Distribution of Vacuolar H+-Pyrophosphatase and a Membrane Integral Protein in a Variety of Green Plants

Vacuolar membranes isolated from several species including fernand moss exhibited pyro-phosphate-dependent H⁺ transport activity.On immunoblot analysis, H⁺ -pyrophosphatase was detected inthe vacuolar membranes. A membrane integral protein of 23,000daltons was not found in the membranes of Chara, Conocephalum,or Kalanchoë. Thus, H⁺-pyrophosphatase may be a universalenzyme among green plants, but the 23-kDa protein is not a commonprotein of central vacuoles. (Received September 10, 1993; Accepted November 29, 1993)     

Microchamber arrays for the identification of individual cells exposed to an X-ray microbeam

To identify individual cells exposed to a X-ray microbeam in a cell population, we developed a biocompatible microchamber-array chip using UV lithography of photopolymer SU-8. The center-to-center distance between microchambers is 50 μm including a wall of 15 μm height. Using the microchamber-array chip, we performed tracking of individual exposed cells. Sample cells loaded in a microchamber array were selectively irradiated with the X-ray microbeam under microscopic observation. All the irradiated cells were indexed by the array arrangement of the microchambers. For about 24 h of post-irradiation incubation, the irradiated cells were identified successfully by time-lapse observation. In addition, the induction of radiation effects was observed in identified cells using immunofluorescence.     

Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.     

Localization of Golgi 58K protein (formiminotransferase cyclodeaminase) to the centrosome

In vertebrate cells, the centrosome consists of a pair of centrioles and surrounding pericentriolar material. Using anti-Golgi 58K protein antibodies that recognize formiminotransferase cyclodeaminase (FTCD), we investigated its localization to the centrosome in various cultured cells and human oviductal secretory cells by immunohistochemistry. In addition to the Golgi apparatus, FTCD was localized to the centrosome, more abundantly around the mother centriole. The centrosome localization of FTCD continued throughout the cell cycle and was not disrupted after Golgi fragmentation, which was induced by colcemid and brefeldin A. Centriole microtubules are polyglutamylated and stable against tubulin depolymerizing drugs. FTCD in the centrosome may be associated with polyglutamylated residues of centriole microtubules and may play a role in providing centrioles with glutamate produced by cyclodeaminase domains of FTCD.     

A novel,potent, and orally active VLA-4 antagonist with good aqueous solubility: trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-7-fluoro-6-benzoxazolyl]acetyl]-(5S)-[methoxy(methyl)amino]methyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid

We have carried out the optimization of substituents at the C-3 or the C-5 position on the pyrrolidine ring of VLA-4 antagonist 3 with 2-(phenylamino)-7-fluorobenzoxazolyl moiety for the purpose of improving in vivo efficacy while maintaining good aqueous solubility. As a result, we successfully increased in vitro activity in the presence of 3% human serum albumin and achieved an exquisite lipophilic and hydrophilic balance of compounds suitable for oral administrative regimen. The modification resulted in the identification of zwitterionic compound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine, which significantly alleviated bronchial hyper-responsiveness to acetylcholine chloride at 12.5 mg/kg, p.o. in a murine asthma model and showed favorable aqueous solubility (JP1, 89 μg/mL; JP2, 462 μg/mL). Furthermore, this compound showed good oral bioavailability (F = 54%) in monkeys.     

Transient neutrophil infiltration after allergen challenge is dependent on specific antibodies and Fc gamma III receptors

Following allergen challenge of sensitized mice, neutrophils are the first inflammatory cells found in bronchoalveolar lavage (BAL) fluid. To determine the underlying mechanism for their accumulation, mice were sensitized to OVA on days 0 and 14, and received, on day 28, a single intranasal challenge (s.i.n.) with either OVA or ragweed. Eight hours after the s.i.n., BAL fluid was obtained. BALB/c mice sensitized and challenged with OVA showed significantly higher total cell counts and numbers of neutrophils in BAL fluid compared to the OVA-sensitized and ragweed-challenged or nonsensitized mice. Levels of neutrophil chemokines in BAL fluid supernatants were markedly elevated in the sensitized and OVA-challenged mice; Fc epsilon RI-deficient mice showed comparable numbers of neutrophils and neutrophil chemokines in BAL fluid after s.i.n. But in sensitized mice lacking the Fc common gamma-chain and B cell-deficient mice, the number of neutrophils and levels of neutrophil chemokines in BAL fluid were significantly lower. Further, mice lacking the FcgammaRIII did not develop this early neutrophil influx. Neutrophil infiltration could be induced in naive mice following intranasal instillation of allergen combined with allergen-specific IgG1. In addition, macrophages from sensitized mice were stimulated with allergen and activated to produce neutrophil chemokines. These results demonstrate that neutrophil influx after allergen challenge requires prior sensitization, is allergen-specific, is mediated through FcgammaRIII, and is dependent on the presence of Ab.     

A suggested role for mitochondria in Noonan syndrome

Noonan syndrome (NS) is an autosomal dominant disorder, and a main feature is congenital heart malformation. About 50% of cases are caused by gain-of-function mutations in the tyrosine phosphatase SHP2/PTPN11, a downstream regulator of ERK/MAPK. Recently it was reported that SHP2 also localizes to the mitochondrial intercristae/intermembrane space (IMS), but the role of SHP2 in mitochondria is unclear. The mitochondrial oxidative phosphorylation (OxPhos) system provides the vast majority of cellular energy and produces reactive oxygen species (ROS). Changes in ROS may interfere with organ development such as that observed in NS patients. Several phosphorylation sites have been found in OxPhos components including cytochrome c oxidase (CcO) and cytochrome c (Cytc), and we hypothesized that OxPhos complexes may be direct or indirect targets of SHP2. We analyzed mitochondrial function using mouse fibroblasts from wild-types, SHP2 knockdowns, and D61G SHP2 mutants leading to constitutively active SHP2, as found in NS patients. Levels of OxPhos complexes were similar except for CcO and Cytc, which were 37% and 28% reduced in the D61G cells. However, CcO activity was significantly increased, as we also found for two lymphoblast cell lines from NS patients with two independent mutations in PTPN11. D61G cells showed lower mitochondrial membrane potential and 30% lower ATP content compared to controls. ROS were significantly increased; aconitase activity, a marker for ROS-induced damage, was decreased; and catalase activity was increased in D61G cells. We propose that decreased energy levels and/or increased ROS may explain, at least in part, some of the clinical features in NS that overlap with children with mitochondrial disorders.     

Psychiatrists' Attitudes toward Metabolic Adverse Events in Patients with Schizophrenia

Background

There is growing concern about the metabolic abnormalities in patients with schizophrenia.

Aims

The aim of this study was to assess the attitudes of psychiatrists toward metabolic adverse events in patients with schizophrenia.

Method

A brief questionnaire was constructed to cover the following broad areas: the psychiatrists'' recognition of the metabolic risk of antipsychotic therapy, pattern of monitoring patients for physical risks, practice pattern for physical risks, and knowledge of metabolic disturbance. In March 2012, the questionnaire was mailed to 8,482 psychiatrists who were working at hospitals belonging to the Japan Psychiatric Hospitals Association.

Results

The overall response rate was 2,583/8,482 (30.5%). Of the respondents, 85.2% (2,200/2,581) reported that they were concerned about prescribing antipsychotics that have a risk of elevating blood sugar; 47.6% (1,201/2,524) stated that their frequency of monitoring patients under antipsychotic treatment was based on their own experiences; and only 20.6% (5,22/2,534) of respondents answered that the frequency with which they monitored their patients was sufficient to reduce the metabolic risks.

Conclusions

Psychiatrists practicing in Japan were generally aware and concerned about the metabolic risks for patients being treated with antipsychotics. Although psychiatrists should monitor their patients for metabolic abnormalities to balance these risks, a limited number of psychiatrists answered that the frequency with which they monitored patients to reduce the metabolic risks was sufficient. Promotion of the best practices of pharmacotherapy and monitoring is needed for psychiatrists treating patients with schizophrenia.     

Productivity and mineral cycling in an oak coppice forest 2. Annual net production of the forest

Annual net production was estimated in the secondary coppice forest near Tokyo, which was dominated by a deciduous oak,Quercus serrata Thunb. Lateral growth of stems and old branches was directly estimated by examining the annual rings for 35 shoots in a clear-cut quadrat of 10m×10m. Phytomasses of current organs were also weighed in the quadrat. Preharvest losses of current organs were determined by twelve 0.5 m² litter traps for fine litter and twelve 6 m² quadrats for woody litter. Branch production was also assessed indirectly by use of the stem-branch allometry and death of branches. The results of the indirect method were in sufficient agreement with the result of the direct one. Grazing loss of leaves from the canopy was estimated directly from the loss in leaf area and indirectly from the animal faeces caught by the litter traps. Net production of the canopy trees was 149 kg a⁻¹ year⁻¹, in which leaf production was 36.9 kg. Animals grazed about 14% of the leaf area by the end of the growing season. True consumption of leaves by animals was 7.6% of leaf production or 10% of leaf mass. Production of undergrowth, mainly a dwarf bamboo,Pleioblastus chino Makino, was 28 kg a⁻¹ year⁻¹, being 15% of the total stand production. Productivity of this forest was significantly higher than that of cool-temperate deciduous broadleaf forests.