Yasuhiro Shinagawa

Phylogenetic Analysis of Spotted Fever Group Rickettsiae Based on gltA, 17‐kDa, and rOmpA Genes Amplified by Nested PCR from Ticks in Japan

In order to understand the natural situation of rickettsiae in the ticks in Japan, the rickettsial genes, gltA gene, rOmpA gene, and 17‐kDa gene, were amplified from the ticks by nested PCR. The prevalences of rickettsial gltA genes among Haemaphysalis formosensis, H. longicornis, H. megaspinosa, Ixodes ovatus, H. flava, H. kitaokai, and I. persulcatus were 62, 57, 24, 24, 19, 13, and 10%, respectively; 26% (186/722) being the average. The gltA genes amplified from the ticks were classified into 9 genotypes (I to IX) by the difference in nucleotide sequences. Genotype I was detected from 7 species of ticks. Genotype II mainly was detected from H. longicornis and H. formosensis. Genotypes III and VII mainly were detected from H. flava and I. ovatus. The polarization in the distribution of genotypes among regions where the ticks were collected was not clear. Based on the phylogenetic analysis of the three genes presented here, genotypes I, III, and IV (detected from H. formosensis, H. hystricia, and I. ovatus) are genetically close with each other, but rickettsiae of the same property still have not been isolated from ticks anywhere in the world. These genotypes should be considered as new species among SFG rickettsiae. Genotype II was identical with strain FUJ‐98, genetically close to R. japonica which has been isolated from ticks in China. Genotype V was identical with R. felis and strain California 2 isolated from the cat flea. This is the first report on the detection of R. felis from ticks. Genotype VI detected from Ixodes sp. did not seem to belong to genus Rickettsia. Based on the previous antigenic data and the phylogenetic analysis presented here, Genotype VII should be considered a variant of R. helvetica and genotype VIII detected from I. ovatus and I. persulcatus were identical with R. helvetica. Genotype IX detected from I. nipponensis was genetically close to the strains IRS3, IRS4, and IrR/Munich isolated from I. ricinus in Slovakia and German.

Identification of genes differentially expressed in a newly isolated human metastasizing esophageal cancer cell line, T.Tn-AT1, by cDNA microarray

We isolated a metastasizing human esophageal squamous cell carcinoma (SCC) cell line, T.Tn‐AT1, from a parental non‐metastasizing cell line, T.Tn, by in vitro selection and by use of a nude mouse orthotopic inoculation model. Then, we compared the expression profiles of 9206 genes in T.Tn‐AT1 and T.Tn by cDNA microarray analysis. The gene expression profiles of T.Tn and T.Tn‐AT1 were very similar, and only 34 genes showed more than 3‐fold differential expression. Among the 34 genes, 29 genes were down‐regulated and only 5 genes were up‐regulated in T.Tn‐AT1 cells. Subsequently, we confirmed the expression levels of 14 of the 34 genes in T.Tn and T.Tn‐AT1 cells by means of reverse transcription‐polymerase chain reaction. The expression of 8 genes (KAL1, HPGD, NDN, REG1A, CXCR4, SPOCK, DIAPH2 and AIF1) was down‐regulated and that of one gene (VNN2) was up‐regulated in T.Tn‐AT1 cells. These 9 genes encoded proteins associated with metastatic processes, such as adhesion, migration, inflammation, proliferation, and differentiation. Thus, these genes might regulate the metastasis of esophageal SCC, and could be predictive markers for lymph node metastasis of esophageal SCC.

Expression of metastasis suppressor gene (KAI1/CD82) in oral squamous cell carcinoma and its clinico-pathological significance

In this study, we investigated the expression of the KAI1/CD82 gene in oral squamous cell carcinoma (oral SCC). We studied 43 oral SCC patients. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to evaluate expression of this gene, and results were compared to the clinico-pathological findings. Twenty-five specimens (58.1%) were KAI1/CD82-positive, and 18 (41.9%) were negative. There were statistically significant relationships between gene expression and both histological malignancy (P=0.0205) and mode of invasion (P=0.0315). But there were no correlations of expression with tumor status, regional lymph node metastasis, pathological lymph node metastasis or histological differentiation. No significant relationship was observed between patient survival and expression of KAI1/CD82 by tumors. The results of this study suggest that the KAI1/CD82 gene may not be a useful predictor of prognosis, although decreased gene expression may be associated with increased invasive ability of oral SCC.