bluuebunny/crossref_metadata_2025_split huggingface dataset:
Altered synthesis of the 26‐kDa heat stress protein family and thermotolerance in cell lines with elevated levels of calcium‐binding proteins
Authors: Evans Douglas P. , Simonette Rebecca A. , Rasmussen Colin D. , Means Anthony R. , Tomasovic Stephen P. 
Published: February 2005

AbstractUsing a bovine papillorna virus‐based vector, mouse mammary adenocarcinoma cells have been transformed to express elevated amounts of functional calmodulin (CaM) (Rasmussen and Means, 1987) and another Ca2+‐binding protein, parvalbumin (PV) (Rasmussen and Means, 1989) that is not normally synthesized in these cells. Parental cells (C127) and cells transformed by the vector alone (BPV‐1), the vector containing a CaM gene (CM‐1), or the vector containing parvalbumin (PV‐1) were used to study the effect of increased synthesis of Ca2+‐binding proteins on heat‐stress protein (HSP) synthesis and cell survival following heating at 43°C. The induction, stability, and repression of the synthesis of most HSPs after 43°C heating was not significantly affected by increased amounts of Ca2+ ‐binding proteins, but the rate of synthesis of all three isoforms of the 26‐kDa HSP (HSP26) was greatly reduced. C127 cells, which have about one half as much CaM as do BPV‐1 cells, synthesized the most HSP26. CM‐1 cells, which have more than fourfold higher levels of CaM than do BPV‐1 cells, had a rate of synthesis of HSP26 approaching that of unheated cells. BPV‐1 cells, with a two‐fold increase in CaM, were intermediate in HSP26 synthesis. This effect on HSP26 synthesis may be largely related to the Ca2+ ‐binding capacity of CaM rather than to a specific CaM‐regulated function, since PV‐1 cells also showed reduced rates of HSP26 synthesis. Survival experiments showed that reduced HSP26 synthesis in cells with increased amounts of Ca2+‐binding proteins did not significantly alter intrinsic resistance to continuous 43°C heating. Thermotolerance was not reduced and appeared to develop more rapidly in CM‐1 and PV‐1 cells. These results suggest that (1) the signal for HSP26 synthesis can be largely abrogated by elevated Ca2+ binding protein levels, and (2) if these HSPs are involved in thermotolerance development, that function may be associated with intracellular Ca2+ homeostasis.
Modulation of Ca2+‐dependent intercellular adhesion in bovine aortic and human umbilical vein endothelial cells by heparin‐binding growth factors
Authors: Bavisotto Linda M. , Schwartz Stephen M. , Heimark Ronald L. 
Published: February 2005

AbstractCultured endothelial cells have been shown to possess two mechanisms of intercellular adhesion: Ca2+‐dependent and Ca2+‐independent. We report here that growth of bovine aortic endothelial cells (BAEC) in complete medium containing purified basic fibroblast growth factor (bFGF, 6 ng/ml) results in loss of Ca2+‐dependent intercellular adhesion. In the presence of heparin (90 μg/ml), this effect is reproduced upon treatment with acidic fibroblast growth factor (aFGF, 6 ng/ml) or endothelial cell growth supplement (ECGS, 100 μg/ml), in both human umbilical vein endothelial cells (HUVEC) and BAEC. Treatment at these doses with aFGF in the absence of heparin or with heparin alone is without significant effect. Loss of Ca2+‐dependent adhesion following treatment of cells with heparin‐binding growth factors (HBGFs) is prevented by pre‐treatment of cell layers with cycloheximide. The Ca2+‐independent adhesion mechanism is unaffected by HBGF treatment. Exposure of endothelial cells to HBGFs, moreover, prevents the eventual establishment of quiescence in growing cultures and restimulates replication in confluent cultures that have reached a final density‐inhibited state. Addition of bFGF alone or aFGF + heparin at these doses results in a 4‐fold increase in DNA synthesis over untreated control cultures at saturation density as reflected by thymidine index. A single addition of bFGF (6 ng/ml) to untreated quiescent confluent BAEC monolayers results in an increase in 3H‐TdR incorporation reaching a peak at 22 hours with a parallel loss of Ca2+‐dependent adhesiveness. Fluorescent staining with rhodamine‐phalloidin demonstrates an altered distribution of polymerized F‐actin in the bFGF‐treated monolayers, marked by disruption of the dense peripheral microfilament bands retained by untreated confluent monolayers. Together, these results indicate that the mitogenic effect of HBGFs in cultured endothelial cells is associated with a “morphogenic” set of responses, perhaps dependent on breakdown of calcium‐dependent cell‐cell contacts.
Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells
Authors: Aronow M. A. , Gerstenfeld L. C. , Owen T. A. , Tassinari M. S. , Stein G. S. , Lian J. B. 
Published: February 2005

AbstractRat calvaria osteoblasts derived from 21‐day‐old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16‐day‐old embryonic chick calvaria‐derived osteoblasts (COB) (Gerstenfeld, et. al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of β‐glycerol‐phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200‐fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.
Immortal phenotype of the HeLa variant D98 is recessive in hybrids formed with normal human fibroblasts
Authors: Pereira‐Smith Olivia M. , Stein Gretchen H. , Robetorye Susan , Meyer‐Demarest Sarah 
Published: February 2005

AbstractNormal human cells such as human diploid fibroblasts (HDF) have a finite pro‐liferative lifespan in culture. Previous studies have shown that the limited lifespan phenotype is dominant in cell hybrids formed by fusion of HDF to at least 23 different kinds of immortal human cells. However, two independent studies reported that hybrid clones formed by the fusion of HDF to the HeLa variant D98 had unlimited division potential. Those results were potentially very important because they implied that a) there is a dominant mechanism for immortalization of human cells in addition to the well‐documented recessive mechanism, and b) a dominant mechanism would lend itself to identification of the immortalizing gene. Consequently, we carried out more detailed studies of the behavior of D98 cells in hybrids. Our results indicate that the majority of D98 x HDF hybrid clones exhibit a clear‐cut finite proliferative lifespan phenotype. In addition, these hybrid cell populations often give rise to an immortal focus of cells that can be seen to take over the population of mortal cells at the end of their lifespan. This phenomenon reconciles our data with the previous reports of immortal D98 x HDF hybrid clones and leads us to conclude that D98 cells do not express a dominant immortalizing gene.
Calcium regulation of growth and differentiation of normal human keratinocytes: Modulation of differentiation competence by stages of growth and extracellular calcium
Authors: Pillai Sreekumar , Bikle Daniel D. , Mancianti Maria‐Laura , Cline Polly , Hincenbergs Mara 
Published: February 2005

AbstractIn this study we examined the different aspects of the pathway leading to the differentiation of keratinocytes as a function of time in culture and calcium concentration of the culture medium. Human neonatal foreskin keratinocytes were grown in a serum‐free, defined medium containing 0.07, 1.2, or 2.4 mM calcium and assayed for the rate of growth and protein synthesis, involucrin content, transglutaminase activity, and cornified envelope formation at preconfluent, confluent, and postconfluent stages of growth. We observed that keratinocytes grown to postconfluence in all calcium concentrations showed an increased protein/DNA ratio and an increased rate of membrane‐associated protein synthesis. Extracellular calcium concentrations did not have a clear influence on these parameters. However, preconfluent and confluent keratinocytes grown in 0.07 mM calcium showed markedly retarded differentiation at all steps, i.e., involucrin synthesis, transglutaminase activity, and cornified envelope formation. Within 1 week after achieving confluence, these keratinocytes began synthesizing involucrin and transglutaminase and developed the ability to form cornified envelopes. Cells grown in 1.2 and 2.4 mM calcium synthesized involucrin and transglutaminase prior to confluence and were fully competent to form cornified envelopes by confluence. Thus external calcium‐regulated keratinocyte differentiation is not an all or none phenomenon, but rather it is the rate at which keratinocytes differentiate that is controlled by calcium. We conclude that either or both higher extracellular calcium concentration and the achievement of cell–;cell contacts lead to a coordinate increase of at least two precursors–;involucrin content and transglutaminase activity–;required for cornified envelope formation. We speculate that a critical level of cytosolic calcium, achieved by increased extracellular calcium or by achievement of Intercellular communication established by cell–cell contact, may trigger mechanisms required for initiation of keratinocyte differentiation.
Precocious induction of tyrosine aminotransferase mRNA by hydrocortisone in cultured fetal rat hepatocytes at different developmental stages
Authors: Hoffmann Birgit , Paul Dieter 
Published: February 2005

AbstractTyrosine‐aminotransferase (TAT) is encoded by a liver‐specific gene known to be expressed perinatally. Fetal rat hepatocytes (gestation day 19) in primary cultures, in which TAT gene expression is normally undetectable, are induced by hydrocortisone to express TAT‐mRNA in a dose‐dependent manner (>10−7M). In hepatocytes incubated with hydrocortisone, TAT‐mRNA levels were marginal after 24 hours, reaching maximal levels at 48 hours. After a pre‐incubation of hepatocytes for 24 hours in the absence of hydrocortisone followed by exposure to hydrocortisone (24–48 hours). TAT‐mRNA levels were high. Hepatocytes derived from fetuses of gestation days 14 and 17 displayed comparable levels of TAT‐mRNA in response to hydrocortisone. These results demonstrate that cultured hepatocytes of gestational stages as early as day 14, which initially do not respond to hydrocortisone by TAT gene induction, undergo a “maturation” process during the initial 24 hours following cultivation, resulting in the acquisition of precocious competence for TAT gene transcription in response to hydrocortisone. This suggests that one or more factor(s), required for hydrocortisone‐inducible TAT gene transcription, and not available in fetal liver until birth (Gluecksohn‐Waelsch: Cell, 18:225–237, 1979) appear in fetal hepatocytes upon cultivation during this “maturation” period, thus permitting precocious TAT gene expression in vitro.
Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress
Authors: Diamond S. L. , Sharefkin J. B. , Dieffenbach C. , Frasier‐Scott K. , McIntire L. V. , Eskin S. G. 
Published: February 2005

AbstractFluid shear stress can stimulate secretion of tissue plasminogen activator (tPA) by cultured human endothelial cells, while plasminogen activator inhibitor type‐1 secretion remains unstimulated. To determine whether hemodynamically induced changes in tPA messenger RNA (mRNA) levels also occur, primary cultures from the same harvst of primary human umbilical vein endothelial cells were either maintained in stationary culture or exposed to arterial levels of shear stress (25 dynes/cm2) for 24 hours. Total cellular RNA was isolated from the shear stressed and stationary cultures and the relative levels of tPA mRNA and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) mRNA were determined using a coupled reverse transcriptase/polymerase chain reaction method. As indicated by the amount of amplification product, tPA mRNA levels were many fold higher (> 10) in endothelial cells subjected to shear stress for 24 hours than in stationary controls. In contrast, mRNA levels for GAPDH were similar in control and shear stressed cells. The constancy of the measured GAPDH signal indicated that the tPA response was a selective effect of fluid shear stress. When a similar polymerase chain reaction method was used, the mRNA levels of basic fibroblast growth factor (bFGF) were found not to vary in comparison to GAPDH mRNA after 24 hours of shear stress. These results indicate that enhancement of the fibrinolytic potential of endothelial cells in response to hemodynamic forces could involve transcriptional events.
Production of insulin‐like growth factor II (IGF‐II) and different forms of IGF‐binding proteins by HT‐29 human colon carcinoma cell line
Authors: Culouscou Jean‐Michel , Remacle‐Bonnet Maryse , Garrouste Françoise , Fantini Jacques , Marvaldi Jacques , Pommier Gilbert 
Published: February 2005

AbstractThe serum‐free medium conditioned by the human colon cancer cell line HT‐29 contains insulin‐like growth factors (IGF) that are entirely complexed to binding proteins (IGF‐BP). Gel filtration in acid conditions of the cell‐conditioned medium permits separation of IGF‐BP from two molecular forms of IGF of 15,000 and 7,500 Mr. As determined by ligand blotting, IGF‐BP are heterogeneous and constituted of three molecular forms of 31,000, 28,000, and 26,000 Mr. Using IGF‐I and IGF‐II radioreceptor assays, IGF‐I radioimmunoassay (RIA), and competitive protein‐binding assay specific for IGF‐II, it is shown that the IGF‐type eluting in 15 K and 7.5 K position from gel filtration is restricted to IGF‐II. Its concentration is ∼6 ng/106 HT‐29 cells with 60% present as a high‐molecular‐weight form of IGF‐II. This large 15 K IGF molecule is devoided of any IGF‐binding activity and might represent incomplete processing of pro‐IGF‐II peptide. By contrast, the level of IGF‐I detected by RIA is barely measurable and considered negligible (0.57 pg/106 HT‐29 cells). Although these IGF‐II‐like peptides exhibit a growth‐promoting activity on FR3T3 fibroblasts, they cannot stimulate, as recombinant IGF‐I or IGF‐II, 3H‐thymidine incorporation into DNA of HT‐29 cells, whatever the experimental conditions used. Finally, we have shown that IGF binding is restricted predominantly to the basolateral domain of the cell membrane by using HT‐29‐D4 clonal cells, derived from the parental HT‐29 cell line, maintained in a differentiated state by culture in a medium in which glucose is replaced by galactose.
Progressive development of the rat osteoblast phenotype in vitro: Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix
Authors: Owen Thomas A. , Aronow Michael , Shalhoub Victoria , Barone Leesa M. , Wilming Laurens , Tassinari Melissa S. , Kennedy Mary Beth , Pockwinse Shirwin , Lian Jane B. , Stein Gary S. 
Published: February 2005

AbstractThe relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods–;proliferation, extracellular matrix maturation, and mineralization–;and 2) two restriction points to which the cells can progress but cannot pass without further signal–;the first when proliferation is down‐regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle‐and cell growth‐regulated genes, produce a fibronectin/type I collagen extracel‐lular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phos‐phatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid‐induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.
Cytomatrix synthesis in MDCK epithelial cells
Authors: Mitchell John J. , Low Robert B. , Woodcock‐Mitchell Janet L. 
Published: February 2005

AbstractDetailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak J. Biol. Chem., 256: 4863–4870, 1981), was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with [14C] leucine over several days and then pulse‐labeled for 4 hours with [3 H] leucine. FSRs (as percent per hour) were calculated from the 3H/14C ratio of cell extracts or individual proteins separated by two‐dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered‐Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of α‐ and β‐tubulin diverged in quiescent cells with α‐tubulin turnover exceeding β‐tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and α‐actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form. The results suggest that metabolic coupling between individual cellular filament systems is not strict. The data are, however, consistent with models that predict that assembly of a subcellular structure influences the turnover of its component proteins.
Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro
Authors: Bethlenfalvay N. C. , White J. C. , Chadwick E. , Lima J. E. 
Published: February 2005

AbstractHigh pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of [G‐3H] hypoxanthine (Hy) into [3H] labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate [3H] ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.
Enhancement of thrombin‐ and lonomycin‐stimulated prostacyclin and platelet‐activating factor production in cultured endothelial cells by a tumor‐promoting phorbol ester
Authors: Zavoico George B. , Hrbolich Janet K. , Gimbrone Michael A. , Schafer Andrew I. 
Published: February 2005

AbstractTumor‐promoting phorbol esters such as 4β‐phorbol 12‐myristate 13‐acetate (PMA) have been shown to act synergistically with Ca2+ ionophores in cell activation, including stimulation of arachidonic acid metabolism. The effects of PMA on unstimulated and Ca2+ ionophore‐ or thrombin‐stimulated PGI2 and platelet‐activating factor (PAF) production in cultured bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were investigated. Incubation of BAEC or HUVEC for 5–10 min with 100 nM PMA alone slightly increased basal PGI2 production. PGI2 production was rapidly stimulated in BAEC and HUVEC treated with the Ca2+ ionophore ionomycin. Preincubation of BAEC of HUVEC with 100 nM PMA for 5–10 min followed by ionomycin for up to 60 min enhanced PGI2 production up to 2.5‐fold. Pretreatment with 100 nM PMA for 5 min also caused a 2‐fold enhancement of thrombin‐stimulated (1 U/ml) PGI2 production in HUVEC. The production of other prostaglandins, PGF2α, PGE2, and PGD2, was also enhanced. In contrast, PMA had no effect on PGI2 synthesized directly from exogenous arachidonic acid or PGH2. The inactive phorbol ester 4α‐phorbol 12,13‐didecanoate was without effect. Since the biosyntheses of both PGI2 and PAF share a common first step, the hydrolysis of their respective phospholipid precursors by phospholipase A2, we investigated whether PMA preincubation could also enhance PAF biosynthesis. Incubation of HUVEC with 100 nM PMA alone had a negligible effect on PAF production. However, thrombin‐stimulated (1 U/ml) PAF production was enhanced 2.6‐fold by preincubation with 100 nM PMA. The protein kinase C inhibitors H‐7 and staurosporine ablated the enhancing effect of PMA on thrombin‐stimulated PGI2 and PAF biosynthesis. These results demonstrate that PMA can significantly alter the production of PGI2 and PAF in vascular endothelial cells, and suggest that protein kinase C activation modulates phospholipase A2 activity in this cell type.