Elucidation and Total Synthesis of the Correct Structures of Tridecapeptides Yaku'amides A and B. Synthesis-Driven Stereochemical Reassignment of Four Amino Acid Residues.

Yaku'amides A (1) and B (2) possess four α,ß-dehydroamino acid residues in their linear tridecapeptide sequence and differ in their residue-3 (Gly for 1 and Ala for 2). The highly unsaturated peptide structure, characteristic cytotoxicity profile, and extreme scarcity from natural sources motivated us to launch synthetic studies of 1 and 2. Here, we report the total synthesis of the originally proposed structure of yaku'amide B (2a) by applying the route to 1a, which was previously established in our group. However, this accomplishment only proved that 2a and natural 2 were structurally different and prompted investigations directed toward determining the true structure of 2. Extensive Marfey's analyses of minute amounts of natural 2 and its degradation products presented us the possible stereoisomers, all of which were synthetically prepared for chromatographic comparison with the authentic fragments of 2. Based on this detective work, we proposed a corrected structure for yaku'amide B (2c), in which the orders of residues-7 and -8 and residues-11 and -12 are reversed. Finally, the total synthesis of 2c led to confirmation of its structural identity. Moreover, the revised structure of yaku'amide A (1c) was constructed by switching Ala-3 to Gly-3 and was found to be chromatographically matched with the re-isolated natural 1. The present work demonstrated the high reliability and sensitivity of the MS- and LC-based structural analyses and the indispensable role of chemical synthesis in structural elucidation of scarce natural products.

Eicosapentaenoic acid is converted via ω-3 epoxygenation to the anti-inflammatory metabolite 12-hydroxy-17,18-epoxyeicosatetraenoic acid.

Eicosapentaenoic acid (EPA) has beneficial effects in many inflammatory disorders. In this study, dietary EPA was converted to 17,18-epoxyeicosatetraenoic acid (17,18-EpETE) by ω-3 epoxygenation in the mouse peritoneal cavity. Mediator lipidomics revealed a series of novel oxygenated metabolites of 17,18-EpETE, and one of the major metabolites, 12-hydroxy-17,18-epoxyeicosatetraenoic acid (12-OH-17,18-EpETE), displayed a potent anti-inflammatory action by limiting neutrophil infiltration in murine zymosan-induced peritonitis. 12-OH-17,18-EpETE inhibited leukotriene B4-induced neutrophil chemotaxis and polarization in vitro in a low nanomolar range (EC50 0.6 nM). The complete structures of two natural isomers were assigned as 12S-OH-17R,18S-EpETE and 12S-OH-17S,18R-EpETE, using chemically synthesized stereoisomers. These natural isomers displayed potent anti-inflammatory action, whereas the unnatural stereoisomers were essentially devoid of activity. These results demonstrate that 17,18-EpETE derived from dietary EPA is converted to a potent bioactive metabolite 12-OH-17,18-EpETE, which may generate an endogenous anti-inflammatory metabolic pathway.

Total Synthesis of Four Stereoisomers of (4Z,7Z,10Z,12E,16Z,18E)-14,20-Dihydroxy-4,7,10,12,16,18-docosahexaenoic Acid and Their Anti-inflammatory Activities.

A novel anti-inflammatory lipid mediator, (4Z,7Z,10Z,12E,14S,16Z,18E,20R)-14,20-dihydroxy-4,7,10,12,16,18-docosahexaenoic acid (1aa), and its three C14,C20 stereoisomers (1ab,ba,bb) were synthesized in a convergent fashion. The carbon backbone of the target compounds was assembled from seven simple fragments by employing two Sonogashira coupling and three SN2 alkynylation reactions. The thus constructed four internal alkynes were chemoselectively reduced to the corresponding (Z)-alkenes by applying a newly developed stepwise protocol: (i) hydrogenation of the three alkynes using Lindlar catalyst and (ii) formation of the dicobalt hexacarbonyl complex from the remaining alkyne and subsequent reductive decomplexation. The synthetic preparation of the stereochemically defined four isomers 1aa,ab,ba,bb permitted determination of the absolute structure of the isolated natural product to be 1aa. Biological testing of the four synthetic 14,20-dihydroxydocosahexaenoic acids disclosed similar anti-inflammatory activities of the non-natural isomers (1ab,ba,bb) and the natural form (1aa).

Goofy coordinates the acuity of olfactory signaling.

The basic scheme of odor perception and signaling from olfactory cilia to the brain is well understood. However, factors that affect olfactory acuity of an animal, the threshold sensitivity to odorants, are less well studied. Using signal sequence trap screening of a mouse olfactory epithelium cDNA library, we identified a novel molecule, Goofy, that is essential for olfactory acuity in mice. Goofy encodes an integral membrane protein with specific expression in the olfactory and vomeronasal sensory neurons and predominant localization to the Golgi compartment. Goofy-deficient mice display aberrant olfactory phenotypes, including the impaired trafficking of adenylyl cyclase III, stunted olfactory cilia, and a higher threshold for physiological and behavioral responses to odorants. In addition, the expression of dominant-negative form of cAMP-dependent protein kinase results in shortening of olfactory cilia, implying a possible mechanistic link between cAMP and ciliogenesis in the olfactory sensory neurons. These results demonstrate that Goofy plays an important role in establishing the acuity of olfactory sensory signaling.

Standalone cell culture microfluidic device-based microphysiological system for automated cell observation and application in nephrotoxicity tests.

Microphysiological systems (MPS) offer an alternative method for culturing cells on microfluidic platforms to model organ functions in pharmaceutical and medical sciences. Although MPS hardware has been proposed to maintain physiological organ function through perfusion culture, no existing MPS can automatically assess cell morphology and conditions online to observe cellular dynamics in detail. Thus, with this study, we aimed to establish a practical strategy for automating cell observation and improving cell evaluation functions with low temporal resolution and throughput in MPS experiments. We developed a versatile standalone cell culture microfluidic device (SCCMD) that integrates microfluidic chips and their peripherals. This device is compliant with the ANSI/SLAS standards and has been seamlessly integrated into an existing automatic cell imaging system. This integration enables automatic cell observation with high temporal resolution in MPS experiments. Perfusion culture of human kidney proximal tubule epithelial cells using the SCCMD improves cell function. By combining the proximal tubule MPS with an existing cell imaging system, nephrotoxicity studies were successfully performed to automate morphological and material permeability evaluation. We believe that the concept of building the ANSI/SLAS-compliant-sized MPS device proposed herein and integrating it into an existing automatic cell imaging system for the online measurement of detailed cell dynamics information and improvement of throughput by automating observation operations is a novel potential research direction for MPS research.

Design and synthesis of skeletal analogues of gambierol: attenuation of amyloid-ß and tau pathology with voltage-gated potassium channel and N-methyl-D-aspartate receptor implications.

Gambierol is a potent neurotoxin that belongs to the family of marine polycyclic ether natural products and primarily targets voltage-gated potassium channels (K(v) channels) in excitable membranes. Previous work in the chemistry of marine polycyclic ethers has suggested the critical importance of the full length of polycyclic ether skeleton for potent biological activity. Although we have previously investigated structure-activity relationships (SARs) of the peripheral functionalities of gambierol, it remained unclear whether the whole polycyclic ether skeleton is needed for its cellular activity. In this work, we designed and synthesized two truncated skeletal analogues of gambierol comprising the EFGH- and BCDEFGH-rings of the parent compound, both of which surprisingly showed similar potency to gambierol on voltage-gated potassium channels (K(v)) inhibition. Moreover, we examined the effect of these compounds in an in vitro model of Alzheimer's disease (AD) obtained from triple transgenic (3xTg-AD) mice, which expresses amyloid beta (Aß) accumulation and tau hyperphosphorylation. In vitro preincubation of the cells with the compounds resulted in significant inhibition of K(+) currents, a reduction in the extra- and intracellular levels of Aß, and a decrease in the levels of hyperphosphorylated tau. In addition, pretreatment with these compounds reduced the steady-state level of the N-methyl-D-aspartate (NMDA) receptor subunit 2A without affecting the 2B subunit. The involvement of glutamate receptors was further suggested by the blockage of the effect of gambierol on tau hyperphosphorylation by glutamate receptor antagonists. The present study constitutes the first discovery of skeletally simplified, designed polycyclic ethers with potent cellular activity and demonstrates the utility of gambierol and its synthetic analogues as chemical probes for understanding the function of K(v) channels as well as the molecular mechanism of Aß metabolism modulated by NMDA receptors.

Effect of gambierol and its tetracyclic and heptacyclic analogues in cultured cerebellar neurons: a structure-activity relationships study.

The polycyclic ether class of marine natural products has attracted the attention of researchers due to their complex and large chemical structures and diverse biological activities. Gambierol is a marine polycyclic ether toxin, first isolated along with ciguatoxin congeners from the dinoflagellate Gambierdiscus toxicus. The parent compound gambierol and the analogues evaluated in this work share the main crucial elements for biological activity, previously described to be the C28=C29 double bond within the H ring and the unsaturated side chain [Fuwa, H., Kainuma, N., Tachibana, K., Tsukano, C., Satake, M., and Sasaki, M. (2004) Diverted total synthesis and biological evaluation of gambierol analogues: Elucidation of crucial structural elements for potent toxicity. Chem. Eur. J. 10, 4894-4909]. With the aim to gain a deeper understanding of the cellular mechanisms involved in the biological activity of these compounds, we compared its activity in primary cultured neurons. The three compounds inhibited voltage-gated potassium channels (Kv) in a concentration-dependent manner and with similar potency, caused a small inhibition of voltage-gated sodium channels (Nav), and evoked cytosolic calcium oscillations. Moreover, the three compounds elicited a "loss of function" effect on Kv channels at concentrations of 0.1 nM. Additionally, both the tetracyclic and the heptacyclic derivatives of gambierol elicited synchronous calcium oscillations similar to those previously described for gambierol in cultured cerebellar neurons. Neither gambierol nor its tetracyclic derivative elicited cell toxicity, while the heptacyclic analogue caused a time-dependent decrease in cell viability. Neither the tetracyclic nor the heptacyclic analogues of gambierol exhibited lethality in mice after ip injection of 50 or 80 µg/kg of each compound. Altogether, the results presented in this work support an identical mechanism of action for gambierol and its tetracyclic and heptacyclic analogues and indicate a "loss of function" effect on potassium channels even after administration of the three compounds at subnanomolar concentrations. In addition, because gambierol is known to stabilize the closed state of Kv3 channels, the results presented in this paper may have implications for understanding of channel functions and for future development of therapies against ciguatera poisoning and potassium channel-related diseases.

A Kinetic Pump Integrated Microfluidic Plate (KIM-Plate) with High Usability for Cell Culture-Based Multiorgan Microphysiological Systems.

Microphysiological systems (MPSs), including organ-on-a-chip (OoC), have attracted attention as a novel method for estimating the effects and side effects of drugs in drug discovery. To reproduce the dynamic in vivo environment, previous MPSs were connected to pump systems to perfuse culture medium. Therefore, most MPSs are not user-friendly and have poor throughput. We aimed to develop a kinetic pump integrated microfluidic plate (KIM-Plate) by applying the stirrer-based micropump to an open access culture plate to improve the usability of MPSs. The KIM-Plate integrates six multiorgan MPS (MO-MPS) units and meets the ANSI/SBS microplate standards. We evaluated the perfusion function of the kinetic pump and found that the KIM-Plate had sufficient agitation effect. Coculture experiments with PXB cells and hiPS intestinal cells showed that the TEER of hiPS intestinal cells and gene expression levels related to the metabolism of PXB cells were increased. Hence, the KIM-Plate is an innovative tool for the easy coculture of highly conditioned cells that is expected to facilitate cell-based assays in the fields of drug discovery and biology because of its usability and high throughput nature.

Coculture with hiPS-derived intestinal cells enhanced human hepatocyte functions in a pneumatic-pressure-driven two-organ microphysiological system.

Examining intestine-liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine-liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs.

[Development of analytical methods for residual N-methyl carbamate pesticides in foods].

One of the major roles of public health agencies is to ensure safe products for consumers through analysis of residual agricultural chemicals and veterinary drugs in foods. The use of agricultural chemicals and veterinary drugs in agriculture is necessary to improve the quality of the food produced. They play a beneficial role in providing a plentiful, low-cost supply of high-quality food. On the other hand, as a consequence of this use, the presence of residues in food that is a critical element of overall public health is unavoidable, and residues in food are of great importance in the evaluation of food quality. N-methyl carbamate pesticides have anticholinesterase activity and are used worldwide. The postcolumn HPLC method is widely used for the detection of N-methyl carbamate pesticides in food. However, this traditional method involves a time-consuming process that requires skillful techniques. Therefore we developed a new analysis method for N-methyl carbamate pesticides as described in detail in this paper. The new method, including sample preparation and determination, is simple and rapid and allows simultaneous determination of pesticides in food within a much shorter analysis time as compared with the traditional method. This should provide high-quality analysis and ensure safe products for consumers.

Synthetic studies on gambieric acids, potent antifungal polycyclic ether natural products: reassignment of the absolute configuration of the nonacyclic polyether core by NMR analysis of model compounds.

A highly stereocontrolled, convergent synthesis of the A/B-ring fragment of gambieric acids (GAs) has been developed on the basis of (i) a Suzuki-Miyaura coupling of the C1-C6 alkylborate and the C7-C17 vinyl iodide and (ii) a diastereoselective haloetherification for the construction of the A-ring tetrahydrofuran as key steps. Inspection of the (1)H and (13)C NMR chemical shifts of the synthesized A/B-ring model compounds led to a stereochemical reassignment of the absolute configuration of the polycyclic ether core of GAs. This structure revision was further supported by a synthesis of the A/BC-ring model compound of gambieric acid B and a comparison of its (1)H and (13)C NMR data with those of the natural product.

Stereocontrolled synthesis of the A/B-ring fragment of gambieric acid B: reassignment of the absolute configuration of the polycyclic ether region.

Stereocontrolled synthesis of the A/B-ring fragment of the originally assigned structure of gambieric acid B and its possible diastereomers has been accomplished. Detailed comparison of their 1H and 13C NMR data with those of the corresponding moiety of the natural product culminated in a stereochemical reassignment of the absolute configuration of the polycyclic ether region of gambieric acid B.

Rapid determination of carbamate pesticides in food using dual counter-current chromatography directly interfaced with mass spectrometry.

Dual counter-current chromatography (dual CCC)-tandem mass spectrometry (MS/MS) was successfully performed with a newly designed spiral column for dual CCC. The small column capacity required for directly coupling with electrospray MS/MS was accomplished by forming a rectangular spiral groove on a plastic disk and sealing it with a PTFE sheet. This novel dual CCC-MS/MS technique was successfully applied for the rapid determination of methomyl, fenobucarb and carbaryl pesticides in food. A two-phase solvent system of n-hexane-acetonitrile-0.1% formic acid (45:45:10) was suitable for both good dual CCC separation and sufficient ionization of pesticides. Recoveries of these three pesticides from mandarin orange and spinach samples fortified at 0.05 mg/kg were in the range of 93-107% with relative standard deviations of 2.4-3.8%.

Differentiation of murine B cells induced by chondroitin sulfate B.

A two-step culture system was used to investigate the role of chondroitin sulfate (CS) B, which is mitogenic to B cells, in differentiation of B cells. Mouse spleen B cells were incubated for 3 days with CSB in the presence of interleukin (IL)-4 and IL-5. After washing, the cells were replated at 10(5) viable cells/well and recultured without CSB in the presence of IL-4 and IL-5. CSB dose-dependently increased IgM production, the greatest enhancement being 450%. Dextran sulfate had a similar effect, whereas other glycosaminoglycans, CSA, CSC, heparin and hyaluronic acid, were marginally effective. Treatment of B cells with CSB resulted in increases in the number of IgM-secreting cells and numbers of CD138-positive cells and CD45R/B220-negative cells. CSB-induced IgM production was inhibited by the protein kinase C (PKC) inhibitor GF109203X but not by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. These results demonstrated that CSB promoted differentiation of B cells in the presence of IL-4 and IL-5 and suggested that PKC but not PI3K is crucial for CSB-induced IgM production.

Diabetes-associated retinal nerve fiber damage evaluated with scanning laser polarimetry.

PURPOSE: To evaluate retinal nerve fiber layer (RNFL) thickness in patients with diabetes mellitus compared with age-matched normal control subjects, to assess the correlation between the RNFL thickness and the severity of retinopathy, and to investigate whether diabetes mellitus is a potential source of abnormal results in glaucoma screening or evaluation with scanning laser polarimetry (SLP). DESIGN: Cross-sectional analysis of normal and diabetic eyes. METHODS: setting: Institutional clinical study. patients: One hundred twenty-eight subjects with type 2 diabetes mellitus and 50 age-matched normal control subjects without glaucoma or glaucoma-suspect. All patients underwent imaging with SLP and repeatable automated perimetry. Subjects with diabetes mellitus were classified into four stages on the basis of the severity of retinopathy that was assessed by dilated funduscopic examination, high-quality fundus color photography, and fluorescein angiography. MAIN OUTCOME MEASURES: The SLP (GDx VCC software, version 5.5.0) parameters. RESULTS: The RNFL thickness in patients with diabetes mellitus was reduced significantly compared with age-matched normal control eyes (P < .01). Moreover, all RNFL thickness parameters of the GDx VCC software decreased significantly as an exacerbation of diabetic retinopathy (P = .0019, P = .0045, P = .0010 for temporal-superior-nasal-inferior-temporal (TSNIT) average, superior average, inferior average, respectively). The nerve fiber indicator also increased significantly (P < .0001), despite an absence of glaucomatous optic nerve damages. CONCLUSION: The RNFL thickness in type 2 diabetes mellitus, which was measured by GDx VCC software, significantly decreased with the severity of diabetic retinopathy. The presence of diabetes mellitus can be a source of false-positive results and overestimation of glaucomatous optic neuropathy when eyes are screened with GDx VCC software.

Application of dual counter-current chromatography for rapid sample preparation of N-methylcarbamate pesticides in vegetable oil and citrus fruit.

Dual counter-current chromatography (dual CCC) has been successfully applied to rapid sample preparation for the simultaneous determination of residual carbaryl, fenobucarb and methomyl in vegetable oil and citrus fruit. The citrus fruit samples were extracted with n-hexane solution containing stable isotopically labeled internal standards (methomyl-d3, fenobucarb-d3 and carbaryl-d9), and applied to dual CCC using a two-phase solvent system of n-hexane-acetonitrile to purify the carbamate pesticides from aliphatic sample matrix. The coiled column was rotated at 420 rpm, the lower mobile phase was introduced through the head toward the tail, and the upper mobile phase in the opposite direction. Due to the high partition efficiency of dual CCC, the lower phase fraction collected from 2 to 5 min after injection could be subjected to flow-injection tandem mass spectrometry directly after concentration. Repetitive sample injection can be performed at high reproducibility without a risk of contamination from the compounds retained in the column.

High-throughput analysis of tetracycline and penicillin antibiotics in animal tissues using electrospray tandem mass spectrometry with selected reaction monitoring transition.

A simple, rapid, and simultaneous analysis method for oxytertracycline, tetracycline, chlortetracycline, penicillin G, ampicillin, and nafcillin in meat has been developed by using electrospray ionization tandem mass spectrometry. The sample preparation was performed by homogenizing with water followed by a centrifugal ultrafiltration, after addition of internal standards (demeclocycline, penicillin G-d5, ampicillin-d5 and nafcillin-d6). The MS/MS analysis involves the combined use of sample enrichment on the short column and a multiple reaction monitoring technique. The overall recoveries from animal (bovine and swine) muscle, kidney, and liver fortified at the levels of 0.05 and 0.1 ppm ranged from 70 to 115% with the coefficients of variation ranging from 0.7 to 14.8% (n = 5). Analysis time, including sample preparation and determination, is only 3h per eight sample and detection limits for all antibiotics are 0.002 ppm. The method is considered to be satisfactory for the rapid screening of the tetracycline and penicillin antibiotic residues in meat.

Abnormalities of scanning laser polarimetry associated with pituitary adenoma.

PURPOSE: To report abnormalities of retinal nerve fiber layer (RNFL) thickness using scanning laser polarimetry in patients with pituitary adenoma. DESIGN: Observational case reports. METHODS: Two patients with pituitary adenoma who had bitemporal visual field abnormalities were examined using scanning laser polarimetry. RESULTS: In Case 1, destruction of the normal two-peak pattern in the RNFL thickness was seen nasally by scanning laser polarimetry; this RNFL pattern corresponded well to visual field abnormalities. In Case 2, abnormalities in the RNFL pattern corresponded well to visual field defects not only in the superotemporal fields but also in the inferotemporal fields where the visual field defects were mild. CONCLUSIONS: Measurement of RNFL thickness using scanning laser polarimetry provides useful information in the diagnosis of chiasmal lesions such as pituitary adenomas.

Application of ion-exchange cartridge clean-up in food analysis. VI. Determination of six penicillins in bovine tissues by liquid chromatography-electrospray ionization tandem mass spectrometry.

A multiresidue analytical method was developed for the quantification of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in bovine tissues using liquid chromatography- electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) with a multiple reaction monitoring technique. Using the deuterated PCG and NFPC as internal standard was effective for improvement of repeatability and accuracy. We chose [M-H-141]- as a monitor ion of MRM analysis and [M-H]- as a precursor ion for each penicillin. Combination of an ion-exchange cartridge clean-up and ion-pair LC enable us to determine the residual penicillins using the standard curves made from standard solutions without the influence of sample matrix on the MS. The average recoveries of PCG, PCV, MPIPC, MCIPC, NFPC and MDIPC from bovine liver, kidney and muscle at the same concentrations as the tolerance levels of PCG (50 microg/kg) ranged from 77 to 101% with the coefficients of variation ranging from 0.7 to 4.2% (n = 5). The limits of quantification for the six penicillins were 2-10 microg/kg in bovine muscle, liver and kidney (S/N ratio >10).

Purification of quinoline yellow components using high-speed counter-current chromatography by stepwise increasing the flow-rate of the mobile phase.

Quinoline yellow (Color Index No. 47005) consists of multiple components that show a large difference in their partition coefficients (K), ranging from 0.03 to 3.3 in the solvent system tert.-butyl methyl ether (MTBE)-1-butanol-acetonitrile-aqueous 0.1 M trifluoroacetic acid (TFA). Consequently, it requires an excessively long elution time for the simultaneous separation of all components by the standard high-speed counter-current chromatography technique, which uses a constant flow-rate of the mobile phase. In order to overcome this problem, we increased the flow-rate of the mobile phase stepwise from 0.1 to 2.0 mL/min. Using this new procedure, six components (0.2-6.1 mg) were successfully isolated from 25 mg of a commercial quinoline yellow preparation in a single run using a two-phase solvent system composed of MTBE-1-butanol-acetonitrile-aqueous 0.1 M TFA (1:3:1:5, v/v). The purified components were analyzed by high-performance liquid chromatography, electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy.