bluuebunny/crossref_metadata_2025 huggingface dataset:
Pyrenesulfonyl azide as a fluorescent label for the study of protein‐lipid boundaries of acetylcholine receptors in membranes
Authors: Gonzalez‐Ros J. M. , Calvo‐Fernandez P. , Šator V. , Martinez‐Carrion M.
Published: May 2005

Acetylcholine receptor (AcChR) enriched membrane fragments from Torpedo californica electroplax were labeled by in situ photogenerated nitrenes from a hydrophobic fluorescent probe, pyrene‐1‐sulfonyl azide. Preferential photolabeling of membrane proteins, mainly AcChR, has been achieved and there is a pronounced exposure of the 48,000 and 55,000 molecular weight subunits of AcChR to the lipid environment of the membrane core.

Covalent attachment of the photogenerated fluorescence probe does not perturb the α‐neurotoxins' binding properties of membrane‐bound AcChR or the desensitization kinetics induced by prolonged exposures to cholinergic agonists. Non‐covalent photoproducts can be conveniently removed from labeled membrane preparations by exchange into lipid vesicles prepared from electroplax membrane lipids. Fluorescence features of model pyrene sulfonyl amide derivatives, such as fine vibrational structure of emission spectra or fluorescence lifetimes, are highly sensitive to the solvent milieu. The covalently bound probe shows similar fluorescence properties in situ. PySA photoproducts have great potential to spectroscopically monitor neurotransmitter induced events on selected AcChR subunits exposed to the hydrophobic environment of membranes.
Effect of vanadate on gill cilia: Switching mechanism in ciliary beat
Authors: Wais‐Steider Jacobo , Satir Peter
Published: May 2005

Lateral (L) cilia of freshwater mussel (Margaritana margaritifera and Elliptio complanatus) gills can be arrested in one of two unique positions. When treated with 12.5 mM CaCl2 and 10−5 M A23187 they arrest in a “hands up” position, ie, pointing frontally. When treated with approximately 10 mM vanadate (V) they arrest in a “hands down” position, ie, pointing abfrontally. L‐cilia treated with 12.5 mM CaCl2 and 1 mM NaN3 also arrest in a “hands down” position; substitution of 20 mM KC1 and 1 mM NaN3 causes cilia to move rapidly and simultaneously to a “hands up” position.

The observations suggest that there are two switching mechanisms for activation of active sliding in ciliary beat one at the end of the recovery stroke and the other at the end of the effective stroke; the first is inhibited by calcium and the second by vanadate or azide. This is consistent with a model of ciliary beating where microtubule doublet numbers 1, 2, 3, and 4 are active during the effective stroke while microtubule doublets numbers 6, 7, 8, and 9 are passive, and the converse occurs during the recovery stroke.
The errors in assembly of MuLV in interferon treated cells
Authors: Pitha Paula M. , Fernie Bruce , Maldarelli Frank , Wivel Nelson A.
Published: May 2005

Interferon treatment of JLSV‐6 cells chronically infected with Rauscher MuLV leads to the formation of noninfectious particles (interferon virions) containing the structural proteins of env and gag genes as well as additional viral polypeptides. In the control virions the major glycoprotein detected is gp71, interferon virions contain in addition to gp71 and 85k dalton (gp85) glucosamine‐containing, fucose‐deficient glycoprotein which is recognized by antiserum to MuLV but not by the gp71 antiserum. The surface iodination of the intact virions indicates that both gp71 and gp85 are the major components of the external virions envelope. However, unlike the control virions in which gp71 associates with p15E (gp90), the gp71‐p15E complex was not detected in interferon virions. The analysis of the iodinated proteins of the disrupted interferon virions revealed the presence of 85k and 65k dalton polypeptides preciptable with antiserum against MuLV, which are not present in the control virions. The difference in the polypeptide pattern of virions produced in the presence of interferon does not seem to be a consequence of the slowdown in the synthesis of viral proteins or their processing in the interferon‐treated cells. Both the structural proteins of env and gag genes seem to be synthesized and processed at a comparable rate in the interferon‐treated and ‐untreated cells. These results indicate an alteration of virus assembly in the presence of interferon.
Structural and functional alterations in the surface of vascular endothelial cells associated with the formation of a confluent cell monolayer and with the withdrawal of fibroblast growth factor
Authors: Vlodavsky Israel , Gospodarowicz Denis
Published: May 2005

Vascular endothelial cells cultured in the presence of fibroblast growth factor (FGF) devide actively when seeded at low or clonal cell densities and upon reachin confluence adopt a morphologic appearance and differentiated properties similar to those of the vascular endothelium in vovi. In this review, we present some of our recent observations regarding the characteristics (both structural and functional) of these endothelial cells and the role of FGF in controlling their proliferation and normal differentation. At confluence the endothelial cells from a monolayer of closely apposed and nondividing cell that have a nonthrombogenic apical surface and can no longer internalize bound ligands such as low‐density lipoprotein (LDL). The adoption of these properties is correlated and possibly causally related to changes in the cell surface such as the appearance of a 60,000 molecular weight protein (CSP‐60); the disappearance of fibronectin from the apical cell surface and its concomitant accumulation in the basal lamina; and a restriction of the lateral mobility of various cell surface receptor sites. In contrast, endothelial cells that are maintained in the absence of FGF undergo within three passages alterations that are incompatible with their in vivo morphologic apperarance and physiologic beharior. They grow at confluence on top of each other and hence can no longer adopt both the structural (CSP‐60, cell surface polarity) and functional (barrier function, nonthrombogenicity) attributes of differentiated endothelial cell. Since these characteristics can be reacquired in response to readdition of FGF, in addition to being a mitogen FGF may also be involved in controlling the differentitation and phenotypic expression of the vascular endothelium.
Polyoma T (tumor) antigen species in abortively and stably transformed cells
Authors: Benjamin Thomas L. , Schaffhausen Brian S. , Silver Jonathan E.
Published: May 2005

Stable neoplastic transformation of cells by polyoma virus requires the participation of two viral genes, designated ts‐a and hr‐t. The effects of mutations in these two genes on the patterns of T‐antigen synthesis during productive infection have been previously described: ts‐ a mutants are affected in the “large” (100K) nuclear T antigen, and hr‐t mutants are affected in the “middle” (36K, 56K, 63K) and “small” (22K) T agtigens. The latter are associated predominantly with the plasma membrane (56K) and cytosol fractions, rrespectively.

Here we examine the expression of the various forms of polyoma T antigen in nonproductive infection (abortive transformation) as well as in stably transformed cell lines of different species. The results on abortive transformation are essentially the same as those described above for productive infection. In stably transformed cells, the middle and small T antigens are seen to various extents. The large T antigen, however, is often absent or present below the level of detection. Clones lacking the large T antigen are found most often among mouse transformants, but are also seen among rat transformants. Retention of the 100K species in transformed cells therefore appears to be, at least in part, an inverse function of the level of permissivity of the host toward productive viral infection. These findings indicate that the induction of the transformed phenotype in both abortively and stably transformed cells generally does not require the large T antigen, but rather the products of the hr‐t gene.
Biosynthesis and maturation of cellular membrane glycoproteins
Authors: Hunt Lawrence A.
Published: May 2005

The biosynthesis and the processing of asparagine‐linked oligosaccharides of cellular membrane glycoproteins were examined in monolayer cultures of BHK21 cells and human diploid fibroblasts after pulse‐and pulse‐chase labeling with [2‐3H] mannose. After pronase digestion, radiolabeled glycopeptides were characterized by high‐resolution gel filtration, with or without additional digestion with various exoglycosidases and endoglycosidases. Pulse‐labeled glycoproteins contained a relatively homogenous population of neutral oligosaccharides (major species: Man9GlcNAc2ASN). The vast majority of these asparagine‐linked oligosaccharides was smaller than the major fraction of lipid‐linked oligosaccharides from the cell and was apparently devoid of terminal glucose. After pulse‐chase or long labeling periods, a significant fraction of the large oligomannosyl cores was processed by removal of mannose units and addition of branch sugars (NeuNAc‐Gal‐GlcNAc), resulting in complex acidic structures containing three and possibly five mannoses. In addition, some of the large oligomannosyl cores were processed by the removal of only several mannoses, resulting in a mixture of neutral structures with 5–9 mannoses. This oligomannosyl core heterogeneity in both neutral and acidic oligosaccharides linked to asparagine in cellular membrane glycoproteins was analogous to the heterogeneity reported for the oligosaccharides of avian RNA tumor virus glycoproteins (Hunt LA, Wright SE, Etchison JR, Summers DF: J Virol 29:336, 1979).
Regulation of dome formation in differentiated epithelial cell cultures
Authors: Lever Julia E.
Published: May 2005

Rat mammary (Rama 25) and dog kidney (MDCK) epithelial cell cultures formed ‘domes’ of cells due to fluid accumulation in focal regions between the culture dish and the cell monolayer. Addition of ouabain caused collapse of domes, suggesting that transport functions were required for maintenance of domes.

Dome formation in both epithelial cell lines was stimulated by a broad spectrum of known inducers of erythroid differentiation in Friend erythroleukemia cells. Among these inducers were: (1) polar solvents such as dimethylsulfoxide, dimethylformamide, and hexamethylene bisacetamide; (2) purines such as hypoxanthine, inosine, and adenosine; (3) low‐molecular‐weight fatty acids such as n‐butyrate; and (4) conditions expected to elevate levels of cyclic AMP. In the latter group were activators of adenylate cyclase such as cholera toxin and prostaglandin E1; cyclic AMP phosphodiesterase inhibitors such as theophylline and 1‐methyl‐3‐isobutylxanthine; and analogs of cyclic AMP.

Induction of domes occurred 15–30 h after addition of inducer to the culture medium. Induction by chemicals was serum‐dependent and required protein synthesis but not DNA synthesis. Induced dome formation was reversible after removal of inducer, requiring the contiuous presence of inducer. Reversal was also observed after either removal of serum or addition of inhibitors of protein synthesis.

These results suggest the hypothesis that domes arise in these epithelial cultures by a process that is similar to cell differentiation and is influenced by cyclic AMP.
Scanning electron microscopy of differentiated liver cells transformed in vitro by chemical carcinogens
Authors: Borek Carmia
Published: May 2005

A scanning electron microscopy study was carried out on differentiated liver cells transformed in vitro by three chemical carcinogens into cells that give rise to carcinomas. The results indicate that the transformed cells grow as a rule in tightly adherent monolayers but differ in topography. There is a tendency toward heterogeneity in cell shape compared to the normal and on the whole toward a larger number of surface microvilli in the malignant cell population. However, both in sparse and confluent cultures the topographic differences are often not striking enough to unequivocally distinguish single neoplastic cells from the normal.
Protein‐RNA interactions during TMV assembly
Authors: Holmes K. C.
Published: May 2005

A review of the structural studies of tobacco mosaic virus (TMV) is given. TMV is essentially a flat helical microcrystal with 16 1/3 subunits per turn. A single strand of RNA runs along the helix and is deeply embedded in the protein. The virus particles form oriented gels from which high‐resolution X‐ray fiber diffraction data can be obtained. This may be interpreted by the use of six heavy‐atom derivatives to give an electron density map at 0.4 nm resolution from which the RNA configuration and the form of the inner part of the protein subunit may be determined. In addition, the protein subunits form a stable 17‐fold two‐layered disk which is involved in virus assembly and which crystallizes. By the use of noncrystallographic symmetry and a single heavy‐atom derivative, it has been possible to solve the structure of the double disk to 0.28 nm resolution. In this structure one sees that an important structural role is played by four alpha‐helices, one of which (the LR helix) appears to form the main binding site for the RNA. The main components of the binding site appear to be hydrophobic interactions with the bases, hydrogen bonds between aspartate groups and the sugars, and arginine salt bridges to the phosphate groups. The binding site is between two turns of the virus helix or between the turns of the double disk. In the disk, the region proximal to the RNA binding site is in a random coil until the RNA binds, whereupon the 24 residues involved build a well‐defined structure, thereby encapsulating the RNA.
β‐Bungarotoxin binding sites (acetylcholine receptors) in denervated mammalian sarcolemma
Authors: Tipnis Ulka R. , Malhotra Sudarshan K.
Published: May 2005

The nonsynaptic sarcolemma of denervated skeletal muscle of rat shows an abundance of ∼15 nm intramembranous particles on the P face. These particles are either singly distributed or are in clusters, and they are essentially lacking from the comparable freeze‐fractures of the innervated sarcolemma. Autoradiographic studies using 125I‐α‐bungarotoxin (BGT) on 1 μ‐thick sections, and freeze‐etch studies using ferritin‐α‐BGT conjugates on membrane fractions, show that the distribution of the label corresponds to the distribution of the 15‐nm particles in the nonsynaptic sarcolemma. On the basis of these results and existing physiologic and biochemical data, it is suggested that the 15‐nm intramembranous particles are components of the α‐BGT binding sites, ie, acetylcholine (Ach) receptors, in the nonsynaptic sarcolemma of denervated muscle and that the two types of distributions represent two spatial manifestations of Ach receptor molecules. The significance of these findings in relation to synapse formation in denervated muscle is discussed.
Alteration of human breast tumor cell membrane functions by chromosome‐mediated gene transfer
Authors: Muneer Razia S. , Gray Peter N.
Published: May 2005

BOT‐2 cells (human breast tumor origin) have an impaired ability to utilize exogenous thymidine. Previous studies revealed this deficiency to be the permeation event rather than phosphorylation, since the cells have active thymidine kinase. Chromosome‐mediated gene transfer was used to transfer genetic information in the form of metaphase chromosomes, from HeLa‐65 cells to the BOT‐2 cells, correcting the permease deficiency. Poly‐L‐ornithine or lipochromes were used for facilitation of chromosome uptake. After selection on HAT medium, transferant clones were isolated at a frequency of 4 X 10−5 and 1 X 10−5, respectively. Transferants MGP‐1 and MGL‐1 are stable after 18 months and have been characterized on the bases of purine and pyrimidine nucleoside uptake, relative thymidine kinase activities, alkaline phosphatase activities, and hydrocortisone‐induced alkaline phosphatase activity. MGP‐1 demonstrates positive thymidine uptake and incorporates radiolabeled thymidine into DNA. MGL‐1 remains thymidine transport‐deficient and survives on HAT by increasing endogenous dihydrofolate reductase activity. Alkaline phosphatase activity in MGL‐1 is similar to HeLa‐65, 2% of that in BOT‐2, and in addition, is inducible 25‐30‐fold by 3 μM hydrocortisone. We have separated, genetically, a thymidine permease function from phosphorylation in cells of human origin and have transferred genetic information for the regulation of alkaline phosphatase.
Tumor‐associated antigenic differences between the primary and the descendant metastatic tumor cell populations
Authors: Gorelik Eliezer , Fogel Mina , Segal Shraga , Feldman Michael
Published: May 2005

The existence of antigenic differences between cell populations in the local growth of the 3LL tumor (L‐3LL) and its lung metastases (M‐3LL) was studied. Normal C57BL/6 spleen cells sensitized in vitro for 5 days against L‐3LL monolayers lysed preferentially L‐3LL targets but not M‐3LL tumor cell targets. Conversely, anti‐M‐3LL‐sensitized lymphocytes killed M‐3LL targets more efficiently than they killed L‐3LL targets. Furthermore, spleen cells from mice bearing subcutaneous L‐3LL tumors were significantly more cytotoxic to L‐3LL targets than to M‐3LL targets and vice versa. M‐3LL cells were found also to be more resistant in vitro and in vivo to natural killer cells than were L‐3LL tumor cells. M‐3LL cells were more resistant than L‐3LL cells to hybrid resistant mechanisms when they were inoculated into F1 (C3Heb X C57BL/6) or F1 (BALB/c X C57BL/6) mice. Anti‐M‐3LL lymphocytes generated both in vitro and in vivo, but not anti‐L‐3LL lymphocytes, admixed with L‐3LL or M‐3LL tumor cells and inoculated into footpads of syngeneic recipients suppressed the development of lung metastases. These results suggest that metastatic cells are indeed phenotypic variants of the local growing tumor cell populations. Presumably, these variants are selected for their capacity to home to and grow in the lungs, and for their resistance to specific immune effects initially evoked against the local tumor and to nonspecific natural killer cells. These data may prove to be of importance with respect to any rational approach to the problem of immunotherapy.
Analysis of transmembrane proteins from eukaryotic cells
Authors: Evans R. M. , Grillo F. G. , Fink L. M.
Published: May 2005

The 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

Frogs (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

The 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

The 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

At 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

Human 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

In 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

Highly 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

Using 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

Cultures 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

Secretory 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

Incubation 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

Plasma 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

The 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

The 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

SJL 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.
Endogenous and exogenous proteolysis of the acetylcholine receptor from torpedo californica
Authors: Huganir Richard L. , Racker Efraim
Published: May 2005

Purified acetylcholine receptor reconstituted into liposomes catalyzes carbamylcholine‐dependent ion flux [10]. An endogenous protease activated by Ca2+ gives rise to an acrylamide gel pattern of the receptor with the 40,000‐dalton subunit apparently as the major component. Exogenous proteases nick the proteins so extensively that the acrylamide gel pattern reveals polypeptides of 20,000 daltons or less. In either case the receptor sediments at 9S, indicating that the polypeptide chains associated. Moreover, the nicked receptors bind α‐bungarotoxin and catalyze carbamylcholine‐dependent ion flux after reconstitution.
The selective effects of charged local anaesthetics on the glucagon‐ and fluoride‐stimulated adenylate cyclase activity of rat‐liver plasma membranes
Authors: Gordon Larry M. , Dipple Irene , Sauerheber Richard D. , Esgate Judy A. , Houslay Miles D.
Published: May 2005

The cationic local anaesthetics carbocaine and unpercaine were found to increase the fluoride‐stimulated adenylate cyclase up to a maximum level; above this maximum level further increases in drug concentration inhibited the enzyme. At concentrations where this activity was stimulated, a fatty acid spin label detected an increase in bilayer fluidity, which, it is suggested, is responsible for the activation of the enzyme. A solubilized enzyme was unaffected by the drugs, a finding consistent with this proposal.

These cationic drugs began to inhibit the glucagon‐stimulated activity at concentrations where they activated the fluoride‐stimulated activity. It is suggested that this is due to their effect on the coupling interaction between the receptor and catalytic unit.

The anionic drugs, phenobarbital, pentobarbital, and salicylic acid, all inhibited the fluoride‐stimulated enzyme. This may be due in part to a direct effect on the protein and in part to the interaction of the drugs with the bilayer. The drugs had small inhibitory effects on the lubrol‐solubilized enzyme.

The glucagon‐stimulated enzyme was initially inhibited by the anionic drugs at low concentrations, then activated, and finally inhibited with increasing drug concentration. The reasons for such changes are complex, but there was no evidence from electron spin resonance studies to suggest that the elevations in activity were due to increases in bilayer fluidity.
Effects of a serum spreading factor on growth and morphology of cells in serum‐free medium
Authors: Barnes David , Wolfe Richard , Serrero Ginette , McClure Don , Sato Gordon
Published: May 2005

A heat‐sensitive, trypsin‐sensitive factor that promoted growth and spreading of cells in serum‐free, hormone‐supplemented medium was partially purified from human serum. The major portion of the proteins in these preparations migrated upon SDS‐polyacrylamide gel electrophoresis with a mobility consistent with molecular weights between 60,000 and 90,000. The spreading activity, which we have termed serum spreading factor, stimulated growth and spreading of a wide variety of cell types. The serum spreading factor was similar to fibronectin in that it showed an affinity for the plastic cell culture substrate but was shown to be distinct from fibronectin by several criteria. This factor may prove useful in studies of cell attachment and spreading and in studies of the relationship of cell shape and cell proliferation.
Enhanced lymphoid and decreased myeloid reconstituting ability of stem cells from long‐term cultures of mouse bone marrow
Authors: Phillips R. A.
Published: May 2005

Mature, functional lymphocytes rapidly disappear from long‐term cultures of mouse bone marrow cells and never reappear. One reason for the loss of B lymphocytes is that the optimal culture conditions for maintenance of myeloid stem cells are suboptimal for lymphocyte survival. However, despite the absence of functional lymphocytes, stem cells from such cultures retain the ability to reconstitute irradiated mice with mitogen‐responsive B and T lymphocytes. In fact, in vitro grown stem cells repopulate the lymphoid system better than the myeloid system; the defective myeloid potential does not result from the absence in the cultures of Thy–1 bearing regulatory cells (TSRC). Although the cultures lack mature lymphocytes, they contain putative T cell precursors detectable with an in vitro colony‐forming assay (CFU‐T). In vitro maintenance of CFU‐T requires an appropriate adherent monolayer. Monolyaters from congenitally anemic mice of genotype S1/S1d fail to support either myeloid precursors or CFU‐T.
H1 Histone and nucleosome repeat length alterations associated with the in vitro differentiation of murine embryonal carcinoma cells to extra‐embryonic endoderm
Authors: Oshima Robert , Curiel Diana , Linney Elwood
Published: May 2005

The histone compositions and average distance between nucleosomes have been determined for F9.22 and PSA1 murine embryonal carcinoma cell lines, for primary extra‐embryonic endoderm derived from the in vitro differentiation of PSA1 embryonal carcinoma cells, and for two long‐term extra‐embryonic endodermal cell lines. A change in the relative proportions of two forms of the H1 histones (H1A and H1B) was found to correlate with the extra‐embryonic endodermal differentiated phenotype. The embryonal carcinoma cells had a ratio of H1A/H1B of 1.49 or greater. In contrast, extra‐embryonic endoderm from either cell lines or freshly isolated from differentiating embryonal carcinoma cell cultures had a ratio of H1A/H1B of less than 0.9. Partial peptide mapping of gel purified H1A and H1B suggest the two proteins differ in primary structure. The nucleosome repeat length of the embryonal carcinoma cell lines was 196 bp of DNA. Primary extra‐embryonic endoderm was found to have a value of 205 bp, but the long‐term extra‐embryonic endodermal cell lines had an average nucleosome repeat length of 187 bp. Since both freshly isolated primary endoderm and the long‐term endodermal cell lines express differentiated functions (basement membrane glycoproteins and plasminogen activator activity), there appears to be no simple correlation between the nucleosome repeat length and the expression of these differentiated functions.