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Biomedical subjects

R J Klebe

Publications and source records attributed to R J Klebe.

At least 37 records · Page 2Linked to original sources
PubMed1992

Immunohistological localization of cell adhesion proteins and integrins in the periodontium.

The distribution of the cell adhesion proteins vitronectin, fibronectin, tenascin, and laminin as well as several integrin subunits, alpha 2, alpha 5, and alpha v, was studied in primate periodontal tissues. Full baboon mandibular sections were analyzed by immunohistochemical methods in order to localize the molecules studied in both soft and hard tissues. Vitronectin was associated with the connective tissue of the marginal gingiva, the periodontal ligament, as well as the endosteum and periosteum. A notable finding was the particularly high staining intensity of vitronectin in the periodontal ligament. Fibronectin was widely distributed in the periodontal connective tissue and was also localized to the pericellular matrix of osteocytes and blood vascular elements. Epithelial basement membranes stained positively for both fibronectin and tenascin. These proteins were also expressed in the periosteal and endosteal connective tissues and the periodontal ligament. The staining intensity for tenascin was higher in zones along the cementum and bone surfaces. Laminin was, characteristically, limited to basement membranes of epithelium and endothelium. The distribution of fibronectin, tenascin, and laminin is related to previous findings in other species. The localization of the several integrin alpha-subunits is also described in full baboon mandibular sections. The vitronectin receptor (alpha v) had a uniquely strong expression in osteoclasts of the alveolar bone and was found, at lesser intensity, on periodontal ligament fibroblasts. The fibronectin receptor alpha subunit, alpha 5, was also observed on osteoclasts, and, in addition, was widely distributed on fibroblasts, cementoblasts, and osteoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar Process↗
PubMed1991

Quantitative assay for morphogenesis indicates the role of extracellular matrix components and G proteins.

A quantitative assay for morphogenesis is described that involves counting the organizing centers (swirling patterns) formed by many cultured fibroblasts. Organizing centers, which are found in vivo, represent one of the smallest units of morphogenesis. We show that macroscopically visible organizing centers form by the merger of smaller organizing centers. Parallel orientation of cells on plastic substrata requires cell-cell contact, but organizing centers can develop without cell-cell contact on collagen gels. On collagen gels, the orientation of collagen fibers determines the orientation of cells with respect to one another. Although organizing centers resemble fingerprints, we have shown that a stochastic process determines the spatial orientation of organizing centers. Treatment of transformed cell lines with agents that increase cAMP levels or alter the activity of guanine nucleotide binding proteins resulted in the generation of organizing centers. Cholesterol precursors involved in protein isoprenylation were found to be potent reverse-transformation agents that could alter the two-dimensional morphogenesis of cells. The simple assay described should permit the analysis of morphogenesis at the molecular and cellular levels.

Animals↗
PubMed1991

The alternative splicing of fibronectin pre-mRNA is altered during aging and in response to growth factors.

The reverse transcription-polymerase chain reaction was used to examine alternative splicing at each of the three fibronectin exons known to undergo alternative splicing, i.e. extra domain A (ED-A), extra domain B (ED-B), and type III connecting sequence (IIICS). Ratios of fibronectin mRNAs with or without a given exon were determined in several rat tissues and human cell lines during aging in vivo and cellular senescence in vitro. We demonstrate that statistically significant shifts in the alternative splicing of fibronectin occur during aging in vivo and in vitro. Since all three alternatively spliced exons are spliced out at a higher frequency in aging tissues and cells, the fibronectin protein produced by old cells should be slightly smaller than that obtained from young cells. The reverse transcription-polymerase chain reaction demonstrates tissue-specific patterns of alternative splicing in several tissues. Whereas fibronectin mRNAs from adult rat tissues were found to range from 0 to 25% ED-A+ and from 0 to 10% ED-B+, fibronectin mRNAs from cultured cell lines were found to be approximately 50-60% ED-A+ and 15-25% ED-B+. We observed similarity in splicing of fibronectin RNA by the different cultured cell lines obtained from many tissues and attribute this observation to the effect of growth factors. We demonstrate that serum deprivation; placement of cells into primary culture; and growth factors such as transforming growth factor beta 1, retinoic acid, and 1,25-dihydroxyvitamin D3 can all change the alternative splicing of fibronectin pre-mRNA in the ED-A, ED-B, and type III connecting sequence exons. Possible mechanisms for the regulation of the alternative splicing of fibronectin RNA by growth factors are discussed.

Aging↗
PubMed1991

IL-1 beta and prostaglandins regulate integrin mRNA expression.

The purpose of this study was to examine the effects of IL-1 beta on integrin expression in MG-63 human osteosarcoma cells. Human recombinant IL-1 beta (rIL-1 beta) produced significant increases in both alpha 2- and alpha 5-subunit mRNA levels, as well as a smaller increase in alpha v-subunit mRNA. In contrast, IL-1 beta decreased alpha 4-subunit mRNA levels by approximately 30% relative to untreated controls. These findings suggest that human IL-1 beta differentially regulates expression of integrins. When cultures were treated with both IL-1 beta and the cyclooxygenase inhibitor, indomethacin, the expression of alpha 2-, alpha 5-, and alpha v-subunit mRNA levels were dramatically increased relative to untreated controls; co-treatment with 0.5 mM prostaglandin E2 (PGE2) partially reversed this effect. Indomethacin alone did not affect integrin mRNA levels. Treatment with IL-1 beta or IL-1 beta + indomethacin also induced significant changes in MG-63 morphology (i.e., increased cell elongation) and increased the ability of cells to contract collagen gels. PGE2 reversed the above effects on cell morphology and gel contraction. These findings indicate that (a) IL-1 beta differentially regulates the expression of integrins and (b) that PGE2, which is induced by IL-1 beta, may provide a negative feedback loop which counteracts the stimulatory effect of IL-1 beta on integrin gene expression. It is suggested that products of inflammation may affect cell behavior by differentially regulating the expression of various integrins.

Actins↗
PubMed1991

Characterization of the extracellular matrix of the primate temporomandibular joint.

The distribution of type I and II collagens, fibronectin and the fibronectin-integrin receptor, tenascin, laminin, link protein, and cartilage-specific glycosaminoglycans was examined in the primate temporomandibular joint complex using an immunohistochemical approach. In general, type I collagen, fibronectin, and the fibronectin-integrin receptor were found to co-distribute throughout the joint complex. Immunostaining for these proteins was notably intense in the prechondroblastic and mineralization zones of the articular cartilages of the joint. Tenascin was identified in several structures of the joint, including the articular cartilages, where intense staining was observed in the prechondroblastic and cartilagenous zones. Laminin was detected only in the adventitia of blood vessels located in the attachment tissues of the disc and joint synovium. Cartilage-specific glycosaminoglycans and type II collagen were observed in the cartilagenous zones of the articular cartilages of the mandibular condyle and temporal bone. In addition, immunostaining for cartilage-specific glycosaminoglycans also was detected throughout the extracellular matrix surrounding "chondrocyte-like" cells located in the joint disc. Despite the localization of cartilage-specific glycosaminoglycans in the disc, type II collagen was not detected in this structure. It is suggested that a fibronectin-integrin receptor mechanism may be involved in the regulation of growth of the articular cartilages of the temporomandibular joint.

Animals↗
PubMed1991

Monoclonal antibodies to periodontal ligament cells.

Ten mouse monoclonal antibodies were prepared against cultured bovine periodontal ligament cells to be used as reagents for the study of periodontal disease and wound healing. Using standard immunohistochemical methods, these antibodies were found to recognize cell surface antigens in formalin-fixed bovine periodontium. Three of the 10 monoclonal antibodies (i.e., PDL-1, PDL-2, and PDL-10) cross-reacted with cells found in primate periodontium. While the isolated monoclonal antibodies appeared to distinguish subpopulations of cells located in the supporting tissues of teeth, immunohistological examination of other organs (dermis, kidney, skeletal muscle, thyroid, and parotid gland) indicated that a number of cell types of mesenchymal origin share an antigen(s) found on periodontal cells. The monoclonal antibodies described in this report should prove to be useful in studies of periodontal disease and guided tissue regeneration by providing both analytical reagents and immunochemical methods for isolating selected cell populations of the periodontium.

Animals↗
PubMed1990

Synthesis of phosphotyrosyl-containing phosphopeptides by solid-phase peptide synthesis.

The synthesis of phosphotyrosine-containing phosphopeptides using solid-phase peptide synthesis (SPPS) techniques is described. We present the synthesis of a Boc-phosphotyrosine derivative, which when used with modifications of the conventional SPPS protocol permits the incorporation of phosphotyrosine into synthetic peptides. The resulting phosphopeptides were authenticated by fast atom bombardment mass spectrometry, amino acid analysis, and phosphate assay. Alkaline phosphatase was found to dephosphorylate synthetic phosphopeptides at different rates, supporting the potential use of these synthetic substrates for studies of phosphoprotein phosphatases. Synthesis of a phosphopeptide using the described protocol has several advantages over the preparation of phosphopeptides via enzymatic phosphorylation.

Alkaline Phosphatase↗
PubMed1990

Cultivation of mammalian cells in heat-sealable pouches that are permeable to carbon dioxide.

Petri plates, 96-well plates, and other unsealed culture vessels are the chief cause of air-borne contamination of cell cultures. In this study, heat-sealable plastic pouches that are permeable to CO2 and other gases are used as a means to avoid contamination. Several applications of heat-sealable pouches are described. First, petri plates can simply be sealed in a wide variety of plastic films that are permeable to CO2. Second, a few CO2-permeable plastic films can be used directly as substrata for mammalian cell growth and can also be cut with a simple hole punch for the isolation of clones. Third, one can grow cells in chemically sterilized carbonic acid solutions and thereby avoid the use of a CO2 incubator entirely.

Animals↗
PubMed1989

Late passage cells display an increase in contractile behavior.

During aging, alterations in the extracellular matrix and cytoskeleton have been noted. Aging affects both the structure and the biological activity of fibronectin as well as the cellular content and organization of f-actin. The purpose of this study was to examine the functional significance of such alterations. Assays for cell migration, cell spreading, collagen gel contraction, and f-actin organization were conducted using diploid cells strains of high and low population doubling level (PDL). First, an assessment of cell spreading behavior revealed that high PDL cells spread more than low PDL cells. Second, analyses performed with a modified phagokinetic track assay indicate that high PDL cells migrate (phagocytize) faster than low PDL cells. Lastly, it was found that high PDL cells contract collagen gels faster than low PDL cells. Therefore, high PDL cells are capable of exerting more force upon their extracellular matrix than low PDL cells. In agreement with previous observations, we found by phalloidin staining that the f-actin content of high PDL cells was substantially greater than that of low PDL cells. The elevated f-actin content of high PDL cells could promote increased cellular contractility thereby leading to the increase in cell spreading, migration, and collagen gel contraction observed here.

Actins↗
PubMed1989

Cells transmit spatial information by orienting collagen fibers.

Parallel orientation of large cell populations occurs when cells are cultured on thin collagen films if and only if a gel is restrained at two or more edges. The direction in which cell bodies orient is determined by the geometry of the gel. It can be shown that tension exerted by cells on a collagen gel leads to reorientation of collagen fibers in a direction determined by the initial geometry of the collagen gel. Hence, tension generated by cells leads to collagen fiber orientation which, in turn, determines the spatial orientation and morphology of cells. Thus, without cell-to-cell contact, cells can communicate with each other by applying tension to collagen fibers in their extracellular matrix. Information concerning the shape of the environment can be transmitted via such long range forces.

Animals↗
PubMed1988

Cytoscribing: a method for micropositioning cells and the construction of two- and three-dimensional synthetic tissues.

Cells may be positioned in precise, predetermined patterns by a technique, termed cytoscribing, in which cell adhesion proteins are deposited on a substratum under computer control. Using standard office equipment, cells may be positioned within a cell diameter with the techniques described. Cytoscribing involves the use of either a computer-controlled ink jet printer or a graphics plotter to deposit cell adhesion proteins and monoclonal antibodies onto a substrate material. By selecting different cell adhesion proteins and methods that permit the formation of positive or negative patterns of cells (cytoscripts), two-dimensional tissues can be constructed. It is also demonstrated that an optical microlithography technique used in the semiconductor industry can be used to position cells with a precision of less than a micron. By utilizing the properties of cell adhesion proteins, both positive and negative cytoscripts of photoengraved images can be obtained. Techniques for the construction of three-dimensional tissues are described. Thin sheets of collagen were obtained by forming collagen heat gels in a mold. Monolayers of cells growing on thin collagen sheets were then attached to one another by gluing the sheets together with collagen. Sheets of cells do not attach to each other readily in the presence of fibronectin and evidence is provided which indicates that this observation is due to a lack of adhesiveness of the upper surface of cells.

Animals↗
PubMed1988

Comments on the clinical application of fibronectin in dentistry.

Several studies have demonstrated that citric acid demineralization of the root surface promotes tissue attachment. Since demineralization exposes collagen to which fibronectin binds, the role of fibronectin in the attachment of cells to the tooth surface has been of considerable interest. It is clear that fibronectin and other cell adhesion proteins can promote cell attachment to the tooth surface; therefore, attempts have been made to utilize these findings in a clinical setting. Using a quantitative ELISA procedure to measure the binding of fibronectin to demineralized bone and tooth, we have found that 1 microgram fibronectin can saturate approximately 1 mg of either demineralized bone or demineralized tooth powder. Since serum contains 300 micrograms fibronectin per mL, the bleeding that occurs during oral surgery should saturate exposed tooth surfaces with amounts of fibronectin adequate for cell adhesion. Thus, exogenous fibronectin would appear to be of little clinical benefit.

Animals↗
PubMed1988

Methods for solid phase peptide synthesis which employ a minimum of instrumentation.

Both automated and manual methods of solid phase peptide synthesis employ three basic steps: (a) Attachment of the first amino acid to a resin, (b) peptide synthesis via successive carbodiimide couplings and (c) cleavage and deblocking of the peptide. Instead of an automated peptide synthesizer, one can manually synthesize peptides with a sintered glass funnel as the only required piece of equipment. Following solid phase synthesis, one can cleave and deblock peptides without the use of anhydrous hydrofluoric acid (HF); hence, the need for specialized equipment required for handling HF can also be eliminated. In the procedure described in this report, cleavage and deblocking is carried out with trifluoromethane sulfonic acid (TFMSA) in glass vessels without the need for high pressure Teflon fittings. Since completion of the coupling reaction can be monitored during each cycle when manual methods are employed, one can avoid repetitive couplings and, thereby economize on reagents. Since TFMSA cleavage and deblocking can be carried out in open glass vessels, one can cleave and deblock large numbers of peptides at the same time. With the methods described, one can satisfactorily prepare large quantities of peptides at minimal cost.

Amino Acid Sequence↗
PubMed1986

Localization of the ganglioside-binding site of fibronectin.

It has been demonstrated via biological assays that fibronectin possesses a receptor for gangliosides that is involved in cell adhesion and restoration of the normal morphology of transformed cells. In this study, fluorescence polarization has been employed to monitor the binding of ganglioside oligosaccharide to fibronectin. Parameters involved in ganglioside oligosaccharide binding to fibronectin are described and compared to the interaction of heparin with fibronectin. A Kd of 1.4 X 10(-8) mol/liter has been calculated, and it is demonstrated that labeled ganglioside oligosaccharides can be eluted from fibronectin with either unlabeled ganglioside oligosaccharides or 4 M urea. Using the fluorescence polarization assay developed in this study for measurement of ganglioside binding to fibronectin, it is demonstrated that gangliosides bind to the 31,000-dalton amino terminal heparin-binding domain of fibronectin. A ganglioside-Sepharose affinity column has been constructed which specifically binds the 31,000-dalton amino terminal fragment of fibronectin. The localization of the ganglioside receptor to the amino terminal domain of fibronectin indicates that the ganglioside receptor is distinct from the putative fibronectin cell surface receptor which is located near the center of the fibronectin molecule.

Binding Sites↗
PubMed1986

Regulation of cell motility, morphology, and growth by sulfated glycosaminoglycans.

Due to the recent observation that heparin binds to several growth factors and cell adhesion molecules, the effect of heparin on biological processes governed by growth factors and cell adhesion molecules was investigated. Pharmacological doses of heparin were found to alter cell growth rate, cellular morphology, and cell motility. Concentrations (microgram/ml) of heparin or dextran sulfate decreased cell growth rate, but not the final cell density attained in plateau phase. The effect of heparin on cell growth rate was most pronounced when cells were cultured in low concentrations of serum. A heparin-induced decrease in cell growth rate could be reversed by addition of platelet-derived growth factor (PDGF), a heparin-binding growth factor. Heparin altered the morphology of all cell lines studied to various degrees. The effect of heparin on cell morphology was quantitated by measuring the heparin-induced change in cell surface area. HT-1080 and HeLa cells nearly doubled in surface area upon exposure to 10 micrograms/ml heparin. Since several heparin-binding cell adhesion proteins mediate both cell spreading and cell migration, the influence of heparin on cell migration was investigated with an improved version of the phagokinetic track technique. Low concentrations of heparin and dextran sulfate were found to increase the rate of cell migration in a dose-dependent fashion. Since the quantitative effect of heparin on cell growth rate, morphology, and migration depends on the cell line studied, it is suggested that three separate phenomena may be involved. The results presented indicate a central role for sulfated glycosaminoglycans in the control of both cell growth and cell-cell interactions.

Animals↗
PubMed1986

Uptake by cells of nucleic acids promoted by compounds sharing the pleiotropic effects of poly(ethylene glycol).

Poly(ethylene glycol) (PEG) is a member of a group of membrane active compounds that have pleiotropic effects on cells, eg, promotion of cell fusion, induction of erythroleukemia cell differentiation, and protection of cells from freezing damage. Since PEG has recently been shown to be an efficient promoter of genetic transformation in bacteria, yeast, and mammalian cells, studies were carried out to determine whether other PEG-related compounds could also promote genetic transformation. In this study, 24 compounds, which behave like PEG in other biological systems, are shown to promote transfection of human cells with isolated poliovirus RNA. That PEG and other commercially important compounds promote transfection indicates that such compounds may represent a biohazard to man.

Drug Synergism↗
PubMed1986

Assignment of the human fibronectin structural gene to chromosome 2.

A cloned human cDNA probe for fibronectin (FN) containing 1.3 kb of the human FN coding region has been used to determine the chromosome that encodes the structural gene in human-mouse somatic cell hybrids. The results show that human chromosome 2 encodes the FN structural gene.

Animals↗