Affinity chromatography of Ankistrodesmus braunii nitrate reductase using blue dextran-sepharose (author's transl)

Blue Dextran has been coupled covalently to Sepharose-4B to purify the enzymatic complex NAD(P)H-nitrate reductase (EC 1.6.6.2) from the green alga Ankistrodesmus braunii by affinity chromatography. The optimum conditions for the accomplishment of the chromatographic process have been determined. The adsorption of nitrate reductase on Blue Dextran Sepharose is optimum when a phosphate buffer of low ionic strength and pH 6.5-7.0 is used. Once the enzyme has been bound to Blue Dextran Sepharose, it can be specifically eluted by addition of NADH and FAD to the washing buffer. However, none of the nucleotides added separately is able to promote the elution of the enzyme from the column. The elution can be also achieved, but not specifically, by increasing the ionic strength of the buffer with KCl. These results have made possible a procedure for the purification of A. braunii nitrate reductase which led to electrophoretic homogeneity, with an overall yield of 70% and a specific activity of 49 units/mg of protein.     

A comparison of ferric-chelate reductase and chlorophyll and growth ratios as indices of selection of quince,pear and olive genotypes under iron deficiency stress

Quince (Cydonia oblonga Mill.), pear (Pyrus communis L.) and olive (Olea europaea L.) genotypes were evaluated for their tolerance to iron deficiency stress by growing young plants in three types of aerated nutrient solutions: (1) with iron, (2) without iron or (3) low in iron and with 10 mM bicarbonate. Plants were obtained either from rooted softwood cuttings or from germination of seeds. The degree of tolerance was evaluated with several indices: (1) the chlorophyll content, (2) the root Fe³⁺ reducing capacity and (3) the whole plant relative growth. Fifteen hours before Fe³⁺ reducing capacity determination, iron was applied to the roots of plants with iron-stress, since this method resulted in increasing the reductase activity. All quince and pear genotypes increased the root Fe³⁺ reducing capacity when grown in the treatments for iron-stress, in relation to control plants of the same genotypes. In olive cultivars, the Fe³⁺ reducing capacity was lower in the iron-stress treatments than in the control one. Studying the relationship between relative growth and chlorophyll content for each genotype under iron-stress, in relation to both indices in control plants, a classification of species and genotypes was established. According to that, most olive cultivars and some pear rootstocks and cultivars appear more iron-efficient than quince rootstocks. Our study shows that in some woody species, determining root Fe³⁺ reducing capacity is not the best method to establish tolerance to iron deficiency stress.     

Characterization of Met-139 as the photolabeled amino acid residue in the steroid binding site of sex hormone binding globulin using delta 6 derivatives of either testosterone or estradiol as unsubstituted photoaffinity labeling reagents.

Immunopurified human sex hormone binding globulin (SHBG) was photoinactivated and photolabeled by radioinert and radioactive photoaffinity labeling steroids delta 6-testosterone (delta 6-T) and delta 6-estradiol (delta 6-E2). The maximal levels of specific incorporation of these two reagents were 0.50 and 0.33 mol of label/mol of SHBG, respectively. Covalently labeled SHBG fractions were citraconylated, reduced, carboxymethylated, and cleaved by trypsin. Separation of tryptic digests by reverse-phase liquid chromatography gave single radioactive peaks at the same retention times with both steroid reagents. However, the two labeled peptidic fractions could be distinguished by capillary electrophoresis and immunodetection with anti-steroid antibodies, whereas the covalent attachment of radioactivity was confirmed by thin-layer chromatography on silica gel. Edman degradation of the two labeled peptides showed a single sequence His-Pro-Ile-([3H]X)-Arg corresponding to the pentapeptide His-Pro-Ile-Met-Arg 136-140 of SHBG sequence. The coincidence, in both cases, of the absence of an identifiable amino acid residue and of the elution of the most intense peak of radioactivity at the fourth cycle of Edman degradation suggests that the same Met-139 residue was labeled by delta 6-[1,2-3H2]T or by delta 6-[17 alpha-3H]E2. Liquid secondary ion mass spectrometry of the two peptides showed [M+H]+ ions at m/z 939.8 or 923.8, corresponding respectively to the addition of delta 6-T or delta 6-E2 to the pentapeptide. The presence of the steroid molecule in the delta 6-[3H]T-pentapeptide conjugate was confirmed by the difference of 2 mass units with the [M+H]+ peak of the delta 6-[4-14C]T-pentapeptide conjugate.     

Gas chromatographic determination of low concentrations of benzoic acid in human plasma and urine

A method for the determination of benzoic acid down to concentrations of 10 ng/ml in plasma or urine is described. After addition of an internal standard, benzoic acid is extracted at acid pH into diethyl ether. Both compounds are derivatized with pentafluorobenzyl bromide. The derivatives are determined by gas chromatography using a ⁴³Ni electron-capture detector. Hippuric acid is hydrolysed in plasma and urine and total benzoic acid is determined by the same technique.     

“A cleaner break”: Genetic divergence between geographic groups and sympatric phenotypes revealed in ballan wrasse (Labrus bergylta)

Capture and long‐distance translocation of cleaner fish to control lice infestations on marine salmonid farms has the potential to influence wild populations via overexploitation in source regions, and introgression in recipient regions. Knowledge of population genetic structure is therefore required. We studied the genetic structure of ballan wrasse, a phenotypically diverse and extensively used cleaner fish, from 18 locations in Norway and Sweden, and from Galicia, Spain, using 82 SNP markers. We detected two very distinct genetic groups in Scandinavia, northwestern and southeastern. These groups were split by a stretch of sandy beaches in southwest Norway, representing a habitat discontinuity for this rocky shore associated benthic egg‐laying species. Wrasse from Galicia were highly differentiated from all Scandinavian locations, but more similar to northwestern than southeastern locations. Distinct genetic differences were observed between sympatric spotty and plain phenotypes in Galicia, but not in Scandinavia. The mechanisms underlying the geographic patterns between phenotypes are discussed, but not identified. We conclude that extensive aquaculture‐mediated translocation of ballan wrasse from Sweden and southern Norway to western and middle Norway has the potential to mix genetically distinct populations. These results question the sustainability of the current cleaner fish practice.     

Sequences related to the major subunit gene fedA of F107 fimbriae in porcine Escherichia coli strains that express adhesive fimbriae

Abstract Porcine Escherichia coli strains isolated from cases fo postweaning diarrhea or edema disease were analysed for the presence of fedA , the major subunit gene of F107 fimbriae. The E. coli isolates were known to contain colonisation factor '8813', or to express F107, 2134P or other fimbriae, different from F4, F5, F6, and F41. PCR with fedA -specific primers, restriction enzyme digestion of the PCR product, and nucleotide sequence analysis demonstrated that 2134P pili, colonisation factor '8813' and fimbriae identified on Australian strains of the O141 serotype belong to one family of F107 fimbrial antigens.     

Identification and functional analysis of NOL7 nuclear and nucleolar localization signals

Background  

NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.