Animal infection models using non‐mammals

The use of non‐human animal models for infection experiments is important for investigating the infectious processes of human pathogenic bacteria at the molecular level. Mammals, such as mice and rabbits, are also utilized as animal infection models, but large numbers of animals are needed for these experiments, which is costly, and fraught with ethical issues. Various non‐mammalian animal infection models have been used to investigate the molecular mechanisms of various human pathogenic bacteria, including Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa. This review discusses the desirable characteristics of non‐mammalian infection models and describes recent non‐mammalian infection models that utilize Caenorhabditis elegans, silkworm, fruit fly, zebrafish, two‐spotted cricket, hornworm, and waxworm

ATP and its metabolite adenosine cooperatively upregulate the antigen-presenting molecules on dendritic cells leading to IFN-gamma production by T cells

Dendritic cells (DCs) present foreign antigens to T cells via the major histocompatibility complex (MHC), thereby inducing acquired immune responses. ATP accumulates at sites of inflammation or in tumor tissues, which triggers local inflammatory responses. However, it remains to be clarified how ATP modulates the functions of DCs. In this study, we investigated the effects of extracellular ATP on mouse bone marrow- derived dendritic cells (BMDCs) as well as the potential for subsequent T cell activation. We found that high concentrations of ATP (1 mM) upregulated the cell surface expression levels of MHC-I, MHC-II, and co-stimulatory molecules CD80 and CD86 but not those of co-inhibitory molecules PD-L1 and PD-L2 in BMDCs. Increased surface expression of MHC-I, MHC-II, CD80, and CD86 was inhibited by a pan-P2 receptor antagonist. In addition, the upregulation of MHC-I and MHC-II expression was inhibited by an adenosine P1 receptor antagonist and by inhibitors of CD39 and CD73, which metabolize ATP to adenosine. These results suggest that adenosine is required for the ATP-induced upregulation of MHC-I and MHC-II. In the mixed leukocyte reaction assay, ATP-stimulated BMDCs activated CD4 and CD8T cells and induced interferon-gamma (IFN-gamma) production by these T cells. Collectively, these results suggest that high concentrations of extracellular ATP upregulate the expression of antigenpresenting and co-stimulatory molecules but not that of coinhibitory molecules in BMDCs. Cooperative stimulation of ATP and its metabolite adenosine was required for the upregulation of MHC-I and MHC-II. These ATP-stimulated BMDCs induced the activation of IFN-gamma-producing T cells upon antigen presentation

Ligation of MHC Class II Induces PKC-Dependent Clathrin-Mediated Endocytosis of MHC Class II

In addition to antigen presentation to CD4(+)T cells, aggregation of cell surface major histocompatibility complex class II (MHC-II) molecules induces signal transduction in antigen presenting cells that regulate cellular functions. We previously reported that crosslinking of MHC-II induced the endocytosis of MHC-II, which was associated with decreased surface expression levels in murine dendritic cells (DCs) and resulted in impaired activation of CD4(+)T cells. However, the downstream signal that induces MHC-II endocytosis remains to be elucidated. In this study, we found that the crosslinking of MHC-II induced intracellular Ca(2+)mobilization, which was necessary for crosslinking-induced MHC-II endocytosis. We also found that these events were suppressed by inhibitors of Syk and phospholipase C (PLC). Treatments with a phorbol ester promoted MHC-II endocytosis, whereas inhibitors of protein kinase C (PKC) suppressed crosslinking-induced endocytosis of MHC-II. These results suggest that PKC could be involved in this process. Furthermore, crosslinking-induced MHC-II endocytosis was suppressed by inhibitors of clathrin-dependent endocytosis. Our results indicate that the crosslinking of MHC-II could stimulate Ca(2+)mobilization and induce the clathrin-dependent endocytosis of MHC-II in murine DCs

Somatosensory Evoked Potential (SEP) in schizophrenics

The differences between schizophrenics and healthy subjects in Somatosensory Evoked Potential (SEP) were studied with 174 schizophrenics (98 male and 76 female) and 200 healthy subjects (100 male and 100 female). SEPs evoked by electric stimuli to the right median nerve were recorded through the two derivations (monopolar : C3’→A1+2; bipolar: C3’→Cz), averaging 100 responses, with 1024 msec of analysis time. Individual SEPs were subjected to the component analysis, and the following statistically significant results were obtained. 1. In male schizophrenics, the peak latencies of SEP components were significantly longer in short-latency component, P1 (bipolar), compared with healthy subjects, and shorter in middle-latency component, P2. In female schizophrenics, those were longer in middle-latency components, N2, P3, N3 (monopolar), and N1, P3, N3 (bipolar). 2. The inter-peak amplitudes in schizophrenics of both sexes were significantly larger in middle-latency components without any changes in short-latency components. 3. A few components with significant differences in latencies and inter-peak amplitudes between the subjects taking neuroleptics more than 600 mg, in chlorpromazine equivalent values, or not, as well as between medicated and unmedicated subjects, coincided with those also between the schizophrenics and healthy subjects. Theses differences in SEPs confirmed in the present study, regardless of schizophrenic subtypes, suggest the dysfunction in somatosensory information processing in schizophrenics, and SEP abnormalities may serve as possible elctrophysiological indices for cognitive dysfunction in schizophrenics

Visual Evoked Potential (VEP) in schizophrenics

The differences between schizophrenics and healthy subjects in Visual evoked potential (VEP) were studied with 174 schizophrenics (98 male and 76 female) and 200 healthy subjects (100 male and 100 female). VEPs evoked by flash stimuli were recorded through the two derivations (monopolar: O1→A1+2; bipolar: O1→Cz), averaging 100 responses, with 1024 msec of analysis time. Individual VEPs were subjected to the component analysis, and the following statistically significant results were obtained. 1. In schizophrenics, the latencies in short-latency components were significantly longer in P2 (male, monopolar), N2, P3, N3 (male, bipolar) and N3 (female, monopolar). Those in middle-latency components were significantly shorter in P4~N5 (male, monopolar) and P6 (female). 2. The inter-peak amplitudes in short-latency components were significantly smaller in P3-N3 (male, mopolar), and larger in P1-N1, N1-P2, P2-N2 (male, bipolar). Those in middle-latency components were significantly smaller in N3-P4 (male, monopolar), and larger in N4-P5 (female, bipolar). 3. A few components with significant differences in latencies and interpeak amplitudes between the subjects taking neuroleptics more than 600 mg, in chlorpromazine equivalent values, or not, as well as between medicated and unmedicated subjects, coincided with those also between the schizophrenics and healthy subjects. Theses differences in VEPs confirmed in the present study, regardless of schizophrenic subtypes, suggest the dysfunction in visual information processing in schizophrenics, and VEP abnormalities may serve as possible elctrophysiological indices for cognitive dysfunction in schizophrenics

Gibberellin Biosynthetic Inhibitors Make Human Malaria Parasite Plasmodium falciparum Cells Swell and Rupture to Death

Malaria remains as one of the most devastating infectious disease, and continues to exact an enormous toll in medical cost and days of labor lost especially in the tropics. Effective malaria control and eventual eradication remain a huge challenge, with efficacious antimalarials as important intervention/management tool. Clearly new alternative drugs that are more affordable and with fewer side effects are desirable. After preliminary in vitro assays with plant growth regulators and inhibitors, here, we focus on biosynthetic inhibitors of gibberellin, a plant hormone with many important roles in plant growth, and show their inhibitory effect on the growth of both apicomplexa, Plasmodium falciparum and Toxoplasma gondii. Treatment of P. falciparum cultures with the gibberellin biosynthetic inhibitors resulted in marked morphological changes that can be reversed to a certain degree under hyperosmotic environment. These unique observations suggest that changes in the parasite membrane permeability may explain the pleiotropic effects observed within the intracellular parasites

ヒスチジン ダツタンサン コウソ ノ ホンヤクゴ プロセシング オ カイスル ヒスタミン ゴウセイ ノ チョウセツ キコウ ニ カンスル ケンキュウ

京都大学0048新制・課程博士博士(薬学)甲第12258号薬博第594号新制||薬||215(附属図書館)24094UT51-2006-J251京都大学薬学研究科生命薬科学専攻(主査)教授 中山 和久, 教授 伊藤 信行, 教授 根岸 学学位規則第4条第1項該当Doctor of Pharmaceutical SciencesKyoto UniversityDA

Knockout of mlaA increases Escherichia coli virulence in a silkworm infection model.

The mlaA gene encodes a lipoprotein to maintain an outer membrane lipid asymmetry in gram-negative bacteria. Although the role of mlaA in bacterial virulence has been studied in several bacterial species, there are no reports of its role in E. coli virulence. In this study, we found that knockout of mlaA in E. coli increased its virulence against silkworms. The mlaA-knockout mutant was sensitive to several antibiotics and detergents, but resistant to vancomycin and chlorhexidine. The mlaA-knockout mutant grew faster than the parent strain in the presence of silkworm hemolymph. The mlaA-knockout mutant also produced a larger amount of outer membrane vesicles than the parent strain. These findings suggest that mlaA knockout causes E. coli resistance to specific antimicrobial substances and increases outer membrane vesicle production, thereby enhancing E. coli virulence properties in the silkworm infection model

Knockout of ribosomal protein RpmJ leads to zinc resistance in Escherichia coli.

Zinc is an essential metal for cells, but excess amounts are toxic. Other than by regulating the intracellular zinc concentration by zinc uptake or efflux, the mechanisms underlying bacterial resistance to excess zinc are unknown. In the present study, we searched for zinc-resistant mutant strains from the Keio collection, a gene knockout library of Escherichia coli, a model gram-negative bacteria. We found that knockout mutant of RpmJ (L36), a 50S ribosomal protein, exhibited zinc resistance. The rpmJ mutant was sensitive to protein synthesis inhibitors and had altered translation fidelity, indicating ribosomal dysfunction. In the rpmJ mutant, the intracellular zinc concentration was decreased under excess zinc conditions. Knockout of ZntA, a zinc efflux pump, abolished the zinc-resistant phenotype of the rpmJ mutant. RNA sequence analysis revealed that the rpmJ mutant exhibited altered gene expression of diverse functional categories, including translation, energy metabolism, and stress response. These findings suggest that knocking out RpmJ alters gene expression patterns and causes zinc resistance by lowering the intracellular zinc concentration. Knockouts of other ribosomal proteins, including RplA, RpmE, RpmI, and RpsT, also led to a zinc-resistant phenotype, suggesting that deletion of ribosomal proteins is closely related to zinc resistance

Knockout of mlaA increases Escherichia coli virulence in a silkworm infection model

The mlaA gene encodes a lipoprotein to maintain an outer membrane lipid asymmetry in gram-negative bacteria. Although the role of mlaA in bacterial virulence has been studied in several bacterial species, there are no reports of its role in E. coli virulence. In this study, we found that knockout of mlaA in E. coli increased its virulence against silkworms. The mlaA-knockout mutant was sensitive to several antibiotics and detergents, but resistant to vancomycin and chlorhexidine. The mlaA-knockout mutant grew faster than the parent strain in the presence of silkworm hemolymph. The mlaA-knockout mutant also produced a larger amount of outer membrane vesicles than the parent strain. These findings suggest that mlaA knockout causes E. coli resistance to specific antimicrobial substances and increases outer membrane vesicle production, thereby enhancing E. coli virulence properties in the silkworm infection model