bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of transmembrane proteins from eukaryotic cells
Authors: Evans R. M. , Grillo F. G. , Fink L. M. 
Published: May 2005

AbstractThe topography and properties of plasma membrane proteins from mouse L‐929 cells are studied by comparing their availability for enzymatic labeling on the external and internal surfaces of the membrane. In order to study the internal surface, phagolysosomes are prepared from cells after they ingest latex particles. The plasma membrane surrounding these seems to have an “inside‐out” orientation. The sugars of the membrane glycoproteins in intact phagolysosomes are not available for interaction with lectins or available for periodate‐borotritide labeling. A comparison of the lectin‐binding proteins lableled by lactoperoxidase‐catalyzed iodination on the external cell surface with those labeled on the internal cell surface suggests that a variety of plasma membrane glycoproteins span the lipid bilayer.Using two‐dimensional gel electrophoresis it has been shown that selected proteins are labeled at both the internal and external faces of the plasma membrane. Analysis of the 2‐D gel electrophoregrams reveals that there are two distinct prominent proteins at 60,000 and 100,000 daltons which are enzymatically iodinated from both sides of the membrane. The partial hydrolysis of the 100,000 dalton protein reveals that different peptides are iodinated when the iodination is performed on intact cells or on the phagolysosomes. These proteins are extensively phosphorylated in cells incubated with inorganic 32P. We conclude that the phagolysosome is probably oriented in an “inside‐out” configuration and that this membrane preparation can be used to study the topographic organization of membrane proteins.The use of oriented membranes, selective labeling of proteins, and affinity separation of proteins in combination with gel electrophoresis to define the position and properties of proteins is discussed.
Rhodopsin phosphorylation suggests biochemical heterogeneities of retinal rod disks
Authors: Shichi Hitoshi , Williams Thomas C. 
Published: May 2005

AbstractFrogs (Rana pipiens) were injected subcutaneously with (3H)‐leucine and allowed to incorporate the radioactive amino acid into newly assembled disks in the retinal rod outer segment. The labeled disks served as a temporal market for following the turnover of rod outer segments. Animals were killed at different times after injection and outer segments were isolated and phosphorylated with ATP in the light. The visual pigment (as isorhodopsin) was regenerated with 9‐cis retinal, extracted, and chromatographed on epichlorohydrin triethanolamine cellulose so that phosphorylated pigment could be separated from unphosphorylated pigment. The ratio of (3H)‐radioactivity of phosphorylated pigment to that of unphosphorylated pigment was then plotted against the time after injection. The ratio was high when (3H)‐labeled disks were largely associated with the basal region of the rod and decreased as the labeled disks moved toward the rod apical region. The results were interpreted as suggesting that newer disks are phosphorylated preferentially to older disks. Papain digestion of (3H)‐labeled disks indicated that rhodopsin in newer disks is more susceptible to proteolysis than that in older disks.
Sequence of the amino‐terminal region of rat liver ribosomal proteins S4, S6, S8, L6, L7a, L18, L27, L30, L37, L37a, and L39
Authors: Wittmann‐Liebold B. , Geissler A. W. , Lin A. , Wool Ira G. 
Published: May 2005

AbstractThe sequence of the amino‐terminal region of eleven rat liver ribosomal proteins–S4, S6, S8, L7a, L18, L27, L30, L37a, and L39 ‐ was determined. The analysis confirmed the homogeneity of the proteins and suggests that they are unique, since no extensive common sequences were found. The N‐terminal regions of the rat liver proteins were compared with amino acid sequences in Saccharomyces cerevisiae and in Escherichia coli ribosomal proteins. It seems likely that the proteins L37 from rat liver and Y55 from yeast ribosomes are homologous. It is possible that rat liver L7a or L37a or both are related to S cerevisiae Y44, although the similar sequences are at the amino‐terminus of the rat liver proteins and in an internal region of Y44. A number of similarities in the sequences of rat liver and E coli ribosomal proteins have been found; however, it is not yet possible to say whether they connote a common ancestry.
Mitogenic effects of murine serum and fibroblast growth factor on EGF nonproliferative variants of 3T3
Authors: Pruss Rebecca M. , Herschman Harvey R. 
Published: May 2005

AbstractThe enhanced ability of murine serum to support growth of 3T3 cells, when compared with fetal calf serum, is also evident on variants of 3T3 cells lacking the ability to bind epidermal growth factor (EGF). Variant 3T3 cell lines unable to bind EGF also retain a mitogenic response to fibroblast growth factor.
Isolation and characterization of the nuclear matrix from the male xenopus laevis following estrogen administration: Kinetics of [3H] uridine incorporation
Authors: Snead Henry W. , McDonald Thomas F. , Baker Mary D. , Lanclos Kenneth D. 
Published: May 2005

AbstractAt various times following estorgen administration, the nuclear matrix was isolated from the liver of male Xenopus laevis by sucrose gradient centrifugation of nuclei treated with a high‐salt buffer and DNase I in the presence of a proteolytic inhibitor (PMSC ‐ phenylmethyl sulfonyl chloride). Electron micrographs of the nuclear matrix demonstrate a sponge‐like network attached to a well‐defined inner envelope with a ribosome‐free outer envelope. Chemical analyses show that the HSB‐DNase‐treated nuclei consist of 16% DNA, 2% RNA, and 82% protein, a composition that is consistent with that of nuclear matrices isolated from other species. The specific activity of the matrix‐associated RNA following estrogen treatment appears to be maximally enhanced after 5 h and decreases until approximately 12 h, when the activity begins to increase again.
Comparison of the structures of human fibronectin and plasma cold‐insoluble globulin
Authors: Balian Gary , Crouch Ed , Click Eva Marie , Carter William G. , Bornstein Paul 
Published: May 2005

AbstractHuman amniotic fluid fibronectin and plasma fibronectin (cold‐incoluble globulin) are indistinguishable both immunologically and by amino acid composition. Cyanogen bromide and tryptic peptides also suggest substantial structural homology. However, carbohydrate analysis has demonstrated additional saccharides in fibronectin and an overall increase in carbohydrate content relative to coldinsoluble globulin. Furthermore, limited proteolytic cleavage of the two proteins indicates differences in primary structure or in conformation. Using affinity‐purified antibodies to cold‐insoluble globulin, a glucosamine‐labeled pronaseresistant component, probably proteoglycan, was found to coprecipitate with fibronectin, suggesting an association between these two macromolecules in the connective tissue matrix.
Genetic studies on mechanisms of protein localization in escherichia coli K‐12
Authors: Hall Michael N. , Emr Scott D. , Silhavy Thomas J. 
Published: May 2005

AbstractIn the last few years, several laboratories have demonstrated that many proteins (both from eukaryotic and prokaryotic organisms) that are destined to be localized in noncytoplasmic locations initially are synthesized as a precursor with a 15–30 amino acid extension at the NH2‐terminal end of the molecule. This extra peptide has been termed the signal sequence, and it has been proposed that this signal plays a role in the localization of the extracytoplasmic protein. We are studying the process by which proteins are exported to the envelope region of Escherichia coli. Our work deals primarily with the outer membrane proteins, λ receptor, the product of the lamB gene, and the major outer membrane (porin) proteins 1a and 1b, products of the ompF and ompC genes.Using techniques of gene fusion, we have demonstrated that information specifying the cellular location of the λ receptor is contained within the lamB gene. Furthermore, we have shown that this information is capable of directing even a normally cytoplasmic protein, β‐galactosidase, to the outer membrane. Some of this information is contained within the signal sequence. Mutations that alter this sequence prevent export of the λ receptor protein. Again using techniques of gene fusion, we have shown that the signal sequence alone is not sufficient to cause export of β‐galactosidase from the cytoplasm. Other information within the lamB gene is required.Selection procedures have been developed to isolate mutations that exhibit a general alteration in the export process. Genetic analysis of these mutations has provided evidence for the involvement of the ribosome in the process of protein localization.The structural genes for the porin proteins, 1a and 1b, are regulated at the transcriptional level by the ompB locus. This has permitted us to extend our studies on outer membrane protein localization to protein 1. With this genetic system, it should be possible to determine if E coli employs more than a single mechanism for the export of proteins to the outer membrane.
Reconstitution of binding protein dependent ribose transport in spheroplasts derived from a binding protein negative escherichia coli K12 mutant and from salmonella typhimurium
Authors: Robb Frank T. , Furlong Clement E. 
Published: May 2005

AbstractHighly purified ribose‐binding protein from Escherichia coli has been used to reconstitute a binding‐protein‐dependent ribose transport in spheroplasts derived from a binding‐protein‐deficient mutant of E coli K 12, and in spheroplasts derived from Salmonella typhimurium. The cross‐species reconstitution was nearly as efficient as the reconstitution of the E coli strain from which the binding protein was derived. Antibody raised against the ribose binding protein completely prevented reconstitution, whereas it had no effect on whole cells. The reconstitution procedure has been improved by generating spheroplasts from cells grown in a rich medium and by reducing the background uptake in spheroplasts through a special washing procedure. Rapid purification of ribose binding protein by high pressure liquid chromatography is also described.
Human T‐cell growth factor: Parameters for production
Authors: Ruscetti Francis W. , Mier James W. , Gallo Robert C. 
Published: May 2005

AbstractUsing conditioned media (CM) from phytohemagglutinin (PHA)‐stimulated peripheral blood lymphocytes (PBL) we observed long‐term selective growth of T‐cells from normal human donors. This T‐cell growth was continuously dependent on addition of a factor called T‐cell growth factor (TCGF). The optimal method for preparing highly active CM from single donor PBL involves the addition of mitomycin C‐treated B‐lymphoblastoid cell lines to the mixture of PBL and PHA. A number of different cell lines greatly augmented the production of TCGF in 18/18 cases. Preparation of plasma membranes from the Daudi cell line could replace the intact cells in the production of TCGF but those from the cell line, Molt‐4, could not. Since the cell surface of Daudi possesses HLA‐D antigens but not HLA‐A, B, and C, and Molt‐4 has HLA‐A and B and not HLA‐D, it is possible that the Ia antigens (HLA‐DRW in man) are important in the release of TCGF. Using this method for growth factor production, an analysis was made concerning the events necessary for lymphocyte activation and the requirements for production and release of TCGF. Removal of PHA 12 hr after incubation had no effect on lymphocyte transformation but decreased TCGF release by 90%. In addition, colchicine and cytosine arabinoside inhibited DNA synthesis but had no effect on TCGF release. Little or no TCGF activity was present after cellular protein synthesis was inhibited by puromycin and cycloheximide. These results suggest that TCGF production: (a) requires protein synthesis; (b) requires binding of the stimulating agent; (c) can occur in a non‐dividing cell, probably a terminally differentiated T‐cell, without the need for cellular proliferation; and (d) needs the assistance of an adherent cell which probably is a monocyte‐macrophage. The ability to produce TCGF from single human donors will allow better understanding of the nature and action of TCGF.
Cell cycle dependent rate of labelling of cellular and secreted glycosaminoglycans in mouse embryonic fibroblasts
Authors: Otto Angela M. , Mühlradt P. F. 
Published: May 2005

AbstractCultures of embryonic fibroblasts from Balb/c or CBA/J mice were given 12‐h pulses of 14C‐galactose, or were double‐labelled with 3H‐galactose and 35H‐sulfate. The time course of the rates of labelling of glycosaminoglycans – galactose label was found in the uronic acid moiety – was studied in synchronously and asynchronously growing cultures. Partial synchrony was achieved by trypsinising quiescent, confluent cells and subsequent transfer of cells to new cultures with fresh medium. Synchrony was monitored by measurement of thymidine uptake in parallel cultures. The distribution of label in the hyaluronic acid, chondroitin sulfate, and heparan sulfate fractions from cells and culture media was determined at each time point.Peaks of DNA synthesis were accompanied by or followed 12 h later by a maximal rate of labelling with galactose of secreted glycosaminoglycans, and – with the exception of hyaluronic acid – also of cellular glycosaminoglycans. The rate of labelling with galactose of glycosphingolipids in parallel cultures followed a different time course. In double‐label experiments the rates of labelling of glycosaminoglycan sulfates with 3H‐galactose and 35S‐sulfate did not go parallel. In older, quiescent cultures the labelling rate with galactose decreased while the sulfation rate increased.It is discussed that the labelling rate with galactose is indicative of the biosynthetic rate of the glycosaminoglycans. The conclusion is reached that glycosaminoglycans are preferentially synthesized and secreted after the S phase of the cell cycle.
ATP‐induced lysis of rat parotid secretory granules: Possible role of ATP in exocytotic release
Authors: Oberg Stephen G. , Robinovitch Murray R. 
Published: May 2005

AbstractSecretory vesicles isolated from a variety of mammalian tissues are known to lyse and thereby release their secretory products when exposed to ATP. This process, which will be termed ATP‐induced lysis, has been studied most extensively using adrenal chromaffin‐granule preparations. We report here that ATP causes the lysis of a highly purified preparation of rat parotid secretory granules. The rate of granule lysis was measured spectrophotometrically, and ATP‐induced lysis was expressed as the increase in the rate of lysis (r = % lysis per min) when ATP was added. This lytic process was characterized with respect to pH, temperature, osmolarity, and the ionic composition of the media ATP‐induced lysis of parotid granules was found to have the following properties in common with the extensively characterized chromaffin‐granule process:

It is a saturable function of ATP with half‐maximal rates observed at 0.5 ± 0.1 mM ATP.
It is temperature dependent, eg, r = 6.1 ± 2.1%/min at 30°C vs 12.2 ± 2.5%/min at 37°C.
It is inhibited in hyperosmotic media, eg, r = 5.3 ± 0.3%/min at 0.3 OsM vs 0.8 ± 0.2%/min at 0.4 OsM.
It shows a nucleotide preference of ATP = GTP > ADP > AMP > CTP = ITP.
It has an anion requirement.
The above findings, combined with reports of ATP‐induced lysis of cholinergeric, insulin, and posterior‐pituitary vesicles, imply that ATP‐induced lysis may reflect an ATP‐dependent property of all secretory vesicles, and as such, this vesicle property could play a similar role in each exocytotic release process. Using a model system, Miller and Racker [22] made a surprising finding that the extent to which liposomes fuse with a black lipid membrane depends on the osmotic gradient across the vesicle membrane. In view of the osmotic dependence of ATP‐induced lysis in this and other secretory‐vesicle preparations, we postulate that ATP may prime secretory vesicles for fusion with the plasma membrane by inducing and/or maintaining an osmotic gradient across the vesicle membrane.
Inhibition of lymphocyte mitogenesis by an arachidonic acid hydroperoxide
Authors: Goodman Michael G. , Weigle William O. 
Published: May 2005

AbstractIncubation of murine spleen cells with the oxidation product of soybean lipoxidase‐treated arachidonic acid results in profound inhibition of induction of proliferation and maturation of these cells. The active entity was shown to be the 15‐hydroperoxide of arachidonic acid (15‐HPAA). Inhibition of the enzymes of the cyclo‐oxygenase pathway fails to disturb this effect, indicating that 15‐HPAA is not a substrate for this series of enzymes. 15‐HPAA produced in this manner interfered with RNA synthesis, DNA synthesis, and blastogenesis, while failing to exert cytotoxic effects on the cells themselves. A variety of lymphocyte subpopulations, distinguished by their responsiveness to a diverse group of mitogens, were all equally inhibited by the addition of 15‐HPAA to culture. Addition of this agent even as late as 24 h after initiation of culture resulted in profound inhibition of the proliferative and differentiative responses of splenic B cells to bacterial lipopolysaccharide (LPS). Exposure of cells to 15‐HPAA for 10–30 min was adequate to initiate inhibition, an event that exhibited marked temperature dependence. The effects of pre‐incubation with 15‐HPAA could not be reversed in its absence in recovery periods of up to 6 h prior to addition of LPS. The implications of these data with reference to cellular activation mechanisms are discussed.
Evidence for a low‐molecular‐weight plasma peptide which stimulates chick chondrocyte metabolism
Authors: Pickart Loren 
Published: May 2005

AbstractPlasma contains a number of insulin‐like activities (ILA) of molecular weights 7,000 to 90,000 (somatomedins and insulin‐like proteins) which stimulate cellular metabolism and may function as growth factors. We have found evidence for the presence of an 800 Dalton peptide in human plasma which markedly stimulates the metabolism of chick chondrocytes.This peptide was extracted from human Cohn fraction IV‐1 by procedures similar to those used for somatomedin isolations. At the Sephadex G‐50 column separation step, the fraction with molecular weights of 300–1,000 was found to markedly stimulate chick chondrocyte metabolism. Rechromatography on Sephadex G‐25 concentrated activity in peptides of molecular weight of about 800. An HPLC separation on a silica C‐18 reverse phase column gave elution of the active peptide at 18% acetonitrile in water. This bioactivity appears to be a peptide which is free of lipids, carbohydrates, nucleic acids, metal ions, and immunoreactive insulin. This factor markedly increased the metabolism of cultured chick chondrocytes, but had only marginal activity on rat chondrocytes. When added at 1 μg/ml to chick chondrocytes cultured in F‐12 medium plus 1.5% fetal calf serum, the HPLC‐purified activity increased DNA synthesis 7.3‐fold, lipid synthesis 10.2‐fold, and lactate production 2.9‐fold after 48 h incubation.However, unlike somatomedins A and C, this factor did not displace insulin from placental membranes. These results suggest that low‐molecular‐weight peptides, which are smaller than the somatomedins, may contribute to the total ILA of human plasma.
Restricted expression of an MHC alloantigen in cells of the erythroid series: A specific marker for erythroid differentiation
Authors: Longenecker B. M. , Mosmann T. R. 
Published: May 2005

AbstractThe spectrum of reactivity with various types of cells of a monoclonal antibody (CH‐4) which detects a private MHC antigen of chickens was analysed. CH‐4 agglutinates only RBCs that possess the B2 (MHC) haplotype. A new rosetteforming cell (RFC) assay was devised to detect individual cells (excluding RBCs) that possess the CH‐4 specificity on their cell surfaces. RBCs that have CH‐4 chemically coupled to their surfaces attach to, and form rosettes with, B2 antigen‐bearing cells. Most non‐RBC RFC were detected in active erythropoietic organs (adult bone marrow and embryonic spleen), and none were found in organs where erythropoiesis does not occur: adult thymus and bursa. Preincubation of bone marrow cells with CH‐4 plus complement almost completely inhibits their capacity to form CFU‐E without affecting their ability to form GM‐CFU. In addition, CH‐4 plus complement does not inhibit the capacity of B2/B2 lymphocytes to induce a graft‐versus‐host reaction under conditions where anti‐B2 lymphocyte alloantisera are completely inhibitory. Our results strongly suggest that CH‐4 monoclonal antibodies detect a private specificity on a gene product of the B‐G locus whose expression is restricted to erythroid stem cells and erythrocytes.
Glucocorticoid‐induced lymphocytolysis: State of the genetic analysis
Authors: Bourgeois Suzanne 
Published: May 2005

AbstractThe glucocorticoid‐induced lysis of lymphoid cell lines offers a genetic approach to steroid hormone action because unresponsive variants can easily be selected as resistant to this lytic effect. The present state of analysis of lymphocytolysis in two murine cell lines, the S49 T‐lymphoma and the W7 thymoma, is reviewed. All glucocorticoid‐resistant variants isolated so far result from various defects in the glucocorticoid receptor. The absence of variants blocked at another step of the lytic mechanism is discussed. The observed hemizygosity of the glucocorticoid receptor locus in the S49 line and the instability of cell hybrids illustrate some of the potential problems encountered in somatic cell genetics.
Production of insulinomimetic antibodies against rat adipocyte membranes by hybridoma cells
Authors: Beachy Joanna C. , Czech Michael P. 
Published: May 2005

AbstractSJL mice were injected intraperitoneally with adipocyte plasma membranes or with intrinsic membrane proteins obtained by extraction of plasma membranes with dimethylmaleic anhydride. Three days after the boost injection, the spleens were removed and fused with NS‐1, a thioguanine‐resistant myeloma cell line derived from P3X63 Ag8 (Balb/c). Following selection for hybrids with hypoxanthine, aminopterin, and thymidine, medium of the hybrid cells was tested for its ability to bind to the plasma membrane of the adipocyte and to stimulate the oxidation of D‐(1‐14C) glucose to 14CO2. Approximately 40% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with plasma membranes produced immunoglobulin that bound to adipocyte plasma membranes. About 30% of these mimicked the ability of insulin to stimulate the oxidation of D‐(1‐14C) glucose to 14CO2 in adipocytes. Media from 51% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with intrinsic membrane proteins produced immunoglobulin that bound to the plasma membrane and 48% of those stimulated glucose oxidation. The bioactivity of the hybrid cell media could be blocked by adsorption with intrinsic membrane proteins or by the removal of immunoglobulins using formalin‐fixed Staphylococcus aureus. The hybrids generated in this study can be divided into three categories: (1) hybrids that secrete antibodies that can bind to plasma membranes and mimic insulin action of glucose transport; (2) hybrids that secrete antibodies that bind to plasma membranes but do not stimulate the oxidation of D‐(1‐14C) glucose to 14CO2; and (3) hybrids that produce no antimembrane antibodies. The data suggest that interaction of immunoglobulins with specific membrane proteins is essential in mimicking the action of insulin on glucose transport and oxidation in the rat adipocyte.