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Biomedical subjects

C Franzblau

Publications and source records attributed to C Franzblau.

At least 55 records · Page 3Linked to original sources
PubMed1988

Glycosaminoglycan content in neonatal rat aortic smooth muscle cell cultures.

In the present study the biosynthesis of glycosaminoglycans (GAGs) by neonatal rat aortic smooth muscle cells in culture was studied. Heparan sulfate (HS) was the predominant GAG of the cell layer accounting for 32-49% of the total GAGs depending on the time in culture. The presence of low sulfated chondroitin sulfate (LSC) in aortic smooth muscle cell cultures is reported here for the first time. The effect of ascorbate on the synthesis and accumulation of these macromolecules resulted in a relative increase of C4S and DS in the cell layer. In contrast, the distribution of the GAGs which were secreted into the medium was not significantly effected by the addition of ascorbate. While HS was always found to be a minor component, the other GAGs were present in about equal concentrations. The total GAG accumulation in the medium was much greater (91-97%) than that of the cell layer (3-9%) indicating that the cells are synthesizing relatively large amounts of GAGs, although incorporation of these macromolecules into the extracellular matrix was consistently low.

Animals↗
PubMed1988

beta-VLDL-induced alterations in growth potentiating activity produced by mononuclear phagocytes.

This report describes the enhancement of growth potentiating activity produced by mononuclear phagocytes that were incubated with beta-migrating very low density lipoproteins (beta-VLDL). Conditioned media harvested from cultured human peripheral blood monocytes incubated in the presence or absence of the lipoprotein were evaluated for their ability to stimulate DNA synthesis ([3H]thymidine incorporation) of sparsely seeded quiescent BHK-21 (BHK) cells as well as neonatal rat aortic smooth muscle cells (NRSMC). Conditioned media from monocytes incubated in the presence of beta-VLDL enhanced [3H]thymidine incorporation into DNA of both BHK and NRSMC, when compared to conditioned media harvested from monocytes incubated in the absence of beta-VLDL. Studying NRSMC, this effect was evident using media collected from monocytes incubated with lipoprotein for 2 days; however, a longer incubation of monocytes plus lipoprotein was necessary to induce changes in growth potentiating activity for BHK cells. Likewise, the effect of beta-VLDL treatment of thioglycollate broth elicited BALB/c mouse peritoneal macrophages was evaluated. Conditioned media from lipoprotein-treated macrophages exhibited greater growth-stimulating activity for both BHK cells and NRSMC when compared to conditioned media from macrophages incubated in the absence of the lipoprotein. beta-VLDL did not affect viability of the mononuclear cells. These findings further implicate the involvement of the monocyte-derived foam cell in the development of atherosclerosis.

Animals↗
PubMed1988

Defenses of the hamster lung against human neutrophil and porcine pancreatic elastase.

Instillation of human neutrophil elastase (HNE) into hamster lungs produces milder emphysema but more pulmonary hemorrhage than an equivalent amount of porcine pancreatic elastase (PPE), whether equivalence is determined by elastolytic units or moles. We undertook a study of the mechanisms of these differences. 125I-HNE or 3H-PPE were instilled intratracheally into hamsters. The partitioning of radioactivity between bronchoalveolar lavage fluid (BAL) and lung tissue was similar for HNE and PPE as were the half-lives, 45 and 51 min, respectively, for uncomplexed, enzymatically active HNE and PPE. In BAL there was preferential binding and inactivation of HNE by the hamsters' alpha-1-protease inhibitor (a-1-PI) whereas PPE was preferentially bound by alpha-2-macroglobulin (a-2-M). This was also observed in vitro when HNE and PPE were incubated with plasma from untreated hamsters. Nevertheless, when the sum of the elastase binding capacity of a-1-PI and a-2-M was considered, hamster plasma had similar binding capacities for HNE and PPE. It is known that the enzymatic activity of elastases is inhibited by formation of a stable complex with a-1-PI. On the other hand, elastases bound to a-2-M are protected against a-1-PI inhibition but can free themselves by proteolysis and exhibit elastolytic activity. Preferential inactivation of HNE by a-1-PI may be one mechanism that accounts for the lesser emphysema-inducing potency of HNE than of PPE.

Animals↗
PubMed1988

Cadmium-chloride-induced air-space enlargement with interstitial pulmonary fibrosis is not associated with destruction of lung elastin. Implications for the pathogenesis of human emphysema.

To determine whether lung elastin is lost during the evolution of cadmium-induced air-space enlargement with pulmonary fibrosis, the lung elastin of 5- to 7-day-old golden Syrian hamster pups was radiolabeled by giving [3H]valine. At maturity, a single intratracheal instillation of 0.5 ml of 0.025% CdCl2 solution was given. Lung mechanics, histologic examination, and biochemistry were studied 5, 10, 21, 42, 105, and 180 days after the cadmium treatment. The animals developed fibrosis and air-space enlargement with decreased lung volumes, compliance, and forced expiratory flow; their functional residual capacity was increased. The total lung collagen and total lung elastin were increased, but there was no loss of radiolabel in lung elastin. We conclude that CdCl2-induced air-space enlargement with pulmonary fibrosis is not accompanied by loss of neonatally formed lung elastic fibers. We hypothesize that air-space enlargement with fibrosis represents a stereotyped response of the lung to fibrosing injuries, which we hypothesize is due to forces from more fibrotic and atelectatic areas causing overdistension of less abnormal air spaces. The air-space enlargement of fibrosing human diseases such as sarcoidosis and eosinophilic granuloma may have a similar basis. Evidence is reviewed that human centrilobular emphysema may be a form of focal air-space enlargement with interstitial fibrosis; there may be mechanisms in addition to elastase-antielastase imbalance that cause human emphysema.

Animals↗
PubMed1988

Repair of protease-damaged elastin in neonatal rat aortic smooth muscle cell cultures.

The objective of this study was to investigate the elastin repair process in the rat aortic smooth muscle cell culture after proteolytic injury. Although little studied in vivo, elastin repair is thought to occur through a sequential process involving enzymatic removal (debridement) of damaged fibers followed by synthesis of tropoelastin, its subsequent processing, and eventual incorporation into new insoluble elastin. A second repair mechanism of proteolytically damaged elastin in a culture system is reported here. Repair in this system relates directly to restoration of resistance to elastin solubilization by hot alkali. As expected, severe injuries were observed with porcine pancreatic elastase (PPE). Using PPE, only 6% of the elastin, relative to control, was resistant to hot alkali immediately after elastase treatment. 4 wk later, resistance to hot alkali had increased dramatically to a mean of 90%. Repair took longer after injury with 75 micrograms of PPE as compared with 50 micrograms of PPE. The limited elastic fiber proteolysis induced by either human neutrophil elastase or porcine trypsin was repaired in culture within 2 wk. Elastin that had been radiolabeled with [3H]lysine 4-5 wk before injury was converted from a hot NaOH-susceptible to a NaOH-resistant elastin fraction during recovery from PPE injury. At the same time, the frayed elastic fibers that were seen with the electron microscope immediately after PPE treatment were replaced by continuous bands of elastin that resembled those in control cultures. Restoration of NaOH resistance did not require a net increase in total cell layer elastin, suggesting that relatively little new tropoelastin incorporation into the cell layer was required for this type of repair. These results suggested a salvage repair mechanism for proteolytically damaged elastin.

Animals↗
PubMed1988

Congo red binding of elastin in aortic smooth muscle cell cultures.

These studies demonstrate that the strong binding capacity of elastin for Congo red can be used to advantage in aortic smooth muscle cell cultures. A fibrous elastin network fluoresces when Congo red is added. Congo red does not alter accumulation of elastin or of total protein, even when the cells are grown in the presence of the dye for long periods of time, indicating that it is not toxic. Porcine pancreatic elastase was used to solubilize elastin in these cultures, to determine the molar ratio of Congo red to elastin, thus making it possible to estimate the amount of elastin solubilized when the cultures are injured. Congo red binding to elastin will be useful in studying elastin accumulation and/or degradation in vitro and in vivo.

Animals↗
PubMed1988

Influence of substratum on corneal epithelial cell growth and protein synthesis.

To evaluate the effect of substratum on corneal epithelial cell growth and on protein synthesis, bacteriological dishes were coated with laminin, fibronectin, Type I collagen or Type IV collagen. Cell growth was enhanced by day 6 on both laminin and Type I collagen substrata. Adhesion sites and parallel microfilament arrays were present only on cells cultured on laminin coated substratum. The synthesis of connective tissue and cytoskeletal proteins was influenced by the four different substrata. Both the time course and the amount synthesized was affected. The results of this study indicate that the substratum plays a significant role in regulating the synthesis of specific proteins.

Animals↗
PubMed1988

Development of a biopolymeric keratoprosthetic material. Evaluation in vitro and in vivo.

Based on the results of in vitro and in vivo experiments, we have determined that the optimal material for the central transparent portion of a perforating keratoprosthesis is a polyvinyl alcohol copolymer hydrogel. The material supports the maintenance and growth of corneal epithelium in vitro as shown by population doublings and transmission electron microscopy. Discs preseeded with epithelial cells were cultured in vitro and transplanted into rabbit corneas. The proliferation of these cells in vivo was demonstrated using 3H-thymidine. Other experiments showed that the preseeded cells not only migrated from the central disc onto the peripheral rim of the host cornea but also that host peripheral epithelial cells migrated onto the anterior surface of the disc. The experiments described in this paper demonstrate that corneal epithelial cells preseeded onto hydrogel discs and transplanted into rabbit corneas remain adherent and are capable of proliferating.

Animals↗
PubMed1988

Tissue-specific posttranslational modification of rat apoE. Synthesis of sialated apoE forms by neonatal rat aortic smooth muscle cells.

We have studied the synthesis, modification, and secretion of rat apoE in primary cultures of neonatal aortic smooth muscle cells and adult rat hepatocytes. The cultures were pulsed with [35S]methionine and the intracellular and secreted apoE were immunoprecipitated and analyzed by two-dimensional isoelectric focusing/polyacrylamide gel electrophoresis and autoradiography. A short pulse (10 min) showed the presence of a major unmodified apoE form in both cultures. This form comigrated on two-dimensional gels with the major rat plasma apoE isoprotein. A longer pulse (15-120 min) resulted in the progressive appearance of intracellularly modified apoE isoproteins in both cultures. The apoE secreted by aortic smooth muscle cells consisted exclusively of sialated apoE isoproteins that were sensitive to neuraminidase treatment. In contrast, the apoE secreted by primary cultures of adult rat hepatocytes, organ cultures of neonatal rat liver, as well as rat plasma apoE, contained several minor modified isoproteins. Nascent apoE secreted by the aortic smooth muscle cell cultures floats in the density range of 1.09 to 1.186 g/ml. We conclude that aortic smooth muscle cells can synthesize and secrete sialated apoE isoproteins associated with nascent lipoproteins floating in the high density lipoprotein region.

Animals↗
PubMed1987

Elastin in a neonatal rat smooth muscle cell culture has greatly decreased susceptibility to proteolysis by human neutrophil elastase. An in vitro model of elastolytic injury.

A neonatal rat aorta smooth muscle cell culture system with a unique elastin-rich extracellular matrix was used as a model substrate for elastases. To study the susceptibility to solubilization of insoluble elastin, cultures were incubated in the presence of human neutrophil elastase (HNE) or porcine pancreatic elastase (PPE) and in the absence of serum for periods up to 45 min. Both the incubation media and cell layers were then assessed for elastin and collagen markers, total protein, and lactate dehydrogenase (LDH). Although HNE and PPE exhibited comparable activity against elastin purified from the cell layer, HNE exhibited a 6.7- to 25-fold reduction in its elastin solubilizing activity using intact cell layers as compared with the purified elastin, whereas PPE exhibited only a 1.5- to 2.5-fold reduction. This effect could not satisfactorily be explained as preferential inhibition of HNE activity in the culture system, because the amount of protein solubilized by HNE was 59% that of PPE. The mean elastin content of PPE-solubilized protein was 110% that of the elastin content of the corresponding cell layer; the value for HNE-solubilized protein was only 16%. Thus, the amount of elastin per microgram of solubilized protein for HNE was 15% that for PPE. Possible explanations for the greatly diminished elastolytic activity of HNE in the culture system include the preference of HNE for other substrates in the cell layer, the inability of HNE to penetrate sufficiently into the cell layer, and the presence of sulfated glycosaminoglycans in the vicinity of the elastin that act in an inhibitory fashion. Although there was extensive proteolytic damage to the extracellular matrix, LDH and DNA measurements indicated that little loss of cells or cell viability occurred. The observed differences in elastolytic activity of HNE and PPE in the culture system parallel the relative emphysema-inducing potency of the elastases in the hamster model of elastase-induced emphysema.

Animals↗
PubMed1986

The effect of prostaglandin E2 (PGE2) on amino acid uptake and protein formation by lung fibroblasts.

The effect of prostaglandin E2 (PGE2) on the utilization of extracellular amino acids by fetal lung fibroblasts was examined. PGE2 decreased the uptake of proline and aminoisobutyric acid (AIB) by quiescent fibroblasts in culture. The uptake of AIB by serum-activated cultures was also dramatically decreased by PGE2. The PGE2-induced decrease in the uptake of AIB was first observed at 4 h following the addition of the effector molecule to the cultures. PGE2 did not affect the uptake of leucine. The addition of cycloheximide also resulted in a decrease in the uptake of proline, similar to that induced by PGE2 at 5 X 10(-8) M. The combination of cycloheximide and PGE2 resulted in a further decrease in proline uptake. Kinetic analysis of AIB uptake following a 24-h PGE2 treatment showed an increase in the apparent Km as compared with untreated cultures. The prostaglandin remained active for at least 72 h after the addition of the molecule. Removal of the PGE2 was followed by an influx of proline into the cells. The decrease in proline uptake was associated with a decrease in the amount of intracellular free proline and an overall decrease in the amount of cell-associated protein. While PGE2 is known to increase intracellular protein degradation, the effect of PGE2 on amino acid uptake was not the result of an increase in the intracellular concentration of amino acids (transinhibition).

Amino Acids↗
PubMed1986

Complex regulation of collagen gene expression in cultured bovine aortic smooth muscle cells.

Cultured bovine aortic smooth muscle cells display an increase in production of type I and type III collagen as a function of the number of days in second passage (Beldekas, J. C., Gerstenfeld, L. C., Sonenshein, G. E., and Franzblau, C. (1982) J. Biol. Chem. 257, 12552-12556). In this study, we report that the regulation of these events is highly complex and relates to the growth state of the cells. Cultures, seeded at 1.5 X 10(6) cells/75-cm2 flask, produced very little collagenous protein early when the cells were proliferating rapidly. As they approached confluence at day 6, collagen synthesis began to increase. Maximal collagen synthesis was observed at day 14. In contrast, the levels of the mRNAs for type I and type III collagen increased only up to the 10th day and thereafter decreased. Cell-free translation analyses indicated that the translational activity of the collagen mRNAs was increasing over the time course. These results suggest that both translational and pretranslational sites are involved in the control of collagen production by aortic smooth muscle cells, and that collagen synthesis is inversely related to the proliferative state of the cells in culture.

Animals↗
PubMed1986

Phospholipid methylation in rabbit aorta.

The formation of phosphatidylcholine by successive methylations of phosphatidylethanolamine using S-adenosylmethionine as the methyl donor was studied in homogenates of rabbit aorta. Addition of phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine, but not phosphatidylethanolamine, stimulated methyltransferase activity and this activity was further stimulated when the phospholipids were dispersed in taurocholate prior to addition to the assay system. No incorporation of radiolabel into sphingomyelin or lysolecithin was detected indicating minimal metabolism of newly formed phosphatidylcholine. The majority of methyltransferase activity was detected in the high-speed pellet of the aortic homogenate; however, since activity was also detected in the high-speed supernatant, the low-speed supernatant preparation was used as the source of enzyme. Methyltransferase activity was characterized in cultured arterial smooth muscle cells using methionine as the radiolabeled precursor. The major product formed was phosphatidylcholine. No difference in enzyme activity was seen as a function of the length of time that cells were in culture or anatomic location of the aortic explant used as a source of cells. Treatment of the cells with cycloheximide did not affect methyltransferase activity. The ability of catecholamine agonists and vasoactive peptides to influence methyltransferase activity was investigated both in the cell-free preparation and in cultured cells. These compounds did not appear to alter methyltransferase activity in rabbit aortic smooth muscle cells.

Animals↗
PubMed1986

Purification and properties of Clostridium difficile cytotoxin B.

Toxin B, a potent cytotoxin produced by Clostridium difficile, was purified to homogeneity from 6-day broth cultures of a toxigenic isolate. Cytotoxin was purified approximately 4000-fold by sequential ammonium sulfate precipitation, DEAE-Sepharose chromatography, and high performance liquid chromatography on a Mono Q anion-exchange column. The molecular weight of reduced purified toxin was 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, compared to 150,000 for unreduced toxin. Dose-response studies indicated that subpicogram concentrations of purified toxin caused rounding of approximately 20,000 IMR-90 fibroblasts. The phenomenon of cell rounding caused by toxin B was correlated with the ratio of globular to filamentous actin in fibroblasts as measured by two techniques. The toxin caused a significant increase in the ratio of globular to filamentous actin which was nearly completed prior to the onset of rounding. We conclude that cell rounding of fibroblasts exposed to toxin B is related to an increase in the ratio of globular to filamentous actin which is produced by small numbers of toxin molecules/cell.

Actins↗
PubMed1986

Elastase effect on the extracellular matrix of rat aortic smooth muscle cells in culture.

The effect of elastase on the extracellular matrix of neonatal rat aortic smooth muscle cell cultures was monitored both chemically and ultrastructurally. Porcine pancreatic elastase was shown to decrease the elastin content in these cultures. Although chemically no distinction could be made between the elastin remaining in the culture matrix after elastase when compared to that in the nontreated cultures, the elastin was dramatically altered morphologically. The elastin assumed a "mottled" appearance after elastase treatment similar to that seen in vivo in emphysema models. A highly sensitive immunogold staining technique was used to detect elastin at the earliest stages of accumulation. Pulse experiments demonstrated an increase in protein synthesis by the cells 20 hr after elastase exposure. The culture system described here provides a model for probing in vivo elastase effects on elastin-containing tissues.

Amino Acid Sequence↗
PubMed1986

Clostridium difficile cytotoxin inhibits protein synthesis in fibroblasts and intestinal mucosa.

The pathophysiology of Clostridium difficile colitis is thought to be mediated by release of toxin A, an enterotoxin, and toxin B, a cytotoxin. We compared the differential effects of toxin B on protein synthesis in IMR-90 fibroblasts and in hamster esophagus, stomach, gallbladder, small intestine, and cecum in organ culture. Toxin B in low concentrations stimulated (p less than 0.001) incorporation of [3H]leucine into fibroblast proteins, whereas at higher dosages it inhibited incorporation (p less than 0.001). This biphasic effect was independent of cell rounding and was not caused by a change in uptake of precursor. Purified toxin B had no effect on protein synthesis in a cell-free rabbit reticulocyte translation system, indicating that inhibition of protein synthesis in intact fibroblast monolayers and intestinal explants is a consequence of toxin B effect on some other cellular target. Toxin B significantly inhibited protein synthesis in hamster cecal explants in a dose-dependent fashion. Again, this inhibition was not mediated by altered precursor uptake. Toxin B significantly inhibited in vitro protein synthesis in hamster terminal ileum, cecum, and sigmoid colon, but not in esophagus, gallbladder, stomach, or duodenum. These results suggest that toxin B-mediated inhibition of protein synthesis may be a generalized toxic effect in tissue culture cells and intestinal epithelium. Inhibition of protein synthesis in the distal intestinal epithelium may contribute to the pathophysiology of colitis caused by this organism.

Cecum↗
PubMed1986

Experimental arthritis in a nonhuman primate. I. Induction by bovine type II collagen.

Six squirrel monkeys immunized with native fetal bovine type II collagen (CII) in complete Freund's adjuvant developed arthritis 3 to 6 weeks later. None of three cebus monkeys given CII plus complete Freund's adjuvant or three control squirrel monkeys immunized with complete Freund's adjuvant alone developed arthritis. In four of the squirrel monkeys, arthritis was symmetrical and involved mainly the interphalangeal and metacarpal phalangeal joints. Two other monkeys had pauciarticular disease. Although three monkeys became cachetic and died, the others regained weight and their arthritis spontaneously remitted with minor residual deformities in digits and larger joints. Each squirrel monkey with arthritis had high titers of CII antibodies whereas the arthritis-resistant cebus monkeys had lower titers of CII antibodies whereas the arthritis-resistant cebus monkeys had lower titers of CII antibodies. As an animal model, experimentally induced arthritis in primates appears to resemble an acute arthropathy in man rather than chronic rheumatoid arthritis.

Acute Disease↗