Mice with cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks.

N-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2 R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 s than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.

Methods for immunoblot detection and electron microscopic localization of septin subunits in mammalian nervous systems.

The minimal functional units of the mammalian septin system are diverse heterooligomers of SEPT1-14 subunits, which are most abundantly and differentially expressed in postmitotic neurons and glia. The subunit compositions of such heterooligomers are thought to differentiate their affinity for other proteins and lipids, and subcellular localization. Thus, high-precision quantification and mapping of each subunit is necessary to understand their subcellular functions and physiological roles. However, systematic information on the localization of individual septin subunits in the mammalian nervous system is limited. Here, we present our experimental workflows for the study of septin expression and localization in the rodent brain by immunoblot and serial section immunoelectron microscopy. Our protocols, based on standard methods, have been rigorously optimized and simplified for universality and reproducibility to aid non-experts in the field.

White Matter Abnormality Correlates with Developmental and Seizure Outcomes in West Syndrome of Unknown Etiology.

BACKGROUND AND PURPOSE: West syndrome is an epileptic encephalopathy characterized by epileptic spasms, a specific pattern on electroencephalography of hypsarrhythmia, and developmental regression. Our aim was to assess white matter abnormalities in West syndrome of unknown etiology. We hypothesized that diffusion tensor imaging reveals white matter abnormalities, especially in patients with poor seizure and developmental outcomes. MATERIALS AND METHODS: We enrolled 23 patients with new-onset West syndrome of unknown etiology. DTI was performed at 12 and 24 months of age. Fractional anisotropy images were compared with those of controls by using tract-based spatial statistics. We compared axial, radial, and mean diffusivity between patients and controls in the fractional anisotropy skeleton. We determined correlations of these parameters with developmental quotient, electroencephalography, and seizure outcomes. We also compared DTI with hypometabolism on fluorodeoxyglucose positron-emission tomography. RESULTS: At 12 months of age, patients showed widespread fractional anisotropy reductions and higher radial diffusivity in the fractional anisotropy skeleton with a significant difference on tract-based spatial statistics. The developmental quotient at 12 months of age correlated positively with fractional anisotropy and negatively with radial and mean diffusivity. Patients with seizure and abnormal findings on electroencephalography after initial treatments had lower fractional anisotropy and higher radial diffusivity. At 24 months, although tract-based spatial statistics did not show significant differences between patients and controls, tract-based spatial statistics in the 10 patients with a developmental quotient of <70 had significant fractional anisotropy reduction. In patients with unilateral temporal lobe hypometabolism on PET, tract-based spatial statistics showed greater fractional anisotropy reduction in the temporal lobe ipsilateral to the side of PET hypometabolism. CONCLUSIONS: Diffuse abnormal findings on DTI at 12 months of age suggest delayed myelination as a key factor underlying abnormal findings on DTI. Conversely, asymmetric abnormal findings on DTI at 24 months may reflect underlying focal pathologies.

Synaptic dysfunction, memory deficits and hippocampal atrophy due to ablation of mitochondrial fission in adult forebrain neurons.

Well-balanced mitochondrial fission and fusion processes are essential for nervous system development. Loss of function of the main mitochondrial fission mediator, dynamin-related protein 1 (Drp1), is lethal early during embryonic development or around birth, but the role of mitochondrial fission in adult neurons remains unclear. Here we show that inducible Drp1 ablation in neurons of the adult mouse forebrain results in progressive, neuronal subtype-specific alterations of mitochondrial morphology in the hippocampus that are marginally responsive to antioxidant treatment. Furthermore, DRP1 loss affects synaptic transmission and memory function. Although these changes culminate in hippocampal atrophy, they are not sufficient to cause neuronal cell death within 10 weeks of genetic Drp1 ablation. Collectively, our in vivo observations clarify the role of mitochondrial fission in neurons, demonstrating that Drp1 ablation in adult forebrain neurons compromises critical neuronal functions without causing overt neurodegeneration.

Consumption of partially hydrolysed guar gum stimulates Bifidobacteria and butyrate-producing bacteria in the human large intestine.

Partially hydrolysed guar gum (PHGG) is a water-soluble dietary fibre that is non-digestible in the upper gastrointestinal tract. It is believed that PHGG benefits the health of hosts by altering the colonic microbiota and stimulating short-chain fatty acid (SCFA) production. However, it remains unclear which bacteria ferment PHGG in the human large intestine. In this study, the effect of PHGG on faecal bacteria was analysed to specify the bacteria that contribute to the fermentation of PHGG in the human large intestine. Ten healthy volunteers consumed PHGG (6 g/day) for 2 weeks. Faeces were collected at 2 weeks prior to consumption, at the end of 2 weeks of consumption, and 2 weeks after consumption of PHGG. Bacterial DNA was extracted from these collected faeces and subjected to real-time PCR using bacterial group- or species-specific primers. The copy number of the butyryl-CoA CoA-transferase gene and the 16S rRNA gene copy numbers of Bifidobacterium, the Clostridium coccoides group, the Roseburia/ Eubacterium rectale group, Eubacterium hallii, and butyrate-producing bacterium strain SS2/1 were significantly increased by the intake of PHGG. Other bacteria and bacterial groups were not significantly influenced by the intake of PHGG. It was believed that the Roseburia/E. rectale group bacteria, Bifidobacterium, the lactate-utilising, butyrate-producing bacteria, E. hallii and bacterium strain SS2/1, would contribute to the fermentation of PHGG in the human large intestine. PHGG may benefit health by stimulating Bifidobacterium and butyrate-producing bacteria in the human large intestine.

PET in infancy predicts long-term outcome during adolescence in cryptogenic West syndrome.

BACKGROUND AND PURPOSE: Developmental and seizure outcomes in patients with cryptogenic West syndrome are variable. Our aim was to clarify the relationship between FDG-PET findings in infancy and long-term seizure and developmental outcome in cryptogenic West syndrome. MATERIALS AND METHODS: From 1991 to 1999, we prospectively performed FDG-PET from the onset of cryptogenic West syndrome in 27 patients. PET was performed at onset and at 10 months of age. In 2012, we evaluated the educational status, psychomotor development, and seizure outcome in 23 of the 27 patients (13-22 years of age). The correlation between PET findings and outcome was evaluated. RESULTS: At onset, PET showed hypometabolism in 13 patients (57%). The second PET after the initial treatment revealed cortical hypometabolism in 7 patients (30%). While hypometabolism at onset disappeared on the second PET in 9 patients, it was newly revealed in 3 patients on the second PET. In 2012, seven patients had persistent or recurrent seizures. Eight patients had intellectual impairment. The first PET did not correlate with seizure or developmental outcome. Five of 7 patients (71%) with hypometabolism seen on the second PET had persistent or recurrent seizures, while 14 of 16 (88%) patients with normal findings on the second PET were free of seizures. Five of 7 patients (71%) showing hypometabolism on the second PET had intellectual impairment. Thirteen of 16 (81%) patients with normal findings on the second PET showed normal intelligence. A significant correlation was found between the second PET and long-term seizure (P = .01) or developmental outcome (P = .03). CONCLUSIONS: Cortical hypometabolism is not permanent; it changes with clinical symptoms. Hypometabolism after initial treatment predicts long-term seizures and poor developmental outcome.

Search for a light pseudoscalar particle in the decay K_{L};{0}-->pi;{0}pi;{0}X.

We performed a search for a light pseudoscalar particle X in the decay K_{L};{0}-->pi;{0}pi;{0}X, X-->gammagamma with the E391a detector at KEK. Such a particle with a mass of 214.3 MeV/c;{2} was suggested by the HyperCP experiment. We found no evidence for X and set an upper limit on the product branching ratio for K_{L};{0}-->pi;{0}pi;{0}X, X-->gammagamma of 2.4x10;{-7} at the 90% confidence level. Upper limits on the branching ratios in the mass region of X from 194.3 to 219.3 MeV/c;{2} are also presented.

Search for the Decay K L0-->pi0nu nu[over].

We performed a search for the K L0-->pi0nu nu[over] decay at the KEK 12-GeV proton synchrotron. No candidate events were observed. An upper limit on the branching ratio for the decay was set to be 6.7 x 10(-8) at the 90% confidence level.

Morning-evening type and stress-related personality in Japanese college students.

An earlier report by Mecacci and Rocchetti showed a significant, weak, and positive correlation between Evening type as measured by the Morningness-Eveningness Questionnaire and Type A personality as measured by the Jenkins Activity Survey and the Bortner Type A Scale in Italian college students. The present study of 503 Japanese college students yielded a significant and weakly negative correlation. The bases for the different results were discussed in a psychophysiological context.

Regulation of human trophoblast proliferation and apoptosis during pregnancy.

In order to elucidate the regulation of human placental growth during pregnancy, we have assessed PCNA expression, apoptosis and Bcl-2 protein expression in placental trophoblasts over the course of pregnancy. PCNA, Bcl-2 protein and Fas antigen expression were examined by the avidin/biotin immunoperoxidase method, while apoptosis was assessed by in situ DNA 3'-end labeling method. Both PCNA expression and apoptotic DNA fragmentation were noted in cytotrophoblasts (C-cells), being most abundant in very early placenta, less abundant in midterm placenta and least abundant in term placenta. In contrast, Bcl-2 protein expression was noted in syncytiotrophoblasts (S-cells), being least abundant in very early placenta, less abundant in midterm placenta and most abundant in term placenta. These results indicate that very early placenta is characterized by highly proliferative activity of C-cells associated with increased occurrence of apoptosis. Since Bcl-2 protein is an apoptosis-inhibiting gene product, the minimal occurrence of apoptosis in term placenta seems likely to be attributable to the increased expression of Bcl-2 protein in S-cell in term placenta. On the other hand, in extravillous trophoblasts on cell columns, both PCNA and Bcl-2 protein expression were pronounced only in the shallower part, while Fas/Fas ligand expression and apoptosis were prominent in the deeper part. Thus, it seems likely that Bcl-2 protein expression also participates in the regulation of extravillous trophoblast apoptosis.

Myocardium extending from the left atrium onto the pulmonary veins: a comparison between subjects with and without atrial fibrillation.

Rapid discharges from the myocardium extendingfrom the left atrium onto the pulmonary vein (PV) have been shown to initiate AF, and AF may be eradicated by the catheter ablation within the PV. However, if there is any difference in the distribution patterns of the myocardial sleeve onto the PV between the subjects with and without AF is to be determined. Twenty-one autopsied hearts were examined. Eleven patients previously had AF before death and another 10 patients had normal sinus rhythm as confirmed from the medical records including ECGs before death. After exposing the heart, the distance to the peripheral end of the myocardium was measured from the PV-atrial junction in each PV. Then, the PVs were sectioned and stained and the distal end of myocardium and the distribution pattern were studied. The anteroposterior diameter of the left atrium was also measured. In 74 of 84 PVs, the myocardium extended beyond the PV-atrial junction. The myocardium was localized surrounding the vascular smooth muscle layerforming a myocardial sleeve. The peripheral end of the myocardial sleeve was irregular and the maximal and minimal distances were measured in each PV. The myocardium extended most distally in the superior PVs compared to the inferior ones and the maximal distance to the peripheral end was similar between the AF and non-AF subjects (8.4 +/- 2.8 vs 8.7 +/- 4.4 mm for the left superior and 6.5 +/- 3.5 vs 5.1 +/- 3.9 mm for the right superior PV, respectively). A significant difference was found in the maximal distance in the inferior PVs: 7.3 +/- 4.6 vs 3.3 +/- 2.8 mm for the left (P < 0.05) and 5.7 +/- 2.4 vs 1.7 +/- 1.9 mm for the right inferior PV (P < 0.001) in the subjects with and without AF, respectively. The diameter of left atrium was slightly dilated in AF patients but insignificantly (4.1 +/- 0.1 vs 3.6 +/- 0.1 cm, P > 0.07). The myocytes on the PV were less uniform and surrounded by more fibrosis in patients with AF compared to those without AF. In conclusion, the myocardium extended beyond the atrium-vein junction onto the PVs. The distribution patterns of the myocardium was almost similar between subjects with and without AF, but the histology suggested variable myocytes in size and fibrosis in patients with AF.

Autophagosome requires specific early Sec proteins for its formation and NSF/SNARE for vacuolar fusion.

Double membrane structure, autophagosome, is formed de novo in the process of autophagy in the yeast Saccharomyces cerevisiae, and many Apg proteins participate in this process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (soluble N-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosome to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23, and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.

Two patients with severe alcoholic hepatitis accompanied by hypercytokinemia and granulocytic hyperelastasemia, successfully treated by intravenous infusion of urinastarine (Miraclid).

Severe alcoholic hepatitis (SAH) is not simply a disease of the liver, but it also causes infection and multiple organ failure, and therefore carries an extremely poor prognosis. We report the successful treatment of two patients with SAH. Case 1: The patient was a 55-year-old man. He was a heavy drinker whose alcohol intake had increased for some 3 years to 1.8 L sake a day. Slight clouding of consciousness, fever, and jaundice were evident on his admission to our hospital. Laboratory data showed leukocytosis with a predominance of polymorphonuclear leukocytes, and a decline in coagulability. He tested negative for various hepatitis virus markers. With the diagnosis of SAH made, steroid pulse therapy and bilirubin adsorption therapy were administered. The jaundice improved and the interleukin-8 (IL-8) level decreased. Continuous intravenous infusion of urinastarine (Miraclid) normalized the granulocyte elastase level. Improvement was also seen in coagulability, ascites, icterus and consciousness. Case 2: The patient was a 49-year-old man. He was a heavy drinker whose alcohol intake had increased for 1 month. Fever, jaundice, ascites, and mild disturbance of consciousness were evident at the time of admission. Examination on admission diagnosed SAH. Bilirubin adsorption and continuous intravenous infusion of urinastarine were initiated. As a result, circulating IL-8 level was decreased and coagulability was improved. Therapy for granulocytic hyperelastasemia and hypercytokinemia supervening on SAH is a new effective approach on improvement in the disease.

Hypocarbia in preterm infants with periventricular leukomalacia: the relation between hypocarbia and mechanical ventilation.

OBJECTIVE: The aim of this study was to elucidate the relationship between mechanical ventilation and hypocarbia in infants with periventricular leukomalacia (PVL). STUDY DESIGN: Matched pair analysis was conducted for 26 infants with PVL and 26 with normal development, who were born between 27 and 32 weeks' gestational age and required mechanical ventilation. The time-averaged carbon dioxide (CO(2)) index, PaCO(2), and pH were calculated every 24 hours for samples obtained from indwelling arterial catheters within the first 72 hours of life. The time-averaged respiratory rate of the ventilator (RR), peak inspiratory pressure (PIP), mean airway pressure (MAP), and ventilator index (VI) were also determined. The time-averaged total respiratory rate (TRR) was determined by observing the movement of the chest wall. The patients' characteristics, antenatal and neonatal variables, and electroencephalographic findings were also compared. RESULTS: The time-averaged CO(2) index was larger, the time-averaged CO(2) lower and the time-averaged pH higher in infants with PVL than in those with normal development on the third day of life. There was no significant difference in the time-averaged RR, PIP, MAP, or VI on any day. TRR was larger in the PVL group than in the control group on each day, but there was no significant difference. No significant difference was observed in the clinical characteristics or neonatal variables. Electroencephalographic abnormalities within 48 hours of life were more frequent in infants with PVL than in those with normal development. CONCLUSION: Hypocarbia was associated with PVL because the time-averaged CO(2) index was larger and the time-averaged PaCO(2) lower in infants with PVL than in those with normal development. However, the ventilator settings were similar among the infants with and without PVL.

Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae.

Vps30p/Apg6p is required for both autophagy and sorting of carboxypeptidase Y (CPY). Although Vps30p is known to interact with Apg14p, its precise role remains unclear. We found that two proteins copurify with Vps30p. They were identified by mass spectrometry to be Vps38p and Vps34p, a phosphatidylinositol (PtdIns) 3-kinase. Vps34p, Vps38p, Apg14p, and Vps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es). Phenotypic analyses indicated that Apg14p and Vps38p are each required for autophagy and CPY sorting, respectively, whereas Vps30p, Vps34p, and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p antibodies and pull-down experiments showed that two distinct Vps34 PtdIns 3-kinase complexes exist: one, containing Vps15p, Vps30p, and Apg14p, functions in autophagy and the other containing Vps15p, Vps30p, and Vps38p functions in CPY sorting. The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3-kinase complex(es). We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.

Characterization of acetic acid bacteria in traditional acetic acid fermentation of rice vinegar (komesu) and unpolished rice vinegar (kurosu) produced in Japan.

Bacterial strains were isolated from samples of Japanese rice vinegar (komesu) and unpolished rice vinegar (kurosu) fermented by the traditional static method. Fermentations have never been inoculated with a pure culture since they were started in 1907. A total of 178 isolates were divided into groups A and B on the basis of enterobacterial repetitive intergenic consensus-PCR and random amplified polymorphic DNA fingerprinting analyses. The 16S ribosomal DNA sequences of strains belonging to each group showed similarities of more than 99% with Acetobacter pasteurianus. Group A strains overwhelmingly dominated all stages of fermentation of both types of vinegar. Our results indicate that appropriate strains of acetic acid bacteria have spontaneously established almost pure cultures during nearly a century of komesu and kurosu fermentation.

A ubiquitin-like system mediates protein lipidation.

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.

Changes in proliferative potential, apoptosis and Bcl-2 protein expression in cytotrophoblasts and syncytiotrophoblast in human placenta over the course of pregnancy.

In order to evaluate placental trophoblast proliferation and apoptosis during pregnancy, we investigated proliferating cell nuclear antigen (PCNA) expression, apoptosis and Bcl-2 protein expression in the human placenta using avidin/biotin immunoperoxidase method to examine PCNA and Bcl-2 protein expression, and TUNEL method to assess apoptosis. The appearance of apoptotic cells in very early term placental trophoblasts was also examined by transmission electron microscopy. PCNA was immunolocalized in the nuclei of cytotrophoblasts (C-cells). Determination of the mean percentage of PCNA-positive nuclei of C-cells revealed that PCNA expression in C-cells was highest in very early term (4th to 5th wk) placentas and significantly decreased with the advance of pregnancy. Bcl-2 protein was immunolocalized in the cytoplasm of syncytiotrophoblast (S-cell), being least abundant in very early term placentas, less abundant in early term and midterm placentas, and most abundant in term placentas. On the basis of TUNEL method, apoptosis was apparent in the nuclei of both C-cells and S-cell. The apoptosis positive rate of C-cell nuclei was highest in very early term 4th to 5th wk placentas, and significantly decreased in early term 7th to 9th wk and midterm placentas, but somewhat increased in term placentas compared to that in midterm placentas. On the other hand, apoptosis positive rate of S-cell nuclei was remarkably higher only in very early term 4th to 5th wk placentas compared to that in early term, midterm and term placentas. Transmission electron microscopy revealed the appearance of apoptotic nucleus in very early term placental trophoblasts. These results demonstrate for the first time that apoptosis in the human normal placenta predominates in both C-cells and S-cell in very early term 4th to 5th wk pregnancy and drastically diminished after 7th wk of pregnancy. An apparent increase in apoptosis in C-cells in term placentas compared to that in midterm placentas may reflect aging of the placenta or parturition-associated biological change. The abundant expression of Bcl-2 protein in S-cell in term placentas may be responsible for the diminished occurrence of apoptosis in S-cell in term placentas.