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J J Sauk

Publications and source records attributed to J J Sauk.

At least 19 recordsLinked to original sources
PubMed1995

Hsp47 and cyclophilin B traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate vesicles. A role for Hsp47 and cyclophilin B in the export of procollagen from the endoplasmic reticulum.

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for concentration of procollagen for ER export.

Amino Acid Isomerases↗
PubMed1995

Osteopontin adhesion receptors on gingival fibroblasts.

Osteopontin (OPN) promotes attachment and spreading of cells in an RGD dependent fashion, suggesting that OPN interacts with integrins on cell surfaces. Here in, we show that LM-609, a monoclonal antibody to the alpha v beta 3 integrin (a vitronectin receptor), inhibited OPN-mediated attachment of gingival fibroblasts. To characterize the cell surface receptors responsible for this interaction, we performed OPN-sepharose affinity chromatography using detergent extracts of 35S-methionine or 125I-surface labeled gingival fibroblasts. Proteins bound to the OPN-matrix were eluted with EDTA and subjected to SDS-PAGE under reducing conditions. EDTA eluates from both 125I-surface labeled and 35S-methionine labeled extracts demonstrated prominent bands in the 90kDa and 50kDa regions, by both autoradiography and fluorography, respectively. These studies suggest that OPN is associated with other cell surface molecules in addition to alpha v beta 3. Furthermore, these as yet to be characterized proteins, may prove to have a stronger affinity for OPN than alpha v beta 3.

Cell Adhesion↗
PubMed1992

Lead inhibits secretion of osteonectin/SPARC without significantly altering collagen or Hsp47 production in osteoblast-like ROS 17/2.8 cells.

In an effort to better understand the consequences of lead (Pb2+) on skeletal growth, the effects of Pb2+ were investigated using ROS 17/2.8 bone-like cells in vitro. These studies revealed that Pb2+ (4.5 x 10(-6) M -4.5 x 10(-7) M) has little or no effect on cell shape except when added immediately following seeding of the cells. However, proliferation of ROS cells was inhibited, in the absence of serum, at concentrations of 4.5 x 10(-6) M Pb2+. Protein production was generally increased, however, the major structural protein of bone, type I collagen, production was only slightly altered. Following treatment of ROS cells with Pb2+, intracellular levels of the calcium-binding protein osteonectin/SPARC were increased. Osteonectin/SPARC secretion into the media was delayed or inhibited. Coincident with retention of osteonectin/SPARC there was a decrease in the levels of osteonectin/SPARC mRNA as determined by Northern analysis. These studies suggest that processes associated with osteonectin/SPARC translation and secretion are sensitive to Pb2+.

Animals↗
PubMed1992

Biological requirements for material integration.

The recognition that synthetic devices can provide functional replacements for failed teeth, or for previously edentulous areas, has resulted in increased emphasis being placed on understanding of the interactions between synthetic materials and host tissues in order for the success of these devices to be optimized. A key to achievement of an optimal biological interface between the implant and the surrounding tissue is through an understanding of host response to materials. This article reviews the biological requirements for implant-tissue integration, with specific focus on the role of adhesion molecules and cytokines (growth factors) in this process. Adhesion molecule/cytokine interactions are discussed, and in particular the possible role for osteopontin, an adhesion molecule as well as a cytokine, is considered in wound healing. Finally, the causes of peri-implantitis are discussed, and methods of decontamination are presented. The decontamination methods focus on enhancement of cell adhesion and integration to the altered implant surface.

Cell Adhesion Molecules↗
PubMed1992

Molecular control of dentinogenesis: a reaction.

This paper represents an invited reaction to three papers presented at the International Conference on Pathobiology of the Dentin/Pulp Complex, June, 1991. Repair dentinogenesis following transplantation into normal and germs free animals are correlated with results elucidating the expression of dentin phosphoproteins, collagen, and osteocalcin. The importance of transcription and translation controls of dentin matrix components are discussed and reviewed. In addition, possible implications of a molecular chaperone protein, Hsp47, in controlling dentinogenesis is introduced. Future research directions are developed and include: (a) identification of odontoblast precursors; (b) delineation of markers for odontoblasts at varying degrees of differentiation; (c) characterization of environmental conditions leading to odontoblast differentiation; (d) determination of the nature of repair and regenerated tissues; (e) elucidation of transcription and translation control factors, and (f) mapping the human genome for dentin matrix constituents.

Collagen↗
PubMed1992

Mineral-associated adhesion proteins are linked to root formation.

There is a general agreement that the extracellular environment plays a critical role in controlling cell behavior. Thus, significant research efforts have focused on understanding the effects of extracellular matrix proteins on cell function. In particular we have focused on determining the role of adhesion proteins in the regulation of root formation. Using an OPN antibody, 2arN (generously provided by Drs Craig and Denhardt), the expression of OPN during root formation was determined. OPN (osteopontin) is a bone-associated adhesion protein. OPN was expressed in the dental follicle region of molars obtained from 3 day old CD-1 mice, but was not expressed in the odontoblast layer. In contrast by day 8, positive staining was noted in the odontoblast layer, as well as in the area of Hertwig's epithelial root sheath. However, at this same time point no positive labeling for 2arN was observed in the enamel organ or in the dental papillae cells. By day 15 positive staining for OPN was seen in the area of the periodontal ligament, as well as the region of primary deposition of extracellular matrix onto dentin. Also determined was the ability of fibronectin, OPN and dentin sialoprotein (DSP) to promote the attachment of dental ectomesenchymal cells, in vitro. Interestingly, these cells attached remarkable well on bacteriological dishes (control) in the absence of an adhesion protein. DSP did not increase cell attachment beyond that observed for control cells. In contrast, both fibronectin and OPN enhanced cell attachment. These studies, while preliminary indicate that OPN is expressed in a unique fashion during root development, thus suggesting a regulatory role for such adhesion proteins during root formation.

Animals↗
PubMed1991

Use of a phosphophoryn-Ca(+2)-collagen composition that mimics a mineralization front in unicortical defects in long bones.

The present study was designed to ascertain if dynamic ionic matrices that mimic the mineralization front could be used as active scaffolds for bone repair. Dentinal phosphophoryn calcium salts were extracted from unerupted bovine dentine using chatopic buffers and EDTA. The phosphophoryns were subsequently isolated following precipitation with CaCl2. The phosphophoryn-Ca+2 salts were then mixed with pepsin solubilized bovine skin collagen and lyophilized into hardened sponges. Three groups of 4 beagle dogs were employed such that one leg served as an experimental test site for a mechanical wound, while the contralateral leg served as a control. Animals were sacrificed at 1,3, and 6 month intervals. The test specimens were harvested, fixed, and processed for routine histology, examined with image histomorphometric analysis, and scored. Tabulation of these data indicated that phosphophoryn-Ca(+2)-collagen enhances the repair of mechanically formed osseous defects in the distal femur of beagle dogs. This enhanced rate of bone repair was manifest by earlier filling of bony voids with osteoid and trabecular bone. Eventually, this process was followed by recortification of the surface defects. These data suggest that components derived from a mineralization front may influence bone formation in unicortical defects within long bones.

Animals↗
PubMed1991

Diverse forms of stress result in changes in cellular levels of osteonectin/SPARC without altering mRNA levels in osteoligament cells.

The osteonectin/SPARC gene has been shown to possess motifs for a heat shock element and metal responsiveness. Also, the expression of the protein has been associated with culture stress in endothelial cells. In the present study, osteoligament (OL) cells derived from the patellar ligament were subjected to diverse forms of stress that included (a) exposure to sodium arsenite, (b) heat shock, (c) cadmium ion, and (d) the amino acid analog, AZC. Osteonectin/SPARC levels in OL cells were determined by Western blot analyses, and immunoprecipitation using antiosteonectin antibodies. Expression of osteonectin/SPARC mRNA was determined by Northern analysis using a 1.5 kb EcoRI restriction fragment of bovine osteonectin cDNA. These studies reveal that osteonectin/SPARC is produced following diverse forms of stress, however, the levels are lower than observed in unchallenged OL cells. In all instances, the mRNA levels were comparable to control cells. These studies indicate that expression of osteonectin/SPARC mRNA is tightly controlled in OL cells and that the protein may be regulated at the level of protein translation.

Animals↗
PubMed1991

Cell attachment activity of cementum: bone sialoprotein II identified in cementum.

Considerable research effort has been directed at preparing root surfaces in a fashion that would promote cell attachment leading to periodontal regeneration; however, no methods have proven to be clinically predictable. Identification of attachment protein(s) associated with the root surface matrix of cementum may prove valuable for developing effective clinical treatments. In this study cementum proteins were extracted from bovine and human teeth by sequential chaotropic extraction using guanidine followed by guanidine/EDTA. The guanidine/EDTA extract, but not guanidine extract, was found to promote attachment of fibroblasts. This attachment activity was inhibitable with synthetic peptide containing the attachment sequence arginine-glycine-aspartic acid (RGD). Fractionation of the guanidine/EDTA extract revealed several fractions with attachment activity. Immunoblot analysis demonstrated that two of these fractions contain the bone-associated RGD containing attachment protein, bone sialoprotein-II (BSP-II). In addition, attachment activity was also noted in other fractions that could not be attributed to BSP-II or fibronectin. These studies indicate that a component of the attachment activity of cementum is likely to be due to BSP-II and that cementum contains additional, as yet undetermined, attachment proteins.

Amino Acid Sequence↗
PubMed1991

Physiology of bone: mineral compartment proteins as candidates for environmental perturbation by lead.

Termine et al. first demonstrated that sequential dissociative extraction and fractionation procedures with protease inhibitors could provide a convenient approach for the study of mineral compartment constituents. The primary extraction regimen used 4 M guanidine HCl to remove most of the protein from the nonmineralized phase of bone. Subsequently, EDTA-guanidine was used to remove the mineral-phase components. These methods discriminate on the basis of physical-chemical association with a mineral phase rather than on the specific gene products of a particular cell. In the present discussion emphasis is directed at a group of divalent cation binding proteins isolated from the mineral compartment of bone. The localization, synthesis, and chemical characteristics of osteonectin, bone sialoproteins I and II, and bone acidic glycoprotein-75 are discussed and offered as possible sites for perturbation by the environment with lead exposure.

Amino Acid Sequence↗
PubMed1991

Human periodontal cells initiate mineral-like nodules in vitro.

A primary objective in the treatment of periodontal diseases is the regeneration of the mineralized and soft connective tissue components of the attachment apparatus. Current theories suggest the cells of the periodontium have the capacity, when appropriately triggered, to actively participate in restoring connective tissues, including mineralized tissues. To evaluate further the role of such cells in periodontal homeostasis, periodontal ligament (PDL) cells and gingival fibroblasts (GF) were cultured and examined for alkaline phosphatase levels and for the ability to produce mineralized nodules in culture. These are two characteristics of osteoblast-like cells in vitro. The levels of alkaline phosphatase produced by these cells were determined by a modified kinetic assay and the ability of these cells to produce mineral-like nodules in vitro was evaluated by Von Kossa staining and light and electron microscopy. PDL cells had significantly higher levels of alkaline phosphatase when compared with gingival fibroblasts obtained from the same patient and the same passage, in vitro. Furthermore PDL cells, but not GF, were capable of producing mineral-like nodules in vitro. These results indicate differences in behavior between PDL cells and GF; such differences may prove important in designing appropriate clinical therapies directed at stimulating periodontal regeneration.

Alkaline Phosphatase↗
PubMed1990

Expression of constitutive and inducible HSP70 and HSP47 is enhanced in cells persistently spread on OPN1 or collagen.

Cells persistently spread on OPN or collagen survive heat shock better than cells transiently spread on fibronectin or tissue culture plates. Thus, a central question is whether constitutively or inducible stress proteins are enhanced in cells grown on adhesive proteins that maintain a persistent spread cell shape. Levels of Hsp 72,73, and colligin/Hsp47 were determined by Western blot analyses. The inducible Hsp 72 was prominently expressed following heat shock in cells grown on OPN or collagen, but not in cells plated on fibronectin coated substratum or on tissue culture plates. Colligin/Hsp 47 and Hsp 73 manifested a similar pattern of expression indicating that these adhesive attachment proteins accommodate cell function through organization of cell architecture.

Cell Adhesion↗
PubMed1990

The expression of colligin/hsp47 after stress in human periodontal fibroblasts in vitro.

Fibroblasts from human periodontal ligaments were grown in vitro. The levels of collagen and total protein in these cells were compared with subject- and passage-matched gingival fibroblasts. In 3 subjects the levels of both were greater in ligament than in gingival fibroblasts. These increased levels were also associated with increased levels of proteins reacting with anti-colligin/hsp47 antibodies on SDS-PAGE. Ligament and gingival fibroblasts were subjected to heat shock, sodium arsenite and the amino acid analogue AZC. These studies showed that (a) sodium arsenite and AZC enhanced the cellular levels of hsp47 in both types of fibroblast, (b) the colligin/hsp47 levels expressed were associated with elevated levels of protein and collagen production and (c) the presence of colligin/hsp47 was decreased under conditions of serum deprivation.

Arsenic↗
PubMed1990

Characteristics of human periodontal ligament cells in vitro.

Periodontal ligament cells may have a role in the regulation of hard and soft periodontal tissues, but their specific function has yet to be determined. To evaluate further their role in periodontal homeostasis, they were examined for osteoblast-like behaviour; in vitro no characteristic osteoblastic responsiveness was found. Periodontal ligament cells gave a PGE2- and isoproterenol-mediated cAMP response, but did not respond in a similar fashion to calcitonin or PTH. When exposed to PGE2, isoproterenol, or 1,25(OH)2 vitamin D3, they did not exhibit an increase in protein production, as measured by [35S]-methionine incorporation. Immunofluorescent localization indicated that periodontal ligament cells produce a bone-associated protein, osteonectin. In addition, mRNA levels for osteonectin and bone proteoglycan I (biglycan) were detected in these cells, in vitro. This information should help to clarify the role such cells play in the regulation of periodontal tissues.

Biglycan↗
PubMed1990

Persistent spreading of ligament cells on osteopontin/bone sialoprotein-I or collagen enhances tolerance to heat shock.

Fibronectin (FN), bone sialoprotein-I (BSP-I), Type I collagen, and a number of synthetic peptides containing the integrin attachment sequence (RDG) were evaluated for their ability to affect stress tolerance in osteo-ligament cells (OL). The attachment and spreading of OL cells was determined by the method of Klebe (1974) and Akiyama et al. (1986). Survival from heat shock was evaluated after the methods of Gerner et al. (1976). These studies showed that FN, BSP-I, and synthetic RGD peptides enhance attachment of OL cells. Increased survival from heat was limited to cells spread on fibronectin, BSP-I, and Type I collagen. OL cells that persistently spread on BSP-I and Type I collagen had more survivors than cells demonstrating transient spreading on FN. These studies indicate that (a) cell spreading is a prerequisite for stress tolerance and (b) enhanced stress tolerance is mediated by protein sequences other than those immediately surrounding the RGD sites in native proteins.

Animals↗
PubMed1990

Expression of attachment proteins during cementogenesis.

There is general agreement that during development the extracellular environment plays a critical role in controlling cell differentiation. Data generated from numerous studies support the possibility that cell attachment proteins and their corresponding cell receptors are possible candidates for this role. In particular, our studies are directed at identifying attachment proteins in mature cementum and establishing the function of these proteins during root formation. Fractionation of guanidine HCL/EDTA extracts of cementum revealed the presence of a bone-associated attachment, BSP, as well as fractions containing as of yet undetermined attachment proteins. Immunofluorescent examination of 1st molar tissues during root formation, obtained from 7 day-old mice neonates, for bone-associated attachment proteins indicated that osteopontin is expressed in the area of Hertwig's epithelial root sheath, but not in the region of the dental papillae. However, dental papillae cells, considered to have the capacity to form cementum, attached to osteopontin coated dishes, in vitro. Thus, unique attachment proteins, as well as those previously identified, were found in mature cementum and during root development. Future studies focused on identifying attachment proteins of mature cementum and determining the spatial and temporal localization of these proteins, pre- and post-cementogenesis, will provide important information necessary for establishing the function of these proteins during root development.

Animals↗
PubMed1989

Cell attachment activity of the 44 kilodalton bone phosphoprotein is not restricted to bone cells.

Proteins that promote cell migration, attachment and spreading are considered to play an important role in the regulation of cell function. Recently, a 44 kilodalton bone phosphoprotein (44K BPP) was shown to enhance the attachment of gingival fibroblasts and osteoblasts in vitro. The potential importance of this attachment protein in the regulation of mineralized tissue homeostasis prompted us to evaluate its ability to promote the attachment and migration of several other cell types. All the fibroblast cell lines and non-transformed calvaria cell lines assayed exhibited enhanced attachment and spreading in response to 44K BPP. Rat osteosarcoma cells (ROS 17/2.8) expressing osteoblast-like features, exhibited enhanced attachment in response to 44K BPP, while non-osteoblast-like cells (ROS 25/1) obtained from the same osteosarcoma did not. Two epithelial cell lines, CCL4 and A431, demonstrated enhanced attachment when exposed to fibronectin or laminin, but not 44K BPP. Another epithelial-like cell line, HT 1080, derived from a fibrosarcoma, showed enhanced attachment in the presence of all three attachment proteins. Fibronectin, but not 44K BPP, promoted the chemotactic migration of fibroblasts. These studies indicate that the role of 44K BPP attachment protein in the regulation of cell behavior is not restricted to bone cells.

Animals↗