bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the tetrahydrobiopterin synthesizing system during maturation of murine reticulocytes
Authors: Kerler F. , Hüultner L. , Ziegler I. , Katzenmaier G. , Bacher A.
Published: February 2005

The enzymes of tetrahydrobiopterin synthesis have been studied in murine bone marow, in spleen, in erythrocytes, and in reticulocytes. Mice with chemically induced and with genetically conditioned reticulocytosis as found in the lactate dehydrogenase deficient strain (Ldh‐1c/Ldh‐1c) were used for analysis of reticulocytic enzyme activities. The activity of the biopterin synthesizing system is highest in bone marrow even though it amounts to only about 10% as compared with liver. The first enzyme of the biosynthetic pathway, GTP‐cyclohydrolase, virtually disappears during the final maturation step of reticulocytes. In contrast, the activities of 6‐pyruvoyltetrahydropterin synthase and of sepiapterin reductase of erythrocytes are only reduced to about one half of the reticulocyte level. The absence of biopterin in erythrocytes is therefore caused by the loss of the enzyme that initiates the pterin biosynthetic pathway.
Effects of transforming growth factor‐β on long‐term human cord blood monocyte cultures
Authors: Orcel Philippe , Bielakoff Josette , De Vernejoul Marie Christine
Published: February 2005

Transforming growth factor‐β (TGF‐β) modulates growth and differentiation in many cel' types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF‐β has been shown to influence bone resorption in organ culture, we have studied the effect of TGF‐β (1‐1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10−9 M 1,25(OH)2D3 TGF‐β, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF‐β of 1 ng/ml, the multinucleated cells were reduced to 2.1% ± 0.3%, compared to 19.3% ± 1.5% in control cultures. TGF‐β inhibited in a dose‐dependent manner the proliferation of cord blood monocytes as assessed by 3H‐thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF‐β Indomethacin did not reverse the inhibitory effects of TGF‐β. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteolasts but not by adult monocytes, and an antibody to HLA‐DR, which is not present on osteoclast. TGF‐β decreased the expression of HLA‐DR and increased in a dose‐dependent manner the proportion of 23C6‐labeled cells; these results suggest that TGF‐β could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF‐β did not induce any 45Ca release from bone cultured with monocytes, suggesting that full osteoclastic differentiation was not achieved. These results emphasize the complex role of TGF‐β in the local regulation of bone cell differentiation and in bone remodeling.
Cyclical binding, processing, and functional interactions of neutrophils with leukotriene B4
Authors: O'Flaherty Joseph T. , Redman Jimmy F. , Jacobson David P.
Published: February 2005

Leukotrene (LT) B4 activates human polymorphonuclear neutrophils. (PMN) by binding to plasmalemmal receptors. It stimulates PMN to raise cytosolic calcium and degranulate. Both responses end within 15–30 sec. However, in < 15 sec, LTB4‐treated PMN lose the ability to respond further to LTB4; decrease the affinity and number of high affinity receptors available for binding LTB4 sequester LTB4 in plasmalemma‐associated sites that are inaccessible to a releasing buffei regi i men; and begin internalizing LTB4. Over the next 90 min, the cells increasingly internalize LTB4 and convert it to less potent metabolites; release the metabolites; recover LTB4 binding sites; and become fully sensitive to LTB4. Contrastingly, during the entire 90 min incubation with LTB4. PMN retained the capacity to bind and respond normally to a second stimulus platelet‐activating factor. We therefore suggest the following model. LTB4 receptors, when ligand‐bound, initiate function but rapidly lose this capacity as they lower their ligand binding affinity and sequester, internalize, or otherwise uncouple from transducing elements. These LTB4 receptor changes contribute to terminating PMN responses and producing a stimulus‐selective state of desensitization. During the desensitization period, PMN progressively process and metabolize LTB4. This removes LTB4 from the environment, thereby allowing PMN to recover functional receptors for and sensitivity to the ligand.
Ultraviolet stimulated melanogenesis by human melanocytes is augmented by di‐acyl glycerol but not TPA
Authors: Friedmann P. S. , Wren F. E. , Matthews J. N. S.
Published: February 2005

Epidermal melanocytes (MC) synthesise melanin in response to ultraviolet radiation (UVR). The mechanisms mediating the UV‐induced activation of melano‐genesis are unknown but since UVR induces turnover of membrane phospholipids generating prostaglandins (PGs) and other products, it is possible that one of these might provide the activating signal. We have examined the effects of prostaglandins (PGs) E1, E2, D2, F2α, and di‐acyl glycerol upon the UV‐induced responses of cultured human MC and the Cloudman S91 melanoma cell line. The PGs had little effect on unirradliated cells and did not alter the response to UVR in either human MC or S91 melanoma cells. However, a synthetic analogue of di‐acyl glycerol, 1‐oleyl 2‐acetyl glycerol (OAG), caused a significant (P<0.0001), dose‐related augmentation of melanin content both in human MC (seven‐fold) and S91 cells (three‐fold). UVR caused a significant augmentation of the OAG‐induced melanognesis of both human MC and S91 cells. Since OAG is known to activate protein kinase C, it was possible that the observed modulation of the UVR signal could be via that pathway. Di‐octanoyl glycerol, another di‐acyl glycerol, which activates kinase C, caused a small (70%) increase in melanogenesis in MC which was not altered by UVR. However, 12‐0 tetradecanoyl phorbol 13‐acetate (TPA), a potent activator of protein kinase C, had no significant effect on either basal or UV‐induced melanin synthesis in either cell type. These data suggest that the UV‐induced signal activating melanogenesis could be mediated by di‐acyl glycerol. Furthermore, they imply that the signal is transduced via an alternative, pathway that might be independent of protein kinase C.
Three distinct effects of SV40 T‐antigen gene transfection on cellular differentiation
Authors: Estervig David N. , Minoo Parviz , Tzen Chin‐Yuan , Scott Robert E.
Published: February 2005

SV40 large T‐antigen‐induced transformation has been reported to block differentiation, but the mechanism(s) of this effect has not been established. The results presented here show that stable transfection of the SV40 T‐antigen gene, via the pSV3neo plasmid, has at least three distinct effects on 3T3T adipocyte differentiation. Cells first show a decreased ability to undergo predifferentiation growth arrest, which is a prerequisite for in vitro 3T3T adipocyte differentiation. However, if predifferentiation growth arrest is accomplished by use of stringent differentiation‐inducing culture conditions, adipocyte differentiation can occur with high frequency. The pSV3neo‐transfected cell clones also show other modifications of the adipocyte differentiation process, including changes in nonterminal (reversible) and terminal (irreversible) steps of adipocyte differentiation. When compared to nontransfected 3T3T cells, the cell clones containing pSV3neo require markedly reduced growth factor concentrations to restimulate proliferation of nonterminally differentiated adipocytes and the terminal step of differentiation is also blocked. These results suggest that integration of the T‐antigen gene, through pSV3neo transfection, has multiple effects on the cellular mechanisms of differentiation. It does not block the differentiation process per se; rather it appears to make cells highly sensitive to proliferation signals, thereby making differentiation more difficult.
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

Using 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

Cultured 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

Rat 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

Normal 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

In 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

Tyrosine‐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

Fluid 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

The 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

The 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

Detailed 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

High 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

Tumor‐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.
Arabinosylcytosine‐induced accumulation of DNA nicks in myotube nuclei detected by in situ nick translation
Authors: Masuck Tony M. , Taylor Andrew R. , Lough John
Published: February 2005

This laboratory has recently reported the occurrence of DNA nicking at the onset of terminal skeletal myogenesis by using the technique of in situ nick translation (Dawon and Lough: Dev. Biol., 727:362–367, 1988). Because 1‐beta‐D‐arabi‐nofuranosylcytosine (araC), a cytocidal agent that is routinely used to remove dividingfibroblasts from myogenic cultures, inhibits DNA repair, it was of interest to determine whether araC treatment resulted in an accumulation of the endogeously created nicks. Thus, we have assessed the accumulation of DNA nicks in myotube cells during a 20 hour araC treatment period at the onset of terminal myogenesis (44–64 hours in vitro) by using three techniques: alkaline sucrose gradient density centrifugation, kinetic in situ nick translation, and cellular in situ nick translation. Although alkaline sucrose gradient centrifugation revealed no detectable nicking after 20 hours, kinetic in situ nick translation analysis revealed subtle but significant increases in DNA nicks caused by araC within 7 hours of drug application, and a 1.5‐fold increase in DNA repair sites after 20 hours of drugtreatment. That theseo bservationsreflectednicking specifically in myotube nuclei was determined by immunocytochemical localization of nicked sites after repair with a biotinyafed nucleotide analog (biotin‐11 ‐dUTP). The effects of araC wereonly incompletely reversible, whetheror not thedrugwas removedfrom the cultures, within 2 days of the treatment period. These results indicate that araC inhibits the repair of endogenously created nicks in the DNA of non‐dividing, terminally differentiating myotube cells, and provide further evidence for DNA nicking and repair as a feature of terminal myogenic differentiation.
Butyrate induces an EctoMg2+‐ATPase activity in Li‐7A human hepatoma cells
Authors: Murray Sandra L. , Knowles Aileen F.
Published: February 2005

The human hepatoma cell line (Li‐7A) possesses a high concentration of epider‐mal growth factor (EGF) receptors and exhibits ectoATPase activity in the presence of either MgATP or CaATP (Knowles: J. Cell. Physiol., 734:109–116, 1988). Growth for 96 hours in the presence of both EGF and cholera toxin or another cyclic AMP elevating agent induced an ectoATPase activity which was more active with CaATP and resistant to inhibition by the sulfhydryl reagent, p‐chloromercuriphenylsulfonate (pCMPS) (Knowles: Arch. Biochem. Biophys., 263: 264–271, 1988). In contrast, treatment of cells with butyrate, a short chain organic acid which can be derived from the analogue, dibutyryl cyclic AMP, resulted in a 4‐7‐fold increase of an ectoATPase which was more active with MgATP and highly sensitive to pCMPS inhibition. Maximal induction by butyrate required 48 hours and was dependent on butyrate concentration, but was independent of EGF and cyclic AMP elevating agents. Of six organic acids tested, butyrate was most effective in the induction of the ectoMg2+‐ATPase. The increase in the ectoMg2+‐ATPase activity could be prevented with actinomycin D and cycloheximide, indicating that both transcription and translation were necessary for induction. In addition to the induction of the ectoMg2+‐ATPase, bu‐tyrate induced alkaline phosphatase activity, but had no effect on a third ectoen‐zyme, 5′‐nucleotidase. These data further support our proposal that two distinct ectoATPases exist in the plasma membranes of Li‐7A hepatoma cells.
Nuclear and cytoplasmic localization of different basic fibroblast growth factor species
Authors: Renko M. , Quarto N. , Morimoto T. , Rifkin D. B.
Published: February 2005

The subcellular distribution of basic fibroblastic growth factor (bFGF) was analyzed by subcellular fractionation and immunofluorescence to gain insight into potential mechanisms for its release from cells. Subcellular fractionation of either SK‐Hep‐1 cells or NIH 3T3 cells transfected with a bFGF cDNA revealed that the 18 kd form of bFGF was found primarily in the cytosolic fraction, whereas the 22 and 24 kd forms of bFGF were found preferentially in ribosomal and nuclear fractions. Analysis of bFGF distribution by immunofluorescence using an antibody that recognized all forms of bFGF indicated both cytoplasmic and nuclear localization but failed to reveal any growth factor in structures representing secretory vesicles. Therefore, bFGF has a distribution inconsistent with that of a secretory protein.
Human bone cell enzyme expression and cellular heterogeneity: Correlation of alkaline phosphatase enzyme activity with cell cycle
Authors: Fedarko N. S. , Bianco P. , Vetter U. , Robey P. Gehron
Published: February 2005

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate‐resistant and ‐sensitive acid phosphatases, and non‐specific esterases were assayed kinetically and histo‐chemically. In asynchronous subconfluent cultures, < 15% of the cells stained positive and assayed activity was 0.04 IU/106 cells/cm2. After 1 week, the percent of alkaline phosphatase positive‐staining cells increased 5‐fold, while activity increased 10‐fold. Non‐specific esterases and tartrate‐sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate‐resistant acid phos‐phatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25–30 h. In synchronous cultures induced by a thymidine‐aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate‐sensitive acid phos‐phatase and non‐specific esterase levels were relatively stable throughout the cell cycle, while tartrate‐resistant acid phosphatase activity was not assavable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.
Colony stimulating factor‐1 Is a negative regulator of the macrophage respiratory burst
Authors: Phillips Wayne A. , Hamilton John A.
Published: February 2005

Several cytokines have previously been shown to prime macrophages for enhanced release of oxygen radicals in response to subsequent stimulation. We now demonstrate that the presence of the macrophage‐specific colony stimulating factor‐1 (CSF‐1) inhibits the priming of murine macrophages by a variety of agents including tumor necrosis factor α, granulocyte/macrophage colony stimulating factor, interferon‐γ, and bacterial lipopolysaccharide. CSF‐1 is also able to reduce the respiratory burst in the absence of priming. Our results indicate that CSF‐1 is a potent negative regulator of the macrophage respiratory burst which acts to oppose the priming (enhancing) action of macrophage activating agents. We propose that CSF‐1 may have a potentially important and previously unrecognized, role as a Physiological regulator which restricts or terminates the activation of macrophages in order to prevent an uncontrolled inflammatory reaction.
Plasminogen activators and their inhibitors in the neuromuscular system: I. Developmental regulation of plasminogen activator isoforms during in vitro myogenesis in two cell lines
Authors: Festoff Barry W. , Rao Jasti S. , Maben Cecilia , Hantaï Daniel
Published: February 2005

Plasminogen activators (PAs), were estimated qualitatively and quantitatively in two different clonal murine skeletal muscle cell lines. Both cell lines produced the two major types of PAs found in mammalian cells, urokinase‐type (uPA) and tissue type (tPA). These two lines are models for the study of myogenesis in vitro, but differ in several growth and differentiation characteristics. Because of their possible involvement in these characteristics we assayed the expression of PAs in both cell systems during development in culture. Utilizing fibrin zymography two isoforms of tPA were detected. One co‐migrated with human tPA at 75 kd and another may represent a tPA:inhibitor complex at 105 Kd. Several isoenzymes of uPA were detected and these changed depending on whether cell homogenates or conditioned medium was analyzed and whether myogenic cells were at single‐cell myoblast or multi‐nucleated myotube stage. Species‐specific antisera to mouse uPA identified 4 uPA bands in muscle cell medium and 5 in cell layers. Antigenic uPA bands also varied depending on stage of myogenesis. Quantitative amidolytic studies using chromogenic substrates showed that maximal PA activity, both uPA and tPA, occurred at the time of myoblast fusion. Furthermore, uPA activity in membranes increased during myogenesis, while both uPA and tPA in medium decreased after fusion. These studies indicate that muscle PA expression is developmentally regulated and may correlate with growth and differentiation in skeletal muscle.
Differences in inositol phosphate production in rat tail artery and thoracic aorta
Authors: Labelle Edward F. , Murray Bonnie M.
Published: February 2005

Pharmacomechanical coupling of vascular smooth muscle is believed to be mediated by inositol trisphosphate (IP3). Numerous studies have demonstrated an increase in inositol phosphates following tissue stimulation using either intact aortic strips or cultured cells from aorta. However, little information is available concerning inositol phosphates in vascular tissue other than in the large conduit vessel, the aorta. This present study was designed to examine the role of inositol phosphate metabolism following adrenergic stimulation of the muscular rat tail artery as compared to the aorta. Segments of thoracic aorta and tail artery from male Sprague Dawley rats were labe ed with [3H]inositol and stimulated with norepinephrine. The norepinephrine concentration that resulted in a half‐maximal stimulation of inositol phosphates was ⌣︁−6 M in both the aorta and tail artery. Although the sensitivity of the two vessels to norepinephrine stimulation were similar, the stimulated levels of IP, IP2, and IP3 were from 1 to 2 orders of magnitude greater in the tail artery than in aorta. IP production in aorta and tail artery was a linear function of time (from 0 to 30 min). Significant levels of IP3 (the 1, 4, 5‐IP3 isomer as determined by HPLC) could only be detected in the tail artery and appeared to be produced optima ly after 5 min of stimulation. The several order of magnitude increase in adrenergic stimulated inositol phosphate production in the tail artery was not due to either an increased magnitude of [3H]inositol incorporated into PI, PIP, and PIP2 or to a greater percentage of smooth muscle cells per unit tissue of the rat tail artery. We believe the resul s of this study demonstrate that the increased inositol phosphate metabolism n the vascular smooth muscle cells of the tail artery is an intrinsic property of the cell. Moreover, due to the significant levels of all inositol phosphates produced in the tail artery, this muscular artery may be a better model, as compared to the aorta, for future studies investigating pharmacomechanical coupling of vascular smooth muscle.
Caffeine, an inhibitor of endocytosis in dictyostelium discoideum amoebae
Authors: Gonzalez Carlos , Klein GéRard , Satre Michel
Published: February 2005

The effect of the trimethylxanthine, caffeine, was examined on the growth and endocytosis pathways of the vegetative amoebae of the cellular slime mold Dic‐tyostelium discoideum. Caffeine at concentrations of 1.5–3 mM was found to inhibit axenic growth, fluid‐phase pinocytosis, and secretion of lysosomal enzymes. Cell viability was unaffected by incubation for 16 hours with 5 mM caffeine but decreased markedly thereafter. Phagocytosis of the bacterium Esch‐erichia coli by Dictyostelium amoebae was also inhibited by caffeine, although at concentrations twofold to threefold higher. Caffeine rapidly entered into amoebae to reach an equilibrium between extracellular and intracellular concentrations, and it was not appreciably metabolized by Dictyostelium. Inhibition of growth and endocytosis was reversible upon removal of the drug and was partially counteracted by 10 mM adenosine. As caffeine discharged intracellular calcium stores in Dictyostelium (Abe et al., 1988), its inhibitory effect on endocytosis could result from the perturbation of calcium homeostasis. In agreement with this hypothesis, the cation La3+ (10 μM), a Ca2+‐transport inhibitor, also strongly reduced fluid‐phase pinocy'osis.
Hybrids from fusion of normal human T lymphocytes with immortal human cells exhibit limited life span
Authors: Pereira‐Smith Olivia M. , Robetorye Susan , Ning Yi , Orson Frank M.
Published: February 2005

A number of normal human cell types have been shown to exhibit cellular senescence in vitro. We and others had found that fusion of normal human fibroblasts with immortal human cells yielded hybrids having limited lifespan. This indicated that the phenotype of cellular senescence is dominant and that immortality results from recessive changes in genes involved in growth control. They also supported the hypothesis that senescence results from genetic mechanisms rather than random damage. Since T lymphocytes are a highly differentiated cell type, in contrast to fibroblasts, it was of interest to determine whether similar mechanisms caused senescence in the T cells. We therefore fused normal human T lymphocytes with an immortal human cell line to determine whether they could restore the senescent, nondividing phenotype in hybrids, as do normal human fibroblasts. Eleven of fifteen hybrid clones studied exhibited limited proliferative potential after achieving a range of population doubling similar to that observed in the cell fusion studies involving normal fibroblasts. These results provide evidence that cellular senescence in T lymphocytes occurs via genetic mechanisms.
Role of the Na+ /K+ /Cl− transporter in the positive inotropic effect of ouabain in cardiac myocytes
Authors: Panet Rivka , Fixler Ruhama , Snyder David , Raz Shmuel , Atlan Henri , Eilam Yael , Hasin Yonathan
Published: February 2005

In this study we have characterized the bumetanide‐sensitive K+ /Na + /Cl− cotransport in cultured rat cardiac myocytes. (1) It carries about 10% of the total K+ influx. (2) It is sensitive to furosemide (Ki0.5 = 10−6M) and bumetanide (Ki0.5 = 10−7M) (3) It is strongly dependent on the extracellular concentrations of Na+ and Cl− (4) It carries out influx of both ions, K+ and Na+. A therapeutic concentration of ouabain (10−7M) stimulated the bumetanide‐sensitive K+ influx (as measured by 86Rb +), in the cultured myocytes, with no effect on the bumetanide‐resistant K+ influx, which was mediated mostly by the Na + /K + pump. Stimulation of the bumetanide‐sensitive Rb+ influx by a low ouabain concentration was strongly dependent on Na+ and Cl− in the extracellular medium. A low concentration of ouabain (10−7M) was found to increase the steady‐state level of cytosolic Na + by 15%. This increase was abolished by the addition of bumetanide or furosemide. These findings suggest that ouabain, at a low (10−7M) concentration, induced its positive inotropic effect in rat cardiac myocytes by increasing Na+ influx into the cells through the bumetanide‐sensitive Na+ / K+ /Cl− cotransporter. In order to examine this hypothesis, we measured the effect of bumetanide on the increased amplitude of systolic cell motion induced by ouabain. Bumetanide or furosemide, added to cultured cardiac myocytes, inhibited the increased amplitude of systolic cell motion induced by ouabain. Neither bumetanide nor furosemide alone has any significant effect on the basal amp itude of systolic cell motion. We propose that stimulation of bumetanidesensitive Na+ influx plays an essential role in the positive inotropic effect in rat cardiac myocytes induced by low concentrations of ouabain.