Fumiko Higashikawa

Improvement of constipation and liver function by plant-derived lactic acid bacteria : A double-blind, randomized trial

OBJECTIVE Lactic acid bacteria (LAB) contribute to human health; however, the probiotic properties vary among strains classified into the same species. The primary objective of this study was to evaluate the effects of yogurts made by different types of LAB on the gastrointestinal system. The yogurts were also evaluated by measuring serum lipid contents and liver functional indicators as a secondary objective. METHODS Healthy human adults (n = 68) with some complaints with regard to intestinal health, including constipation and diarrhea, were randomly assigned to receive one of three types of yogurt in a double-blind manner: type A, a yogurt made by plant-derived LAB (mainly Lactobacillus [Lb.] plantarum SN35N); type B, a yogurt made by plant-derived LAB (mainly Lb. plantarum SN13T); and type C, a yogurt made by animal-derived LAB (mainly Lactococcus lactis A6 and Streptococcus thermophilus 510) as a control. The subjects consumed 100 g of yogurt daily for 6 wk. Data were collected from clinical visits at 2-wk intervals and by diaries used to record defecation and health conditions. RESULTS Drastic and constant increments of defecation frequency in subjects with constipation were observed with type A and B yogurts but not with type C yogurt. Type B and C yogurts resulted in decreases in total and low-density lipoprotein cholesterol. The serum concentrations of liver functional parameters were improved by the type B yogurt (12-25% reduction). CONCLUSION The present study suggests that Lb. plantarum SN13T exhibits a superior probiotic effect on constipation in addition to improving the serum lipid contents and liver function.

Dose-dependent Intestinal and Hepatic First-pass Metabolism of Midazolam, a Cytochrome P450 3A Substrate with Differently Modulated Enzyme Activity in Rats

The dose‐dependent first‐pass metabolism of midazolam, a cytochrome P450 (CYP) 3A substrate, was separately estimated in the intestine and liver after administration into a jejunal loop of rats with differently modulated enzyme activity. Modulation of CYP3A enzyme activity of Sprague‐Dawley rats was performed by pretreating the rats with inducers such as dexamethasone or by co‐administering ketoconazole (an inhibitor) with midazolam.

Enhanced biological activity of polymeric osteopontin

Osteopontin is a multifunctional glycoprotein with roles in immunomodulation, inflammatory response, tissue mineralization, and tissue remodeling, which are mediated primarily through integrins. Transglutaminase 2 selectively cross‐links proteins by isopeptide bonding. Osteopontin is one of the substrates of this enzyme and undergoes polymerization; however, the biological meaning of this polymerization remains unknown. Using recombinant osteopontin polymerized with purified transglutaminase 2, we examined cell adhesion, spreading, focal contact formation, and migration of SW480 or HUVE cells. All of these cellular behaviors were dramatically enhanced with polymeric osteopontin. These enhancements of cellular functions imply that polymerization might modulate physiological and pathological functions of osteopontin.

A molecular mechanism for copper transportation to tyrosinase that is assisted by a metallochaperone, caddie protein

The Cu(II)-soaked crystal structure of tyrosinase that is present in a complex with a protein, designated “caddie,” which we previously determined, possesses two copper ions at its catalytic center. We had identified two copper-binding sites in the caddie protein and speculated that copper bound to caddie may be transported to the tyrosinase catalytic center. In our present study, at a 1.16–1.58 Å resolution, we determined the crystal structures of tyrosinase complexed with caddie prepared by altering the soaking time of the copper ion and the structures of tyrosinase complexed with different caddie mutants that display little or no capacity to activate tyrosinase. Based on these structures, we propose a molecular mechanism by which two copper ions are transported to the tyrosinase catalytic center with the assistance of caddie acting as a metallochaperone.

Polymeric Osteopontin Employs Integrin α9β1 as a Receptor and Attracts Neutrophils by Presenting a de Novo Binding Site

Osteopontin (OPN) is a cytokine and ligand for multiple members of the integrin family. OPN undergoes the in vivo polymerization catalyzed by cross-linking enzyme transglutaminase 2, which consequently increases the bioactivity through enhanced interaction with integrins. The integrin α9β1, highly expressed on neutrophils, binds to the sequence SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence appears to be cryptic in intact OPN because α9β1 does not recognize intact OPN. Because transglutaminase 2-catalyzed polymers change their physical and chemical properties, we hypothesized that the SVVYGLR site might also be exposed on polymeric OPN. As expected, α9β1 turned into a receptor for polymeric OPN, a result obtained by cell adhesion and migration assays with α9-transfected cells and by detection of direct binding of recombinant soluble α9β1 with colorimetry and surface plasmon resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a ligand for α9β1, has been reported to attract neutrophils, we next examined migration of neutrophils to polymeric OPN using time-lapse microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which was clearly inhibited by anti-α9β1 antibody. Unexpectedly, mutagenesis studies showed that α9β1 bound to polymeric OPN independently of the SVVYGLR sequence, and further, SVVYGLR sequence of polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize polymeric OPN. These results demonstrate that polymerization of OPN generates a novel α9β1-binding site and that the interaction of this site with the α9β1 integrin is critical to the neutrophil chemotaxis induced by polymeric OPN.

The Obesity and Fatty Liver Are Reduced by Plant-Derived Pediococcus pentosaceus LP28 in High Fat Diet-Induced Obese Mice

We evaluated the effect of an oral administration of a plant-derived lactic acid bacterium, Pediococcus pentosaceus LP28 (LP28), on metabolic syndrome by using high fat diet-induced obese mice. The obese mice were divided into 2 groups and fed either a high fat or regular diet for 8 weeks. Each group was further divided into 3 groups, which took LP28, another plant-derived Lactobacillus plantarum SN13T (SN13T) or no lactic acid bacteria (LAB). The lean control mice were fed a regular diet without inducing obesity prior to the experiment. LP28 reduced body weight gain and liver lipid contents (triglyceride and cholesterol), in mice fed a high fat diet for 8 weeks (40%, 54%, and 70% less than those of the control group without LAB, and P = 0.018, P<0.001, and P = 0.021, respectively), whereas SN13T and the heat treated LP28 at 121°C for 15 min were ineffective. Abdominal visceral fat in the high fat diet mice fed with LP28 was also lower than that without LAB by 44%, although it was not significant but borderline (P = 0.076). The sizes of the adipocytes and the lipid droplets in the livers were obviously decreased. A real-time PCR analyses showed that lipid metabolism-related genes, such as CD36 (P = 0.013), SCD1 encoding stearoyl-CoA desaturase 1 (not significant but borderline, P = 0.066), and PPARγ encoding peroxisome proliferator-activated receptor gamma (P = 0.039), were down-regulated by taking LP28 continuously, when compared with those of the control group. In conclusion, LP28 may be a useful LAB strain for the prevention and reduction of the metabolic syndrome.

In‐vivo and In‐vitro Metabolic Clearance of Midazolam, a Cytochrome P450 3A Substrate, by the Liver under Normal and Increased Enzyme Activity in Rats

The metabolic clearance of midazolam, a cytochrome P450 (CYP) 3A substrate, by the liver under normal and increased enzyme activity in rats was determined in‐vivo and in‐vitro to elucidate the reproducibility of the in‐vivo hepatic extraction ratio of midazolam from the in‐vitro study. The hepatic enzyme activity was modified by pretreating rats with a CYP inducer such as dexamethasone and clotrimazole.

Association of Single Nucleotide Polymorphisms in the Eosinophil Peroxidase Gene with Japanese Cedar Pollinosis

Background: Japanese cedar pollinosis is the most common form of hay fever in spring in Japan. We have previously demonstrated that single nucleotide polymorphism Pro358Leu of exon 7 in the eosinophil peroxidase (EPO) gene is associated with cedar pollinosis, although the association has not been confirmed by analysis of the whole gene in a different population. Methods: We sequenced all exons of the EPO gene in 60 children with pollinosis and their parents using the PCR-restriction fragment length polymorphism method. Results: We found 8 polymorphisms, Ile40Met, Gln122His, Arg202Arg (A660G), Asn303Asn (C909T), Arg326Pro, Arg326His, Pro358Leu, and Asn572Ty, in the EPO gene. As a result of the transmission disequilibrium test, we recognized significant transmissions of 202Arg (660G) in exon 6 in addition to 358Leu of exon 7 in the EPO gene of affected children. Conclusions: Our results might indicate that polymorphisms of the EPO gene are associated with Japanese cedar pollinosis.

Genotypes and Haplotypes of CCR2 and CCR3 Genes in Japanese Cedar Pollinosis

Whole genome scan analyses have revealed that the chromosomal region 3p21.3, which contains a gene cluster of the CC chemokine receptor, is possibly critical for the pathogenesis of allergic inflammation. Japanese cedar pollinosis is mediated by a type I allergy and induces seasonal rhinitis and conjunctivitis in humans as the most common form of hay fever in spring in Japan, although the candidate genes for cedar pollinosis remain to be elucidated. We sequenced CCR1, CCR2, CCR3, CCR5, and CCXCR1 using the PCR restriction fragment length polymorphism method in subjects with cedar pollinosis and controls. We found 8 polymorphisms of A111G, Arg127Cys and Arg252Gln in CCXCR1, T885C in CCR1, Val64Ile and T780C in CCR2, T51C in CCR3 and Arg223Gln in CCR5. The transmission disequilibrium test using 60 children with pollinosis and their parents and an association study using unrelated adult subjects (151 patients and 157 controls) showed a significant association of 64Ile in CCR2 and 51C in CCR3 with cedar pollinosis. The frequency of haplotype 64Ile/780C/51C in pollinosis was significantly higher than in controls. Our results suggest that CCR2 and CCR3 genes are candidate genes for Japanese cedar pollinosis.

Reduction of serum lipids by the intake of the extract of garlic fermented with Monascus pilosus: a randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND & AIMS A dietary supplement containing garlic fermented with Monascus pilosus (MGFE) may be useful to decrease the lipid concentrations in serum without serious adverse effects. The aim of the study was to assess whether MGFE decreases serum lipid contents in volunteers with mild hyperlipidemia in a randomized, double-blind, placebo-controlled, parallel-group trial. METHODS Healthy subjects (n = 55) with serum triglyceride concentrations of 120-200mg/dL were randomly assigned to take either MGFE or placebo capsules for 12 wk. RESULTS A borderline, but not significant effect, to decrease triglyceride concentrations in serum (approximately 15% reduction at maximum, P = 0.062, in time × treatment interaction effect) was observed by the MGFE intake for 12 wk. The decreased reading exhibited a significant difference at wk-8 between the MGFE and placebo groups (unpaired t test, P = 0.007). The total cholesterol (P = 0.003) and LDL cholesterol (P = 0.001) contents in the serum and the LDL/HDL ratio (P < 0.001) were significant in time × treatment interaction effects by the MGFE intake. However, no alteration in the body fat percentage and abdominal circumference was observed. CONCLUSIONS The intake of MGFE decreased triglyceride and cholesterol in serum with no appreciable adverse effects in normal to mildly hyperlipidemic individuals, suggesting that it may be effective to improve and prevent the metabolic syndrome. This clinical trial was registered with Clinicaltrials.gov as NCT00938249.