Effective Mechanism for Synthesis of Neurotransmitter Glutamate and its Loading into Synaptic Vesicles.

Glutamate accumulation into synaptic vesicles is a pivotal step in glutamate transmission. This process is achieved by a vesicular glutamate transporter (VGLUT) coupled to v-type proton ATPase. Normal synaptic transmission, in particular during intensive neuronal firing, would demand rapid transmitter re-filling of emptied synaptic vesicles. We have previously shown that isolated synaptic vesicles are capable of synthesizing glutamate from α-ketoglutarate (not from glutamine) by vesicle-bound aspartate aminotransferase for immediate uptake, in addition to ATP required for uptake by vesicle-bound glycolytic enzymes. This suggests that local synthesis of these substances, essential for glutamate transmission, could occur at the synaptic vesicle. Here we provide evidence that synaptosomes (pinched-off nerve terminals) also accumulate α-ketoglutarate-derived glutamate into synaptic vesicles within, at the expense of ATP generated through glycolysis. Glutamine-derived glutamate is also accumulated into synaptic vesicles in synaptosomes. The underlying mechanism is discussed. It is suggested that local synthesis of both glutamate and ATP at the presynaptic synaptic vesicle would represent an efficient mechanism for swift glutamate loading into synaptic vesicles, supporting maintenance of normal synaptic transmission.

Cucumis sativus secretes 4'-ketoriboflavin under iron-deficient conditions.

A new compound in cucumber, Cucumis sativus, nutrient solution that appears under iron-deficient conditions, but not under ordinary culture conditions, has been revealed by HPLC analysis. The chemical structure of this compound was identified using LC-MS and NMR techniques as that of 4'-ketoriboflavin. This is the first report to show that 4'-ketoriboflavin can be found in metabolites from organisms.

Synthesis of telechelic olefin polymers via catalyzed chain growth on multinuclear alkylene zinc compounds.

Multinuclear alkylene zinc (MAZ) compounds of the type EtZn-(R″-Zn)n-Et (R″ = ethyl and propyl branched alkylene groups) were synthesized by a simple one-step procedure in nonpolar hydrocarbon solvents from α,ω-dienes (e.g., 1,7-octadiene or 1,9-decadiene) and diethylzinc using a bis(salicylaldiminato)Zr(IV) complex, [(2-methylcyclohexyl)N═CH(2-O-C6H3-3,5-di-tert-butyl)]2ZrMe2, as a catalyst. The MAZ serves as a divalent reversible chain-transfer agent for olefin polymerization, resulting in telechelic Zn-metalated polyolefins whose molecular weights are controllable over a wide range. The Zn-terminated telechelics serve as a polymer precursor for further reactions and can be converted into a variety of telechelic functionalized polyolefins in high yield.

Synaptic vesicles are capable of synthesizing the VGLUT substrate glutamate from α-ketoglutarate for vesicular loading.

Synaptic vesicle loading of glutamate is a pivotal step in glutamate synaptic transmission. The molecular machinery responsible for this step is comprised of v-type proton-pump ATPase and a vesicular glutamate transporter. Recent evidence indicates that synaptic vesicles are endowed with glycolytic ATP-synthesizing enzymes, providing energy for immediate use by vesicle-bound proton-pump ATPase. In this study, we provide evidence that synaptic vesicles are also capable of synthesizing the vesicular glutamate transporter substrate glutamate, from α-ketoglutarate and l-aspartate (as the amino group donor); glutamate thus produced is taken up into vesicles. We also report a finding that α-ketoglutarate-derived glutamate uptake into synaptic vesicles and aspartate aminotransferase are inhibited by 2,3-pyrazinedicarboxylate. Evidence is given that this is a selective inhibitor for aspartate aminotransferase. These observations provide insight into understanding the nerve endings' mechanism for high efficiency in glutamate transmission. Finding this inhibitor may have implications for further experimentation on the role of α-ketoglutarate-derived glutamate in glutamate transmission.

Enhanced glutamate uptake into synaptic vesicles fueled by vesicle-generated ATP from phosphoenolpyruvate and ADP. Proposed role of a novel enzyme.

Glycolytic ATP synthesis by synaptic vesicles provides an efficient mechanism for fueling vesicular loading of the neurotransmitter glutamate. This is achieved in part by vesicle-bound pyruvate kinase. However, we have found that vesicular glutamate uptake, in the presence of the pyruvate kinase substrates ADP and phosphoenolpyruvate (PEP), substantially exceeds that caused by exogenous ATP. We propose that this much enhanced uptake is in part due to extra ATP produced via a mechanism involving a novel enzyme, PEP-dependent ADP synthase. We discuss implications for this enzyme in energy homeostasis and pathophysiology, as well as in efficient synaptic glutamate transmission.

MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling.

Reactive oxygen species (ROS) mediate abscisic acid (ABA) signaling in guard cells. To dissect guard cell ABA-ROS signaling genetically, a cell type-specific functional genomics approach was used to identify 2 MAPK genes, MPK9 and MPK12, which are preferentially and highly expressed in guard cells. To provide genetic evidence for their function, Arabidopsis single and double TILLING mutants that carry deleterious point mutations in these genes were isolated. RNAi-based gene-silencing plant lines, in which both genes are silenced simultaneously, were generated also. Mutants carrying a mutation in only 1 of these genes did not show any altered phenotype, indicating functional redundancy in these genes. ABA-induced stomatal closure was strongly impaired in 2 independent RNAi lines in which both MPK9 and MPK12 transcripts were significantly silenced. Consistent with this result, mpk9-1/12-1 double mutants showed an enhanced transpirational water loss and ABA- and H(2)O(2)-insensitive stomatal response. Furthermore, ABA and calcium failed to activate anion channels in guard cells of mpk9-1/12-1, indicating that these 2 MPKs act upstream of anion channels in guard cell ABA signaling. An MPK12-YFP fusion construct rescued the ABA-insensitive stomatal response phenotype of mpk9-1/12-1, demonstrating that the phenotype was caused by the mutations. The MPK12 protein is localized in the cytosol and the nucleus, and ABA and H(2)O(2) treatments enhance the protein kinase activity of MPK12. Together, these results provide genetic evidence that MPK9 and MPK12 function downstream of ROS to regulate guard cell ABA signaling positively.

[A case of ruptured cerebral aneurysm associated with coarctation of the aorta].

A 15-year-old boy with subarachnoid hemorrhage was planned for emergency cerebral aneurysm clipping under general anesthesia. He had different blood pressure between the upper limbs and we found coarctation of the aorta at left subclavian artery bifurcation in the preoperative angiography. To prevent re-rupture of cerebral aneurysm and ischemia of abdominal organs, we monitored arterial blood pressure in bilateral radial arteries and non-invasive blood pressure in the left thigh, and his blood pressure was maintained within 120-150 mmHg of systolic pressure in the right radial artery and 50-70 mmHg of mean arterial pressure in the left radial artery and the left thigh during general anesthesia. The preoperative period elapsed uneventfully and the patient was planned for repair of coarctation of the aorta after discharge.

Synechocystis ferredoxin-NADP(+) oxidoreductase is capable of functioning as ferric reductase and of driving the Fenton reaction in the absence or presence of free flavin.

We purified free flavin-independent NADPH oxidoreductase from Synechocystis sp. PCC6803 based on NADPH oxidation activity elicited during reduction of t-butyl hydroperoxide in the presence of Fe(III)-EDTA. The N-terminal sequencing of the purified enzyme revealed it to be ferredoxin-NADP(+) oxidoreductase (FNR( S )). The purified enzyme reacted with cytochrome c, ferricyanide and 2,6-dichloroindophenol (DCIP). The substrate specificity of the enzyme was similar to the known FNR. DNA degradation occurring in the presence of NADPH, Fe(III)-EDTA and hydrogen peroxide was potently enhanced by the purified enzyme, indicating that Synechocystis FNR( S ) may drive the Fenton reaction. The Fenton reaction by Synechocystis FNR( S ) in the presence of natural chelate iron compounds tended to be considerably lower than that in the presence of synthetic chelate iron compounds. The Synechocystis FNR( S ) is considered to reduce ferric iron to ferrous iron when it evokes the Fenton reaction. Although Synechocystis FNR( S ) was able to reduce iron compounds in the absence of free flavin, the ferric reduction by the enzyme was enhanced by the addition of free flavin. The enhancement was detected not only in the presence of natural chelate iron compounds but also synthetic chelate iron compounds.

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction.

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP(+) reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k(cat)/K(m) value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k(cat)/K(m) value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.

Chlorella vulgaris aldehyde reductase is capable of functioning as ferric reductase and of driving the fenton reaction in the presence of free flavin.

The free flavin-dependent Fenton reaction was detected in cell-free extracts of Chlorella. The corresponding enzyme was purified to homogeneity, and its N-terminal sequence was highly homologous to those of aldo-keto reductase family enzymes. The purified enzyme displayed aldehyde reductase activity in the presence of NADPH. Additionally, it showed ferric reductase activity and drove the Fenton reaction in the presence of free FAD and NADH.

Free flavins accelerate release of ferrous iron from iron storage proteins by both free flavin-dependent and -independent ferric reductases in Escherichia coli.

Ferredoxin NADP+ oxidoreductase (Fpr) and oxygen-insensitive NAD(P)H nitroreductase (NfnB) are purified from Escherichia coli JM109 (E. coli JM109) as a predominant free flavin-independent ferric reductase. In the present study, we prepared natural iron storage proteins, E. coli ferritin A (FtnA) and bacterioferritin (Bfr), to show the effective ferrous iron release from these proteins by Fpr and NfnB in the presence of free flavins. Fpr and NfnB showed flavin reductase activity for flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and riboflavin, and their ferrous iron release activities were positively associated with the catalytic efficiencies (kcat/Km) for individual flavins. The ferrous iron release activity of E. coli cell-free extracts was affected by flavin reductase activity of the extracts. The Butyl TOYOPEARL column chromatography of the extracts, on the basis of NAD(P)H-dependent flavin reductase activity, resulted in the separation of six active fractions containing Fpr, NfnB, NAD(P)H-quinone oxidoreductase (QOR), flavin reductase (Fre) or alkyl hydroperoxide reductase subunit F (AhpF) as major components. Like Fpr and NfnB, recombinant QOR, Fre, and AhpF showed flavin reductase activity and ferrous iron release activity in the presence of free flavins, indicating an association of flavin reductase activity with ferrous iron releasing activity. Taken together, both free flavin-dependent and free flavin-independent ferric reductases in E. coli require free flavins to mediate an electron transfer from NAD(P)H to ferric iron in the iron storage proteins for the effective ferrous iron release.

Intracellular free flavin and its associated enzymes participate in oxygen and iron metabolism in Amphibacillus xylanus lacking a respiratory chain.

Amphibacillus xylanus is a recently identified bacterium which grows well under both aerobic and anaerobic conditions and may prove useful for biomass utilization. Amphibacillus xylanus, despite lacking a respiratory chain, consumes oxygen at a similar rate to Escherichia coli (130-140 µmol oxygen·min-1·g-1 dry cells at 37 °C), suggesting that it has an alternative system that uses a large amount of oxygen. Amphibacillus xylanus NADH oxidase (Nox) was previously reported to rapidly reduce molecular oxygen content in the presence of exogenously added free flavin. Here, we established a quantitative method for determining the intracellular concentrations of free flavins in A. xylanus, involving French pressure and ultrafiltration membranes. The intracellular concentrations of flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and riboflavin were estimated to be approximately 8, 3, and 1 µm, respectively. In the presence of FAD, the predominant free flavin species, two flavoproteins Nox (which binds FAD) and NAD(P)H oxidoreductase (Npo, which binds FMN), were identified as central free flavin-associated enzymes in the oxygen metabolic pathway. Under 8 µm free FAD, the catalytic efficiency (kcat/Km) of recombinant Nox and Npo for oxygen increased by approximately fivefold and ninefold, respectively. Nox and Npo levels were increased, and intracellular FAD formation was stimulated following exposure of A. xylanus to oxygen. This suggests that these two enzymes and free FAD contribute to effective oxygen detoxification and NAD(P)+ regeneration to maintain redox balance during aerobic growth. Furthermore, A. xylanus required iron to grow aerobically. We also discuss the contribution of the free flavin-associated system to the process of iron utilization.

Synechocystis DrgA protein functioning as nitroreductase and ferric reductase is capable of catalyzing the Fenton reaction.

In order to identify an enzyme capable of Fenton reaction in Synechocystis, we purified an enzyme catalyzing one-electron reduction of t-butyl hydroperoxide in the presence of FAD and Fe(III)-EDTA. The enzyme was a 26 kDa protein, and its N-terminal amino acid sequencing revealed it to be DrgA protein previously reported as quinone reductase [Matsuo M, Endo T and Asada K (1998) Plant Cell Physiol39, 751-755]. The DrgA protein exhibited potent quinone reductase activity and, furthermore, we newly found that it contained FMN and highly catalyzed nitroreductase, flavin reductase and ferric reductase activities. This is the first demonstration of nitroreductase activity of DrgA protein previously identified by a drgA mutant phenotype. DrgA protein strongly catalyzed the Fenton reaction in the presence of synthetic chelate compounds, but did so poorly in the presence of natural chelate compounds. Its ferric reductase activity was observed with both natural and synthetic chelate compounds with a better efficiency with the latter. In addition to small molecular-weight chemical chelators, an iron transporter protein, transferrin, and an iron storage protein, ferritin, turned out to be substrates of the DrgA protein, suggesting it might play a role in iron metabolism under physiological conditions and possibly catalyze the Fenton reaction under hyper-reductive conditions in this microorganism.

[A case of sarcoidosis who showed rapid swelling of mediastinal and abdominal lymph nodes].

A 49-year-old woman noticed hoarseness and facial palsey three months prior to her visit to our hospital. Chest radiograph and CT scanning revealed bilateral mediastinal and hilar lymphadenopathy. Bronchofiberoptic biopsy showed sarcoidosis. Her symptoms improved under no treatment. However, she showed rapid increase of mediastinal and abdominal lymph nodes swelling and elevation of serum level of sIL-2R during observation. Therefore, we must discriminate sarcoidosis-lymphoma syndrome from exacerbation of sarcoidosis. Mediastinoscopic biopsy was conducted for diagnosis, and it revealed exacerbation of sarcoidosis. We reported this rare case of rapid increase of mediastinal and abdominal lymph node swelling due to sarcoidosis.

Low-frequency and very low-intensity ultrasound decreases blood pressure in hypertensive subjects with type 2 diabetes.

BACKGROUND: Despite lifestyle interventions and various types of anti-hypertension agents, hypertension remains difficult to control in some patients with type 2 diabetes. As a noninvasive device-based approach for the treatment of clinic hypertension, we examined the effects of low-frequency and low-intensity ultrasound (500 or 800kHz, 25mW/cm(2)) applied to the forearm on blood pressure (BP) and pulse rate in Japanese subjects with type 2 diabetes and hypertension. METHODS: We examined the effects of low-frequency and low-intensity ultrasound (500 or 800kHz, 25mW/cm(2)) applied to the forearm on BP, pulse rate, and pulse pressure in 212 Japanese subjects (82 men and 130 women; mean age±SE, 65±1years) with type 2 diabetes and hypertension (systolic BP>140mmHg). The subjects were treated with anti-hypertension agents. RESULTS: Systolic and diastolic BP, pulse rate, pulse pressure in the 800-kHz ultrasound treatment group were significantly lower than the baseline values in hypertensive subjects with type 2 diabetes, and lower than those of placebo controls. In addition, systolic and diastolic BP, pulse rate, and pulse pressure in the 500-kHz ultrasound treatment group were significantly lower than the baseline values in hypertensive subjects with type 2 diabetes, and systolic BP, pulse rate, and pulse pressure were significantly lower than those of placebo controls. CONCLUSIONS: Low-frequency (800kHz or 500kHz) and low-intensity (25mW/cm(2)) ultrasound irradiation to the forearm might have potential usefulness as a therapeutic application for clinic hypertension in subjects with type 2 diabetes.

Isolation of lactic acid bacteria exhibiting high scavenging activity for environmental hydrogen peroxide from fermented foods and its two scavenging enzymes for hydrogen peroxide.

To obtain lactic acid bacteria that scavenge environmental hydrogen peroxide, we developed a specialized enrichment medium and successfully isolated Pediococcus pentosaceus Be1 strain from a fermented food. This strain showed vigorous environmental hydrogen peroxide scavenging activity over a wide range of hydrogen peroxide concentrations. High Mn-catalase and NADH peroxidase activities were found in the cell-free extract of the P. pentosaceus Be1 strain, and these two hydrogen peroxide scavenging enzymes were purified from the cell-free extract of the strain. Mn-catalase has been purified from several microorganisms by several researchers, and the NADH peroxidase was first purified from the original strain in this report. After cloning the genes of the Mn-catalase and the NADH peroxidase, the deduced amino acid sequences were compared with those of known related enzymes.

Border control--a membrane-linked interactome of Arabidopsis.

Cellular membranes act as signaling platforms and control solute transport. Membrane receptors, transporters, and enzymes communicate with intracellular processes through protein-protein interactions. Using a split-ubiquitin yeast two-hybrid screen that covers a test-space of 6.4 × 10(6) pairs, we identified 12,102 membrane/signaling protein interactions from Arabidopsis. Besides confirmation of expected interactions such as heterotrimeric G protein subunit interactions and aquaporin oligomerization, >99% of the interactions were previously unknown. Interactions were confirmed at a rate of 32% in orthogonal in planta split-green flourescent protein interaction assays, which was statistically indistinguishable from the confirmation rate for known interactions collected from literature (38%). Regulatory associations in membrane protein trafficking, turnover, and phosphorylation include regulation of potassium channel activity through abscisic acid signaling, transporter activity by a WNK kinase, and a brassinolide receptor kinase by trafficking-related proteins. These examples underscore the utility of the membrane/signaling protein interaction network for gene discovery and hypothesis generation in plants and other organisms.

A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2.

Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein-protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases (RLKs), 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions, and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 386) pPPIs between 179 proteins, yielding a scale-free network (r(2) = 0.863). Eighty of 142 transmembrane RLKs tested positive, identifying 3 homomers, 63 heteromers, and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G-protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

Response of the microaerophilic Bifidobacterium species, B. boum and B. thermophilum, to oxygen.

We investigated the effects of O2 on Bifidobacterium species using liquid shaking cultures under various O2 concentrations. Although most of the Bifidobacterium species we selected showed O2 sensitivity, two species, B. boum and B. thermophilum, demonstrated microaerophilic profiles. The growth of B. bifidum and B. longum was inhibited under high-O2 conditions accompanied by the accumulation of H2O2 in the medium, and growth was restored by adding catalase to the medium. B. boum and B. thermophilum grew well even under 20% O2 conditions without H2O2 accumulation, and growth was stimulated compared to anoxic growth. H2O-forming NADH oxidase activities were detected dominantly in cell extracts of B. boum and B. thermophilum under acidic reaction conditions (pH 5.0 to 6.0).