Hidetaka Yamamoto

Effects of ALDH2 gene polymorphisms and alcohol-drinking behavior on micronuclei frequency in non-smokers

Alcohol abuse is a serious health problem, leading to life-threatening damage to most of the important organ systems. Genotoxic damage is used as an early effect indicator in the surveillance of human exposure to genotoxic substances. Intra- and inter-individual variations of baseline frequencies of micronuclei (MN) in peripheral blood lymphocytes of human populations have been reported previously. Polymorphisms in a few metabolic enzyme genes seem to account for a proportion of this variability, but the impact of specific genetic variants on MN frequencies has not yet been clarified. In 42 healthy Japanese non-smoking men, we investigated the relationship between the MN frequency levels and genetic polymorphisms in three different genes: aldehyde dehydrogenase 2 (ALDH2), X-ray repair cross-complementing group 1 (XRCC1) and excision repair cross-complementing group 2 (ERCC2). Genotyping was performed by PCR-RFLP analysis. The ALDH2 variant (deficient-type) was significantly associated with increased MN frequency levels in subjects with drinking more than three times per week, whereas the XRCC1 and ERCC2 variants seemed to be unrelated to the MN frequency. The ALDH2-deficient habitual drinkers had an average MN frequency of 5.88+/-0.58 (+/- S.E.) compared with 3.20 +/- 0.80 in the ALDH2-proficient habitual drinkers (P<0.05). The ALDH2-proficient non-habitual drinkers had the lowest MN frequency (1.56 +/- 0.41). Furthermore, subjects with highest levels of mean MN frequency, who consumed more than 100g of alcohol per week and more than three times per week, had A2 genotype of ALDH2. A significant odds ratio (12.25, P<0.05) for the MN frequency levels above the 50th percentile value was observed for the ALDH2-deficient individuals versus the ALDH2-proficient individuals after adjustment for several confounders. These results strongly suggest that human early genotoxic effect studies based on the cytogenetic markers of MN should take into account both the individual ALDH2 polymorphism and the potential confounding effect of the drinking behavior.

Estimation of stress in child neglect from thymic involution

It is difficult to evaluate the extent of stress in cases of suspected child abuse/neglect in a medico-legal autopsy. We have previously reported that stress due to abuse/neglect was found to have led to thymic involution. To elucidate the influence upon thymocytes differentiation, we compared the proportion of the thymocyte subpopulation in the thymus of a neglected child with one in an age-matched control obtained from cardiac surgery. We found that the relative number of CD4+ CD8+ double positive (DP) thymocytes decreased in the neglected child. It was presumed that the selective decrease in the number of the immature DP thymocytes with CD3- to low bcl-2low caused the thymic involution in the neglected child. It was suggested that an alteration in the proportion of thymocytes subpopulation might be used as an index of stress in cases of child abuse/neglect.

Effects of Prednisolone and Complex of Vitamin B1, B2, B6 and B12 on Organophosphorus Compound-Induced Delayed Neurotoxicity

Effects of Complex of Prednisolone and Vitamin B1, B2, B6 and B12 on Organophosphorus Compound‐Induced Delayed Neurotoxicity: Fengyuan Piao, et al. Department of Public Health, School of Medicine, Mie University—Protective effects of prednisolone as a synthetic adrenal cortical hormone and complex of vitamin B1, B2, B6 and B12 on organophosphorus compound‐induced delayed neurotoxicity (OPIDN) caused by leptophos and tri‐ocresyl phosphate (TOCP) as organophosphates (OPs) were examined. Nine groups of hens (six for each) were used. Eight groups received intravenous injection of 30 mg/kg of leptophos or 40 mg/kg of TOCP (four groups in each). Among them, three groups which received leptophos were given (p.o.) predonisolone (2 mg/body), vitamin B complex (25 mg/body) or both 3 h after OPs injection and then every day for 15 d (one group for each); the same treatment was performed on three groups which received TOCP. The remaining one group served as controls. It was observed that delayed neuropathy induced by OPs could not be resisted completely by the treatment with prednisolone or vitamin B complex, but clinical signs of OPIDN and pathological changes in hens that received these two protective agents after OPs were less severe than those in hens that received only OPs. Of these groups, the improvement in clinical signs was best shown in hens that received the both two protective agents. In addition, improvement in clinical signs among the hens that did not deteriorate to paralysis was observed. In particular, those which developed mild ataxia recovered well. It is indicated that combining administration of prednisolone and vitamin B complex early before clinical signs are manifest is effective in alleviating neuropathy. It is also suggested that recovery or good prognosis will be expected, as long as progression of the clinical signs is prevented before paralysis develops in delayed neuropathy.

Legume Lectins Inhibit Human Parainfluenza Virus Type 2 Infection by Interfering with the Entry

Three lectins with different sugar binding specificities were investigated for anti-viral activity against human parainfluenza virus type 2 (hPIV-2). The lectins, concanavalin A (Con A), lens culinaris agglutinin (LCA) and peanut agglutinin (PNA), inhibited cell fusion and hemadsorption induced by hPIV-2. Virus nucleoprotein (NP) gene synthesis was largely inhibited, but fusion (F) and hemagglutinin-neuraminidase (HN) gene syntheses were not. An indirect immunofluorescence study showed that Con A inhibited virus NP, F and HN protein syntheses, but LCA did not completely inhibit them, and that PNA inhibited only NP protein synthesis. Using a recombinant green fluorescence protein-expressing hPIV-2, without matrix protein (rghPIV-2ΔM), it was found that virus entry into the cells was not completely prevented. The lectins considerably reduced the number of viruses released compared with that of virus infected cells. The lectins bound to cell surface within 10 min, and many aggregates were observed at 30 min. Con A and LCA slightly disrupted actin microfilaments and microtubules, but PNA had almost no effect on them. These results indicated that the inhibitory effects of the lectins were caused mainly by the considerable prevention of virus adsorption to the cells by the lectin binding to their receptors.

Completion of the full-length genome sequence of human parainfluenza virus types 4A and 4B: sequence analysis of the large protein genes and gene start, intergenic and end sequences

We have already reported the nucleotide sequences of the NP, P/V, M, F and HN genes of human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B). Here, we have determined the sequences of the L protein genes as well as the gene start, intergenic and end sequences, thereby completing the full-length genome sequence of hPIV-4A and 4B. hPIV-4A and 4B have 17,052 and 17,304 nucleotides, respectively. The end sequence of hPIV-4, especially 4B, was extraordinarily long. In a comparison with members of the genus Rubulavirus, the hPIV-4 L proteins were closely related to those of mumps virus (MUV) and hPIV-2, less closely related to those of Menangle virus and Tioman virus, and more distantly related to those of Mapuera virus and porcine rubulavirus.

Fatal acute alcohol intoxication in an ALDH2 heterozygote: a case report

On an evening in November, a 25-year-old man was found dead in his bedroom. There were many empty snap-out sheets for flunitrazepam tablets in the trash at his bedside. He had been beaten by a gang of young people earlier in the morning of the same day. At the medico-legal autopsy, although there were many bruises and/or abrasions on the whole body, only slight subdural hemorrhage was observed, and none of them was thought to be the cause of death. Flunitrazepam and its metabolites were not detected in his body fluid by gas chromatography-mass spectrometry (GC-MS). Marked lung edema and a severe congestion of organs were observed. His blood alcohol concentration from the femoral vein was 2.00 mg/ml. Fatal cases of acute alcohol intoxication usually have shown higher alcohol concentration (2.25-6.23 mg/ml). Although the genotype of aldehyde dehydrogenase 2 (ALDH2) has not previously been mentioned as a contributing factor in determining the cause of death, in this case the genotype of ALDH2 was ALDH2*1/2 and thus is important. Those who possess the ALDH2*2 gene show high concentrations of acetaldehyde (AcH) at even comparatively lower alcohol levels. Consequently, the cause of death was considered to be acute alcohol intoxication including AcH poisoning.

Micronucleus Assay of Human Lymphocytes: A Comparison of Cytokinesis-block and Human Capillary Blood Lymphocytes Methods

Micronucleus (MN) expression in Peripheral blood lymphocytes is well established as a method to monitor chromosome damage in human populations. The use of MN as a measure of chromosome damage in peripheral blood lymphocytes was first proposed by Countryman and Heddle and subsequently improved with the development of the cytokinesis-block micronucleus (CBMN) method, which has been extensively used to evaluate chromosome damage in human populations. Nevertheless, the CBMN method requires more blood collected by venous puncture, and the procedure detecting MN in cultured lymphocytes is time-consuming. In 1992, a method for MN testing of lymphocytes with human capillary blood collected by skin puncture was developed. The procedure was simple and fast because MNs were detected in non-cultured lymphocytes, and only 1 or 2 drops of capillary blood were needed, so it was called the human capillary blood lymphocytes MN (HCBL-MN) method. The present study gives the comparison of the results of MN frequency in human lymphocytes using the two methods in the same subjects in order to find a simple and sensitive method, to clarify the correlation between the two methods and to provide information on baseline MN frequency in human lymphocytes detected by the two methods.

Immunodeficiency induced by child abuse

We report the case of a 9-year-old girl who died from sepsis from cellulitis of the neck caused by a right ear injury. The autopsy findings showed severe involution of the thymus and atrophy of lymphoid tissues. The impairment of T- and B-cell functions was demonstrated both histologically and immunohistologically. Thymic involution caused by child abuse might lead to secondary immunodeficiency.

Tissue-specific expression of Le(Y) antigen in high endothelial venules of human lymphoid tissues.

In this study, we demonstrated that the anti-LeYantibody (BM-1) especially reacted with high endothelial venules (HEVs) in peripheral lymph nodes of blood group O individuals. The LeYexpression on HEVs showed a unique tissue-specific pattern, i.e., a large amount of the LeYexpression in peripheral lymph nodes and no or small amounts in mesenteric lymph node. Statistical analysis showed that there was the significant difference between the percentage of LeY-positive HEVs in peripheral lymph nodes and mesenteric lymph nodes. No expression of LeYwas observed in vessels of Payers patch, thymus, spleen and other non-lymphoid organs. In blood group A or B individuals, the reactivity between HEVs and anti-LeYantibody increased after enzyme digestion with α-N-acetylgalactosaminidase or α-galactosidase. These findings show that the expression of difucosylated blood group ABH antigens are especially expressed on HEVs in peripheral lymph nodes. Furthermore, the tissue-specific pattern suggests that these antigens may be related to intercellular adhesion between lymphocytes and HEVs.

Ribavirin inhibits human parainfluenza virus type 2 replication in vitro.

The antiviral activities of eight nucleoside analog antiviral drugs (ribavirin, acyclovir, lamivudine, 3′‐azido‐3′‐deoxythymidine, emtricitabine, tenofovir, penciclovir and ganciclovir) against human parainfluenza virus type 2 (hPIV‐2) were investigated. Only ribavirin (RBV) inhibited both cell fusion and hemadsorption induced by hPIV‐2. RBV considerably reduced the number of viruses released from the cells. Virus genome synthesis was inhibited by RBV, as determined by real time PCR. An indirect immunofluorescence study showed that RBV largely inhibited viral protein synthesis. mRNAs of the proteins were not detected, indicating that inhibition of protein synthesis was caused by transcription inhibition by RBV. Using a recombinant green fluorescence protein‐expressing hPIV‐2 without matrix protein, it was found that RBV did not completely inhibit virus entry into the cells; however, it almost completely blocked multinucleated giant cell formation. RBV did not disrupt actin microfilaments and microtubules. These results indicate that the inhibitory effect of RBV is caused by inhibition of both virus genome and mRNA synthesis, resulting in inhibition of virus protein synthesis, viral replication and multinucleated giant cell formation (extensive cell‐to‐cell spreading of the virus).