bluuebunny/crossref_metadata_2025_split huggingface dataset:
Vasopressin rapidly stimulates protein kinase C in digitonin‐permeabilized swiss 3T3 cells: Involvement of a pertussis toxin‐insensitive guanine nucleotide binding protein
Authors: Erusalimsky Jorge D. , Rozengurt Enrique 
Published: February 2005

AbstractGuanine nucleotides and pertussis toxin were used to test for the involvement of a guanine nucleotide binding protein in the vasopressin V1 receptor‐mediated stimulation of protein kinase C activity in Swiss 3T3 cells. Addition of vasopressin in the presence of [γ‐32P]ATP and digitonin caused a marked and rapid increase (8 ± 1‐fold after 1 min) in the phosphorylation of an Mr = 80,000 cellular protein (80K), a specific marker for protein kinase C activation. This phosphorylation was selectively blocked by the V1 receptor antagonist Pmp1‐0‐Me‐Tyr2 [Arg8]vasopressin, indicating that the effect was mediated through the vasopressin V1 receptor. Down regulation of protein kinase C by prior prolonged pretreatment of intact cells with phorbol 12, 13‐dibutyrate (PBt2) blocked the ability of vasopressin to stimulate the phosphorylation of 80K in digitonin‐permeabilized cells. Addition of a submaximal concentration of vasopressin together with the GTP analogue GTP‐γ‐S caused a synergistic stimulation of 80K phosphorylation. The GDP analogue GDP‐β‐S caused a 50% inhibition of the phosphorylation of 80K induced by a saturating concentration of vasopressin and shifted the vasopressin dose‐response curve to the right. GDP‐β‐S had no effect on the dose‐response for the stimulation of 80K phosphorylation induced by PBt2. Prior incubation of intact quiescent cultures of Swiss 3T3 cells with pertussis toxin did not impair either vasopressin‐induced increase in cytosolic [Ca2+] or activation of protein kinase C. These findings provide functional evidence for the involvement of a pertussis toxin‐insesitive G protein in the vasopressin V1 receptor‐mediated stimulation of protein kinase C in Swiss 3T3 cells.
Evidence for increased αMSH receptor binding in the F1 variant of B16 melanoma cells grown in dialyzed fetal calf serum
Authors: McQuillan A. , Hutchinson M. , Panasci L. C. 
Published: February 2005

AbstractThe specific binding of an αMSH analogue (Ac‐[Nle4, D‐Phe7] αMSH4–11 NH2) was enhanced in the presence of 10% dialyzed fetal calf serum (FCS) as compared with 10% FCS (nondialyzed) in the F1 variant of B16 melanoma cells. The replenishment of dialyzed serum with adrenocorticotrophic hormone (ACTH) or insulin had no effect on the increased level of αMSH receptor binding in these cells. However, 10 nM αMSH or 1 μM ACTH under identical conditions significantly decreased the level of αMSH binding. Competitive binding studies involving the αMSH analogue revealed that the specificity of the receptor was restricted to the complete molecule of αMSH, our analogue, βMSH and ACTH1–24. ACTH4–10, which contains the amino acid sequence responsible for biological activity, showed a very low affinity for the receptor. Furthermore, we observed an interesting phenomenon unique to dialyzed FCS in that once the cells were grown to confluence and melanin was produced, the cells were no longer viable. However, in McCoy's medium, which is deficient in tyrosine, the cells did not produce melanin and remained viable.
Kinetics of insulin internalization and processing in adipocytes: Effects of insulin concentration
Authors: Jochen Albert , Hays Judith , Lee Martha 
Published: February 2005

AbstractWe have studied the effect of insulin concentration on the kinetics of insulin internalization and efflux in isolated rat adipocytes. To determine internalization rates adipocytes were incubated with 125l‐insulin at 37°C; and at frequent, early time points surface‐bound and intracellular insulin were quantitated. Surface‐bound and intracellular insulin were discriminated by the sensitivity of the former to rapid dissociation by a pH 3.0 buffer at 4°C. From this data the endocytotic (internalization) rate constant (ke) was calculated for six insulin concentrations ranging from 0.3 to 100 ng/ml. Ke was found to decrease in an insulin concentration‐dependent manner (P < .001). Thus, values for ke were 0.121 ± 0.006 min−1 versus 0.074 ± 0.011 min−1 at 0.3 ng/ml and 100 ng/ml, respectively. The decrease in ke did not parallel insulin concentration‐dependent changes in insulin receptor affinity indicating it was not the result of an inability of low affinity receptors to be internalized. The kinetics of insulin efflux were determined by loading various concentrations of 125l‐insulin into the adipocyte interior, washing away surface‐bound and extracellular insulin, and then monitoring the subsequent efflux of pre‐loaded insulin into medium that contained the same concentration of insulin used in the loading step. The overall rate of efflux was independent of insulin concentration. In summary, these results show that at high insulin concentrations the efficiency of insulin internalization is impaired. In contrast, the rate of insulin efflux is unaffected.
Cell cycle dependent growth factor regulation of gene expression
Authors: Morgan Claudia J. , Pledger W. J. 
Published: February 2005

AbstractThe expression of the proto‐oncogenes c‐fos and c‐myc is a rapid response of G0‐arrested fibroblasts to serum and peptide growth factors; however, the role of the c‐fos and c‐myc gene products in subsequent cell cycle transit is not understood. We examined the expression of c‐fos and c‐myc mRNA in Balb/c 3T3 murine fibroblasts in response to platelet‐derived growth factor (PDGF) and platelet‐poor plasma, using arrest points associated with density dependent growth inhibition or metabolic inhibition to synchronize cells in S phase of the cell cycle. The expression of c‐fos and c‐myc mRNA in Balb/c 3T3 cells was differentially regulated with respect to growth factor dependence and cell cycle dependence, c‐fos expression was induced in the presence of PDGF and was unaffected by plasma. The induction of c‐fos expression in response to PDGF was cell cycle independent, occurring in cells transiting S phase and G2 as well as in G0 arrest. In contrast, c‐myc expression was both growth factor and cell cycle dependent. In G0 arrested cells, c‐myc expression was PDGF‐dependent and plasma‐independent, and PDGF was required for maintenance of elevated c‐myc levels during G1 transit. In cells transiting S phase, c‐myc mRNA was induced in response to PDGF, but was also plasma‐dependent in S phase cells that had been “primed” by exposure to PDGF during S phase.
Activation and proliferation signals in murine macrophages: Relationships among c‐fos and c‐myc expression, phosphoinositide hydrolysis, superoxide formation, and DNA synthesis
Authors: Hamilton John A. , Veis Nurin , Bordun Anne‐Marie , Vairo Gino , Gonda Thomas J. , Phillips Wayne A. 
Published: February 2005

AbstractMurine bone marrow‐derived macrophages (BMM) undergo DNA synthesis in response to growth factors such as colony stimulating factor‐1 (CSF‐1) and granulocyte‐macrophage CSF (GM‐CSF). These macrophages can also be “activated,” but without subsequent DNA synthesis, by a number of other agents, including lipopolysaccharide (LPS), concanavalin A, zymosan, formyl‐methionyl‐leucylphenylalanine (FMLP), and the Ca2+ ionophore, A23187. When BMM are treated with a range of stimuli, there is some, although not perfect, correlation between transient elevations in both c‐myc mRNA and c‐fos mRNA levels and increases in DNA synthesis. However, enhanced DNA synthesis and oncogene expression are readily dissociated from rises in inositol phosphates and, by implication, phospholipase C‐mediated hydrolysis of phosphatidyl inositol 4,5‐bisphosphate. Superoxide formation in BMM can also be dissociated from the other responses and does not necessarily depend on protein kinase C activation.
Appearance of basic fibroblast growth factor receptors upon differentiation of rat mammary epithelial to myoepithelial‐like cells in culture
Authors: Fernig David G. , Smith John A. , Rudland Philip S. 
Published: February 2005

AbstractThe binding of [125I]‐epidermal growth factor (EGF) and [125I]‐basic fibroblast growth factor (bFGF) to a number of single‐cell cloned rat mammary cell lines was measured using a saturation assay. Similar numbers of high‐affinity [125I]‐EGF binding sites (KD 1.3 nM) were found in epithelial and myoepithelial‐like cell lines. In contrast, high‐affinity (KD 35–276 pM) [125I]‐bFGF binding sites were present on fibroblastic and myoepithelial‐like cell lines but were not detectable on epithelial cell lines. A series of cell lines representing stages in the differentiation pathway of epithelial cells to an elongated myoepithelial‐like morphology showed a graded increase in the number of bFGF receptors. The sensitivity of a cell line to stimulation of DNA synthesis by bFGF correlated with the level of expression of bFGF receptors on the cellular surface. Complexes of cell surface receptors affinity‐cross‐linked to [125I]‐bFGF were analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). In each case two distinct complexes having apparent molecular weights of 180 kDa and 160 kDa were observed.
Male germ cell specific sulfogalactoglycerolipid is recognized and degraded by mycoplasmas associated with male infertility
Authors: Lingwood Clifford , Schramayr Susan , Quinn Patricia 
Published: February 2005

AbstractSulfogalactosylglycerolipid (SGG) is the major mammalian male germ cell glycolipid and has been implicated in sperm/egg binding. Mycoplasma pulmonis, a species of Mollicutes, is associated with male infertility in rodents.Purified SGG incubated in the presence of M. pulmonis was enzymatically degraded by both desulfation and deacylation. Desulfation occurred primarily at alkaline pH, and deacylation also increased with increased pH, indicating that these represent novel enzymatic activities. Digestion was facilitated, but not dependent on, the presence of detergent. Rat spermatozoa exposed to M. pulmonis showed a reduction in SGG content which was particularly marked for cauda (mature) spermatozoa. With the aid of tic overlay binding procedure, intact M. pulmonis were found to bind specifically to sulfated glycolipids and thus SGG may provide the cell membrane receptor for this organism. The topology of mycoplasma binding to rat sperm was consistent with the known topology of sperm SGG. The reduced binding (and subsequent digestion) of caput spermatozoan SGG correlates with the membrane colocalization of SGG and its endogenous binding protein at this stage. Separation of SGG and its binding protein during epididymal sperm maturation appears to facilitate M. pulmonis binding to and digestion of cauda sperm SGG.The binding and degradation of the sperm SGG by M. pulmonis may play a role in the induction of infertility which follows infection with these organisms by interfering in sperm/egg receptor recognition.
Morphology of the differentiation and maturation of LLC‐PK1 epithelia
Authors: Pfaller Walter , Cstraunthaler Gerhard , Loidl Peter 
Published: February 2005

AbstractIn the present study, a stereologic approach was utilized to quantitatively assess morphological changes during the differentiation of LLC‐PK1 cells into an epithelial membrane. This renal epithelial cell line has been described to undergo morphological changes during differentiation and maturation from subconfluent culture to a confluent epithelial layer. An increase in the number of apical microvilli, interpreted as an areal increase in this membrane domain, was reported. This morphological differentiation was found to be accompanied by an increase in the expression of apical Na+‐dependent hexose transport and the activities of certain brush border enzymes. Since no data are available that quantify the morphologic changes during LLC‐PK1 differentiation, a quantitative morphologic—stereologic—investigation was performed for an early (6 days) and a late (12 days) state of confluence of LLC‐PK1 monolayer cultures. The following morphological parameters were determined by light and electron microscopic morphometry: volume fractions (Vv) of nuclei, mitochondria, and lysosomes, and surface densities(Sv) of the apical and basolateral cell membrane domains. For the apical membrane surface, the microvillous fraction has been measured separately. Since the stereologic approach used in the present study allows the determination of absolute cell volumes, the absolute measures of organelle volumes (V)and membrane surfaces (S) per average cell can be calculated from volume and surface densities. Although no changes in cell density were found for 6 and 12 day old LLC‐PK1 monolayers, indicating ceased cell proliferation due to contact inhibition, remarkable changes were found concerning the absolute cell volume and apical membrane surface. The observed increase in the apical cell surface was exclusively due to the enlarged microvillous surface fraction. This finding is in good agreement with the increased number of Na+‐dependent hexose transporters as well as with the increased expression of apical membrane marker enzymes observed during the differentiation of LLC‐PK1 monolayers.
Tumor promoters retard the loss of a transient subpopulation of cells in low passage Syrian hamster cell cultures
Authors: Ueo Hiroaki , Nakano Shuji , Paul O. P. Ts'O , Bruce Sarah A. 
Published: February 2005

AbstractEarly passage normal diploid Syrian hamster (SH) fetal cell cultures contain a transient subpopulation of contact‐insensitive (CS−) cells which lack density‐dependent inhibition of cell division. The size of this CS− subpopulation decreases during in vitro passage by conversion of the CS− cells to contact‐sensitive (CS+) cells. Approximately 10—15 population doublings after the frequency of the CS− cells has declined to below 0.001%, mass cultures cease proliferating and exhibit cellular senescence. Cultures with higher initial numbers of CS− cells exhibit longer in vitro proliferative life spans than cultures with smaller initial numbers of CS− cells. Active tumor promoting phorbol esters (12‐O‐tetra‐decanoyl‐phorbol‐13‐acetate [TPA] and phorbol‐12, 13‐didecanoate [PDD]) retard the decline in the proportion of CS− cells during in vitro passage, while the inactive tumor promoting phorbol ester, 4α‐phorbol‐12, 13‐didecanoate (4αPDD) has no effect on the rate of loss of the CS− cells. In addition, continuous treatment from secondary culture with TPA or PDD extends by approximately twofold the in vitro proliferative life span of SH fetal cell cultures. Treatment must, however, begin at passage 1 or 2 when the CS− cells are still present. After the proportion of the CS− cells has decreased to <0.001%, as in passage 6 cultures, promoters have no effect on the life span of the culture. This finding that exposure to promoters results in both a prolonged maintenance of the CS− cellular subpopulation, as well as an extention of in vitro proliferative life span, suggests that the conversion of CS− cells to CS+ cells is involved in the mechanism of in vitro senescence.
Chloride action potentials and currents in embryonic skeletal muscle of the chick
Authors: Steele Joy A. 
Published: February 2005

AbstractChloride‐dependent action potentials were elicited from embryonic skeletal muscle fibers of the chick during the last week of in ovo development. The duration of the action potentials was extremely long (>8 sec). The action potentials were reversibly blocked by the stilbene derivative, SITS, a specific blocker of chloride permeability. Using patch clamp pipettes, in which the intracellular chloride concentration was controlled and with other types of ion channels blocked, the membrane potential at the peak of the action potential closely coincided with the chloride equilibrium potential calculated from the Nernst equation. These data indicate that activation of a chloride‐selective conductance underlies the long duration action potential. The occurrence of the chloride‐dependent action potential was found to increase during embryonic development. The percentage of fibers that displayed the action potential increased from ∼︁20% at embryonic day 13 to ∼︁ 70% at hatching. Chloride‐dependent action potentials were not found in adult fibers. The voltage and time‐dependent currents underlying the action potential were recorded under voltage clamp using the whole‐cell version of the patch pipette technique. The reversal potential of the currents was found to shift with the chloride concentration gradient in a manner predicted by the Nernst equation, and the currents were blocked by SITS. These data indicate that chloride ions were the charge carriers. The conductance was activated by depolarization and exhibited very slow activation and deactivation kinetics.
Differentiation of simian virus 40 transformed human mammary epithelial stem cell lines to myoepithelial‐like cells is associated with increased expression of viral large T antigen
Authors: Rudland Philip S. , Barraclough Roger 
Published: February 2005

AbstractCloned simian virus 40 (SV40)‐transformed human breast epithelial cell lines can differentiate to myoepithelial‐like cells, and these can be isolated as clonal cell lines. Immunofluorescent and immunocytochemical analysis of such cell lines growing on plastic surfaces, collagen gels, and as tumor‐nodules in nude mice indicate that all the cell lines produce SV40 large T antigen, but that the production of this antigen is qualitatively increased in the myoepithelial‐like cells and cell lines. The myoepithelial‐like cell lines produce 4—6 times more immunoprecipitable large T antigen than the parental epithelial cells. The amount of mRNA for large T antigen is also increased by 3.5—5‐fold in the myoepithelial‐like cell lines when analysed by dot‐blot or by Northern hybridisations. Thus, differentiation along the myoepithelial‐like cell pathway is associated in these SV40‐transformed cells with increased expression of the viral large T antigen. It is suggested that immortalization of primary breast epithelial cell cultures may be, in part, due to the expression of large T antigen preventing processes of terminal keratinization.
Extracellular calcium affects the membrane currents of cultured human keratinocytes
Authors: Mauro Theodora M. , Pappone Pamela A. , Isseroff R. Rivkah 
Published: February 2005

AbstractElectrophysiologic properties of cultured human keratinocytes were studied using the patch voltage‐clamp technique. Undifferentiated, proliferative keratinocytes grown in low Ca2+ medium had an average resting membrane potential of −24 mV. Voltage‐clamp experiments showed that these cells had two membrane ionic currents: a large voltage‐independent leak conductance, and a smaller voltage‐dependent Cl− current that activated with depolarization. Increasing the extra‐cellular Ca2+ concentration from 0.15 to 2 mM resulted in a doubling of the magnitude of the voltage‐gated current and a shift in current activation to more negative potentials. Since levels of extracellular Ca2+ can alter the morphology and differentiation state of keratinocytes, the finding of a Ca2+ ‐activated Cl− current in these cells suggests a role for this conductance in the initiation of differentiation.
Bradykinin induces superoxide anion release from human endothelial cells
Authors: Holland James A. , Pritchard Kirkwood A. , Pappolla Miguel A. , Wolin Michael S. , Rogers Nancy J. , Stemerman Michael B. 
Published: February 2005

AbstractThe time‐dependent release of superoxide anion (O) from bradykinin (Bk)‐stimulated human umbilical vein endothelial cells (EC) was measured as the superoxide dismutase‐inhibitable reduction of ferricytochrome C employing a novel application of microspectrophotometry. In the absence of Bk, O release by EC was not detectable. EC exposure to Bk (10−6 to 10−5 M) resulted in a rapid release of O. The release of O occurred within 5 minutes of exposure. O release was partially inhibited by indomethacin (63 ± 6%), thus suggesting that arachidonic acid metabolism, through cyclooxygenase, contributes to EC O production. EC O release may be an important component in the pathophysiologic actions of Bk on vascular function.
Contraction of vascular smooth muscle in cell culture
Authors: Murray Thomas R. , Marshall Bryan E. , Macarak Edward J. 
Published: February 2005

AbstractThe use of cultured vascular smooth muscle cells for the study of events related to excitation and contraction of smooth muscle has been limited by the inability to reliably induce contractile responses after subculturing of the cells. This limitation has been overcome by the cell culture preparation described herein. We demonstrate that appropriate responses to both smooth muscle agonists and vasodilators were preserved in cells that were serially subcultured. Fetal bovine pulmonary artery and aortic cell cultures were established following enzymatic dispersion of the medial portion of freshly harvested vessels. At various times after isolation, cells were transferred to microscope coverslips coated with a polymerized silicone preparation (polydimethyl siloxane). Tension forces generated by the cells were manifested as wrinkles and distortions of this flexible growth surface. Visual evidence of cell contraction in the form of increased wrinkling was documented for cells exposed to angiotensin II, carbachol, and KCl. Decreases in cell tension occurred following treatment with isoproterenol, and those relaxing effects were overcome by subsequent treatment with the agonist carbachol. The contractile responses did not diminish with prolonged maintenance in culture or repeated subculturing. Phosphorylation of the light chains on the contractile protein myosin was also measured as a biochemical index of agonist‐induced contraction. Cells depolarized with KCl or exposed to carbachol showed increased myosin phosphorylation when analyzed by 2‐dimensional gel electrophoresis. The responses remained intact through 7 passages and 9 weeks in culture. These results show that cultured vascular smooth muscle cells do not necessarily undergo a phenotypic modulation with loss of contractility under prolonged maintenance in culture.
Reprogramming of gene expression in postischemic rat liver: Induction of proto‐oncogenes and hsp 70 gene family
Authors: Schiaffonati Luisa , Rappocciolo Emilia , Tacchini Lorenza , Cairo Gaetano , Bernelli‐Zazzera Aldo 
Published: February 2005

AbstractSteady‐state levels of messenger RNA (mRNA) for different members of the heatshock protein 70 gene family were studied in rat livers reperfused after nonnecrogenic ischemia. The expression of constitutive hsc 73 gene decreases during ischemia, returns to normal upon reperfusion, and increases 4 hr after restoration of blood flow. Reperfusion induces the expression of another hsp 70 gene family member (the so‐called inducible hsp 70 gene), which remains at high levels for at least 7 hr. The induction of hsp 70 family genes is preceded by activation of the cellular oncogene c‐fos, the most prompt change in gene expression detected in reperfused liver. Run‐on experiments demonstrate that the increased expression of these genes is largely dependent on activation of transcription. Changes in the amount of c‐myc and ornithine decarboxylase mRNA are not evident, while the level of the mRNA for glucose‐regulated protein GRP 78 increases later, concurrent with the onset of the acute phase response to surgical trauma. Analysis of polysomal and nonpolysomal fractions from sucrose gradients indicates that in postischemic liver, hsp 70 and hsc 73 mRNA are rapidly engaged on light polysomal or nonpolysomal complexes and are later shifted to polysomes. Albumin mRNA displays the same behavior, indicating that hsp 70 mRNA are not preferentially translated and that increased transcription is the major mechanism for enhanced hsp synthesis in postischemic liver. Damage by active oxygen species, pressure overload, and derangements of protein synthesis is likely to include the causative factors of increased expression of c‐fos and the hsp 70 gene family in postischemic reperfused liver.