bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface architecture of the plant cell: Biogenesis of the cell wall, with special emphasis on the role of the plasma membrane in cellulose biosynthesis
Authors: Montezinos David , Brown R. Malcolm
Published: May 2005

Cell wall structure and biogenesis in the unicellular green alga, Oocystis apiculata, is described. The wall consists of an outer amourphous primary layer and an inner secondary layer of highly organized cellulosic microfibrils. The primary wall is deposited immediately after cytokinesis. Golgi‐derived products contribute to this layer. Cortical microtubules underlie the plasma membrane immediately before and during primary wall formation. They function in maintaining the elliptical cell shape. Following primary wall synthesis, Golgi‐derived materials accumulate on the cell surface to form the periplasmic layer. This layer functions in the deposition of coating and cross‐linking substances which associate with cellulosic microfibrils of the incipient secondary wall. Secondary wall microfibrils are assembled in association with the plasma membrane. Freeze‐etch preparations of untreated, living cells reveal linear terminal complexes in association with growing cellulosic microfibrils. These complexes are embedded in the EF fracture face of the plasma membrane. The newly synthesized microfibril lies in a groove of the outer leaflet of the plasma membrane. The groove is decorated on the EF fracture face by perpendicular structures termed “ridges.” The ridges interlink with definitive rows of particles associated with the PF fracture face of the inner leaflet of the plasma membrane. These particles are termed “granule bands,” and they function in the orientation of the newly synthesized microfibrils. Microfibril development in relation to a coordinated multienzyme complex is discussed. The process of cell wall biogenesis in Oocystis is compared to that in higher plants.
Membrane protein alterations during the early stages of sporulation of bacillus subtilis
Authors: Goldman Robert C.
Published: May 2005

Membrane protein alterations during the early stages of sporuloation were examined by polyacrylamide gel electrophoresis. Solubilized samples of the vegetative cell membrane (VCM), sporulation membrane fraction (SMF), and inner forespore membranes (IFM) were compared with respect to their protein compositions. The VCM contained 39 protein components, distinguishable as separate bands on gel electrophoresis, and these ranged in molecular weight from 16,000 to greater than 100,000. During the first 5 hr of sporulation, 6 of these 39 protein bands disappeared, 8 increased and 12 decreased in concentration, and 13 showed no discernible change. In addition, 15 new protein components were identified in the SMF during the fireist 5 hr. The new components consisted of 7 protein bands that were transiently associated with the SMF, and 8 proteins that persisted in the SMF from their time of appearance until at least T5 of sporulation. Comparison of the protein composition of the IFM with those of the VCM and SMF revealed that membrane protein alterations occur during sporulation.

The turnover of H3‐tryptophan‐labeleld membrane protein was followed during growth and sporulation. During the 30 min of growth following a simple chase with excess unlabeled tryptophan, membrane protein appeared stable, whereas 5–10% of the nonmembrane protein turned over to acid‐soluble material. However, manipulation of the cells by dilution ito fresh medium, or centrifugation, as part of the chase procedure, resulted in elution of membrane protein to the cytoplasm. In contrast, proteins labeled during vegetative growth were always eluted to the cytoplasm during the first 2 hr of sporulation, and this was followed by a period of reassociation with the membrane fraction. The results are discussed with respect to membrane differentiation as it relates to spore development.
Ricinus communis toxin‐mediated inhibition of protein synthesis in cell‐free extracts of a toxin‐resistant variant mouse lymphoma cell line
Authors: Robbins James C. , Hunter Tony R. , Nicolson Garth L.
Published: May 2005

Ricinus communis agglutinin II (RCAII, ricin, toxin) at low concentrations inhibits protein synthesis in cell‐free extracts, but not in intact cells, of an RCAII‐resistant mouse lymphoma variant cell line. The concentration dependence of the inhibition by RCAII was the same in cell‐free extracts of both RCAII‐resistant variant and RCAII‐sensitive parental cells, while intact parental cells are 250 times more sensitive to RCAII toxicity. The onset of RCAII inhibition of cell‐free protein synthesis was extremely rapid in both cases, being complete in a few minutes. Under these condidtions RCAII inhibits protein synthesis in intact RCAII‐sensitive parental cells, but maximal inhibition requires several hours to occur. These results support our previous electron microscopic observations that the variant cells are defective in the uptake of RCAII by endocytosis at low toxin concentrations.
Two general classes of cytoplasmic actin filaments in tissue culture cells: The role of tropomyosin
Authors: Lazarides Elias
Published: May 2005

In the assembly of actin filaments that takes place during the spreading of a polulation of human lung cells, after trypsin detachment off the substratum and replating, tropomyosin exhibits a considrable lag in its association with the newly forming filament bundles; it begins to associate with them during the later stages of cell spreading as the actin filament bundles normally seen in interphase cells begin to organize. This lag is evident in a number of cell types that are spreading onto a substratum; it does not appear to be due to a selective degradation of this molecule during rounding up of the cells, since tropmyosin associates with the actin filament bundles after this lag even under conditions where the protein synthetic activity of the cell is inhibited to more than 95% by cycloheximide. The preferential binding of tropomyosin to fully assembled filament bundles but not to newly formed bundles of actin filaments suggests therefore the existence of two classes of action filaments: those that bind tropomyosin and those that do not. This selective localization of tropomyosin and those that do not. This selective localization of tropomyosin on actin filaments was further pursued by examining the localization of this molecule in membrane ruffles. The immunofluorescent results indicate that ruffling is an actin‐filament‐dependent, microtubule‐independent phenomenon. Tropomyosin is absent from membrane ruffles under a variety of circumstances where ruffling is expressed and, more generally, from any other cellular activity where actin filaments are expected to be in a dynamic state of reorganization or are required to be in a flexible configuraion. It is concluded that in tissue culture cells tropomyosin binds preferentially to actin filaments involved in structural support to confer rigidity upon them as well as aid them in maintaining a stretched phenotype. The absence of tropomyosin from certain motile phenomena where actin filaments are involved indicates that these classes of actin filaments are regulated by cytoplasmic mechanisms distinct from that by which tropomyosin (and troponin) mediates contractility in skeletal mulscle; it opens the possibility that different types of actin filaments enagaged in different cellular motile phenomenon in tissue culture cells may be regulated by a host of coexisting regulatory mechanisms, some as yet undetermined.
Isolation and purification of bacterial membrane proteins by the use of organic solvents: The lactose permease and the carbodiimide‐reactive protein of the adenosinetriphosphatase complex of escherichia coli
Authors: Altendorf Karlheinz , Lukas Margot , Kohl Brigitte , Müuller Clemens R. , Sandermann Heinrich
Published: May 2005

Techniques for the solubilization and fractionation of integral membrane proteins have been developed in recent years. A small portion of membrane protein (about 2%, proteolipid fraction) will partition into chloroform or 1‐butanol, and, in several cases, these proteins retain functional activity. A virtually complete solubilization can be achieved at neutral pH by use of aprotic solvents, like hexamethylphosphoric triamide or N‐methylpyrrolidone.

At relatively low concentrations (< 3 M) aprotic solvents inhibited β‐D‐galactoside transport by whole cells and the derivative membrane vesicles of Escherichia coli, but this inhibition could be largely reversed by a simple washing procedure. At higher concentrations of aprotic solvent (5–6 M), 50–80% of the total protein of lactose transport‐positive membrane vesicles was solubilized. When these extracts were added to intact lactose transport‐negative membrane vesicles, lactose transport was reconstituted, the required energy being provided by either respiration (e.g., addition of D‐lactate) or by a K+ diffusion potential established with the aid of valinomycin.

The dicyclohexylcarbodiimide (DCCD)‐reactive subunit of the E. coli ATPase complex was found to partition into chloroform, and to be amenable to further purification in organic solvent. Ether precipitation and chromatography on DEAE‐cellulose and hydroxypropyl‐Sephadex G‐50 yielded an homogeneous polypeptide of an apparent molecular weight of 9,000.

The purified and unlabeled DCCD‐reactive protein was incorporated into K+‐loaded liposomes, and a membrane potential was generated by the addition of valinomycin. There are indications that the DCCD‐reactive protein alone made the membrane specifically permeable for protons.
Isolation and partial characterization of the heterophile antigen of infectious mononucleosis from bovine erythrocytes
Authors: Merrick J. M. , Schifferle R. , Zadarlik K. , Kano K. , Milgrom F.
Published: May 2005

The heterophile antigen (Paul‐Bunnell antigen, PBA) of infectious mononucleosis was isolated by extraction of an aqueous suspension of bovine erythrocyte stromata with chloroform‐methanol (2:1). The upper aqueous layer contained gangliosides, PBA, and a high‐molecular‐weight glycoprotein. PBA and gangliosides were separated from the high‐molecular‐weight glycoprotein by extraction of lyophilized upper layer with chloroform‐methanol solvents. Separation of PBA from gangliosides was carried out by chromatography on DEAE‐cellulose with chloroform‐methanol solvents. PBA appeared to be a minor glycoprotein component of the erythrocyte membrane and had both hydrophobic and hydrophilic properties. It was soluble in either organic or aqueous solvents. On SDS‐polyacrylamide gel electrophoresis, it migrated as a single component that stained for protein with Coomassie blue, for carbohydrate with periodic acid‐Schiff reagent, and for lipid with oil red 0; it had an apparent molecular weight of 26,000. It was composed of 62% protein with major amino acids: glutamic acid, proline, glycine, isoleucine, leucine, and threonine (158, 116, 98, 90, 85, and 82 residues per 1,000 residues, respectively). Carbohydrate content was 9.2% with major sugar constituents: sialic acid, galactosamine, and galactose. Serologic activity of PBA was destroyed by pronase but not by trypsin.
Studies related to the head‐maturation pathway of bacteriophages T4 and T2: II. Nuclear disruption, protein synthesis and particle formation with the mutant 43−·30−·46−
Authors: Wunderli H. , Couture E. , Vince D. A. , Kellenberger E.
Published: May 2005

We describe the aberrant phage multiplication of the triple conditional lethal mutant 43−(polymerase)· 30−(ligase)·46−(exonuclease) of bacteriophage T4D in which phage DNA replication is arrested but some late protein synthesis occurs (33). The nuclear disruption is indistinguishable from wild type. Forty‐five empty small and empty large particles are assembled per cell when the multiplicity of infection (m.o.i.) is 100. This number corresponds closely to the 38 phage equivalents of cleaved major head protein determined biochemically. By reducing the m.o.i. the number of observable particles decreases, reaching 1–5 per cell at an m.o.i. of 5(+5).

The total synthesis of phage related proteins is not significantly dependant on the m.o.i. The synthesis of late proteins is about 10% of that of wild type at high m.o.i. and decreases with the m.o.i. The different early and late proteins do not show the same relative proportions as in wild type and respond differently to an increased m.o.i. These and other results are discussed with respect to the role of phage DNA in prehead assembly and head maturation.
Plant lectins detect age and region specific differences in cell surface carbohydrates and cell reassociation behavior of embryonic mouse cerebellar cells
Authors: Hatten Mary E. , Sidman Richard L.
Published: May 2005

When plated at high cell density in a microwell culture system, freshly dissociated embryonic mouse cerebellar cells assemble into reproducible, 3‐dimensional patterns. The addition of the dimeric lectin Succinyl Concanavalin A blocks reversibly the formation of the microwell pattern, suggesting that cell surface carbohydrates affect the reassociation behavior of embryonic mouse cerebellar cells.

Agglutination studes of dissociated cell populations harvested from different regions of the embryonic brain reveal that different lectins agglutinate cell populations from different embryonic brain regions. Cells from E13 cerebellum are agglutinated with Concanavalin A, wheat germ agglutinin, Ricinus communis agglutinin, mol wt 60,000, Ricinus communis agglutinin, mol wt 120,000, and Lens culinaris, but not by soybean agglutinin or a fucose‐binding protein. Cells from the midbrain are agglutinated only with Concanavalin A, Ricinus communis agglutinin, mol wt 60,000 and Ricinus communis agglutinin, mol wt 120,000; those from the cerebral cortex are agglutinated only with Lens culinaris; and those from the medulla are agglutinated only with Ricinus communis agglutinin, mol wt 60,000, and Ricinus communis agglutinin, mol wt 120,000. In addition, agglutination of cerebellar cells with Concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin is diminished over the course of development from embryonic day 13 to postnatal day 7. These studies suggest regional differences in the cell surfaces of the developling brain that are further modulated during the differentiation of the tissues.

On a poly(D‐lysine) treated substrate in microwell cultures, cell migration is unique to the cerebellum of the 4 brain regions studied. Surfaces treated with carbohydrate‐derivatized poly(D‐lysine) are currently being tested for their efficacy as substrates for differential cell migration.
Membrane assembly: Synthesis and intracellular processing of the vesicular stomatitis viral glycoprotein
Authors: Katz Flora N. , Rothman James E. , Knipe David M. , Lodish Harvey F.
Published: May 2005

The glycoprotein (G) of vesicular stomatitis virus (VSV) is synthesized on membrane‐bound polyribosomes. Approximately 30 min after its synthesis, it reaches the surface plasma membrane where it is incorporated into budding virus. The first part of this paper focuses on the 2 intracellular, mimbrane‐bound, glycosylated forms of the glycoprotein which are intermediates in its biogenesis. All glycosylation and processing is completed in the smooth microsome fraction before the protein reaches the surface.

Next, we turn to the mechanism by which G is synthesized on membrane‐bound polyribosomes. All of the G mRNA is bound to membranes, and studies with puromycin suggest that this attachment of G mRNA is mediated by the nascent glycoprotein chain. After its synthesis G is a transmembrane protein with about 30 amino acids at the carboxyl terminus remaining on the cytoplasmic side of the endoplasmic reticulum. Since 95% of the glycoprotein, containing the carbohydrate residues, is resistant to attack by external proteases, it appears to be within the lumen of the endoplasmic reticulum or embedded within the lipid bilayer. Finally, we show that synthesis, glycosylation, and proper asymmetric insertion of G into the ER can be achieved in cell‐free extracts. Both glycosylation of G and proper insertion into the ER membrane in this cell‐free system require concomitant protein synthesis.
Comparison of the carbohydrate of sindbis virus glycoproteins with the carbohydrate of host glycoproteins
Authors: Keegstra Kenneth , Burke David
Published: May 2005

The carbohydrate portions of the Sindbis virus glycoproteins were compared with the carbohydrate portions of cell surface glycoproteins from uninfected host cells. Comparisons of the size of glycopeptides were made using gel filtrations. Comparisons of sugar linkages were made by methylation analysis. The conclusion was that the Sindbis carbohydrate is similar to a portion of the host carbohydrate. Thus, the Sindbis carbohydrate structures appear to be structures normally made in the uninfected host cell, but which are added to the Sindbis glycoproteins in virus‐infected cells.
Synthesis, secretion, and attachment of lets glycoprotein in normal and transformed cells
Authors: Hynes Richard O. , Destree Antonia T. , Mautner Vivien M. , Ali Iqbal U.
Published: May 2005

LETS glycoprotein is a surface glycoprotein which is absent or greatly diminished on the surfaces of transformed cells. Normal cells secrete large amounts of this protein into the medium; transformed cell medium contains much less. The difference is not due to degradation of the soluble LETS protein. Biosynthesis of LETS protein can be studied by analysis of cell extracts by detergent extraction and immune precipitation and appears to proceed in transformed cells at a reduced rate compared with normal cells. Addition of inhibitors of protein synthesis to transformed cell cultures causes the small amount of LETS protein in the medium to attach to the cells. Addition of normal conditioned medium, which contains LETS protein, to transformed cells alters their morphology towards normal. Addition of purified LETS protein to transformed cells causes the cells to attach, spread, align with one another, and regain actin cables. The results indicate that LETS protein can exchange between cell surface and medium and that it affects cellular adhesion, morphology, and cytoskeleton.
Enzymatic conversion of proteins to glycoproteins by lipid‐linked saccharides: A study of potential exogenous acceptor proteins
Authors: Kronquist Kathryn E. , Lennarz William J.
Published: May 2005

Previous studies have shown that a membrane preparation from hen oviduct catalyzes transfer of oligosaccharide from oligosaccharide‐P‐P‐dolichol to denatured RNase and α‐lactalbumin. To gain further insight into the structural requirements of a protein that allow it to serve as a substrate for glycosylation, the acceptor ability of a variety of other modified proteins containing the tripeptide sequence ‐ASN‐X‐(SER/THR)‐ has been investigated. Of 7 proteins tested, 2 (ovine prolactin and rabbit muscle triosephosphate isomerase) could be enzymatically glycosylated by a particulate preparation from hen oviduct. The remaining 5 proteins, assayed as either S‐carboxy‐methylated or S‐aminoethylated derivatives, were inactive as carbohydrate acceptors. However, cyanogen bromide treatment of 2 of the inactive proteins, bovine catalase and concanavalin A from jack bean, yielded peptide fragments which served as substrates for glycosylation. These results suggests that for some proteins, disruption of the tertiary structure is sufficient to allow attachment of carbohydrate. Other denatured proteins may possess additional restrictions imposed by their secondary structure. In certain cases, these restrictions are removed when the polypeptide chain is fragmented.
Inhibition of the binding of low‐density lipoprotein to its cell surface receptor in human fibroblasts by positively charged proteins
Authors: Brown Michael S. , Deuel Thomas F. , Basu Sandip K. , Goldstein Joseph L.
Published: May 2005

A group of proteins and polyamino acids with positively charged domains were shown to inhibit the binding of 125I‐LDL to its receptor on the surface of human fibroblasts. The list of inhibitory proteins included platelet factor 4 (which has a cluster of lysine residues at its carboxyl terminus), two lysinerich histones, poly‐L‐lysines of chain length greater than 4, and protamine. These proteins were effective in the concentration range of 5–50 μg/ml. Two other positively charged proteins, lysozyme and avidin, did not inhibit 125I‐LDL binding. Kinetic studies suggested that protamine was not acting simply as a competitive inhibitor with regard to the LDL receptor. In light of previous data showing that polyanions such as heparin and polyphosphates also inhibit 125I‐LDL binding to its cell surface receptor, the current findings suggest that charge interactions are important in this binding reaction. In a related series of studies, a number of glycoproteins and their asialo derivatives as well as a number of sugar phosphates failed to inhibit 125I‐LDL binding to its receptor in fibroblasts.
Physical‐chemical studies of spectrin
Authors: Ralston G. B.
Published: May 2005

In recent years considerable progress has been made in the understanding of the structure and function of the red blood cell membrane. The protein spectrin, of high molecular weight and propensity for self‐association, appears to play a major role, in concert with actin, in maintaining the shape and integrity of the membrane. A study of the physical‐chemical properties of spectrin, and its size, shape, self‐association pattern, and its interaction with other components, leads to a plausible model for the way this protein performs its biological role. The evidence from the structure and interactions of spectrin suggests a structure which is relatively symmetrical yet highly expanded, and which allows extensive, two‐dimensional network formation with actin. In these respects, the structure of spectrin is quite different from that of myosin, to which it has often been likened.
The exchangeability of human erythrocyte membrane cholesterol
Authors: Lange Yvonne , D'Alessandro Josephine S.
Published: May 2005

A new method has been used to determine what fraction of human erythrocyte cholesterol is available for exchange with plasma unesterified cholesterol. Erythrocytes labeled with 3H‐cholesterol by this exchange process were incubated with sonicated phosphatidylcholine vesicles, giving rise to a net movement of cholesterol out of the cells. The specific activity of cholesterol taken up by the vesicles depended on the length of time of incubation. Initially the specific activity in the vesicles was greater than that in the cells, but after approximately 10% of cell cholesterol had been removed, the specific activity of subsequently removed cholesterol was equal to that of the remaining erythrocyte cholesterol. We conclude from these data that (a) all of the cholesterol in the erythrocyte is exchangeable with plasma, and (b) approximately 10% of erythrocyte cholesterol is in a more rapidly exchangeable pool than the remainder.
Beta‐adrenergic receptors: Regulatory role of agonists
Authors: Lefkowitz Robert J. , Limbird Lee E. , Williams Lewis T. , Wessels Michael
Published: May 2005

Direct radioligand binding studies have been used to probe the molecular mechanisms whereby agonist catecholamines regulate the function of betaadrenergic receptors in a model system, the frog erythrocyte. The unique characteristics of agonist as opposed to antagonist action are first, the ability to stimulate the adenylate cyclase through the receptor and second, the ability to desensitize the system by alterations induced in beta‐adrenergic receptors. These properties of agonist are not shared by antagonist despite the high affinity and specificity of antagonist binding to the beta‐adrenergic receptors. Agonist and antagonist receptor complexes may be distinguished in a variety of ways including differences in their sensitivity to regulatory guanine nucleotides and also by gel chromatography on AcA 34 Ultragel. The agonist receptor complex appears to elute from the columns with an apparently increased size. A “dynamic receptor affinity model” of beta‐adrenergic receptor action is proposed which features several distinct conformational states of the receptor. Agonists have much higher affinity for the physiologically active or coupled state of the receptor, whereas antagonists have equal affinity for both. In addition, a third “desensitized” state of the receptor is also postulated to exist.
Effects of abnormal cation transport on deformability of desiccytes
Authors: Clark Margaret R. , Mohandas Narla , Caggiano Vincent , Shohet Stephen B.
Published: May 2005

We have studied the deformability of subpopulations of red cells from a patient with “desiccytosis”, a disorder characterized by increased membrane permeability to potassium and associated with a probable increase in sodium‐sodium exchange. Cells become increasingly dehydrated after maturation because of continued potassium loss without compensatory sodium gain, and they exhibit a progressive increase in mean cell hemoglobin concentration (MCHC). This increase in MCHC causes the cells to become undeformable at shear stress values which result in extensive deformation of normal cells. Reduction of MCHC to approximately normal levels by suspending the cells in hypotonic medium restores normal deformability to all but 0.1–0.2% of the cells. These results suggest that the major factor leading to premature destruction in this disorder is whole cell rigidity conferred by increased intracellular hemoglobin concentrations, rather than any associated membrane rigidity.
Erythrocyte troponin inhibitor‐like protein: Isolation and characterization
Authors: Maimon Jonathan , Puszkin Saul
Published: May 2005

A protein was isolated from a human erythrocyte lysate with an apparent molecular weight of 23,000–24,000 daltons. This protein was purified by batch DEAE cellulose followed by column DEAE cellulose chromatography and a gradient of NaCl. On sodium dodecyl sulfate acrylamide electrophoresis, the erythrocyte protein comigrated with muscle troponin inhibitor. An isoelectric precipitation (pH 9.25) was used for the separation of muscle troponin inhibitor from a complex with another troponin component. Both the erythrocyte protein and the muscle troponin inhibitor partially inhibited muscle myosin Ca2+ and K+‐EDTA ATPase activity. Furthermore, they inhibited actin‐activated Mg2+‐ATPase of muscle myosin. The inhibitory effects were absent in the presence of muscle troponin calcium‐binding component. Muscle troponin inhibitor and the erythrocyte troponin inhibitor‐like protein bound to muscle myosin when myosin was precipitated twice at low ionic strength. The presence of a troponin inhibitor‐like protein in erythrocytes suggests that it may be a component in the regulation of contractile activity.
Spin label studies on rat liver and heart plasma membranes: Effects of temperature, calcium, and lanthanum on membrane fluidity
Authors: Gordon Larry M. , Sauerheber Richard D. , Esgate Judy A.
Published: May 2005

The structures of rat liver and heart plasma membranes were studied with the 5‐nitroxide stearic acid spin probe, I(1 2,3). The polarity‐corrected order parameters (S) of liver and heart plasma membranes were independent of probe concentration only if experimentally determined low I(1 2,3)/lipid ratios were employed. At higher probe/lipid ratios, the order parameters of both membrane systems decreased with increasing probe concentration, and these effects were attributed to enhanced nitroxide radical interactions. Examination of the temperature dependence of approximate and polarity‐corrected order parameters indicated that lipid phase separations occur in liver (between 19° and 28°C) and heart (between 21° and 32°C) plasma membranes. The possibility that a wide variety of membrane‐associated functions may be influenced by these thermotropic phase separations is considered.

Addition of 3.9 mM CaCl2 to I(1 2,3)‐labeled liver plasma membrane decreased the fluidity as indicated by a 5% increase in S at 37°C. Similarly, titrating I(1 2,3)‐labeled heart plasma membranes with either CaCl2 or LaCl3 decreased the lipid fluidity at 37°C, although the magnitude of the La3+ effect was larger and occurred at lower concentrations than that induced by Ca2+; addition of 0.2 mM La3+ or 3.2 mM Ca2+ increased S by approximately 7% and 5%, respectively. The above cation effects reflected only alterations in the membrane fluidity and were not due to changes in probe–probe interactions. Ca2+ and La3+ at these concentrations decrease the activities of such plasma membrane enzymes as Na+, K+‐ATPase and adenylyl cyclase, and it is suggested that the inhibition of these enzymes may be due in part to cation‐mediated decreases in the lipid fluidity.
Transmembrane interactions and the mechanisms of transport of proteins across membranes
Authors: Singer S. J. , Ash J. F. , Bourguignon Lilly Y. W. , Heggeness Michael H. , Louvard Daniel
Published: May 2005

We have made observations, by double fluorescence staining of the same cell, of the distributions of surface receptors, and of intracellular actin and myosin, on cultured normal fibroblasts and other flat cells, and on lymphocytes and other rounded cells. The binding of multivalent ligands (a lectin or specific antibodies) to a cell surface receptor on flat cells clusters the cell receptors into small patches, which line up directly over the actin‐ and myosin‐containing stress fibers inside the cell. Similar ligands binding to rounded cells can cause their surface receptors to be collected into caps on the surface, and these caps are invariably found to be associated with concentrations of actin and myosin under the capped membrane. Although these ligand‐induced surface phenomena appear to be different on flat and rounded cells, we propose that in both cases clusters of receptors become linked across the membrane to actin‐ and myosin‐containing structures. In flat cells these structures are very long stress fibers; therefore, when clusters of receptors become linked to these fibers, the clusters are immobilized. In round cells, membrane‐associated actin‐ and myosin‐containing structures are apparently much less extensive than in flat cells; therefore, clusters of receptors linked to these structures are still mobile in the plane of the membrane. We suggest that in this case the clusters are then actively collected into a cap by an analogue of the muscle sliding filament mechanism.

To explain the transmembrane linkage, we propose that actin is associated with the plasma membrane as a peripheral protein which is directly or indirectly bound to an integral protein (or proteins) X of the membrane. Individual molecules of any receptor are not bound to X, but after they are specifically clustered into patches, a patch of receptors then becomes bound to S and hence to actin/myosin.

Patching and capping of specific receptors on rounded cells is often accompanied by a specific endocytosis of the ligand‐receptor complexes. This represents one common transport mechanism of a protein (the ligand) across the plasma membrane. The possibility is discussed that this type of endocytosis is mediated by a transmembrane linkage of the clustered receptor to actin/myosin. Another mechanism of endocytosis involves the “coated pit” structures that are observed by electron microscopy of plasma membranes. The possible relationships between an actin/myosin and a coated pit mechanism of endocytosis are explored.
Bilayer balance and regulation of red cell shape changes
Authors: Mohandas Narla , Greenquist Alfred C. , Shohet Stephen B.
Published: May 2005

Discocytic human red cells undergo discocyte‐echinocyte and discocytestomatocyte transformations under the action of a wide variety of lipid‐soluble anionic and cationic agents respectively. These shape transformations are explained by the bilayer couple hypothesis of Sheetz and Singer to be the result of preferential distribution of the anionic agents in the outer half of the bilayer and the cationic agents in the inner half of the bilayer. We demonstrate that echinocytogenic effects indeed occur when the naturally occurring phospholipid lysophosphatidylcholine (LPC) is localized in the outer half of the bilayer, and stomatocytogenic effects occur when LPC is in the inner half. However, in contrast to the bilayer couple hypothesis, our results show that simple equivalent membrane surface area expansion on each layer is insufficient to maintain the discocytic shape and there exists a differential concentration effect of LPC on the two halves of the bilayer.
Properties of (Mg2+ + Ca2+)‐ATPase of erythrocyte membranes prepared by different procedures: Influence of Mg2+, Ca2+, ATP, and protein activator
Authors: Katz Sidney , Roufogalis Basil D. , Landman Amiram D. , Ho Larry
Published: May 2005

Erythrocyte membranes prepared by three different procedures showed (Mg2+ + Ca2+)‐ATPase activities differing in specific activity and in affinity for Ca2+. The (Mg2+ + Ca2+)‐ATPase activity of the three preparations was stimulated to different extents by a Ca2+‐dependent protein activator isolated from hemolystes. The Ca2+ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2–200 μM or lowering the Mg:ATP ratio to less than one shifted the (Mg2+ + Ca2+)‐ATPase activity in stepwise hemolysis membranes from mixed “high” and “low” affinity to a single high Ca2+ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strengh, or membranes prepared by the EDTA (1–10 mM) procedure, did not undergo the shift in the Ca2+ affinity with changes in ATP and MgCl2 concentrations. The EDTA‐wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg2+ + Ca2+)‐ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.
Characterization of antigenic sialoglycoprotein subunits of the placental brush border membranes: Comparison with liver and kidney membrane subunits by two‐dimensional electrophoresis
Authors: Wada H. Garrett , Hass Philip E. , Sussman Howard H.
Published: May 2005

The sialoglycoprotein subunits of human placental brush border membranes were labeled by sequential treatment with periodate and (3H)‐sodium borohydride, which trititates sialic acid, and by lactoperoxidase‐catalyzed (125I) iodination of tyrosine residues. The labeled subunits were characterized with respect to their affinity for antisera raised against Triton X‐100 extracts of placental brush border membranes. The immunochemically reactive components were analyzed by two‐dimensional electrophoresis according to a modification of the O'Farrell technique [20] enabling the assignment of estimated Mr̄ and pI. Of the 33 3H‐labeled brush border subunits present in Triton X‐100‐solubilized membrane preparations, 18 subunits reacted with antiplacental brush border antisera insolubilized on CNBr‐activated Sepharose or in immunoprecipitates. Fourteen of these tritiated subunits were also labeled with 125I, confirming that these are glycoproteins.

The plasma membranes of normal human liver and microsomes from kidney were examined for the placental brush border glycoprotein subunits by reaction with insolubilized antiplacental brush border antisera and two‐dimensional electrophoresis of the reacting tritium‐labeled subunits. Comparison of the two‐dimensional electrophoretic maps of the immunochemically reacting glycoproteins from liver, kidney, and placenta resulted in the identification of seven placental subunits in common with liver and kidney on the basis of antigenic cross‐reactivity, Mr̄, and pI. Four placental glycoproteins were not found in the other tissues and are potentially specific to the placenta. Three of the placental subunits were only seen in placenta and kidney. Three of the subunits ran at the dye front and could not be assigned molecular weights. One of the subunits was poorly labeled by tritiation of sialic acid and was not considered.
Regulation of the cell cycle of 3T3 cells in culture by a surface membrane‐enriched cell fraction
Authors: Whittenberger Brock , Raben Daniel , Glaser Luis
Published: May 2005

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G1 protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.
Ribosome crystallization in homogenates and cell extracts of chick embryos
Authors: Barbieri Marcello
Published: May 2005

Ribosome microcrystals have been obtained for the first time in homogenates and extracts of chick embryos mainly in the form of P422 stacks that have average linear dimensions some 40% greater than those obtained in vivo.
Structure of functional a salina – E Coli hybrid ribosome by electron microscopy
Authors: Boublik M. , Wydro R. M. , Hellmann W. , Jenkins F.
Published: May 2005

Small 40S Artemia salina and large 50S Escherichia coli ribosomal subunits can be assembled into 73S hybrid monosomes active in model assays for protein synthesis. The reciprocal combination–small 30S E coli and large 60S A salina–fails to form hybrids. The 73S hybrid particles strongly resemble homologous 70S E coli and 80S A salina monosomes. The morphologic differences between the corresponding eukaryotic and prokaryotic ribosomal particles, established by electron microscopy, do not significantly affect the assembly and mutual orientation of 40S A salina and 50S E coli subunits in the heterologous monosome. The fact that the structure of the interface, the supposed site of protein synthesis, is preserved in the active hybrid implies that retention or loss of biologic activity of hybrid ribosomes is determined by the extent of conformational changes in the interface.
The accessibility of antigenic determinants of ribosomal protein s4 in situ
Authors: Winkelmann Donald , Kahan Lawrence
Published: May 2005

Antibodies to Escherichia coli ribosomal protein S4 react with S4 in subribosomal particles, eg, the complex of 16S RNA with S4, S7, S8, S15, S16, S17, and S19 and the RI* reconstitution intermediate, but they do not react with intact 30S subunits. Antibodies were isolated by three different methods from antisera obtained during the immunization of eight rabbits. Some of these antibody preparations, which contained contaminant antibodies directed against other ribosomal proteins, reacted with subunits, but this reaction was not affected by removal of the anti‐S4 antibody population. Other antibody preparations did not react with subunits. It is concluded that the antigenic determinants of S4 are accessible in some protein deficient subribosomal particles but not in intact 30S subunits.
Extracellular lectin and its glycosaminoglycan inhibitor in chick muscle cultures
Authors: Ceri Howard , Shadle Paula J. , Kobiler David , Barondes Samuel H.
Published: May 2005

Embryonic chick muscle contains two developmentally regulated lectins, which may be involved in cell interactions. These endogenous lectins are assayed as agglutinins of appropriate test erythrocytes. One of these, called lectin‐2, interacts with specific glycosaminoglycans, especially heparin and dermatan sulfate. Lectin‐2 is present at constant levels in both chick fibroblast and chick muscle cells throughout 14 days of culture but is released into the medium of cultured embryonic muscle after 7‐8 days of culture, soon after myoblast fusion. Lectin‐2 interacts strongly with a component of substrate‐attached material in embryonic muscle cultures which is extractable from the culture dishes with alkali after the cells have been removed with ethylediaminetetraacetic acid. The active component in the substrate‐attached material appears to be a glycosaminoglycan that is a more potent inhibitor of lectin‐2 agglutination activity than any of the known glycosaminoglycans that we have tested. The active material is degraded by chondroitinase ABC but not by chondrotinase AC, hyaluronidase, or proteolytic enzymes and thus appears to be similar to dermatan sulfate. The results of these studies raise the possibility that lectin‐2 functions by interacting with glycosaminoglycans, either associated with the cell surface or with the extracellular matrix.
Cellular and metabolic specificity in the interaction of adhesion proteins with collagen and with cells
Authors: Kleinman H. K. , Hewitt A. T. , Murray J. C. , Liotta L. A. , Rennard S. I. , Pennypacker J. P. , McGoodwin E. B. , Martin G. R. , Fishman P. H.
Published: May 2005

Fibronectin mediates the adhesion of fibroblasts to collagen substrates, binding first to the collagen and then to the cells. We report here that the interaction of the cells with the fibronectin‐collagen complex is blocked by specific gangliosides, GD1 a and GT1, and that the sugar moieties of these gangliosides contain the inhibitory activity. The gangliosides act by binding to fibronectin, suggesting that they may be the cell surface receptor for fibronectin. Evidence is presented that other adhesion proteins or mechanisms of attachment exist for chondrocytes, epidermal cells, and transformed tumorigenic cells, since adhesion of these cells is not stimulated by fibronectin. Chondrocytes adhere via a serum factor that is more temperature‐sensitive and less basic than fibronectin. Unlike that of fibroblasts chondrocyte adhesion is stimulated by low levels of gangliosides. Epidermal cells adhere preferentially to type IV (basement membrane) collagen but at a much slower rate than fibroblasts or chondrocytes. This suggests that these epidermal cells synthesize their own specific adhesion factor. Metastatic cells cultured from the T241 fibrosarcoma adhere rapidly to type IV collagen in the absence of fibronectin and do not synthesize significant amounts of collagen or fibronectin. Their growth, in contrast to that of normal fibroblasts, is unaffected by a specific inhibitor of collagen synthesis. These data indicate the importance of specific collagens and adhesion proteins in the adhesion of certain cells and suggest that a reduction in the synthesis of collagen and of fibronectin is related to some of the abnormalities observed in transformed cells.
Cell surface fibronectin and oncogenic transformation
Authors: Hynes Richard O. , Destree Antonia T. , Perkins Margaret E. , Wagner Denisa D.
Published: May 2005

Fibronectin is a large glycoprotein at the cell surface of many different cell types; a related protein is present in plasma. Fibronectin is a dimer of 230,000‐dalton subunits and also occurs in larger aggregates; it forms fibrillar networks at the cell surface, between cells and substrata and between adjacent cells, and it is not a typical membrane protein. Cell surface fibronectin is reduced in amount or absent on transformed cells and in many cases its loss correlates with acquisition of tumorigenicity and, in particular, metastatic ability. Exceptions to the correlations with transformation and tumorigenicity exist. Loss of fibronectin and the resulting reduced adhesion appear to be involved in pleiotrpoic alterations in cell behavior and may be responsible for several aspects of the transformed phenotype in vitro. Fibronectin interacts with other macromolecules (collagen/gelatin, fibrin/fibrinogen, proteoglycans) and is apparently connected to microfilaments inside the cell.
Tumor metastases and cell‐mediated immunity in a model system in DBA/2 mice. VIII. Expression and shedding of Fcγ receptors on metastatic tumor cell variants
Authors: Schirrmacher Volker , Jacobs Wolfgang
Published: May 2005

The expression of receptors for the Fc portion of IgG immunoglobin molecules was studied on tumor cell lines with high and low metastatic capacity. Two tumor cell lines from DBA/2 mice that had high metastatic activity, ESb and MDAY‐D2, contained a high percentage of Fc receptor positive cells, as detected in a rosette assay with IgG antibody‐coated erythrocytes (EA). In contrast, the low metastatic parental line Eb, from which ESb was derived, contained only a low percentage of EA‐rosette‐forming cells. ESb ascites tumor cells adapted to tissue culture in the presence of 2‐mercaptoethanol (2ME) had a high expression of Fc receptors, whereas a cell line adapted to tissue culture in the absence of 2ME had a low expression of Fc receptors.

“Soluble” Fc receptors were detectable by their ability to bind to EA and to cause blocking of rosette formation. They were found to be present in fluids from tumor‐bearing animals, such as serum and cell‐free ascites. Even animals with an ascites tumor of the low‐metastatic line Eb contained “soluble” Fc receptors.

The results are discussed with regard to their possible significance for tumor metastasis.
The serum‐free growth of Balb/c 3T3 cells in medium supplemented with bovine colostrum
Authors: Klagsbrun M. , Neumann J.
Published: May 2005

Bovine milk contains growth promoting factors that stimulate DNA synthesis and cell division in confluent monolayers of quiescent Balb/c 3T3 cells. The growth factor activity was highest in colostrum obtained within 24 hours after birth of a calf. Samples of milk obtained 32 hours and 60 hours after birth were 20% and 1% as active respectively as was a sample obtained 8 hours after birth in stimulating DNA synthesis. No activity was detectable 3 days after birth or thereafter. A similar temporal dependence was found in sheep's milk. Bovine colostrum obtained on the day of a calf's birth can be substituted for serum and will support the growth of sparse Balb/c 3T3 cells to confluence. In Dulbecco's modified Eagle's medium (DMEM) supplemented with 2.5% (vol/vol) bovine colostrum, the number of Balb/c 3T3 cells in a dish increased 35‐fold, from 2.0 × 104 cells to 7 × 105 cells. The generation time was approximately 38 hours. Proliferation of cells was characterized by formation of clusters of confluent Balb/c 3T3 cells which were smaller in size and more tightly packed than were Balb/c 3T3 cells grown to confluence in serum. No proliferation was detected in DMEM supplemented with milk obtained 10 days after birth of a calf or in DMEM supplemented with bovine serum albumen.