bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell surface carbohydrates of preimplantation embryos as assessed by lectin binding
Authors: Brownell Anna G. 
Published: May 2005

AbstractPreimplantation embryos were obtained from the uteri and oviducts of 2 strains of mice, Swiss CD‐1 and B6 CBA. After removal of the zona pellucida by treatment with pronase, FITC‐lectins were bound to the embryonic cell surfaces at either 4°C or 37°C. Both morula and blastocyst stage embryos bound the following lectins, FITC‐ConA, FITC‐WGA, FITC‐RCAII and FITC‐RCAI. No difference in binding was observed between the morula stage and the blastocyst stage within each mouse strain for each specific lectin. However B6 CBA embryos bound less FITC‐ConA and FITC‐WGA than the corresponding Swiss CD‐1 embryos. The topographical arrangement of the lectin receptors was observed to differ between 4°C and 37°C for FITC‐Con A, FITC‐RCAII, and FITC‐RCAI. While lectins bound at 4°C showed a pattern of continuous labeling, the same lectin at 37°C showed aggregation of lectin receptors into patches indicating lateral mobility of these receptors within the embryonic cell membranes. In contrast FITC‐WGA bound at 4°C and 37°C demonstrated continuous labeling of embryos at both temperatures. FITC‐fucose binding protein did not bind to Swiss CD‐1 embryos.The invasiveness of trophoblastic cells of mouse blastocysts was studied by culturing isolated embryos without prior enzyme treatment on reconstituted collagen gels. After 4 days in BME containing only glutamine and bovine serum albumin as supplements, the embryos shed their zona pellucida and implanted into the collagen gel as indicated by zones of lysis in proximity to the embryonic cells when analyzed by scanning electron microscopy.
Interaction between cytoplasmic (Ca2+—Mg2+) ATPase activator and the erythrocyte membrane
Authors: Vincenzi Frank F. , Farrance Martha L. 
Published: May 2005

AbstractHuman red blood cells (RBC) contain a cytoplasmic, nonhemoglobin protein which activates the (Ca2+‐Mg2+) ATPase of isolated RBC membranes. Results presented in this paper confirm that activation of (Ca2+‐Mg2+)ATPase is associated with binding of the cytoplasmic activator to the membrane. Binding of the cytoplasmic activator is reversible and dependent on ionic strength and Ca2+. Cytoplasmic activator is sensitive to trypsin but is not degraded when intact RBC are exposed to trypsin. Cytoplasmic activator does not modify the (Ca2+‐Mg2+)‐ATPase of membranes from RBC exposed to activator prior to hemolysis. Thus, the activator is located in the cell and appears to act by binding to the inner membrane surface.
Organization and polysaccharides of sponge aggregation factor
Authors: Humphreys Susie , Humphreys Tom , Sano James 
Published: May 2005

AbstractAggregation factor, the macromolecular complex which mediates species‐specific aggreagation of dissociated sponge cells, was isolated from several species, partially characterized, and visualized by electron microscopy. All factors were large fibrous complexes with a backbone and side chains or arms. In some factors, the backbone is linear. In others it is circular and the complex appears as a sunburst with arms extending like rays from the circle. The size and location of the polysaccharide chains have been studied using purified preparations of Microciona prolifera. “Sunbursts” treated with ethylenediaminetraacetate (EDTA) for 4 weeks at 0°C dissociate into 3 protein‐ and polysaccharide‐containing components. Sodium dodecyl sulfate does not cause the sunburst to dissociate nor does it inhibit dissociation in the presence of EDTA suggesting that dissociation is not due to hydrolytic enzymes. The dissociation products were tractionated on a 977‐Å pore size micropore glass column. Fifteen percent of the material is excluded and appears in the electron microscope as the central circle of the sunburst. Digestion of the circles with 10−3 M dithiothreitol (DTT) and 0.5 mg/ml proteinase K for 72 h at 37°C produces 2 polysaccharide chanis of 65,000 and 6,000 daltons as fractionated and sized on a 233‐Å pore size micropore glass column using Pharmacia dextrans as standards. The included fractions of the EDTA‐treated material are subunits of the arms which contain 70% of the polysaccharide. A single polysaccharide of 6,000 daltons as measured on 233‐Å size glass beads and Sephadex G‐75 is released from these subunits by proteinase digestion. Pharmacia dextrans are used as standard on both columns. We calculate that there would be four 65,000‐dalton chains and one hundred 6,000‐dalton chains per circle and fifty 6,000‐dalton chains per arm. The third component of the EDTA‐treated preparation is partially included on the column. It appears as linear fibrils in the electron microscope and contains polydisperse polysaccharides of several‐hundred‐thousand daltons. It may be an impurity since there is apparently less than 1 of the large polysaccharide chains per sunburst.
The biosynthesis of mannolipids and mannose‐containing complex glycans by the retina
Authors: Kean Edward L. 
Published: May 2005

AbstractLarge‐scale incubations were carried out with homogenates of the retinas of the 15–16‐day‐old chick embryo in the presence of GDP[U‐14C] mannose, from which there were isolated a mannolipid (Lipid I), oligosaccharide‐lipids (Lipid II), and glycoprotein (residue). These incubations were performed in the presence of endogenous acceptors as well as dolichyl phosphate. [14C] Mannolipid I was subjected to chromatography on DEAE cellulose and silicic acid. The response to these, as well as TLC, enzymatic, and chemical treatments, were consistent with the product being dolichyl phosphomannose. [14C] Lipid II was purified by DEAE cellulose chromatography and gel filtration on LH‐20. Responses to these treatments, as well as TLC and paper chromatography, were consistent with this product being of the class of the oligosaccharide‐pyrophosphate‐lipids. The residue remaining after removal of the lipids was shown to contain glycoproteins by conversion of high‐molecular‐weight radioactive material to low‐molecular‐weight [14C] mannose‐containing glycopeptides by the action of pronase. These reactions and their products are consistent with there being in the retina, the pathway for glycoprotein synthesis involving the participation of the lipid‐activated carbohydrates.When the incubations were performed in the presence of ATP or ADP there was a decrease in the labeling of Lipid I, accompanied by an increase in the labeling of Lipid II and glycoprotein. When incubated in the presence of dolichyl phosphate and deergent, however, the stimulatory effect of ATP did not occur. The effect on these activities of a variety of other nucleotide phosphates was also examined.
The turnover of a tissue specific cell surface ligand which inhibits lectin induced capping
Authors: McDonough James , Lilien Jack 
Published: May 2005

AbstractTen‐day‐old embryonic chick neural retina release into the environment glycoprotein ligands which bind to homologous cells, inhibiting the lectin‐induced redistribution of cell surface receptors. Material with identical activity is released from trypsin‐dissociated neural retina cells that are allowed to repair in culture for 2 h and are then transferred to fresh medium. Release of ligand is inhibited by cytosine arabinoside, hydroxyurea, UDP, and EDTA, and is potentiated by MnCl2. These data suggest that a glycosyltransferase reaction plays a critical role in the turnover of the cell surface ligand. Reactivation of enzymatically deglycosylated ligand solutions by intact cells provides further support for this hypothesis.Release of ligand is also accompanied by a loss of the agglutinability of the cells by a tissue‐specific component which accumulates in monolayer conditioned medium. Conditions which inhibit release maintain maximal agglutinability suggesting similar mechanisms mediate both processes.
The structure of intrinsic membrane proteins
Authors: Guidotti Guido 
Published: May 2005

AbstractIntrinsic membrane proteins are embedded in the lipid bilayer so that the polypeptides come in contact with the non‐polar region of the bilayer. There are two major types of intrinsic proteins: those with most of their mass outside the cytoplasm (Type I) and those with most of their mass inside the cytoplasm (Type II). In the latter group are the membrane transport systems. The anion exchange system of the human erythrocyte is a dimer of band 3 polypeptides. These polypeptides span the bilayer, have most of their mass in the cytoplasm, and are glycosylated. About 20–25% of the polypeptide, however, is in the bilayer. Arguments are presented to support the view that the intramembrane segments of the protein are α‐helical and that the major protein‐protein interactions between the subunits are in the cytoplasmic portion of the protein.
Regulation of glucose carriers in chick fibroblasts
Authors: Amos Harold , Musliner Thomas A. , Asdourian Hovan 
Published: May 2005

AbstractThe derepression of glucose transport initiated by removing glucose from the incubation medium requires both protein and RNA synthesis. The synthesis and accumulation of putative mRNA for the carrier protein(s) can be demonstrated by inhibiting protein synthesis with cycloheximide (2 μg/ml). Release from inhibition with simulataneous addition of actionmycin D (1–5 μg/ml) results in a burst of carrier synthesis that achieves virtually maximal derepression in 4–6 h. An external energy source provided by a “nonrepressive” sugar (D‐fructose, D‐xylose) or by pyruvate is required to accomplish carrier synthesis. Previous failure to demonstrate mRNA accumulation was due to the depletion of energy in the starved cells. Glucose acts as a repressor at a posttranscriptional step, probably at the level of turnover of formed carrier.The protection of formed carrier in the absence of glucose and by inhibitors of protein synthesis even in the presence of glucose has encouraged conjecture that a protease is activated by a metabolic product of glucose that is analogous to a co‐repressor. The glucose metabolite either activates the protease by direct interaction with it or alters the conformation of the carrier to expose a critical region to protease attack. Indeed the regulation of carrier density in the membrane of chick fibroblasts may be achieved entirely by carrier inactivation, the rate of which is a function of glucose concentration in the culture medium.
Cell surface glycosyltransferases—do they exist?
Authors: Deppert Wolfgang , Walter Gernot 
Published: May 2005

AbstractThe presence of glycosyltransferases on surfaces of mammalian cells has been reported by many investigators and a biological role for these enzymes in cell adhesion and cell recognition has been postulated. Critical analysis, however, showed 2 major complications regarding the assay for cell surface glycosyltransferases: (1) hydrolysis of the nucleotide sugar by cell surface enzymes and subsequent intracellular use of the free sugar and (2) loss of cell integrity if trypsinized or EDTA‐treated cells were used in suspension asays. We have assayed intact, viable cells in monolayer for cell surface glycosyltransferases using conditions under which intracellular utilization of free sugars generated by hydrolysis of the nucleotide sugar was prevented. Our data demonstrate that the presence of galactosyltransferases on the surface of a variety of cells, including established (normal and virally transformed) as well as nonestablished cells, is unlikely. No evidence for the existence of cell surface fucosyl‐and sialyltransferases could be obtained, but our data do not exclude the possibility that low levels of these enzymes are present.
Direct modification of the glycocalyx of a cultured muscle cell line by incorporation of foreign gangliosides and an integral membrane glycoprotein
Authors: Barratt Dwight G. , Rogers Jackilynn D. , Sharom Frances J. , Grant Chris W. M. 
Published: May 2005

AbstractAs part of a program to better understand the cause‐or‐effect nature of the relationship between cell surface carbohydrate and cell properties and behaviour, experiments have been carried out on direct modification of the glycocalyx of cultured cells. Modification was by incorporation of gangliosides and an integral membrane glycoprotein chosen to be dissimilar to species occurring naturally in the cell line. Two methods of incorporation were investigated: simple addition of the new components to the culture medium for various times, or assembly of the components into the walls of lipid vesicles which were subsequently fused with cells. Gangliosides from beef brain and glycophorin, the major human erythrocyte sialoglycoprotein, were successfully added to the surface of myoblasts in quantities sufficient to represent a significant perturbation. Changes in cell adhesion, morphology, and viability were observed which seem to be a direct result of glycocalyx modification.
Temperature‐dependent morphological changes in membranes of bacillus stearothermophilus
Authors: Halverson Craig A. , Esser Alfred F. , Souza Kenneth A. 
Published: May 2005

AbstractBacillus stearothermophilus cells vary the lipid fatty acid composition of cytoplasmic membranes with growth temperature. Spin label studies of such membranes have been interpreted to indicate lateral lipid phase separations at the growth temperature. We have now used freeze‐fracture electron microscopy to confirm the spin label studies. Freeze‐fracture faces of protoplasts indicate slight but distinct protein aggregation at the growth temperature. Aggregation increases rapidly with decreasing quench temperature in wild‐type cells. In contrast we were unable to demonstrate extended protein segregation in membranes of a temperature‐sensitive mutant that contains more than 58% branched fatty acids.Storage of protoplasts for prolonged times below the lipid phase transition results in the appearance of corrugated fracture faces with 300‐ to 500‐Å repeat patterns, although this organism does not synthesize lecithins.
Biochemical and ultrastructural studies of ectoglycosyltransferase systems of murine L1210 leukemic cells
Authors: Bernacki Ralph J. , Porter Carl W. 
Published: May 2005

AbstractIntact murine L1210 leukemic cells incorporated significant quantities of [3H]‐N‐acetylneuraminic acid directly from CMP‐N‐acetylneuraminic acid. When pretreated with Vibrio cholerae neuraminidase, incorporation increased sixfold to tenfold. Biochemical studies comparing incorporation of N‐acetyl‐neuraminic acid from the nucleotide sugar with that from free sugar demonstrated that the relatively high levels of incorporation from CMP‐N‐acetyl‐neuraminic acid could not be due to the incorporation of free sugar generated by extracellular degradation of the nucleotide sugar. Very little N‐acetylneuraminic acid was taken up or incorporated by L 1210 cells from free sugar and this incorporation was not increased by neuraminidase pretreatment. Moreover, extracellular breakdown of CMP‐N‐acetylneuraminic acid during incubations with L 1210 cells was rather insignificant.Electron microscope autoradiography of cells incubated with CMP‐N‐acetylneuraminic acid demonstrated that greater than 84% of the incorporated radioactivity was associated with the plasma membrane and less than 1% with the Golgi apparatus. These findings are consistent with the conclusion that incroporation of N‐acetylneuraminic acid from CMP‐N‐acetylneuraminic acid is the consequence of a cell surface sialytransferase system. Pretreatment of cells with the nonpenetrating reagent, diazonium salt of sulfonilic acid, significantly inhibited this ectoenzyme system while only marginally affecting galactose uptake and incorporation at the Golgi apparatus. Interestingly, incorporation from CMP‐N‐acetylneuraminic acid declined as the viability of the cell population declined. When taken together, the above evidence develops a rigorous argument for the presence of a sialyltransferase enzyme system at the cell surface of L 1210 cells.Studies directed towards the detection of a similar ectogalactosyltransferase system were also undertaken. Cells incubated in the presence of UDP‐[3H]‐galactose incorporated radioactivity into a macromolecular fraction. The presence of excess unlabeled galactose in the incubation medium significantly reduced this incorporation. Electron microscope autoradiographs of cells incubated with UDP‐[3H]‐galactose, demonstrated that incorporation occurred primarily at the Golgi apparatus. The grain distribution in these autoradiographs was similar to that for free galactose. Thus, the incorporation observed for L‐1210 cells incubated in UDP‐[3H]‐galactose was due primarily to the intracellular utilization of free galactose generated by extracellular degradation of the nucleotide sugar. Inability t o demonstrate an ectogalacto‐syltransferase system on L1210 cells does not rule out the possibility that the enzyme is present but undetectable due t o the absence of appropriate cell surface acceptor molecules.
Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins
Authors: Fowler Velia , Bennett Vann 
Published: May 2005

AbstractInteractions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water‐soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate‐labeled integral membrane proteins (> 70% of label in band 3 and PAS‐1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus‐fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside‐out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000‐dalton fragment binds to spectrin in solution, competitively inhibits association of 32P‐spectrin with inside‐out vesicles with a Ki of 10−7M, and causes rapid dissociation of 32P‐spectrin from vesicles. Both acid‐treated 72,000‐dalton fragment and the 45,000 dalton‐cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.
Retention of insulin‐stimulated D‐glucose transport activity by adipocyte plasma membranes following extraction of extrinsic proteins
Authors: Pillion Dennis J. , Shanahan Michael F. , Czech Michael P. 
Published: May 2005

AbstractPlasma membrane vesicles prepared from adipocytes incubated with insulin exhibited accelerated D‐glucose transport activity characteristic of insulin action on intact fat cells. Both control and insulin‐stimulated D‐glucose transport activities were inhibited by cytochalasin B and thiol reagents. Extraction of plasma membranes with dimethylmaleic anhydride eluted 80% of the protein from plasma membrane vesicles. The two major glycoprotein bands (94,000 and 78,000 daltons) and small amounts of a 56,000‐dalton band were retained in dodecyl sulfate gels of the extracted membranes. Both control and insulin‐activated D‐glucose transport activities were retained by plasma membrane vesicles extracted with dimethylmaleic anhydride. Cytochalasin B binding activity was also retained by extracted membrane vescles and D‐glucose uptake into extracted vescles derived from untreated or insulin‐treated fat cells was inhibited by cytochalasin B. These results suggest that the modification of the adipocyte hexose transport system elicited by insulin action is not altered by a major purification step which involves quantitative extraction of extrinsic membrane proteins.
Characterization of the ribosomal binding site in rat liver rough microsomes: Ribophorins I and II, two integral membrane proteins related to ribosome binding
Authors: Kreibich Gert , Czakó‐Graham Maria , Grebenau Ruth , Mok Winnie , Rodriguez‐Boulan Enrique , Sabatini David D. 
Published: May 2005

AbstractRat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: (i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; (ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detegent Kyro EOB; (iii) in intact rough microsomes ribophorins can be crosslinked chemically to the ribosomes and therefore are in close proximity to them.Treatment of rough microsomes with a low Triton X‐100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton‐treated microsomes containing invaginations led to the recovery of smooth and “rough‐inverted” vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosome‐binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents sugest that ribophorins are necessary for in vitro ribosome binding. Ribophorin‐like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross‐react with antisera against them.
Membrane permeability of bifunctional, amino site‐specific, cross‐linking reagents
Authors: Miyakawa Tokichi , Takemoto Larry J. , Fox C. Fred 
Published: May 2005

AbstractThe membrane permeability of a series of reversible cross‐linking reagents which are diazide tartarate derivatives has been compared with that of dimethyl‐3,3′‐dithiobispropionimidate (DTBP). The diazide tartarate derivatives tested include tartryl‐diazide (TDA), tartryl‐di(glycylazide) (TDGA), tartryl‐di(β‐alanylazide) (TDAA), tartryl‐di‐(γ‐aminobutyrylazide) (TDBA), tartryl‐di (ϵ‐aminocaproylazide) (TDCA). TDA, which has the shortest chain length of the diazide tartarate derivatives tested, proved to be readily permeable through the erythrocyte membrane. When added at equal concentration to unsealed ghosts, TDGA was at least as reactive as DTBP in its ability to cross link the internally displayed proteins 1, 2, 4.1, 4.2, and 6. Treatment of resealed ghosts by DTBP produced oligomeric complexes of these proteins plus apparent homooligomeric complexes of hemoglobin. TDGA at the same concentrations did not cross‐link any of these components, indicating its membrane‐impermeable nature. As the chain length of the homologous series increased from TDGA to TDCA, the cross‐linkers became increasingly permeable through the erythrocyte membrane.