Relationship between the Number of Pharmacists per Pharmacy and the Provision of Home Healthcare Services in Japan.

Home healthcare services provided by community pharmacists are essential for maintaining community care, especially in Japan's aging population. Personnel shortage in pharmacies is occasionally cited as the reason why pharmacies are unable to provide home healthcare services. This study examined the relationship between the number of pharmacists in each pharmacy and the provision of home healthcare services. The number of full-time and part-time pharmacists per pharmacy has a positive impact on the provision of home healthcare services. Moreover, the larger the number of pharmacists per pharmacy, the easier it is for the pharmacy to provide home healthcare services. With regard to pharmacies with one full-time pharmacist, there are more pharmacies that provide home healthcare services when the population density of municipalities where the pharmacy is located is high. However, the impact of the number of pharmacists on population density became obscure when the number of full-time pharmacists per pharmacy was three or more. Taken together, these findings indicate that the provision of home healthcare services by pharmacies is related to the number of pharmacists per pharmacy and the population density of the area. This could have implications for widening regional disparities in home healthcare services.

Impact of the increase in the number of community pharmacists on their geographical distribution in Japan: a retrospective survey.

BACKGROUND: Appropriate distribution of health care resources is required to adjust regional disparities in the quality of health care. Besides, the number of community pharmacists in Japan has increased recently, but the impact of this increase on the distribution of community pharmacists is unknown. Thus, we aimed at investigating the effect of the increase in the number of community pharmacists on the distribution per population and per area of inhabitable land. METHODS: Data from 2008 to 2018 were used. Equity among municipalities in the number of community pharmacists per population and per area of inhabitable land was assessed using the Gini coefficient. A mosaic plot was used to demonstrate the relationship between the population density and increase in the number of community pharmacists per municipality. RESULTS: The number of community pharmacists increased by approximately 1.3-fold from 2008 to 2018 in Japan. The Gini coefficient per population decreased gradually, whereas that per area increased slightly, with no change in distribution per area of inhabitable land. The number of community pharmacists per population increased regardless of the population density, but this increase per area was smaller for lower population density groups and larger for higher population density groups. CONCLUSION: The increase in the number of community pharmacists has improved the distribution of community pharmacists per population, but not that per area of inhabitable land. The maldistribution of community pharmacists per area implies an imbalance in the distance between pharmacies and residents. Thus, there is need for measures to improve the distribution of community pharmacists.

[Survey of Label Information of Foods with Function Claims].

The consumption of health food products, such as Foods with Function Claims, has grown in Japan. Significant information, such as possible side effects or drug interactions, are expected to be described on the packaging to help consumers to make an informed choice about products. In this study, we checked the items described on the packaging of Foods with Function Claims containing eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA), Salacinol/Fagomine/Neokotalanol, or Varyl-Tyrosine/Lactotripeptide. We found that the label information on the package have issues that need to be addressed; for example, the description about a warning for concomitant use with antithrombotic drugs was found in only 29.7% of EPA and/or DHA containing products (44 out of 148). Providing information for safe usage of products to consumers is pivotal. Therefore, improving product labeling, and further pharmaceutical support in case of taking health foods, should be considered.

Passive immunization with anti-ActA and anti-listeriolysin O antibodies protects against Listeria monocytogenes infection in mice.

Listeria monocytogenes is an intracellular pathogen that causes listeriosis. Due to its intracellular niche, L. monocytogenes has evolved to limit immune recognition and response to infection. Antibodies that are slightly induced by listerial infection are completely unable to protect re-infection of L. monocytogenes. Thus, a role of antibody on the protective effect against L. monocytogenes infection has been neglected for a long time. In the present study, we reported that passive immunization with an excessive amount of antibodies against ActA and listeriolysin O (LLO) attenuates severity of L. monocytogenes infection. Combination of these antibodies improved survival of L. monocytogenes infected mice. Bacterial load in spleen and liver of listerial infected mice and infected RAW264.7 cells were significantly reduced by administration of anti-ActA and anti-LLO antibodies. In addition, anti-LLO antibody neutralized LLO activity and inhibited the bacterial escape from the lysosomal compartments. Moreover, anti-ActA antibody neutralized ActA activity and suppressed actin tail formation and cell-to-cell spread. Thus, our studies reveal that passive immunization with the excessive amount of anti-ActA and -LLO antibodies has potential to provide the protective effect against listerial infection.

Fibronectin-binding protein, FbpA, is the adhesin responsible for pathogenesis of Listeria monocytogenes infection.

The role of fibronectin binding protein A (FbpA) in Listeria monocytogenes infection and its pathogenesis were studied in vivo and in vitro by constructing a fbpA-deficient mutant of L. monocytogenes (ΔfbpA). In vivo, ΔfbpA was less pathogenic in mutant mice than was wild-type L. monocytogenes. FbpA did not affect the amounts of various virulence-determining factors, including internalin B and listeriolysin O. However, adherence to, and invasion of, mouse hepatocytes by the ΔfbpA mutant were reduced. In contrast, adherence to, but not invasion of, the ΔfbpA mutant to macrophages was attenuated. Fibronectin contributed to the efficient adherence and invasion of wild-type L. monocytogenes, but not to those of the ΔfbpA mutant. Attenuation of adhesion and uptake of the ΔfbpA mutant were reversed by overexpression of FbpA in it. FbpA was not involved in intracellular growth, autophagy induction or actin tail formation. Thus, the present findings clearly show that FbpA acts as an important adhesion molecule of L. monocytogenes, especially regarding hepatocytes, without modulating the expression of other virulence factors that have been implicated in the pathogenesis of L. monocytogenes infection.

Caenorhabditis elegans avoids staphylococcal superantigenic toxins via 5-hydroxytryptamine-dependent pathway.

Avoidance behavior of Caenorhabditis elegans, a nematode, towards Staphylococcus aureus, a pathogenic bacterium, was studied. Caenorhabditis elegans avoided S. aureus cultures and also their culture supernatants, suggesting that secretory molecules are involved in the repellent activity. We demonstrated that toxic shock syndrome toxin 1 (TSST-1) and staphylococcal enterotoxin C (SEC), the superantigenic toxins produced by S. aureus, are responsible for the nematode avoidance. By using TSST-1 and SEC mutants, the results indicated that the repellent activity of these toxins is independent of their superantigenic activity. The TSST-1 and SEC were found to locate at chemosensory neurons that are responsible for the recognition of repellents and avoidance of pathogenic bacteria. When mutants of C. elegans deficient in Toll/interleukin-1 receptor (TIR-1) and 5-hydroxytryptamine (5-HT) biosynthesis were used, avoidance behavior was attenuated. In the 5-HT biosynthesis deficient mutant nematodes, the avoidance activity was recovered when exogenous 5-HT was added. tph-1 expression and 5-HT production were upregulated when the nematodes were treated with TSST-1 or SEC. These results suggest that C. elegans avoids S. aureus by recognizing secretory molecules including TSST-1 and SEC and this avoidance is dependent on TIR and production of 5-HT.

Crystal structure of mitochondrial quinol-fumarate reductase from the parasitic nematode Ascaris suum.

In the anaerobic respiratory chain of the parasitic nematode Ascaris suum, complex II couples the reduction of fumarate to the oxidation of rhodoquinol, a reverse reaction catalyzed by mammalian complex II. In this study, the first structure of anaerobic complex II of mitochondria was determined. The structure, composed of four subunits and five co-factors, is similar to that of aerobic complex II, except for an extra peptide found in the smallest anchor subunit of the A. suum enzyme. We discuss herein the structure-function relationship of the enzyme and the critical role of the low redox potential of rhodoquinol in the fumarate reduction of A. suum complex II.

Autolysin amidase of Listeria monocytogenes promotes efficient colonization of mouse hepatocytes and enhances host immune response.

Listeria monocytogenes is an intracellularly growing pathogen which is able to infect and to spread from cells to cells. It produces several virulence factors required for invasion and intracellular niche colonization. Endogenous peptidoglycan hydrolases which are important for survival of bacteria have been shown to be involved in pathogenesis. An autolysin amidase (Ami)-deficient mutant of L. monocytogenes (Δami) is attenuated in virulence as evidenced by a reduction in mortality of infected mice. We showed that Ami is not essential for bacterial growth and protein secretion. Histopathological analysis suggests that Ami promotes bacterial colonization of hepatocytes. By using cultured eukaryotic cells, we present evidence that a critical function of Ami in pathogenesis is to promote an efficient listerial adherence and internalization into mouse hepatocytes. Simultaneously, the peptidoglycan hydrolase activity of Ami linked to the release of immunologically active cell wall components enhances production of tumor necrosis factor (TNF)-α and interleukin 6. In the early phase of infection, interferon-γ and TNF-α production of Δami-infected mice is significantly less than that of wild-type controls, suggesting a contribution of Ami to enhance the host innate immune response to listerial infection.

Mouse peptidoglycan recognition protein PGLYRP-1 plays a role in the host innate immune response against Listeria monocytogenes infection.

The role of mouse peptidoglycan recognition protein PGLYRP-1 in innate immunity against Listeria monocytogenes infection was studied. The recombinant mouse PGLYRP-1 and a polyclonal antibody specific to PGLYRP-1 were prepared. The mouse PGLYRP-1 showed antibacterial activities against L. monocytogenes and other Gram-positive bacteria. PGLYRP-1 mRNA expression was induced in the spleens and livers of mice infected with L. monocytogenes. The viable bacterial number increased, and the production of cytokines such as gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) was reduced in mice when mice had been injected with anti-PGLYRP-1 antibody before infection. The levels of IFN-γ and TNF-α titers in the organs were higher and the viable bacterial number was reduced in mice injected with recombinant mouse PGLYRP-1 (rmPGLYRP-1) before infection. PGLYRP-1 could directly induce these cytokines in spleen cell cultures. The elimination of intracellular bacteria was upregulated in NMuLi hepatocyte cells overexpressing PGLYRP-1. The enhancement of the elimination of L. monocytogenes from the organs was observed in IFN-γ(-/-) mice by rmPGLYRP-1 administration but not in TNF-α(-/-) mice. These results suggest that PGLYRP-1 plays a role in innate immunity against L. monocytogenes infection by inducing TNF-α.

Crystallization of mitochondrial rhodoquinol-fumarate reductase from the parasitic nematode Ascaris suum with the specific inhibitor flutolanil.

In adult Ascaris suum (roundworm) mitochondrial membrane-bound complex II acts as a rhodoquinol-fumarate reductase, which is the reverse reaction to that of mammalian complex II (succinate-ubiquinone reductase). The adult A. suum rhodoquinol-fumarate reductase was crystallized in the presence of octaethyleneglycol monododecyl ether and n-dodecyl-beta-D-maltopyranoside in a 3:2 weight ratio. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 123.75, b = 129.08, c = 221.12 A, and diffracted to 2.8 A resolution using synchrotron radiation. The presence of two molecules in the asymmetric unit (120 kDa x 2) gives a crystal volume per protein mass (V(M)) of 3.6 A(3) Da(-1).

An evolutionary 'intermediate state' of mitochondrial translation systems found in Trichinella species of parasitic nematodes: co-evolution of tRNA and EF-Tu.

EF-Tu delivers aminoacyl-tRNAs to ribosomes in the translation system. However, unusual truncations found in some animal mitochondrial tRNAs seem to prevent recognition by a canonical EF-Tu. We showed previously that the chromadorean nematode has two distinct EF-Tus, one of which (EF-Tu1) binds only to T-armless aminoacyl-tRNAs and the other (EF-Tu2) binds to D-armless Ser-tRNAs. Neither of the EF-Tus can bind to canonical cloverleaf tRNAs. In this study, by analyzing the translation system of enoplean nematode Trichinella species, we address how EF-Tus and tRNAs have evolved from the canonical structures toward those of the chromadorean translation system. Trichinella mitochondria possess three types of tRNAs: cloverleaf tRNAs, which do not exist in chromadorean nematode mitochondria; T-armless tRNAs; and D-armless tRNAs. We found two mitochondrial EF-Tu species, EF-Tu1 and EF-Tu2, in Trichinella britovi. T.britovi EF-Tu2 could bind to only D-armless Ser-tRNA, as Caenorhabditis elegans EF-Tu2 does. In contrast to the case of C.elegans EF-Tu1, however, T.britovi EF-Tu1 bound to all three types of tRNA present in Trichinella mitochondria. These results suggest that Trichinella mitochondrial translation system, and particularly the tRNA-binding specificity of EF-Tu1, could be an intermediate state between the canonical system and the chromadorean nematode mitochondrial system.

Paragonimus westermani and some rare intestinal trematodes recovered from raccoon dogs (Nyctereutes procyonoides viverrinus) introduced recently on Yakushima Island, Japan.

Helminth parasites were collected from 9 raccoon dogs (Nyctereutes procyonoides viverrinus) and 2 Japanese weasels (Mustela itatsi sho) on Yakushima Island, Japan. The former carnivore was introduced to this World Natural Heritage Area presumably within the last two decades, expanding its population thence, although detailed process(es) of the introduction is unknown. The collected trematodes from raccoon dogs included the triploid form of Paragonimus westermani, Brachylaima tokudai, Maritrema eroliae, and Pseudocryptotropa sp. Simultaneously, Paragonimus ohirai was found in one weasel killed by a traffic accident. Although the triploid form of P. westermani and P. ohirai are known to be distributed in some river-mouth areas of Yakushima Island based on previous surveys on crab hosts, natural infection was detected for the first time in wild final hosts. Particularly, the raccoon dog infected with P. westermani was caught in a mountainous area, distant from human residence or river-mouth areas. Although it is possible that the infected raccoon dog moved from a river-mouth area endemic with P. westermani after infection, the alternative scenario remains to be pursued; the endemic area of this zoonosis is expanding along with the recent expansion of raccoon dogs or feral cats (Felis catus), that became prevalent recently on this island including the mountainous areas. Maritrema eroliae taking a variety of shorebirds as its natural final hosts, and a minute trematode, Pseudocryptotropa sp., taking unknown natural final host(s) were recorded for the first time in raccoon dogs.

Identification and characterization of the threadworm, Strongyloides procyonis, from feral raccoons (Procyon lotor) in Japan.

Strongyloides procyonis Little, 1966 was detected about 45 years ago in raccoons (Procyon lotor) of southern Louisiana, U.S.A., and was demonstrated experimentally to cause creeping eruption and a short-lived intestinal infection in a healthy human volunteer. After its description and demonstration of its pathogenicity in humans, S. procyonis has not been found in raccoons in North America despite repeated surveys. During a survey on feral raccoons in Japan, S. procyonis parasitic females were identified in 66 (28.3%) of 233 raccoons collected between May 2004 and January 2005. The number of parasitic females recovered from individual raccoons was 1-197 (geomean, 3.2). Both the morphological features and the nucleotide sequences of the small and large subunit ribosomal RNA genes (SSU/LSU rDNA) of S. procyonis closely resembled those of zoonotic Strongyloides stercoralis. The sequences of internal transcribed spacer (ITS)1 and 28S rDNA could differentiate clearly these 2 species. Awareness of S. procyonis in raccoons in North America and other places worldwide where raccoons are introduced and naturalized is important to assess the epidemiological significance of this potentially zoonotic helminth species.

Gizzard spirurid nematode Acuaria skrjabini in Japanese tree sparrows and a gray starling from Tokyo.

Gastrointestinal helminths were collected from 49 Japanese tree sparrows (Passer montanus saturatus) in Tokyo, Japan. In 16 sparrows, 1-9 (average, 3.5) gizzard spirurid nematodes (Acuaria skrjabini Ozerskaya, 1926) were found embedded in the mucosa of the gizzard. In addition, Capillaria sp., Platynosomum passeri Yamashita et Tsumura, 1962, and a hymenolepidid cestode were collected from 1, 2, and 1 sparrows, respectively. A sexually mature A. skrjabini female and 3 males were found also in a young gray starling (Sturnus cineraceus) that was found dead in the same area after failure to leave the nest. Starlings are a new host record for this spirurid species. Until this study, this gizzard spirurid species has not been recorded in this country or the Far East region.

Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate.

Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD-orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 A resolution using synchrotron radiation (lambda = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 x 34 kDa) gives a crystal volume per protein weight (VM) of 2.2 A3 Da(-1) and a solvent content of 44%.

Larva migrans by Baylisascaris transfuga: fatal neurological diseases in Mongolian jirds, but not in mice.

Raccoon roundworms (Baylisascaris procyonis) and other Baylisascaris species cause patent or latent larva migrans (LM) in a variety of mammals and birds, including humans. It is not clear whether LM by Baylisascaris transfuga, roundworms of bears, is associated with clinical neurological disorders. To clarify this issue, ICR and BALB/c mice as well as Mongolian jirds (Meriones unguiculatus) were orally inoculated with 2,000-5,000 embryonated eggs of B. transfuga. In mice, the ascarid caused symptomatic LM of limited extent and duration, whereas the infection was fatal in jirds; i.e., they exhibited general signs such as severe depression and emaciation on days 8-11 postinfection (PI) and died, or they developed progressive and fatal neurological disorders after day 14 PI. Histological examination showed B. transfuga larvae in the brain of all mice and jirds examined, and the larvae collected from them developed to a size comparable with that of B. procyonis. There existed, however, critical differences in host reactions against larvae localized in the brain of mice and jirds; B. transfuga larvae found in mice were surrounded by granulomatous reactions and immobilized, whereas larvae found in jirds were free from any host reaction and mobile, causing extensive malacia.

Rhodoquinone reaction site of mitochondrial complex I, in parasitic helminth, Ascaris suum.

The components and organization of the respiratory chain in helminth mitochondria vary remarkably depending upon the stage of the life cycle. Mitochondrial complex I in the parasitic helminth Ascaris suum uses ubiquinone-9 (UQ(9)) and rhodoquinone-9 (RQ(9)) under aerobic and anaerobic conditions, respectively. In this study, we investigated structural features of the quinone reduction site of A. suum complex I using a series of quinazoline-type inhibitors and also by the kinetic analysis of rhodoquinone-2 (RQ(2)) and ubiquinone-2 (UQ(2)) reduction. Structure-activity profiles of the inhibition by quinazolines were comparable, but not completely identical, between NADH-RQ(2) and NADH-UQ(2) oxidoreductase activities. However, the inhibitory mechanism of quinazolines was competitive and partially competitive against RQ(2) and UQ(2), respectively. The pH profiles of both activities differed remarkably; NADH-RQ(2) oxidoreductase activity showed an optimum pH at 7.6, whereas NADH-UQ(2) oxidoreductase activity showed two optima pH at 6.4 and 7.2. Our results indicate that although A. suum complex I uses both RQ(2) and UQ(2) as an electron acceptor, the manner of reaction (or binding) of the two quinones differs.

Transmission electron microscopy of Schistosoma mansoni cercariae treated with hinokitiol (beta-thujaplicin), a compound for potential skin application against cercarial penetration.

Since skin is the only route of entry of the parasite in schistosomiasis patients, intervention at the level of skin penetration should control the infection. Several compounds were screened for their ability to protect against cercarial penetration. Hinokitiol (beta-thujaplicin) was found to have a significant cercaricidal effect in vitro, although there is no information on its cercaricidal mechanisms. To study the kinetics of morphological changes in Schistosoma mansoni associated with exposure to hinokitiol in vitro, cercariae were incubated in media containing hinokitiol at different concentrations and examined by transmission electron microscopy (TEM). TEM revealed that ultrastructural changes occurred by 15 minutes post exposure, at a concentration of 25 microg/ml. Degenerative changes involving both tegument and deeper parenchymal structures were progressive with duration of exposure at the concentration of 50 microg/ml. These structural changes may account for the inability of hinokitiol-treated cercariae to infect the host.

Isolation and characterization of the stage-specific cytochrome b small subunit (CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria.

We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.