[Evaluation of Pharmacists' Active Intervention to Reduce Potentially Inappropriate Medications in Special Older Adult Nursing Home].

Currently, elderly care facilities that do not offer long-term care are not required to employ pharmacists, and duties such as the dispensing and distribution of medicines are entrusted to external pharmacies. Pharmacists seldom spend sufficient time at the facilities for elderly people requiring special care. Thus, in many cases, the pharmacists have insufficient knowledge of the residents' medication status, leading to their inability in determining whether the residents are receiving a suitable drug therapy. We previously documented various problems in the practices adopted by nursing staff (with negligible intervention by pharmacists) for assisting residents in taking their medications. In the present pilot study, we attempted to eliminate the use of potentially inappropriate medications by stationing a pharmacist at a nursing home for 24 h every week (3 d/week). We proactively collected information from nurses and other nursing staff and observed the residents' actual living conditions and medication use. As a result of this intervention, 56 prescriptions were changed. However, only two of these were changed exclusively based on the prescription information. Most prescriptions were able to change based on the information obtained by the pharmacist present at the facility. Therefore, pharmacists' presence at the facility (at least for a few hours) is necessary, as they can actively intervene and collaborate with other staff to prevent the use of potentially inappropriate medications.

Telepharmacy in mountainous depopulated areas of Japan: an exploratory interview study of patients' perspectives.

Japan has an ageing population and geographical impediments to healthcare access, so an experimental trial of telepharmacy has recently been implemented in remote islands or remote areas of Japan prior to the formal implementation. This exploratory study was conducted to understand patients' perspectives on telepharmacy in a mountainous depopulated area away from urban areas of Japan. Semi-structured interviews were conducted with four elderly patients, who were all of the patients receiving telepharmacy in Toyone village, Japan, at the time of the survey. The transcribed interview data were qualitatively analyzed by coding and categorization. The subjects thought telepharmacy would be advantageous to overcome poor access to a clinic and to improve convenience in processes ranging from medical examination to obtaining prescribed medicines. However, they pointed out the low digital literacy of the elderly. Also, they had low expectations for pharmacists, because they had previously had no relationship with pharmacists due to lack of pharmacies in the area. To promote telepharmacy, efforts to eliminate resistance to smartphones and to provide support for smartphone operations are needed among the elderly. Work is also needed to establish how pharmacists should best be involved in patient care and health support in remote areas. Our findings suggest that telepharmacy is useful in remote areas of Japan, but in locations where there is no existing relationship with pharmacists, it would be desirable for pharmacists to be actively involved with the community to maximize its effectiveness.

Structural basis for the high thermal stability and optimum pH of sphingomyelinase C from Streptomyces griseocarneus.

Sphingomyelinase C (SMC) hydrolyzes sphingomyelin to ceramide and phosphocholine. Prokaryotic SMCs share sequence homology with mammalian SMCs that have enzymatic pH optima at neutral pH. SMC from the nonpathogenic prokaryote Streptomyces griseocarneus shows notable enzymatic features such as higher optimum pH and thermostability than other prokaryotic SMCs. Determination of the three-dimensional structure of S. griseocarneus-SMC (Sg-SMC) and comparison with other SMC structures represents a promising strategy to elucidate the unique enzymatic features of Sg-SMC on a structural basis. Therefore, we determined the crystal structure of Sg-SMC at 2.0 Å resolution by X-ray crystallography. Comparison of the Sg-SMC structure with three other structurally known SMCs from Listeria ivanovii, Bacillus cereus, and Staphylococcus aureus indicated that Sg-SMC is more diverse in sequence and that structural differences in the main chain between these SMCs are primarily located on the molecular surface distant from the active site. Comparison of the surface area of the four SMCs revealed that Sg-SMC has the most compact structure, which may contribute to the enhanced thermostability of Sg-SMC. Regarding the hydrogen bond network in the active site of Sg-SMC, a basic amino acid, Arg278, is involved, whereas the corresponding residue in other SMCs (Ser or Asn) does not form hydrogen bonds with metal-coordinating water molecules. Hydrogen bond formation between Arg278 and a Mg2+ ion-coordinating water molecule may be responsible for the higher optimal pH of Sg-SMC compared to that of other SMCs.

Molecular cloning, heterologous expression, and enzymatic characterization of lysoplasmalogen-specific phospholipase D from Thermocrispum sp.

Lysoplasmalogen (LyPls)-specific phospholipase D (LyPls-PLD) is an enzyme that catalyses the hydrolytic cleavage of the phosphoester bond of LyPls, releasing ethanolamine or choline, and 1-(1-alkenyl)-sn-glycero-3-phosphate (lysoplasmenic acid). Little is known about LyPls-PLD and metabolic pathways of plasmalogen (Pls). Reportedly, Pls levels in human serum/plasma correlate with several diseases such as Alzheimer's disease and arteriosclerosis as well as a variety of biological processes including apoptosis and cell signaling. We identified a LyPls-PLD from Thermocrispum sp. strain RD004668, and the enzyme was purified, characterized, cloned, and expressed using pET24a(+)/Escherichia coli with a His tag. The enzyme's preferred substrate was choline LyPls (LyPlsCho), with only modest activity toward ethanolamine LyPls. Under optimum conditions (pH 8.0 and 50 °C), steady-state kinetic analysis for LyPlsCho yielded Km and kcat values of 13.2 µm and 70.6 s-1, respectively. The ORF of LyPls-PLD gene consisted of 1005 bp coding a 334-amino-acid (aa) protein. The deduced aa sequence of LyPls-PLD showed high similarity to those of glycerophosphodiester phosphodiesterases (GDPDs); however, the substrate specificity differed completely from those of GDPDs and general phospholipase Ds (PLDs). Structural homology modeling showed that two putative catalytic residues (His46, His88) of LyPls-PLD were highly conserved to GDPDs. Mutational and kinetic analyses suggested that Ala55, Asn56, and Phe211 in the active site of LyPls-PLD may participate in the substrate recognition. These findings will help to elucidate differences among LyPls-PLD, PLD, and GDPD with regard to function, substrate recognition mechanism, and biochemical roles. DATA ACCESSIBILITY: Thermocrispum sp. strain RD004668 and its 16S rDNA sequence were deposited in the NITE Patent Microorganisms Depositary (NPMD; Chiba, Japan) as NITE BP-01628 and in the DDBJ database under the accession number AB873024. The nucleotide sequences of the 16S rDNA of strain RD004668 and the LyPls-PLD gene were deposited in the DDBJ database under the accession numbers AB873024 and AB874601, respectively. ENZYME: EC number EC 3.1.4.4.

Substrate recognition mechanism of Streptomyces phospholipase D and enzymatic measurement of plasmalogen.

The substrate recognition mechanism of phospholipase D and enzymatic measurement of choline plasmalogen were investigated. A phospholipase D (PLD684) from Streptomyces sp. strain NA684 was purified 184-fold from the culture supernatant with 23.7% recovery. Maximum activity for l-α-lysophosphatidylcholine (LPC) hydrolysis was found at pH 5.0 and 80°C. The hydrolytic activity was remarkably affected by the concentration of Triton X-100 in the reaction mixture. In the presence of 0.05-0.5% and 0.1-0.2% (wt/vol) Triton X-100, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and choline plasmalogen were efficiently hydrolyzed by PLD684, respectively. Hydrolysis of LPC and choline lysoplasmalogen did not require Triton X-100; rather, the hydrolytic activity was inhibited by more than 0.05% (wt/vol) Triton X-100. The enzyme preferred mixed micelle substrates to liposomal substrates and hydrolyzed 98.4% of mixed micelle POPC in 1 h. Kinetic analysis showed that the rate-limiting steps of hydrolysis of mixed micelle POPC and emulsified LPC by PLD684 were the bulk step and the surface step, respectively. These results suggest that PLD684 has at least two substrate recognition mechanisms to recognize various phospholipids that have considerably different physical properties derived from their head and tail groups. Understanding of how PLD684 recognizes substrate forms will be useful for elucidating roles of lipolytic proteins in nature. Moreover, we report an enzymatic measurement of choline plasmalogen using PLD684 and phospholipase B. This is the first enzymatic method for measuring choline plasmalogen.

A novel phospholipase B from Streptomyces sp. NA684--purification, characterization, gene cloning, extracellular production and prediction of the catalytic residues.

A novel metal ion-independent phospholipase B (PLB684) from Streptomyces sp. strain NA684 was purified 264-fold from the culture supernatant with 2.85% recovery (6330 U·mg protein⁻¹). The enzyme functions as a monomer with a molecular mass of 38.9 kDa. Maximum activity was found at pH 8.4 and 50 °C. The substrate specificity was in the order: phosphatidylcholine ≥ phosphatidic acid ≥ lysophosphatidylcholine > phosphatidylserine > phosphatidylinositol > phosphatidylglycerol. The enzyme did not hydrolyze phosphatidylethanolamine, tristearin and dipalmitin. PLB684 hydrolyzed lysophosphatidylcholine and diacylphosphatidylcholine, and lysophosphatidylcholine was primarily produced during the early stages of phosphatidylcholine hydrolysis. The apparent K(m), V(max) and k(cat) for hydrolysis of dimyristoyl phosphatidic acid were 14.5 mm, 15.8 mmol·min⁻¹·mg protein⁻¹ and 1.02 × 104 s⁻¹, respectively. The positional specificity of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine hydrolysis was investigated using GC. In the reaction equilibrium, the molar ratio of released fatty acids (sn-1: sn-2) was 45 : 55. The ORF of the gene is 1239 bp in length and codes for a 30-amino acid signal peptide and a 382-amino acid mature enzyme. The deduced amino acid sequence of PLB684 shows 60% identity to a uncharacterized protein of Streptomyces auratus AGR0001(UniProt accession number: J1RQY0). The extracellular production of PLB684 was achieved using a pUC702 expression vector and Streptomyces lividans as the host. Mutagenesis analysis showed that Ser12 is essential for the catalytic function of PLB684 and that the active site may include residues Ser330 and His332.

Crystal structure of phospholipase A1 from Streptomyces albidoflavus NA297.

The metal-independent lipase from Streptomyces albidoflavus NA297 (SaPLA1) is a phospholipase A1 as it preferentially hydrolyzes the sn-1 acyl ester in glycerophospholipids, yielding a fatty acid and 2-acyl-lysophospholipid. The molecular mechanism underlying the substrate binding by SaPLA1 is currently unknown. In this study, the crystal structure of SaPLA1 was determined at 1.75Å resolutions by molecular replacement. A structural similarity search indicated the highest structural similarity to an esterase from Streptomyces scabies, followed by GDSL family enzymes. The SaPLA1 active site is composed of a Ser-His dyad (Ser11 and His218), whereby stabilization of the imidazole is provided by the main-chain carbonyl oxygen of Ser216, a common variation of the catalytic triad in many serine hydrolases, where this carbonyl maintains the orientation of the active site histidine residue. The hydrophobic pocket and cleft for lipid binding are adjacent to the active site, and are approximately 13-15Å deep and 14-16Å long. A partial polyethylene glycol structure was found in this hydrophobic pocket.

Kinetic characterization and Mg2+ enhancement of Streptomyces griseocarneus sphingomyelinase C produced by recombinant Streptomyces lividans.

Sphingomyelinase C (SMC) of the actinomycete, Streptomycesgriseocarneus NBRC13471, was constitutively expressed to high levels using Streptomyces lividans host and thereafter was extracellularly secreted into the cell culture. Purified SMC had a high specific activity (approximately 550-950 U/mg) and was obtained in high yields (approximately 120 mg/L of culture). SMC activity was enhanced by MgCl(2), and the maximum activity (542±25 U/mg) was observed in the presence of 1.5 mol/L (M) MgCl(2). Dynamic light scattering analysis proved that the highest specific SMC activity was obtained with the smallest mixed micelles of sphingomyelin (SM) and Triton X-100. The turnover rate (k(cat)), K(m) and k(cat)/K(m) values for SM were 346 s(-1), 0.458 mM, and 756 mM(-1)s(-1), respectively, in the presence of 1M MgCl(2). The k(cat) was strongly influenced by the MgCl(2) concentration. By contrast, the K(m) value was independent of the MgCl(2) concentration and was almost constant. Circular dichroism spectroscopy indicated that MgCl(2) did not cause local structural changes in SMC. From these results, we concluded that the SMC activity enhancement by MgCl(2) was caused by the increased specific surface area of the mixed micelles composed of substrate, SM, and Triton X-100.

Purification, characterization, molecular cloning and extracellular production of a phospholipase A1 from Streptomyces albidoflavus NA297.

A novel metal ion-independent phospholipase A1 of Streptomyces albidoflavus isolated from Japanese soil has been purified and characterized. The enzyme consists of a 33-residue N-terminal signal secretion sequence and a 269-residue mature protein with a deduced molecular weight of 27,199. Efficient and extracellular production of the recombinant enzyme was successfully achieved using Streptomyces lividans cells and an expression vector. A large amount (25 mg protein, 14.7 kU) of recombinant enzyme with high specific activity (588 U/mg protein) was purified by simple purification steps. The maximum activity was found at pH 7.2 and 50 °C. At pH 7.2, the enzyme preferably hydrolyzed phosphatidic acid and phosphatidylserine; however, the substrate specificity was dependent on the reaction pH. The enzyme hydrolyzed lysophosphatidylcholine and not triglyceride and the p-nitrophenyl ester of fatty acids. At the reaction equilibrium, the molar ratio of released free fatty acids (sn-1:sn-2) was 63:37. The hydrolysis of phosphatidic acid at 50 °C and pH 7.2 gave apparent V max and k cat values of 1389 µmol min(-1) mg protein(-1) and 630 s(-1), respectively. The apparent K m and k cat/K m values were 2.38 mM and 265 mM(-1) s(-1), respectively. Mutagenesis analysis showed that Ser11 is essential for the catalytic function of the enzyme and the active site may include residues Ser216 and His218.

Extracellular production of a sphingomyelinase from Streptomyces griseocarneus using Streptomyces lividans.

The structural gene for sphingomyelinase (SMase) from Streptomyces griseocarneus, was introduced into Streptomyces lividans using a shuttle vector, pUC702, for Escherichia coli/S. lividans. High-level secretory production of SMase was achieved using the promoter, signal sequence and terminator regions of phospholipase D from Streptoverticillium cinnamoneum. The transformant constitutively expressed a high specific activity of SMase extracellularly during batch culture. Maximum SMase activity (555 ± 114 U/mg protein) was with 1.75 M MgCl(2) which was about 50-fold more than that with 10 mM MgCl(2).