Binding of adenosine 3',5'-monophosphate dependent protein kinase regulatory subunit to immobilized cyclic nucleotide derivatives.

Several cyclic nucleotide derivatives with aminoalkyl side chains attached to the purine ring were synthesized and their interactions with adenosine 3',5'-monophosphate (cAMP) dependent protein kinase were studied before and after immobilization to CNBr-activated Sepharose 4B. The soluble N6-substituted derivatives were as effective as cAMP itself in activating protein kinase and were more effective than 8-substituted cAMP derivatives, whereas the 2-substituted cAMP derivatives and the cGMP derivatives were the least effective. All of the synthetic derivatives tested were poor substrates for beef heart phosphodiesterase being hydrolyzed at rates less than 2% for that of cAMP itself. Utilizing methodology developed to evaluate the affinity of protein kinase for immogilized cyclic nucleotides it was found that all of the immobilized cyclic nucleotides interacted with protein kinase in a biospecific manner as judged by the following criteria: (1) the immobilized cyclic nucleotides competed with cAMP for the binding sites on protein kinase; (2) the analogous spacer-arm did not compete; and (3) the effects of enzyme concentration, MgATP, and cleavage of the cyclic phosphate ring on the interactions of protein kinase with the immobilized cyclic nucleotides were the same as previously shown for free cAMP. In addition, the immobilized ligands were bound with the same order of effectiveness as the analogous soluble ligand. The observed Ka for the activation of 0.005 muM protein kinase by N6-H2N(CH2)2-cAMP was increased from 0.23 to 3 muM by the process of immobilization. This increase was unaffected by the coupling density and spacer-arm length. The observed Kb for 0.10 muM protein kinase binding to immobilized N6-H2N(CH2)2-cAMP was increased as the molecular sieving exclusion limit of the matrix used was decreased indicating that at least part of this decrease in apparent affinity upon immobilization is due to exclusion of the enzyme from a portion of the matrix and therefore of the immobilized ligand molecules.     

Microbiological leaching of a chalcopyrite concentrate by Thiobacillus ferrooxidans

The microbiological leaching of a chalcopyrite concentrate has been investigated using a pure strain of Thiobacillus ferrooxidans. The optimum leaching conditions regarding pH, temperature, and pulp density were found to be 2.3, 35°C, and 22%, respectively. The energy of activation was calculated to be 16.7 kcal/mol. During these experiments the maximum rate of copper dissolution was about 215 mg/liters/hr and the final copper concentration was as high as 55 g/liter. This latter value is in the range of copper concentrations which may be used for direct electrorecovery of copper. Jarosite formation was observed during the leaching of the chalcopyrite concentrate. When the leach residue was reground to expose new substrate surface, subsequent leaching resulted in copper extractions up to about 80%. On the basis of this experimental work, a flow sheet has been proposed for commercial scale biohydrometallurgical treatment of high-grade chalcopyrite materials.     

Properties of purified hydrogenase from the particulate fraction of Desulfovibrio vulgaris, Miyazaki.

The properties of purified hydrogenase [EC 1.12.2.1] solubilized from particulate fraction of sonicated Desulfovibrio vulgaris cells are described. The enzyme was a brownish iron-sulfur protein of molecular weight 89,000, composed of two different subunits (mol. wt.: 28,000 and 59,000), and it contained 7-9 iron atoms and 7-8 labile sulfide ions. Molybdenum was not detected in the preparation. The absorption spectrum of the enzyme was characteristic of iron-sulfur proteins. The millimolar absorbance coefficients of the enzyme were about 164 at 280nm, and 47 at 400nm. The absorption spectrum of the enzyme in the visible region changed upon incubating the enzyme under H2 in the presence of cytochrome c3, but not in its absence. This spectral change was due to the reduction of the enzyme. The absorbance ratio at 400nm of the reduced and the oxidized forms of the enzyme was 0.66. The activity of the enzyme was hardly affected by metal-complexing agents such as cyanide, azide, 1,10-phenanthroline, etc., except for CO, which was a strong inhibitor of the enzyme. The activity was inhibited by SH-reagents such as p-chloromercuribenzenesulfonate. The enzyme was significantly resistant to urea, but susceptible to sodium dodecyl sulfate. These properties were very similar to those of clostridial hydrogenase [EC 1.12.7.1], in spite of differences in the acceptor specificity and subunit structure.     

Solaniol, a Toxic Metabolite of Fusarium solani

Fusarium solani M-1-1 isolated from moldy bean hulls produces T-2 toxin, diacetoxyscirpenol, and a new toxic trichothecene, solaniol, in Czapek-Dox-peptone medium.     

Plant reproductive intervals and pollinators in the aseasonal tropics: a new model

What factors determine reproductive intervals and modes of pollination in plants of the aseasonal tropics? To answer this general question, we present a new explanation for some community patterns of plant reproductive intervals and pollinators observed in a lowland dipterocarp forest in Sarawak, Malaysia, using a mathematical model featuring different display effects for different types of pollinators. Predictions from the model matched with the following observed patterns: (i) flowering intervals were different among forest strata (forest floor < understorey < canopy < subcanopy and emergent), and not in the exact order of stratum height; (ii) among generalist pollinators, the proportion of social foragers was maximum in intermediate forest stratum; and (iii) plants pollinated by specialist pollinators were found on the forest floor and in gaps.     

Correlation between sequence conservation of the 5' untranslated region and codon usage bias in Mus musculus genes

The codon adaptation index (CAI) values of all protein-coding sequences of the full-length cDNA libraries of Mus musculus were computed based on the RIKEN mouse full-length cDNA library. We have also computed the extent of consensus in flanking sequences of the initiator ATG codon based on the 'relative entropy' values of respective nucleotide positions (from -20 to +12 bp relative to the initiator ATG codon) for each group of genes classified by CAI values. With regard to the two nucleotides positions (-3 and +4) known to be highly conserved in Kozak's consensus sequence, a clear correlation between CAI values and relative entropy values was observed at position -3 but this was not significant at position +4, although a significant correlation was found at position -1 of the consensus sequence. Further, although no correlation was observed at any additional positions, relative entropy values were very high at positions -4, -6, and -8 in genes with high CAI values. These findings suggest that the extent of conservation in the flanking sequence of the initiator ATG codon including Kozak's consensus sequence was an important factor in modulation of the translation efficiency as well as synonymous codon usage bias particularly in highly expressed genes.     

Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION? sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.     

Purification and characterization of thermostable H<Subscript>2</Subscript>O<Subscript>2</Subscript>-forming NADH oxidase from 2-phenylethanol-assimilating <Emphasis Type="Italic">Brevibacterium</Emphasis> sp. KU1309

A cytoplasmic NADH oxidase (NOX) was purified from a soil bacteria, Brevibacterium sp. KU1309, which is able to grow in the medium containing 2-phenylethanol as the sole source of carbon under an aerobic condition. The enzyme catalyzed the oxidation of NADH to NAD+ involving two-electron reduction of O2 to H2O2. The molecular weight of the enzyme was estimated to be 102 kDa by gel filtration and 57 kDa by SDS-PAGE, which indicates that the NOX was a homodimer consisting of a single subunit. The enzyme was stable up to 70 degrees C at a broad range of pH from 7 to 11. The enzyme activity increased about ten-fold with the addition of ammonium salt, while it was inhibited by Zn2+ (39%), Cu2+ (41%), Hg2+ (72%) and Ag+ (37%). The enzyme acts on NADH, but not on NADPH. The regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or L: -phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling.