Analysis of a pair of END+ and END? viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production

The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N^pro. Reportedly, the amino acid residues in the zinc-binding TRASH motif of N^pro determine the difference in characteristics between END-phenomenon-positive (END⁺) and END-phenomenon-negative (END⁻) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END⁺ and END⁻ viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END⁺ and END⁻ viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N^pro was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.     

Two phases of intracellular reactive oxygen species production during victorin-induced cell death in oats

Reactive oxygen species (ROS) are thought to be involved in various forms of programmed cell death (PCD) in animal and plant cells. PCD, along with the production of ROS, occurs during plant–pathogen interactions. Here we show that victorin, a host-specific toxin produced by Cochliobolus victoriae, which causes victoria blight of oats, induces two phases of intracellular ROS production in victorin-sensitive oat mesophyll cells. The initial production of ROS is restricted at mitochondria and not accompanied with cellular oxidative damage. Later production of ROS is dispersed into cells concomitant with lipid peroxidation, chloroplast dysfunction, and cell death. Superoxide dismutase can clearly suppress the initial ROS production and delay the progression of cell death. These data indicate that the initial ROS production may be involved in the cell death induction process, and the later ROS production may play important roles in events leading to cellular disruption.     

A novel phenomenon predicting the entry into a state of hibernation in Syrian hamsters (Mesocricetus auratus)

When Syrian hamsters (Mesocricetus auratus) are bred in a cold and short-day environment, most animals go into hibernation after a certain period of time. However, to date it has not been possible to predict which hamster will enter hibernation. In this study, we subcutaneously implanted thermo-loggers in hamsters bred in the cold environment, and recorded the subcutaneous temperature at short intervals until they went into hibernation. A time series analysis of temperature disclosed that a fall of 0.4 to 0.8 degrees C in subcutaneous temperature was seen 5 to 16 days before entering hibernation, and this phenomenon continued for three days or more. No hamster went into the hibernation without displaying this signal. Although the mechanism by which this phenomenon takes place is not clear, it is a sign from the body, which is useful for indicating if a hamster will enter hibernation shortly.     

Survey of coccidial oocysts and parasite eggs in feces of free-ranging Grus japonensis

Fecal survey of Eimeria oocysts and parasite eggs was conducted for 219 fecal samples of free-ranging Grus japonensis in Kushiro district in Hokkaido in April 2003. Positive rate and mean oocysts (or eggs) per gram in positive samples were 26% (57/219) and 8.8 (0.2-136) in oocysts of Eimeria reichenowi, 18.3% (40/219) and 320 (100-1,000) in trematode eggs, 0.1% (2/219) and 0.2 (0.2-0.3) in eggs of Nematoda A, and 4.1% (9/219) and 0.8 (0.2-3.6) in eggs of Nematoda B, respectively.     

Autophagy defends cells against invading group A Streptococcus

We found that the autophagic machinery could effectively eliminate pathogenic group A Streptococcus (GAS) within nonphagocytic cells. After escaping from endosomes into the cytoplasm, GAS became enveloped by autophagosome-like compartments and were killed upon fusion of these compartments with lysosomes. In autophagy-deficient Atg5-/- cells, GAS survived, multiplied, and were released from the cells. Thus, the autophagic machinery can act as an innate defense system against invading pathogens.     

Rapid detection of chitosanase activity in chitosanase gene-transformed strains of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> by lytic infection of specific bacteriophages

 In combination with lytic infection by virulent phages, a simple method for monitoring transgenic strains of Enterobacter cloacae was developed in this study. First, 15 strains of E. cloacae were used as indicator bacteria to isolate virulent phages with different host ranges. Of the phages isolated, five isolates (EcP-22, -35, -45, -55, and -70) were used to construct a set of virulent phages corresponding to all strains of E. cloacae. Using this phage set, a rhizosphere strain (KRM-055E) of E. cloacae was effectively screened from field soil. KRM-055E was transformed with a prokaryotic chitosanase gene csnSM1 and infected with the phage EcP-03, which can lyse the strain most effectively. The lysis of KRM-055E/csn occurred 2 h after inoculation, and the chitosanase activity was simply detected by dropping the lysate onto an agar plate containing glycol chitosan. The positive signal for chitosanase activity was detected in the 2-h lysates, and the signal intensity reached a maximum in the 5-h lysate. The present assay was simple, rapid, inexpensive, easy to perform, and applicable to another strains. Received: August 2, 2002 / Accepted: October 31, 2002 Acknowledgments This work was supported in part by a grant (no. 99L01205) from the “Research for the Future” program of the Japan Society for the Promotion of Science. We are grateful to Dr. M. Sato, National Institute of Agrobiological Science, Dr. H. Okamoto, Fukui Agricultural Experiment Station, and Dr. K. Tsuda, Kyoto Prefectural Institute of Agricultural Biotechnology, for kindly providing E. cloacae strains. We thank Dr. P. Park, Kobe University, for technical support with the electron microscopic observations.     

Pathogenicity, Mating Ability and DNA Restriction Fragment Length Polymorphisms of Pyricularia Populations Isolated from Gramineae, Bambusideae and Zingiberaceae Plants

Eighty-five Pyricularia isolates were collected from 29 host species of Gramineae, Bambusideae and Zingiberaceae plants sampled in Brazil, Uganda, Ivory Coast, India, Nepal, China, Indonesia and Japan. These isolates were compared on the basis of pathogenicity, mating ability and restriction fragment length polymorphisms with single-copy DNA probes. Based on the pathogenicity to eight differential gramineous plants, these isolates were classified into seven pathotypes: finger millet type, foxtail millet type, common millet type, rice type, crabgrass type, Italian ryegrass/ weeping lovegrass type, and non-cereal/grass type. Genetic variation among these isolates was assessed by RFLP analysis with two restriction enzymes and nine single-copy DNA probes isolated from a finger millet strain. An UPGMA dendrogram based on the RFLPs revealed that the 85 isolates could be classified into seven major groups. Isolates from cereal crops (finger millet, foxtail millet, common millet, wheat and rice) and a grass, Brachiaria plantaginea, were clustered into a single group. They were further divided into six subgroups corresponding to the pathotypes. Among cereal crop isolates only an isolate from pearl millet was located into a different group. The remaining isolates were clustered into five groups designated as the crabgrass group, the buffelgrass and jungle rice group, the rice cutgrass, knotroot bristlegrass and Setaria tomentosa group, the bamboo and bamboo grass group and the Zingiber mioga group. The isolates from cereal crops were generally capable of mating with finger millet strains and constituted a closed mating compatibility group. These results suggested that the isolates from cereal crops form a single group with a common ancestor although they are pathogenic to taxonomically diverse plants. A combined analysis of the pathogenicity and genetic similarity suggested that the transmission of M. grisea isolates occurs in natural agroecosystems between finger millet and Eleusine africana, goosegrass or Bambusa arundinacea, between foxtail millet and green bristlegrass, and between rice and tall fescue, Italian ryegrass, sweet vernalgrass, reed canarygrass or Oryza longistaminata. Received 9 August 1999/ Accepted in revised form 12 November 1999     

New method for ethephon ((2-chloroethyl)phosphonic acid) residue analysis,and detection of residual levels in the fruit and vegetables of Western Japan

A new method for the detection and quantification of ethephon residues in fruit and vegetables was developed. The present study indicates that fruit and vegetables require a rapid and simple cleanup step before using gas chromatograph/mass spectrometry. The recovery and precision of the new method were evaluated by spiking the fruit and vegetable samples with 0.01-0.1 microg/g of ethephon. The amount of ethephon residue can be determined with good accuracy (recovery, 78.6-109%; coefficient variation, 2.65-6.41%), and the detection limit, defined as the amount of ethephon equivalent to three standard deviations (SD) of the noise level in observations at the baseline level of the selected ion (m/z 110), was 4 pg. The determination limit, defined as the equivalent to 8 SD of the noise level, was 11 pg. The working range was between 10 and 1000 ng/mL, and the correlation coefficient was 0.999 in the five experiments. Ethephon residues were determined between <2 and 97 ng/g in commercial pineapples from Western Japan.     

Acknowledgment to Reviewers

Acknowledgment to Reviewers

Acknowledgment to Reviewers