Isolation of levoglucosan-utilizing thermophilic bacteria.

We previously developed an industrial production process for novel water-soluble indigestible polysaccharides (resistant glucan mixture, RGM). During the process, an anhydrosugar-levoglucosan -is formed as a by-product and needs to be removed to manufacture a complete non-calorie product. Here, we attempted to isolate thermophilic bacteria that utilize levoglucosan as a sole carbon source, to establish a removing process for levoglucosan at higher temperature. Approximately 800 natural samples were used to isolate levoglucosan-utilizing microorganisms. Interestingly, levoglucosan-utilizing microorganisms-most of which were filamentous fungi or yeasts-could be isolated from almost all samples at 25°C. We isolated three thermophilic bacteria that grew well on levoglucosan medium at 60°C. Two of them and the other were identified as Bacillus smithii and Parageobacillus thermoglucosidasius, respectively, by 16S rDNA sequence analysis. Using B. smithii S-2701M, which showed best growth on levoglucosan, glucose and levoglucosan in 5% (wt/vol) RGM were completely diminished at 50°C for 144 h. These bacteria are known to have a biotechnological potential, given that they can ferment a range of carbon sources. This is the first report in the utilization of levoglucosan by these thermophiles, suggesting that our results expand their biotechnological potential for the unutilized carbon resources.

Digestion-Resistant Dextrin Derivatives Are Moderately Digested in the Small Intestine and Contribute More to Energy Production Than Predicted from Large-Bowel Fermentation in Rats.

Background: Digestion-resistant dextrin derivatives (DRDDs), including resistant maltodextrin (RM), polydextrose, and resistant glucan (RG), have been developed as low-energy foods. However, data on the resistance of DRDDs to small-intestinal digestion are scarce.Objective: We sought to determine the site and extent of DRDD breakdown in the rat intestine and to predict its energy contributions.Methods: In vitro small-intestinal resistance of DRDDs was evaluated by the AOAC method for dietary fiber measurement and by artificial digestion with the use of pancreatic α-amylase and brush-boarder membrane vesicles. In vivo small-intestinal resistance of DRDDs was determined from the feces of male ileorectostomized Sprague-Dawley rats fed a control diet or a diet containing one of the DRDDs at 50 g/kg for 9 d (period 1) and then for 10 d (period 2), during which they received 1 g neomycin/L in their drinking water. Separately, male Sprague-Dawley rats were fed the same diets for 4 wk, and the whole-gut recoveries of DRDDs were determined from feces at days 8-10.Results: Small-intestinal resistances determined in vitro by artificial digestion (RM: 70%; polydextrose: 67%; RG: 69%) were lower than those measured by the AOAC method (RM: 92%; polydextrose: 80%; RG: 82%). In the ileorectostomized rats, fecal dry-matter excretions were consistently greater in the DRDDs than in the control. The small-intestinal resistances of the DRDDs were 68%, 58%, and 62% in period 1 and 66%, 61%, and 67% during period 2 for RM, polydextrose, and RG, respectively. The resistances did not differ among the DRDDs at either time. In the normal rats, food intakes and body weight gains did not differ among the groups. The whole-gut recovery of RM (13%) was lower than that of polydextrose (33%) and RG (29%), which did not differ.Conclusions: DRDDs were more digestible in the rat small intestine than the AOAC method. The energy contribution from small-intestine digestibility, not just large-bowel fermentability, must be considered in determining the energy contribution of DRDDs. Whether humans respond similarly needs to be tested.

Deuterodechlorination of Aryl/Heteroaryl Chlorides Catalyzed by a Palladium/Unsymmetrical NHC System.

The catalytic deuterodechlorination of aryl/heteroaryl chlorides was developed with a palladium/unsymmetrical NHC system, and the precisely controlled introduction of deuterium into a variety of aryl/heteroaryl compounds was achieved with a high level of efficiency, selectivity, and deuteration degree. This method was also successfully applied to the transformation of bioactive agents even in a gram-scale synthesis. The crystal structure analysis of Pd-NHC complexes led to the observation of Pd-arene interaction.

Effects of Resistant Glucan Mixture on Bowel Movement in Female Volunteers.

Resistant glucan (RG) is a water-soluble polysaccharide resistant to hydrolysis by digestive enzymes in the human gastrointestinal system. RG mixture (RGM) contains more than 75% RG as dietary fiber and other saccharides. The effects of ingestion of 3.3, 6.6, and 13.2 g/d of RGM (containing of 2.5, 5.0, and 10.0 g/d RG as dietary fiber) on the fecal properties and the frequency of defecation were investigated in 60 female volunteers with constipation. The study was designed as a randomized, single-blinded, and placebo-controlled parallel-group trial. Each subject consumed RGM or a placebo (digestible maltodextrin) for 2 wk. Questionnaire data on the effects on bowel movement were analyzed according to defecation days, defecation frequency, fecal volume, fecal shapes, fecal color, fecal odor, and fecal excretory feeling. The results showed significant increases in defecation frequency (p<0.05), defecation days (p<0.05), and fecal volume (p<0.05) during the 13.2 g/d RGM ingestion period. The effects of RGM on defecation days and frequency showed a dose-dependent increase (p<0.05). These results suggested that the intake of RGM increased defecation days, defecation frequency, and fecal volume. In the gastrointestinal tract, RGM is useful as a water-soluble dietary fiber for the improvement of bowel movements.

Metabolism and bioavailability of newly developed dietary fiber materials, resistant glucan and hydrogenated resistant glucan, in rats and humans.

BACKGROUND: Resistant glucan (RG) and hydrogenated resistant glucan (HRG) are new dietary fiber materials developed to decrease the risk of metabolic syndrome and lifestyle-related diseases. We investigated the metabolism and bioavailability of RG and HRG using rats and humans. METHODS: Purified RG and HRG were used as test substances. After 25 Wistar male rats (270 g) were fed with an experimental diet (AIN93M diet with the cellulose replaced by ß-corn starch) ad libitum for 1 week, they were used for the experiment involving blood collection and circulating air collection. Ten participants (5 males, 22.5 y, BMI 20.4 kg/m(2); 5 females, 25.8 y, BMI 20.9 kg/m(2)) voluntarily participated in this study. The study was carried out using a within-subject, repeated measures design. Effects of RG and HRG on the response for blood glucose and insulin and hydrogen excretion were compared with those of glucose and a typical nondigestible and fermentable fructooligosaccharide (FOS) in rats and humans. Available energy was evaluated using the fermentability based on breath hydrogen excretion. RESULTS: When purified RG or HRG (400 mg) was administered orally to rats, blood glucose and insulin increased slightly, but less than when glucose was administration (P < 0.05). Hydrogen started to be excreted 120 min after administration of RG with negligibly small peak at 180 min, thereafter excreted scarcely until 1440 min. Hydrogen excretion after HRG administration showed a larger peak than RG at 180 min, but was markedly less than FOS. RG and HRG were excreted in feces, but not urine. When purified RG or HRG (30 g) were ingested by healthy humans, blood glucose and insulin levels increased scarcely. Breath hydrogen excretion increased slightly, but remarkably less than FOS. Ingestion of purified RG or HRG (5 g) to evaluate available energy, increased scarcely glucose and insulin levels and breath hydrogen excretion. Available energy was evaluated as 0 kcal/g for purified RG and 1 kcal/g for HRG. CONCLUSION: The bioavailability was very low in both humans and rats, because oligosaccharide of minor component in purified RG and HRG was metabolized via intestinal microbes but major components with higher molecular weight were metabolized scarcely. Moreover, the ingestion of 30 g of RG or HRG did not induce apparent acute side effects in healthy adults. RG and HRG might potentially be used as new dietary fiber materials with low energy.

Safety evaluation of a newly-developed dietary fiber: resistant glucan mixture.

Resistant glucan mixture (RGM), a water-soluble dietary fiber produced by the random polymerization of glucose with activated carbon as a catalyst at a high temperature, has been recently developed by our group. There has been little physiological and safety research into RGM and therefore we now present our research into its safety. A reverse mutation assay indicated that RGM is not mutagenic either with or without metabolic activation. We conducted a 90-day subchronic oral toxicity study in rats. Male and female rats fed either a 3% or 5% w/w RGM diet had no muddy or watery stools, and there was no RGM-related death in any group. Although some parameters in the 3% and 5% w/w groups were significantly different from those in the control group, these changes were not due to any toxicity from RGM. The results indicated that the No Observed Adverse Effect Level (NOAEL) of RGM was 3.3 and 3.9 g/kg body weight (BW) per day in male and female rats, respectively. We then studied the gastrointestinal effects of RGM in healthy adult humans. Gastrointestinal symptoms, such as gurgling sounds, flatus and tenesmus, were mild and transient. In men and women, the maximum no-effect dose for diarrhea was more than 0.9 g RGM /kg BW. The results of our current safety assessment studies suggest that RGM is safe for human consumption.

Digestibility of new dietary fibre materials, resistant glucan and hydrogenated resistant glucan in rats and humans, and the physical effects in rats.

Resistant glucan (RG) and hydrogenated resistant glucan (HRG) are newly developed non-digestible carbohydrate materials that decrease lifestyle-related diseases. The bioavailability of RG and HRG was investigated by in vitro experiments using human and rat small intestinal enzymes and by in vivo experiments using rats in the present study. Oligosaccharides, which are minor components of RG and HRG, were hydrolysed slightly by small intestinal enzymes of humans and rats, and the hydrolysing activity was slightly higher in rats than in humans. The amount of glucose released from HRG was greater than that from RG. However, the high-molecular-weight carbohydrates of the main components were hardly hydrolysed. Furthermore, neither RG nor HRG inhibited disaccharidase activity. When rats were raised on a diet containing 5 % of RG, HRG, resistant maltodextrin or fructo-oligosaccharide (FOS) for 4 weeks, all rats developed loose stools and did not recover during the experiment, except for the FOS group. Body weight gain was normal in all groups and was not significantly different compared with the control group. Caecal tissue and content weights were significantly increased by feeding RG or HRG, although other organ and tissue weights were not significantly different among the groups. In conclusion, RG and HRG consist of small amounts of glucose and digestible and non-digestible oligosaccharides, and large amounts of glucose polymers, which were hardly hydrolysed by α-amylase and small intestinal enzymes. RG and HRG, which were developed newly as dietary fibre materials, had no harmful effects on the growth and development of rats.

Palladium-catalyzed synthesis of heterocycle-containing diarylmethanes through Suzuki-Miyaura cross-coupling.

The heterocycle-containing diarylmethane synthesis from chloromethyl(hetero)arenes with (hetero)arylboron reagents was attained using the palladium/ether-imidazolium chloride system. This coupling process tolerated a diverse range of heteroaromatic moieties with sufficient catalytic activity to achieve the efficient synthesis of various diheteroarylmethanes in good to excellent yields.

Pleckstrin-2 selectively interacts with phosphatidylinositol 3-kinase lipid products and regulates actin organization and cell spreading.

Pleckstrin-2 (PLEK2) has been implicated to be regulated by phosphatidylinositol (PI) 3-kinase, while pleckstrin1 (PLEK1) has been suggested to be a major PKC substrate in platelets. In this paper, we confirmed that PLEK2 specifically bound to the PI 3-kinase products in vitro and explored its behavior. PLEK2 was found to be expressed in various adherent cell lines, while PLEK1 expression was restricted to non-adherent cells in the protein level. Expression of PLEK2 in COS1 cells induced formation of protrusive F-actin structure and enhanced the actin rearrangements induced on collagen- or fibronectin-coated plates. A PLEK2 mutant incapable of binding to the PI 3-kinase products did not show any effect on actin rearrangement. Knockdown of PLEK2 by shRNA inhibited spreading of HCC2998 adenocarcinoma cells. PLEK2 colocalized with Rac and was suggested to be oligomerized. These results suggest that PLEK2 is involved in actin rearrangement in a PI 3-kinase dependent manner.

Identification of ribosomal protein S3a as a candidate for a novel PI 3-kinase target in the nucleus.

Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) is an important lipid second messenger that mediates various cell responses. We have searched for the nuclear PIP(3) binding proteins using PIP(3) analogue beads. A 33 kD protein was detected in this method, which was identified as ribosomal protein S3a by the mass spectrometric analysis. The recombinant S3a protein bound specifically to PIP(3). S3a localized not only in the cytosol but also in the nucleus. Interestingly, not cytosolic but nuclear S3a bound to PIP(3), suggesting different roles of S3a in the cytosol and the nucleus.