Essential role of Cav3.2 T-type calcium channels in butyrate-induced colonic pain and nociceptor hypersensitivity in mice.

Given the role of Cav3.2 isoform among T-type Ca2+ channels (T-channels) in somatic and visceral nociceptive processing, we analyzed the contribution of Cav3.2 to butyrate-induced colonic pain and nociceptor hypersensitivity in mice, to evaluate whether Cav3.2 could serve as a target for treatment of visceral pain in irritable bowel syndrome (IBS) patients. Mice of ddY strain, and wild-type and Cav3.2-knockout mice of a C57BL/6J background received intracolonic administration of butyrate twice a day for 3 days. Referred hyperalgesia in the lower abdomen was assessed by von Frey test, and colonic hypersensitivity to distension by a volume load or chemicals was evaluated by counting nociceptive behaviors. Spinal phosphorylated ERK was detected by immunohistochemistry. Cav3.2 knockdown was accomplished by intrathecal injection of antisense oligodeoxynucleotides. Butyrate treatment caused referred hyperalgesia and colonic hypersensitivity to distension in ddY mice, which was abolished by T-channel blockers and/or Cav3.2 knockdown. Butyrate also increased the number of spinal phosphorylated ERK-positive neurons following colonic distension in the anesthetized ddY mice. The butyrate-treated ddY mice also exhibited T-channel-dependent colonic hypersensitivity to intracolonic Na2S, known to enhance Cav3.2 activity, and TRPV1, TRPA1 or proteinase-activated receptor 2 (PAR2) agonists. Wild-type, but not Cav3.2-knockout, mice of a C57BL/6J background, after treated with butyrate, mimicked the T-channel-dependent referred hyperalgesia and colonic hypersensitivity in butyrate-treated ddY mice. Our study provides definitive evidence for an essential role of Cav3.2 in the butyrate-induced colonic pain and nociceptor hypersensitivity, which might serve as a target for treatment of visceral pain in IBS patients.

[Identification and determination of the isomer and subsidiary color in food blue no. 2 (indigo carmine) by LC/MS and HPLC].

CFR and JECFA specify that the total color in FD&C Blue No. 2 (B2; Indigo Carmine, Indigotine, Food Blue No. 2) is not less than 85%, its isomer (B2iso) in B2 is not more than 18%, and its subsidiary color (B2sub) in B2 is not more than 2% (CFR) or 1% (JECFA). Japan's Specifications and Standards for Food Additives, 7th Edition, specifies that the total color in B2 is not less than 85.0% and other color materials in B2 are not detected by paper chromatography. LC/MS and HPLC were employed to identify and determine the main component (B2m) of B2, B2iso, and B2sub. The pseudo molecular ions (B2m and B2iso: [M-2Na+H]-, m/z=421; B2sub: [M-Na]-, m/z=341) of each color material were obtained and identified by LC/MS based on their absorptions and mass spectra. The contents of B2iso and B2sub in B2 samples (certified samples from fiscal year 1998 to fiscal year 2002) were determined by HPLC using calibration curves for the standards of B2m and B2iso. The contents of B2iso in most samples were less than 10%, and the contents of B2sub in all samples were not more than 1%. All of them were within the regulatory limits set by the CFR and JECFA.

[Studies on the contents of water-soluble chlorides and water-soluble sulfates in food color aluminum lakes].

Japan's Specifications and Standards for Food Additives, 7th Edition (JSFA-VII) does not set limits for total contents of water-soluble chlorides and water-soluble sulfates (water-soluble inorganic salts) in food color aluminum lakes (FC-Als). However, the regulatory limit is 2% in JECFA and CFR. We used column switching suppressor-type ion chromatography (CSS-IC) for determination of anions. The total contents of water-soluble inorganic salts in FC-Als (112 qualified samples) were determined using the modified CSS-IC from fiscal year 1998 to fiscal year 2003. Total contents of water-soluble inorganic salts in four samples exceeded 2%. From an international point of view, it is desirable that the total content of water-soluble inorganic salts in FC-Al should again be regulated in Japan.

[Estimated production by the official inspection of tar colors (including aluminum lakes) in fiscal year 2003].

There were 197 official inspections of tar colors and their lakes in fiscal year 2003, the two of their samples were rejected, and the other were qualified. Total production amount of tar colors that passed inspection in Japan in fiscal year 2003 reached 147.9 tons. Tar color production amounts were described by month and by manufacturer. The food tar color produced in the largest amount was Food Yellow No. 4, accounting for 41.7% during this period.

[Estimated production by the official inspection of tar colors (including aluminum lakes) in fiscal year 2002].

There were 157 official inspections of tar colors and their lakes in fiscal year 2002, and all of their samples were qualified. Total production amount of tar colors that passed inspection in Japan in fiscal year 2002 reached 113.6 tons. Tar color production amounts were described by month and by manufacturer. The food tar color produced in the largest amount was Food Yellow No. 4, accounting for 42.5% during this period.

[Studies on flavianic acids as color reference standard for thin-layer chromatography].

Various flavianic acids were prepared and examined for the preparation for color reference standard for thin-layer chromatography (TLC). Their analytical data were: IR spetra, their specific absorptions were same; no impurities were detected; specific odor-free. It was clear that the prepared flavianic acids were useable as color reference standards for TLC.

[Studies on eight color reference standards for thin-layer chromatography].

Eight color NIHS reference standards were studied on IR spectra and thin-layer chromatography (TLC). Their IR spectra and TLCs were determined. They were also authorized as reference standards for TLC.

Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10.

A gene encoding a cholesterol oxidase from Brevibacterium sterolicum nov. sp. ATCC21387 was isolated by an expression cloning method and highly expressed by a recombinant strain Escherichia coli MM294/pnH10. The purified cholesterol oxidase was a typical flavoprotein with a molecular mass of 46.5 kDa, absorption peaks at 280, 360, and 450 nm. Optimum pH and temperature were found at pH 6.5 and 55 degrees C, respectively. The enzyme acted on 3beta-hydroxysteroids such as cholesterol, pregnenolone, and beta-sitosterol at high rates, but on dehydro-epi-androsterone to a lesser degree. The molecular and catalytic properties were different from those of cholesterol oxidase I, which was initially discovered in B. sterolicum nov. sp. ATCC21387. The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (K(m)=30 microM).