Phosphorylation of plasma membrane H+-ATPase Thr881 participates in light-induced stomatal opening.

Plasma membrane (PM) H+-ATPase is crucial for light-induced stomatal opening and phosphorylation of a penultimate residue, Thr948 (pen-Thr, numbering according to Arabidopsis AHA1) is required for enzyme activation. In this study, a comprehensive phosphoproteomic analysis using guard cell protoplasts from Vicia faba shows that both red and blue light increase the phosphorylation of Thr881, of PM H+-ATPase. Light-induced stomatal opening and the blue light-induced increase in stomatal conductance are reduced in transgenic Arabidopsis plants expressing mutant AHA1-T881A in aha1-9, whereas the blue light-induced phosphorylation of pen-Thr is unaffected. Auxin and photosynthetically active radiation induce the phosphorylation of both Thr881 and pen-Thr in etiolated seedlings and leaves, respectively. The dephosphorylation of phosphorylated Thr881 and pen-Thr are mediated by type 2 C protein phosphatase clade D isoforms. Taken together, Thr881 phosphorylation, in addition of the pen-Thr phosphorylation, are important for PM H+-ATPase function during physiological responses, such as light-induced stomatal opening in Arabidopsis thaliana.

Development of a HPLC system using a phase-separation multiphase flow as an eluent coupled to a silica-particle packed column.

The study reports the development of a high-performance liquid chromatography (HPLC) system in which a phase-separation multiphase flow as the eluent and a silica-particle based packed column as the separation column were combined to form the phase separation mode. Twenty-four types of mixed solutions of water/acetonitrile/ethyl acetate and water/acetonitrile were applied as eluents to the system at 20 °C. 2,6-Naphthalenedisulfonic acid (NDS) and 1-naphthol (NA) were injected as model analytes into the system. They showed separation tendency in organic solvent-rich eluents in normal-phase mode and NA was detected earlier than NDS. Subsequently, seven types of the ternary mixed solutions were examined as eluents in the HPLC system at 20 °C and 0 °C. These mixed solutions worked as a two-phase separation mixed solution, providing a phase-separation multiphase flow at 0 °C in the separation column. In the organic solvent-rich eluent, the mixture of analytes was separated at both 20 °C (normal-phase mode) and 0 °C (phase-separation mode), with NA being detected earlier than NDS. The separation at 0 °C was more efficient than at 20 °C. In the water-rich eluent, the mixture of NDS and NA was not separated at 20 °C but was separated at 0 °C (phase-separation mode), with NDS being detected earlier than NA. We also discussed the separation mechanism of phase-separation mode in HPLC together with the computer simulation for the multiphase flow in the cylindrical tube having sub-µm inner diameter.

Development of HPLC system that uses phase-separation multiphase flow as an eluent.

We developed a new type of HPLC system that uses phase-separation multiphase flow as an eluent. A commercially available HPLC system with a packed separation column filled with octadecyl-modified silica (ODS) particles was used. First, as preliminary experiments, 25 kinds of mixed solutions of water/acetonitrile/ethyl acetate and water/acetonitrile were supplied to the system to act as eluents at 20 °C. 2,6-Naphthalenedisulfonic acid (NDS) and 1-naphthol (NA) mixture was used as a model and mixed analyte was injected into the system. Roughly speaking, they were not separated in organic solvent-rich eluents and well separated in water-rich eluents, in which NDS eluted faster than NA. This means that HPLC worked under a reverse-phase mode for separation at 20 °C. Next, the separation of the mixed analyte was examined on HPLC at 5 °C, and then after judging the results, four kinds of ternary mixed solutions were in detail as eluents on HPLC at 20 °C and 5 °C. Based on their volume ratio, the ternary mixed solutions acted as a two-phase separation mixed solution, leading to a phase-separation multiphase flow. Consequently, the solutions flowed homogeneously and heterogeneously in the column at 20 °C and 5 °C, respectively. For example, the ternary mixed solutions containing water/acetonitrile/ethyl acetate at volume ratios of 20:60:20 (organic solvent-rich) and 70:23:7 (water-rich) were delivered into the system as eluents at 20 °C and 5 °C. In the organic solvent-rich eluent, the mixture of NDS and NA was not separated at 20 °C but was separated at 5 °C, the elution of NA being faster than the one of NDS (phase-separation mode). In the water-rich eluent, the mixture of analytes was separated at both 20 °C and 5 °C, the elution of NDS being faster than the one of NA. The separation at 5 °C was more effective than at 20 °C (reverse-phase mode and phase-separation mode). This separation performance and elution order can be attributed to the phase-separation multiphase flow at 5 °C.

Photosynthetic-Product-Dependent Activation of Plasma Membrane H+-ATPase and Nitrate Uptake in Arabidopsis Leaves.

Plasma membrane (PM) proton-translocating adenosine triphosphatase (H+-ATPase) is a pivotal enzyme for plant growth and development that acts as a primary transporter and is activated by phosphorylation of the penultimate residue, threonine, at the C-terminus. Small Auxin-Up RNA family proteins maintain the phosphorylation level via inhibiting dephosphorylation of the residue by protein phosphatase 2C-D clade. Photosynthetically active radiation activates PM H+-ATPase via phosphorylation in mesophyll cells of Arabidopsis thaliana, and phosphorylation of PM H+-ATPase depends on photosynthesis and photosynthesis-related sugar supplementation, such as sucrose, fructose and glucose. However, the molecular mechanism and physiological role of photosynthesis-dependent PM H+-ATPase activation are still unknown. Analysis using sugar analogs, such as palatinose, turanose and 2-deoxy glucose, revealed that sucrose metabolites and products of glycolysis such as pyruvate induce phosphorylation of PM H+-ATPase. Transcriptome analysis showed that the novel isoform of the Small Auxin-Up RNA genes, SAUR30, is upregulated in a light- and sucrose-dependent manner. Time-course analyses of sucrose supplementation showed that the phosphorylation level of PM H+-ATPase increased within 10 min, but the expression level of SAUR30 increased later than 10 min. The results suggest that two temporal regulations may participate in the regulation of PM H+-ATPase. Interestingly, a 15NO3- uptake assay in leaves showed that light increases 15NO3- uptake and that increment of 15NO3- uptake depends on PM H+-ATPase activity. The results opened the possibility of the physiological role of photosynthesis-dependent PM H+-ATPase activation in the uptake of NO3-. We speculate that PM H+-ATPase may connect photosynthesis and nitrogen metabolism in leaves.

RNA-seq analysis, targeted long-read sequencing and in silico prediction to unravel pathogenic intronic events and complicated splicing abnormalities in dystrophinopathy.

Dystrophinopathy is caused by alterations in DMD. Approximately 1% of patients remain genetically undiagnosed, because intronic variations are not detected by standard methods. Here, we combined laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal DMD transcript generation. DMD transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequencing. Genome sequencing captured intronic variants and their effects were interpreted using in silico tools. Targeted long-read sequencing was applied in cases with suspected structural genomic abnormalities. Abnormal DMD transcripts were detected in 19 of 20 cases; Exonization of intronic sequences in 15 cases, exon skipping in one case, aberrantly spliced and polyadenylated transcripts in two cases and transcription termination in one case. Intronic single nucleotide variants, chromosomal rearrangements and nucleotide repeat expansion were identified in DMD gene as pathogenic causes of transcript alteration. Our combined analysis approach successfully identified pathogenic events. Detection of diseasing-causing mechanisms in DMD transcripts could inform the therapeutic options for patients with dystrophinopathy.

A win-win scenario for photosynthesis and the plasma membrane H+ pump.

In plants, cytosolic and extracellular pH homeostasis are crucial for various physiological processes, including the uptake of macronutrients and micronutrients, cell elongation, cell expansion, and enzyme activity. Proton (H+) gradients and the membrane potential are generated by a H+ pump consisting of an active primary transporter. Plasma membrane (PM) H+-ATPase, a PM-localized H+ pump, plays a pivotal role in maintaining pH homeostasis in plant cells and extracellular regions. PM H+-ATPase activity is regulated by protein abundance and by post-translational modifications. Several stimuli have been found to activate the PM H+-ATPase through phosphorylation of the penultimate threonine (Thr) of the carboxy terminus. Light- and photosynthesis-induced phosphorylation of PM H+-ATPase are conserved phenomena among various plant species. In this work, we review recent findings related to PM H+-ATPase regulation in the photosynthetic tissues of plants, focusing on its mechanisms and physiological roles. The physiological roles of photosynthesis-dependent PM H+-ATPase activation are discussed in the context of nitrate uptake and cytoplasmic streaming in leaves.

Novel separation mode of HPLC based on phase-separation multiphase flow.

A novel separation mode for high-performance liquid chromatography (HPLC) is proposed based on phase-separation multiphase flow. A commercially available HPLC system was used with a packed-separation column of octadecyl-silica (ODS)-modified particles. Water/acetonitrile/ethyl acetate ternary mixed solutions, (a) 1:8:1, (b) 1:3:1, and (c) 16:3:1 (v/v/v), were delivered into the system as an eluent at 20 and 5 °C. The ternary mixed solution acted as a two-phase separation solution leading to phase-separation multiphase flow. The solution flowed in the column homogeneously and heterogeneously at 20 and 5 °C, respectively. 1-Naphthol (NA) and 2,6-naphthalenedisulfonic acid (NDS) were injected into the system as model analytes. At 20 °C, the analyte mixture did not separate in solutions (a) and (b) while it separated in solution (c) with the elution order of NDS followed by NA. At 5 °C, it did not separate in solution (a), while it separated in solution (b) with elution order of NA followed by NDS and solution (c) with elution order of NDS followed by NA more effectively than 20 °C. The separation behavior and elution order are possibly caused by the phase-separation multiphase flow.

Lack of Vacuolar H+ -Pyrophosphatase and Cytosolic Pyrophosphatases Causes Fatal Developmental Defects in Arabidopsis thaliana.

The cytosolic level of inorganic pyrophosphate (PPi) is finely regulated, with PPi hydrolyzed primarily by the vacuolar H+-pyrophosphatase (H+-PPase, VHP1/FUGU5/AVP1) and secondarily by five cytosolic soluble pyrophosphatases (sPPases; PPa1-PPa5) in Arabidopsis thaliana. Loss-of-function mutants of H+-PPase (fugu5s) have been reported to show atrophic phenotypes in their rosette leaves when nitrate is the sole nitrogen source in the culture medium. For this phenotype, two questions remain unanswered: why does atrophy depend on physical contact between shoots and the medium, and how does ammonium prevent such atrophy. To understand the mechanism driving this phenotype, we analyzed the growth and phenotypes of mutants on ammonium-free medium in detail. fugu5-1 showed cuticle defects, cell swelling, reduced ß-glucan levels, and vein malformation in the leaves, suggesting cell wall weakening and cell lethality. Based on the observation in the double mutants fugu5-1 ppa1 and fugu5-1 ppa4 of more severe atrophy compared to fugu5-1, the nitrogen-dependent phenotype might be linked to PPi metabolism. To elucidate the role of ammonium in this process, we examined the fluctuations of sPPase mRNA levels and the possibility of alternative PPi-removing factors, such as other types of pyrophosphatase. First, we found that both the protein and mRNA levels of sPPases were unaffected by the nitrogen source. Second, to assess the influence of other PPi-removing factors, we examined the phenotypes of triple knockout mutants of H+-PPase and two sPPases on ammonium-containing medium. Both fugu5 ppa1 ppa2 and fugu5 ppa1 ppa4 had nearly lethal embryonic phenotypes, with the survivors showing striking dwarfism and abnormal morphology. Moreover, fugu5 ppa1+/- ppa4 showed severe atrophy at the leaf margins. The other triple mutants, fugu5 ppa1 ppa5 and fugu5 ppa2 ppa4, exhibited death of root hairs and were nearly sterile due to deformed pistils, respectively, even when grown on standard medium. Together, these results suggest that H+-PPase and sPPases act in concert to maintain PPi homeostasis, that the existence of other PPi removers is unlikely, and that ammonium may suppress the production of PPi during nitrogen metabolism rather than stimulating PPi hydrolysis.

Early phase 2 trial of TAS-205 in patients with Duchenne muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a progressive muscular disease characterized by chronic cycles of inflammatory and necrotic processes. Prostaglandin D2 (PGD2 ) is produced by hematopoietic PGD synthase (HPGDS), which is pathologically implicated in muscle necrosis. This randomized, double-blind, placebo-controlled early phase 2 study (NCT02752048) aimed to assess the efficacy and safety of the novel selective HPGDS inhibitor, TAS-205, with exploratory measures in male DMD patients aged ≥5 years. METHODS: Patients were randomized 1:1:1 to receive low-dose TAS-205 (6.67-13.33 mg/kg/dose), high-dose TAS-205 (13.33-26.67 mg/kg/dose), or placebo. The primary endpoint was the change from baseline in a 6-minute walk distance (6MWD) at Week 24. RESULTS: Thirty-six patients were enrolled, of whom 35 patients were analysed for safety. The mean (standard error) changes from baseline to Week 24 in 6MWD were -17.0 (17.6) m in the placebo group (n = 10), -3.5 (20.3) m in the TAS-205 low-dose group (n = 11), and -7.5 (11.2) m in the TAS-205 high-dose group (n = 11). The mean (95% confidence interval) difference from the placebo group was 13.5 (-43.3 to 70.2) m in the TAS-205 low-dose group and 9.5 (-33.3 to 52.4) m in the TAS-205 high-dose group. No obvious differences were observed in the incidences of adverse events between treatment groups. No adverse drug reactions specific to TAS-205 treatment were observed. INTERPRETATION: The HPGDS inhibitor TAS-205 showed a favorable safety profile in DMD patients. Further research is required to examine the effectiveness of TAS-205 in a larger trial.

Vacuolar H+-Pyrophosphatase and Cytosolic Soluble Pyrophosphatases Cooperatively Regulate Pyrophosphate Levels in Arabidopsis thaliana.

Inorganic pyrophosphate (PPi) is a phosphate donor and energy source. Many metabolic reactions that generate PPi are suppressed by high levels of PPi. Here, we investigated how proper levels of cytosolic PPi are maintained, focusing on soluble pyrophosphatases (AtPPa1 to AtPPa5; hereafter PPa1 to PPa5) and vacuolar H+-pyrophosphatase (H+-PPase, AtVHP1/FUGU5) in Arabidopsis thaliana In planta, five PPa isozymes tagged with GFP were detected in the cytosol and nuclei. Immunochemical analyses revealed a high abundance of PPa1 and the absence of PPa3 in vegetative tissue. In addition, the heterologous expression of each PPa restored growth in a soluble PPase-defective yeast strain. Although the quadruple knockout mutant plant ppa1 ppa2 ppa4 ppa5 showed no obvious phenotypes, H+-PPase and PPa1 double mutants (fugu5 ppa1) exhibited significant phenotypes, including dwarfism, high PPi concentrations, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Altered cell arrangements and weakened cell walls in the root tip were particularly evident in fugu5 ppa1 and were more severe than in fugu5 Our results indicate that H+-PPase is essential for maintaining adequate PPi levels and that the cytosolic PPa isozymes, particularly PPa1, prevent increases in PPi concentrations to toxic levels. We discuss fugu5 ppa1 phenotypes in relation to metabolic reactions and PPi homeostasis.

Biochemical, Structural and Physiological Characteristics of Vacuolar H+-Pyrophosphatase.

Proton-translocating inorganic pyrophosphatase (H+-PPase) actively translocates protons across membranes coupled with the hydrolysis of inorganic pyrophosphate (PPi). H+-PPase, which is composed of a single protein and uses a simple compound as a substrate, has been recognized as a new type of ion pump in addition to the P-, F- and V-type ion-translocating ATPases. H+- and Na+-PPases are distributed in various organisms including plants, parasitic protozoa, Archaebacteria and bacteria, but are not present in animals or yeast. Vacuolar H+-PPase has dual functions in plant cells: hydrolysis of cytosolic PPi to maintain the levels of PPi, and translocation of protons into vacuoles to maintain the acidity of the vacuolar lumen. Acidification performed with the vacuolar-type H+-ATPase and H+-PPase is essential to maintain acidic conditions, which are necessary for vacuolar hydrolytic enzymes and for supplying energy to secondary active transporters. Recent studies using loss-of-function mutant lines of H+-PPase and complementation lines with soluble PPases have emphasized the physiological importance of the scavenging role of PPi. An overview of the main features of H+-PPases present in the vacuolar membrane is provided in terms of tissue distribution in plants, intracellular localization, structure-function relationship, biochemical potential as a proton pump and functional stability.

[Development of a Real-Time Reverse Transcription PCR Assay System for Detection of Three Subtypes of Influenza A and Influenza B Viruses].

We developed the initial real-time reverse transcription PCR assay system for seasonal influenza viruses in 2011. This prototype assay system could detect and identify specific influenza A virus subtypes[H1N1, H3N2 and (H1N1) pdm09] and influenza B virus. In the 2012-2013 season, our prototype PCR assay didn't work well because of point mutations occurred in the neuraminidase (NA) gene of the A (H3N2) strain. We improved the prototype assay by changing the target gene for A (H3N2) strain (2013 improved PCR assay). Moreover, we added the measurement system for the matrix (M) gene that was well conserved and common to all influenza A subtypes. In the 2013-2014 season, point mutations in the hemagglutinin (HA) gene of the A (H1N1) pdm09 strain lowered the sensitivity of the 2013 improved PCR assay, so that we changed the target gene for A (H1N1)pdm09 strain (2014 improved PCR assay). We analyzed swab samples from 1,721 patients in total by at least one of the three PCR assays we developed, and demonstrated that the PCR assays had excellent sensitivity and specificity compared with those of the commercially available rapid immunochromatography kit we used. In this study, the M gene was positive in all patients who were finally diagnosed as influenza A positive by 2013 or 2014 improved PCR assay. Therefore, measurement of the M gene, which is hardly to be affected by antigenic drift of influenza viruses, is thought to be useful in clinical practice.

[Mutations in the Neuraminidase Gene of the Epidemic Influenza Virus Strain Isolated in the 2012-2013 Season and Improvement of a PCR Assay for the Detection of Influenza Virus].

In 2011, we developed a real-time RT-PCR method to rapidly and sensitively detect three subtypes of influenza A virus [H1N1, H3N2, and influenza (H1N1) 2009] and influenza B virus (the conventional PCR method). This method was useful during the 2011-2012 epidemic season. However, epidemic influenza A virus strain H3N2 in the 2012-2013 season was undetectable by this method, possibly due to mutation in the neuraminidase (NA) gene of epidemic influenza A virus strain H3N2. Therefore, we improved the method by using the hemagglutinin (HA) gene instead of the NA gene as the target for the detection of influenza A virus strain H3N2. In addition, this improved PCR method also included a PCR detection system for the matrix (M) gene, well conserved and common to all influenza A virus strains. As a result, influenza A virus strain H3N2, which was undetectable by the conventional PCR method, was positive by the improved PCR method. Testing of specimens from 219 influenza-like illness patients during the 2012-2013 season by the influenza antigen immunochromatographic assay and conventional and improved PCR methods showed influenza virus A-positive rates of 24.2, 1.8, and 28.3%, respectively. All influenza A virus strains were positive for the M gene (in 62 [28.3%] of the 219 patients). These results suggest that the improved PCR method can determine the presence or absence of influenza A virus infection, even if a mutation in the HA or NA gene occurs in the future.

Abnormal glucose metabolism in aromatic L-amino acid decarboxylase deficiency.

We report sibling cases of aromatic L-amino acid decarboxylase (AADC) deficiency, which is a very rare congenital metabolic disorder. These patients were born to healthy and non-consanguineous parents, and presented oculogyric crises, paroxysmal dystonic attacks, and severe psychomotor retardation since early infancy. In cerebrospinal fluid the levels of homovanilic acid and 5-hydroxyindoleacetic acid were very low and the level of L-dopa was very high. The diagnosis was confirmed by the lack of AADC activity in plasma, and a point mutation in the AADC gene. MRI revealed a slightly small volume of the prefrontal areas and normal myelination in both patients. Positron emission tomography using 2-deoxy-2[(18)F] fluoro-D-glucose was performed in one patient, which revealed hypometabolism in the prefrontal cortex and bilateral basal ganglia with a little laterality. These findings suggested that the severe dystonic features were caused by abnormal function of bilateral basal ganglia and severe psychomotor retardation could be due to abnormalities in prefrontal cortical activity.

[Enzyme replacement therapy in a patient with Pompe disease].

Pompe disease is a rare autosomal recessive disease caused by the deficiency of acid alpha-glucosidase (GAA), which is required for the degradation of lysosomal glycogen. Glycogen accumulation in heart, muscle and liver eventually leads to muscle weakness, hepatomegaly and cardiomegaly. Although an approved therapy does not exist, the efficacy of enzyme replacement therapy (ERT) has recently been reported in multinational trials in Europe and the US. Here, we present data on the efficacy of recombinant human acid alpha-glucosidase (rhGAA) (provided by Genzyme Corporation) in a patient with Pompe disease. At 5 months of age, motor delay (could not raise his head) and cardiomegaly were observed. A definite diagnosis of Pompe disease was made at 8 months of age after the accumulation of glycogen in a muscle biopsy specimen was observed. This was confirmed by low GAA activity. Since then, motor delay predominated and he was unable to sit independently by age 2.5 years. Every 2 weeks, 20 mg/kg of rhGAA was infused intravenously. To assess the effectiveness, chest X-ray, echocardiography and auditory brain response were recorded. The patient was administered rhGAA for 26 months from 2 years and 8 months of age. Following the initiation of ERT, hepatomegaly and cardiac function (ejection fraction) were rapidly improved and motor function was gradually improved. At 4 years and 10 months, the patient could walk with support. No adverse event has been observed. It can be concluded that ERT with rhGAA is an effective and safe regimen for this case.

Influenza vaccination for severely multiply handicapped persons/children in the 2005-2006 season.

In this study, we report the effectiveness of trivalent inactivated influenza vaccination (TIV) for severely multiply handicapped persons/children (SMHPs) in the 2005-2006 season. In 77 SMHPs, A/New York/55/2004 (H3N2) which was the changed vaccine-strain showed significant differences in the geometric mean titers (P<0.05) and seroprotection rates (P<0.01) between pre- and post-vaccination. A/New Caledonia/20/99 (H1N1) and B/Shanghai/361/2002, which were the unchanged vaccine-strains, showed no significant differences. We defined the potential responders as those who can achieve 1:40 or more hemagglutination inhibition (HAI) titer after vaccination with any vaccine-strain. Therefore, the rate of potential responders is equivalent to the rate of seroprotection, estimated to be 40-60% among the SMHPs and >80% among the control group in this study. In the SMHPs, even potential responders could only achieve limited HAI titers (1:40-80) even after repeated vaccination. In contrast, the control group showed higher HAI titers compared to the SMHPs for the unchanged vaccine-strains caused by the priming effect. These data suggest that it might be difficult for SMHPs (including potential responders) to achieve the priming effect by the current TIV. Consequently, they cannot obtain a booster effect.

Clinical aspects of hemimegalencephaly by means of a nationwide survey.

We surveyed Japanese patients with hemimegalencephaly by means of a questionnaire. Clinical findings, including intellectual and motor function levels and epileptic symptoms, were investigated. All 44 patients (28 males and 16 females) with hemimegalencephaly were sporadic. Sixteen patients had underlying neurocutaneous syndromes. The number of patients with right-sided hemimegalencephaly (n = 29) was almost twice that of patients with left-sided hemimegalencephaly (n = 15). Forty-one patients had mental retardation and hemiparesis and 14 patients were bedridden. All patients had epileptic seizures, which first appeared within a month in 18 cases and within 6 months in 11 cases. In 42 patients, magnetic resonance imaging revealed both cortical and white-matter abnormalities in the affected hemisphere. Antiepileptic drugs were not very effective. Fifteen patients were surgically treated. Eleven patients underwent functional hemispherectomy, which resulted in fairly good seizure control and improved development. There is a correlation between the onset of epilepsy and the degree of clinical severity of motor deficit and intellectual level. Neither underlying disorders nor laterality of the affected side was related to the degree of clinical severity.