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At least 91 records · Page 5Linked to original sources
PubMed1984

Membrane patches and whole-cell membranes: a comparison of electrical properties in rat clonal pituitary (GH3) cells.

A comparison has been made between the electrical properties of excised 'outside-out' patches and whole-cell membranes of GH3 cells using the patch-pipette technique. Despite a complicated surface morphology, which includes numerous microvilli, ruffles and blebs, high-resistance seals (typically greater than 10(11) omega) were consistently formed between patch pipettes and GH3 cell membranes. When the internal solution contained 120 mM-CsF, outward currents through K channels were blocked and large Na channel currents were consistently observed in the whole-cell recording mode. Using the same solutions, single Na channel currents were readily observed in outside-out patches. Averaging patch currents yielded macroscopic currents showing the same voltage-dependent kinetics as those observed for the whole-cell membrane. The current vs. voltage and inactivation time constant vs. voltage relationships for the Na channel shifted towards more negative potentials (25 mV or more) within approximately 30 min after going into the whole-cell recording mode. These same relationships could be measured for outside-out patches and their positions along the voltage axis coincided with the asymptotic values measured in the whole-cell mode. When the internal solution contained 120 mM-N-methylglucamine fluoride and the external solution contained 120 mM-Tris chloride, no ionic channel currents could be observed either for whole-cell or outside-out patch membranes. Under these conditions, displacement currents induced by tetraphenyl borate (TPB) were recorded in both types of membranes. The total charge moved showed a sigmoidal dependence upon the applied voltage for both whole-cell and outside-out patch membranes. The charge vs. voltage relationship showed a shift along the voltage axis similar to that observed for Na channels except that the magnitude of the shift was larger. A shift in this relationship was also observed for excised patches but the observable magnitude of the shift was smaller than that in the whole-cell recording mode. The asymptotic values of the charge vs. voltage relationship were similar for whole-cell and outside-out patches, as were the asymptotic values for the translocation time constants. It is concluded that there are no fundamental differences in the properties of ionic channel and displacement currents between whole-cell membranes and excised membrane patches.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

LPS inhibits endothelin-1-mediated eNOS translocation to the cell membrane in sinusoidal endothelial cells.

OBJECTIVE: The objectives of this study were to develop a model for studying endothelin-1-mediated eNOS regulation in cultured sinusoidal endothelial cells and determine the effect of endothelin-1 and endotoxin (LPS) on eNOS localization. METHODS: Changes in caveolin-1, calmodulin, and eNOS expression were determined by western blot and densitometric analysis. Endothelin receptor expression and localization and the intracellular localization of eNOS and caveolin-1 were assessed by confocal microscopy. RESULTS: Sinusoidal endothelial cells expressed caveolin-1 and calmodulin, and expression was altered in cultured and passaged cells. eNOS expression decreased significantly in 24-h cultured cells, with expression dropping below the level of detection in passaged cells. Both endothelin A and endothelin B receptors were expressed on the cell surface after 24 h in culture. In 24-h cultured cells, caveolin-1 was localized in the perinuclear region and cell membrane, while eNOS was predominantly localized in the perinuclear region, where it co-localized with caveolin-1. Endothelin-1 stimulated eNOS translocation to the cell membrane. Pretreatment with LPS markedly inhibited the endothelin-1-mediated eNOS translocation. CONCLUSIONS: These studies demonstrate an LPS-mediated uncoupling of endothelin receptor activation and eNOS translocation. This functional uncoupling may, in part, account for the hyperconstrictive effects of endothelin-1 during inflammatory conditions.

Animals↗
PubMed1975

T-cell membrane characteristics of "mycosis cells" in the skin and lymph node.

In some patients with mycosis fungoides atypical cells ("mycosis cells") are found in the blood. Recently the T-cell membrane characteristics of these atypical cells have been described. In this paper the results of a study of the atypical cells isolated from the lymph nodes and the skin lesions of three patients with mycosis fungoides are presented. Using electron microscopy, it could be demonstrated that the atypical cells formed rosettes with uncoated sheep red blood cells, but not with antibody-complement-coated sheep erythrocytes, indicating the T-cell membrane characteristics of the atypical cells.

Aged↗
PubMed1992

In vitro binding of Campylobacter jejuni/coli outer membrane preparations to INT 407 cell membranes.

Outer membrane fractions (OMs) of nine Campylobacter (C.) jejuni and two C. coli strains belonging to different serovars, from human and various animal origins, were extracted by treatment with sodium N-lauryl sarcosinate. Using n-octyl-beta-D-glucopyranoside a 42-kDa protein and a flagella-enriched fraction were obtained. The capacity of the crude bacterial OM preparations, the purified 42-kDa protein and the flagella to bind to membranes of the human embryonic intestinal cell line INT 407 was tested by an enzyme-linked immunosorbent assay. The crude OM and the 42-kDa-enriched fraction were found to bind very well to the cell membranes, whereas the flagella preparation showed only a weak binding. Using monoclonal antibodies (mAbs) with HS 2-lipopolysaccharide (LPS) specificity, binding of crude HS 2 strain OM preparations to cell membranes was detected in a significant range, whereas with flagellin-specific mAbs binding of OMs and flagella to cell membranes was only detected to a very low extent. Binding of OMs to cell membranes was inhibited by preincubation of OMs with serovar-specific mouse hyperimmune serum, whereas on preincubation with mAbs directed against LPS or flagella binding was practically not inhibited. OMs extracted after pretreatment of the bacteria with proteinase K showed an altered SDS-PAGE pattern especially for the 42-kDa protein subunit and and their capacity to bind to cell membranes was significantly reduced. The binding was also reduced by preincubation of the OMs with L-fucose or D-mannose.

Animals↗
PubMed1975

Feline oncornavirus-associated cell membrane antigen. V. Humoral immune response to virus and cell membrane antigens in cats inoculated with Gardner-Arnstein feline sarcoma virus.

Tumor growth responses in 5- to 6-week-old kittens inoculated with the Gardner-Arnstein strain of feline sarcoma virus exhibited three distinct pattern: 1) complete tumor regression or no detectable tumor growth in approximately one-third of 43 inoculated kittens, 2) rapid tumor progression which led to debilitation and death within 16.2 +/- 4.2 weeks following infection in an additional one-third, and 3) slow tumor growth or temporary regressions in the remaining third. The feline oncornavirus-associated cell membrane antigen (FOCMA) antibody response was closely correlated with tumor progression; rapid progressors had the lowest antibody titers, whereas those in the "no tumor or permanent regression" categories had the highest titers. These results agreed with those previously observed with another virus strain, the Snyder-Theilen feline sarcoma virus. Cats in the intermediate categories of tumor growth also had intermediate levels of FOCMA antibody. The presence of virus-neutralizing (VN) activity was not always correlated with anti-FOCMA activity. Animals in the rapid-progressor category, compared to the regressors or slow progressors, were more likely to have detectable VN antibody during early periods. Conversely, animals in the regressor group or group with no tumors were more likely to show an early rise in detectable anti-FOCMA activity than animals in either of the progressor groups.

Animals↗
PubMed1994

Cell membrane fluidity in K562 cells and its relation to receptor expression.

Cell membrane fluidity (CMF) and transferrin receptor (Tf-R) expression were investigated in K562 cells, a human chronic myelocytic leukemia cell line, treated by gamma-interferon (IFN-gamma). CMF was increased using spin-labeled electron spin resonance techniques, and Tf-R expression was measured by flow cytometric analysis with an EPICS-750 flow cytometer/cell sorter. Treatment of K562 cells in suspension culture with IFN-gamma for as long a time as 6 hr caused an increase in CMF, and then returned to the level of control cells at 12 hr. Conversely, by 24 hr after the beginning of treatment, the rigidity of CMF was increased. Thus, the changes of IFN-gamma-induced CMF was biphasic. While the early change of CMF is related to signal generation and transmission, the later change may reflect changes in lipid compositions and/or cytoskeletal complexes of the plasma cell membrane. A significant increase of Tf-R after 6 hr and 24 hr in number was obtained by treatment of K562 cells with IFN-gamma, but at 12 hr the number of Tf-R did not differ from the control. These results suggested that the early phase of upregulation of Tf-R induced by IFN-gamma was caused by increased CMF, and the late phase of upregulation of Tf-R was due to increased rigidity of CMF. In conclusion, the state of CMF associated with a certain receptor expression in cells is not rigid and can be modulated to some extent by exogenous influences. This may open possibilities of some adjuvant therapeutic measures in malignant diseases by increasing the antigenicity of tumor cells.

Cell Membrane↗
PubMed1991

Antigenicity and immunogenicity of an extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells.

In order to identify T-cell antigens from Histoplasma capsulatum yeast cells, we prepared a detergent extract of the cell wall and cell membrane of yeast-phase H. capsulatum G217B and analyzed its antigenicity and immunogenicity. Mice injected with viable H. capsulatum yeast cells or with 500 or 1,000 micrograms of the extract mounted a delayed-type hypersensitivity response to solubilized cell wall and cell membrane. Vaccination with this antigenic preparation conferred a protective immune response in mice that were challenged intravenously with H. capsulatum yeast cells. The extract induced in vitro proliferation by splenocytes from mice injected with either viable yeast cells or the soluble cell wall and cell membrane preparation. We also examined the profile of in vitro responses by a murine T-cell line and by cloned T cells to soluble cell wall and cell membrane by employing the technique of T-cell immunoblotting. Two prominent regions that stimulated the T-cell line and cloned T cells were identified. Fractions encompassing an area between 53 and 64 kDa caused proliferation by a T-cell line and five of six clones. Antigens recognized by the T-cell line and by three of six clones were contained in another area that extended from 69 to 82 kDa. The data demonstrate that this soluble extract from cell wall and cell membrane contains antigens recognized by T cells and mediates protective immunity. Moreover, T-cell immunoblotting provides a useful technique for mapping immunoreactive molecules from H. capsulatum yeast cells.

Animals↗
PubMed1986

Functional heterogeneity in the early distal tubule of the Amphiuma kidney: evidence for two modes of Cl- and K+ transport across the basolateral cell membrane.

Measurements of cell volume and basolateral cell membrane potential difference (Vbl) were made simultaneously and on the same cell to study chloride and potassium transport across the basolateral cell membrane of individual cells from the Amphiuma early distal tubule or diluting segment. Two distinct types of responses of Vbl are evident when either chloride is lowered or potassium is raised in the basolateral solution. In one cell type (high basolateral conductance, HBC), these maneuvers cause large depolarizations in Vbl, suggesting that the basolateral cell membrane has significant partial conductances to both chloride and to potassium. In contrast, Vbl of another group (low basolateral conductance, LBC) is much less sensitive to the same maneuvers, indicating that the partial conductance of the basolateral cell membrane is quite low. Consistent with the difference in partial conductance is the ratio of resistances, which is immeasurably high in HBC cells and averages 1.2 +/- 0.4 in LBC cells. Increasing basolateral potassium to 98 mM causes both types to swell, with HBC cells swelling approximately two times faster than LBC cells. In both cell types, potassium-induced cell swelling is inhibited by removing chloride from the perfusion solutions. In animals kept in a low-potassium environment, the number of HBC cells is approximately equal to the number of LBC cells. The proportion of LBC cells increases following potassium adaptation. This data suggests that there are at least two types of cells present in Amphiuma early distal tubule, one cell type in which both chloride and potassium move across the basolateral cell membrane primarily via a conductive pathway and another in which the movements are mostly electroneutral, probably involving a K-Cl cotransport system, and that this heterogeneity may change in response to the metabolic state of the animal.

Animals↗
PubMed1996

Red cell membrane remodeling in sickle cell anemia. Sequestration of membrane lipids and proteins in Heinz bodies.

In red cells from patients with sickle cell anemia, hemoglobin S denatures and forms Heinz bodies. Binding of Heinz bodies to the inner surface of the sickle cell membrane promotes clustering and colocalization of the membrane protein band 3, outer surface-bound autologous IgG and, to some extent, the membrane proteins glycophorin and ankyrin. Loss of transbilayer lipid asymmetry is also found in certain populations of sickle red cells. The lateral distribution of sickle cell membrane lipids has not been examined, however. In this report, we examine by fluorescence microscopy the incorporation and distribution of the fluorescent phospholipid analogues 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-phosphatidylserine and NBD-phosphatidylcholine in sickle red cells. Both phospholipid analogues are observed to accumulate prominently at sites of Heinz bodies. Accumulation at sites of Heinz bodies is also shown by 1,'1-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, a fluorescent lipid analogue that readily crosses membranes, but not by fluorescein-phosphatidylethanolamine, an analogue that is localized to the outer leaflet of the membrane. Double labeling and confocal microscopy techniques show that NBD-lipids, band 3 protein, protein 4.1, ankyrin, and spectrin are all sequestered within sickle red cells and colocalized at sites of Heinz bodies. We propose that Heinz bodies provide a hydrophobic surface on which sickle red cell membrane lipids and proteins are sequestered.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

Reconstitution of human 5-hydroxytryptamine5A receptor--G protein coupling in E. coli and Sf9 cell membranes with membranes from Sf9 cells expressing mammalian G proteins.

The human 5-hydroxytryptamine5A (h5-ht5A) receptor was expressed in Escherichia coli (h5-ht5A-E. coli) to verify its pharmacological profile in the absence of G proteins. In addition, the ability of the h5-ht5A receptor to interact with mammalian Gi/o and Gs proteins was investigated by a new reconstitution approach. Agonists displayed lower affinities for h5-ht5A-E. coli than for stably transfected h5-ht5A-HEK 293 cells, due to the absence of G protein coupling in E. coli. Lysergic acid diethylamide behaved as a neutral antagonist, showing equal affinities for the G protein-coupled and the uncoupled receptor. To analyze the G protein coupling behavior of the h5-ht5A receptor, h5-ht5A-E. coli membranes or h5-ht5A-Sf9 insect cell membranes were fused by vortexing to membranes from baculovirus-infected Sf9 cells expressing mammalian G proteins. The ability of the h5-ht5A receptor to differentiate between Gi/Go/Gz and Gs proteins was explored by investigation of agonist binding affinities and agonist-induced stimulation of [35S]GTP gamma S binding. The h5-ht5A receptor failed to interact with Gz and Gs proteins and coupled equally well to Gj and Go proteins to form a complex with high affinity for agonists. Under the applied conditions, however, Gi proteins were found to be better activated than Go proteins in the [35S]GTP gamma S binding assay.

Animals↗
PubMed1985

Immunofluorescence detection of liver cell membrane autoantibodies using PLC/PRF/5 cells.

Detection of liver cell membrane autoantibodies is routinely performed by immunofluorescence testing of patient sera on rabbit hepatocyte suspensions. We have investigated the possible use of cells from the PLC/PRF/5 human hepatoma cell-line. These cells were employed as substrate in an immunofluorescence test which was compared with the conventional rabbit hepatocyte assay. We found a close correlation between the results obtained with these different substrates on visual reading. We furthermore compared visual reading of immunofluorescence preparations with flow-cytometrical analysis of immunostained cell suspensions. The results with these different methods were largely confirmatory. The PLC/PRF/5 cells are easily available and should therefore be regarded as a highly valuable new substrate for detection of liver cell membrane autoantibodies. Flow cytometry appears to be a technically simple and reliable method for quantitative analysis.

Animals↗
PubMed2005

Critical illness with hyponatraemia and impaired cell membrane integrity--the "sick cell syndrome" revisited.

OBJECTIVE: To determine whether impaired cell membrane permeability exists in critically ill patients with "sick cell" type hyponatraemia. DESIGN AND METHODS: A 36 year old male patient was identified in an intensive care unit (ICU) with liver disease and multi-organ failure. His initial serum sodium (Na) was 101 mmol/L and osmolar gap + 35 mmol/L. A flow cytometric system was used to assess lymphocyte membrane integrity using fluorescein diacetate (FDA) and propidium iodide (PI). Following this, similar studies were carried out in 17 hyponatraemic (Na < 130 mmol/L) and 19 normonatraemic (Na > 136 mmol/L) ICU patients. RESULTS: Flow cytometry in the index patient showed two clear populations of cells-one was normal (with identical characteristics to a healthy control) and the other had dysfunctional cell membrane integrity. The extended patient series, however, revealed only 2 other patients with similar flow cytometric patterns-one hyponatraemic and one normonatraemic. CONCLUSIONS: Cell membrane studies in the index patient demonstrated supportive evidence for the "sick cell syndrome" in critically ill patients. The extended series revealed that 3/37 (8%) had this abnormality, which was however not consistently associated with hyponatraemia.

Adult↗
PubMed1967

Effect of some proteins on the yeast cell membrane.

Yeast cells, Candida utilis, in water suspension and in the absence of electrolytes were found to be very sensitive to several proteins of moderate size, including ribonuclease, protamine, lysozyme, bovine serum albumin, cytochrome c, and myoglobin. Viability ceases rapidly, and ultraviolet-absorbing compounds (260 mmu) and the amino acid pool are released into the medium. The ultraviolet-absorbing material appears to be the nucleotide and coenzyme fraction usually extracted by 0.2 n perchloric acid at low temperature. The ribonucleic acid fraction remains in the cell ghosts and can be released by ribonuclease. The enzymatic properties of some of these proteins have no relation to their damaging effect on the cell membrane. Poly-l-lysine shows the same activity.

Amino Acids↗