Inactivation of Bacteriophage ϕX174 by d-Fructose 6-Phosphate

The inactivation of bacteriophage ?X174 by d-fructose 6-phosphate was investigated. This inactivation was inhibited by EDTA or reducing agents, and stimulated by Cu²⁺ but other metal ions could not be substituted for Cu²⁺. The reaction was also inhibited by superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and various free radical scavengers.

No detectable changes were observed in adsorption capacity of phage and in the conformation of the virion. The viral DNA in the virion was, however, found to be cleaved. This strand scission was also enhanced by Cu²⁺ and protected by catalase. Similar results were obtained when ?X174 DNA was directly treated with d-fructose 6-phosphate.

It is concluded that the inactivation of ?X174 is due to DNA strand scission in the virion by the free radical of d-fructose 6-phosphate or oxygen radicals generated during autoxidation of d-fructose 6-phosphate.     

Effect of Rifampicin and Uracil Depletion on Bacteriophage ϕX174 DNA Synthesis in an E. coli dnaHts Mutant

The fluorescent antibody technique was used to trace an inoculated Nocardia erythropolis strain which was capable of rapidly degrading phthalate esters in soil column and activated sludge systems. The reaction of antibody to Nocardia erythropolis S-1 was highly strain specific, i. e., only one of twelve other strain of N. erythropolis was stained with this fluorescent antibody. All other species of Nocardia and other genera of bacteria and a strain of Candida were not stained. Using this technique it was demonstrated that N. erythropolis S-1 inoculated into activated sludge and soil column systems was successfully distinguished from many other microorganisms in mixed culture systems, and the distribution of this strain was appreciated.     

Mechanism of Action of Bacillus thuringiensis Insecticidal Delta-endotoxin on Insect Cells in Vitro

Cultured insect cells, TN-368 from the cabbage looper, swelled and burst upon treatment with the enzyme-digested delta-endotoxin of Bacillus thuringiensis var. aizawai. The cytotoxic sweelling was depended upon the amount of the delta-endotoxin added and the concentration of NaCl or KC1 in the isotonic solution. The concentration of Na⁺ in the swollen cells approximately doubled in isotonic NaCl, while that of K⁺ decreased to 10% of the original cellular concentration. The cell swelling was inhibited by tetrodotoxin and also by ouabain in only KC1 isotonic solution. On the other hand, 4-aminopyridine stimulated the swelling in the isotonic KC1 solution, These results indicate that the delta-endotoxin induces the stimulation of Na+ influx and K+ efflux in the isotonic NaCl solution, and also stimulates the Na⁺, K⁺-ATPase in the isotonic KC1 solution. The cytotoxic swelling was also blocked by cAMP, AMP, ATP, GTP, and NAD, but not by adenosine and GMP. These results suggest the participation of nucleotide derivatives in the action of delta-endotoxin.     

Effect of Chloramphenicol on the DNA Synthesis of Bacteriophage ϕX174

In bacteriophage ?X174 infection, the net synthesis of replicative form DNA ceased between 15 and 20 min after infection. When 30 μg of chloramphenicol/ml was added, net RF synthesis, however, continued beyond the normal time and level of turn-off. Experiments with ?X174 mutants unable to synthesize single-stranded DNA showed that a protein synthesis was required for the cessation of net RF synthesis and the protein was synthesized between 10 and 15 min after infection.     

Inhibition of malaria parasite growth by quinomycin A and its derivatives through DNA-intercalating activity

Quinomycin A and its derivatives were identified as potent antimalarial (Plasmodium falciparum) agents in a screen of the RIKEN NPDepo chemical library. IC₅₀ values of quinomycin A and UK-63,598 were approximately 100 times lower than that of the antimalarial drug chloroquine. This activity was mitigated by the addition of plasmid DNA, suggesting that these compounds act against parasites by intercalating into their DNA.     

Glycine Activating Enzyme in the Posterior Silkgland of Silkworm

1. Aminoacyl tRNA synthetase was extracted from the silkgland of silkworm (Bombyxmori Linné) and fractionated on a DEAE-cellulose column. Activities were estimated by ATP-PP_i exchange reaction as well as glycyl tRNA formation.

2. Two peaks, A and B, having ATP-PP_i exchange activity were found in the separated fractions, respectively. There was also observed a marked difference between the both peaks with respect to the pH optimum and activity dependence on MgCl₂ concentration.

3. Peak A showed no activity of glycyl tRNA formation. Only a part of peak B coincided with the activity of glycyl tRNA formation. The activities of both the ATP-PP_i exchange reaction and glycyl tRNA formation were found to be dependent on MgCl₂ concentration, and the optimum concentration was different between two peaks.

4. It also seemed to exist two peaks of activities, a and b, in glycyl tRNA formation which could be separated with a DEAE-cellulose column.     

Characterization of a Degraded Product Derived from Serratia piscatorum Polysaccharide by Ultrasonication

Acidic polysaccharide, PLS F–II, was prepared from Serratia piscatorum polysaccharide, PLS N–I, by a sequence of ultrasonication and gel filtration and was examined for chemical composition and biological activity.

The purified PLS F–II preparation was shown to be homogeneous by ultracentrifugation, zone electrophoresis and column chromatography. The molecular weight was estimated to be about 2 × 10⁴ by the Archibald method. PLS F–II was composed of l-rhamnose, d-galactose and d-galacturonic acid in the molar ratio of 2: 1: 1 and was partially acylated on the galacturonic acid residues.

PLS F–II was found to enhance the antibody formation in mice, although it showed no anti-inflammatory activity.     

Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea

Capnocytophaga ochracea is present in the dental plaque biofilm of patients with periodontitis. Biofilm cells change their phenotype through quorum sensing in response to fluctuations in cell-population density. Quorum sensing is mediated by auto-inducers (AIs). AI-2 is involved in intercellular signaling, and production of its distant precursor is catalyzed by LuxS, an enzyme involved in the activated methyl cycle. Our aim was to clarify the role of LuxS in biofilm formation by C. ochracea. Two luxS-deficient mutants, TmAI2 and LKT7, were constructed from C. ochracea ATCC 27872 by homologous recombination. The mutants produced significantly less AI-2 than the wild type. The growth rates of these mutants were similar to that of the wild-type in both undiluted Tryptic soy broth and 0.5 × Tryptic soy broth. However, according to crystal violet staining, they produced significantly less biofilm than the wild type. Confocal laser scanning microscopy and scanning electron microscopy showed that the biofilm of the TmAI2 strain had a rougher structure than that of the wild type. Complementation of TmAI-2 with extrinsic AI-2 from the culture supernatant of wild-type strain did not restore biofilm formation by the TmAI2 strain, but complementation of LKT7 strain with luxS partially restored biofilm formation. These results indicate that LuxS is involved in biofilm formation by C. ochracea, and that the attenuation of biofilm formation by the mutants is likely caused by a defect in the activated methyl cycle rather than by a loss of AI-2.     

Genomic Motifs as a Novel Indicator of the Relationship between Strains Isolated from the Epidemic of Porcine Epidemic Diarrhea in 2013-2014

Porcine epidemic diarrhea virus (PEDV) is a positive-sense RNA virus that causes infectious gastroenteritis in pigs. Following a PED outbreak that occurred in China in 2010, the disease was identified for the first time in the United States in April 2013, and was reported in many other countries worldwide from 2013 to 2014. As a novel approach to elucidate the epidemiological relationship between PEDV strains, we explored their genome sequences to identify the motifs that were shared within related strains. Of PED outbreaks reported in many countries during 2013–2014, 119 PEDV strains in Japan, USA, Canada, Mexico, Germany, and Korea were selected and used in this study. We developed a motif mining program, which aimed to identify a specific region of the genome that was exclusively shared by a group of PEDV strains. Eight motifs were identified (M1–M8) and they were observed in 41, 9, 18, 6, 10, 14, 2, and 2 strains, respectively. Motifs M1–M6 were shared by strains from more than two countries, and seemed to originate from one PEDV strain, Indiana12.83/USA/2013, among the 119 strains studied. BLAST search for motifs M1–M6 revealed that M3–M5 were almost identical to the strain ZMDZY identified in 2011 in China, while M1 and M2 were similar to other Chinese strains isolated in 2011–2012. Consequently, the PED outbreaks in these six countries may be closely related, and multiple transmissions of PEDV strains between these countries may have occurred during 2013–2014. Although tools such as phylogenetic tree analysis with whole genome sequences are increasingly applied to reveal the connection between isolates, its interpretation is sometimes inconclusive. Application of motifs as a tool to examine the whole genome sequences of causative agents will be more objective and will be an explicit indicator of their relationship.