Methyl chloride metabolism of the strictly anaerobic,methyl chloride-utilizing homoacetogen strain MC

The methyl chloride metabolism of the homoacetogenic, methyl chloride-utilizing strain MC was investigated with cell extracts and cell suspensions of the organism. Cell extracts were found to contain all enzyme activities required for the conversion of methyl chloride or of H₂ plus CO₂ to acetate. They catalyzed the dechlorination of methyl chloride with tetrahydrofolate as the methyl acceptor at a rate of 20 nmol/min × mg of cell protein. Also, the O-demethylation of vanillate with tetrahydrofolate could be measured at a rate of 40 nmol/min × mg. Different enzyme systems appeared to be responsible for the dehalogenation of CH₃Cl and for the O-demethylation of methoxylated aromatic compounds, since cells grown with methoxylated aromatic compounds exhibited a significantly lower activity of CH₃Cl conversion than methyl chloride grown cells and vice versa. In addition, ammonium thiocyanate (5 mM) completely inhibited CH₃Cl dechlorination, whereas the consumption of vanillate was not affected significantly. The data were taken to indicate, that the methyl chloride dehalogenation is catalyzed by a specific, inducible enzyme present in strain MC, and that tetrahydrofolate rather than the corrinoid-protein involved in acetate formation is the primary acceptor of the methyl group in the dechlorination reaction.     

The use of the nonradioactive digoxigenin chemiluminescent technology for plant genomic Southern blot hybridization: A comparison with radioactivity

A nonradioactive labelling and detection method for plant genomic DNA analysis is compared to the radioactive method. The radioisotopes are replaced by a nucleotide, digoxigenin-11-dUTP, and the signal detection is accomplished by the enzymatic reaction of alkaline phosphatase, conjugated to anti-digoxigenin antibodies, with the chemiluminescent substrate AMPPD (3-(2-spiroadamantane)-4-methoxy-4(3 phosphorytoxy) phenyl-1, 2-dioxetane). The sensitivity of the radioactive and nonradioactive methods are directly compared using identical Southern blots subjected to the radioactive and nonradioactive detection. The advantages of this nonradioactive method are discussed.     

Stereoselective HPLC bioanalysis of atenolol enantiomers in plasma: Application to a comparative human pharmacokinetic study

An enantioselective HPLC bioassay has been developed relying on extraction of (R)- and (S)-atenolol from alkalinized plasma or serum (pH > 12) into dichloromethane containing 5% (v/v) 1-butanol followed by an achiral derivatization of the drug with phosgene leading to (R)- and (S)-oxazolidine-2-one derivatives. Under these conditions there was quantitative conversion of the acetamido group to the corresponding nitrile. These stable derivatives were separated on a (R,R)-diaminocylohexane-dinitrobenzoyl chiral stationary phase [(R,R)-DACH-DNB] using dichloromethane/methanol 98/2 as mobile phase. Determination limits of 0.5 ng for (R)- and 0.6 ng for (S)-atenolol could be achieved using fluorimetric detection. The assay was applied to a human pharmacokinetic study which was performed in a randomized cross-over, double-blind fashion in 12 healthy volunteers, administering single oral doses of 100 mg (R,S)-, 50 mg (R)-, and 50 mg (S)-atenolol AUC_0–24 and C_max values of (R)-atenolol were slightly but significant higher than those of (S)-atenolol. The R/S ratios were 1.09 for AUC(R)/AUC(S) and 1.03 for C_max (R)/C_max(S) (P < 0.01) respectively after administration of the racemic drug. However, there were no differences between AUC, C_max, and t_½ values of each enantiomer, whether they were administered as single enantiometers or in the form of its racemic mixture. © 1993 Wiley-Liss, Inc.     

The mating-type locus B alpha 1 of Schizophyllum commune contains a pheromone receptor gene and putative pheromone genes.

Analysis of the multispecific B alpha mating-type locus of Schizophyllum commune provided evidence that pheromones and pheromone receptors govern recognition of self versus non-self and sexual development in this homobasidiomycetous fungus. Four subclones of an 8.2 kb genomic fragment carrying B alpha 1 specificity induced B-regulated sexual morphogenesis when introduced into a strain with one of the eight compatible B alpha specificities that are known to exist in nature. One of these clones, which activated all other B alpha specificities, contains a gene termed bar1. The predicted protein product of bar1, as well as that of bar2, a homologous gene isolated from a B alpha 2 strain, has significant homology to known fungal pheromone receptor proteins in the rhodopsin-like superfamily of G protein-linked receptors. The other three active B alpha 1 clones were subcloned further to identify the minimal active element in each clone. Every active subclone contains a putative pheromone gene ending in a signal for possible isoprenylation. A message of approximately 600 bp was observed for one of these genes, bap1(1). This paper presents the first evidence for a system of multiple pheromones and pheromone receptors as a basis for multispecific mating types in a fungus.     

Engineering of zinc finger and MHC motifs to locked-in tertiary folds

The assembly of helical and -sheet peptide blocks containing reactive chain ends results inhighly branched chain architectures (locked-in folds) mimicking native tertiary structures.This molecular kit strategy allows to bypass the protein folding problem in protein de novodesign and gives access to protein mimetics of high thermodynamic stability. The validity ofthis concept is exemplified for the design and synthesis of locked-in folds mimicking the zincfinger and MHC folding motifs.     

Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat

More than 100 resistance genes against wheat rust pathogens have been described in wheat and its relatives. Although many of them have been extensively used in wheat resistance breeding, none of these resistance loci has yet been analyzed at the molecular level. By screening a set of near-isogenic lines carrying different leaf rust resistance genes with a wheat probe encoding a serine/threonine protein kinase, we detected a polymorphic DNA fragment in the line with the Lr10 resistance gene. This fragment mapped to the Lr10 disease resistance locus and encodes a receptor-like protein kinase which we called LRK10. LRK10 contains a new type of extracellular domain not found in known plant or animal receptor kinases. Several conserved amino acids in S-domain glycoproteins and receptor-like kinases were also found in LRK10, suggesting that LRK10 and S-domain proteins belong to the same superfamily of specific recognition molecules in plants. Lrk10 was expressed at low levels in young seedlings and belongs to a gene family. Analysis of wheat lines with and without the Lr10 gene demonstrated that Lrk10 and Lr10 belong to the same genetic locus. We conclude that gene isolation based on protein kinase homology can identify new receptor domains and provide candidates for disease resistance genes in the complex wheat genome.     

Immunological studies on lysosomal sphingomyelinase: Identification of a 28 000-Da component deficient in urine from patients with Niemann-Pick disease types A and B

The immunoblotting technique was used to identify sphingomyeJinase protein in samples of tissue and urine after subjection to poIyacrylamide-gel etectrophoresis in the presence of sodium dodecyl sulphate. In a sphingomyelinase preparation purified from control urine a prominent band was seen with an M_r of 28 000 Da. Glycoprotein fractions from urine and placenta, a membrane extract from spleen, and a partially purified sphingomyelinase preparation from placenta contained the 28 000-Da band plus additional, higher-M_r bands. The 28 000-Da band was detectable in urine from a patient with Niemann-Pick disease type C, but not in urine from patients with Niemann-Pick disease types A and B. It is concluded t h a t sphingomyeJinase is composed of at least one polypeptide with an M_r of 28 000 Da and that this polypeptide is deficient in the urine of patients with Niemann-Pick disease types A and B.