Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.     

Expression and Regulation of the Arsenic Resistance Operon of Acidiphilium multivorum AIU 301 Plasmid pKW301 in Escherichia coli

The arsenic resistance (ars) operon from plasmid pKW301 of Acidiphilium multivorum AIU 301 was cloned and sequenced. This DNA sequence contains five genes in the following order: arsR, arsD, arsA, arsB, arsC. The predicted amino acid sequences of all of the gene products are homologous to the amino acid sequences of the ars gene products of Escherichia coli plasmid R773 and IncN plasmid R46. The ars operon cloned from A. multivorum conferred resistance to arsenate and arsenite on E. coli. Expression of the ars genes with the bacteriophage T7 RNA polymerase-promoter system allowed E. coli to overexpress ArsD, ArsA, and ArsC but not ArsR or ArsB. The apparent molecular weights of ArsD, ArsA, and ArsC were 13,000, 64,000, and 16,000, respectively. A primer extension analysis showed that the ars mRNA started at a position 19 nucleotides upstream from the arsR ATG in E. coli. Although the arsR gene of A. multivorum AIU 301 encodes a polypeptide of 84 amino acids that is smaller and less homologous than any of the other ArsR proteins, inactivation of the arsR gene resulted in constitutive expression of the ars genes, suggesting that ArsR of pKW301 controls the expression of this operon.     

Detection of Escherichia coli O157:H7 in the Beef Marketed in Malaysia

Twelve strains of Escherichia coli O157:H7 were isolated from 9 of 25 beef samples purchased from retail stores in Malaysia. These strains produced Shiga toxin 2 with or without Shiga toxin 1 and had the eae gene and a 60-MDa plasmid. The antibiograms and the profiles of the arbitrarily primed PCR of the strains were diverse, suggesting that the strains may have originated from diverse sources.     

Quercetin metabolites inhibit copper ion-induced lipid peroxidation in rat plasma

The oxidative susceptibility of plasma obtained from rats after intragastric administration of quercetin was studied to know whether or not quercetin acts as an in vivo antioxidant after metabolic conversion. Quercetin was raised in the rat blood plasma essentially as glucuronide and/or sulfate conjugates. The plasma obtained from rats after quercetin administration was more resistant against copper sulfate-induced lipid peroxidation than the control plasma on the basis of the accumulation of cholesteryl ester hydroperoxides and the consumption of α-tocopherol. The results strongly suggest that some conjugated metabolites of quercetin act as effective antioxidants when plasma is subject to metal ion-induced lipid peroxidation.     

Introduction of the cDNA for shape Arabidopsis glycerol-3-phosphate acyltransferase (GPAT) confers unsaturation of fatty acids and chilling tolerance of photosynthesis on rice

The chilling sensitivity of several plant species is closely correlated with the levels of unsaturation of fatty acids in the phosphatidylglycerol (PG) of chloroplast membranes. Plants with a high proportion of unsaturated fatty acids, such as Arabidopsis thaliana, are resistant to chilling, whereas species like squash with only a low proportion are rather sensitive to chilling. The glycerol-3-phosphate O-acyltransferase (GPAT) enzyme of chloroplasts plays an important role in determining the levels of PG fatty acid desaturation.A cDNA for oleate-selective GPAT of Arabidopsis under the control of a maize Ubiquitin promoter was introduced into rice (Oryza sativa L.) using the Agrobacterium-mediated gene transfer method. The levels of unsaturated fatty acids in the phosphatidylglycerol of transformed rice leaves were found to be 28% higher than that of untransformed controls. The net photosynthetic rate of leaves of transformed rice plants was 20% higher than that of the wild type at 17°C. Thus, introduction of cDNA for the Arabidopsis GPAT causes greater unsaturation of fatty acids and confers chilling tolerance of photosynthesis on rice.     

Porcine α-1,3-galactosyltransferase: full length cDNA cloning, genomic organization, and analysis of splicing variants

Full length cDNA and genomic DNA of porcine -1,3-galactosyltransferase were isolated, and their structures were analysed. The coding region was encoded by six exons as in the mouse, and the length of each exon was conserved between the two species. The porcine exons were designated Exon 4–9, since in the mouse coding exons started from Exon 4. Introns tended to be longer in the porcine gene; the distance from Exon 4 to the 3-end of Exon 9 was 24 kb, while this region was 18 kb in the mouse gene. The cDNA structure was extended from the previous data to the 3-end and to the 5 side of the cDNA. In addition to a cDNA clone with all coding exons, clones lacking parts of these exons were isolated and their structures were determined. One variant lacked Exon 5; the second, Exons 5 and 6; and the third, Exons 5, 6 and 7. The last variant was not found in the mouse, and cDNA transfection of this variant yielded scarcely any enzymatic activity using asialo 1-acid glycoprotein as a substrate, and decreased activity using N-acetyllactosamine as a substrate.     

Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.