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The two main components from a Nelumbo nucifera leaf extract (NnEx) were investigated for their ability to prevent triglyceride accumulation and promoting lipolysis. Sun-dried Nelumbo nucifera leaves were immersed in hot water to extract the soluble components, and the resulting solution was analyzed by LC-MS and nuclear magnetic resonance. The results showed that quercetin-3-O-s-glucuronide (Q3GA) and quercetin were the key components of the NnEx. In vitro experiments confirmed that quercetin and Q3GA functioned in lipid metabolism by promoting triglyceride degradation through inhibition of the cAMP pathway. In vivo experiments showed that NnEx ingestion inhibited the accumulation of neutral fats in ICR mice and transitioned the hepatocytes of type II diabetic KK-Ay mice out of glycogenosis. These results highlight the ability of NnEx to control metabolism by modulating fat and sugar absorption and may provide an interesting novel treatment for obesity and related lifestyle diseases such as type II diabetes.

BACKGROUND Dietary supplementation with carotenoids can have beneficial health effects, but carotenoids are poorly absorbed. OBJECTIVES We aimed to evaluate how milk fermented by lactic acid bacteria affects dietary carotenoid bioavailability in humans and rats and to investigate mechanisms by which active components in milk fermented by Lactobacilli enhance dietary carotenoid absorption. METHODS Male rats (n = 8/group) were administered β-carotene or β-carotene + fermented milk. Rats (n = 6/group) were also pretreated with ezetimibe, a cholesterol absorption inhibitor, to investigate β-carotene transport mechanisms. In humans, 3 studies were conducted using a randomized crossover method. Subjects (n = 16/study) consumed a vegetable (carrot, tomato, or spinach) drink alone or with a fermented milk drink. Blood samples were collected at various time points after consumption. RESULTS In rats, the serum β-carotene area under the concentration-time curve (AUC) was significantly higher for the β-carotene + fermented milk than for β-carotene only. A significant correlation (r = 0.83, P

Objective: The purpose of this study was to clarify the influence of consuming yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (1073R-1-yogurt) on influenza virus-bound s...

Background Chronic inflammation and insulin resistance are factors that are related to obesity. We have suggested that the administration of heat-treated Lactobacillus plantarum OLL2712 (OLL2712) cells can improve glucose and lipid metabolism by suppressing chronic inflammation in mouse models and a preliminary clinical study. Objective The aim of this study was to investigate whether ingesting OLL2712 cells can reduce body fat accumulation and improve metabolic risk factors, in overweight, healthy adults. Methods This study was a randomized, double-blind, placebo-controlled, parallel-group trial conducted at a single center in Japan. The study participants included 100 overweight (BMI range, ≥25 to 5 × 109 heat-treated OLL2712 cells, respectively, daily for 12 wk. The primary outcome was the 12-wk change in the abdominal fat area, as assessed by computed tomography, and the secondary outcomes were glucose and lipid metabolism-related parameters and chronic inflammation markers, which were analyzed using a linear mixed model. Results The 12-wk change of abdominal fat area (difference: 8.5 cm2; 95% CI: 0.3, 16.6 cm2; P = 0.040) and fasting plasma glucose (difference: 3.2 mg/dL; 95% CI: 0.8, 5.6 mg/dL; P = 0.021) were significantly less in the OLL2712 group than the placebo group. The overall trend of serum IL-6 was significantly decreased in the OLL2712 group compared with baseline and the placebo group. Conclusions The ingestion of heat-treated OLL2712 cells reduces body fat accumulation and the deterioration of glycemic control and chronic inflammation, in overweight, healthy adults. We hypothesize that OLL2712 cells may prevent obesity by regulating chronic inflammation. This trial was registered at the University Hospital Medical Information Network Clinical Trials Registry as UMIN000027709.

The two main components from a Nelumbo nucifera leaf extract (NnEx) were investigated for their ability to prevent triglyceride accumulation and promoting lipolysis. Sun-dried Nelumbo nucifera leaves were immersed in hot water to extract the soluble components, and the resulting solution was analyzed by LC-MS and nuclear magnetic resonance. The results showed that quercetin-3-O-ß-glucuronide (Q3GA) and quercetin were the key components of the NnEx. In vitro experiments confirmed that quercetin and Q3GA functioned in lipid metabolism by promoting triglyceride degradation through inhibition of the cAMP pathway. In vivo experiments showed that NnEx ingestion inhibited the accumulation of neutral fats in ICR mice and transitioned the hepatocytes of type II diabetic KK-Ay mice out of glycogenosis. These results highlight the ability of NnEx to control metabolism by modulating fat and sugar absorption and may provide an interesting novel treatment for obesity and related lifestyle diseases such as type II diabetes.

The gut is an extremely complicated ecosystem where micro-organisms, nutrients and host cells interact vigorously. Although the function of the intestine and its barrier system weakens with age, some probiotics can potentially prevent age-related intestinal dysfunction. Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, which are the constituents of LB81 yogurt, are representative probiotics. However, it is unclear whether their long-term intake has a beneficial influence on systemic function. Here, we examined the gut microbiome, fecal metabolites and gene expression profiles of various organs in mice. Although age-related alterations were apparent in them, long-term LB81 yogurt intake led to an increased Bacteroidetes to Firmicutes ratio and elevated abundance of the bacterial family S24-7 (Bacteroidetes), which is known to be associated with butyrate and propanoate production. According to our fecal metabolite analysis to detect enrichment, long-term LB81 yogurt intake altered the intestinal metabolic pathways associated with propanoate and butanoate in the mice. Gene ontology analysis also revealed that long-term LB81 yogurt intake influenced many physiological functions related to the defense response. The profiles of various genes associated with antimicrobial peptides-, tight junctions-, adherens junctions- and mucus-associated intestinal barrier functions were also drastically altered in the LB81 yogurt-fed mice. Thus, long-term intake of LB81 yogurt has the potential to maintain systemic homeostasis, such as the gut barrier function, by controlling the intestinal microbiome and its metabolites.

The ingestion of Lactobacillus plantarum OLL2712 (OLL2712) cells improved glucose metabolism by suppressing chronic inflammation in mouse models and in a preliminary clinical study. We aimed to clarify the effect of OLL2712 on glucose metabolism and chronic inflammation for healthy adults. Prediabetic adults (n = 130, age range: 20&ndash, 64 years) were randomly assigned to either the placebo or OLL2712 groups (n = 65 each) and were administered conventional yogurt or yogurt containing more than 5 &times, 109 heat-treated OLL2712 cells, respectively, daily for 12 weeks. Reduced HbA1c levels after 12 weeks of treatment were observed in both groups compared to those at baseline, however, the 12-week reduction of HbA1c levels was significantly greater in the OLL2712 group than in the placebo group. Increased chronic inflammation marker levels and insulin-resistant index (HOMA-IR) occurred in the placebo group but not in the OLL2712 group. Fasting blood glucose (FBG) levels did not change significantly in both groups, however, in subgroup analyses including participants with higher FBG levels, FBG levels were significantly reduced only in the OLL2712 group compared to baseline. These results suggest that OLL2712 cell ingestion can reduce HbA1c levels and can prevent the aggravation of chronic inflammation and insulin resistance.

Background Chronic inflammation and insulin resistance are factors that are related to obesity. We have suggested that the administration of heat-treated Lactobacillus plantarum OLL2712 (OLL2712) cells can improve glucose and lipid metabolism by suppressing chronic inflammation in mouse models and a preliminary clinical study. Objective The aim of this study was to investigate whether ingesting OLL2712 cells can reduce body fat accumulation and improve metabolic risk factors, in overweight, healthy adults. Methods This study was a randomized, double-blind, placebo-controlled, parallel-group trial conducted at a single center in Japan. The study participants included 100 overweight (BMI range, ≥25 to 5 × 109 heat-treated OLL2712 cells, respectively, daily for 12 wk. The primary outcome was the 12-wk change in the abdominal fat area, as assessed by computed tomography, and the secondary outcomes were glucose and lipid metabolism-related parameters and chronic inflammation markers, which were analyzed using a linear mixed model. Results The 12-wk change of abdominal fat area (difference: 8.5 cm2; 95% CI: 0.3, 16.6 cm2; P = 0.040) and fasting plasma glucose (difference: 3.2 mg/dL; 95% CI: 0.8, 5.6 mg/dL; P = 0.021) were significantly less in the OLL2712 group than the placebo group. The overall trend of serum IL-6 was significantly decreased in the OLL2712 group compared with baseline and the placebo group. Conclusions The ingestion of heat-treated OLL2712 cells reduces body fat accumulation and the deterioration of glycemic control and chronic inflammation, in overweight, healthy adults. We hypothesize that OLL2712 cells may prevent obesity by regulating chronic inflammation. This trial was registered at the University Hospital Medical Information Network Clinical Trials Registry as UMIN000027709.

Background: Although there is evidence about the beneficial effects of probiotics, their effects on aspirin-induced small bowel injuries have not been well examined. We evaluated the effects of the probiotic Lactobacillus gasseri OLL2716 (LG) on aspirin-induced small intestinal lesions, such as ulcers, erosions, reddened lesions, and bleeding. Summary: This study enrolled 64 patients who received aspirin for more than 1 month and provided written informed consent to be part of the study. The patients received 112 ml of yogurt containing LG or placebo twice daily for 6 weeks. Small bowel injuries were evaluated by capsule endoscopy before and after consuming the yogurt. The effect of LG on patient symptoms was also assessed using the Frequency Scale for the Symptoms of Gastroesophageal Reflux Disease (FSSG) and Gastrointestinal Symptom Rating Scale (GSRS) questionnaires before and after 6 weeks of treatment. There was no significant difference in any baseline characteristics and the number of small bowel mucosal breaks between the 2 groups. In contrast with the placebo group, the LG group had significantly fewer small bowel mucosal breaks and reddened lesions after 6 weeks (p < 0.01). The FSSG and GSRS scores were also significantly improved in the LG group but not in the placebo group. Key Messages: This double-blind, placebo-controlled study found that LG may be useful in reducing aspirin-induced small bowel injuries and in mitigating gastrointestinal symptoms.

Yogurt is generally recognized as a beneficial food for our health, but research into its physiological effects has focused mainly on intestinal dysfunctions such as constipation and diarrhea. We previously found yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (hereafter OLL1073R-1) could reduce risks of catching the common cold and flu in human trials. It was assumed that immunostimulatory exopolysaccharide (EPS) produced from OLL1073R-1 play an important role in this context. However, few studies have examined the immunostimulatory effects of traditional Bulgarian yogurts fermented with different strains of lactobacilli and their metabolites. Therefore, we screened 139 L. delbrueckii ssp. bulgaricus strains and identified OLL1073R-1 as the most robust producer of EPS. This strain was also the only strain that induced the production of IFN-γ in vitro. Oral administration of the EPS or yogurt fermented with OLL1073R-1 and Streptococcus thermophilus OLS3059 (OLL1073R-1 yogurt) augmented natural killer (NK) cell activity and induced IFN-γ production in spleen cells in mice, whereas 2 other yogurts fermented with other strains had no effect on NK cell activity. Cellular preparations of the OLL1073R-1 strain also slightly augmented NK cell activity, but were less effective than EPS itself. The EPS-dependent stimulation of NK cell activity was abrogated in IFN-γ knockout mice and in myeloid differentiation factor 88 knockout mice. Furthermore, IFN-γ production from spleen cells stimulated with EPS was completely blocked with both anti-IL-12 and anti-IL-18 antibodies in vitro. These findings suggest that NK cell activation by OLL1073R-1 yogurt is EPS-dependent, occurs via IL-12- and IL-18-mediated IFN-γ production, and requires myeloid differentiation factor 88. We showed that traditional Bulgarian yogurt could exert immunostimulatory effects by selecting starter strains and part of the mechanisms depend on IFN-γ inducible EPS produced from L. delbrueckii ssp. bulgaricus. Further investigations on processes of fermentation to increase of the EPS may lead to the development of new functional foods that keep our immune functions stable.

Chronic inflammation is involved in the development of colon cancer by inducing mutations and aberrant DNA methylation in colon epithelial cells. Furthermore, there is growing evidence that colonic microbiota modulates the inflammation response in the host and influences colon tumorigenesis. However, the influence of colonic microbiota on aberrant DNA methylation remains unknown. Here, we show the effect of colonic microbes on DNA methylation and tumorigenicity using a mouse model of human ulcerative colitis. Mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS) showed an increase in degree of colitis, as estimated by body weight, occult blood, and stool consistency/diarrhea at 2 weeks after treatment, but treatment with antibiotics markedly reduced the severity of the colitis. Although mucosal hyperplasia and increased inflammation‐related genes were observed in the colonic epithelial cells of the AOM/DSS‐treated mice, treatment with antibiotics abrogated these changes. In addition, treatment with antibiotics significantly decreased the number of mucosal nodules from 5.9 ± 5.3 to 0.2 ± 0.6 (P

Fatigue caused by summer heat is a typical indefinite complaint along with anorexia, loss of sleep, stress, lack of motivation and, in some cases, catching a cold. Yogurt fermented with Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 has been shown to stimulate the immune system and reduce the risk of catching colds. Here, we conducted a randomized, double-blinded, placebo-controlled trial to investigate whether ingesting this yogurt could ameliorate summer heat fatigue in 49 healthy males (median age 40.0 &plusmn, 6.0 years, 30&ndash, 49 years) who felt fatigued every summer. Fatigue was evaluated by visual analogue scales (VAS) and the balance of sympathetic/parasympathetic nervous systems. After 12 weeks of ingestion in early autumn, the VAS fatigue scores in the yogurt group were lower than those of the placebo group. These results indicate that yogurt fermented with L. bulgaricus OLL1073R-1 can ameliorate summer heat fatigue lasting up to early autumn.

Objective Previously, we demonstrated that the administration of heat-killed OLL2712 cells suppressed chronic inflammation and improved hyperglycemia in a mouse model of obesity and diabetes. The aim of this study was to preliminarily examine the effect of OLL2712 supplementation on glucose metabolism and chronic inflammation in prediabetic subjects. Methods This study was a prospective, 12-wk, single-arm, open trial, followed by a 4-wk posttreatment period. Inclusion criteria were fasting plasma glucose levels of 105 to 130 mg/dL in an age range of 35 to 65 y. Thirty individuals consumed a dairy beverage containing ∼1 × 1010 heat-killed OLL2712 cells for 12 wk. Results The ingestion of the OLL2712 beverage significantly improved fasting plasma glucose levels, serum glycoalbumin levels, and insulin resistance indexes compared with baseline levels. The intervention also suppressed serum monocyte chemotactic protein-1 and interleukin-6 levels, which are proinflammatory cytokines involved in the development of insulin resistance and hyperglycemia. Furthermore, stratified analysis by these proinflammatory cytokine levels revealed that the beneficial effects of OLL2712 beverage were observed particularly in individuals with chronic inflammation at baseline. Conclusion The results of this study suggested that heat-killed OLL2712 cells have the potential to improve insulin resistance and glucose metabolism by suppressing chronic inflammation.

Lactobacillus gasseri PA-3 (PA-3) has been previously shown to decrease serum uric acid (SUA) levels in subjects with increased SUA. In this study, we investigated whether PA-3 is also capable of decreasing SUA levels in patients with hyperuricaemia and/or gout.Twenty-five patients with hyperuricaemia and/or gout completed this study. Urate-lowering drugs were discontinued for 12 weeks (week -4 to week 8). After flushing of urate-lowering drugs for 4 weeks (week 0), patients were randomised equally to receive diets containing yoghurt beverages with PA-3 or without PA-3 for a duration of 8 weeks (week 8). The intention to treat (ITT) population included all subjects who were randomised, and the per-protocol (PP) population included subjects who completed the experiment with compliance. We evaluated SUA levels at the end of the study as well as changes in SUA levels in comparison to week 0.In both ITT and PP analyses, there were no significant differences in SUA levels or in the changes in SUA levels compared to week 0 between the two groups. However, in a sub-population whose SUA levels at week 0 were within one SD of the mean of the whole PP population, changes in SUA levels in the group consuming PA-3-containing yoghurt were significantly lower than those of the control group (p = .0378).PA-3-containing yoghurt improves SUA levels, even in patients with hyperuricaemia and/or gout.

Excessive intake of purine-rich foods elevates serum levels of uric acid. Animal and fish meats contain high amounts of inosine and its related purines, and the reduction of taking those purines is crucial for the improvement of serum uric acid levels. We previously showed that Lactobacillus gasseri PA-3 (PA-3) incorporates adenosine and its related purines and that oral treatment with PA-3 reduced adenosine absorption in rats. This study investigated whether PA-3 also incorporates IMP (inosine 5′-monophosphate), inosine, and hypoxanthine, and whether it reduces their absorption in rats. PA-3 was incubated in vitro with radioisotope (RI)-labeled IMP, inosine, and hypoxanthine, and the incorporation of these compounds by PA-3 was evaluated. In addition, rats were orally administered PA-3 along with RI-labeled inosine 5′-monophosphate, inosine, or hypoxanthine, and the ability of PA-3 to attenuate the absorption of these purines was determined. PA-3 incorporated all three purines and displayed greater proliferation in the presence than in the absence of these purines. Oral administration of PA-3 to rats reduced the absorption of IMP, inosine, and hypoxanthine. These results indicate that PA-3 reduces the absorption of purines contained in foods and it is expected that PA-3 contributes attenuation of the excessive intake of dietary purines.

Although there is evidence about the beneficial effects of probiotics, their effects on aspirin-induced small bowel injuries have not been well examined. We evaluated the effects of the probiotic Lactobacillus gasseri OLL2716 (LG) on aspirin-induced small intestinal lesions, such as ulcers, erosions, reddened lesions, and bleeding.This study enrolled 64 patients who received aspirin for more than 1 month and provided written informed consent to be part of the study. The patients received 112 ml of yogurt containing LG or placebo twice daily for 6 weeks. Small bowel injuries were evaluated by capsule endoscopy before and after consuming the yogurt. The effect of LG on patient symptoms was also assessed using the Frequency Scale for the Symptoms of Gastroesophageal Reflux Disease (FSSG) and Gastrointestinal Symptom Rating Scale (GSRS) questionnaires before and after 6 weeks of treatment. There was no significant difference in any baseline characteristics and the number of small bowel mucosal breaks between the 2 groups. In contrast with the placebo group, the LG group had significantly fewer small bowel mucosal breaks and reddened lesions after 6 weeks (p0.01). The FSSG and GSRS scores were also significantly improved in the LG group but not in the placebo group. Key Messages: This double-blind, placebo-controlled study found that LG may be useful in reducing aspirin-induced small bowel injuries and in mitigating gastrointestinal symptoms.

Dendritic polymer-modified porous silica particles prepared by a poly(amidoamine) (PAMAM) dendrimer synthesis reaction were characterized by elementary analysis (EA) and size exclusion chromatography (SEC). EA demonstrated the successful alternate growth of dendritic layers on the particles. The phased growth of polymer moieties by cascade reactions filled mesopores on the silica particles and decreased the exclusion limit molecular weight (Mlim) step by step. The density of the dendritic polymer moieties was affected by the reaction conditions, such as reaction time, temperature, and generation. The mesopore size on the particles, indicated as Mlim, was controlled by the grafting ratio, which could be adjusted by the reaction conditions. Furthermore, the elution behaviors of SEC standard samples with a Mw lower than the Mlim were governed by the local microstructure of the dendritic polymer moieties on the silica particles, most notably by polymer density. It was found that the local microstructure of th...

Exposure to ultraviolet-B (UV-B) irradiation causes skin barrier defects. Based on earlier findings that milk phospholipids containing high amounts of sphingomyelin (SM) improved the water content of the stratum corneum (SC) in normal mice, here we investigated the effects of dietary milk SM on skin barrier defects induced by a single dose of UV-B irradiation in hairless mice. Nine week old hairless mice were orally administrated SM (146 mg/kg BW/day) for a total of ten days. After seven days of SM administration, the dorsal skin was exposed to a single dose of UV-B (20 mJ/cm2). Administration of SM significantly suppressed an increase in transepidermal water loss and a decrease in SC water content induced by UV-B irradiation. SM supplementation significantly maintained covalently-bound ω-hydroxy ceramide levels and down-regulated mRNA levels of acute inflammation-associated genes, including thymic stromal lymphopoietin, interleukin-1 beta, and interleukin-6. Furthermore, significantly higher levels of loricrin and transglutaminase-3 mRNA were observed in the SM group. Our study shows for the first time that dietary SM modulates epidermal structures, and can help prevent disruption of skin barrier function after UV-B irradiation.

近年, 無水フタル酸製造において, その原料事情によりナフタレンおよびo-キシレンに共用できる高機能触媒の開発が望まれている。この目的に適用できる耐摩耗性TiO2担持V2O5系流動触媒の開発例はこれまで報告されていない。本報では, 耐摩耗性に優れた微小球形の高活性, 高寿命の流動触媒の開発のため, スプレードライ法を用いる300°Cで焼成したときのアナターゼTiO2 (2θ=25.3°) の結晶子径が10nm以下となる水酸化チタンゲルを担体として用いる新規な触媒製造法を開発し, さらに第3成分の添加により流動触媒の組成および物性の適正化を検討した。その結果, 既報のSiO2担持V2O5系触媒の結果と異なり, 無水フタル酸生成にはK2SO4/V2O5

Background Dietary milk phospholipids (MPLs) increase hydration of the stratum corneum and reduced transepidermal water loss (TEWL) in hairless mice fed a standard diet. However, the mechanism by which MPLs improve skin barrier functions has yet to be established. Objective This study was designed to examine the mechanism by which MPLs may affect covalently bound ceramides and markers of skin inflammation and improve the skin barrier defect in hairless mice fed a magnesium-deficient (HR-AD) diet. Methods Four-week-old female hairless mice were randomized into four groups (n = 10/group), and fed a standard (control) diet, the HR-AD diet, the HR-AD diet supplemented with either 7.0 g/kg MPLs (low [L]-MPL) or 41.0 g/kg MPLs (high [H]-MPL). Results Dietary MPLs improved the dry skin condition of hairless mice fed the HR-AD diet. MPLs significantly increased the percentage of covalently bound ω-hydroxy ceramides in the epidermis, and significantly decreased both thymus and activation-regulated chemokine (TARC) mRNA and thymic stromal lymphopoietin (TSLP) mRNA levels in skin, compared with the HR-AD diet. Furthermore, the MPL diets significantly decreased serum concentrations of immunoglobulin-E, TARC, TSLP, and soluble P-selectin versus the HR-AD diet. Conclusion Our study showed for the first time that dietary MPLs may modulate epidermal covalently bound ceramides associated with formation of lamellar structures and suppress skin inflammation, resulting in improved skin barrier function.

流動層ナフタレン酸化触媒として微小粒径シリカゾルをシリカ源とする耐摩耗性シリカ担持V2O5-K2SO4-SO3系共融組成触媒の製造法を検討し, 優れた流動触媒物性と無水フタル酸収率を与える触媒を開発した。本触媒を実装置に適用した結果, 初期には320°Cの低温で従来触媒を大幅に上回る無水フタル酸収率の向上 (約4%) を達成した。しかし, この低温条件下で使用中, 経時的に触媒活性の低下と流動不良現象が発生した。本研究では本融液組成触媒の触媒劣化原因を触媒組成の変化の面よりSEM-EPMA分析技術を用いて究明した。この流動不良現象は低温反応および原料ナフタレン負荷による触媒活性成分 (V2O5/K2O/SO3) の粒子外表面への移動および触媒中活性成分組成比の変化に起因し, それにより粒子表面に析出した高融点活性成分 (K2O/V2O5モル比2以下) により促進されることが判明した。酸化排ガス中SO2濃度 (触媒からのSO3揮散に相当する) は, ナフタレン酸化活性の低下および流動不良の発生に重要な役割を果たしていることが確認された。Cs2SO4添加により低融点化した共融混合物からなる新改良触媒を用いた実装置での評価テストにおいて, 安定操業と収率向上が確認された。触媒中のV5+濃度と実装置での無水フタル酸収率との間に良好な相関性が得られた。

Human midkine is expressed and secreted in the medium under the control of an AOX1 gene promoter in Pichia pastoris using its own secretion signal. The midkine precursor is properly processed to yield the correct amino-terminus of mature midkine. However, more than half of the product receives yeast specific mannosylations. The sites for the mannosylations were determined to be the three threonine residues in the carboxy-terminal region of human midkine. In order to obtain non-mannosylated midkine, alanine residues were substituted for the three threonine residues by site specific mutagenesis. HPLC and mass spectrometry confirmed that the mutant midkine contained almost no mannose residues. Despite the amino acid substitutions in the carboxy-terminal region, mutant human midkine, promoted CHO cell proliferation as well as normal midkine.

Recombinant human midkine was expressed in the cells of Pichia pastoris under the control of the AOX1 gene promoter. The expression of midkine was efficiently induced by methanol in a high cell density fermentation. Approximately 0.3 g/l culture of midkine accumulated in the cells by 72 h after induction. When the cells were disrupted, midkine was recovered in an insoluble form, and was insoluble even in the presence of 7 M urea. The precipitate was dissolved in the buffer solution (pH 8) containing 8 M guanidine hydrochloride, 10 mM dithiothreitol, 1 mM EDTA and 50 mM Tris-Cl, and then, midkine was renatured by dialysis at high concentration against the buffer solution (pH 8) containing 0.5 M sodium chloride and 20 mM Tris-Cl. The renatured midkine was recovered using a SP-Sepharose column, and purified further by Heparin-Sepharose column chromatography. Approximately 64 mg/l culture of the purified midkine was obtained. The amino acid sequence of amino-terminus and the amino acid composition of midkine were the same as those of Met-midkine that has a methionine residue at the amino-teminus. Mass spectrometry of purified Met-midkine showed a mass of 13370.7 Da (average), almost the theoretical mass for it. The Met-midkine enhanced the proliferation of Chinese hamster ovary (CHO) cells.

It is known that the stomach is colonized by indigenous lactobacilli in mice. The aim of this study was to examine the role of such lactobacilli in the development of the stomach. For a DNA microarray analysis, germ-free BALB/c mice were orally inoculated with 10 9 CFU lactobacilli, and their stomachs were excised after 10 days to extract RNA. As a result, lactobacillus-associated gnotobiotic mice showed dramatically decreased expression of the gastrin gene in comparison to germ-free mice. The mean of the log 2 fold change in the gastrin gene was −4.3. Immunohistochemistry also demonstrated the number of gastrin-positive (gastrin + ) cells to be significantly lower in the lactobacillus-associated gnotobiotic mice than in the germ-free mice. However, there was no significant difference in the number of somatostatin + cells in these groups of mice. Consequently, gastric acid secretion also decreased in the mice colonized by lactobacilli. In addition, an increase in the expression of the genes related to muscle system development, such as nebulin and troponin genes, was observed in lactobacillus-associated mice. Moreover, infection of germ-free mice with Helicobacter pylori also showed the down- and upregulation of gastrin and muscle genes, respectively, in the stomach. These results thus suggested that indigenous lactobacilli in the stomach significantly affect the regulation of gastrin-mediated gastric acid secretion without affecting somatostatin secretion in mice, while H. pylori also exerts such an effect on the stomach.

Approximately 260 mg/l of authentic recombinant human pleiotrophin (rhPTN) was expressed into the medium of high-cell density fermentation using a Pichia pastoris protein expression system. The prepro-sequence of yeast alpha-mating factor was used successfully. The recombinant hPTN was efficiently recovered from the medium by expanded bed adsorption, and purified using successive column chromatography steps. In the purified rhPTN preparation, modified rhPTN were scarcely detected. Circular dichroism measurement of the purified PTN showed the presence of the characteristic beta-structures in the protein.

Recombinant human bile salt-stimulated lipase (rhBSSL) was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris . Human BSSL has 16 successively repeated sequences in the carboxy terminal region. The sequence consists of 11 amino acid residues. The coding sequence for the middle 11 of the 16 repeats was removed from hBSSL cDNA to facilitate efficient secretory expression. The clone used for fermentation was a transformant of GS115 ( his4 ) integrated with four copies of the expression cassette containing the modified hBSSL cDNA. Unique fermentation conditions were required for efficient expressions of rhBSSL in the high cell-density fermentation. A sufficient glycerol feed at 30°C and pH 4 under an adequate concentration of dissolved oxygen in the growth phase make the cells active over a long induction period of approximately 15 days. On methanol induction, the concentration of dissolved oxygen should be maintained very low in the presence of sorbitol and skimmed milk at 20°C and pH 5.7. Under these conditions, 0.8–1 g of rhBSSL was secreted in 1 liter of the medium. By immunoelectron microscopy, rhBSSL-tagged gold particles were located in secretion microbodies after the beginning of methanol induction. The secreted rhBSSL was efficiently captured and purified by expanded bed adsorption chromatography.

Experiments were carried out to study the relationship between binding affinity to the benzodiazepine receptor and pharmacological activity, especially anti-anxiety activity, of clinically useful benzodiazepines. In the in vitro experiments, fludiazepam showed the highest affinity to the benzodiazepine receptor with 4 times more potency than that of diazepam, which paralleled the in vivo activity. Diazepam and nimetazepam also bound with high affinities as expected from their in vivo activities. On the contrary, medazepam and cloxazolam showed extremely low affinities and oxazolam showed no affinity, although they showed moderate in vivo activity. However, their metabolites were found to have both high affinity and in vivo activities. These results strongly suggest that in the case of medazepam, cloxazolam and oxazolam, their metabolites may bind to receptor sites in the brain and then elicit pharmacological action. This conclusion was supported by the fact that a good correlation between the binding affinity and the anti-anxiety activity of the tested compounds was observed.

エチルベンゼンからスチレンの製造に対する酸化鉄触媒の Al2O3 と K2O による二重促進効果について調べた。酸化鉄触媒へ 6.6wt% 以下の Al2O3 を合体させるとスチレン収率が増大した。Al2O3 6.6wt% 含有酸化鉄触媒へ K2O 2.0wt% を添加するとその触媒選択性は 82% から 96% まで増大した。上記結果はピリジン化学吸着値とよく関係づけられた。比較実験から, Al2O3 と K2O を適当の割合にいれた本酸化鉄触媒は活性・安定性の点で Cr2O3 を含有する二種類の現用工業触媒に劣らないことがわかった。

The vapor phase sqtalyt-ic-methylation of ev-picoline with methanol has been studied in a flow system using solid acid, base and acid-base catalysts over a range of temperatures fr6n1400 to 530 C under atmospheric pressure.On Shes. e cat. a.1. ystsAthg 14. ajor. liquid-produ. cts obtained were 2, 6-lutidine along with small amounts of pyridine, B-picoline, 2, 4 and 2, 5-lutidines and 2, 4, 6-collidine. The Torder of the gatalytic qctivity wqs silica Tnagnesuzia silica-alumina kk T-alumina fluorinated rm T: al miKaV magnesium phosphate. Silica gel, magnesia and calcia were virtually inactive.

The vapor phase catalytic synthesis of xylenes from toluene and formaldehyde in the presence of hydrogenC6H5CH3+HCHO+H2=C6H4(CH3)2+H2Ohas been studied over some catalysts in a flow system under an atmospheric pressure.Among several catalysts examined, a silica-alumina catalyst containing 0.1wt% of platinum was found to be the most active and selective for the production of xylenes rich in p-isomer: The concentration of p-xylene in their isomers produced at a temperature of 455°C was approximately two times higher than that of the equilibrium composition. It was confirmed that the catalytic stability of silica-alumina containing platinum for this reaction was far better than that of silica-alumina alone.

The kinetics of gas-phase catalytic isomerization of o-xylene has been studied with a flow reactor under normal pressure at a temperature range between 340 and 420°C.The hydrogen/feed molar ratio has been kept at 1 to 3 and that of W/F at 5.9 to 20.4hr•g-cat./mole over a silica-alumina catalyst exchanged with nickel-ion.The rate of reaction has been correlated satisfactorily with a mechanism which assumed the single-site surface reaction, and the rate expressionr=kiKo-x(po-x-pm-x/Kp)/1+Ko-xpo-x+Km-xpm-xhas agreed fairly well with the data, in accordance with the kinetics of the same reaction over a silica-alumina catalyst without nickel-ion.These results have been discussed in the light of the cation-exchange effects on the acid property of silica-alumina which had been suggested in the previous papers.

The vapor phase catalytic synthesis of aniline from phenol and ammonia over some catalysts has been studied in a flow reactor under atmospheric pressure. Activity tests were carried out at a temperature range of 380-530°C, a W/F ratio of 3.7g cat•hr/mol total-feed and 3 mole ratio of ammonia/phenol. The order of catalyst activities at 450°C was found to be as follows:SiO2-Al2O3(25%Al2O3)>>SiO2-Al2O3(13%Al2O3)>5%F-SiO2-Al2O3(13%Al2O3)>7.5%F-SiO2-Al2O3(25%Al2O3)>CaY zeolite>>γ-Al2O3.SiO2-MgO, Ca phosphate and NaY zeolite were virtually inactive. The activities of these catalysts were reasonably correlated with the heat of adsorption of phenol on their surfaces determined gas-chromatographically, showing that the adsorption of phenol on the catalyst plays an important role in this reaction. A kinetic experiment on the most effective catalyst SiO2-Al2O3 (25%Al2O3) at 500°C using a differential reactor technique indicated that the rate of the production of aniline was proportional to pNH3 at a partial pressure range of phenol of 0.4-0.6atm and to pNH3•p1/2C_??_H_??_O_??_ at that of phenol of 0.04-0.09atm. The partial pressure dependency on the rate was explained according to the Rideal-Eley mechanism, involving a surface reaction between phenol chemisorbed and ammonia physically adsorbed or in bulk gas phase.

The role of acid catalysts, such as platinum on silica-alumina, in the formation of xylenes rich in p-isomer from CH3C6H5 and HCHO in the presence of hydrogen was investigated using the extended Huckel molecular orbital theory. We made several findings. Firstly, the reactivity of CH3C6H5 increased remarkably on the basis of its chemisorption on the catalyst by means of the pseudo σ-orbital of CH3, while that of HCHO stayed almost constant, with or without chemisorption on the catalyst. Secondly, there was some evidence that p- or o-CH3C6H4CH2OH was a reaction intermediate. An assumption was made that CH3C6H4CH2OH resulted from the reaction between chemisorbed CH3C6H5 and physically adsorbed HCHO. Thirdly, the catalytic decomposition of the intermediate made available CH3C6H4CH2 radicals, which coupled with H radicals formed by the partial decomposition of HCHO and so provided the desired product, p-CH3C6H4CH3, with higher selectivity. Finally, detailed discussion was made with respect to the modes of the chemisorption of the reactants on the catalyst, together with the reaction mechanism, in accordance with our particular interests.

Catalytic properties of nickel supported on stoichiometric titanium carbide in acetone hydrogenation have been studied at 150C.Over the catalyst (I) which was rcduced with hydrogen after calcination in nitrogen isobutyl methyl ketone was formed as a minor product, whereas over the catalyst (II) which was reduced after calcination in air the ketone was formed predominantly along with a small amount of 2-propanol. X-Ray analysis showed the existence of nickel oxide in I in addition to metallic nickel and stoichiometric titanium carbide, whereas in asmall amount of titanium dioxide in rutile form existed in addition to the above species. Both nickel oxide on the carbide (III) and nickel on rutile (IV) exhibited some activity for the acetone hydrogenation; the main product was the alcohol over and the ketone over IV. Over unsupported nickel the ketone was not formed.It is concluded from the experimental results that the catalyst systems which consisted of nickel with or without its oxide and titanium carbide with or without titanium dioxide caused the simultaneous formation of the alcohol and the ketone in the hydrogenation of acetone.