bluuebunny/crossref_metadata_2025_split huggingface dataset:
The common α subunit of bovine glycoprotein hormones: Limited formation of native structure by the totally nonglycosylated polypeptide chain
Authors: Strickland Thomas W. , Thomason Arlen R. , Nilson John H. , Pierce John G. 
Published: December 2004

AbstractThe folding of the bovine glycoprotein hormone α subunit, synthesized in bacteria following insertion of the nucleotide sequence coding for this polypeptide, has been studied to determine the effect that a complete lack of carbohydrate has on this process. The bacterially derived α polypeptide (bac‐α), extracted from E. coli in the presence of reductant and denaturant, had an estimated 0.2% native structure as determined by a conformationally sensitive radioimmunoassay. Upon reduction of disulfide bonds and reoxidation in air, the amount of native structure increased about 18‐fold. Approximately 2% of the refolded bac‐α preparation combines with the β subunit of human chorionic gonadotropin (hCGβ) to form a complex that binds to the gonadotropin receptor and elicits a biological response. Since the correct folding (by immunological criteria) of bac‐α (ca 3%) is significantly greater than expected from a random formation of disulfide bonds (0.1 %), it appears that correct folding of α subunit can occur in the complete absence of carbohydrate, though in very low yield. Native bovine lutropin α subunit (LHα) and chemically deglycosylated LHα (which retains two asparagine‐linked N‐acetyl glucosamine residues per α oligosaccharide) were subjected to the same reduction/reoxidation regimen as the bacterially produced a subunit. As has been reported previously [Giudice LC, Pierce, JG, J Biol Chem 251: 6392, 1976] intact LHα fully regained its native structure. The partially deglycosylated LHα also refolds to a native‐like structure in high yield as assessed by immunological assays and by its ability to combine with HGCβ to form a biologically active complex. The data show that carbohydrate, while not obligatory for correct folding, greatly facilitates the formation of functional α subunit.
Angiogenesis is stimulated by a tumor‐derived endothelial cell growth factor
Authors: Shing Yuen , Folkman Judah , Haudenschild Christian , Lund Dennis , Crum Rosa , Klagsbrun Michael 
Published: December 2004

AbstractA growth factor mitogenic for BALB/C 3T3 cells and capillary endothelial cells was isolated from a rat chondrosarcoma and purified to homogeneity. Purification was accomplished by a combination of BioRex 70 cation exchange chromatography and heparin affinity chromatography. The pure chondrosarcoma‐derived growth factor (ChDGF) had a molecular weight of about 18.000. The angiogenesis activity of pure ChDGF was tested by measuring its ability to vascularize the chorioallantoic membrane (CAM) and yolk sac membrane of the developing chick. The ability of ChDGF to induce the growth of limbal vessels in the rat cornea was also measured. To quantitate the angiogenesis response, a unit system based on the growth factor activity of ChDGF for 3T3 cells was adopted. ChDGF was found to have a specific activity of about 5 units/ng when applied to 3T3 cells. About 300–600 units of ChDGF in the two types of developing chick membrane and 30–50 units of ChDGF in the rat cornea were found to stimulate noninflammatory angiogenesis.
Immunoreactive fibroblast growth factor (FGF) in a transplantable chondrosarcoma: Inhibition of tumor growth by antibodies to FGF
Authors: Baird Andrew , Mormède Pierre , Böhlen Peter 
Published: December 2004

AbstractUsing a radioimmunoassay for bovine pituitary fibroblast growth factor (FGF), we have established the presence of the immunoreactive mitogen in extracts of a transplantable mouse chondrosarcoma. Both neutral and acidic extracts of the tumor contain an immunoreactive FGF (ir‐FGF) that cross‐reacts in a parallel and dose‐dependent fashion in the radioimmunoassay. The ir‐FGF is retained on heparin‐Sepharose affinity columns and can be detected in the same molecular weight forms as rat pituitary FGF. Mice (C57/B1) inoculated with the tumor (10 mg, im) show a decreased rate of tumor growth when passively immunized with the antiserum to FGF. The results establish the presence of FGF in this tumor and implicate its role in the etiology of its development.
Role of nonenzymatic glycosylation in atherogenesis
Authors: Cerami Anthony , Vlassara Helen , Brownlee Michael 
Published: December 2004

AbstractThis review summarizes progress in nonenzymatic glycosylation research of potential relevance to atherosclerosis using a hypothetical model based on current concepts of atherogenesis. Recently, new information has been presented showing that the initial Amadori product undergoes a series of further reactions and rearrangements to form adducts, called advanced glycosylation end products (AGE). These products are irreversible and accumulate indefinitely on long‐lived molecules. These AGE covalently trap soluble plasma proteins, act as signals for macrophage recognition and uptake, and induce mutations in double‐stranded plasmid DNA. Covalent trapping of low‐density lipoprotein (LDL) by AGE on collagen or elastin could promote lipid accumulation in the arterial wall, whereas AGE trapping of von Willebrand factor would increse platelet adhesion and aggregation leading to intimal smooth muscle cell proliferation. Recognition and uptake of AGE‐proteins by scavenging macrophages could further contribute to the process of atherogenesis by stimulating release of macrophage secretory products such as macrophage‐derived growth factor. Accumulation of AGE on smooth muscle cell DNA might also enhance arterial smooth muscle cell proliferation by increasing the rate of mutations affecting growth controls. This model should provide the basis for future experiments.
Refolding of serine proteinases
Authors: Light Albert , Duda Chester T. , Odorzynski Thomas W. , Moore William G. I. 
Published: December 2004

AbstractBovine trypsinogen and chymotrypsinogen were successfully refolded as the mixed disulfide of glutathione using cysteine as the disulfide interchange catalyst. The native structures were regenerated with yields of 40%–50% at pH 8.6 and 4 °C, and the half‐time for the refolding was approximately 60–75 min. We then refolded threonine‐neochymotrypsinogen, which is a two‐chain structure held together by disulfide bonds and produced on cleavage of Tyr 146‐Thr 147 in native chymotrypsinogen [Duda CT, Light A, J Biol Chem 257 9866–9871, 1982]. Neochymotrypsinogen was denatured and fully reduced, and the thiols were converted to the mixed disulfide of glutathione. The two polypeptide fragments, representing the amino‐ and carboxyl‐terminal domains, were separated on Sephadex G‐75. Mixtures of the polypeptide fragments varying in the ratio of their concentration from 1 : 5 to 5 : 1 were refolded with yields of 21–28%. The lack of dependence on the concentration of either fragmènt and the relatively high yields suggest independent folding of the amino‐ and carboxyl‐terminal domains. When the globular structures of the domains formed, they then interacted with one another and produced the native intermolecular disulfide bridge and the proper geometry of the active site.
The bacterial phosphotransferase system: Kinetic characterization of the glucose, mannitol, glucitol, and N‐acetylglucosamine systems
Authors: Grenier Frank C. , Waygood E. Bruce , Saier Milton H. 
Published: December 2004

AbstractThe kinetic mechanisms by which the glucose, glucitol, N‐acetylglucosamine, and mannitol enzymes II catalyze sugar phosphorylation have been investigated in vitro. Lineweaver‐Burk analyses indicate that the glucose and glucitol enzymes II catalyze sugar phosphorylation by a sequential mechanism when the two substrates are phospho‐enzyme III and sugar. The N‐acetylglucosamine and mannitol enzymes II, which do not function with an enzyme III, catalyze sugar phosphorylation by a ping‐pong mechanism when the two substrates are phospho‐HPr and sugar. These results, as well as previously published kinetic characterizations, suggest a common kinetic mechanism for all enzymes II of the system. It is suggested that all enzymes II and enzyme II‐III pairs arose from a single (fused) gene product containing two sites of phosphorylation and that phosphoryl transfer from the second phosphorylation site to sugar can only occur when the enzyme II‐III pair is present in the associated state.
Expression of epidermal growth factor receptor and associated glycoprotein on cultured human brain tumor cells
Authors: Steck Peter A. , Gallick Gary E. , Maxwell Steve A. , Moser Richard P. , Gutterman Jordan U. , Yung W. K. Alfred , Kloetzer William S. , Arlinghaus Ralph B. 
Published: December 2004

AbstractThe expression of epidermal growth factor (EGF‐R) in normal glial and glioma cells grown in culture was examined by using several independent assays. Immunoprecipitation with the monoclonal antibody R1 of extracts from metabolically labeled glial and glioma cells revealed a protein of Mr ∼ 170,000, with a migration in sodium dodecyl sulfate‐polyacrylamide gels identical to the EGR‐R of A431 epidermal carcinoma cells. Furthermore, in the majority of glioma extracts, a protein of Mr ∼ 190,000 was specifically immunoprecipitated by this antibody. Similar results were obtained by immunoblotting with a second antibody directed against a synthetic peptide in the sequence of the V‐erb‐B oncogene. In cell lines expressing both proteins, each was specifically phosphorylated on tyrosine in immune complex kinase assays. The majority of glioma cells bound between 40,000 to 80,000 125I‐labeled epidermal growth factor molecules per cell. These results suggest that the expression of EGF‐R is common in cultured human glioma cells. In addition, a structurally related protein, is expressed in some of these cells.
The versatility of proteolytic enzymes
Authors: Neurath Hans 
Published: December 2004

AbstractThe growing realization of their physiological importance has generated renewed interest in the study of proteolytic enzymes. Modern methods of protein chemistry and molecular biology have revealed new insights into the protein and gene structure of a variety of protein precursors and their processing by limited proteolysis. Examples are given in this review for transmembrane processes and the role of signal peptidases of both eukaryotic and prokaryotic origin, the processing of prohormones and precursors of growth factors, protein components of blood coagulation, fibrinolysis, and of the complement system, and a group of granulocyte proteases, including the mast cell serine proteases. The relationship of homologous domains found in many of these proteases and their zymogens to protein evolution is a recurrent theme of this discussion.
Characterization of elastase associated with granulomatous tissue remodeling
Authors: Izaki Seiichi , Hsu Pair‐Shin , Okamoto Mitsuse , Epstein William L. , Fukuyama Kimie 
Published: December 2004

AbstractElastases have been reported to be involved in various types of tissue injury. In this study we detected hydrolytic activities for [3H]‐elastin and Suc‐Ala‐Ala‐Ala‐pNA (SLAPN) in hepatic granulomas which became elevated in parallel with enlargement of the granulomas and disappearance of aldehyde‐fuchsin‐stained filaments in the lesions of mice infected with Schistosoma mansoni. The elastase was partially purified by gel filtration followed by anion‐exchange chromatography. This enzyme has a molecular weight of 20‐25k and hydrolyzed denatured collagen (azocoll), Glu‐Pro‐Val‐pNA, SLAPN, and [3H]‐elastin. Optimal pH was 7‐8.5. It is a serine proteinase and distinct in its inhibitor profile from murine peritoneal macrophage elastase, which has been reported by others. Digestion of elastic fibers in vessel walls and fine fibrils in newly developed granulomas by the granuloma elastase was histochemically identified with aldehyde‐fuchsin stain. These results indicate that a serine proteinease functions as a major elastase in granulomatous tisssue remodeling and may account for the disappearance of elastic fibers and other elements of the matrix in fully developed granulomas.
Enzymes of phospholipid metabolism in rat pancreatic islets: Subcellular distribution and the effect of glucose and calcium
Authors: Rana Rajendra S. , Kowluru Anjaneyulu , MacDonald Michael J. 
Published: December 2004

AbstractThe effect of glucose and calcium on the activities of the phosphatidylinositol cycle enzymes, CDP‐diglyceride inositol transferase, diacylglycerokinase, and lysophosphatidylcholine 2‐acyltransferase in rat pancreatic islets was studied. Calcium inhibited the activity of CDP‐diglyceride inositol transferase but had no effect on lysophosphatidylcholine 2‐acyltransferase and diacylglycerokinase activites. Upon preincubation of islets in a concentration of glucose known to stimulate insulin release, the activity of lysophosphatidylcholine 2‐acyltransferase, but not that of diacylglycerokinase or the CDP‐diglyceride inositol transferase, was stimulated. Subcellular fractionation of pancreatic islets showed that secretory granule membranes were enriched in CDP‐diglyceride inositol transferase, whereas lyso‐phosphatidylcholine 2‐acyltransferase activity was highest in the microsomal membranes. The activation of 2‐acyltransferase by incubating islets in insulinotropic glucose, and the calcium sensitivity of CDP‐diglyceride inositol transferase, suggest that these enzymes may have roles in regulation of insulin secretion.
Structure and function of human tissue‐type plasminogen activator (t‐PA)
Authors: van Zonneveld Anton‐Jan , Veerman Harry , MacDonald Marcy E. , Pannekoek Hans , van Mourik Jan A. 
Published: December 2004

AbstractFull‐length tissue‐type plasminogen activator (t‐PA) cDNA served to construct deletion mutants within the N‐terminal “heavy” (H)‐chain of the t‐PA molecule. The H‐chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins (“finger,” “epidermal growth factor,” “kringles”). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon‐intron junctions of the chromosomal t‐PA gene. Recombinant t‐PA deletion mutant proteins were obtained after transient expression in mouse Ltk−, cells, transfected with SV40‐pBR322‐derived t‐PA cDNA plasmids. It is demonstrated that the serine protease moiety of t‐PA and its substrate specificity for plasminogen is entirely contained within the C‐terminal “light” (L)‐chain of the protein. The presence of cDNA, encoding the t‐PA signal peptide preceding the remaining portion of t‐PA, suffices to achieve secretion of (mutant) t‐PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t‐PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H‐chain, kringle Kl, and the epidermal growth‐factor‐like domain, do not contribute to this property of t‐PA. These findings correlate well with the fibrin‐binding properties of the rt‐PA deletion‐mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t‐PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L‐chain of t‐PA. Deleting specific segments of t‐PA H‐chain cDNA and subsequent transient expression in mouse Ltk− cells of t‐PA deletion‐mutant proteins did not affect the formation of a stable complex between mutant t‐PA and PAI.
Somatic events unmask recessive cancer genes to initiate malignancy
Authors: Gallie Brenda L. , Worton Ronald G. 
Published: December 2004

AbstractA heritable mutation predisposes an individual to certain childhood malignancies, such as retinoblastoma and Wilms' tumor. The chromosomal locations of the genes responsible for the predisposition are known by linkage with chromosomal deletions and enzyme markers. A study of these tumors in comparison to the normal constitutional cells of the patients, using enzyme and DNA markers near the predisposing genes, has shown that these genes are recessive to normal wild‐type alleles at the cellular level. Expression of the recessive phenotype (malignancy) involves the same genetic events that were observed in Chinese hamster cell hybrids carrying recessive drug resistance genes. In both the experimental and clinical situations, the wild‐type allele is‐most commonly eliminated by chromosome loss with duplication of the mutant chromosome. Simple chromosome loss and mitotic recombination have been documented in both systems. In the remaining 30% of cases, inactivation or microdeletion of the wild‐type allele are assumed to be responsible for expression of the recessive phenotype. Osteo‐sarcoma is a common second tumor in patients who have had retinoblastoma. Studies with markers in osteosarcoma show that these tumors also result from unmasking of the recessive phenotype by loss of the normal allele at the retinoblastoma locus, whether or not the patient had retinoblastoma. Subsequent chromosomal rearrangements and amplification of oncogenes that occur in these homozygous tumors provide progressive growth advantage. In other malignancies, in which studies have so far focused on oncogene amplification and chromosomal rearrangements, unmasking of recessive mutations may also be the critical initiating events.
Intracellular signalling of initiation of DNA synthesis: Effect of cell‐free extracts from anti‐receptor‐stimulated B lymphocytes on spleen cell nuclei
Authors: Ullah Sibghat , Kern Milton 
Published: December 2004

AbstractTo study the relatively late intracellular signals involved in the proliferative response of B lymphocytes to antibodies specific for surface membrane immuno‐globulins, extracts from antibody activated cells were mixed with Xenopus laevis splenic nuclei, and the incorporation of thymidine 5′‐triphosphate into DNA was assessed. The slight incorporation observed with either nuclei or extract alone was markedly enhanced upon mixing the two entities when the extract was derived from cells cultured with but not without anti‐receptor antibody. The appearance of active extract correlated well with the culture requirements necessary for the induction of B lymphocyte proliferation and, as revealed by time course studies, the active component arises relatively late in the activation process. Moreover, the appearance of active extracts is independent of DNA synthesis but is dependent on protein synthesis as judged from studies with metabolic inhibitors. Appropriate homogenization of activated cells yielded nuclei and cytoplasm with 85% of the activity confined to nuclei. In addition, purified active extracts exhibited DNA binding although the active component was readily distinguishable from polymerase α by chromatographic techniques. It is tentatively concluded that the active component represents either some replication protein other than polymerase or some earlier signal necessary to induce the formation or utilization of replicating proteins.
Viral P21 Ki‐RAS protein: A potent intracellular mitogen that stimulates adenylate cyclase activity in early G1 phase of cultured rat cells
Authors: Franks Douglas J. , Whitfield James F. , Durkin Jon P. 
Published: December 2004

AbstractRat kidney (NRK) cells infected with a temperature‐sensitive mutant of the Kirsten sarcoma virus were arrested in the G0/G1phase of their cell cycle by incubation in serum‐deficient medium at a p21‐inactivating temperature of 41°C. These quiescent ts K‐NRK cells were then stimulated to transit g1 and initiate DNA replication by lowering the temperature to 36°C, which rapidly reactivated p21 Reactivating the viral Ki‐RAS protein by temperature shift led to an increase in adenylate cyclase activity in early G1 phase. The Ki‐RAS protein increased the sensitivity of adenylate cyclase to guany1 nucleotides by a mechanism that seemed to involve inactivation of the enzyme's inhibitory G1regulatory protein.
Inhibition of T‐lymphocyte activation by amiloride
Authors: Baliga B. Surendra , Sindel Lawrence J. , Jenkins Lucy D. , Rashatwar Subash , Artman Michael 
Published: December 2004

AbstractThe T‐lymphocyte activation process involves a series of coordinately coupled biochemical events occuring in response to antigen or mitogen. These events have not been completely characterized. The present studies investigate the mechanism of protein synthesis during the initial phase of T‐cell activation. Among the early biochemical changes, induction of protein synthesis was observed as early as 10 minutes after mitogen stimulation of T‐lymphocytes. This early protein synthesis was inhibited by cycloheximide but was insensitive to actinomycin‐D, indicating the presence of preformed mRNA in resting lymphocytes. Since early protein synthesis parallels the increase in protein kinase C activity in activated T‐lymphocytes, these two biochemical events may be related. In the present report, amiloride, an inhibitor of Na+/H+antiport and protein kinase C, significantly inhibited [3H‐]leucine and [3H‐]thymidine incorporation in a dose‐dependent manner into phytohemagglutinin (PHA)‐stimulated T‐lymphocytes. Furthermore, when T‐lymphocytes were stimulated by phorbol myristate acetate, a known activator of protein kinase C, a similar inhibition of protein and DNA synthesis by amiloride was observed. The partially purified cytosol fraction isolated from PHA‐activated T‐lymphocytes showed a 75% decrease in protein kinase C‐mediated [32P] incorporation from ATP in the presence of 100 μM amiloride. These results suggest that the T‐cell activation process following exposure to mitogens involves early protein synthesis, which may be mediated by protein kinase C.
ATP‐dependent protein kinase activities in the oral pathogen Streptococcus mutans
Authors: Mimura Carol S. , Poy Florence , Jacobson Gary R. 
Published: December 2004

AbstractATP‐dependent protein kinase activities were detected in both membrane and cytoplasmic fractions from the oral pathogen Streptococcus mutans. Different polypeptides were phosphorylated by endogenous kinase(s) in the two fractions. In membranes, five phosphoproteins were detected with apparent masses of 82, 37, 22, 12, and 10 kilodaltons (KD). In cytoplasm, two major acid‐stable phosphoproteins were found. One was identified as HPr of the, phosphoenolpyruvate (PEP)‐dependent phosphotransferase system (PTS), while the other had an apparent mass of 61 KD. Both of these proteins were phosphorylated on a seryl residue. Fructose 1,6‐bisphosphate stimulated phosphorylation of HPr by the kinase and inhibited phosphorylation of the 61‐KD protein. In contrast, fructose 1‐phosphate, 2‐phosphoglycerate, 3‐phosphoglycerate, and dihydroxyacetone phosphate inhibited phosphorylation of HPr and stimulated phosphorylation of the 61‐KD protein. Several other glycolytic intermediates as well as inorganic phosphate inhibited phosphorylation of either or both proteins. Preincubation of cytoplasm with PEP prior to incubation with ATP reduced the amount of phospho‐(seryl)‐HPr formed, but not that of the 61‐KD phosphoprotein. The latter protein has not yet been identified but has properties that suggest that it may be the protein kinase itself. These results provide evidence for one or more soluble ATP‐dependent protein kinases in S mutans that are regulated by glycolytic intermediates and that may play a role in the modulation of carbohydrate uptake and metabolism in this organism. A model for feedback regulation of sugar transport in S mutans, mediated by an allosterically regulated kinase is presented.