bluuebunny/crossref_metadata_2025_split huggingface dataset:
Understanding bone cell biology requires an integrated approach: Reliable opportunities to study osteoclast biology in vivo
Authors: Cielinski Matthew J. , Marks Sandy C. 
Published: May 2005

AbstractThe relative simplicity of all in vitro methods to study bone cell biology will at best result in oversimplification of the development and functional capacity of the skeleton in vivo. We have shown this to be true for selected aspects of bone cell biology, but numerous other examples are available.One alternative is to undertake skeletal research in vivo. It is important that those in bone research be willing to move increasingly in this direction not only to understand the true complexitities of skeletal versatility, but also to avoid repetition and perpetuation of erroneous or irrelevant conclusions which waste resources.Toward this end we have described two situations, osteopetrosis and tooth eruption, in which reproducible abrogations or local activations of bone resorption can be examined in vivo. The application of emerging molecular and morphological techniques that permit the subcellular dissection of metabolic pathways and their precise cellular localization, such as a combination of the variety of in situ hybridzation technologies with PCR, antisense probes, and antibody blockase, will allow the investigator greater control of variables in vivo. We expect that these technologies, largely worked out in vitro, combined with highly selected, appropriate models, as we have oulined here for osteoclast biology worked out in vitro, combined with highly selected, appropriate models, as we have ourlined here for osteoclast biology, will make research in vivo less intimidating and increase the frequency with which the real biology is studied directly.
Nongenomic regulation of extracellular matrix events by vitamin D metabolites
Authors: Boyan Barbara D. , Dean D. D. , Sylvia V. L. , Schwartz Z. 
Published: May 2005

AbstractVitamin D metabolites appear to regulate chondrocytes and osteoblasts via a combination of genomic and nongenomic mechanisms. Specificity of the nongenomic response to either 1,25‐(OH)2D3 or 24, 25‐(OH)2D3 may be conferred by the chemical composition of the target membrane and its fluid mosaic structure, by the presence of specific membrane receptors, or by the interaction with classic Vitamin D receptors. Nongenomic effects have been shown to include changes in membrane fluidity, fatty acid acylation and reacylation, arachidonic acid metabolism and prostaglandin production, calcium ion flux, and protein kinaase C activity. Chondrocytes metabolize 25‐(OH)D3 to 1,25‐(OH)2D3 and 24,25‐(OH)2D3; production of these metabolites is regulated by both growth factors and hormones and is dependent on the state of cell maturation. 1,25‐(OH)2D3 and 24,25‐(OH)2D3 may interact directly with extracellular matix vesicles to regulate their function in the matrix, including protease activity, resulting in matrix modefication and calcification. Isolated matrix vesicles, produced by growth zone chondrocytes, can activate latent transforming growth factor‐β when incubated with exogenous 1,25‐(OH)2D3. These observations suggest that nongenomic regulation of martix vesicle structure and function may be a mechanism by which mesenchymal cells, like osteoblasts and chndrocytes, may modulate events in the extracellular matrix at sites distant from the cell surace.
Underlying mechanisms at the bone–biomaterial interface.
Authors: Schwartz Zvi , Boyan B. D. 
Published: May 2005

AbstractIn order to understand how biomaterials influence bone formation in vivo, it is necessary to examine Cellular response to materials in the context of wound healing. Four interrelated properties of biomaterials (chemical composition, surface energy, surface roughness, and surface topography) affect mesenchymal cells in vitro. Attachment, proliferation, metabolism, matrix synthesis, and differentiation of osteoblast‐like cell lines and primary chondrocytes are sensitive to one or more of these properties. The nature of the response depends on cell maturation state. Rarely do differentiated osteoblasts or chondrocytes see a mateial prior to its modification by biological fluids, immune cells and less differentiated mesenchymal cells in vivo. Studies using the rat marrow ablation model of endosteal wound healing indicate that ability of osteoblasts to synthesize and calcify their extracellular matrix is affected by the local presence of the material. Changes in the morphology and biochemistry of matriix vesicles, extracellular organelles associated with matrix maturation and calcification, seen in normal endosteal healing, are altered by implants. Moreover, the material exerts a systemic effect on endosteal healing as well. This may be due to local effects on gwoth factor production and secretion into the circulation, as well as to the fact that the implant may serve as a bioreactor.
Cytosolic phospholipase C activity: I. Evidence for coupling with cytosolic guanine nucleotide‐binding protein, Giα
Authors: Akompong Thomas , Spencer Robert L. , McEwen Bruce S. 
Published: May 2005

AbstractIn a previous report we shwed that glucocorticoed inhibition of cytosolic PLC activity correlated with a reduction in cytosolic Giα levels, suggesting that there may be a functional relationship between cytosolic PLC and cytosolic Giα. In order to establish the nature of the coupliing between cytosolic Giα and cytosolic PLC we examined the effects of Protein activators, and inhibitors on cytosolic PLC activity from rat spenocytes and the rat lymphoma cell line Nb 2, with [3H] PI and [3H]PIP2 as substrates. (1) Neither GTP nor its nonhydrolyzable analogue, GTPγS, at 100 μm had any effect on the calcium stimulated as well as the basal PLC activity. (2) Howevr, affinity purified antibodies to Giα1 and Giα2 inhibited soluble PLC activity, by 85% and 55%, respectively, with PI as substrate; with PIP2 as substrate, soluble PLC activity was inhibited 50–70% by antibodies to Gi1, whereas antibodies to Gi2 had little effect. (3)Administration of Giα1 antisense oligonucleotides to splenocytes for 48 h produced 25–40% decrease in cytosolic Giα1 levels compared to control. The soluble PLC activity with both PI and PIP2 as substrates was also reduced by 25–50% compared to control conditions. This suggest that cytosolic Giα is associated with the activation of splenocyte soluble PLC. (4) Pertussis toxin administered in vivo sugnificantly reduced cytosolic Giα immunoreactivity and soluble PLC activiry when PI was used as substrate, providing additional evidence that cytosolic Giα is associated with the activation of splencyte soluble PLC. (5) Another agent that has beeen used extensively to define G‐protein coupled processes is NaF/AlCl3. NaF(4mM; with or without AlCl3 inhibited soluble PLC activity with PIP2 as substrate, in contrast ot the stimulatory effect that has been reported in the activation of membrane PLC. 6) because NaF can act as a protein phosphatase inhibitor, we also tested the effects of trifluoperzine (50 μm, TFP), an inhibitor of protein phosphatase 2B; TFP (50 μm) signigicantly inhibited soluble PLC activity PI was used as substrate. These results suggest a direct involvement of cytosolic Giα in the activation of soluble PLC form splenocytes. Other questions pertaining to the functional significance, the nature, and possible substrate preference of the splenocyte Giα coupled PLC is addressed in the second paper.
Alterations in cellular gene expression without changes in nuclear matrix protein content
Authors: Macoska Jill , Hoover Carol N. , Pienta Kenneth J. 
Published: May 2005

AbstractCell metabolism and function are modulated in part by cell and nuclear shape. Nuclear shape is controlled by the nuclear matrix, the RNA‐protein skeleton the nucleus, and its interactions with cytoskeletal systems such as intermediate filaments and actin microfilaments. The nuclear matrix plays an important role in cell function and gene expression because active genes are bound to the nuclear matrix whereas inactive genes are not. It is unknown, however, how genes move on and off the matrix, and whether these events require compositional protein changes, i.e., alterations in protein content of the nuclear matrix, or other, more subtle alterations and/or modificatins. The purpose of this investigation was to begin to determine how nuclear matrix protein composition is related to gene expression. We demonstrate that gene expression can change without apparent changes in the protein composition of the nuclear matrix in MCF10A breast epithelial cells.
Expression of α1‐chimaerin (rac‐1 GAP) alters the cytoskeletal and adhesive properties of fibroblasts
Authors: Herrera Roman , Shivers Brenda D. 
Published: May 2005

AbstractThe small GTP‐binding protein rac‐1, a member of the ras gene superfamily of GTPases, is thought to be a key component of a signal transduction pathway that mediates cell membrane ruffling and actin stress fiber formation induced by growth factors. rac‐1 protein is regulated by the interplay of several activities:proteins that enhance GDP dissociation (GDP Dissociation Stimulator, GDS), inhibit nucleotide exchange (GDP Dissociation Inhibitor, GDI), or accelerate GTP hydrolysis (GTPase Activating Protein, GAP). We have assessed the relative contribution of the rac‐1/GAP interactions to the overall activity of rac‐1 by expressing α1‐chimaerin, a rac‐1‐specific GAP, in fibroblasts. NIH 3T3 cells were transfected with (α1)‐chimaerin‐expressing cells showed rac‐1 GAP activity that was regulated by phosphatidylserine and phorbol ester.The cells expressing α1‐chimaerin showed a distinct phenotype. They had altered adhesive properties as measured by their ability to bind to a fibronection‐coated glass surface, suggesting that the expression of a rac‐1 GAP alters the assembly of integrin receptors, actin and cytoskeletal proteins such as vinculin and talin. Direct demonstration of this phenomenon was achieved by studying the organization of actin stress fiber and formation of focal adhesions in the α1‐chimaerin expressing cells following stimulation by growth factors. Mock transfected cells, upon serum or lysophospatidic acid stimulation, organize actin as a dense array of parallel fibers running the length of the cell. This process did not take place in the cells expressing rac‐1 GAP. Similarly, the formation of focal adhesions as measured by the appearance of vinculin clusters was imparied in the α1‐chimaerin expressing cells. These results demonstrate that expression of a GAP for rac‐1 in fibroblasts produces profound changes in the cytoskeletal organization and suggest that GAP activity negatively regulates rac‐1 function.
Cell surface control of differentiation in acanthamoeba
Authors: Yang Shiguang , Villemez Clarence 
Published: May 2005

AbstractAcanthamoeba castellanii (Neff) is a free‐living soil amoeba with close relatives that are opportunistic pathogens. Trophozoites differentiatite into cysts when deprived of nutrients; cysts convert into trophozoites, leaving the well behind, in the presence of nutrients. The data presented here, which includes immunoaffinity purification of the receptor, indicate that cell surface molecular signals also control Acanthamoeba differentiation in both directions. Monoclonal antibodies that bind specifically to a 40 kD trophozoite protein initiate the encystment of trophozoites. When bound to cysts the same monoclonal antibodies prevent excystment. Washing away the antibody allows both trophozoites and cysts to resume normal activity. One of these monoclonal antibodies inhibits pinocytosis, while another has effect on pinocytosis.
Changes in the sulfation extent of membrane‐associated proteoglycans produced by sertoli cells in culture
Authors: Rodríguez Juan Pablo 
Published: May 2005

AbstractSertoli cells in culture synthesize two different membrane‐associated proteoglycans (MA‐PG): a proteoglycan containing heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycan (GAG) chains and a CS‐PG containing only CS‐GAG chains. The structure of these molecules is regulated by the presence of fetal calf serum (FCS) in the culture medium. Changes in the concentration of FCS resulted in changes in the total 35SO4 incorporation into MA‐PG and a shift in the elution profile of each component subjected to ion‐exchange chromatography. Thus, without FCS, the incorporation was low, while in 1% and 10% FCS, the uptake of the precursor was 1.7 and 4.5 times higher, respectively. MA‐PG synthesized by Sertoli cells cultured in 10% FCS eluted from DEAE‐Sephacel columns at higher salt concentration than the MA‐PG synthesized by cells cultured in 0% or 1% FCS. Double‐labeled experiments showed that the 35SO4/3H‐glucosamine ratio incorporated into MA‐PG produced by Sertoli cells, increased from 17.6 to 23.6 and 50.9 in cells cultured at 0, 1, and 10% FCS, respectively. However, the presence of FCS affected neither the hydrodynamic size nor the chemical nature of GAG chains of MA‐PG. These results show that changes in the FCS concentration promote changes in the sulfation extent of MA‐PG molecules produced by Sertoli cells.
Platelet talin is phosphorylated by calyculin A
Authors: Murata Kohei , Sakon Masato , Kambayashi Jun‐ichi , Okuyama Masaki , Hase Toshiharu , Mori Takesada 
Published: May 2005

AbstractCalyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin‐induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane‐cytoskeleton interaction through dephosphorylation of talin.
Generation of phosphorylcholine as an essential event in the activation of Raf‐1 and MAP‐kinases in growth factors‐induced mitogenic stimulation
Authors: Jiménez Benilde , del Peso Luis , Montaner Silvia , Esteve Pilar , Lacal Juan Carlos 
Published: May 2005

AbstractCell proliferation is regulated by an appropriate combination of intracellular signals involving activation of kinases and the generation of phospholipid metabolites. We report here that growth factors induce a biphasic generation of phosphorylcholine (PCho) in quiescent NIH 3T3 cells, resulting in an early and transient increase at 100 s and a larger and sustained increase after 3 h of stimulation. Generation of PCho at both early and late times of growth factors stimulation results from the consecutive activation of phospholipase D (PLD) and choline kinase (ChoK). Production of PCho by specific growth factors seems an essential requirement for the early signals associated to activation of Raf‐1 and MAP kinases, since blockage of choline kinase completely inhibited activation of Raf‐1 and MAP kinases by PDGF or FGF. Both the transient early increase and the late sustained increase in PCho are required for the induction of DNA‐synthesis, besides completion of the activation of the serine/threonine kinases cascade. Thus, our results strongly suggest that generation of PCho by the PLD/choline kinase pathway is one of the critical steps in regulating cell growth in NIH 3T3 stimulated by growth factors.
Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: Evidence for cellular processing of Ca2+ and Pi prior to matrix mineralization
Authors: Wu Licia N. Y. , Ishikawa Yoshinori , Sauer Glenn R. , Genge Brian R. , Mwale Fackson , Mishima Hiroyuki , Wuthier Roy E. 
Published: May 2005

AbstractAdvances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone‐related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca2+ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca2+ in their periphery and X‐ray microanalytical mapping revealed the presence of many Ca2+‐ and Pi‐rich cell surface structures ranging from filamentous processes 0.14 ± 0.02 μm by 0.5–2.0 μm, to spherical globules 0.70 ± 0.27 μm in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 ± 0.19 μm) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca2+‐rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca2+ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca2+ and Pi is directly involved in endochondral calcification.
SPARC mediates focal adhesion disassembly in endothelial cells through a follistatin‐like region and the Ca2+‐binding EF‐hand
Authors: Murphy‐Ullrich Joanne E. , Lane Timothy F. , Pallero Manuel A. , Sage E. Helene 
Published: May 2005

AbstractSPARC is a one of a group of extracellular matrix proteins that regulate cell adhesion through a loss of focal adhesion plaques from spread cells. We previously reported that SPARC reduced the number of bovine aortic endothelial (BAE) cells positive for focal adhesions [Murphy‐Ullrich et al. (1991): J Cell Biol 115:1127–1136]. We have now characterized the effect of SPARC on the cytoskeleton of BAE cells. Addition of SPARC to spread BAE cells caused a dose‐dependent loss of focal adhesion‐positive cells, that was maximal at ∼ 1 μg/ml (0.03 μM). Consistent with the loss of adhesion plaques as detected by interference reflection microscopy, vinculin appeared diffuse and F‐actin was redistributed to the periphery of cells incubated with SPARC. However, the distribution of the integrin αvβ3 remained clustered in a plaque‐like distribution. These data, and the observation that SPARC binds to BAE cells but not to the extracellular matrix, indicate that SPARC acts via interactions with cell surface molecules and not by steric/physical disruption of integrin‐extracellular matrix ligands. To determine the region(s) of SPARC that mediate a loss of focal adhesions, we tested peptides from the four distinct regions of SPARC. The cationic, cysteine‐rich peptide 2.1 (amino acids 54–73) and the Ca2+‐binding EF‐hand‐containing peptide 4.2 (amino acids 254–273) were active in focal adhesion disassembly. Furthermore, antibodies specific for these regions neutralized the focal adhesion‐labilizing activity of SPARC. These results are consistent with previous data showing that peptide 2.1 and 4.2 interact with BAE cell surface proteins and indicate that the loss of focal adhesions from endothelial cells exposed to SPARC is a receptor‐mediated event.
Increased PKC activity in cultured human keratinocytes and fibroblasts after treatment with 1α,25‐dihydroxyvitamin D3
Authors: Hanafin Nancy M. , Persons Kelly Scott , Holick Michael F. 
Published: May 2005

Abstract1α,25‐Dihydroxyvitamin D3 (10−12 M to 10−8 M) caused a dose dependent increase in PKC activity in the solubilized membrane fractions of cultured human keratinocytes and in the cytosolic fractions of cultured human fibroblasts. Maximum activity was induced by 1α,25‐dihydroxyvitamin D3 at 24 h. Sphingosine, which is believed to inhibit PKC mediated biological responses, blunted 1α,25(OH)2D3′s inducement of PKC activity in both keratinocytes and fibroblasts. Identical hormone treatment of vitamin D receptor deficient fibroblasts did not increase PKC activity. Treatment of keratinocytes and fibroblasts with 1β,25‐dihydroxyvitamin D3, which is believed to be ineffective in inducing genomic responses, did not induce PKC activity.
Osteocalcin cluster: Implications for functional studies
Authors: Desbois Christelle , Karsenty Gerard 
Published: May 2005

AbstractOsteocalcin is a skeletal member of the family of extracellular mineral binding Gla protein. Osteocalcin is synthesized only by the osteoblast and it is secreted into the bone matrix at the time of bone mineralization. The mineral binding properties of osteocalcin as well as its spatial and temporal pattern of expression suggest that it plays a role during bone mineralization, however until now its biological function is unclear. To understand osteocalcin function during skeletogenesis we mutated the two osteocalcin genes by homologous recombination in embryonic stem (ES) cells. Eight targeted clones were identified by Southern analysis using external probes. One of these clones contributed to the germ line of mouse chimera. Interbreeding of heterozygotes is currently in progress. Mutant mice will be useful to understand osteocalcin function in vivo.
Bone cell function, regulation, and communication: A role for nitric oxide
Authors: Collin‐Osdoby Patricia , Nickols G. Allen , Osdoby Philip 
Published: May 2005

AbstractA large array of factors serve as vital communication links between cells and the characterization, regulation, and mechanisms of action of such factors are topics of intense research efforts. Most intercellular messenger molecules which have been described over the years are represented by proteins, small peptides, amino acids or their derivatives, ions, lipid metabolites, or steroids. However, a small uncharged free radical, nitric oxide, has recently garnered much attention as a potent multifunctional signal molecule with widespread actions within and between diverse tissues. Biochemical, molecular, and regulatory studies of the family of enzymes responsible for nitric oxide synthesis, nitric oxide synthases, have established that there are at least three distinct isoforms of this enzyme which are differentially expressed and regulated in various cells or tissues. Modulation of these isoenzyme levels or activities by diverse signals is mediated via transcriptional, translational, and/or post‐translational mechanisms, and consequently, alterations in such control may influence normal or pathological processes. Nitric oxide appears to exert pronounced effects on skeletal physiology and its production by various bone cells, elicited target cell responses, modulation by other signalling molecules (e.g., cytokines, hormones, fatty acid derivatives), and chemical interactions with other free radicals (e.g., superoxide anions, hydroxyl radicals) may form one important facet of the many complicated communication pathways controlling bone cell physiology and remodeling. Further cell and molecular studies are needed to address the precise roles that nitric oxide plays in bone development and in the formation and degradation of bone during ordinary bone metabolism. In addition, alterations in the regulation and action of the bone nitric oxide system as a function of certain bone disorders may be manifested by perturbations in bone integrity or mineral homeostasis. In this article, we review the current evidence implicating nitric oxide as an important messenger molecule in bone intercellular communication, speculate on potential roles for this radical in bone biology, and discuss possible future directions for advanced research into the function of nitric oxide in skeletal physiology.