Yukihisa Kurono

Esterase-Like Activity of Serum Albumin: Characterization of Its Structural Chemistry Using p-Nitrophenyl Esters as Substrates

AbstractPurpose. To elucidate the catalytic mechanism of the esterase-like activity of serum albumin (SA), the reactivity of SA from six species was investigated using p-nitrophenyl esters as model substrates. Methods. The effect of pH and the energetic and thermodynamic profiles of SA were determined for all species for p-nitrophenyl acetate (PNPA). Then, kinetic and thermodynamic studies using a series of p- and o-nitrophenyl esters with different side chains and human SA (HSA) were carried out. The influence of deuterium oxide was also evaluated. Finally, the information gained was used to construct a computer model of the structural chemistry of the reaction. Results. The pH profiles suggest that the nucleophilic character of the catalytic residue (Tyr-411 in the case of HSA) is essential for activity. This kcat-dependent activity was found to increase with a decrease in the activation free energy change (ΔG). Hence, the magnitude of ΔG, which is dependent on activation entropy change (ΔS), as calculated from the thermodynamic analysis, can be regarded as an indicator of hydrolytic activity. It indicates that p-nitrophenyl propionate (PNPP) is the best substrate by evaluating the reactions of nitrophenyl esters with HSA. The findings here indicate that deuterium oxide has no significant effect on the rate of hydrolysis of PNPA by HSA. Conclusions. The results are consistent with a scenario in which HSA becomes acylated due to a nucleophilic attack by Tyr-411 on the substrate and then is deacylated by general acid or base catalysis with the participation of water.

PEN‐2 enhances γ‐cleavage after presenilin heterodimer formation

The presenilin (PS) complex, including PS, nicastrin, APH‐1 and PEN‐2, is essential for γ‐secretase activity, which is required for amyloid β‐protein (Aβ) generation. However, the precise individual roles of the three cofactors in the PS complex in Aβ generation remain to be clarified. Here, to distinguish the roles of PS cofactors in γ‐secretase activity from those in PS endoproteolysis, we investigated their roles in the γ‐secretase activity reconstituted by the coexpression of PS N‐ and C‐terminal fragments (NTF and CTF) in PS‐null cells. We demonstrate that the coexpression of PS1 NTF and CTF forms the heterodimer and restores Aβ generation in PS‐null cells. The generation of Aβ was saturable at a certain expression level of PS1 NTF/CTF, while the overexpression of PEN‐2 alone resulted in a further increase in Aβ generation. Although PEN‐2 did not enhance PS1 NTF/CTF heterodimer formation, PEN‐2 expression reduced the IC50 of a specific γ‐secretase inhibitor, a transition state analogue, for Aβ generation, suggesting that PEN‐2 expression enhances the affinity or the accessibility of the substrate to the catalytic site. Thus, our results strongly suggest that PEN‐2 is not only an essential component of the γ‐secretase complex but also an enhancer of γ‐cleavage after PS heterodimer formation.

An efficient synthesis of C20–C25 building blocks for calyculin A

Abstract The C 20 –C 25 building blocks 2 and 3a for calyculin A, a protein phosphatase inhibitor, have been efficiently prepared from L-malic acid utilizing the Grignard reaction of the Weinreb amides 8 and 14 , followed by stereoselective reduction of the ketones 9 and 15 , respectively, as the key steps.

Synthesis of new sugar derivatives and evaluation of their antibacterial activities against Mycobacterium tuberculosis.

A series of sugar derivatives (1-13) were synthesized and evaluated for antibacterial activity against Mycobacteriumtuberculosis (MTB), especially multi-drug resistant (MDR) MTB, and the structure-activity relationships of these compounds were studied. The results showed that the compound OCT313 (2-acetamido-2deoxy-beta-D-glucopyranosyl N,N-dimethyldithiocarbamate) (4) exhibited significant in vitro bactericidal activity, and that the dithiocarbamate group at C-1 position of the glucopyranoside ring was requisite for the antibacterial activity.

Molecular assembly of covalently-linked mesoporphyrin dimers with light-harvesting polypeptides

Abstract Light-harvesting (LH)-α and -β polypeptides separately isolated from photosynthetic bacteria, R. rubrum , organized mesoporphyrin dimers ( Scheme 1 ) in the n -octyl-β- d -glucopyranoside (OG) micelle, depending upon the porphyrin structure and temperature. An efficient energy transfer from Zn porphyrin to Ni porphyrin in 7 due to the presence of the LH polypeptides was observed.

Esterase-like activity of human serum albumin: Enantioselectivity in the burst phase of reaction with p-nitrophenyl α-methoxyphenyl acetate

Enantioselectivity in the burst phase of the reactions of D- and L-p-nitrophenyl alpha-methoxyphenyl acetates with human and bovine serum albumin was investigated kinetically in pH 7.4 phosphate buffer and at 25 degrees C. The burst phase was measured under the conditions of excess albumin over the enantiomer. Both albumins reacted with D enantiomer about threefold faster than L enantiomer, mainly due to the catalytic step and not due to the binding step. The reactivity of human serum albumin toward the enantiomers was four- to fivefold higher than that of bovine serum albumin.

Evaluation of valproate effects on acylcarnitine in epileptic children by LC-MS/MS

BACKGROUND Valproate (VPA) is a simple fatty acid and a substrate for the fatty acid β-oxidation pathway. Previous data suggested that the toxicity of VPA may be provoked by carnitine deficiency and the inhibition of mitochondrial β-oxidation. OBJECTIVE The aim of the present study was to elucidate the effect of VPA treatment on carnitine and isomer-differentiated acylcarnitine disposition, and determined the relationships between acylcarnitines and blood VPA levels in long-term treated patients with VPA and/or other antiepileptic drugs. METHODS Serum samples were obtained from children aged 1-15 years old treated for at least 6 months with VPA alone (n=28) or VPA combined with other anticonvulsants (n=23) and untreated controls (n=23). Serum acylcarnitines were separated from their isomers and quantified using high-performance liquid chromatography-tandem mass spectrometry. RESULTS We found higher 3-hydroxyisovalerylcarnitine levels and trace amounts of valproylcarnitine in both VPA monotherapy and polytherapy patients. Other acylcarnitines, hexanoylcarnitine, C12, C14:1-carnitines and the ratio of long-chain acylcarnitine to free carnitine were also higher in VPA polytherapy individuals than in controls. VPA monotherapy does not result in decreases in free carnitine or in the accumulation of long-chain acylcarnitines. Blood VPA concentrations correlated positively with hexanoylcarnitine, C12, C14:1, C16:1, C18:1-carnitines in all VPA-treated children (n=51). CONCLUSION Long-term VPA treatment in pediatric patients could affect some specific acylcarnitines, which is enhanced by the concomitant use of other anticonvulsants, and the formation of valproylcarnitine alone seems insufficient to develop severe carnitine deficiency at therapeutic doses of VPA.

Reconstitution of γ-secretase by truncated presenilin (PS) fragments revealed that PS C-terminal transmembrane domain is critical for formation of γ-secretase complex

The presenilin (PS) complex, including PS, nicastrin (NCT), APH‐1 and PEN‐2, is essential for γ‐secretase activity. Previously, the PS C‐terminal tail was shown to be essential for γ‐secretase activity. Here, to further understand the precise mechanism underlying the activation of γ‐secretase regulated by PS cofactors, we focused on the role of the PS1 C‐terminal region including transmembrane domain (TM) 8 in γ‐secretase activity. For this purpose, we co‐expressed C‐terminally truncated PS1 (PS1ΔC) completely lacking γ‐secretase activity and the PS1 C‐terminal short fragment in PS‐null cells, because the successful reconstitution of γ‐secretase activity in PS‐null cells by the co‐expression of PS1ΔC and the PS1 C‐terminal short fragment would allow us to investigate the role of the PS1 C‐terminal region in γ‐secretase activity. We found that the exogenous expression of the PS1 C‐terminal short fragment with NCT and APH‐1 completely rescued a defect of the γ‐secretase activity of PS1ΔC in PS‐null cells. With this reconstitution system, we demonstrate that both TM8 and the PS1 C‐terminal seven‐amino‐acid‐residue tail are involved in the formation of the active γ‐secretase complex via the assembly of PS1 with NCT and APH‐1.

High Doses of Oseltamivir Phosphate Induce Acute Respiratory Arrest in Anaesthetized Rats

It has been reported that one of the serious adverse events after the treatment of oseltamivir phosphate (OP) for influenza patients is sudden death resulting from cardiorespiratory arrest. To investigate the aetiology of such an adverse consequence, we examined effects of OP (expressed as free base) on blood pressure and ventilation in anaesthetized rats with vagotomy. Intravenous OP (30–200 mg/kg) caused dose‐dependent hypotension and bradycardia in spontaneously breathing animals. Concomitantly with changes in blood pressure, the tracheal airflow increased. The ventilatory rate hastened during the injection and then transiently slowed around 1 min. after the administration (transient hypopnea). Thereafter, it gradually returned to control. The hypopnea increased with increasing dose and ventilatory arrest occurred at 200 mg/kg. Intraduodenal OP (500–1000 mg/kg) provoked cardioventilatory arrest 72–218 min. after the injection. Oseltamivir carboxylate (100–200 mg/kg, i.v.), an active metabolite of OP, had no significant effect on ventilation and blood pressure. In artificially ventilated animals, intravenous OP caused slowing of the respiratory rate around 1 min. after the injection in a dose‐dependent manner. This effect of OP waned in 5 min. after the administration. The amplitude of phrenic nerve discharge was not changed at lower doses (30–100 mg/kg). The phrenic nerve stopped to discharge immediately after higher doses (150–200 mg/kg). We demonstrated that OP causes central suppression of the respiratory function in rats and suggest a relationship between the OP‐induced cardiorespiratory arrest and sudden death observed in influenza patients after taking OP.

Novel mutations in the cytochrome P450 2C19 gene: a pitfall of the PCR-RFLP method for identifying a common mutation

AbstractCYP2C19 is a clinically important enzyme involved in the metabolism of therapeutic drugs such as (S)-mephenytoin, omeprazole, proguanil, and diazepam. Individuals can be characterized as either extensive metabolizers (EM) or poor metabolizers (PM) on the basis of CYP2C19 enzyme activity. The PM phenotype occurs in 2-5% of Caucasian populations, but at higher frequencies (18-23%) in Asians. CYP2C19*2 and CYP2C19*3, which are single-nucleotide polymorphisms of CYP2C19, are the main cause of PM phenotyping in homozygotes or compound heterozygotes. We report two novel mutations in the CYP2C19 gene identified by direct sequencing and subcloning procedures. One of these mutations was considered to be CYP2C19*3 by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). This result suggests that mutations classed as CYP2C19*3 might include other mutations. Further studies are needed to clarify the relationship between these novel mutations and enzyme activity.