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The mechanosensory hair cells of the inner ear are required for hearing and balance and have a distinctive apical structure, the hair bundle, that converts mechanical stimuli into electrical signals. This structure comprises a single cilium, the kinocilium, lying adjacent to an ensemble of actin-based projections known as stereocilia. Hair bundle polarity depends on kinociliary protocadherin-15 (Pcdh15) localization. Protocadherin-15 is found only in hair-cell kinocilia, and is not localized to the primary cilia of adjacent supporting cells. Thus, Pcdh15 must be specifically targeted and trafficked into the hair-cell kinocilium. Here we show that kinocilial Pcdh15 trafficking relies on cell type-specific coupling to the generic intraflagellar transport (IFT) transport mechanism. We uncover a role for fibroblast growth factor receptor 1 (FGFR1) in loading Pcdh15 onto kinociliary transport particles in hair cells. We find that on activation, FGFR1 binds and phosphorylates Pcdh15. Moreover, we find a previously uncharacterized role for clathrin in coupling this kinocilia-specific cargo with the anterograde IFT-B complex through the adaptor, DAB2. Our results identify a modified ciliary transport pathway used for Pcdh15 transport into the cilium of the inner ear hair cell and coordinated by FGFR1 activity.

In addition to well-known curcuminoids, three colored metabolites were isolated from cultured cell clumps that had been induced from buds on turmeric rhizomes. The isolated compounds were identified as dihydro derivatives of curcuminoids, dihydrocurcumin (dihydroCurc), dihydrodesmethoxycurcumin-a (dihydroDMC-a), and dihydrobisdesmethoxycurcumin (dihydroBDMC). The cell clumps did not contain dihydroDMC-b, an isomer of dihydroDMC-a. A comparison of the distribution profiles of curcuminoids and dihydrocurcuminoids in the cell clumps with those in the rhizomes, leaves, and roots revealed the following differences: Unlike rhizomes, the cell clumps, leaves, and roots contained dihydrocurcuminoids as the major colored constituents. Whereas dimethoxy compounds, curcumin and dihydrocurcumin, respectively, were most abundant in the rhizomes and leaves, one of the monomethoxy derivatives, dihydroDMC-a, was found most abundantly in the cell clumps and roots. While both dihydroDMC-a and b were detected in the rhizomes, dihydroDMC-b was not detectable in the cell clumps, leaves, or roots. The occurrence of only one of the two possible isomers of dihydroDMC suggests biosynthetic formation of dihydrocurcuminoids in turmeric.

Curcuminoids found in the rhizome of turmeric, Curcuma longa, possess various biological activities. Despite much attention regarding the biosynthesis of curcuminoids because of their pharmaceutically important properties and biosynthetically intriguing structures, no enzyme systems have been elucidated. Here we propose a pathway for curcuminoid biosynthesis in the herb C. longa, which includes two novel type III polyketide synthases. One of the type III polyketide synthases, named diketide-CoA synthase (DCS), catalyzed the formation of feruloyldiketide-CoA by condensing feruloyl-CoA and malonyl-CoA. The other, named curcumin synthase (CURS), catalyzed the in vitro formation of curcuminoids from cinnamoyldiketide-N-acetylcysteamine (a mimic of the CoA ester) and feruloyl-CoA. Co-incubation of DCS and CURS in the presence of feruloyl-CoA and malonyl-CoA yielded curcumin at high efficiency, although CURS itself possessed low activity for the synthesis of curcumin from feruloyl-CoA and malonyl-CoA. These findings thus revealed the curcumin biosynthetic route in turmeric, in which DCS synthesizes feruloyldiketide-CoA, and CURS then converts the diketide-CoA esters into a curcuminoid scaffold.

In order to investigate the biosynthesis of curcuminoid in rhizomes of turmeric (Curcuma longa), we established an in vitro culture system of turmeric plants for feeding 13C-labeled precursors. Analyses of labeled desmethoxycurcumin (DMC), an unsymmetrical curcuminoid, by 13C-NMR, revealed that one molecule of acetic acid or malonic acid and two molecules of phenylalanine or phenylpropanoids, but not tyrosine, were incorporated into DMC. The incorporation efficiencies of the same precursors into DMC and curcumin were similar, and were in the order malonic acid > acetic acid, and cinnamic acid > p-coumaric acid >> ferulic acid. These results suggest the possibility that the pathway to curcuminoids utilized two cinnamoyl CoAs and one malonyl CoA, and that hydroxy- and methoxy-functional groups on the aromatic rings were introduced after the formation of the curcuminoid skeleton.

We study transport properties of the orbital Kondo effect via single quantum dot systems with plural electrons by applying the noncrossing approximation to the Anderson impurity model with orbitals. Investigating the case for the half filling of the quantum dot with three orbitals, we discuss the effects of the orbital level splitting on the conductance and the thermopower.

We study the orbital Kondo effect due to two electrons occupying three orbital levels in quantum dot (QD) systems. By applying the non-crossing approximation to the three-orbital Anderson impurity model, we calculate the conductance and the thermopower. We consider the realistic situation in QD systems, where the correlation effect induces the orbital-level splitting even at zero field. It is found that the level splitting at zero field larger than temperature revives the maximum structure in the conductance clearly, which characterizes the singlet-triplet Kondo effect. We also show that the enhancement of the Kondo temperature due to the singlet-triplet mechanism gives rise to the characteristic behavior in the thermopower as well. This is in contrast with the two-orbital case, where the thermopower is zero irrespective of the level splitting. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Objectives: This study aimed to investigate the efficacy of encapsulating therapeutic molecules in poly lactic/glycolic acid (PLGA) nanoparticles for drug delivery to the cochlea. Study Design: An experimental study. Methods: We examined the distribution of rhodamine, a fluorescent dye, in the cochlea, liver, and kidney of guinea pigs. Intravenous injection of rhodamine or rhodamine-encapsulated PLGA nanoparticles was used to target the fluorescent dye systemically to the liver, kidney, and cochlea, and these molecules were applied locally to the round window membrane (RWM) of the cochlea. The localization of rhodamine fluorescence in each region was quantitatively analyzed. Results: After systemic application of rhodamine nanoparticles, fluorescence was identified in the liver, kidney, and cochlea. The systemic application of nanoparticles had a significant effect on targeted and sustained delivery of rhodamine to the liver but not the kidney or cochlea. Rhodamine nanoparticles placed on the RWM were identified in the scala tympani as nanoparticles, indicating that the PLGA nanoparticles can permeate through the RWM. Furthermore, the local application of rhodamine nanoparticles to the RWM was more effective in targeted delivery to the cochlea than systemic application. Conclusions: These findings indicate that PLGA nanoparticles can be an useful drug carrier to the cochlea via local application.

The multidrug transporter, MDR1-mediated interaction of digoxin with antiarrhythmic or antianginal drugs was examined in vitro by using the MDR1-overexpressing LLC-GA5-COL150 cells, which were established by transfection with human MDR1 cDNA into porcine kidney epithelial LLC-PK(1) cells. Amiodarone, its active metabolite monodesethyl-amiodarone (DEA), and quinidine markedly inhibited the basal-to-apical transport (renal secretion) of [(3)H]digoxin and increased the apical-to-basal transport (reabsorption), but cibenzoline and lidocaine showed slight inhibition of the transport, and disopyramide and mexiletin had no such effects. The IC(50) values for amiodarone, DEA and quinidine on [(3)H]digoxin transport in LLC-GA5-COL150 cells were 5.48 microM, 1.27 microM and 9.52 microM, respectively. These were comparable to, or only several times the achievable concentration in clinical use, suggesting that MDR1 could be responsible for the drug interaction between digoxin and amiodarone found in clinical reports and that DEA contributes the elevation of digoxin serum concentration. Similarly, dipyridamole altered the transport, but isosorbide showed only slight modification of the transport. The IC(50) value for dipyridamole was 40.0 microM, also only several times the achievable concentration in clinical use, indicating a risk of interaction.

In anti-Helicobacter pylori therapy using omeprazole and antimicrobials, the efficacy can be related to the CYP2C19 genotype groups; the eradication rates were 83% in extensive metabolizers and 100% in poor metabolizers. The present study was undertaken to help predict the optimal dosage of omeprazole for extensive metabolizers in this therapy. Seven healthy Japanese subjects, classified based on the CYP2C19 genotype into extensive metabolizers (n=4) and poor metabolizers (n=3), participated in this study. Each subject received a single oral dose of omeprazole 20, 40, and 80 mg, with at least a 1-week washout period between each dose. Plasma concentrations of omeprazole and its two metabolites were monitored for 12 h after each dose of medication. After each dose was administered, the pharmacokinetic profiles of omeprazole and its two metabolites were significantly different between extensive metabolizers and poor metabolizers. The area under the plasma concentration-time curve (AUC) of omeprazole in extensive metabolizers was disproportionally increased 3.2- or 19.2-fold with dose escalation from 20 to 40 or 80 mg omeprazole, respectively. In contrast, the AUC of omeprazole was proportionally increased with the higher dose in poor metabolizers. The AUC of omeprazole after 20 mg administration to poor metabolizers was almost equal to the AUC in extensive metabolizers after 80 mg administration. In anti-H. pylori therapy, the recommended dose of omeprazole for extensive metabolizers is suggested to be a maximum of 80 mg x 2/d based on pharmacokinetic considerations.

Genotyping of polymorphic drug metabolizing enzymes may be useful to estimate the blood concentration, efficacy, and toxicity of drugs before administration. Blood samples are most generally used for genotyping; however, sampling is invasive and complicated by handling and transport. Therefore, the authors developed genotyping methods using nonblood specimens, and then each genotype was compared with that from blood. Healthy Japanese volunteers provided hairs (n = 50), buccal cell swabs (n = 50), and fingernails (n = 30) for N-acetyltransferase 2 and CYP2C19 genotyping. Recovery of genomic DNA from each nonblood specimen was lower than that from 0.5 mL blood. Using a modification of the DNA extraction and polymerase chain reaction amplification method, genotypes were diagnosed without failure, even for those with very low levels of DNA. Both genotypes from these specimens completely matched the genotypes from the blood of the same subject. These nonblood specimens can be convenient, accessible, and economical alternatives to blood as a source of DNA for genotyping.

Purpose. To predict the CYP2C19 genotype-dependence in anti-Helicobacter pylori(H. pylori) therapy when lansoprazole or rabeprazole was used instead of omeprazole as a proton pump inhibitor (PPI).

Sulfapyridine (SP) is metabolized by polymorphic N-acetyltransferase 2 (NAT2) [EC 2.3.1.5]. In this study, the correlation between the NAT2 genotype and the pharmacokinetics of SP after multiple oral dosing of sulfasalazine (SASP) was examined to elucidate the effect of multiple dosing on the predictability of the phenotype by NAT2 genotyping. Seven healthy subjects were classified into two groups; the homozygotes for the wild-type allele, NAT2*4/*4 (Group I) and the compound heterozygotes for the mutant allele (NAT2*4/*6A or NAT2*4/*7B) (Group II). All received once-daily 1 g of SASP (Salazopyrin®) orally for 8 d. Plasma concentrations and urinary recoveries of SASP, SP and N-acetylsulfapyridine (AcSP) were monitored for 8 d. At 24 h on Day 1, the plasma concentration of SASP was lower and those of SP and AcSP were higher in Group II compared with Group I, but there was no significant difference. The plasma concentration ratio of AcSP to SP (AcSP/SP) tended to be lower in Group II. Urinary recoveries of SP and AcSP were increased in Group II, and their ratio was slightly reduced in Group II. Multiple dosing for 8 d resulted in an increase in the plasma concentrations of SASP, SP and AcSP. The difference between Group I and II was marked compared with single dosing, resulting in a significant difference in the plasma concentration of SP and the ratio of AcSP/SP. The simple input-output pharmacokinetic model applied for the analysis of plasma concentrations and urinary recoveries of SP and AcSP suggested the acetylation of SP into AcSP was 2.7-fold reduced in Group II (p=0.064).

Purpose. A combination of proton pump inhibitors and antimicrobials has been applied as an anti–Helicobacter pylori (H. pylori) therapy. Omeprazole, one of the proton pump inhibitors, is metabolized by CYP2C19, which exhibits genetic polymorphism. It was reported previously that the overall anti–H. pylori efficacy can be related to the CYP2C19 genotype. The main aim of the present study was to obtain a rational explanation for the relationship between the overall anti–H. pylori efficacy and the CYP2C19 genotype.

Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in our hospital. The HQ ointments were highly effective in the treatment of various types of skin pigmentations; however, various problems have emerged including chromatic aberration of the ointments, a relatively large variability of efficacy, and mild side effects. Chromatic aberration is expected to induce non-compliance, and this may be the reason for the relatively large variability in efficacy. In this paper, the effects of various storage conditions on the chromatic aberration and HQ content of HQ ointments were evaluated, and it was suggested that the chromatic aberration was accelerated by exposure to high temperature, air and light, although these had no effect on the HQ content. In addition, various types of HQ ointments were prepared to find a formulation to minimize chromatic aberration, and it was found that the concentrations of antioxidants, Na(2)SO(3) and L(+)-ascorbic acid (AsA), seemed to be too high, and that the protective effect of AsA on chromatic aberration was mainly due to its acidifying effect.

Two different samples of serum were prepared from a blood specimen by using two types of serum-separation tubes. Each serum sample was diluted by 100 times with deionized and sub-boiling distilled water. Thirty-six elements in the serum and exudate, from devices used for the serum preparation, were determined by inductively coupled plasma mass spectrometry (ICP-MS). From the comparison of elemental concentrations in two groups of 11 serum samples, it was found that the exudate from serum-separation tubes, as well as from disposable stainless steel needles, had serious effects on the elemental concentrations in the serum. Means for the concentrations of Li, Co, Ga, Cd, Sn, Sb, Ba, La, and Ce in the two serum groups were statistically different from each other. The concentrations of Cr, Mn, Fe, and Mo in both groups were apparently higher than those reported in the literature, suggesting contamination from the disposable stainless steel needle and spectral interferences due to molecular species produced in the argon plasma.

The generation of superoxide has been implicated as a cause of cochlear damage from excessive noise. Cu/Zn superoxide dismutase (SOD1) generally will protect against superoxide-mediated tissue injury but protection by this enzyme against noise trauma is controversial. This study assessed auditory function in C57BL/6 mice overexpressing SOD1 or treated with lecithinized SOD1 (PC-SOD1). Noise exposure caused significantly higher threshold shifts in PC-SOD1-treated animals than physiological saline-treated animals. Cochlear tissues of PC-SOD1-treated animals exhibited significant elevation of the levels in the SOD activity, not in the catalase activity, in comparison with those of saline-treated animals. Likewise, transgenic mice overexpressing SOD1 tended to suffer higher threshold shifts than nontransgenic littermates from noise exposure. The findings indicate that increasing SOD1 enhances auditory dysfunction following noise exposure.

Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in our hospital. The HQ ointments were highly effective in the treatment of various types of skin pigmentations; however, various problems have emerged including chromatic aberration of the ointments, a relatively large variability of efficacy, and mild topical side effects including irritation. In this paper, the cytotoxicity of HQ was assessed in vitro using rat skin fibroblasts as the concentration with 50% survival after 24 h exposure to be 16.5 microM. The intradermal concentrations at 2 h after application of the HQ ointments was also estimated to be 358 mM and 51.7 mM in stratum corneum and viable tissue (viable epidermis+dermis), respectively, by an in vitro rat skin permeation study with rat full-thickness abdominal skin and Franz-type diffusion cells. It was demonstrated that the intradermal concentration of HQ was much higher than that eliciting cytotoxicity, suggesting that the topical side effects after application of HQ ointment were due to the cytotoxicity of HQ.

A series of clinical studies on the cytochrome P450 2C19 (CYP2C19) genotype and the pharmacokinetics and pharmacodynamics of three proton pump inhibitors (PPIs), omeprazole, lansoprazole and rabeprazole, have been conducted to establish the individualized pharmacotherapy based on the CYP2C19 genotyping, and in the present study, the CYP2C19 genotype-dependency was more pronounced for omeprazole than the other two. Herein, to validate further the difference among 3 PPIs in CYP2C19 genotype-dependency on the phenotype, a comparative in vitro study was conducted using the human liver microsomes and newly developed anti-human CYP antibodies. The residual concentrations of omeprazole and lansoprazole in 5 lots of human liver microsomes were dependent on the CYP2C19 activities, however, for rabeprazole, there was no correlation. The hydroxylation of omeprazole was more inhibited by anti-CYP2C19 antibody than lansoprazole, whereas anti-CYP3A4 antibody showed similar inhibition. In conclusion, the relative contribution of CYP2C19 on total metabolism of 3 PPIs elucidated herein coincided with the CYP2C19 genotype-dependent pharmacokinetics.

Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in Japan by imitating skin lightening creams commercially available in the U.S.A. and European Union. In our hospital, HQ ointments consisting of 5 or 10% HQ, 1.6% L(+)-ascorbic acid (AsA), 0.5% (w/w) Na2SO3, 10% (v/w) glycerin and hydrophilic ointment have been prepared. However, various problems have been observed including chromatic aberration of HQ ointments, relatively large variability of efficacy, and undesirable side effects although they were mild. Herein, the pharmaceutical and clinical properties of the HQ ointments were evaluated. HQ ointments were highly effective for treatment of various types of skin pigmentation. Chromatic aberration occurred during 3 months of storage, but this could be suppressed by storage at 4 degrees C. Chromatic aberration was independent of prescribed HQ content, and was not explained by alterations of HQ or p-benzoquinone (p-BQ) contents. Unexpectedly, removal of both antioxidants resulted in suppression of chromatic aberration, but an increase in p-BQ content. Acidification by removal of Na2SO3 only was further effective for the suppression of chromatic aberration, but with a decrease of p-BQ content except in the initial period. Chromatic aberration was due to water soluble material and insoluble material both formed by co-existence of HQ and p-BQ at a molecular ratio of 5:3 to 1:1. 1H-NMR analysis elucidated that the water soluble material was not HQ or p-BQ, and the insoluble material was a complex of HQ and p-BQ with non-covalent binding.

The serum levels of 18 constituents of 153 small cetaceans of both sexes from North-Western Pacific waters were measured. No sex differences were observed in any biochemical parameters in mature animals, but the serum levels of alkaline phosphatase (Alp), P, Na, and Cl were found to be age-related. The increase in serum Alp activity in younger animals seemed to originate from bone. Comparing the levels with those in humans there was (1) no difference in the levels of total protein, albumin, creatinine, total cholesterol, and γ-glutamyltranspeptidase, (2) a lower level of uric acid, and (3) higher levels of serum urea nitrogen, aspartate aminotransferase, alanine aminotransferase, lactic dehydrogenase, Alp, creatine kinase, Na, K, Cl, Ca, and P.

ICP-AESにより得られた男性透析患者及び男性健常者の頭髪中の元素濃度の解析を行った.頭髪中の元素濃度が非常に多くの因子により影響を受けるため,平均値の有意差検定から得られる情報が必ずしもその目的としている集団の特徴を示さない場合のあることを示唆した.又,元素間の相関を詳しく調べ,透析患者と健常者の両方でMg-Ca,Mg-Sr,Ca-Srの相関(いずれもp

Six patients were found to have increased serum chloride concentrations when these concentrations were determined with an ion-selective electrode, but not when determined by continuous flow mercuric thiocyanate colorimetry or amperometric-coulometric titration. Their serum bromide levels of 1.8-8.0 mmol/l were much higher than those of 0.07-0.13 mmol/l in normal controls. The urinary bromide excretion, measured in two of these patients, was higher than that in normal subjects. No common symptoms or abnormalities in laboratory findings except hyperbromidaemia were found in these patients, who claimed not to have taken any drugs containing bromide. For determination of the incidence of subclinical hyperbromidaemia, the serum bromide concentrations were measured in sera of 1,323 outpatients sent to Tokushima University Hospital for routine measurements of blood chemistry over a one-month period. Five samples showed abnormally high bromide levels. It is concluded that subclinical hyperbromidaemia is not as rare as generally thought, though the aetiology of this state is unknown. Chloride determination with an ion-selective electrode can be used to screen for hyperbromidaemia, since increased levels of bromide ion result in apparently high chloride values.

ICP-AESを用いて,血清中の9元素(Mg, Ca,, Fe,Cu, Zn, P, Si, Na, K)を同時測定するための前処理法として除タンパク法を検討した.除タンパク試薬としては,血清と混合後の終濃度が,硝酸は0.28M,トリクロロ酢酸は5.0%になるような混液が適当であることが分かった.本法による前処理は,血清0.5mlに精製水1.5mlを加えて混合し,これに除タンパク試薬4.0mlを加えてかき混ぜ,放置後遠心分離し,その上清をICP-AESの検液とする.前処理法として,よく使用されているテフロン密閉容器を用いた分解法との比較,又,自動化学分析機における測定値との比較を行い,本法は,ICP-AESで測定する血清試料の前処理法として有用な方法であることが分かった.本法を用いて,健常者や透析患者血清の前処理を行い,満足すべき結果を得た.

Curcuminoids are pharmaceutically important compounds isolated from the herb Curcuma longa. Two additional type III polyketide synthases, named CURS2 and CURS3, that are capable of curcuminoid synthesis were identified and characterized. In vitro analysis revealed that CURS2 preferred feruloyl-CoA as a starter substrate and CURS3 preferred both feruloyl-CoA and p-coumaroyl-CoA. These results suggested that CURS2 synthesizes curcumin or demethoxycurcumin and CURS3 synthesizes curcumin, bisdemethoxycurcumin and demethoxycurcumin. The availability of the substrates and the expression levels of the three different enzymes capable of curcuminoid synthesis with different substrate specificities might influence the composition of curcuminoids in the turmeric and in different cultivars.