bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells
Authors: Cooper Richard A.
Published: May 2005

Cholesterol and phospholipid are the two major lipids of the red cell membrane. Cholesterol is insoluble in water but is solubilized by phospholipids both in membranes and in plasma lipoproteins. Morever, cholesterol exchanges between membranes and lipoproteins. An equilibrium partition is established based on the amount of cholesterol relative to phospholipid (C/PL) in these two compartments.

Increases in the C/PL of red cell membranes have been studied under three conditions: First, spontaneous increases in vivo have been observed in the spur red cells of patients with severe liver disease; second, similar red cell changes in vivo have been induced by the administration of cholesterol‐enriched diets to rodents and dogs; third, increases in membrane cholesterol have been induced in vitro by enriching the C/PL of the lipoprotein environment with cholesterol‐phospholipid dispersions (liposomes) having a C/PL of >1.0. In each case, there is a close relationship between the C/PL of the plasma environment and the C/PL of the red cell membrane. In vivo, the C/PL mole ratio of red cell membranes ranges from a normal value of 0.9–1.0 to values which approach but do not reach 2.0. In vitro, this ratio approaches 3.0.

Cholesterol enrichment of red cell membranes directly influences membrane lipid fluidity, as assessed by the rotational diffusion of hydrophobic fluorescent probes such as diphenyl hexatriene (DPH). A close correlation exists between increases in red cell membrane C/PL and decreases in membrane fluidity over the range of membrane C/PL from 1.0 to 2.0; however, little further change in fluidity occurs when membrane C/PL is increased to 2.0–3.0.

Cholesterol enrichment of red cell membranes is associated with the transformation of cell contour to one which is redundant and folded, and this is associated with a decrease in red cell filterability in vitro. Circulation in vivo in the presence of the slpeen further modifies cell shape to a spiny, irregular (spur) form, and the survival of cholesterol‐rich red cells is decreased in the presence of the spleen. Although active Na‐K transport is not influenced by cholesterol enrichment of human red cells, several carrier‐mediated transport pathways are inhibited. We have demonstrated this effect for the cotransport of Na + K and similar results have been obtained by others in studies of organic acid transport and the transport of small neutral molecules such as erythritol and glycerol.

Thus, red cell membrane C/PL is sensitive to the C/PL of the plasma environment. Increasing membrane C/PL causes a decrease in membrane fluidity, and these changes are associated with a reduction in membrane permeability, a distortion of cell contour and filterability and a shortening of the survival of redcells in vivo.
Characterization of the glucose transporter from human erythrocytes
Authors: Sogin David C. , Hinkle Peter C.
Published: May 2005

The D‐glucose transporter from human erythrocytes has been purified and reconstituted by Kasahara and Hinkle (J Biol Chem 252:7394–7390). Using a similar purification scheme, we have isolated the protein with 65% of the extracted phospholipid at a lipid‐protein ratio of 14:1 by weight. The KD (0.14 μM) and extent (11 nmoles/mg protein) for binding of 3H‐cytochalasin B was determined by equilibrium dialysis. Glucose was a linear competitive inhibitor of binding of cytochalasin B, with an inhibition constant of 30 mM. To further characterize the protein, samples were filtered in the presence of sodium dodecyl sulfate (SDS) through Sepharose 6B to remove 95% of the lipid followed by filtration of Sephadex G150 to remove the remaining lipid and a contaminating amount of a minor, lower‐molecular‐weight protein. This preparation contains only 24% acidic and basic amino acids. The protein also contains 5% neutral sugars (of which 3% is galactose), 7% glucosamine, and 5% sialic acid.
Regulation of the receptor‐mediated cyclic AMP response of kidney to parathyroid hormone in the vitamin D‐deficient rat
Authors: Forte Leonard R. , Carnes David L. , Nickols G. Allen , Anast Constantine S.
Published: May 2005

Rats fed a diet deficient in vitamin D were found to exhibit a refractory cyclic AMP response of kidney slices to parathyroid hormone and a marked decrease in membrane parathyroid hormone‐dependent adenylate cyclase activity. Both the characteristic calcium deficiency (hypocalcemia) and secondary elevation of circulating parathyroid hormone appeared before the first noticeable decrease in hormone‐dependent enzyme activity. After repletion of D‐deficient rats with vitamin D2, we found that serum calcium and parathyroid hormone were both restored to normal levels before the depressed enzyme response to the hormone was reversed. Moreover, infusion of parathyroid hormone into vitamin D‐replete rats led to a marked reduction in parathyroid hormone‐dependent adenylate cyclase activity, which was partly restored to control level 3 hours after discontinuing the hormone infusion. Taken as a whole, this study suggests that the elevated endogenous parathyroid hormone in the vitamin D‐deficient rat is involved in the “down‐regulation” of renal cyclic AMP responsiveness to the hormone. However, these experiments do not rule out the possibility that calcium deficiency and/or vitamin D per se participate in the regulation of the renal cyclic AMP response to parathyroid hormone.
The Escherichia coli adenylate cyclase complex: Activation by phosphoenolpyruvate
Authors: Peterkofsky Alan , Gazdar Celia
Published: May 2005

A model for the regulation of the activity of Escherichia coli adenylate cyclase is presented. It is proposed that Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) interacts in a regulatory sense with the catalytic unit of adenylate cyclase. The phosphoenolpyruvate (PEP)‐dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state of adenylate cyclase. The pyruvate or sugar‐dependent dephosphorylation of Enzyme I is correlated with a low activity state of adenylate cyclase. Evidence in support of the proposed model involves the observation that Enzyme I mutants have low cAMP levels and that PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase, Kinetic studies indicate that various ligands have opposing effects on adenylate cyclase. While PEP activates the enzyme, either glucose or pyruvate inhibit it. The unique relationships of PEP and Enzyme I to adenylate cyclase activity are discussed.
Modulator binding protein antagonizes activation of (Ca2+ + Mg2+)‐ATPase and Ca2+ transport of red blood cell membranes
Authors: Larsen Fred L. , Raess Beat U. , Hinds Thomas R. , Vincenzi Frank F.
Published: May 2005

Red blood cells contain a protein that activates membrane‐bound (Ca2+ + Mg2+)‐ATPase and Ca2+ transport. The red blood cell activator protein is similar to a modulator protein that stimulates cyclic AMP phosphodiesterase. Wang and Desai [Journal of Biological Chemistry 252:4175–4184, 1977] described a modulator‐binding protein that antagonizes the activation of cyclic AMP phosphodiesterase by modulator protein. In the present work, modulator‐binding protein was shown to antagonize the activation of (Ca2+ + Mg2+)‐ATPase and Ca2+ transport by red blood cell activator protein. The results further demonstrate the similarity between the activator protein from human red blood cells and the modulator protein from bovine brain.
Formation of γ‐glutamyl‐ϵ‐lysine bridges between membrane proteins by a Ca2+‐regulated enzyme in intact erythrocytes
Authors: Lorand L. , Siefring G. E. , Lowe‐Krentz L.
Published: May 2005

A rise in the intracellular concentration of Ca2+‐ions in human erythrocytes causes the formation of high‐molecular‐weight membrane protein polymers, cross‐linked by γ‐glutamyl‐ϵ‐lysine side chain bridges. Cross‐linking involves proteins at the cytoplasmic side of the membrane (band 4.1, spectrin, and band 3 materials) and the reaction is catalyzed by the intrinsic transglutaminase. This enzyme is regulated by Ca2+‐ions and it exists in a latent form in normal cells. The protein polymer, isolated from the membranes of Ca2+‐loaded intact human red cells, is heterogeneous in size and may contain as many as 6 moles of γ‐glutamyl‐ϵ‐lysine cross‐links per 100,000 gm of protein.

Synthetic compounds, which either compete against the ϵ‐lysine cross‐linking functionalities of the protein substrates (eg, histamine, aminoacetonitrile, cystamine) or directly inactivate the transamidase (eg, cystamine), inhibit the membrane polymerization reaction in intact human erythrocytes. They also interfere with the Ca2+‐induced irreversible shape change from discocyte to echinocyte and inhibit the irreversible loss of membrane deformability. Thus, the transamidase‐catalyzed production of γ‐glutamyl‐ϵ‐lysine cross‐links in the membrane may be a common denominator in these cellular manifestations.
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

In 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

Initial 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

It 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

Vasopressin 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

The 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

Human 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

Control 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

We 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

Cold‐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

The 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

The 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

The 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

Shearing 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

The 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

Macrophages 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.
Erythropoietic enhancing activity (EEA) secreted by the human cell line, GCT
Authors: Abboud Camille N. , Dipersio John F. , Brennan James K. , Lichtman Marshall A.
Published: May 2005

Medium conditioned by the monocyte‐like cell line GCT contains colony‐stimulating activity (CSA), a mediator of in vitro granulopoiesis. Also, the conditioned medium (CM) contains erythroid‐enhancing activity (EEA), which can be demonstrated in a system utilizing either nonadherent marrow or blood mononuclear cells, erythropoietin (1–2 units/ml), and 20 ml/dl fetal calf serum. Under these conditions, GCT CM enhances the growth of CFU‐E and BFU‐E. Attempts were made to characterize the molecular features of EEA. Serum‐free GCT cell CM was fractionated on Sephacryl S200 and Ultrogel AcA54. EEA and CSA cochromatographed with apparent molecular weights of ∼ 40,000 daltons on Sephacryl and ∼ 30,000 daltons on Ultrogel. Fractionation on DEAE Sephacel led to an apparent separation of CSA from EEA; however, when diluted, the fractions containing CSA had EEA. Undiluted fractions containing potent CSA inhibited erythropoiesis; however, dilution of these fractions resulted in marked EEA. Diluted crude GCT CM and DEAE Sephacel fractions enriched in EEA were also capable of sustaining BFU‐E in liquid culture and mediating erythropoietin‐independent colony growth. CSA could not be unequivocally separated from EEA on concanavalin A‐Sepharose, since the diluted void volume containing CSA also had EEA. EEA was present in CM boiled for 60 minutes, whereas CSA was markedly reduced but not abolished. The inverse relationship between CSA concentration and EEA mandates dilution of fractions when bioassayed for these two activities. Although CSA and EEA are similar in molecular weight, they appear to be partially separable by ion‐exchange chromatography and heat stability.
The extracellular matrix and the control of proliferation of vascular endothelial and vascular smooth muscle cells
Authors: Gospodarowicz D. , Vlodavsky I. , Savion N.
Published: May 2005

In this short review we describe the observations which have led us to conclude that one of the most important components involved in modulating cell proliferation in vitro, and probably in vivo as well, may be the extrac‐cellular matrix upon which cells rest.
Inhibition of cell proliferation and protease activity by cartilage factors and heparin
Authors: Hatcher Victor B. , Tsien Grace , Oberman Martin S. , Burk Peter G.
Published: May 2005

Proliferating rat smooth muscle cells and fibroblasts have membrane‐associated protease activity. High concentrations of heparin inhibited membrane‐associated protease activity and cell proliferation, while low concentration of heparin promoted smooth muscle cell proliferation. The inhibition of protease activity and proliferation was abolished when heparin was treated with protamine sulfate or when acid treated fetal calf serum was used. Heparin required the presence of an acid labile factor(s) in serum for the inhibition of protease activity and proliferation. Heparin and antithrombin III in the presence of acid‐treated fetal calf serum did not inhibit cell proliferation or protease activity. Cartilage factors isolated from bovine nasal cartilage containing trypsin inhibitory activity, but not papain inhibitory activity, inhibited rat smooth muscle and fibroblast proliferation and surface associated protease activity. The cartilage factors did not require acid‐labile components in the fetal calf serum for the inhibitory activity. The inhibitory activity due to heparin and cartilage factors was not permanent under our experimental condition. Protein synthesis was not inhibited by heparin or the cartilage factors. In rat smooth muscle cells and fibroblasts, the expression of surface‐associated protease activity was related to the proliferative state of the cells. Surface protease activity was only present on proliferating cells. When surface protease activity was inhibited by high concentrations of heparin in the presence of an acid‐labile serum component(s) or cartilage factors, cell proliferation was also inhibited.
In vitro characteristics of metastatic variant subclones of restricted genetic origin
Authors: Talmadge James E. , Starkey Jean R. , Stanford David R.
Published: May 2005

We have studied several metastatic variant cell lines derived from a common clonal origin and their transformed and untransformed parental cell lines. A number of in vitro characteristics were examined for each tumor line and these properties were correlated with the ability of the tumor cells to form pulmonary nodules in an experimental metastasis assay.

Direct correlations with metastatic behavior in the lung colony assay were found to exist with the amount of cell‐bound Concanavalin A and the procoagulant activities of cell lysates. In vitro parameters that did not correlate with the metastatic phenotype were: population doubling times in culture, saturation density achieved in culture, the number of colony‐forming cells shed from confluent cultures, rates of cellular attachment to homotypic or heterotypic cell monolayers, plasminogen‐activator production and procoagulant activity produced in serum‐free conditioned medium.
Spin label studies of erythrocytes with abnormal lipid composition: comparison of red cells in a hereditary hemolytic syndrome and lecithin: Cholesterol acyltransferase deficiency
Authors: Godin David V. , Herring F. Geoffrey
Published: May 2005

Erythrocytes from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency have been shown to exhibit an increase in membrane fluidity which is surprisingly small in view of the extensive alterations both in membrane lipicl composition (namely, an elevation in cholesterol and phosphatidylcholine contents as well as a decrease in phosphatidylethanolamine) and in the functional properties of these cells. In the hope of deriving some information concerning the interrelationship between the structural and functional abnormalities, we have used the spin probe 5‐doxyl stearic acid to investigate the temperature‐dependent fluidity properties of red cells from two patients with a hereditary hemolytic syndrome (HHS) whose red cells are also characterized by qualitatively similar alterations in phosphatidylcholine and phosphatidylethanolamine but, unlike those in LCAT deficiency, have relatively normal levels of membrane cholesterol. A small increase in membrane fluidity of HHS erythrocytes equivalent to that previously observed in LCAT deficiency was found, indicating that membrane cholesterol level does not exert an important modulatory influence on membrane fluidity in these cells. It is concluded that while the distinct patterns of structural and functional erythrocyte alterations in these two disorders cannot be explained on the basis of differences in bulk membrane fluidity, the marginally increased fluidity may underlie the abnormalities in osmotic fragility and membrane p‐nitrophenylphosphatase activity which are shared in common by both types of modified red cells.
Normal and neoplastic lymphocyte maturation
Authors: Weissman I. L. , McGrath M. S. , Pillemer E. , Hollander N. , Rouse R. V. , Jerabek L. , Stevens S. K. , Scollay R. G. , Butcher E. G.
Published: May 2005

Lymphocytes arc cells that are responsible for processes of specific antigen recognition and for those aspects of the immune response that characterize adaptive immunity. In this respect adaptive immunity can be characterized as antigen‐induced immune memory and effector functions as compared to native immunity – the nonspecific phagocytic and humoral protective elements in lower vertebrates. In vertebrates both B and T lymphocytes apparently express self‐synthesized receptors that (1) are involved in the recognition of antigens, and (2) mediate the interactions between various important cells in the hematolymphoid system. There are three major subclasses of T lymphocytes – those involved with helper/inducer functions, those involved with suppressor functions, and those involved in direct cytotoxicity of antigenic target cells [1,2].
I‐A Mutation resulted in a selective loss of an antigen‐specific Ir gene function
Authors: Lin C. Shirley , Rosenthal Alan S. , Hansen Ted H.
Published: May 2005

The immune responses to several antigens were compared in the I‐A mutant mouse strain B6.C‐H‐2bm12 and the wild‐type strain C57BL/6. With a lymph node cell proliferation assay, the response to two of these antigens, beef insulin and (TG)A‐L, was demonstrated to be controlled by a gene in the I‐Ab region. B6.C‐H‐2bm12 mice failed to respond to beef insulin, while their responses to (TG)A‐L, DNP‐OVA and PPD were comparable with those of the wild‐type strain C57BL/6. Taken together with previous studies, these data suggest that the product of a single pleiotropic I‐A gene, an la molecule, functions as a histocompatibility, la, and MLR antigen, as well as a necessary component for Ir gene function. Furthermore, the data reported here demonstrate that la molecules have multiple functional “Ir determinants,” one of which has been altered in the B6.C‐H‐2bm12 mutant. The B6.C‐H‐2bm12 mice, therefore, represent a powerful analytical tool for the understanding of the cellular and molecular basis for Ir gene control of the immune response.
Ligatin binds phosphohexose residues on acidic hydrolases
Authors: Jakoi E. R. , Kempe K. , Gaston S.M.
Published: May 2005

Ligatin, a receptor that recognizes phosphorylated sugars, was isolated from plasma membranes of mouse macrophages, rat ileum, and rat brain. Several acidic hydrolases including N‐acetyl β‐D‐glucosaminidase (β‐NAG) were solubilized with this receptor. The solubilized β‐NAG bound to ligatin in vitro as demonstrated by affinity chromatography using the immobilized receptor. β‐N‐Acetyl D‐glucosaminidase‐ligatin complexes were dissociated by low concentrations of mannose 6‐phosphate (Man6P) and/or glucose 1‐phosphate (Glc 1P). The effectiveness of these two phosphomonosaccharides varied depending on the source of the enzyme: ileal β‐NAG‐ligatin complexes showed a four‐fold preferential dissociation with Man6P; macrophage complexes showed a 160‐fold preferential dissociation with Glc 1P. Brain complexes dissociated with nearly equal preference for Man6P and Glc 1P. Heterologous complexes displayed the specificity characteristic of the source of the enzyme regardless of the source of the ligatin. Treatment of the solubilized hydrolases with endoglucosaminidase H released phosphorous‐32 label from these enzymes and prevented binding of β‐NAG to ligatin. However, treatment of the solubilized hydrolases with alkaline phosphatase reduced the binding of β‐NAG to ligatin by no more than 30%. This apparent resistance of β‐NAG to dephosphorylation was consistent with the chromatographic behavior on QAE of 3H‐labeled acidic oligosaccharides isolated from the solubilized hydrolases. The oligosaccharides that contain phosphorylated hexose were less acidic than phosphomonoesters and were insensitive to alkaline phosphatase until subjected to acid hydrolysis. These results suggested the presence of a phosphodiester on β‐NAG analogous to the NAC glucosamine 1 P6 mannose present on β‐glucuronidase isolated from mouse lymphoma cells (Tabas I, Kornfeld, S: J Biol Chem 255: 6633, 1980).
Aggregation of sponge cells: Immunological characterization of the species‐specific geodia aggregation factor
Authors: Conrad Jürgen , Zahn Rudolf K. , Kurelec Branko , Uhlenbruck Gerd , Müller Werner E. G.
Published: May 2005

Antibodies were raised against the purified aggregation factor from Geodia cydonium in order to clarify its function during cell aggregation in the homologous and heterologous system. These antibodies inhibited only cell aggregation in the homologous Geodia system and were inactive in the heterologous Tethya lyncurium system. These findings directly indicated that the species‐specific reaggregation of sponge cells was initiated by the soluble aggregation factor as already assumed in earlier studies. The amount of neutralizing antibodies was determined by a precipitation reaction with the antigen in capillaries and by microdiffusion. By using the latter technique we got evidence that the Geodia aggregation factor contained a component that was antigenetically related to a galactose‐specific lectin present in Geodia cydonium.
Praktische Erfahrungen über das Korrosionsschutzverhalten der Anstrichbezinkung
Authors: Sagel Hans
Published: May 2005

Allgemeines über Zinkanstrichmittel; Verarbeitungstechnische Erfordernisse; Vorbehandlung der Eisenoberfläche.

Vergleichende Gegenüberstellung von Testergebnissen mit unterschiedlichen Zinkstaubfarben nach verschiedenen Testverfahren.

Praktische Erfahrungen mit Zinkstaubfarben im allgemeinen Stahlhochbau, bei Freiluftkonstruktionen und in der Stahlblech‐verarbeitenden Industrie.
Untersuchungen über das Korrosionsverhalten von Aluminium und Aluminiumlegierungen gegenüber organischen und anorganischen Basen
Authors: Bukowiecki A.
Published: May 2005

In der vorliegenden Abhandlung wird auf Grund von bei 20°C durchgeführten 73stündigen Korrosionsversuchen mit verschiedenen Aluminiumwerkstoffen die aluminiumangreifende Wirkung organischer Basen mit der anorganischer verglichen. Die Aggressivität der untersuchten Basen nimmt in der anorganischer verglichen. Die Aggressivität der untersuchten Basen nimmt in der Reihenfolge Natriumhydroxyd, Methylamin, Aethylamin, n‐Butylamin, Ammoniak, Triäthanolamin, Natriumcarbonat, Calciumhydroxyd, Hydrazin ab. Die Korrosionsfälligkeit einzelner Werkstoffe vermindert sich entsprechend der Reihe Reinaluminium Al 99,3, Reinstaluminium Raffinal Raf W (über 99,99% Al), Anticorodal Ac B, Avional Av‐23 V, Perunal Pu V und Peraluman Pe‐30 W. Bei jedem Gemisch Base/Wasser besteht eine kritische Konzentration, bei welcher die Intensität des Angriffs auf Aluminium ein Maximum erreicht. Sehr konzentrierte Basen wirken nur ganz schwach, wasserfreie Basen kaum aluminiumangreifend. Zwischen den Korrosionseigenschaften der einzelnen Basen und ihrem pH‐Wert bzw. ihrer elektrischen Leitfähigkeit bestehen keine eindeutigen Zusammenhänge.
Untersuchungen an einer zahnärztlichen Gold‐Platin‐Legierung
Authors: Yamaguchi S. , Tsuchiya T. , Nishikawa M.
Published: May 2005

Die Legierung, die auch eine gewisse Menge Silicium enthält, besteht aus Mischkristallen mit Einschlüssen intermetallischer Verbindungen. Bei der Wärmebehandlung (zur Entladung) entsteht an der Oberfläche eine SiO2‐Schicht, die einerseits als Haftmittel für das Dentalporzellan dient und andererseits bei Kontakt mit Speichel in einen kolloidalen Zustand übergeht, so daß ein physiologisch einwandfreies Verhalten erreicht wird.