Genotyping and quantitation of noroviruses in oysters from two distinct sea areas in Japan

Norovirus (NV) is a causative agent of acute gastroenteritis in humans, and shellfishes including oysters act as major vehicles of the virus. To investigate the genetic characteristics of NVs, we collected 1,512 oysters for raw consumption between October 2002 and March 2005 from two distinct areas (area A: the Sanriku Sea area; area B: the Setouchi Sea area). We detected the capsid gene and subjected it to phylogenetic analysis. By further quantification of the copy number of the genome by using real-time PCR, the NV capcid gene was detected in approximately 5% of the oysters, and they showed wide diversity. Two percent of the oysters from area B showed relatively large number of NVs, i.e., over 100 copies of capsid gene/oyster, whereas this was not observed in area A. Most of the detected NVs from oysters and humans were genetically related when the capsid region was compared. These results suggested that NVs obtained from humans and those obtained from oysters showed a potential relationship to each other and that some populations of Japanese oysters accumulated a relatively large number of NVs.     

Characterization of enzymes involved in the ethanol production of <Emphasis Type="Italic">Moorella</Emphasis> sp. HUC22-1

Since the thermophilic bacterium Moorella sp. HUC22-1 produces 120 mM acetate and 5.2 mM ethanol from H₂–CO₂, several candidate genes, which were predicted to code for three alcohol dehydrogenases (AdhA, B, C) and one acetaldehyde dehydrogenase (Aldh), were cloned from HUC22-1. The cloned genes were subcloned into a His-tagged expression vector and expressed in Escherichia coli. Recombinant AdhA and B were both dependent on NADP(H) but independent of NAD(H), and their reduction activities from aldehyde to alcohol were higher than their oxidation activities. In contrast with AdhA and B, no activity of AdhC was observed in either reaction. On the other hand, Aldh was active toward both NADP(H) and NAD(H). The enzyme activity of Aldh was directed toward the thioester cleavage and the thioester condensation. When 50 μg of AdhA and 50 μg Aldh were added to the buffer solution (pH 8.0) containing NADPH, NADH and acetyl-CoA at 60°C, 1.6 mM ethanol was produced from 3 mM acetyl-CoA after 90 min. Expression analysis of the mRNAs revealed that the expression level of aldh was threefold higher in the H₂–CO₂ culture than that in the fructose culture, but levels of adhA, B and C were decreased.     

A segmental duplication encompassing S-haplotype triggers pollen-part self-compatibility in Japanese pear (Pyrus pyrifolia)

Self-compatible mutants of self-incompatible crops have been extensively studied for research and agricultural purposes. Until now, the only known pollen-part self-compatible mutants in Rosaceae subtribe Pyrinae, which contains many important fruit trees, were polyploid. This study revealed that the pollen-part self-compatibility of breeding selection 415-1, a recently discovered mutant of Japanese pear (Pyrus pyrifolia) derived from γ-irradiated pollen, is caused by a duplication of an S-haplotype. In the progeny of 415-1, some plants had three S-haplotypes, two of which were from the pollen parent. Thus, 415-1 was able to produce pollen with two S-haplotypes, even though it was found to be diploid: the relative nuclear DNA content measured by flow cytometry showed no significant difference from that of a diploid cultivar. Inheritance patterns of simple sequence repeat (SSR) alleles in the same linkage group as the S-locus (LG 17) showed that some SSRs closely linked to S-haplotypes were duplicated in progeny containing the duplicated S-haplotype. These results indicate that the pollen-part self-compatibility of 415-1 is not caused by a mutation of pollen S factors in either one of the S-haplotypes, but by a segmental duplication encompassing the S-haplotype. Consequently, 415-1 can produce S-heteroallelic pollen grains that are capable of breaking down self-incompatibility (SI) by competitive interaction between the two different S factors in the pollen grain. 415-1 is the first diploid pollen-part self-compatible mutant with a duplicated S-haplotype to be discovered in the Pyrinae. The fact that 415-1 is not polyploid makes it particularly valuable for further studies of SI mechanisms.     

The use of species-specific DNA markers for assessing alien chromosome transfer in Brassica rapa and Brassica oleracea-monosomic additions of Raphanus sativus

Monosomic addition lines (MALs) are useful materials not only for cytogenetic and molecular genetic studies but also for plant breeding as gene sources. In our previous study, two MALs in the tribe Brassiceae were developed, one being Raphanus sativus lines with alien chromosomes of Brassica rapa (B. rapa-monosomic addition lines; BrMALs) and the second being those with alien chromosomes of Brassica oleracea (B. oleracea-monosomic addition lines; BoMALs). We developed species-specific DNA markers from the genomic sequences of B. rapa and B. oleracea comparing them with those of R. sativus, and identified chromosomes added in BrMALs and BoMALs using these markers. It was revealed that eight types of BrMALs have seven chromosomes of B. rapa and seven types of BoMALs have six chromosomes of B. oleracea. Furthermore, chromosome breakage and homoeologous recombination were suggested to have occurred in some MALs. The developed species-specific DNA markers are considered to be useful for producing MALs and also for assessing chromosome abnormality in MALs.     

Development of On-Chip Multi-Imaging Flow Cytometry for Identification of Imaging Biomarkers of Clustered Circulating Tumor Cells

An on-chip multi-imaging flow cytometry system has been developed to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross-sectional area, number of nuclei and size of clusters as “imaging biomarkers”, with simultaneous acquisition and analysis of both bright-field (BF) and fluorescent (FL) images at 200 frames per second (fps); by using this system, we examined the effectiveness of using imaging biomarkers for the identification of clustered circulating tumor cells (CTCs). Sample blood of rats in which a prostate cancer cell line (MAT-LyLu) had been pre-implanted was applied to a microchannel on a disposable microchip after staining the nuclei using fluorescent dye for their visualization, and the acquired images were measured and compared with those of healthy rats. In terms of the results, clustered cells having (1) cell area larger than 200 µm² and (2) nucleus area larger than 90 µm² were specifically observed in cancer cell-implanted blood, but were not observed in healthy rats. In addition, (3) clusters having more than 3 nuclei were specific for cancer-implanted blood and (4) a ratio between the actual perimeter and the perimeter calculated from the obtained area, which reflects a shape distorted from ideal roundness, of less than 0.90 was specific for all clusters having more than 3 nuclei and was also specific for cancer-implanted blood. The collected clusters larger than 300 µm² were examined by quantitative gene copy number assay, and were identified as being CTCs. These results indicate the usefulness of the imaging biomarkers for characterizing clusters, and all of the four examined imaging biomarkers—cluster area, nuclei area, nuclei number, and ratio of perimeter—can identify clustered CTCs in blood with the same level of preciseness using multi-imaging cytometry.     

Discovery of new chemotype dipeptidyl peptidase IV inhibitors having (R)-3-amino-3-methyl piperidine as a pharmacophore

Structures containing the (R)-3-amino-3-methyl piperidine unit as a new pharmacophore moiety have been shown to possess moderate inhibitory activity for DPP-4 with good pharmacokinetics profile. One of these compounds was found to have good oral bioavailability and PK/PD profile in ZF-rat.     

Gene conversion from SLG to SRK resulting in self-compatibility in Brassica rapa

Self-compatible S-54 homozygotic plants were found in progenies of an F(1) hybrid cultivar in Chinese cabbage. Pollination tests revealed that this self-compatibility is controlled by the S locus and caused by the loss of the recognition function of the stigma. SRK, the gene for the recognition molecule in the stigma, was normally transcribed and translated in the self-compatible plants. The 1034-bp region in the receptor domain of SRK in the self-compatible plants was 100% identical to SLG in S-54, while that in self-incompatible S-54 homozygotic plants was 95.1% identical. These results suggest that the self-compatibility of the S-54 homozygotes is due to amino-acid changes caused by gene conversion from SLG to SRK.     

A childhood-onset intestinal toxemia botulism during chemotherapy for relapsed acute leukemia

Background

Botulism is a potentially fatal infection characterized by progressive muscle weakness, bulbar paralysis, constipation and other autonomic dysfunctions. A recent report suggested that cancer chemotherapy might increase the risk for the intestinal toxemia botulism in both adults and children.

Case presentation

We report a 5-year-old boy, who developed general muscle weakness, constipation, ptosis and mydriasis during the third induction therapy for relapsed acute myeloid leukemia. He had recent histories of multiple antibiotic therapy for bacteremia and intake of well water at home. Repeated bacterial cultures identified Clostridium botulinum producing botulinum neurotoxin A. Botulinum toxin A was isolated from his stools at 17, 21, and 23 days after the onset. Symptoms were self-limiting, and were fully recovered without anti-botulinum toxin globulin therapy.

Conclusion

This is the second report of a pediatric case with cancer chemotherapy-associated intestinal toxemia botulism. Our case provides further evidence that the immunocompromised status due to anti-cancer treatments increases the risk for the development of botulism at all ages in childhood.