Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

18-Substituted steroids. Part 15. 6 beta-Hydroxylation of aldosterone by liver

6 beta-Hydroxyaldosterone and 6 beta-hydroxy-17-isoaldosterone, characterized by high-field NMR studies, are among the major polar metabolites formed from aldosterone by incubation with rat liver slices or microsomal fraction. It is uncertain at present whether the 17-iso product results from an enzymatic or a chemical inversion of configuration. Periodate degradation of the 6 beta-hydroxyaldosterone gave 6 beta-hydroxyaldosterone gamma-lactone, identical with a synthetic sample.     

Identification of psychrotrophic pseudomonads from goats' milk by computer-assisted analysis of carbon source assimilation data

The results of carbon source assimilation tests on a group of psychrotrophic pseudomonas were compared with published data for established Pseudomonas taxa, using computer-assisted numerical taxonomic analysis and a modified diagnostic computer program. Several phenons were not grouped at the biovar level by numerical taxonomic analysis. Identification of strains by the diagnostic program revealed heterogeneity among those in the unassigned phenons, and supported a continuum concept among the fluorescent pseudomonads.     

A numerical taxonomic study of proteolytic and lipolytic psychrotrophs isolated from caprine milk

Lipolytic and proteolytic psychrotrophs were isolated from raw and pasteurized goats' milk, which had been stored at 5 degrees C for 7 d. The 241 strains isolated and 20 reference strains were examined by 149 biochemical, physiological, and morphological tests. The results yielded 195 characters suitable for taxonomic analysis. Computer-assisted complete linkage analysis, using the Jaccard coefficient, produced 22 phenons at 75% S. The results showed that Pseudomonas fluorescens was the predominant psychrotrophic bacterium, but that Pseudomonas fragi was dominant in some milk samples. Strains of Serratia liquefaciens and Flavobacterium balustinum were also identified.     

Non-histone chromatin proteins in beef thyroid: distinct phosphorylation patterns of several protein kinases

Summary Non-histone chromatin protein (NHCP) fractions were extracted from purified beef thyroid nuclear preparations and tested for the presence of protein kinase activities using several known mediators of thyroid regulation, as well as potential phosphotransferase substrates using purified or partially purified protein kinase activities. The addition of cAMP/3-isobutyl-l-methylxanthine had no effect on NHCP historic kinase activity; the addition of 10 g of the heat-stable cAMP-dependent protein kinase A inhibitor, however, resulted in a 47% reduction in histone H₂ kinase activity. Nuclear casein kinase II activity was present in the NHCP fractions as evidenced by the capacity of spermine to stimulate (ED₅₀ = 0.19 mM) and heparin to inhibit (ID₅₀ = 0.09 g/ml) the phosphorylation of casein; further, the phosphotransferase activity could be purified by sequential casein-agarose and spermine-agarose affinity chromatography. Neither calcium-calmodulin nor calcium/phosphatidylserine/diolein had an effect on NHCP casein kinase or histone kinase activities, respectively. The addition of cAMP-dependent protein kinase A catalytic subunit, nuclear casein kinase II, calcium-activated calmodulin-dependent protein kinase and diacylglycerol-activated calcium/phospholipid-dependent protein kinase C activities exhibited distinct phosphorylation patterns when NHCP were used as substrates and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. We conclude that NHCP fraction from beef thyroid: 1) contains both cAMP-dependent protein kinase A catalytic subunit and nuclear casein kinase II and 2) substrates for cAMP-dependent protein kinase A, calcium-activited calmodulin-dependent protein kinase, protein kinase C, and nuclear casein kinase II.Abbreviations NHCP Non-Histone Chromatin Proteins - PK-A cAMP-Dependent Protein Kinase - CAMPK Calcium-Activated Calmodulin-Dependent Protein Kinase - PK-C Diacylglycerol-Activated Calcium/phospholipid-dependent Protein Kinase - NK-11 Nuclear Casein Kinase 11 - CK-G Cytosolic Casein Kinase G or 11 - PMSF Phenylmethyl Sulfonyl Fluoride - PKI the Heat Stable PK-A Inhibitor (Walsh inhibitor) - SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis - EDTA Ethylenediamine Tetraacetic Acid - EGTA Ethyleneglycol bis- (B-aminoethyl ether) N,N,N,N,-Tetraacetic Acid - PS Phosphatidylserine - DO 1,2-Diolein     

Activation of galactose-containing glycoprotein and solid supports by galactose oxidase in presence of catalase for immobilization purposes

Summary Specific oxidation of D-galactose present in the carbohydrate moiety of glucose oxidase from Aspergillus niger by galactose oxidase in the presence of catalase (48% efficiency) did not change the activity of the enzyme. Oxidized enzyme was coupled to hydrazide derivatives of O--D-galactosyl Separon H 1000 or of Sepharose 4B. Both solid supports were modified with adipic acid dihydrazide after their activation with galactose oxidase. Each immobilized preparation of glucose oxidase showed higher activity than was achieved by other immobilizing procedures.     

Synthesis and degradation of collagens in skin of healthy and protein-malnourished rats in vivo, studied by 18O2 labelling.

To explore the effects of growth retardation, caused by restricted protein intake, on collagen turnover in the whole skin, Sprague-Dawley rats (n = 20) were labelled with 18O2 and fed on either an adequate (18%) or a low (3%) lactalbumin diet. Skin biopsies were obtained at intervals during the following 6 months. Independent groups of animals (n = 186) were used to determine the size of the 0.5 M-acetic acid-soluble and -insoluble collagen pools in the entire skin of healthy and malnourished rats. Collagen was estimated by measurement of hydroxyproline. Soluble-collagen synthesis rates were equivalent to 99 +/- 8 mumol of hydroxyproline/day in healthy animals and 11 +/- 2 mumol/day in malnourished rats. Insoluble-collagen synthesis rates were 32 and 5 mumol of hydroxyproline/day in the healthy and protein-depleted rats respectively. The degradation of soluble collagen amounted to 37 +/- 8 and 6 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Efflux of collagen from the soluble collagen, defined as the sum of the rate of soluble collagen that is degraded plus that which matures into insoluble collagen, was 70 +/- 8 and 11 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Insoluble collagen was not degraded in either group. The fraction of soluble collagen leaving the pool that was converted into insoluble collagen was 0.46 in both diet groups. It is concluded that the turnover of soluble collagen is markedly decreased with malnutrition, but degradation and conversion into insoluble collagen account for the same proportions of efflux from the soluble-collagen pool as in rapidly growing rats.     

Development of the esophageal muscles in embryos of the sea urchin Strongylocentrotus purpuratus

Summary Development of the esophageal muscles in embryonic sea urchins is described using light- and electron microscopy. The muscles develop from processes of about 14 cells of the coelomic epithelium that become immunore-active to anti-actin at about 60 h (12–14° C). Initially, eachmyoblast extends a single process with numerous fine filopodia around the esophagus. By 72 h the processes have reached the midline and fused with those from cells of the contralateral coelomic sac. Myoblasts begin to migrate out of the coelomic epithelium between 72 and 84 h. By 72 h the processes stain with the F-actin specific probe NBD-phallacidin. The contractile apparatus is not evident in transmission electron-microscopic preparations of embryos at 70 h, but by 84 h the contractile apparatus is present and the muscle cells are capable of contraction. Because the myoblasts migrate free of the coelomic epithelium and are situated on the blastocoelar side of the basal lamina, it is suggested that that they should be considered as a class of mesenchymal cells.