Stimulation of collagen synthesis in fibroblast cultures by superoxide.

Exposure of diploid fetal human fibroblasts (IMR-90) to superoxide generated by dihydroxyfumarate resulted in increased collagen synthesis. The synthesis of type III collagen was stimulated to a greater extent than the synthesis of type I collagen. The stimulation of collagen synthesis was abolished by superoxide dismutase. Our observations suggest that superoxide may play a role in the regulation of collagen synthesis and may modulate differential collagen gene expression. These observations may explain the increased synthesis of collagen in tissues following inflammation or exposure to oxidant conditions.     

Influence of two cell harvesting methods on intracellular ATP and amino acid concentrations in human fibroblast cultures

The intracellular ATP and amino acid concentrations were determined in human fibroblast cultures reaching confluence. The values obtained were very different, depending on the cell harvesting method: trypsinization or scraping. Trypsinization appeared to be the better method for measuring the ATP concentrations (21.25 +/- 0.96 nmol per mg cell protein), this level being much lower with scraping. On the contrary, scraping was the most appropriate method for amino acid measurement. This work underlines the importance of harvesting methods for metabolic studies in human cell cultures.     

A quantitative assay for collagen synthesis in microwell fibroblast cultures.

A new method for estimating collagen synthesis in microwell cultures of fibroblasts is presented, ³H-Labeled-proline-collagen was purified by successive salt precipitations at acid and neutral pH in the presence of carrier collagen. Variability between replicates was less than 10% (standard deviation) and recovery of labeled collage internal standards was greater than 90%. More than 90% of recoverable radioactivity was in collagen as demonstrated by polyacrylamide gel electrophoresis and carboxymethyl cellulose chromatography. The culture system is highly reproducible and allows use of a large number of separate cultures with uniformity of culture conditions and economy of reagents.     

氨基酸前体对棘白霉素B发酵合成的影响

以构巢曲霉(Aspergillus nidulans)发酵生产酯肽类化合物棘白霉素B,研究棘白霉素B己肽环结构的前体性氨基酸对产物的代谢影响,显示出脯氨酸、鸟氨酸、苏氨酸在发酵48 h 补入,对发酵代谢有促进作用.利用响应面统计学方法进行前体及有机氮源配方优化,得出优化配方:脯氨酸4.55 mg/mL,鸟氨酸1.85 mg/mL,苏氨酸0.97 mg/mL,棉籽粉2.62％,摇瓶发酵水平达到3 270μg/mL,较原水平提高30.2％.新工艺在50 L罐上放大,发酵水平进一步提高至3 520 μg/mL,显示出良好的工业应用前景.     

Intensification of the initial stage in amino acid metabolism in germinating cereal seeds with exogenous glutamine and proline

The initial stage of amino acid metabolism was intensified in germinating wheat seeds with exogenous glutamine and proline. Exogenous glutamine and proline accumulated over 2 h at 4°C in swelling seeds were spent at different rates over the following 2 h at 20°C, thus compensating for insufficiency of these amino acids during the initial stage of development. Creation of additional pools of glutamine and proline during the initial stage of amino acid metabolism had positive effects on the seed germination and vital activity of the plants.     

Effects of heparin and dextran sulphate on the production of collagen and protein in diabetic and non-diabetic human skin fibroblast cultures

Addition of heparin and dextran sulphate to human skin fibroblasts in cell cultures caused an increase in [3H]-proline incorporation into collagen and total protein in the culture medium by cells derived from nondiabetics. Cells from type 2 diabetic subjects were significantly less affected by dextran sulphate addition, suggesting altered regulatory mechanisms for collagen production in these cells. Addition of chondroitin sulphate caused a dose-dependent increase in labelled collagen, indicating a possible role for this glycosaminoglycan as modulator of collagen deposition.     

Intensification of the initial stage of amino acid metabolism in germinating cereal seeds by exogenous glutamine and proline

The initial stage of amino acid metabolism was intensified in germinating wheat seeds with exogenous glutamine and proline. Exogenous glutamine and proline accumulated over 2 h at 4 degrees C in swelling seeds were spent at different rates over the following 2 h at 20 degrees C, thus compensating for insufficiency of these amino acids during the initial stage of development. Creation of an additional store of glutamine and proline during the initial stage of amino acid metabolism had positive effects on the seed germination and vital activity of the plants.     

Effect of water stress on proline synthesis from radioactive precursors

Barley (Hordeum vulgare L. var. Prior) leaves converted more ¹⁴C-glutamic acid to free proline when water-stressed than when turgid; neither decreased protein synthesis nor isotope trapping by the enlarged free proline pools found in wilted tissue seemed to account for the result. This apparent stimulation of proline biosynthesis in wilted leaves was not observed when radioactive ornithine or P5C (Δ¹-pyrroline-5-carboxylate, an intermediate following glutamate in proline synthesis) were used as proline precursors unless proline levels were high as a result of previous water stress. We interpret this to mean that any stimulation of proline synthesis by water stress must act on P5C formation rather than its reduction to proline. Experiments showing greater apparent conversion of ¹⁴C-glutamate to proline do not unequivocally prove that proline synthesis is stimulated by water stress, as P5C feeding studies show that proline oxidation is inhibited under comparable conditions. This inhibition could account, at least in part, for increased proline labeling, and must be considered an alternate possibility.     

Biosynthesis of hyperforin and adhyperforin from amino acid precursors in shoot cultures of Hypericum perforatum

Hyperforin and adhyperforin contribute to the antidepressant effects of Hypericum perforatum. The involvement of branched-chain amino acids in the biosynthesis of hyperforin and adhyperforin was demonstrated in H. perforatum shoot cultures. L-[U-(13)C(5)]Valine and L-[U-(13)C(6)]isoleucine, upon administration to the shoot cultures, were incorporated into acyl side chain of hyperforin and adhyperforin, respectively. Feeding the shoot cultures with unlabelled L-isoleucine at a concentration of 2mM induced a 3.7-fold increase in the production of adhyperforin. The addition of 3mM L-threonine, a precursor of isoleucine, stimulated a 2.0-fold increase in the accumulation of adhyperforin. The administration of L-valine at concentrations of 0-5mM had no stimulating effect on the hyperforin production in H. perforatum shoot cultures.     

The effects of lead upon collagen synthesis and proline hydroxylation in the Swiss mouse 3T6 fibroblast.

The effects of lead upon collagen synthesis and proline hydroxylation were examined in the Swiss mouse 3T6 fibroblast. The results indicate that lead reduces proline hydroxylation in stationary phase cultures of 3T6 cells, resulting in increased cellular retention of unhydroxylated procollagen. Inhibition of proline hydroxylation by lead was prevented by increasing the extracellular $\text{Fe}{}^{\mathrm{2+}}\text{Pb}{}^{\mathrm{2+}}$ molar ratio. Interference by lead in the hydroxylation of proline in logarithmic phase cultures of 3T6 cells resulted in increases in the 0.5 n HClO₄ soluble/insoluble hydroxyproline ratio. This was attributed to an increase in the rate of breakdown of lead-induced unhydroxylated procollagen. Kinetic analysis of the lead-iron interaction with proline hydroxylase suggests that the mechanism is competitive.     

Influence of cell density on collagen biosynthesis in fibroblast cultures.

Characteristic features of collagen metabolism in human skin fibroblasts were studied in relation to cell density. Measuring peptide-bound hydroxyproline we found that collagen synthesis per cell decreased when cultures approached confluency. On the other hand, the relative rate of collagen synthesis (collagen/total protein) was higher in quiescent than in proliferating cultures. With increasing cell density the proportion of type III collagen in comparison with type I was found to be slightly increased. In addition, in low-density cultures [alpha I(I)]3 collagen trimers were produced in considerable amounts, whereas they were no longer detected in cultures with a high cell density. Although hydroxylation of proline residues was normal in all cell stages, conversion of procollagen into collagen was found to depend strongly on the density at which the cells were investigated. Almost no cleavage of procollagen peptides was observed in rapidly growing cells, whereas highly confluent cell cultures converted most of the newly synthesized procollagen molecules.     

The effect of amphotericin b-deoxycholate on proliferation and protein synthesis in human skin fibroblast cultures

Summary Cultures of adult human skin fibroblasts were grown in the presence of the recommended antifungal dose (3 μg per ml) of amphotericin B-deoxycholate. A reduction in cell culture growth, measured as DNA content and protein content per culture, was observed. However, radioisotope incorporation into noncollagen protein and, to a lesser extent, collagen protein was enhanced. These effects were due to amphotericin B, not to deoxycholate. These observations were made under several growth conditions and indicate that cell proliferation or isotope-labeling studies in fibroblasts in the presence of amphotericin B-deoxycholate are susceptible to errors in interpretation. Supported by PHS Grants AM-02456, AM-15312 and AM-17047, by the Kroc Foundation, and by the American Diabetes Association, Washington Affiliate. Recipient of Research Career Development Award AM-47142 from NIAMDD, and to whom requests for reprints should be addressed.     

Effects of hyperlipoproteinemic serum and exogenous proline concentration on collagen synthesis by isolated rabbit aortas.

Collagen synthesis was measured in segments of normal rabbit aorta, incubated in vitro, by monitoring the formation of peptidyl-14C-hydroxyproline from [U-14C]-L-proline added to the incubation medium. The effect of hyperlipoproteinemic rabbit serum on the rate of collagen synthesis was compared with the effect of normal rabbit serum. No differences in the rates of synthesis were detected between the two batches of serum, despite a 60-fold difference in serum cholesterol concentration. Increases in free proline concentration in the incubation medium resulted in changes in proline flux between medium and tissue pools of free proline, but medium proline concentration had no effect on the rate of collagen synthesis.     

Differentiation between senescence (M1) and crisis (M2) in human fibroblast cultures

Normal human fibroblasts undergo only a limited number of divisions in culture and eventually enter a nonreplicative state designated senescence or mortality stage 1 (M1). Expression of certain viral oncogenes, such as the SV40 large T antigen (SV40 T-Ag), can elicit a significant extension of replicative life span, but these cultures eventually also cease dividing. This proliferative decline has been designated crisis or mortality stage 2 (M2). BrdU incorporation assays are commonly used to distinguish between senescence (<5% labeling index) and crisis (>30% labeling index). It has not been possible, however, to ascertain whether the high labeling index, indicative of ongoing DNA replication, was caused by the presence of T-Ag. We used gene targeting to knock out both copies of the p21(CIP1/WAF1) gene in presenescent human fibroblasts. p21 -/- cells displayed an extended life span but eventually entered a nonproliferative state. In their terminally nonproliferative state both p21 +/+ and p21 -/- cultures were positive for the senescence-associated beta-galactosidase (SA-beta-gal) activity; in contrast, the labeling index of p21 +/+ cells was low (<5%) whereas the labeling index of p21 -/- cells was high (>30%). The observation that p21 -/- and SV40 T-Ag-expressing cells behave identically with respect to life span extension as well as the high labeling index in the terminally nonproliferative state indicates that crisis is not a phenomenon induced solely by viral oncogenes, but a physiological state resulting from the bypass of normal senescence mechanisms. The widely used biomarker for senescence, SA-beta-gal, cannot distinguish between senescence and crisis. We propose that all SA-beta-gal-positive cultures should be further examined for their BrdU labeling index.