bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of divalent‐cation chelators and chloramphenicol on the spatial relationship of the nuclear envelope to chromatin in micronuclei of chinese hamster cells
Authors: Chai Lee S. , Weinfeld Herbert , Sandberg Avery A. 
Published: May 2005

AbstractIn the presence of the spindle poison Colcemid in the culture medium to prevent anaphase, approximately 20% of Chinese hamster metaphase cells were converted to micronucleated cells during 7 h. In the micronuclei the chromosomes had become enclosed by a nuclear envelope (NE). In the light‐microscope the micronuclei were of two kinds: with either visible chromatids or with decondensed chromosomes. In the electron microscope (EM) the spatial relationship of the NE to the chromatin was of two kinds only in the presence of Colcemid. In about 90% of the micronucleated cells the spatial relationship was normal, ie, the NE was immediately adjacent to the chromatin. In the remaining cells, the NE was distended so that the outer NE was separated from the inner one. In the presence of the drivalent cation chelator, (ethylenedinitrilo) tetraacetic acid (EDTA) or the Ca2+‐chelator [ethylenebis (oxyethylenenitrilo)] tetraacetic acid (EGTA), in addition to Colcemid, the amount of cells with micronuclei increased to 40%. The light‐microscope appearance was the same as that found in the absence of the chelating agents. However, after Colcemid plus EGTA, EM revealed that only about 50% of the micronucleated cells had NE that was immediately adjacent to the chromatin and about 10% of them had distended outer NE. In the remaining 40% a third kind of spatial relationship was seen: the NE was intact but most of it was not adjacent to the chromatin. Furthermore, this type of micronucleus often contained mitochondria within the confines of NE. Thus, Ca2+ and possibly Mg2+ may regulate the rate of formation of the NE and also its ultrastructural relation to the chromatin. Mitochondrial function also appears to be involved in this relationship. In the presence of chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, in addition to Colcemid, only about 50% of the micronucleated cells exhibited the normal relationship. The outer NE was separated from the inner NE in about 46% of the micronucleated cells and the third kind of NE‐chromatin relationship was observed only in 2%. In the case of the third kind of relationship produced by CAP, inclusion of mitochondria within the micronuclei was not observed, in contrast to the finding with EGTA.
Gap junctions and ACTH sensitivity in Y‐1 adrenal tumor cells
Authors: Decker R. S. , Donta S. T. , Larsen W. J. , Murray S. A. 
Published: May 2005

AbstractInitial studies of adrenocorticotropin‐sensitive (ACTH‐sensitive) and ACTH‐insensitive Y‐1 adrenal cortical tumor cell lines suggest a relationship between responsiveness to ACTH and the presence of gap junctions. An ACTH‐sensitive clone of Y‐1 cells possesses gap junctions and these junctions appear to enlarge with ACTH treatment. Gap junctions have not been observed, however, in an ACTH‐insensitive clone of Y‐1 tumor cells even when stimulated to produce cyclic adenosine monophosphate and steroids with cholera toxin.
Membrane protein organization in ATP‐depleted and irreversibly sickled red cells
Authors: Palek J. , Liu S. C. 
Published: May 2005

AbstractIt has been proposed that the spectrin‐actin submembrane network participates in control of red cell shape and deformability. We have examined ATP‐ and calcium‐dependent changes in organization of spectrin in the membrane employing cross‐linking of the nearest membrane protein neighbors by spontaneous or catalyzed (CuSO4, O‐phenanthroline) intermolecular disulfide couplings and two‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis.Cross‐linking of fresh red cells resulted in the formation of spectrin and actin dimers and tetramers. ATP‐depleted red cells differed from fresh cells in the presence of an additional reducible polymer of MW > 1 × 106 selectively enriched in spectrin. This polymer formed spontaneously when red cells were depleted of ATP under aerobic conditions. After anaerobic ATP depletion, the polymer formed in ghosts after cross‐linking by catalytic oxidation. Polymerization was prevented by maintenance of ATP and coincided with an ATP‐dependent discocyte‐echinocyte transformation. This suggests that, in ATP‐depleted red cells, spectrin is rearranged to establish closer contacts, and that this may contribute to the discocyte‐echinocyte transformation.The introduction of greater than 0.5 mM Ca++ into ghosts by inclusion in hemolysis buffer or into fresh red cells (but not ATP‐depleted red cells) by treatment with ionophore A23187 spontaneously produced a nonreducible polymer which others have attributed to transamidative cross‐linking of spectrin, band 3, and other proteins. Spontaneous formation of both polymer types (reducible in aerobically ATP‐depleted red cells and nonreducible in fresh, Ca++ enriched red cells) resulted in stabilization (“autocatalytic fixation”) of spheroechinocytic shape.Irreversibly sickled cells, which have increased calcium and decreased ATP, and exhibit a permanent membrane deformation, failed to form any of the above polymers. This suggests that in contrast to normal cells depleted of ATP in vitro, fixation of ISC shape in vivo is not related to Ca‐ and ATP‐dependent membrane protein polymerization. However, ISCs had an increased propensity to form the reducible, spectrin‐rich polymer during a subsequent metabolic depletion in vitro. This was associated with transformation of ISCs into spheroechinocytes. Similar echinocytic ISCs were found to constitute 5–10% of the densest fractions of freshly separated ISCs. ISCs then exhibit sphero‐echniocyte transformation, both in vitro and in vivo. We propose that this is due to spectrin reorganization that presumably results from the progressively increasing calcium and decreasing ATP of ISCs.These data provide evidence of altered spectrin organization in membranes of ATP‐depleted, calcium‐enriched red cells in vitro and in vivo.
Effect of metabolic inhibitors on vasopressin‐stimulated transport systems in the toad bladder
Authors: Hays Richard M. , Franki Nicholas , Ross Lawrence S. 
Published: May 2005

AbstractVasopressin increases the permeability of receptor cells to water and, in tissues such as toad bladder, to solutes such as urea. While cyclic AMP appears to play a major role in mediating the effects of vasopressin, there is evidence that activation of the water permeability system and the urea permeability system involves separate pathways. In the present study, we have shown that inhibitors of oxidative metabolism (rotenone, dinitrophenol, and methylene blue) selectively inhibit either vasopressin‐stimulated water flow or vasopressin‐stimulated urea transport. There was no inhibition, however, when exogenous cyclic AMP was substituted for vasopressin, and little to no inhibition when the potent analogue 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) was employed. Rotenone had no effect on adenylate cyclase activity or cyclic AMP levels within the cell; dinitrophenol decreased adenylate cyclase activity minimally.Additional studies with vinblastine and nocodazole, inhibitors of microtubule assembly, demonstrated an inhibition of vasopressin and cyclic AMP‐stimulated water flow but showed no effect on urea transport.We would conclude that water and urea transport, as examples of hormone‐stimulated processes, have different links to cell metabolism, and that in addition to cyclic AMP, a non‐nucleotide pathway may be involved in the action of vasopressin.
Substructure of human erythrocyte spectrin
Authors: Hsu C. J. , Lemay A. , Eshdat Y. , Marchesi V. T. 
Published: May 2005

AbstractThe human erythrocyte structural protein spectrin and its subunits I, II were isolated in the presence of Na‐dodecyl‐sulfate by gel filtration and preparative gel electrophoresis. After removal of the detergent, spectrin alpha‐helical content is comparable to spectrin isolated without detergent. Subunits I and II formed single bands in isoelectric focusing (pI = 5.6) and in Ornstein‐Davis disc gel electrophoresis systems, indicating the individual subunits are homogenous in nature. The molecular weights of the subunits I and II, determined by Ferguson plot, are 237,500 and 238,600, respectively, which is in good agreement with values obtained by the standard SDS gel relative mobility method. Limited tryptic digestion of spectrin and two‐dimensional peptide maps of the individual subunits cleaved by S‐cyanylation reaction showed dissimilar patterns, suggesting differences in primary structure between the two subunits.
Properties of epithelial cells cultured from human carcinomas and nonmalignant tissues
Authors: Smith Helene S. , Hackett Adeline J. , Riggs John L. , Mosesson Michael W. , Walton Judie R. , Stampfer Martha R. 
Published: May 2005

AbstractHuman epithelial cell cultures were examined for expression of plasminogen activator and fibronectin matrix. All of the cells examined showed ultrastructural evidence suggesting their epithelial origin, including microvilli and specialized junctions. The nonmalignant cells were also negative for endothelial cell markers (ie, they lacked factor VIII antigen, a nonthrombogenic surface and Weibel‐Palade bodies). The nonmalignant lines all produced large amounts of plasminogen activator, whereas the tumor‐derived lines showed a gradation of activities, ranging from lines having as much activity as the nonmalignant lines to lines having little or no activity above background. For both normal and malignant cells, addition of dexamethesone only slightly decreased the levels of plasminogen activator. By immunofluorescence microscopy, normal bladder and fetal intestine epithelial cells showed fibronectin in a globular and fibrillar matrix. In contrast, normal mammary epithelial cells had a much diminished amount of fibronectin with a punctate distribution.
Growth control by cell to cell contact
Authors: Bunge Richard , Glaser Luis , Lieberman Michael , Raben Dan , Salzer James , Whittenberger Brock , Woolsey Thomas 
Published: May 2005

AbstractControl of cell growth by cell to cell contact is reviewed with particular emphasis on two systems – contact inhibition of growth observed with Swiss 3T3 cells and the mitogenic stimulation of Schwann cells by dorsal root ganglia neurites. In both cases the biological effect can be reproduced by the addition of surface membranes to the corresponding cells. In the case of contact inhibition of 3T3 cells, biological activity appears to correlate with membrane binding to the cells. An octylglucoside extract of 3T3 plasma membranes retains the biological activity (growth inhibition) of the original membranes.
Isolation and immunological characterization of an iron‐regulated, transformation‐sensitive cell surface protein of normal rat kidney cells
Authors: Fernandez‐Pol J. A. 
Published: May 2005

AbstractWe have analyzed the surface proteins of cultured normal rat kidney (NRK) cells and virus‐transfromed NRK cells subjected to iron deprivation. Such a treatment specifically induces two transformation‐sensitive plasma membrane‐associated glycoproteins with a subunit molecular wegiht of 160,000 (160 K) and 130,000 (130 K) daltons in NRK cells. In these cells the 160 K glycoprotein is readily available to lactoperoxidase‐mediated iodination, and the 130 K is apparently inaccessible to iodination. Major differences were revealed when iodinated membrane proteins of normal and virus‐transformed cells subjected to iron deprivation were compared. In Kirsten sarcoma virus‐transformed NRK cells the 160 K glycoprotein was weakly labeled. In two clones of simian virus 40‐transformed NRK cells the 160 K glycoprotein was weakly labeled or not at all. The 130 K glycoprotein was inaccessible to iodination in all the virus‐transformed cell lines.The 160 K and 130 K glycoproteins were isolated form plasma membranes of NRK cells using preparative SDS gel electrophoresis. Antibodies generated against these glycoproteins stained the external surfaces of NRK cells and induced antigen redistribution. Evidence presented suggests that 160 K and 130 K are plasma membrane‐associated procollagen molecules. A possible interaction of these proteins with transferrin is also described. The data suggest that these proteins may have an important role in the sequence of events leading to transformation.
Carbohydrate structure of the major glycopeptide from human cold‐insoluble globulin
Authors: Fisher Susan J. , Laine Roger A. 
Published: May 2005

AbstractCold‐insoluble globulin (CIg) is a member of a group of circulating and cell‐associated, high‐molecular‐weight glycoproteins termed fibronectins. CIg was isolated from human plasma by affinity chromatography on gelatin‐Sepharose. SDS‐polyacrylamide gel electrophoresis of the purified glycoprotein gave a double band that migrated near myosin. The CIg glycopeptides were released by pronase digestion and isolated by chromatography on Sephadex G‐50. Affinity chromatography of the major G‐50 peak on Con A‐Sepharose resulted in two fractions: one‐third of the glycopeptides were unbound and two‐thirds were weakly bound (WB). Sugar composition analysis of the unbound glycopeptides by GLC of the trimethylsilyl methyl glycosides gave the following molar ratios: sialic acid, 2.5; galactose, 3.0; N‐acetylglucosamine, 4.9; and mannose, 3.0. Sugar composition analysis of the WB glycopeptides gave the following molar ratios: sialic acid, 1.7; galactose, 2.0; N‐acetylglucosamine, 4.1; and mannose, 3.0. The WB CIg glycopeptides cochromatographed on Sephadex G‐50 with WB transferrin glycopeptides giving an estimated molecular weight of 2,800. After degradation with neuraminidase alone or sequentially with β‐galactosidase the CIg and transferrin glycopeptides again cochromatographed. Methylation linkage analysis of the intact and the partially degraded glycopeptides indicated that the carbohydrate structure of the major human CIg glycopeptide resembles that of the major glycopeptide from transferrin.
Evidence of microfilament‐associated mitochondrial movement
Authors: Bradley Timothy J. , Satir Peter 
Published: May 2005

AbstractThe mitochondria in the lower Malpighian tubule of the insect Rhodnius prolixus can be stimulated by feeding in vivo and by 5‐hydroxytryptamine in vitro, to move from a position below the cell cortex to one inside the apical microvilli. During and following their movement into the microvilli, the mitochondria are intimately associated with the microfilaments of the cell cortex and microvillar core bundle. Bridges approximately 14 nm in length and 4 nm in diameter are observed connecting the microvillar microfilaments to the outer mitochondrial membrane and microvillar plasma membrane. Depolymerization of all visible microtubules with colchicine does not inhibit 5‐HT‐stimulated mitochondrial movement. On the other hand, treatment with cytochalasin B does block mitochondrial movement, suggesting that microfilaments play a role in the mitochondrial motility. We have labeled the microvillar microfilaments, which are 6 nm in diameter, with heavy meromyosin, which supports the contention that they contain actin. A model of the mechanism of mitochondrial movement is presented in which mitochondria slide into position in the microvilli along actin‐containing microfilaments in a manner analogous to the sliding actin‐myosin model of skeletal muscle.
Isolation and properties of gamma protein, the major transmembrane sialoglycoprotein of the HeLa cell
Authors: Kramer Randall H. , Canellakis E. S. 
Published: May 2005

AbstractThe surface of the HeLa cell is composed of a heterogeneous population of sialogly coproteins which undergo lectin‐mediated endocytosis (Kramer and Canellakis, Biochim Biophys Acta 551:328, 1979). One such sialoglyco‐protein, gamma protein, is the major periodate‐Schiff‐reactive and [3H]‐glucosamine‐labeled component of the plasma membrane; it has an apparent molecular weight of 165,000. Gamma protein is also the major [125I]‐wheat germ agglutinin‐binding component in sodium dodecyl sulfate gels. Neuraminidase digestion of HeLa cells abolishes binding of [125I]‐wheat germ agglutinin to gamma protein, and pretreatment of cells with wheat germ agglutinin protects gamma protein from desialation by neuraminidase. suggesting that wheat germ agglutinin binds to the sialic acid residues of gamma protein at the cell surface. Gamma protein can be extracted with various detergents but not with high‐salt, chelating, or chaotropic agents. Intact inside‐out plasma membrane vesicles have been prepared from HeLa cells that had phagocytosed latex particles. Treatment of these isolated vesicles with trypsin reduces the molecular weight of gamma protein. These results suggest that gamma protein is an integral membrane protein that spans the plasma membrane. Gamma protein can be purified to homogeneity by sequential lithium diiodosalicylate‐phenol extraction, wheat germ agglutinin‐agarose affinity chromatography, and preparative sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.
Isolation and characterization of human placental chorionic villar extracellular matrix
Authors: Ohno M. , Nagle R. B. , Meezan E. , Brendel K. 
Published: May 2005

AbstractThe cell‐free extracellular matrix of human placental chorionic villi has been prepared by a procedure employing extraction of the terminal villar fragments with the detergents Triton X‐100 and sodium deoxycholate. The isolated human placental extracellular matrix retains an intact, but collapsed, histoarchitecture, as observed by scanning and transmission electron microscopy. It remains intact, in large part because of the presence of continuous sheets of villar basement membranes and associated interstitial collagen fibers and scattered patches of fibrin. The staining characteristics and chemical composition of the isolated human placental extracellular matrix are similar to those reported for basement membranes in several tissues and indicate the presence of collagen‐like and glycoprotein components in this preparation. Gel electrophoresis of urea‐SDS‐mercaptoethanol extracts of the matrix showed that it consists of several polypeptide components of various molecular weights, some of which are associated into high molecular weight complexes by disulfide bonds.
Analysis of human erythrocyte membrane vesicles produced by shearing
Authors: Schrier S. L. , Junga I. 
Published: May 2005

AbstractShearing of ghosts in a French pressure cell produces three classes of microvesicles that differ from endocytic vacuoles, exocytic vacuoles, and inside‐out vesicles. It was thought that an analysis of these vesicles might provide some clues about the assembly of proteins within the human erythrocyte membrane. The microvesicles were separated into three visible bands, labeled top, middle, and bottom, and assayed for activity of Mg++‐ATPase, Na+, K+‐ATPase, acetylcholinesterase, glyceraldehyde‐phosphate dehydrogense, and NADH oxidoreductase. Their proteins were also characterized by polyacrylamide gel electrophoresis with both Coomassie blue staining, to assess total protein content and distribution, and PAS‐staining, to characterize sialoglycopeptides. In order to minimize problems inherent in ghost preparation, Dodge or hypotonic ghosts and glycol or isotonic ghosts were used in all studies. Middle membrane vesicles most resembled intact ghosts. Top vesicles had reduced levels of NADH oxidoreductase and more PAS‐2 at the expense of PAS‐1. The bottom vesicle class was very much enriched with PAS‐1 at the expense of PAS‐2, and PAS‐3 was completely absent. In addition bottom vesicles had highest NADH oxidoreductase activity but lowest activity of all the other enzymes measured. These vesicle classes could not have been produced by tangential shearing through the membrane, nor could radial shearing through a membrane in which all proteins were free to move laterally have accounted for the three discrete vesicle classes or for their different patterns of enzymes and proteins. The analysis of the microvesicles produced by shearing is most consistent with radial shearing through membranes where there may be fixed domains superimposed on the basic fluid‐mosaic structure.
The association of α‐actinin with the plasma membrane
Authors: Burridge Keith , McCullough Lois 
Published: May 2005

AbstractThe role of α‐actinin in the attachment of actin to plasma membranes has been investigated. Specific antibody staining of SDS gels has indicated that α‐actinin is a major component in isolated plasma membranes prepared from three different cell types by two different procedures. Using specific extraction conditions, most of the α‐actinin can be selectively extracted from the membranes with relatively little parallel release of actin. This selective dissociation of α‐actinin from the plasma membrane leads us to conclude that α‐actinin is present in these membrane preparations, because it is bound to actin, and that α‐actinin does not form a direct link between actin and the membrane.
The scavenger cell pathway for lipoprotein degradation: Specificity of the binding site that mediates the uptake of negatively‐charged LDL by macrophages
Authors: Brown Michael S. , Basu Sandip K. , Falck J. R. , Ho Y. K. , Goldstein Joseph L. 
Published: May 2005

AbstractMacrophages isolated from a variety of organs in several animal species exhibit high affinity binding sites that recognize chemically modified proteins. One of these binding sites recognizes human plasma low density lipoprotein (LDL) in which the positive charges on the epsilon‐amino groups of lysine have been removed or neutralized by chemical modification, thus giving the protein an enhanced negative charge. Effective treatments include reaction of LDL with organic acid anhydrides (acetylation or maleylation) and reaction with aldehydes, such as treatment with malondialdehyde. After the negatively‐charged LDL binds to the surface receptor sites, it is rapidly internalized by the macrophages by endocytosis and hydrolyzed in lysosomes. The liberated cholesterol is reesterified in the cytoplasm, producing massive cholesteryl ester deposition. The binding site for negatively‐charged LDL has been demonstrated so far only on macrophages and other scavenger cells. It is not expressed in cultured fibroblasts, smooth muscle cells, lymphocytes, or adrenal cells. In addition to its affinity for acetylated LDL and malondialdehyde‐treated LDL, the macrophage site binds a variety of polyanions. It exhibits a particularly high affinity for certain sulfated polysaccharides (dextran sulfate and fucoidin), certain polynucleotides (polyinosinic acid and polyguanylic acid), polyvinyl sulfate, and maleylated albumin. It is possible that the site that binds negatively‐charged LDL may be responsible for the massive accumulation of cholesteryl esters that occurs in vivo in macrophages and other scavenger cells in patients with high levels of circulating plasma LDL.