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Hiroshi Ihara

Publications and source records attributed to Hiroshi Ihara.

11 recordsLinked to original sources
PubMed2006

In vitro selection of zinc finger DNA-binding proteins through ribosome display.

DNA-binding proteins with sequence specificities have a variety of applications. To create novel functional DNA-binding proteins, in vivo selection methods have been developed. There are, however, crucial problems with such methods, e.g., limitation of library size and difficulty of expression of toxic proteins for the host cells. In order to overcome these problems, we developed a novel way to select DNA-binding proteins using an in vitro ribosome display technique. The three zinc finger DNA-binding protein libraries, based on a Zif268 containing randomized sequence in each finger, were prepared and transcribed to mRNA in vitro. The ternary ribosomal complexes, formed by mRNA, ribosome, and translated DNA-binding protein during translation in a rabbit reticulocyte in vitro translation system, were selected with biotinylated target DNA fragments bound to streptavidin magnetic beads. The extracted mRNAs from the selected complexes were amplified using reverse transcription PCR and then sequenced. This is the first report of the selection of DNA-binding proteins involving an in vitro ribosome display technique.

Animals↗
PubMed2005

Assay values for thiamine or thiamine phosphate esters in whole blood do not depend on the anticoagulant used.

We compared the whole blood, plasma, and erythrocyte (red blood cell (RBC)) concentrations of thiamine and thiamine phosphate esters in the presence of heparin or EDTA as anticoagulants. Three blood specimens were collected from each of 24 healthy volunteers into evacuated collection tubes containing the following anticoagulants: heparin, Na2EDTA, or K2EDTA. The concentrations of nonphosphorylated free thiamine (T), thiamine monophosphate (TMP), thiamine diphosphate (TDP), and thiamine triphosphate (TTP) were determined by the NH2-column HPLC method. The anticoagulant used had no effect on the concentrations obtained in whole blood and plasma of thiamine or any of the above thiamine compounds (P>0.05). RBCs were isolated by centrifugation and washed with isotonic saline, and the cell counts of the washed cells were adjusted to their whole blood values. In the washed RBCs with any anticoagulant, the concentrations of T, TMP, and TDP expressed either as nmol/L of whole blood or a ratio to hemoglobin were significantly lower (P<0.05) than those in whole blood.

Adult↗
PubMed2005

Values of water-soluble vitamins in blood and urine of Japanese young men and women consuming a semi-purified diet based on the Japanese Dietary Reference Intakes.

We investigated the levels of water-soluble vitamins except for vitamin B6 in the blood and urine of Japanese college male (n = 10) and female (n = 10) students. They consumed for 7 d a semi-purified diet based on Japanese Dietary Reference Intakes to assess the Recommended Dietary Allowances (RDA) for water-soluble vitamins and to present some new normal values for blood and urine levels of water-soluble vitamins in Japanese. The blood and the 24-h urine samples were collected on the last day of the experiment and measured. The values of total vitamin B1 in whole blood, total vitamin B2 in whole blood, total cyanocobalamin in serum, total nicotinamide in whole blood, total pantothenic acid in whole blood, total folates in serum, total biotin in serum, and ascorbic acid in plasma were 104+/-17 pmol/mL (mean+/-SD), 216+/-25 pmol/mL, 0.34+/-0.05 pmol/mL, 59.1+/- 5.0 nmol/ mL, 2.45+/-0.37 nmol/mL, 15.6+/-4.6 pmol/mL, 8.3+/-0.5 pmol/mL, and 62+/-10 nmol/mL, respectively, in males, and 90+/-23, 234+/-18, 0.67+/-0.20, 61.9+/-6.0, 2.48+/-0.30, 30.2+/-8.6, 8.4+/-0.3, and 67+/-14, respectively, in females. There was a significant difference in the values of cyanocobalamin and total folates between men and women. The urinary excretion of vitamin B1, vitamin B2, cyanocobalamin, sum of the catabolic metabolites of nicotinamide, pantothenic acid, folates, biotin, and ascorbic acid were 665+/-114 nmol/d, 562+/-325 nmol/d, 93+/-31 pmol/d, 84+/-26 micromol/d, 9.3+/-2.3 micromol/d, 19.4+/-2.8 nmol/d, 83+/-18 pmol/d, and 148+/-51 micromol/d, respectively, in males, and 495+/-212, 580+/-146, 145+/-49, 83+/-19, 16.9+/-1.3, 22.7+/-2.7, 83+/-23, and 140+/-51, respectively, in females. There was a significant difference in the urinary excretion of cyanocobalamin, pantothenic acid and total folates between men and women. These values will be useful for the nutritional assessment of water-soluble vitamins for Japanese, although the examination period was short. In future, an experiment with various age groups and re-evaluation by repeated experiments will provide more accurate values.

Adult↗
PubMed2004

Antioxidant capacities of ascorbic acid, uric acid, alpha-tocopherol, and bilirubin can be measured in the presence of another antioxidant, serum albumin.

Human serum contains several antioxidants. The total antioxidant capacity (AOC) is the sum of all the antioxidant activities present in serum. The aim of our study was to investigate whether the AOC of bilirubin (BR), alpha-tocopherol (TOH), ascorbic acid (AA), and uric acid (UA) could be measured with good precision and recovery in the presence of human serum albumin (HSA). We measured the AOC of each antioxidant using a Cobas Mira S instrument (Roche Diagnostic Systems, Montclair, NJ) by measuring the inhibitory effect of a given compound on the oxidation of the radical cations of 2,2'-azino-di-(3-ethylbenzthiazoline 6-sulphonate) (ABTS) incubated with metmyoglobin and H(2)O(2). The assay had a linear AOC response range of 27-2,000 micromol/L. The within- and between-day coefficients of variation (CVs) did not exceed 3.4% and 4.2%, respectively. The AOC of albumin in serum is much greater than that of BR, TOH, AA, or UA owing to the substantially greater concentration of HSA in serum. An aliquot of a solution of AA, UA, BR, or TOH was added to HSA or distilled water, and the AOC was determined. The AOC of BR, TOH, AA, and UA increased in a linear way with increasing concentrations. However, we found that the magnitude of increase in the AOC of a mixture of HSA and any of these antioxidants was lower than the sum of the AOC of HSA and any one of the following: AA, UA, BR, or TOH (all expressed in micromol/L).

Antioxidants↗
PubMed2004

Stability of fat-soluble and water-soluble vitamins in artificially prepared, vitamin-enriched, lyophilized serum.

Vitamin-enriched, lyophilized serum (VES) was prepared for an inter-laboratory study to compare vitamin assays. The VES contained water-soluble vitamins (vitamin B1, vitamin B12, vitamin C, and folate), fat-soluble vitamins (vitamin A and vitamin E), and cholesterol. We performed stability studies and determined vitamin concentrations and total cholesterol in VES stored at -20 degrees C for 12 months. Our recovery of the water-soluble vitamins in reconstituted VES was 70-142%, but we recovered only 33-45% of the fat-soluble vitamins. Physicochemical properties, such as specific gravity and viscosity of the reconstituted VES did not affect manual or automated measurements of these vitamins. Vial-to-vial differences found for the VES were the same as the within-day analytical variations. There was no evidence of degradation of vitamin A, vitamin B1, vitamin B12, vitamin C, folate, and cholesterol over 12 months in VES stored at -20 degrees C. Following deproteinization, vitamin C concentration was found to be lower than when not deproteinated. Vitamin E was less stable in VES, however, and the degradation during 12 months was lower than the between-day analytical variation of the assay. Our VES is the first preparation of lyophilized control serum that contains water-soluble and fat-soluble vitamins.

Ascorbic Acid↗
PubMed2004

Recommended dietary allowance for vitamin C in the United States is also applicable to a population of young Japanese women.

The recommended dietary allowance (RDA) for ascorbic acid (AA) in Canada and the United States has been set for several years at 75 mg/day for women 19-30 years old. Recently this level was questioned, and an increase to 90 mg/day was suggested. For Japanese women in the same age group, we found that the RDA for AA is currently 100 mg/day. Our goal was to determine which RDA is sufficient for maintaining a serum concentration of AA in young Japanese women above the lower reference limit of 7.0 mg/L. We measured serum AA concentrations by an ascorbate oxidase method in 176 healthy Japanese women (19-26 years old). We also performed an ROC analysis to estimate the optimal cutoff value for oral dosage to distinguish individuals with hypovitaminosis-C (<7.0 mg/L) from those with a normal serum AA. We evaluated the Japanese RDA using the 75 or 90 mg/day U.S. RDA and the weight ratio between Japanese and U.S. women, and discovered that the RDA value ranged between 66 and 79 mg/day. From the ROC analysis, we found that the optimal daily dosage of AA is approximately 75 mg/day. This value gave the highest efficiency, sensitivity, negative predictive value, and positive likelihood ratio, and the lowest negative likelihood ratio. Therefore, an RDA of 100 mg/day may be unnecessarily high for young Japanese women.

Adult↗
PubMed2003

Selective use of transthyretin and retinol-binding protein as markers in the postoperative assessment of protein nutritional status.

We evaluated several markers to judge the postoperative state of protein nutriture in eight patients following surgery for cancer. Seven patients had a good prognosis and had no evidence of infections or other complications. Following surgery, all of the patients showed a shift toward abnormal values for the serum concentrations of albumin, transthyretin (TT), retinol-binding protein (RBP), and the amino acid (AA) ratio of nonessential to essential amino acids. In patients without complications, the AA ratio returned to normal first. When blood specimens were collected at 7-day intervals, concentrations of RBP and TT were revealed to be decreased and recovered at the same time, or TT was recovered after RBP was normalized. RBP and TT were usually abnormal until the AA ratio became normal. Although albumin moved toward normal concentrations after RBP and TT, the albumin concentrations in some patients were slightly above the lower reference value, whereas RBP and TT were significantly below their lower reference limits. In these patients, assessments over the next 7-14 days showed persistently low values for albumin, TT, and RBP. We recommend the selective use of TT and RBP for the postoperative assessment of protein nutriture in surgical patients.

Aged↗
PubMed2003

Removal of protein-bound and unbound unconjugated bilirubin by perfusion of plasma through an anion-exchange resin in a case of Crigler-Najjar syndrome type I.

BACKGROUND: Patients with Crigler-Najjar syndrome, type I (CNS-I) have an inherited absence of hepatocellular bilirubin uridine diphosphate-glucuronosyltransferase activity, which results in severe unconjugated hyperbilirubinaemia, often causing kernicterus and death in infancy or childhood. METHODS: Our patient is a 19-year-old Japanese man with CNS-I diagnosed by the complete absence of the hepatocellular enzyme in a liver biopsy and genotyping. The efficacies of the removal of protein-bound (PBB) and unbound (UB) unconjugated bilirubin by phototherapy, plasma perfusion and liver transplantation were compared in the patient. RESULTS: At the age of 5 years, phototherapy treatment reduced the patient's PBB by 21% and UB by 34%, and 98% of the bilirubin produced daily was removed. At the age of 16 years, plasma perfusion combined with nightly phototherapy completely removed the daily production of bilirubin; however, by 24 h post-treatment, the PBB and UB were again increased. Apparently, these treatments were effective in reducing PBB and UB, but the effect was only temporary. Following liver transplantation, PBB and UB decreased to normal concentrations. CONCLUSIONS: Liver transplantation as a potential cure should be performed at a younger age, particularly in confirmed CNS-I cases for which reliable effects of phototherapy cannot be guaranteed.

Adolescent↗
PubMed2003

The assay of ascorbic acid in serum is not affected by physiological concentrations of transferrin and hemoglobin.

Transferrin and hemoglobin have been reported to oxidize L-ascorbic acid (AA) in vitro. The aim of this study was to determine whether or not physiological concentrations of serum transferrin (reference range 22-45 micromol/L) and hemoglobin (reference range 0-3.0 micromol/L) interfer with the measurement of AA in the serum. Transferrin (33 to 41 micromol/L) and hemoglobin (1.9 micromol/L) added to freshly pooled serum significantly decreased measured AA in the serum (p < 0.05). However, we found that the magnitude of the decrease in AA due to transferrin at concentrations within the reference range or up to 80 micromol/L was inconsequential, and had no clinical importance in diagnosing a low AA concentration. Hemoglobin at concentrations within the reference range had little affect on the serum AA measurement. However, when serum specimens were stored at 4 degrees C for more than 1.5 h, the magnitude of the decrease in AA due to hemoglobin at physiological concentrations may cause a misleading clinical diagnostic evaluation of low AA concentration.

Ascorbic Acid↗