Determination of Anthraquinone-Tagged Amines Using High-Performance Liquid Chromatography with Online UV Irradiation and Luminol Chemiluminescence Detection

Quinones are frequently used as derivatization reagents in HPLC analysis to improve detection sensitivity. In the present study, a simple, sensitive, and selective chemiluminescence (CL) derivatization strategy for biogenic amines, prior to their HPLC-CL analysis, was developed. The novel CL derivatization strategy was established based on using anthraquinone-2-carbonyl chloride as derivatizing agent for amines and then using the unique property of the quinones’ moiety to generate reactive oxygen species (ROS) in response to UV irradiation. Typical amines such as tryptamine and phenethylamine were derivatized with anthraquinone-2-carbonyl chloride and then injected into an HPLC system equipped with an online photoreactor. The anthraquinone-tagged amines are separated and then UV-irradiated when they pass through a photoreactor to generate ROS from the quinone moiety of the derivative. Tryptamine and phenethylamine can be determined by measuring the chemiluminescence intensity produced by the reaction of the generated ROS with luminol. The chemiluminescence disappears when the photoreactor is turned off, suggesting that ROS are no longer generated from the quinone moiety in the absence of UV irradiation. This result indicates that the generation of ROS could be controlled by turning the photoreactor on and off. Under the optimized conditions, the limits of detection for tryptamine and phenethylamine were 124 and 84 nM, respectively. The developed method is successfully applied to determine the concentrations of tryptamine and phenethylamine in wine samples

Isolation of TCR genes with tumor-killing activity from tumor-infiltrating and circulating lymphocytes in a tumor rejection cynomolgus macaque model

To develop effective adoptive cell transfer therapy using T cell receptor (TCR)-engineered T cells, it is critical to isolate tumor-reactive TCRs that have potent anti-tumor activity. In humans, tumor-infiltrating lymphocytes (TILs) have been reported to contain CD8+PD-1+ T cells that express tumor-reactive TCRs. Characterization of tumor reactivity of TILs from non-human primate tumors could improve anti-tumor activity of TCR-engineered T cells in preclinical research. In this study, we sought to isolate TCR genes from CD8+PD-1+ T cells among TILs in a cynomolgus macaque model of tumor transplantation in which the tumors were infiltrated with CD8+ T cells and were eventually rejected. We analyzed the repertoire of TCRα and β pairs obtained from single CD8+PD-1+ T cells in TILs and circulating lymphocytes and identified multiple TCR pairs with high frequency, suggesting that T cells expressing these recurrent TCRs were clonally expanded in response to tumor cells. We further showed that the recurrent TCRs exhibited cytotoxic activity to tumor cells in vitro and potent anti-tumor activity in mice transplanted with tumor cells. These results imply that this tumor transplantation macaque model recapitulates key features of human TILs and can serve as a platform toward preclinical studies of non-human primate tumor models

TRPM2 Contributes to Inflammatory and Neuropathic Pain through the Aggravation of Pronociceptive Inflammatory Responses in Mice.

慢性痛の原因となる神経炎症応答の増悪機構を解明－新しい鎮痛薬開発の可能性－. 京都大学プレスリリース. 2012-03-15.Accumulating evidence suggests that neuroimmune interactions contribute to pathological pain. Transient receptor potential melastatin 2 (TRPM2) is a nonselective Ca(2+)-permeable cation channel that acts as a sensor for reactive oxygen species. TRPM2 is expressed abundantly in immune cells and is important in inflammatory processes. The results of the present study show that TRPM2 plays a crucial role in inflammatory and neuropathic pain. While wild-type and TRPM2 knock-out mice showed no difference in their basal sensitivity to mechanical and thermal stimulation, nocifensive behaviors in the formalin test were reduced in TRPM2 knock-out mice. In carrageenan-induced inflammatory pain and sciatic nerve injury-induced neuropathic pain models, mechanical allodynia and thermal hyperalgesia were attenuated in TRPM2 knock-out mice. Carrageenan-induced inflammation and sciatic nerve injury increased the expression of TRPM2 mRNA in the inflamed paw and around the injured sciatic nerve, respectively. TRPM2 deficiency diminished the infiltration of neutrophils and the production of chemokine (C-X-C motif) ligand-2 (CXCL2), a major chemokine that recruits neutrophils, but did not alter the recruitment of F4/80-positive macrophages in the inflamed paw or around the injured sciatic nerve. Microglial activation after nerve injury was suppressed in the spinal cord of TRPM2 knock-out mice. Furthermore, CXCL2 production and inducible nitric oxide synthase induction were diminished in cultured macrophages and microglia derived from TRPM2 knock-out mice. Together, these results suggest that TRPM2 expressed in macrophages and microglia aggravates peripheral and spinal pronociceptive inflammatory responses and contributes to the pathogenesis of inflammatory and neuropathic pain

First Case Report of Sepsis Due to Rothia aeria in a Neonate▿

Rothia aeria, a gram-positive coccoid- to rod-shaped bacterium with irregular morphology, is an extremely rare causative organism of infections in humans. We report the first case of R. aeria sepsis clinically manifested in a female neonate soon after birth

Inhibition of Aflatoxin Production by Citrinin and Non-Enzymatic Formation of a Novel Citrinin-Kojic Acid Adduct

Screening for microorganisms that inhibit aflatoxin production from environments showed that Penicillium citrinum inhibited aflatoxin production by Aspergillus parasiticus. The inhibitory substance in the culture medium of P. citrinum was confirmed to be citrinin (CTN). RT-PCR analyses showed that CTN did not inhibit expressions of aflatoxin biosynthetic genes (aflR, pksL1, and fas-1) of A. parasiticus, whereas feeding experiments using A. parasiticus showed that CTN inhibited the in vivo conversion of dihydrosterigmatocystin to AFB2·AFG2. These results suggest that CTN inhibits a certain post-transcriptional step in aflatoxin biosynthesis. CTN in the culture medium of A. parasiticus was found to be decreased or lost with time, suggesting that a certain metabolite produced by A. parasiticus is the cause of the CTN decrease; we then purified, characterized, and then analyzed the substance. Physico-chemical analyses confirmed that the metabolite causing a decrease in CTN fluorescence was kojic acid (KA) and the resulting product was identified as a novel substance: (1R,3S,4R)-3,4-dihydro-6,8-dihydroxy-1-(3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl)-3,4,5-trimethyl-1H-isochromene-7-carboxylic acid, which was named “CTN-KA adduct”. Our examination of the metabolites’ toxicities revealed that unlike CTN, the CTN-KA adduct did not inhibit aflatoxin production by A. parasiticus. These results indicate that CTN’s toxicity was alleviated with KA by converting CTN to the CTN-KA adduct

Detoxication of Citrinin with Kojic Acid by the Formation of the Citrinin-Kojic Acid Adduct, and the Enhancement of Kojic Acid Production by Citrinin via Oxidative Stress in <i>Aspergillus parasiticus</i>

Our previous work showed that citrinin (CTN) produced bay Penicillium citrinum inhibited the production of aflatoxin by Aspergillus parasiticus. We also reported that CTN was non-enzymatically converted to a novel CTN-KA adduct with kojic acid (KA) in aqueous condition. We herein observed that unlike CTN, the CTN-KA adduct does not show antimicrobial activity against Escherichia coli or Bacillus subtilis or any cytotoxic effect on HeLa cells, suggesting that CTN was detoxified by KA by the formation of the CTN-KA adduct. To examine the function of KA production by fungi, we isolated A. parasiticus mutants with impaired KA production. When the mutants were incubated in either liquid or agar medium supplemented with CTN, they were more susceptible to CTN than the wild KA-producing strain. The same results were obtained when we used the A. oryzae KA-producing strain RIB40 and KA-non-producing strains. When KA was added to the CTN-containing agar medium, the inhibition of growth by CTN was remarkably mitigated, suggesting that the production of KA protected the fungal growth from CTN’s toxicity. We also observed that CTN enhanced the production of KA by A. parasiticus as well as A. oryzae strains. Reverse transcription-PCR showed that CTN enhanced the expression of KA biosynthetic genes (kojA, kojR, and kojT) of A. parasiticus. However, the enhancement of KA production with CTN was repressed by the addition of α-tocopherol or butylated hydroxy anisole, suggesting that KA production is enhanced by oxidative stress via the formation of reactive oxygen species caused by CTN. In contrast, α−tocopherol did not affect inhibition of AF production as well as fungal growth by CTN, suggesting that the regulation of these inhibitions with CTN might be different from that of KA production. We propose a regulation scheme of CTN for each of KA production, AF production, and fungal growth in A. parasiticus