bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cellular expression of bone‐related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures
Authors: Malaval Luc , Modrowski Dominique , Gupta Ashwani K. , Aubin Jane E. 
Published: February 2005

AbstractRat bone marrow stromal cells comprise a heterogeneous mixture of cell lineages including osteoblastic cells. When grown in the presence of ascorbic acid, β‐glycerophosphate and 10−8M dexamethasone, osteoprogenitor cells within the population divide and differentiate to form bone nodules (Maniatopoulos et al., 1988, Cell Tissue Res., 254:317–330; Aubin et al., 1990, J. Bone Miner. Res., 5:S81) providing a useful model to investigate temporal and spatial changes in expression of osteoblastic markers. Immunocytochemistry was combined with Northern blotting, enzymatic assay, and radioimmunoassay to analyze the expression of bonerelated proteins during the growth and differentiation sequence. By mRNA levels, protein production and/or enzymatic activity, expression of osteocalcin, bone sialoprotein, and alkaline phosphatase increased concomitantly with the development of bone nodules, while osteopontin mRNA levels decreased and those of SPARC/osteonectin did not change significantly. In older cultures with mineralizing nodules, mRNA levels for alkaline phosphatase and bone sialoprotein, but not osteocalcin, declined. Immunolabeling revealed that cells in early cultures stained poorly for SPARC/osteonectin and strongly for thrombospondin. Later, SPARC/osteonectin staining increased in most cells, while thrombospondin staining could be seen in both matrix and in cells, but with marked intercellular variability in intensity. At all time points studied, osteoblasts within bone nodules stained homogeneously for thrombospondin and alkaline phosphatase, and with marked heterogeneity of intensity amongst cells for SPARC/osteonectin and osteocalcin. Labelling with RCC455.4, a monoclonal antibody raised against rat calvaria cells which Intensely labels osteoblasts and osteocytes (Turksen et al., 1992, J. Histochem. Cytochem., 40:1339–1352), co‐localized with osteocalcin. Alkaline phosphatase activity and the amount of osteocalcin determined by both radioimmunoassay and immunolabelling decreased in very late cultures, a time corresponding to appearance of fully mineralized nodules. These studies indicate that the bone marrow stromal cell system is a useful model to study the temporal and spatial expression of bone‐related proteins during osteogenesis and formation, mineralization, and maturation of bone nodules. Further, immunolabelling at the individual cell and single bone nodule level allowed discrimination of marked variability of expression of osteoblast markers during the differentiation sequence. © 1994 Wiley‐Liss, Inc.
Activation of serpins and their cognate proteases in muscle after crush injury
Authors: Festoff Barry W. , Reddy Rajendra B. , Vanbecelaere Marnie , Smirnova Irina , Chao Julie 
Published: February 2005

AbstractDirect muscle injury was induced in rats in order to evaluate alterations in the balance of serine proteases and inhibitors (serpins) as a response to tissue damage. It was previously found that certain proteases, specifically urokinase‐like plasminogen activator (uPA) and others, required activation in order to effect regeneration. We hypothesized that the magnitude and temporal sequence of serpin activation would follow, pari passu, activation of their cognate proteases. In addition to uPA, tissue PA (tPA) and tissue kallikrein were the proteases studied. The serpins we analyzed were protease nexin I (PNI), PA inhibitor 1 (PAI‐1), and the kallikrein‐binding protein (KBP). uPA nearly doubled 48 h after injury, while there was no change in amidolytic activity after addition of fibrin monomer as an estimation of tPA activity. Tissue kallikrein activity, barely detectable in normal muscle, slowly increased, nearly tripling at 7 days after injury. Greater magnitude and more rapid changes in muscle serpins occurred over the same post‐injury time course. By 24 h PNI increased threefold, while PAI‐1 increased more slowly, reaching double the control values by 5 days after injury. Surprisingly, KBP, the serpin‐class inhibitor of tissue kallikrein, had the most robust response, increasing tenfold over control 48 h after crush injury of muscle. These results further implicate the serpin:protease balance in tissue injury. Participation of complex receptors, such as the α2‐macroglobulin receptor/low density lipoprotein receptor‐related protein (LRP), various growth factors, cytokines, and other molecules, in regulating this balance is implicated by these data. © 1994 wiley‐Liss, Inc.
Stimulation of hepatocyte DNA synthesis by prostaglandin E2 and prostaglandin F2α additivity with the effect of norepinephrine, and synergism with epidermal growth factor
Authors: Refsnes Magne , Thoresen G. Hege , Dajani Olav F. , Christoffersen Thoralf 
Published: February 2005

AbstractPrevious data obtained in vivo and in vitro suggest that both prostaglandins (PGs) and catecholamines may have a role in promoting hepatocyte proliferation, and PGE2 and PGFF2α have also been implicated as mediators of the mitogenic actions of epidermal growth factor (EGF) (and transforming growth factor alpha [TGFα]). We have studied the effects of PGs and norepinephrine on DNA synthesis in serum‐free primary cultures of rat hepatocytes, and compared the PG effects with those of norepinephrine. PGE2, PGF2α, PGD2, and the synthetic analog dimethyl‐PGE2 markedly enhanced the DNA synthesis. A more quantitative analysis of the effects of PGE2 and PGF2α on the DNA synthesis, in the presence and absence of EGF, indicated that these PGs interacted in an essentially multiplicative manner with the effect of EGF. The effects of PGE2 and PGF2α showed almost complete additivity with the stimulation of DNA synthesis produced by maximally effective concentrations of norepinephrine. The data suggest (a) that PGE2 and PGF2α facilitate and synergize with, rather than mediate, the actions of EGF in hepatocytes, and (b) that this effect of the PGs occurs by mechanisms that are at least partly distinct from those of norepinephrine. © 1994 wiley‐Liss, Inc.
Coordinate and independent regulation of αB‐crystallin and HSP27 expression in response to physiological stress
Authors: Head Mark W. , Corbin Elaine , Goldman James E. 
Published: February 2005

Abstractα‐Crystallins share structural and functional properties with the stress protein hsp27. These polypeptides are expressed at low constitutive levels in many tissues including brain, and αB‐crystallin and hsp27 can accumulate in central nervous system glia in a variety of neurological conditions. We report here that heat shock and exposure to transition metals result in an increase in the steady state mRNA level of αB‐crystallin and hsp27 in primary cultures of rat forebrain astrocytes. Both exposure to tumour necrosis factor‐α and hypertonic conditions result in αB‐crystallin mRNA accumulation but no change in the hsp27 mRNA level. Under some of these conditions increased synthesis and accumulation of αB‐crystallin and hsp27 protein are also evident. We are unable to detect αA‐crystallin mRNA in resting or stressed astrocytes. A novel phenomenon involving a transitory change in stress protein mRNA mobility in Northern blots during induction is reported, which is stress type and cell type independent. The results demonstrate multiple stress regulation of αB‐crystallin and hsp27 in cultured astrocytes, suggesting that they can legitimately be regarded as stress proteins in the central nervous system. © 1994 wiley‐Liss, Inc.
Dependence of collagen remodelling on α‐smooth muscle actin expression by fibroblasts
Authors: Arora Pamela D. , McCulloch Christopher A. G. 
Published: February 2005

AbstractTo study the relation between expression of the putative myofibroblast marker α‐smooth muscle actin and the remodelling of extracellular matrix, immunocytochemical, gel electrophoresis, and collagen gel contraction studies were performed on two human fibroblast subtypes. Double immunolabelling for total actins and α‐smooth muscle (sm) actin as well as affinity labelling of filamentous and monomeric actins in gingival fibroblasts demonstrated that α‐sm was colocalized in stress fibres and in regions with high levels of monomeric actin throughout the cytoplasm. α‐sm comprised up to 14% of total cellular actin as assessed by 2D gel electrophoresis. Thirteen different gingival and seven different periodontal ligament fibroblast lines constitutively expressed on α‐sm actin. These cells exhibited up to 60% inter‐line variations of fluorescence due to α‐sm actin and up to 70% and 45% inter‐line variation in the rate of collagen gel contraction. Quantitative, single cell fluorimetry of α‐sm actin immunoreactivity demonstrated a linear relation between gel contraction and α‐sm actin (correlation coefficients of 0.71 for gingival and 0.61 for periodontal ligament cells), but there was no detectable relationship between total actin content and gel contraction. In contrast, flow cytometry demonstrated that 99% of the total gated cells from cell lines exhibiting rapid gel contraction showed α‐sm actin staining above background fluorescence as compared to only 35% of cells with slow rates of gel contraction. Contracting collagen gels stained with FITC‐phalloidin showed cells with well‐developed stress fibres that were progressively more compact and elongated during the time of maximal gel contraction. To examine the dependence of gel contraction on assembly of monomeric actin into actin filaments, cells were electroporated in the presence of phalloidin or cytochalasin D. Collagen gels exhibited up to 100% inhibition of gel contraction that was dose‐dependent. Gel contraction was inhibited 93% by electroinjection of cells with α‐sm actin antibody prior to incubation, but the antibody did not inhibit actin assembly after attachment and spreading on substrates. These data indicate that gel contraction is dependent on α‐sm actin expression and that α‐sm actin is a functional marker for a fibroblast subtype that rapidly remodels the extracellular matrix. © 1994 wiley‐Liss, Inc.
Electrogenic NaK pump current in rat skeletal myoballs
Authors: Li Kai‐Xun , Sperelakis Nicholas 
Published: February 2005

AbstractCatecholamines and insulin have been reported to hyperpolarize skeletal muscle fibers via stimulation of the electrogenic Na‐K pump (Flatman and Clausen, 1979, Nature, 281:580–581). Therefore, the electrogenic Na‐K pump current was investigated in cultured colcemid‐treated rat skeletal myoballs using whole‐cell voltage clamp. Skeletal muscles were taken from newborn rat hindlegs, trypsin digested, and cultured. By day 7, all myoblast cells fused into myotubes. After treatment with the microtubule disrupter colcemid (10−7 M) for 2 days, some of the myotubes became transformed into spherical myoballs, having an average diameter of 41.2 ± 1.5 μm (n = 21). The resting membrane potential averaged ‐56.8 ± 1.7 mV (n = 40). Ouabain (1 mM) quickly depolarized the myoballs to ‐51.1 ± 1.1 mV (n = 27), showing the existence of an electrogenic Na‐K pump in the skeletal myoball preparation. The values for the specific membrane resistance and capacitance were 5.5 ± 1.0 KΩ‐cm2 (n = 21) and 3.7 ± 0.3 μF/cm2 (n = 21), respectively. The pump current averaged 0.28 ± 0.03 pA/pF (n = 10), with the membrane potential at ‐60 mV and 10 mM intrapipette Na+. The Na‐K pump contribution to resting membrane potential was calculated to be 5.7 mV, matching the ouabain‐induced rapid depolarization. When the Na‐K pump was stimulated with 50 mM intrapipette Na+, the pump current was about doubled (0.52 ± 0.08 pA/pF; n = 10). Isoproterenol (1 μM) and 8‐Br‐cAMP (1 mM) also significantly increased pump current by 50% (0.42 ± 0.04 pA/pF; n = 9) and 64% (0.46 ± 0.09 pA/pF; n = 7), respectively. In contrast, although insulin and phorbol ester also increased pump current, this increase was not statistically significant. The ineffectiveness of insulin and phorbol ester may be due to colcemid interfering with Na‐K pump translocation from internal vesicles to the sarcolemma. © 1994 wiley‐Liss, Inc.
Role of membrane surface potential and other factors in the uptake of non‐transferrin‐bound iron by reticulocytes
Authors: Quail Elizabeth A. , Morgan Evan H. 
Published: February 2005

AbstractReticulocytes suspended in low ionic strength media such as isotonic sucrose solution efficiently take up non‐transferrin‐bound iron and utilize it for heme synthesis. The present study was undertaken to determine how such media facilitate iron utilization by the cells. The effects of changes in membrane surface potential, membrane permeability, cell size, transmembrane potential difference, oxidation state of the iron, and lipid peroxidation were investigated. Iron uptake to heme, cytosol, and stromal fractions of cells was measured using rabbit reticulo‐cytes incubated with 59Fe‐labelled Fe(II) in 0.27 M sucrose, pH 6.5. Suspension of the cells in sucrose led to increased membrane permeability, loss of intracellular K+, decreased cell size, and increased transmembrane potential difference. However, none of these changes could account for the high efficiency of iron uptake which was observed. The large negative membrane surface potential which occurs in sucrose was modified by the addition of mono‐, di‐, tri‐, and polyvalent cations to the solution. This inhibited iron uptake to a degree which for many cations varied with their valency. Other cations (Mn2+, Co2+, Ni2+, Zn2+) were also very potent inhibitors, probably due to direct action on the transport process. Ferricyanide inhibited iron uptake, while ferrocyanide and ascorbate increased the uptake of Fe(III) but not Fe(II). It is concluded that the high negative surface potential of reticulocytes suspended in sucrose solution facilitates iron uptake by aiding the approach of iron to the transport site on the cell membrane. The iron is probably transported into the cell in the ferrous form. © 1994 wiley‐Liss, Inc.
Nitric oxide and energy production in articular chondrocytes
Authors: Stefanovic‐Racic M. , Stadler J. , Georgescu H. I. , Evans C. H. 
Published: February 2005

AbstractAddition of human, recombinant interleukin‐1β (hrIL‐1β) to cultures of lapine articular chondrocytes provoked a delayed increase in the production of both nitric oxide (NO) and lactate. These two phenomena followed a similar time course and shared a parallel dose‐response sensitivity to hrIL‐1β. A causal relationship is suggested by the ability of N‐monomethyl‐L‐arginine (NMA), an inhibitor of NO synthase, to blunt the glycolytic response to hrIL‐1β. Furthermore, addition of S‐nitroso‐N‐acetylpenicillamine (SNAP), which spontaneously generates NO in culture, increased lactate production to the same degree as IL‐1. However, 8‐Br‐cGMP and isobutylmethylxanthine (IBMX) had no effect either in the presence or absence of IL‐1. Even under standard, aerobic, cell culture conditions, chondrocytes consumed little oxygen, either in the presence or absence of IL‐1 or NMA. Furthermore, cyanide at concentrations up to 100 μM had no effect upon NO synthesis or lactate production. Thus, the increases in glycolysis under study were not secondary to reduced mitochondrial activity. Although cells treated with IL‐1 had increased rates of glycolysis, their concentrations of ATP fell below those of untreated chondrocytes in a time‐dependent, but NMA‐independent, manner. Transforming growth factor‐β (TGF‐β) and synovial cytokines (CAF) also increased lactate production. However, TGF‐β failed to induce NO, and its effect on glycolysis was independent of NMA. Furthermore, cells treated with TGF‐β were not depleted in ATP. These data are consistent with hypotheses that rates of proteoglycan synthesis are, in part, regulated by the intracellular concentration of ATP or by changes in pericellular pH. These two possibilities are not mutually exclusive. © 1994 wiley‐Liss, Inc.
Changes in mitochondrial shape and distribution induced by ethacrynic acid and the transient formation of a mitochondrial reticulum
Authors: Soltys Bohdan J. , Gupta Radhey S. 
Published: February 2005

AbstractWe have examined the effect of ethacrynic acid on mitochondrial morphology and distribution as well as on cellular toxicity in cultured human fibroblasts, African Green Monkey B‐SC‐1 kidney cells, and Chinese hamster ovary cells. Treatment of the above cells with 66 μM ethacrynic acid causes no reduction in cell viability after 2 h but is cytotoxic upon prolonged (6–7 days) exposure. Ethacrynic acid treatment for up to 2 h is found to cause novel shape changes and redistribution of mitochondria, as assessed by immunofluorescence and electron microscopy. Early effects include the transient formation of a mitochondrial reticulum involving the majority of mitochondria, and these reticula are aligned along microtubules. At later times within 2 h, mitochondrial distributions become disoriented (show no association with microtubules), and an aggregation and final positioning of mitochondria around the nucleus is observed. Whole mount electron microscopy shows that mitochondria in treated cells increase in length and form junctions, indicating reticula result from mitochondrial fusion. Electron microscopy of sections through ethacrynic acid induced reticula demonstrates structural continuity in mitochondria at branch points and the presence of regular cristae. Staining of endoplasmic reticulum and mitochondria in intact cells with the cyanine dye 3,3′‐dihexyloxacarbocyanine iodide provides evidence of concurrent aggregation of endoplasmic reticulum. Rhodamine 123 staining of living cells followed by immunofluorescent labeling of mitochondria in the same cells indicates that all mitochondria retain a transmembrane potential during the druginduced shape changes and redistributions. The described effects of ethacrynic acid on mitochondrial morphology as well as on cellular toxicity are completely prevented by 0.5 mM dithiothreitol, indicating that ethacrynic acid is acting as a sulfhydryl reagent to produce the observed effects. The above observations also indicate that ethacrynic acid effects on mitochondrial morphology are an early event in the drug‐induced cytotoxicity. The generation of varied mitochondrial morphologies by fusion and fission of mitochondria and its modulation by agents such as ethacrynic acid are discussed. © 1994 wiley‐Liss, Inc.
Staurosporine, a non‐specific PKC inhibitor, induces keratinocyte differentiation and raises intracellular calcium, but Ro31–8220, a specific inhibitor, does not
Authors: Jones K. T. , Sharpe G. R. 
Published: February 2005

AbstractThe responsiveness of normal human keratinocytes to different modulators of protein kinase C (PKC) was investigated. The PKC agonist TPA, staurosporine (a non‐specific inhibitor), and Ro31–8220 (a specific inhibitor) were studied for effect on cell morphology, growth rate, involucrin expression, and intracellular calcium levels. Surprisingly the response to nanomolar concentrations of staurosporine was similar to TPA and induced a fusiform morphology, inhibited growth, increased involucrin levels, and raised intracellular calcium. Staurosporine also increased the number of cornified envelopes, and its action therefore appeared identical to TPA. In contrast, Ro31–8220 had little effect on morphology or growth and blocked both the TPA‐induced growth inhibition and calcium rise. Ro31–8220 had no effect on staurosporine‐induced growth inhibition but partially reduced its associated calcium rise. These results suggest PKC activation is required for keratinocyte differentiation and that staurosporine acts like a PKC agonist to give a similar effect as TPA. Specific inhibition of PKC by Ro31–8220 inhibits TPA‐induced differentiation. © 1994 wiley‐Liss, Inc.
Role of cell density/cell‐cell contact, and growth state in expression of differentiated properties by the LLC‐PK1 Cell
Authors: Amsler Kurt 
Published: February 2005

AbstractPopulations of the renal epithelial cell line, LLC‐PK,1, acquire many properties characteristic of the proximal tubular cell at confluence. At confluence cells both enter a nonproliferative state and develop extensive cell‐cell contacts. To determine if one or both factors is responsible for acquisition of the differentiated phenotype, growth arrest was initiated in populations of varying densities by two procedures (serum deprivation and thymidine block) and expression of several differentiated properties (Na‐hexose symport activity, gamma‐glutamyl transpeptidase activity, alkaline phosphatase activity, and villin protein) was examined. Induction of growth arrest resulted in expression of all differentiated properties even in subconfluent populations. The level of expression in a population was proportional to cell density at the initiation of growth arrest; higher density was associated with increased expression. Evidence indicated the existence of some minimal density below which cells could not express detectable levels of differentiated properties in response to induction of growth arrest. The procedure used to initiate growth arrest did not affect this behavior, indicating that initiation of cell growth arrest rather than hormone deprivation was the inducing factor. These results indicate that both cell growth state and cell density independently modulate expression of differentiated properties by the LLC‐PK1 cell. These results are incorporated into a model in which cells in the absence of “appropriate” cell‐cell contact arrest at a differentiation‐incompetent cell cycle point. In the presence of appropriate cell‐cell contact (as yet undefined) cells arrest at a distinct differentiation‐competent cell cycle point and initiate expression of the differentiated phenotype. © 1994 wiley‐Liss, Inc.
Engagement of the T‐cell antigen receptor by anti‐CD3 monoclonal antibody causes a rapid increase in lymphocyte F‐actin
Authors: Phatak Pradyumna D. , Packman Charles H. 
Published: February 2005

AbstractActivation of protein kinase C (PKC) causes a rapid and sustained increase in the F‐actin of T lymphocytes. Because the phosphatidylinositol pathway and the cytoskeleton play a role in lymphocyte activation, we examined the relationship between signal transduction and the F‐actin increase in human blood T cells. Anti‐CD3 monoclonal antibodies (mAbs) initiate signals which result in activation of T lymphocytes through the T‐cell receptor (TCR), involving the phosphatidylinositol pathway, activation of PKC, and increasing intracellular calcium (Cai2+). The fluorescent probe NBD‐phallacidin was used to examine the conformational state of actin following stimulation of T lymphocytes with anti‐CD3 mAb. Each of three different murine anti‐CD3 mAbs caused rapid increases in lymphocytic F‐actin content, which was enhanced by cross‐linking with a goat anti‐mouse IgG. A maximally effective dose of the mAb Leu 4 caused a rise in cellular F‐actin of 1.8‐fold at 2 minutes and a three‐fold increase in Cai2+. Ionomycin, 100 nM, caused a Cai2+ rise similar in magnitude to that caused by anti‐CD3 mAb but had no effect on F‐actin content. Inhibitors of PKC, 1(5‐isoquinolinylsulfonyl)‐2methylpiperazine (H7), sphingosine, and sphinganine lowered the resting cellular F‐actin and partially blocked the increase in F‐actin caused by either anti‐CD3 mAb or ionomycin; however, they had no effect on the rise in Cai2+. Cells leached of Ca2+ with EGTA and ionomycin exhibited no Cai2+ increase in response to anti‐CD3 mAb or ionomycin; such cells retained the F‐actin increase caused by anti‐CD3 mAb. We conclude that stimulation of human T lymphocytes via the TCR causes an early rapid increase in F‐actin content. Activation of PKC may play a role but the concomitant Cai2+ increase is neither sufficient nor necessary for the F‐actin increase. © 1994 wiley‐Liss, Inc.