The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth.

The presence of thiols in living systems is critical for the maintenance of cellular redox homeostasis, the maintenance of protein thiol-disulfide ratios and the protection of cells from reactive oxygen species. In addition to the well studied tripeptide glutathione (γ-Glu-Cys-Gly), a number of compounds have been identified that contribute to these essential cellular roles. Many of these molecules are of great clinical interest due to their essential role in the biochemistry of a number of deadly pathogens, as well as their possible role as therapeutic agents in the treatment of a number of diseases. A series of studies were undertaken using theoretical, chemical and biochemical approaches on a selection of thiols, ergothioneine, the ovothiols and mycothiol, to further our understanding of these necessary biological components. Ergothioneine is present at significant physiological levels in humans and other mammals; however, a definitive role for this thiol has yet to be determined. It has been implicated in radical scavenging in vivo and shows promise as a therapeutic agent against disease states caused by oxidative damage. Given the clinical importance of this intracellular thiol, further investigation into the behaviour of ergothioneine appeared warranted. A high level theoretical study was performed to determine the thermodynamic driving force behind the instability of the ergothioneine disulfide, as well as the thermodynamics of the reactions of ergothioneine with a selection of biologically relevant reactive oxygen species. These results were compared to those determined for a glutathione model compound, as well as the related ovothiols. The latter are believed to act as hydrogen peroxide scavengers in vivo and are currently under review as possible therapeutics against oxidative damage. The structural differences between the ovothiols and ergothioneine dramatically affect their reactivity and this study investigates the thermodynamic driving forces behind these differences. Mycothiol is the major thiol found in the Actinomycetales bacteria, which include the causative agent of tuberculosis, and the enzymes which use mycothiol have been identified as important targets for the development of novel antimicrobials. To better understand the in vivo behaviour of mycothiol, a thorough conformational search was performed to determine what, if any, trends exist among the low energy conformers expected to be present in solution. Knowledge of the conformations preferred by mycothiol may aid in the design of substrate-based inhibitors targeted at mycothiol-dependent enzymes. In addition, the efforts towards the identification of a mycothiol-dependent glyoxalase system are described. The glyoxalase system is essential for the detoxification of methylglyoxal, a toxic by-product of glycolysis, and this system would serve as a target for the design of new therapeutics against tuberculosis and other pathogenic Actinomycetales bacteria. In addition to the study of intracellular thiols, this work details a preliminary theoretical study of the thermodynamics of the phosphorylation of proteinaceous serine residues by inositol pyrophosphates in eukaryotic cell-free extracts. It has been postulated that this observed activity may represent a novel signalling pathway in eukaryotes. This study focused on the effect of inositol pyrophosphate structure and overall charge on the thermodynamics of these reactions. This information should contribute to our understanding of this novel cellular phosphorylation process.

Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

"Uniaxial Cyclic Stretch-Stimulated Glucose Transport Is Mediated by a Ca2+-Dependent Mechanism in Cultured Skeletal Muscle Cells" Pathobiology, v.74, n.3, pp.159-168を、博士論文として提出したもの。 / 名古屋大学博士学位論文 学位の種類:博士（リハビリテーション療法学）（課程）学位授与年月日:平成19年3月23日

Presenilin (PS) is the catalytic subunit of the gamma-secretase which is responsible for the cleavage of amyloid precursor protein to form beta amyloid (Aβ). Mutations in PS cause familial Alzheimer’s disease (FAD) by increasing the Aβ plaques formation in the brain and thereby induce neurodegeneration. Apart from this, FAD-linked PS mutations have been demonstrated to disrupt cellular calcium (Ca2+) homeostasis. Ca2+is a vital secondary messenger that involved in various neurophysiological functions, including memory, learning, and neuroplasticity and mounting evidence suggesting that Ca2+dysregulation associated with PS mutations may play a proximal role in the AD pathogenesis. Yet, the molecular mechanism for Ca2+dysregulation in AD remains debatable. It has been reported that cellular Ca2+homeostatsis can be disrupted in various ways. On the one hand, mutant PS has been demonstrated to exaggerate Ca2+release from the endoplasmic reticulum (ER) through different pathways. On the other hand, attenuatedCa2+influx from the extracellular medium through the capacitative Ca2+entry (CCE) pathway has also been reported to bring about cellular Ca2+disruption. However, the molecular mechanism for the PS mutation-mediated CCE deficits is largely unknown. For this reason, the objective of the current study is to elucidate the underlying molecular mechanism for attenuated CCE in AD. In this study, human neuronal cell line SH-SY5Y is employed as a cellular model to investigate the effect of wild-type or FAD-linked PS1 mutation on CCE pathway. Using single cell Ca2+imaging technique, significant CCE deficits was observed in SH-SY5Y stably expressing FAD-linked PS1mutation, PS1M146L. Interestingly, this CCE attenuation in PS1 mutant expressing cells was not mediated by the down-regulation of STIM1 and Orai1 expression, the known essential molecular players in the CCE pathway. Instead, co-immunoprecipitation and proximity ligation assay have suggested a physical interaction between PS1 and STIM1 proteins. Moreover, a putative gamma-secretase mediated STIM1 cleavage was discovered by western blotting. In addition, confocal imaging showed that PS1M146L significantlyreduceSTIM1 puncta formation and ER translocation followed by the activation of CCE pathway by ER Ca2+store depletion with thapsigargin. This indicated that mutant PS1 attenuates CCE by affecting STIM1 oligomerization or its recruitment with Orai1. Taken together, our results suggested the negative regulatory role of PS on CCE pathway and hypothesized the molecular mechanism of CCE where FAD-linked PS mutation is perceived as a gain-of-function mutation and enhanced the negative impact on STIM1 to inhibit Ca2+entry.This hypothetic model provides new insights into the molecular regulation for CCE pathway and the identification of the interacting domains between PS1 and STIM1 may suggest novel targets for the development of therapeutic agents that help to treat the disease. / published_or_final_version / Physiology / Master / Master of Philosophy

Forces are increasingly recognized as major regulators of cell physiology and function, and the mechanical properties of cells are essential to the mechanisms by which cells sense forces, transmit them to the cell interior or to other cells, and transduce them into chemical signals that impact a spectrum of cellular responses. Furthermore, cells can sense their extracellular environment and regulate their own mechanics and biology. Due to limitation of methodology, the cortical property of cells has been extensively characterized; however, the mechanics and dynamics of cytoplasm which consists all key cellular organelles, remains poorly understood. Moreover, a basic understanding of cell mechanics, such as which parameters correlates with cell stiffness and therefore impact cell biology is unknown. In this thesis, we firstly present a thorough investigation of the mechanical and dynamic properties of the cytoplasm, including direct measurement of cytoplasmic material property using optical tweezers, and visualization of intracellular dynamics by tracer particles. By combining these two measurements we obtain a directly characterization of the cytoplasmic forces; we further apply this method to study cancer cells and cells without vimentin intermediate filament, and find that cancer cells have significantly stronger intracellular forces, which vimentin intermediate filament does not have effect on the force generation. Secondly, we present our result on the role of cell volume in cell mechanics and cell biology. We show that the volume of a cell changes upon the property of the extracellular environment; the change in cell volume directly induces change in the mechanical property of both cytoplasm and cell cortex. We further show that the change in cell volume is due to intracellular water influx/efflux, and this has significant impact on cell biology, such as stem cell differentiation. Finally, we present a direct characterization of the equation of state of living cells by measuring cell volume under increasing osmotic pressure. We show that a living cell, under osmotic compression, behaves as Van der Waals gas with a hard sphere excluded volume; the minimum volume of cells is determined by cellular proteins, which the equation of state of living cells is dominated by intracellular ions. / Engineering and Applied Sciences

The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth.

The lungs and upper airways are mucosal surfaces that are common site for infection with an enormous variety of inhaled pathogens. Therefore, induction of immune responses in the respiratory tract is crucial for protection against respiratory diseases. One of the pathogens infecting the host via the respiratory tract is Mycobacterium Tuberculosis. The reported efficacy of the currently used Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis is highly variable, ranging from 50% against pulmonary tuberculosis to 80% against disseminated tuberculosis. Recently, the current route of vaccination (intradermal) has been considered as a possible factor influencing the protective capacity of the BCG vaccine. In this regard, intradermal route most likely induces protective systemic responses while it fails to induce optimal responses in the lungs. Therefore, our working hypothesis is that vaccination should be directed towards the respiratory mucosal immunity in order to improve the degree of host protection in the lungs. In this thesis we studied the effect of the route of immunization as well as of different mucosal adjuvants on the induction of mucosal immune responses against the mycobacterial surface antigen PstS-1. We found that, the intranasal (i.n.) route of immunization was a more favorable route inducing strong local immune responses, than intraperitoneal (i.p.) route. Indeed, i.n. route immunization, unlike the i.p. route, elicited strong IgA responses in the lungs accompanied by a major influx of CD4+ T cells and a significant local production of IFN-gamma. IgA, being the predominant Ig isotype at mucosal tissues, is considered a major effector molecule involved in defense mechanisms against viral and bacterial pathogens at these sites. Therefore, we investigated the possible role of IgA in the protection of the respiratory mucosa against mycobacterial infections, using mice deficient in IgA and in the polymeric Ig receptor. We show that, deficient mice are more susceptible to mycobacterial infections than wild type mice, thereby demonstrating a role for IgA in protection against mycobacteria. Importantly, our studies revealed a reduced production of protective factors, such as INF-gamma and TNF-alpha in the lungs of deficient mice that was associated with the higher susceptibility seen in these mice compared to wild-type mice. We also conducted challenge experiments against another respiratory pathogen, Chlamydia pneumoniae, using IgA deficient mice. Likewise to mycobacteria, our data support a role for IgA in the protection of the respiratory tract against C. pneumoniae infection. Finally, we investigated the possible mechanisms explaining the reduced pro-inflammatory responses in IgA deficient mice. Our data indicated that IgA deficient mice present a defective response to stimulation with LPS or 19kDa which appears to be both, essentially due to suboptimal stimulation of macrophages and restricted to the lungs.

The cochlear nucleus (CN), as the first brain centre in the auditory system is responsible for sorting the neural signals received from the cochlea, into parallel processing streams for transmission to the assorted higher auditory nuclei. A commissural connection formed between cochlear nuclei through direct projections, thereby provides the first site in the central auditory system at which binaural information is able to influence the ascending auditory signal. This thesis investigates the nature of commissural projections and the impact of their input upon neurons of the ventral CN (VCN) through in vivo intracellular and extracellular electrophysiological recordings together with both acoustic and electrical stimulation of the contralateral CN.

No description available.