bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunocytochemical localization of annexin V (CaBP33), a Ca2+‐dependent phospholipid‐and membrane‐binding protein, in the rat nervous system and skeletal muscles and in the porcine heart
Authors: Spreca Antonio , Rambotti Maria Grazia , Giambanco Ileana , Pula Grazia , Bianchi Roberta , Ceccarelli Paolo , Donato Rosario 
Published: February 2005

AbstractWe investigated the ultrastructural localization of annexin V a Ca2+‐dependent phospholipid‐ and membrane‐binding protein in the nervous system, heart, and skeletal muscles. The results indicate that in the cerebellum the protein is restricted to glial cells, where it is found diffusely in the cytoplasm as well as associated with plasma membranes. Bergmann glial cell bodies and processes and astrocytes in the cerebellar cortex and oligodendrocytes in the cerebellar white matter displayed an intense immune reaction product. In sciatic nerves, the protein was exclusively found in Schwann cells with a subcellular localization similar to that seen in glial cells in the cerebellum. Pituicytes in the neurohypophysis were intensely immunostained, whereas axons were not. In the heart, annexin V was restricted to the sarcolemma, transverse tubules, and intercalated discs. In skeletal muscles the protein was localized to the sarcolemma and transverse tubules. No evidence for the presence of the protein in the sarcoplasm or in association with mitochondria, the sarcoplasmic reticulum, or contractile elements was obtained. The observation that plasma membranes in cells expressing annexin V have the protein associated with them is in agreement with previous data on Ca2+‐dependent binding of the protein to brain and heart membranes, and on existence of both EGTA‐ and Triton X‐100‐extractable and resistant fractions of annexin V in these membranes. The present data support the hypothesis that annexin V might be involved in membrane trafficking and suggest a role for this protein in the regulation of cytoplasmic activities in glial cells. © 1992 Wiley‐Liss, Inc.
Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers
Authors: Stasek Jerome E. , Patterson Carolyn E. , Garcia Joe G. N. 
Published: February 2005

AbstractCytoskeletal protein (CSP) interactions are critical to the contractile response in muscle and non‐muscle cells. Current concepts suggest that activation of the contractile apparatus occurs through selective phosphorylation by specific cellular kinase systems. Because the Ca2+‐phospholipid‐dependent protein kinase C (PKC) is involved in the regulation of a number of key endothelial cell responses, the hypothesis that PKC modulates endothelial cell contraction and monolayer permeability was tested. Phorbol myristate acetate (PMA), a direct PKC activator, and α‐thrombin, a receptor‐mediated agonist known to increase endothelial cell permeability, both induced rapid, dose‐dependent activation and translocation of PKC in bovine pulmonary artery endothelial cells (BPAEC), as assessed by γ‐[32P]ATP phosphorylation of H1 histone in cellular fractions. This activation was temporally associated with evidence of agonist‐mediated endothelial cell contraction as demonstrated by characteristic changes in cellular morphology. Agonist‐induced activation of the contractile apparatus was associated with increases in BPAEC monolayer permeability to albumin (∼200% increase with 10−6 M PMA, ∼400% increase with 10−8 M α‐thrombin). To more closely examine the role of PKC in activation of the contractile apparatus, PKC‐mediated phosphorylation of two specific CSPs, the actin‐ and calmodulin‐binding protein, caldesmon77, and the intermediate filament protein, vimentin, was assessed. In vitro phosphorylation of both caldesmon and vimentin was demonstrated by addition of exogenous, purified BPAEC PKC to unstimulated BPAEC homogenates, to purified bovine platelet caldesmon77, or to purified smooth muscle caldesmon150. Caldesmon77 and vimentin phosphorylation were observed in intact [32P]‐labeled BPAEC monolayers stimulated with either PMA or α‐thrombin, as detected by immunoprecipitation. In addition, BPAEC pretreatment with the PKC inhibitor, staurosporine, prevented α‐thrombin‐ and PMA‐induced phosphorylation of both cytoskeletal proteins, attenuated morphologic evidence of contraction, and abolished agonist‐induced barrier dysfunction. These results demonstrate that agonist‐stimulated PKC activity results in cytoskeletal protein phosphorylation in BPAEC monolayers, an event which occurs in concert with agonist‐mediated endothelial cell contraction and resultant barrier dysfunction. © 1992 Wiley‐Liss, Inc.
Heat induced protein denaturation in the particulate fraction of hela S3 cells: Effect of thermotolerance
Authors: Burgman P. W. J. J. , Konings A. W. T. 
Published: February 2005

AbstractIn this study we investigated the effect of heat on the proteins of the particulate fraction (PF) of HeLa S3 cells using electron spin resonance (ESR) and thermal gel analysis (TGA). ESR detects overall conformational changes in proteins, while TGA detects denaturation (aggregation due to formation of disulfide bonds) in specific proteins. For ESR measurements the ‐SH groups of the proteins were labelled with a maleimido bound spin label (4‐maleimido‐tempo). The sample was heated inside the ESR spectrometer at a rate of 1°/min. ESR spectra were made every 2–3° between 20° and 70°. In the PF of untreated cells conformational changes in proteins were observed in three temperature streches: between 38 and 44° (transition A, TA); between 47 and 53° (transition B, TB); and above 58° (transition C, TC). With TGA, using the same heating rate, we identified three proteins (55, 70, and 90kD) which denatured during TB. No protein denaturation was observed during TA, while during TC denaturation of all remaining proteins in the PF occurred. When the ESR and TGA measurements were done with the PF of (heat‐induced) thermotolerant ceils, TA was unchanged while TB and TC started at lated with the degree of thermotolerance that was induce in the cells. These results suggest that protection against heat‐induced denaturation of proteins in the PF in involved in heat induced thermotolerance. © 1992 Wiley‐Liss, Inc © 1992 Wiley‐Liss, Inc.
Basic fibroblast growth factor increases junctional communication and connexin 43 expression in microvascular endothelial cells
Authors: Pepper M. S. , Meda P. 
Published: February 2005

AbstractWe have analyzed the effect of basic fibroblast growth factor (bFGF) on junctional communication (coupling) and connexin 43 (Cx43) expression in bovine microvascular endothelial (BME) cells. In control confluent culturers, the incidence of coupling, as assessed by the intercellular transfer of microinjected Lucifer Yellow, was limited to 13% of injected cells, and decreased to 0% with time in culture. After exposure to bFGF (3ng/ml), the incidence of coupling was increased in a time‐dependent manner, reaching a maximum of 38% of microinjected cells after 10–12 hours. The extent of coupling, as assessed by scrape loading, was maximally increased 2.1‐fold 8–9 hours after addition of bFGF. bFGF also induced a 2‐fold increase in Cx43 as assessed by Western blotting and increased Cx43 immunolabelling at contacting interfaces of adjacent BME cells. Cx43 mRNA was likewise increased after exposure to bFGF in a time‐and dose‐dependent manner, with a maximal 6–7 fold increase after a 4 hour exposure to 3–10ng/ml. Finally, the increase in coupling and Cx43 mRNA expression observed after mechanically wounding a confluent monolayer of BME cells was markedly reduced by antibodies to bFGF, which have previously been shown to inhibit migration. Taken together, these results indicate that exogenous and endogenous bFGF increase intercellular communication and Cx43 expression in microvascular endothelial cells. We propose that the bFGF‐mediated increase in coupling is necessary for the coordination of endothelial cells during angiogenesis and other vessel wall functions. © 1992 Wiley‐Liss, Inc.
Methylation of atypical protein aspartyl residues during the stress response of HeLa cells
Authors: Ladino Cynthia A. , O'Connor Clare M. 
Published: February 2005

AbstractA protein carboxyl methyltransferase (PCMT), which specifically modifies atypical protein L‐isoaspartyl and D‐aspartyl residues, is widely distributed in eucaryotic cells, but the factors that regulate its activity in vivo have not been identified. It has been proposed that the PCMT initiates the repair of structurally damaged proteins. To test the possibility that the concentration of structurally abnormal cellular proteins affects PCMT activity, protein carboxyl methylation reactions were studied in HeLa cells exposed to various stresses that increase the extent of protein unfolding in cells. Protein carboxyl methylation rates increased 70–80% during incubations at 42°C and remained elevated for periods of up to 8 hr. This sustained increase was greater than that predicted from thermal effects on the enzyme alone and may relect the exposure of atypical aspartyl sites as proteins unfold as well as increased rates of protein deamidation and isomerization at elevated temperatures. Methylation rates showed no increases following 12 hr incubations with the amino acid analogs L‐azetidine‐2‐carboxylic acid or L‐canavanine. Northern blot analysis of RNA preparations from control and stressed cells revealed three major transcripts for the PCMT in HeLa cells, which are 1.6, 2.6, and 4.5 kb in length. The concentrations of all three transcripts decreased by ∼ 20% from control levels during heat shock. No changes in PCMT transcript concentrations were observed during incubation with the amino acid analogs. By contrast, large increases in the concentrations of hsp70 and ubiquitin trascripts were observed following either heat or chemical stresses. The results demonstrate that the PCMT is a constitutive component of cells whose function is required under normal conditions as well as during stress conditions, which accelerate structural damage to cellular proteins. © 1992 Wiley‐Liss, Inc.
Constitutive HSP70: Oligomerization and its dependence on ATP binding
Authors: Kim Dooha , Lee Yong J. , Corry Peter M. 
Published: February 2005

AbstractThe constitutive HSP70 purified from CHO cells, which indicated a single band in SDS‐polyacrylamide gel electrophoresis, showed multiple bands in native‐polyacrylamide gel electrophoresis. These results indicate that the protein may exist in oligomeric forms. After crosslinking the oligomers with glutaraldehyde, SDS‐polyacrylamide gel electrophoresis showed three protein bands of molecular weight 70 kDa, 153 kDa, and 200 kDa corresponded to monomer, dimer, and trimer, respectively. The relative amount of oligomeric forms was dependent upon ATP concentrations: it increased upon hydrolysis of ATP or decreased upon incubation with high concentrations of ATP (1–10 mM). Autoradiographic analysis of the native polyacrylamide gel electrophoresis of HSP70 following incubation with [γ‐32P]ATP revealed that ATP bound to only monomer. These results suggest that the equilibrium between oligomeric forms is dependent on ATP concentrations. Nonetheless, during heat shock, both monomer and oligomer might be indistinguishably associated with some proteins, probably denatured proteins. © 1992 Wiley‐Liss, Inc.
Ligand‐induced protein tyrosine kinase activity in living cells coexpressing intact EGF receptors and receptors with an extensive cytosolic deletion
Authors: Dou Yi , Hoffman Patricia , Hoffman Brian L. , Carlin Cathleen 
Published: February 2005

AbstractA population of stable NIH 3T3 transfectants with two molecular weight classes of membrane‐bound EGF receptors encoded by a human EGF receptor cDNA has been identified and characterized. In addition to intact EGF receptors, these cells also express a molecule with an extensive cytosolic deletion. This deletion includes the ligand‐activated intrinsic protein tyrosine kinase catalytic domain. Treatment with EGF caused dimerization of intact and truncated receptors, allowing us to assess protein tyrosine kinase activity in the heterodimer isolated from living cells. In contrast to homodimeric complexes with intact EGF receptor only, heterodimers were deficient in protein tyrosine kinase activity. Moreover, physical association between intact and truncated molecules suppressed receptor autophosphorylation by EGF receptor protein tyrosine kinase activated by antibody binding in vitro. Evidence presented here supports the idea that protein tyrosine kinase activation is facilitated by interaction between adjacent receptor molecules with intact catalytic domains. Furthermore, molecules with cytoplasmic deletions that are physically associated with kinase‐active EGF receptors appear to behave as dominant negative mutations. The HerC cl cells used in this study were selected with methotrexate to amplify the EGF receptor cDNA, and in that sense may resemble certain tumor‐derived cells characterized by overexpressed and rearranged EGF receptor genes. © 1992 Wiley‐Liss, Inc.
Localization of p35 (annexin I, lipocortin I) in normal adult rat kidney and during recovery from ischemia
Authors: McKanna James A. , Chuncharunee Aporn , Munger Karen A. , Breyer Julia A. , Cohen Stanley , Harris Raymond C. 
Published: February 2005

AbstractThe 35‐kDa protein (p35, lipocortin I, annexin I), originally discovered as a Ca++‐dependent substrate for the EGF receptor tyrosine kinase, binds Ca++ and phospholipids, is developmentally regulated in embryos and has restricted expression in adults. Immunohistochemistry of normal rat kidney shows that p35 is enriched in epithelia of Bowman's capsule, the macula densa, and medullary/papillary collecting ducts, suggesting that p35 is related to specialized renal functions. Light staining is observed in the thick ascending limb; elsewhere, immunoreactivity is nil. Since renal recovery from ischemia involves both hyperplasia and hypertrophy and reportedly is accelerated by EGF, we examined p35 distribution during this process. After 48 hours of recovery, both the distribution and amount of renal p35 are altered. Immunoblots show p35 levels increased at least threefold in whole‐kidney homogenates. The expression of p35 is still highly restricted in recovering kidneys; however, the thick ascending limb now stains heavily. This is the first documentation of alterations in annexin levels during a pathophysiologic response. However, our attempts to discern effects of exogenous EGF on the recovery from ischemia were negative for both mitotic index and renal function assays. © 1992 Wiley‐Liss, Inc.
Isolation of rat bone marrow mast lineage cells using Thy 1.1 and rat stem cell factor
Authors: Medlock Eugene S. , Mineo Chrysa , Housman Jerry M. , Elliott Gary S. , Trebasky Lisa D. , Langley Keith E. , Zsebo Krisztina M. 
Published: February 2005

AbstractRecent reports have shown that various marrow‐derived cell populations respond vigorously to recombinant rat stem cell factor (rrSCF164), one form of the kitligand. In the present study, we isolated cell populations from rat bone marrow using the Thy 1.1 antigen (an antigen that in the rat is differentially expressed on primitive hemopoietic progenitor cells) and fluorescently conjugated rrSCF164 (rrSCF164‐PE). We show that rrSCF164 only stimulates cells that are enriched in the brightest Thy 1.1 populations (Thy 1.1bright). Numerous cell lines were generated by serial passage in rrSCF164 containing medium, and the prototypic cell lines have been designated SRT002 and SRT003. Each cell line retains the Thy 1.1bright phenotype and does not respond to interleukins (IL) 1–8, IL‐10, granulocyte (G) colony‐stimulating factor (CSF), granulocyte macrophage (GM) CSF, M‐CSF, or crude preparations of mitogen‐stimulated T‐cell supernatants. The Thy 1.1bright population of rat marrow was subdivided into a subset that binds rrSCF164‐PE (Thy 1.1bright, rrSCF164+). The majority of these cells possess certain characteristics in common with marrow‐derived mast cells and the Thy 1.1bright, rrSCF164 responsive cell lines, having similar granule morphology, being metachromatic, and reacting positively with alcian blue. Moreover, rats treated with rrSCF164 displayed significant increases in Thy 1.1bright, rrSCF164+ cells in the bone marrow. These studies show that the combination of Thy 1.1 and rrSCF164 makes possible the isolation of a unique subset of rat bone marrow cells that differentially express the Thy 1.1 antigen and the cell surface receptor c‐kit, the majority of which are morphologically similar to marrow‐derived mast cells. © 1992 Wiley‐Liss, Inc.
Cycloheximide protection against actinomycin D cytotoxicity
Authors: Borrelli Michael J. , Stafford Diane M. , Rausch Cynthia M. , Ofenstein John P. , Cosenza Steven C. , Soprano Kenneth J. 
Published: February 2005

AbstractPretreatment plus concomitant treatment with 10 μg/ml cycloheximide protected Chinese hamster ovary cells and Swiss 3T3 cells against the cytotoxicity of actinomycin D. The cycloheximide treatment reduced the intracellular concentration of actinomycin D by reducing the level of actinomycin D bound to the acid precipitable fraction of the cell. Levels of unbound actinomycin D were unaffected by cycloheximide, indicating that the plasma membrane permeability to AD was not reduced. Actinomycin D inhibited total transcription but did not reduce cytoplasmic levels of rRNA nor of most tested mRNA; however, cytoplasmic levels of c‐myc mRNA were reduced below detectability. Cycloheximide treatment further inhibited total transcription and had no effect on cytoplasmic levels of rRNA nor of most tested mRNA. Cytoplasmic levels of c‐myc were elevated by cycloheximide and remained so even in the presence of actinomycin D. These data suggested that a reduction in cytoplasmic levels of short lived, essential mRNA, such as c‐myc mRNA, was one lethal lesion of actinomycin D. Furthermore, cycloheximide's protection may result, in part, from its ability to stabilize and/or elevate cytoplasmic levels of these mRNA, thus counteracting their depletion by actinomycin D. Protection may also result from the cycloheximide‐induced reduction of actinomycin D bound to the acid precipitable fraction of the cells. © 1992 Wiley‐Liss, Inc.
Alterations in cellular calcium handling as a result of systemic calcium deficiency in the developing chick embryo: I. Erythrocytes
Authors: Koide Masafumi , Smith Carol A. , Miyahara Takashi , Tuan Rocky S. 
Published: February 2005

AbstractChick embryos rendered calcium (Ca) deficient by shell‐less (SL) culture develop hypertension and tachycardia. Since hypocalcemia is accompanied by hypernatremia systemically but not by lower cellular Ca (Koide and Tuan, 1989), we speculate that cellular Ca handling may be altered in the SL embryo, perhaps involving Na transport. Using erythrocytes (RBC) from day‐14 SL and normal (NL) embryos as the experimental cell, cellular Ca handling was studied under varying extracellular osmotic and ionic conditions by analyzing 45Ca uptake and cell volume regulation. Two agents, p‐chloromercuriphenylsulfonate (PCM), and inosine/iodoacetamide (INI) were used to treat the RBCs to modify plasma membrane ion permeability and to deplete cellular ATP, respectively. Other cellular functions and activities related to Ca homeostasis, including ATP content and Ca2+‐ATPase activity, were also analyzed. These analyses showed: (1) in NaCl, Ca uptake was similar in NL and SL cells, except after INI treatment, which resulted in slower Ca uptake by the SL cells, (2) in choline and sucrose, Ca uptake by SL RBCs was higher, (3) Ca uptake by RBCs of both embryos changed depending on the osmotic agent (Na < K ≤ choline < sucrose), (4) Ca2+‐ATPase activity was higher in SL RBC, although there was no change in the size or charge of the enzyme, and (5) in any osmotic agent, cellular Na was significantly lower, whereas cellular K was higher, in SL RBC. Based on these results, three features of RBC Ca handling were apparent: (1) Na‐Ca exchange was functional and was more active in SL RBCs, (2) Ca uptake was dependent on the total ionic electrochemical gradient but not on bulk H2O movement, and (3) Ca pumping out capacity was directly correlated with Ca2+‐ATPase activity. Elevated Ca uptake in sucrose‐treated SL RBC is therefore indicative of its greater ion permeability. Taken together, these findings indicate that cellular Ca handling of the RBCs of SL chick embryos is characterized by a more active Na‐Ca exchange system, greater ion permeability, and higher Ca pumping out capacity, thereby suggesting an up‐regulated Ca handling function in the SL RBCs. The abnormal cellular Ca handling may be a direct result of the systemic Ca deficiency of the SL chick embryo and may be functionally related to its hypertension and tachycardia. © 1992 Wiley‐Liss, Inc.
Different relationships between cellular adenosine or 3′‐phosphorylation and cellular adenine ribonucleotide catabolism may be obtained
Authors: Overgaard‐Hansen Kay , Klenow Hans 
Published: February 2005

AbstractTreatment of BALB/c‐3T3 mouse fibroblasts with 3′‐led to a rapid accumulation of 3′‐phosphates and the kinetics of this process has been determined. Concomitant with accumulation of these compounds, the adenine ribonucleotide pool was reduced. The kinetics of the two processes suggested that they were tightly coupled. The inhibitory effect of relatively high concentrations of coformycin indicated that IMP was an intermediate in the catabolic pathway. Similar experiments with Ehrlich ascites tumor cells were performed in Ringer‐Hepes solution at pH 6.5 or 7.5 and with varying concentrations of orthophosphate. The experiments were performed with cells where ATP was [3H]‐. This allowed the determination of the catabolism of adenine ribonucleotides to labeled nucleosides under conditions where added adenosine was phosphorylated. The results showed that at low phosphate concentration (5.8 mM) at pH 6.5 adenosine may be phosphorylated at a rate that was completely balanced to the concomitant catabolism of adenine ribonucleotides; that is, there was apparently a tight kinetic coupling between anabolism of adenosine and catabolism of adenine ribonucleotides. With 3′‐a corresponding effect was obtained although the apparent coupling between phosphorylation of 3′‐and catabolism of adenine ribonucleotides was not complete. When experiments were performed at the same pH but at high concentration of phosphate (45 mM) there was in contrast no coupling between the two processes; that is, ATP was present in constant amounts while 3′‐phosphates accumulated at a high rate. In experiments with adenosine under these conditions there was still some although a relatively limited degree of apparent coupling between phosphorylation of adenosine and catabolism of adenine ribonucleotides. In both lines of cells used and with both adenosine and 3′‐, the main products of the catabolism of adenine ribonucleotides were inosine and hypoxanthine. With 3′‐there was in addition (about 20%) formation of xanthosine, suggesting that IMP dehydrogenase had also been activated. These results lead to the suggestion that adenosine (or 3′‐) may be phosphorylated in two ways. 1) Phosphorylation may depend on an adenosine kinase unrelated to catabolism of adenine ribonucleotides. 2) Phosphorylation may be tightly coupled to catabolism of adenine ribonucleotides. A nucleoside phosphotransferase may catalyze the transfer of a phosphoryl group from IMP to adenosine (or 3′‐) to form AMP (or 3′‐) and inosine, a process that may be tightly coupled to an AMP deaminase reaction. The IMP formed in the latter reaction may not be released but transferred to the phosphotransferase. In contrast, the AMP formed in the phosphotransferase reaction should be in equilibrium with soluble AMP. It is assumed that a physical complex may exist, possibly in a membrane bound form, between AMP deaminase and the nucleoside phosphotransferase. © 1993 Wiley‐Liss, Inc.