One of the hallmarks of chronic cardiac disease is the excessive formation of fibrous extracellular matrix. This inappropriate remodeling is mediated in large part by cardiac fibroblasts and phenoconverted myofibroblasts. The protooncoprotein Ski has previously been described as possessing anti-fibrotic properties within the myocardium, in addition to triggering apoptosis when overexpressed. In the current study, we found that overexpression of Ski results in a set of distinct morphological and biochemical changes within primary cardiac myofibroblasts that is indicative of apoptosis. Its upregulation is associated with the expression of pro-apoptotic factors such as Bax and Bak, as well as caspase-9 and -7. In all, our results indicate that Ski triggers a pro-death mechanism in primary rat cardiac myofibroblasts that is mediated through the intrinsic apoptotic pathway. The survival of these cells appears to be prolonged by a pro-survival autophagic response as apoptosis is hastened when autophagy is inhibited. The observed cell death response is likely working in parallel with the previously observed anti-fibrotic properties of Ski within this cell type. As myofibroblast cells are the engines of matrix expansion in heart failure, we suggest that using Ski or a projected Ski-mimetic to induce graded apoptosis in myofibroblasts within the failing heart may be a novel therapeutic mechanism of controlling cardiac fibrosis. / October 2015

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Our study shows that the secretion of a major glycosylated, phosphoprotein with a molecular weight of 69kDa (pp69) is a specific marker for non-transformed NRK-49F cells. Antibody raised against pp69 recognizes, in addition to pp69, another major phosphoprotein with a molecular weight of 62kDa (pp62) secreted by RR1022 and spontaneously transformed NRK-49F cells (spt-NRK-49F). Immunoprecipitation of total cell lysates from both NRK-49F and RR1022 cells with anti-pp69 antibody detected only pp69. Treatments with: epidermal growth factor (EGF), transforming growth factor-$ beta$ (TGF-$ beta$) retinoic acid (RA), and TPA modulate the levels of pp69 present in the conditioned media. Furthermore, TPA and EGF induce the synthesis of 3 internal peptides with molecular weights of 58, 54, and 44 kDa which appear to be pre-processed forms of pp69. / Treatment of NRK-49F cells with insulin, EGF, TGF-$ beta$, PPA, levamisole and spermine clearly demonstrate alterations in the phosphorylation of pp69, concomitant with changes in extracellular phosphatase activity.

Prolidase deficiency (McKusick 26413) is a rare autosomal recessive disorder characterized by iminodipeptiduria, skin lesions and mental retardation. The enzyme prolidase hydrolyzes dipeptides containing C-terminal proline or hydroxyproline. / The results presented here indicate that prolidase plays a major role in the recycling of dipeptide bound proline. Control fibroblasts were able to use iminodipeptides in lieu of proline to sustain normal growth and protein synthesis whereas prolidase deficient cells were not. / Iminodipeptides added to the media of control and mutant cells showed no adverse effects on protein synthesis or cell growth. These results are consistent with a mechanism of biochemical pathology in which proline deprivation caused by the enzyme deficit is the cause of damage to skin cells. / Prolidase regulation by product and substrate was studied. A two fold decrease of prolidase activity was observed in fibroblasts grown in excess proline. However, cells grown in medium in which iminodipeptides replaced proline showed no significant difference in prolidase activity.

A system was developed to study membrane transport in isolated human fibroblast plasma membrane vesicles, avoiding potential complications of intracellular binding and metabolism in the intact cell. Ten-fold enrichment of plasma membranes after subcellular fractionation was confirmed with appropriate markers. Transport competence was established by the following criteria: osmotic sensitivity, stereospecificity, temperature dependence, sodium gradient stimulation, response to ions and ionophores, saturability, and exchange properties. Methotrexate uptake was osmotically sensitive, temperature sensitive, saturable, and inhibited by folinic acid and phosphate. Measurement of lysine uptake was complicated by binding and lack of sodium dependence, but was stimulated by an interior-negative membrane potential, and intravesicular lysine or arginine. Exchange properties of the lysine carrier were exploited to assess its function in fibroblasts from patients with the Mendelian phenotype, lysinuric protein intolerance (LPI). LPI vesicles were not different from controls in their lysine transport phenotype.

No description available.

The aim of this study was to examine the claim that a single, mutant oncogene can transform NIH 3T3 mouse fibroblasts into transformed, tumorigenic cells, acting in a genetically dominant fashion. A c.Ha-ras 1 oncogene, cloned from the EJ human bladder carcinoma cell line, was inserted into a shuttle vector carrying the selectable marker gene gpt, which encodes the enzyme XPRT (xanthine-guanine phosphoribosyl transferase). This construct, pSV2gptEJ, was transfected into NIH 3T3 cells by the calcium phosphate precipitation method and cells which had incorporated the plasmid were selected by growth in mycophenolic acidcontaining medium, to which gpt confers resistance. A number of clonal lines were established and their tumorigenicity tested. Tumour cell lines derived from these transfectants were back-selected using 2-thioxanthine, a cytotoxic analogue of the xanthine-guanine phosphoribosyl transferase substrate, to isolate clones which no longer contained functional pSV2gptEJ sequences. Six sub-clones which did not express detectable levels of the ras oncogene product, p21^H.ras , were obtained. All were judged to be less transformed than the transfected parent cells: they appeared morphologically normal, were more serum-sensitive, showed clear saturation densities and were more anchorage-dependent. Three of these sub-clones were found to be tumorigenic at all sites tested. Cytological examination of the NIH 3T3 transfectants revealed that significant perturbation of their chromosome complement accompanied transfection. The transfection process, in the absence of DNA or with pSV2gpt alone, was found to be capable of transforming NIH 3T3 cells. Finally, a brief investigation of the effect of a "functional EJ-ras gene upon the differentiated phenotypes of these cell lines was attempted by comparing their ability to produce an extracellular matrix.

Prolidase deficiency (MIM 26413), an autosomal recessive phenotype, is caused by rare alleles at a locus on chromosome 19cent.-q13.2. The clinical phenotype is pleiotropic (affecting skin, brain, etc.) and of variable expressivity (benign to early death). I established skin fibroblast cultures from 6 homozygous probands and 6 obligate heterozygotes, purified prolidase (E.C. 3.4.13.9, a homodimer) from normal human fibroblasts, raised a monospecific rabbit antiserum to the subunit, and studied its biosynthesis. Pulse-chase immunoprecipitation experiments showed that the subunit is synthesized in the cytosol as a 58 KDa. polypeptide and not processed further. Homozygous prolidase-deficient cell strains expressed 3 classes of mutant alleles which by complementation analysis mapped to one locus. The alleles were designated CRM$-$ (nul), CRM+ activity/size variant, and CRM+ activity variant. Heterozygotes carrying CRM$-$ alleles have heat stable prolidase (50$ sp circ$C, 1hr); heterozygotes carrying CRM+ variant alleles have heat labile enzyme. The finding implies that variant CRM+ allele(s) can confer negative allelic complementation on the dimeric enzyme (dominant relative phenotype). CRM$-$ homozygous cells contain varying amounts of an alternative imidodipeptidase-like activity. The variant prolidase allele (major gene) and amount of alternative "prolidase" activity (modifier gene) are apparently both determinants of the associated clinical phenotype in prolidase deficiency. I obtained and sequenced a tryptic peptide from human kidney prolidase for synthesis of oligonucleotide probes in the future.

Knee injuries are a major cause of orthopedic disabilities in the United States. Current reconstruction techniques for torn anterior cruciate ligaments (ACL) require extensive surgery and long physical rehabilitation times since the tissue does not heal upon injury. A common ACL injury occurs where the gap at the rupture site remains open after injury and fails to heal, which can lead to premature osteoarthritis and disability. Hydrogels are a popular material used for tissue engineering applications due to their ability to retain water and good biocompatibility. Previous work has shown that hydrogels can be made through the mixed-mode reaction of radically crosslinked thiol groups and acrylate end groups. This project explores mixed-mode oligo[poly(ethylene glycol) fumarate] (OPF)-based hydrogels as alternate carriers for regeneration of partial tear ligament defects. The main purpose of this project was to determine the degradative properties of and cell response to thiol-PEG-thiol (PEG-diSH), a novel hydrogel material. The swelling and degradative properties of hydrogels containing three components OPF, PEG-diacrylate (PEG-DA), and PEG-diSH were characterized by their fold swelling. In addition, cell viability, morphology changes, proliferation and collagen production were analyzed in tri-ratio hydrogels with and without the presence of RGD over three weeks. Results showed that the hydrogels containing PEG-diSH demonstrated significantly larger fold swelling and promoted cell clustering (as shown by increased area of clusters), probably due to the larger mesh size and possibly due to the presence of free thiol functional groups present in the network from the mixed-mode reaction. However, an increase in cell number was not found in these gels up to eight days, suggesting that cell migration may play a role in the appearance of clusters. Additionally, increased cell spreading in response to RGD was observed inside gels containing PEG-diSH; no spreading was seen in the non PEG-diSH gels (± RGD), possibly because the mesh size was too small to allow for clustering or spreading within the matrix. Results from this work suggest that the presence of PEG-diSH could promote cell-cell contact within the clusters which could be useful in systems where direct contact promotes tissue formation or cell differentiation.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.