bluuebunny/crossref_metadata_2025_split huggingface dataset:
Membrane alterations in irreversibly sickled cells: Hemoglobin–membrane interaction
Authors: Lessin Lawrence S. , Kurantsin‐Mills Joseph , Wallas Charles , Weems Henri 
Published: May 2005

AbstractIrreversibly sickled cells (ISCs) are sickle erythrocytes which retain bipolar enlongated shapes despite reoxygenation and owe their biophysical abnormalities to acquired membrane alterations. Freeze‐etched membranes both of ISCs produced in vitro and ISCs isolated in vivo reveal microbodies fixed to the internal (PS) surface which obscure spectrin filaments. Intramembranous particles (IMPs) on the intramembrane (PF) surface aggregate over regions of subsurface microbodies. Electron microscopy of diaminobenzidine‐treated ISC ghosts show the microbodies to contain hemoglobin and/or hemoglobin derivatives. Scanning electron microscopy and freeze‐etching demonstrate that membrane–hemoglobin S interaction in ISCs enhances the membrane loss by microspherulation. Membrane‐bound hemoglobin is five times greater in in vivo ISCs than non‐ISCs, and increases during ISC production, paralleling depletion of adenosine triphosphate. Polyacrylamide gel electrophoresis of ISC membranes shows the presence of high‐molecular‐weight heteropolymers in the pre–band 1 region, a decrease in band 4.1 and an increase in bands 7, 8, and globin. The role of cross‐linked membrane protein polymers in the generation of ISCs is discussed and is synthesized in terms of a unified concept for the determinants of the genesis of ISCs.
Tight junction development between cultured hepatoma cells: Possible stages in assembly and enhancement with dexamethasone
Authors: Porvaznik Martin , Johnson Ross G. , Sheridan Judson D. 
Published: May 2005

AbstractFreeze‐fracture and thin‐section methods were used to study tight junction formation between confluent H4‐II‐E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: (1) “formation zones” (smooth P‐fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; (2) linear strands and aggregates of 9–11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E‐face depressions; (3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specialization may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 × 10−6 M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.
Solubilization and conversion of hepatic adenylate cyclase to a form requiring MnATP as substrate
Authors: Londos Constantine , Lad Pramod M. , Nielsen Thor B. , Rodbell Martin 
Published: May 2005

AbstractA general feature of membrane‐bound adenylate cyclase systems is the “lability” of the basal enzyme to dispersion by detergents. A stable form of the detergentsolubilized enzyme is obtained only if the membrane‐bound enzyme is first pretreated with fluoride or Gpp(NH)p. However, we have found with the basal hepatic enzyme that the lability is evident primarily when MgATP is used as substrate; substitution of MnATP for MgATP reveals that substantial basal activity survives detergent treatment. This effect is independent of the detergent; it is seen with either Lubrol PX or with deoxycholate. In addition to the altered substrate requirement, the membrane‐bound and solubilized forms of the basal enzyme exhibit other differences. In contrast to the membrane‐bound form, the solubilized enzyme shows (1) weak stimulation by Gpp(NH)p; (2) little inhibition by adenosine, (3) strong inhibition by Pi or PPi, and (4) and apparent loss of the Me2+‐reactive regulatory site. Such dissimilarities between membranebound and solubilized cyclase are not seen if the membranes are pretreated with Gpp(NH)p prior to exposure to detergents. The characteristics of the solubilized basal hepatic enzyme are similar to those of the naturally occurring soluble adenylate cyclase found in mature rat testes. It would appear that separation of adenylate cyclase from components that confer regulation by divalent cation and guanine nucleotides produces a form of the enzyme that will turnover only MnATP; this may represent the free catalytic moiety. Such preparations could be useful in reconstructing some of the regulatory functions of adenylate cyclase seen in its membrane‐bound form.
Interaction between glycophorin and phospholipids in recombined systems
Authors: Romans Alice Y. , Yeagle Philip L. , O'connor Sally E. , Grisham Charles M. 
Published: May 2005

AbstractBoth the MN‐glycoprotein from human erythrocytes and the hydrophobic fragment from the protein isolated with trypsin treatment, T(is), have been recombined with egg phosphatidylcholine in bilayers at various phospholipid/protein ratios. In order to investigate the effect of the protein on the phospholipid headgroups, 31P nuclear magnetic resonance spectra were obtained with the MN‐glycoprotein recombined with egg phosphtidylcholine, which revealed two classes of phospholipid enviroments, one immobilized and one not immobilized. Electron spin resonance (ESR) of fatty acid methyl ester spin labels provided supporting evidence.Computer analysis of the ESR spectra indicate that 4–5 moles of phospholipid are immobilized per mole of protein over a wide range of lipid‐to‐protein ratios. The immobilization of the phospholipids appears mediated by both the polar headgroups and the hydrocarbon tails of the phospholipid.
Role of bound water in biological membrane structure: Fluorescence and infrared studies
Authors: Schneider Allan S. , Middaugh C. Russell , Oldewurtel Mary D. 
Published: May 2005

AbstractBound water is a major component of biological membranes and is required for the structural stability of the lipid bilayer. It has also been postulated that it is involved in water transport, membrane fusion, and mobility of membrane proteins and lipids. We have measured the fluorescence emission of membrane‐bound 1‐anilino‐8‐naphthalenesulfonate (ANS) and the infrared spectra of membranes, both as a function of hydration. ANS fluorescence is sensitive to polarity and fluidity of the membrane‐aqueous interface, while infrared absorption is sensitive to the hydrogen bonding and vibrational motion of water and membrane proteins and lipids. The fluorescence results provide evidence of increasing rigidity and/or decreasing polarity of the membrane‐aqueous interface with removal of water. The membrane infrared spectra show prominent hydration‐dependent changes in a number of bands with possible assignments to cholesterol (vinyl CH bend, OH stretch), protein (amide A, II, V), and bound water (OH stretch). Further characterization of the bound water should allow its incorporation into current models of membrane structure and give insight into the role of membrane hydration in cell surface function.
Ribosome crystallization in nuclei of chick embryos
Authors: Barbieri Marcello 
Published: May 2005

AbstractRibosome crystallization within nuclei has been studied in chick embryos with procedures which increase its frequency by various orders of magnitude as compared to previous findings. The extrusion of ribosome microcrystals from nuclei is reported for the first time, and a model for the transfer of ribosomes from nucleus to cytoplasm is proposed.
Comparison of the major polypeptides of the erythrocyte nuclear envelope
Authors: Cochran David L. , Egle Patsy M. , Shelton Keith R. , Yeoman Lynn C. 
Published: May 2005

AbstractThe three most abundant nonhistone polypeptides (molecular weights 75,000, 71,000 and 61,000) of the avian erythrocyte nucleus have previously been isolated in the nuclear envelope fraction. They have been separated by sodium dodecylsulfate‐polyacrylamide gel electrophoresis and peptide‐mapped after limited enzymatic digestion. Three enzymes–chymotrypsin, papain and Staphylococcus aureus protease–were used. Results obtained with each enzyme indicate strong similarities between the three nuclear envelope polypeptides. The amino acid compositions of the two most abundant polypeptides (P75 and P71) have been determined and found to be similar. Further, they readily yield large fragments upon brief alkaline hydrolysis. For both P75 and P71 the degree and the pattern of alkaline fragmentation are almost identical. A 61,000‐dalton polypeptide which appears to be P61 is obtained from P75 and P71 by mild acid hydrolysis. These results establish the close chemical similarity of these predominant polypeptides in the erythrocyte nucleus and suggest that they serve related functions.
Reconstituted low density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes to cells
Authors: Krieger Monty , Anderson Richard G. W. , Goldstein Joseph L. , Brown Michael S. , Smith Louis C. , Kao Yin J. , Pownall Henry J. , Gotto Antonio M. 
Published: May 2005

AbstractPrevious studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3‐pyrenemethyl‐23, 24‐dinor‐5‐cholen‐22‐oate‐3β‐yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane‐extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor‐mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence‐activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.
Human fibrosarcoma cells produce fibronectin‐releasing peptides
Authors: Keski‐Oja Jorma , Marquardt Hans , De Larco Joseph E. , Todaro George J. 
Published: May 2005

AbstractA sensitive radioimmunoassay, specific for human fibronectin, was used to measure the ability of certain biologically active polypeptides to release fibronectin from cultured human lung fibroblasts into their culture media. Concentrated, serum‐free culture supernatant from a human fibrosarcoma cell line was fractionated by gel filtration chromatography in the presence of acetic acid. Various polypeptides with molecular weights between 46,000 and 6,000 were tested for their ability to release fibronectin from cells. The column fraction, containing polypeptides with an apparent molecular weight of 10,000, exhibited the ability to rapidly release fibronectin from target cells. The activity could be inhibited by phenylmethyl sulphonylfluoride. Several other hormonal factors, tested in parallel with the column fractions, failed to show this effect. The 10,000 dalton molecular weight polypeptides may represent a family of cellular gene products responsible for maintenance of low levels of surface associated fibronectin in fibrosarcoma cells and thus be related to their infiltrating properties by preventing the formation of the extracellular matrix.
Fibronectin associated with the glial component of embryonic brain cell cultures
Authors: Kavinsky Clifford J. , Garber Beatrice B. 
Published: May 2005

AbstractIn a basic approach to investigations of neuronal–glial interactions during both normal brain development and its pathogenesis, embryonic brain cell populations were fractionated into purified neuronal and glial components. Using separation procedures based on differential adhesion and cytotoxicity, the isolated neuronal and glial phenotypes could be identified by distinct morphological and biochemical characteristics, including the visualization of glial fibrillary acid protein (GFA) within glial cells in immunohistochemical assays with monospecific anti‐GFA serum.When unfractionated cerebrum cells dissociated from 10‐day chick or 14‐day mouse embryos were plated as monolayers and cultured for 1‐14 days, monospecific antiserum against fibronectin (LETS glycoprotein) was found to react with many, but not all, of the cells as revealed by indirect immunofluorescence microscopy. The isolated neuronal and glial components of these populations were used to determine whether the appearance of membrane‐associated fibronectin was characteristic of one cell type or the other, or both, and if neuronal–glial cell interaction was required for its expression. It was found that the surfaces of glial cells, completely isolated from neurons, showed an intense fluorescent reaction to the anti‐fibronectin serum. In contrast, the purified neuronal cultures showed no fluorescence with either the anti‐GFA or anti‐fibronectin sera. These results demonstrate fibronectin as a cell surface protein associated primarily with glial cells and independent of neuronal–glial cell interaction for its expression. Furthermore, the results indicate that the fibronectin observed on glial cell surfaces in these cultures is produced endogenously and is not due to the preferential binding of fibronectin present in the culture medium. The role of fibronectin as an adhesive molecule in neuronal–glial interactions is discussed.
Stimulation of human vascular endothelial cell growth by a platelet‐derived growth factor and thrombin
Authors: Zetter Bruce R. , Antoniades Harry N. 
Published: May 2005

AbstractRepair of a vascular wound is mediated by migration and subsequent replication of the endothelial cells that form the inner lining of blood vessels. We have measured the growth response of human umbilical vein endothelial cells (HuE) to two polypeptides that are transiently produced in high concentrations at the site of a wound; the platelet‐derived growth factor (PDGF) and the protease thrombin. When 104 HuE cells are seeded as a dense island (2‐mm diameter) in the center of a 16‐mm tissue culture well in medium containing 20% human serum derived from platelet‐poor plasma (PDS), no increase in cell number or colony size is observed. With the addition of 0.5 ng/ml partially purified PDGF, colony size increases and the number of cells after 8 days is 4.8 × 104. When human thrombin (1 μg/ml) is added along with the PDGF, the cell number rises to 9.2 × 104. Thrombin alone stimulates no increase in cell number. Although partially purified PDGF stimulates endothelial cells maintained in PDS as well as those maintained in whole blood serum (WBS), pure PDGF is active only when assayed in medium that contains WBS and is supplemented with thrombin. These results suggest the existence of a second class of platelet‐derived factors that enable HuE cells to respond to the mitogenic activity of the purified platelet mitogen and thrombin.
Effect of thiourea and substituted thioureas on dynein ATPase and on the turbidity response of tetrahymena cilia
Authors: Blum J. J. , Hayes A. 
Published: May 2005

AbstractThe effects of thioura and of several substituted thioureas–phenylthiourea, α‐naphtylthiourea, metiamide, and burimamide–on dynein ATPase have been studied. The substituted thioureas are over 30 times more potent than thiourea in causing enhancement of 30S dynein ATPase activity and inhibition of 14S dynein ATPase activity. The effects of thiourea and phenylthiourea can be prevented by very low concentrations of β‐mercaptoethanol or dithiotheritol. Axonemal ATPase is also enhanced by the thioureas, but the reaction proceeds more slowly than for solubilized 30S dynein. Enhancement of 30S dynein ATPase by metiamide is prevented by low (∼ 1 μM) concentrations of ATP and, less effectively, by AMP‐PNP, but not by AMP‐PCP even though the latter is a stronger inhibitor of 30S dynein ATPase than is AMP‐PNP.The thioureas inhibit the ATP‐induced decrease in turbidity (measured as ΔA350) of axonemal suspensions. Inhibition of the turbidity response is also prevented by low concentrations of β‐mercaptoethanol, but, in contrast to the irreversible enhancement of ATPase activity, inhibition of the turbidity response is largely reversible. The ability of 30S dynein to rebind onto twice extracted axonemes is not changed by treatment with phenylthiourea or metiamide.These observations indicate that the thioureas react with at least two sets of SH or S–S groups on axonemes. Reaction with the group(s) on the 30S dynein causes an apparently irreversible enhancement of ATPase activity. Reaction with another group(s) causes a reversible inhibition of the turbidity response.
Membrane dynamics of differentiating cultured embryonic chick skeletal muscle cells by fluorescence microscopy techniques
Authors: Elson Hannah Friedman , Yguerabide Juan 
Published: May 2005

AbstractChanges in membrane fluidity during myogenesis have been studied by fluorescence microscopy of individual cells growing in monolayer cultures of embryonic chick skeletal muscle cells. Membrane fluidity was determined by the techniques of fluorescence photobleaching recovery (FDR), with the use of a lipidsoluble carbocyanine dye, and by fluorescence depolarization (FD), with perylene used as the lipid probe. The fluidity of myoblast plasma membranes, as determined from FPR measurements in membrane areas above nuclei, increased during the period of myoblast fusion and then returned to its initial level. The membrane fluidity of fibroblasts, also found in these primary cultures, remained constant. The fluidity in specific regions along the length of the myoblast membrane was studied by FD, and it was observed that the extended arms of the myoblast have the highest fluidity on the cell and that the tips at the ends of the arms had the lowest fluidity. However, since the perylene probe used in the FD experiments appeared to label cytoplasmic components, changes in fluidity measured with this probe reflect changes in membrane fluidity as well as in cytoplasmic fluidity. The relative change in each of these compartments cannot yet be ascertained. Tips have specialized surface structures, filopodia and lamellipodia, which may be accompanied by a more immobile membrane as well as a more rigid cytoplasm. Rounded cells, which may also have a more convoluted surface structure, show a lower apparent membrane fluidity than extended cells.
Ehrlich ascites tumor cell surface labeling and kinetics of glycocalyx release
Authors: Smith Thomas C. , Levinson Charles 
Published: May 2005

AbstractEhrlich ascites tumor cells spontaneously release cell surface material (glycocalyx) into isotonic saline medium. Exposure of these cells to tritium‐labeled 4,4′‐diisothiocyano‐1,2′‐dihenylethane‐2,2′‐disulfonic acid (3H2DIDS) at 4°C leads to preferential labeling of the cell surface coat. We have combined studies of the kinetics of 3H2DIDS‐label release, the effects of enzymatic treatment, and cell electrophoretic mobility to characterize the 3H2DIDS‐labeled components of the cell surface. Approximately 73% of the cell‐associated radioactivity is spontaneously released from the cells after 5 h at 23°C. The kinetics of release is consistent with the first‐order loss of two fractions; a slow (τ½ = 360 min) component representing 33% of the radioactivity, and a fast (τ½ = 20 min) component representing 26%. The remaining 14% of the labile binding may reflect mechanically induced surface release. Trypsin (1 μ/ml) also removes approximately 73% of the labeled material within 30 min and converts the kinectics of release to that of a single component (τ½ = 5.5 min). The specific activity (SA) of material released by trypsin immediately after labeling is 83% of the SA of the material spontaneously los in 1 h. However, trypsinization following a 2‐h period of spontaneous release yields material of reduced (43%) SA. Neither 3H2DIDS labeling nor the initial spontaneous loss of labeled material alters cell electrophoretic mobility. However, extended spontaneous release is accompanied by a significant decrease in surface charge density. Trypsinization immediately following labeling or after spontaneous release (2 h) reduces mobility by 32%. We have tentatively identified the slowly released compartment as contributing to cell surface negativity.
7‐Acetylcytochalasin B: Differential effects on sugar transport and cell motility
Authors: Lees Andrew , Lin Shin 
Published: May 2005

AbstractCytochalasin B (CB) is a potent inhibitor of sugar transport and cell motility in animal cells. We have synthesized and characterized the CB derivative 7‐acetylcytochalasin B (CBAc) and have found that it has differential effects on transport and motile processes in fibroblasts. The derivative inhibited sugar transport in human red cells, 3T3 cells, and chicken embryo fibroblasts at micromolar concentrations, although it was less potent than its parent compound. Unlike CB, which causes fibroblasts to round up and arborize at less than 10 μM, CBAc had no effect on fibroblast morphology and membrane ruffling at concentrations as high as 90 μM. Competitive binding experiments using [3H] CB showed that the affinity of CBAc for sites related to sugar transport in the red cell membrane is about one‐fourth of that of CB. In contrast, similar experiments using [3H] dihydrocytochalasin B (a derivative which inhibits cell motility but not sugar transport) showed that the affinity of CBAc for sites associated with red cell spectrin and actin is only about 1/20 of that dihydrocytochalasin B. This study demonstrates that acetylation of the C‐7 hydroxyl group of CB reduces its effect on cell morphology and motility much more than its ability to inhibit sugar transport. This observation, together with our earlier work with dihydrocytochalasin B, establishes that the pharmacologic effects of CB on fibroblasts result from the binding of the drug to two distinct classes of receptors and that these receptors interact with different parts of the cytochalasin molecule.