Collagen biosynthesis by human skin fibroblasts III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. The results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased tha amount of [³H]hydroxyproline syntehsized. Ascorbic acid, however, did not increase the synthesis of ³H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and selection of procollagen.     

Membrane targeting of RecA during genetic transformation

Recombination in prokaryotes and eukaryotes is mediated by the RecA family of proteins. Although the interactions between RecA and DNA are well studied, the cellular location of these interactions is not known. Using genetic transformation of Streptococcus pneumoniae as a model system, there was increased expression of a protein, colligrin, and RecA, products of the rec locus during genetic transfer. These proteins formed a complex and were found associated with the membranes of genetically competent cells. With immunoelectron microscopy and subcellular fractionation, we showed that the induction of competence led to the translocation of RecA and colligrin to the membrane and to the formation of clusters of RecA in a colligrin-dependent step. Based on the behaviour of colligrin and RecA during genetic exchange and the numerous proteins in prokaryotes and eukaryotes with domains similar to colligrin, we suggest that there may exist a family of proteins, which gathers macromolecules at specific sites in biological membranes.     

REPRODUCTIVE CAPACITY AND SEED SIZE IN LUPINUS TEXENSIS

The average number of ovules produced per individual of Lupinus texensis is much greater than the average number of seeds per plant. Each plant produces approximately 2,000 ovules but only 2.5% develop into seeds. One fourth of the seeds is lost due to abortion and 0.3% is lost due to predation on the plant. Mature seeds from this population exhibit a five-fold range in weight, from 10 to 56 mg. The distribution of seed weights in the field population is skewed and leptokurtic. Seed wt is positively correlated with both seed germination and seedling survivorship. Heritability of seed wt is 0.09. There is no correlation between average seed wt per plant and total number of seeds per plant, seeds per pod, or legumes per plant.     

Reversible covalent immobilization of ligands and proteins on polyacrylamide gels

Ligands and proteins were covalently but reversibly immobilized on polyacrylamide gels using novel acrylic monomers whose syntheses are reported here. These reagents have an acrylyl group at one end for copolymerization into gels, an N-succinimidyl ester at the other allowing rapid immobilization of molecules having an available primary amino group, and a cleavable disulfide bond in the middle. Two immobilization methods were developed using these reagents. In the first method, a ligand with a primary amino group was treated with the immobilization reagent in anhydrous ethanol and the resulting amide derivative was purified and copolymerized with acrylamide and bisacrylamide resulting in the desired reversible immobilization. In the second method, the immobilization reagents (at densities up to 50 mumol/ml) were directly copolymerized with acrylamide and bisacrylamide to form activated gels of the desired shape and porosity. Proteins or other ligands in aqueous buffers were then added to the activated gels resulting in their covalent immobilization. Ligands or proteins immobilized using the methods reported here remained stably bound even when gels were subjected to boiling in detergents or high-ionic-strength buffers. Immobilized ligands were readily released (greater than 97%) from gels by treatment with quantitative amounts of aqueous dithiothreitol (DTT) under mild conditions. Immobilized proteins were also released (up to 87%) from the gels by DTT treatment. Small ligands (e.g., aminohexyl glycosides), active enzymes, and glycoproteins were immobilized, and then recovered, using these reagents.     

The synergistic effect of carbon concentration and high temperature on lipid peroxidation in Peridinium gatunense

Lipid peroxidation in Peridinium samples taken from two differentdepths in Lake Kinneret fluctuated throughout the spring withan overall increasing trend. Samples from 0.5 and 5 m showeda similar peroxidation pattern, which was maximal after thefall off in algal biomass. The rapid decline in Peridinium biomasscoincided with ambient lake temperatures of 21–23C. Fattyacid composition profiles were similar at both depths, althoughafter the peak of the bloom, a significant increase in polyunsaturatedfatty acids and oleic acid was only found at 0.5 m, togetherwith a decrease in the percentage of polyunsaturated fatty acids.These effects were related to ambient light stress rather thana result of lipid peroxidation. Lake samples taken at differentperiods of the bloom and incubated at various temperatures showeddifferential peroxidation. Higher temperatures caused increasedlipid peroxidation, but this appeared to be dependent on thesampling period. Samples withdrawn from the lake at the beginningof the bloom showed little peroxidation after a 5 day incubationat 14C, room temperature (25C) or ambient lake temperature(16C) compared to mid-bloom samples in which there was a significantincrease in peroxidation when they were incubated at room temperature(25C) or ambient lake temperature (22C). Incubation at 14Cinhibited peroxidation; however, samples from mid-bloom againshowed enhanced peroxidation compared with those from the beginningof the bloom. These in situ results suggested a relationshipbetween temperature, another environmental variable during thebloom and lipid peroxidation in Peridinium. As total dissolvedinorganic carbon (DIC) concentrations fall significantly duringthe progress of the bloom and represent an important sourceof environmental stress, laboratory experiments were establishedto investigate the synergistic effect of temperature and carbonnutrition on lipid peroxidation in Peridinium cultures. Increasedtemperature alone caused a slight increase in lipid peroxidation,but this was greatly augmented by carbon limitation. Althoughcarbon limitation induced increased catalase activity, at highertemperatures activity declined after 48 h, allowing for thesubstantial increase in lipid peroxidation.     

Identification of a male-specific (H-Y) antigen on the flagellar plasma membrane of ram epididymal spermatozoa

Summary H-Y (male-specific) antigen has been detected on the plasma membranes of both caput and caudal ram spermatozoa using both immunoperoxidase and immunofluorescence labelling techniques. In these spermatozoa the distribution of H-Y antigen appears to be confined to both the posterior region of the head and the mid-piece region of the flagellum. In addition, caput spermatozoa also exhibit intense immunoperoxidase staining of the cytoplasmic droplet which is situated on the flagellum at the base of the head. Western blot analyses of purified plasma membranes from the flagella of caudal spermatozoa have revealed the presence of a malespecific protein with an estimated molecular weight of 25,000–27,000.