Paenibacillus sp. W-61のキシラン分解・資化における菌体表層キシラナーゼの機能解明

Tohoku University伊藤 義

Recent Research Progress in the Applied Microbiology Laboratory : Enzymes, Toxins and Cell-Envelope Structure of Environmental and Pathogenic Bacteria(Recent Topics of the Agricultunal Biological Science in Tohoku University)

Here we describe the current status of our studies of xylanases of Paenibacillus sp. W-61, poly (D-lactic acid) hydrolases of Amycolatopsis sp. K104-1 and Brevibacterium sp. 93, pectin lyase of plant pathogenic Pectobacterium carotovora and bi-component toxins of Staphylococcus aureus. Paenibacillus sp. W-61 utilizes xylan as a carbon source via three enzymes, of which Xyn5 plays a crucial role both in xylan utilization and in the synthesis of the other two xylanases. Understanding the roles of each xylanase in xylan degradation will help to develop an enzymatic preparation of xylose from xylan. The poly (D-lactic acid) [PLA] hydrolases of Amycolatopsis sp. K104-1 and Brevibacterium sp. 93 appear to be novel enzymes with potential for PLA recycling. We recently purified and characterized the PLA hydrolases produced by these bacteria. The plant-soft rotting bacterium P. carotovora produces pectin lyase, a potential pathogenic factor, and a bacteriocin when exposed to DNA-damaging agents. We recently determined the cis-acting regulatory sequences responsible for the DNA-damage-inducible synthesis of the enzyme and bacteriocin. Leukocidin and γ-hemolysin are bi-component toxins that lyse leukocytes and erythrocytes respectively. We describe the molecular structure and pore-forming mechanism of the toxin components encoded by the chromosome and by a phage genome. Finally, we briefly describe the cell-envelope structure of the rumen bacterium Selenomonas ruminantium and its outer membrane protein Mep45 that possibly bridges the outer membrane and the peptidoglycan. The objects of our studies include environmental bacteria (xylanolytic Paenibacillus sp. W-61 and poly (D-lactic acid)-hydrolyzing bacteria Amycolatopsis sp. K104-1 and Brevibacterium sp. 93), as well as the plant and animal pathogens, Pectobacterium carotovora and Staphylococcus aureus, respectively. Here we briefly describe the current status of our investigations into these bacteria, their extracellular enzymes (or toxins), and an outer membrane protein in the rumen bacterium Selenomonas ruminantium subsp. lactilytica. Our research also includes polyglutamate biosynthesis and the degradation enzymes of a Bacillus subtilis Natto starter, acetan (a polysaccharide produced by Acetobacter and Gluconobacter)-degrading enzymes of Paenibacillus sp. and catabolic enzymes of amino acid and polyamine in Pseudomonas aeruginosa PAOl. The topics of these projects are available on our Web site

Nurses' Practical Wisdom for the Support of Dementia Patients Among Hospital Outpatients

Purpose: To establish and understand nurses' practical wisdom and interventions of support for dementia and possible dementia patients at hospital outpatient wards. Methods: A qualitative design was used to collect data through semi-structured focus group interviews. The participants were 13 female nurses working at hospital outpatient wards.Data were analyzed using the KJ Method. Results: Seven themes symbolizing the properties of the final label were extracted as follows:‘Observation of patients with focused awareness, and are continuously engaged with their patients',‘Approach to the problems of the patients, and sensitively work to understand the worries of patients based on past cases of problems',‘Looking out for simple ways patients can look after themselves, implicitly and thoroughly, making the best use of the ways that patients are familiar with and which they are able to understand',‘Preparations for scheduled consultations by developing a network to assist with problem prevention and recording episodes about problems involving the patients',‘Requests for cooperation to continue treatment by choosing intermediaries/resources appropriately as based on the importance of the medical treatment',‘Responses that do not conflict with the feelings of the family by considering the possible reluctance of accepting that a family member has dementia', and‘Attitude not to blame matters on dementia by reflecting on how the environment and care ought to be'.’ Conclusion:Nurses' practical wisdom is a type of support provided for patients in a natural manner without being noticed as special or particular by the patients

Procyanidin B3 Prevents Articular Cartilage Degeneration and Heterotopic Cartilage Formation in a Mouse Surgical Osteoarthritis Model

Osteoarthritis (OA) is a common disease in the elderly due to an imbalance in cartilage degradation and synthesis. Heterotopic ossification (HO) occurs when ectopic masses of endochondral bone form within the soft tissues around the joints and is triggered by inflammation of the soft tissues. Procyanidin B3 (B3) is a procyanidin dimer that is widely studied due to its high abundance in the human diet and antioxidant activity. Here, we evaluated the role of B3 isolated from grape seeds in the maintenance of chondrocytes in vitro and in vivo. We observed that B3 inhibited H2O2-induced apoptosis in primary chondrocytes, suppressed H2O2- or IL-1ß−induced nitric oxide synthase (iNOS) production, and prevented IL-1ß−induced suppression of chondrocyte differentiation marker gene expression in primary chondrocytes. Moreover, B3 treatment enhanced the early differentiation of ATDC5 cells. To examine whether B3 prevents cartilage destruction in vivo, OA was surgically induced in C57BL/6J mice followed by oral administration of B3 or vehicle control. Daily oral B3 administration protected articular cartilage from OA and prevented chondrocyte apoptosis in surgically-induced OA joints. Furthermore, B3 administration prevented heterotopic cartilage formation near the surgical region. iNOS protein expression was enhanced in the synovial tissues and the pseudocapsule around the surgical region in OA mice fed a control diet, but was reduced in mice that received B3. Together, these data indicated that in the OA model, B3 prevented OA progression and heterotopic cartilage formation, at least in a part through the suppression of iNOS. These results support the potential therapeutic benefits of B3 for treatment of human OA and heterotopic ossification

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution

Nurses\u27 Practical Wisdom for the Support of Dementia Patients Among Hospital Outpatients

Purpose: To establish and understand nurses\u27 practical wisdom and interventions of support for dementia and possible dementia patients at hospital outpatient wards.\nMethods: A qualitative design was used to collect data through semi-structured focus group interviews. The participants were 13 female nurses working at hospital outpatient wards.Data were analyzed using the KJ Method.\nResults: Seven themes symbolizing the properties of the final label were extracted as follows:‘Observation of patients with focused awareness, and are continuously engaged with their patients\u27,‘Approach to the problems of the patients, and sensitively work to understand the worries of patients based on past cases of problems\u27,‘Looking out for simple ways patients can look after themselves, implicitly and thoroughly, making the best use of the ways that patients are familiar with and which they are able to understand\u27,‘Preparations for scheduled consultations by developing a network to assist with problem prevention and recording episodes about problems involving the patients\u27,‘Requests for cooperation to continue treatment by choosing intermediaries/resources appropriately as based on the importance of the medical treatment\u27,‘Responses that do not conflict with the feelings of the family by considering the possible reluctance of accepting that a family member has dementia\u27, and‘Attitude not to blame matters on dementia by reflecting on how the environment and care ought to be\u27.’\nConclusion:Nurses\u27 practical wisdom is a type of support provided for patients in a natural manner without being noticed as special or particular by the patients

The Membrane Lipoprotein LppX of Paenibacillus sp. Strain W-61 Serves as a Molecular Chaperone for Xylanase of Glycoside Hydrolase Family 11 during Secretion across the Cytoplasmic Membrane▿ †

Paenibacillus sp. strain W-61, which can utilize xylan as the sole source of carbon and energy, produces extracellular xylanases 1 and 3 (Xyn1 and Xyn3) and cell surface xylanase 5. In this study we found that lppX, immediately downstream of xyn1, encodes a lipoprotein located on the outer layer of the cytoplasmic membrane and that the LppX lipoprotein is essential for the secretion of active Xyn1 across the cytoplasmic membranes. In Escherichia coli, wild-type LppX was destined for the inner layer of the outer membrane. Mutant LppX(C19A), in which Cys-19, a possible lipomodification residue, is replaced with Ala, was located in the periplasm without being anchored to the membranes. Another mutant, LppX(S20D S21D), with substitutions of Asp for Ser-20 and Ser-21 (conversion to an Asp-Asp signal for sorting to the inner membrane), resided on the outer layer of the inner membrane, demonstrating that LppX has the sorting property of a lipoprotein. E. coli harboring both xyn1 and lppX secreted active Xyn1 into the periplasm. In contrast, E. coli carrying xyn1 alone failed to do so, accumulating inactive Xyn1 in the cytoplasmic membranes. Exogenous LppX(C19A) liberated the inactive Xyn1, which had been stagnating in the inner membrane, into the medium as an active enzyme. Thus, we propose that LppX is a novel type of lipoprotein that assists Xyn1 in making the proper fold necessary for traveling across the cytoplasmic membranes to be secreted as an active enzyme

Cell Surface Xylanases of the Glycoside Hydrolase Family 10 Are Essential for Xylan Utilization by Paenibacillus sp. W-61 as Generators of Xylo-Oligosaccharide Inducers for the Xylanase Genes ▿

Paenibacillus sp. W-61 is capable of utilizing water-insoluble xylan for carbon and energy sources and has three xylanase genes, xyn1, xyn3, and xyn5. Xyn1, Xyn3, and Xyn5 are extracellular enzymes of the glycoside hydrolase (GH) families 11, 30, and 10, respectively. Xyn5 contains several domains including those of carbohydrate-binding modules (CBMs) similar to a surface-layer homologous (SLH) protein. This study focused on the role of Xyn5, localized on the cell surface, in water-insoluble xylan utilization. Electron microscopy using immunogold staining revealed Xyn5 clusters over the entire cell surface. Xyn5 was bound to cell wall fractions through its SLH domain. A Δxyn5 mutant grew poorly and produced minimal amounts of Xyn1 and Xyn3 on water-insoluble xylan. A Xyn5 mutant lacking the SLH domain (Xyn5ΔSLH) grew poorly, secreting Xyn5ΔSLH into the medium and producing minimal Xyn1 and Xyn3 on water-insoluble xylan. A mutant with an intact xyn5 produced Xyn5 on the cell surface, grew normally, and actively synthesized Xyn1 and Xyn3 on water-insoluble xylan. Quantitative reverse transcription-PCR showed that xylobiose, generated from water-insoluble xylan decomposition by Xyn5, is the most active inducer for xyn1 and xyn3. Luciferase assays using a Xyn5-luciferase fusion protein suggested that xylotriose is the best inducer for xyn5. The cell surface Xyn5 appears to play two essential roles in water-insoluble xylan utilization: (i) generation of the xylo-oligosaccharide inducers of all the xyn genes from water-insoluble xylan and (ii) attachment of the cells to the substrate so that the generated inducers can be immediately taken up by cells to activate expression of the xyn system

Expression of ICAM-1 and Acute Inflammatory Cell Infiltration in the Early Phase of Radiation Colitis in Rats

Inflammatory cell infiltration of the colon is observed at an early stage of radiation-induced colitis. The emigration of inflammatory cells from the circulation requires interactions between cell adhesion molecules on the vascular endothelium and molecules on the surface of leukocytes. To elucidate this process, the present work analyzes the kinetics of the expression of intercellular adhesion molecule-1 (ICAM-1) and the accumulation of inflammatory myeloperoxidase (MPO)-positive cells in relation to the appearance of acute radiation colitis prior to an overt radiation-induced ulcer. Colon tissues were obtained from Wistar Kyoto rats at various times after 22.5 Gy irradiation to the rectum. Histologically, crypt depletion and numerous inflammatory cells were observed 4 days after irradiation, and mucosal ulcer 6 days after irradiation. ICAM-1 immunopositivity was present in the endothelial cells of small vessels in the mucosa of both control and irradiated rats. ICAM-1 mRNA expression was detected in normal colon and irradiated colon by reverse transcription-PCR. In Northern blotting, ICAM-1 mRNA levels were found to increase markedly in the irradiated colon compared to the normal colon. In Western blotting, ICAM-1 protein expression also increased with a peak one day after irradiation, and remained elevated up to 6 days thereafter. The number of MPO-positive cells in lamina propria mucosa increased in a time-dependent fashion from 6 h to 6 days after irradiation. These data suggest that up-regulation of ICAM-1 in endothelial cells and accumulation of MPO positive cells play important roles in the development of radiation-induced colonic ulcer