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Biomedical subjects

R J Klebe

Publications and source records attributed to R J Klebe.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of integrin gene expression by substrate adherence.

Under substrate adherent conditions, integrin gene expression can be regulated by transforming growth factor-beta, interleukin-1 beta, and prostaglandins. This report demonstrates a new mechanism that can differentially control the expression of several integrins. When MG-63 osteosarcoma cells are maintained in suspension, up-regulation of several integrin alpha-subunits takes place. Within as little as 4 h, the mRNA levels for both the alpha 2- and alpha 4-subunits are increased 4- and 6-fold, respectively. It was found that mRNA levels for the alpha 2-, alpha 4-, and alpha v-subunits were markedly increased in several differentiated cell lines under nonadherent conditions; however, cells that did not express a given integrin under substrate adherent conditions also did not express this integrin when maintained in suspension. The alpha 5-subunit did not upregulate during suspension growth. By immunocytochemistry, changes in integrin mRNA levels were confirmed at the protein level. Both cytochalasin B and a phorbol ester were found to induce the expression of the alpha 2-subunit, but not the alpha 4- and alpha 5-subunits, in a dose-dependent fashion. Many investigators have documented changes in gene expression that result from changes in "cell shape." These phenomena may result from up-regulation of integrin gene expression induced by the lack of substrate adherence.

Base Sequence↗
PubMed1992

Alpha 5 integrin subunit expression changes during myogenesis.

Fibronectin and its cellular receptor, the alpha 5 beta 1 integrin, are involved in the transmembrane signalling events that control muscle cell differentiation. In this study, the expression of the alpha 5 integrin subunit was followed by reverse transcription-polymerase chain reaction (RT-PCR) to determine alterations during myogenesis. In studies of murine muscle, we found a 90% reduction in the level of the alpha 5 integrin subunit mRNA during early postnatal development. Concurrently, the fibronectin alternative splicing pattern changed markedly in the EIIIB and V exons. In-vitro analyses of these molecules during myoblast differentiation revealed changes that followed trends similar to those observed in vivo, although of lesser magnitude. These observations imply an important role of fibronectin and the alpha 5 integrin subunit in muscle development.

Alternative Splicing↗
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↗
PubMed1979

Bioautographic visualization of aminoacylase-1: assignment of the structural gene ACY-1 to chromosome 3 in man.

A bioautographic assay was developed for the visualization of aminoacylase-1 (N-acylamino acid aminohydrolase, ACY-1; EC 3.5.1.14) after zone electrophoresis. Bioautography and species differences in electrophoretic mobility of ACY-1 made it possible to investigate the chromosome assignment of the gene for human ACY-1 using human--mouse somatic cell hybrids. Human ACY-1 segregated concordantly with beta-galactosidase-A (beta GALA; EC 3.2.1.23) but showed discordant segregation with 32 other markers representing 23 linkage groups. The beta GALA gene has been previously assigned to chromosome 3. From this evidence and confirming chromosome analyses, ACY-1 has been assigned to chromosome 3. A genetic polymorphism in the electrophoretic mobility of ACY was observed in mouse strains, demonstrating that this enzyme can be mapped in genetic crosses of Mus musculus.

Amidohydrolases↗
PubMed1979

Characterization of naturally occurring auxotrophic mammalian cells.

In a previous study, several cultured cell lines were detected which are naturally occurring auxotrophs. In this investigation, the enzyme deficienceis involved are described. It is demonstrated that the Chinese hamster cell lines CHO(K1), YH21, RJK-36, and CHW-1102 are deficient in cystathionase and argininosuccinate synthetase. In addition, CHO (K1) and CHW-1102 were found to lack argininosuccinate lyase. CHW-1102 cells were also found to be unable to proliferate in medium containing branched-chain alpha-keto acids in place of the corresponding L-amino acids since CHW-1102 cells lack branched-chain aminotransferase.

Amino Acid Isomerases↗
PubMed1978

Localization of the binding site for cell attachment in the alpha1(I) chain of collagen.

Certain cells such as CHO (Chinese hamster ovary) and fibroblasts attach to a substrate of type I collagen via proteins that link the cell surface to collagen. Serum contains a glycoprotein, c-CAP (collagen cell attachment protein), that can mediate this adhesion. Previously, we established that alpha1(I)-CB7 (cyanogen bromide peptide 7), which lies within residues 552 to 822 of collagen, contains a major binding site for c-CAP(alpha1(I)-CB7 has been renumbered to account for an additional triplet recently identified at residue 613 (P. P. Fietzek and R. W. Glanville, manuscript in preparation). Now we have examined the ability of various peptides derived by proteolytic digestion of alpha1(I)-CB7 to bind to c-CAP based upon their ability to inhibit cell attachment to collagen. The binding site lies within residues 757 to 791. It is likely that this is the sole binding site in the alpha1(I) chain since cleavage of the bond between residues 775 and 776 in the alpha1(I) chain destroys cell attachment activity.

Amino Acid Sequence↗
PubMed1978

Argininosuccinic aciduria: assignment of the argininosuccinate lyase gene to the pter to q22 region of human chromosome 7 by bioautography.

Argininosuccinic aciduria, an autosomal recessive disorder of the urea cycle in humans, is associated with a deficiency of argininosuccinate lyase (ASL; L-argininosuccinate arginine-lyase, EC 4.3.2.1). ASL activity was visualized on gels after electrophoresis by a new method, termed bioautography. Bioautography involves the use of mutant bacteria to visualize the location of mammalian enzymes after zone electrophoresis. By this technique, human ASL migrated to a position different from mouse ASL, while a survey of mouse strains, tissues, and tissue culture cell extracts demonstrated the same electrophoretic form and no genetic variants of mouse ASL. Identifying human ASL, by bioautography in human-mouse somatic cell hybrids has made it possible to regionally locate the ASL gene on human chromosome 7. The human ASL phenotype segregated concordantly with the human enzyme beta-glucoronidase (GUS; beta-D-glucoronide glucuronosohydrolase, EC 3.2.1.31) in cell hybrids, but showed discordant segregation with 32 other enzyme markers representing 23 linkage groups. The gene for GUS has been assigned to chromosome 7 in humans, and cosegregation (synteny) of ASL and GUS demonstrates the assignment of ASL to chromosome 7. Regional location of ASL and GUS to the pter to q22 region of chromosome 7 was achieved in hybrids segregating a 7/9 translocation.

Amino Acid Metabolism, Inborn Errors↗
PubMed1977

Bioautography: a general method for the visualization of isozymes.

A new method has been developed for visualization of isozymes which are difficult or impossible to detect with standard histochemical or autoradiographic methods. The principle of this method, bioautography, is the use of a microbial reagent to locate an enzyme after gel electrophoresis. When bioautography was compared to other staining procedures, the bioautographic method yielded identical results to those observed by the histochemical method for lactate dehydrogenase (LDH) or by the autoradiographic method for the adenine phosphoribosyltransferase (APRT). Using the bioautographic method, stains for enzymes which could not be visualized by any other procedure have been developed: argininosuccinate lyase and branched-chain aminotransferase. By employing appropriately genetically marked bacterial strains, it should be possible to develop new isozyme stains for a large number of unstudied isozymes.

Adenine Phosphoribosyltransferase↗