Impact of habitual chewing on gut motility via microbiota transition.

The gut environment, including the microbiota and its metabolites and short-chain fatty acids (SCFA), is essential for health maintenance. It is considered that functional recovery treatment for masticatory dysphagia affects the composition of the gut microbiota, indicating that habitual mastication, depending on the hardness of the food, may affect the gut microbiota and environment. However, the impact of chronic powdered diet feeding on the colonic condition and motility remains unclear. Here, we evaluated various colonic features in mice fed with powdered diets for a long-term and a mouse model with masticatory behavior. We observed a decreased abundance of the SCFA-producing bacterial genera in the ceca of the powdered diet-fed mice. Based on the importance of SCFAs in gut immune homeostasis and motility, interestingly, powdered diet feeding also resulted in constipation-like symptoms due to mild colitis, which were ameliorated by the administration of a neutrophil-depleting agent and neutrophil elastase inhibitors. Lastly, the suppressed colonic motility in the powdered diet-fed mice was significantly improved by loading masticatory activity for 2 h. Thus, feeding habits with appropriate masticatory activity and stimulation may play a key role in providing a favorable gut environment based on interactions between the gut microbiota and host immune system.

Pharmaceutical studies on and clinical application of olanzapine suppositories prepared as a hospital preparation.

BACKGROUND: A new formulation of olanzapine available for terminally ill patients is needed. Rectal administration using suppositories is an alternative for patients for whom administration via the oral route is not feasible. In the present study, we prepared olanzapine suppositories, and confirmed using pharmaceutical tests. Furthermore, we demonstrated the efficacy and safety of olanzapine suppositories in terminally ill patients. METHODS: We prepared olanzapine suppositories using bases consisting of different compositions of Witepsol H-15, Witepsol S-55, and Witepsol E-75. The suppository release test was performed, and the olanzapine suppository with the best dissolution rate was selected. The suppository was assessed using the content uniformity test, content test in suppositories, hardness test, stability test, and clinical efficacy and safety. RESULTS: The dissolution rate at 360 min of olanzapine suppositories with Witepsol H-15 was the best (77.0 ± 3.3 %). The suppositories prepared had a uniform weight (2.47 ± 0.02 g) and content (2.11 ± 0.07 mg). The power required to break suppositories was 7.96 ± 0.55 kgf. When olanzapine suppositories were stored with protection from light, their contents were maintained regardless of whether the temperature was at 4 °C or room temperature. The numbers of patients administered 2.5 mg, 5 mg, and 10 mg of olanzapine suppositories were 4, 19, and 1. The percentages of patients with delirium or nausea and vomiting cured with olanzapine suppositories were 82 and 57 %, respectively. CONCLUSION: We suggest that olanzapine suppositories prepared in the hospital by pharmacists will improve the quality of life of terminally ill patients. TRIAL REGISTRATION: UMIN000022172. May 2, 2016 retrospectively registered.

Characterization of the membrane-targeting C1 domain in Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some forms of pasteurellosis. The toxin activates G(q)- and G(12/13)-dependent pathways through the deamidation of a glutamine residue in the alpha-subunit of heterotrimeric GTPases. We recently reported the crystal structure of the C terminus (residues 575-1285) of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine protease-like catalytic triad. In this study, we analyzed the membrane-targeting function of the C1 domain in detail. The C1 domain consists of seven helices of which the first four (residues 590-670), showing structural similarity to the N terminus of Clostridium difficile toxin B, were found to be involved in the recruitment of C-PMT to the plasma membrane. C-PMT lacking these helices (C-PMT DeltaC1(4H)) neither localized to the plasma membrane nor stimulated the G(q/12/13)-dependent signaling pathways. When the membrane-targeting property was complemented by a peptide tag with an N-myristoylation motif, C-PMT DeltaC1(4H) recovered the PMT activity. Direct binding between the C1 domain and liposomes containing phospholipids was evidenced by surface plasmon resonance analyses. These results indicate that the C1 domain of C-PMT functions as a targeting signal for the plasma membrane.

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT), one of the virulence factors produced by the bacteria, exerts its toxicity by up-regulating various signaling cascades downstream of the heterotrimeric GTPases Gq and G12/13 in an unknown fashion. Here, we present the crystal structure of the C-terminal region (residues 575-1,285) of PMT, which carries an intracellularly active moiety. The overall structure of C-terminal region of PMT displays a Trojan horse-like shape, composed of three domains with a "feet"-,"body"-, and "head"-type arrangement, which were designated C1, C2, and C3 from the N to the C terminus, respectively. The C1 domain, showing marked similarity in steric structure to the N-terminal domain of Clostridium difficile toxin B, was found to lead the toxin molecule to the plasma membrane. The C3 domain possesses the Cys-His-Asp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying Cys-His-Asp. PMT toxicities on target cells were completely abrogated when one of the amino acids constituting the triad was mutated. Our results indicate that PMT is an enzyme toxin carrying the cysteine protease-like catalytic triad dependent on the redox state and functions on the cytoplasmic face of the plasma membrane of target cells.

Crystallization and preliminary X-ray analysis of human MTH1 complexed with two oxidized nucleotides, 8-oxo-dGMP and 2-oxo-dATP.

Human MutT homologue 1 (hMTH1) hydrolyzes a variety of oxidized purine nucleoside triphosphates, including 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP and 8-oxo-dATP, to their corresponding nucleoside monophosphates, while Escherichia coli MutT possesses prominent substrate specificity for 8-oxoguanine nucleotides. Three types of crystals were obtained corresponding to the following complexes: selenomethionine-labelled hMTH1 with 8-oxo-dGMP (SeMet hMTH1-8-oxo-dGMP), hMTH1 with 8-oxo-dGMP (hMTH1-8-oxo-dGMP) and hMTH1 with 2-oxo-dATP (hMTH1-2-oxo-dATP). Crystals of the SeMet hMTH1-8-oxo-dGMP complex belong to space group P4(1)2(1)2, with unit-cell parameters a = b = 45.8, c = 153.6 A, and diffracted to 2.90 A. Crystals of hMTH1-8-oxo-dGMP and hMTH1-2-oxo-dATP belong to space groups P2(1) and P2(1)2(1)2(1), with unit-cell parameters a = 34.0, b = 59.0, c = 65.9 A, beta = 90.7 degrees and a = 59.2, b = 67.3, c = 80.0 A, respectively. Their diffraction data were collected at resolutions of 1.95 and 2.22 A, respectively.

Crystallization and preliminary crystallographic studies of the Pasteurella multocida toxin catalytic domain.

The C-terminal catalytic domain of Pasteurella multocida toxin, which is the virulence factor of the organism in P. multocida, has been expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data to 1.9 A resolution were obtained at the BL44XU beamline of SPring-8 from a flash-frozen crystal at 100 K. The crystals belong to space group C2, with unit-cell parameters a = 111.0, b = 150.4, c = 77.1 A, beta = 105.5 degrees, and are likely to contain one C-PMT (726 residues) per asymmetric unit.

Mammalian enzymes for preventing transcriptional errors caused by oxidative damage.

8-Oxo-7,8-dihydroguanine (8-oxoGua) is produced in cells by reactive oxygen species normally formed during cellular metabolic processes. This oxidized base can pair with both adenine and cytosine, and thus the existence of this base in messenger RNA would cause translational errors. The MutT protein of Escherichia coli degrades 8-oxoGua-containing ribonucleoside di- and triphosphates to the monophosphate, thereby preventing the misincorporation of 8-oxoGua into RNA. Here, we show that for human the MutT-related proteins, NUDT5 and MTH1 have the ability to prevent translational errors caused by oxidative damage. The increase in the production of erroneous proteins by oxidative damage is 28-fold over the wild-type cells in E.coli mutT deficient cells. By the expression of NUDT5 or MTH1 in the cells, it is reduced to 1.4- or 1.2-fold, respectively. NUDT5 and MTH1 hydrolyze 8-oxoGDP to 8-oxoGMP with V(max)/K(m) values of 1.3 x 10(-3) and 1.7 x 10(-3), respectively, values which are considerably higher than those for its normal counterpart, GDP (0.1-0.5 x 10(-3)). MTH1, but not NUDT5, possesses an additional activity to degrade 8-oxoGTP to the monophosphate. These results indicate that the elimination of 8-oxoGua-containing ribonucleotides from the precursor pool is important to ensure accurate protein synthesis and that both NUDT5 and MTH1 are involved in this process in human cells.