Yuki Kobayashi

Genetic dissection of medial habenula–interpeduncular nucleus pathway function in mice

The habenular complex linking forebrain and midbrain structures is subdivided into the medial (mHb) and the lateral nuclei (lHb). The mHb is characterized by the expression of specific nicotinic acetylcholine receptor isoforms and the release of acetylcholine to the interpeduncular nucleus (IPN), the sole output region of the mHb. The specific function of this circuit, however, is poorly understood. Here we generated transgenic mice in which mHb cells were selectively ablated postnatally. These lesions led to large reductions in acetylcholine levels within the IPN. The mutant mice exhibited abnormalities in a wide range of behavioral domains. They tended to be hyperactive during the early night period and were maladapted when repeatedly exposed to new environments. Mutant mice also showed a high rate of premature responses in the 5-choice serial reaction time task (5-CSRTT), indicating impulsive and compulsive behavior. Additionally, mice also exhibited delay and effort aversion in a decision-making test, deficits in spatial memory, a subtle increase in anxiety levels, and attenuated sensorimotor gating. IntelliCage studies under social housing conditions confirmed hyperactivity, environmental maladaptation, and impulsive/compulsive behavior, delay discounting, deficits in long-term spatial memory, and reduced flexibility in complex learning paradigms. In 5-CSRTT and adaptation tasks, systemic administration of nicotine slowed down nose-poke reaction and enhanced adaptation in control but not mutant mice. These findings demonstrate that the mHb–IPN pathway plays a crucial role in inhibitory control and cognition-dependent executive functions.

Cognitive deficits in single App knock-in mouse models

Transgenic mouse models of Alzheimers disease (AD) with nonphysiologic overexpression of amyloid precursor protein (APP) exhibit various unnatural symptoms/dysfunctions. To overcome this issue, mice with single humanized App knock-in (KI) carrying Swedish (NL), Beyreuther/Iberian (F), and Arctic (G) mutations in different combinations were recently developed. The validity of these mouse models of AD from a behavioral viewpoint, however, has not been extensively evaluated. Thus, using an automated behavior monitoring system, we analyzed various behavioral domains, including executive function, and learning and memory. The App-KI mice carrying NL-G-F mutations showed clear deficits in spatial memory and flexible learning, enhanced compulsive behavior, and reduced attention performance. Mice carrying NL-F mutations exhibited modest abnormalities. The NL-G-F mice had a greater and more rapid accumulation of Aβ deposits and glial responses. These findings reveal that single pathologic App-KI is sufficient to produce deficits in broad cognitive domains and that App-KI mouse lines with different levels of pathophysiology are useful models of AD.

Association analysis of adenosine A1 receptor gene (ADORA1) polymorphisms with schizophrenia in a Japanese population

Objective The human adenosine A1 receptor gene (ADORA1) localizes to chromosome 1q32 is 76.8 kbp in length and contains six exons. ADORA1 is ubiquitously expressed in the central nervous system and clinical and pharmacological evidence suggest the involvement of adenosine neurotransmission in the pathogenesis of schizophrenia. Therefore, we investigated the contribution of genetic variations of ADORA1 to the pathophysiological mechanisms of Japanese schizophrenia patients. Methods We performed genetic analysis of 29 polymorphic markers in 200 schizophrenic patients and 210 healthy controls from the Kyushu region of Japan. In statistical analysis, we performed the univariate analysis with genotypes and allele frequencies, linkage disequilibrium (LD) analyses, multivariate analysis, haplotype analysis, and sliding window haplotype analysis. Results In univariate analysis, no statistical difference was shown, after Bonferroni correction. By LD analysis, however, we could not find any LD blocks. In haplotype analysis, a total of 359 haplotypes were estimated. In multivariate analysis, we found three statistically different markers. In sliding window haplotype analysis, there were four statistically different haplotypes. Conclusion This is the first study describing the involvement of ADORA1 polymorphisms in the pathophysiological mechanisms of schizophrenia in a Japanese population. These results corroborate our previous pharmacological and neurochemical studies in the rat that have suggested an association between ADORA1 neurotransmission and the schizophrenic effects of the N-methyl-D-aspartate receptor antagonist phencyclidine. Thus, ADORA1 polymorphisms may represent good candidate markers for schizophrenia research and ADORA1 may be involved in the pathophysiological mechanisms of schizophrenia in Japanese populations.

MgB2 Thin Films Fabricated by a Precursor and Post-annealing Method Have a High Jc in High Magnetic Fields

The critical current densities (Jc) of MgB2 thin films deposited on α-Al2O3 fabricated by a precursor and post-annealing process have been investigated in high magnetic fields of up to 30 T. Jc values of 2.8×105, 3.0×104, and 3.7×103 A/cm2 were obtained at 4.2 K in external magnetic fields of 10, 20, and 26 T respectively, applied parallel to the film surface. These values were comparable to those seen for Nb3Sn in fields up to 18 T, and surpassed them in fields over 18 T. The superconducting transition temperature was Tconset=29.6 K and Tczero=28.5 K. Grain connectivity was estimated to be 38.4%. The Hc2(0) estimated from the resistivity measurements was 45 T. Transmission electron microscope (TEM) observations of the microstructures suggested that grains 10–20 nm in size exist that show no epitaxial growth relationship to the sapphire substrate. Jc enhancements in fields up to ~26 T are due to the strong grain boundary pinning associated with the small grains and the small size of MgO precipitates.

Properties of MgB/sub 2/ films with very high transport critical current densities

Magnesium diboride, MgB/sub 2/, thin films are fabricated through two different kinds of process. One is an in-situ process by using electron beam deposition and the other is an ex-situ process by using the combination of pulsed laser deposition and heat treatment. The critical current density, J/sub c/, is investigated as a function of external magnetic field in the range of 0-7 T and/or temperatures ranging from 4.2 K to the critical temperature by using dc 4-probe transport method. The in-situ processed film shows very high J/sub c/, e.g., 7.1/spl times/10/sup 6/, 1.2/spl times/10/sup 6/, and 1.4/spl times/10/sup 5/ A/cm/sup 2/ in 0, 4, and 7 T (perpendicular fields), respectively. Angular dependence of J/sub c/ is much different between the two kinds of films. J/sub c/-angle (magnetic field) curves of the in-situ processed film show two peaks; one is around the field perpendicular to the film surface and the other is around the parallel field. On the contrary, the curves of the ex-situ processed film have only one peak around the parallel field. Microstructure analyses show that the in-situ film has columnar grains aligned perpendicular to the film surface and that the ex-situ one has granular grains with random orientations. These results indicate that the grain boundaries between columnar grains act as effective pinning centers and enhance J/sub c/ in the perpendicular fields.

The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen

Background: Factor C is autocatalytically activated on lipopolysaccharides. Results: The N-terminal Arg of factor C is essential for the autocatalytic activation. Conclusion: The N-terminal Arg of factor C is required to be in sufficiently close vicinity for the autocatalytic activation. Significance: The recombinant factor C with restricted and homogeneous N-glycans may be useful for further structure-function studies. Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules.

Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade

Background: It is not known whether LPS is required for the activation of coagulation factor B. Results: Factor B is an LPS-binding zymogen activated by α-factor C in an LPS-dependent manner. Conclusion: The clip domain of factor B has an important role in localizing factor B to LPS. Significance: Horseshoe crab coagulation is an ideal model for understanding proteolytic cascades. Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to α-factor C on LPS and is artificially converted to β-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B. Here we found that wild-type factor B expressed in HEK293S cells is activated by α-factor C, but not by β-factor C, in an LPS-dependent manner and that β-factor C loses the LPS binding activity of factor C through additional cleavage by chymotrypsin within the N-terminal LPS-binding region. Surface plasmon resonance and quartz crystal microbalance analyses revealed that wild-type factor B binds to LPS with high affinity comparable with that of factor C, demonstrating that factor B is the second LPS-binding zymogen in the cascade. An LPS-binding site of wild-type factor B was found in the N-terminal clip domain, and the activation rate of a clip domain deletion mutant was considerably slower than that of wild-type factor B. Moreover, in the presence of LPS, Triton X-100 inhibited the activation of wild-type factor B by α-factor C. We conclude that the clip domain of factor B has an important role in localizing factor B to the surface of Gram-negative bacteria or LPS released from bacteria to initiate effective proteolytic activation by α-factor C.

Genetic engineering approach to develop next-generation reagents for endotoxin quantification

The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources.

Flux Pinning Centers in MgB2 Thin Films Prepared by an Electron Beam Evaporation Technique

We prepared MgB2 thin films by an electron beam evaporation technique and found that either vacancies and/or B substitutions on Mg sites worked as effective pinning centers in MgB2. We also found that the grain boundaries formed by the columnar grains work as effective pinning centers, by measuring transport J cs by changing the relative angle between grain boundaries and the applied magnetic field. We also found that introduced MgO nano-particles in MgB2 work as effective pinning centers.

The Influence of Pre-analytical Factors on the Analysis of Circulating MicroRNA

Background: MicroRNAs (miRNA) are expected as useful biomarkers for various diseases. We studied the pre-analytical factors causing variation in the analysis of miRNA. Material and Methods: Blood samples were collected from 25 healthy subjects. Plasma and serum were obtained from the same samples. The levels of miR-451, -16, -126, and -223 were analyzed using RT-qPCR. Cel-miR-39 was added as a spiked-in control in each sample. Results: With the exception of miR-451, the levels of the miRNAs in plasma were higher than in serum. After high-speed centrifugation, the levels of miRNAs were almost equal between plasma and serum except for miR-451. Membrane filtration with 0.45 µm pore size reduced the levels of plasma miRNAs. The coagulation accelerators for serum processing did not affect the analysis of miRNA. The use of fraction containing particles of > 0.45 µm in size showed the inhibitory effect on the analysis of plasma miR-451. The RNase inhibitor was effective for protecting against the degradation of miRNAs. Conclusion: Plasma contains factors modifying miRNA profiles. The immediate processing of plasma with membrane filtration and RNase inhibitor may be a relevant method for achieving the stable analysis of miRNA