A Simple Colorimetrie Assay for Aspartate Aminotransferase

γ-Glutamylmethylamide (γ-GMA) synthetase was detected in crude extracts of Methylophaga sp. AA-30, but neither methylamine dehydrogenase nor N-methylglutamate dehydrogenase was observed. A large amount of γ-GMA was accumulated in the cells when the growth on methanol-methylamine was inhibited with iodoacetate, but the accumulation was not observed in the cells grown on methanol-(NH₄)₂SO₄. It is thought that γ-GMA is a metabolic intermediate of the methylamine-dissimilating pathway in the bacterium. In addition, γ-GMA-dissimilating enzymes were found in methylamine-grown cells. The enzymes, which consisted of H protein and L protein, required α-ketoglutaric acid, Mg²⁺ or Mn²⁺, and ammonia as a cofactor. Although the enzyme catalyzed the formation of glutamate from γ-GMA, it did not catalyze the formation of N-methylglutamate. Consequently, in this bacterium, methylamine seems to be metabolized through a different pathway from the N-methylglutamate pathway.     

Isolation and Characterization of PDE-I and II,the Inhibitors of Cyclic Adenosine-3′,5′-monophosphate Phosphodiesterase

In the screening for inhibitors of cyclic adenosine-3′,5′-monophosphate phosphodiesterase, two compounds, PDE-I (C₁₃H₁₃N₃O₅) and PDE-II (C₁₄H₁₄N₂O₅), were isolated from culture filtrates of a Streptomyces. Concentrations for 50% inhibitions of PDE-I and PDE-II against the high Km enzyme were 15 µm and 13 µm, and those against the low Km enzyme were 65 µm and 130 µm, respectively. Production, isolation and characterization of these compounds are described.     

Glucosyl Glycerophosphoric Acid and its Polymer Excreted by α-Amylase-forming Bacillus subtilis

α-Amylase formation by washed cell suspensions of Bac. subtilis was found to be accompanied by the excretion of a compound consisting of glucose, glycerol and phosphoric acid. It was excreted as a polymer and a monomer. The former, a kind of teichoic acid, was significantly dominant in quantity when the cells were incubated under the conditions suitable for α-amylase formation. On the other hand, the monomer prevailed when the bacterial cells were under the unfavorable conditions for the enzyme formation.

Both compounds were purified by ion exchange column chromatography. Chemical and enzymatic investigations revealed the following structures: 2-O-α-d-glucopyranosyl-glycerol-3-monophosphoric acid for the monomer, and a polymerized form of the monomer through phosphodiester linkages involving the hydroxyl groups on C3 of the glycerol, for the polymer.     

Excretion of 5′-Nucleotides from Bacterial Cells

Incubating the dried cells of Brevibacterium sojae No. 425-40 in alkaline buffer, the excretion of 5′-nucleotides accompanying with the decrease of intracellular RNA was observed. Then the determination of the optimum condition of the excretion and the investigation on the enzyme responsible for the degradation of endogenous RNA were carried out.

In the experiments using sonicate and disrupted cells, it appeared that orthophos-phate and Mg⁺⁺ might be accelerative or essential for the degradation of endogenous RNA and, in addition to four 5′-nucleotides (AMP, GMP, UMP and CMP), each nucleoside 5′-diphosphate was also contained in its degraded products. Nucleoside 2′- or 3′-monophos-phates were not detected. Although it was not clear whether phosphodiesterase concerned with the degradation of intracellular RNA or not, it was suggested that polynucleotide phos-phorylase acted mainly on the degradation.

The maximal excretion of 5′-nucleotides from dried cells was obtained by suspending 1 to 2% of dried cells in 0.05 M carbonate-bicarbonate buffer (pH 10) and incubating it at 60°C for two to three hours. Orthophosphate and Mg⁺⁺ were not required for the excretion.     

α-Amylase Formation and Nucleic Acid Metabolism of Bacillus subtilis

The nucleic acid metabolism in washed cells of Bacillus subtilis was investigated with special reference to amylase formation of the bacterium. On incubation of the suspension of the washed cells, purines, pyrimidines and their related compounds were observed in the medium. However, in the medium of the cells incubated with a calcium chelater, where no amylase formation occurred, were detected adenosine- and guanosine-monophosphate in addition to those described above. The addition of a calcium chelater was also found to decrease the quantity of the nucleic acids being involved in the lysozyme-sensitive fraction of the bacterial cells, suggesting the possibility that the metabolism of nucleic acids in this fraction is closely related to amylase formation of the cells.     

Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids

The ¹H–¹³C HMQC signals of the ¹³CH₃ moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ₁-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically ¹³CH₃-labeled [U–²H;¹⁵N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G.     

An Atypical Tubulin Kinase Mediates Stress-Induced Microtubule Depolymerization in Arabidopsis

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No Association of Coffee Consumption with Gastric Ulcer,Duodenal Ulcer,Reflux Esophagitis,and Non-Erosive Reflux Disease: A Cross-Sectional Study of 8,013 Healthy Subjects in Japan

Probably due to caffeine-induced gastric acid secretion, negative effects of coffee upon various upper-gastrointestinal diseases have been precariously accepted, despite the inadequate epidemiological evidence. Our aim is to evaluate the effect of coffee consumption on four major acid-related diseases: gastric ulcer (GU), duodenal ulcer (DU), reflux esophagitis (RE), and non-erosive reflux disease (NERD) based on the large-scale multivariate analysis. Of the 9,517 healthy adults, GU, DU, and RE were diagnosed by endoscopy, and NERD was diagnosed by the symptoms of heartburn and regurgitation without esophageal erosion. Associations between coffee consumption and the four disorders were evaluated, together with age, gender, body mass index (BMI), Helicobacter pylori (HP) infection status, pepsinogen I/II ratio, smoking, and alcohol. We further performed meta-analysis using the random effects model to redefine the relationship between coffee intake and peptic ulcer disease. The eligible 8,013 study subjects comprised of 5,451 coffee drinkers and 2,562 non-coffee drinkers. By univariate analysis, age, BMI, pepsinogen I/II ratio, smoking, and alcohol showed significant associations with coffee consumption. By multiple logistic regression analysis, positively correlated factors with significance were HP infection, current smoking, BMI, and pepsinogen I/II ratio for GU; HP infection, pepsinogen I/II ratio, and current smoking for DU; HP non-infection, male, BMI, pepsinogen I/II ratio, smoking, age, and alcohol for RE; younger age, smoking, and female for NERD. The meta-analyses could detect any association of coffee consumption with neither GU nor DU. In conclusion, there are no significant relationship between coffee consumption and the four major acid-related upper gastrointestinal disorders.     

Background Factors of Reflux Esophagitis and Non-Erosive Reflux Disease: A Cross-Sectional Study of 10,837 Subjects in Japan

Background

Despite the high prevalence of gastroesophageal reflux disease (GERD), its risk factors are still a subject of controversy. This is probably due to inadequate distinction between reflux esophagitis (RE) and non-erosive reflux disease (NERD), and is also due to inadequate evaluation of adjacent stomach. Our aim is therefore to define background factors of RE and NERD independently, based on the evaluation of Helicobacter pylori infection and gastric atrophy.

Methods

We analyzed 10,837 healthy Japanese subjects (6,332 men and 4,505 women, aged 20–87 years) who underwent upper gastrointestinal endoscopy. RE was diagnosed as the presence of mucosal break, and NERD was diagnosed as the presence of heartburn and/or acid regurgitation in RE-free subjects. Using GERD-free subjects as control, background factors for RE and NERD were separately analyzed using logistic regression to evaluate standardized coefficients (SC), odds ratio (OR), and p-value.

Results

Of the 10,837 study subjects, we diagnosed 733 (6.8%) as RE and 1,722 (15.9%) as NERD. For RE, male gender (SC = 0.557, OR = 1.75), HP non-infection (SC = 0.552, OR = 1.74), higher pepsinogen I/II ratio (SC = 0.496, OR = 1.64), higher BMI (SC = 0.464, OR = 1.60), alcohol drinking (SC = 0.161, OR = 1.17), older age (SC = 0.148, OR = 1.16), and smoking (SC = 0.129, OR = 1.14) are positively correlated factors. For NERD, HP infection (SC = 0.106, OR = 1.11), female gender (SC = 0.099, OR = 1.10), younger age (SC = 0.099, OR = 1.10), higher pepsinogen I/II ratio (SC = 0.099, OR = 1.10), smoking (SC = 0.080, OR = 1.08), higher BMI (SC = 0.078, OR = 1.08), and alcohol drinking (SC = 0.076, OR = 1.08) are positively correlated factors. Prevalence of RE in subjects with chronic HP infection and successful HP eradication denotes significant difference (2.3% and 8.8%; p<0.0001), whereas that of NERD shows no difference (18.2% and 20.8%; p = 0.064).

Conclusions

Significantly associated factors of NERD are considerably different from those of RE, indicating that these two disorders are pathophysiologically distinct. Eradication of Helicobacter pylori may have disadvantageous effects on RE but not on NERD.