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Biomedical subjects

C Franzblau

Publications and source records attributed to C Franzblau.

At least 91 records · Page 5Linked to original sources
PubMed1984

Mammalian cell growth on collagen-hydrogels.

Hydroxyethylmethacrylate (HEMA) hydrogels have a peculiar crater-like topography which renders them ideal for studying cell-to-substrate contact formation. Cultured rabbit aortic smooth muscle cells (SMC) were grown on collagen-HEMA hydrogels, and the ultrastructure of developing cell attachment sites was studied. By 3 hours after cell seeding, both the rounded and spreading SMC appeared anchored to the hydrogel via extra-cellular connective tissue-like material. The fully formed attachment site present at 5-8 day was characterized by large bundles of intracellular myofilaments inserting onto areas of increased electron density along the plasmalemmal membrane. Large amounts of extracellular connective tissue-like material also appeared attached to the areas of increased electron density. Fully formed cell substratum attachment sites were not observed when either elastin-HEMA or hydrogels polymerized in the absence of protein were employed. The growth and collagen synthesis by human embryonic lung fibroblasts (IMR-90) and endothelial cells on collagen-HEMA hydrogels and tissue culture plastic were also examined. Although the two cell types grew equally well on the two surfaces, differences in protein synthesis were observed. The procollagen Type I and III ratio synthesized by the fibroblasts remained the same while the ratio synthesized by the endothelial cells varied even though their morphology appeared similar. The fibroblasts synthesized less collagen when grown on collagen-HEMA hydrogels (this effect may be related to age), while the amount of collagen synthesized by the endothelial cells grown on the two surfaces was similar.

Animals↗
PubMed1983

Ultrastructural effects of Clostridium difficile toxin B on smooth muscle cells and fibroblasts.

The mechanism by which Clostridium difficile toxin B causes cells in culture to round was investigated. Cultured human lung fibroblasts and rabbit aortic smooth muscle cells were treated with partially purified or purified toxin B and monitored by light and transmission electron microscopy (TEM). Both preparations caused progressive cell rounding which correlated with disorganization of actin-containing myofilament bundles. Thin myofilaments became fragmented and finally disappeared (after 24 h) and dense bodies became more prominent, while all other organelles appeared unaffected.

Animals↗
PubMed1983

Cell-free translation of calf type III collagen. Effect of magnesium on ribosome movement during elongation.

Calf aortic smooth muscle cell cultures produce both type III and type I collagen. Polyadenylated mRNA species purified from these cells direct the synthesis of prepro-alpha 1(III), prepro-alpha 1(I), and prepro-alpha 2(I) in a rabbit reticulocyte cell-free system. These polypeptides were identified by specific immunoprecipitation, cyanogen bromide peptide mapping, and bacterial collagenase digestion. Lower molecular weight collagenase susceptible polypeptides were also produced in translation reactions incubated under conditions optimized for incorporation of radiolabeled amino acids. Their presence did not appear to result from ribonuclease or protease involvement or from premature termination. Increasing the Mg2+ concentration in the translation system significantly reduced the production of these lower molecular weight species. Pulse-chase experiments indicate that the time required for completion of full length preprocollagen at the high Mg2+ concentration is greatly decreased compared to the low concentration. Additional experiments suggest that the incomplete collagen polypeptides result from pausing of ribosome movement during elongation. The relative synthesis of type III and type I chains was examined as a function of mRNA concentration in the cell-free system. At levels of RNA above saturation, the relative production of type III decreased with respect to type I. These data suggest that the ability of the alpha 1(III) mRNA to initiate translation is less efficient than the mRNAs of alpha 1(I) and alpha 2(I).

Animals↗
PubMed1983

Functional alpha 1-protease inhibitor in the lower respiratory tract of cigarette smokers is not decreased.

Cigarette smoking is the major risk factor for the development of pulmonary emphysema, a disorder that may result from an imbalance between the elastase and antielastase levels in the lungs. Decreased functional alpha 1-protease inhibitor, an inhibitor of neutrophil elastase, might render smokers susceptible to elastase-catalyzed destruction of pulmonary elastic fibers and the development of emphysema. Binding and inactivation of isotopically labeled porcine pancreatic elastase and human neutrophil elastase by alpha 1-protease inhibitor were measured in fluid obtained by bronchoalveolar lavage of volunteers. The inhibition of elastase-catalyzed solubilization of elastin and a tripeptide substrate were also determined. The mean level of functional alpha 1-protease inhibitor in the bronchoalveolar lavage fluid of smokers was found to be equal to or greater than that of nonsmokers, contradicting reports by other investigators. Increased elastase derived from pulmonary neutrophils, rather than decreased functional alpha 1-protease inhibitor, appears to be the main factor in the genesis of emphysema in smokers.

Adult↗
PubMed1983

Autoradiographic study of 3H-methylated elastase in hamster lungs.

An emphysemalike condition can be induced in animal lungs by the instillation of a single dose of elastase. Autoradiography was used to determine the location of 3H-methylated porcine pancreatic elastase in hamster lungs at four time points. Six hours after instillation of radiolabeled enzyme the distribution of silver grains was very patchy, but in heavily labeled areas grains were concentrated over macrophages, connective tissue areas and over some fibroblasts. By 24 hr the labeling of connective tissue areas was no longer evident and almost all silver grains were associated with macrophages or with the edema fluid that filled many alveoli at this time. By 4 days only macrophages exhibited concentrations of silver grains. The labeling of macrophages was still evident at 7 days. Elastase inactivated by N-acetyl-(L-alanyl)3-L-alanine chloromethyl ketone showed a different distribution 6 hr after instillation. Silver grains were concentrated over macrophages and alveolar type II cells but showed no affinity for connective tissue areas or fibroblasts. By 24 hr almost all grains were located over heavily labeled macrophages.

Animals↗
PubMed1983

Radioimmunoassay for methotrexate using hydroxyethylmethacrylate hydrogel.

A sensitive, rapid method for the measurement of methotrexate in biologic fluids has been developed using a solid-phase radioimmunoassay (RIA). Hydroxyethylmethacrylate monomer was polymerized in the presence of rabbit antimethotrexate antisera and the resultant gels lyophilized, ground to a fine powder, and aliquoted into 3-ml syringes fitted with a fritted filter disc. Control gels without incorporated antibodies did not bind radiolabeled methotrexate, whereas a dose-response curve expressing percent bound methotrexate against antiserum concentration could be readily constructed for appropriate gels. Drug concentrations of less than 1 ng/ml can be measured. The fate of a bolus IV injection of methotrexate (100 mg) into an adult female baboon was evaluated by the HEMA-RIA system and compared with a previously published enzyme assay for the drug. These studies describe a stable and efficient system for rapid separation of bound antigen by simple filtration.

Animals↗
PubMed1983

Effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells.

The use of protein hydroxy ethylmethacrylate (HEMA) hydrogels to control cell morphology and growth, as well as the synthesis of extracellular matrix components, is described in this communication. HEMA hydrogels prepared with collagen support growth of embryonic lung fibroblasts (IMR-90), as well as bovine aortic and pulmonary artery endothelial cells at a level comparable to the respective cells grown on tissue culture surfaces. On the other hand, HEMA hydrogels prepared with solubilized elastin inhibit the fibroblast growth and prevent both types of endothelial cell cultures from achieving their normal morphology. These morphologically altered endothelial cells resume a normal cobblestone-like appearance when subcultivated from the elastin-HEMA hydrogels to tissue culture plastic. When pulsed with [14C]proline, the procollagens synthesized by the endothelial cells on the different surfaces vary, as shown by immunoprecipitation and polyacrylamide gel electrophoresis. On the standard tissue culture plastic, the confluent cells produce mainly type III procollagen in the medium, whereas those endothelial cells grown on collagen and elastin-HEMA hydrogels synthesize primarily type I procollagen (much like sprouting cells on tissue culture plastic), regardless of their morphology.

Acrylates↗
PubMed1983

Preservation of ultrastructure of cells cultured on protein-hydroxyethylmethacrylate hydrogels.

A method for studying the ultrastructure of cells grown on hydroxyethylmethacrylate (HEMA) hydrogels is described. Under normal conditions, HEMA hydrogels tend to swell when placed in hypotonic solutions and to shrink during alcohol dehydration. To overcome this severe swelling and/or shrinking, all solutions used during the fixation procedure are made in Puck's saline G, and dehydration is accomplished with a graded series of ethanol solutions prepared with Puck's saline G and polyethylene glycol. Infiltration of the sample with embedding material is achieved with the aid of a vacuum oven. Our findings suggest that the cells cultured on collagen hydrogels are ultrastructurally indistinguishable from those cultured on tissue-culture plastic. In addition, the unusual crater-like topography of the hydrogel can be utilized as an experimental aid in the study of cell attachment and spreading.

Acrylates↗
PubMed1983

The fate of neutrophil elastase incorporated by human alveolar macrophages.

Although protease-antiprotease imbalance is widely thought to contribute to the genesis of emphysema, the involvement of the alveolar macrophage in this process is poorly defined. We have quantified the uptake of radioiodinated human neutrophil elastase by human alveolar macrophages in monolayer culture and assessed the enzyme's fate during periods as long as 48 h after uptake using molecular sieve chromatography. Approximately half of the radiolabel eluted with enzymatically inactive material of molecular sizes corresponding to either degraded or alpha1-protease-inhibitor-bound elastase. The remainder of the radiolabel eluted at 29,000 daltons, in fractions containing enzyme that solubilized particulate elastin, and likely represented intact neutrophil elastase. Macrophages from smokers and nonsmokers showed similar characteristics of incorporation and disposition of neutrophil elastase. Lysates of uncultured alveolar macrophages from smokers and nonsmokers contained (mean +/- SE, n = 4) 7.4 +/- 0.8 and 3.3 +/- 1.4 ng of neutrophil elastase activity per 10(6) cells, respectively (not significant). However, as smokers have 11-fold more cells obtainable by lavage, the total lavaged elastase loads in alveolar macrophages were 561.7 +/- 72.3 and 21.3 +/- 6.8 (p less than 0.01) in smokers and nonsmokers, respectively. We conclude that the alveolar macrophage may clear and inactivate neutrophil elastase in the lung. In addition, by conserving some ingested elastase in an enzymatically active form, the macrophage may serve as an elastase reservoir. In areas of high macrophage density, as around the respiratory bronchioles of smokers, the macrophage could release a portion of the incorporated elastase and damage lung elastin.

Cell Survival↗
PubMed1983

Human neutrophil elastase does not bind to alpha 1-protease inhibitor that has been exposed to activated human neutrophils.

The effect of leukocyte-derived oxidants on the elastase-inhibitory capacity of alpha 1-protease inhibitor was examined in an in vitro system using cells and purified proteins from human sources. The exposure of alpha 1-protease inhibitor to the myeloperoxidase-hydrogen peroxide-halide system resulted in a nearly complete loss of its ability to bind and inactivate purified human neutrophil elastase. A similar loss of binding to and inactivation of human neutrophil elastase was observed on exposure of alpha 1-protease inhibitor to human neutrophils in the presence of a halide and the neutrophil-activating agent, phorbol myristate acetate. This loss of elastase binding activity was abrogated by the addition of azide or catalase but not superoxide dismutase or heated catalase. The data suggest oxidative inactivation of alpha 1-protease inhibitor by secreted myeloperoxidase and hydrogen peroxide. Thus, the reported effects of leukocyte oxidants, especially the myeloperoxidase system, on alpha 1-protease inhibitor have been confirmed using the most pathophysiologically relevant protease, human neutrophil elastase, as the test enzyme. The role of the neutrophil in the pathogenesis of emphysema may, therefore, include the secretion of both elastase and oxidants that impair antielastase defenses.

Blood Proteins↗
PubMed1983

The balance of lung connective tissue elements in elastase-induced emphysema.

Elastase-induced emphysema is associated with changes in all components of connective tissue, including elastin. The abnormal restructuring of lung parenchyma that occurs after elastase administration in hamsters might reflect an abnormal balance of elastin, collagen, and glycosaminoglycan in lung parenchyma. To test this hypothesis, we measured total amounts and levels of accumulation over 24 hr of connective tissue elements in lung explants at several points over a 1-year period after a single elastase treatment in hamsters. We found acute, early changes in the metabolism of elastin, collagen, and glycosaminoglycan consistent with degradation of elastin and increased turnover of collagen and glycosaminoglycan. By day 21 after elastase treatment, lung elastin had returned to control values, then rose and stayed elevated throughout the remainder of the study period. Total collagen levels rose in both control and elastase-treated lungs by the same amount over the year period. However, incorporation of 14C-proline into collagen hydroxyproline was elevated only in elastase-treated lungs over the period of 21 to 360 days. Incorporation of 14C-glucosamine into glycosaminoglycan was greatest over the period of 1 to 5 days after treatment and total levels also peaked at this time. By day 21 both incorporation of glucosamine and total levels of glycosaminoglycan had returned to normal, where they remained. The ratios of glycosaminoglycan to elastin and glycosaminoglycan to collagen were calculated over the period of 21 to 360 days to determine whether the long-term relative balance of these components had changed in elastase-treated lungs. We found that a steady-state imbalance existed between heparan sulfate and collagen and heparan sulfate and elastin, suggesting that an inappropriate amount of heparan sulfate was present relative to the amounts of collagen and elastin. We conclude that administration of elastase results in changes in the long-term balance of lung connective tissue components.

Animals↗
PubMed1982

The appearance of free hydroxyproline as the major product of degradation of newly synthesized collagen in cell culture.

Embryonic lung fibroblasts and rabbit vascular smooth muscle cells have the ability to degrade newly synthesized collagen. Analysis of 24-h pulse media from cultures given [14C]proline demonstrates that greater than 90% of the degraded collagen is represented by free hydroxyproline rather than the peptide-bound imino acid. The addition of cycloheximide or alpha-alpha-dipyridyl to the culture medium during the pulse period severely diminished the formation of the free hydroxyproline demonstrating its enzymatic and protein (collagen) origin. It is proposed that assessment of free hydroxyproline formation may allow us to distinguish between intracellular and extracellular collagen degradation.

Animals↗
PubMed1982

Cell density and estradiol modulation of procollagen type III in cultured calf smooth muscle cells.

We have reported previously that 17 beta-estradiol reduces the amount of procollagen type III produced in cultured bovine aortic smooth muscle cells. The effect of cell density and estradiol on the production of collagen in second passage cultures has been further evaluated. In agreement with our previous findings, at day 14, significant differences in [14C]hydroxyproline production were detected between hormone-treated and control cultures. To further relate this observation to cell culture density and estradiol treatment, collagen production was evaluated after a 1-h pulse period. Quantities of assembled procollagen molecules and the individual collagenous polypeptides were determined by DEAE-cellulose chromatography or by immunoprecipitation. As cell numbers increased with time in culture, an augmented production of both procollagen types I and III was observed. The increased production of total type III collagen and procollagen molecules lagged behind type I production. Thus, it was found that the ratio of procollagen type I to type III decreased with days in culture from 2.7 to 1.0, and the ratio of the total of each type decreased from 7.5 to 3.4. In the presence of 1.0 x 10(-8) M estradiol, the level of type III collagen was reduced by approximately 50% at all culture times. The production of procollagen type I was not affected and remained equivalent in both hormone-treated and control cultures. The different ratios obtained by the two methods indicates that procollagen type I is processed more rapidly than procollagen type III in the 1-hour pulse period.

Animals↗
PubMed1982

Effects of ascorbate on insoluble elastin accumulation and cross-link formation in rabbit pulmonary artery smooth muscle cultures.

Cultured pulmonary artery smooth muscle cells derived from the medial vessel layer of weanling rabbits were grown in the presence or absence of sodium ascorbate. The connective tissue elements insoluble elastin and collagen were identified and quantified. Formation and accumulation of alpha-aminoadipic acid gamma-semialdehyde (allysine) and the intermolecular cross-links desmosine (Des), isodesmosine (Ides), and aldol condensation product (Aldol) were evaluated from [14C]lysine pulse-chase experiments. [14C]Des, [14C]Ides, peptide-bound [14C]lysine, [14C]allysine, and [14C]Aldol were determined from amino acid analysis. The latter two components were determined after reduction with NaBH4. [14C]Proline conversion to hydroxy[14C]proline and collagenase susceptibility were used to identify and quantify collagen synthesis. Ascorbate dramatically affects insoluble elastin synthesis, accumulation, and cross-link formation. Cells grown in the presence of ascorbate synthesize and accumulate significantly less insoluble elastin than non-ascorbate cultures. Those elastin molecules which do become incorporated into the extracellular matrix in the presence of ascorbate contain a slightly elevated content of hydroxyproline and lysine and, most importantly, are turned over more rapidly.

Animals↗