Comprehensive end-of-life care practices for older patients with heart failure provided by specialized nurses: a qualitative study.

BACKGROUND: The context of end-of-life care for older heart failure patients with a complex clinical course provided by certified nurse specialists in gerontology (GCNSs) and Certified nurses in chronic heart failure (CNCHFs) is unclear; therefore, this study aims to describe comprehensive nursing practice for older patients with heart failure at their end of life. METHODS: This study adopts a qualitative descriptive design using content analysis. Five GCNSs, and five CNCHFs were interviewed using a web app from January to March 2022. RESULTS: Thirteen categories of nursing practices for older patients with heart failure were generated: (1) Provide thorough acute care by a multidisciplinary team to alleviate dyspnea, (2) Assess psychiatric symptoms and use a suitable environment to perform treatment, (3) Explain the progression of heart failure with the doctor, (4) Build a trusting relationship with the patient and family and implement advance care planning (ACP) early during the patient's recovery, (5) Involve multiple professions to help patients to achieve their desired life, (6) Perform ACP always in collaboration with multiple professionals, (7) Provide lifestyle guidance according to patients' feelings so that they can continue living at home after discharge from the hospital, (8) Provide palliative and acute care in parallel with multiple professions, (9) Achieve end-of-life care at home through multidisciplinary cooperation, (10) Provide basic nursing care to the patient and family until the moment of death, (11) Provide concurrent acute and palliative care as well as psychological support to alleviate physical and mental symptoms, (12) Share the patient's prognosis and future wishes with multiple professionals, and (13) Engage in ACP from early stages, through several conversations with patients and their families. CONCLUSIONS: Specialized nurses provide acute care, palliative care, and psychological support to alleviate physical and mental symptoms throughout the different stages of chronic heart failure. In addition to nursing care by specialized nurses at each stage shown in this study, it is important to initiate ACP early in the end-of-life stage and to provide care for patients with multiple professionals.

The role of calcium binding to the EF-hand-like motif in bacterial solute-binding protein for alginate import.

Gram-negative Sphingomonas sp. A1 incorporates acidic polysaccharide alginate into the cytoplasm via a cell-surface alginate-binding protein (AlgQ2)-dependent ATP-binding cassette transporter (AlgM1M2SS). We investigated the function of calcium bound to the EF-hand-like motif in AlgQ2 by introducing mutations at the calcium-binding site. The X-ray crystallography of the AlgQ2 mutant (D179A/E180A) demonstrated the absence of calcium binding and significant disorder of the EF-hand-like motif. Distinct from the wild-type AlgQ2, the mutant was quite unstable at temperature of strain A1 growth, although unsaturated alginate oligosaccharides stabilized the mutant by formation of substrate/protein complex. In the assay of ATPase and alginate transport by AlgM1M2SS reconstructed in the liposome, the wild-type and mutant AlgQ2 induced AlgM1M2SS ATPase activity in the presence of unsaturated alginate tetrasaccharide. These results indicate that the calcium bound to EF-hand-like motif stabilizes the substrate-unbound AlgQ2 but is not required for the complexation of substrate-bound AlgQ2 and AlgM1M2SS.

Structural studies on bacterial system used in the recognition and uptake of the macromolecule alginate.

Alginate is an acidic heteropolysaccharide produced by brown seaweed and certain kinds of bacteria. The cells of Sphingomonas sp. strain A1, a gram-negative bacterium, have several alginate-degrading enzymes in their cytoplasm and efficiently utilize this polymer for their growth. Sphingomonas sp. strain A1 cells can directly incorporate alginate into their cytoplasm through a transport system consisting of a "pit" on their cell surface, substrate-binding proteins in their periplasm, and an ATP-binding cassette transporter in their inner membrane. This review deals with the structural and functional aspects of bacterial systems necessary for the recognition and uptake of alginate.

A solute-binding protein in the closed conformation induces ATP hydrolysis in a bacterial ATP-binding cassette transporter involved in the import of alginate.

The Gram-negative bacterium Sphingomonas sp. A1 incorporates alginate into cells via the cell-surface pit without prior depolymerization by extracellular enzymes. Alginate import across cytoplasmic membranes thereby depends on the ATP-binding cassette transporter AlgM1M2SS (a heterotetramer of AlgM1, AlgM2, and AlgS), which cooperates with the periplasmic solute-binding protein AlgQ1 or AlgQ2; however, several details of AlgM1M2SS-mediated alginate import are not well-understood. Herein, we analyzed ATPase and transport activities of AlgM1M2SS after reconstitution into liposomes with AlgQ2 and alginate oligosaccharide substrates having different polymerization degrees (PDs). Longer alginate oligosaccharides (PD ≥ 5) stimulated the ATPase activity of AlgM1M2SS but were inert as substrates of AlgM1M2SS-mediated transport, indicating that AlgM1M2SS-mediated ATP hydrolysis can be stimulated independently of substrate transport. Using X-ray crystallography in the presence of AlgQ2 and long alginate oligosaccharides (PD 6-8) and with the humid air and glue-coating method, we determined the crystal structure of AlgM1M2SS in complex with oligosaccharide-bound AlgQ2 at 3.6 Å resolution. The structure of the ATP-binding cassette transporter in complex with non-transport ligand-bound periplasmic solute-binding protein revealed that AlgM1M2SS and AlgQ2 adopt inward-facing and closed conformations, respectively. These in vitro assays and structural analyses indicated that interactions between AlgM1M2SS in the inward-facing conformation and periplasmic ligand-bound AlgQ2 in the closed conformation induce ATP hydrolysis by the ATP-binding protein AlgS. We conclude that substrate-bound AlgQ2 in the closed conformation initially interacts with AlgM1M2SS, the AlgM1M2SS-AlgQ2 complex then forms, and this formation is followed by ATP hydrolysis.

Binding mode of metal ions to the bacterial iron import protein EfeO.

The tripartite EfeUOB system functions as a low pH iron importer in Gram-negative bacteria. In the alginate-assimilating bacterium Sphingomonas sp. strain A1, an additional EfeO-like protein (Algp7) is encoded downstream of the efeUOB operon. Here we show the metal binding mode of Algp7, which carries a M_75 metallopeptidase motif. The Algp7 protein was purified from recombinant E. coli cells and was subsequently characterized using differential scanning fluorimetry, fluorescence spectrometry, atomic absorption spectroscopy, and X-ray crystallography. The fluorescence of a dye, SYPRO Orange, bound to denatured Algp7 in the absence and presence of metal ions was measured during heat treatment. The fluorescence profile of Algp7 in the presence of metals such as ferric, ferrous, and zinc ions, shifted to a higher temperature, suggesting that Algp7 binds these metal ions and that metal ion-bound Algp7 is more thermally stable than the ligand-free form. Algp7 was directly demonstrated to show an ability to bind copper ion by atomic absorption spectroscopy. Crystal structure of metal ion-bound Algp7 revealed that the metal ion is bound to the cleft surrounded by several acidic residues. Four residues, Glu79, Glu82, Asp96, and Glu178, distinct from the M_75 motif (His115xxGlu118), are coordinated to the metal ion. This is the first report to provide structural insights into metal binding by the bacterial EfeO element.

Metabolic fate of unsaturated glucuronic/iduronic acids from glycosaminoglycans: molecular identification and structure determination of streptococcal isomerase and dehydrogenase.

Glycosaminoglycans in mammalian extracellular matrices are degraded to their constituents, unsaturated uronic (glucuronic/iduronic) acids and amino sugars, through successive reactions of bacterial polysaccharide lyase and unsaturated glucuronyl hydrolase. Genes coding for glycosaminoglycan-acting lyase, unsaturated glucuronyl hydrolase, and the phosphotransferase system are assembled into a cluster in the genome of pathogenic bacteria, such as streptococci and clostridia. Here, we studied the streptococcal metabolic pathway of unsaturated uronic acids and the structure/function relationship of its relevant isomerase and dehydrogenase. Two proteins (gbs1892 and gbs1891) of Streptococcus agalactiae strain NEM316 were overexpressed in Escherichia coli, purified, and characterized. 4-Deoxy-l-threo-5-hexosulose-uronate (Dhu) nonenzymatically generated from unsaturated uronic acids was converted to 2-keto-3-deoxy-d-gluconate via 3-deoxy-d-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD). DhuI and DhuD enzymatically corresponded to 4-deoxy-l-threo-5-hexosulose-uronate ketol-isomerase (KduI) and 2-keto-3-deoxy-d-gluconate dehydrogenase (KduD), respectively, involved in pectin metabolism, although no or low sequence identity was observed between DhuI and KduI or between DhuD and KduD, respectively. Genes for DhuI and DhuD were found to be included in the streptococcal genetic cluster, whereas KduI and KduD are encoded in clostridia. Tertiary and quaternary structures of DhuI and DhuD were determined by x-ray crystallography. Distinct from KduI ß-barrels, DhuI adopts an α/ß/α-barrel structure as a basic scaffold similar to that of ribose 5-phosphate isomerase. The structure of DhuD is unable to accommodate the substrate/cofactor, suggesting that conformational changes are essential to trigger enzyme catalysis. This is the first report on the bacterial metabolism of glycosaminoglycan-derived unsaturated uronic acids by isomerase and dehydrogenase.

Structural determinants in bacterial 2-keto-3-deoxy-D-gluconate dehydrogenase KduD for dual-coenzyme specificity.

Short-chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2-keto-3-deoxy-d-gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG-producing SDR enzymes (NADPH-dependent A1-R and NADH-dependent A1-R') involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes-KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)-prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5-diketo-3-deoxy-d-gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X-ray crystallography. Both PcaKduD and SpyDhuD consist of a three-layered structure, α/ß/α, with a coenzyme-binding site in the Rossmann fold; similar to enzymes A1-R and A1-R', both arrange the two short and long loops close to the coenzyme-binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1-R' but not A1-R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2' coenzyme region were determined to be structurally crucial for dual-coenzyme specificity in PcaKduD by structural comparison of the NADH- and NADPH-specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual-coenzyme specificity. Proteins 2016; 84:934-947. © 2016 Wiley Periodicals, Inc.

Lateral-typed flagellin responsible for formation of a polar flagellum but not of lateral flagella in Sphingomonas sp. strain A1.

Alginate-assimilating Sphingomonas sp. strain A1 is the Gram-negative bacterium first identified to form a single polar flagellum containing lateral-typed flagellin (p6) in the filament. In addition to the p6 flagellin, two polar-typed flagellins (p5 and p5') are also included in the flagellum. Here we show the significant role of p6 as well as p5/p5' in flagellum formation and cell motility towards alginate. A p6 gene disruptant significantly reduced flagellum formation and it showed no cell motility, whereas each mutant with a disruption in the p5 or p5' gene exhibited cell motility through the formation of a polar flagellum containing p6. The ratio of p6 to p5 decreased in proportion to cell growth, suggesting that strain A1 changes flagellin ratios in the filament depending on the external environment. Each of purified recombinant p5 and p6 proteins formed the filament by in vitro self-assembly and an anti-p5 antibody reacted with the p5 filament but not with the p6 filament. Immunoelectron microscopy using an anti-p5 antibody indicated that strain A1 formed two types of the filament in a single polar flagellum: p6 alone in the entire filament and p5 elongation filament subsequent to the p6 proximal end. Immunoprecipitation with an anti-p5 antibody directly demonstrated that p5 and p6 coexist in a single filament. Strain A1 cells were also found to exhibit a chemotactic motility in response to alginate. This is the first report on function/location of the lateral-typed flagellin in a single polar flagellum and the bacterial chemotaxis towards alginate.

Formation of a single polar flagellum by two distinct flagellar gene sets in Sphingomonas sp. strain A1.

Gram-negative Sphingomonas sp. strain A1, originally identified as a non-motile and aflagellate bacterium, possesses two sets of genes required for flagellar formation. In this study, we characterized the flagellar genes and flagellum formation in strain A1. Flagellar gene cluster set I contained 35 flagellar genes, including one flagellin gene (p6), where the gene assembly structure resembled that required for the formation of lateral flagella in gammaproteobacteria. The set II flagellar genes were arranged in eight shorter clusters with 46 flagellar genes, including two flagellin genes (p5 and p5') and flhF, which is required for polar flagella. Our molecular phylogenetic analysis of the bacterial flagellins also demonstrated that, in contrast to p5 and p5', p6 was categorized as a lateral flagellin group. The motile phenotype appeared in strain A1 cells when they were subcultured on semisolid media. The motile strain A1 cells produced a single flagellum at the cell pole. DNA microarray analyses using non-motile and motile strain A1 cells indicated that flagellar formation was accompanied by increased transcription of both flagellar gene sets. The two flagellins p5 and p6 were major components of the flagellar filaments isolated from motile strain A1 cells, indicating that the polar flagellum is formed by lateral and non-lateral flagellins.

Alginate-dependent gene expression mechanism in Sphingomonas sp. strain A1.

Sphingomonas sp. strain A1, a Gram-negative bacterium, directly incorporates alginate polysaccharide into the cytoplasm through a periplasmic alginate-binding protein-dependent ATP-binding cassette transporter. The polysaccharide is degraded to monosaccharides via the formation of oligosaccharides by endo- and exotype alginate lyases. The strain A1 proteins for alginate uptake and degradation are encoded in both strands of a genetic cluster in the bacterial genome and inducibly expressed in the presence of alginate. Here we show the function of the alginate-dependent transcription factor AlgO and its mode of action on the genetic cluster and alginate oligosaccharides. A putative gene within the genetic cluster seems to encode a transcription factor-like protein (AlgO). Mutant strain A1 (ΔAlgO mutant) cells with a disrupted algO gene constitutively produced alginate-related proteins. DNA microarray analysis indicated that wild-type cells inducibly transcribed the genetic cluster only in the presence of alginate, while ΔAlgO mutant cells constitutively expressed the genetic cluster. A gel mobility shift assay showed that AlgO binds to the specific intergenic region between algO and algS (algO-algS). Binding of AlgO to the algO-algS intergenic region diminished with increasing alginate oligosaccharides. These results demonstrated a novel alginate-dependent gene expression mechanism. In the absence of alginate, AlgO binds to the algO-algS intergenic region and represses the expression of both strands of the genetic cluster, while in the presence of alginate, AlgO dissociates from the algO-algS intergenic region via binding to alginate oligosaccharides produced through the lyase reaction and subsequently initiates transcription of the genetic cluster. This is the first report on the mechanism by which alginate regulates the expression of the gene cluster.

Crystal structure of Pedobacter heparinus heparin lyase Hep III with the active site in a deep cleft.

Pedobacter heparinus (formerly known as Flavobacterium heparinum) is a typical glycosaminoglycan-degrading bacterium that produces three heparin lyases, Hep I, Hep II, and Hep III, which act on heparins with 1,4-glycoside bonds between uronate and amino sugar residues. Being different from Hep I and Hep II, Hep III is specific for heparan sulfate. Here we describe the crystal structure of Hep III with the active site located in a deep cleft. The X-ray crystallographic structure of Hep III was determined at 2.20 Å resolution using single-wavelength anomalous diffraction. This enzyme comprised an N-terminal α/α-barrel domain and a C-terminal antiparallel ß-sheet domain as its basic scaffold. Overall structures of Hep II and Hep III were similar, although Hep III exhibited an open form compared with the closed form of Hep II. Superimposition of Hep III and heparin tetrasaccharide-bound Hep II suggested that an active site of Hep III was located in the deep cleft at the interface between its two domains. Three mutants (N240A, Y294F, and H424A) with mutations at the active site had significantly reduced enzyme activity. This is the first report of the structure-function relationship of P. heparinus Hep III.

Recognition of heteropolysaccharide alginate by periplasmic solute-binding proteins of a bacterial ABC transporter.

Alginate is a heteropolysaccharide that consists of ß-D-mannuronate (M) and α-L-guluronate (G). The Gram-negative bacterium Sphingomonas sp. A1 directly incorporates alginate into the cytoplasm through the periplasmic solute-binding protein (AlgQ1 and AlgQ2)-dependent ABC transporter (AlgM1-AlgM2/AlgS-AlgS). Two binding proteins with at least four subsites strongly recognize the nonreducing terminal residue of alginate at subsite 1. Here, we show the broad substrate preference of strain A1 solute-binding proteins for M and G present in alginate and demonstrate the structural determinants in binding proteins for heteropolysaccharide recognition through X-ray crystallography of four AlgQ1 structures in complex with saturated and unsaturated alginate oligosaccharides. Alginates with different M/G ratios were assimilated by strain A1 cells and bound to AlgQ1 and AlgQ2. Crystal structures of oligosaccharide-bound forms revealed that in addition to interaction between AlgQ1 and unsaturated oligosaccharides, the binding protein binds through hydrogen bonds to the C4 hydroxyl group of the saturated nonreducing terminal residue at subsite 1. The M residue of saturated oligosaccharides is predominantly accommodated at subsite 1 because of the strict binding of Ser-273 to the carboxyl group of the residue. In unsaturated trisaccharide (ΔGGG or ΔMMM)-bound AlgQ1, the protein interacts appropriately with substrate hydroxyl groups at subsites 2 and 3 to accommodate M or G, while substrate carboxyl groups are strictly recognized by the specific residues Tyr-129 at subsite 2 and Lys-22 at subsite 3. Because of this substrate recognition mechanism, strain A1 solute-binding proteins can bind heteropolysaccharide alginate with different M/G ratios.

Structural determinants in streptococcal unsaturated glucuronyl hydrolase for recognition of glycosaminoglycan sulfate groups.

Pathogenic Streptococcus agalactiae produces polysaccharide lyases and unsaturated glucuronyl hydrolase (UGL), which are prerequisite for complete degradation of mammalian extracellular matrices, including glycosaminoglycans such as chondroitin and hyaluronan. Unlike the Bacillus enzyme, streptococcal UGLs prefer sulfated glycosaminoglycans. Here, we show the loop flexibility for substrate binding and structural determinants for recognition of glycosaminoglycan sulfate groups in S. agalactiae UGL (SagUGL). UGL also degraded unsaturated heparin disaccharides; this indicates that the enzyme released unsaturated iduronic and glucuronic acids from substrates. We determined the crystal structures of SagUGL wild-type enzyme and both substrate-free and substrate-bound D175N mutants by x-ray crystallography and noted that the loop over the active cleft exhibits flexible motion for substrate binding. Several residues in the active cleft bind to the substrate, unsaturated chondroitin disaccharide with a sulfate group at the C-6 position of GalNAc residue. The sulfate group is hydrogen-bonded to Ser-365 and Ser-368 and close to Lys-370. As compared with wild-type enzyme, S365H, S368G, and K370I mutants exhibited higher Michaelis constants toward the substrate. The conversion of SagUGL to Bacillus sp. GL1 UGL-like enzyme via site-directed mutagenesis demonstrated that Ser-365 and Lys-370 are essential for direct binding and for electrostatic interaction, respectively, for recognition of the sulfate group by SagUGL. Molecular conversion was also achieved in SagUGL Arg-236 with an affinity for the sulfate group at the C-4 position of the GalNAc residue. These residues binding to sulfate groups are frequently conserved in pathogenic bacterial UGLs, suggesting that the motif "R-//-SXX(S)XK" (where the hyphen and slash marks in the motif indicate the presence of over 100 residues in the enzyme and parentheses indicate that Ser-368 makes little contribution to enzyme activity) is crucial for degradation of sulfated glycosaminoglycans.

Induced-fit motion of a lid loop involved in catalysis in alginate lyase A1-III.

The structures of two mutants (H192A and Y246F) of a mannuronate-specific alginate lyase, A1-III, from Sphingomonas species A1 complexed with a tetrasaccharide substrate [4-deoxy-L-erythro-hex-4-ene-pyranosyluronate-(mannuronate)(2)-mannuronic acid] were determined by X-ray crystallography at around 2.2 Šresolution together with the apo form of the H192A mutant. The final models of the complex forms, which comprised two monomers (of 353 amino-acid residues each), 268-287 water molecules and two tetrasaccharide substrates, had R factors of around 0.17. A large conformational change occurred in the position of the lid loop (residues 64-85) in holo H192A and Y246F compared with that in apo H192A. The lid loop migrated about 14 Šfrom an open form to a closed form to interact with the bound tetrasaccharide and a catalytic residue. The tetrasaccharide was bound in the active cleft at subsites -3 to +1 as a substrate form in which the glycosidic linkage to be cleaved existed between subsites -1 and +1. In particular, the O(η) atom of Tyr68 in the closed lid loop forms a hydrogen bond to the side chain of a presumed catalytic residue, O(η) of Tyr246, which acts both as an acid and a base catalyst in a syn mechanism.

Crystallization and preliminary X-ray analysis of alginate importer from Sphingomonas sp. A1.

Sphingomonas sp. A1 directly incorporates alginate polysaccharides through a 'superchannel' comprising a pit on the cell surface, alginate-binding proteins in the periplasm and an ABC transporter (alginate importer) in the inner membrane. Alginate importer, consisting of four subunits, AlgM1, AlgM2 and two molecules of AlgS, was crystallized in the presence of the binding protein AlgQ2. Preliminary X-ray analysis showed that the crystal diffracted to 3.3 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 72.5, b = 136.8, c = 273.3 Å, suggesting the presence of one complex in the asymmetric unit.

Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10Å resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

Working condition of nurses in Japan: awareness of work-life balance among nursing personnel at a university hospital.

AIM: The aim of this study was to examine the awareness of work-life balance (WLB) among the nursing personnel at a university hospital in Japan. DESIGN: Survey. METHODS: A questionnaire was sent to 1236 nursing personnel working at a university hospital and 1081 (87·5%) responses received. The questions concerned the following: (1) respondent demographical characteristics, (2) living background, (3) wishes for working environments and (4) motivation to work and health condition. The data were analysed by simple and cross-tabulations. RESULTS: The results can be summarised as follows: (1) the concept and significance of WLB has not yet been established among nurses in Japan, (2) three factors were found, which nurses need as working environment, 1st quality-of-life benefits, 2nd flexible working style, 3rd lifelong learning and (3) nurses who reported good WLB also reported higher job satisfaction and job motivation. CONCLUSION: The concept and significance of WLB has not yet been established among nurses. Thus, it is needed to enlighten nurses on the concept and significance of WLB as well as how they should practise WLB. Approximately half of the nurses gave their jobs first priority in reality, although they had various needs for their private lives and household affairs, as well as jobs. WLB cannot be achieved only by individual attitudes; thus, organisational efforts are necessary. In the correlation between WLB and 'job satisfaction' and 'job motivation,' nurses who were satisfied with their job and those who were highly motivated showed higher WLB. There was a significant correlation between satisfaction and motivation. RELEVANCE TO CLINICAL PRACTICE: It is necessary that nurses understand and have any information about working policy to improve working conditions to get better conditions which they need.

Substrate specificity of streptococcal unsaturated glucuronyl hydrolases for sulfated glycosaminoglycan.

Unsaturated glucuronyl hydrolase (UGL) categorized into the glycoside hydrolase family 88 catalyzes the hydrolytic release of an unsaturated glucuronic acid from glycosaminoglycan disaccharides, which are produced from mammalian extracellular matrices through the beta-elimination reaction of polysaccharide lyases. Here, we show enzyme characteristics of pathogenic streptococcal UGLs and structural determinants for the enzyme substrate specificity. The putative genes for UGL and phosphotransferase system for amino sugar, a component of glycosaminoglycans, are assembled into a cluster in the genome of pyogenic and hemolytic streptococci such as Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes, which produce extracellular hyaluronate lyase as a virulent factor. The UGLs of these three streptococci were overexpressed in Escherichia coli cells, purified, and characterized. Streptococcal UGLs degraded unsaturated hyaluronate and chondroitin disaccharides most efficiently at approximately pH 5.5 and 37 degrees C. Distinct from Bacillus sp. GL1 UGL, streptococcal UGLs preferred sulfated substrates. DNA microarray and Western blotting indicated that the enzyme was constitutively expressed in S. agalactiae cells, although the expression level increased in the presence of glycosaminoglycan. The crystal structure of S. agalactiae UGL (SagUGL) was determined at 1.75 A resolution by x-ray crystallography. SagUGL adopts alpha(6)/alpha(6)-barrel structure as a basic scaffold similar to Bacillus UGL, but the arrangement of amino acid residues in the active site differs between the two. SagUGL Arg-236 was found to be one of the residues involved in its activity for the sulfated substrate through structural comparison and site-directed mutagenesis. This is the first report on the structure and function of streptococcal UGLs.

Mutational studies of the peptidoglycan hydrolase FlgJ of Sphingomonas sp. strain A1.

The flagellar protein FlgJ, a member of glycoside hydrolase family 73, has N- and C-terminal domains that are responsible for flagellar rod assembly and peptidoglycan hydrolysis, respectively. The crystal structure of the C-terminal domain of SPH1045 (SPH1045-C), the FlgJ from Sphingomonas sp. strain A1, showed a long cleft formed by two lobes, alpha and beta. In this study, seven site-specific mutants of residues in the cleft were prepared and analyzed. Enzyme activity was reduced most significantly in mutants E185A and Y281A, followed by E224A. A comparison of the crystal structure of the inactive mutant E185A with that of other related enzymes revealed that Glu185 is structurally reasonable as the proton donor and that Tyr281 is close to Glu185. Glu224 is, however, far from the catalytic site, which is inconsistent with the decreased activity exhibited by E224A. The structural flexibility of Glu224 and its neighboring residues observed in SPH1045-C may indicate that this region is able to change its conformation upon substrate binding.

Crystal structure of the glycosidase family 73 peptidoglycan hydrolase FlgJ.

Glycoside hydrolase (GH) categorized into family 73 plays an important role in degrading bacterial cell wall peptidoglycan. The flagellar protein FlgJ contains N- and C-terminal domains responsible for flagellar rod assembly and peptidoglycan hydrolysis, respectively. A member of family GH-73, the C-terminal domain (SPH1045-C) of FlgJ from Sphingomonas sp. strain A1 was expressed in Escherichia coli, purified, and characterized. SPH1045-C exhibited bacterial cell lytic activity most efficiently at pH 6.0 and 37 degrees C. The X-ray crystallographic structure of SPH1045-C was determined at 1.74 A resolution by single-wavelength anomalous diffraction. The enzyme consists of two lobes, alpha and beta. A deep cleft located between the two lobes can accommodate polymer molecules, suggesting that the active site is located in the cleft. Although SPH1045-C shows a structural homology with family GH-22 and GH-23 lysozymes, the arrangement of the nucleophile/base residue in the active site is specific to each peptidoglycan hydrolase.