Fine mapping and DNA fiber FISH analysis locates the tobamovirus resistance gene L3 of Capsicum chinense in a 400-kb region of R-like genes cluster embedded in highly repetitive sequences.

The tobamovirus resistance gene L(3) of Capsicum chinense was mapped using an intra-specific F2 population (2,016 individuals) of Capsicum annuum cultivars, into one of which had been introduced the C. chinense L(3) gene, and an inter-specific F2 population (3,391 individuals) between C. chinense and Capsicum frutescence. Analysis of a BAC library with an AFLP marker closely linked to L(3)-resistance revealed the presence of homologs of the tomato disease resistance gene I2. Partial or full-length coding sequences were cloned by degenerate PCR from 35 different pepper I2 homologs and 17 genetic markers were generated in the inter-specific combination. The L(3) gene was mapped between I2 homolog marker IH1-04 and BAC-end marker 189D23M, and located within a region encompassing two different BAC contigs consisting of four and one clones, respectively. DNA fiber FISH analysis revealed that these two contigs are separated from each other by about 30 kb. DNA fiber FISH results and Southern blotting of the BAC clones suggested that the L(3) locus-containing region is rich in highly repetitive sequences. Southern blot analysis indicated that the two BAC contigs contain more than ten copies of the I2 homologs. In contrast to the inter-specific F2 population, no recombinant progeny were identified to have a crossover point within two BAC contigs consisting of seven and two clones in the intra-specific F2 population. Moreover, distribution of the crossover points differed between the two populations, suggesting linkage disequilibrium in the region containing the L locus.

The methylation status of cytosines in a tau gene promoter region alters with age to downregulate transcriptional activity in human cerebral cortex.

Changes with age in the methylation status of cytosines in a promoter region of the tau gene were investigated in autopsy human cerebral cortex, using the bisulfite method, polymerase chain reaction (PCR) and direct sequencing of PCR products. While the total number of methylcytosines decreased with age, the changes in methylation status differed among transcription factor binding sites. Cytosines in the AP2-binding sites were never methylated in any of the cases studied at any age. Methylcytosines in the binding sites for Sp1, a transcriptional activator, significantly increased with age, whereas those in the binding sites for GCF, a repressor of GC-rich promoters, significantly decreased with age. These findings suggest that the methylation status of cytosines in the promoter region of the tau gene alters with age to decrease its transcriptional activity in the human cerebral cortex.

Changes with aging and ischemia in nicotinic acetylcholine receptor subunit alpha7 mRNA expression in postmortem human frontal cortex and putamen.

Age-related changes in nicotinic acetylcholine receptor (nAChR) subunit alpha7 messenger RNA (mRNA) expression in postmortem human frontal cortex and putamen of controls and status lacunaris patients were investigated using nonradioactive reverse transcription(RT)-PCR. In the frontal cortex of control brains, alpha7 subunit mRNA significantly decreased with age (P < 0.05). In the putamen, alpha7 subunit mRNA expression was significantly lower than that in the frontal cortex (P < 0.0001), and showed no significant correlation with age. However, in cases with status lacunaris in the putamen, alpha7 subunit mRNA expression was significantly higher compared with controls (P < 0.001). The reduction in alpha7 nAChR in the frontal cortex with age may decrease functional cholinergic synapses and cortical activity, and play a role in the cognitive impairments associated with normal aging. The functional significance of the upregulation of alpha7 nAChR mRNA in ischemic conditions remains to be determined.

Reduction with age in methylcytosine in the promoter region -224 approximately -101 of the amyloid precursor protein gene in autopsy human cortex.

Methylation status of cytosines and its changes with age in the promoter region (-226 approximately -101) of the amyloid precursor protein (APP) was analyzed using bisulfite genomic sequencing in the cerebral cortex of human autopsy brain. Cytosines at 13 locations were methylated in at least one of the cases studied. Methylcytosines at these locations was more frequent in cases 70 years old (8%) (p<0.05). Cytosines at -207, -204, -200, and -182 are frequently methylated, and the frequency of methylcytosine in these locations was significantly higher in cases 70 years old (5%) (p<0.01). These cytosines constituted one of the 9-bp-long GC-rich elements (GGGCGC G/A GG) or an 11-bp inverted repeat (GGCCGT CGGCC). The present findings indicate that some cytosines, particularly those at -207 approximately -182, in the promoter region of the APP gene are frequently methylated and suggest that their demethylation with age may have some significance in the development of Abeta deposition in the aged brain. The relative importance of these elements in the total promoter activity of the APP gene remains to be definitively established.

Decrease with age in methylcytosines in the promoter region of receptor for advanced glycated end products (RAGE) gene in autopsy human cortex.

Changes with age in the methylation status of cytosines in the promoter region of the receptor for advanced glycated end products (RAGE) in autopsy human cortex were investigated, using the bisulfite method, polymerase chain reaction (PCR), and direct sequencing of PCR products. The total number of methylcytosines significantly decreased with age. While the number of methylated cytosines at CpG dinucleotides was stable throughout adult life, that at sites other than CpG dinucleotides significantly decreased with age in cases >/=70 years old. Of 13 transcription factor binding sites, cytosines in CpG doublets in NF-IL6 and SP-1 binding sites were methylated in all cases, suggesting that these sites are repressed throughout adulthood. In contrast, the number of methylcytosines in AP-2 or SP-1 binding sites located at CpC, CpA, or CTG was significantly lower or at least tended to be lower in cases >/=70 years than <70 years old. These reductions in the number of methylcytosines at transcription factor binding sites may increase expression of RAGE, which may in turn play a role in aging of the brain.

Thirteen-week repeated oral dose toxicity study of ecabapide, a gastroprokinetic drug, in dogs and rats.

Thirteen-week oral repeated dose toxicity of ecabapide, a gastroprokinetic drug, was investigated in dogs at dosage levels of 50, 175 or 600 mg/kg, and in rats at dosage levels of 25, 100, 400 or 1600 mg/kg. In dogs, vomiting, aqueous salivation, body weight gain inhibition, and hemolytic anemia, together with an increase in Heinz body formation, were observed at 175 and/or 600 mg/kg. Histological examination revealed enhanced hemosiderin deposition in the liver and spleen, retention of erythrocytes in the splenic sinus and enhanced erythropoiesis in bone marrow at 175 and/or 600 mg/kg. In the rat study, although increases in serum total protein, albumin and calcium, as well as increased liver and kidney weights, were observed at 400 and/or 1600 mg/kg, no obvious morphological changes were seen. The hemolytic anemia and an increased Heinz body formation were not observed in rats, indicating a species difference. On the basis of these results, the non-toxic dose of ecabapide was considered to be 50 mg/kg in dogs and 100 mg/kg in rats.

[Intravenous single dose toxicity of iodixanol, a new nonionic iso-osmolar contrast medium, in mice, rats and monkeys].

The single-dose intravenous toxicities of iodixanol, a new nonionic iso-osmolar contrast medium, were investigated in mice, rats and monkeys. The LD50 values were estimated to be 17.9 gI/kg for male mice and 16.2 gI/kg for female mice, 18.8 gI/kg for male rats and 22.0 gI/kg for female rats, and more than 10.0 gI/kg for monkeys. There was no marked sex difference in mice or rats, nor any significant difference observed between these two rodent species. Decrease in spontaneous locomotor activity, ptosis, respiratory depression and abdominal posture were observed in many mice and rats. These signs disappeared mostly by 8 days after dosing in surviving animals. Death occurred between immediately and 4 days after dosing in mice, and between immediately and 14 days after dosing in rats. Transient depression of body weight gain was observed in the surviving mice and rats by 7 days after dosing. Histological examinations revealed congestion or hemorrhage in the renal medulla, vacuolation or necrosis of the renal proximal tubular epithelium in mice and rats that died and vacuolation of the renal proximal tubular epithelium in surviving rats. There were no significant treatment-related changes in the laboratory and pathological examinations in monkeys.

[Toxicity of iodixanol, a new nonionic iso-osmolar contrast medium, to cynomolgus monkeys by repeated intravenous administration for 4 weeks with a 4-week recovery period].

Groups of 3 male and 3 female wild-caught cynomolgus monkeys were given iodixanol, a radiographic contrast medium, by intravenous injection at dosage levels of 100, 300 or 1,000 mgI/kg/day for four weeks to evaluate its toxicity. An extra 2 animals of each sex were given 1,000 mgI/kg/day for 4 weeks and then retained for 4 weeks without treatment to assess recovery. There were no deaths during the treatment period. Bruising at the injection sites was noted clinically and macroscopically was caused in part by the injection procedure itself and in part by the viscosity of the test formulation and the size of the dose. This finding is therefore, of no toxicological significance. No effects of treatment with iodixanol on body weights, food consumption, laboratory investigations, ophthalmoscopic examinations, organ weights or bone myelograms were noted. Cytoplasmic vacuolation of the kidney proximal tubules was noted with incidence and severity increasing with dosage level among animals given 100, 300 or 1,000 mgI/kg/day. This effect was noted not to be fully reversible at 1,000 mgI/kg/day after 4 weeks off-dose although reduced severity was noted and recovery was apparent in some animals.