Biomedical subjects

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↗

PubMed1994

Hsp47 and the translation-translocation machinery cooperate in the production of alpha 1(I) chains of type I procollagen.

Hsp47, an endoplasmic reticulum resident protein, has gelatin-binding and procollagen-binding properties and has been hypothesized to function as a molecular chaperone in regulating procollagen folding and/or assembly. In this report, we further investigate the interaction of Hsp47 with polysome-associated alpha 1(I) procollagen chains following antisense treatment of 3T6 cells. For these studies, we employed phosphorothioate oligodeoxynucleotides directed to the first five codons of Hsp47 that straddle the predicted translation initiation site of mouse Hsp47. Cells depleted of Hsp47 in this manner were observed to produce diminished amounts of fully elongated nascent alpha 1(I) procollagen while accumulating shorter procollagen peptides associated with peptidyl-tRNA. Pulse-labeling of cells with [35S]methionine followed by treatment with puromycin and immunoprecipitation with anti-Hsp47 and anti-procollagen antibodies revealed that Hsp47 is associated with alpha 1(I) procollagen at a very early point during translocation of the nascent procollagen chains. Although Hsp47 appears to possess properties similar to grp78/BiP, Hsp47 binding early during translocation favors a more specialized specific function relative to chain selection or completion of stable folding in type I procollagen.

Animals↗

PubMed1994

Association of Hsp47, Grp78, and Grp94 with procollagen supports the successive or coupled action of molecular chaperones.

Hsp47, Grp78, and Grp94 have been implicated with procollagen maturation events. In particular, Hsp47 has been shown to nascent procollagen alpha 1(I) chains in the course of synthesis and/or translocation into the endoplasmic reticulum (ER). Although, Hsp47 binding to gelatin and collagen has previously been suggested to be independent of ATP. Grp78 and Grp94 are known to dissociate from its substrates by an ATP-dependent release mechanism. The early association of Hsp47 with procollagen and its relatively late release suggested that other chaperones, Grp78 and Grp94, interact successively or concurrently with Hsp47. Herein, we examined how these events occur in cells metabolically stressed by depletion of ATP. In cells depleted of ATP, the release of Hsp47, Grp78, and Grp94 from maturing procollagen is delayed. Thus, in cells experiencing metabolic stress, newly synthesized procollagen unable to properly fold became stably bound to a complex of molecular chaperones. In that Hsp47, Grp78, and Grp94 could be recovered with nascent procollagen and as oligomers in ATP depleted cells suggests that these chaperones function in a series of coupled or successive reactions.

2,2'-Dipyridyl↗

PubMed1994

Decrease of heat shock protein 27/28 with heat stress in HTLV-I-transformed cells.

We have previously reported an increase of human T-lymphotropic leukemia/lymphoma virus type I (HTLV-I) replication after heat treatment of MT-2 cells, an HTLV-I-transformed human lymphoid cell line. In this study, we investigated the effect of heat stress on the expression of Hsp27/28 in MT-2 cells. In contrast with previous studies of other cell types, a decrease of Hsp27/28 expression in MT-2 cells and an increase of Hsp70 family proteins in both MT-2 and uninfected lymphoid CEM cells were found following heat treatment at 42 degrees C. Furthermore, heat treatment resulted in an early rapid increase in the phosphorylated form of Hsp27/28 in both MT-2 and CEM cells. The results suggest that early post-translational phosphorylation of HSP27/28 could be a determinant of the ability of MT-2 cells to survive hyperthermia.

Blotting, Northern↗

PubMed1994

Dynamic variations in the expression of type I collagen and its molecular chaperone Hsp47 in cells of the mouse dental follicle during tooth eruption.

Tooth eruption is a precisely timed and sequenced event that brings the tooth from within bone into a functional position in the mouth. Every part of the developing tooth has been theoretically implicated as a primary factor in this process, but it now appears that eruption is multifactorial, with the dental follicle and type I collagen playing an important part. Immunological probes were used here to investigate in vivo and in vitro the temporal and spatial expression of type I collagen and its molecular chaperone Hsp47 in the dental follicle during eruption. Mandibles were dissected from 2-, 5-, 9- and 11-day-old neonatal mice and fixed in 95% ethanol overnight. Sections of 7 microns were obtained and reacted with antibodies directed against type I collagen. Dental follicles were isolated from 2-, 5-, 9- and 11-day-old neonates and cells were grown in culture for 8 days. Slides were then reacted with antibodies directed against type I collagen and Hsp47. The production of type I collagen and Hsp47 in the follicle varied with the stage of dental development and eruption. There was a progressive decrease of type I collagen in the coronal part of the follicle, leading to an arrest of its production in these areas. These findings support the notion that cells of the coronal portion of the dental follicle stop producing type I collagen as a prerequisite to the initiation of tooth eruption and that this phenotype persists in vitro.

Animals↗

PubMed1994

Loss of heterozygosity involving the APC gene in oral squamous cell carcinomas.

The tumor suppressor gene adenomatous polyposis coli has been shown to be altered in colon and esophageal cancers. Because of similar causes of oral and esophageal cancers, we investigated allelic deletion of the adenomatous polyposis coli gene in oral cancers by examining tumor cells of persons normally heterozygous at a polymorphic restriction site in adenomatous polyposis coli. Deoxyribonucleic acid was extracted from 20 formalin-fixed microdissected sections and one fresh specimen of oral squamous cell carcinomas and amplified with the use of the polymerase chain reaction. The amplified deoxyribonucleic acid was digested with Rsa I, subjected to polyacrylamide gel electrophoresis, and examined for loss of heterozygosity in adenomatous polyposis coli alleles. Samples from nine persons were homozygous for the adenomatous polyposis coli restriction site in both tumor and normal tissues and thus were uninformative. Three of the 12 samples from heterozygous persons showed loss of one adenomatous polyposis coli allele in tumor tissues. The loss of an adenomatous polyposis coli gene allele in 25% of the carcinomas examined suggests that inactivation of adenomatous polyposis coli or another neighboring gene on chromosome 5q may be involved in carcinogenesis in the oral cavity.

Adult↗

PubMed1993

Loss of heterozygosity of p53 in oral cancers demonstrated by the polymerase chain reaction.

BACKGROUND: Alterations in the tumor suppressor gene p53 are the most frequently detected genetic abnormalities in human cancers. Inactivated tumor suppressor genes, including p53, often are suggested by loss of heterozygosity (LOH) studies. p53 gene inactivation has been reported in esophageal cancers. Because the etiologic factors for esophageal and intraoral carcinomas often are the same, corresponding molecular events may occur in oral squamous cell carcinoma (SCC) development. METHODS: The authors investigated LOH of the p53 gene in DNA from 27 primary oral cancers using a polymerase chain reaction (PCR)-based restriction fragment length polymorphism assay. DNA from fixed specimens of SCC and normal tissues was isolated and amplified at two p53 gene polymorphic restriction sites. RESULTS: In heterozygous individuals, 10 of 14 (71%) intraoral SCC demonstrated loss of p53 heterozygosity at one polymorphic restriction site. Two of five carcinomas showed LOH at a second site. CONCLUSIONS: These results suggest that inactivation of p53 is involved in the development or progression of SCC of the oral cavity.

Base Sequence↗

PubMed1993

Hsp 47 is localized to regions of type I collagen production in developing murine femurs and molars.

To determine whether the proposed molecular chaperone Hsp47 is associated with the production of heterotrimeric procollagen, the distribution of anti-Hsp47 and anti-collagen antibodies were examined in developing murine femurs and molars of 22-23-day CD-1 mice. In addition, the expression of Hsp47, and collagen mRNAs were assessed by in situ hybridization using oligonucleotide probes. These studies revealed that Hsp47 was developmentally expressed and produced in regions that are coincident with type I collagen. Hsp47 was not localized in cartilaginous zones of developing femurs or in the regions of developing molars producing type III collagen. These results support the hypothesis that Hsp47 is necessary for the assurance of type I procollagen and is not expressed with other homotrimeric procollagen molecules.

Animals↗

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↗