bluuebunny/crossref_metadata_2025_split huggingface dataset:
A novel succinyl dipeptide stimulates directed cell migration by modulating protein kinase C activity
Authors: Mascardo Renato N. , Thompson Wayne , Gallo Mary Ann , Chambers Timothy C. , Eilon Gabriel 
Published: February 2005

AbstractIn determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E‐64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl‐leucyl‐agmatine (SLA), an analogue of E‐64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on en‐dothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetato (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P‐labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.
Effect of inflammatory cytokines on human endothelial cell proliferation
Authors: Saegusa Yasuhiro , Ziff Morris , Welkovich Linda , Cavender Druie 
Published: February 2005

AbstractNeovascularization, a common occurrence in chronic inflammatory lesions, requires endothelial cell (EC) proliferation. Because this form of inflammation is often mediated by immunologically generated cytokines, the effects of such cytokines on human umbilical vein EC proliferation in vitro were investigated. Low concentrations of recombinant interferon gamma (rIFN‐γ) (10—100 U/ml), but not a higher concentration (1,000 U/ml), enhanced both basal and endothelial cell growth factor (ECGF)‐stimulated EC proliferation. Recombinant interleukin 1 (rIL‐1) and recombinant tumor necrosis factor‐α (rTNF) had minor effects on basal EC proliferation, but significant inhibition was observed in the presence of ECGF. A combination of rIFN‐γ and rTNF induced marked suppression of EC proliferation, which appeared to be due to a cytotoxic effect on the EC, as demonstrated by51Cr release. In contrast, the combination of rIFN‐γ and rIL‐1 had only an additive effect on EC proliferation, with no evidence of cytotoxicity. These results suggest that cytokines have important regulatory roles in local vascular proliferation. These effects varied not only with the individual cytokine, but also with the combination of cytokines used. The most striking effects were (1) the stimulation of proliferation by IFN‐γ at a low concentration and (2) the inhibition by both rIL‐1 and rTNF of ECGF‐stimulated proliferation.
Dexamethasone‐induced killing of neoplastic cells of lymphoid derivation: Lack of early calcium involvement
Authors: Bansal Neelam , Houle Antoinette G. , Melnykovych George 
Published: February 2005

AbstractThe role of calcium influx in dexamethasone‐induced fragmentation of DNA was studied in the glucocorticoid‐sensitive human lymphoid line of T cell derivation (CEM‐C7). Reduction of calcium content in the medium or the use of EGTA increased DNA fragmentation and appeared to slightly enhance the effect of dexamethasone. Incubation of isolated nuclei in the presence of high concentrations of calcium did not bring about significant DNA fragmentation. Calmida‐zolium, an antagonist of calmodulin dependent reactions did not reduce the sensitivity of CEM‐C7 cells to dexamethasone nor did it modify the response to dexamethasone of the resistant CEM‐C1 line. It appears that in contrast to rodent thymocytes, massive calcium influx is not per se responsible for the initiation of directed cell killing (apoptosis).
Cytoskeleton as a target in menadione‐induced oxidative stress in cultured mammalian cells. I. Biochemical and immunocytochemical features
Authors: Bellomo G. , Mirabelli F. , Vairetti M. , Iosi F. , Malorni W. 
Published: February 2005

AbstractCytoskeletal abnormalities occurring during oxidative stress generated by the metabolism of the redox cycling compound 2‐methyl‐1,4‐naphtoquinone (menadione) have been investigated in different mammalian cells in culture. Extraction of the whole cytoskeleton as well as the intermediate filament‐ and the microtubule‐enriched fractions from menadione‐treated cells revealed a marked depletion of protein sulfhydryl groups. The analysis of the whole cytoskeletal fraction by PAGE showed a menadione‐dependent and thiol‐sensitive oxidation of actin, leading to the formation of high‐molecular‐weight aggregates. In addition, the extraction of this fraction with high concentrations of KCl entailed only a partial solubilization of actin. The comparative cytochemical analysis performed on treated cells showed a menadione‐dependent clustering of actin microfilaments. The metabolism of menadione induced microtubule depolymerization and inhibition of GTP‐induced microtubule assembly from soluble cytosolic components. The latter phenomenon was prevented by previously treating the cytosolic fraction with thiol reductants such as dithiothreitol. Menadione increased the protein content of the intermediate‐size filament fraction, partially purified by one or more cycles of disassembly/assembly, and particularly enriched in polypeptides reacting with antikeratin antibodies. Furthermore, a reversible and oxidation‐dependent change of the electrophoretic mobility of some polypeptides in this fraction was detected. The immunocytochemical investigation of intermediate‐size filament distribution in menadione‐treated cells, however, revealed only minor modifications mainly consisting of perinuclear condensation of cytokeratin structures. These findings suggest that cytoskeletal structures (actin microfilaments, microtubules, and intermediate‐size filaments) are actually significant targets in quinone‐induced oxidative stress.
Growth‐associated modifications of low‐molecular‐weight thiols and protein sulfhydryls in human bronchial fibroblasts
Authors: Atzori Luigi , Dypbukt Jeannette M. , Sundqvist Kristina , Cotgreave Ian , Edman Charlotte C. , Moldéus Peter , Grafström Roland C. 
Published: February 2005

AbstractThe thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol‐reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin pertubs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein‐bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low‐molecular‐weight thiols and protein‐bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol‐depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein‐bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cystine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low‐molecular‐weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.
Regulation of Na+/H+ and Cl−/HCO3− antiports in vero cells
Authors: Tønnessen Tor Inge , Aas Alf Tore , Ludt Jannikke , Blomhoff Heidi Kiil , Olsnes Sjur 
Published: February 2005

AbstractThe effect of serum, phorbol‐12‐myristate‐13‐acetate (TPA), and forskolin on the activity Na+/H+ antiport and the Na+‐coupled and Na+‐independent Cl−/ HCO3− antiport was studied in Vero cells by measuring 22Na+ and 36Cl− fluxes and changes in cytosolic pH (pHi). The Na+‐independent Cl−/HCO3− antiport, which acts as an acidifying mechanism, is strongly pH‐sensitive. In serum‐starved cells it is activated at alkaline cytosolic pH, with a half‐maximal activity at pHi ∼7.20. Incubation with serum increased the activity of the Na+‐independent Cl−/HCO3− antiport at pHi values from 6.8 to 7.2. Thus serum appeared to alter the pHi sensitivity of this antiporter such that the threshold value for activation of the antiport was shifted to a more acidic value. Na+ /H+ antiport was somewhat stimulated initially by addition of serum, but further incubation with serum (> 45 min) decreased its activity. The activity of the Na+ ‐coupled Cl−/HCO3− antiport, which is the major alkalinizing antiport in Vero cells, was not altered by short‐term incubation with serum (< 10 min) but decreased after prolonged incubation (> 45 min). Our findings with TPA and forskolin indicate that the effect of serum is partly mediated by the protein kinase C pathway, whereas the cyclic adenosine monophosphate pathway does not appear to play an important role. The net effect of serum on the pHi‐regulating antiports was a slight decrease in intracellular pH.
Fibroblast growth factor stimulates protein kinase C in quiescent 3T3 cells without Ca2+ mobilization or inositol phosphate accumulation
Authors: Nånberg Eewa , Morris Clive , Higgins Theresa , Vara Francisco , Rozengurt Enrique 
Published: February 2005

AbstractTo elucidate the transmembrane signalling processes initiated by fibroblast growth factor (FGF), we have studied the effect of recombinant basic FGF (bFGF) on various early events associated with mitogenesis in Swiss 3T3 fibroblasts. bFGF, at mitogenic concentrations, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. In contrast, bFGF stimulated the phosphorylation of the Mr 80,000 (80K) cellular protein which is a major substrate of protein kinase C. This effect was potentiated by the diacylglycerol kinase inhibitor R59022. Two‐dimensional polyacrylamide gel electrophoresis and phosphopeptide mapping showed that the 80K phosphoproteins generated in response to bFGF, bombesin, and phorbol 12,13‐dibutyrate were indistinguishable. Down‐regulation of protein kinase C prevented bFGF stimulation of 80K phosphorylation. Other protein kinase C‐dependent early events such as transmodulation of the epidermal growth factor receptor, cytoplasmic alkalinization, inhibition of vasopressin induced increase in cytosolic [Ca2+], and enhancement of cAMP accumulation in response to forskolin were also induced by bFGF. Similar results were obtained when bFGF was added to quiescent cultures of tertiary mouse embryo fibroblasts. We conclude that bFGF stimulates protein kinase C through a signal transduction pathway distinct from inositol phospholipid turnover and Ca2+ mobilization.
Small intestinal differentiation in human colon carcinoma HT29 cells has distinct effects on the lateral diffusion of lipids (ganglioside GM1) and proteins (HLA Class 1, HLA Class 2, and neoplastic epithelial antigens) in the apical cell membrane
Authors: Magnusson Karl‐Eric , Gustafsson Mikael , Holmgren Kajsa , Johansson Birgitta 
Published: February 2005

AbstractWe have studied the effect of maturation to small intestinal‐like epithelial cells of the human colonic calcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP). Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21‐day period. Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM‐Gal. In comparison to control cells grown in DMEM‐Glu medium, the more small intestinal‐like cells grown in DMEM‐Gal displayed no alteration of the lateral mobility of either cholera toxin (B subuni)‐labelled ganglioside GM1 (diffusion coefficient, D [x 108] = 0.8–0.9 cm2s−1; mobile fraction, R = 50‐60%) or antibody‐stained Class 2 histocompatibility (HLA‐DR) antigen (D [x 109] = 2 cm2s−1; R = 60–70%). However, antibody‐labelled β2‐microglobulin of HLA Class 1 antigen displayed increased mobility in HT29‐Gal cells; D was × 1.4 and R × 1.8 larger in the HT29‐Gal cells. By contrast, the mobility of a neoplastic antigen was reduced; D and R were × 0.60 and × 0.69 of the values seen in HT29‐Glu cells. It is thus concluded that DMEM‐Gal‐induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobil‐ity of different membrane components.
Regulation of polyamine transport in Chinese hamster ovary cells
Authors: Byers Timothy L. , Pegg Anthony E. 
Published: February 2005

AbstractControl Chinese hamster ovary (CHO) cells and mutant CHO cells lacking ornithine decarboxylase activity (CHODC−) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyamines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with α‐difluoromethylornithine in CHO cells or via polyamine starvation of CHODC− cells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyamine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyamine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyamine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl) polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylgiyoxal bis(guanylhydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.
Intracellular distribution of the c‐fos antigen during the cell cycle
Authors: Rahm Magnus , Hultgardh‐Nilsson Anna , Jiang Wei‐Qin , Sejersen Thomas , Ringertz Nils R. 
Published: February 2005

AbstractThe subcellular localization of the c‐fos proto‐oncogene product was studied in the G1, S, G2, and mitotic phases of the cell cycle by indirect immunofluores‐cence. For these analyse c‐fos transfected L6J1 rat skeletal myoblasts and adult rat aortic smooth muscle cells in secondary culture, and c‐fos‐ and c‐myc co‐transfected mouse Swiss 3T3 fibroblasts were used. During G1, S, and G2, the c‐fos protein was evenly distributed in the nucleus, with exclusion of the nucleoli. In mitotic prophase the c‐fos antigen was dissociated from the condensed chromosomes and became diffusely distributed in the cell cytoplasm, where it remained until telophase, when, again, it appeared to be associated with chromatin in the re‐assembling nucleus. When comparing the subnuclear distribution of the c‐fos product with that of densely packed DNA, stained with the fluorochrome Hoechst, an inverse relationship was found. Dispersed chromatin regions with weak Hoechst DNA fluorescence showed a stronger fos immunofluorescence than regions that contained a higher concentration of DNA. The localization of c‐fos antigen partially overlapped with that of antigens typical of small nuclear ribonucleoprotein complexes participating in transcription and splicing.To examine if the c‐fos protein would bind preferentially to specific interphase chromosomes the nucleus was fragmented into micronuclei containing single, or groups of, chromosomes. Immunofluorescence analysis showed that the majority of micronuclei were fos‐positive. Possible roles of the c‐fos proto‐oncogene product are discussed in relation to other nuclear antigens.
Sodium‐dependent phosphate transport in primary cultures of renal tubule cells from young and adult rats
Authors: Chen Ming L. , King Rebecca S. , Armbrecht H. James 
Published: February 2005

AbstractThe transport of phosphate by primary cultures of renal cells from young (5‐6 weeks) and adult (10–12 months) rats was studied. Renal tubule cells isolated from young and adult groups exhibited typical epithelial morphology and similar growth rates. The Na‐dependent phosphate uptake was saturable with a Km of 5–7 μM over a substrate range of 1–500 μM. A decrease in Na‐dependent phosphate uptake in adult cells (30%) was found compared to that of young cells. The Na‐independent component of phosphate uptake did not vary with age. In addition, the inhibition of phosphate uptake by a variety of compounds (ouabain, gramicidin, 2,4‐dinitrolphenol, KCN, and arsenate) were similar in both age groups. Kinetic analysis showed that a significant reduction in Vmax (4.4 ± 0.4 vs. 3.1 ± 0.2 nmol Pi/mg protein/10 min in young and adult cells, respectively), but not Km, resulted in this decreased uptake of phosphate in adult groups. There was no difference in the efflux of phosphate from both age groups. When cells were preincubated in a phosphate‐free medium for 24 hours, the uptake of phosphate was increased to 46% and 24% of their corresponding controls in young and adult cells, respectively. The decreased phosphate uptake and limited adaptation to a phosphate‐free medium by the adult renal cells may account for the hypophosphatemia and phosphaturia seen in adult and old animals in vivo.
Membrane potential of rat calvaria bone cells: Dependence on temperature
Authors: Massas Ruth , Korenstein Rafi , Bincmann Dieter , Tetsch Peter 
Published: February 2005

AbstractThe membrane potentials of bone cells derived from calvaria of new born rats was shown to be strongly dependent on temperature. When we lowered the temperature from 36°C to 26°C, cells with spontaneous resting membrane potentials (MP) of ‐80 to‐50 mV depolarized (mean amplitude 8 mV; n = 33), and the membrane resistance increased by ∼ 80% (n = 20). The temperature response depended on the actual MP, the reversal potential being in the range of −80 to −90 mV. With the application of ouabain (0.1−1 mmol/liter; n = 12), cells depolarized. Simultaneously, the reversal potential of the temperature response was shifted towards more positive values and approached the actual MP level of the cells. Consequently, the depolarization amplitudes induced by lowering temperature were reduced at spontaneous MP levels. The rise of the membrane resistance during cooling was unaffected. When the extracellular chloride concentration was reduced from 133 to 9 mmol/liter, temperature‐de pendent depolarizations persisted at spontaneous MP values (n = 5). The findings indicate that the marked effects of temperature changes on the MP of bone‐derived cells are mainly determined by changes of the potassium conductance.
Effect of butylated hydroxytoluene on biliineage differentiation of the human HL‐60 myeioblastic leukemia cell
Authors: Burns C. Patrick , Petersen Eric S. 
Published: February 2005

AbstractButylated hydroxytoluene (BHT), which has both antioxidant and membrane active properties, has been reported to affect cellular differentiation. We studied its effect on the bipotent lineage differentiation of the important HL‐60 human my‐eloblastic leukemia cell line using reduction of nitroblue tetrazolium, cell cycle analysis, population growth rote, monoclonal antibodies, and morphology. BHT markedly accelerated retinoic acid‐induced myelocytic differentiation and dihydroxyvitamin D3‐induced monocvtic differentiation in a concentration and time‐dependent manner. Butylated hydroxyanisole (BHA) had a comparable effect. Preincubation with the compounds was not necessary to evoke the acceleration. Other antioxidants and inhibitors of eicosanoid synthesis were inactive. We conclude that the important food preservatives BHT and BHA accelerate the kinetics of terminal differentiation of human leukemia and that this effect is likely due at least in part to their membrane active properties.
Lipopolysaccharide induces competence genes JE and KC in Balb/C 3T3 cells
Authors: Tannenbaum Charles S. , Major Jennifer A. , Poptic Earl J. , DiCorleto Paul E. , Hamilton Thomas A. 
Published: February 2005

AbstractThe expression of the early genes JE and KC has been examined in Balb/c 3T3 cells treated with bacterial lipopolysaccharide (LPS). Previous studies showed that JE and KC mRNAs are induced in murine peritoneal macrophages treated with LPS, suggesting a role for these genes in inflammatory responses. Consistent with this possibility are recently published cDNA sequences which document that both genes are members of a superfamily of inflammation‐ and/or growth‐related cytokines. In the present study, we provide evidence that the mRNAs for JE and KC are specifically induced by LPS treatment of Balb/c 3T3 cells. The LPS‐stimulated expression of JE and KC was dose dependent, and exhibited a transient time course; message levels were maximal between 2 and 4 hr and declined by 8 hr. The LPS‐augmented accumulation of JE and KC occurred even in the presence of cyclohexamide, which additionally had a superinducing effect on the expression of both genes. Cyclohexamide alone, in the absence of LPS, also induced JE and KC mRNA accumulation. LPS‐stimulated JE and KC mRNA expression was dependent upon the stimulation of transcription as determined by nuclear “run‐on” studies. Comparative analyses indicated that, under the conditions employed, LPS was a somewhat less effective stimulant of JE expression than PDGF or EGF, and was more effective than PDGF and equivalent to EGF in its ability to augment KC accumulation. Unlike PDGF and EGF, LPS did not stimulate DNA synthesis by Balb/c 3T3 cells at any time over the 72 hr period examined. The ability of the inflammatory, non‐mitogenic stimulus LPS to selectively induce JE and KC mRNA expression by fibroblasts may reflect their participation in inflammation and wound healing as secretory cells.