bluuebunny/crossref_metadata_2025_split huggingface dataset:
Maturation‐dependent regulation of protein kinase C activity by vitamin D3 metabolites in chondrocyte cultures
Authors: Sylvia V. L. , Schwartz Z. , Schuman L. , Morgan R. T. , Mackey S. , Gomez R. , Boyan B. D. 
Published: February 2005

AbstractVitamin D3 metabolites regulate the differentiation of chondrocytes isolated from the growth zone or resting zone of rat costochondral cartilage. Since some of the direct membrane effects of vitamin D metabolites are nongenomic, we hypothesized that protein kinase C (PKC) plays a role in signal transduction for these chondrocyte differentiation factors and that the regulation of PKC by the vitamin D metabolites is cell maturation dependent. Confluent, fourth passage cultures of growth zone and resting zone chondrocytes were treated with vitamin D3 metabolites for up to 24 h, lysed, and cell extracts assayed for kinase activity using a specific PKC substrate peptide. The addition of 1,25‐(OH)2D3 to growth zone cell cultures resulted in a rapid dose‐dependent stimulation of PKC, significant at 10−9‐10−7 M, beginning at 3 min and sustained until 90 min; 1,25‐(OH)2D3 had no effect on PKC activity in resting zone chondrocyte cultures. The addition of 24,25‐(OH)2D3 to resting zone cultures showed a slower PKC activation, with significant stimulation seen at 90–360 min for 10−8‐10−7 M 24,25‐(OH)2D3. However, 24,25‐(OH)2D3 had no effect on PKC activity in growth zone cell cultures at all times and concentrations examined. The specificity of PKC stimulation by the vitamin D3 metabolites was verified using a specific pseudosubstrate region peptide inhibitor, which reduced PKC activity when included in the reaction mixture. Pretreatment of the cultures with U73,122, a phospholipase C inhibitor, decreased 1,25‐(OH)2D3—stimulated PKC activity but had no effect upon 24,25‐(OH)2D3–induced activity. The tyrosine kinase inhibitor, genistein, did not inhibit the PKC response in either vitamin D3 metabolites‐treated culture. Neither actinomycin D nor cycloheximide affected 1,25‐(OH)2D3–induced PKC activity in growth zone chondrocyte cultures, while both compounds inhibited 24,25‐(OH)2D3—indiuced activity in resting zone chondrocyte cultures. The results of this study indicate that vitamin D metabolites stimulate PKC activity in a metabolite‐ and cell‐maturation‐specific manner. Effects of 1,25‐(OH)2D3 appear to be nongenomic, whereas the effects of 24,25‐(OH)2D3 probably involve a genomic mechanism. © 1993 Wiley‐Liss, Inc.
Heparin‐like molecules bind differentially to prion‐proteins and change their intracellular metabolic fate
Authors: Gabizon Ruth , Meiner Zeev , Halimi Michele , Ben‐Sasson Shmuel A. 
Published: February 2005

AbstractPrPSc is the only known component of the scrapie prion. The difference between PrPSc and its normal isoform PrPc is probably conformational, since no difference has been found in the amino acid sequence or postranslational modifications between both proteins. Heparan sulfate (HS) has been shown to be a component of amyloid plaques in a number of diseases including the prion diseases. We now present evidence that PrP can specifically bind to heparin‐like compounds and that this interaction might have a physiological significance. HS can increase the concentration of PrP in normal neuroblastoma cells, whereas low molecular weight heparin (LMWH) does not. In contrast, LMWH and other heparin‐like molecules, excluding HS, can inhibit the synthesis of PrPSc in scrapie infected cells and reverse their phenotype back to normal as judged by measurement of PrPSc by immunoblotting and by infectivity experiments. Whether an interaction between PrP and glycosaminoglycans plays a direct role in the conversion of PrPc into PrPSc remains to be established. © 1993 Wiley‐Liss, Inc.
Specific simple sugars promote chemotaxis and chemokinesis of corneal endothelial cells
Authors: Vogel Tikva , Blake Diane A. , Whikehart David R. , Guo Neng‐Hua , Zabrenetzky Vivian S. , Roberts David D. 
Published: February 2005

AbstractBovine corneal endothelial cells showed a strong migratory response to specific simple sugars (D‐glucose and sucrose, but not L‐glucose, sorbitol, lactose, or D‐galactose) at concentrations above 10 mM. Checkerboard analysis of the migratory responses in modified Boyden chambers indicated both chemotactic and chemokinetic effects. Serum starvation of the cultures increased the chemotaxis towards D‐glucose and 2‐deoxy‐D‐glucose, but not towards sucrose. Migration to sucrose and glucose was inhibited by chelation of extracellular calcium or by inhibition of Na+,K+ ATPase with ouabain. To date, this migratory response has been found only in corneal endothelial cells. Neither human melanoma cells, human breast carcinoma cells, bovine aortic endothelial cells, nor bovine microvascular endothelial cells migrated towards simple sugars, although all cell types migrated toward fibronectin in chemotaxis assays. After 16–19 passages in culture, bovine corneal endothelial cells retained their ability to migrate towards fibronectin, but lost their ability to migrate towards sugars. This loss of migratory response was accompanied by a sevenfold decrease in Na+,K+ ATPase activity. Although loss of Na+,K+ ATPase activity accompanied the loss of migratory response, pretreatment of cell cultures with 25 mM glucose did not stimulate, but rather lowered Na+,K+ ATPase activity in low or high passage cultures. © 1993 Wiley‐Liss, Inc.
Coupling of glucose transport and phosphorylation in Xenopus oocytes and cultured cells: Determination of the rate‐limiting step
Authors: Whitesell Richard R. , Aboumrad Mireille K. , Powers Alvin C. , Regen David M. , Le Chinh , Beechem Joseph M. , May James M. , Abumrad Nada A. 
Published: February 2005

AbstractThe initial events in glucose metabolism by all cells are the transport and phosphorylation of glucose. To quantify the relative contributions of these two processes to overall glucose utilization, we have developed an experimental approach for their in situ measurement as parallel processes. The method is based on the use of intracellular [2‐3H]glucose as a substrate for both the transporter and hexokinase, and involves simultaneous measurement of [2‐3H]glucose efflux and of 3H2O released by phosphorylation. The Xenopus oocyte expression system was used to test the method, since in these cells transport and phosphory lation activities can be regulated by expression of mRNA or injection of foreign protein. Oocytes microinjected with [2‐3H]glucose showed no release of injected glucose, but did have saturable phosphorylation kinetics, with a Km of 40 7μM and a Vmax of 0.1 nmol/min/oocyte. Co‐injection of yeast hexokinase increased glucose phosphorylation by five‐fold. Expression of human glucose transporter (GLUT1) mRNA resulted in a 25‐30‐fold increase in the rate of saturable efflux of microinjected glucose compared to control oocytes. The kinetics of transport and phosphorylation of [2‐3H]glucose were analyzed by a multiple curve‐fitting program that provided estimates of kinetic coefficients for both processes from a single time course. The analysis showed that expression of GLUT1 shifted the rate‐limiting step in glucose utilization from transport to phosphorylation. A similar shift occurred at a three‐fold lower extracellular concentration of 2‐deoxyglucose. In a pancreatic beta cell line both transport and phosphorylation showed high Km values, with phosphorylation as the limiting step. The in situ measurement of glucose transport and phosphorylation as parallel processes should be useful in defining the relative contributions of each step to overall glucose metabolism in other cell and tissue models. © 1993 Wiley‐Liss, Inc.
Response of stratified cultures of human keratinocytes to disruption of proteoglycan synthesis by p‐nitrophenyl‐β‐D‐xylopyranoside
Authors: Haggerty John G. , Bretton Randolph H. , Milstone Leonard M. 
Published: February 2005

AbstractProteoglycans play a role in regulating proliferation and adhesion of cells to each other and to the basal lamina. Synthesis of proteoglycans is disrupted by β‐xylosides, which serve as alternate substrate sites for glycosaminoglycan chain attachment and therefore prevent glycosylation of the core protein. We have investigated the effects of p‐nitrophenyl‐β‐D‐xylopyranoside (PNP‐xyloside) on cultured human keratinocytes. Stratified cultures were incubated for 7 days with PNP‐xyloside (0.05–2.0 mM). Concentrations as low as 0.05 mM increased the secretion of free chondroitin sulfate by 10–15‐fold over untreated cultures. Cellassociated proteoglycan decreased as PNP‐xyloside concentration increased. At 2 mM PNP‐xyloside, heparan sulfate as well as chondroitin sulfate addition to core proteins was disrupted: the core protein of epican, a heparan sulfate form of CD44 found on keratinocytes, was detected immunologically but lacked heparan sulfate. 2.0 mM PNP‐xyloside reduced the number of attached cells by 20–25% after 7 days, but had little effect on morphology or protein synthesis. These results indicate that intact proteoglycans are not critical for maintaining epidermal keratinocyte stratification, cell‐cell adhesion, or growth. © 1994 Wiley‐Liss, Inc.
Injuries to cultivated BJA‐B cells by ajoene, a garlic‐derived natural compound: Cell viability, glutathione metabolism, and pools of acidic amino acids
Authors: Scharfenberg Klaus , Ryll Thomas , Wagner Roland , Wagner Karl G. 
Published: February 2005

AbstractAjoene (4,5,9‐trithiadodeca‐1,6,11‐triene‐9‐oxide), a garlic‐derived natural compound, which had been shown to have cytostatic/cytotoxic properties, was tested with a B cell lymphoma‐derived cell line (BJA‐B cells) in order to elucidate its mechanism of cytotoxic action. Viability of the cells was determined by the Trypan blue exclusion test and the colorimetric tetrazolium (MTT) assay, whereas metabolic disturbance was evaluated by measuring the pools of reduced (GSH), oxidized glutathione (GSSG) and the acidic amino acids, Glu and Asp. Fast uptake of ajoene was accompanied by an immediate reduction of the CSH and increase in the GSSG levels. The extent of these changes, as well as the further development of the metabolite pools, depended on the ajoene dose per cell. At a sublethal ajoene dose the GSH and GSSG pools rose at the later stages to levels much higher than in the control experiment. Bleb formation at the cytoplasmic membrane was a further rapid phenomenon, although injuries detected by Trypan blue exclusion developed only at a later stage. The MTT assay, performed in a parallel experiment (48 h after ajoene addition), showed, however, that reduction of cell viability was established at the very beginning of ajoene exposure. Altogether, the action of ajoene strongly resembled oxidative stress (i.e., interference with SH homeostasis and its pleiotropic consequences to cell physiology and metabolism). © 1994 Wiley‐Liss, Inc.
Transrepression of type II collagen by TGF‐β and FGF is protein kinase C dependent and is mediated through regulatory sequences in the promoter and first intron
Authors: Bradham Douglass M. , Wiesche Beatrix In Der , Precht Patricia , Balakir Richard , Horton Walter 
Published: February 2005

AbstractTransforming growth factor beta and basic fibroblast growth factor are multipotential factors found in bone and cartilage that may be involved in both the proliferation and differentiation of chondrocytes. It was previously reported that TGF‐β plus FGF caused a modulation of chondrocyte phenotype that included the downregulation of steady‐state level of the collagen II transcript. In this report, the results of nuclear run‐off data indicate that repression of transcript initiation from the collagen II gene is the primary mechanism involved in the growth factor induced inhibition. Transient transfection assays with CAT expression vectors containing portions of the collagen II gene show that the TGF‐β/FGF induced transrepression requires a region in the first intron previously reported to have transcriptional enhancer activity and to bind chondrocyte nuclear proteins. In addition, silencer elements in the promoter also appear to play a role. Protein data as well as transient transfection experiments indicate that the activation of protein kinase C is necessary for the growth factor–induced down‐regulation of collagen II expression. These studies suggest that a cascade initiating with PKC activation is responsible for modifying transcription factors that interact with regulatory sequences in the collagen II gene. A detailed understanding of the factors involved in cartilage‐specific gene regulation in chondrocytes would facilitate development of therapeutic protocols for the repair of degenerated cartilage in diseases such as osteoarthritis. © 1994 Wiley‐Liss, Inc.
Regulation of insulin‐like growth factor binding protein synthesis and secretion in human retinal pigment epithelial cells
Authors: Feldman Eva L. , Randolph Ann E. 
Published: February 2005

AbstractCultured human retinal pigment epithelial cells (RPE) secrete insulin‐like growth factor binding proteins (IGFBPs), a family of polypeptides which modulate the actions of the insulin‐like growth factors. RPE cells secrete two IGFBPs with Mr estimates of 34,000 and 46,000, respectively. Treatment of RPE cells with IGF‐I markedly stimulated the secretion of the 46,000 Mr form. This stimulation occurred via an IGF‐I receptor independent mechanism because both [QAYL]IGF‐I (an IGF‐I analogue with decreased affinity for the IGFBPs but normal affinity for the IGF‐I receptor) and α‐IR3 (a blocking monoclonal antibody against the IGF‐I receptor) had no effect on IGF‐I stimulated increases in IGFBPs. Additionally, [QAYL]IGF‐I enhanced RPE cell proliferation to the same magnitude as IGF‐I. Treatment with IGF‐I, [QAYL]IGF‐I, or α‐IR3 had no effect on steady‐state levels of the 2.5 kb IGFBP‐3 or the 1.3 kb IGFBP‐6 mRNA transcripts as measured by Northern blotting and quantitative autoradiography. Forskolin and a group of candidate growth factors, including platelet‐derived growth factor, epidermal growth factor, and acidic and basic fibroblast growth factor, modestly increased IGFBP secretion when compared to untreated cells, but these effects were small when compared to IGF‐I treatment. Fetal calf serum enhanced the presence of the 2.5 kb IGFBP‐3 mRNA transcript in a dose‐dependent fashion but had no effect on the 1.3 kb IGFBP‐6 mRNA transcript. IGF‐I, forskolin, and the candidate growth factors had no effect on either IGFBP‐3 or IGFBP‐6 mRNA. These data suggest that the production of IGFBPs in human RPE cells is regulated by distinct mechanisms which include (1) an IGF‐I receptor independent interaction of IGF‐I with secreted IGFBPs and (2) de novo synthesis of IGFBPs by serum‐containing factors. © 1994 Wiley‐Liss, Inc.
Invadopodia promote proteolysis of a wide variety of extracellular matrix proteins
Authors: Kelly Thomas , Mueller Susette C. , Yeh Yunyun , Chen Wen‐Tien 
Published: February 2005

AbstractChicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus invade the extracellular matrix (ECM) using plasma membrane protrusions, termed invadopodia, that contact and dissolve the matrix. NormalThroughout this manuscript normal cells refers to chicken embryo fibroblasts and transformed cells refers to these cells infected with Rous sarcoma virus.
cells neither form invadopodia nor degrade the ECM. Here we show that cells expressing invadopodia degrade and enter into a fibronectin‐rich matrix produced by normal fibroblasts. Within 6 h after seeding onto the matrix, the invasive cells create an area devoid of matrix fibrils surrounding the cell body. Proteolysis mediates this matrix clearing because sevenfold more radiolabeled matrix is released into the growth media by the transformed cells relative to the normal cells. In addition to this assembled matrix, transformed cells were grown on thin layers of purified ECM proteins, revealing that invadopodia can degrade fibronectin, collagen type I, collagen type IV, and laminin. A 160 kDa protease that is extracted from transformed cells by Triton X‐114 partitions into the detergent phase and is prominent in ventral plasma membranes that contact the ECM suggesting that it is a membrane associated protease. © 1994 Wiley‐Liss, Inc.
DNA strand breakage during physiological apoptosis of the embryonic chick lens: Free 3' OH end single strand breaks do not accumulate even in the presence of a cation‐independent deoxyribonuclease
Authors: Chaudun E. , Arruti C. , Courtois Y. , Ferrag F. , Jeanny J. C. , Patel B. N. , Skidmore C. , Torriclia A. , Counis M. F. 
Published: February 2005

AbstractEpithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosmes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single‐strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP‐ribose)n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP‐ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double‐strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation. © 1994 Wiley‐Liss, Inc.
Activated platelet‐derived growth factor autocrine pathway drives the transformed phenotype of a human glioblastoma cell line
Authors: Vassbotn Flemming S. , Östman Arne , Langeland Nina , Holmsen Holm , Westermark Bengt , Heldin Carl‐Henrik , Nistér Monica 
Published: February 2005

AbstractHuman glioblastoma cells (A172) were found to concomitantly express PDGF‐BB and PDGF β‐receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up‐regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U343MGa 31L) or on Ki‐ras‐transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transtormed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists. © 1994 Wiley‐Liss, Inc.
Involvement of alkaline phosphatase in the modulation of receptor signaling in osteoblasts: Evidence for a difference between human parathyroid hormone‐related protein and human parathyroid hormone
Authors: Fukayama Shoichi , Tashjian Armen H. 
Published: February 2005

AbstractWe have previously reported that alkaline phosphatase (ALPase) is functionally involved in calcium uptake by several osteoblast‐like cell lines. We have extended these studies to investigate the actions of ALPase on the cAMP response to and the receptor binding of human parathyroid hormone (hPTH) and human parathyroid hormone‐related protein (hPTHrP). Pretreatment of human osteoblast‐like SaOS‐2 cells with human placental ALPase (hpALPase) inhibited the cAMP response to hPTH(1‐34) but had no effect on the actions of hPTHrP(1‐34) or vasoactive intestinal peptide. The inhibitory effect was reversed by L‐Phe‐Gly‐Gly, an inhibitor of hpALPase. Treatment of SaOS‐2 cells with hpALPase modestly reduced the binding of hPTH to 70% of control values, with little or no effect on the binding of hPTHrP. Bovine kidney and calf intestine ALPases were without effect on either the cAMP response or binding of hPTH or hPTHrP in SaOS‐2 cells. In rat osteoblast‐like ROS 17/2.8 cells, hpALPase had no effect on cAMP production stimulated by hPTH(1‐34) or hPTHrP(1‐34), arguing against a nonspecific effect of hpALPase. We suggest that, in SaOS‐2 cells, the common PTH/PTHrP receptor can differentiate between the agonist activities of hPTH and hPTHrP by a mechanism that is sensitive to hpALPase. © 1994 Wiley‐Liss, Inc.
Distinct protein tyrosine phosphorylation during mitogenesis induced in quiescent sv40‐transformed 3T3 T cells by insulin or vanadate
Authors: Wang Hanlin , Scott Robert E. 
Published: February 2005

AbstractInsulin and vanadate selectively induce mitogenesis in quiescent SV40 large T antigen‐transformed 3T3 T cells (CSV3–1) but not in quiescent nontransformed 3T3 T cells. Insulin and vanadate mediate this effect in CSV3–1 cells by distinct signal transduction mechanisms that involve protein tyrosine kinase activity. To further study these processes, changes in protein tyrosine phosphorylation induced by insulin and vanadate were investigated. Using immunoprecipitation and Western blotting techniques with antiphosphotyrosine antibodies, we report distinct protein phosphorylation characteristics in insulin‐ and vanadate‐stimulated CSV3–1 cells. The insulin receptor β‐subunit is phosphorylated within 2 min after insulin stimulation of transformed CSV3–1 cells. Insulin also stimulates a rapid increase in tyrosine phosphorylation of the 170 kDa insulin receptor substrate‐1 and complex formation between the phosphorylated insulin receptor substrate‐1 and the 85 kDa subunit of phosphatidylinositol 3'‐kinase. In contrast, vanadate does not initially increase detectable phosphorylation of any proteins, including neither the insulin receptor nor the insulin receptor substrate‐1. After 60 min, however, a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins is observed in vanadate‐treated CSV3–1 cells. Furthermore, treatment of CSV3–1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation but only minimally inhibits the effects of insulin. Finally, insulin stimulates the phosphorytion of a 33 kDa protein, whereas vanadate does not. By comparison, in nontransformed 3T3 T cells, insulin induces a delayed and weaker tyrosine phosphorylation of the insulin receptor β‐subunit and vanadate does not enhance the tyrosine phosphorylation of the 55 and 64 kDa proteins. These data together indicate that the mitogenic effects of insulin and vanadate are associated with distinct protein phosphorylation patterns that appear to be differentially regulated in SV40‐transformed and nontransformed 3T3 T cells. © 1994 Wiley‐Liss, Inc.