bluuebunny/crossref_metadata_2025 huggingface dataset:
Decreased levels of alpha 1(I) procollagen mRNA in dermal fibroblasts grown on fibrin gels and in response to fibrinopeptide B
Authors: Pardes Jeffrey B. , Takagi Hajime , Martin Theresa A. , Ochoa M. Sofia , Falanga Vincent
Published: February 2005

We have investigated human neonatal fibroblast synthetic activity in response to fibrin substrates and components of fibrin formation and degradation. Greater than threefold downregulation of procollagen mRNA levels was seen 24 hours after fibroblasts were grown on fibrin gels as compared to tissue culture plastic. This downregulation occurred in both reptilase‐generated fibrin (retention of fibrinopeptide B) and thrombin‐generated fibrin (loss of both fibrinopeptide A and B). However, fibroblasts grown on fibrin retained their capacity to respond to the stimulatory action of transforming growth factor (TGF)‐beta 1. Fibroblasts seeded on reptilase‐generated fibrin displayed an abnormal morphology manifested by dendritic appearance and cell rounding, while fibroblast attachment was enhanced by 30% on thrombin‐generated fibrin substrate (P < 0.02). Fibrinopeptides A and B, which are generated during fibrin formation, increased and decreased procollagen mRNA levels, respectively. Tissue plasminogen activator (t‐PA) increased procollagen mRNA and TGF‐beta 1 levels as early as 6 hours after cells were grown on tissue culture plastic, but this stimulation did not occur in cells cultured on a fibrin substrate. We conclude that alpha 1(I) procollagen mRNA levels in cultures of human dermal fibroblasts are consistently down‐regulated by a fibrin substrate and are directly and profoundly influenced by complex interactions between components involved in the formation and removal of fibrin. © 1995 Wiley‐Liss, Inc.
Epidermal growth factor rapidly impairs activation of p34cdc2 protein kinase in HeLa cells at the G2‐M boundary
Authors: Barth Holger , Kinzel Volker
Published: February 2005

Epidermal growth factor (EGF) has been shown rapidly to inhibit the transition from G2 phase to mitosis; beyond this transition point the cells are refractory to EGF (Kinzel et al., 1990, Cancer Res., 50:7932–7936). Using synchronized HeLa cells, EGF has now been shown to induce an overall decrease of the histone H1 kinase activity of p34cdc2 after 20 min of treatment, a time course which correlates with the number of cells in metaphase. The kinase level of actively mitotic cells is not altered by EGF. Neither the amount of p34cdc2 protein present nor that of Cyclin B is influenced by EGF, and the formation of the p34cdc2/Cyclin B complex is also unaffected. The use of antiphosphotyrosine antibodies, however, showed that p34cdc2 from cultures treated with EGF was more intensely stained than that of control cells, indicating that EGF treatment prevents the tyrosine dephosphorylation which is required for expression of the protein kinase activity of the complex. Taken together, the results show that EGF in HeLa cells very rapidly prevents the p34cdc2/Cyclin B complex from expressing kinase activity at the G2‐M boundary, which appears to be the cause for the inhibition in G2 phase. © 1995 Wiley‐Liss, Inc.
Effects of mutations in cAMP‐dependent protein kinase on chloride efflux in Caco‐2 human colonic carcinoma cells
Authors: Krolczyk Arkadiusz J. , Bear Christine E. , Lai Patrick F. H. , Schimmer Bernard P.
Published: February 2005

In order to evaluate the importance of cAMP and cAMP‐dependent protein kinase (cAMPdPK) in the regulation of chloride efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, Caco‐2, human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of regulator subunit of cAMPdPK under control of the mouse metallothionein 1 promoter. Four stable transformants were isolated that expressed the mutant subunit in a Zn2+‐inducible manner and exhibited Zn2+‐inducible inhibition of cAMPdPK activity. The parental and transformed Caco‐2 cells were examined for their abilities to regulate chloride efflux in response to various secretagogues using a radioactive iodide‐efflux assay. In the transformants, induction of the protein kinase mutation with ZnSo4 markedly decreased chloride efflux in response to forskolin, the 8‐(4‐chlorophenylthio) analog of cAMP, vasoactive intestial polypeptide, prostaglandin E2 and isoproterenol, whereas Zn2+‐treated parental cells remained responsive to these secretagogues. Treatment with carbachol, calcium ionophores or phorbol ester did not acutely affect chloride efflux. Together, these studies indicate that cAMP and cAMPdPK are essential components of secretagogue‐regulated chloride channel activity in the Caco‐2 cell line. In whole cell patch clamp recordings, induction of the cAMPdPK mutation inhibited anionic conductances indicative of the CFTR chloride channel, whereas purified catalytic subunit of cAMPdPK, added intracellularly, reversed the inhibition. These latter results demonstrate that the CFTR chloride channels in the protein kinase‐defective transformants are normal and that the protein kinase mutation specifically affects their regulation, presumably by direct phosphorylation. © 1995 Wiley‐Liss, Inc.
Analysis of EPC‐1 growth state–dependent expression, specificity, and conservation of related sequences
Authors: Pignolo Robert J. , Rotenberg Mitch O. , Cristofalo Vincent J.
Published: February 2005

The transcipt for EPC‐1 (early population doubling level (PDL) cDNA‐1) is induced under conditions of growth arrest due to density‐dependent contact inhibition and/or serum deprivation in early‐passage but not in senescent WI‐38 fibroblasts. We have characterized the EPC‐1 transcript with respect to its cell‐cycle regulation, tissue specificity, and interspecies conservation of related genomic sequences. In low density, quiescent (serum‐deprived), early‐passage fibroblasts that are stimulated to proliferate with fresh serum, steady‐state EPC‐1 transcript levels are steadily reduced until they reach a basal level approximately 24 h after stimulation. However, when early‐passage fibroblasts are made quiescent by both serum deprivation and density‐dependent contact inhibition and then stimulated with serum, steady‐state EPC‐1 transcript levels remain relatively constant throughout a 36 h period following serum stimulation. Senescent WI‐38 cells (>95% life span completed) do not express EPC‐1 under these conditions. We show that differences in the regulation of EPC‐1 transcript levels in early‐passage cells are due to differences in growth state rather than changes in cell densityor contact. We also show that expression of the EPC‐1 transcript is limited to specific cell types and that related genomic sequences are found in all mammalian species examined as well as in the chicken. © 1995 Wiley‐Liss, Inc.
Mevalonate dependency of the early cell cycle mitogenic response to epidermal growth factor and prostaglandin F2α in Swiss mouse 3T3 cells
Authors: Ortiz Marcela B. , Goin Mercedes , de Alzaga Maria B. Gomez , Hammarstrom Swen , de Asua Luis Jimenez
Published: February 2005

Lovastatin (LOV), a hydroxy‐methylglutaryl‐coenzyme A (HMGCoA) reductase competitive inhibitor, blocks epidermal growth factor (EGF)— or prostaglandin F2α (PGF2α)—induced mitogenesis in confluent resting Swiss 3T3 cells. This inhibition occurs even in the presence of insulin, which potentiates the action of these mitogens in such cells. LOV exerts its effect in a 2–80 μM concentration range, with both mitogens attaining 50% inhibition at 7.5 μM. LOV exerted its effect within 0–8 h following mitogenic induction. Mevanolactone (10–80 μM) in the presence of LOV could reverse LOV inhibition within a similar time period. LOV‐induced blockage of PGF2α response is reflected in a decrease in the rate of cell entry into S phase. Neither cholesterol, ubiquinone, nor dolichols of various lengths could revert LOV blockage. In EGF‐ or PGF2α‐stimulated cells, LOV did not inhibit [3H]leucine or [3H]mannose incorporation into proteins, while tunicamycin, an inhibitor of N′ glycosylation, prevented this last phenomenon. Thus, it appears that LOV exerts its action neither by inhibiting unspecific protein synthesis nor by impairing the N′ glycosylation process. These findings strongly suggest that either EGF or PGF2α stimulations generate early cell cycle signals which induce mevalonate formation, N′ glycoprotein synthesis, and proliferation. The causal relationship of these events to various mechanisms controlling the onset of DNA synthesis is also discussed. © 1995 Wiley‐Liss, Inc.
Expression of tissue‐type plasminogen activator and its inhibitor couples with development of capillary network by human microvascular endothelial cells on matrigel
Authors: Ito Ken‐Ichi , Ryuto Masahiro , Ushiro Shin , Ono Mayumi , Sugenoya Akira , Kuraoka Akio , Shibata Yosaburo , Kuwano Michihiko
Published: February 2005

Human omental microvascular endothelial (HOME) cells seeded on Matrigel begin to migrate within 1 h, forming honeycomb‐like structures and capillary‐like networks within 18 h. Cross‐sections of the capillary networks show them to be tube‐like structures. Northern blot analysis showed that tissue‐type plasminogen activator (t‐PA) mRNA synthesis increased from the initial state at 0 h after seeding on Matrigel, reaching a steady state after 4 h. This elevated cellular t‐PA mRNA level decreased markedly at 24 h. In contrast, the cellular plasminogen activator inhibitor‐1 (PAI‐1) mRNA level demonstrated biphasic curves during the 24 h after seeding on Matrigel: the PAI‐1 mRNA level was increased eightfold initially at 4 h over that at O h, then declined, and again secondarily increased to greater than tenfold at 18 h. Cellular levels of both 72 kD type IV collagenase and tissue inhibitor of metalloproteinase (TIMP‐2) mRNA were increased only a slightly within 2–4 h. These elevated mRNA levels were maintained for 18 h, while the TIMP‐1 mRNA level increased up to 18 h, reaching around three times the level at O h. However, on collagen‐coated dishes, cellular levels of t‐PA, PAI‐1, 72 kD type IV collagenase, TIMP‐1, and TIMP‐2 mRNA were not greatly changed during incubation for 24 h. On Matrigel, the cellular t‐PA mRNA level at 18 h after seeding was greatly increased when treated with specific anti‐transforming growth factor‐β (TGF‐β) antibody. In contrast, both PAI‐1 and TIMP‐1 mRNA levels at 18 h were reduced in the presence of anti‐TGF‐β antibody. Development of the capillary network on Matrigel was inhibited in the presence of anti‐t‐PA antibody. Epidermal growth factor (EGF) enhanced t‐PA gene expression and TGF‐β inhibited its expression in HOME cells cultured on collagen‐coated dishes. On the other hand, TGF‐β enhanced cellular expression of the PAI‐1 gene. The formation of a capillary network by HOME cells on Matrigel appears to be balanced by angiogenic EGF and anti‐angiogenic TGF‐β through modulation of PA activity. © 1995 Wiley‐Liss, Inc.
Activation of a transfected FGFR‐1 receptor in Madin‐Darby epithelial cells results in a reversible loss of epithelial properties
Authors: Migdal Michal , Soker Shay , Yarden Yosef , Neufeld Gera
Published: February 2005

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types derived from mesoderm and neuroectoderm. The activity of bFGF is mediated by several types of closely related receptors belonging to the tyrosine‐kinase family of receptors. We have found that Madin‐Darby epithelial cells (MDCK) do not seem to produce bFGF or bFGF receptors. High level expression of human bFGF cDNA in these cells did not produce any mitogenic or morphological effects. Expression of the mouse‐derived cDNA encoding FGF receptor‐1 (FGFR‐1) in MDCK cells resulted in the acquisition of a fibroblast‐like morphology when the transfected cells were cultured at low density in the presence of 0.6% fetal calf serum and 20 ng/ml bFGF. Acidic fibroblast growth factor (aFGF) also induced these morphological changes but not keratinocyte growth factor. The morphological effect was not accompanied by increased bFGF‐induced cell proliferation and did not result in the loss of epithelial cell markers such as cytokeratins. However, the morphological transition was accompanied by changes in the intracellular distribution of actin. In spite of these changes the transfected cells formed monolayers even in the presence of bFGF. Coexpression of bFGF and FGFR‐1 in the MDCK cells resulted in similar morphological effects that were not dependent upon exogenous bFGF. These morphological effects were mimicked by exposure of MDCK cells to either orthovanadate or phorbol ester. Parental and FGFR‐1 ‐expressing MDCK cells formed monolayers tht displayed high electrical resistance. Incubation of monolayers of FGFR‐1‐transfected cells with bFGF resulted in the loss of trans‐epithelial resitance. Monolayers of parental MDCK cells did not lose their trans‐epithelial resistance in response to bFGF, although exposure to phorbol ester did result in the loss of their trans‐epithelial resistance, indicating that the effects on the trans‐epithelial resistance are mediated by protein kinase C activation. Interestingly, orthovanadate did not cause a loss of transepithelial resistance, suggesting that the loss of trans‐epithelial resistance is separable from the morphological transition. © 1995 Wiley‐Liss, Inc.
Receptor binding of PDGF‐AA and PDGF‐BB, and the modulation of PDGF receptors by TGF‐β, in human periodontal ligament cells
Authors: Oates T. W. , Kose K. N. , Xie J. F. , Graves D. T. , Collins J. M. , Cochran D. L.
Published: February 2005

The growth factors PDGF‐AA and PDGF‐BB have previously been shown to be potent mitogens for human periodontal ligament (hPDL) cells in vitro. Additionally, the mitogenic response to PDGF‐AA has been shown to be specifically inhibited by TGF‐β. The purpose of the present investigation was to examine the binding of PDGF‐AA and PDGF‐BB, and the modulation of PDGF binding by TGF‐β, in hPDL cells. Scatchard analysis identified an average of 32,000 PDGF‐AA high‐affinity binding sites per cell with a dissociation constant (Kd) of 0.66 nM and an average of 36,000 PDGF‐BB binding sites per cell with a dissociation constant (kd) of 0.44 nM. After treatment with TGF‐β, the receptor number for PDGF‐AA was found to specifically decrease by approximately 50%, with no change in binding affinity. This reduced number of binding sites was shown to correlate with both a decrease in levels of receptor tyrosine phosphorylation and a decreased number of α receptor subunits. Northern blot analysis identified the TGF‐β‐mediated decrease in PDGF α receptor subunit mRNA levels. PDGF‐BB showed little change in the number of binding sites or in the binding affinity with TGF‐β treatment, and the data were consistent with an increase in the number of β receptor subunits. These results demonstrate nearly equivalent numbers of receptors for both PDGF‐AA and PDGF‐BB in hPDL cells. Also, modulation of PDGF binding, by TFG‐β, was shown to result in a reduced number of α receptor subunits with an increase in the number of β receptor subunits. © 1995 Wiley‐Liss, Inc.
Zinc inhibits turnover of labile mRNAs in intact cells
Authors: Taylor Gregory A. , Blackshear Perry J.
Published: February 2005

For immediate early genes such as the c‐fos proto‐oncogene, mRNA breakdown is very rapid and is largely responsible for the transient nature of mRNA accumulation after transcription is stimulated. We found that in several types of cultured cells and in mice, Zn++ caused marked accumulation of c‐fos mRNA and that of another labile mRNA, that encoding the tristetraprolin (TTP) protein. Exposure of TK‐L cells to 100 μM ZnSO4 caused an increase of c‐fos and TTP mRNA levels within 1 h that reached peak levels in 4–8 h and remained constant to 12 h. Increases in fos protein accumulation were also noted. When the cells were exposed to Zn++ for 4 h and then exposed to actinomycin D, both c‐fos and TTP mRNA levels remained constant for up to 10 h, indicating that Zn++ was preventing the breakdown of both c‐fos and TTP mRNA. Also, 100 μM ZnSO4 inhibited protein synthesis in TK‐L cells, suggesting that the effect on mRNA accumulation © 1995 Wiley‐Liss, Inc.
Cell density—dependent regulation of PLCγ1 tyrosine phosphorylation and catalytic activity in an intestinal cell line (IEC‐6)
Authors: Polk D. Brent , McCollum Gary W. , Carpenter Graham
Published: February 2005

Administration of epidermal growth factor (EGF) to rats has been shown to induce both mitogenic and nonmitogenic responses in the intestine. The mechanisms to describe a multiplicity of hormonal responses within a single tissue are unclear but likely involve selectivity among receptor substrates. A nontransformed rat jejunal crypt intestinal epithelial cell line (IEC‐6) was studied to determine if the regulation of receptor tyrosine kinase substrates is affected by cell population physiology. EGF stimulated a rapid increase in inositol trisphosphate in confluent but not subconfluent cells. Similarly, treatment of confluent IEC‐6 cells with EGF provoked a significant increase in the hydrolysis of Ptdlns 4,5‐P2 by immunoisolated PLCγ1. The tyrosine phosphorlation state of PLCγ1 and the association of PLCγ1 with the EGF receptor were increased by EGF in confluent cells only. In contrast, the autophosphorylation state of the EGF receptor and the tyrosine phosphorylation state of another SH2‐containing EGF receptor substrate SHC were increased by EGF regardless of cell density. Western blot analysis revealed equal protein expression of PLCγ1 in confluent and subconfluent cells. EGF receptor protein expression and ligand binding capacity were slightly increased in confluent compared to subconfluent cells. EGF regulation of PLCγ1, therefore, is regulated by physiological factors dependent on cell density in IEC‐6 cells. © 1995 Wiley‐Liss, Inc.
Down‐regulation of statin, a nonproliferation‐specific nuclear protein, and up‐regulation of c‐myc after initiation of programmed cell death in mouse fibroblasts
Authors: Wang Eugenia , Pandey Siyaram
Published: February 2005

Deprivation of growth factors has been shown to induce programmed cell death in many cell types, including mouse 3T3 fibroblasts. Programmed cell death (apoptosis) is an active process of self‐destruction which is thought to require the expression of unique genes. Recently, the expression of cell cycle genes such as c‐fos and c‐myc, and re‐entrance to cell cycle traverse, are thought to be necessary to induce programmed cell death. Previous work in this laboratory has shown that statin is a nonproliferation‐specific nuclear protein present in the nuclei of young quiescent or senescent human fibroblasts, as well as in growth‐arrested mouse 3T3 fibroblasts; we have reported that statin disappears rapidly after the blockage of growth arrest is removed and cells are allowed to resume cell cycle traverse. In this report we address the question of whether cells induced to enter the programmed cell death process also lose the expression of statin. We studied density‐arrested quiescent mouse 3T3 cells, which undergo rapid cell death by apoptosis upon serum deprivation. Our results suggest that c‐myc expression is induced, as previously reported in other systems of apoptotic death. Interestingly, we also find that statin indeed disappears after the induction of programmed cell death is initiated. These results further support the notion that when apoptosis is induced, cells behave as though released from replication arrest, and experience some part of the G1 phase of the cell cycle. The difference between this event and normal cell cycle traverse is that this experience of the G1 phase in the apoptotic process is an abortive one, with the end result of cell demise. © 1995 Wiley‐Liss, Inc.
Adrenocorticotropin‐resistant mutants of the Y1 adrenal cell line fail to express the adrenocorticotropin receptor
Authors: Schimmer Bernard P. , Kwan Wai King , Tsao Jennivine , Qiu Rong
Published: February 2005

This report examines the basis for adrenocorticotropin (ACTH) resistance in two mutant clones (Y6 and OS3) derived from the ACTH‐responsive Y1 mouse adrenocortical tumor cell line. These two mutants were originally characterized by their failure to respond to ACTH with increased adenylyl cyclase activity and as a consequence were resistant to the steroidogenic effects of the hormone. We now demonstrate that ACTH resistance in the Y6 and OS3 mutants results from the failure to express the gene encoding the ACTH receptor. Whereas parental Y1 cells express ACTH receptor transcripts at low levels and are stimulated by ACTH or 8‐bromo‐cAMP to increase the accumulation of ACTH receptor transcripts approximately twofold, the Y6 and OS3 mutants do not express receptor transcripts either in the presence or absence of 8‐bromo‐cAMP. The gene encoding the ACTH receptor appears to be present in the Y6 and OS3 mutants, as determined by Southern blot hybridization analysis. Moreover, in the Y6 mutant the ACTH receptor gene appears to be silenced by a modification that is reversed following the growth of the cells as tumors in mice. Clonal isolates of Y6 cells grown as tumors recover the ability to express ACTH receptor transcripts at low but detectable levels and acquire the ability to respond to ACTH with increased adenylyl cyclase activity. Finally, Y6 and OS3 cells transformed with a gene encoding the mouse β2‐adrenergic receptor respond to the β‐adrenergic agonist, isoproterenol, in a manner that is indistinguishable from the similarly transformed parent Y1 cell line. These latter results demonstrate the functional integrity of the adenylyl cyclase system in the ACTH‐resistant mutants and indicate that the failure to express ACTH receptor transcripts limits the responsiveness of these clones. © 1995 Wiley‐Liss, Inc.
Multiple second messenger pathways regulate IL‐β‐induced expression of PGHS‐2 mRNA in normal human skin fibroblasts
Authors: Warnock Lorna Jane , Hunninghake Gary W.
Published: February 2005

Very little is known about the specific regulation of PGHS‐2 mRNA compared with PGHS‐1 mRNA. Using normal human fibroblasts, we show that at baseline there is constitutive expression of PGHS‐1 mRNA and barely detectable amounts of PGHS‐2 mRNA. There was a marked increase in PGHS‐2 mRNA transcription following exposure to IL‐β. Maximal expression of PGHS‐2 mRNA occurred with concentrations of IL‐β ≥ 1 ng/ml at 3 hours after stimulation. Downregulation of protein kinase C (PKC) activity by pretreating fibroblast cultures with PMA inhibited IL‐1–induced PGHS‐2 mRNA expression without affecting the constitutive expression of PGHS‐1 mRNA. The addition of various PKC inhibitors also blocked the IL‐β induction of PGHS‐2 mRNA but did not alter PGHS‐1 mRNA expression; inhibitors of protein kinase A (PKA) or tyrosine kinase (TK) had only a limited effect on IL‐β‐induced PGHS‐2 mRNA expression. These findings show that IL‐β increases PGHS‐2 mRNA, at least in part, via activation of PKC. Activation of PKA or TK appears to have a more limited role in this process. © 1995 Wiley‐Liss, Inc.
Shear stress modulates endothelial cell morphology and F‐actin organization through the regulation of focal adhesion‐associated proteins
Authors: Girard Peggy R. , Nerem Robert M.
Published: February 2005

Flow‐related shear stress has been shown to modulate endothelial cell structure and function including F‐actin microfilament organization. Focal adhesion‐associated proteins such as vinculin, talin, and specific integrins may play a role in the modulation of these cytoskeletal and morphological changes. Double‐label immunofluorescence studies indicated that, in static culture, α5β1 fibronectin receptors (α5β1 FNRs) and αvβ3 vitronectin receptors (αvβ3 VNRs) were found predominantly in the peripheral regions of bovine aortic endothelial cells (BAECs) corresponding to the localization of vinculin, talin, and actin microfilament terminations. In response to shear stress, concomitant with cell elongation and the appearance of stress fibers aligned with the direction of flow, there was a prominent localization of vinculin and αvβ3 VNRs as the “upstream” end of the cells. Stress fiber terminations were clearly evident at these concentrations of focal adhesion‐associated proteins. These data suggest that the upstream concentration of these proteins may direct shear stress‐induced stress fiber formation and may function in the alignment of the fibers in the direction of flow. Levels of surface αvβ3 VNRs were found to decrease in response to flow, possibly reflecting the decrease in numbers of “downstream” receptors. Unlike the arrangement of vinculin and αvβ3 VNRs observed following exposure to flow, talin and α5β1 FNRs, in addition to being localized at the upstream end of the cell, were also evenly distributed throughout the rest of the cell. Surface levels of α5β1 FNRs increased in response to shear stress, perhaps providing an increased adherence of BAECs to the extracellular matrix through these receptors. These data suggest that focal adhesion‐associated proteins play specific roles in the response of BAECs to shear stress. © 1995 Wiley‐Liss, Inc.
Thermotolerant cells possess an enhanced capacity to repair heat‐induced alterations to centrosome structure and function
Authors: Vidair Charles A. , Doxsey Stephen J. , Dewey William C.
Published: February 2005

To study the mechanism of thermotolerance, the adaptive response by which cells become transiently resistant to killing by heat shock, we have focused on the centrosome, an organelle whose disorganization is closely correlated with thermal killing in Chinese hamster ovary (CHO) cells. Centrosome structure was studied by use of antisera directed against pericentrin, a 220 Kd protein of the pericentriolar material (PCM). Centrosome function was measured in intact cells by performing microtubule regrowth following exposure to the drug nocodazole. Immediately following heating at 45°C for 4–18 min, centrosomal staining by antipericentrin decreased. Thereafter, staining gradually recovered, although abnormal configurations of staining appeared in heated cultures 10–20 h later. In contrast, abnormal patterns of staining rarely developed in thermotolerant cultures. Centriole number was not perturbed by heat, indicating that the heat effect was specific for the PCM. Heat also caused an immediate reduction in the number of microtubules nucleated by the PCM. As for staining by antipericentrin, microtubule nucleation recovered during 3–20 h at 37°C after heating. The immediate, heat‐induced decrease in antipericentrin staining or microtubule nucleation was similar in thermotolerant and nontolerant cells. In contrast, the inhibition for both endpoints recovered to control levels much more quickly in thermotolerant cells than in nontolerant cells. Furthermore, new protein synthesis was not required for the recovery of microtubule nucleation. These data show that thermotolerant cells have an enhanced capacity to repair thermal damage to centrosome structure and function, and suggest that a faster rate of recovery prevents disorganization of the PCM that is observed in nontolerant cells several hours after heating. © 1995 Wiley‐Liss, Inc.
Histone H4 proximal promoter mediates a complex transcriptional response during differentiation of 3T3L1 adipocytes
Authors: Ramsey‐Ewing Anna L. , Bortell Rita , Stein Gary S. , Stein Janet L.
Published: February 2005

We have investigated the promoter element(s) required by the cell cycle regulated FO108 human histone H4 gene for control of gene expression during adipocyte proliferation and differentiation. Stable 3T3L1 cell lines were established that express fusion genes in which the histone H4 promoter is joined to chloramphenicol acetyltransferase (cat) as a reporter gene. Expression of the H4CAT fusion genes was monitored in proliferating and confluent 3T3L1 preadipocytes and in differentiating 3T3L1 adipocytes. The results indicate that the H4 cell cycle element (CCE), which mediates S phase‐specific stimulation of H4 gene transcription, is not required for transcriptional regulation during differentiation. Instead, a minimal H4 promoter (nucleotides −46 to −11) is sufficient to mediate the complex transcriptional response of H4 gene expression observed during the process of adipocyte differentiation of 3T3L1 cells. In addition, the data suggest that down‐regulation of histone gene expression during cellular differentiation may be mediated by passive inactivation of the promoter due to loss of positive regulatory factor(s). © 1995 Wiley‐Liss, Inc.
Identification of insulin‐like growth factor binding proteins from cultured human epidermal keratinocytes
Authors: Murashita Mari M. , Russo Vincenzo C. , Edmondson Stephanie R. , Wraight Christopher J. , Werther George A.
Published: February 2005

The role and mechanisms of action of insulin‐like growth factors (IGFs) in skin remain unclear. Epidermal keratinocytes possess IGF‐I receptors and are responsive to IGF‐I, which is primarily derived from underlying dermal fibroblasts. IGF binding proteins (IGFBPs), also synthesized by fibroblasts, may be involved in paracrine targeting of IGF‐I to its receptors. We therefore examined whether human keratinocytes synthesize IGFBPs and their mRNAs. Following culture in complete medium (containing bovine pituitary extract and epidermal growth factor) Western ligand blotting (WLB) of cell conditioned medium revealed a major band of 32 kD, a less abundant IGFBP of 24 kD at all passages, and a 37–42 kD IGFBP which increased in abundance in late passage. Immunoprecipitation followed by WLB confirmed that the predominant 32 kD band was IGFBP‐2. Radioimmunoassay of IGFBP‐1, ‐3, and ‐6 revealed detectable levels of IGFBP‐3 and significant levels of IGFBP‐6, but not IGFBP‐1. Northern analysis following culture in complete medium revealed that at early passage IGFBP‐1, ‐2, ‐4, and ‐6 mRNAs were detectable. IGFBP‐3 and ‐5 mRNAs were not detectable. Following culture in growth factor‐free medium a 37–42 kD band, consistent with IGFBP‐3, was predominant and a 24 kD band consistent with IGFBP‐4 was also present. These data demonstrate the expression of a distinct pattern of IGFBPs by cultured human keratinocytes dependent on culture conditions. Keratinocyte‐derived IGFBPs are likely to play a role in the transport and targeting of IGF‐I from dermally derived fibroblasts to the epidermis. © 1995 Wiley‐Liss, Inc.
Ascorbic acid stimulates barrier function of cultured endothelial cell monolayer
Authors: Utoguchi Naoki , Ikeda Kenji , Saeki Kazuhiko , Oka Naomi , Mizuguchi Hiroyuki , Kubo Kazuyosi , Nakagawa Shinsaku , Mayumi Tadanori
Published: February 2005

The macromolecular permeability of cultured bovine aortic, bovine venous, and human umbilical vein endothelial cell monolayers was decreased significantly in culture medium containing L‐ascorbic acid (Asc Acid; 0.01–0.1 mM) and L‐ascorbic acid 2‐phosphate (Asc 2‐P). Dithiothreitol, which shows reducing activity equivalent to that of Asc Acid, did not affect endothelial permeability. Asc Acid induced a sixfold increase in collagen synthesis by the endothelial cells. The coexistence of L‐azetidine 2‐carboxylic acid, an inhibitor of collagen synthesis, attenuated the effect of Asc 2‐P in a dose‐dependent manner. Another collagen synthesis inhibitor, ethyl‐3,4‐dihydroxybenzoate, also inhibited collagen synthesis and increased endothelial permeability. The decrease in permeability of the endothelial monolayer was dependent on a reduction of the permeability coefficient of the endothelial monolayer. These findings indicate that endothelial barrier function is stimulated by Asc Acid via an increase in collagen synthesis. © 1995 Wiley‐Liss, Inc.
Matrix protein synthesis by glomerular mesangial cells in culture: Effects of transforming growth factor β (TGFβ) and platelet‐derived growth factor (PDGF) on fibronectin and collagen type IV mRNA
Authors: Hänsch Gertrud Maria , Wagner Christof , Bürger Antje , Dong Wenjie , Staehler G. , Stoeck Michael
Published: February 2005

The pathogenesis of glomerular scarring is multifactional; recent evidence suggests that transforming growth factor β (TGFβ), a pleiotropic cicatricial mediator, may promote mesangial sclerosis by enhancing the production of extracellular matrix proteins. We studied the effect of TGFβ1 and TFGβ2 on collagen type IV and fibronectin (FN) synthesis in human glomerular mesangial cells in culture (GMC). Two hours after addition of TGFβ, an up to twofold increase in abundance of collagen type IV mRNA was found, which further increased up to fivefold within 24 h. Addition of cycloheximide did not inhibit the TGFβ effect, but caused by itself an up to twofold increase in the abundance of collagen type IV mRNA after 2 h. Together with collagen mRNA, the mRNA for FN and for platelet‐derived growth factor (PDGF) was also enhanced. PDGF was found to enhance abundance of the collagen type IV and fibronectin mRNA in GMC. A neutralizing antibody to PDGF or a PDGF‐antisense oligonucleotide partly inhibited the TGFβ‐induced increase of collagen type IV mRNA, suggesting that TGFβ can affect the collagen type IV synthesis not only directly but also indirectly via the synthesis of PDGF. © 1995 Wiley‐Liss, Inc.
Effects of the overexpression of the small heat shock protein, HSP27, on the sensitivity of human fibroblast cells exposed to oxidative stress
Authors: Arata Satoru , Hamaguchi Shino , Nose Kiyoshi
Published: February 2005

The role of the human small heat shock protein (HSP27) in oxidative stress was examined using stable transformants of an immortalized human fibroblast cell line (KMST‐6) isolated by transfection of HSP27 expression vectors. Several stable transformants that expressed high or low levels of HSP27 protein were obtained. Clones expressing high levels of HSP27 were more sensitive to growth inhibition by a low dose of hydrogen peroxide (0.1 mM) than those expressing low levels. Clones expressing high levels of HSP27 did not acquire obvious resistance to hyperthermy and cytotoxic agents, except for one (#13), in which resistance to cytotoxic agents was increased. The level of phosphorylated HSP27 in clones expressing high levels of this protein increased at 30 min and was sustained even 4 hours after exposing the cells to 0.1 mM of hydrogen peroxide. On the other hand, the levels in clones expressing low levels of HSP27 were reduced within 4 hours after exposure to hydrogen peroxide. Furthermore, overexpression of nonphosphorylatable mutant HSP27 did not affect sensitivity to oxidative stress. These results suggested that constitutively high expression of HSP27 in KMST‐6 cells make them susceptible to oxidative stress resulting in growth arrest, and this mechanism could involve the phosphorylation of HSP27. © 1995 Wiley‐Liss, Inc.
Immunochemical characterisation of megakaryocyte potentiator activity from mouse bone marrow
Authors: Banu Naheed , Williams Neil
Published: February 2005

Megakaryocyte potentiator derived from mouse bone marrow has been shown to be immunologically similar to Interleukin‐6 (IL‐6). In this study the activity has been characterised by biochemical and immunochemical techniques. The activity is described as a O‐linked glycosylated molecule with an apparent MW of 15 Kd and pl of the range pH 5.9–6.35. The data show that mouse bone marrow potentiator activity is a variant of IL‐6 and with the potential to enhance megakaryocyte growth. © 1995 Wiley‐Liss, Inc.
Growth of mouse hepatocytes is stimulated by gastrin
Authors: Yao Chong Zheng , Bold Richard J. , Ishizuka Jin , Townsend Courtney M. , Thompson James C.
Published: February 2005

Hepatocyte growth is regulated by various growth factors, including epidermal growth factor (EGF) and insulin. Recently, several additional peptide hormones have been shown to stimulate growth of hepatocyte only in the presence of EGF or insulin and are thus termed secondary mitogens. Gastrin regulates growth of normal and neoplastic gastrointestinal tissues, but the effect on growth of hepatocyte is unknown. We examined the effect of gastrin on growth of a normal mouse hepatocyte (NMH) line established in our laboratory. Effect of gastrin‐17 (G‐17) (10−8 to 10−6 M) on growth of NMH cells was examined in either the presence or absence of EGF in the culture medium. Growth of NMH cells was evaluated by incorporation of either bromodeoxyuridine (BrdU) or 3H‐thymidine and by counting cells. Presence of a cell‐surface receptor for G‐17 was determined by Scatchard analysis using 125I‐G‐17. In the presence of EGF, gastrin stimulated growth of NMH cells; in the absence of EGF, gastrin did not affect growth. The stimulatory effect of gastrin on NMH cells was blocked by JMV 320, a CCK‐B type receptor antagonist. NMH cells possess a single, high affinity binding site for gastrin (Kd = 1.2 nM); EGF increased the gastrin binding capacity compared to non‐treated cells (3.5 ± 0.4 vs. 2.2 ± 0.6 fmol/106 cells). G‐17 stimulated growth of NMH cells through a single high affinity receptor for G‐17 which pharmcologically appears to be the CCK‐B type only in the presence of EGF and thus can be considered a secondary mitogen. © 1995 Wiley‐Liss, Inc.
Isolation of nonsedimentable lipid‐protein particles from insect intestine
Authors: Desantis T. A. , Paliyath G. , Thompson J. E. , Downer R. G. H.
Published: February 2005

Nonsedimentable lipid‐protein particles have been isolated from intestinal tissue of the American cockroach, Periplaneta americana. Most of the particles were within the range 30–50 nm in diameter and appear to originate from larger structures. Lipid analysis of the particles showed them to be enriched in neutral lipid components relative to microsomal membranes. Specifically, there is a decline in the amounts of phosphatidylcholine and phosphatidylethanolamine in the nonsedimentable particles compared with the microsomal membranes. Also, in contrast to microsomal membranes, the particles have a higher content of phosphatidic acid along with 1,2‐ and 1,3‐diacyglycerols, free fatty acids and an unidentified lipid that co‐migrates with sterol ester, wax ester and hydrocarbon standards in thin layer chromatograms. The cytosol, separated from the particles by ultrafiltration, contained phosphatidic acid, free fatty acids and the unidentified lipid. By contrast, the composition of neutral lipids in the cytosol resembles that of the particles. SDS—PAGE analysis of microsomal membranes, the particles and particle free cytosol shows an enrichment of low molecular weight proteins in the particles and cytosol. The particles and cytosol appear to possess proteolytic activity that is distinguishable from that of corresponding microsomal membranes since the incubation of these components with BSA resulted in the formation of distinct polypeptides. Many characteristics of these particles resemble those of the deteriosomes that have been isolated from plant tissue. © 1995 Wiley‐Liss, Inc.
Cyclic‐AMP inhibits cell growth and negatively interacts with platelet membrane glycoprotein expression on the Dami human megakaryoblastic cell line
Authors: Vittet Daniel , Duperray Christophe , Chevillard Claude
Published: February 2005

Intracellular signaling processes by which hematopoietic growth factors regulate megakaryocytopoiesis remain uncompletely understood. Cyclic AMP (cAMP) has been shown to be implicated in the regulation of growth and differentiation in various normal and malignant cell types. Since a few studies have suggested the possible involvement of the cAMP pathway as one of the intracellular mechanisms whereby megakaryocytopoiesis may be regulated, we investigated the functional effects of cAMP on the human megakaryoblastic Dami cell line. We observed that exposure of Dami cells to cAMP analogs or to agents elevating intracellular cAMP levels yielded dose‐dependent cell growth inhibition. Cell cycle progression analysis of cells predominantly synchronized at the G1/S boundary by prior treatment with hydroxyurea revealed that cAMP transiently accumulated cells in the G2/M phase, then slowing down cell cycle. On the other hand, immunofluorescence and Northern blot analysis of megakaryocytic differentiation marker expression showed that probes we have used significantly inhibited GPlb expression. Moreover, although these agents used alone did not affect GPllb/llla expression, they markedly reversed phorbol ester‐induced GPllb/llla expression increase. These inhibitory cAMP actions on glycoprotein expression were not the result of cell cycle perturbation since we observed that GPlb and GPllb/llla expression were not cell cycle dependent. All these data may then be consistent with a potential negative regulatory role of the cAMP intracellular signaling pathway during megakaryocytopoiesis. © 1995 Wiley‐Liss, Inc.
Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide
Authors: Betz Natalie A. , Westhoff Brenda A. , Johnson Terry C.
Published: February 2005

Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS‐18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS‐18‐induced growth inhibition in BALB‐c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS‐18. Calcium did not alter CeReS‐18 binding to its cell surface receptor and CeReS‐18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS‐18‐inhibited 3T3 cells resuts in reentry into the cell cycle. A greater than 3‐hour exposure to increased calcium is required for escape from CeReS‐18‐induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10‐fold resulted in an approximately 7‐fold increase in total cell‐associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS‐18 did not affect total cell‐associated calcium or the increase in total cell‐associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS‐18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium‐sensitive step exists in the recovery from CeReS‐18‐induced growth inhibition. CeReS‐18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression. © 1995 Wiley‐Liss, Inc.
Effect of herparin on bovine epithelial lens cell proliferation induced by heparin affin regulatory peptide
Authors: Delbé J. , Vacherot F. , Laaroubi K. , Barritault D. , Courty J.
Published: February 2005

HARP (heparin affin regultory peptide) is an 18 kDa heparin binding protein, also known as HB‐GAM or pleiotrophin (PTN) which has been primarily isolated from brain and uterus, and displays neurite outgrowth, angiogenic and mitogenic activities. Previously, we have expressed the human cDNA encoding human HARP in NIH 3T3 cells. Purified recombinant HARP displayed mitogenic activity for endothelial cells. Its NH2‐terminal sequence indicates that the HARP molecule possesses a three amino acid extension from the signal peptide more than the NH2‐terminal described. For HB‐GAM or PTN, these three amino acids may be essential for the stability and the mitogenic activity of this growth factor. In an attempt to further study the mode of action of this growth factor, we have investigated the mitogenic effect of HARP on various cell types. In contrast to FGF‐2, HARP failed to induce stimulation of DNA synthesis on a CCL39 cell line. However, we found that in quiescent bovine epithelial lens (BEL) cells, the stimulation of DNA synthesis induced by HARP is dose‐dependent (EC50: 2.5 ng/ml) and maximal stimulation is as potent as that induced by FGF‐2 (EC50: 25 pg/ml). Interestingly, when BEL cells were allowed to quiesce in the presence of serum, the stimulation induced by HARP is considerably less potent. In this highly responsive cell system, heparin could potentiate the mitogenic activity of HARP at very low doses (0.1‐1 m̈g/ml) and inhibit this activity at concentrations of 10 m̈g/ml. In contrast to its protective effect on FGF‐1 and ‐2, heparin was unable to preserve HARP from tryptic and chymotryptic degradations. © 1995 Wiley‐Liss, Inc.
Apoptosis during HL‐60 cell differentiation is closely related to a G0/G1 cell cycle arrest
Authors: Terui Yasuhito , Furukawa Yusuke , Kikuchi Jiro , Saito Masaki
Published: February 2005

The cell cycle‐associated differences in the susceptibility to apoptosis were examined in HL‐60 cells before and after differentiation with phorbol 12, 13‐dibutyrate (PDBu). HL‐60 cells in various phases of the cell cycle were separated by the counterflow centrifugal elutriation and the susceptibility to apoptosis was measured by the morphological examination and by DNA fragmentation assay. Undifferentiated HL‐60 cells in S phase showed a significantly higher susceptibility to apoptosis than those in G0/G1 or G2/M phase either in the absence or presence of apoptosis‐inducing reagents such as A23187, actinomycin D (Act D), and cycloheximide (CHX). In contrast, PDBu‐treated HL‐60 cells preferentially underwent apoptosis in G0/G1 phase. When untreated HL‐60 cells enriched for G0/G1 phase were recultured in a complete medium, the percentage of apoptotic cells increased after 6‐12 h in correlation with the increase in S‐phase cells. When the same experiment was performed with PBDu‐treated cells, spontaneous increase of apoptotic cells was observed while almost all cells remained in G0/G1 phase. Northern blot analysis revealed that undifferentiated cells expressed the same amounts of bcl‐2 mRNA in each cell cycle phase, whereas G0/G1‐predominant reduction of bcl‐2 mRNA was noted in PDBu‐treated cells. There was no difference in the amounts of CD11b mRNA between G0/G1 fraction and S + G2/M fraction of differentiated HL‐60 cells. BCL‐2 overexpression could almost completely abrogate the G0/G1‐predominant induction of apoptosis in differentiated HL‐60 cells. These results suggest that G0/G1 cell cycle arrest and down‐regulation of bcl‐2 mRNA in G0/G1 phase might be associated with apoptosis in differentiated HL‐60 cells whereas the weakness of chromatin structure in S phase might be related to apoptosis in undifferentiated HL‐60 cells. © 1995 Wiley‐Liss, Inc.
Evidence for a pertussis toxin sensitive calcium entry pathway in thyroid FRTL‐5 cells
Authors: Törnquist Kid , Ekokoski Elina
Published: February 2005

Receptor‐mediated calcium entry was investigated in Fura 2 loaded FRTL‐5 cells. The purinergic agonist ATP activated the release of sequestered calcium and the entry of extracellular calcium. Downregulation of protein kinase C (PKC) substantially enhanced the ATP‐evoked calcium entry. Pretreatment of the cells with pertussis toxin (Ptx) decreased the ATP‐evoked calcium entry by 56% and the release of sequestered calcium by 34%. In PKC‐downregulated cells, the effect of Ptx treatment on the ATP‐evoked increase in [Ca2+]iwas 73% and 44%, respectively. Phorbol myristic acetate (PMA) decreased the ATP‐evoked calcium entry to the same extent as Ptx. In Ptx‐treated cells, the ATP‐evoked influx of 45Ca2+ was attenuated. Stimulation of the cells with P2p‐purinergic agonist GTP evoked no entry of calcium, although GTP released the same amount of sequestered calcium as did ATP. PKC downregulation or pretreatment with Ptx had no effects on the GTP‐evoked responses, whereas PMA decreased the GTP‐evoked release of calcium. We conclude that the ATP‐activated rapid calcium entry pathway is a second messenger‐operated calcium channel. © 1995 Wiley‐Liss, Inc.