bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production of a monoclonal antibody directed against the nerve growth factor receptor from sympathetic membranes
Authors: Morgan Claudia J. , Bradshaw Ralph A. 
Published: December 2004

AbstractSpleen cells from BALB/c mice immunized with a plasma membrane‐enriched fraction from rabbit sympathetic ganglia were fused with the mouse myeloma NS1. A hybrid clone was obtained that produced monoclonal antibody directed against the receptor for nerve growth factor (NGF). The antibody, identified as IgG, was able to immunoprecipitate solubilized NGF receptor in the presence or absence of bound NGF. The antibody bound specifically to sympathetic membranes with high affinity but did not affect the binding of 125I‐NGF to its receptor in sympathetic or sensory neurons or PC12 cells.
The differential distribution of beta tubulin mRNAs in individual mammalian brain cells
Authors: Griffin W. Sue T. , Alejos Michael A. , Cox Erica J. , Morrison Marcelle R. 
Published: December 2004

AbstractIt has been shown by in vitro translation of polyadenylated messenger RNAs (poly(A)+ mRNAs) that the mRNAs encoding both alpha and beta tubulin isotypes are present at much higher relative levels in the developing rat brain than they are in the adult [1], suggesting that the requirements for tubulin subunits vary with cell type and/or with the developmental stages of a particular cell type. The postnatally developing rat cerebellum, with its readily identifiable cell populations that perform the gamut of developmental tasks, is a suitable model for analyzing specific cellular mRNA distributions during development. In this report, by in situ hybridization techniques it is shown that, by comparison to total cellular poly(A)+ mRNA levels, there is relatively more of the total beta tubulin mRNAs in mitotically active external granule layer cells than in those in the internal granule layer. These results show that migration and differentiation of these granule cells is accompanied by a decrease in their beta tubulin mRNA levels relative to the levels in granule cells of the external granule cell layer. Furthermore, the relative levels of beta tubulin mRNA both in the prenatally formed Purkinje cells and the postnatally formed stellate cells are two to fourfold less than in the granule cells of the internal granule cell layer.
Purification and partial characterization of a liver cell proliferation factor called hepatopoietin
Authors: Goldberg Michel 
Published: December 2004

AbstractThe purification and partial characterization of a liver cell proliferation factor called hepatopoietin are described. Hepatopoietin was isolated from remnant livers or blood plasma of partially hepatectomized rats and purified approximately 13,000‐fold. The stokes radius was 2.65 ± 0.2 nm and the apparent molecular weight was calculated to be 38,000 ± 5,000 D. Hepatopoietin is a heat‐stable glycoprotein and is organ specific but species nonspecific. In vivo it stimulates about three to four times the DNA synthesis as well as the mitotic rate of the liver of normal rats after i.p. injection. Hepatopoietin is inactivated upon incubation with galactosidase or trypsin‐chymotrypsin.
Regulation of discoidin I gene expression in dictyostelium discoideum by cell‐cell contact and cAMP
Authors: Berger Edward A. , Bozzone Donna M. , Berman Marcia B. , Morgenthaler Jennifer A. , Clark Judy M. 
Published: December 2004

AbstractWe have previously presented evidence that cell‐cell contact is the normal developmental signal to deactivate discoidin I gene expression in D discoideum [Berger EA, Clark JM: Proc Natl Acad Sci USA 80:4983, 1983]. Here we provide genetic evidence to support this hypothesis by examining gene expression in a cohesion‐defective mutant, strain EB‐21, which enters the developmental program but is blocked at the loose mound stage, When this strain was developed in suspension, the cells remained almost entirely as single amoebae, unlike the wild type, which formed large multicellular aggregates. In both strains, discoidin I mRNA levels were low in vegetative cells but rose sharply during the first few hours of development. However, the peak level reached at 8 hr in EB‐21 exceeded that observed in wild type, and while the level declined markedly over the next few hours in wild type, it remained highly elevated in the mutant. Thus, there was a correlation between the inability of EB‐21 to form normal cell‐cell contacts and its deficiency in inactivating discoidin I gene expression.Previous studies from several laboratories, including this one, have demonstrated that exogenously added cAMP can block or reverse the changes in gene expression normally seen upon cell disaggregation. This has led us to propose that cAMP serves as a second messenger regulating the expression of contact‐regulated genes. Here we provide additional support for this hypothesis. Intracellular cAMP levels rapidly dropped several‐fold when wild type tight cell aggregates were disaggregated and remained low as the cells were cultured in the disaggregated state, Furthermore, overexpression of discoidin I mRNA late in development in EB‐21 was corrected by addition of high concentrations of cAMP. These results are consistent with a second messenger function for cAMP in the contact‐mediated regulatory response, and they indicate that the cAMP response machinery for discoidin I gene expression is capable of functioning in the cohesion‐defective EB‐21 strain.
Deposition of type X collagen in the cartilage extracellular matrix
Authors: Cancedda Ranieri , Capasso Olga , Castagnola Patrizio , Descalzi‐Cancedda Fiorella , Quarto Natalina 
Published: December 2004

AbstractIn cultured chick embryo chondrocytes, type X collagen is preferentially deposited in the extracellular matrix, the ratio between type II and type X collagen being about 5 times higher in the culture medium than in the cell layer. When the newly synthesized collagens deposited in slices from the epiphyseal cartilage of 17‐day‐old embryo tibiae were isolated, type X collagen was always the major species. In agreement with this result the mRNA for type X collagen was the predominant mRNA species purified from the same tissue. When the total collagen (unlabeled) deposited in the epiphyseal cartilage was analyzed, it was observed that type X collagen represented only 1/15 of the type II collagen recovered in the same preparation. The possible explanations for these differences are discussed.
Interaction of the insulin receptor kinase with serine/threonine kinases in vitro
Authors: Haring Hans U. , White Morris F. , Kahn C. Ronald , Ahmad Zafeer , DePaoli‐Roach Anna A. , Roach Peter J. 
Published: December 2004

AbstractInsulin causes rapid phosphorylation of the β subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threoine kinases known to phosphorylate glycogen synthase. No insulin‐dependent phosphorylation was ob served when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin‐dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin‐dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulindependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin‐dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.
Phenazine methosulfate stimulation of ouabain‐sensitive Rb+ uptake by HeLa cells: Effects of respiratory inhibitors, anaerobiosis, and ascorbate
Authors: Ikehara Toshitaka , Yamaguchi Hisao , Hosokawa Keiko , Kaku Masayuki , Miyamoto Hiroshi 
Published: December 2004

AbstractPhenazine methosulfate (PMS) stimulates ouabain‐sensitive Rb+ uptake by HeLa cells. This stimulation cannot be attributed to the effect of the dye on the intracellular Na+ or ATP content. Respiratory inhibitors, such as 5 mM NaCN and 5 mM rotenone, and anaerobic conditions enhance the stimulation of Rb+ uptake by PMS. Cellular respiration is stimulated, but lactate production is reduced in the presence of PMS, irrespective of the presence of respiratory inhibitors. Cellular NADH is oxidized markedly on addition of PMS plus inhibitors, but it is not affected by addition of the inhibitors only. In the presence of a high concentration of PMS, PMS‐stimulated ouabain‐sensitive Rb+ uptake is inhibited by addition of ascorbate. From these results it is concluded that Na/K‐pump activity is closely related to the cellular redox state.
Role of protein kinase C in transmembrane signaling
Authors: Takai Yoshimi , Kaibuchi Kozo , Tsuda Terutaka , Hoshijima Masahiko 
Published: December 2004

AbstractMany extracellular signals elicit Ca2+ mobilization and diacylglycerol formation in their target cells. Diacylglycerol is derived from the receptor‐linked phosphoinositide turnover and serves as a second messenger for the activation of protein kinase C in the presence of Ca2+ and phosphatidylserine. Unique diacylglycerols such as 1‐oleoyl‐2‐acetyl‐glycerol, which activate intracellular protein kinase C when added to intact cells, have been synthesized. Tumor‐promoting phorbol esters substitute for such diacylglycerols and directly activate protein kinase C in both intact cell and cell‐free systems. Under appropriate conditions, the synthetic diacylglycerols and phorbol esters induce protein kinase C activation without Ca2+ mobilization, whereas Ca2+ ionophore A23187 induces Ca2+ mobilization without protein kinase C activation. Using these substances, we have obtained evidence that both protein kinase C and Ca2+ are involved in and play a synergistic role in exocytosis, cell division, and other cellular functions. In this article, the role of protein kinase C in transmembrane signaling is discussed.
Molecular mechanisms of cytotoxicity mediated by Entamoeba histolytica: Characterization of a pore‐forming protein (PFP)
Authors: Young John Ding‐E , Cohn Zanvil A. 
Published: December 2004

AbstractEntamoeba histolytica is the human pathogen responsible for amebiasis. This infection is characterized by an invasive enteric illness that may spread to multiple organs. The parasite E histolytica may now be cultivated in vitro, with Diamond's axenic medium [1]. In culture, this protozoan remains cytolytic to a variety of cell types, including neutrophils and macrophages (reviewed in [2]). The mechanism(s) involved in the expression of this potent cytolysis remain(s) unclear. Previous studies from several laboratories have suggested that the cell killing mediated by amoeba is dependent on intimate contact of amoeba with the target cell membrane [3–9]. Following contact, the amoeba readily ingests the target cell (Fig. 1A). However, more recent cinemicroscopic and kinetic studies [8,9] (see also Fig. 1B) indicate that the cytolysis mediated by amoeba may occur prior to phagocytosis, raising the possibility of an extracellular cytolytic event triggered by surface contact.Previous work from this laboratory has described the properties of a highly enriched plasma membrane fraction and surface polypeptides from axenically grown E histolytica [10,11]. Studies from this laboratory on its extensive vacuolar apparatus and its role in pinocytosis and intracellular acidification have also been published [12,13]. Here, we review some recent evidence for the isolation and characterization of a pore‐forming protein (PFP) extracted from this protozoan, which may be closely related to the killing of target cells mediated by pore‐forming proteins of the immune system (the complement cascade as well as proteins extracted from granules of different immune cells [Young et al, in preparation]), which may thereby suggest a very similar and general mechanism of cell‐mediated cytotoxicity.
Dissecting the roles of individual interactions in protein stability: Lessons from a circularized protein
Authors: Goldenberg David P. 
Published: December 2004

AbstractA circular form of bovine pancreatic trypsin inhibitor (BPTI) has been prepared by introducing a peptide bond between the N‐ and C‐termini, which are in close proximity in the native conformation. The pathway and energetics of the disulphide‐coupled folding transition of the circular protein have been studied using methods applied previously to the unmodified protein. The cross‐link between the termini was found not to significantly stabilize the native state in spite of the expected reduction in entropy of the unfolded protein. This unexpected result has led to a reexamination of the stabilization expected from a cross‐link, considering effects on the native, as well as unfolded, states of the protein. The greatest stabilization is expected when the cross‐linked groups are held rigidly in the native protein in the optimum orientation for forming the cross‐link. Similar analyses, utilizing thermodynamic cycles, can be applied to other interactions that stabilize native proteins, including disulphide bonds, salt bridges, and hydrogen bonds and to modifications to the protein that remove them. In general, the contribution of an individual interaction to the stability of the native state depends on the extent to which the interaction is favored in the native conformation, which can vary greatly depending on the local environment of the interacting groups.
Purification of human DNA (cytosine‐5‐)‐methyltransferase
Authors: Zucker Keith E. , Riggs Arthur D. , Smith Steven S. 
Published: December 2004

AbstractWe have developed a facile procedure for the purification of DNA methyltransferase activity from human placenta. The procedure avoids the isolation of nuclei and the dialysis and chromatography of large volumes. A purification of 38,000‐fold from the whole cell extract has been achieved. The procedure employs ion exchange, affinity, and hydrophobic interaction chromatography coupled with preparative glycerol gradient centrifugation. A protein of 126,000 daltons was found to copurify with the activity and was the major band seen in the most highly purified material after SDS gel electrophoresis. This observation, coupled with an observed sedimentation coefficient of 6.3S, suggests that the enzyme is composed of a single polypeptide chain of this molecular weight. Hemimethylated DNA was found to be the preferred substrate for the enzyme at each stage in the purification. The ratio of the activity of the purified product on hemimethylated to that on unmethylated M13 duplex DNA was about 12 to 1. Thus, the purified activity has the properties postulated for a maintenance methyltransferase. The availability of highly purified human DNA methyltransferase should facilitate many studies on the structure, function, and expression of these activities in both normal and transformed cells.
12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA)‐induced gene sequences in human primary diploid fibroblasts and their expression in SV40‐transformed fibroblasts
Authors: Angel Peter , Rahmsdorf Hans Jobst , Pöting Annette , Lücke‐Huhle Christine , Herrlich Peter 
Published: December 2004

AbstractWe have isolated cDNA sequences from TPA‐treated primary human fibroblasts, which indicate RNA species that are coordinately regulated after treatment of these cells with either ultraviolet light, mitomycin C, the UV‐induced factor EPIF, or TPA. The levels of RNA are elevated in Bloom syndrome (cells of two out of three patients). After transformation with SV40 one of the sequences is overexpressed while another one is reduced. Both genes maintain their inducibility by the agents mentioned.
The relation between adenosine diphosphate‐ribosylation and mammary gland differentiation
Authors: Bolander Franklyn F. 
Published: December 2004

AbstractPoly(adenosine diphosphate [ADP]‐ribosyl)ation, although associated with differentiation in many systems, exhibited a reciprocal relationship with mammary gland differentiation, and both the synthetic and degradatory pathways complemented each other in this regard. Poly(ADP‐ribosyl)synthetase activity declined during pregnancy and lactation, while poly(ADP‐ribose) degradatory activity rose late in pregnancy and peaked during lactation. In explant cultures, similar changes occurred and appeared to be under separate hormonal control; prolactin suppressed the synthetase activity, whereas insulin stimulated the poly(ADP‐ribosyl)glycohydrolase activity. This latter effect may be mediated by a decline in cAMP levels for the following reasons: the glycohydrolase is known to be inhibited by cAMP, insulin decreased cAMP concentrations in mammary explants by 70%, and cholera toxin blocked the effects of insulin on poly(ADP‐ribose) degradation. This reciprocal relationship between poly(ADP‐ribosyl)ation and mammary gland differentiation is further supported by pharmacological studies: in the presence of insulin, cortisol, and prolactin, an inhibitor of the synthetase stimulated α‐lactalbumin three‐fold over hormone stimulation alone. However, this inhibitor was unable to induce differentiation in the absence of prolactin. Therefore, although there is a close association between a decline in enzyme activity and mammary differentiation, the data are insufficient to support a causal relationship.
Synthetic potential of Staphylococcus aureus V8‐protease: An approach toward semisynthesis of covalent analogs of α‐chain of hemoglobin S
Authors: Seetharam Ramnath , Acharya A. Seetharama 
Published: December 2004

AbstractEnzyme‐catalyzed reformation of peptide bonds in the noncovalent fragment systems of proteins has been emerging as a convenient procedure for the semisynthesis of covalent analogs of the respective proteins. Limited proteolysis of the α‐chain of hemoglobin S with Staphylococcus aureus V8‐protease converts the chain into a fragment‐complementing system by hydrolyzing the peptide bond Glu(30)‐Arg(31) of the chain. Therefore, it is conceivable that semisynthesis of covalent analogs of α‐chain could be achieved if conditions for the V8‐protease catalyzed formation of peptide bonds could be established. The synthetic potential of V8‐protease has been now investigated by incubating V8‐protease‐derived fragments of α‐chain, namely α1–30 and α31–47 with the enzyme at pH 6.0 in the presence of n‐propanol as the organic cosolvent. RP high performance liquid chromatography analysis showed that a new chromatographically distinct component is generated on incubation, and this has been identified as α1–47 by amino acid analysis, redigestion with V8‐protease (in the absence of n‐propanol), and tryptic peptide mapping. Optimal conditions for the synthesis of α1–47 is at pH 6.0, 4°C, and 24 hr of incubation with 25% n‐propanol as organic cosolvent. This stereospecific condensation of the fragments proceeded to a high level of about 50% in 24 hr. Further incubation up to 72 hr did not increase the yield of α1–47, suggesting that an equilibration of synthesis and hydrolysis reactions has been attained. The demonstration of the synthetic potential of V8‐protease and the fact that α1–30 and α31–141 interact to form a native‐like complex, opens up an approach for the semisynthesis of covalent analogs of α‐chain of hemoglobin S.
Copper ions and hydrogen peroxide form hypochlorite from NaCl thereby mimicking myeloperoxidase
Authors: Frenkel Krystyna , Blum Fredric , Troll Walter 
Published: December 2004

AbstractSea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H2O2) prevents polyspermy in Arbacia punctulata. We found that it is not H2O2 but probably hypochlorous acid/hypochlorite (HOCl/OCl−) derived from H2O2 that is toxic to the supernumerary sperm. The spermicidal activity of H2O2 is potentiated by at least one order of magnitude by cupric ions (Cu2+). This increased toxicity is not due to the formation of hydroxyl radicals (·OH) because ·OH scavengers did not counteract the activity of Cu2+. More‐over, substitution of Cu2+ by ferrous ions (Fe2+), which are known to cause formation of ·OH from H2O2, had no effect on fertilization even at 102−103 times higher concentrations. In contrast, 3‐amino‐1,2,4‐triazole (AT), an HOCl/OCl− scavenger, totally reversed the toxic effects of Cu2+. Furthermore, we found that HOCl/OCl− is generated in solutions of H2O2 and Cu2+ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl−. HOCl/OCl− generated by Cu2+ from H2O2 and Cl−, a low concentration of exogenously added HOCl/OCl−, or increased concentrations of H2O2 has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl− from the Cl− present in sea water through reaction with H2O2 generated by the newly fertilized egg.
Guanidine hydrochloride denaturation studies of mutant forms of staphylococcal nuclease
Authors: Shortle David 
Published: December 2004

AbstractSeveral mutant forms of Staphylococcal nuclease with one or two defined amino acid substitutions have been purified, and the effects of the altered amino acid sequence on the stability of the folded conformation have been analyzed by guanidine hydrochloride denaturation. Two nuc‐ mutations, which greatly reduced the level of enzyme activity accumulated in Ecoli colonies carrying a recombinant plasmid with the mutant nuc gene (i.e., a NUC‐phenotype), both result in protein unfolding at significantly lower guanidine hydrochloride concentrations than the wild‐type protein, whereas three sup mutations isolated on the basis of their ability to suppress partially the NUC‐phenotype of the above two mutations result in unfolding at significantly higher guanidine hydrochloride concentrations. Characterization of nuclease molecules with two different amino acid substitutions, either nuc − + sup pairs or sup + sup pairs, suggests that the effect of an amino acid substitution on the stability of the native conformation, as measured by the value of ΔΔGD, may not be a constant, but rather a variable that is sensitive to the presence of other substitutions at distant sites in the same molecule. Surprisingly, the slopes of the log Kapp vs guanidine hydrochloride concentration plots vary by as much as 35% among the different proteins.