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Biomedical subjects

R A Berg

Publications and source records attributed to R A Berg.

At least 163 records · Page 9Linked to original sources
PubMed1981

Regulation of collagen production by the beta-adrenergic system.

The suppression of collagen production by increasing the cyclic (c) AMP content of cultured cells was examined vis-à-vis the beta-adrenergic system. Cultured human fetal lung fibroblasts incubated for 6 h with the beta-agonists isoproterenol or epinephrine produced approximately 30% less collagen per cell than in the absence of the hormones. To demonstrate that the beta-agonists were operating by their interaction with the beta-receptor to stimulate adenylate cyclase to increase the intracellular content of cAMP, d- and l-isoproterenol were incubated separately with the cultured cells. Only l-isoproterenol increased intracellular cAMP and decreased collagen production. While 20 nM l-isoproterenol was effective, the d-isomer was ineffective even at 2muM. An increase in cAMP from 40 to 73 pmol/mg protein was effective in suppressing collagen production; increasing the cAMP content to much higher levels had little additional effect on collagen production. 3-Isobutyl-1-methylxanthine, an analog of theophylline that inhibits phosphodiesterase, potentiated the effect of isoproterenol in suppressing collagen production. Further support for the concept that isoproterenol suppressed collagen production by acting through the beta-receptor was provided by the finding that only the l-isomer of propranolol, a beta-blocker, was effective in blocking both the increase in intracellular cAMP and the suppression of collagen production caused by isoproterenol. These results demonstrate that collagen production in human fibroblasts can be regulated by the beta-adrenergic system and indicate that when the cAMP content is increased beyond a threshold value, collagen production is suppressed. Since collagen production is sensitive to the small changes of cAMP content of cells brought about by beta-stimulation in cultured cells, the results point to a possibly important mechanism for the regulation of collagen production in the body.

Cells, Cultured↗
PubMed1980

The assembly of tetrameric prolyl hydroxylase in tendon fibroblasts from newly synthesized alpha-subunits and from preformed cross-reacting protein.

Embryonic-chick tendon cells were incubated in suspension for 4h with (14)C-labelled amino acids, cell extracts were subjected to gel filtration, and the effluent was examined by rocket immunoelectrophoresis by using antibodies specific for the beta-subunit of chick prolyl hydroxylase. Two peaks of immunoreactive protein were found. The first peak contained 40% of the immunoreactive protein eluted from the column and 100% of the enzyme activity. Polyacrylamide-slab-gel electrophoresis in sodium dodecyl sulphate of an immunoprecipitate of this peak demonstrated that it consisted of the tetrameric form of prolyl hydroxylase, subunit composition alpha(2)beta(2) where alpha and beta are non-identical subunits. Only the alpha-subunits were labelled, indicating that they were synthesized during the 4h labelling period. The beta-subunits were unlabelled, indicating that they had been synthesized before the labelling period. The second peak eluted from the gel-filtration column contained 60% of the immunoreactive protein eluted from the column and was enzymically inactive. Polyacrylamide-slab-gel electrophoresis of an immunoprecipitate of this peak indicated that it consisted of a single labelled polypeptide chain, identified as cross-reacting protein, which was related to, but not identical with, the beta-subunit of prolyl hydroxylase. Pulse-chase experiments were performed on cultured chick tendon cells to demonstrate that alpha-subunits and cross-reacting protein had half-lives of about 60h. The half-life of beta-subunits was considerably longer, and the kinetic pattern was consistent with their being derived from a labelled precursor such as cross-reacting protein. The data presented here indicate that the active tetrameric form of prolyl hydroxylase in cells is assembled from alpha-subunits which are newly synthesized, and from beta-subunits which are derived from cross-reacting protein.

Animals↗
PubMed1980

Control of collagen production by human diploid lung fibroblasts.

The fibroblast is a differentiated mesenchymal cell which produces and exports collagen, a macromolecule that plays a critical structural role in the function of most organs. To evaluate the control soft tissue fibroblasts have over collagen production, HFL-1, a diploid human lung cell strain, was studied during periods of rapid cell growth and relatively slow growth over 25 population doublings. To minimize environmental influences, the extracellular milieu of the cells was kept constant throughout the study period. Rates of collagen production per cell per unit time were quantitated by labeling HFL-1 with [14C]proline and measuring the production of [14C]hydroxyproline after taking into consideration the specific activity of [14C]proline within the free intracelllular proline pool and the per cent hydroxylation of proline residues in newly synthesized collagen. Although the specific activity of intracellular free proline and the per cent hydroxylation of proline in collagen varied considerably depending on the growth rates of the cells, collagen production by HFL-1 was constant, even during periods of rapid cell growth. Thus, under conditions of a stable environment, populations of soft tissue fibroblasts rigidly control their collagen production. In cultures that maintained a constant doubling time, this stability was maintained over at least 25 population doublings, suggesting that on the average, collagen production appears to be tightly controlled and dissociated from the events and sequelae of cell division.

Cell Division↗
PubMed1980

Purification and characterization of the amino-terminal propeptide of Pro alpha 1(I) chains from embryonic chick tendon procollagen.

A peptide with an apparent molecular weight of 23,000 was isolated from the medium of cultured chick embryo tendons. Comparison of tryptic peptides derived from the medium peptide and from the amino-terminal, bacterial collagenase resistant portion of Type I procollagen indicated that the medium peptide represented the amino-terminal precursor-specific region of the pro alpha 1 (I) chain of procollagen. This conclusion was supported by the demonstration that antibodies against the medium peptide reacted with Type I procollagen in a radioimmune assay but did not react with a peptide derived from the carboxy-terminal propeptide of Type I procollagen. In addition, the reaction with Type I procollagen was inhibited with the purified amino-terminal, collagenase-resistant portion of pro alpha 1 (I) chains. Finally, amino acid sequencing demonstrated that the amino propeptide of dermatosparactic calf pN alpha 1 (I) chains and the medium peptide have similar amino-terminal sequences. Carbohydrate analysis established the presence of one residue of N-acetylglucosamine and a trace of mannose and galactose.

Amino Acid Sequence↗
PubMed1980

New enzyme immunoassays for measurement of influenza A/Victoria/3/75 virus in nasal washes.

Nasopharyngeal (NP) washes from 12 patients experimentally infected with influenza A/Victoria/3/75 wild-type virus were tested in 2 newly developed enzyme-immunoassay (EIA) systems. Results were compared with infectivity titres of the NP wash specimens in rhesus monkey kidney-tissue culture. EIA detected A/Victoria/3/75 haemagglutinin antigen for more days than did culture. EIA is also rapid and easy to do in large numbers; hence it may have advantages over culture methods for the diagnosis of influenza infections and the investigation of influenza epidemiology.

Animals↗
PubMed1980

Effect of cyclic AMP on the intracellular degradation of newly synthesized collagen.

Prostaglandin E1 and cholera toxin increased the intracellular levels of cyclic AMP of human lung fibroblasts. With prostaglandin E1, the increase in cyclic AMP occurred within 10 min followed by a decline to less than one-half of peak values in 6 h. With cholera toxin, the increase occurred within 60 min but the level of cyclic AMP remained increased for 6 h. Both agents caused a decrease in collagen production as expressed as the proportion of newly synthesized protein represented by collagen. The increase in cyclic AMP levels was accompanied by a marked increase in the proportion of newly synthesized collagen which was degraded intracellularly prior to secretion. Analysis of the degraded collagen showed it to be predominantly less than 1000 daltons in molecular mass, but still in peptide linkage. The data are consistent with the hypothesis that cyclic AMP levels in diploid fibroblasts regulate the amount of collagen produced by fibroblasts, at least in part, by modulating the level of intracellular collagen degradation.

Animals↗
PubMed1980

Friedreich's ataxia with acute cardiomyopathy.

Friedreich's ataxia (FA) is a progressive, spinocerebellar degenerative disease. Onset is generally in the second decade of life, occurring as a neurologic degenerative process. Most, if not all, patients have an associated cardiomyopathy, which is frequently the cause of death. We studied two siblings who had FA with acute cardiomyopathy at 3 and 5 years of age, respectively, and in whom the classic nervous system signs developed, only later. The diagnosis of FA should be considered in patients of any age who have unexplained cardiomyopathy.

Child↗
PubMed1980

Regulation of the production of secretory proteins: intracellular degradation of newly synthesized "defective" collagen.

Confluent cultures of human fetal lung fibroblasts degrade approximately 10% of their newly synthesized collagen within the cell prior to secretion. This basal level of intracellular degradation could not be inhibited by colchicine or cytochalasin B, inhibitors of microtubular and microfilament function, respectively, or by N(alpha)-p-tosyl-L-lysine chloromethyl ketone, chloroquine, or NH(4)Cl, inhibitors of lysosomal enzymes. In contrast, cells in early logarithmic growth degrade approximately 30% of their newly synthesized collagen. This enhanced degradation of collagen in rapidly growing cells could be suppressed by inhibitors of lysosomal proteases and partially inhibited by disrupters of microtubular and microfilament function. A significant proportion of the collagen synthesized by these cultures contained prolyl residues that were incompletely hydroxylated. Because such collagen is "defective" (i.e., not capable of assuming a triple helical conformation), the results suggest that enhanced intracellular degradation may be a mechanism by which cells control the quality of collagen they produce. To test this hypothesis, confluent cells were incubated with the proline analog cis-4-hydroxyproline; such cells demonstrated enhanced collagen degradation that could be inhibited by agents that interfere with lysosomal, microtubular, or microfilament function. Because collagen containing cis-4-hydroxyproline cannot form a perfect triple helix, the data are consistent with the concept that defective collagen is recognized by cells and degraded prior to secretion. Thus, the proportion of newly synthesized collagen that undergoes intracellular degradation seems to be modulated, in part, by the conformation of the collagen molecule. Intracellular proteolysis may represent a means by which collagen-producing cells regulate the quality and quantity of collagen available for extracellular function. Although the exact mechanism of intracellular collagen degradation is unknown, the data presented here are consistent with a role for lysosomal proteases in this process.

Cell Line↗
PubMed1980

Prevention of collagen deposition following pulmonary oxygen toxicity in the rat by cis-4-hydroxy-L-proline.

Exposure of rats to high oxygen tensions causes increased collagen content of lungs and alveolar enlargement in 3-6 wk. We tested whether cis-hydroxyproline, a proline analogue that inhibits collagen synthesis, could prevent the collagen accumulation and alveolar enlargement. Rats were exposed to hyperoxia for 60 h and then to room air and hyperoxia for alternate 24-h periods for 11.5 d. Treated oxygen-exposed rats received 200 mg/kg cis-hydroxyproline twice daily over the 14-d exposure period. Control rats breathed room air. Examination of lungs on day 14 showed collagen content of oxygen-exposed lungs to be 48% greater than control (P < 0.05). The collagen content of the treated oxygen-exposed lungs was -12% of control (NS). Total lung volume was 16% greater than control in oxygen-exposed rats (P < 0.05) and 8% greater than control in treated oxygen-exposed rats (NS). Morphometric studies showed alveolar size was greater than control in oxygen-exposed rats (188+/-11 [SE] vs. 143+/-6 mumul [P < 0.05]). Oxygen-exposed, treated rats had a mean alveolar volume of 150+/-7 mumul. Lung pressure-volume curves were significantly shifted to the left of control in the oxygen-exposed rats, whereas the curves of the oxygen-exposed, treated group were identical to control. These data suggest that cis-hydroxyproline prevented the accumulation of collagen in the lungs in pulmonary oxygen toxicity. In addition, there was apparent protection from airspace dilatation and decreased lung elasticity, suggesting that alveolar enlargement after oxygen toxicity is linked to the deposition in lung tissue of new connective tissue fibers.

Animals↗
PubMed1980

Aplasia cutis congenita complicated by sagittal sinus hemorrhage.

An infant with aplasia cutis congenita complicated by sagittal sinus hemorrhage is described. In cases of large defects, plastic surgical repair has been recommended for prevention of hemorrhage and infection. The complication of sagittal sinus hemorrhage should be considered in patients with large midline scalp defects, especially when associated with absence of the underlying cranium.

Cranial Sinuses↗
PubMed1979

Cell density-dependent increase in prolyl hydroxylase activity in cultured L-929 cells requires de novo protein synthesis.

Prolyl hydroxylase activity in cultured L-929 cells was found to increase when cells grew from log phase to stationary phase and when cells were harvested at the mid-log phase and replated at higher cell densities. Cycloheximide and actinomycin D inhibited the cell density-dependent increase in prolyl hydroxylase activity indicating that the increase in prolyl hydroxylase activity required de novo synthesis of protein and RNA. Prolyl hydroxylase was purified from cultured L-929 cells and antibodies against the protein were raised in rabbits. The antibodies were used to demonstrate that L-929 cells contained two forms of prolyl hydroxylase: an enzymatically active, tetrameric form consisting of two alpha and two beta polypeptide chains and an enzymatically inactive form containing immunologically cross-reacting protein. The polypeptide chains alpha, beta and cross-reacting protein were obtained by immunoadsorption. Peptide map analysis indicated that cross-reacting protein was similar if not identical to beta in primary structure, and alpha was different from both beta and cross-reacting protein. The results suggested that the prolyl hydroxylase levels in cells or tissues may be regulated by new protein and/or RNA synthesis.

Animals↗
PubMed1979

Kinetics for the secretion of nonhelical procollagen by freshly isolated tendon cells.

Fibroblasts isolated by enzymic digestion of chick embryo tendons have previously been used to examine the kinetics for the secretion of procollagen (Kao, W. W.-Y., Berg, R. A., and Prockop, D. J. (1977) J. Biol. Chem. 252, 8391-8397). The results indicated that the kinetics approximated the sum of two first order processes with half-times of 14 and 115 min. Here, the same fibroblasts were incubated in the presence of 1.53 mM cis-4-hydroxyproline, an analogue of proline, or in the presence of 0.3 mM alpha,alpha'-dipyridyl, an inhibitor of prolyl hydroxylase, so that the cells synthesized procollagen which could not assume a triple helical conformation characteristic of procollagen. Measurements of the secretion of nonhelical procollagen indicated that the kinetics for secretion differed from the kinetics for the secretion of procollagen and approximated a single first order process with a half-time of approximately 130 min. The nonhelical procollagen synthesized and secreted in the presence of either cis-4-hydroxyproline or alpha,alpha'-dipyridyl consisted of disulfide-bonded pro gamma chains of type I procollagen. The results suggested that the intracellular nonhelical procollagen was present in a single metabolic pool and secretion from this pool occurred with a different rate-limiting step than for helical procollagen. Further results indicated that nonhelical procollagen had a high affinity for prolyl hydroxylase and the affinity for the enzyme was greatly reduced if the procollagen was allowed to assume the triple helical conformation characteristic of normal procollagen. The results are consistent with the hypothesis that the secretion of procollagen is influenced by its conformation-dependent interaction with prolyl hydroxylase or other post-translational enzymes.

2,2'-Dipyridyl↗
PubMed1979

Multisite proteins and randomly coiled polymeric ligands. Chain length dependence of binding constants.

A model mechanism was developed for the binding of a rigid multisite protein with a randomly coiled multivalent ligand. Probabilities of the formation of chain loops between sites located at given distances at the protein were calculated by an extension of the concept of ring closure in coiled chain molecules. Expressions were derived for the dependence of overall equilibrium quantities, such as the binding constant between the protein and the ligand, on intrinsic parameters such as intrinsic binding constants, number of sites at the protein and their distances and on the chain length of the polymeric ligand. A pronounced chain length dependence of the overall binding constant was predicted even at chain lengths much longer than the size of the protein. Such a dependence was previously observed for the enzyme prolyl hydroxylase which acts on polymeric substrates like (ProProGly)n. This so far unexplained feature is quantitatively described by the model mechanism which is believed to be applicable to many other interactions of biological importance.

Enzymes↗
PubMed1979

Detection of Candida antigen in sera of patients with candidiasis by an enzyme-linked immunosorbent assay-inhibition technique.

A total of 37 serum samples from 27 cancer patients were tested by an enzyme-linked immunosorbent assay-inhibition technique for the detection of Candida antigen. In 20 randomly chosen sera from patients without clinical evidence of candidiasis and in 10 sera from patients proven by autopsy not to have candidiasis, the inhibition ranged up to 17%; in contrast, inhibition ranged from 22 to 56% in all seven patients proven by autopsy to have systemic candidiasis, indicating the presence of Candida antigen in the sera of these patients. This technique appears promising in diagnosing disseminated candidiasis in cancer patients.

Antigens, Fungal↗