Application of cDNA Macroarray for Simultaneous Detection of 12 Potato Viruses.

A complementary DNA (cDNA) macroarray was developed for simultaneous detection of 12 different potato viruses. A suitable region in the viral genome for each was selected for Alfalfa mosaic virus, Cucumber mosaic virus, Potato aucuba mosaic virus, Potato leafroll virus, Potato mop-top virus, Potato virus A, Potato virus M, Potato virus S, Potato virus X, Potato virus Y, Tomato ringspot virus, and Tomato spotted wilt virus, and their respective cDNAs were cloned into plasmid vectors. Capture probes for each virus ranging from 290 to 577 bp were generated by polymerase chain reaction (PCR) and immobilized on a nylon membrane. Total RNAs were extracted from each of these virus infected-plants, and cDNAs were synthesized from the RNA extracts using a random 9-mer primer. Subsequently, PCR reactions were performed using one primer pair for each of the 12 viruses. During PCR, amplified cDNAs were labeled with biotin and used as a target for hybridization analyses on a macroarray membrane. Hybridization signals between capture probes for the 12 viruses and their respective target cDNAs were observed using chemiluminescent or colorimetric detection. In all viruses, hybridization signals with capture probes were detected only when homologous virus targets were examined, and no hybridization to healthy plant extract was observed, facilitating identification of each virus. The results by colorimetric detection agreed with those obtained using chemiluminescence. The macroarray method developed was 5 × 102 to 4 × 106 times more sensitive than enzyme-linked immunosorbent assay and 5 to 5 × 104 times more sensitive than reverse-transcription PCR, except for Alfalfa mosaic virus. Colorimetric detection and substantial reduction in cross-hybridization signals much improved the method compared with other array-based detection methods for practical use.

Evaluation of functional relationships for predicting mainstream smoke constituent machine yields for conventional cigarettes from the Japanese market.

The objective of this study was to predict mainstream smoke constituent yields for conventional filter cigarette brands on sale in Japan between 2004 and 2005. Mainstream smoke was generated under ISO machine smoking conditions. Developed functional relationships indicate the validity of benchmarking even for a market which is characterized by a diversity of tobacco blend types. Smoke yields were in general well predicted by linear regression with "tar" (R2>or=0.7). Blend-type-sensitive analytes like tobacco-specific nitrosamines showed improved prediction relationships after blend stratification or regressing against nitric oxide (NO, R(2)>0.7). Relationships calculated from 83 exploratory brands were validated with a subset of 23 validation brands. Seventy-five to one-hundred percent of the validation brand yields were inside the 95% prediction intervals. The mean-relative prediction error over all analytes was 24% after stratification. Smoke constituents yields analyzed in 2002 from 96 Japanese market products were well predicted and indicate the model validity over time. Similar relationships were observed when comparing American blended filter cigarette yields from Japan and worldwide markets. Consistent with reported results from previous benchmark studies and market surveys mainstream smoke constituent yields are well predictable when "tar" (and NO) yields are known.

High-throughput array production using precision glass syringes.

The advantages of using 1, 96, or 384 precision glass syringes in automated high-throughput microdispensers in creating highly uniform and reproducible DNA, protein, and organic compound array filters and slides are described. Using the Hydra Microdispenser and Tango Liquid Handling system, 0.1-5 ng (in 50-300 nL) PCR-amplified, human cancer-related genes and housekeeping genes were spotted onto nylon membranes and coated slides. Protein solutions of 50 microg/mL to 1 mg/mL were spotted onto coated slides or onto MaxiSorp 96-well plates. Up to 6144 spots/membrane and up to 1000 spots/slide were printed. The size of the spots created by glass syringes was uniform and reproducible (precision variation of less than 5%) from spot to spot and membrane to membrane. Using a Tango 384 system, a total of ten 6144-spot filters can be produced in approximately 25 min, translating into a spotting speed of 2.5 min/membrane.

Comparison of pollutant runoff in Lake Biwa tributaries, Japan and the Brunette River watershed, Canada.

This study is aimed at verifying runoff pollutant loadings from urban areas. Urban runoff has been considered an important source of diffuse pollution especially during storm events. This paper describes the pollutant runoff during storm events, mainly in terms of effects of watershed characteristics. Data collected from Lake Biwa tributaries, Japan, have shown fundamental information to control pollutant runoff into receiving water. Also, data from the Brunette River watershed, Canada, which is a highly urbanized watershed in the Vancouver region, have been used for a comparative analysis. In the results, available information for the environmental management of urban storm water runoff was obtained by comparing the data on pollutant runoff in both watersheds.

Phosphorylation of histones triggers DNA fragmentation in thymocyte undergoing apoptosis induced by protein phosphatase inhibitors.

The treatment of thymocytes with protein phosphatase inhibitors such as calyculin A and okadaic acid resulted in apoptosis with a concomitant increase in phosphorylation of nuclear proteins. The phosphorylated protein in the thymocyte nuclei induced by protein phosphatase inhibitors was identified as histones by the use of two-dimensional polyacrylamide gel electrophoresis. These compounds accelerated the phosphorylation of histone H2A, H3, and H1. On the other hand, little phosphorylation of H2B and H4 by these compounds was observed. The effect of these compounds on the level of nuclear histones was also examined using high-performance capillary electrophoresis. No significant changes in the level of histones were seen in the nuclei of thymocytes treated with calyculin A and okadaic acid. Thus, the induction of thymocyte apoptosis is involved in the chemical modification of histones but not the change in their quantity. Moreover, the treatment of thymocytes with calyculin A increased the sensitivity toward endogenous DNase in the nuclei. These results suggest that phosphorylation of histones, especially H2A, H3, and H1, is an early step of triggering DNA fragmentation in thymocyte apoptosis.

Suppressive effects of novel ferulic acid derivatives on cellular responses induced by phorbol ester, and by combined lipopolysaccharide and interferon-gamma.

A total of 23 ferulic acid (FA) derivatives were synthesized, and investigated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus (EBV) activation and superoxide (O(2)(-)) generation. Most of the derivatives showed significant EBV activation suppression or cytotoxicity at a concentration of 100 microM, with FA15 as the most potent suppressor. In both assays, FA6-FA17, bearing straight- or branched-alkyl side chains, exhibited marked suppression of O(2)(-) generation, with both FA16 and FA17 being highly active, while FA itself was virtually inactive. The activity differences seen between FA16/FA17 and FA are attributable, at least in part, to their cellular incorporating efficiencies. Further, both FA15 and FA21 attenuated the expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins, while FA did not. Our results suggest that these novel FA derivatives are effective chemopreventive agents.

Preparation of ferulic acid and its application for the synthesis of cancer chemopreventive agents.

We suggest that chemical raw materials can best be obtained from natural resources. Ferulic acid is easily prepared in large quantities from rice bran pitch, a blackish brown waste oil with high viscosity, discharged in the process of the rice bran oil production. As an application of ferulic acid, potential cancer chemopreventive agents could be synthesized using organic synthetic methods.

PTCH gene mutations in invasive transitional cell carcinoma of the bladder.

LOH analysis suggests that multiple tumor suppressor genes play a role in the development of human TCC. The human homolog of the Drosophila PTCH was recently cloned and mapped to the BCNS locus on 9q22.3, a chromosomal region commonly deleted in TCCs. We first examined the steady state mRNA transcription of the PTCH, SMOH and GLI3 genes of the HH signal transduction pathway in TCC cell lines and normal urothelium. Normal urothelium and TCC cell lines express these three genes within the PTCH signal transduction pathway. We then screened for PTCH mutations in 'hot spot' exons 6, 8, 13 and 16 by PCR/SSCP analysis of genomic DNAs from 54 TCC tumor samples and control autologous peripheral blood lymphocytes. DNA sequence analysis confirmed TCC-specific mutations in two of 54 patients (3.7%). These mutations resulted a single amino acid substitution and two frame shifts. One tumor had PTCH mutations in exon 16 as well as exon 13 and one tumor had a mutation in exon 13 alone. Both TCC tumors that contained PTCH mutations had a loss of heterozygosity at 9q. Although the PTCH protein has an unknown function in urothelial cells, the detection of the PTCH, SMOH and GLI3 transcripts in normal urothelium and TCC cell lines and rare PTCH mutations in tumor samples suggest that the HH pathway may have a role in controlling the proliferation of urothelial cells and that PTCH mutations may contribute to the development of a subset of TCCs.

Novel p21WAF1/CIP1 mutations in superficial and invasive transitional cell carcinomas.

Alterations in the p53 gene are a predominant component in the development of transitional cell carcinoma (TCC), but the particular pathways distal to p53 alterations which contribute to urothelial transformation are not defined. Here, the p21WAF1/CIP1 gene, a p53 inducible and p53 independent gene product, was studied in TCC. p21WAF1/CIP1 expression was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) from five cell lines and 28 tumor specimens (14 superficial, 14 muscle invasive). This was expressed as a ratio of the gene product to L7, a ribosomal housekeeping gene. In addition, exons 4 through 8 of the p53 gene as well as exon 2 of the p21WAF1/CIP1 gene were assayed for mutations by polymerase chain reaction/single stranded conformation polymorphism analysis (PCR/SSCP). Candidate mutations were verified by sequencing. p21WAF1/CIP1/L7 expression was significantly decreased in invasive lesions compared to superficial lesions (P<0.002). p53 mutations were detected by PCR/SSCP in seven tumors [25%] (one superficial, six invasive) and p21WAF1/CIP1/L7 expression was significantly decreased in all tumors that had p53 mutations (P<0.007). PCR/SSCP analysis of exon 2 in p21WAF1/CIP1 detected band shifts in four/28 tumor specimens (two superficial, two invasive), which sequencing and comparison to autologous normal matched DNA revealed as novel mutations.

p53 wild-type and p53 nullizygous Big Blue transgenic mice have similar frequencies and patterns of observed mutation in liver, spleen and brain.

Transgenic mouse mutation detection systems offer a powerful tool for analysis of spontaneous and induced mutations in vivo. Mice doubly transgenic for a null mutation of the p53 tumor suppressor gene and a lambda shuttle vector harboring the lacI gene were utilized to examine the rate and pattern of spontaneous somatic mutation of the lacI transgene in vivo. Three somatic tissues were examined: liver, spleen and brain. At 6 weeks of age, three p53 wild type (+/+) and three p53 nullizygous (-/-), lacI (+/-) male mice were analysed. The mutation frequencies in the two genotypes were similar. The mutant frequencies for wild type (+/+) and nullizygous (-/-) p53 genotypes were, respectively, 4.2 x 10(-5) and 3.6 x 10(-5) in the liver, 4.3 x 10(-5) and 3.4 x 10(-5) in the spleen and 2.8 x 10(-5-) and 3.0 x 10(-5) in the brain. When the data from the three tissues were combined, the mutant frequency was 3.7 x 10(-5) for the (+/+) genotype and 3.3 x 10(-5) for the (-/-) genotype. By sequencing both strands in the DNA-binding region of the lacI gene, 91 mutations were found. When recurrent mutations in the same mouse were excluded, a total of 67 definitely independent mutations were found. No statistically significant differences were found in the mutational spectra between the two genotypes when the three tissues were analysed individually or combined (P = 0.58). These findings suggest a need to reconsider the general form of hypothesis that the p53 gene serves as the 'guardian of the genome'.

Antimicrobial stress compounds from Hypochoeris radicata.

The exudates of Hypochoeris radicata leaves stressed with cupric chloride afforded two eudesmane- and guaiane-type sesquiterpenes, and two alkenals, (2E,4E)-6-hydroxyhexadienal and (2E,4E)-hexadienedial (mucondialdehyde). Their structures were determined by spectroscopic and synthetic methods.

Familial occurrence of congenital bile duct dilatation.

The occurrence of congenital bile duct dilatation (CBD) in both a mother and her daughter was recently experienced at Niigata University Hospital and Niigata Shimin Hospital. Bile duct dilatation with anomalous pancreaticobiliary ductal junction (AP-BDJ) was disclosed in both. Intrahepatic bile duct dilatation was only in the mother. Removal of dilated bile duct, cholecystectomy, and hepaticojejunostomy were performed in both. Fourteen cases of CBD from seven families were collected from literature and discussed.

Fructose 2,6-bisphosphate changes in rat brain during ischemia.

Brain ischemia was produced by bilateral ligation of the common carotid arteries of spontaneously hypertensive rats. The concentrations of fructose 2,6-bisphosphate and other glycolytic intermediates as well as of pyridine and adenine nucleotides were measured in frozen brain samples. In contrast to the decrease reported in hepatocytes under anoxic conditions, the fructose 2,6-bisphosphate content was increased by 20-30% during the early stages of ischemia. Elevation in fructose 1,6-bisphosphate level and lactate formation followed the rise in fructose 2,6-bisphosphate content, a finding suggesting that this compound plays a key role in the compensatory acceleration of glycolysis under ischemic conditions in vivo.

[Follow-up study of bone grafts to the alveolar clefts].

Clinical follow-up study after the grafting with fresh autogenous iliac bone to the alveolar clefts in cleft lip and palate patients was carried out up to 2 years post operation. Twenty two patients were examined, to whom grafts of iliac cortical or iliac marrow bone was carried out by the First or the Second Department of Oral Surgery, Kagoshima University Dental School from July, 1985 to April, 1988. Their cleft types were classified as 18 unilateral and 4 bilateral cleft lips and palates. The ages at bone grafting were ranged from 8 years 9 months to 17 years 6 months, and the average of which was 12 years 3 months. Nine of these patients were grafted with the cortical bone, and the other 13 were grafted with the marrow bone. The perforations at alveolar clefts were covered with the tongue flap in 10 clefts of 10 patients, while they were covered with the local mucous flap in 15 clefts of 12 patients. Among these patients, dental casts, X-ray radiograms and oral pictures of 15 patients were surveyed postoperatively. Results were as follows: 1) Contours of the alveolar clefts were well corrected in 9 patients, especially in those cases of pre-operatively expanded unilateral clefts, grafted with the marrow bone and covered with the local mucous flaps. 2) The viable rate of grafted bone was 100.0% for the marrow bone, while it was 62.5% for the cortical bone.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurinoma of the trigeminal root presenting as atypical trigeminal neuralgia: diagnostic values of orbicularis oculi reflex and magnetic resonance imaging. A case report.

A case of trigeminal root neurinoma presenting as atypical trigeminal neuralgia was reported. The orbicularis oculi reflex was absent on the affected side, but reappeared after operation. The demonstration of the tumor was much clearer on magnetic resonance imaging. The usefulness of these electrophysiological and radiological studies in differentiating varied entities of trigeminal neuralgia is stressed.