bluuebunny/crossref_metadata_2025 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

The 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

A 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

Using 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

This 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

Bovine 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

The 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

The 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

The 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

Elastases 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

The 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

Full‐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

A 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

To 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

Rat 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

The 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

ATP‐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.
Cellular signal transduction and the reversal of malignancy
Authors: Lockwood Arthur H. , Murphy Suzanne K. , Borislow Steven , Lazarus Adam , Pendergast Maryanne
Published: December 2004

Animal cells contain only a few defined molecular systems that transduce hormonal and growth signals from the external environment to the intracellular milieu to regulate cellular growth and differentiation. Among the most ubiquitous of these “second messenger” pathways are those utilizing cyclic AMP and phosphatidylinositide turnover. The former activates protein kinase A, while the latter leads to the activation of protein kinase C and mobilization of intracellular calcium. Lesions induced by oncogenes in signal transduction systems may be responsible for the cancerous transformation of cells. In many tumor cell lines, including some transformed by the ras and sis oncogenes, activation of protein kinase A by elevation of cyclic AMP or activation of protein kinase C by addition of phorbol esters can restore many normal aspects of growth and morphology. Such “reverse transformation” is accompanied by the phosphorylation of unique cellular proteins and alterations in the phosphoinositide cycle. Molecular mechanisms by which activation of signal transduction systems can attenuate the malignant phenotype are considered in the context of cellular growth and differentiation.
Estradiol‐dependent trans‐acting factor binds preferentially to a dyad‐symmetry structure within the third intron of the avian vitellogenin gene
Authors: Jost Jean‐Pierre , Saluz Hanspeter , Jiricny Josef , Moncharmont Bruno
Published: December 2004

The secondary activation of the avian vitellogenin II gene in isolated liver nuclei by cytoplasmatic liver extracts of estradiol‐treated chicks is accompanied by the binding of a protein from the extract to the structural part of the cloned gene. Both the DNA‐binding and gene‐stimulatory activities, which cochromatograph on heparin‐Sepharose, are apparently present only in the cytoplasmatic liver extracts of estradiol‐treated roosters and in the oviduct extracts of egg‐laying hens. DNA‐binding competition assays combined with exonuclease III footprinting showed that the factor binds to the imperfect dyad‐symmetry structure 5‘GTCTTGTTCCAAAC3’ within the third intron of the gene. The factor is sequence specific and binds equally well to both single‐ and double‐stranded DNA with an estimated dissociation constant of 3.5 × 10−10M.
Isolation of a calcium‐sensitive, 35,000‐dalton microfilament‐ and liposome‐binding protein from ascites tumor cell microvilli: Identification as monomeric calpactin
Authors: Liu Yuecheung , Brew Keith , Carraway Kermit L. , Carraway Coralie A. Carothers
Published: December 2004

Microvilli isolated from the MAT‐C1 ascites subline of the 13762 rat mammary adenocarcinoma contain a major calcium‐sensitive microfilament‐binding protein, AMV‐p35 (ascites microvillar p35). Association of AMV‐p35 with microfilament cores during Triton X‐100 extraction of the microvilli is half‐maximal at 0.1‐0.2 mM calcium. The protein, which comprises 6% of the total microvillar protein, can be isolated from microfilament cores prepared in the presence of calcium by extraction with EGTA and purification by ion‐exchange chromatography. Alternatively, the protein can be isolated from Triton extracts of microvilli prepared in the absence of calcium by precipitation with calcium, solubilization of the precipitate with EGTA, and chromatography on an ion‐exchange column. AMV‐p35 binds to phosphatidylserine liposomes and F‐actin with half‐maximal calcium concentrations of about 10 μM and 0.2 mM, respectively. Treatment of AMV‐p35 with chymotrypsin yields a 33,000‐dalton fragment, behavior similar to the tyrosine kinase substrates calpactins I and II and lipocortins I and II. Immunoblot analyses using antibodies directed against calpactin I, lipocortin I, and lipocortin II showed strong reactivity of AMV‐p35 with anti‐calpactin I and anti‐lipocortin II, but little reactivity toward anti‐lipocortin I. The close relationship between AMV‐p35 and calpactin I was verified by amino acid sequence analyses of peptides isolated from cyanogen bromide digests of AMV‐p35. By gel filtration and velocity sedimentation analyses purified AMV‐p35 is a 35,000‐dalton monomer. Moreover, AMV‐p35 extracted directly from microvilli in Triton/EGTA also behaves as a 35,000‐dalton menomer. These findings indicate that AMV‐p35 is closely related to the pp60src kinase substrate calpactin I (p36). However, AMV‐p35 occurs in the microvilli as a monomer rather than as the heterotetrameric calpactin found in several other cell types.
Hormone‐dependent processing of the avian progesterone receptor
Authors: Sullivan William P. , Smith David F. , Beito Thomas G. , Krco Christopher J. , Toft David O.
Published: December 2004

Avian progesterone receptor exists as two forms, A and B, with molecular weights of 79,000 and 110,000 daltons, respectively. The origin and significance of these two forms is an area of active investigation and debate. Monoclonal antibodies produced against these two forms were used to examine receptor stability in cytosol and changes in the receptor forms induced by hormone binding.

The lability of hormone binding at elevated temperatures is well documented. Analysis by Western blotting showed the receptor was stable in freshly prepared oviduct cytosol for 2 hr at 37°C, while hormone binding was lost within 30 min. However, loss of receptor through degradation was seen when cytosol was prepared from frozen tissue or when homogenization was excessive.

Progesterone was injected into diethylstilbestrol‐stimulated chicks to examine, in vivo, effects of hormone treatment on receptor forms in the cytosol and nuclear fractions. Progesterone treatment caused a time‐ and dose‐dependent conversion of the A receptor to a form (A′) with a slower electrophoretic mobility. The cytosolic progesterone receptor was divided equally between the B and A forms, while the nuclear receptor was predominantly A′. The amount of nuclear receptor was consistently less than cytosolic receptor. Receptor phosphorylation was analyzed by incubating tissue minces with [32P]orthophosphate with or without progesterone followed by immune isolation of receptor forms. Progesterone treatment caused a time‐dependent increase in cytosol receptor phosphorylation which was evident after 5 min of treatment. This phosphorylation was observed with both the A and B receptor forms. The results indicate that receptor phosphorylation is a very early event during progesterone action.
Swiss 3T3 mouse embryo fibroblasts transfected with a human prepro‐GRP gene synthesize and secrete pro‐GRP rather than GRP
Authors: Lebacq‐Verheyden Anne‐Marie , Segal Shoshana , Cuttitta Frank , Battey James F.
Published: December 2004

A prepro‐gastrin‐releasing peptide (GRP) gene was introduced into Swiss 3T3 mouse embryo fibroblasts by DNA transfection in an attempt to establish autocrine growth stimulation. Clonal transfectants expressed varying amounts of GRP encoding mRNA. They synthesized and secreted a ∼ 15‐kd pro‐GRP hormone but not fully processed 2.8‐kd GRP. Accordingly, no changes in growth properties were associated with GRP gene expression. We postulate that Swiss 3T3 fibroblasts lack the enzymes necessary to process significantly pro‐GRP into biologically active peptides and that this deficiency may be responsible for the failure to establish autocrine growth stimulation in the transfected cells.
Effect of mutations affecting Na+:H+ antiport activity on tumorigenic potential of hamster lung fibroblasts
Authors: Lagarde Alain E. , Franchi Arlette J. , Paris Sonia , Pouysségur Jacques M.
Published: December 2004

Mutants unable to regulate intracellular pH through the Na+:H+ antiport system were found to evolve tumors less frequently than wild‐type CCL39 hamster lung fibroblasts, after transplantation in athymic nude mice. When rare tumors arose, they comprised cells which were transformed in vitro, but which upon retransplantation grew at a lower rate than tumor cells originating from CCL39 cells. Both parental and mutant cells became transformed after transfection of the activated Harvey ras oncogene, but transfectants derived from the mutants had a weaker tumorigenic potential. These results suggest that transformed characteristics can be acquired independently from the Na+:H+ antiporter. However, the presence of this system provides a selective growth advantage when cells are confronted with natural environments, as it occurs during the expansion of tumors in a host.
ADP‐ATP Carrier of Saccharomyces cerevisiae contains a mitochondrial import signal between amino acids 72 and 111
Authors: Smagula Cynthia S. , Douglas Michael G.
Published: December 2004

The ADP‐ATP carrier (also referred to as the adenine nucleotide translocator) of Saccharomyces cerevisiae is encoded by a nuclear gene, translated in the cytosol, and imported into the mitochondrial inner membrane. In order to study the determinants of mitochondrial import, a series of fusion proteins, consisting of the first 21, 72, and 111 amino acids of the ADP‐ATP carrier, joined to mouse dihydrofolate reductase were generated. Dihydrofolate reductase is a cytosolic protein that does not bind mitochondria. The reticulocyte lysate reaction containing the 35S‐methionine‐labeled protein was incubated with mitochondria in a buffer containing 3% BSA. Following incubation for import, the reactions were treated with 1 mM PMSF or 25 μg/ml proteinase K; mitochondria were reisolated and analyzed by gel electrophoresis. The 21 and 72 amino acid hybrid proteins showed a low level of binding to mitochondria: the bound form was entirely protease accessible. The 111 amino acid hybrid protein was imported to a protease‐protected location within mitochondria. It is concluded that the first 72 amino acids of the ADP‐ATP carrier do not suffice to import the protein into mitochondria and that the region between amino acids 72 and 111, a region that contains a transmembrane‐spanning domain, constitutes at least part of the mitochondrial import signal.
Monoclonal antibodies to carbohydrate antigens in autologous bone marrow transplantation
Authors: Ball Edward D. , Howell Alexandra L.
Published: December 2004

Normal and malignant myeloid cells express a highly immunogenic oligosaccharide, lacto‐n‐fucopentaose‐III (LNF‐III), that has been identified by numerous monoclonal antibodies (MoAb). We have been interested in the use of a particular monoclonal antibody to LNF‐III, PM‐81, in the treatment of patients with acute myelogenous leukemia using the antibody to treat bone marrow in vitro. Following in vitro treatment of bone marrow with PM‐81 and another MoAb, AML‐2–23, the remaining cells are used as an autograft in a patient treated with high‐dose chemotherapy and radiotherapy. In order to enhance the ability of the MoAb to lyse leukemic cells in the remission bone marrow, we have explored the effect of neuraminidase treatment on leukemia cells. In this paper we describe that myeloid leukemia cells expressing low levels of LNF‐III by immunofluorescence can be shown to have high levels of LNF‐III after neuraminidase treatment. In addition, we show that normal bone marrow progenitor cells do not have cryptic LNF‐III antigen, thus allowing the application of this finding to the clinical setting. Moreover, we have shown that leukemia colony‐forming cells from one patient with acute myelogenous leukemia express cryptic LNF‐III and that after exposure to neuraminidase there was an increased ability of PM‐81 in the presence of complement to eliminate these colony forming cells. These data indicate that the LNF‐III moiety is almost universally expressed on myeloid leukemia cells and their progenitors but not expressed on normal progenitors. Thus, it may be possible to enhance leukemia cell kill in vitro by neuraminidase treatment of bone marrow.
Cell surface expression of 4β‐galactosyltransferase accompanies rat parotid gland acinar cell transition to growth
Authors: Marchase Richard B. , Kidd Vincent J. , Rivera Angel A. , Humphreys‐Beher Michael G.
Published: December 2004

Rat parotid gland acinar cells stimulated to divide by a chronic regimen of isoproterenol demonstrate a dramatic increase in the synthesis of the glycosyltransferase 4β‐galactosyltransferase. A plasma membrane localization for much of the increase in 4β‐galactosyltransferase was determined by density gradient membrane fractionation. Golgi‐enriched fractions showed no increase in specific activity, while plasma membrane activity increased 40‐fold. This selective increase at the cell surface was confirmed by immunofluorescence of intact, nonpermeabilized cells from treated glands, using a monospecific antibody prepared against the purified bovine milk transferase. In detergent‐permeabilized cells staining of nontreated cells was seen only as groups of perinuclear vesicles, presumed to be Golgi apparatus. In isoproterenol‐treated and permcabilized cells both presumptive Golgi and cell surface staining was apparent. Enzyme assays performed on intact cells established that the enzyme's active site was oriented to the exterior of the cells. The transferase could be detected as early as 3 hr after the primary challenge with isoproterenol. Pretrcatment of rats with cycloheximide prevented its appearance.
Inhibition of the proliferation of Nb2 cells by femtomolar concentrations of cholera toxin and partial reversal of the effect by 12‐O‐tetradecanoyl‐phorbol‐13‐acetate
Authors: Pines M. , Ashkenazi A. , Cohen‐Chapnik N. , Binder L. , Gertler A.
Published: December 2004

One hour of exposure to cholera toxin is sufficient to elicit a significant delay in the initiation of DNA synthesis and cell division in lactogenic hormone‐dependent Nb2‐11C lymphoma cells. The inhibitory effect occurs already at very low concentrations of cholera toxin (5–50 fM), at which it is not accompanied by a detectable increase in intracellular cAMP, or ADP‐ribosylation of the alpha subunit of Gs, the stimulatory guanine nucleotide binding protein of adenylate cyclase; IBMX, the phosphodies‐terase inhibitor, acts synergistically to cholera toxin, indicating that a minute increase in cAMP may be sufficient for the inhibition. This indication is substantiated by the finding that dibutyryl cAMP also inhibits cell proliferation. Phorbol diester reverses partially the inhibitory activity of cholera toxin. It is most likely that this effect does not result from blocking the increase in cAMP, but rather from some subsequent, yet unidentified, events. The inhibitory effect of cholera toxin is not dependent on the concentration of the proliferation‐stimulating lactogenic hormone and cannot be abolished or reduced by excess of the hormone. Cholera toxin also inhibits the autonomous proliferation of a lactogenic hormone‐independent cell line (Nb2‐SP); however, in this case the inhibition is not affected by TPA.
Major 56,000‐dalton, soluble phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP‐dependent protein kinase
Authors: Paupard Marie‐Christine , MacLeod Janet , Wasco Wilma , Orr George A.
Published: December 2004

It has been shown that cAMP‐dependent phosphorylation of a soluble sperm protein is important for the initiation of flagellar motion. The suggestion has been made that this motility initiation protein, named axokinin, is the major 56,000‐dalton phos‐phoprotein present in both dog sperm and in other cells containing axokinin‐like activity. Since the regulatory subunit of a type II cAMP‐dependent protein kinase is a ubiquitous cAMP‐dependent phosphoprotein of similar subunit molecular weight as reported for axokinin, we have addressed the question of how many soluble 56,000‐dalton cAMP‐dependent phosphoproteins are present in mammalian sperm. We report that in bovine sperm cytosol, the ratio of the type I to type II cAMP‐dependent protein kinase is approximately 1:1. The type II regulatory subunit is related to the non‐neural form of the enzyme and undergoes a phosphorylation‐dependent electrophoretic mobility shift. The apparent subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 daltons, respectively. When bovine sperm cytosol or detergent extracts are phosphorylated in the presence of catalytic subunits, two major proteins are phosphorylated and have subunit molecular weights of 56,000 and 40,000 daltons. If, however, the type II regulator subunit (RII) is quantitatively removed from these extracts using cither immobilized cAMP or an anti‐RII monoclonal affinity column, the ability to phosphorylate the 56,000‐ but not 40,000‐dalton polypeptide is lost. These data suggest that the major 56,000 dalton cAMP‐dependent phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP‐dependent protein kinase and not the motility initiator protein, axokinin.
Oligosaccharide heterogeneity of glycoproteins sulfated during the vegetative growth of Dictyostelium discoideum
Authors: Davis Simon J.
Published: December 2004

Macromolecules are sulfated during the vegetative growth of Dictyostelium discoideum. A characterisation of the structures of sulfated oligosaccharides associated with these macromolecules indicates that the oligosaccharides are heterogeneous. Endoglycosidase and pronase digestion were used with gel‐filtration chromatography to obtain two different Oligosaccharide fractions and a glycopeptide fraction; these were further characterised by ion‐exchange and lectin‐affinity chromatography and by acid hydrolysis. The data indicate that up to 43% of the sulfate is associated with typical N‐linked oligosaccharides, that up to 5% is associated with N‐linked oligosaccharides that are either very large or extremely highly charged, and that the remaining sulfate is associated with oligosaccharides non‐N‐linked to protein. Each fraction was also shown to be heterogeneous at most other structural levels. Electrophoretic analyses following the endoglycosidasc and pronase treatments indicated that all of the macromolecules are glycoproteins and suggested further that at least two of the Oligosaccharide fractions are located on different groups of glycoproteins.
Comparative study of nuclear and cytoplasmic glycogen isolated from mutant HD33 ascites cells
Authors: Kopun Marijana , Granzow Christof , Krisman Clara R.
Published: December 2004

Mutant cells of the HD33 subline of the Ehrlich‐Lettré ascites tumor synthesize and store glycogen mainly intranuclearly, when growing in vivo, and exclusively in the cytoplasm, when permanently cultivated as a suspension cell strain. To investigate whether there exist differences between glycogen of nuclear and cytoplasmic origin, the ultrastructure and the biophysical and biochemical properties of glycogen from in vivo and in vitro grown HD33 ascites cells were compared. Pronounced heterogeneity and differences in glycogen particle ultrastructure were evident in situ and after isolation of the native, high‐molecular polysaccharide. Nuclear glycogen contains a fraction of heavier molecules (up to 2 × 109) and larger particles (up to 340 nm) which could not be found in the cytoplasmic preparations, which contained only particles smaller than 140 nm. The subparticles of β‐type are similar in both nuclear and cytoplasmic glycogen. The absorption spectra and glucose analysis after degradation with phosphorylase and debranching enzyme indicate that nuclear glycogen has a higher degree of branching, associated with a decrease in the average chain length between the branching points, and shorter external polyglucosidic chains than cytoplasmic glycogen. This is the first report about the analysis and properties of isolated nuclear glycogen.
Type I protein kinase C isozyme in the visual‐information‐processing pathway of monkey brain
Authors: Huang Freesia L. , Yoshida Yasuyoshi , Nakabayashi Hiroki , Friedman David P. , Ungerleider Leslie G. , Young W. Scott , Huang Kuo‐Ping
Published: December 2004

Previously using PKC isozyme‐specific antibodies for immunoblot analysis, we demonstrated the heterogeneous distribution of PKC isozymes in various regions of monkey and rat brains and that type I PKC was most abundant in cerebellum, hippocampus, amygdala, and cerebral cortex (Huang et al.: J Biol Chem 262:15714–15720, 1987). Using these antibodies, we have also demonstrated that type I, II, and III PKC are products of PKC genes γ, β, and α, respectively (Huang et al.: Biochem Biophys Res Commun 149:946–952, 1987). By immunocytochemical analysis, type I PKC‐specific antibody showed strong reactivity in various types of neuron in hippocampal formation, amygdala, cerebellum, and neocortex. In hippocampal formation, granule cells of dentate gyrus and pyramidal cells of hippocampus were heavily stained. By immunoblot analysis, relative levels of PKC isozymes in several areas of monkey cerebral cortex involved in the visual information processing and storage were determined. Both type II and III PKCs appeared to be evenly distributed and at moderate levels, type I PKC formed a gradient of increasing concentration rostral along the cerebral cortex of occipital to temporal and then to the limbic areas. Neurobehavioral studies have demonstrated that the neocortical and limbic areas of the anterior and medial temporal regions participate more directly than the striate, prestriate, and posterior temporal regions in the storage of visual representations and that both hippocampus and amygdala are important in the memory formation. As type I PKC is present at high levels in hippocampus, amygdala, and anterior temporal lobe, we predict that the type I protein kinase C may participate in the plastic changes important for mnemonic function.
Identification of HTLV‐I gag protease and its sequential processing of the gag gene product
Authors: Hatanaka Masakazu , Nam Seok Hyun
Published: December 2004

The full‐length provirus of human T‐cell leukemia virus type I (HTLV‐I) was isolated from MT‐2, a lymphoid cell line producing HTLV‐I. In transfected cells, structural proteins of HTLV‐I, the gag and env products, were formed and processed in the same manner as observed in MT‐2 cells. The nucleotide sequence was determined for a region between the gag and pol genes of the proviral DNA clone containing an open‐reading frame. The deduced amino acid sequences show that this open‐reading frame encodes a putative HTLV‐I protease.

The protease gene (pro) of HTLV‐I was investigated using a vaccinia virus expression vector. Processing of 53k gag precursor polyprotein into mature p19, p24, and p15 gag structural proteins was detectable with a recombinant plasmid harboring the entire gag‐ and protease‐coding sequence. We demonstrated that the protease processed the gag precursor polyprotein in a trans‐action. A change in the sequence Asp(64)‐Thr‐Gly, the catalytic core sequence among aspartyl proteases, to Gly‐Thr‐Gly was shown to abolish correct processing, suggesting that HTLV‐I protease may belong to the aspartyl protease group. The 76k gag‐pro precursor polyprotein was identified, implying that a cis‐acting function of HTLV‐I protease may be necessary to trigger the initial cleavage event for its own release from a precursor protein, followed by the release of p53 gag precursor protein. The p53 gag precursor protein is then processed by the trans‐action of the released protease to form p19, p24, and p15.
In vivo treatment with anti‐CD8 and anti‐CD5 monoclonal antibodies alters induced tolerance to adjuvant arthritis
Authors: Larsson Per , Holmdahl Rikard , Klareskog Lars
Published: December 2004

Resistance (low dose tolerance) to adjuvant arthritis was induced by intradermal immunization with 10 μg Mycobacterium tuberculosis administered 5 and 3 weeks before induction of arthritis. With the purpose of determining phenotypes of cells which participate in the maintenance of the induced resistance to adjuvant arthritis, tolerized rats were treated with two different anti‐T‐cell monoclonal antibodies. In tolerized rats, it was shown that anti‐CD8 (OX8) antibodies, which caused an elimination of CD8+ lymphoid cells as determined by immunofluoresnce analysis, made the rats responsive to an arthritogenic challenge with mycobacteria. Nine of 19 (47.4%) rats developed the disease as compared with 2 of 18 (11.1%) (P < 0.05) in the control antibody‐treated group. Also, in vivo treatment with anti‐CD5 (OX19) monoclonal antibodies made the rats responsive to an arthritogenic challenge with mycobacteria. Nine of 15 (60%) anti‐CD5‐treated rats developed the disease as compared with 2 of 18 (11.1%) (P < 0.01) rats in the control group. Immunofluorescence analysis performed after anti‐CD5 treatment showed a reduction of staining of CD5+ cells as well as a down‐regulation of the staining intensity of CD5 cell surface receptors on the remaining CD5+ cells. These data indicate that CD8+‐ as well as CD5+ cells participate in the maintenance of low dose tolerance to adjuvant arthritis.