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1.
Mucosal Immunol ; 10(5): 1351-1360, 2017 09.
Article in English | MEDLINE | ID: mdl-28120848

ABSTRACT

Obesity is associated with multiple comorbidities such as cardiovascular diseases and has a huge economic impact on the health-care system. However, the treatment of obesity remains insufficient in terms of efficacy, tolerability, and safety. Here we created a nasal vaccine against obesity for the first time. To avoid the injectable administration-caused pain and skin-related adverse event, we focused on the intranasal route of antigen delivery. We developed a vaccine antigen (ghrelin-PspA (pneumococcal surface protein A)), which is a recombinant fusion protein incorporating ghrelin, a hormone that stimulates food intake and decreases energy expenditure, and PspA, a candidate of pneumococcal vaccine as a carrier protein. Ghrelin-PspA antigen was mixed with cyclic di-GMP adjuvant to enhance the immunogenicity and incorporated within a nanometer-sized hydrogel for the effective antigen delivery. Intranasal immunization with ghrelin-PspA vaccine elicited serum immunoglobulin G antibodies against ghrelin and attenuated body weight gain in diet-induced obesity mice. This obesity-attenuating effect was caused by a decrease in fat accumulation and an increase in energy expenditure that was partially due to an increase in the expression of mitochondrial uncoupling protein 1 in brown adipose tissue. The development of this nasal vaccine provides a new strategy for the prevention and treatment of obesity.


Subject(s)
Bacterial Proteins/genetics , Gels/administration & dosage , Ghrelin/genetics , Nanoparticles/administration & dosage , Obesity/immunology , Recombinant Fusion Proteins/administration & dosage , Vaccines/immunology , Administration, Intranasal , Animals , Antibody Formation , Body Weight , Diet Therapy , Disease Models, Animal , Ghrelin/immunology , Humans , Immunoglobulin G/blood , Male , Mice , Mice, Inbred C57BL
3.
Dis Esophagus ; 27(8): 737-43, 2014.
Article in English | MEDLINE | ID: mdl-24917486

ABSTRACT

The survival benefit of second-line chemotherapy with docetaxel in platinum-refractory patients with advanced esophageal cancer (AEC) remains unclear. A retrospective analysis of AEC patients with Eastern Cooperative Oncology Group performance status (PS)≤2 was performed, and major organ functions were preserved, who determined to receive docetaxel or best supportive care (BSC) alone after failure of platinum-based chemotherapy. The post-progression survival (PPS), defined as survival time after disease progression following platinum-based chemotherapy, was analyzed by multivariate Cox regression analysis using factors identified as significant in univariate analysis of various 20 characteristics (age, sex, PS, primary tumor location, etc) including Glasgow prognostic score (GPS), which is a well-known prognostic factor in many malignant tumors. Sixty-six and 45 patients were determined to receive docetaxel and BSC between January 2007 and December 2011, respectively. The median PPS was 5.4 months (95% confidence interval [CI] 4.8-6.0) in the docetaxel group and 3.3 months (95% CI 2.5-4.0) in the BSC group (hazard ratio [HR] 0.56, 95% CI 0.38-0.84, P=0.005). Univariate analysis revealed six significant factors: treatment, PS, GPS, number of metastatic organs, liver metastasis, and bone metastasis. Multivariate analysis including these significant factors revealed three independent prognostic factors: docetaxel treatment (HR 0.62, 95% CI 0.39-0.99, P=0.043), better GPS (HR 0.61, 95% CI 0.46-0.81, P=0.001), and no bone metastasis (HR 0.31, 95% CI 0.15-0.68, P=0.003). There was a trend for PPS in favor of the docetaxel group compared with patients who refused docetaxel treatment in the BSC group (adjusted HR 0.61, 95% CI 0.29-1.29, P=0.20). Docetaxel treatment may have prolonged survival in platinum-refractory patients with AEC.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/mortality , Platinum/therapeutic use , Taxoids/therapeutic use , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Disease Progression , Docetaxel , Female , Humans , Male , Middle Aged , Platinum/administration & dosage , Prognosis , Retrospective Studies , Survival Analysis , Taxoids/administration & dosage
4.
Clin Pharmacol Ther ; 88(6): 809-13, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20980997

ABSTRACT

Despite the outstanding results generally obtained with imatinib mesylate (IM) in the treatment of chronic myeloid leukemia (CML), some patients show a poor molecular response. To evaluate the relationship between steady-state trough plasma IM concentration (IM-C(min)) and clinical response in CML patients, we integrated data from six independent Japanese studies. Among 254 CML patients, the mean IM-C(min) was 1,010.5 ng/ml. Importantly, IM-C(min) was significantly higher in patients who achieved a major molecular response (MMR) than in those who did not (P = 0.002). Multivariate analysis showed that an MMR was associated with both age (odds ratio (OR) = 0.97 (0.958-0.995); P = 0.0153) and with IM-C(min) (OR = 1.0008 (1.0003-1.0015); P = 0.0044). Given that patients with IM-C(min) values >1,002 ng/ml had a higher probability of achieving an MMR in our large cohort (P = 0.0120), the data suggest that monitoring of IM levels in plasma may improve the efficacy of IM therapy for CML patients.


Subject(s)
Asian People , Leukemia, Myeloid, Chronic-Phase/drug therapy , Leukemia, Myeloid, Chronic-Phase/metabolism , Piperazines/pharmacokinetics , Piperazines/therapeutic use , Pyrimidines/pharmacokinetics , Pyrimidines/therapeutic use , Adolescent , Adult , Aged , Aged, 80 and over , Benzamides , Cohort Studies , Female , Humans , Imatinib Mesylate , Male , Middle Aged , Treatment Outcome , Young Adult
5.
Neuroscience ; 147(3): 639-51, 2007 Jul 13.
Article in English | MEDLINE | ID: mdl-17560726

ABSTRACT

We have assessed amyloid beta protein (Abeta)-induced neurotoxicity, with and without added tunicamycin (TM), an inhibitor of N-glycosylation in the endoplasmic reticulum (ER), in rat organotypic hippocampal slice cultures (OHCs). In the rat OHCs cultured for 3 weeks, there was little neurotoxicity after treatment with Abeta(25-35) (25 microM) alone for 48 h. However, with TM alone, concentration-dependent neuronal death was observed at concentrations between 20 and 80 microg/mL. When amyloid-beta protein was combined with tunicamycin (Abeta+TM), cell death was more acute than with TM alone. Western blot analysis revealed that calpain activity and the active forms of caspase-12 and caspase-3 was increased after exposure to Abeta+TM as compared with exposure to TM alone. In contrast, the levels of glucose regulated protein (GRP)94, GRP78 and C/EBP homologous protein (CHOP) were not changed in the presence of Abeta. Abeta potentiation of TM neurotoxicity was reversibly blocked by S-allyl-L-cysteine (SAC), an organosulfur compound purified from aged garlic extract, and the L-type calcium channel blocker, nifedipine, in a restricted neuronal area of the OHCs. Simultaneously applied SAC also reversed the increases in calpain activity and the active forms of caspase-12 and caspase-3 by Abeta+TM with no change in the increased levels of GRP94, GRP78 and CHOP. These data indicate that Abeta facilitates the calpain-caspase-12-caspase-3 pathway, thus potentiating TM-induced neuronal death in the hippocampus.


Subject(s)
Amyloid beta-Peptides/pharmacology , Antiviral Agents/pharmacology , Hippocampus/cytology , Neurons/drug effects , Peptide Fragments/pharmacology , Tunicamycin/pharmacology , Animals , Animals, Newborn , Cell Count , Cell Death/drug effects , Dose-Response Relationship, Drug , Drug Synergism , Gene Expression Regulation/drug effects , Molecular Chaperones/metabolism , Organ Culture Techniques , Rats , Rats, Wistar
6.
J Neural Transm (Vienna) ; 114(9): 1135-42, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17533511

ABSTRACT

Microdialysis technique was used to study the effects of the locally applied alpha adrenoceptor agonist phenylephrine and antagonist phentolamine on the basal noradrenaline efflux as well as on the noradrenaline uptake inhibitor desipramine-elicited noradrenaline efflux in the nucleus accumbens (NAc) of freely moving rats. Tetrodotoxin reduced basal noradrenaline efflux by 72%, whereas desipramine increased it by 204%. Phenylephrine reduced the basal noradrenaline efflux by 32% and phentolamine blocked this effect. Phentolamine elevated the basal noradrenaline efflux by 150% and phenylephrine counteracted this effect. The desipramine-elicited noradrenaline efflux was not affected by phenylephrine, but enhanced by phentolamine. Desipramine counteracted the effects of phenylephrine and potentiated those of phentolamine. These results indicate that the accumbal noradrenaline efflux is under inhibitory control of alpha adrenoceptors that are suggested to be presynaptically located on adrenergic nerve terminals in the NAc. Furthermore, this study suggests that the conformational state of alpha adrenoceptors varies across the available amount of noradrenaline. The clinical impact of these data is discussed.


Subject(s)
Microdialysis , Norepinephrine/metabolism , Nucleus Accumbens/physiology , Receptors, Adrenergic, alpha/physiology , Adrenergic Agonists/pharmacology , Adrenergic Antagonists/pharmacology , Animals , Exploratory Behavior/drug effects , Exploratory Behavior/physiology , Male , Microdialysis/methods , Nucleus Accumbens/drug effects , Rats , Rats, Sprague-Dawley
7.
J Neural Transm (Vienna) ; 111(1): 37-45, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14714214

ABSTRACT

Ginsenosides Rb1 and Rg1 are the main active ingredients of Panax ginseng C.A. Meyer (Araliaceae). They appear to exert protection against ischaemia and anoxic damage in animal models, suggesting an antioxidative and cytoprotective role. In our study, primary cultures from embryonic mouse mesencephalon are applied to examine the effects of these two ginsenosides on neuritic growth of dopaminergic cells and their survival affected by 1-methyl-4-phenylpyridinium-iodide (MPP(+)). Ginsenoside Rb1 (at 10 microM) enhanced the survival of dopaminergic neurons by 19% compared to untreated control. MPP(+) (at 1 microM) significantly reduced the number of dopaminergic neurons and severely affected neuronal processes. Both ginsenosides counteracted these degenerations and significantly protected lengths and numbers of neurites of TH(+) cells. Both compounds however could not prevent the cell loss caused by MPP(+). Our study thus indicates partial neurotrophic and neuroprotective actions of ginsenosides Rb1 and Rg1 in dopaminergic cell culture.


Subject(s)
1-Methyl-4-phenylpyridinium/toxicity , Dopamine/metabolism , Ginsenosides/pharmacology , Mesencephalon/drug effects , Animals , Cell Division/drug effects , Cell Division/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Female , Mesencephalon/metabolism , Mesencephalon/pathology , Mice , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Pregnancy
8.
Neuroscience ; 122(4): 885-95, 2003.
Article in English | MEDLINE | ID: mdl-14643758

ABSTRACT

S-allyl-L-cysteine (SAC), one of the organosulfur compounds found in aged garlic extract, has been shown to possess various biological effects including neurotrophic activity. In our previous experiments, we found that SAC could protect against amyloid beta-protein (Abeta)- and tunicamycin-induced cell death in differentiated PC12 cells. In the study described here, we characterized the neuronal death induced by Abeta, 4-hydroxynonenal (HNE), tunicamycin, and trophic factor deprivation, and investigated whether and how SAC could prevent this in cultured rat hippocampal neurons. Treatment with SAC protected these cells against Abeta- and tunicamycin-induced neuronal death, which is mediated predominantly through caspase-12-dependent pathway in a concentration-dependent manner. In contrast, it afforded no protection against HNE- and trophic factor-deprivation-induced cell death, which has been shown to be mediated by caspase-3-dependent pathway. SAC also attenuated the Abeta-induced increase of intracellular reactive oxygen species in hippocampal neurons. SAC had no effect on Abeta-induced cell death in cultured cerebellar granule neurons, which was prevented by a caspase-3 inhibitor. These results suggest that SAC could protect against the neuronal cell death that is triggered by ER dysfunction in the hippocampus, and that it has no effect on neuronal cell death that is dependent upon the caspase-3 mediated pathway.


Subject(s)
Amyloid beta-Peptides/toxicity , Cysteine/analogs & derivatives , Cysteine/pharmacology , Hippocampus/drug effects , Neurons/drug effects , Tunicamycin/toxicity , Animals , Cell Death/drug effects , Cell Death/physiology , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Hippocampus/physiology , Neurons/physiology , Neuroprotective Agents/pharmacology , Rats , Rats, Wistar
9.
Biosci Biotechnol Biochem ; 65(8): 1736-40, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11577711

ABSTRACT

A novel method for synthesizing CMP-NeuAc was established. We first confirmed that the putative neuA gene of Haemophilus influenzae, identified by its whole genome sequence project, indeed encodes CMP-NeuAc synthetase (EC 2.7.7.43). The enzyme requires CTP as a cytidylyl donor for cytidylylation of NeuAc. The enzyme was coupled with an enzymatic CTP-generating system from CMP and inorganic polyphosphate as a sole phospho-donor driven by the combination of polyphosphate kinase and CMP kinase, where phosphorylation of CMP is done by the combined activity expressed by both enzymes, and subsequent phosphorylation of CDP by polyphosphate kinase itself occurred efficiently. When CMP-NeuAc synthetase of H. influenzae, polyphosphate kinase, and CMP kinase were added to the reaction mixture containing equimolar concentrations (15 mM) of CMP and NeuAc, and polyphosphate (150 mM in terms of phosphate), CMP-NeuAc was synthesized up to 10 mM in 67% yield.


Subject(s)
Cytidine Monophosphate/analogs & derivatives , Cytidine Monophosphate/chemical synthesis , N-Acylneuraminate Cytidylyltransferase/chemical synthesis , Sialic Acids/chemical synthesis , Cytidine Triphosphate/chemical synthesis , Escherichia coli/genetics , Escherichia coli/metabolism , Haemophilus influenzae/enzymology , Haemophilus influenzae/genetics , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Phosphotransferases (Phosphate Group Acceptor)/genetics , Plasmids/genetics , Recombinant Proteins/chemical synthesis
10.
J Neurosci ; 21(16): 6069-76, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11487630

ABSTRACT

Oxidative stress is thought to be the cause of nerve cell death in many CNS pathologies, including ischemia, trauma, and neurodegenerative disease. Glutamate kills nerve cells that lack ionotropic glutamate receptors via the inhibition of the cystine-glutamate antiporter x(c)(-), resulting in the inhibition of cystine uptake, the loss of glutathione, and the initiation of an oxidative stress cell death pathway. A number of catecholamines were found to block this pathway. Specifically, dopamine and related ligands inhibit glutamate-induced cell death in both clonal nerve cell lines and rat cortical neurons. The protective effects of dopamine, apomorphine, and apocodeine, but not epinephrine and norepinephrine, are antagonized by dopamine D4 antagonists. A dopamine D4 agonist also protects, and this protective effect is inhibited by U101958, a dopamine D4 antagonist. Although the protective effects of some of the catecholamines are correlated with their antioxidant activities, there is no correlation between the protective and antioxidant activities of several other ligands. Normally, glutamate causes an increase in reactive oxygen species (ROS) and intracellular Ca(2+). Apomorphine partially inhibits glutamate-induced ROS production and blocks the opening of cGMP-operated Ca(2+) channels that lead to Ca(2+) elevation in the late part of the cell death pathway. These data suggest that the protective effects of apomorphine on oxidative stress-induced cell death are, at least in part, mediated by dopamine D4 receptors via the regulation of cGMP-operated Ca(2+) channels.


Subject(s)
Apomorphine/analogs & derivatives , Cell Death/physiology , Receptors, Dopamine D2/metabolism , Animals , Antioxidants/metabolism , Antioxidants/pharmacology , Apomorphine/pharmacology , Binding, Competitive/drug effects , Calcium/metabolism , Cell Death/drug effects , Cell Line , Cyclic GMP/metabolism , Dopamine/metabolism , Dopamine/pharmacology , Dopamine Agonists/pharmacology , Dopamine Antagonists/pharmacology , Dopamine D2 Receptor Antagonists , Epinephrine/pharmacology , Glutamic Acid/metabolism , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/metabolism , Mice , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Neuroprotective Agents/pharmacology , Norepinephrine/pharmacology , Oxidative Stress/drug effects , Rats , Reactive Oxygen Species/metabolism , Receptors, Dopamine D2/agonists , Receptors, Dopamine D4
11.
Biochem Biophys Res Commun ; 281(3): 821-6, 2001 Mar 02.
Article in English | MEDLINE | ID: mdl-11237733

ABSTRACT

In Pseudomonas aeruginosa PAO1, we have found massive polyphosphate:AMP phosphotransferase activity and polyphosphate:ADP phosphotransferase activity known as the reverse catalytic activity of polyphosphate kinase which participates in polyphosphate synthesis in the bacterium. Biochemical analysis using the partially purified polyphosphate:ADP phosphotransferase has revealed that it is independent of polyphosphate kinase and can function as polyphosphate-dependent nucleoside diphosphate kinase which most prefers GDP to the other three nucleoside diphosphates as a phospho-acceptor. It has been also demonstrated that polyphosphate:AMP phosphotransferase activity marked in the bacterium mainly originates from the combined action of the polyphosphate:ADP phosphotransferase described above and adenylate kinase. Both of the polyphosphate-utilizing activities require short polyP as a phospho-donor whose chain length is <75.


Subject(s)
Phosphotransferases (Phosphate Group Acceptor)/metabolism , Pseudomonas aeruginosa/enzymology , Base Sequence , Blotting, Western , DNA Primers
12.
Free Radic Biol Med ; 30(4): 433-46, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11182299

ABSTRACT

Flavonoids are a family of antioxidants found in fruits and vegetables as well as in popular beverages such as red wine and tea. Although the physiological benefits of flavonoids have been largely attributed to their antioxidant properties in plasma, flavonoids may also protect cells from various insults. Nerve cell death from oxidative stress has been implicated in a variety of pathologies, including stroke, trauma, and diseases such as Alzheimer's and Parkinson's. To determine the potential protective mechanisms of flavonoids in cell death, the mouse hippocampal cell line HT-22, a model system for oxidative stress, was used. In this system, exogenous glutamate inhibits cystine uptake and depletes intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species (ROS) and an increase in Ca(2+) influx, which ultimately causes neuronal death. Many, but not all, flavonoids protect HT-22 cells and rat primary neurons from glutamate toxicity as well as from five other oxidative injuries. Three structural requirements of flavonoids for protection from glutamate are the hydroxylated C3, an unsaturated C ring, and hydrophobicity. We also found three distinct mechanisms of protection. These include increasing intracellular GSH, directly lowering levels of ROS, and preventing the influx of Ca(2+) despite high levels of ROS. These data show that the mechanism of protection from oxidative insults by flavonoids is highly specific for each compound.


Subject(s)
Antioxidants/pharmacology , Flavonoids/pharmacology , Neurons/drug effects , Oxidative Stress/drug effects , Vitamin E/analogs & derivatives , Vitamin E/pharmacology , Animals , Calcium/metabolism , Cell Death/drug effects , Cell Line , Flavonoids/antagonists & inhibitors , Free Radicals , Glutamic Acid/toxicity , Glutathione/metabolism , Hippocampus , Mice , Neurons/metabolism , Reactive Oxygen Species , Vitamin E/antagonists & inhibitors
13.
Neurosci Lett ; 297(3): 207-10, 2001 Jan 19.
Article in English | MEDLINE | ID: mdl-11137764

ABSTRACT

To characterize seizure-associated increases in cerebral cortical and thalamic cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities in lethargic (lh/lh) mice, a genetic model of absence seizures, we examined the effects of ethosuximide and CGP 46381 on these DNA-binding activities. Repeated administration (twice a day for 5 days) of ethosuximide (200 mg/kg) or CGP 46381 (60 mg/kg) attenuated both seizure behavior and the increased DNA-binding activities, and was more effective than a single administration of these drugs. These treatments did not affect either normal behavior or basal DNA-binding activities in non-epileptic control (+/+) mice. Gel supershift assays revealed that the increased CRE-binding activity was attributable to activation of the binding activity of CREB, and that the c-Fos-c-Jun complex was a component of the increased AP-1 DNA-binding activity.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Epilepsy, Absence/metabolism , Ethosuximide/administration & dosage , Phosphinic Acids/administration & dosage , Transcription Factor AP-1/metabolism , Animals , Anticonvulsants/administration & dosage , Cerebellum/metabolism , Cerebral Cortex/metabolism , DNA/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Administration Schedule , Epilepsy, Absence/drug therapy , GABA Antagonists/administration & dosage , GABA-B Receptor Antagonists , Hippocampus/metabolism , Mice , Sleep Stages/drug effects , Thalamus/metabolism , Treatment Outcome
14.
J Biosci Bioeng ; 91(6): 557-63, 2001.
Article in English | MEDLINE | ID: mdl-16233039

ABSTRACT

Polyphosphate-AMP phosphotransferase (PAP) and polyphosphate kinase (PPK) were used for designing a novel ATP regeneration system, named the PAP-PPK ATP regeneration system. PAP is an enzyme that catalyzes the phospho-conversion of AMP to ADP, and PPK catalyzes ATP formation from ADP. Both enzymes use inorganic polyphosphate [poly(P)] as a phosphate donor. In the PAP-PPK ATP regeneration system, ATP was continuously synthesized from AMP by the coupling reaction of PAP and PPK using poly(P). Poly(P) is a cheap material compared to acetyl phosphate, phosphoenol pyruvate and creatine phosphate, which are phosphate donors used for conventional ATP regeneration systems. To achieve efficient synthesis of ATP from AMP, an excessive amount of poly(P) should be added to the reaction solution because both PAP and PPK consume poly(P) as a phosphate donor. Using this ATP generation reaction, we constructed the PAP-PPK ATP regeneration system with acetyl-CoA synthase and succeeded in synthesizing acetyl-CoA from CoA, acetate and AMP. Since too much poly(P) may chelate MG2+ and inhibit enzyme activity, the Mg2+ concentration was optimized to 24 mM in the presence of 30 mM poly(P) in the reaction. In this reaction, ATP was regenerated 39.8 times from AMP, and 99.5% of CoA was converted to acetyl-CoA. In addition, since the PAP-PPK ATP regeneration system can regenerate GTP from GMP, it could also be used as a GTP regeneration system.

15.
Proc Natl Acad Sci U S A ; 97(26): 14168-71, 2000 Dec 19.
Article in English | MEDLINE | ID: mdl-11106368

ABSTRACT

Polyphosphate kinase (PPK), responsible for the processive synthesis of inorganic polyphosphate (polyP) from ATP in Escherichia coli, can transfer in reverse the terminal phosphate residue of polyP to ADP to yield ATP. PolyP also serves as a donor in a polyP:AMP phosphotransferase (PAP) activity observed in extracts of Acinetobacter johnsonii and Myxococcus xanthus. We have found that overexpression of the gene encoding PPK results in a large enhancement of PAP activity in E. coli. The PAP activity requires both PPK and adenylate kinase in equimolar amounts. PPK and adenylate kinase form a complex in the presence of polyphosphate. We discuss a phosphotransfer mechanism that involves both enzymes and enables polyP to be a phospho-donor to AMP.


Subject(s)
Adenylate Kinase/metabolism , Escherichia coli/enzymology , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Catalysis , Phosphates/metabolism , Phosphotransferases (Phosphate Group Acceptor)/biosynthesis
16.
Biosci Biotechnol Biochem ; 64(2): 386-92, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10737197

ABSTRACT

Uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) has been synthesized by a yeast-based method from 5'-UMP and glucosamine, in which yeast cells catalyze the conversion of 5'-UMP to 5'-UTP and provide enzymes involved in UDP-GlcNAc synthesis using 5'-UTP and glucosamine as substrates. However, this conventional method is not suitable for practical production of UDP-GlcNAc because of the low yield of the product. We found that the yqgR gene product of Bacillus subtilis, which has been identified as a glucokinase, can catalyze the phosphorylation of N-acetylglucosamine (GlcNAc) to give GlcNAc-6-phosphate, an intermediate of UDP-GlcNAc biosynthesis. The addition of the yqgR gene product to the yeast-based reaction system enabled us to synthesize UDP-GlcNAc using GlcNAc in place of glucosamine. The addition of two enzymes, GlcNAc-phosphate mutase and UDP-GlcNAc pyrophosphorylase, increased the yield of UDP-GlcNAc. Using this novel method, UDP-GlcNAc was produced at an amount of 78 mM from 100 mM 5'-UMP and 100 mM GlcNAc.


Subject(s)
Uridine Diphosphate N-Acetylglucosamine/genetics , Amino Acid Sequence , Bacillus subtilis/enzymology , Base Sequence , Cloning, Molecular , DNA Primers , DNA, Recombinant , Glucokinase/metabolism , Molecular Sequence Data , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid
17.
Biochemistry (Mosc) ; 65(3): 315-23, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10739474

ABSTRACT

In this review, we discuss the following two subjects: 1) the physiological function of polyphosphate (poly(P)) as a regulatory factor for gene expression in Escherichia coli, and 2) novel functions of E. coli polyphosphate kinase (PPK) and their applications. With regard to the first subject, it has been shown that E. coli cells in which yeast exopolyphosphatase (poly(P)ase), PPX1, was overproduced reduced resistance to H2O2 and heat shock as did a mutant whose polyphosphate kinase gene is disrupted. Sensitivity to H2O2 and heat shock evinced by cells that overproduce PPX1 is attributed to depressed levels of rpoS expression. Since rpoS is a central element in a regulatory network that governs the expression of stationary-phase-induced genes, poly(P) affects the expression of many genes through controlling rpoS expression. Furthermore, poly(P) is also involved in expression of other stress-inducible genes that are not directly regulated by rpoS. The second subject includes the application of novel functions of PPK for nucleoside triphosphate (NTP) regeneration. Recently E. coli PPK has been found to catalyze the kination of not only ADP but also other nucleoside diphosphates using poly(P) as a phospho-donor, yielding NTPs. This nucleoside diphosphate kinase-like activity of PPK was confirmed to be available for NTP regeneration essential for enzymatic oligosaccharide synthesis using the sugar nucleotide cycling method. PPK has also been found to express a poly(P):AMP phosphotransferase activity by coupling with adenylate kinase (ADK) in E. coli. The ATP-regeneration system consisting of ADK, PPK, and poly(P) was shown to be promising for practical utilization of poly(P) as ATP substitute.


Subject(s)
Escherichia coli/enzymology , Phosphotransferases (Phosphate Group Acceptor)/physiology , Polyphosphates/metabolism , Adenosine Triphosphate/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Phosphotransferases (Phosphate Group Acceptor)/genetics
18.
Neurosci Res ; 38(4): 357-63, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11164562

ABSTRACT

The present study investigated the effect of morphine on synaptic transmission and long-term potentiation (LTP) in the dentate gyrus using rat hippocampal slice preparations. Field excitatory postsynaptic potential (fEPSP) and population spike (PS), evoked by stimulation of the perforant path, were recorded from the dentate molecular layer and the stratum granulosum, respectively. Following application of 10 microM morphine, PS amplitude increased gradually in 10 min and was eventually potentiated by approximately 50%. The phenomenon showed a concentration-dependent manner and was completely canceled by naloxone, a mu opioid receptor antagonist. Furthermore, morphine-induced PS augmentation was not detected in disinhibited hippocampal slices, which suggests that the inhibitory input to the dentate granule cells was required for the facilitatory effect of morphine. Neither fEPSP nor tetanus-induced LTP of PS was altered by morphine application. The data support the hypothesis that mu opioid receptor activity modulates inhibitory recurrent circuits in the dentate gyrus and thereby, indirectly plays a regulatory role for hippocampal excitatory neurotransmission.


Subject(s)
Dentate Gyrus/drug effects , Excitatory Postsynaptic Potentials/drug effects , Morphine/pharmacology , Narcotics/pharmacology , Neural Inhibition/drug effects , Synaptic Transmission/drug effects , gamma-Aminobutyric Acid/metabolism , Animals , Dentate Gyrus/metabolism , Dose-Response Relationship, Drug , Excitatory Postsynaptic Potentials/physiology , Interneurons/drug effects , Interneurons/metabolism , Long-Term Potentiation/drug effects , Long-Term Potentiation/physiology , Male , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Neural Inhibition/physiology , Rats , Rats, Wistar , Receptors, GABA-A/drug effects , Receptors, GABA-A/metabolism , Receptors, Opioid, mu/drug effects , Receptors, Opioid, mu/metabolism , Synaptic Transmission/physiology
19.
Yakugaku Zasshi ; 119(7): 510-8, 1999 Jul.
Article in Japanese | MEDLINE | ID: mdl-10434803

ABSTRACT

Convulsive seizures caused by many different stimuli have been shown to induce activator protein-1 (AP-1) transcription factors in the brain, particularly in the hippocampus. Previous results from our laboratory demonstrated that thalamic and cerebral cortical AP-1 DNA- and cyclic AMP responsive element (CRE)- binding activities in the absence seizure model mice were significantly higher than those in nonepileptic control mice. In order to characterize further a correlation between convulsive seizures and inducible transcription factors, we investigated convulsive seizure-dependent increases in AP-1 DNA- and CRE-binding activities in various brain regions of the mice. Administration of pentylentetrazole and kainic acid provoked clonic and limbic type seizures, respectively, and increased AP-1 DNA- and CRE- binding activities in the cerebral cortex and hippocampus but not in other regions. Maximal electric shock (MES) induced tonic convulsions and increased hippocampal and cerebral cortical AP-1 DNA- and CRE- binding activities. Sodium phenobarbital (50 mg/kg, i.p.), an anticonvulsant, suppressed both convulsions and increases in these DNA-binding activities induced by MES. In contrast, ethosuximide, an antiabsence drug, did not affect MES-induced convulsions or increases in these DNA-binding activities. These data suggest that convulsive seizures increase not only AP-1 DNA-binding but also CRE-binding activities in the cerebral cortex and hippocampus. These data combined with our previous results also suggest that regional differences in increases in CRE- and AP-1 DNA-binding activities between convulsive seizures and absence seizures are attributable to differences in the regions and pathways which are responsible for the genesis and spreading seizure activities in the central nervous system.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , DNA/metabolism , Epilepsy/metabolism , Transcription Factor AP-1/metabolism , Animals , Anticonvulsants/pharmacology , Cerebral Cortex/metabolism , Disease Models, Animal , Hippocampus/metabolism , Male , Mice , Mice, Inbred Strains , Protein Binding/drug effects
20.
DNA Res ; 6(2): 103-8, 1999 Apr 30.
Article in English | MEDLINE | ID: mdl-10382967

ABSTRACT

In Pseudomonas aeriginosa, a gene, ppx, that encodes exopolyphosphatase [exopoly(P)ase; EC 3.6.1.11] of 506 amino acids (56,419 Da) was found downstream of the gene for polyphosphate kinase, ppk. Since ppx is located in the opposite direction of the ppk gene, they do not constitute an operon. The predicted amino acid sequence of PPX is 41% identical with Escherichia coli PPX. The gene product of ppx (paPPX) was overproduced in E. coli, and its activity was evaluated. Orthophosphate (Pi) is released from polyphosphate [poly(P)], the average chain lengths of which are 79 and 750, respectively. The amount of Pi released matched the amount of poly(P) lost. Thus ppx encodes an enzyme that has exopoly(P)ase activity.


Subject(s)
Acid Anhydride Hydrolases/genetics , Pseudomonas aeruginosa/genetics , Amino Acid Sequence , Base Sequence , DNA, Bacterial , Molecular Sequence Data , Pseudomonas aeruginosa/enzymology , Sequence Homology, Amino Acid
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