New alkaloids from Banisteriopsis caapi

Three new alkaloids isolated from Banisteriopsis caapi, were identified as harmic amide (1-carbamoyl-7-methoxy β-carboline), acetyl norharmine (1-acetyl-7-methoxy β-carboline) and ketotetrahydronorharmine (7-methoxy-1,2,3,4-tetrahydro-1-oxo-β-carboline)     

Oxidative Degradation of Squalene by Arthrobacter Species

An organism isolated from soil and identified as Arthrobacter sp. was studied for its squalene degradation. The degradation product from squalene, which accumulated in the culture broth, was isolated and identified as trans-geranylacetone by mass spectrometry, gas chromatography, infrared spectrometry, and nuclear magnetic resonance spectrometry. Addition of a high concentration of K₂HPO₄ to the culture medium resulted in accumulation of fairly large amounts of carboxylic acids in addition to geranylacetone. These carboxylic acids were identified as isovaleric, β,β′-dimethylacrylic, geranic, and (+)-(R)-citronellic acids. Among these acids, α,β-saturated carboxylic acids were found to be predominant in quantity.     

Incorporation of methionine by a soluble enzyme system from Escherichia coli

A soluble fraction from Escherichia coli B was found to incorporate methionine into 95°C CCl₃COOH-insoluble fraction. The incorporation required methionyl-tRNA synthetase, methionine tRNA, ATP, Mg²⁺ and bovine milk casein. The casein could be replaced by arginylated bovine serum albumin and arginylated bovine α-lactalbumin. A mixture of 19 amino acids other than methionine and GTP had no effect on the incorporation. KCl was rather inhibitory. Puromycin, RNase A and trypsin inhibited the incorporation, while DNase I did not. The soluble fraction also incorporated the methionyl moiety of methionyl-tRNA. This incorporation was not affected by the addition of free methionine.     

Genes affecting the productivity of alpha-amylase in Bacillus subtilis Marburg.

Genetic control of alpha-amylase (alpha-1,4-glucan glucanohydrolase, EC 3.2.1.1.) production by Bacillus subtilis 168 was studied from the standpoint that alpha-amylase production by bacteria is dependent on a long-lived messenger ribonucleic acid and obeys the following equation: E = kappa integral of X-DT where x = cell mass at time t, E = alpha amylase produced, t = culture time, and kappa = productivity constant. So a productivity constand (kappa) is obtained from the slope of the straight line plot of alpha-amylase formed versus the total mass of cells accumulated over that time during the culture process. The following results were obtained. (i) Two sequential mutants, derived from the 168(kappa = 20) strain and having improved alpha-amylase productivity (168 leads to 196), were analyzed for their serine and metal protease production. Strain 128 (kappa = 40) produced half the amount of both proteases, but strain 196 (kappa = 60 similar to 80) produced 20 times that in the original strain. (ii) Amy+ transformants, using the 196 strain as the other three had higher productivity (kappa = 37 similar to 46). These transformants (J71, J47, groups. Seventy-one of 74 Amy+ transformants had a kappa value of 21.0 plus or minus 2.1 and the other three had higher productivity (kappa = 37 similar to 46). These transformants (J71,J47, and J10) produced levels of serine and metal proteases 20 times higher than the other transformants. (iii) Strains 196, J71, J47, and J10 were found to be nonmotile and resistant to phage PBS1, whereas other strains, including strains 168, 128, 3 revertants of strain J71 and 2 revertants of strain 196, were all motile and sensitive to the phage. (iv) Strains 196 and J71 were nonflagellated under electron microscopic observation but strain 168, 128 and a revertant of J71 were flagellated. From the above experimental results, the existence of a quality controlling gene (amyB) was deduced, which is loosely linked to the structural gene and controls productivities of alpha-amylase and proteases, and flagellation. The probable existence of another regulatory gene, amyC, is also discussed.     

New organic bases from amazonian Banisteriopsis caapi

Three new bases were isolated from Banisteriopsis caapi; they are harmine N-oxide, harmic acid methyl ester (methyl 7-methoxy-β-carboline 1-carboxylate) and harmalinic acid (7-methoxy-3,4-dihydro-β-carboline 1-carboxylic acid).     

Effects of the combined substitutions of amino acid residues on thermal properties of cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria

In the two cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea (CmIDH and CpIDH, respectively), the combined substitutions of amino acid residues between the Leu693, Leu724 and Phe735 residues of CmIDH and the corresponding Phe693, Gln724 and Leu735 residues of CpIDH were introduced by site-directed mutagenesis. A double mutant of CmIDH substituted its Leu724 and Phe735 residues by the corresponding ones of CpIDH, CmL724Q/F735L, and the triple mutant of CpIDH, CpF693L/Q724L/L735F, showed the most decrease and increase of activity, respectively, of each wild-type and its all mutated enzymes. In the case of CmIDH, the substitutions of these three amino acid residues resulted in the decrease of catalytic activity and thermostability for activity, but the combined substitutions of amino acid residues did not necessarily exert additive effects on these properties. On the other hand, similar substitutions in CpIDH had quite opposite effects to CmIDH, and the effects of the combined substitutions were additive. All multiple mutants of CmIDH and CpIDH showed lower and higher catalytic efficiency (k _cat/K _m) values than the respective wild-type enzymes. Single and multiple mutations of the substituted amino acid residues in the CmIDH and CpIDH led to the increase and decrease of sensitivity to tryptic digestion, indicating that the stability of protein structure was decreased and increased by the mutations, respectively.     

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp⁰ strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp⁰ strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp⁰ suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp⁰ backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.     

Osmotic Stress Responses and Plant Growth Controlled by Potassium Transporters in Arabidopsis

Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K⁺ uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K⁺ channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using ⁸⁶radioactive rubidium ion (⁸⁶Rb⁺) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABA-mediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses.     

Identification of ABCG2 Dysfunction as a Major Factor Contributing to Gout

The ATP-binding cassette, subfamily G, member 2 gene ABCG2/BCRP locates in a gout-susceptibility locus (MIM 138900) on chromosome 4q. Recent genome-wide association studies also showed that the ABCG2 gene relates to serum uric acid levels and gout. Since ABCG2 is also known as a transporter of nucleotide analogs that are structurally similar to urate, and is an exporter that has common polymorphic reduced functionality variants, ABCG2 could be a urate secretion transporter and a gene causing gout. To find candidate mutations in ABCG2, we performed a mutation analysis of the ABCG2 gene in 90 Japanese patients with hyperuricemia and found six non-synonymous mutations. Among the variants, ATP-dependent urate transport was reduced or eliminated in five variants, and two out of the five variants (Q126X and Q141K) were frequently detected in patients. Haplotype frequency analysis revealed that there is no simultaneous presence of Q126X and Q141K in one haplotype. As Q126X and Q141K are a nonfunctional and half-functional haplotype, respectively, their genotype combinations are divided into four estimated functional groups. The association study with 161 male gout patients and 865 male controls showed that all of those who had dysfunctional ABCG2 had an increased risk of gout, and that a remarkable risk was observed in those with ≤1/4 function (OR, 25.8; 95% CI, 10.3–64.6; p = 3.39 × 10^?21). In 2,150 Japanese individuals, the frequency of those with dysfunctional ABCG2 was more than 50%. Our function-based clinicogenetic analysis identified the combinations of dysfunctional variants of ABCG2 as a major contributing factor in Japanese patients with gout.