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Excessive activation of CD4

Macrophages can initiate innate immune responses against microbes and cancer. The aim of this study was to elucidate the effects of Brassica rapa L. on macrophages. The production of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon-γ induced by the insoluble fraction of B. rapa L. was decreased in macrophage-depleted spleen cells compared with controls. The insoluble fraction of B. rapa L. induced expression of H-2Kb, I-Ab, CD40, and CD86, production of cytokines and nitric oxide, and phagocytic activity in RAW264 cells. After treatment with the insoluble fraction, IL-6 and TNF-α production was significantly decreased by anti-Toll-like receptor (TLR)2 mAb or polymyxin B compared with the control. Furthermore, insoluble fraction-mediated cytokine production was significantly lower in peritoneal macrophages from TLR2−/− and TLR4−/− mice compared with wild-type mice. These results suggest that B. rapa L. is a potentially effective immunomodulator for activating macrophages to prevent infections.

Aging is associated with a decrease in the function of the immune system, a phenomenon known as immunosenescence, which results in reduced resistance to infection. Caloric restriction (CR) is known to prolong lifespan and to regulate immune function. However, whether and how CR affects immunosenescence remains unclear. Here, we evaluated the effect of long- and short-term CR on immunosenescence by subjecting wild-type mice to CR between 6 and 18 months of age or between 17 and 18 months of age, respectively. Compared with a normal diet or short-term CR, long-term CR induced marked or complete attenuation of age-related decreases in the frequency of spleen NK cells and NKT cells; naïve CD4

Our previous studies have elucidated that oral administration of Brassica rapa L. extract, known as Nozawana in Japan, alters immune responses and gut microbiota composition, increasing the numbers of butyrate-producing bacteria. Therefore, further investigation would help elucidate the mechanism attributable for the changes and health-promoting effects observed after B rapa L. extract ingestion. To reveal the modulation effects of fermented B. rapa L. on immune function and intestinal bacterial community structure, we conducted an intervention study with healthy volunteers followed by a mouse feeding study. The pilot intervention study was conducted for healthy volunteers aged 40–64 years under the hypothesis that the number of subjects exhibiting any change in gut microbiota in response to fermented B. rapa L. consumption may be limited. In total, 20 volunteers consumed 30 g of fermented B. rapa L. per day for 4 weeks. The fecal bacterial community composition of the volunteers was characterized using terminal-restriction fragment length polymorphism patterning followed by clustering analysis. To evaluate the detailed changes in the immune responses and the gut bacterial composition, assessed by high-throughput sequencing, we fed healthy mice with freeze-dried, fermented B. rapa L. for 2 weeks. The fecal bacterial community composition of the volunteers before the intervention was divided into three clades. Regardless of the clade, the defecation frequency significantly increased during the intervention weeks compared with that before the intervention. However, this clustering detected a specific increase of Prevotella in one cluster (low to zero Prevotella and high occupation of Clostridium at clusters IV and XIVa) post-ingestion. The cytokine production of spleen cells significantly increased due to feeding fermented B. rapa L. to the mice. This supplementary in vivo trial provided comparable results to the volunteer study regarding the effects of ingestion of the material given the compositional change complying with that of dietary fiber, particularly in the increase of genera Prevotella, Lachnospira, and genera in the Ruminococcaceae family, and the increase in daily defecation amount during 2 weeks of administration. We conclude that feeding fermented B. rapa L. may be responsible for the observed modulation in gut microbiota to increase fiber-degrading bacteria and butyrate-producing bacteria which may be relevant to the improvement in bowel function such as defecation frequency.

Abnormal T helper 17 (Th17) responses promote inflammation and cause inflammatory diseases. Natural components that modulate Th17 functions can be effective for the amelioration of inflammatory diseases. Procyanidin B2 3,3''-di-O-gallate (PCB2DG) contained in grape seeds markedly suppressed interleukin (IL)-17 production from spleen cells but not CD4+ T cells. The aim of this study was to elucidate the mechanisms by which PCB2DG suppresses IL-17. Our results showed that PCB2DG suppressed the production of IL-17, tumor necrosis factor (TNF)-α, IL-1β, and IL-6 with the suppression of transcription factors expression. In addition, we revealed that TNF-α and IL-1β were required to induce IL-17 production in this experimental condition, and PCB2DG suppressed these cytokines from dendritic cells (DCs). Furthermore, CD4-DC co-culture experiments showed that the production of IL-17, TNF-α, and IL-1β was markedly inhibited in co-cultures of PCB2DG-pretreated CD4+ T cells and DCs. These results suggested that PCB2DG first modulated TNF-α production by CD4+ T cells and then suppressed IL-1β secretion from DCs, resulting in decreased IL-17 production. Thus, PCB2DG can control the cytokine network associated with Th17 cells, providing a novel mechanism underlying the immunosuppressive effects of polyphenols.

Caloric restriction (CR) has long been known to increase median and maximal lifespans and to decrease mortality and morbidity in short-lived animal models, likely by altering fundamental biological processes that regulate aging and longevity. However, the detailed mechanisms of immunomodulation by CR remain unclear. In this study, we established a mouse model for CR and analyzed the changes of immune cells in these mice. The CR mice fed a calorie-restricted diet for 4 weeks had lower body weight and fat mass compared with control mice. The proportions of CD4+, CD8+, and naive CD4+ T cells in spleen cells from CR mice were higher than those in of control mice. Additionally, the proportion of CD8+ T cells was significantly decreased and the mRNA expression of proinflammatory cytokines in the colon of CR mice was significantly decreased compared with those of control mice. To determine the effect of CR on microRNA (miRNA) expression, serum and tissues were collected from mice and the expression level of miRNA was analyzed by real-time RT-PCR. As a result, the expressions of miR-16-5p, miR-196b-5p, and miR-218-5p in serum from CR mice were higher than those in control mice. The expression of miR-16-5p increased in the spleen, thymus, colon, and stomach of CR mice compared with expression in control mice. Furthermore, RAW264 cells transfected with a miR-16-5p mimic significantly decreased the mRNA expression of IL-1β, IL-6, and TNF-α under LPS stimulation. These results suggested that miR-16-5p might be a critical factor involving the anti-inflammatory effects of calorie-restricted feeding.

Our previous studies have elucidated that oral administration of Brassica rapa L. extract, known as Nozawana in Japan, alters immune responses and gut microbiota composition, increasing the numbers of butyrate-producing bacteria. Therefore, further investigation would help elucidate the mechanism attributable for the changes and health-promoting effects observed after B rapa L. extract ingestion. To reveal the modulation effects of fermented B. rapa L. on immune function and intestinal bacterial community structure, we conducted an intervention study with healthy volunteers followed by a mouse feeding study. The pilot intervention study was conducted for healthy volunteers aged 40–64 years under the hypothesis that the number of subjects exhibiting any change in gut microbiota in response to fermented B. rapa L. consumption may be limited. In total, 20 volunteers consumed 30 g of fermented B. rapa L. per day for 4 weeks. The fecal bacterial community composition of the volunteers was characterized using terminal-restriction fragment length polymorphism patterning followed by clustering analysis. To evaluate the detailed changes in the immune responses and the gut bacterial composition, assessed by high-throughput sequencing, we fed healthy mice with freeze-dried, fermented B. rapa L. for 2 weeks. The fecal bacterial community composition of the volunteers before the intervention was divided into three clades. Regardless of the clade, the defecation frequency significantly increased during the intervention weeks compared with that before the intervention. However, this clustering detected a specific increase of Prevotella in one cluster (low to zero Prevotella and high occupation of Clostridium at clusters IV and XIVa) post-ingestion. The cytokine production of spleen cells significantly increased due to feeding fermented B. rapa L. to the mice. This supplementary in vivo trial provided comparable results to the volunteer study regarding the effects of ingestion of the material given the compositional change complying with that of dietary fiber, particularly in the increase of genera Prevotella, Lachnospira, and genera in the Ruminococcaceae family, and the increase in daily defecation amount during 2 weeks of administration. We conclude that feeding fermented B. rapa L. may be responsible for the observed modulation in gut microbiota to increase fiber-degrading bacteria and butyrate-producing bacteria which may be relevant to the improvement in bowel function such as defecation frequency.

Procyanidins are polyphenols with antioxidant, anti-obesity, and anti-inflammatory properties. Procyanidin B2 (PCB2) gallate; specifically, PCB2 3,3″-di-O-gallate (PCB2DG), inhibits cytokine production in T cells. However, the molecular interactions and partners of PCB2DG underlying this suppression of cytokine production are unclear. The present study aimed to elucidate mechanisms underlying regulation of tumor necrosis factor (TNF)-α production by PCB2DG. We found that production of TNF-α and glycolytic activity in activated CD4+ T cells were suppressed by PCB2DG treatment. The inhibition of TNF-α production was found to be mediated by mammalian target of rapamycin (mTOR) and hypoxia inducible factor 1 (HIF-1) pathway, as PCB2DG suppressed the expression of HIF-1α, p-mTOR, and p-p70S6K (a downstream of the mTOR complex, mTORC1). Moreover, suppression of TNF-α production was mediated by regulation of the glycolytic enzyme lactate dehydrogenase at the posttranscriptional level. These results suggest that PCB2DG regulates TNF-α production by inhibiting glycolytic activity via the mTOR-HIF-1 pathway.

A novel synthetic path to enantiomerically enriched 5-hydroxycyclohex-2-enone, a versatile chiral building block, is developed by a two-step sequence, where an enantioselective deprotonation of a 3,5-dihydroxycyclohexanone derivative with lithium (S,S′)-α,α′-dimethyldibenzylamide in the presence of TMSCl, and subsequent treatment of the silyl enol ether with TBAF in THF at room temperature are employed as the key reactions.

Laccase is a member of the multi-copper oxidase family and a promising for hair coloring. In this study, we isolated a novel alkaline-induced laccase from the white-rot fungus Flammulina velutipes and studied the possibility to apply the enzyme for hair coloring. Laccase activity detected in the culture supernatant of F. velutipes was found to significantly increase when exchanging the medium to laccase inducing one whose pH was adjusted to 9.0. Three isozymes were detected by activity staining on non-denaturing SDS-PAGE. The major isozyme, Flac1, was purified from the culture supernatant after being induced at pH 9.0 by ion-exchange column chromatography. The N-terminal peptide sequence of Flac1 was determined, revealing clear homology with laccases from other white-rot fungi. Optimum pH of oxidation was found to be around pH 5.0-6.5 regardless of several different substrates used. Oxidation activities of Flac1 to several hair dye agents as substrate showed the higher activity at pH 6.5 than that at pH 9.0. Oxidation activity was also detected at pH 9.0 which was suitable for hair coloring. When the purified Flac1 was applied for hair coloring system without using hydrogen peroxide, effective coloring was observed at the protein amount of 0.25mg/1g of hair used. These results indicated that this alkaline-induced novel laccase isolated from the culture supernatant of F. velutipes might be a useful enzyme for hair color.

3種の生薬を主成分としたペーストタイプの歯肉炎, 歯周炎治療薬「アセス」と同一処方である含嗽剤LA -1の歯周疾患に対する臨床的検討を行った。臨床所見および各種診査により歯肉炎, 歯周炎と診断された96症例を対象としだ。ブラッシングは患者が従来行っていた方法で歯磨剤を使用しないで行わせ, ブラッシング終了後本剤を1日, 朝, 夕2回4週間使用させ以下の結果を得た。1. 歯肉炎の諸症状に対して改善が認められ有効率は92.3%であった2. 歯周炎の諸症状に対して改善が認められ有効率は2週目で55.7%, 4週目で77.1%であった。3. 試験期間中LA-1が原因と考えられる副作用は1例も認められなかった。4. LA-1の歯周疾患に対する効果が認められ, 一般用医薬品として有用な薬剤であると考えられた。

歯周外科手術の際に用いられる縫合糸の種類が創傷治癒に与える影響を検索する一助として勿碗zo, 伽oにおいて縫合糸へのプラーク内微生物の付着状態をSEMを用いて観察した。in vitroではA. viscosus, S. viv sanguis, S. mutansの3菌種を使用し, 太さ5-0のマルチフィラメントである絹糸, ポリェステル糸, モノフィラメントであるナイロン糸の3種類の糸に対し静菌系, 増菌系において付着試験を行った。また歯周外科手術時に前述の3種の縫合糸を用いて縫合を行い, 抜糸時に試料として採取し, SEMを用いて観察を行った。その結果, 2種のマルチフィラメントの縫合糸はモノフィラメントの縫合糸に比較しプラーク内微生物が付着しやすく, またin vitroの系では毛細管現象によると思われる微生物の縫合糸上での移動も認められ, in vivoでは, 組織を貫通viしtてroいた縫合糸上にも細菌のコロニー形成が存在した。

ヒトの歯槽骨由来の骨芽細胞様細胞の特性を調べるために, 細菌性コラゲナーゼで処理した歯槽骨片から増殖してくる細胞群 (E-AB) と無処理の骨片から増殖してくる細胞群 (N-AB) とに分けて培養し, 両細胞群のタンパク質合成を14C-prolineの取込みで比較検討した。両細胞群の培養上清に分泌するタンパク質に占めるコラーゲン性タンパク質の割合は, E-ABで12.5%, N-ABで10.4%と異なり, その結果放射能 (14C) の取込量はE-ABの方がN-ABよりわずかに高い値を示したが, 両細胞群の全タンパク質合成能には差異を認めなかった。両細胞群の分泌タンパク質をコラゲナーゼあるいは臭化シアンで処理したものをSDS-ポリアクリルアミドゲル電気泳動で分離して比較すると, 両細胞群が合成する各タンパク質の量的比率には相違はあるが, 種類は類似していた。また, 両細胞群ともにI型コラーゲンの他に少量のIII型コラーゲンを分泌し, 型別合成比率もほぼ同程度であった。索

Preparation of 6-substituted 4-hydroxytetrahydro-2H-pyran-2-one (12), a key intermediate for the synthesis of (-)-tetrahydrolipstatin, was achieved in an optically pure form by employing an enantioselective deprotonation reaction of the prochiral bicyclic derivative (5) as a key step.

数mg以下の試料中の0.5～2mg 程度の炭化ケイ素(SiC)を薄層試料としてXRD定量する場合の問題点について研究した.SiCは高硬度で粉砕が困難なので,種々の粒度のSiC粉末を用いて回転試料台で回転させた場合と静止状態で,回折図形の再現性を調べ,定量に必要な粒度の限界を明らかにした.又,各種粒度のSiC粉末に内標準ケイ素粉末を加えた薄層試料のSiC/Si回折線強度比とSiC量との関係式を比較した.その結果,2本のSiC回折線を併用すれば,配向性や構造型混合比の変化が多少あっても,#3000～#400の粒度のSiCを同一検量線で定量できることが分かった.

試料5~10gを用い,フッ化水素酸その他の酸による化学処理と2段階の重液分離によって炭化ケイ素を単離しひょう量する.次いでX線回折分析,蛍光X線分析及び顕微鏡観察をして炭化ケイ素の同定,どんな構造型のものから成るか,妨害物質が含まれていないこと,粒度などを調べる.構造多形をもつ炭化ケイ素の構造型や粒度のような情報はその出所,その他の議論に役立つ.定量下限は約0.02% である.同定に対する妨害物質,回収率及び実際試料の分析例も示してある.3種の試料の分析例からすると一般の土壌試料に応用できると思われる.