A soil yeast Rhodotorula glutinis increased torularhodin, that is one of the final products of carotenoid biosynthesis, when loading oxygen stress. In addition, a mutant of this yeast, which produces torularhodin more than β-carotene, gained resistance to oxygen stress. In this study, changes in carotenoids biosynthesis due to addition of 2, 2'-azobis(2-amidinopropane)-dihydrochloride (AAPH) as a peroxyl radicals generator were examined, and scavenging peroxyl radicals by torularhodin was also compared with that by β-carotene. When 0.01mM AAPH was added to the culture medium, the amount of carotenoids in yeast was increased. With increase in load by AAPH addition, torularhodin was consumed faster than β-carotene. After torularhodin decreased to the same concentration as AAPH free condition, β-carotene began to decrease. These results suggest that torularhodin was important in defense against oxidation loading by peroxyl radicals in R. glutinis. Scavenging of peroxyl radicals by torularhodin was evaluated using electron spin resonance. In the reaction of cumene hydroperoxide and 5, 10, 15, 20-tetraphenyl-21H, 23H-prophine iron (III) [TPP-Fe(III)], torularhodin exhibited concentration-dependent inhibition and its activity reached to 60% at 2.5mM. On the other hand, α-tocopherol exhibited strong activity at low concentrations with a maximum at 0.25mM, but its activity decreased above this concentration. Scavenging activity by torularhodin was greater than that of α-tocopherol at 2.5mM. In the pyrolysis of 2, 2′-azobisisobutyronnitrile (AIBN), both torularhodin and α-tocopherol exhibited concentration-dependent inhibition, but the scavenging activity of α-tocopherol was stronger. In addition, torularhodin inhibited lipid peroxide formation of rat brain homogenate in a concentration-dependent manner, and its inhibitory effect was stronger than that of α-tocopherol at concentration of 1μM and above. Torularhodin thus appears to be an effective scavenger of peroxyl radicals and to have strong antiperoxidative activity. The strong inhibition of lipid peroxidation by torularhodin at low concentration suggests that torularhodin has other activities in addition to peroxyl radical scavenging. R. glutinis tenders to increase the production of torularhodin by load oxygen stress, but this was because of its strong peroxyl radicals scavenging.
Oxidized low-density lipoproteins have a strong cytotoxic effect on cultured endothelial cells that can result in massive apoptosis. We studied the characteristics and mechanism of an effect of an angiotensin-converting enzyme (ACE) inhibitor against oxysterol-induced apoptosis in human aortic endothelial cells. Low concentrations of oxysterols increased apoptosis, but not necrosis. An ACE inhibitor, temocaprilat, significantly and dose-dependently decreased this apoptosis. Addition of HOE 140, a kinin B2 receptor antagonist, or N-nitro-L-arginine methylester (L-NAME), a nitric oxide synthase inhibitor, countered this effect of temocaprilat. The ACE inhibitor lessened increases in caspase-3 activity and intracellular oxygen radical production induced by oxysterols; again, the amelioration was blocked by HOE140 or L-NAME. These findings demonstrate that oxysterols caused endothelial apoptosis via an increase in intracellular reactive oxygen species and activation of caspase-3. Temocaprilat ameliorated apoptosis through increase of nitric oxide production via an increase in bradykinin, with a resulting increase in nitric oxide.
The Epstein-Barr (EB) virus early antigen expression inhibition test on Raji cells is accepted worldwide as a useful assay system for screening of cancer chemo-preventive agents, and the results obtained with it correlate somewhat with those of in vivo screening by the mouse skin test. In this study, to investigate the mechanism of cancer chemoprevention by retinoic acid, we demonstrated the effect of retinoic acid on gene expression induced in Raji cells by 12-O-tetra-decanoylphorbol-13-acetate by using DNA chip technology. We found significant changes in the levels of 322 mRNA's in the cells. Down-regulated genes numbered 109, and up-regulated ones 213. In addition, we evaluated a large number of genes expressed in Raji cells in response to retinoic acid by using a Cell Signaling Network Database (CSNDB) and thereby extracted 21 interesting genes. The retinoic acid responsiveness of these 21 genes in Raji cells is a new finding. We also found three candidates of signaling pathways through examining the enormous gene expression data. This trial may be invaluable for DNA chip/microarray users because of our new methodology using combined array data and database information.
We constructed isogenic Shiga (-like) toxin (Stx-1 and/or Stx-2) gene-deletion mutants of enterohemorrhagic Escherichia coli (EHEC) GPU96MM (O157:H7). A vector with temperature-sensitive replication origin was used for the construction. The parts of stx-1 and stx-2 on the GPU96MM genome were replaced with kanamycin and chloramphenicol resistance genes, respectively. The mutants deficient in stx-1, stx-2, and both of them were named GPU993, GPU994, and GPU995, respectively. Each mutation was confirmed by the polymerase chain reaction, enzyme-linked immunosorbent assay using antibodies to B subunits of Stx-1 and Stx-2, and the cytotoxic activity of the bacterial culture supernatants toward HeLa cells was detected for GPU96MM and the mutants except for GPU995. These results indicate that GPU993 and GPU994 lack productivity for the respective toxins and GPU995, for both of them.
This was conducted to examine the effects of brown seaweed (wakame) on blood pressure and serum biochemical parameters in hypertensive subjects. Of the 37 elderly out-patients with hypertension who started the study, 36 of them completed it. This study was a randomly assigned, case-controlled one. Nineteen patients received a daily dose of 5g of dried wakame powder packed in 12 capsules. Eighteen gender-matched subjects with age difference±2 years, and starting time of participation within±2 weeks, were selected as the control group. Patients visited the clinic every 4 weeks. The observation period was 8 weeks. In the wakame group, the average amount of wakame ingested was 3.3g. The systolic blood pressure (SBP) in this group dropped 13mmHg below the baseline (p<0.01) after 4 weeks, and 8mmHg (p<0.05) after 8 weeks. The diastolic blood pressure (DBP) decreased by 9mmHg (p<0.01) after 4 weeks and by 8mmHg (p<0.05) after 8 weeks. In the control group, no significant changes were seen in either SBP or DBP. However, the differences in reduction in SBP and DBP were significant between the wakame and control groups. Regarding clinical chemistry data, hypercholesterolemia in the wakame group decreased by 8% after 4 weeks. No other abnormal changes were observed in either group. We conclude that wakame has beneficial effects as a supplemental regimen in the treatment of hypertension.
D-Psicose (D-ribo-2-hexulose), a C-3 epimer of D-fructose, is present in small quantities in commercial carbohydrates and agricultural products. To evaluate the possibility of D-psicose as low-energy sugar substitute, we studied the effects on body fat accumulation of D-psicose compared with cellulose or D-fructose in rats. Wistar male rats were fed experimental diets including 5% D-psicose, cellulose or D-fructose for 21 days. Abdominal adipose tissue weight was significantly lower (p<0.05) in rats fed D-psicose than in those fed D-fructose. Gene expression of uncoupling protein-1 and β3-adrenergic receptor in the brown adipose tissue did not differ among the dietary groups. Fatty acid synthase and glucose 6-phosphate dehydrogenase activities in the liver were significantly lower (p<0.05) in rats fed D-psicose, whereas lipoprotein lipase activities in the heart, soleus muscle, perirenal adipose tissue, and subcutaneous adipose tissue did not differ. These results suggest that supplementation of D-psicose in the diet suppresses hepatic lipogenic enzyme activities. The lower abdominal fat accumulation in rats fed D-psicose might have resulted from lower lipogenesis in the liver. D-Psicose could prove to be a superior sugar substitute.
Highly sensitive and rapid assay procedures for human mitochondrial the pyruvate dehydrogenase (PDH) complex, the 2-oxoglutarate dehydrogenase (OGDH) complex and their 5 component enzymes were established for use with crude tissue extracts, and their enzymatic activities were determined. Overall reaction activities of the PDH and OGDH complexes were very high, in order of heart>abdominal skeletal muscle>kidney. Full-length cDNA encoding the precursor of theporcine dihydrolipoamide acetyltransferase (E2p) subunit was isolated and characterized. It contained an open reading frame encoding a leader peptide (86 residues) and a mature enzyme protein (561 residues, Mr=59, 699). The reduced enzyme protein showed 91% identity with the human enzyme. All genes encoding the component enzymes of the 2 human complexes were expressed in a variety of organs, but most strongly in heart and skeletal muscle (abdominal muscle) in a similar order as found for their enzymatic activities.