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Biomedical subjects

P Christner

Publications and source records attributed to P Christner.

At least 19 recordsLinked to original sources
PubMed1996

Alteration in the conformational stability of collagen caused by the incorporation of the lysine analogue S-2-aminoethylcysteine.

We studied the effects of the lysine analogue S-2-aminoethylcysteine on the activation of lysyl tRNA and on the secretion and conformational stability of newly synthesized type I collagen in embryonic chick tendon fibroblasts. The analogue competed efficiently with lysine for activation onto tRNA without affecting significantly the activation of other amino acids (Km for lysine: 1.6 microM; Ki for S-2-aminoethylcysteine: 1.4 microM). The analogue also profoundly inhibited the synthesis and secretion of [14C]procollagen but did not affect the synthesis or secretion of non-collagenous proteins. Although the [14C]proline-labeled procollagen synthesized in the presence of S-2-aminoethylcysteine contained normal levels of hydroxyproline, it was susceptible to digestion with pepsin at 25 degrees C, indicating that incorporation of the analogue altered the conformational stability of the collagen triple helix. This analogue should be a powerful tool to further study the role of lysine on collagen structure and to determine how altered collagen structure affects its synthesis and secretion. Furthermore, this analogue may be a potent and selective inhibitor of collagen accumulation in pathologic conditions accompanied by tissue fibrosis.

Amino Acids↗
PubMed1993

The tight skin (Tsk) mutation in the mouse, a model for human fibrotic diseases, is tightly linked to the beta 2-microglobulin (B2m) gene on chromosome 2.

The Tsk mutation in the mouse is characterized by the excessive accumulation of collagen in skin and various internal organs, including the heart and lungs. These connective tissue abnormalities are similar to those present in human systemic sclerosis or scleroderma. The Tsk mutation provides an opportunity to investigate, at the molecular level, the pathogenesis of tissue fibrosis. As a first step to cloning the Tsk gene, we report the localization of the Tsk mutation with respect to known molecular markers on mouse chromosome 2. N2 progeny carrying the Tsk mutation were obtained from an intersubspecific backcross of [(C57BL/6-pa +/+ Tsk x Mus castaneus)F1 x M. castaneus] mice. Genomic DNA from each N2 mouse was subjected to Southern and PCR analyses to identify restriction fragment length polymorphisms and simple sequence length polymorphisms, respectively. Our results refine the location of Tsk to a 3-cM region, eliminate several genes from consideration as the Tsk mutation, identify molecular probes tightly linked with Tsk, and suggest candidate genes responsible for the Tsk phenotype.

Animals↗
PubMed1985

Collagenase in the lower respiratory tract of patients with adult respiratory distress syndrome.

Collagenase activity in the bronchoalveolar lavage (BAL) of patients with adult respiratory distress syndrome (ARDS) was measured against Type I collagen (17 patients) and against Type III collagen (13 patients). Serine protease activity was also measured against Type III collagen (13 patients). Type I collagenase activity was detectable in 12 of 17 and Type III collagenase was detectable in 12 of 13 patients with ARDS. The 10 control subjects had no detectable Types I or III collagenase activity. Total and differential white cell counts were analyzed in the lavage fluid. Although the total counts did not differ between patients with ARDS and control subjects, the percentage of neutrophils was increased more than 25-fold and the percentage of macrophages was reduced almost 10-fold in the ARDS patients. Serial collagenase activity was followed in 1 ARDS survivor. In this patient Type III collagenase activity peaked before the Type I collagenase activity or serine protease activity reached their maximums. Both the latter enzyme activities paralleled the total recoverable cells in the BAL.

Adult↗
PubMed1985

Utilization of a peroxidase antiperoxidase complex in an enzyme-linked immunosorbent assay of elastin-derived peptides in human plasma.

Chronic obstructive pulmonary disease (COPD), a major cause of morbidity and death in the smoking population, develops insidiously over many years, and significant impairment of lung function usually occurs before the disease is diagnosed. Because lung elastin degradation appears to be a prerequisite for the development of the disease, immunologic detection of elastin-derived peptides in the blood might be an effective approach to the early detection and monitoring of the disease. We here report an improved enzyme-linked immunosorbent assay for elastin peptides using a peroxidase-antiperoxidase complex as the reporter group. The assay is sensitive to 2 ng/ml elastin peptides. We show that for optimal, reproducible results the assay should be carried out at 16 degrees C rather than at room temperature and that determinations should be made on plasma containing protease inhibitors rather than on serum. The levels of elastin-derived peptides appeared to remain relatively constant when multiple samples were taken during a 5- to 10-wk period from individual subjects. In addition, patients with COPD had elevated elastin peptide levels (127 +/- 47 ng/ml) compared with levels in normal nonsmokers (58 +/- 17 ng/ml), whereas normal smokers had values intermediate between the 2 groups (mean peptide levels of 76 +/- 42 ng/ml). A small group of normal smokers (20%) had elevated elastin peptide levels similar to those in the emphysema group and may represent that group of smokers who are at risk of developing obstructive lung disease.

Animals↗
PubMed1984

Secretion of elastin in the embryonic chick aorta as visualized by immunoelectron microscopy.

Recently, significant advances have been made in characterizing the pathway of elastin biosynthesis from the biochemical point of view and a 70,000 dalton protein, designated tropoelastin, appears to be the primary translation product and soluble intermediate of the insoluble elastin. However, relatively little is known concerning the intracellular secretory pathway of tropoelastin. We previously developed an electron microscopic technique using elastin-specific antibody and ferritin-conjugated secondary antibody to identify intracellular elastin and to identify, provisionally, intracellular vesicles containing elastin ( Damiano et al., Conn. Tiss . Res. 8: 185-188, 1981). However, the method did not permit localization of elastin in other intracellular organelles. We now describe an improved post-embedding technique using the peroxidase-antiperoxidase method to detect the primary elastin antibody and have localized elastin in both the endothelial and medial cells of the embryonic chick aorta. Specific staining was visualized in the cisternae of the endoplasmic reticulum, in the Golgi apparatus, and in vesicles forming on the trans side of the Golgi. Some of these smaller vesicles appeared to fuse, forming larger vesicles which may have a storage function. Both types of vesicles were seen fusing with the cell plasma membrane, suggesting that elastin is secreted by an exocytotic process. These results suggest that tropoelastin follows the classical pathway for protein secretion.

Animals↗
PubMed1983

Immunologic measurement of elastin-derived peptides in human serum.

Chronic obstructive pulmonary disease (COPD), a major cause of morbidity and death in the smoking population, develops insidiously over many years, and usually significant impairment of lung function has occurred before the disease is diagnosed. It is likely that destruction of the elastic fiber is a prerequisite for the development of the disease, and it is possible that immunologic identification in the serum of peptides derived from lung elastin degradation might be an effective approach to the early detection and monitoring of the disease. We prepared antibodies to peptides derived from human lung parenchymal elastin and used these antibodies in an enzyme-linked immunosorbant assay to quantitate elastin-derived peptides in the serum of 39 normal control nonsmokers, 33 smokers with normal lung function, and 40 patients with COPD. On average, statistically significant higher levels of elastin-derived peptides were found in the normal smokers and COPD patients compared to the controls. Further work with larger numbers of subjects is necessary to determine whether such a test is effective in identifying those individuals who are at risk of developing COPD.

Antibodies↗
PubMed1982

Purification of human neutrophil collagenase and production of a monospecific antiserum.

Although there is good evidence for the presence of human neutrophil (PMN) collagenase, only moderate purification has been reported. The probable explanation for this fact is that most assays used to specifically measure collagenase activity are not reliable if high levels of several different proteases are also present in the assay mixture. The PMN granule is just such a concentrated mixture. Therefore, polyacrylamide gel electrophoresis was used to identify and quantitate the alpha 1 3/4 and alpha 2 3/4 cleavage products diagnostic for mammalian collagenase. White cells (85% PMN's) were lysed in 0.34 M sucrose and granules were obtained. The granules were lysed by sonication, and the lysate was chromatographed on a Sephadex G-200 column followed by a Trasylol-Sepharose 4B column. This procedure resulted in a 1350-fold purification and a yield of 75 micrograms of enzyme/unit of blood. The collagenase was inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid but not by sulfhydryl or serine protease inhibitors. The preparation was free of elastase, which has been shown to cleave type III collagen into alpha 1 3/4 and alpha 1 1/4 pieces. The pI of collagenase was shown to be 4.7 by isoelectric focusing, and the enzyme lost activity below a pH of 6.5 if collagen was absent. Antiserum was produced by 100-micrograms injections of the purified collagenase into rabbits. Titers were measured by the enzyme-linked immunosorbent assay. For determination of the specificity, collagenase and PMN extract were isoelectrically focused and blotted onto nitrocellulose. The antibody recognized only one band of protein in the PMN extract, which comigrated with the purified collagenase.

Animals↗
PubMed1981

An analysis of the organ and species immunospecificity of elastin.

The connective tissue protein elastin is largely responsible for maintaining the elasticity of major blood vessels and lung tissue. Comparatively few studies have been made of the immunologic properties of elastin because of its high degree of insolubility and because of its apparent limited capacity to elicit precipitating antibodies. Recent studies have focused on the possible role of elastolytic enzymes, particularly leukocyte elastase, in the pathogenesis of chronic obstructive lung disease, but there are no reports in the literature concerning the antigenic properties of peptides released by digestion of insoluble elastin with leukocyte elastase. In the present study, we have obtained antibodies in rabbits to peptides prepared by digestion of dog and human lung and aortic elastin with either oxalic acid or leukocyte elastase. By hemagglutination assays, we have shown generally that: (1) within a given species the peptides from aorta and lung cross-react strongly with one another, (2) there was rather poor cross-reactivity between peptides from different species (3) the peptides obtained by oxalic acid digestion cross-react poorly with those obtained by elastase digestion, (4) the antibodies to the peptides cross-reacted with the insoluble elastin from which the peptides were derived. These elastin-specific antibodies may be useful for ultrastructural localization of elastin and identification of fragments derived from elastin in the sera of humans and experimental animals.

Animals↗
PubMed1980

Immunohistochemical localization of amelogenins in enameloid of lower vertebrate teeth.

The indirect method of immunofluorescence was used to demonstrate the presence of amelogenins in the enameloid of teeth and dermal denticles of Chondrichthyes; in the enameloid of Teleostei and Amphibia; and in the enamel of Reptilia. Nonmammalian amelogenins are formed in the ectodermal cells of tooth organs and chemically are so similar to mammalian amelogenins that they interact with antiserum prepared from bovine enamel matrix.

Amelogenesis↗
PubMed1980

Immunologic identification of elastin-derived peptides in the serums of dogs with experimental emphysema.

Pulmonary emphysema is a disease in which peptides formed by the enzymatic degradation of the amorphous component of interstitial elastin may be release from the lung. In order to provide a test that can monitor the destruction of lung elastin invivo, we developed a hemagglutination inhibiton assay that specifically and quantitatively measure elastin-derived peptides in serum. Using this method, was measured the concentration of elastin-derived peptides in the serums of dogs developing emphysema through the administration of porcine pancreatic elastase. Elastin-derived peptides could be detected in the serums for a period of 12 days after the administration of a single 25- or 50-mg dose of elastase, and for at least 40 days after a 100-mg dose. There was a good correlation between the maximal concentration of elastin derived peptides found in the serums and the amount of elastase administered to the animal. This immunologic method may be useful in following the progression of experiment emphysema.

Animals↗
PubMed1980

Collagenase in the human periodontal ligament.

Collagenase activity was demonstrated by direct incubation to be present in human periodontal ligament. This activity was found in only one of two populations of ligament, i.e., those ligaments taken from teeth in which their attachment site was at least 2.5 mm apical to the cementoenamel junction. The collagenase was demonstrated to be of host origin because it degraded collagen into 3/4 and 1/4 alpha chain fragments characteristic of mammalian collagenases. The enzyme was shown to be inhibited in the presence of EDTA and to have a pH optimum of 7.5.

Collagen↗
PubMed1979

Immunofluorescent evidence for the similarity of amelogenins in calf, mouse and pig teeth.

Antiserum was prepared to fetal bovine enamel matrix and was used to localize the amelogenins in developing bovine molars by immunofluorescent microscopy. Amelogenins could be identified to preameloblasts, secretory ameloblasts, stratum intermedium cells, and the newly deposited enamel matrix. Mature enamel matrix did not fluoresce except in a thin line along the DEJ and adjacent to the ameloblasts. Immature enamel matrix of murine and porcine teeth fluoresced when treated with antiserum to bovine enamel matrix. No other portions of tooth buds or other tissues reacted with the specific antiserum.

Animals↗
PubMed1979

Immunologic localization of elastin by electron microscopy.

Research on the pathogenesis of experimental emphysema has involved studies of the distribution of and destruction of elastin in the alveolar interstitium. The ill-defined organization of elastin in the alveolar interstitium makes it difficult to identify the elastin specifically by staining procedures ordinarily used for electron microscopy. This problem becomes more significant when the elastic tissue is fragmented during emphysema development and localization of the elastin fragments is essential. Therefore, a specific technique using high-titer antibodies against purified canine lung elastin was developed. The primary antibody was used on preembedded or etched postembedded sections. Localization of the antielastin IgG was accomplished with ferritin-labeled rabbit antisheep IgG as the secondary antibody. Treatment with the preimmune serum gave negligible ferritin background staining. The antielastin antibody did not react with lung connective tissue proteins such as the microfibrillar component of elastin or collagen or proteoglycan. The antielastin antibody appeared to be species specific. The method may be useful for studies of experimental emphysema.

Animals↗
PubMed1977

Preferential usage of tRNA isoaccepting species in collagen synthesis.

A new double label technique is described for determining the usage of individual isoaccepting tRNA species by in vitro protein-synthesizing systems. Employing this method we show that of four major species of glycyl-tRNA one which is cognate to GGU and GGC is used predominantly in collagen synthesis by polysomes isolated from embryonic chick calvaria. A similar preferential usage was found for one of two alanyl-tRNA species. No preference for any particular isoaccepting tRNA species was observed in the synthesis of noncollagenous proteins by either calvaria or liver polysomes except for the disproportionate usage of one lysyl-tRNA species. Whether such preferential usage permits translational control of collagen synthesis in vivo remains to be determined.

Alanine↗