bluuebunny/crossref_metadata_2025_split huggingface dataset:
Temporal studies on the tissue compartmentalization of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during bone formation In Vitro
Authors: Kasugai Shohei , Nagata Toshihiko , Sodek Jaro 
Published: February 2005

AbstractTo study the role of noncollagenous proteins in bone formation, the synthesis and tissue distribution of BSP (bone sialoprotein), OPN (osteopontin) and SPARC (secreted protein acidic and rich in cysteine) were analyzed using pulse‐chase and continuous labeling protocols during bone formation by cultures of rat calvarial cells. Following a 1 h labeling period with [35S]methionine or [35SO4], radiolabeled BSP was rapidly lost from the cells and appeared transiently in the culture medium and in a 4 M GuHCI extract (G1) of the mineralized tissue. Coinciding with the loss of BSP from these compartments, radiolabeled BSP increased in demineralizing, 0.5 M EDTA extracts (E) of the bone, in a subsequent GuHCI extract (G2), and in a bacterial collagenase digest (CD fraction) of the extracted tissue, over a 24 h chase period. In comparison, the 55 kDa form of OPN, with a small amount of the 44 kDa OPN, was secreted almost entirely into the culture medium. Most of the 44 kDa OPN, together with some 55 kDa OPN, accumulated rapidly in the E extract but could not be detected in either G extract or in the CD fraction. SPARC appeared transiently in the G1 extract, but was otherwise quantitatively secreted into the culture medium from where it was lost by complexing and/or degradation. When cultures were continuously labeled over a 12 day period with [35S]methionine, radiolabeled BSP and 44 kDa OPN accumulated in the E extract together with a small amount of SPARC. Some radiolabeled BSP also accumulated in the G2 extract. From the relative incorporation of [35SO4] over the same time period, a time‐dependent loss in sulphate from the BSP was evident. Using a 24 h pulse‐labeling protocol, the amount of radiolabeled BSP and OPN in the E extract and the BSP in the G2 extract were not altered significantly over a 12‐day chase period. These studies demonstrate that the 44 kDa OPN and most of the BSP are rapidly bound to the hydroxyapatite crystals where they may regulate crystal formation and growth during bone formation. Some BSP is deposited in the osteoid and appears to become masked by the formation of hydroxyapatite, indicating a potential role for this protein in epitactic nucleation of hydroxyapatite crystal formation. © 1992 Wiley‐Liss, Inc.
Macrophage membrane glycoprotein binding of Griffonia simplicifolia I‐B4 induces TNF‐alpha production and a tumoricidal response
Authors: Tabor Dale R. , Theus S. A. , Barnett John B. , Jacobs Richard F. 
Published: February 2005

AbstractThioglycollate‐elicited macrophages (mϕ), upon binding the lectin Griffonia simplicifolia IB4 (GSIB4) at the plasma membrane, are induced to secrete several low molecular weight proteins. In this investigation, results from specific ELISA and immunoprecipitation analysis of these molecules confirmed that the cytokine, tumor necrosis factor‐α (TNF‐α), belongs to the group of elicited proteins. This specific mϕ response is directly influenced by the dose of GSIB4 used and the time in contact with the cells. At 40 μg/ml GSIB4, the maximum dose of lectin used, the mϕ activity was equal to that achieved when the cells were incubated with an interferon‐γ/lipopolysaccaride (IFN/LPS) stimulus alone. Moreover, the data showed that TNF‐mediated tumoricidal activity was significantly influenced by GSIB4 binding to the mε membrane. When the lectin was incubated alone or in sequence with IFN/LPS, this ligand‐receptor binding promoted the lysis of WEHI 164 tumor target cells. However, concurrent incubation of both IFN/LPS and GSIB4 with mϕ significantly diminished the tumoricidal response. This suggested that one of the metabolic pathways utilized subsequent to receptor‐ligand binding was altered by these interactions. When cyclic AMP (cAMP) and inositol triphosphate (IP3) levels were examined, the results showed that the concentration of cAMP was unchanged despite the fact that IP3 levels were significantly enhanced upon mϕ‐GSIB4 binding. Collectively, the data show that GSIB4 binding to specific glycoproteins in the mϕ membrane induces TNF‐α production and facilitates TNF‐α dependent tumoricidal responses. It also appears that the transduction of the signal, in part, at least utilizes the phosphatidyl inositol pathway. Finally, it is noteworthy that mϕ activity is influenced by the sequence in which GSIB4 is presented to the mϕ relative to the IFN/LPS treatment. © 1992 Wiley‐Liss, Inc.
C‐Fos expression is hypersensitive to serum‐stimulation in cultured cystic kidney cells from the C57BL/6J‐cpk mouse
Authors: Rankin Carolyn A. , Grantham Jared J. , Calvet James P. 
Published: February 2005

AbstractCystic kidneys of the C57BL/6J‐cpk murine model of polycystic kidney disease show a marked overexpression of the proto‐oncogenes c‐fos, c‐myc, and c‐ki‐ras, consistent with an increased rate of cell proliferation and an altered state of differentiation. To determine if cystic cells have increased responsiveness to stimulation with mitogenic agents, quiescent primary cultures from normal and cystic cpk kidneys were treated with fetal bovine serum (FBS), 8‐bromo‐cAMP (cAMP), or epidermal growth factor (EGF). The level of c‐fos induction following stimulation by FBS was found to be dramatically higher in cystic cells than in normal cells; whereas induction by cAMP or EGF was essentially the same in both cell types and much less than that seen in FBS‐stimulated cells. To determine if this serum hypersensitivity reflects an increased proliferative state in vivo, c‐fos induction was examined in cultures derived from normal kidneys stimulated to regenerate by folic acid‐induced acute renal injury. As with cystic kidneys, the folic acid‐injured kidneys showed increased c‐fos responsiveness to FBS in cell culture. These experiments suggest that cystic and regenerating kidneys have an altered phenotypic state in vivo that is manifested in cell culture by serum hypersensitivity. However, whereas the folic acid‐injured kidneys ultimately reestablish normal kidney function, cystic kidneys further progress to renal failure, suggesting that cystic epithelial cells are locked in this altered state of differentiation. © 1992 Wiley‐Liss, Inc.
Relationship between culture conditions and the dependency on mitochondrial function of mammalian cell proliferation
Authors: Van Den Bogert C. , Spelbrink J. N. , Dekker H. L. 
Published: February 2005

AbstractIn cultured mammalian cells, the relationship was investigated between mitochondrial function and proliferation under various culture conditions. Continuous inhibition of the expression of the mitochondrial genome was used to reduce the activity of enzymes involved in oxidative phosphorylation by 50% at every cell division. Under these conditions, culturing in relatively poor media resulted in arrest of the proliferation of most cell lines after 1 cell division. This was preceded by decreasing levels of ATP and increasing levels of ADP, suggesting that the ATP‐generating capacity of the cells was limiting. Culturing in richer media led to arrest of the proliferation after 5 to 6 divisions, but accumulation of ADP was not observed. Addition of pyruvate to rich culture media and, at least for 1 cell line, increasing the CO2 levels, completely prevented proliferation arrest. Inability to synthesise metabolic precursors via mitochondrial intermediary metabolism probably explains growth arrest of cells cultured in rich media. Pyruvate and CO2 were, however, without effect on the proliferation arrest of cells cultured in relatively poor media. Therefore, pyruvate dependency for growth of cells without functional mitochondria holds true only under culture conditions where the ATP‐generating capacity of the cells is not limiting. © 1992 Wiley‐Liss, Inc.
Proliferation‐associated oxygen consumption and morphology of tumor cells in monolayer and spheroid culture
Authors: Bredel‐Geissler A. , Karbach U. , Walenta S. , Vollrath L. , Mueller‐Klieser W. 
Published: February 2005

AbstractThe oxygen consumption rate, proliferative activity, and morphology of EMT6/Ro mouse mammary sarcoma cells in monolayer and multicellular spheroid culture have been investigated in a comparative study. During the transition of monolayer cells from the exponential into the plateau growth phase, there is a distinct decrease in the cellular volume that is associated with a corresponding decrease in the proliferative and respiratory activity of the cells. The decline in cell volume is mainly due to a decrease in the content of cytoplasm, whereas the size of the nucleus is only slightly reduced. A concomitant decrease in the number of mitochondria per cell obviously accounts for the reduction in cellular oxygen uptake. Despite a continuous decrease of cell proliferation from the surface to interior regions of EMT6 spheroids reflected by a gradient in tritiated thymidine labeling, volume‐related oxygen consumption is rather uniform in viable regions of these aggregates. The finding can be explained by the results of the morphometric evaluation showing a uniform volume density of mitochondria, i.e., of oxygenconsuming sites within these spheroids. © 1992 Wiley‐Liss, Inc.
Apoptosis of murine BW 5147 thymoma cells induced by cold shock
Authors: Kruman Inna I. , Gukovskaya Anna S. , Petrunyaka Victor V. , Beletsky Igor P. , Trepakova Elena S. 
Published: February 2005

AbstractExposure of thymoma BW 5147 cells to cold (0–2°C) followed by rewarming at 37°C (cold shock) resulted in internucleosomal DNA cleavage. Sensitivity to cold shock‐induced cell death was critically dependent on the serum concentrations in the medium and limited to serum‐deficient medium (2% serum concentration), whereas cells in the complete growth medium (10%) were completely resistant. RNA/protein‐synthesis inhibitors (cycloheximide and actinomicin D) had no effect on cold shock‐induced DNA cleavage in BW 5147 cells. The DNA fragmentation seems to be independent of increase in the cytosolic Ca2+ level. Moreover, reduction in the calcium content of the external medium by EGTA induced DNA cleavage. Incubation of BW 5147 cells in the presence of colchicine and cytochalasin B led to the apoptosis. The latter suggest that the internucleosomal DNA cleavage induced by cold shock may be concerned with the disruption of some cytoskeletal network caused by cooling. The results are discussed in relation to cell proliferation. © 1992 Wiley‐Liss, Inc.
Basic calcium phosphate crystals cause coordinate induction and secretion of collagenase and stromelysin
Authors: McCarthy Geraldine M. , Mitchell Peter G. , Struve Janine A. , Cheung Herman S. 
Published: February 2005

AbstractSynovial fluid basic calcium phosphate crystals (BCP) are often found in severely degenerated joints. Crystalline BCP is a growth factor stimulating fibroblast mitogenesis and acting as a compentence factor similar to platelet‐derived growth factor. In human fibroblasts (HF), the synthesis of collagenase and stromelysin is coordinately induced after stimulation with a variety of cytokines and growth factors. We sought to determine whether BCP, like other growth factors, might induce protease that would damage articular tissue. Northern blot analysis of mRNA for collagenase and stromelysin in HF stimulated with BCP was performed. Secreted enzymes were analysed by immunoblot using a monoclonal antibody to collagenase and by immunoprecipitation using a polyclonal antibody to stromelysin. Stromelysin activity was confirmed using casein substrate gels. A significant, dose‐dependent accumulation of collagenase and stromelysin message was evident after 4 h had continued for at least 24 h in BCP‐stimulated cultures. Fortynine and 54 kD proteins immunoreacting with collagenase antibody were identified in the conditioned media (CM) from BCP‐stimulated cultures while 50 and 55 kD proteins were identified by immunoprecipitation with stromelysin antibody. Collagenase activity was increased significantly in the CM from BCP treated cells; casein substrate gels showed casein degrading bands at molecular weights consitent with stomelysin. BCP stimulates coordinate induction of collagenase and stromelysin which may mediate the joint destruction associated with these crystals. © 1992 Wiley‐Liss, Inc.
Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: II. Effects of calmodulin and calmodulin inhibitors
Authors: Stout Marguerite A. , Silver Paul J. 
Published: February 2005

AbstractThe role of calmodulin (CaM) in modulating calcium (Ca) uptake by sarcoplasmic reticulum (SR) of vascular smooth muscle was studied in saponin skinned strips of rat caudal artery. Exogenous CaM concentrations ranging 0.3–1.8 μM did not statistically change the steady state MgATP‐dependent Ca content, the MgATP‐independent Ca content, or the oxalate‐stimulated Ca influx. Calmidazolium (CDZ), W‐7, and trifluoperazine (TFP) were used to examine the potential effect of an endogenous CaM pool on inward Ca transport. The IC50 of these antagonists for inhibiton of Ca‐CaM‐stimulated phosphodiesterase activity and Ca‐activated superprecipitiation of canine aortic actomyosin was measured and found to be be in the low micromolar range with a rank order of potency of inhibition of CDZ > TFP > W‐7. In skinned tissues, micromolar concentrations of antagonists that inhibited CaM‐mediated reactions in isolated enzyme systems did not reduce Ca content or oxalate‐stimulated Ca influx. At higher concentration of 100–200 μM, the MgATP‐dependent Ca content was significantly reduced by TFP and W‐7 but not by CDZ. The order of potency for inhibition of Ca uptake was TFP > W‐7 > CDZ. The MgATP‐independent Ca content was significantly decreased only by 200 μM TFP. Although none of these inhibitors significantly altered Ca efflux at concentrations up to 100 μM, Ca release was significantly stimulated by all three at 200 μM. The TFP‐stimulated Ca release was partially inhibited by ruthenium red. The results indicate that neither exogenous CaM nor an endogenous CaM pool directly modulates inward Ca transport by the SR of saponin skinned caudal artery. The inhibition of Ca uptake produced by hundred micromolar concentrations of CaM antagonists fails to correlate with the order of and with the potency of inhibition measured in isolated enzyme systems. This suggests that the inhibition of Ca uptake produced by high concentrations of these antagonists may be independent of a specific interaction with CaM. The activation of Ca release by high concentrations of CaM antagonists may involve a nonspecific increase in membrane permeability as well as modulation of a membrane Ca Chanel. © 1992 Wiley‐Liss, Inc.
Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone‐responsive adenylate cyclase in clonal osteoblast‐like cell by down‐regulating parathyroid hormone receptors
Authors: Katz Michael S. , Gutierrez Gloria E. , Mundy Gregory R. , Caulfield Michael P. , Hymer Tazuko K. , McKee Roberta L. 
Published: February 2005

AbstractThe effects of the monokines tumor necrosis factor α (TNF) and interleukin 1 (IL 1) on parathyroid hormone (PTH)‐responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR‐106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR‐106 cells for 48 hr each produced concentration‐dependent inhibition of PTH‐sensitive adeylate cyclase, with maximal inhibition of PTH response (40% for TNF. 24% for IL 1) occuring at 10−8 M of either monikine. Both monokines also decreased adenylate cyclase stimulation by the tumor‐derived PTH‐related Protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor‐mediated stimulation of adenylate cyclase by isoproterenol and nonreceptors‐mediated enzyme activation by cholera toxin forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radiodinated agonistt mono‐[125I]‐[Nle3, 18, Tyr34]bPTH‐(1–34)NH2 to UMR‐106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH‐(1–34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertusis toxin increased PTH‐sensitive adenylate cyclase activity but did not attenuate monokine‐induced inhibiton of PTH response. In time course studies, brief (1hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results and indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time protein synthesis‐dependent down‐regulation of PTH receptors linked to adenylate cyclase. © 1992 Wiley‐Liss, Inc.
Potassium transport in nonpigmented epithelial cells of ocular ciliary body: Inhibition of a Na+, K+, Cl− cotransporter by protein kinase C
Authors: Crook Richard B. , Von Brauchitsch Douglas K. , Polansky Jon R. 
Published: February 2005

AbstractThe mechanism by which 86Rb+ (used as a tracer for K+) enters human nonpigmented ciliary epithelial cells were investigated. Ouabain‐inhibitable bumetanide‐insensitive 86Rb+ transport accounted for ∼70–80% of total, whereas bumetanide‐inhibitable ouabain‐insensitive uptake accounted for 15–25% of total. K+ channel blockers such as BaCl2 reduced uptake by ∼5%. Bumetanide inhibited 86Rb+ uptake with an IC50 of 0.5 μM, while furosemide inhibited with an IC50 of about 20 μM. Bumetanide‐inhibitable 86Rb+ uptake was reduced in Na+‐free or Cl−‐free media, suggesting that Na+ and Cl− were required for optimal uptake via this mechanism. These characteristics are consistent with a Na+, K+, Cl− contransporter in NPE cells. Treatment of NPE cells for 15 min with phorbol 12‐myristate, 13‐acetate (PMA), an activator of protein kinase C, caused a 50–70% decrease in 86Rb+ uptake via the Na+, K+, Cl− cotransporter. Other 86Rb+ uptake mechanisms were not affected. 86Rb+ uptake via the Na+, K+, Cl− contransporter could be inhibited by other phorbol esters and by dioctanoylglycerol, an analog of diacylglycerol, but not by 4α phorbol didecanoate, an ineffective activator of protein kinase C. Staurosporine, a protein kinase C inhibitor, blocked phorbol ester inhibition of 86Rb+ uptake. These data suggest that a Na+, K+, Cl− cotransporter in NPE cells is inhibited by activation of protein kinase C. © 1992 Wiley‐Liss, Inc.
Transforming growth factor β1 and adrenocorticortropin differentially regulate the synthesis of adrenocortical cell heparan sulfate proteoglycans and their binding of basic fibroblast growth factor
Authors: Jiang Zhiwen , Savona Catherine , Chambaz Edmond M. , Feige Jean‐Jacques 
Published: February 2005

AbstractAdrenocortical differentiated functions are under the control of both endocrine hormones such as ACTH and local factors such as transforming growth factor β (TGFβ) or basic fibroblast growth factor (bFGF). Besides their regulatory actions on the synthesis of corticosteroids, these two classes of factors also exert some important effects on the cellular environment. We have examined here the regulation by ACTH and TGFβ of adrenocortical cell proteoglycan synthesis and secretion. Under basal conditions, adrenocortical cells synthesized and secreted several species of sulfated proteoglycans, 80% of them being recovered in solution in the culture medium. When analyzed by ion exchange chromatography, the cell extracts and the media from cells metabolically labeled with 35S‐suifate were found to contain two and three species of radioactive sulfated proteoglycans, respectively. All species were proteoheparan‐sulfates. Treatment of adrenocortical cells with TGFβ1 or ACTH resulted in a significant increase of the incorporation of 35S into both secreted and cell‐associated proteoglycans. ACTH stimulated more than three times the amount of secreted proteoglycans eluting from DEAE‐Trisacryl as peak B, whereas TGFβ preferentially increased the amount of peak C. No important modification of the size of the synthesized proteoglycans was observed. The subpopulation of heparan sulfate proteoglycans capable to bind bFGF was also largely increased after ACTH or TGFβ treatment and paralleled the variation in overall proteoheparan sulfate synthesis. Thus those effects of TGFβ and ACTH on proteoglycan synthesis may participate in an increased ability of adrenocortical cells to bind and respond to bFGF. © 1992 Wiley‐Liss, Inc.
Cellular and molecular regulation of factor V expression in human megakaryocytes
Authors: Gewirtz Alan M. , Shapiro Charles , Shen Yu Min , Boyd Robert , Colman Robert W. 
Published: February 2005

AbstractThe mechanisms responsible for regulating FV expression in normal human megakaryocytes are unknown. To test the hypothesis that they are related to cell maturation events, we correlated human megakaryocyte FV antigen content with several putative maturation markers including cell size, morphologic stage of development, and ploidy level. Mature megakaryocytes were isolated from normal marrow by counterflow centrifugal elutriation. The cells were immunofluorescently labeled with a monoclonal antibody probe (B10) directed against the FV connecting peptide (150 kDa) and then reacted with Chromomycin A3 to allow for simultaneous DNA quantitation in the same cell. After processing, individual cells were stages and sized. FV antigen and nuclear DNA levels (ploidy) were measured by microphotometric measurements of total cytoplasmic or nuclear fluorescence. A total of 1,006 cells were examined, of which 12% were stage I, 8% were stage II, 35% were stage III, and 45% were stage IV. The geometric mean diameter (± SD) of cells in these stages was 48.3 ± 11.8 μm2, 54.9 ± 14.4 μm2, 61.7 ± 20.02 μm2, and 56.7 ± 13.2 μm2, respectively. Respective ploidy values in arbitrary fluorescence units, where 2 N = 5%, were 28.2 ± 18.2%, 31.4 ± 19.3%, 54.3 ± 26.6%, and 33.2 ± 22.7%. Calculated correlation coefficients (r) and coefficient of determination (r2) values suggested that FV antigen levels varied independently of any of the maturation markers studied. However, FV antigen levels could be upregulated by 24 h exposure to 8 nM phorbol myristate acetate (PMA). Presence of FV mRNA in a pure population of megakaryocytes was demonstrated by in situ hybridization and the polymerase chain reaction. Relative levels of megakaryocyte FV mRNA, as assessed by in situ hybridization, failed to reveal a detectable change after PMA exposure in spite of an increase in detectable protein. These data suggest that FV synthesis may be regulated at the post‐transcriptional level and that it is subject to regulatory influences which are not coordinately linked to development of other terminal maturation markers.