Nobuko Shimizu

Sequence analysis and expression of the P450 aromatase and estrogen receptor genes in the Xenopus ovary

Recent studies point to a key role for the estrogen synthesizing enzyme P450 aromatase (P450 arom) in ovary determination in fish, birds and reptiles. It is unclear whether estrogen synthesis is important in sex determination of Xenopus gonad. To determine whether the aromatase gene is transcribed in the gonads of Xenopus tadpoles during the sex determination, we cloned a P450 arom cDNA and examined the level of P450 arom and estrogen receptor (ER) gene expression in association with estrogen activity. cDNA clones for P450 arom were isolated from a Xenopus ovarian cDNA library. There was an open reading frame (ORF) of 1500 bp from the ATG start to TAA stop codons encoding 500 predicted amino acids. cDNAs for P450 arom have previously been cloned from various vertebrates. The homology between the Xenopus P450 aromatase and the human P450 arom was higher. The expression of the P450 arom gene was mainly limited to reproductive organs. To determine the beginning of estrogen activity in gonads of embryos, expression of the aromatase and ER gene was also examined by RQ-RT-PCR. Both Xenopus aromatase and ER mRNA was detected at stage 51 in gonads. These observations are consistent with estrogens having a key role in ovarian development in various other vertebrates.

Enzymatic resolution of cis- and trans-1,2,3,4,6,7,8,8a-octahydro-8a-methyl-6-oxo-naphthyl acetate derivatives

Abstract Extensive screenings using commercially available enzymes were performed in relation to the asymmetric hydrolysis reactions of the (±)- cis- ( 1 , 3 and 5 ) and (±)- trans -1,2,3,4,6,7,8,8a-octahydro-8a-methyl-6- oxo -naphthyl acetates ( 7 , 9 and 11 ). The combinations of enzymes from plants and cis -equatorial compounds as well as the combinations of lipases from microorganisms and trans -axial-5-methoxycarbonyl acetate showed high enantiomeric ratio in asymmetric hydrolysis. However, the enantiomeric ratio of the combinations of lipases from microorganisms and trans -axial isomers except trans -axial-5-methoxycarbonyl derivative was less satisfactory. High E values (>200) were observed when β-amylase from wheat and lipase from Candida cylindracea were used for the (±)- cis -acetate 1 and the (±)- trans -isomer 7 , respectively, to obtain the corresponding chiral products with high enantiomeric ratios (>99:1).

Discordance between Two Interferon-Gamma Release Assays in the Diagnosis of Latent Tuberculosis Infection in Healthcare Workers

Background: Interferon-gamma release assays (IGRAs) more accurately diagnose Mycobacterium tuberculosis (Mtb) infection than the tuberculin skin test. To prevent outbreaks in medical facilities, early detection and treatment of Mtb infection (including latent tuberculosis infection (LTBI)) is important in healthcare workers. Therefore, the IGRAs have considerable utility for Mtb infection control in medical facilities. In Japan, two IGRAs are commercially available, QuantiFERON®-TBGold In-Tube assay (QFT-GIT) and T-SPOT®.TB (T-SPOT). However, it remains unclear if diagnostic yields of LTBI by both IGRAs are equivalent in healthcare workers. Methods: We performed both QFT-GIT and T-SPOT simultaneously in healthcare workers with a high risk of LTBI (excluding active tuberculosis) between December 2012 and February 2013. Results: Among 313 subjects (excluding 2 cases with indeterminate T-SPOT), 6 (1.9 %) and 12 (3.8 %) were QFT-GIT positive and T-SPOT positive, respectively. There was no significant concordance of results between the QFT-GIT and the T-SPOT (p=0.064 and Kappa=0.43, 95% confidence interval 0.082-0.78). Among 10 discordant cases between two IGRAs, 8 cases had IGRAs’ results near the cutoff values. Conclusion: Without a diagnostic gold standard for LTBI, it is difficult for us to further assess which test is more accurate and more suitable for the diagnosis of LTBI. However, to diagnose LTBI of healthcare workers with IGRAs’ results near the cutoff values, we should consider clinical context, such as contact level, as well as the results of IGRAs.

Unusual bromination of tetrahydro-(–)-α-santonins and new santonin isomers: X-ray crystal and molecular structure of 2β,14-dibromo-4α,5β,6β,11βH-tetrahydrosantonin

A novel bromination–dehydrobromination reaction of tetrahydro-(–)-α-santonins is reported which has led, via the 2α,14- and 2β, 14-dibromo ketones (2a,b), to two new dienone isomers of (–)-α-santonin whose structures were established by X-ray diffraction and c.d. studies of (2b).

Evaluation of anticoagulant effects of direct thrombin inhibitors, dabigatran and argatroban, based on the Lineweaver–Burk plot applied to the Clauss assay

Direct thrombin inhibitors (DTIs) represented by dabigatran were expected to be available for therapeutic use without the need for routine monitoring, in contrast to warfarin. However, clinical concerns regarding impacts of plasma dabigatran concentrations on the rate of major bleeding have been raised.1 Clinical and basic aspects of DTIs aid in studying how best to address concerns regarding bleeding risk in therapeutic use. Based on enzymatic examinations with a synthetic substrate, S-2238, it has been well recognised that dabigatran and another DTI argatroban reversibly and competitively inhibit thrombin reaction.2 ,3 However, it still remains unclear whether the type of inhibition by DTIs of thrombin reaction with S-2238 can be applied to the anticoagulant effects. The Clauss assay is classical but most popular even now for quantification of plasma fibrinogen concentrations.4 This is based on the quantitative relationship between fibrinogen concentrations and time for fibrin clot formation by thrombin. As a high concentration of thrombin ranging from 40.25 to 115 IU/mL is added to dilute test plasma, the clotting time depends exclusively on concentrations of fibrinogen but not of other coagulation factors. The Lineweaver–Burk plot, which is one of classical linear transformations of the Michaelis–Menten equation for enzymatic reaction kinetics, presents curve-linearity between the reciprocal of the substrate concentration versus the reciprocal of the rate of reaction.5 When used for determining the type of enzyme inhibition, this plot can distinguish competitive, uncompetitive, non-competitive and mixed inhibitors. To assess how DTIs affect in vitro clotting from the enzymatic perspective, the Lineweaver–Burk plot was …