Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy.

Understanding metabolism in live microalgae is crucial for efficient biomaterial engineering, but conventional methods fail to evaluate heterogeneous populations of motile microalgae due to the labelling requirements and limited imaging speeds. Here, we demonstrate label-free video-rate metabolite imaging of live Euglena gracilis and statistical analysis of intracellular metabolite distributions under different culture conditions. Our approach provides further insights into understanding microalgal heterogeneity, optimizing culture methods and screening mutant microalgae.

Potent production of capsaicinoids and capsinoids by Capsicum peppers.

The fundamental structure of capsinoids is a fatty acid ester with vanillyl alcohol, whereas in capsaicinoids, a fatty acid amide is linked to vanillylamine. To clarify the relationship between their biosynthesis in Capsicum plants, we carried out an in vivo tracer experiment using stable isotopically labeled putative precursors. Liquid chromatography-tandem mass spectrometry was used to measure the uptake of isotopes into metabolites after injection of the labeled precursors into intact fruits of a pungent cultivar, Peru, and a non-pungent cultivar, CH-19 Sweet. Labeled vanillylamine was incorporated into capsaicinoids in both cultivars. While labeled vanillyl alcohol was incorporated into capsinoids in both cultivars, the accumulation of intact capsaicinoids in Peru was suppressed by over 60% after administration of vanillyl alcohol. In Peru, labeled vanillin was converted to both vanillylamine and, in 5-fold excess, vanillyl alcohol. Moreover, labeled vanillin was converted exclusively to vanillyl alcohol in CH-19 Sweet. These data are consistent with the incorporation of labeled vanillin into capsaicinoids and capsinoids in both cultivars. We conclude that pungent cultivars are highly potent producers of vanillyl alcohol that is incorporated into capsinoids and that biosynthesis of capsinoids is catalyzed by capsaicin synthase.

Synthesis of stable isotope-labeled precursors for the biosyntheses of capsaicinoids, capsinoids, and capsiconinoids.

Stable isotope-labeled precursors were synthesized for an analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate the biosynthetic flow of capsaicinoids, capsinoids, and capsiconinoids. [1'-(13)C][5-(2)H]-Vanillin was prepared by the condensation of guaiacol with [(13)C]-chloroform and a D(2)O treatment. Labeled vanillylamine, vanillyl alcohol, ferulic acid, and coniferyl alcohol were prepared from the labeled vanillin. The labeled vanillylamine was converted to labeled capsaicinoid in a crude enzyme solution extracted from pungent Capsicum fruits.

Regiospecific [2 + 2] photocyclodimerization of trans-4-styrylpyridines templated by cucurbit[8]uril.

Addition of HCl accelerated the photocyclodimerization of trans-4-styrylpyridine 1a in methanol and increased the yield of syn-head-to-tail (syn-HT) dimer 2a through the effect of cation-π interactions between the pyridinium ion of one molecule and the phenyl group of the other. We examined the photoirradiation products of derivatives of 1a having alkyl substituents on the phenyl group (1b-1f). The effect of the alkyl substituent on product distribution was rather limited for the photoreaction in MeOH solutions. However, the substituents had a distinct effect on the product distribution for the photoreaction of the inclusion complexes of hydrochloride salts of trans-4-styrylpyridines with cucurbit[8]uril in aqueous solutions. Introducing an alkyl group at the 2- or 3-position of the phenyl group completely shifted the major product from the syn-HT dimer to the syn-head-to-head (syn-HH) dimer. By adjusting the balance of host-guest interactions and cation-π interactions between guest molecules through systematic changes in the substituents on the phenyl ring of trans-4-styrylpyridine, we could change the orientation of the reactant molecules in the host cavity, resulting in a change of the major regioisomer of the photocyclodimerization products.

Cysteine protease inhibitors suppress the development of tolerance to morphine antinociception.

The effects of various protease inhibitors on the development of antinociceptive tolerance to morphine were examined in mice. Intrathecal (i.t.) administration of morphine (0.01-1 nmol) produced a dose-dependent and significant antinociceptive effect in the 0.5% formalin test. When the doses of morphine (mg/kg, s.c. per injection) were given as pretreatment twice daily for two days [first day (30) and second day (60)], i.t. administration of morphine (0.1 nmol) was inactive due to antinociceptive tolerance on the third day. Tolerance to i.t. morphine was significantly suppressed by the i.t. injection of N-ethylmaleimide or Boc-Tyr-Gly-NHO-Bz, inhibitors of cysteine proteases involved in dynorphin degradation, as well as by dynorphin A, dynorphin B and (-) U-50,488, a selective kappa-opioid receptor agonist. On the other hand, amastatin, an aminopeptidase inhibitor, phosphoramidon, an endopeptidase 24.11 inhibitor, lisinopril, an angiotensin-converting enzyme inhibitor, and phenylmethanesulfonyl fluoride, a serine protease inhibitor, were inactive. These results suggest that cysteine protease inhibitors suppress the development of morphine tolerance presumably through the inhibition of dynorphin degradation.

S-(+)-fenfluramine-induced nociceptive behavior in mice: Involvement of interactions between spinal serotonin and substance P systems.

Intrathecal (i.t.) administration into mice of S-(+)-fenfluramine (0.01-0.1nmol), a serotonin (5-hydroxytryptamine, 5-HT) releaser, produced a behavioral response consisting of scratching, biting and licking. Here, we report the behavioral characteristics and the involvement of interactions between 5-HT and substance P (SP) systems in the S-(+)-fenfluramine-induced behavioral response. The S-(+)-fenfluramine-induced behavioral response peaked at 5-15min and almost disappeared at 20min after injection. The behavior induced by S-(+)-fenfluramine (0.1nmol) was dose-dependently inhibited by an intraperitoneal injection of morphine (0.02-0.5mg/kg), suggesting that the behavioral response is related to nociception. The S-(+)-fenfluramine-induced nociceptive behavior was significantly inhibited by pretreatment with 5-HT antiserum and co-administration of ketanserin, a selective 5-HT2 receptor antagonist. However, WAY-100635, a selective 5-HT1A receptor antagonist, and ramosetron, a selective 5-HT3 receptor antagonist, were not active. On the other hand, SP antiserum and RP67580, a selective neurokinin-1 (NK1) receptor antagonist, significantly inhibited S-(+)-fenfluramine-induced nociceptive behavior. These results suggest that i.t.-administered S-(+)-fenfluramine releases SP through the activation of 5-HT2 receptors subsequent to 5-HT release, and, as a result, produces nociceptive behavior.