bluuebunny/crossref_metadata_2025_split huggingface dataset:
The role of duplication in the expression of a variable surface glycoprotein gene of trypanosoma brucei
Authors: Young John R. , Turner Mervyn J. , Williams Richard O. 
Published: December 2004

AbstractThe variable surface glycoprotein (VSG) genes of Trypanosoma brucei have been classified into two groups depending upon whether or not duplication of the genes is observed when they are expressed. We report here the observation of duplication apparently linked to espression of the ILTaT 1.3 gene in the ETaR 1 trypanosome stock. In the ILTaR 1 stock, expression of the ILTaT 1.3 VSG did not involve a new duplication, but instead activation of a preexisting gene copy that had been apparently generated earlier by a duplication event analogous to that directly observed in the ETaR 1 trypanosomes. The results suggest that the well‐characterised gene duplications found with other VSG genes are common to all VSG genes but are not directly responsible for controlling expression. All currently available data can be accomodated by a model that assumes that gene duplication and replacement occurs independently of antigenic switching.
Differential regulation of the accumulation of the light‐harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves
Authors: Bennett John , Jenkins Gareth I. , Hartley Martin R. 
Published: December 2004

AbstractThe photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5‐bisphosphate carbox‐ylase/oxygenase (RUBISCO), and the light‐harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de‐etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40‐fold, at least 200‐fold, and about 25‐fold, respectively, while the total RNA content per apical bud increased only 3.5‐fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60‐, 100‐, and 200‐fold, respectively. The LHCP increased steadily in concentration during de‐etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24‐hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far‐red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far‐red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady‐state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.
Partial characterisation of the high and low molecular weight forms of P388D1‐derived interleukin 1
Authors: Prestidge R. L. , Koopman W. J. , Bennett J. C. 
Published: December 2004

AbstractThe murine macrophage‐derived cell line P388D1 secretes the lymphokine inter‐leukin 1 (IL‐1) when stimulated by a variety of agents. When stimulated by bacterial lipopolysaccharide (LPS) the cells release IL‐1 in both high and low molecular weight (m.w.) forms. The proportion of high m.w. IL‐1 is reduced when IL‐1‐containing supernatants are concentrated by ammonium sulfate precipitation subsequent to hollow‐fiber filtration. The high m.w. form can be converted to the low m.w. form by proteolysis, reduction and alkylation, or chromatography in a dissociating solvent. The low m.w. form remains as such, even when reconcentrated in fetal calf serum‐containing medium. The high m.w. form thus likely consists of a complex between low m.w. IL‐1 and another protein secreted by the P388D1 cell line.
Active proteinase inhibitors associated with human breast epithelial cells
Authors: Gendler Sandra J. , TőKés Zoltán A. 
Published: December 2004

AbstractThe major glycoproteins synthesized by human breast epithelial cells have been characterized [6,8]. The most consistently observed and prominent component in supernatants of organ cultures of breast surgical specimens and of MCF‐7 cells was gp 68 which has been immunologically identified as α‐1‐antichymotrypsin (Achy). In the present study we demonstrate that this glycoprotein can form an irreversible complex with chymotrypsin, which indicates that it is a functional inhibitor. The 14C‐glucosamine‐labeled gp 68 forms a stable, 88,000‐dalton. enzyme‐inhibitor complex with chymotrypsin. The molecule is secreted continuously for 9 days into a chemically defined, serum‐free medium. In addition to the de novo synthesized inhibitor, another component is adsorbed from fetal bovine scrum and subsequently released into serum‐free medium. This component also forms an irreversible, 88,000‐dalton complex with enzyme. The observations establish that two types of inhibitors are associated with human breast epithelial cells, one actively synthesized and the other derived from serum. Both of these molecules may have significant roles in stabilizing cell surface components and in protecting extracellular matrices from untimely degradation.
Dimethyl sulfoxide decreases specific EGF binding
Authors: Earp H. Shelton , Blaisdell Joyce , Lin Qixiong 
Published: December 2004

AbstractDimethyl sulfoxidc (DMSO) stimulates tyrosine phosphorylation of the hepatic EGF receptor in isolated membrane preparations. To determine whether DMSO affects EGF binding, primary cultures of rat hepatocytes were incubated with 1–10% DMSO for 30 min prior to the addition of 125I‐EGF. DMSO (1–2%) reduced specific 125I‐EGF binding; the effect was maximal (a 40–60% reduction) at 5–7.5% DMSO and was reversed by removing the DMSO. Scatchard analysis showed that the reduction in binding was due to a change in receptor affinity. The decrease in binding was not seen when other, slightly less polar, solvents (eg, acetone and ethanol) were tested. DMSO also reduced 125I‐EGF binding to purified rat liver plasma membranes. This reduction was seen in the absence of added ATP and in membranes that had been pretreated with TLCK, a tyrosine kinase inhibitor. Thus, completion of the receptor autophosphorylation reaction was not necessary to effect the change. The data arc consistent with a DMSO‐induced alteration of receptor conformation that rcversibly reduces receptor affinity.
Evidence for the evolutionary relatedness of the proteins of the bacterial phosphoenolpyruvate:Sugar phosphotransferase system
Authors: Saier Milton H. , Grenier Frank C. , Lee Catherine A. , Waygood E. Bruce 
Published: December 2004

AbstractThe phosphoenolpyruvate:sugar phosphotransferase system (PTS) found in enteric bacteria is a complex enzyme system consisting of a non‐sugar‐specific phospho‐transfer protein called Enzyme I, two small non‐sugar‐specific phosphocarrier substrates of Enzyme I, designated HPr and FPr, and at least 11 sugar‐specific Enzymes II or Enzyme II‐III pairs which are phosphorylated at the expense of phospho‐HPr or phospho‐FPr. In this communication, evidence is presented which suggests that these proteins share a common evolutionary origin and that a fructose‐specific phosphotransferase may have been the primordial ancestor of them all. The evidence results from an evaluation of (1) PTS protein sequence data; (2) structural analysis of operons encoding proteins of the PTS; (3) genetic regulatory mechanisms controlling expression of these operons; (4) enzymatic characteristics of the PTS systems; (5) immunological cross reactivities of these proteins; (6) comparative studies of phosphotransferase systems from evolutionarily divergent bacteria; (7) the nature of the phosphorylated protein intermediates; (8) molecular weight comparisons among the different Enzymes II and Enzyme II‐III pairs; and (9) interaction studies involving different PTS protein constituents. The evidence leads to a unifying theory concerning the evolutionary origin of the system, explains many structural, functional, and regulatory properties of the phosphotransferase system, and leads to specific predictions which should guide future research concerned with genetic, biochemical, and physiological aspects of the system.
PDGF Stimulates transient phosphorylation of 180,000 dalton protein
Authors: Harrington Maureen A. , Estes John E. , Leof Edward , Pledger W. Jack 
Published: December 2004

AbstractCell‐free extracts of platelet‐derived growth factor (PDGF) treated, density‐arrested, quiescent BALB/c‐3T3 cells are capable of phosphorylating a 180,000 dalton protein (PP180). The phosphorylation of PP180 was observed in SDS polyacrylamide gel electrophoresis profiles of Nonidet P‐40 solubilized cell preparations that had been incubated with [γ‐32P]ATP. When quiescent BALB/c‐3T3 cell cultures were incubated at 37°C with PDGF, phosphorylation of PP180 in cell extracts could be detected after a 3‐min exposure of the intact cells to PDGF, which was maximal after 10–15 minutes and had diminished by 30–60 min. PDGF stimulation of PP180 phosphorylation also was observed in extracts of cells that had been incubated with PDGF at 4°C: however, in contrast to PDGF exposure at 37°C, the ability of cell extracts to phosphorylate PP180 did not decrease even after 4 hr of cell exposure to PDGF at 4°C. When cells exposed to PDGF at 4°C were transferred to 37°C for 30 min, the ability of cell extracts to phosphorylate PP180 decreased to a nonstimulated level. After cells stimulated by PDGF showed a diminished ability to phosphorylate PP180, immediate restimulation with PDGF did not induce the ability to phosphorylate PP180. Incubation for 11 hr at 37° C was required before readdition of PDGF allowed observable phosphorylation of PP180 in cell extracts, but maximum PDGF stimulation of the phosphorylation of PP180 was found after the cells were incubated for 24 hr in culture conditions.The amount of the stimulation PP180 phosphorylation was dependent on the concentration of PDGF. The stimulation of DNA synthesis by PDGF was correlated to the phosphorylation of PP180. This phosphorylation activity was not observed in extracts of cells that had been treated with epidermal growth factor (EGF), somatomedin C, insulin, plasma, or fibroblast growth factor (FGF). This novel experimental approach allows the investigation of a PDGF‐stimulated phosphorylation activity in relation to the cell cycle and growth regulation.
The structure of a small collagenous fragment isolated from chicken hyaline cartilage
Authors: Mayne Richard , van der Rest Michel , Weaver Darrel C. , Butler William T. 
Published: December 2004

AbstractIn previous experiments, two collagenous fragments were isolated from pepsin digests of chicken hyaline cartilage and called the high molecular weight. (HMW) and low molecular weight (LMW) fractions [3]. In the present experiments, the chains of LMW were isolated after denaturation and subsequent reduction and alkylation of interchain disulfide bridges and were further fractionated by carboxymethyl‐cellulose chromatography. Four peaks were resolved during chromatography and were designated LMW 1, 2A, 2B, and 3. Amino acid analyses and peptide mapping after cleavage with trypsin, V8 protease, and cyanogen bromide showed that three genetically distinct chains must be present in LMW. Fractions 2A and 2B were very similar, but not identical, in structure. LMW 1, 2A plus 2B, and 3 were consistently isolated in approximately equal proportions, suggesting that the probable chain organization of LMW is [1][2A + 2B][3]. This suggestion was supported further by experiments that attempted to fractionate LMW by carboxymethyl‐cellulose chromatography after denaturation but without reduction and alkylation of interchain disulfide bridges. No fractionation of LMW was achieved, the single peak subsequently being shown to contain LMW 1, 2A plus 2B, and 3.
Stimulation of pp60c‐src tyrosyl kinase activity in polyoma virus–infected mouse cells is closely associated with polyoma middle tumor antigen synthesis
Authors: Bolen Joseph B. , Lewis Andrew M. , Israel Mark A. 
Published: December 2004

AbstractWe have examined the effect of polyoma virus infection of primary mouse embryo cells on the tyrosyl kinase activity associated with the cellular src gene product. pp60c‐src. The results of our studies demonstrate that infection of mouse cells with wild‐type polyoma virus or viral mutants capable of transforming rodent cells in culture and inducing tumors in animals results in the stimulation of pp60c‐src tyrosyl kinase activity. The level of pp60c‐src kinase stimulation in infected cells was found to be proportional to both the oncogenic potential of the virus strain used for infection and the characteristic phenotype of rodent cells transformed by the various strains of polyoma virus. Stimulation of pp60c‐src kinase activity was not observed in mouse cells infected with transformation‐defective strains of polyoma virus. In examining the kinetics of pp60c‐src kinase stimulation in mouse cells at various times following wild‐type polyoma virus infection, we found that the level of pp60c‐src kinase activity correlated directly with the synthesis of polyoma virus‐encoded tumor antigens. By comparing wild‐type polyoma virus with other viral mutants in these experiments, we conclude that the stimulation of pp60c‐src kinase activity in mouse cells following polyoma virus infection is associated with the synthesis of middle tumor antigen.
Direct evidence for the interaction of platelet surface membrane proteins GPIIb and III with cytoskeletal components: Protein crosslinking studies
Authors: Painter Richard G. , Gaarde William , Ginsberg Mark H. 
Published: December 2004

AbstractWhen intact platelets are incubated at 37°C with Concanavalin A (ConA), the two major surface membrane proteins GPIIb and III become associated with the Triton‐insoluble cytoskeleton [4]. Preincubation of platelets with a variety of metabolic inhibitors, including cytochalasin B, 2‐deoxy‐D‐glucose, and antimycin A or lidocaine, had no effect on the ability of ConA to produce this effect. These results suggested that the ConA‐induced anchorage of GPIIb and III to the Triton‐insoluble cytoskeleton is a passive process requiring clustering of GPIIb‐III molecules but not requiring the metabolic energy of an intact cell. This was supported by experiments that showed that ConA binding to plasma membrane‐rich fractions at 37°C could induce association of GPIIb and III with a sedimentable actin‐rich, Triton‐insoluble membrane matrix. Similar results were obtained when membranes were first isolated from ConA‐treated cells. Adding DNAse I, an actin depolymcrizing agent, into the Triton extraction buffer inhibited the ConA‐induced sedimentation of GPIIb‐III and actin by 50% in the presence of Mg2+‐ATP. Treatment of ConA‐treated membranes with dimethyl‐3,3′‐dithiobispropiomidate, a bifunctional, reducible protein crosslinking agent, produced Triton‐insoluble crosslinked species of discrete molecular weights. When these crosslinked species were analyzed by SDS‐PAGE in the presence of β‐mercaptoethanol, they were found to be composed of a 180–200 K dalton protein, GPIIb, GPIII, and actin. Crosslinking of these components was equally effective after Triton treatment and indicated as well that the species crosslinked in the intact membrane was stable after Triton extraction.Addition of crosslinker to membranes not treated with ConA produced similar crosslinked species. Analysis of their composition on reduced gels revealed that the amounts of GPIIb and III were reduced greatly ( < 10% of the total input GPIIb and III) but that the 180–200 k dalton protein and actin content were similar to that seen with ConA‐treated membranes. These results are consistent with the notion that ConA clusters mobile, unanchored molecules of GPIIb‐III ( ∼ 90–95% of the total) around a small fraction of IIb‐III that is associated with a submembranous cytoskeleton.
Binding of antibodies in liposomes to extracellular matrix antigens
Authors: Torchilin V. P. , Klibanov A. L. , Ivanov N. N. , Gluckhova M. A. , Koteliansky V. E. , Kleinman H. K. , Martin G. R. 
Published: December 2004

AbstractWe have incorporated antibodies against fibronectin or laminin into liposomes and studied their interaction with insoluble forms of these antigens. The antibodies, after modification by palmitoylchloride, were incorporated into the lipid bilayer by the cholate dialysis method. The antibodies in the liposomes recognized their specific antigen with little reaction to the alternative attachment protein or to albumin (<2%). The binding of antibody‐containing liposomes to insoluble antigen was inhibited by soluble antibodies to the respective antigens but not by antibodies to other antigens. The affinity constant of the liposome‐antibody complex with the antigen was estimated at 1–10 × 10−9 M liposomes. Thus, antibodies in liposomes retain their reactivity and specificity, and the reaction constant is comparable to that observed for immune complexes.
Limited proteolytic digestion of coated vesicle assembly polypeptides abolishes reassembly activity
Authors: Zaremba Sam , Keen James H. 
Published: December 2004

AbstractWe have previously identified a fraction containing several assembly polypeptides (AP) that promotes reassembly of clathrin into vesicle‐free coat structures [Zaremba S, Keen JH: J Cell Biol 97:1339, 1983]. The AP are prepared from purified bovine brain‐coated vesicles by extraction with 0.5 M TRIS‐HCl followed by Sepharose CL‐4B column chromatography. Centrifugation in sucrose gradients under nonassembly conditions supports earlier observations suggesting that four active polypeptides in the AP preparation, of Mr ∼ 110,000, 100,000, 50,000, and 16,500 are present in a discrete complex that is incorporated as a unit into reassembled coats. The 16,500‐dalton polypeptide does not coelectrophorese with authentic bovine brain calmodulin and does not exhibit calmodulin's Ca2+‐induced shift in electrophoretic mobility. When the partially purified AP fraction was digested with elastase, the Mr ∼ 110,000 and 100,000 polypeptides were rapidly degraded with little or no effect on the Mr ∼ 50,000 and 16,500 bands. This treatment abolished the in vitro coat‐forming ability of the AP fraction and the loss of activity closely parallels the loss of the Mr ∼ 100,000 band. Disappearance of the Mr ∼ 110,000 and 100,000 bands is accompanied by the generation of new bands at Mr ∼ 76,000 and 65,000. When the elastase‐treated AP is examined by sucrose gradient sedimentation in nonassembly buffers, the new bands continue to cosediment with the Mr ∼ 50,000 and 16,500 polypeptides. This indicates that the elastase digestion has cleaved off a fragment of the Mr ∼ 110,000 and 100,000 bands, leaving behind a truncated, inactive AP complex. A protein kinase activity has been detected in coated vesicle preparations that utilizes the 50,000‐dalton AP as its preferred substrate [Keen JH, Zaremba S: J Cell Biol 97:174a, 1983]. Elastase treatment does not abolish this activity, indicating that the kinase by itself is not sufficient for maintaining reassembly activity.
Studies with digitonin‐treated rat hepatocytes (nude cells)
Authors: Katz Joseph , Wals P. A. 
Published: December 2004

AbstractIsolated rat hepatocytes were treated with digitonin to strip the plasma membrane. The effect of digitonin concentration and exposure time on the recovery of marker enzymes for cell organelles was examined. Hepatocytes treated at room temperature for 1–2 min with 1 mg/ml of digitonin lose some 40% of their protein but retain over 95% of their intact mitochondria and peroxisomes, 90–95% of their endoplasmic reticulum, and about 80% of their lysosomal enzymes. There is little loss of the mitochondrial intermembrane content, and both oxygen uptake and phosphorylation are unimpaired by the treatment.Electron microscopy reveals a complete loss of the plasma membrane, in spite of limited loss of marker enzymes for this membrane. Scanning electron microscopy revealed the interior of the cells to be made up of a dense network of fibers and lamellae attached to the nucleus, mitochondria, and small organelles. The treated cells were stable for many hours when kept in 0.25 M sucrose containing 25 mM monovalent salts. In salt‐free sucrose the cells broke up very rapidly into nuclei and other single organelles. Addition of 5 mM NaCl or KCl retards breakup, and 15–20 min were required for dissolution. Intermediate stages, illustrated by scanning electron micrographs, show structure and chains made up mainly of mitochondria held together by a lamellar network. The rapid breakdown occurred at a pH above 7.5 in an oxygen atmosphere and in the presence of phosphate and apparently is an energy‐requiring process. It is slow below a pH of 7.2, and at a pH of 6.8 the treated cells remain completely stable in salt‐free sucrose. Our results suggest that endoplastic reticulum is a major component of the cytostructure holding together nuclei and organelles.
Actin‐associated cell‐surface glycoprotein from ascites cell microvilli: A disulfide‐linked multimer
Authors: Jung Goeh , Andrews David M. , Carraway Kermit L. , Carraway Coralie A. Carothers 
Published: December 2004

AbstractIsolated microvilli of the MAT‐Cl subline of the 13762 rat mammary adenocarcinoma contain a transmembrane complex composed of a cell surface, cytoskeleton‐associated glycoprotein (CAG), actin, and a 58,000‐dalton polypeptide (58K). The behavior of CAG has been studied by differential centrifugation and velocity sedimentation gradient centrifugation of detergent extracts of microvilli. CAG can be pelleted along with a fraction of the microvillar actin even in the presence of ionic detergents and under microfilament‐depolymerizing conditions. By velocity sedimentation analysis CAG in Triton/PBS extracts sediments as a large, hetero‐geneous species (sedimentation coefficient > 25S). In Sarkosyl and sodium dodecyl sulfate (SDS) the size and heterogeneity are somewhat reduced. In SDS CAG sediments as a 20S species in the absence of mercaptoethanol and as a 5S species in the presence of mercaptoethanol. These results indicate that CAG is a disulfide‐linked multimer in the microvillus membrane. We suggest that the stable multimeric structure of CAG permits it to act as the membrane association site for several microfilaments and plays an important role in the formation and stabilization of the microvillus structure.
Transforming growth factor (TGF) activity in human urine: Synergism between TFG‐beta and urogastrone
Authors: Twardzik Daniel R. , Sherwin Stephen A. 
Published: December 2004

AbstractHuman urine contains an acid and heat stable peptide with an apparent molecular weight of 8,000–10,000 that, in the presence of urogastrone (EGF), induces the anchorage‐independent growth of nontransformed cells in semisolid media. This nonmitogenic growth‐modulating activity does not compete with EGF for binding to EGF membrane receptor sites and can be resolved from EGF by high‐performance liquid chromatography. The urine‐derived growth factor has been purified more than 10,000‐fold and shares many biochemical properties with and is functionally related to the B class of TGFs isolated from transformed cells and nonneoplastic tissues. The low molecular weight anchorage‐independent growth‐, stimulating activity universally present in human urine is a result of the synergistic interaction of this urine‐derived TGF‐beta and urogastrone.