Kayoko Saiki

Molecular Identification and Characterization of Novel Membrane-bound Metalloprotease, the Soluble Secreted Form of Which Hydrolyzes a Variety of Vasoactive Peptides

One class of zinc metalloproteases, represented by neutral endopeptidase 24.11 and endothelin-converting enzyme, has been shown to be involved in proteolytic activation or inactivation of many regulatory peptides. Here, we report molecular cloning and characterization of a novel member of this type II membrane-bound metalloprotease family, termed soluble secreted endopeptidase (SEP). Alternative splicing results in the generation of another transcript, SEPΔ, which lacks a 69-base pair nucleotide segment following the transmembrane helix. Both SEP and SEPΔmRNA are detected in all mouse tissues examined. Transfection of an SEP cDNA expression construct resulted in the expression of the membrane-bound form of SEP in the early secretory pathway as well as the soluble secreted form of the enzyme in the culture medium. In contrast, transfection of the SEPΔ cDNA only results in the expression of the membrane-bound form. In vitroenzymological analysis of the recombinant soluble form of SEP demonstrated that it hydrolyzes a variety of vasoactive peptides, including endothelin-1, atrial natriuretic peptide, and angiotensin I. This activity of SEP was inhibited by phosphoramidon and the neutral endopeptidase 24.11 specific inhibitor thiorphan, but it was only partially inhibited by the endothelin-converting enzyme specific inhibitor FR901533. These findings suggest that SEP is a novel metalloprotease that possesses a broad substrate specificity and that it may be involved in the metabolism of biologically active peptides intracellulary as well as extracellularly.

Circadian variation of the urinary 6β-hydroxycortisol to cortisol ratio that would reflect hepatic CYP3A activity

AbstractObjectives: Chronopharmacokinetics of drugs metabolized by cytochrome P450 3A (CYP3A) has been reported recently; however, little is studied on intra-individual circadian variation in CYP3A activity in human. The aim of this study was to assess the intra-individual diurnal variation and day-to-day variation of the urinary 6β-hydroxycortisol to cortisol ratio, a noninvasive index of human CYP3A activity. Methods: Urine samples from ten healthy Japanese men were collected over four time intervals (0900 hours to 1300 hours, 1300 hours to 1700 hours, 1700 hours to 2100 hours and 2100 hours to 0900 hours) on days 1, 5 and 14 to verify diurnal variation, and 24-h urine was collected to study day-to-day variation over 2 weeks. Urinary 6β-hydroxycortisol and cortisol were determined by means of high-performance liquid chromatography/atmospheric pressure chemical ionization–mass spectrometry. Results: The ratio of urinary 6β-hydroxycortisol to cortisol exhibited noteworthy diurnal variation intra-individually; 2.8-fold on average. However, day-do-day intra-individual variation of the ratio was not observed over 2 weeks; the coefficient of variation was 11.9 ± 3.0%. Conclusion: The result indicates that imprudent use of random urine has a great risk of false evaluation in assessment of the 6β-hydroxycortisol to cortisol ratio and that the ratio in 24-h urine samples provides a more robust measure of the inter-individual difference of this metabolic ratio, which to a certain but not complete extent represents the CYP3A activity.

Specific determination of urinary 6β-hydroxycortisol and cortisol by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

The urinary 6beta-hydroxycortisol to cortisol ratio is believed to be a noninvasive index of cytochrome P450 3A activity. For precise assessment of the ratio in human urine, we have developed a reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry method. The selective method was accurate and reproducible with intra- and inter-day precision of variation coefficients of less than 8%. The 6beta-hydroxycortisol to cortisol ratio ranged from 3.0 to 12.4 in healthy Japanese 24-h urine. With the recent popularization of LC-MS, our LC-MS method will be advantageous to detect human in vivo CYP3A activity for clinical investigation and routine measurement in various laboratories.

Co-occurrence of mutations in both dystrophin- and androgen-receptor genes is a novel cause of female Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder. Here, we report a novel mechanism for the occurrence of DMD in females. In a Vietnamese DMD girl, conventional PCR amplification analysis disclosed a deletion of exons 12–19 of the dystrophin gene on Xp21.2, with a karyotype of 46, XY. Furthermore, a novel mutation in the androgen-receptor gene on Xq11.2-q12 was identified in this girl, which led to male pseudohermaphroditism. Co-occurrence of mutations of these two genes constitutes a novel mechanism underlying female DMD.

Time-dependent Changes in the Carotenoid Composition and Preferential Binding of Spirilloxanthin to the Reaction Center and Anhydrorhodovibrin to the LH1 Antenna Complex in Rhodobium marinum ¶

Carotenoids were isolated from the cells of Rhodobium marinum, and their structures were determined by mass spectrometry and 1H nuclear magnetic resonance spectroscopy; the carotenoids include lycopene, rhodopin, anhydrorhodovibrin, rhodovibrin and spirilloxanthin. Time‐dependent changes in the carotenoid composition in the reaction center (RC) and the light‐harvesting complex 1 (LH1) were traced by high‐performance liquid chromatography analysis of the extracts. The carotenoid composition changed according to the spirilloxanthin biosynthetic pathway. However, spirilloxanthin having the longest conjugated chain was always preferentially bound to the RC, and anhydrorhodovibrin and other precursors to the LH1.

Changes in regional cerebral blood volume in frontal cortex during mental work with and without caffeine intake: functional monitoring using near-infrared spectroscopy

Near-infrared spectroscopy (NIRS) was used to measure frontal regional cerebral blood volume (rCBV) in a person whose brain was under the influence of pharmacological agents while the person was performing a complex task. Fourteen healthy participants were administered Uchida-Kraepelin psychodiagnostic (UKP) tests before and after caffeine intake, and the concentration of caffeine in the urine was measured. The average number of answers and the average number of correct answers given by the participants improved significantly following caffeine intake. During the UKP testing, changes in the rCBV in the inferior frontal cortex were continuously measured using NIRS. The volume during the rest periods decreased as a result of caffeine-induced constriction of the cerebral arteriola. The volume increased during the mental work, but the degree of the increase was the same before and after caffeine intake. Although the performance of the mental work improved following caffeine intake, the improvement was not reflected in the rCBV in the inferior frontal cerebral cortex. These results suggest that caffeine helps to protect the brain from excessive hyperemia in addition to activating the neurons in the prefrontal cortex.

Palladium-catalyzed carbonylative coupling of iodobenzene and 2-methyl-3-butyn-2-ol under biphasic conditions: Formation of furanones

Abstract Carbonylative coupling of iodobenzene and 2-methyl-3-butyn-2-ol ( 4 ) in aqueous NaOH/benzene was carried out by using Pd(OAc) 2 /Ph 3 P/Bu 4 PBr ( A ) and Pd(OAc) 2 /Ph 2 P(m-C 6 H 4 SO 3 Na) ( B ) as catalyst. In sharp contrast with a homogeneous Et 3 N solution, this biphasic solvent system gives 3-isopropylidene-5-phenyl-2(2H)-furanone ( 1 ) in a moderate yield. The other carbonylated products are 2,2-dimethyl-5-phenyl-3(2H)-furanone ( 2 ) and benzoic acid. The formation of 1 is explained by the following sequential reactions; carbonylative coupling of iodobenzene with 4 to form 4-hydroxy-4-methyl-1-phenyl-2-pentyn-1-one ( 6 ), hydrogenolysis of 6 to 4-methyl-1-phenyl-2,3-pentadien-1-one ( 8 ) and its cyclocarbonylation to 1 . Catalyst A gives 1 in a higher yield than B .

In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G→A mutations in introns of the dystrophin gene

Background: Splicing patterns are critical for assessing clinical phenotype of mutations in the dystrophin gene. However, it is still unclear how to predict alternative splicing pathways in such cases of splice-site mutation in the dystrophin gene. Objective: To identify elements determining alternative splicing pathways in intron +1G→A mutations of the dystrophin gene. Results: We found that exon 25 is spliced out in the +1G→A mutation in intron 25, resulting in mild Becker muscular dystrophy, and that a cryptic splice site within exon 45 was activated in severe Duchenne muscular dystrophy with a mutation of +1G→A mutation in 45. Furthermore, in vitro splicing analysis using a pre-constructed expression vector showed that the mutant intron 25 produced one transcript that lacked exon 25. In contrast, the same splice-site mutation in intron 45 produced three splicing products. One product used the same cryptic donor splice site within exon 45 as the in vivo donor site and another product used a cryptic splice site within the vector sequence. Notably, the available cryptic splice site was not activated by the same G→A mutation of intron 25. Conclusion: It was concluded that sequences inserted into the in vitro splicing assay minigene contain cis-elements that determine splicing pathways. By taking other +1G→A mutations in the introns of the dystrophin gene reported in the literature into consideration, it seems that cryptic splice-site activation is seen only in strong exons. This finding will help to elucidate the molecular pathogenesis of dystrophinopathy and to predict efficiency of induction of exon skipping with antisense oligonucleotides for treatment of Duchenne muscular dystrophy.

Blood Lysophosphatidylcholine (LPC) Levels and Characteristic Molecular Species in Neonates: Prolonged Low Blood LPC Levels in Very Low Birth Weight Infants

Lysophosphatidylcholine (LPC) has various stimulatory effects on many types of immune cells. The purpose of our study was to characterize blood LPC levels and to determine the composition of LPC molecular species (LPCs) in the neonatal period. Thirty-six neonates were enrolled in this study and then grouped according to birth-weight as follows: non-very low birth weight (NVLBW); ≥1,500 g (n = 17), and very low birth weight (VLBW); <1,500 g (n = 19). Sixteen healthy normal adults were used as controls. Levels of total blood LPC and LPCs (16:0-, 18:0-, 18:1-, 18:2-, and 20:4-LPC species) were measured using HPLC coupled with tandem mass spectrometry. Total blood LPC levels at birth in neonates in both groups (NVLBW and VLBW) were significantly lower than those of adult levels. In NVLBW infants, LPC levels reached adult levels at postnatal day 3 compared with VLBW infants, who attained adult levels after postnatal day 57 (around full-term). The composition of the LPCs was different not only between neonates and adults, but between NVLBW and VLBW infants. These findings may be associated with the difference of immunity among adults, NVLBW, and VLBW infants.

Determination of 1-methyl-1,2,3,4-tetrahydroisoquinoline in mouse brain after treatment with haloperidol by gas chromatography–selected ion monitoring

The content of the endogenous amine, 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ), in mouse brain, treated with the antipsychotic agent haloperidol (HP) was determined by GC-SIM (selected ion monitoring) system. 1-MeTIQ in brain was extracted with chloroform at pH 11-12 and was detected as PFP derivative by GC-SIM. The 1-MeTIQ contents in mouse brains following intraperitoneal administration of HP or its dehydrated product, HPTP (1 and 4 mg/kg per day, for four days), were markedly reduced compared with control groups. This result agrees well with the findings in human idiopathic parkinsonianism and in MPTP-treated mouse brain. In addition, this finding suggests that the change of the endogenous amine 1-MeTIQ content in the brain plays an important role in the pathogenesis of toxin-induced parkinsonism.