gamma radiation have different impacts on the erythrocyte membrane fluidity, involving, probably, the different rate of energy deposition in the samples and the direct interaction of electrons with the erythrocyte membranes. (authors)

Removal properties of human enteric viruses in a pilot-scale membrane bioreactor (MBR) process.

Science.gov (United States)

Miura, Takayuki; Okabe, Satoshi; Nakahara, Yoshihito; Sano, Daisuke

2015-05-15

In order to evaluate removal properties of human enteric viruses from wastewater by a membrane bioreactor (MBR), influent, anoxic and oxic mixed liquor, and membrane effluent samples were collected in a pilot-scale anoxic-oxic MBR process for 16 months, and concentrations of enteroviruses, norovirus GII, and sapoviruses were determined by real-time PCR using murine norovirus as a process control. Mixed liquor samples were separated into liquid and solid phases by centrifugation, and viruses in the bulk solution and those associated with mixed liquor suspended solids (MLSS) were quantified. Enteroviruses, norovirus GII, and sapoviruses were detected in the influent throughout the sampling period (geometrical mean, 4.0, 3.1, and 4.4 log copies/mL, respectively). Enterovirus concentrations in the solid phase of mixed liquor were generally lower than those in the liquid phase, and the mean log reduction value between influent and anoxic mixed liquor was 0.40 log units. In contrast, norovirus GII and sapovirus concentrations in the solid phase were equal to or higher than those in the liquid phase, and higher log reduction values (1.3 and 1.1 log units, respectively) were observed between influent and anoxic mixed liquor. This suggested that enteroviruses were less associated with MLSS than norovirus GII and sapoviruses, resulting in lower enterovirus removal in the activated sludge process. Enteroviruses and norovirus GII were detected in the MBR effluent but sapoviruses were not in any effluent samples. When MLSS concentration was reduced to 50-60% of a normal operation level, passages of enteroviruses and norovirus GII through a PVDF microfiltration membrane were observed. Since rejection of viruses by the membrane was not related to trans-membrane pressure which was monitored as a parameter of membrane fouling, the results indicated that adsorption to MLSS plays an important role in virus removal by an MBR, and removal properties vary by viruses reflecting different

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.

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...

Radiation effects on cell membranes

International Nuclear Information System (INIS)

Koeteles, G.J.

1982-01-01

The recent developments in the field of membrane biology of eukaryotic cells result in revival of relevant radiobiological studies. The spatial relations and chemical nature of membrane components provide rather sensitive targets. Experimental data are presented concerning the effects of relatively low doses of X-irradiation and low concentration of tritiated water (HTO) on various receptor functions - concanavalin A, cationized ferritin, poliovirus - of plasma membranes of animal and human cells which point to early and temporary disturbances of the composite structures and functions of membranes. References are given to the multifold roles of radiationinduced membrane phenomena on the development and regeneration of radiation injuries. (orig.)

Radiation effects on cell membranes

Energy Technology Data Exchange (ETDEWEB)

Koeteles, G.J.

1982-11-01

The recent developments in the field of membrane biology of eukaryotic cells result in revival of relevant radiobiological studies. The spatial relations and chemical nature of membrane components provide rather sensitive targets. Experimental data are presented concerning the effects of relatively low doses of X-irradiation and low concentration of tritiated water (HTO) on various receptor functions - concanavalin A, cationized ferritin, poliovirus - of plasma membranes of animal and human cells which point to early and temporary disturbances of the composite structures and functions of membranes. References are given to the multifold roles of radiationinduced membrane phenomena on the development and regeneration of radiation injuries.

Radiation effects on cell membranes

International Nuclear Information System (INIS)

Koeteles, G.J.

1982-01-01

Experimental data are presented concerning the effects of relatively low doses of x radiation and low concentration of tritiated water (HTO) on various receptor functions - concanavalin A, cationized ferritin, poliovirus of plasma membranes of animal and human cells which point to early and temporary disturbances of the composite structures and functions of membranes. References are given to the manifold influence of radiation-induced membrane phenomenon on the development and regeneration of radiation injuries. (author)

Development of a human corneal epithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals.

Science.gov (United States)

Takezawa, Toshiaki; Nishikawa, Kazunori; Wang, Pi-Chao

2011-09-01

The brief TEER (trans-epithelial electrical resistance) assay after exposing chemicals to corneal epithelium in vivo is known as a suitable method for evaluating corneal irritancy and permeability quantitatively and continuously. A collagen vitrigel membrane we previously developed is a thin (about 20 μm thick) and transparent membrane composed of high density collagen fibrils equivalent to connective tissues in vivo, e.g. corneal Bowman's membrane. To develop such a TEER assay system in vitro utilizing a human corneal epithelial model, HCE-T cells (a human corneal epithelial cell line) were cultured on the collagen vitrigel membrane substratum prepared in a Millicell chamber suitable for TEER measurement. Human corneal epithelium model possessing 5-6 cell layers sufficient for TEER assay was successfully reconstructed on the substratum in the Millicell chamber by culturing the cells in monolayer for 2 days and subsequently in air-liquid interface for 7 days. The exposure of chemicals to the model induced the time-dependent relative changes of TEER in response to the characteristic of each chemical within a few minutes. These results suggest that the TEER assay using the human corneal epithelial model is very useful for an ocular irritancy evaluation as an alternative to the Draize eye irritation test. Copyright © 2011 Elsevier Ltd. All rights reserved.

Permeability of human placenta and fetal membranes to thyrotropin-stimulating hormone in vitro.

Science.gov (United States)

Bajoria, R; Fisk, N M

1998-05-01

We determined the placental transfer of TSH in an in vitro model of dually perfused isolated lobule in 28 human term placentas by adding varying concentrations (5-60 microIU mL(-1)) of TSH as a single bolus dose to the closed maternal circulation. Transmembrane transfer of TSH was also studied by adding 45 microIU mL(-1) to the maternal or fetal compartment of a dual chamber of fetal membranes in culture. Passage of freely diffusible markers creatinine and antipyrine were also studied in this model. TSH concentration was measured by third generation chemiluminescence assay with a sensitivity of 10 mIU mL(-1). In the perfusion experiments, at physiologic concentrations the slow decline of TSH in the maternal circulation was associated with a small linear increase in fetal levels to 0.11 +/- 0.04% of initial dose at 2 h. The placental transfer rate was 0.08 microIU min(-1). Increasing maternal concentrations of TSH were associated with proportional increases in transfer rate (y = 0.002x; R2 = 0.99) and placental uptake (y = 0.01x; R2 = 0.97). The placental permeability of TSH was 2.4 x 10(-4) mL min(-1) g(-1) and was proportional to its coefficients of diffusion in water and molecular size. The transmembrane transfer and permeability of TSH was comparable to those of the placenta. We conclude that TSH crosses the human term placenta and fetal membranes sparingly.

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.

Substrate Specificity, Membrane Topology, and Activity Regulation of Human Alkaline Ceramidase 2 (ACER2)*

OpenAIRE

Sun, Wei; Jin, Junfei; Xu, Ruijuan; Hu, Wei; Szulc, Zdzislaw M.; Bielawski, Jacek; Obeid, Lina M.; Mao, Cungui

2010-01-01

Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Δypc1Δydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed...

The human periodontal membrane: focusing on the spatial interrelation between the epithelial layer of Malassez, fibers, and innervation

DEFF Research Database (Denmark)

Kjaer, Inger; Nolting, Dorrit

2009-01-01

OBJECTIVE: The purpose of the present study was to map the spatial interrelation of fibers, peripheral nerves, and epithelial layer of Malassez in human periodontal membrane in areas close to the root surfaces. MATERIAL AND METHODS: Four healthy permanent teeth extracted from four patients during...

Enhancement of the blood compatibility of dialyzer membranes by the physical adsorption of human thrombomodulin (ART-123).

Science.gov (United States)

Omichi, Masaaki; Matsusaki, Michiya; Kato, Shinya; Maruyama, Ikuro; Akashi, Mitsuru

2010-11-01

ART-123 is a recombinant soluble human thrombomodulin (hTM) with excellent anticoagulant activity. We focused on improving the blood compatibility of the polysulfone-polyvinylpyrrolidone dialyzer surface by the physical adsorption of ART-123 onto the surface. The blood compatibility of the dialyzer with the hTM adsorbed membrane was evaluated by measuring the differential pressure between the arterial and the venous pressures and by blood parameters during blood circulation. The hTM adsorbed dialyzer membrane inhibited blood clot formation without heparin administration due to the anticoagulant activity of hTM for over 4 h. The physically adsorbed hTM was stable during blood circulation, and it did not affect activated clotting time, which is significant drawback of heparin administration, and blood cell counts of RBC, WBC, or platelets. The physical adsorption of hTM onto the dialyzer membrane will be a simple and safe method to prevent blood coagulation during dialysis instead of heparin administration. © 2010 Wiley Periodicals, Inc.

Factors Determining the Oxygen Permeability of Biological Membranes: Oxygen Transport Across Eye Lens Fiber-Cell Plasma Membranes.

Science.gov (United States)

Subczynski, Witold Karol; Widomska, Justyna; Mainali, Laxman

2017-01-01

Electron paramagnetic resonance (EPR) spin-label oximetry allows the oxygen permeability coefficient to be evaluated across homogeneous lipid bilayer membranes and, in some cases, across coexisting membrane domains without their physical separation. The most pronounced effect on oxygen permeability is observed for cholesterol, which additionally induces the formation of membrane domains. In intact biological membranes, integral proteins induce the formation of boundary and trapped lipid domains with a low oxygen permeability. The effective oxygen permeability coefficient across the intact biological membrane is affected not only by the oxygen permeability coefficients evaluated for each lipid domain but also by the surface area occupied by these domains in the membrane. All these factors observed in fiber cell plasma membranes of clear human eye lenses are reviewed here.

Biotransformation of endorphins by a synaptosomal plasma membrane preparation of rat brain and by human serum

NARCIS (Netherlands)

Burbach, J.P.H.; Loeber, J.G.; Verhoef, J.; Kloet, E.R. de; Wied, D. de

1979-01-01

β-Endorphin (β-LPH 61–91), γ-endorphin (61–77), des-tyrosine-γ-endorphin (62–77), α-endorphin (61–76), and β-LPH 61–69 either labeled with [125I] at the N-terminal 61-tyrosine residue or unlabeled were incubated with a crude synaptosomal plasma membrane fraction of rat brain or in human serum. At

Electron microscopic study of the myelinated nerve fibres and the perineurial cell basement membrane in the diabetic human peripheral nerves

International Nuclear Information System (INIS)

ElBarrany, Wagih G.; Hamdy, Raid M.; AlHayani, Abdulmonem A.; Jalalah, Sawsan M.

2009-01-01

To study the quantitative and ultrastructural changes in myelinated nerve fibers and the basement membranes of the perineurial cells in diabetic nerves. The study was performed at the Department of Anatomy, Faculty of Medicine, King Abdul-Aziz University, Jeddah, Saudi Arabia from 2003 to 2005. Human sural nerves were obtained from 15 lower limbs and 5 diabetic nerve biopsies. The total mean and density of myelinated nerve fibers per fascicle were calculated, with density of microtubules and mitochondria in the axoplasm. The number of the perineurial cell basement membrane layers was counted, and thickness of the basement membrane was measured. Among the 15 diabetic and 5 normal human sural nerves, the average diameters, number and surface area of myelinated nerve fibers and axonal microtubules density were found to be less in diabetic nerves. Mitochondrial density was higher in diabetic axons. Thickness of the perineurial cell basement membrane had a greater mean, but the number of perineurial cell layers was less than that of the diabetic group. The inner cellular layer of the perineurium of the diabetic nerves contained large vacuoles containing electron-dense degenerated myelin. A few specimens showed degenerated myelinated nerve fibers, while others showed recovering ones. Retracted axoplasms were encountered with albumin extravasation. Diabetes caused an increase in perineurial permeability. The diabetic sural nerve showed marked decrease in the myelinated nerve fibres, increase degenerated mitochondria, and decreased microtubules. (author)

Structure of a membrane-attack complex/perforin (MACPF) family protein from the human gut symbiont Bacteroides thetaiotaomicron

International Nuclear Information System (INIS)

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.; Bedem, Henry van den; 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.

2010-01-01

The crystal structure of a novel MACPF protein, which may play a role in the adaptation of commensal bacteria to host environments in the human gut, was determined and analyzed. 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

Progesterone increases nitric oxide synthesis in human vascular endothelial cells through activation of membrane progesterone receptor-α.

Science.gov (United States)

Pang, Yefei; Dong, Jing; Thomas, Peter

2015-05-15

Progesterone exerts beneficial effects on the human cardiovascular system by inducing rapid increases in nitric oxide (NO) production in vascular endothelial cells, but the receptors mediating these nongenomic progesterone actions remain unclear. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that progesterone binds to plasma membranes of HUVECs with the characteristics of membrane progesterone receptors (mPRs). The selective mPR agonist Org OD 02-0 had high binding affinity for the progesterone receptor on HUVEC membranes, whereas nuclear PR (nPR) agonists R5020 and medroxyprogesterone acetate displayed low binding affinities. Immunocytochemical and Western blot analyses confirmed that mPRs are expressed in HUVECs and are localized on their plasma membranes. NO levels increased rapidly after treatment with 20 nM progesterone, Org OD 02-0, and a progesterone-BSA conjugate but not with R5020, suggesting that this progesterone action is at the cell surface and initiated through mPRs. Progesterone and Org OD 02-0 (20 nM) also significantly increased endothelial nitric oxide synthase (eNOS) activity and eNOS phosphorylation. Knockdown of mPRα expression by treatment with small-interfering RNA (siRNA) blocked the stimulatory effects of 20 nM progesterone on NO production and eNOS phosphorylation, whereas knockdown of nPR was ineffective. Treatment with PI3K/Akt and MAP kinase inhibitors blocked the stimulatory effects of progesterone, Org OD 02-0, and progesterone-BSA on NO production and eNOS phosphorylation and also prevented progesterone- and Org OD 02-0-induced increases in Akt and ERK phosphorylation. The results suggest that progesterone stimulation of NO production in HUVECs is mediated by mPRα and involves signaling through PI3K/Akt and MAP kinase pathways. Copyright © 2015 the American Physiological Society.

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...

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...... not be suited for sophisticated studies of GlyR pharmacology and kinetics. However, the assay offers several advantages in studies of ligand-receptor interactions. Furthermore, the assay could be highly useful in the search for structurally novel ligands acting at GlyRs.......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...... ion did not appear to potentiate GlyR function at lower concentrations. Analogously, whereas pregnenolone sulphate inhibited alpha1 GlyR function, the potentiation of alpha1 GlyR by pregnenolone in electrophysiological studies could not be reproduced in the assay. In conclusion, the FMP assay may...

Influence of some DNA-alkylating drugs on thermal stability, acid and osmotic resistance of the membrane of whole human erythrocytes and their ghosts.

Science.gov (United States)

Ivanov, I T; Gadjeva, V

2000-09-01

Human erythrocytes and their resealed ghosts were alkylated under identical conditions using three groups of alkylating antitumor agents: mustards, triazenes and chloroethyl nitrosoureas. Osmotic fragility, acid resistance and thermal stability of membranes were changed only in alkylated ghosts in proportion to the concentration of the alkylating agent. All the alkylating agents decreased acid resistance in ghosts. The clinically used drugs sarcolysine, dacarbazine and lomustine all decreased osmotic fragility and thermal stability of ghost membranes depending on their lipophilicity. DM-COOH did not decrease osmotic fragility and thermal stability of ghost membranes, while NEM increased thermal stability of membranes. The preliminary but not subsequent treatment of ghosts with DM-COOH fully abolished the alkylation-induced thermal labilization of ghost membrane proteins while NEM had a partial effect only. The present study gives direct evidence that alkylating agents, having a high therapeutic activity against malignant growth, bind covalently to proteins of cellular membranes.

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...

Retinoid inhibition of in vitro invasion of human amnion basement membrane by human tumor cells

International Nuclear Information System (INIS)

Fazely, F.

1988-01-01

The effects measured were the inhibition of tumor cell migration through the basement membrane (BM) and tumor cell degradative enzyme activity on 3 H-proline labeled collagenous and non collagenous components of the BM. The human lung carcinoma A549 or the human Ewing's sarcoma TC-106 cell lines treated with retinoids for two days were incubated on the BM in the absence of retinoids. A dose-dependent inhibition of cell invasion was produced by retinoids. Among the retinoids tested the most powerful was retinol acetate which inhibited invasion by 50% of A549 cells at a concentration of 0.09 μg/ml, and TC-106 cells at 0.08 μg/ml. Retinol acetate inhibited A549 and TC-106 cell growth by approximately 50% at levels almost 100-fold higher than those needed for antiinvasive activity. Retinol acetate was about 20 times more potent than retinoic acid and 30 times more than retinol palmitate. Furthermore, A549 cells treated with retinol acetate, under conditions whereby an anti-invasive state was induced,showed an increase in the number of cellular retinoic acid binding proteins (CRABP), a decrease in the activity of type IV collagenase and ectosialyltransferase, and no change in the activity of transglutaminase

Immunochemical and ultrastructural assessment of the nature of the pericellular basement membrane of human decidual cells

DEFF Research Database (Denmark)

Wewer, U M; Faber, M; Liotta, L A

1985-01-01

Human decidual cells of early and late pregnancy were studied immunochemically and ultrastructurally with respect to the presence and nature of pericellular basement membrane material. The most prominent cell type in decidual tissue of both early and late pregnancy were large, mature epithelioid......-linked immunosorbent assay. Biosynthesis of laminin was shown by [35S]methionine labeling of short term organ cultures of decidual tissue followed by immunoprecipation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. The laminin chains migrated with the apparent molecular weights of 300...... and 200 kilodaltons under reducing conditions. Two other separate populations of cells were apparent in the decidual tissue of early pregnancy. A smaller group of rounded intermediate sized (15 to 25 micron) decidual cells had focal deposits basement membrane immunoreactive material scattered at the cell...

The Human ABCG1 Transporter Mobilizes Plasma Membrane and Late Endosomal Non-Sphingomyelin-Associated-Cholesterol for Efflux and Esterification

Directory of Open Access Journals (Sweden)

Edward B. Neufeld

2014-12-01

Full Text Available We have previously shown that GFP-tagged human ABCG1 on the plasma membrane (PM and in late endosomes (LE mobilizes sterol on both sides of the membrane lipid bilayer, thereby increasing cellular cholesterol efflux to lipid surfaces. In the present study, we examined ABCG1-induced changes in membrane cholesterol distribution, organization, and mobility. ABCG1-GFP expression increased the amount of mobile, non-sphingomyelin(SM-associated cholesterol at the PM and LE, but not the amount of SM-associated-cholesterol or SM. ABCG1-mobilized non-SM-associated-cholesterol rapidly cycled between the PM and LE and effluxed from the PM to extracellular acceptors, or, relocated to intracellular sites of esterification. ABCG1 increased detergent-soluble pools of PM and LE cholesterol, generated detergent-resistant, non-SM-associated PM cholesterol, and increased resistance to both amphotericin B-induced (cholesterol-mediated and lysenin-induced (SM-mediated cytolysis, consistent with altered organization of both PM cholesterol and SM. ABCG1 itself resided in detergent-soluble membrane domains. We propose that PM and LE ABCG1 residing at the phase boundary between ordered (Lo and disordered (Ld membrane lipid domains alters SM and cholesterol organization thereby increasing cholesterol flux between Lo and Ld, and hence, the amount of cholesterol available for removal by acceptors on either side of the membrane bilayer for either efflux or esterification.

Inflammatory Response of Human Gestational Membranes to Ureaplasma parvum Using a Novel Dual-Chamber Tissue Explant System.

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Potts, Lauren C; Feng, Liping; Seed, Patrick C; Jayes, Friederike L; Kuchibhatla, Maragatha; Antczak, Brian; Nazzal, Matthew K; Murtha, Amy P

2016-05-01

Preterm premature rupture of membranes (PPROM) is often associated with intra-amniotic inflammation and infection. Current understanding of the pathogenesis of PPROM includes activation of pro-inflammatory cytokines and proteolytic enzymes leading to compromise of membrane integrity. The impact of exposure to bacterial pathogens, including Ureaplasma parvum, on gestational membranes is poorly understood. Our objective was to develop a dual-chamber system to characterize the inflammatory response of gestational membranes to U. parvum in a directional nature. Full-thickness human gestational membrane explants, with either choriodecidua or amnion oriented superiorly, were suspended between two washers in a cylindrical device, creating two distinct compartments. Brilliant green dye was introduced into the top chamber to assess the integrity of the system. Tissue viability was evaluated after 72 h using a colorimetric cell proliferation assay. Choriodecidua or amnion was exposed to three doses of U. parvum and incubated for 24 h. Following treatment, media from each compartment were used for quantification of U. parvum (quantitative PCR), interleukin (IL)-8 (enzyme-linked immunosorbent assay), and matrix metalloproteinase (MMP)-2 and MMP-9 activity (zymography). We observed that system integrity and explant viability were maintained over 72 h. Dose-dependent increases in recovered U. parvum, IL-8 concentration, and MMP-2 activity were detected in both compartments. Significant differences in IL-8 concentration and MMP-9 activity were found between the choriodecidua and amnion. This tissue explant system can be used to investigate the inflammatory consequences of directional bacterial exposure for gestational membranes and provides insight into the pathogenesis of PPROM and infectious complications of pregnancy. © 2016 by the Society for the Study of Reproduction, Inc.

Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells

International Nuclear Information System (INIS)

Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Musarrat, Javed

2012-01-01

Highlights: ► Butachlor exhibited strong binding affinity with DNA and produced 8-oxodG adducts. ► Butachlor induced DNA strand breaks and micronuclei formation in PBMN cells. ► Butachlor induced ROS and dissipation of mitochondrial membrane potential in cells. ► Butachlor resulted in cell cycle arrest and eventually caused cellular necrosis. -- Abstract: Butachlor is a systemic herbicide widely applied on rice, tea, wheat, beans and other crops; however, it concurrently exerts toxic effects on beneficial organisms like earthworms, aquatic invertebrates and other non-target animals including humans. Owing to the associated risk to humans, this chloroacetanilide class of herbicide was investigated with the aim to assess its potential for the (i) interaction with DNA, (ii) mitochondria membrane damage and DNA strand breaks and (iii) cell cycle arrest and necrosis in butachlor treated human peripheral blood mononuclear (PBMN) cells. Fluorescence quenching data revealed the binding constant (Ka = 1.2 × 10 4 M −1 ) and binding capacity (n = 1.02) of butachlor with ctDNA. The oxidative potential of butachlor was ascertained based on its capacity of inducing reactive oxygen species (ROS) and substantial amounts of promutagenic 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) adducts in DNA. Also, the discernible butachlor dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) and increased fluorescence intensity of 2′,7′-dichlorodihydro fluorescein diacetate (DCFH-DA) in treated cells signifies decreased mitochondrial membrane potential (ΔΨm) due to intracellular ROS generation. The comet data revealed significantly greater Olive tail moment (OTM) values in butachlor treated PBMN cells vs untreated and DMSO controls. Treatment of cultured PBMN cells for 24 h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). The flow

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

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Vidyadharan, Arun Kumar; Ravindran, Anjana

2014-01-01

Patient wanted to restore her lost teeth with implants in the lower left first molar and second premolar region. Cone beam computerized tomography (CBCT) revealed inadequate bone width and height around future implant sites. The extraction socket of second premolar area revealed inadequate socket healing with sparse bone fill after 4 months of extraction. To evaluate the clinical feasibility of using a collagen physical resorbable barrier made of human pericardium (HP) to augment localized alveolar ridge defects for the subsequent placement of dental implants. Ridge augmentation was done in the compromised area using Puros® demineralized bone matrix (DBM) Putty with chips and an HP allograft membrane. Horizontal (width) and vertical hard tissue measurements with CBCT were recorded on the day of ridge augmentation surgery, 4 month and 7 months follow-up. Intra oral periapical taken 1 year after implant installation showed minimal crestal bone loss. Bone volume achieved through guided bone regeneration was a gain of 4.8 mm horizontally (width) and 6.8 mm vertically in the deficient ridge within a period of 7 months following the procedure. 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.

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.

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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

Blood-group-Ii-active gangliosides of human erythrocyte membranes

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Feizi, T.; Childs, R.A.; Hakomori, S.-I.; Powell, M.E.

1978-01-01

More than ten new types of gangliosides, in addition to haematoside and sialosylparagloboside, were isolated from human erythrocyte membranes. These were separated by successive chromatographies on DAEA-Sephadex, on porous silica-gel columns and on thin-layer silica gel as acetylated compounds. Highly potent blood-group-Ii and moderate blood-group-H activities were demonstrated in some of the ganglioside fractions. The gangliosides incorporated into chlolesterol/phosphatidylcholine liposomes stoicheiometrically inhibited binding of anti-(blood-group-I and i) antibodies to a radioiodinated blood-group-Ii-active glycoprotein. The fraction with the highest blood-group-I activity, I(g) fraction, behaved like sialosyl-deca- to dodeca-glycosylceramides on t.l.c. Certain blood-group-I and most of the i-determinants were in partially or completely cryptic form and could be unmasked by sialidase treatment. Thus the I and i antigens, which are known to occur on internal structures of blood-group-ABH-active glycoproteins in secretions, also occur in the interior of the carbohydrate chains of erythrocyte gangliosides. (author)

Time Average Holography Study of Human Tympanic Membrane with Altered Middle Ear Ossicular Chain

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Cheng, Jeffrey T.; Ravicz, Michael E.; Rosowski, John J.; Hulli, Nesim; Hernandez-Montes, Maria S.; Furlong, Cosme

2009-02-01

Computer-assisted time average holographic interferometry was used to study the vibration of the human tympanic membrane (TM) in cadaveric temporal bones before and after alterations of the ossicular chain. Simultaneous laser Doppler vibrometer measurements of stapes velocity were performed to estimate the conductive hearing loss caused by ossicular alterations. The quantified TM motion described from holographic images was correlated with stapes velocity to define relations between TM motion and stapes velocity in various ossicular disorders. The results suggest that motions of the TM are relatively uncoupled from stapes motion at frequencies above 1000 Hz.

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

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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).

The Effect of Scala Tympani Morphology on Basilar Membrane Contact With a Straight Electrode Array: A Human Temporal Bone Study.

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Verberne, Juul; Risi, Frank; Campbell, Luke; Chambers, Scott; O'Leary, Stephen

2017-01-01

Scala tympani morphology influences the insertion dynamics and intra-scalar position of straight electrode arrays. Hearing preservation is the goal of cochlear implantation with current thin straight electrode arrays. These hug the lateral wall, facilitating full, atraumatic insertions. However, most studies still report some postoperative hearing loss. This study explores the influence of scala tympani morphology on array position relative to the basilar membrane and its possible contribution to postoperative hearing loss. Twenty-six fresh-frozen human temporal bones implanted with a straight electrode array were three-dimensionally reconstructed from micro-photographic histological sections. Insertion depth and the proximity between the array and basilar membrane were recorded. Lateral wall shape was quantified as a curvature ratio. Insertion depths ranged from 233 to 470 degrees. The mean first point of contact between the array and basilar membrane was 185 degrees; arrays tended to remain in contact with the membrane after first contacting it. Eighty-nine and 93% of arrays that reached the upper basal (>240-360 degrees) and second (>360-720 degrees) turns respectively contacted the basilar membrane in these regions. Scalar wall curvature ratio decreased significantly (the wall became steeper) from the basal to second turns. This shift correlated with a reduced distance between the array and basilar membrane. Scala tympani morphology influences the insertion dynamics and intra-scalar position of a straight electrode array. In addition to gross trauma of cochlear structures, contact between the array and basilar membrane and how this impacts membrane function should be considered in hearing preservation cases.

Dried Human Amniotic Membrane Does Not Alleviate Inflammation and Fibrosis in Experimental Strabismus Surgery

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Bo Young Chun

2013-01-01

Full Text Available Purpose. The purpose of this study was to evaluate the efficacy of dried human amniotic membrane (AM in reducing the postoperative inflammatory response and scarring after strabismus surgery. Methods. The inflammatory response at the extraocular muscle reattachment site was analyzed after superior rectus (SR resection in 12 rabbits. Dried human AM (Ambiodry2 was applied between the resected SR muscle plane and Tenon’s capsule of the left eyes of rabbits. As a control, the right eyes of rabbits underwent SR resection only. The surgeon randomly ordered which eye gets operated first during the experiment. Two weeks later, enucleation was performed. Six sagittal sections were made for each eye at the insertion of the SR muscle. The grade of postoperative inflammation and the presence of fibrosis were evaluated in histological examinations. Results. There was no statistically significant difference in the intensity of inflammation and fibrous proliferation between the eyes treated with dried human AM after SR resection and those treated with SR resection only. Conclusions. The use of dried human AM was not effective in controlling the postoperative inflammation and scarring in rabbit eyes after extraocular muscle surgery. However, this may be due to the devitalized dry preparation of human AM (Ambiodry2, which may have lost the expected anti-inflammatory and anti-scarring properties, and further studies on humans may be necessary.

The Peri-islet Basement Membrane, a Barrier to Infiltrating Leukocytes in Type 1 Diabetes in Mouse and Human

DEFF Research Database (Denmark)

Korpos, Eva; Kadri, Nadir; Kappelhoff, Reinhild

2013-01-01

We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data demonstr...... IM are reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies.......We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data...... demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte...

Transplantation with cultured stem cells derived from the human amniotic membrane for corneal alkali burns: an experimental study.

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Zeng, Wei; Li, Yanwei; Zeng, Guangwei; Yang, Bo; Zhu, Yu

2014-01-01

Amniotic membranes (AM) have been used in a wide range of clinical applications. We successfully extracted mesenchymal stem cells (MSCs) from human AM, but little is known about the use and efficacy of human amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) for the treatment of alkali burns. We utilized hAM-dMSCs transplantation, AM grafting, and their combined use in the treatment of alkali burns. An experimental model in rabbits was devised to analyze the use of these techniques with immunocytochemistry and ELISA. The survival and migration of hAM-dMSCs labeled by SPION in the host were assessed with Prussian blue staining. Compared with the control group, the treated groups demonstrated faster reconstruction of the corneal epithelium, and lower levels of corneal opacification and neovascularization within corneal alkali burns. Furthermore, dark blue-stained particles were detected in the limbus corneae at day 28. These results demonstrated the ability of hAM-dMSCs to enhance epithelial healing and reduce corneal opacification and neovascularization in corneal alkali wounds.

Schneiderian membrane detachment using transcrestal hydrodynamic ultrasonic cavitational sinus lift: a human cadaver head study and histologic analysis.

Science.gov (United States)

Troedhan, Angelo; Kurrek, Andreas; Wainwright, Marcel; Jank, Siegfried

2014-08-01

Recent studies have suggested the osteogenic layer of the periosteum at the base of the sinus membrane to play a key role in bone regeneration after sinus lift procedures. Thus, atraumatic detachment of the sinus membrane with an intact periosteum seems mandatory. The present histologic study of fresh human cadaver heads investigated the detachment behavior and histologic integrity of the detached periosteum after application of the transcrestal hydrodynamic ultrasonic cavitational sinus lift (tHUCSL-INTRALIFT). A total of 15 sinuses in 8 fresh human cadaver heads were treated using tHUCSL-INTRALIFT. After surgery, they were checked macroscopically for damage to the sinus membrane and then processed for histologic inspection under light microscopy. A total of 150 histologic specimens, randomly selected from the core surgical sites, were investigated using hematoxylin-eosin (HE), Azan, and trichrome staining. None of the 150 inspected specimens showed any perforation or dissection of the periosteum from the subepithelial connective tissue and respiratory epithelium and were fully detached from the bony antrum floor. The connecting Sharpey fibers revealed to be cleanly separated from the sinus floor in all specimens. The results of the present study suggest tHUCSL-INTRALIFT should be used to perform predictable and safe detachment of the periosteum from the bony sinus floor as a prerequisite for undisturbed and successful physiologic subantral bone regeneration. Copyright © 2014 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

International Nuclear Information System (INIS)

Stevenson, K.B.; Nauseef, W.M.; Clark, R.A.

1987-01-01

The glucoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3 H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody. Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove adsorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils

Spontaneous transfer of stearic acids between human serum albumin and PEG:2000-grafted DPPC membranes.

Science.gov (United States)

Pantusa, Manuela; Stirpe, Andrea; Sportelli, Luigi; Bartucci, Rosa

2010-05-01

Electron spin resonance (ESR) spectroscopy is used to study the transfer of stearic acids between human serum albumin (HSA) and sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Protein/lipid dispersions are considered in which spin-labelled stearic acids at the 16th carbon atom along the acyl chain (16-SASL) are inserted either in the protein or in the SSL. Two component ESR spectra with different rotational mobility are obtained over a broad range of temperature and membrane composition. Indeed, superimposed to an anisotropic protein-signal, appears a more isotropic lipid-signal. Since in the samples only one matrix (protein or membranes) is spin-labelled, the other component accounts for the transfer of 16-SASL between albumin and membranes. The two components have been resolved and quantified by spectral subtractions, and the fraction, f (p) (16-SASL), of spin labels bound non-covalently to the protein has been used to monitor the transfer. It is found that it depends on the type of donor and acceptor matrix, on the physical state of the membranes and on the grafting density of the polymer-lipids. Indeed, it is favoured from SSL to HSA and the fraction of stearic acids transferred increases with temperature in both directions of transfer. Moreover, in the presence of polymer-lipids, the transfer from HSA to SSL is slightly attenuated, especially in the brush regime of the polymer-chains. Instead, the transfer from SSL to HSA is favoured by the polymer-lipids much more in the mushroom than in the brush regime.

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.

Fabrication and evaluation of novel zeolite membranes to control the neoplastic activity and anti-tumoral drug treatments in human breast cancer cells. Part 1: Synthesis and characterization of Pure Zeolite Membranes and Mixed Matrix Membranes for adhesion and growth of cancer cells

International Nuclear Information System (INIS)

Tavolaro, Palmira; Martino, Guglielmo; Andò, Sebastiano; Tavolaro, Adalgisa

2016-01-01

Novel pure and hybrid zeolite membranes were prepared with appropriate different physicochemical characteristics such as frameworks, hydrophilicity, crystal size, chemical composition, acid-base properties (Point of Zero Charge, PZC) and surface morphology and used in inorganic cell/scaffold constructs. Because the control of cell interactions, as the adhesion, proliferation, remodelling and mobility, is important for differentiation and progression of tumors, this work focused on response of cancer cells adhered and grown on synthesized zeolite surfaces in order to study the influence of these scaffolds in controlled conditions. We have selected the MCF-7 and MDA-MB-231 human breast cancer cell line as model tumor cell lines. This study showed that all the zeolite membranes synthesized are excellent scaffolds because they are very selective materials to support the adhesion and growth of neoplastic cells. All zeolite scaffolds were characterized by FESEM, FTIR ATR, XRD, AFM, PZC and contact angle analyses. Cell adhesion, viability and morphology were measured by count, MTT assay and FESEM microphotography analysis, at various incubation times. - Highlights: • Novel pure and hybrid zeolite scaffolds were developed. • PZMs and MMMs were characterized and used with human cancer cells. • A systematic study of zeolite scaffolds influence on cell adhesion and morphology was performed. • The PZC value of zeolite membranes controls the cell-cell and scaffold-cell interactions.

Fabrication and evaluation of novel zeolite membranes to control the neoplastic activity and anti-tumoral drug treatments in human breast cancer cells. Part 1: Synthesis and characterization of Pure Zeolite Membranes and Mixed Matrix Membranes for adhesion and growth of cancer cells

Energy Technology Data Exchange (ETDEWEB)

Tavolaro, Palmira, E-mail: p.tavolaro@unical.it [Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Cubo 4/c, 87036 Rende (Italy); Martino, Guglielmo [Department Di.B.E.S.T. (Biologia, Ecologia, Scienze della Terra), Unit of Physiology, University of Calabria, Cubo 4/c, 87036 Rende (Italy); Andò, Sebastiano [Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Cubo 4/c, 87036 Rende (Italy); Tavolaro, Adalgisa [Research Institute on Membrane Technology, Unit of Zeolite Membranes, ITM-CNR, University of Calabria, Cubo 17/c, 87036 Rende (Italy)

2016-12-01

Novel pure and hybrid zeolite membranes were prepared with appropriate different physicochemical characteristics such as frameworks, hydrophilicity, crystal size, chemical composition, acid-base properties (Point of Zero Charge, PZC) and surface morphology and used in inorganic cell/scaffold constructs. Because the control of cell interactions, as the adhesion, proliferation, remodelling and mobility, is important for differentiation and progression of tumors, this work focused on response of cancer cells adhered and grown on synthesized zeolite surfaces in order to study the influence of these scaffolds in controlled conditions. We have selected the MCF-7 and MDA-MB-231 human breast cancer cell line as model tumor cell lines. This study showed that all the zeolite membranes synthesized are excellent scaffolds because they are very selective materials to support the adhesion and growth of neoplastic cells. All zeolite scaffolds were characterized by FESEM, FTIR ATR, XRD, AFM, PZC and contact angle analyses. Cell adhesion, viability and morphology were measured by count, MTT assay and FESEM microphotography analysis, at various incubation times. - Highlights: • Novel pure and hybrid zeolite scaffolds were developed. • PZMs and MMMs were characterized and used with human cancer cells. • A systematic study of zeolite scaffolds influence on cell adhesion and morphology was performed. • The PZC value of zeolite membranes controls the cell-cell and scaffold-cell interactions.

Construction and characterization of human oral mucosa equivalent using hyper-dry amniotic membrane as a matrix.

Science.gov (United States)

Qi, Fangfang; Yoshida, Toshiko; Koike, Takeshi; Aizawa, Hitoshi; Shimane, Tetsu; Li, Yinghui; Yamada, Shinichi; Okabe, Motonori; Nikaido, Toshio; Kurita, Hiroshi

2016-05-01

Human amniotic membrane(HAM) as a graft material has been used in various fields. Hyper-dry amniotic membrane (HD-AM) is a novel dried amniotic membrane that is easy to handle and can be preserved at room temperature without time limitation. The purpose of this study was to investigate the useful properties of HD-AM in reconstruction of the oral mucosa. Human oral keratinocytes were isolated and seeded on HD-AM in serum-free culture system. Oral mucosa equivalent (OME) was developed and transplanted onto full-thickness wound on athymic mice. The wound healing was analyzed and the OME both before and after transplantation was analyzed with hematoxylin-eosin staining and immunohistochemical staining for Cytokines 10 (CK10), Cytokines 16 (CK16), and Ivolucrin (IVL). Oral keratinocytes spread and proliferated well on HD-AM. Two weeks after air-lifting, OME had formed with good differentiation and morphology. We confirmed immunohistochemically that the expression of CK10 was positive in all suprabasal layers, as was CK16 in the upper layers, while IVL was present in all cell layers. Three weeks after transplantation to athymic mice, the newly generated tissue had survived well with the smallest contraction. The epithelial cells of newly generated tissue expressed CK10 throughout in all suprabasal layers, IVL was mainly in the granular layer, and CK16 positive cells were observed in all spinous layer and granular layer but were not expressed in the mouse skin, all of which were similar to native gingival mucosa. The OME with HD-AM as a matrix revealed a good morphology and stable wound healing. This study demonstrates that HD-AM is a useful and feasible biomaterial for oral mucosa reconstruction. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts.

Science.gov (United States)

Reisner, P D; Brandt, P C; Vanaman, T C

1997-01-01

It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.

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.

Regenerative and Antibacterial Properties of Acellular Fish Skin Grafts and Human Amnion/Chorion Membrane: Implications for Tissue Preservation in Combat Casualty Care.

Science.gov (United States)

Magnusson, Skuli; Baldursson, Baldur Tumi; Kjartansson, Hilmar; Rolfsson, Ottar; Sigurjonsson, Gudmundur Fertram

2017-03-01

Improvised explosive devices and new directed energy weapons are changing warfare injuries from penetrating wounds to large surface area thermal and blast injuries. Acellular fish skin is used for tissue repair and during manufacturing subjected to gentle processing compared to biologic materials derived from mammals. This is due to the absence of viral and prion disease transmission risk, preserving natural structure and composition of the fish skin graft. The aim of this study was to assess properties of acellular fish skin relevant for severe battlefield injuries and to compare those properties with those of dehydrated human amnion/chorion membrane. We evaluated cell ingrowth capabilities of the biological materials with microscopy techniques. Bacterial barrier properties were tested with a 2-chamber model. The microstructure of the acellular fish skin is highly porous, whereas the microstructure of dehydrated human amnion/chorion membrane is mostly nonporous. The fish skin grafts show superior ability to support 3-dimensional ingrowth of cells compared to dehydrated human amnion/chorion membrane (p fish skin is a bacterial barrier for 24 to 48 hours. The unique biomechanical properties of the acellular fish skin graft make it ideal to be used as a conformal cover for severe trauma and burn wounds in the battlefield. Reprint & Copyright © 2017 Association of Military Surgeons of the U.S.

Nanomaterials for membrane fouling control: accomplishments and challenges.

Science.gov (United States)

Yang, Qian; Mi, Baoxia

2013-11-01

We report a review of recent research efforts on incorporating nanomaterials-including metal/metal oxide nanoparticles, carbon-based nanomaterials, and polymeric nanomaterials-into/onto membranes to improve membrane antifouling properties in biomedical or potentially medical-related applications. In general, nanomaterials can be incorporated into/onto a membrane by blending them into membrane fabricating materials or by attaching them to membrane surfaces via physical or chemical approaches. Overall, the fascinating, multifaceted properties (eg, high hydrophilicity, superparamagnetic properties, antibacterial properties, amenable functionality, strong hydration capability) of nanomaterials provide numerous novel strategies and unprecedented opportunities to fully mitigate membrane fouling. However, there are still challenges in achieving a broader adoption of nanomaterials in the membrane processes used for biomedical applications. Most of these challenges arise from the concerns over their long-term antifouling performance, hemocompatibility, and toxicity toward humans. Therefore, rigorous investigation is still needed before the adoption of some of these nanomaterials in biomedical applications, especially for those nanomaterials proposed to be used in the human body or in contact with living tissue/body fluids for a long period of time. Nevertheless, it is reasonable to predict that the service lifetime of membrane-based biomedical devices and implants will be prolonged significantly with the adoption of appropriate fouling control strategies. Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Is there a stepwise increase in neonatal morbidities according to histological stage (or grade) of acute chorioamnionitis and funisitis?: effect of gestational age at delivery.

Science.gov (United States)

Lee, Yeri; Kim, Hyun-Joo; Choi, Suk-Joo; Oh, Soo-Young; Kim, Jung-Sun; Roh, Cheong-Rae; Kim, Jong-Hwa

2015-03-01

To test if there is a stepwise difference in neonatal outcomes according to the stage (or grade) of histological inflammatory response in the chorioamniotic membranes and umbilical cords of preterm premature rupture of membranes (PPROM). This retrospective study included singleton pregnancies diagnosed as PPROM and delivered prior to 34 weeks of gestation (n=339). Acute histological chorioamnionitis and funisitis were subdivided into stages (or grade) as defined by Redline et al. Neonatal composite morbidities and mortality were also monitored. Univariate and multivariate analyses were conducted. Increasing stage (or grade) of acute histological chorioamnionitis and funisitis was significantly associated with an earlier gestational age at membrane rupture and delivery. Among neonatal outcomes, respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), intraventricular hemorrhage, retinopathy of prematurity, and composite morbidity showed incremental incidence according to increased stage (or grade) of acute chorioamnionitis, while periventricular leukomalacia and necrotizing enterocolitis did not. Only RDS, BPD, and composite morbidity showed similar incremental incidences associated with severity of funisitis stage. However, the incremental trends of each neonatal outcome were found to be nonsignificant by multivariate analysis adjusting confounding variables including gestational age at delivery. Higher incidences of neonatal morbidity according to increased stage (or grade) of either acute histological chorioamnionitis or funisitis were due to an earlier gestational age at delivery.

Evaluation of Human Amniotic Membrane as a Wound Dressing for Split-Thickness Skin-Graft Donor Sites

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Denys J. Loeffelbein

2014-01-01

Full Text Available Human amniotic membrane (HAM has been used as a biomaterial in various surgical procedures and exceeds some qualities of common materials. We evaluated HAM as wound dressing for split-thickness skin-graft (STSG donor sites in a swine model (Part A and a clinical trial (Part B. Part A: STSG donor sites in 4 piglets were treated with HAM or a clinically used conventional polyurethane (PU foil (n=8 each. Biopsies were taken on days 5, 7, 10, 20, 40, and 60 and investigated immunohistochemically for alpha-smooth muscle actin (αSMA: wound contraction marker, von Willebrand factor (vWF: angiogenesis, Ki-67 (cell proliferation, and laminin (basement membrane integrity. Part B: STSG donor sites in 45 adult patients (16 female/29 male were treated with HAM covered by PU foam, solely by PU foam, or PU foil/paraffin gauze (n=15 each. Part A revealed no difference in the rate of wound closure between groups. HAM showed improved esthetic results and inhibitory effects on cicatrization. Angioneogenesis was reduced, and basement membrane formation was accelerated in HAM group. Part B: no difference in re-epithelialization/infection rate was found. HAM caused less ichor exudation and less pruritus. HAM has no relevant advantage over conventional dressings but might be a cost-effective alternative.

ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast

International Nuclear Information System (INIS)

Fisher, G.J.; Kelley, L.K.; Smith, C.H.

1987-01-01

As a first step in understanding the cellular basis of maternal-fetal calcium transfer, the authors examined the characteristics of calcium uptake by a highly purified preparation of the syncytiotrophoblast basal (fetal facing) plasma membrane. In the presence of nanomolar concentrations of free calcium, basal membranes demonstrated substantial ATP-dependent calcium uptake. This uptake required magnesium, was not significantly affected by Na + or K + (50 mM), or sodium azide (10 mM). Intravesicular calcium was rapidly and completely released by the calcium ionophore rapidly and completely released by the calcium ionophore A23187. Calcium transport was significantly stimulated by the calcium-dependent regulatory protein calmodulin. Placental membrane fractions enriched in endoplasmic reticulum (ER) and mitochondria also demonstrated ATP-dependent calcium uptake. In contrast to basal membrane, mitochondrial calcium uptake was completely inhibited by azide. The rate of calcium uptake was completely inhibited by azide. The rate of calcium uptake by the ER was only 20% of that of basal membranes. They conclude that the placental basal plasma membrane possesses a high-affinity calcium transport system similar to that found in plasma membranes of a variety of cell types. This transporter is situated to permit it to function in vivo in maternal-fetal calcium transfer

Effects of ionizing radiation on glycerolated amniotic membranes as a substract for cultured human epithelium

International Nuclear Information System (INIS)

Paggiaro, Andre Oliveira

2011-01-01

The amniotic membrane (AM) is a biomaterial with biological properties that are beneficial to tissue repair. It has been used as a temporary coverage to threat burns and chronic wounds. Recently, it has been served as a substrate for keratinocytes culture to construct a living skin equivalent. However, MA is a biological material, and its transplantation could cause infectious disease for receptors. So, it must be preserved and sterilized before clinical use. The aim of this study was to evaluate the radiation effects on glycerol-preserved MA, considering its compatibility to support human keratinocytes culture. Four MA were stored in high concentrations of glycerol (> 85%) and half of them were radio sterilized with a dose of 25 kGy. Then, we established two groups: nonirradiated MA (MA-ni) and irradiated MA (MA-i). Both groups was deepithelialized by a standardized protocol and was investigated morphologically, immunohistochemical and ultrastructural. Subsequently human keratinocytes were cultivated immersed and in air-liquid interface on denuded surface of MA-i and MA-ni. The results were compared at 14 and 21 days of culture by light and electron microscopy. After epithelial denudation, analyses demonstrated the continuity of the basement membrane in MA-ni group, whereas in the irradiated group, there was no indication of the basement membrane’s presence on the surface of MA. The cell cultures showed that in the non-irradiated group, there was growth of a multi-layered and differentiated epithelium, with a stratum corneum’s formation in air-liquid interface. In the irradiated group, the epithelium had only two or three layer, little cell differentiation, with the same results immersed or air-liquid interface system. Glycerol-preserved MA was biocompatible with the growth of a cultivated epithelium, showing its potential as a skin substitute. Irradiation at 25 kGy cause structural damage to the tissue, making changes in basement membrane, that facilitates

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

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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.

Membrane fusion and exocytosis.

Science.gov (United States)

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Effects of cobalt-60 ionizing radiation on human erythrocyte and its membrane proteins

International Nuclear Information System (INIS)

Amancio, Francisco Fernandes

1998-01-01

Ionizing radiation has several uses, as sterilization and radiotherapy, by its effects on living beings. recently, it has been used, at relatively lower doses (25 Gy), on blood for transfusions, mainly to eliminate undesirable graft host reactions, for use in multi transfused or immunocompromised patients. Here, we study the effect of larger doses of cobalt-60 ionizing radiation (25-1600 Gy) on human erythrocytes, by cytometric, physiologic, biochemical and immunological methods, looking for its effects and its detection. The red cells presented a clear dose-dependent increase in this volume, when irradiated in doses higher than 200 Gy, more significant in stored blood, but without hemolysis. Osmotic fragility was increased only after irradiation of more than 400 Gy. By ektacytometry, there was a lower deformability of irradiated red cells, at low stress (0.3 Pa), similar to capillary flow, but without alteration in higher stress (3 Pa), found in cardiac chambers. By SDS-PAGE, it was demonstrated that irradiated isolated erythrocyte membranes had aggregation of spectrin molecules, and decay of bands with lower molecular mass. This effect could be attributed to the radiation-induced hydroxyl radical, by specific scavenger studies. Those modifications were both antigenic and immunogenic in experimental animals, and the induced antibodies recognizes, by ELISA and immunoblot, both native or irradiated membrane proteins. They recognize rather irradiated whole erythrocyte than native ones, by hemagglutination, indirect immunofluorescence or flow cytometry assays. Our data suggests that human red cells could be irradiated at higher doses than those usually employed, with possible effect on other contaminant pathogens, without loss of viability of its use in transfusions. After improvements, irradiation induced epitopes detection could be a new tool in biological dosimetry. (author)

Molecular cloning of human protein 4.2: A major component of the erythrocyte membrane

International Nuclear Information System (INIS)

Sung, L.A.; Chien, Shu; Lambert, K.; Chang, Longsheng; Bliss, S.A.; Bouhassira, E.E.; Nagel, R.L.; Schwartz, R.S.; Rybicki, A.C.

1990-01-01

Protein 4.2 (P4.2) comprises ∼5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. The authors now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-air insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of ∼77 and ∼80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates

Membrane Inlet Mass Spectrometry for Homeland Security and Forensic Applications

Science.gov (United States)

Giannoukos, Stamatios; Brkić, Boris; Taylor, Stephen; France, Neil

2015-02-01

A man-portable membrane inlet mass spectrometer has been built and tested to detect and monitor characteristic odors emitted from the human body and also from threat substances. In each case, a heated membrane sampling probe was used. During human scent monitoring experiments, data were obtained for inorganic gases and volatile organic compounds emitted from human breath and sweat in a confined space. Volatile emissions were detected from the human body at low ppb concentrations. Experiments with compounds associated with narcotics, explosives, and chemical warfare agents were conducted for a range of membrane types. Test compounds included methyl benzoate (odor signature of cocaine), piperidine (precursor in clandestine phencyclidine manufacturing processes), 2-nitrotoluene (breakdown product of TNT), cyclohexanone (volatile signature of plastic explosives), dimethyl methylphosphonate (used in sarin and soman nerve agent production), and 2-chloroethyl ethyl sulfide (simulant compound for sulfur mustard gas). Gas phase calibration experiments were performed allowing sub-ppb LOD to be established. The results showed excellent linearity versus concentration and rapid membrane response times.

Tissue eosinophilia induced by recombinant human interleukin-5 in the hamster cheek pouch membrane

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M. Minnicozzi

1995-01-01

Full Text Available Interleukin-5 (IL-5 is a cytokine that preferentially effects the development and function of eosinophils, and is considered important in the pathophysiology of allergic inflammation. In this study, we evaluated the ability of recombinant human IL-5 (rHu IL-5 to promote tissue eosinophilia and the importance of this eosinophilia to pathological alterations in vascular function. Repetitive subcutaneous administration for 18 days of rHu IL-5 resulted in a 7-fold increase in the number of eosinophils found in the ipsilateral hamster cheek pouch membrane. The contralateral cheek pouch membrane and peritoneum of these animals showed lesser but significant elevations in the number of eosinophils. In contrast, denatured rHu IL-5 did not elevate eosinophils in these tissues. Through the use of intravital microscopy and fluorometric analysis, rHu IL-5 treated hamster cheek pouch membranes were evaluated for alterations in microvascular permeability, using plasma clearance of FITC-dextran 150 as an index. Despite promoting a prominent tissue eosinophilia, the repetitive subcutaneous injections of rHu IL-5 did not alter the clearance of FITC-dextran 150. Topical application of rHu IL-5 to the cheek pouch, also, had no effect on the clearance of FITC-dextran 150. Immunofluorescence observations using an antibody to the granule protein, eosinophil peroxidase, indicated that the recruited cells had not degranulated. Our results support the importance of IL-5 in the recruitment of tissue eosinophils, but further stimulation is probably required to cause degranulation of these cells and the ensuing tissue damage.

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

of the CR1 binding site with the monoclonal antibody 3D9 also resulted in a minor reduction in MAC deposition, while FE8 and 3D9, in combination, markedly reduced deposition of both C3 fragments (91 +/- 5%) and C9 (95 +/- 3%). The kinetics of C3-fragment and MAC deposition, as well as the dependence of both......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...

Immunohistochemical studies of the periodontal membrane in primary teeth

DEFF Research Database (Denmark)

Bille, Marie-Louise Bastholm; Nolting, Dorrit; Kjær, Inger

2009-01-01

Objectives. To describe the periodontal membrane of human primary teeth immunohistochemically, while focusing on the epithelial layer of Malassez, fibers, and peripheral nerves, and to compare the findings with those of a previous study of human permanent teeth. Material and methods. Nineteen human...... could be identical to those in regions with no resorption. Conclusion. In regions without resorption, spatial organization of the periodontal membrane of primary teeth was similar to that of permanent teeth, although the number and distribution of epithelial cells and fibers differed. In regions...

From PPROM to caul: The evolution of membrane rupture in mammals

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Gregory Stempfle

2013-12-01

Full Text Available Rupture of the extraembryonic membranes that form the gestational sac in humans is a typical feature of human parturition. However, preterm premature rupture of membranes (PPROM occurs in approximately 1% of pregnancies, and is a leading cause of preterm birth. Conversely, retention of an intact gestational sac during parturition in the form of a caul is a rare occurrence. Understanding the molecular and evolutionary underpinnings of these disparate phenotypes can provide insight into both normal pregnancy and PPROM. Using phylogenetic techniques we reconstructed the evolution of the gestational sac phenotype at parturition in 55 mammal species representing all major viviparous mammal groups. We infer the ancestral state in therians, eutherians, and primates, as in humans, is a ruptured gestational sac at parturition. We present evidence that intact membranes at parturition have evolved convergently in diverse mammals including horses, elephants, and bats. In order to gain insight into the molecular underpinnings of the evolution of enhanced membrane integrity we also used comparative genomics techniques to reconstruct the evolution of a subset of genes implicated in PPROM, and find that four genes (ADAMTS2, COL1A1, COL5A1, LEPRE1 show significant evidence of increased nonsynonymous rates of substitution on lineages with intact membranes as compared to those with ruptured membranes. Among these genes, we also discovered that 17 human SNPs are associated with or near amino acid replacement sites in those mammals with intact membranes. These SNPs are candidate functional variants within humans, which may play roles in both PPROM and/or the retention of the gestational sac at birth.

Age-dependent effects of He-Ne laser irradiation on the membrane fluidity of human erythrocytes

Science.gov (United States)

Kovacs, Eugenia; Savopol, Tudor; Pologea-Moraru, Roxana; Makropoulou, Mersini I.; Serafetinides, Alexander A.

1997-12-01

The low power He-Ne laser radiation has been extensively used in past decades as medical device to relieve pain, accelerate wound healing as well as aiming beam in invisible laser beam in invisible laser beam applications. It is not known however if there are any secondary, undesirable effects of He-Ne laser radiation on the irradiated tissue. In this paper we investigate the changes induced in membrane fluidity of human erythrocyte during/upon the interaction with the He-Ne laser beam having the parameters currently used for target aiming in laser surgery.

Effects of human low and high density lipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes

International Nuclear Information System (INIS)

Brissette, L.; Nol, S.P.

1986-01-01

Upon incubation with rat liver membranes, radioiodinated rat intermediate density lipoproteins (IDL) interacted with at least two binding sites having a low and a high affinity as demonstrated by the curvilinear Scatchard plots obtained from the specific binding data. The purpose of our work was to identify the nature of these binding sites. Human low density lipoproteins (LDL), contain apolipoprotein B only, and human high density lipoproteins (HDL3), containing neither apolipoprotein B nor E, were both capable of decreasing the specific binding of rat 125 I-IDL. The Scatchard analysis clearly revealed that only the low affinity component was affected by the addition of these human lipoproteins. In fact, the low affinity binding component gradually decreased as the amount of human LDL or HDL3 increased in the binding assay. At a 200-fold excess of human LDL or HDL3, the low affinity binding was totally masked, and the Scatchard plot of the specific 125 I-IDL binding became linear. Only the high affinity binding component was left, enabling a precise measurement of its binding parameters. In a series of competitive displacement experiments in which the binding assay contained a 200-fold excess of human LDL or HDL3, only unlabeled rat IDL effectively displaced the binding of rat 125 I-IDL. We conclude that the low affinity binding of rat IDL to rat liver membranes is due to weak interactions with unspecified lipoprotein binding sites. The camouflage of these sites by human lipoproteins makes possible the study of IDL binding to the high affinity component which likely represents the combined effect of IDL binding to both the remnant and the LDL receptors

An Integrated Framework Advancing Membrane Protein Modeling and Design.

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Rebecca F Alford

2015-09-01

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

Near-ultraviolet radiation-induced lipid peroxidation and membrane effects in Escherichia coli and human skin fibroblasts

International Nuclear Information System (INIS)

Chamberlain, J.

1987-01-01

The first part of this thesis examines the response of an unsaturated fatty acid auxotroph, Escherichia coli K1060 to broad-band near-UV radiation. Sensitivity, lipid peroxidation and leakage of rubidium from irradiated cells were found to increase with increasing unsaturation of membrane fatty acids. The involvement of singlet oxygen was implicated by an increase in sensitivity, lipid peroxidation and leakage of rubidium following irradiation in deuterium oxide. Some factors influencing survival following irradiation were investigated, where lower growth rates were shown to enhance survival. In the second part, the study was extended to human fibroblasts where a normal human skin fibroblast strain, GM730 and a strain derived from an actinic reticuloid patient, AR6LO, are compared. Lipid peroxidation was measured in both cell lines following broad-band near-UV irradiation. Membrane activity, as assessed by the pinocytic uptake of 14 C-sucrose and its subsequent release from the cell, was measured. Near-UV irradiation was found to increase such activity in both strains. Vitamin E and Trolox-C were found to decrease this response in AR6LO but not GM730 cells. The final part consists of preliminary investigations into the near-UV induced peroxidation of fatty acids and liposomes, and the subsequent increase in the level of hydroperoxides in the hours following irradiation. (author)

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

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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.

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

OpenAIRE

Orsini, F.; Santacroce, M.; Cremona, A.; Gosvami, N. N.; Lascialfari, A.; Hoogenboom, B. W.

2014-01-01

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...

Facile preparation of salt-tolerant anion-exchange membrane adsorber using hydrophobic membrane as substrate.

Science.gov (United States)

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

2017-03-24

In this study, a polyvinylidene fluoride (PVDF) hydrophobic membrane with high mechanical property was used as substrate to prepare salt-tolerant anion-exchange (STAE) membrane adsorber. Effective hydrophilization and functionalization of PVDF membrane was realized via polydopamine (PDA) deposition, thus overcoming the drawbacks of hydrophobic substrates including poor water permeability, inert property as well as severe non-specific adsorption. The following polyallylamine (PAH) coupling was carried out at pH 10.0, where unprotonated amine groups on PAH chains were more prone to couple with PDA. This membrane adsorber could remain 75% of protein binding capacity when NaCl concentration increased from 0 to 150mM, while its protein binding capacity was independent of flow rate from 10 to 100 membrane volume (MV)/min due to its high mechanical strength (tensile strength: 43.58±2.30MPa). With 200mM NaCl addition at pH 7.5, high purity (above 99%) and high recovery (almost 100%) of Immunoglobulin G (IgG) were obtained when using the STAE membrane adsorber to separate IgG/human serum albumin (HSA) mixture, being similar to that without NaCl at pH 6.0 (both under the flow rate of 10-100MV/min). Finally, the reliable reusability was confirmed by five reuse cycles of protein binding and elution operations. In comparison with commercial membrane adsorber, the new membrane adsorber exhibited a better mechanical property, higher IgG polishing efficiency and reusability, while the protein binding capacity was lower due to less NH 2 density on the membrane. The outcome of this work not only offers a facile and effective approach to prepare membrane adsorbers based on hydrophobic membranes, but also demonstrates great potential of this new designed STAE membrane adsorbers for efficient monoclonal antibody (mAb) polishing. Copyright © 2017 Elsevier B.V. All rights reserved.

A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia

International Nuclear Information System (INIS)

Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.; Guetarni, D.; Bachir, D.; Colonna, P.; Beuzard, Y.

1989-01-01

The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with [ 3 H]N-ethylmaleimide. This pool of soluble alpha chains was 0.067 ± 0.017% of hemoglobin in blood of normal adult, 0.11 ± 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [ 3 H]N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups

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

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...

The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization.

Science.gov (United States)

Brast, Sabine; Grabner, Alexander; Sucic, Sonja; Sitte, Harald H; Hermann, Edwin; Pavenstädt, Hermann; Schlatter, Eberhard; Ciarimboli, Giuliano

2012-03-01

Human organic cation transporter 2 (hOCT2) is involved in transport of many endogenous and exogenous organic cations, mainly in kidney and brain cells. Because the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes, and if so, which part of the transporter is involved in the oligomerization. A yeast 2-hybrid mating-based split-ubiquitin system (mbSUS), fluorescence resonance energy transfer, Western blot analysis, cross-linking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium were applied to human embryonic kidney 293 (HEK293) cells transfected with hOCT2 and partly also to freshly isolated human proximal tubules. The role of cysteines for oligomerization and trafficking of the transporter to the plasma membranes was investigated in cysteine mutants of hOCT2. hOCT2 formed oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutation of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for correct trafficking of the transporter to the plasma membrane and for its oligomerization.

Biogenesis of plasma membrane cholesterol

International Nuclear Information System (INIS)

Lange, Y.

1986-01-01

A striking feature of the molecular organization of eukaryotic cells is the singular enrichment of their plasma membranes in sterols. The authors studies are directed at elucidating the mechanisms underlying this inhomogeneous disposition. Cholesterol oxidase catalyzes the oxidation of plasma membrane cholesterol in intact cells, leaving intracellular cholesterol pools untouched. With this technique, the plasma membrane was shown to contain 95% of the unesterified cholesterol of cultured human fibroblasts. Cholesterol synthesized from [ 3 H] acetate moved to the plasma membrane with a half-time of 1 h at 37 0 C. They used equilibrium gradient centrifugation of homogenates of biosynthetically labeled, cholesterol oxidase treated cells to examine the distribution of newly synthesized sterols among intracellular pools. Surprisingly, lanosterol, a major precursor of cholesterol, and intracellular cholesterol both peaked at much lower buoyant density than did 3-hydroxy-3-methylglutaryl-CoA reductase. This suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. The cholesterol in the buoyant fraction eventually moved to the plasma membrane. Digitonin treatment increased the density of the newly synthesized cholesterol fractions, indicating that nascent cholesterol in transit is associated with cholesterol-rich membranes. The authors are testing the hypothesis that the pathway of cholesterol biosynthesis is spatially organized in various intracellular membranes such that the sequence of biosynthetic steps both concentrates the sterol and conveys it to the plasma membrane

Environment-sensitive ion-track membranes

International Nuclear Information System (INIS)

Yoshida, Masaru

1996-01-01

Development of an environment-sensitive porous membrane from ion-track membranes may realize by combining the techniques of ion beam radiation and those of molecular designing and synthesis for intelligent materials. Now, the development of such membrane is progressing with an aim at selecting some specific substances and accurately control its pore size in response to any small environmental stimulus such as temperature change. The authors have been studying the molecular design, synthesis and functional expression of intelligent materials, which are called here as environment-sensitive gels. In this report, the outlines of the apparatus for the production of such porous membrane was described. An organic polymer membrane was irradiated with an ion beam and followed by chemical etching to make ion track pores. Scanning electron microscopic observation for the cross section of the membrane showed that the pore shape varies greatly depending on the ion nuclide used. The characteristics of newly produced porous membranes consisting of CR-30/A-ProDMe and polyethylene-telephtharate were investigated in respect of pore size change responding to temperature. These studies of design, synthesis and functions of such gels would enable to substitute artificial materials for the functions of human sensors. (M.N.). 54 refs

Peripheral Protein Unfolding Drives Membrane Bending.

Science.gov (United States)

Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian

2018-06-20

Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions

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Sandy Azzi

2015-06-01

Full Text Available Intrarenal interleukin-15 (IL-15 participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15 isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC to peritumoral (ptumTEC, tumoral (RCC, and cancer stem cells (CSC/CD105+. RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15 isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα. This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression.

Mitofilin complexes: conserved organizers of mitochondrial membrane architecture.

Science.gov (United States)

Zerbes, Ralf M; van der Klei, Ida J; Veenhuis, Marten; Pfanner, Nikolaus; van der Laan, Martin; Bohnert, Maria

2012-11-01

Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, thereby generating contact sites between the two membrane systems of mitochondria. Within the inner membrane, mitofilins are part of hetero-oligomeric protein complexes that have been termed the mitochondrial inner membrane organizing system (MINOS). MINOS integrity is required for the maintenance of the characteristic morphology of the inner mitochondrial membrane, with an inner boundary region closely apposed to the outer membrane and cristae membranes, which form large tubular invaginations that protrude into the mitochondrial matrix and harbor the enzyme complexes of the oxidative phosphorylation machinery. MINOS deficiency comes along with a loss of crista junction structures and the detachment of cristae from the inner boundary membrane. MINOS has been conserved in evolution from unicellular eukaryotes to humans, where alterations of MINOS subunits are associated with multiple pathological conditions.

Erythrocyte Membrane Failure by Electromechanical Stress

Directory of Open Access Journals (Sweden)

E Du

2018-01-01

Full Text Available We envision that electrodeformation of biological cells through dielectrophoresis as a new technique to elucidate the mechanistic details underlying membrane failure by electrical and mechanical stresses. Here we demonstrate the full control of cellular uniaxial deformation and tensile recovery in biological cells via amplitude-modified electric field at radio frequency by an interdigitated electrode array in microfluidics. Transient creep and cyclic experiments were performed on individually tracked human erythrocytes. Observations of the viscoelastic-to-viscoplastic deformation behavior and the localized plastic deformations in erythrocyte membranes suggest that electromechanical stress results in irreversible membrane failure. Examples of membrane failure can be separated into different groups according to the loading scenarios: mechanical stiffening, physical damage, morphological transformation from discocyte to echinocyte, and whole cell lysis. These results show that this technique can be potentially utilized to explore membrane failure in erythrocytes affected by other pathophysiological processes.

Assembly factors for the membrane arm of human complex I.

Science.gov (United States)

Andrews, Byron; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

2013-11-19

Mitochondrial respiratory complex I is a product of both the nuclear and mitochondrial genomes. The integration of seven subunits encoded in mitochondrial DNA into the inner membrane, their association with 14 nuclear-encoded membrane subunits, the construction of the extrinsic arm from 23 additional nuclear-encoded proteins, iron-sulfur clusters, and flavin mononucleotide cofactor require the participation of assembly factors. Some are intrinsic to the complex, whereas others participate transiently. The suppression of the expression of the NDUFA11 subunit of complex I disrupted the assembly of the complex, and subcomplexes with masses of 550 and 815 kDa accumulated. Eight of the known extrinsic assembly factors plus a hydrophobic protein, C3orf1, were associated with the subcomplexes. The characteristics of C3orf1, of another assembly factor, TMEM126B, and of NDUFA11 suggest that they all participate in constructing the membrane arm of complex I.

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.

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

Science.gov (United States)

Logez, Christel; Berger, Sylvie; Legros, Céline; Banères, Jean-Louis; Cohen, William; Delagrange, Philippe; Nosjean, Olivier; Boutin, Jean A; Ferry, Gilles; Simonin, Frédéric; Wagner, Renaud

2014-01-01

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.

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.

Metagenomic identification of a novel salt tolerance gene from the human gut microbiome which encodes a membrane protein with homology to a brp/blh-family β-carotene 15,15'-monooxygenase.

Directory of Open Access Journals (Sweden)

Eamonn P Culligan

Full Text Available The human gut microbiome consists of at least 3 million non-redundant genes, 150 times that of the core human genome. Herein, we report the identification and characterisation of a novel stress tolerance gene from the human gut metagenome. The locus, assigned brpA, encodes a membrane protein with homology to a brp/blh-family β-carotene monooxygenase. Cloning and heterologous expression of brpA in Escherichia coli confers a significant salt tolerance phenotype. Furthermore, when cultured in the presence of exogenous β-carotene, cell pellets adopt a red/orange pigmentation indicating the incorporation of carotenoids in the cell membrane.

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.

Genotoxicity assessment of membrane concentrates of landfill leachate treated with Fenton reagent and UV-Fenton reagent using human hepatoma cell line

International Nuclear Information System (INIS)

Wang, Guifang; Lu, Gang; Yin, Pinghe; Zhao, Ling; Jimmy Yu, Qiming

2016-01-01

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.

Membrane Stabilizing Activity And Phytochemistry Of Hibiscus rosa ...

African Journals Online (AJOL)

The human erythrocyte membrane stabilizing activity of saline extract of Hibiscus rosa-sinensis leaves was investigated as part of efforts at validating its use as anti-arthritic and anti-inflammatory agent. The results of the membrane stabilizing activity of the extract, when compared to two non-steroidal anti-inflammatory drugs ...

Ridge Preservation Comparing a Nonresorbable PTFE Membrane to a Resorbable Collagen Membrane: A Clinical and Histologic Study in Humans.

Science.gov (United States)

Arbab, Hussain; Greenwell, Henry; Hill, Margaret; Morton, Dean; Vidal, Ricardo; Shumway, Brian; Allan, Nicholas D

2016-02-01

The primary aim of this randomized, controlled, blinded clinical trial was to compare the effect of a resorbable collagen membrane (CM group) versus a nonresorbable high-density polytetrafluoroethylene membrane (PTFE group) on the clinical and histologic outcomes of a ridge preservation procedure. All 24 sites received an intrasocket cancellous allograft and a buccal overlay bovine derived xenograft. The change in horizontal crestal ridge width was -1.4 ± 1.2 mm for the CM group, whereas the PTFE group lost -2.2 ± 1.5 mm, which was not statistically significant between groups (P > 0.05). Vertical ridge height change was -1.2 ± 1.5 for the CM group, whereas the PTFE group lost -0.5 ± 1.6, which was not significantly different between groups (P > 0.05). The percent vital bone was similar and not significantly different between groups. Primary closure was not obtained and the exposed membrane portion over the socket opening healed with keratinized tissue. The choice of a resorbable versus a nonresorbable barrier membrane did not affect the clinical or the histologic outcome of ridge preservation treatment.

Inositol phosphates influence the membrane bound Ca2+/Mg2+ stimulated ATPase from human erythrocyte membranes

International Nuclear Information System (INIS)

Kester, M.; Ekholm, J.; Kumar, R.; Hanahan, D.J.

1986-01-01

The modulation by exogenous inositol phosphates of the membrane Ca 2+ /Mg 2+ ATPase from saponin/EGTA lysed human erythrocytes was determined in a buffer (pH 7.6) containing histidine, 80 mM, MgCl 2 , 3.3 mM, NaCl, 74 mM, KCl, 30 mM, Na 2 ATP, 2.3 mM, ouabain, 0.83 mM, with variable amounts of CaCl 2 and EGTA. The ATPase assay was linear with time at 44 0 C. The inositol phosphates were commercially obtained and were also prepared from 32 P labeled rabbit platelet inositol phospholipids. Inositol triphosphate (IP 3 ) elevated the Ca 2+ /Mg 2+ ATPase activity over basal levels in a dose, time, and calcium dependent manner and were increased up to 85% of control values. Activities for the Na + /K + -ATPase and a Mg 2+ ATPase were not effected by IP 3 . Ca 2+ /Mg 2+ APTase activity with IP 2 or IP 3 could be synergistically elevated with calmodulin addition. The activation of the ATPase with IP 3 was calcium dependent in a range from .001 to .02 mM. The apparent Km and Vmax values were determined for IP 3 stimulated Ca 2+ /Mg 2+ ATPase

Reactive membrane technology: Two case studies

DEFF Research Database (Denmark)

Zeuner, Birgitte; Luo, Jianquan; Pinelo, Manuel

2014-01-01

investigated the effect of applied pressure, enzyme concentration, pH, and membrane properties on fouling-induced enzyme immobilization. In another study, the production of the human milk oligosaccharide 3’-sialyllactose by an engineered sialidase from Trypanosoma rangeli (Tr6) was significantly improved......Enzymatic processes are generally sustainable processes that use mild conditions and natural substrates. Membrane technology can be employed for enzyme immobilization as well as for recycling free enzymes. Using alcohol dehydrogenase (ADH) as part of a process to recycle CO2 to methanol, we...... in an enzymatic membrane reactor. The entire process can be improved by employing a series of ultra- and nanofiltrations....

Adsorption and Orientation of Human Islet Amyloid Polypeptide (hIAPP Monomer at Anionic Lipid Bilayers: Implications for Membrane-Mediated Aggregation

Directory of Open Access Journals (Sweden)

Guanghong Wei

2013-03-01

Full Text Available Protein misfolding and aggregation cause serious degenerative diseases, such as Alzheimer’s and type II diabetes. Human islet amyloid polypeptide (hIAPP is the major component of amyloid deposits found in the pancreas of type II diabetic patients. Increasing evidence suggests that β-cell death is related to the interaction of hIAPP with the cellular membrane, which accelerates peptide aggregation. In this study, as a first step towards understanding the membrane-mediated hIAPP aggregation, we investigate the atomic details of the initial step of hIAPP-membrane interaction, including the adsorption orientation and conformation of hIAPP monomer at an anionic POPG lipid bilayer by performing all-atom molecular dynamics simulations. We found that hIAPP monomer is quickly adsorbed to bilayer surface, and the adsorption is initiated from the N-terminal residues driven by strong electrostatic interactions of the positively-charged residues K1 and R11 with negatively-charged lipid headgroups. hIAPP binds parallel to the lipid bilayer surface as a stable helix through residues 7–22, consistent with previous experimental study. Remarkably, different simulations lead to the same binding orientation stabilized by electrostatic and H-bonding interactions, with residues R11, F15 and S19 oriented towards membrane and hydrophobic residues L12, A13, L16 and V17 exposed to solvent. Implications for membrane-mediated hIAPP aggregation are discussed.

Biocompatibility of epoxidized styrene-butadiene-styrene block copolymer membrane

International Nuclear Information System (INIS)

Yang, Jen Ming; Tsai, Shih Chang

2010-01-01

Styrene-butadiene-styrene block copolymer (SBS) membrane was prepared by solution casting method and then was epoxidized with peroxyformic acid generated in situ to yield the epoxidized styrene-butadiene-styrene block copolymer membrane (ESBS). The structure and properties of ESBS were characterized with infrared spectroscopy, Universal Testing Machine, differential scanning calorimetry (DSC), and thermogravimetry analysis (TGA). The performances of contact angle, water content, protein adsorption, and water vapor transmission rate on ESBS membrane were determined. After epoxidation, the hydrophilicity of the membrane increased. The water vapor transmission rate of ESBS membrane is similar to human skin. The biocompatibility of ESBS membrane was evaluated with the cell culture of fibroblasts on the membrane. It revealed that the cells not only remained viable but also proliferated on the surface of the various ESBS membranes and the population doubling time for fibroblast culture decreased.

Membrane vesicles from multidrug-resistant human carcinoma cells contain a specific 150,000-170,000 dalton protein detected by photoaffinity labeling

International Nuclear Information System (INIS)

Cornwell, M.M.; Safa, A.R.; Felsted, R.L.; Gottesman, M.M.; Pastan, I.

1986-01-01

The authors have selected multidrug-resistant human KB carcinoma cells in high levels of colchicine (KB-C4) or vinblastine (KB-V1) which are cross-resistant to many other structurally unrelated chemotheraputic agents. To determine the mechanism of reduced drug accumulation, they measured 3 H-vinblastine ( 3 H-VBL) association with membrane vesicles made from parental drug sensitive, drug-resistant and revertant cells. Membrane vesicles from highly multidrug resistant cells exhibited increased specific and saturable binding of vinblastine, (Kd = 1 μM) that was temperature dependent and trypsin sensitive. To identify the molecules which bind vinblastine, membrane vesicles were exposed to two photo-activatable analogs of vinblastine, (N-P-(azido-3,5,-[ 3 H]-benzoyl)-N'-β-aminoethylvindisine ( 3 H-NAB) and N-P-(azido-3-[ 125 I]-solicyl)-N'-β-aminoethylvindesine ( 125 I-NASV). The specific labeling of a 150,000-170,000 dalton protein in membrane vesicles from multidrug-resistant KB-C4 and KB-V1 cells was found. 125 I-NASV labeling was inhibited by vinblastine, vincrinstine and verapamil but not by colchicine or dexamethasone. The 150,000-170,000 dalton protein may have an important role in the multidrug resistance phenotype

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.

Lipopolysaccharide Membranes and Membrane Proteins of Pseudomonas aeruginosa Studied by Computer Simulation

Energy Technology Data Exchange (ETDEWEB)

Straatsma, TP

2006-12-01

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium with high metabolic versatility and an exceptional ability to adapt to a wide range of ecological environments, including soil, marches, coastal habitats, plant and animal tissues. Gram-negative microbes are characterized by the asymmetric lipopolysaccharide outer membrane, the study of which is important for a number of applications. The adhesion to mineral surfaces plays a central role in characterizing their contribution to the fate of contaminants in complex environmental systems by effecting microbial transport through soils, respiration redox chemistry, and ion mobility. Another important application stems from the fact that it is also a major opportunistic human pathogen that can result in life-threatening infections in many immunocompromised patients, such as lung infections in children with cystic fibrosis, bacteraemia in burn victims, urinary-tract infections in catheterized patients, hospital-acquired pneumonia in patients on respirators, infections in cancer patients receiving chemotherapy, and keratitis and corneal ulcers in users of extended-wear soft contact lenses. The inherent resistance against antibiotics which has been linked with the specific interactions in the outer membrane of P. aeruginosa makes these infections difficult to treat. Developments in simulation methodologies as well as computer hardware have enabled the molecular simulation of biological systems of increasing size and with increasing accuracy, providing detail that is difficult or impossible to obtain experimentally. Computer simulation studies contribute to our understanding of the behavior of proteins, protein-protein and protein-DNA complexes. In recent years, a number of research groups have made significant progress in applying these methods to the study of biological membranes. However, these applications have been focused exclusively on lipid bilayer membranes and on membrane proteins in lipid

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

Energy Technology Data Exchange (ETDEWEB)

Lisenko, S A; Kugeiko, M M [Belarusian State University, Minsk (Belarus)

2013-07-31

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. (laser applications in biology and medicine)

An Improved 2-Dimensional Gel Electrophoresis Method for Resolving Human Erythrocyte Membrane Proteins.

Science.gov (United States)

Kumar, Manoj; Singh, Rajendra; Meena, Anil; Patidar, Bhagwan S; Prasad, Rajendra; Chhabra, Sunil K; Bansal, Surendra K

2017-01-01

The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for

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

Indian Academy of Sciences (India)

Unknown

SLP, stomatin-like-protein-2; TX-100, Triton X-100. ... via electrostatic interactions that can be disrupted by the simultaneous increase in pH and ionic strength of the solubilization .... dentified components of the DRMs and the membrane ..... Analysis of proteins and cholesterol in 6 ... Band 3, the main integral protein of the.

Restored in vivo-like membrane lipidomics positively influence in vitro features of cultured mesenchymal stromal/stem cells derived from human placenta.

Science.gov (United States)

Chatgilialoglu, Alexandros; Rossi, Martina; Alviano, Francesco; Poggi, Paola; Zannini, Chiara; Marchionni, Cosetta; Ricci, Francesca; Tazzari, Pier Luigi; Taglioli, Valentina; Calder, Philip C; Bonsi, Laura

2017-02-07

The study of lipid metabolism in stem cell physiology has recently raised great interest. The role of lipids goes beyond the mere structural involvement in assembling extra- and intra-cellular compartments. Nevertheless, we are still far from understanding the impact of membrane lipidomics in stemness maintenance and differentiation patterns. In the last years, it has been reported how in vitro cell culturing can modify membrane lipidomics. The aim of the present work was to study the membrane fatty acid profile of mesenchymal stromal cells (MSCs) derived from human fetal membranes (hFM-MSCs) and to correlate this to specific biological properties by using chemically defined tailored lipid supplements (Refeed®). Freshly isolated hFM-MSCs were characterized for their membrane fatty acid composition. hFM-MSCs were cultivated in vitro following a classical protocol and their membrane fatty acid profile at different passages was compared to the profile in vivo. A tailored Refeed® lipid supplement was developed with the aim of reducing the differences created by the in vitro cultivation and was tested on cultured hFM-MSCs. Cell morphology, viability, proliferation, angiogenic differentiation, and immunomodulatory properties after in vitro exposure to the tailored Refeed® lipid supplement were investigated. A significant modification of hFM-MSC membrane fatty acid composition occurred during in vitro culture. Using a tailored lipid supplement, the fatty acid composition of cultured cells remained more similar to their in vivo counterparts, being characterized by a higher polyunsaturated and omega-6 fatty acid content. These changes in membrane composition had no effect on cell morphology and viability, but were linked with increased cell proliferation rate, angiogenic differentiation, and immunomodulatory properties. In particular, Refeed®-supplemented hFM-MSCs showed greater ability to express fully functional cell membrane molecules. Culturing hFM-MSCs alters their

Two-layer membranes of calcium phosphate/collagen/PLGA nanofibres: in vitro biomineralisation and osteogenic differentiation of human mesenchymal stem cells

Science.gov (United States)

Hild, Nora; Schneider, Oliver D.; Mohn, Dirk; Luechinger, Norman A.; Koehler, Fabian M.; Hofmann, Sandra; Vetsch, Jolanda R.; Thimm, Benjamin W.; Müller, Ralph; Stark, Wendelin J.

2011-02-01

The present study evaluates the in vitro biomedical performance of an electrospun, flexible, anisotropic bilayer with one layer containing a collagen to mineral ratio similar to that in bone. The double membrane consists of a poly(lactide-co-glycolide) (PLGA) layer and an amorphous calcium phosphate (a-CaP)/collagen (Col)/PLGA layer. In vitro biomineralisation and a cell culture study with human mesenchymal stem cells (hMSC) were conducted to characterise such membranes for possible application as biomaterials. Nanofibres with different a-CaP/Col/PLGA compositions were synthesised by electrospinning to mimic the actual composition of bone tissue. Immersion in simulated body fluid and in cell culture medium resulted in the deposition of a hydroxyapatite layer. Incubation of hMSC for 4 weeks allowed for assessment of the proliferation and osteogenic differentiation of the cells on both sides of the double membrane. Confocal laser scanning microscopy was used to observe the proper adhesion of the cells. Calcium and collagen content was proven by Alizarin red S and Sirius red assays. Acute cytotoxic effects of the nanoparticles or the chemicals used in the scaffold preparation could be excluded based on viability assays (alamarBlue and alkaline phosphatase activity). The findings suggest possible application of such double membranes is in treatment of bone defects with complex geometries as wound dressing material.The present study evaluates the in vitro biomedical performance of an electrospun, flexible, anisotropic bilayer with one layer containing a collagen to mineral ratio similar to that in bone. The double membrane consists of a poly(lactide-co-glycolide) (PLGA) layer and an amorphous calcium phosphate (a-CaP)/collagen (Col)/PLGA layer. In vitro biomineralisation and a cell culture study with human mesenchymal stem cells (hMSC) were conducted to characterise such membranes for possible application as biomaterials. Nanofibres with different a

Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane.

Science.gov (United States)

Groffen, A J; Ruegg, M A; Dijkman, H; van de Velden, T J; Buskens, C A; van den Born, J; Assmann, K J; Monnens, L A; Veerkamp, J H; van den Heuvel, L P

1998-01-01

Agrin is a heparan sulfate proteoglycan (HSPG) that is highly concentrated in the synaptic basal lamina at the neuromuscular junction (NMJ). Agrin-like immunoreactivity is also detected outside the NMJ. Here we show that agrin is a major HSPG component of the human glomerular basement membrane (GBM). This is in addition to perlecan, a previously characterized HSPG of basement membranes. Antibodies against agrin and against an unidentified GBM HSPG produced a strong staining of the GBM and the NMJ, different from that observed with anti-perlecan antibodies. In addition, anti-agrin antisera recognized purified GBM HSPG and competed with an anti-GBM HSPG monoclonal antibody in ELISA. Furthermore, both antibodies recognized a molecule that migrated in SDS-PAGE as a smear and had a molecular mass of approximately 200-210 kD after deglycosylation. In immunoelectron microscopy, agrin showed a linear distribution along the GBM and was present throughout the width of the GBM. This was again different from perlecan, which was exclusively present on the endothelial side of the GBM and was distributed in a nonlinear manner. Quantitative ELISA showed that, compared with perlecan, the agrin-like GBM HSPG showed a sixfold higher molarity in crude glomerular extract. These results show that agrin is a major component of the GBM, indicating that it may play a role in renal ultrafiltration and cell matrix interaction. (J Histochem Cytochem 46:19-27, 1998)

Airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors

Energy Technology Data Exchange (ETDEWEB)

Li, E L.F.; Perdue, J F [Lady Davis Institute, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec, Canada

1980-10-01

A method is described in which high-speed centrifugation of membranes through an oil phase is used to separate membrane-bound and detergent-solubilized polypeptide receptor-iodinated ligand complexes from unbound ligands. Three centrifuges, the Brinkmann Eppendorf (5412), the Beckman Microfuge B and the Beckman Airfuge were evaluated for this capability. Under the conditions described, the Beckman Airfuge surpassed the others in recovering previously /sup 125/I- and /sup 32/P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific (/sup 125/I)insulin and (/sup 125/I)thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, and found to be 4 to 5 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 ..mu..g of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating /sup 125/I-labelled ligand-soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure.

Radiation-induced structural changes in membrane proteins of human erythrocytes and ghosts and the relation to cellular morphology

Energy Technology Data Exchange (ETDEWEB)

Schuurhuis, G.J.; Hommes, J.; Vos, J.; Molenaar, I.; Konings, A.W.T. (Rijksuniversiteit Groningen (Netherlands))

1984-02-01

Isolated human erythrocytes and ghosts were irradiated with X-rays under different experimental conditions and the effect examined with regard to the structure of membrane proteins and morphology of whole cells and ghosts. From sodium dodecyl sulphate/polyacrylamide gel electrophoresis it is concluded that spectrin (band 1 and 2) is the most radiosensitive of the membrane proteins examined. X-irradiation of cells and ghosts induced covalent cross-linking of a small fraction of membrane proteins. In the protein aggregates thus formed spectrin was found to be the major component. Molecular disulphide (-SS-) bridges seemed to account for part of the cross-links observed. Some nondisulphide cross-links were found, especially when ghosts were irradiated. Significant amounts of spectrin aggregates were formed during post-irradiation incubation at 37/sup 0/C but not at 4/sup 0/C. In the intact cell a transformation in shape from discocyte to echinocyte accompanied the process of post-irradiation spectrin aggregation. The characteristics of both processes, such as their reversibility with adenosine, point to a metabolic involvement. It is shown that there is no causal relationship between the two phenomena observed. Possible causes of the post-irradiation effects and the parallelism with similar processes in non-irradiated metabolically depleted cells are discussed.

Radiation-induced structural changes in membrane proteins of human erythrocytes and ghosts and the relation to cellular morphology

International Nuclear Information System (INIS)

Schuurhuis, G.J.; Hommes, J.; Vos, J.; Molenaar, I.; Konings, A.W.T.

1984-01-01

Isolated human erythrocytes and ghosts were irradiated with X-rays under different experimental conditions and the effect examined with regard to the structure of membrane proteins and morphology of whole cells and ghosts. From sodium dodecyl sulphate/polyacrylamide gel electrophoresis it is concluded that spectrin (band 1 and 2) is the most radiosensitive of the membrane proteins examined. X-irradiation of cells and ghosts induced covalent cross-linking of a small fraction of membrane proteins. In the protein aggregates thus formed spectrin was found to be the major component. Molecular disulphide (-SS-) bridges seemed to account for part of the cross-links observed. Some nondisulphide cross-links were found, especially when ghosts were irradiated. Significant amounts of spectrin aggregates were formed during post-irradiation incubation at 37 0 C but not at 4 0 C. In the intact cell a transformation in shape from discocyte to echinocyte accompanied the process of post-irradiation spectrin aggregation. The characteristics of both processes, such as their reversibility with adenosine, point to a metabolic involvement. It is shown that there is no causal relationship between the two phenomena observed. Possible causes of the post-irradiation effects and the parallelism with similar processes in non-irradiated metabolically depleted cells are discussed. (author)

Effect of membrane protein concentration on binding of 3H-imipramine in human platelets

International Nuclear Information System (INIS)

Barkai, A.I.; Kowalik, S.; Baron, M.

1985-01-01

Binding of 3 H-imipramine to platelet membranes has been implicated as a marker for depression. Comparing 3 H-IMI binding between depressed patients and normal subjects we observed an increase in the dissociation constant Kd with increasing membrane protein. This phenomenon was studied more rigorously in five normal subjects. Platelet membranes were prepared and adjusted to four concentrations of protein ranging from 100 to 800 micrograms/ml. The 3 H-IMI binding parameters of maximum binding sites number (Bmax) and Kd were obtained by Scatchard analysis at each membrane concentration. A positive linear relationship was found between K/sub d/ values and the concentration of membrane protein in the assay, but no change was observed in Bmax. The variability in Kd values reported in the literature may be accounted for in part by the different concentrations of membrane protein used in various studies

Radiosterilization of freeze-dried human amniotic Membrane and its use in the treatment of burn wound. Algerian experience

International Nuclear Information System (INIS)

Djefal, A.; Mahlous, M.; Nacer Khodja, A.; Larbi, M.; Larbi Daho Bachir, M.

2001-01-01

The present study evaluates the usefulness of human amniotic membrane as biological dressing and its efficacy in the treatment of burns comparatively to the conventional dressing. We reported the practical methods of preparation, preservation and radiation sterilisation of amnion, and the clinical results of its successful use in the treatment of 80 cases of superficial and intermediate depth dermal burns. The increased rate of healing, pain relief, good adhesion to the bed wound and absence of infection were observed

Effects of Three Kinds of Curcuminoids on Anti-Oxidative System and Membrane Deformation of Human Peripheral Blood Erythrocytes in High Glucose Levels

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Wei Yang

2015-01-01

Full Text Available Background/Aims: Curcuminoids are the main bioactive constituents of the rhizome of turmeric. Erythrocytes lesions in diabetes are probably related to hyperglycemia and protein glycation. It has been reported that curcumin prevent lipid peroxidation. However, reports on the effects of demethoxycurcumin and bis-demethoxycurcumin on human erythrocytes at high glucose levels are scarce. Our aim is to investigate the effect of curcuminoids on oxidative stress and membrane of erythrocytes exposed to hyperglycemic condition. Methods: In this study, the different blood samples were treated with two doses of glucose (10 or 30 mM to mimic hyperglycemia in the presence or absence of three kinds of curcuminoids (5 or 10 μM in a medium at 37 °C for 24 h (Each experiment consists of 20 blood samples from 10 male and 10 female volunteers. The malondialdehyde was checked by HPLC, antioxidase (GSH and GSSG were measured by LC/MS, SOD was checked by WST-1 kit, morphology and phospholipid symmetry were detected by flow cytometry, confocal scanning microscope and scanning electron microscope. Results: The results illustrated that all three curcuminoids reduce oxidative stress damage on the membrane and maintain a better profile for erythrocytes. Furthermore, three curcuminoids had benefit effects on antioxidase. Conclusion: The three kinds of curcuminoids supplementation may prevent lipid peroxidation at different intensity and membrane dysfunction of human erythrocytes in hyperglycemia.

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...... (FMP) assay. The K(m) and K(i) values obtained for 12 standard EAAT ligands at EAAT1, EAAT2 and EAAT3 in the FMP assay correlated well with the K(i) values obtained in the [(3) H]-d-aspartate assay (r(2) values of 0.92, 0.92, and 0.95, respectively). Furthermore, the pharmacological characteristics...

Caveolae as plasma membrane sensors, protectors and organizers.

Science.gov (United States)

Parton, Robert G; del Pozo, Miguel A

2013-02-01

Caveolae are submicroscopic, plasma membrane pits that are abundant in many mammalian cell types. The past few years have seen a quantum leap in our understanding of the formation, dynamics and functions of these enigmatic structures. Caveolae have now emerged as vital plasma membrane sensors that can respond to plasma membrane stresses and remodel the extracellular environment. Caveolae at the plasma membrane can be removed by endocytosis to regulate their surface density or can be disassembled and their structural components degraded. Coat proteins, called cavins, work together with caveolins to regulate the formation of caveolae but also have the potential to dynamically transmit signals that originate in caveolae to various cellular destinations. The importance of caveolae as protective elements in the plasma membrane, and as membrane organizers and sensors, is highlighted by links between caveolae dysfunction and human diseases, including muscular dystrophies and cancer.

Enzymatic Oxidation of Cholesterol: Properties and Functional Effects of Cholestenone in Cell Membranes

DEFF Research Database (Denmark)

Neuvonen, M.; Manna, M.; Mokkila, S.

2014-01-01

of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human...... fibroblasts, cholestenone was released from membranes to physiological extracellular acceptors more avidly than cholesterol, but without acceptors it remained in cells over a day. To address the functional effects of cholestenone, we studied fibroblast migration during wound healing. When cells were either...... similarly to control cells. Thus, cholesterol oxidation produces long-term functional effects in cells and these are in part due to the generated membrane active cholestenone....

The effect of membrane diffusion potential change on anionic drugs ...

African Journals Online (AJOL)

The effect of membrane potential change on anionic drugs Indomethacin and barbitone induced human erythrocyte shape change and red cell uptake of drug has been studied using microscopy and spectrophotometry techniques respectively. The membrane potential was changed by reducing the extracellular chloride ...

Pediatric medical device development by surgeons via capstone engineering design programs.

Science.gov (United States)

Sack, Bryan S; Elizondo, Rodolfo A; Huang, Gene O; Janzen, Nicolette; Espinoza, Jimmy; Sanz-Cortes, Magdalena; Dietrich, Jennifer E; Hakim, Julie; Richardson, Eric S; Oden, Maria; Hanks, John; Haridas, Balakrishna; Hury, James F; Koh, Chester J

2018-03-01

There is a need for pediatric medical devices that accommodate the unique physiology and anatomy of pediatric patients that is increasingly receiving more attention. However, there is limited literature on the programs within children's hospitals and academia that can support pediatric device development. We describe our experience with pediatric device design utilizing collaborations between a children's hospital and two engineering schools. Utilizing the academic year as a timeline, unmet pediatric device needs were identified by surgical faculty and matched with an engineering mentor and a team of students within the Capstone Engineering Design programs at two universities. The final prototypes were showcased at the end of the academic year and if appropriate, provisional patent applications were filed. All twelve teams successfully developed device prototypes, and five teams obtained provisional patents. The prototypes that obtained provisional patents included a non-operative ureteral stent removal system, an evacuation device for small kidney stone fragments, a mechanical leech, an anchoring system of the chorio-amniotic membranes during fetal surgery, and a fetal oxygenation monitor during fetoscopic procedures. Capstone Engineering Design programs in partnership with surgical faculty at children's hospitals can play an effective role in the prototype development of novel pediatric medical devices. N/A - No clinical subjects or human testing was performed. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of neurotensin-related peptides in human thymic epithelial cell membranes and relationship with major histocompatibility complex class I molecules.

Science.gov (United States)

Vanneste, Y; Thome, A N; Vandersmissen, E; Charlet, C; Franchimont, D; Martens, H; Lhiaubet, A M; Schimpff, R M; Rostène, W; Geenen, V

1997-06-01

This study shows the expression at the cell surface of human thymic epithelial cells (TEC) of a neurotensin (NT)-like immunoreactivity. NT radio-immunoassay (RIA) revealed that cultured human TEC contain +/-5 ng immunoreactive (ir) NT/10(6) cells, of which 5% is associated with plasma cell membranes. HPLC analysis of NT-ir present in human TEC showed a major peak of NT-ir corresponding to NT1-13. NT-ir was not detected in the supernatant of human TEC cultures. Using an affinity column prepared with a anti-MHC class I monoclonal antibody, NT-ir-related peptides were retained on the column and eluted together with MHC class I-related proteins. According to the elution time on HPLC of these peptides, they correspond to intact NT1-13, as well as to smaller fragments of NT1-13.

OCTN3 is a mammalian peroxisomal membrane carnitine transporter

International Nuclear Information System (INIS)

Lamhonwah, Anne-Marie; Ackerley, Cameron A.; Tilups, Aina; Edwards, Vernon D.; Wanders, Ronald J.; Tein, Ingrid

2005-01-01

Carnitine is a zwitterion essential for the β-oxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes. We now demonstrate expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (K m 20 μM), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrate expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast with two peroxisomal biogenesis disorders, we show reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane. Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for the intermediate-affinity carnitine/organic cation transporter, OCTN3, on peroxisomal membranes now shown to be expressed in both human and murine peroxisomes. This Octn3 localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism

Testing of Synthetic Biological Membranes for Forward Osmosis Applications

Science.gov (United States)

Parodi, Jurek; Mangado, Jaione Romero; Stefanson, Ofir; Flynn, Michael; Mancinelli, Rocco; Kawashima, Brian; Trieu, Serena; Brozell, Adrian; Rosenberg, Kevan

2016-01-01

Commercially available forward osmosis membranes have been extensively tested for human space flight wastewater treatment. Despite the improvements achieved in the last decades, there is still a challenge to produce reliable membranes with anti-fouling properties, chemical resistance, and high flux and selectivity. Synthetic biological membranes that mimic the ones present in nature, which underwent millions of years of evolution, represent a potential solution for further development and progress in membrane technology. Biomimetic forward osmosis membranes based on a polymeric support filter and coated with surfactant multilayers have been engineered to investigate how different manufacturing processes impact the performance and structure of the membrane. However, initial results of the first generation prototype membranes tests reveal a high scatter in the data, due to the current testing apparatus set up. The testing apparatus has been upgraded to improve data collection, reduce errors, and to allow higher control of the testing process.

Structure of the human vitreoretinal border region

DEFF Research Database (Denmark)

Heegaard, Steffen

1994-01-01

Øjenpatologi, vitreoretinal border region, inner limiting membrane, retina, topographical variation, human......Øjenpatologi, vitreoretinal border region, inner limiting membrane, retina, topographical variation, human...

Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins

DEFF Research Database (Denmark)

Elortza, Felix; Nühse, Thomas S; Foster, Leonard J

2003-01-01

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains...... unclear, they have attracted attention because they act as enzymes and receptors in cell adhesion, differentiation, and host-pathogen interactions. GPI-APs may represent potential diagnostic and therapeutic targets in humans and are interesting in plant biotechnology because of their key role in root...... and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date....

The human skin/chick chorioallantoic membrane model accurately predicts the potency of cosmetic allergens.

Science.gov (United States)

Slodownik, Dan; Grinberg, Igor; Spira, Ram M; Skornik, Yehuda; Goldstein, Ronald S

2009-04-01

The current standard method for predicting contact allergenicity is the murine local lymph node assay (LLNA). Public objection to the use of animals in testing of cosmetics makes the development of a system that does not use sentient animals highly desirable. The chorioallantoic membrane (CAM) of the chick egg has been extensively used for the growth of normal and transformed mammalian tissues. The CAM is not innervated, and embryos are sacrificed before the development of pain perception. The aim of this study was to determine whether the sensitization phase of contact dermatitis to known cosmetic allergens can be quantified using CAM-engrafted human skin and how these results compare with published EC3 data obtained with the LLNA. We studied six common molecules used in allergen testing and quantified migration of epidermal Langerhans cells (LC) as a measure of their allergic potency. All agents with known allergic potential induced statistically significant migration of LC. The data obtained correlated well with published data for these allergens generated using the LLNA test. The human-skin CAM model therefore has great potential as an inexpensive, non-radioactive, in vivo alternative to the LLNA, which does not require the use of sentient animals. In addition, this system has the advantage of testing the allergic response of human, rather than animal skin.

Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane.

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Leilismara Sousa

Full Text Available Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1, iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 μM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5% than in women and was associated with an increase (446% in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS and an increase (327% in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 μM FeCl3 for 24 h showed an increase (132% in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels.

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.

The Effects of Lipid Membranes, Crowding and Osmolytes on the Aggregation, and Fibrillation Propensity of Human IAPP

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Mimi Gao

2015-01-01

Full Text Available Type 2 diabetes mellitus (T2DM is an age-related and metabolic disease. Its development is hallmarked, among others, by the dysfunction and degeneration of β-cells of the pancreatic islets of Langerhans. The major pathological characteristic thereby is the formation of extracellular amyloid deposits consisting of the islet amyloid polypeptide (IAPP. The process of human IAPP (hIAPP self-association, and the intermediate structures formed as well as the interaction of hIAPP with membrane systems seem to be, at least to a major extent, responsible for the cytotoxicity. Here we present a summary and comparison of the amyloidogenic propensities of hIAPP in bulk solution and in the presence of various neutral and charged lipid bilayer systems as well as biological membranes. We also discuss the cellular effects of macromolecular crowding and osmolytes on the aggregation pathway of hIAPP. Understanding the influence of different cellular factors on hIAPP aggregation will provide more insight into the onset of T2DM and help to develop novel therapeutic strategies.

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.

Human Lactoferricin Is Partially Folded in Aqueous Solution and Is Better Stabilized in a Membrane Mimetic Solvent

Science.gov (United States)

Hunter, Howard N.; Demcoe, A. Ross; Jenssen, Håvard; Gutteberg, Tore J.; Vogel, Hans J.

2005-01-01

Lactoferricins are highly basic bioactive peptides that are released in the stomach through proteolytic cleavage of various lactoferrin proteins. Here we have determined the solution structure of human lactoferricin (LfcinH) by conventional two-dimensional nuclear magnetic resonance methods in both aqueous solution and a membrane mimetic solvent. Unlike the 25-residue bovine lactoferricin (LfcinB), which adopts a somewhat distorted antiparallel β sheet, the longer LfcinH peptide shows a helical content from Gln14 to Lys29 in the membrane mimetic solvent but a nonexistent β-sheet character in either the N- or C-terminal regions of the peptide. The helical characteristic of the LfcinH peptide resembles the conformation that this region adopts in the crystal structure of the intact protein. The LfcinH structure determined in aqueous solution displays a nascent helix in the form of a coiled conformation in the region from Gln14 to Lys29. Numerous hydrophobic interactions create the basis for the better-defined overall structure observed in the membrane mimetic solvent. The 49-residue LfcinH peptide isolated for these studies was found to be slightly longer than previously reported peptide preparations and was found to have an intact peptide bond between residues Ala11 and Val12. The distinct solution structures of LfcinH and LfcinB represent a novel difference in the physical properties of these two peptides, which contributes to their unique physiological activities. PMID:16048952

Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

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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.

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.

The human adenovirus E4-ORF1 protein subverts discs large 1 to mediate membrane recruitment and dysregulation of phosphatidylinositol 3-kinase.

Directory of Open Access Journals (Sweden)

Kathleen Kong

2014-05-01

Full Text Available Adenoviruses infect epithelial cells lining mucous membranes to cause acute diseases in people. They are also utilized as vectors for vaccination and for gene and cancer therapy, as well as tools to discover mechanisms of cancer due to their tumorigenic potential in experimental animals. The adenovirus E4-ORF1 gene encodes an oncoprotein that promotes viral replication, cell survival, and transformation by activating phosphatidylinositol 3-kinase (PI3K. While the mechanism of activation is not understood, this function depends on a complex formed between E4-ORF1 and the membrane-associated cellular PDZ protein Discs Large 1 (Dlg1, a common viral target having both tumor suppressor and oncogenic functions. Here, we report that in human epithelial cells, E4-ORF1 interacts with the regulatory and catalytic subunits of PI3K and elevates their levels. Like PI3K activation, PI3K protein elevation by E4-ORF1 requires Dlg1. We further show that Dlg1, E4-ORF1, and PI3K form a ternary complex at the plasma membrane. At this site, Dlg1 also co-localizes with the activated PI3K effector protein Akt, indicating that the ternary complex mediates PI3K signaling. Signifying the functional importance of the ternary complex, the capacity of E4-ORF1 to induce soft agar growth and focus formation in cells is ablated either by a mutation that prevents E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug. These results demonstrate that E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1 hijacks the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for constitutive activation. This novel mechanism of Dlg1 subversion by adenovirus to dysregulate PI3K could be used by other pathogenic viruses, such as human papillomavirus, human T-cell leukemia virus type 1, and influenza A virus, which also target Dlg1 and activate PI3K in cells.

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.

Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment.

Science.gov (United States)

Yu, Yuanjie; Böing, Anita N; Hau, Chi M; Hajji, Najat; Ruf, Wolfram; Sturk, Auguste; Nieuwland, Rienk

2018-06-01

 Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored.  This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains.  Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined.  Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density ( ρ ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI.  Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity. Schattauer GmbH Stuttgart.

NMR studies of human blood cells in health and disease. I. Alterations of the plasma membrane water permeability of erythrocytes

International Nuclear Information System (INIS)

Katona, Eva; Doaga, I. O.; Radulet, Diana; Caplanusi, A.; Negreanu, Cezarina; Mihele, Denisa

1999-01-01

Alterations in pathological cases of the human erythrocyte membrane water permeability were investigated by using a Mn 2+ -doping 1 H nuclear magnetic resonance (NMR) technique. The temperature dependence of the apparent water diffusional exchange through erythrocyte membranes in chronic hepatitis, diabetes, dyslipidemia and essential hypertension was measured and compared to healthy controls. Using moderate manganese concentrations (9-18 mM) and Carr-Purcell-Meiboom-Gill pulse sequences with a large number of refocusing π pulses and short interpulse delay (100 μs) our values of the water exchange times (τ e ) across erythrocyte membranes, obtained within a 10 min time period following the moment of doping, were independent of the actual manganese concentration and the Arrhenius plot for water exchange was linear over the range of 22-42 deg C. A marked increase of the water exchange times values was observed in all studied disease states. In case of chronic hepatitis, diabetes and dyslipidemia the changes observed in transmembrane water exchange time were associated with significant increase in the apparent activation energy of the diffusional water permeability thus, pointing out alterations in the function of the erythrocyte water channel. (author)

NMR studies of human blood cells in health and disease. I. Alterations of the plasma membrane water permeability of erythrocytes

Energy Technology Data Exchange (ETDEWEB)

Katona, Eva; Doaga, I O; Radulet, Diana [Department of Biophysics, Carol Davila University of Medicine and Pharmaceutics, 8 Blvd. Eroilor Sanitari, POB 15-205, RO-76241 Bucharest (Romania); Caplanusi, A [Medical Biochemistry Department, Carol Davila University of Medicine and Pharmaceutics, 8 Blvd. Eroilor Sanitari, POB 15-205, RO-76241 Bucharest (Romania); Negreanu, Cezarina [Division of New Energy Conversion Methods, Institute of Research and Design for Thermoenergetic Equipment, ICPET-CERCETARE, Bucharest (Romania); Mihele, Denisa [Clinical Laboratory Department, Carol Davila University of Medicine and Pharmaceutics, 8 Blvd. Eroilor Sanitari, POB 15-205, RO-76241 Bucharest (Romania)

1999-07-01

Alterations in pathological cases of the human erythrocyte membrane water permeability were investigated by using a Mn{sup 2+}-doping {sup 1}H nuclear magnetic resonance (NMR) technique. The temperature dependence of the apparent water diffusional exchange through erythrocyte membranes in chronic hepatitis, diabetes, dyslipidemia and essential hypertension was measured and compared to healthy controls. Using moderate manganese concentrations (9-18 mM) and Carr-Purcell-Meiboom-Gill pulse sequences with a large number of refocusing {pi} pulses and short interpulse delay (100 {mu}s) our values of the water exchange times ({tau}{sub e}) across erythrocyte membranes, obtained within a 10 min time period following the moment of doping, were independent of the actual manganese concentration and the Arrhenius plot for water exchange was linear over the range of 22-42 deg C. A marked increase of the water exchange times values was observed in all studied disease states. In case of chronic hepatitis, diabetes and dyslipidemia the changes observed in transmembrane water exchange time were associated with significant increase in the apparent activation energy of the diffusional water permeability thus, pointing out alterations in the function of the erythrocyte water channel. (author)

Fabrication of Greener Membranes from Ionic Liquid Solutions

KAUST Repository

Kim, DooLi

2017-06-01

Membrane technology plays a crucial role in different separation processes such as biotechnology, pharmaceutical, and food industries, drinking water supply, and wastewater treatment. However, there is a growing concern that solvents commonly used for membrane fabrication, such as dimethylformamide (DMF), dimethylacetamide (DMAc), and 1-methyl-2-pyrrolidone (NMP), are toxic to the environment and human health. To explore the possibility of substituting these toxic solvents by less toxic or safer solvents, polymers commonly used for membrane fabrication, such as polyacrylonitrile (PAN), cellulose acetate (CA), polyethersulfone (PES), and poly(ether imide sulfone) (EXTEMTM), were dissolved in ionic liquids. Flat sheet and hollow fiber membranes were then fabricated. The thermodynamics of the polymer solutions, the kinetics of phase inversion and other factors, which resulted in significant differences in the membrane structure, compared to those of membranes fabricated from more toxic solvents, were investigated. Higher water permeance with smaller pores, unique and uniform morphologies, and narrower pore size distribution, were observed in the ionic liquid-based membranes. Furthermore, comparable performance on separation of peptides and proteins with various molecular weights was achieved with the membranes fabricated from ionic liquid solutions. In summary, we propose less hazardous polymer solutions to the environment, which can be used for the membrane fabrication with better performance and more regular morphology.

Membranes, methods of making membranes, and methods of separating gases using membranes

Science.gov (United States)

Ho, W. S. Winston

2012-10-02

Membranes, methods of making membranes, and methods of separating gases using membranes are provided. The membranes can include at least one hydrophilic polymer, at least one cross-linking agent, at least one base, and at least one amino compound. The methods of separating gases using membranes can include contacting a gas stream containing at least one of CO.sub.2, H.sub.2S, and HCl with one side of a nonporous and at least one of CO.sub.2, H.sub.2S, and HCl selectively permeable membrane such that at least one of CO.sub.2, H.sub.2S, and HCl is selectively transported through the membrane.

N-3 fatty acids and membrane microdomains: from model membranes to lymphocyte function.

Science.gov (United States)

Shaikh, Saame Raza; Teague, Heather

2012-12-01

This article summarizes the author's research on fish oil derived n-3 fatty acids, plasma membrane organization and B cell function. We first cover basic model membrane studies that investigated how docosahexaenoic acid (DHA) targeted the organization of sphingolipid-cholesterol enriched lipid microdomains. A key finding here was that DHA had a relatively poor affinity for cholesterol. This work led to a model that predicted DHA acyl chains in cells would manipulate lipid-protein microdomain organization and thereby function. We then review how the predictions of the model were tested with B cells in vitro followed by experiments using mice fed fish oil. These studies reveal a highly complex picture on how n-3 fatty acids target lipid-protein organization and B cell function. Key findings are as follows: (1) n-3 fatty acids target not just the plasma membrane but also endomembrane organization; (2) DHA, but not eicosapentaenoic acid (EPA), disrupts microdomain spatial distribution (i.e. clustering), (3) DHA alters protein lateral organization and (4) changes in membrane organization are accompanied by functional effects on both innate and adaptive B cell function. Altogether, the research over the past 10 years has led to an evolution of the original model on how DHA reorganizes membrane microdomains. The work raises the intriguing possibility of testing the model at the human level to target health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization of the 3,3',5-triiodo-L-thyronine-binding site on plasma membranes from human placenta

International Nuclear Information System (INIS)

Alderson, R.; Pastan, I.; Cheng, S.

1985-01-01

The binding of [ 125 I]T3 to sites on human placenta plasma membranes was characterized, and the binding site was solubilized after affinity labeling with N-bromoacetyl-[ 125 I]T3 (BrAc[ 125 I]T3). Two classes of T3-binding sites were detected. One class has a high affinity (K /sub d/ = 2.0nM) and a low capacity (approximately 320 fmol/mg protein); the other has a low affinity (K /sub k/ = 18.5 microM) and a high capacity (approximately 2.2 pmol/mg protein). The binding sites were found to be specific for T3 in that other thyroid hormone analogs (D-T3, rT3, D-T4, and L-T4) were less effective or ineffective in displacing the bound [ 125 I]T3. The affinity labeling ligand BrAc[ 125 I]T3 was found to specifically label a protein with an apparent mol wt of 65,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The BrAc[ 125 I]T3-labeled protein was solubilized with 2 mM 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate. The apparent mol wt of the labeled protein was between 140,000 and 150,000 by Sephadex-G-200 gel filtration. These data demonstrate that a high affinity binding site specific for T3 is present on plasma membranes from human placenta and that the binding site is a protein, most likely a dimer, with a native mol wt between 140,000 and 150,000

Cryopreserved Ultra-Thick Human Amniotic Membrane for Conjunctival Surface Reconstruction After Excision of Conjunctival Tumors.

Science.gov (United States)

Tanaka, Thais S; Demirci, Hakan

2016-04-01

Cryopreserved ultra-thick human amniotic membrane (AM) is used for glaucoma surgery. We evaluated the use of cryopreserved ultra-thick human AM for conjunctival surface reconstruction after excision of a conjunctival tumor. We retrospectively reviewed the medical records of 28 patients who underwent conjunctival surface reconstruction with cryopreserved ultra-thick human AM after excision of the tumor. The AM was secured to the surrounding conjunctiva and underlying sclera with interrupted 8-0 Vicryl sutures. Clinical data regarding demographics, diagnosis, size and location of conjunctival tumors, patient outcome, and complications were gathered. Of 28 patients, 6 (21.4%) had malignant melanoma, 4 (14.3%) had squamous cell carcinoma, 6 (21.4%) had conjunctival intraepithelial neoplasia, 1 (3.6%) had sebaceous carcinoma, 1 (3.6%) had mucoepidermoid carcinoma, 1 (3.6%) had conjunctival intraepithelial dysplasia, 5 (17.9%) had pterygium, 2 (7.1%) had compound nevus, 1 (3.6%) had a large epithelial inclusion cyst, and 1 (3.6%) patient had a granuloma. The mean area of graft size was 156 ± 120 mm2. Postoperatively, the graft was well tolerated with no failure, discomfort, or dehiscence. During the 17-month mean follow-up, symblepharon, which was clinically nonsignificant, developed in 3 (11%) patients and partial stem cell deficiency was noted in 5 (18%) patients. Cryopreserved ultra-thick human AM is a well-tolerated, effective graft material that is easy to handle. It is a viable alternative for conjunctival surface reconstruction after excision of a conjunctival tumor.

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

Science.gov (United States)

Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

2015-09-01

Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American

Application of dynamic membranes in anaerobic membranes in anaerobic membrane bioreactor systems

NARCIS (Netherlands)

Erşahin, M.E.

2015-01-01

Anaerobic membrane bioreactors (AnMBRs) physically ensure biomass retention by the application of a membrane filtration process. With growing application experiences from aerobic membrane bioreactors (MBRs), the combination of membrane and anaerobic processes has received much attention and become

Niemann-Pick C2 protein regulates sterol transport between plasma membrane and late endosomes in human fibroblasts

DEFF Research Database (Denmark)

Berzina, Zane; Solanko, Lukasz M; Mehadi, Ahmed S

2018-01-01

/LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous...... but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM...

Isolation of plasma membranes from cultured glioma cells and application to evaluation of membrane sphingomyelin turnover

International Nuclear Information System (INIS)

Cook, H.W.; Palmer, F.B.; Byers, D.M.; Spence, M.W.

1988-01-01

A rapid and reliable method for the isolation of plasma membranes and microsomes of high purity and yield from cultured glioma cells is described. The procedure involves disruption by N2 cavitation, preliminary separation by centrifugation in Tricine buffer, and final separation on a gradient formed from 40% Percoll at pH 9.3. Enzyme and chemical markers indicated greater than 60% yield with six- to eightfold enrichment for plasma membranes and greater than 25% yield with three- to fourfold enrichment for a microsomal fraction consisting mainly of endoplasmic reticulum. The final fractions were obtained with high reproducibility in less than 1 h from the time of cell harvesting. Application of this procedure to human fibroblasts in culture is assessed. The isolation procedure was applied to investigations of synthesis and turnover of sphingomyelin and phosphatidylcholine in plasma membranes of glioma cells following incubation for 4-24 h with [methyl- 3 H]choline. These studies indicated that radioactivity from phosphatidylcholine synthesized in microsomes from exogenous choline may serve as a precursor of the head-group of sphingomyelin accumulating in the plasma membrane

The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3.

Science.gov (United States)

Sirena, Dominique; Lilienfeld, Benjamin; Eisenhut, Markus; Kälin, Stefan; Boucke, Karin; Beerli, Roger R; Vogt, Lorenz; Ruedl, Christiane; Bachmann, Martin F; Greber, Urs F; Hemmi, Silvio

2004-05-01

Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.

Effect of gamma irradiation on membranes of normal and pathological erythrocytes (beta-thalassemia)

International Nuclear Information System (INIS)

Sportelli, L.; Bonincontro, A.; Cametti, C.; Consiglio Nazionale delle Ricerche, Rome

1987-01-01

The influence of ionizing radiation on the membrane of human normal erythrocytes has extensively been studied and a variety of effects including changes in the cation fluxes or in non-electrolytes permeability, in membrane fluidity, in peroxidation of unsaturated lipids as well as chemical composition or structural modifications has been observed. However, only few studies deal with the effects of ionizing radiation on pathological red blood cells. In this work, we have investigated by means of electron spin resonance (ESR) spectroscopy the effects of 60 Co γ-radiation on the normal and homozygous β-thalassemic human erythrocyte membranes. (orig.)

Membrane-Active Epithelial Keratin 6A Fragments (KAMPs) Are Unique Human Antimicrobial Peptides with a Non-αβ Structure

Science.gov (United States)

Lee, Judy T. Y.; Wang, Guangshun; Tam, Yu Tong; Tam, Connie

2016-01-01

Antibiotic resistance is a pressing global health problem that threatens millions of lives each year. Natural antimicrobial peptides and their synthetic derivatives, including peptoids and peptidomimetics, are promising candidates as novel antibiotics. Recently, the C-terminal glycine-rich fragments of human epithelial keratin 6A were found to have bactericidal and cytoprotective activities. Here, we used an improved 2-dimensional NMR method coupled with a new protocol for structural refinement by low temperature simulated annealing to characterize the solution structure of these kerain-derived antimicrobial peptides (KAMPs). Two specific KAMPs in complex with membrane mimicking sodium dodecyl sulfate (SDS) micelles displayed amphipathic conformations with only local bends and turns, and a central 10-residue glycine-rich hydrophobic strip that is central to bactericidal activity. To our knowledge, this is the first report of non-αβ structure for human antimicrobial peptides. Direct observation of Staphylococcus aureus and Pseudomonas aeruginosa by scanning and transmission electron microscopy showed that KAMPs deformed bacterial cell envelopes and induced pore formation. Notably, in competitive binding experiments, KAMPs demonstrated binding affinities to LPS and LTA that did not correlate with their bactericidal activities, suggesting peptide-LPS and peptide-LTA interactions are less important in their mechanisms of action. Moreover, immunoprecipitation of KAMPs-bacterial factor complexes indicated that membrane surface lipoprotein SlyB and intracellular machineries NQR sodium pump and ribosomes are potential molecular targets for the peptides. Results of this study improve our understanding of the bactericidal function of epithelial cytokeratin fragments, and highlight an unexplored class of human antimicrobial peptides, which may serve as non-αβ peptide scaffolds for the design of novel peptide-based antibiotics. PMID:27891122

Membrane-Active Epithelial Keratin 6A Fragments (KAMPs Are Unique Human Antimicrobial Peptides with a Non-αβ Structure

Directory of Open Access Journals (Sweden)

Judy Tsz Ying Lee

2016-11-01

Full Text Available Antibiotic resistance is a pressing global health problem that threatens millions of lives each year. Natural antimicrobial peptides and their synthetic derivatives, including peptoids and peptidomimetics, are promising candidates as novel antibiotics. Recently, the C-terminal glycine-rich fragments of human epithelial keratin 6A were found to have bactericidal and cytoprotective activities. Here, we used an improved 2-dimensional NMR method coupled with a new protocol for structural refinement by low temperature simulated annealing to characterize the solution structure of these kerain-derived antimicrobial peptides (KAMPs. Two specific KAMPs in complex with membrane mimicking sodium dodecyl sulfate (SDS micelles displayed amphipathic conformations with only local bends and turns, and a central 10-residue glycine-rich hydrophobic strip that is central to bactericidal activity. To our knowledge, this is the first report of non-αβ structure for human antimicrobial peptides. Direct observation of Staphylococcus aureus and Pseudomonas aeruginosa by scanning and transmission electron microscopy showed that KAMPs deformed bacterial cell envelopes and induced pore formation. Notably, in competitive binding experiments, KAMPs demonstrated binding affinities to LPS and LTA that did not correlate with their bactericidal activities, suggesting peptide-LPS and peptide-LTA interactions are less important in their mechanisms of action. Moreover, immunoprecipitation of KAMPs-bacterial factor complexes indicated that membrane surface lipoprotein SlyB and intracellular machineries NQR sodium pump and ribosomes are potential molecular targets for the peptides. Results of this study improve our understanding of the bactericidal function of epithelial cytokeratin fragments, and highlight an unexplored class of human antimicrobial peptides, which may serve as non-αβ peptide scaffolds for the design of novel peptide-based antibiotics.

Identification of the glucose transporter in mammalian cell membranes using an 125(I)-forskolin photoaffinity label

International Nuclear Information System (INIS)

Ruoho, A.; Wadzinski, B.; Shanahan, M.

1987-01-01

The glucose transporter has been identified in a variety of mammlian cell membranes using a carrier-free photoactivatable radioiodinated derivative of forskolin, 3-iodo-4-azidophenethylamido-7-0-succinyldeacetyl-forskolin, [I-125]IAPS-Fsk, at 1-10 nM. The membranes which have been photolabeled with [I-125]IAPS-Fsk are: rat cardiac sarcolemmal membranes, rat cortex and cerebellum synaptic membranes, human placental membranes, and wild type S49 lymphoma cell membranes. The glucose transporter in rat cardiac sarcolemmal membranes and rat cortex and cerebellum synaptic membranes was determined to be 45 kDa by SDS-PAGE. Photolysis of human placental membranes and S49 lymphoma membranes with [I-125]IAPS-Fsk followed by SDS-PAGE indicated specific derivatization of a broad band (45-55 kDa) in placental membranes and a narrower band (45 kDa) in the S49 lymphoma membranes. Digestion of the [I-125]IPAS-Fsk labelled placental and S49 lymphoma membranes with endo-B-galactosidase showed a reduction in the apparent molecular weight of the radiolabelled band to 40 kDa. Trypsinization of labelled placental and lymphoma membranes produced an 18 kDa radiolabelled proteolytic fragment. [I-125]IAPS-Fsk is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues

Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

Science.gov (United States)

Faundez, Victor; Koval, Michael; Mattheyses, Alexa L.; Kowalczyk, Andrew P.

2014-01-01

Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV. PMID:24498201

Protein receptor-independent plasma membrane remodeling by HAMLET

DEFF Research Database (Denmark)

Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.

2015-01-01

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This "protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains...... in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range...... of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a "receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET...

Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5'-O-(3-thiotriphosphate)

International Nuclear Information System (INIS)

Baldassare, J.J.; Henderson, P.A.; Fisher, G.J.

1989-01-01

The effects of thrombin and GTPγS on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous [ 3 H]inositol-labeled membranes or with lipid vesicles containing either [ 3 H]phosphatidylinositol or [ 3 H]phosphatidylinositol 4,5-bisphosphate. GTPγS (1 μM) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP 3 ), inositol bisphosphate (IP 2 ), or inositol phosphate (IP) from [ 3 H]inositol-labeled membranes. IP 2 and IP 3 , but not IP, from [ 3 H]inositol-labeled membranes were, however, stimulated 3-fold by GTPγS (1 μM) plus thrombin (1 unit/mL). A higher concentration of GTPγS (100 μM) alone also stimulated IP 2 and IP 3 , but not IP, release. In the presence of 1 mM calcium, release of IP 2 and IP 3 was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by platelet membrane associated PLC was also markedly enhanced by GTPγS (100 μM) or GTPγS (1 μM) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP 2 was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTPγS (100 μM) or calcium (1 mM) dependent PIP 2 breakdown, while TPA inhibited GTPγS-dependent but not calcium-dependent phospholipase C activity

Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.

Science.gov (United States)

Ge, Yan; Yoshiie, Kiyotaka; Kuribayashi, Futoshi; Lin, Mingqun; Rikihisa, Yasuko

2005-01-01

The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. bfl-1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to approximately 5-25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl-1 mRNA levels by A. phagocytophilum. Differences in mcl-1, bax, bcl-w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10-12 h incubation, whereas A. phagocytophilum-infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner in A. phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest that A. phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl-1 and inhibition of mitochondria-mediated activation of caspase 3.

Changes in the Fatty Acid Profile and Phospholipid Molecular Species Composition of Human Erythrocyte Membranes after Hybrid Palm and Extra Virgin Olive Oil Supplementation.

Science.gov (United States)

Pacetti, D; Gagliardi, R; Balzano, M; Frega, N G; Ojeda, M L; Borrero, M; Ruiz, A; Lucci, P

2016-07-13

This work aims to evaluate and compare, for the first time, the effects of extra virgin olive oil (EVOO) and hybrid palm oil (HPO) supplementation on the fatty acid profile and phospholipid (PL) molecular species composition of human erythrocyte membranes. Results supported the effectiveness of both HPO and EVOO supplementation (3 months, 25 mL/day) in decreasing the lipophilic index of erythrocytes with no significant differences between HPO and EVOO groups at month 3. On the other hand, the novel and rapid ultraperformance liquid chromatography-tandem mass spectrometry method used for PL analysis reveals an increase in the levels of phosphatidylcholine and phosphatidylethanolamine species esterified with polyunsaturated fatty acids. This work demonstrates the ability of both EVOO and HPO to increase the degree of unsaturation of erythrocyte membrane lipids with an improvement in membrane fluidity that could be associated with a lower risk of developing cardiovascular diseases.

Smart membranes for monitoring membrane based desalination processes

KAUST Repository

Laleg-Kirati, Taous-Meriem

2017-10-12

Various examples are related to smart membranes for monitoring membrane based process such as, e.g., membrane distillation processes. In one example, a membrane, includes a porous surface and a plurality of sensors (e.g., temperature, flow and/or impedance sensors) mounted on the porous surface. In another example, a membrane distillation (MD) process includes the membrane. Processing circuitry can be configured to monitor outputs of the plurality of sensors. The monitored outputs can be used to determine membrane degradation, membrane fouling, or to provide an indication of membrane replacement or cleaning. The sensors can also provide temperatures or temperature differentials across the porous surface, which can be used to improve modeling or control the MD process.

H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen

International Nuclear Information System (INIS)

Collier, I.E.; Wilhelm, S.M.; Eisen, A.Z.

1988-01-01

H-ras transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on this ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors. Type IV collagenase consists of three domains. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin

GSL-enriched membrane microdomains in innate immune responses.

Science.gov (United States)

Nakayama, Hitoshi; Ogawa, Hideoki; Takamori, Kenji; Iwabuchi, Kazuhisa

2013-06-01

Many pathogens target glycosphingolipids (GSLs), which, together with cholesterol, GPI-anchored proteins, and various signaling molecules, cluster on host cell membranes to form GSL-enriched membrane microdomains (lipid rafts). These GSL-enriched membrane microdomains may therefore be involved in host-pathogen interactions. Innate immune responses are triggered by the association of pathogens with phagocytes, such as neutrophils, macrophages and dendritic cells. Phagocytes express a diverse array of pattern-recognition receptors (PRRs), which sense invading microorganisms and trigger pathogen-specific signaling. PRRs can recognize highly conserved pathogen-associated molecular patterns expressed on microorganisms. The GSL lactosylceramide (LacCer, CDw17), which binds to various microorganisms, including Candida albicans, is expressed predominantly on the plasma membranes of human mature neutrophils and forms membrane microdomains together with the Src family tyrosine kinase Lyn. These LacCer-enriched membrane microdomains can mediate superoxide generation, migration, and phagocytosis, indicating that LacCer functions as a PRR in innate immunity. Moreover, the interactions of GSL-enriched membrane microdomains with membrane proteins, such as growth factor receptors, are important in mediating the physiological properties of these proteins. Similarly, we recently found that interactions between LacCer-enriched membrane microdomains and CD11b/CD18 (Mac-1, CR3, or αMβ2-integrin) are significant for neutrophil phagocytosis of non-opsonized microorganisms. This review describes the functional role of LacCer-enriched membrane microdomains and their interactions with CD11b/CD18.

Recent advances on polymeric membranes for membrane reactors

KAUST Repository

Buonomenna, M. G.; Choi, Seung Hak

2012-01-01

. The successful use of membranes in membrane reactors is primary the result of two developments concerning: (i) membrane materials and (ii) membrane structures. The selection of a suited material and preparation technique depends on the application the membrane

The biological activities of (1,3)-(1,6)-{beta}-d-glucan and porous electrospun PLGA membranes containing {beta}-glucan in human dermal fibroblasts and adipose tissue-derived stem cells

Energy Technology Data Exchange (ETDEWEB)

Woo, Yeon I; Park, Bong Joo; Kim, Hye-Lee; Lee, Mi Hee; Kim, Jungsung; Park, Jong-Chul [Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Yang, Young-Il [Department of Pathology, School of Medicine, Paik Institute for Clinical Research, Inje University, 633-165 Gae-dong, Busan-jin-gu, Busan 614-735 (Korea, Republic of); Kim, Jung Koo [Department of Biomedical Engineering, College of Biomedical Science and Engineering, Inje University, Kimhae 621-749 (Korea, Republic of); Tsubaki, Kazufumi [R and D division, Asahi Denka Co. Ltd, 7-2-35 Higashi-ogu, Arakawa-ku, Tokyo 116-8554 (Japan); Han, Dong-Wook, E-mail: parkjc@yuhs.a [Department of Nanomedical Engineering, College of Nanoscience and Nanotechnology, Pusan National University, geumjeong-gu, Busan 609-735 (Korea, Republic of)

2010-08-01

In this study, we investigated the possible roles of (1,3)-(1,6)-{beta}-d-glucan ({beta}-glucan) and porous electrospun poly-lactide-co-glycolide (PLGA) membranes containing {beta}-glucan for skin wound healing, especially their effect on adult human dermal fibroblast (aHDF) and adipose tissue-derived stem cell (ADSC) activation, proliferation, migration, collagen gel contraction and biological safety tests of the prepared membrane. This study demonstrated that {beta}-glucan and porous PLGA membranes containing {beta}-glucan have enhanced the cellular responses, proliferation and migration, of aHDFs and ADSCs and the result of a collagen gel contraction assay also revealed that collagen gels contract strongly after 4 h post-gelation incubation with {beta}-glucan. Furthermore, we confirmed that porous PLGA membranes containing {beta}-glucan are biologically safe for wound healing study. These results indicate that the porous PLGA membranes containing {beta}-glucan interacted favorably with the membrane and the topical administration of {beta}-glucan was useful in promoting wound healing. Therefore, our study suggests that {beta}-glucan and porous PLGA membranes containing {beta}-glucan may be useful as a material for enhancing wound healing.

Proteomic analysis of GPI-anchored membrane proteins

DEFF Research Database (Denmark)

Jung, Hye Ryung; Jensen, Ole Nørregaard

2006-01-01

Glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs) represent a subset of post-translationally modified proteins that are tethered to the outer leaflet of the plasma membrane via a C-terminal GPI anchor. GPI-APs are found in a variety of eukaryote species, from pathogenic microorganisms...... to humans. GPI-APs confer important cellular functions as receptors, enzymes and scaffolding molecules. Specific enzymes and detergent extraction methods combined with separation technologies and mass spectrometry permit proteomic analysis of GPI-APs from plasma membrane preparations to reveal cell...

Differential effects of inhibitors and detergents on the Ca2+-ATPase and Mg2+-ATPase activities of the plasma membrane of a human oat cell carcinoma

International Nuclear Information System (INIS)

Knowles, A.F.; Lawrence, C.M.

1986-01-01

Plasma membranes of human oat cell carcinoma possess Mg 2+ - and Ca 2+ -dependent ATPase activities of similar magnitude. These activities exhibit the unusual characteristic of being inactiviated by prolonged incubation of the membrane with 1-2 mM dithiothreitol (DTT). Inactivation by DTT was prevented by lowering the incubation temperature, elevation of the membrane protein concentration, and addition of ATP. Fluorosulfonylbenzoyl adenosine (FSBA), an affinity ATP analog, also inactivates these activities. The Ca 2+ -ATPase activity appears to be more sensitive to both DTT and FSBA. The Ca 2+ -ATPase activity is more easily inactivated by Triton X-100, while the Mg 2+ -ATPase is preferentially activated by digitonin. These differential effects of inhibitors and detergents suggest that the Ca 2+ -ATPase and Mg 2+ -ATPase are separate enzymes. Incubation of oat cell carcinoma plasma membrane with [ 3 H]FSBA resulted in the labeling of several proteins. A labelled 35,000 dalton protein corresponds to the molecular weight of the oat cell carcinoma plasma membrane Ca 2+ -ATPase previously purified in this laboratory. The identity of one or more of the other labelled proteins with the Mg 2+ -ATPase has not been demonstrated, but is presently under investigation

Recent advances on polymeric membranes for membrane reactors

KAUST Repository

Buonomenna, M. G.

2012-06-24

Membrane reactors are generally applied in high temperature reactions (>400 °C). In the field of fine chemical synthesis, however, much milder conditions are generally applicable and polymeric membranes were applied without their damage. The successful use of membranes in membrane reactors is primary the result of two developments concerning: (i) membrane materials and (ii) membrane structures. The selection of a suited material and preparation technique depends on the application the membrane is to be used in. In this chapter a review of up to date literature about polymers and configuration catalyst/ membranes used in some recent polymeric membrane reactors is given. The new emerging concept of polymeric microcapsules as catalytic microreactors has been proposed. © 2012 Bentham Science Publishers. All rights reserved.

Robust performance of a membrane bioreactor for removing antibiotic resistance genes exposed to antibiotics: Role of membrane foulants.

Science.gov (United States)

Zhu, Yijing; Wang, Yayi; Zhou, Shuai; Jiang, Xuxin; Ma, Xiao; Liu, Chao

2018-03-01

Antibiotic resistance genes (ARGs) are an emerging concern in wastewater treatment plants (WWTPs), as dissemination of ARGs can pose a serious risk to human health. Few studies, however, have quantified ARGs in membrane bioreactors (MBRs), although MBRs have been widely used for both municipal and industrial wastewater treatment. To reveal the capacity of MBRs for removal of ARGs and the response of membrane fouling after antibiotic exposure, five typical ARG subtypes (sulI, sulII, tetC, tetX and ereA) and int1 were quantified affiliated by systematic membrane foulants analysis in a laboratory-scale anoxic/aerobic membrane bioreactor (A/O-MBR). Sulfamethoxazole and tetracycline hydrochloride additions increased ARG abundances by 0.5-1.4 orders of magnitude in the activated sludge, while the ARG removal performance of the membrane module remained stable (or even increased with ARG absolute abundance in several cases), with the abundance of removed ARGs ranging from 0.6 to 5.6 orders of magnitude. Specifically, the distribution of ARGs in membrane foulants accounted for 13%-25% of the total absolute abundance of all tested MBR samples. Indeed, substantial fouling occurred after the antibiotic additions, with the mean concentrations of soluble microbial product (SMP) and extracellular polymeric substance (EPS) increasing by 340% and 220%, respectively, in a membrane fouling cycle; moreover, the contents of EPS and SMP in the membrane foulants were significantly correlated with the ARG absolute abundance of membrane foulants (p removal of ARGs in MBR systems, and highlight the contribution of membrane fouling to ARG removals in terms of the potential of MBR as an effective strategy to reduce ARG levels in WWTP effluent. Copyright © 2017 Elsevier Ltd. All rights reserved.

CO2 Acquisition Membrane (CAM)

Science.gov (United States)

Mason, Larry W.; Way, J. Douglas; Vlasse, Marcus

2003-01-01

The objective of CAM is to develop, test, and analyze thin film membrane materials for separation and purification of carbon dioxide (CO2) from mixtures of gases, such as those found in the Martian atmosphere. The membranes are targeted toward In Situ Resource Utilization (ISRU) applications that will operate in extraterrestrial environments and support future unmanned and human space missions. A primary application is the Sabatier Electrolysis process that uses Mars atmosphere CO2 as raw material for producing water, oxygen, and methane for rocket fuel and habitat support. Other applications include use as an inlet filter to collect and concentrate Mars atmospheric argon and nitrogen gases for habitat pressurization, and to remove CO2 from breathing gases in Closed Environment Life Support Systems (CELSS). CAM membrane materials include crystalline faujasite (FAU) zeolite and rubbery polymers such as silicone rubber (PDMS) that have been shown in the literature and via molecular simulation to favor adsorption and permeation of CO2 over nitrogen and argon. Pure gas permeation tests using commercial PDMS membranes have shown that both CO2 permeance and the separation factor relative to other gases increase as the temperature decreases, and low (Delta)P(Sub CO2) favors higher separation factors. The ideal CO2/N2 separation factor increases from 7.5 to 17.5 as temperature decreases from 22 C to -30 C. For gas mixtures containing CO2, N2, and Ar, plasticization decreased the separation factors from 4.5 to 6 over the same temperature range. We currently synthesize and test our own Na(+) FAU zeolite membranes using standard formulations and secondary growth methods on porous alumina. Preliminary tests with a Na(+) FAU membrane at 22 C show a He/SF6 ideal separation factor of 62, exceeding the Knudsen diffusion selectivity by an order of magnitude. This shows that the membrane is relatively free from large defects and associated non-selective (viscous flow) transport

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.

Membrane-lipid therapy: A historical perspective of membrane-targeted therapies - From lipid bilayer structure to the pathophysiological regulation of cells.

Science.gov (United States)

Escribá, Pablo V

2017-09-01

Our current understanding of membrane lipid composition, structure and functions has led to the investigation of their role in cell signaling, both in healthy and pathological cells. As a consequence, therapies based on the regulation of membrane lipid composition and structure have been recently developed. This novel field, known as Membrane Lipid Therapy, is growing and evolving rapidly, providing treatments that are now in use or that are being studied for their application to oncological disorders, Alzheimer's disease, spinal cord injury, stroke, diabetes, obesity, and neuropathic pain. This field has arisen from relevant discoveries on the behavior of membranes in recent decades, and it paves the way to adopt new approaches in modern pharmacology and nutrition. This innovative area will promote further investigation into membranes and the development of new therapies with molecules that target the cell membrane. Due to the prominent roles of membranes in the cells' physiology and the paucity of therapeutic approaches based on the regulation of the lipids they contain, it is expected that membrane lipid therapy will provide new treatments for numerous pathologies. The first on-purpose rationally designed molecule in this field, minerval, is currently being tested in clinical trials and it is expected to enter the market around 2020. However, it seems feasible that during the next few decades other membrane regulators will also be marketed for the treatment of human pathologies. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017. Published by Elsevier B.V.

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

Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

International Nuclear Information System (INIS)

Zambetti, G.; Stein, J.; Stein, G.

1987-01-01

The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

One-way membrane trafficking of SOS in receptor-triggered Ras activation.

Science.gov (United States)

Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T

2016-09-01

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.

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 u...

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

Membrane fouling mechanism of biofilm-membrane bioreactor (BF-MBR): Pore blocking model and membrane cleaning.

Science.gov (United States)

Zheng, Yi; Zhang, Wenxiang; Tang, Bing; Ding, Jie; Zheng, Yi; Zhang, Zhien

2018-02-01

Biofilm membrane bioreactor (BF-MBR) is considered as an important wastewater treatment technology that incorporates advantages of both biofilm and MBR process, as well as can alleviate membrane fouling, with respect to the conventional activated sludge MBR. But, to be efficient, it necessitates the establishment of proper methods for the assessment of membrane fouling. Four Hermia membrane blocking models were adopted to quantify and evaluate the membrane fouling of BF-MBR. The experiments were conducted with various operational conditions, including membrane types, agitation speeds and transmembrane pressure (TMP). Good agreement between cake formation model and experimental data was found, confirming the validity of the Hermia models for assessing the membrane fouling of BF-MBR and that cake layer deposits on membrane. Moreover, the influences of membrane types, agitation speeds and transmembrane pressure on the Hermia pore blocking coefficient of cake layer were investigated. In addition, the permeability recovery after membrane cleaning at various operational conditions was studied. This work confirms that, unlike conventional activated sludge MBR, BF-MBR possesses a low degree of membrane fouling and a higher membrane permeability recovery after cleaning. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fibrous membranes in diabetic retinopathy and bevacizumab.

Science.gov (United States)

Pattwell, David M; Stappler, Theodor; Sheridan, Carl; Heimann, Heinrich; Gibran, Syed K; Wong, David; Hiscott, Paul

2010-01-01

The purpose of this study was to determine the histopathologic characteristics of bevacizumab-treated human proliferative diabetic retinopathy (PDR) membranes with particular regard to membrane vasculature as a step toward addressing the effects of the drug on PDR membranes. Intravitreous injection of bevacizumab, an antivascular endothelial growth factor monoclonal antibody, has recently been advocated as an adjunct in surgery for PDR. In this context, a clinically observed decrease in PDR epiretinal membrane vascularity (vascular regression) occurs from 24 hours to 48 hours after injection, but the exact mechanisms of drug action are unknown. A consecutive series of seven PDR membrane specimens that had been removed sequentially from seven bevacizumab-treated patients were studied retrospectively. The membrane specimens were examined using light microscopic methods, including immunohistochemistry. Five of the seven membranes were clinically avascular (one contained "ghost" vessels) and did not hemorrhage during excision. Of these 5 specimens, which included 1 removed 7 days after a total of 6 intravitreous injections of 1.25 mg bevacizumab, 4 contained histologically detectable capillaries (1 did not). These blood vessels were lined by endothelial cells as determined by immunohistochemistry for the endothelial markers CD31 and CD34. The two remaining membranes were clinically and histologically still vascularized despite bevacizumab treatment. All the specimens also contained smooth muscle actin-containing fibroblastic cells within the collagenous stroma. The findings do not support the concept that the clinical phenomenon of vascular regression in PDR membranes after bevacizumab injection in the vitreous is resulting from obliteration of the membrane blood vessels. Another mechanism appears to be involved in at least some patients, possibly a vasoconstrictive response. Such a mechanism might explain reversal of the effects of bevacizumab that has been reported

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

International Nuclear Information System (INIS)

Gaihede, Michael; Liao Donghua; Gregersen, Hans

2007-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Gaihede, Michael [Department of Otolaryngology, Head and Neck Surgery, Aalborg Hospital, Aarhus University Hospital, Aalborg (Denmark); Liao Donghua [Centre of Excellence in Visceral Biomechanics and Pain, Aalborg Hospital, Aarhus University Hospital, Aalborg (Denmark); Gregersen, Hans [Centre of Excellence in Visceral Biomechanics and Pain, Aalborg Hospital, Aarhus University Hospital, Aalborg (Denmark)

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{sup -1}, respectively) compared to the younger group (mean = 1.066 and 1.206 kN m{sup -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.

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.

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.

Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells.

Science.gov (United States)

Bacakova, Marketa; Pajorova, Julia; Stranska, Denisa; Hadraba, Daniel; Lopot, Frantisek; Riedel, Tomas; Brynda, Eduard; Zaloudkova, Margit; Bacakova, Lucie

2017-01-01

Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide- co -glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types.

Energy efficient reconcentration of diluted human urine using ion exchange membranes in bioelectrochemical systems.

Science.gov (United States)

Tice, Ryan C; Kim, Younggy

2014-11-01

Nutrients can be recovered from source separated human urine; however, nutrient reconcentration (i.e., volume reduction of collected urine) requires energy-intensive treatment processes, making it practically difficult to utilize human urine. In this study, energy-efficient nutrient reconcentration was demonstrated using ion exchange membranes (IEMs) in a microbial electrolysis cell (MEC) where substrate oxidation at the MEC anode provides energy for the separation of nutrient ions (e.g., NH4(+), HPO4(2-)). The rate of nutrient separation was magnified with increasing number of IEM pairs and electric voltage application (Eap). Ammonia and phosphate were reconcentrated from diluted human urine by a factor of up to 4.5 and 3.0, respectively (Eap = 1.2 V; 3-IEM pairs). The concentrating factor increased with increasing degrees of volume reduction, but it remained stationary when the volume ratio between the diluate (urine solution that is diluted in the IEM stack) and concentrate (urine solution that is reconcentrated) was 6 or greater. The energy requirement normalized by the mass of nutrient reconcentrated was 6.48 MJ/kg-N (1.80 kWh/kg-N) and 117.6 MJ/kg-P (32.7 kWh/kg-P). In addition to nutrient separation, the examined MEC reactor with three IEM pairs showed 54% removal of COD (chemical oxygen demand) in 47-hr batch operation. The high sulfate concentration in human urine resulted in substantial growth of both of acetate-oxidizing and H2-oxidizing sulfate reducing bacteria, greatly diminishing the energy recovery and Coulombic efficiency. However, the high microbial activity of sulfate reducing bacteria hardly affected the rate of nutrient reconcentration. With the capability to reconcentrate nutrients at a minimal energy consumption and simultaneous COD removal, the examined bioelectrochemical treatment method with an IEM application has a potential for practical nutrient recovery and sustainable treatment of source-separated human urine. Copyright © 2014

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...

The potential of immobilized artificial membrane chromatography to predict human oral absorption.

Science.gov (United States)

Tsopelas, Fotios; Vallianatou, Theodosia; Tsantili-Kakoulidou, Anna

2016-01-01

The potential of immobilized artificial membrane (IAM) chromatography to estimate human oral absorption (%HOA) was investigated. For this purpose, retention indices on IAM stationary phases reported previously by our group or measured by other authors under similar conditions were used to model %HOA data, compiled from literature sources. Considering the pH gradient in gastrointestinal tract, the highest logkw(IAM) values were considered, obtained either at pH7.4 or 5.5, defined as logkw(IAM)(best). Non linear models were established upon introduction of additional parameters and after exclusion of drugs which are substrates either to efflux or uptake transporters. The best model included Abraham's hydrogen-bond acidity parameter, molecular weight as well as the positively and negatively charged molecular fractions. For reasons of comparison between IAM chromatography and traditional lipophilicity, corresponding models were derived by replacing IAM retention factors with octanol-water distribution coefficients (logD). An overexpression of electrostatic interactions with phosphate anions was observed in the case of IAM retention as expressed by the negative contribution of the positively charged fraction F(+). The same parameter is statistically significant also in the logD model, but with a positive sign, indicating the attraction of basic drugs in the negatively charged inner membrane. To validate the obtained models a blind test set of 22 structurally diverse drugs was used, whose logkw(IAM)(best) values were determined and analyzed in the present study under similar conditions. IAM retention factors were further compared with MDCK cell lines permeability data taken from literature for a set of validation drugs. The overexpression of electrostatic interactions with phosphate anions on IAM surface was also evident in respect to MDCK permeability. In contrast to the clear classification between drugs with high and poor (or intermediate) absorption provided by MDCK

Guanine nucleotides stimulate hydrolysis of phosphatidyl inositol bis phosphate in human myelin membranes

International Nuclear Information System (INIS)

Boulias, C.; Moscarello, M.A.

1989-01-01

Phosphodiesterase activity was stimulated in myelin membranes in the presence of guanine nucleotide analogues. This activity was reduced in myelin membranes which had been adenosine diphosphate ribosylated in the presence of cholera toxin which ADP-ribosylated three proteins of Mr 46,000, 43,000 and 18,500. Aluminum fluoride treatment of myelin had the same stimulatory effects on phosphodiesterase activity as did the guanine nucleotides

Palmitoylation of POTE family proteins for plasma membrane targeting

International Nuclear Information System (INIS)

Das, Sudipto; Ise, Tomoko; Nagata, Satoshi; Maeda, Hiroshi; Bera, Tapan K.; Pastan, Ira

2007-01-01

The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane

Smart membranes for monitoring membrane based desalination processes

KAUST Repository

Laleg-Kirati, Taous-Meriem; Karam, Ayman M.

2017-01-01

Various examples are related to smart membranes for monitoring membrane based process such as, e.g., membrane distillation processes. In one example, a membrane, includes a porous surface and a plurality of sensors (e.g., temperature, flow and

[Effect of 3-bromopyruvate on mitochondrial membrane potential and apoptosis of human breast carcinoma SK-BR-3 cells].

Science.gov (United States)

Zhang, Yuanyuan; Liu, Zhe; Zhang, Qianwen; Chao, Zhenhua; Zhang, Pei; Xia, Fei; Jiang, Chenchen; Liu, Hao; Jiang, Zhiwen

2013-09-01

To study the effect of glycolysis inhibitor 3-bromopyruvate (3-BrPA) in inducing apoptosis of human breast carcinoma cells SK-BR-3 and the possible mechanism. MTT assay was used to detect the growth inhibition induced by 3-BrPA in breast cancer cells SK-BR-3. The apoptotic cells were detected by flow cytometry with propidium iodide (PI). ATP levels in the cells were detected by ATP assay kit, and DHE fluorescent probe technique was used to determine superoxide anion levels; the mitochondrial membrane potential was assessed using JC-1 staining assay. MTT assay showed that the proliferation of SK-BR-3 cells was inhibited by 3-BrPA in a time- and concentration-dependent manner. Exposure to 80, 160, and 320 µmol·L(-1) 3-BrPA for 24 h resulted in cell apoptosis rates of 6.7%, 22.3%, and 79.6%, respectively, and the intracellular ATP levels of SK-BR-3 cells treated with 80, 160, 320 µmol·L(-1) 3-BrPA for 5 h were 87.7%, 60.6%, and 23.7% of the control levels. 3-BrPA at 160 µmol·L(-1) increased reactive oxygen levels and lowered mitochondrial membrane potential of SK-BR-3 cells. 3-BrPA can inhibit cell proliferation, reduce the mitochondrial membrane potential and induce apoptosis in SK-BR-3 cells, the mechanism of which may involve a reduced ATP level by inhibiting glycolysis and increasing the reactive oxygen level in the cells.

Flow cytometry analysis of FITC-labeled concanavalin A binding to human blood cells as an indicator of radiation-induced membrane alterations

International Nuclear Information System (INIS)

Donnadieu-Claraz, M.; Paillole, N.; Voisin, P.

1995-01-01

The 3 H concanavalin-A binding to human blood cells have been described as a promising biological indicator of radiation overexposure. Flow cytometry adaptation of this technique using fluorescein-labelled concanavalin-A were performed to estimate time-dependent changes in binding on human blood cells membranes after in vitro γ irradiation ( 60 Co). Result revealed significant enhanced lectin-binding to platelets and erythrocytes in a dose range of 0,5-5 Gy, 1 and 3 hours after irradiation. However for both platelets and erythrocytes, it was impossible to discriminate between the different doses. Further studies are necessary to confirm the suitability of lectin-binding as a biological indicator for radiation dose assessment. (authors). 5 refs., 1 fig

Biomimetic membranes and methods of making biomimetic membranes

Science.gov (United States)

Rempe, Susan; Brinker, Jeffrey C.; Rogers, David Michael; Jiang, Ying-Bing; Yang, Shaorong

2016-11-08

The present disclosure is directed to biomimetic membranes and methods of manufacturing such membranes that include structural features that mimic the structures of cellular membrane channels and produce membrane designs capable of high selectivity and high permeability or adsorptivity. The membrane structure, material and chemistry can be selected to perform liquid separations, gas separation and capture, ion transport and adsorption for a variety of applications.

A Novel Soluble Peptide with pH-Responsive Membrane Insertion.

Science.gov (United States)

Nguyen, Vanessa P; Alves, Daiane S; Scott, Haden L; Davis, Forrest L; Barrera, Francisco N

2015-11-03

Several diseases, such as cancer, are characterized by acidification of the extracellular environment. Acidosis can be employed as a target to specifically direct therapies to the diseased tissue. We have used first principles to design an acidity-triggered rational membrane (ATRAM) peptide with high solubility in solution that is able to interact with lipid membranes in a pH-dependent fashion. Biophysical studies show that the ATRAM peptide binds to the surface of lipid membranes at pH 8.0. However, acidification leads to the peptide inserting into the lipid bilayer as a transmembrane α-helix. The insertion of ATRAM into membranes occurs at a moderately acidic pH (with a pK of 6.5), similar to the extracellular pH found in solid tumors. Studies with human cell lines showed a highly efficient pH-dependent membrane targeting, without causing toxicity. Here we show that it is possible to rationally design a soluble peptide that selectively targets cell membranes in acidic environments.

Formation of carrageenan-CaCO{sub 3} bioactive membranes

Energy Technology Data Exchange (ETDEWEB)

Nogueira, Lucas F.B.; Maniglia, Bianca C.; Pereira, Lourivaldo S.; Tapia-Blácido, Delia R.; Ramos, Ana P., E-mail: anapr@ffclrp.usp.br

2016-01-01

The high biocompatibility and resorbability of polymeric membranes have encouraged their use to manufacture medical devices. Here, we report on the preparation of membranes consisting of carrageenan, a naturally occurring sulfated polysaccharide that forms helical structures in the presence of calcium ions. We incorporated CaCO{sub 3} particles into the membranes to enhance their bioactivity and mechanical properties. Infrared spectroscopy and X-ray diffraction data confirmed CaCO{sub 3} incorporation into the polymeric matrix. We tested the bioactivity of the samples by immersing them in a solution that mimics the ionic composition and pH of the human body fluid. The hybrid membranes generated hydroxyapatite, as attested by X-ray diffraction data. Scanning electron and atomic force microscopies aided investigation of membrane topography before and after CaCO{sub 3} deposition. The wettability and surface free energy, evaluated by contact angle measures, increased in the presence of CaCO{sub 3} particles. These parameters are important for membrane implantation in the body. Moreover, membrane stiffness was up to 110% higher in the presence of the inorganic particles, as revealed by Young's modulus. - Highlights: • Hybrid kappa and iota carrageenan-CaCO{sub 3} membranes were formed. • The hybrid membrane's origin hydroxyapatite after exposure to simulated body fluid • The carrageenan's specificity to bind Ca{sup 2+} ions tailors the surface properties.

Gel layer formation on membranes in Membrane Bioreactors

NARCIS (Netherlands)

Van den Brink, P.F.H.

2014-01-01

The widespread application of membrane bioreactors (MBRs) for municipal wastewater treatment is hampered by membrane fouling. Fouling increases energy demand, reduces process performance and creates the need for more frequent (chemical) membrane cleaning or replacement. Membrane fouling in MBRs is

The NMR structure of human obestatin in membrane-like environments: insights into the structure-bioactivity relationship of obestatin.

Science.gov (United States)

Alén, Begoña O; Nieto, Lidia; Gurriarán-Rodríguez, Uxía; Mosteiro, Carlos S; Álvarez-Pérez, Juan C; Otero-Alén, María; Camiña, Jesús P; Gallego, Rosalía; García-Caballero, Tomás; Martín-Pastor, Manuel; Casanueva, Felipe F; Jiménez-Barbero, Jesús; Pazos, Yolanda

2012-01-01

The quest for therapeutic applications of obestatin involves, as a first step, the determination of its 3D solution structure and the relationship between this structure and the biological activity of obestatin. On this basis, we have employed a combination of circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, and modeling techniques to determine the solution structure of human obestatin (1). Other analogues, including human non-amidated obestatin (2) and the fragment peptides (6-23)-obestatin (3), (11-23)-obestatin (4), and (16-23)-obestatin (5) have also been scrutinized. These studies have been performed in a micellar environment to mimic the cell membrane (sodium dodecyl sulfate, SDS). Furthermore, structural-activity relationship studies have been performed by assessing the in vitro proliferative capabilities of these peptides in the human retinal pigmented epithelial cell line ARPE-19 (ERK1/2 and Akt phosphorylation, Ki67 expression, and cellular proliferation). Our findings emphasize the importance of both the primary structure (composition and size) and particular segments of the obestatin molecule that posses significant α-helical characteristics. Additionally, details of a species-specific role for obestatin have also been hypothesized by comparing human and mouse obestatins (1 and 6, respectively) at both the structural and bioactivity levels.

The NMR structure of human obestatin in membrane-like environments: insights into the structure-bioactivity relationship of obestatin.

Directory of Open Access Journals (Sweden)

Begoña O Alén

Full Text Available The quest for therapeutic applications of obestatin involves, as a first step, the determination of its 3D solution structure and the relationship between this structure and the biological activity of obestatin. On this basis, we have employed a combination of circular dichroism (CD, nuclear magnetic resonance (NMR spectroscopy, and modeling techniques to determine the solution structure of human obestatin (1. Other analogues, including human non-amidated obestatin (2 and the fragment peptides (6-23-obestatin (3, (11-23-obestatin (4, and (16-23-obestatin (5 have also been scrutinized. These studies have been performed in a micellar environment to mimic the cell membrane (sodium dodecyl sulfate, SDS. Furthermore, structural-activity relationship studies have been performed by assessing the in vitro proliferative capabilities of these peptides in the human retinal pigmented epithelial cell line ARPE-19 (ERK1/2 and Akt phosphorylation, Ki67 expression, and cellular proliferation. Our findings emphasize the importance of both the primary structure (composition and size and particular segments of the obestatin molecule that posses significant α-helical characteristics. Additionally, details of a species-specific role for obestatin have also been hypothesized by comparing human and mouse obestatins (1 and 6, respectively at both the structural and bioactivity levels.

N-acylated peptides derived from human lactoferricin perturb organization of cardiolipin and phosphatidylethanolamine in cell membranes and induce defects in Escherichia coli cell division.

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Dagmar Zweytick

Full Text Available Two types of recently described antibacterial peptides derived from human lactoferricin, either nonacylated or N-acylated, were studied for their different interaction with membranes of Escherichia coli in vivo and in model systems. Electron microscopy revealed striking effects on the bacterial membrane as both peptide types induced formation of large membrane blebs. Electron and fluorescence microscopy, however demonstrated that only the N-acylated peptides partially induced the generation of oversized cells, which might reflect defects in cell-division. Further a different distribution of cardiolipin domains on the E. coli membrane was shown only in the presence of the N-acylated peptides. The lipid was distributed over the whole bacterial cell surface, whereas cardiolipin in untreated and nonacylated peptide-treated cells was mainly located at the septum and poles. Studies with bacterial membrane mimics, such as cardiolipin or phosphatidylethanolamine revealed that both types of peptides interacted with the negatively charged lipid cardiolipin. The nonacylated peptides however induced segregation of cardiolipin into peptide-enriched and peptide-poor lipid domains, while the N-acylated peptides promoted formation of many small heterogeneous domains. Only N-acylated peptides caused additional severe effects on the main phase transition of liposomes composed of pure phosphatidylethanolamine, while both peptide types inhibited the lamellar to hexagonal phase transition. Lipid mixtures of phosphatidylethanolamine and cardiolipin revealed anionic clustering by all peptide types. However additional strong perturbation of the neutral lipids was only seen with the N-acylated peptides. Nuclear magnetic resonance demonstrated different conformational arrangement of the N-acylated peptide in anionic and zwitterionic micelles revealing possible mechanistic differences in their action on different membrane lipids. We hypothesized that both peptides kill

Cationic nanoparticles induce nanoscale disruption in living cell plasma membranes.

Science.gov (United States)

Chen, Jiumei; Hessler, Jessica A; Putchakayala, Krishna; Panama, Brian K; Khan, Damian P; Hong, Seungpyo; Mullen, Douglas G; Dimaggio, Stassi C; Som, Abhigyan; Tew, Gregory N; Lopatin, Anatoli N; Baker, James R; Holl, Mark M Banaszak; Orr, Bradford G

2009-08-13

It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes.

Nanodisc-solubilized membrane protein library reflects the membrane proteome.

Science.gov (United States)

Marty, Michael T; Wilcox, Kyle C; Klein, William L; Sligar, Stephen G

2013-05-01

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.

Deletion of the basement membrane heparan sulfate proteoglycan type XVIII collagen causes hypertriglyceridemia in mice and humans.

Directory of Open Access Journals (Sweden)

Joseph R Bishop

2010-11-01

Full Text Available Lipoprotein lipase (Lpl acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism.We examined mutant mice defective in collagen XVIII (Col18, a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia.This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.

[Binding of the antileukemia drug Escherichia coli L-asparaginase to the plasma membrane of normal human mononuclear cells].

Science.gov (United States)

Mercado-Vianco, L; Arenas-Díaz, G

1999-06-01

To demonstrate that the enzyme L-asparaginase from Escherichia coli (EcA) binds to the plasma membranes of normal human lymphocytes and monocytes. Lymphocytes and monocytes were isolated from heparinized blood samples which came from healthy volunteer donors. The cells were incubated with EcA to detect a possible binding of the enzyme to the mononuclear cells by indirect immunofluorescence using confocal microscopy. Meanwhile, ultracentrifugation was used to obtain the erythrocyte ghost microsomal fraction (P100) which was then analyzed by Western blotting to determine if EcA binds the lipid bilayer unspecifically. For the immunoassays, monospecific polyclonal antibodies were obtained from ascitic tumors developed in mice immunized with commercial L-asparaginase. EcA bins the lymphocyte and monocyte plasma membranes. In monocytes, there occurs a capping phenomenon, that is, the accumulation of fluorescent marker in one region. The image analyzer highlights it clearly at a depth of 3.8 microns. This binding would be unspecific, that is, there is no mediation of a specific receptor that binds EcA. This arises from the ability of the enzyme to bind to the membranes of erythrocyte ghost, as evidenced by the ability of the molecule to associate with a hydrophobic medium. The antibodies against EcA obtained from ascitic tumours developed in mice do not show cross reactivity with Na+/K+ ATPase, aspartate aminotransferase, nor with extracts of blood cells, which would make it a specific tool for the detection of EcA in whole cells and in homogenates electrotransfered to nitrocellulose membranes. L-asparaginase from E. coli behaves as a lipoprotein due to its ability to insert itself into hydrophobic environments, in which it resembles an isozyme present in T. pyriformis. The binding of this enzyme to lymphocytes and monocytes, demonstrated in this work, would permit the modification of the antileukemic treatment injecting doses of EcA bound to patient's own isolated immune

HIV-1 matrix dependent membrane targeting is regulated by Gag mRNA trafficking.

Directory of Open Access Journals (Sweden)

Jing Jin

Full Text Available Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV post-transcriptional regulatory element (PRE mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

Hemocompatibility of poly(vinylidene fluoride) membrane grafted with network-like and brush-like antifouling layer controlled via plasma-induced surface PEGylation.

Science.gov (United States)

Chang, Yung; Shih, Yu-Ju; Ko, Chao-Yin; Jhong, Jheng-Fong; Liu, Ying-Ling; Wei, Ta-Chin

2011-05-03

In this work, the hemocompatibility of PEGylated poly(vinylidene fluoride) (PVDF) microporous membranes with varying grafting coverage and structures via plasma-induced surface PEGylation was studied. Network-like and brush-like PEGylated layers on PVDF membrane surfaces were achieved by low-pressure and atmospheric plasma treatment. The chemical composition, physical morphology, grafting structure, surface hydrophilicity, and hydration capability of prepared membranes were determined to illustrate the correlations between grafting qualities and hemocompatibility of PEGylated PVDF membranes in contact with human blood. Plasma protein adsorption onto different PEGylated PVDF membranes from single-protein solutions and the complex medium of 100% human plasma were measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Hemocompatibility of the PEGylated membranes was evaluated by the antifouling property of platelet adhesion observed by scanning electron microscopy (SEM) and the anticoagulant activity of the blood coagulant determined by testing plasma-clotting time. The control of grafting structures of PEGylated layers highly regulates the PVDF membrane to resist the adsorption of plasma proteins, the adhesion of platelets, and the coagulation of human plasma. It was found that PVDF membranes grafted with brush-like PEGylated layers presented higher hydration capability with binding water molecules than with network-like PEGylated layers to improve the hemocompatible character of plasma protein and blood platelet resistance in human blood. This work suggests that the hemocompatible nature of grafted PEGylated polymers by controlling grafting structures gives them great potential in the molecular design of antithrombogenic membranes for use in human blood.

Flux Enhancement in Membrane Distillation Using Nanofiber Membranes

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T. Jiříček

2016-01-01

Full Text Available Membrane distillation (MD is an emerging separation technology, whose largest application potential lies in the desalination of highly concentrated solutions, which are out of the scope of reverse osmosis. Despite many attractive features, this technology is still awaiting large industrial application. The main reason is the lack of commercially available membranes with fluxes comparable to reverse osmosis. MD is a thermal separation process driven by a partial vapour pressure difference. Flux, distillate purity, and thermal efficiency are always in conflict, all three being strictly connected with pore size, membrane hydrophobicity, and thickness. The world has not seen the ideal membrane yet, but nanofibers may offer a solution to these contradictory requirements. Membranes of electrospun PVDF were tested under various conditions on a direct contact (DCMD unit, in order to determine the optimum conditions for maximum flux. In addition, their performance was compared to commonly available PTFE, PE, and PES membranes. It was confirmed that thinner membranes have higher fluxes and a lower distillate purity and also higher energy losses via conduction across the membrane. As both mass and heat transfer are connected, it is best to develop new membranes with a target application in mind, for the specific membrane module and operational conditions.

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

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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.

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

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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.

Fabrication of electrospun nanofibrous membranes for membrane distillation application

KAUST Repository

Francis, Lijo

2013-02-01

Nanofibrous membranes of Matrimid have been successfully fabricated using an electrospinning technique under optimized conditions. Nanofibrous membranes are found to be highly hydrophobic with a high water contact angle of 130°. Field emission scanning electron microscopy and pore size distribution analysis revealed the big pore size structure of electrospun membranes to be greater than 2 μm and the pore size distribution is found to be narrow. Flat sheet Matrimid membranes were fabricated via casting followed by phase inversion. The morphology, pore size distribution, and water contact angle were measured and compared with the electrospun membranes. Both membranes fabricated by electrospinning and phase inversion techniques were tested in a direct contact membrane distillation process. Electrospun membranes showed high water vapor flux of 56 kg/m2-h, which is very high compared to the casted membrane as well as most of the fabricated and commercially available highly hydrophobic membranes. ©2013 Desalination Publications.

Sensing inhomogeneous mechanical properties of human corneal Descemet's membrane with AFM nano-indentation.

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Di Mundo, Rosa; Recchia, Giuseppina; Parekh, Mohit; Ruzza, Alessandro; Ferrari, Stefano; Carbone, Giuseppe

2017-10-01

The paper describes a highly space-resolved characterization of the surface mechanical properties of the posterior human corneal layer (Descemet's membrane). This has been accomplished with Atomic Force Microscopy (AFM) nano-indentation by using a probe with a sharp tip geometry. Results indicate that the contact with this biological tissue in liquid occurs with no (or very low) adhesion. More importantly, under the same operating conditions, a broad distribution of penetration depth can be measured on different x-y positions of the tissue surface, indicating a high inhomogeneity of surface stiffness, not yet clearly reported in the literature. An important contribution to such inhomogeneity should be ascribed to the discontinuous nature of the collagen/proteoglycans fibers matrix tissue, as can be imaged by AFM when the tissue is semi-dry. Using classical contact mechanics calculations adapted to the specific geometry of the tetrahedral tip it has been found that the elastic modulus E of the material in the very proximity of the surface ranges from 0.23 to 2.6 kPa. Copyright © 2017 Elsevier Ltd. All rights reserved.

Alternative energy efficient membrane bioreactor using reciprocating submerged membrane.

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Ho, J; Smith, S; Roh, H K

2014-01-01

A novel membrane bioreactor (MBR) pilot system, using membrane reciprocation instead of air scouring, was operated at constant high flux and daily fluctuating flux to demonstrate its application under peak and diurnal flow conditions. Low and stable transmembrane pressure was achieved at 40 l/m(2)/h (LMH) by use of repetitive membrane reciprocation. The results reveal that the inertial forces acting on the membrane fibers effectively propel foulants from the membrane surface. Reciprocation of the hollow fiber membrane is beneficial for the constant removal of solids that may build up on the membrane surface and inside the membrane bundle. The membrane reciprocation in the reciprocating MBR pilot consumed less energy than coarse air scouring used in conventional MBR systems. Specific energy consumption for the membrane reciprocation was 0.072 kWh/m(3) permeate produced at 40 LMH flux, which is 75% less than for a conventional air scouring system as reported in literature without consideration of energy consumption for biological aeration (0.29 kWh/m(3)). The daily fluctuating flux test confirmed that the membrane reciprocation is effective to handle fluctuating flux up to 50 LMH. The pilot-scale reciprocating MBR system successfully demonstrated that fouling can be controlled via 0.43 Hz membrane reciprocation with 44 mm or higher amplitude.

Toxins and antimicrobial peptides: interactions with membranes

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Schlamadinger, Diana E.; Gable, Jonathan E.; Kim, Judy E.

2009-08-01

The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of resistant pathogens.

Unbound fraction of fluconazole and linezolid in human plasma as determined by ultrafiltration: Impact of membrane type.

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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.

Scaling laws for oxygen transport across the space-filling system of respiratory membranes in the human lung

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Hou, Chen

Space-filling fractal surfaces play a fundamental role in how organisms function at various levels and in how structure determines function at different levels. In this thesis, we develop a quantitative theory of oxygen transport to and across the surface of the highly branched, space-filling system of alveoli, the fundamental gas exchange unit (acinar airways), in the human lung. Oxygen transport in the acinar airways is by diffusion, and we treat the two steps---diffusion through the branched airways, and transfer across the alveolar membranes---as a stationary diffusion-reaction problem, taking into account that there may be steep concentration gradients between the entrance and remote alveoli (screening). We develop a renormalization treatment of this screening effect and derive an analytic formula for the oxygen current across the cumulative alveolar membrane surface, modeled as a fractal, space-filling surface. The formula predicts the current from a minimum of morphological data of the acinus and appropriate values of the transport parameters, through a number of power laws (scaling laws). We find that the lung at rest operates near the borderline between partial screening and no screening; that it switches to no screening under exercise; and that the computed currents agree with measured values within experimental uncertainties. From an analysis of the computed current as a function of membrane permeability, we find that the space-filling structure of the gas exchanger is simultaneously optimal with respect to five criteria. The exchanger (i) generates a maximum oxygen current at minimum permeability; (ii) 'wastes' a minimum of surface area; (iii) maintains a minimum residence time of oxygen in the acinar airways; (iv) has a maximum fault tolerance to loss of permeability; and (v) generates a maximum current increase when switching from rest to exercise.

Membrane order in the plasma membrane and endocytic recycling compartment.

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Iaea, David B; Maxfield, Frederick R

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia.

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Wang, Wei; Yang, Lianwei; Huang, Xiumin; Fu, Wenkun; Pan, Dequan; Cai, Linli; Ye, Jianghui; Liu, Jian; Xia, Ningshao; Cheng, Tong; Zhu, Hua

2017-12-01

Syncytia formation has been considered important for cell-to-cell spread and pathogenesis of many viruses. As a syncytium forms, individual nuclei often congregate together, allowing close contact of nuclear membranes and possibly fusion to occur. However, there is currently no reported evidence of nuclear membrane fusion between adjacent nuclei in wild-type virus-induced syncytia. Varicella-zoster virus (VZV) is one typical syncytia-inducing virus that causes chickenpox and shingles in humans. Here, we report, for the first time, an interesting observation of apparent fusion of the outer nuclear membranes from juxtaposed nuclei that comprise VZV syncytia both in ARPE-19 human epithelial cells in vitro and in human skin xenografts in the SCID-hu mouse model in vivo. This work reveals a novel aspect of VZV-related cytopathic effect in the context of multinucleated syncytia. Additionally, the information provided by this study could be helpful for future studies on interactions of viruses with host cell nuclei. Copyright © 2017 Elsevier Inc. All rights reserved.

Membrane-bound human orphan cytochrome P450 2U1: Sequence singularities, construction of a full 3D model, and substrate docking.

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Ducassou, Lionel; Dhers, Laura; Jonasson, Gabriella; Pietrancosta, Nicolas; Boucher, Jean-Luc; Mansuy, Daniel; André, François

2017-09-01

Human cytochrome P450 2U1 (CYP2U1) is an orphan CYP that exhibits several distinctive characteristics among the 57 human CYPs with a highly conserved sequence in almost all living organisms. We compared its protein sequence with those of the 57 human CYPs and constructed a 3D structure of a full-length CYP2U1 model bound to a POPC membrane. We also performed docking experiments of arachidonic acid (AA) and N-arachidonoylserotonin (AS) in this model. The protein sequence of CYP2U1 displayed two unique characteristics when compared to those of the human CYPs, the presence of a longer N-terminal region upstream of the putative trans-membrane helix (TMH) containing 8 proline residues, and of an insert of about 20 amino acids containing 5 arginine residues between helices A' and A. Its N-terminal part upstream of TMH involved an additional short terminal helix, in a manner similar to what was reported in the crystal structure of Saccharomyces cerevisiae CYP51. Our model also showed a specific interaction between the charged residues of insert AA' and phosphate groups of lipid polar heads, suggesting a possible role of this insert in substrate recruitment. Docking of AA and AS in this model showed these substrates in channel 2ac, with the terminal alkyl chain of AA or the indole ring of AS close to the heme, in agreement with the reported CYP2U1-catalyzed AA and AS hydroxylation regioselectivities. This model should be useful to find new endogenous or exogenous CYP2U1 substrates and to interpret the regioselectivity of their hydroxylation. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

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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

Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

Science.gov (United States)

Otsuki, Tetsuji; Ota, Toshio; Nishikawa, Tetsuo; Hayashi, Koji; Suzuki, Yutaka; Yamamoto, Jun-ichi; Wakamatsu, Ai; Kimura, Kouichi; Sakamoto, Katsuhiko; Hatano, Naoto; Kawai, Yuri; Ishii, Shizuko; Saito, Kaoru; Kojima, Shin-ichi; Sugiyama, Tomoyasu; Ono, Tetsuyoshi; Okano, Kazunori; Yoshikawa, Yoko; Aotsuka, Satoshi; Sasaki, Naokazu; Hattori, Atsushi; Okumura, Koji; Nagai, Keiichi; Sugano, Sumio; Isogai, Takao

2005-01-01

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

Giant plasma membrane vesicles: models for understanding membrane organization.

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Levental, Kandice R; Levental, Ilya

2015-01-01

The organization of eukaryotic membranes into functional domains continues to fascinate and puzzle cell biologists and biophysicists. The lipid raft hypothesis proposes that collective lipid interactions compartmentalize the membrane into coexisting liquid domains that are central to membrane physiology. This hypothesis has proven controversial because such structures cannot be directly visualized in live cells by light microscopy. The recent observations of liquid-liquid phase separation in biological membranes are an important validation of the raft hypothesis and enable application of the experimental toolbox of membrane physics to a biologically complex phase-separated membrane. This review addresses the role of giant plasma membrane vesicles (GPMVs) in refining the raft hypothesis and expands on the application of GPMVs as an experimental model to answer some of key outstanding problems in membrane biology. Copyright © 2015 Elsevier Inc. All rights reserved.

Structure and physical properties of bio membranes and model membranes

International Nuclear Information System (INIS)

Tibor Hianik

2006-01-01

Bio membranes belong to the most important structures of the cell and the cell organelles. They play not only structural role of the barrier separating the external and internal part of the membrane but contain also various functional molecules, like receptors, ionic channels, carriers and enzymes. The cell membrane also preserves non-equilibrium state in a cell which is crucial for maintaining its excitability and other signaling functions. The growing interest to the bio membranes is also due to their unique physical properties. From physical point of view the bio membranes, that are composed of lipid bilayer into which are incorporated integral proteins and on their surface are anchored peripheral proteins and polysaccharides, represent liquid s crystal of smectic type. The bio membranes are characterized by anisotropy of structural and physical properties. The complex structure of bio membranes makes the study of their physical properties rather difficult. Therefore several model systems that mimic the structure of bio membranes were developed. Among them the lipid monolayers at an air-water interphase, bilayer lipid membranes, supported bilayer lipid membranes and liposomes are most known. This work is focused on the introduction into the physical word of the bio membranes and their models. After introduction to the membrane structure and the history of its establishment, the physical properties of the bio membranes and their models are stepwise presented. The most focus is on the properties of lipid monolayers, bilayer lipid membranes, supported bilayer lipid membranes and liposomes that were most detailed studied. This lecture has tutorial character that may be useful for undergraduate and graduate students in the area of biophysics, biochemistry, molecular biology and bioengineering, however it contains also original work of the author and his co-worker and PhD students, that may be useful also for specialists working in the field of bio membranes and model

Polyurethane Nanofiber Membranes for Waste Water Treatment by Membrane Distillation

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T. Jiříček

2017-01-01

Full Text Available Self-sustained electrospun polyurethane nanofiber membranes were manufactured and tested on a direct-contact membrane distillation unit in an effort to find the optimum membrane thickness to maximize flux rate and minimize heat losses across the membrane. Also salt retention and flux at high salinities up to 100 g kg−1 were evaluated. Even though the complex structure of nanofiber layers has extreme specific surface and porosity, membrane performance was surprisingly predictable; the highest flux was achieved with the thinnest membranes and the best energy efficiency was achieved with the thickest membranes. All membranes had salt retention above 99%. Nanotechnology offers the potential to find modern solutions for desalination of waste waters, by introducing new materials with revolutionary properties, but new membranes must be developed according to the target application.

Isolation and identification of java race amniotic membrane secretory leukocyte protease inhibitor gene

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Elly Munadziroh

2008-09-01

Full Text Available Background: Secretory leukocyte protease inhibitor (SLPI has been found to facilitate epithelialization, maintain a normal epithelial phenotype, reduce inflammation, secrete growth factors such as IL-4, IL-6, IL-10, EGF, FGF, TGF, HGFand 2-microbulin. SLPI is serine protease inhibitor, which found in secretions such as whole saliva, seminal fluid, cervical mucus, synovial fluid, breast milk, tears, amniotic fluid and amniotic membrane. Impaired healing states are characterized by excessive proteolysis and oftenbacterial infection, leading to the hypothesis that SLPI may have a role in the healing process in oral inflammation and contributes to tissue repair in oral mucosa. The oral wound healing response is impaired in the SLPI sufficient mice since matrix synthesis and collagen deposition delayed. The objective of this research is to isolate and identify the amniotic membrane of Java Race SLPI Gene. Methods: SLPI RNA was isolated from Java Race amniotic membrane and the cDNA was amplified by polymerase chain reaction (PCR. Result: Through sequence analyses, SLPI cDNA was 530 nucleotide in length with a predicted molecular mass about 12 kDa. The nucleotide sequence showed that human SLPI from sample was 98% identical with human SLPI from gene bank. PCR analysis revealed that the mRNA of SLPI was highly expressed in the amniotic membrane from Java Race sample. Conclusion: it is demonstrated that human SLPI are highly conserved in sequence content as compared to the human SLPI from gene.

Fouling in Membrane Distillation, Osmotic Distillation and Osmotic Membrane Distillation

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Mourad Laqbaqbi

2017-03-01

Full Text Available Various membrane separation processes are being used for seawater desalination and treatment of wastewaters in order to deal with the worldwide water shortage problem. Different types of membranes of distinct morphologies, structures and physico-chemical characteristics are employed. Among the considered membrane technologies, membrane distillation (MD, osmotic distillation (OD and osmotic membrane distillation (OMD use porous and hydrophobic membranes for production of distilled water and/or concentration of wastewaters for recovery and recycling of valuable compounds. However, the efficiency of these technologies is hampered by fouling phenomena. This refers to the accumulation of organic/inorganic deposits including biological matter on the membrane surface and/or in the membrane pores. Fouling in MD, OD and OMD differs from that observed in electric and pressure-driven membrane processes such electrodialysis (ED, membrane capacitive deionization (MCD, reverse osmosis (RO, nanofiltration (NF, ultrafiltration (UF, microfiltration (MF, etc. Other than pore blockage, fouling in MD, OD and OMD increases the risk of membrane pores wetting and reduces therefore the quantity and quality of the produced water or the concentration efficiency of the process. This review deals with the observed fouling phenomena in MD, OD and OMD. It highlights different detected fouling types (organic fouling, inorganic fouling and biofouling, fouling characterization techniques as well as various methods of fouling reduction including pretreatment, membrane modification, membrane cleaning and antiscalants application.

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

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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.

HAMLET interacts with lipid membranes and perturbs their structure and integrity.

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Mossberg, Ann-Kristin; Puchades, Maja; Halskau, Øyvind; Baumann, Anne; Lanekoff, Ingela; Chao, Yinxia; Martinez, Aurora; Svanborg, Catharina; Karlsson, Roger

2010-02-23

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

In vitro evaluation of various bioabsorbable and nonresorbable barrier membranes for guided tissue regeneration

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Smeets Ralf

2008-10-01

Full Text Available Abstract Background Different types of bioabsorbable and nonresorbable membranes have been widely used for guided tissue regeneration (GTR with its ultimate goal of regenerating lost periodontal structures. The purpose of the present study was to evaluate the biological effects of various bioabsorbable and nonresorbable membranes in cultures of primary human gingival fibroblasts (HGF, periodontal ligament fibroblasts (PDLF and human osteoblast-like (HOB cells in vitro. Methods Three commercially available collagen membranes [TutoDent® (TD, Resodont® (RD and BioGide® (BG] as well as three nonresorbable polytetrafluoroethylene (PTFE membranes [ACE (AC, Cytoplast® (CT and TefGen-FD® (TG] were tested. Cells plated on culture dishes (CD served as positive controls. The effect of the barrier membranes on HGF, PDLF as well as HOB cells was assessed by the Alamar Blue fluorometric proliferation assay after 1, 2.5, 4, 24 and 48 h time periods. The structural and morphological properties of the membranes were evaluated by scanning electron microscopy (SEM. Results The results showed that of the six barriers tested, TD and RD demonstrated the highest rate of HGF proliferation at both earlier (1 h and later (48 h time periods (P P ≤ 0.001. In HOB cell culture, the highest rate of cell proliferation was also calculated for TD at all time periods (P Conclusion Results from the present study suggested that GTR membrane materials, per se, may influence cell proliferation in the process of periodontal tissue/bone regeneration. Among the six membranes examined, the bioabsorbable membranes demonstrated to be more suitable to stimulate cellular proliferation compared to nonresorbable PTFE membranes.

Physiologically based pharmacokinetics of radioiodinated human beta-endorphin in rats. An application of the capillary membrane-limited model

Energy Technology Data Exchange (ETDEWEB)

Sato, H.; Sugiyama, Y.; Sawada, Y.; Iga, T.; Hanano, M.

1987-07-01

In order to simulate the distribution and elimination of radioiodinated human beta-endorphin (/sup 125/I-beta-EP) after iv bolus injection in rats, we proposed a physiologically based pharmacokinetic model incorporating diffusional transport of /sup 125/I-beta-EP across the capillary membrane. This model assumes that the distribution of /sup 125/I-beta-EP is restricted only within the blood and the tissue interstitial fluid, and that a diffusional barrier across the capillary membrane exists in each tissue except the liver. The tissue-to-blood partition coefficients were estimated from the ratios of the concentration in tissues to that in arterial plasma at the terminal (pseudoequilibrium) phase. The total body plasma clearance (9.0 ml/min/kg) was appropriately assigned to the liver and kidney. The transcapillary diffusion clearances of /sup 125/I-beta-EP were also estimated and shown to correlate linearly with that of inulin in several tissues. Numerically solving the mass-balance differential equations as to plasma and each tissue simultaneously, simulated concentration curves of /sup 125/I-beta-EP corresponded well with the observed data. It was suggested by the simulation that the initial rapid disappearance of /sup 125/I-beta-EP from plasma after iv injection could be attributed in part to the transcapillary diffusion of the peptide.

Basally activated nonselective cation currents regulate the resting membrane potential in human and monkey colonic smooth muscle

Science.gov (United States)

Dwyer, Laura; Rhee, Poong-Lyul; Lowe, Vanessa; Zheng, Haifeng; Peri, Lauren; Ro, Seungil; Sanders, Kenton M.

2011-01-01

Resting membrane potential (RMP) plays an important role in determining the basal excitability of gastrointestinal smooth muscle. The RMP in colonic muscles is significantly less negative than the equilibrium potential of K+, suggesting that it is regulated not only by K+ conductances but by inward conductances such as Na+ and/or Ca2+. We investigated the contribution of nonselective cation channels (NSCC) to the RMP in human and monkey colonic smooth muscle cells (SMC) using voltage- and current-clamp techniques. Qualitative reverse transcriptase-polymerase chain reaction was performed to examine potential molecular candidates for these channels among the transient receptor potential (TRP) channel superfamily. Spontaneous transient inward currents and holding currents were recorded in human and monkey SMC. Replacement of extracellular Na+ with equimolar tetraethylammonium or Ca2+ with Mn2+ inhibited basally activated nonselective cation currents. Trivalent cations inhibited these channels. Under current clamp, replacement of extracellular Na+ with N-methyl-d-glucamine or addition of trivalent cations caused hyperpolarization. Three unitary conductances of NSCC were observed in human and monkey colonic SMC. Molecular candidates for basally active NSCC were TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 in human and monkey SMC. Comparison of the biophysical properties of these TRP channels with basally active NSCC (bINSCC) suggests that TRPM4 and specific TRPC heteromultimer combinations may underlie the three single-channel conductances of bINSCC. In conclusion, these findings suggest that basally activated NSCC contribute to the RMP in human and monkey colonic SMC and therefore may play an important role in determining basal excitability of colonic smooth muscle. PMID:21566016

Flow and fouling in membrane filters: Effects of membrane morphology

Science.gov (United States)

Sanaei, Pejman; Cummings, Linda J.

2015-11-01

Membrane filters are widely-used in microfiltration applications. Many types of filter membranes are produced commercially, for different filtration applications, but broadly speaking the requirements are to achieve fine control of separation, with low power consumption. The answer to this problem might seem obvious: select the membrane with the largest pore size and void fraction consistent with the separation requirements. However, membrane fouling (an inevitable consequence of successful filtration) is a complicated process, which depends on many parameters other than membrane pore size and void fraction; and which itself greatly affects the filtration process and membrane functionality. In this work we formulate mathematical models that can (i) account for the membrane internal morphology (internal structure, pore size & shape, etc.); (ii) fouling of membranes with specific morphology; and (iii) make some predictions as to what type of membrane morphology might offer optimum filtration performance.

The memory formalism in the diffusion of drugs through skin membrane

Energy Technology Data Exchange (ETDEWEB)

Caputo, Michele; Cametti, Cesare, E-mail: cesare.cametti@roma1.infn.i [Dipartimento di Fisica, Universita di Roma ' La Sapienza' , Piazzale A. Moro 2, I-00185 Rome (Italy) and CNR-INFM-SOFT, Unita di Roma1 Rome (Italy)

2009-06-21

The diffusion of drugs across a composite structure such as a biological membrane is a rather complex phenomenon, where the assumptions on which the Fick equations are based are not always true, because of the inhomogeneous nature of the lipid membrane, the diffusion rate and the solubility of the drug being strongly dependent on the local position across the membrane. These problems are particularly strengthened in composite structures of a considerable thickness such as the human skin, where the high heterogeneity provokes the transport through different simultaneous pathways. In this note, we generalize the diffusion model based on Fick's second equation by the introduction of a space-dependent diffusion constant within the memory formalism (diffusion with memory) approach. The model predictions have been compared with experimental results concerning the permeation of two different compounds through human skin in vivo, such as piroxicam, an anti-inflammatory drug and 4-cyanophenol, a test chemical model compound. In both cases, reasonably good agreement has been found.

The memory formalism in the diffusion of drugs through skin membrane

International Nuclear Information System (INIS)

Caputo, Michele; Cametti, Cesare

2009-01-01

The diffusion of drugs across a composite structure such as a biological membrane is a rather complex phenomenon, where the assumptions on which the Fick equations are based are not always true, because of the inhomogeneous nature of the lipid membrane, the diffusion rate and the solubility of the drug being strongly dependent on the local position across the membrane. These problems are particularly strengthened in composite structures of a considerable thickness such as the human skin, where the high heterogeneity provokes the transport through different simultaneous pathways. In this note, we generalize the diffusion model based on Fick's second equation by the introduction of a space-dependent diffusion constant within the memory formalism (diffusion with memory) approach. The model predictions have been compared with experimental results concerning the permeation of two different compounds through human skin in vivo, such as piroxicam, an anti-inflammatory drug and 4-cyanophenol, a test chemical model compound. In both cases, reasonably good agreement has been found.

Autogenous bone graft and ePTFE membrane in the treatment of peri-implantitis. II. Stereologic and histologic observations in cynomolgus monkeys

DEFF Research Database (Denmark)

Schou, Søren; Holmstrup, Palle; Skovgaard, Lene Theil

2003-01-01

autogenous bone graft; guided bone regeneration; histology; membrane; non-human primates; oral implants; osseointegration; pathalogy; peri-implantitis; stereology; treatment......autogenous bone graft; guided bone regeneration; histology; membrane; non-human primates; oral implants; osseointegration; pathalogy; peri-implantitis; stereology; treatment...

Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase

International Nuclear Information System (INIS)

Roberts, W.L.; Rosenberry, T.L.

1986-01-01

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase

Kir2.1 channels set two levels of resting membrane potential with inward rectification.

Science.gov (United States)

Chen, Kuihao; Zuo, Dongchuan; Liu, Zheng; Chen, Haijun

2018-04-01

Strong inward rectifier K + channels (Kir2.1) mediate background K + currents primarily responsible for maintenance of resting membrane potential. Multiple types of cells exhibit two levels of resting membrane potential. Kir2.1 and K2P1 currents counterbalance, partially accounting for the phenomenon of human cardiomyocytes in subphysiological extracellular K + concentrations or pathological hypokalemic conditions. The mechanism of how Kir2.1 channels contribute to the two levels of resting membrane potential in different types of cells is not well understood. Here we test the hypothesis that Kir2.1 channels set two levels of resting membrane potential with inward rectification. Under hypokalemic conditions, Kir2.1 currents counterbalance HCN2 or HCN4 cation currents in CHO cells that heterologously express both channels, generating N-shaped current-voltage relationships that cross the voltage axis three times and reconstituting two levels of resting membrane potential. Blockade of HCN channels eliminated the phenomenon in K2P1-deficient Kir2.1-expressing human cardiomyocytes derived from induced pluripotent stem cells or CHO cells expressing both Kir2.1 and HCN2 channels. Weakly inward rectifier Kir4.1 or inward rectification-deficient Kir2.1•E224G mutant channels do not set such two levels of resting membrane potential when co-expressed with HCN2 channels in CHO cells or when overexpressed in human cardiomyocytes derived from induced pluripotent stem cells. These findings demonstrate a common mechanism that Kir2.1 channels set two levels of resting membrane potential with inward rectification by balancing inward currents through different cation channels such as hyperpolarization-activated HCN channels or hypokalemia-induced K2P1 leak channels.

Fluorescent and radiolabelling of pepsin-digested human glomerular basement membrane with a newly developed hydroxy-coumarin derivative (CASE)

International Nuclear Information System (INIS)

Rand-Weaver, M.; Abuknesha, R.A.; Price, R.G.

1985-01-01

The labelling of pepsin-digested human glomerular basement membrane (pHGBM) with a newly developed fluorescent iodine acceptor 7-hydroxy-coumarin-3-acetic acid N-hydroxysucciniimydyl ester (CASE) is described. The binding of a monoclonal antibody to pHGBM was assessed by radiobinding assays, and when directly iodinated pHGBM was used there was no apparent binding. When CASE was conjugated to pHGBM prior to iodination 11% binding was achieved. CASE acting as an iodine acceptor may be useful for proteins containing few or inaccessible tyrosine residues or which are destroyed by introduction of 125 I. Since CASE is fluorescent, small amounts of material can be detected during isolation prior to iodination. (orig.)

Fundamentals of membrane bioreactors materials, systems and membrane fouling

CERN Document Server

Ladewig, Bradley

2017-01-01

This book provides a critical, carefully researched, up-to-date summary of membranes for membrane bioreactors. It presents a comprehensive and self-contained outline of the fundamentals of membrane bioreactors, especially their relevance as an advanced water treatment technology. This outline helps to bring the technology to the readers’ attention, and positions the critical topic of membrane fouling as one of the key impediments to its more widescale adoption. The target readership includes researchers and industrial practitioners with an interest in membrane bioreactors.

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.

Structure of the Human Mitochondrial Ribosome Studied In Situ by Cryoelectron Tomography.

Science.gov (United States)

Englmeier, Robert; Pfeffer, Stefan; Förster, Friedrich

2017-10-03

Mitochondria maintain their own genome and its corresponding protein synthesis machine, the mitochondrial ribosome (mitoribosome). Mitoribosomes primarily synthesize highly hydrophobic proteins of the inner mitochondrial membrane. Recent studies revealed the complete structure of the isolated mammalian mitoribosome, but its mode of membrane association remained hypothetical. In this study, we used cryoelectron tomography to visualize human mitoribosomes in isolated mitochondria. The subtomogram average of the membrane-associated human mitoribosome reveals a single major contact site with the inner membrane, mediated by the mitochondria-specific protein mL45. A second rRNA-mediated contact site that is present in yeast is absent in humans, resulting in a more variable association of the human mitoribosome with the inner membrane. Despite extensive structural differences of mammalian and fungal mitoribosomal structure, the principal organization of peptide exit tunnel and the mL45 homolog remains invariant, presumably to align the mitoribosome with the membrane-embedded insertion machinery. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interactions of Ras proteins with the plasma membrane and their roles in signaling.

Science.gov (United States)

Eisenberg, Sharon; Henis, Yoav I

2008-01-01

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

Adaptive silicone-membrane lenses: planar vs. shaped membrane

CSIR Research Space (South Africa)

Schneider, F

2009-08-01

Full Text Available Engineering, Georges-Koehler-Allee 102, Freiburg 79110, Germany florian.schneider@imtek.uni-freiburg.de ABSTRACT We compare the performance and optical quality of two types of adaptive fluidic silicone-membrane lenses. The membranes feature either a...-membrane lenses: planar vs. shaped membrane Florian Schneider1,2, Philipp Waibel2 and Ulrike Wallrabe2 1 CSIR, Materials Science and Manufacturing, PO Box 395, Pretoria 0001, South Africa 2 University of Freiburg – IMTEK, Department of Microsystems...

Radiation Interaction with Therapeutic Drugs and Cell Membranes

International Nuclear Information System (INIS)

Martin, Diana I.; Manaila, Elena N.; Matei, Constantin I.; Iacob, Nicusor I.; Ighigeanu, Daniel I.; Craciun, Gabriela D.; Moisescu, Mihaela I.; Savopol, Tudor D.; Kovacs, Eugenia A.; Cinca, Sabin A.; Margaritescu, Irina D.

2007-01-01

This transient permeabilized state of the cell membrane, named the 'cell electroporation' (CE) can be used to increase cells uptake of drugs that do not readily pass cell membrane, thus enabling their cytotoxicity. The anticancer drugs, such as bleomycin (BL) and cisplatin, are the most candidates for the combined use with ionizing and non-ionizing radiation fields. The methods and installations for the cell electroporation by electron beam (EB) and microwave (MW) irradiation are presented. The viability tests of the human leukocytes under EB and MW exposure with/without the BL in the cell cultures are discussed

Degradation of Polypropylene Membranes Applied in Membrane Distillation Crystallizer

Directory of Open Access Journals (Sweden)

Marek Gryta

2016-03-01

Full Text Available The studies on the resistance to degradation of capillary polypropylene membranes assembled in a membrane crystallizer were performed. The supersaturation state of salt was achieved by evaporation of water from the NaCl saturated solutions using membrane distillation process. A high feed temperature (363 K was used in order to enhance the degradation effects and to shorten the test times. Salt crystallization was carried out by the application of batch or fluidized bed crystallizer. A significant membrane scaling was observed regardless of the method of realized crystallization. The SEM-EDS, DSC, and FTIR methods were used for investigations of polypropylene degradation. The salt crystallization onto the membrane surface accelerated polypropylene degradation. Due to a polymer degradation, the presence of carbonyl groups on the membranes’ surface was identified. Besides the changes in the chemical structure a significant mechanical damage of the membranes, mainly caused by the internal scaling, was also found. As a result, the membranes were severely damaged after 150 h of process operation. A high level of salt rejection was maintained despite damage to the external membrane surface.

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....

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

Science.gov (United States)

Riedl, Sabrina; Leber, Regina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

2015-11-01

Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity. Copyright © 2015. Published by Elsevier B.V.

Introducing Membrane Charge and Membrane Potential to T Cell Signaling

Directory of Open Access Journals (Sweden)

Yuanqing Ma

2017-11-01

Full Text Available While membrane models now include the heterogeneous distribution of lipids, the impact of membrane charges on regulating the association of proteins with the plasma membrane is often overlooked. Charged lipids are asymmetrically distributed between the two leaflets of the plasma membrane, resulting in the inner leaflet being negatively charged and a surface potential that attracts and binds positively charged ions, proteins, and peptide motifs. These interactions not only create a transmembrane potential but they can also facilitate the formation of charged membrane domains. Here, we reference fields outside of immunology in which consequences of membrane charge are better characterized to highlight important mechanisms. We then focus on T cell receptor (TCR signaling, reviewing the evidence that membrane charges and membrane-associated calcium regulate phosphorylation of the TCR–CD3 complex and discuss how the immunological synapse exhibits distinct patterns of membrane charge distribution. We propose that charged lipids, ions in solution, and transient protein interactions form a dynamic equilibrium during T cell activation.

Polyazole hollow fiber membranes for direct contact membrane distillation

KAUST Repository

Maab, Husnul; Alsaadi, Ahmad Salem; Francis, Lijo; Livazovic, Sara; Ghaffour, NorEddine; Amy, Gary L.; Nunes, Suzana Pereira

2013-01-01

Porous hollow fiber membranes were fabricated from fluorinated polyoxadiazole and polytriazole by a dry-wet spinning method for application in desalination of Red Sea water by direct contact membrane distillation (DCMD). The data were compared with commercially available hollow fiber MD membranes prepared from poly(vinylidene fluoride). The membranes were characterized by electron microscopy, liquid entry pressure (LEP), and pore diameter measurements. Finally, the hollow fiber membranes were tested for DCMD. Salt selectivity as high as 99.95% and water fluxes as high as 35 and 41 L m -2 h-1 were demonstrated, respectively, for polyoxadiazole and polytriazole hollow fiber membranes, operating at 80 C feed temperature and 20 C permeate. © 2013 American Chemical Society.

Polyazole hollow fiber membranes for direct contact membrane distillation

KAUST Repository

Maab, Husnul

2013-08-07

Porous hollow fiber membranes were fabricated from fluorinated polyoxadiazole and polytriazole by a dry-wet spinning method for application in desalination of Red Sea water by direct contact membrane distillation (DCMD). The data were compared with commercially available hollow fiber MD membranes prepared from poly(vinylidene fluoride). The membranes were characterized by electron microscopy, liquid entry pressure (LEP), and pore diameter measurements. Finally, the hollow fiber membranes were tested for DCMD. Salt selectivity as high as 99.95% and water fluxes as high as 35 and 41 L m -2 h-1 were demonstrated, respectively, for polyoxadiazole and polytriazole hollow fiber membranes, operating at 80 C feed temperature and 20 C permeate. © 2013 American Chemical Society.

Effect of pinacidil on norepinephrine- and potassium-induced contractions and membrane potential in rat and human resistance vessels and in rat aorta

International Nuclear Information System (INIS)

Videbaek, L.M.; Aalkjaer, C.; Mulvany, M.J.

1988-01-01

The effect of pinacidil on contractile responses to norepinephrine, potassium, and membrane potential was examined in rat and human resistance vessels. In some experiments rat aorta was also used. Pinacidil (0.1-30 microM) caused a concentration-dependent relaxation of norepinephrine-induced contractions in all vessels studied. In the same concentration range, pinacidil had only little effect on potassium (125 mM) activated rat mesenteric and femoral resistance vessels. In denervated rat mesenteric resistance vessels, a depolarization with potassium (125 mM) before superimposing a norepinephrine tone markedly diminished the effect of pinacidil. In resting rat mesenteric resistance vessels, pinacidil (1-10 microM) caused a hyperpolarization of 10-15 mV. In rat aorta, pinacidil (10 microM) caused a significant (p less than 0.001) increase in 86 Rb+ efflux rate constant whereas 1 microM had no effect. The results of these experiments indicate that the vasodilating effect may be caused by a hyperpolarization of the vascular smooth muscle cell membrane

Pichia pastoris: a recombinant microfactory for antibodies and human membrane proteins.

Science.gov (United States)

Gonçalves, A M; Pedro, A Q; Maia, C; Sousa, F; Queiroz, J A; Passarinha, L A

2013-05-01

During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

International Nuclear Information System (INIS)

Hu Chuan; Hardee, Deborah; Minnear, Fred

2007-01-01

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins

The effect of natural and synthetic fatty acids on membrane structure, microdomain organization, cellular functions and human health.

Science.gov (United States)

Ibarguren, Maitane; López, David J; Escribá, Pablo V

2014-06-01

This review deals with the effects of synthetic and natural fatty acids on the biophysical properties of membranes, and on their implication on cell function. Natural fatty acids are constituents of more complex lipids, like triacylglycerides or phospholipids, which are used by cells to store and obtain energy, as well as for structural purposes. Accordingly, natural and synthetic fatty acids may modify the structure of the lipid membrane, altering its microdomain organization and other physical properties, and provoking changes in cell signaling. Therefore, by modulating fatty acids it is possible to regulate the structure of the membrane, influencing the cell processes that are reliant on this structure and potentially reverting pathological cell dysfunctions that may provoke cancer, diabetes, hypertension, Alzheimer's and Parkinson's disease. The so-called Membrane Lipid Therapy offers a strategy to regulate the membrane composition through drug administration, potentially reverting pathological processes by re-adapting cell membrane structure. Certain fatty acids and their synthetic derivatives are described here that may potentially be used in such therapies, where the cell membrane itself can be considered as a target to combat disease. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

TiO2-Based Phosphoproteomic Analysis of the Plasma Membrane and the Effects of Phosphatase Inhibitor Treatment

DEFF Research Database (Denmark)

Thingholm, Tine; Larsen, Martin Røssel; Ingrell, Christian

2008-01-01

Phosphorylation of plasma membrane proteins frequently initiates signal transduction pathways or attenuate plasma membrane transport processes. Because of the low abundance and hydrophobic features of many plasma membrane proteins and the low stoichiometry of protein phosphorylation, studies...... of the plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO 2-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing....... We used sucrose centrifugation in combination with sodium carbonate extraction to achieve efficient and reproducible purification of low microgram levels of plasma membrane proteins from human mesenchymal stem cells (hMSCs, 10 (7) cells), achieving more than 70% yield of membrane proteins...

Plasma membrane proteomics and its application in clinical cancer biomarker discovery

DEFF Research Database (Denmark)

Leth-Larsen, Rikke; Lund, Rikke; Ditzel, Henrik J

2010-01-01

Plasma membrane proteins that are exposed on the cell surface have important biological functions, such as signaling into and out of the cells, ion transport, and cell-cell and cell-matrix interactions. The expression level of many of the plasma membrane proteins involved in these key functions...... targeted by protein drugs, such as human antibodies, that have enhanced survival of several groups of cancer patients. The combination of novel analytical approaches and subcellular fractionation procedures has made it possible to study the plasma membrane proteome in more detail, which will elucidate...... cancer biology, particularly metastasis, and guide future development of novel drug targets. The technical advances in plasma membrane proteomics and the consequent biological revelations will be discussed herein. Many of the advances have been made using cancer cell lines, but because the main goal...

Protective Effects of Ferulic Acid on High Glucose-Induced Protein Glycation, Lipid Peroxidation, and Membrane Ion Pump Activity in Human Erythrocytes.

Directory of Open Access Journals (Sweden)

Weerachat Sompong

Full Text Available Ferulic acid (FA is the ubiquitous phytochemical phenolic derivative of cinnamic acid. Experimental studies in diabetic models demonstrate that FA possesses multiple mechanisms of action associated with anti-hyperglycemic activity. The mechanism by which FA prevents diabetes-associated vascular damages remains unknown. The aim of study was to investigate the protective effects of FA on protein glycation, lipid peroxidation, membrane ion pump activity, and phosphatidylserine exposure in high glucose-exposed human erythrocytes. Our results demonstrated that FA (10-100 μM significantly reduced the levels of glycated hemoglobin (HbA1c whereas 0.1-100 μM concentrations inhibited lipid peroxidation in erythrocytes exposed to 45 mM glucose. This was associated with increased glucose consumption. High glucose treatment also caused a significant reduction in Na+/K+-ATPase activity in the erythrocyte plasma membrane which could be reversed by FA. Furthermore, we found that FA (0.1-100 μM prevented high glucose-induced phosphatidylserine exposure. These findings provide insights into a novel mechanism of FA for the prevention of vascular dysfunction associated with diabetes.

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

Science.gov (United States)

Quon, Evan; Beh, Christopher T.

2015-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

Liver plasma membranes: an effective method to analyze membrane proteome.

Science.gov (United States)

Cao, Rui; Liang, Songping

2012-01-01

Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

Membrane Biophysics

CERN Document Server

Ashrafuzzaman, Mohammad

2013-01-01

Physics, mathematics and chemistry all play a vital role in understanding the true nature and functioning of biological membranes, key elements of living processes. Besides simple spectroscopic observations and electrical measurements of membranes we address in this book the phenomena of coexistence and independent existence of different membrane components using various theoretical approaches. This treatment will be helpful for readers who want to understand biological processes by applying both simple observations and fundamental scientific analysis. It provides a deep understanding of the causes and effects of processes inside membranes, and will thus eventually open new doors for high-level pharmaceutical approaches towards fighting membrane- and cell-related diseases.

Rapid and simple pretreatment of human body fluids using electromembrane extraction across supported liquid membrane for capillary electrophoretic determination of lithium.

Science.gov (United States)

Strieglerová, Lenka; Kubáň, Pavel; Boček, Petr

2011-05-01

Electromembrane extraction was used for simultaneous sample cleanup and preconcentration of lithium from untreated human body fluids. The sample of a body fluid was diluted 100 times with 0.5 mM Tris solution and lithium was extracted by electromigration through a supported liquid membrane composed of 1-octanol into 100 mM acetic acid acceptor solution. Matrix compounds, such as proteins, red blood cells, and other high-molecular-weight compounds were efficiently retained on the supported liquid membrane. The liquid membrane was anchored in pores of a short segment of a polypropylene hollow fiber, which represented a low cost, single use, disposable extraction unit and was discarded after each use. Acceptor solutions were analyzed using capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C(4) D) and baseline separation of lithium was achieved in a background electrolyte solution consisting of 18 mM L-histidine and 40 mM acetic acid at pH 4.6. Repeatability of the electromembrane extraction-CE-C(4) D method was evaluated for the determination of lithium in standard solutions and real samples and was better than 0.6 and 8.2% for migration times and peak areas, respectively. The concentration limit of detection of 9 nM was achieved. The developed method was applied to the determination of lithium in urine, blood serum, blood plasma, and whole blood at both endogenous and therapeutic concentration levels. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fast iodide-SAD phasing for high-throughput membrane protein structure determination.

Science.gov (United States)

Melnikov, Igor; Polovinkin, Vitaly; Kovalev, Kirill; Gushchin, Ivan; Shevtsov, Mikhail; Shevchenko, Vitaly; Mishin, Alexey; Alekseev, Alexey; Rodriguez-Valera, Francisco; Borshchevskiy, Valentin; Cherezov, Vadim; Leonard, Gordon A; Gordeliy, Valentin; Popov, Alexander

2017-05-01

We describe a fast, easy, and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide-single-wavelength anomalous diffraction (I-SAD). The potential universality of the method is based on a common feature of membrane proteins-the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts. We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins, including a human G protein-coupled receptor (GPCR), and we show that I-SAD can be applied using data collection strategies based on either standard or serial x-ray crystallography techniques.

Human islet viability and function is maintained during high density shipment in silicone rubber membrane vessels

Science.gov (United States)

Kitzmann, Jennifer P; Pepper, Andrew R; Lopez, Boris G; Pawlick, Rena; Kin, Tatsuya; O’Gorman, Doug; Mueller, Kathryn R; Gruessner, Angelika C; Avgoustiniatos, Efstathios S; Karatzas, Theodore; Szot, Greg L; Posselt, Andrew M; Stock, Peter G; Wilson, John R; Shapiro, AM; Papas, Klearchos K

2014-01-01

The shipment of human islets from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function in transit is critically important. Gas-permeable silicone rubber membrane (SRM) vessels reduce the risk of hypoxia-induced death or dysfunction during high-density islet culture or shipment. SRM vessels may offer additional advantages: they are cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment). Human islets(IE) were isolated from two manufacturing centers and shipped in 10cm2 surface area SRM vessels in temperature and pressure controlled containers to a distant center following at least two days of culture (n = 6). Three conditions were examined: low density (LD), high density (HD), and a micro centrifuge tube negative control (NC). LD was designed to mimic the standard culture density for human islet preparations (200 IE/cm2), while HD was designed to have a 20-fold higher tissue density, which would enable the culture of an entire human isolation in 1–3 vessels. Upon receipt, islets were assessed for viability, measured by oxygen consumption rate normalized to DNA content (OCR/DNA), and quantity, measured by DNA, and, when possible, potency and function with dynamic glucose-stimulated insulin secretion (GSIS) measurements and transplants in immunodeficient B6 rag mice. Post-shipment OCR/DNA was not reduced in HD versus LD, and was substantially reduced in the NC condition. HD islets exhibited normal function post-shipment. Based on the data we conclude that entire islet isolations (up to 400,000 IE) may be shipped using a single, larger SRM vessel with no negative effect on viability and ex vivo and in vivo function. PMID:25131090

Polyurethane Nanofiber Membranes for Waste Water Treatment by Membrane Distillation

OpenAIRE

Jiříček, T.; Komárek, M.; Lederer, T.

2017-01-01

Self-sustained electrospun polyurethane nanofiber membranes were manufactured and tested on a direct-contact membrane distillation unit in an effort to find the optimum membrane thickness to maximize flux rate and minimize heat losses across the membrane. Also salt retention and flux at high salinities up to 100 g kg−1 were evaluated. Even though the complex structure of nanofiber layers has extreme specific surface and porosity, membrane performance was surprisingly predictable; the highest ...

Direct Cytoskeleton Forces Cause Membrane Softening in Red Blood Cells

Science.gov (United States)

Rodríguez-García, Ruddi; López-Montero, Iván; Mell, Michael; Egea, Gustavo; Gov, Nir S.; Monroy, Francisco

2015-01-01

Erythrocytes are flexible cells specialized in the systemic transport of oxygen in vertebrates. This physiological function is connected to their outstanding ability to deform in passing through narrow capillaries. In recent years, there has been an influx of experimental evidence of enhanced cell-shape fluctuations related to metabolically driven activity of the erythroid membrane skeleton. However, no direct observation of the active cytoskeleton forces has yet been reported to our knowledge. Here, we show experimental evidence of the presence of temporally correlated forces superposed over the thermal fluctuations of the erythrocyte membrane. These forces are ATP-dependent and drive enhanced flickering motions in human erythrocytes. Theoretical analyses provide support for a direct force exerted on the membrane by the cytoskeleton nodes as pulses of well-defined average duration. In addition, such metabolically regulated active forces cause global membrane softening, a mechanical attribute related to the functional erythroid deformability. PMID:26083919

Nanodisc-solubilized membrane protein library reflects the membrane proteome

OpenAIRE

Marty, Michael T.; Wilcox, Kyle C.; Klein, William L.; Sligar, Stephen G.

2013-01-01

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membr...

Impact of sludge flocs on membrane fouling in membrane bioreactors

DEFF Research Database (Denmark)

Christensen, Morten Lykkegaard; Niessen, Wolfgang; Jørgensen, Mads Koustrup

Membrane bioreactors (MBR) are widely used for wastewater treatment, but membrane fouling reduces membrane performance and thereby increases the cost for membranes and fouling control. Large variation in filtration properties measured as flux decline was observed for the different types of sludges....... Further, the flux could partly be reestablished after the relaxation period depending on the sludge composition. The results underline that sludge properties are important for membrane fouling and that control of floc properties, as determined by the composition of the microbial communities...... and the physico-chemical properties, is an efficient method to reduce membrane fouling in the MBR. High concentration of suspended extracellular substances (EPS) and small particles (up to 10 µm) resulted in pronounced fouling propensity. The membrane fouling resistance was reduced at high concentration...

G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

Science.gov (United States)

Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

2017-09-01

G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

Physiologically-Relevant Modes of Membrane Interactions by the Human Antimicrobial Peptide, LL-37, Revealed by SFG Experiments

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Ding, Bei; Soblosky, Lauren; Nguyen, Khoi; Geng, Junqing; Yu, Xinglong; Ramamoorthy, Ayyalusamy; Chen, Zhan

2013-05-01

Antimicrobial peptides (AMPs) could become the next generation antibiotic compounds which can overcome bacterial resistance by disrupting cell membranes and it is essential to determine the factors underlying its mechanism of action. Although high-resolution NMR and other biological studies have provided valuable insights, it has been a major challenge to follow the AMP-membrane interactions at physiologically-relevant low peptide concentrations. In this study, we demonstrate a novel approach to overcome this major limitation by performing Sum Frequency Generation (SFG) vibrational spectroscopic experiments on lipid bilayers containing an AMP, LL-37. Our results demonstrate the power of SFG to study non-linear helical peptides and also infer that lipid-peptide interaction and the peptide orientation depend on the lipid membrane composition. The observed SFG signal changes capture the aggregating process of LL-37 on membrane. In addition, our SFG results on cholesterol-containing lipid bilayers indicate the inhibition effect of cholesterol on peptide-induced membrane permeation process.

Pretreatment and Membrane Hydrophilic Modification to Reduce Membrane Fouling

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Huaqiang Chu

2013-09-01

Full Text Available The application of low pressure membranes (microfiltration/ultrafiltration has undergone accelerated development for drinking water production. However, the major obstacle encountered in its popularization is membrane fouling caused by natural organic matter (NOM. This paper firstly summarizes the two factors causing the organic membrane fouling, including molecular weight (MW and hydrophilicity/hydrophobicity of NOM, and then presents a brief introduction of the methods which can prevent membrane fouling such as pretreatment of the feed water (e.g., coagulation, adsorption, and pre-oxidation and membrane hydrophilic modification (e.g., plasma modification, irradiation grafting modification, surface coating modification, blend modification, etc.. Perspectives of further research are also discussed.

Molecular cloning of complementary DNAs encoding the heavy chain of the human 4F2 cell-surface antigen: a type II membrane glycoprotein involved in normal and neoplastic cell growth

International Nuclear Information System (INIS)

Quackenbush, E.; Clabby, M.; Gottesdiener, K.M.; Barbosa, J.; Jones, N.H.; Strominger, J.L.; Speck, S.; Leiden, J.M.

1987-01-01

Complementary DNA (cDNA) clones encoding the heavy chain of the heterodimeric human membrane glycoprotein 4F2 have been isolated by immunoscreening of a λgt11 expression library. The identity of these clones has been confirmed by hybridization to RNA and DNA prepared from mouse L-cell transfectants, which were produced by whole cell gene transfer and selected for cell-surface expression of the human 4F2 heavy chain. DNA sequence analysis suggest that the 4F2 heavy-chain cDNAs encode an approximately 526-amino acid type II membrane glycoprotein, which is composed of a large C-terminal extracellular domain, a single potential transmembrane region, and a 50-81 amino acid N-terminal intracytoplasmic domain. Southern blotting experiments have shown that the 4F2 heavy-chain cDNAs are derived from a single-copy gene that has been highly conserved during mammalian evolution

Recent developments on ion-exchange membranes and electro-membrane processes.

Science.gov (United States)

Nagarale, R K; Gohil, G S; Shahi, Vinod K

2006-02-28

Rapid growth of chemical and biotechnology in diversified areas fuels the demand for the need of reliable green technologies for the down stream processes, which include separation, purification and isolation of the molecules. Ion-exchange membrane technologies are non-hazardous in nature and being widely used not only for separation and purification but their application also extended towards energy conversion devices, storage batteries and sensors etc. Now there is a quite demand for the ion-exchange membrane with better selectivities, less electrical resistance, high chemical, mechanical and thermal stability as well as good durability. A lot of work has been done for the development of these types of ion-exchange membranes during the past twenty-five years. Herein we have reviewed the preparation of various types of ion-exchange membranes, their characterization and applications for different electro-membrane processes. Primary attention has been given to the chemical route used for the membrane preparation. Several general reactions used for the preparation of ion-exchange membranes were described. Methodologies used for the characterization of these membranes and their applications were also reviewed for the benefit of readers, so that they can get all information about the ion-exchange membranes at one platform. Although there are large number of reports available regarding preparations and applications of ion-exchange membranes more emphasis were predicted for the usefulness of these membranes or processes for solving certain type of industrial or social problems. More efforts are needed to bring many products or processes to pilot scale and extent their applications.

A sacrificial process for fabrication of biodegradable polymer membranes with submicron thickness.

Science.gov (United States)

Beardslee, Luke A; Stolwijk, Judith; Khaladj, Dimitrius A; Trebak, Mohamed; Halman, Justin; Torrejon, Karen Y; Niamsiri, Nuttawee; Bergkvist, Magnus

2016-08-01

A new sacrificial molding process using a single mask has been developed to fabricate ultrathin 2-dimensional membranes from several biocompatible polymeric materials. The fabrication process is similar to a sacrificial microelectromechanical systems (MEMS) process flow, where a mold is created from a material that can be coated with a biodegradable polymer and subsequently etched away, leaving behind a very thin polymer membrane. In this work, two different sacrificial mold materials, silicon dioxide (SiO2 ) and Liftoff Resist (LOR) were used. Three different biodegradable materials; polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and polyglycidyl methacrylate (PGMA), were chosen as model polymers. We demonstrate that this process is capable of fabricating 200-500 nm thin, through-hole polymer membranes with various geometries, pore-sizes and spatial features approaching 2.5 µm using a mold fabricated via a single contact photolithography exposure. In addition, the membranes can be mounted to support rings made from either SU8 or PCL for easy handling after release. Cell culture compatibility of the fabricated membranes was evaluated with human dermal microvascular endothelial cells (HDMECs) seeded onto the ultrathin porous membranes, where the cells grew and formed confluent layers with well-established cell-cell contacts. Furthermore, human trabecular meshwork cells (HTMCs) cultured on these scaffolds showed similar proliferation as on flat PCL substrates, further validating its compatibility. All together, these results demonstrated the feasibility of our sacrificial fabrication process to produce biocompatible, ultra-thin membranes with defined microstructures (i.e., pores) with the potential to be used as substrates for tissue engineering applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1192-1201, 2016. © 2015 Wiley Periodicals, Inc.

Clustering on Membranes

DEFF Research Database (Denmark)

Johannes, Ludger; Pezeshkian, Weria; Ipsen, John H

2018-01-01

Clustering of extracellular ligands and proteins on the plasma membrane is required to perform specific cellular functions, such as signaling and endocytosis. Attractive forces that originate in perturbations of the membrane's physical properties contribute to this clustering, in addition to direct...... protein-protein interactions. However, these membrane-mediated forces have not all been equally considered, despite their importance. In this review, we describe how line tension, lipid depletion, and membrane curvature contribute to membrane-mediated clustering. Additional attractive forces that arise...... from protein-induced perturbation of a membrane's fluctuations are also described. This review aims to provide a survey of the current understanding of membrane-mediated clustering and how this supports precise biological functions....

Human myosin VIIA responsible for the Usher 1B syndrome: a predicted membrane-associated motor protein expressed in developing sensory epithelia.

Science.gov (United States)

Weil, D; Levy, G; Sahly, I; Levi-Acobas, F; Blanchard, S; El-Amraoui, A; Crozet, F; Philippe, H; Abitbol, M; Petit, C

1996-04-16

The gene encoding human myosin VIIA is responsible for Usher syndrome type III (USH1B), a disease which associates profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. The reconstituted cDNA sequence presented here predicts a 2215 amino acid protein with a typical unconventional myosin structure. This protein is expected to dimerize into a two-headed molecule. The C terminus of its tail shares homology with the membrane-binding domain of the band 4.1 protein superfamily. The gene consists of 48 coding exons. It encodes several alternatively spliced forms. In situ hybridization analysis in human embryos demonstrates that the myosin VIIA gene is expressed in the pigment epithelium and the photoreceptor cells of the retina, thus indicating that both cell types may be involved in the USH1B retinal degenerative process. In addition, the gene is expressed in the human embryonic cochlear and vestibular neuroepithelia. We suggest that deafness and vestibular dysfunction in USH1B patients result from a defect in the morphogenesis of the inner ear sensory cell stereocilia.

Immunohistochemical evaluation of fibrovascular and cellular pre-iridal membranes in dogs.

Science.gov (United States)

Bauer, Bianca S; Sandmeyer, Lynne S; Hall, Riley B; Grahn, Bruce H

2012-03-01

Histologically, two morphologically distinct types of pre-iridal membranes appear to occur in diseased canine globes: fibrovascular and cellular. Cellular pre-iridal membranes of corneal endothelial origin exist in iridocorneal endothelial (ICE) syndrome in humans and arise through metaplastic transformation of corneal endothelial cells into epithelial-like cells.(1) The purpose of this study was to (i) evaluate immunohistochemical staining of these two types of membranes in diseased canine globes, (ii) determine whether endothelial cell metaplasia or iridal vascular budding plays a role in cellular membrane formation and (iii) compare the primary histopathologic diagnosis between the two groups. Hematoxylin and eosin (H&E)-stained slides of 28 enucleated canine specimens with pre-iridal membranes were randomly selected and examined with light microscopy. The globes were divided into two groups based on the appearance of the membrane: fibrovascular or cellular, and the histopathologic diagnoses were recorded. Immunohistochemical staining for vimentin, cytokeratin AE1/AE3, and Von Willebrand's factor (Factor VIII) was completed on the slides of each globe. The histopathologic diagnoses were compared between the two groups. The fibrovascular and cellular membranes stained positive for vimentin and negative for cytokeratin AE1/AE3. All fibrovascular membranes stained positive for Factor VIII compared with the cellular membranes which stained negative. In the cellular membrane group, primary glaucoma was a common histologic diagnosis. Immunohistochemical evaluation in this study does not support the hypothesis of metaplastic transformation of endothelial cells into epithelial-like cells in the canine globes with cellular membranes. The cellular membranes in this study do not represent a canine version of ICE syndrome and are not of vascular endothelial origin. © 2012 American College of Veterinary Ophthalmologists.

Analysis of proton exchange membrane fuel cell performance with alternate membranes

Energy Technology Data Exchange (ETDEWEB)

Wakizoe, Masanobu; Velev, O A; Srinivasan, S [Texas A and M Univ., College Station, TX (United States). Texas Engineering Experiment Station

1995-02-01

Renewed interest in proton exchange membrane fuel cell technology for space and terrestrial (particularly electric vehicles) was stimulated by the demonstration, in the mid 1980s, of high energy efficiencies and high power densities. One of the most vital components of the PEMFC is the proton conducting membrane. In this paper, an analysis is made of the performances of PEMFCs with Dupont`s Nafion, Dow`s experimental, and Asahi Chemical`s Aciplex-S membranes. Attempts were also made to draw correlations between the PEMFC performances with the three types of membranes and their physico-chemical characteristics. Practically identical levels of performances (energy efficiency, power density, and lifetime) were achieved in PEMFCs with the Dow and the Aciplex-S membranes and these performances were better than in the PEMFCs with the Nafion-115 membrane. The electrode kinetic parameters for oxygen reduction are better for the PEMFCs with the Aciplex-S and Nafion membranes than with the Dow membranes. The PEMFCs with the Aciplex-S and Dow membranes have nearly the same internal resistances which are considerably lower than for the PEMFC with the Nafion membrane. The desired membrane characteristics to obtain high levels of performance are low equivalent weight and high water content. (Author)

Erythrocyte membrane stabilization effect and antioxidant activity of methyl methacrylate

International Nuclear Information System (INIS)

Popov, B.

2004-01-01

Methyl methacrylate (MMK) is a synthetic product with mild impact on human health that is not well studied on cellular basis. Here, human erythrocytes were used to investigate the effects MMK exerts on acid and heat-induced hemolysis. Biphasic effect of MMK was observed for acid-induced hemolysis; i.e., protection at low (0 - 0.05% v/v) and stimulation at higher (0.1- 0.4% v/v) concentrations. The maximal protective effect was produced at 0.03% (v/v). At this concentration MMK increased the temperatures of heat denaturation of erythrocyte membrane proteins, spectrin and integral proteins, by about 2 0 C and inhibited the heat-induced hemolysis by 20 %. This membrane stabilization effect of MMK is similar to that produced by some anti-inflammatory and antirheumatic drugs. The increased acid resistance possibly indicated anti-oxidant properties of MMK. The nonenzymatic antioxidant activity test evidenced that MMK has no superoxide dismutase-like activity but demonstrates strong catalase-like activity (about 900 kU/mmol at 0.05-0.1 mmol/l concentration). The results indicate that at low concentration MMK exerts benign effect on cellular membrane that could find therapeutic usage. (author)

Helicobacter pylori Outer Membrane Protein-Related Pathogenesis

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Yuichi Matsuo

2017-03-01

Full Text Available Helicobacter pylori colonizes the human stomach and induces inflammation, and in some cases persistent infection can result in gastric cancer. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and outer membrane proteins (OMPs play a pivotal role in binding to human cells. Some OMP interaction molecules are known in H. pylori, and their associated host cell responses have been gradually clarified. Many studies have demonstrated that OMPs are essential to CagA translocation into gastric cells via the Type IV secretion system of H. pylori. This review summarizes the mechanisms through which H. pylori utilizes OMPs to colonize the human stomach and how OMPs cooperate with the Type IV secretion system.

Electrospun superhydrophobic membranes with unique structures for membrane distillation.

Science.gov (United States)

Liao, Yuan; Loh, Chun-Heng; Wang, Rong; Fane, Anthony G

2014-09-24

With modest temperature demand, low operating pressure, and high solute rejection, membrane distillation (MD) is an attractive option for desalination, waste treatment, and food and pharmaceutical processing. However, large-scale practical applications of MD are still hindered by the absence of effective membranes with high hydrophobicity, high porosity, and adequate mechanical strength, which are important properties for MD permeation fluxes, stable long-term performance, and effective packing in modules without damage. This study describes novel design strategies for highly robust superhydrophobic dual-layer membranes for MD via electrospinning. One of the newly developed membranes comprises a durable and ultrathin 3-dimensional (3D) superhydrophobic skin and porous nanofibrous support whereas another was fabricated by electrospinning 3D superhydrophobic layers on a nonwoven support. These membranes exhibit superhydrophobicity toward distilled water, salty water, oil-in-water emulsion, and beverages, which enables them to be used not only for desalination but also for other processes. The superhydrophobic dual-layer membrane #3S-N with nanofibrous support has a competitive permeation flux of 24.6 ± 1.2 kg m(-2) h(-1) in MD (feed and permeate temperate were set as 333 and 293 K, respectively) due to the higher porosity of the nanofibrous scaffold. Meanwhile, the membranes with the nonwoven support exhibit greater mechanical strength due to this support combined with better long-term performance because of the thicker 3D superhydrophobic layers. The morphology, pore size, porosity, mechanical properties, and liquid enter pressure of water of these superhydrophobic composite membranes with two different structures are reported and compared with commercial polyvinylidene fluoride membranes.

Helicobacter pylori Disrupts Host Cell Membranes, Initiating a Repair Response and Cell Proliferation

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Hsueh-Fen Juan

2012-08-01

Full Text Available Helicobacter pylori (H. pylori, the human stomach pathogen, lives on the inner surface of the stomach and causes chronic gastritis, peptic ulcer, and gastric cancer. Plasma membrane repair response is a matter of life and death for human cells against physical and biological damage. We here test the hypothesis that H. pylori also causes plasma membrane disruption injury, and that not only a membrane repair response but also a cell proliferation response are thereby activated. Vacuolating cytotoxin A (VacA and cytotoxin-associated gene A (CagA have been considered to be major H. pylori virulence factors. Gastric cancer cells were infected with H. pylori wild type (vacA+/cagA+, single mutant (ΔvacA or ΔcagA or double mutant (ΔvacA/ΔcagA strains and plasma membrane disruption events and consequent activation of membrane repair components monitored. H. pylori disrupts the host cell plasma membrane, allowing localized dye and extracellular Ca²⁺ influx. Ca²⁺-triggered members of the annexin family, A1 and A4, translocate, in response to injury, to the plasma membrane, and cell surface expression of an exocytotic maker of repair, LAMP-2, increases. Additional forms of plasma membrane disruption, unrelated to H. pylori exposure, also promote host cell proliferation. We propose that H. pylori activation of a plasma membrane repair is pro-proliferative. This study might therefore provide new insight into potential mechanisms of H. pylori-induced gastric carcinogenesis.

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.

Microporous silica membranes

DEFF Research Database (Denmark)

Boffa, Vittorio; Yue, Yuanzheng

2012-01-01

Hydrothermal stability is a crucial factor for the application of microporous silica-based membranes in industrial processes. Indeed, it is well established that steam exposure may cause densification and defect formation in microporous silica membranes, which are detrimental to both membrane...... permeability and selectivity. Numerous previous studies show that microporous transition metal doped-silica membranes are hydrothermally more stable than pure silica membranes, but less permeable. Here we present a quantitative study on the impact of type and concentration of transition metal ions...... on the microporous structure, stability and permeability of amorphous silica-based membranes, providing information on how to design chemical compositions and synthetic paths for the fabrication of silica-based membranes with a well accessible and highly stabile microporous structure....

Electrokinetic migration across artificial liquid membranes. New concept for rapid sample preparation of biological fluids.

Science.gov (United States)

Pedersen-Bjergaard, Stig; Rasmussen, Knut Einar

2006-03-24

Basic drug substances were transported across a thin artificial organic liquid membrane by the application of 300 V d.c. From a 300 microl aqueous donor compartment (containing 10 mM HCl), the drugs migrated through a 200 microm artificial liquid membrane of 2-nitrophenyl octyl ether immobilized in the pores of a polypropylene hollow fiber, and into a 30 microl aqueous acceptor solution of 10 mM HCl inside the lumen of the hollow fiber. The transport was forced by an electrical potential difference sustained over the liquid membrane, resulting in electrokinetic migration of drug substances from the donor compartment to the acceptor solution. Within 5 min of operation at 300 V, pethidine, nortriptyline, methadone, haloperidol, and loperamide were extracted with recoveries in the range 70-79%, which corresponded to enrichments in the range 7.0-7.9. The chemical composition of the organic liquid membrane strongly affected the permeability, and may serve as an efficient tool for controlling the transport selectivity. Water samples, human plasma, and human urine were successfully processed, and in light of the present report, electrokinetic migration across thin artificial liquid membranes may be an interesting tool for future isolation within chemical analysis.

Composite Membrane with Underwater-Oleophobic Surface for Anti-Oil-Fouling Membrane Distillation.

Science.gov (United States)

Wang, Zhangxin; Hou, Deyin; Lin, Shihong

2016-04-05

In this study, we fabricated a composite membrane for membrane distillation (MD) by modifying a commercial hydrophobic polyvinylidene fluoride (PVDF) membrane with a nanocomposite coating comprising silica nanoparticles, chitosan hydrogel and fluoro-polymer. The composite membrane exhibits asymmetric wettability, with the modified surface being in-air hydrophilic and underwater oleophobic, and the unmodified surface remaining hydrophobic. By comparing the performance of the composite membrane and the pristine PVDF membrane in direct contact MD experiments using a saline emulsion with 1000 ppm crude oil (in water), we showed that the fabricated composite membrane was significantly more resistant to oil fouling compared to the pristine hydrophobic PVDF membrane. Force spectroscopy was conducted for the interaction between an oil droplet and the membrane surface using a force tensiometer. The difference between the composite membrane and the pristine PVDF membrane in their interaction with an oil droplet served to explain the difference in the fouling propensities between these two membranes observed in MD experiments. The results from this study suggest that underwater oleophobic coating can effectively mitigate oil fouling in MD operations, and that the fabricated composite membrane with asymmetric wettability can enable MD to desalinate hypersaline wastewater with high concentrations of hydrophobic contaminants.

Substrate specificity within a family of outer membrane carboxylate channels.

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Elif Eren

2012-01-01

Full Text Available Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.

Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

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Marc Lenoir

2015-10-01

Full Text Available The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH and Tec homology (TH domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.