A Method of Fractionation of Bacterial Cytoplasmic Membrane

A study was made of thermogravimetric analyses of microcrystalline cellulose, (Avicell), over a temperature range from 240°C to 300°C under air and nitrogen by means of a thermal balance. For comparative purpose, cellobiose and glucose were also used. The volatilization rate of cellulose was related to the amount of pyrolytic residue and accelerated owing to the oxidation in the presence of atmospheric oxygen. The apparent activation energies of pyrolysis were obtained from weight loss data.

Quantities of carbonyl and carboxyl groups in pyrocellulose increased linearly against degradation stages of cellulose irrespective of pyrolytic temperatures and times.     

Studies on the Autolysis of Aspergillus oryzae

Mode of action of crystalline nuclease O obtained from autolyzed Aspergillus oryzae on RNA and synthetic homopolymers was examined. Crystalline nuclease O had no strict base specificity, although the velocity of hydrolysis was poly A > poly U > RNA > poly C. This enzyme did not degrade poly G. Digestion of high molecular weight RNA with an excess of this enzyme produced mono-, di- and trinucleotides with 5′-terminal phosphate. The amount of mono-, di- and trinucleotides was, respectively, 13.6, 70.0 and 16.4% of total degradation products. All the four bases were detected in mononucleotide fraction and 3′-terminals and 5′-terminals of oligonucleotides.     

Milk Clotting Enzyme from Microorganisms

The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.

The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.

Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.

The enzyme activity was inhibited by Hg⁺⁺ or Fe⁺⁺⁺.

Trypsinogenkinase activity was not contained in this enzyme.

The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.

The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.

Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin.     

Mechanism of Antimycin A Resistance in Bacillus subtilis

The site of action of antimycin A is known to lie between cytochrome b and c in the respiratory chain of mammalian cells. But in general, bacteria, even those which have cytochromes similar to those of mammalian cells such as Bacillus subtilis, are naturally resistant to this antibiotic.

The mechanism of this natural resistance is studied using a strain of B. subtilis. Succinoxidase activity of the intact cells of this bacterium showed very low sensitivity to the antibiotic, but on disruption of the cells, the sensitivity increased 7.5 times. Moreover, the activity of the intact cells could be sensitized by treatment with cationic detergent. In addition to the permeability barrier suggested by the above results, it was found that the electron transport system of this bacterium contained antimycin A insensitive by-path.     

Identification of Residues Essential for the Activity and Substrate Affinity of l-Carnitine Dehydrogenase

Recently, two l-carnitine dehydrogenases from soil isolates Rhizobium sp. (Rs-CDH) and Xanthomonas translucens (Xt-CDH) have demonstrated to exhibit mutually differing affinities toward l-carnitine. To identify residues important for affinity to the substrate, we compared the primary structure of Xt-CDH and Rs-CDH with the recognized 3D structure of 3-hydroxyacyl-CoA dehydrogenase (PDB code: 1F0Y). Then, six residues of Xt-CDH (Phe143, Gly188, Ile190, Ala191, Gly223, and Ala224) and the corresponding residues of Rs-CDH (Tyr140, Ala185, Val187, Gly188, Ser220, and Phe221) were selected for further mutagenesis. The residues of Xt-CDH were replaced with that of Rs-CDH at the corresponding position and vice versa. All Rs-CDH mutants exhibited slight effects on substrate affinity, except for the double mutants Rs-V187I/G188A, which was devoid of enzyme activity. All Xt-CDH mutants showed different K _m values. Xt-F143Y caused a higher increase in the K _m value. Furthermore, the kinetic parameters of 10 mutants at Xt-F143 and Rs-Y140 were investigated. All Rs-Y140 mutants, except aromatic residues (Phe, Trp), produced proteins that were almost entirely devoid of enzyme activity and with disrupted affinity to l-carnitine. All Xt-F143 variants showed a marked reduction (P ≤ 0.05) in enzyme activity. Overall, our results suggest that the aromatic rings of Tyr140 in Rs-CDH and Phe143 of Xt-CDH are essential for substrate recognition.     

Low Ambient Temperature and Intracerebral Hemorrhage: The INTERACT2 Study

Background

Rates of acute intracerebral hemorrhage (ICH) increase in winter months but the magnitude of risk is unknown. We aimed to quantify the association of ambient temperature with the risk of ICH in the Intensive Blood Pressure Reduction in Acute Cerebral Haemorrhage Trial (INTERACT2) participants on an hourly timescale.

Methods

INTERACT2 was an international, open, blinded endpoint, randomized controlled trial of patients with spontaneous ICH (<6h of onset) and elevated systolic blood pressure (SBP, 150–220 mmHg) assigned to intensive (target SBP <140 mmHg) or guideline-recommended (SBP <180 mmHg) BP treatment. We linked individual level hourly temperature to baseline data of 1997 participants, and performed case-crossover analyses using a distributed lag non-linear model with 24h lag period to assess the association of ambient temperature and risk of ICH. Results were presented as overall cumulative odds ratios (ORs) and 95% CI.

Results

Low ambient temperature (≤10°C) was associated with increased risks of ICH: overall cumulative OR was 1.37 (0.99–1.91) for 10°C, 1.92 (1.31–2.81) for 0°C, 3.13 (1.89–5.19) for -10°C, and 5.76 (2.30–14.42) for -20°C, as compared with a reference temperature of 20°C.There was no clear relation of low temperature beyond three hours after exposure. Results were consistent in sensitivity analyses.

Conclusions

Exposure to low ambient temperature within several hours increases the risk of ICH.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00716079","term_id":"NCT00716079"}}NCT00716079     

Analysis of novel disease-related genes in bronchial asthma

Bronchial asthma is a complex disease characterized by airway inflammation involving interleukin (IL)-4 and IL-13. We have applied microarray analyses to human bronchial epithelial cultures to probe for genes regulated by these cytokines and have identified a subset of disease-relevant genes by comparison with cDNA libraries derived from normal and asthmatic bronchial biopsies. Squamous cell carcinoma antigen-1 (SCCA1) and SCCA2, the cysteine and serine protease inhibitors, respectively, showed the highest expression by IL-4 and IL-13, and particularly, SCCA1 was significantly increased in the asthmatic cDNA library. STAT6 was shown to be involved in expression of SCCA1 and SCCA2 in vitro. Furthermore, serum levels of SCCA were also elevated in asthmatic patients. Taken together, it was supposed that SCCA may play some role in the pathogenesis of bronchia asthma, and measuring its serum level may be relevant for diagnosing or monitoring the status of bronchial asthma. In a complex disorder such as asthma, this combination of in vitro and in vivo genomic approaches is a powerful discriminatory method enabling identification of novel disease-related genes and their mechanisms of regulation.     

Isolation of Zeanic Acid,a Natural Plant Growth-regulator from Corn Steep Liquor and Its Chemical Structure

Corn steep liquor markedly inhibited the growth of the grass, alfalfa, as previously reported. The active principles from corn steep liquor were isolated. From the acidic fraction of corn steep liquor, white needles and yellow needles were isolated. The compound of white needles, which inhibited the germination of alfalfa seeds at the concentrations above 500 ppm, was found to be identical with ferulic acid known as germination inhibitor of plant seeds. The compound of yellow needles did not inhibit the germination of alfalfa seeds, but stimulated the growth of the plant. It has a structure related to β-acid which has been isolated from rice-bran and has been identified as 2,6-dihydroxycinchoninic acid. This active substance is a new quinoline, 2,8-dihydroxycinchoninic acid and was named zeanic acid.     

Bacterial p-Hydroxybenzoate Hydroxylase

Screening for p-hydroxybenzoate-utilizing pseudomonads was carried out. Species belonging to Fluorescent group could grow at the expense of p-hydroxybenzoate as sole carbon source. Two species in Chromogenic group and three species in Achromogenic group could grow in the same manner. Specic and total activities of crude extracts differed from each other strain under cultural conditions employed. Ps. desmolytica IAM 1123 and Ps. desmolytica 4-B were selected as superior strains for the sources of p-hydroxybenzoate hydroxylase. The type and the activity of protocatechuate oxygenase were also investigated. Protocatechuate 3,4-oxygenase was found in the cells belonging to Fluorescent and Chromogenic groups whereas 4,5-oxygenase was found in Achromogenic group.