A proenzyme from chicken plasma similar to human plasma prekallikrein

We report the isolation of a specific protease zymogen from chicken plasma. The purification procedure involves barium citrate precipitation, ammonium sulfate fractionation, removal of plasminogen and plasmin on lysine-Sepharose, followed by anion and cation exchange, and gel permeation chromatography. Based on quantitative radioimmunoassay the zymogen is present in plasma at a concentration of 160 mg/liter, and it is obtained by our procedure in highly purified form with a yield of 1.4%. The single polypeptide chain contains an NH2-terminal alanine residue. The native molecule migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 84,000 under reducing conditions. It can be identified as an inactive proenzyme because it has very low amidolytic activity, does not react with the fluorescent active site titrant 4-methyl-lumbelliferyl p-guanidinobenzoate, and does not incorporate radioactive [3H]diisopropylfluorophosphate. It is very susceptible to limited proteolysis which converts it to an active enzyme with trypsin-like specificity. The active enzyme, likewise a single polypeptide chain, migrates as a doublet with apparent molecular weights of 39,000 and 40,000. Its amidolytic activity with synthetic peptide substrates is at least 40-fold higher than that of the proenzyme, it reacts efficiently with 4-methylumbelliferyl p-guanidinobenzoate, and incorporates [3H]diisopropylfluorophosphate while undergoing irreversible inactivation. The enzyme appears to be a reasonably efficient plasminogen activator in zymographic gels, but not in solution. With human high molecular weight kininogen as substrate the enzyme was about 25% as efficient as human plasma kallikrein. It lacks any plasminogen-independent proteolytic activity with other protein substrates, and it hydrolyzes small peptide substrates designed for both human kallikrein and urinary urokinase, respectively. Inhibition studies with peptide chloromethyl ketones indicate enzymatic properties closer to human plasma kallikrein than to the human plasminogen activator urokinase (EC 3.4.21.31). The chicken plasma enzyme and the plasminogen activator from the conditioned media of Rous sarcoma virus-transformed chick embryo fibroblasts treated with tumor promoter are different by criteria of tryptic peptide maps, and amino acid composition and enzymatic specificity. The designations chicken plasma prekallikrein plasminogen proactivator and chicken plasma kallikrein plasminogen activator are proposed for the zymogen and enzyme forms, respectively. Using rabbit antibodies against the proenzyme we developed a solid phase immunoadsorption procedure that allowed us to isolate the protein with an overall yield of 11.4%.     

DEGRADATION OF RIBONUCLEIC ACID IN MOUSE FIBROBLASTS TREATED WITH ACTINOMYCIN

The cellular RNA content of mouse fibroblasts incubated with actinomycin decreases at a rate of about 1 to 1.5 per cent per hour, while DNA and protein content remain unchanged. This degradation affects nuclear and cytoplasmic RNA, ribosomal and soluble RNA. The breakdown products appear quantitatively in the acid-soluble fraction of the cells and the medium. Polynucleotides synthesized a short period (120 minutes) prior to exposure to actinomycin are degraded before those synthesized 8 to 12 hours previously.     

Differences in the repertoires of basement membrane degrading enzymes in two carcinoma sublines with distinct patterns of site-selective metastasis.

Basement membrane-degrading enzymes of two clonal sublines of the murine Lewis lung carcinoma with distinct patterns of organ-selective metastasis were analyzed. Subline M-27 is highly metastatic to the lung and does not form liver metastases, while subline H-59 is highly metastatic to lymph nodes and liver, but not to lung. Qualitative and quantitative differences in the enzymatic profiles were found. H-59 cells which were significantly more invasive in vitro in the Matrigel invasion assay were found by zymogram analysis to secrete high levels of a 72 kDa gelatinase, while M-27 cells produced low levels of this gelatinase and of a higher molecular weight species which migrated in the 107 kDa region. On the other hand, M-27 cells produced significantly higher levels of urokinase type plasminogen activator (uPA) as indicated by a fibrinolysis assay and by Western blot analysis. Northern blot assays revealed an increase of approx. 3-fold in mRNA for cathepsin B in tumor M-27 which was reflected in a quantitative difference in plasma membrane cathepsin B levels as detected by Western blot analysis. H-59 cells on the other hand expressed approx. 8.5-fold more mRNA for cathepsin L. The quantitative differences in the levels of basement membrane degrading proteinases released by these tumor cells suggest that invasion by these cells is differentially regulated--a possible factor in their distinct patterns of dissemination.     

Growth hormone stimulates hepatic lipid mobilization in rainbow trout,Oncorhynchus mykiss

Rainbow trout were used to characterize the direct influence of growth hormone on hepatic lipid mobilization in vitro. Liver was removed from fish fasted 72 h, sliced into 1-mm³ pieces and incubated in Hanks' medium containing ovine or salmon growth hormone (0.28 ng·ml^-1–28 g·ml^-1). Lipid mobilization, resulting from triacylglycerol hydrolysis, was evaluated by fatty acid and glycerol release into culture medium. Control rates of fatty acid release and glycerol release were 0.95±0.16 (mean ± SE) and 0.88±0.28 mol·l^-1·mg fresh weight, respectively. Both ovine growth hormone (28 ng·ml^-1) and salmon growth hormone (28 ng·ml^-1) stimulated fatty acid release into culture medium, increasing rates by 112% and 97%, respectively, during the course of a 24-h incubation. Glycerol release was similarly augmented by growth hormone treatment. Growth hormone-stimulated metabolite release became evident within 12 h and persisted for up to 72 h. The presence of amino acids in the culture medium potentiated the lipolytic action of growth hormone. Ovine growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 108% increase in glycerol, release over rates observed in the absence of amino acids. Salmon growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 44% increase in glycerol, release over rates observed in the absence of amino acids. Ovine growth hormone (28 ng·ml^-1) also stimulated gluconeogenesis, as indicated by a 75% increase in phosphoenolpyruvate carboxykinase activity in liver pieces incubated in the presence of amino acids. These results indicate that growth hormone directly stimulates lipid breakdown in the liver of trout and that amino acids potentiate growth hormone-stimulated lipolysis.Abbreviations AA amino acid(s) - dGDP deoxy-guanosine diphosphate - ED ₅₀ 50% effective dose - FA fatty acid(s) - fw fesh weight - GH growth hormone - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - MS-222 tricaine methanesulfonate - MEM minimum essential medium - oGH ovine growth hormone - PEPCK phosphoenolpyruvate carboxykinase - PK_c protein kinase C - rpm revolutions per minute - sGH salmon growth hormone - TG triacylglycerol - w/v weight per volume This paper was presented in abstract form at the Annual Meeting of the American Society of Zoologists, Dec. 26–30, 1991, Atlanta, GA     

Mechanisms responsible for the positive diversity–productivity relationship in Minnesota grasslands

Species’ extinctions have spurred debate on whether interactions among few or among many species cause a positive diversity–productivity relationship in experimentally assembled grasslands. We addressed this question by quantifying the productivity of 14 species across an experimental diversity gradient in Minnesota. We found that interspecific interactions leading to coexistence and competitive displacement both determine which species overyield; i.e. are more productive at high diversity. Overyielding species were either superior N competitors (C4 grasses) or N fixers (legumes). Surprisingly, these species were not most productive in monoculture, thus, the ‘selection’ of productive species in diverse plots did not cause the positive diversity–productivity relationship. Both positive (with legumes) and negative interspecific interactions (with C4 grasses) determined whether individual species overyielded. Foliar pathogens did not cause overyielding, although other natural enemies may be responsible. Overyielding species are not displacing underyielding species over time, implying that other diversity‐promoting interactions also operate in this experiment.     

Reliability and stability of mothers' reports about their pregnancies with twins.

The objective of this study was to determine if mothers' retrospective reports about events in their pregnancies with twins are reliable and stable. Six hundred and twenty-four mothers completed psychiatric interviews about their twins. These interviews also contained questions about the mothers' pregnancies, the perinatal period, and the child's early development. The mothers reported first on one twin and then on the other with interviews spaced from 3 days to 2 weeks apart. Thus mothers reported on the same pregnancy twice. Of these mothers, 47 were re-interviewed 6 to 18 months later by raters blind to the results of the initial interview. The twin design allowed us to compare the short-term reliability of the 624 mothers' reports of the same pregnancy. The re-interview of the 47 mothers enabled us to compare the stability of reports over a longer time period. Agreement between the reports was measured with the kappa statistic. Kappas were good to excellent for the short-term reports of pregnancy for each twin for the 624 mothers. Kappas were equally high for the 47 mothers that were re-interviewed 6 to 18 months later. Mothers show good reliability and stability of reporting about events during pregnancy.     

On the statistical assessment of similarities in DNA sequences

The statistical behavior of the similarity score for unrelated DNA sequences calculated as letter-by-letter comparison or from various forms of optimal alignment was studied. It was found that natural DNA-sequences from a data base and true random sequences show the same statistical behavior in terms of such scores. This makes it possible to adopt a simple criterion for the rejection of fortuitous similarity. It is based on the mean and standard deviation of chance scores whose expected values, depending on chain length, gap penalty and probability of letter coincidence, may be calculated from formulae given in the paper.