Biomedical subjects
Publications and source records attributed to Mikako Ito.
Dengue virus type 1 (DENV-1), which was responsible for the dengue fever outbreak in Yap State, Micronesia, in 2004, was isolated from serum samples of 4 dengue patients in Japan. Genome sequencing demonstrated that this virus belonged to genotype IV and had a 29-nucleotide deletion in the 3 noncoding region.
In many flaviruses, first 50-400 nucleotides of 3' non-translated region (3' NTR) exhibit lower conservation level than other regions and are called "variable region". Two dengue type 1 virus (DENV-1) strains, which have 17- and 29-nt deletion in the variable region, were recently isolated from Japanese dengue fever patients. The effect of a small deletion in the 3' NTR was analyzed using two DENV-1 viruses which were prepared from a newly developed infectious cDNA clone. These included a recombinant virus rDENV-1(02-20), without any deletion in 3' NTR, and rDENV-1m10, with 19-nt deletion in the variable region of rDENV-1(02-20). These two viruses were compared for growth kinetics and plaque morphology in Vero, Huh-7 and C6/36 cells. No apparent difference was detected between rDENV-1(02-20) and rDENV-1m10 in replication efficiency and plaque size in these cell lines. The results suggest that the complete variable region of DENV-1 is dispensable for virus replication and propagation in vitro.
The variations in nuclear DNA content from 2 x 10(2) to 2.5 x 10(5) Mbp are reported in higher plants. The major finding so far is that the genome size of plant species differs by three orders of magnitude, which are more variable than the other organisms. Investigations pertaining to the manner in which DNA is packaged in the nucleus provide us with basic information on the made of DNA existence in the plant nucleus. However, the fundamentals on nuclear DNA content and nuclear size, which underlie and enable the flexible containment of such large differences in nuclear DNA content, remain unknown. We analyzed the nuclear volumes of plants with 2C value DNA contents ranging from 3.2 x 10(2) to 1.0 x 10(5) Mbp. As a result, we obtained a constant ratio between the DNA volume and nuclear volume, which does not exceed 3%. Furthermore, we also demonstrate that the nuclear Rabl model of chromatin organisation is not a common 3-D structure, even in plants with large nuclear DNA contents. The existence of an upper limit of DNA volume ratio would present a basal parameter for the future insight into the nuclear organisation in higher plants.
Cerebrospinal fluid specimens from 57 patients diagnosed with meningitis were tested for Japanese encephalitis virus. Total RNA was extracted from the specimens and amplified. Two products had highest homology with Nakayama strain and 2 with Ishikawa strain. Results suggest that Japanese encephalitis virus causes some aseptic meningitis in Japan.
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The fluorogenic TaqMan reverse transcriptase PCR (RT-PCR) assay was developed for detecting each of the dengue virus (DV) types 1 to 4. DV genome was detected in all the 35 serum samples from confirmed dengue cases by the TaqMan RT-PCR, although it was not detected in 13 and 21% by conventional type-specific and cross-reactive RT-PCR, respectively.
Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis.
The effect of mouse brain-derived, inactivated Japanese encephalitis (JE) vaccine on West Nile virus (WNV) infection was examined using a murine model. Mice were immunized with JE vaccine twice and challenged with lethal doses of WNV. When mice were intracranially challenged with WNV, none of the immunized mice were protected. When mice were intraperitoneally challenged, the immunized mice were protected at higher immunization levels. Cross-reactive neutralizing antibodies to WNV were induced by immunization with JE vaccine; however, the levels were much lower than those to JEV. These results indicate that the currently available mouse brain-derived inactivated JE vaccine can induce partial protective immunity to WNV in mice.
The 5-methylcytosines (m5C) play a critical role in epigenetic control, often being recognized by proteins containing a methyl-CpG-binding domain (MBD). Database screening has identified at least 12 putative methyl-CpG-binding proteins from Arabidopsis; we have isolated corresponding cDNAs for seven of them. Despite variation in size and amino acid sequence, all seven proteins exclusively migrate into the nucleus as revealed by green fluorescent protein fusion protein assay, suggesting a relationship with chromatin structure. However, DNA-binding assays using bacterially expressed proteins and synthetic oligonucleotides containing m5C in CpGs showed only one to specifically bind, designated AtMBD5. Further analysis showed that AtMBD5 efficiently binds to m5C in CpNpN (N is A, T, or C) but not in CpNpG sequences, both frequently found in plant DNA. The other six proteins showed either nonspecific DNA binding or no ability to recognize m5C. RNA-blot hybridization and immunoblot analysis indicated AtMBD5 to be present essentially in all tissues. Using green fluorescent protein driven by the authentic promoter, AtMBD5 was found to be actively expressed in pistils and root tips. Because m5Cs in CpG and CpNpN are considered to function in gene expression and gene silencing, respectively, the present results suggest that AtMBD5 may have distinct functions in regulation and/or self defense of genes in actively proliferating cells.
BACKGROUND: There is a geographical overlap between the two main rabies epidemiological cycles maintained by dogs and vampire bats in Latin America. The geographical and temporal coincidence of rabies outbreaks of respective origins is not unusual in rural areas of Latin America. These circumstances make it difficult to discriminate the intraspecies and interspecies transmission pathways of rabies. OBJECTIVE: This study was conducted to develop techniques to discriminate dog-related and vampire bat-related rabies virus isolates (DRRV and VRRV, respectively) in Brazil. STUDY DESIGN: The 1396 nucleotides of the nucleoprotein gene of a total of 27 DRRV and VRRV were sequenced. Strain-specific (SS) primers were developed based on these sequences. Forty-nine rabies virus strains isolated from animals and humans in several parts of Brazil were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) with SS primers. These rabies viruses were also amplified by RT-PCR with general rabies primers and the PCR products were cut by three restriction enzymes, Blp I, Bsu36 I and BspE I. RESULTS: All the DRRV and VRRV were distinguished by RT-PCR with SS primers. The PCR products obtained from DRRV were cut at one site by Blp I, but not by Bsu36 I. The PCR products obtained from VRRV were cut at one or two sites by Bsu36 I, but not by Blp I. Blp I and Bsu36 I clearly discriminated DRRV and VRRV in restriction fragment length polymorphysim (RFLP) assays. The results of SS RT-PCR and RFLP were consistent. CONCLUSION: SS RT-PCR and RFLP assays have been developed for determining the origins of rabies virus isolates in Brazil. These assays are simple and rapid, and will be useful for identifying the rabies virus reservoirs of field isolates in Brazil, especially when used together.
When maize seedlings were exposed to cold stress, a genome-wide demethylation occurred in root tissues. Screening of genomic DNA identified one particular fragment that was demethylated during chilling. This 1.8-kb fragment, designated ZmMI1, contained part of the coding region of a putative protein and part of a retrotransposon-like sequence. ZmMI1 was transcribed only under cold stress. Direct methylation mapping revealed that hypomethylated regions spanning 150 bases alternated with hypermethylated regions spanning 50 bases. Analysis of nuclear DNA digested with micrococcal nuclease indicated that these regions corresponded to nucleosome cores and linkers, respectively. Cold stress induced severe demethylation in core regions but left linker regions relatively intact. Thus, methylation and demethylation were periodic in nucleosomes. The following biological significance is conceivable. First, because DNA methylation in nucleosomes induces alteration of gene expression by changing chromatin structures, vast demethylation may serve as a common switch for many genes that are simultaneously controlled upon environmental cues. Second, because artificial demethylation induces heritable changes in plant phenotype (Sano, H., Kamada, I., Youssefian, S., Katsumi, M., and Wabilko, H. (1990) Mol. Gen. Genet. 220, 441-447), altered DNA methylation may result in epigenetic inheritance, in which gene expression is modified without changing the nucleotide sequence.
Chloroplast DNA of the green alga Chlamydomonas reinhardtii is maternally inherited. Methylation mapping directly revealed that, before mating, chloroplast DNA of maternal (mating type plus; mt(+)) gametes is heavily methylated whereas that of paternal (mating type minus; mt(-)) gametes is not. Indirect immunofluorescence analyses with anti-5-methylcytosine mAbs visually showed methylation to occur exclusively in chloroplast DNA of mt(+) gametes, and not in mt(-) gametes or nuclear DNA of either mt. To clarify the relationship between methylation and maternal inheritance of chloroplast DNA, we have isolated and characterized a cDNA encoding a DNA methyltransferase. The deduced protein, CrMET1, consists of 1,344 aa and contains a conserved catalytic domain at the C terminal and a nonconserved N-terminal region. The predicted N-terminal region has an arginine-rich domain, suggesting CrMET1 is transferred to chloroplasts. This finding could be directly shown by green fluorescent protein epifluorescence microscopy analyses. CrMET1 transcripts were found to be absent in both mt(+) and mt(-) vegetative cells. Upon gametogenesis, however, transcript levels clearly increased in mt(+) but not mt(-) cells. These experiments suggest that the CrMET1 protein is located in chloroplasts and that it specifically methylates cytosine residues of chloroplast DNA in mt(+) gametes. This conclusion was further strengthened by the observation that, during gametogenesis, CrMET1 is expressed in a mt(-) mutant, mat-1, whose chloroplast DNA is heavily methylated in gametes and paternally inherited. The results provide evidence that cytosine methylation plays a critical role in maternal inheritance of chloroplast genes in C. reinhardtii.
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