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Kenji Omori

Publications and source records attributed to Kenji Omori.

16 recordsLinked to original sources
PubMed2005

PfPDE1, a novel cGMP-specific phosphodiesterase from the human malaria parasite Plasmodium falciparum.

This is the first report of molecular characterization of a novel cyclic nucleotide PDE (phosphodiesterase), isolated from the human malaria parasite Plasmodium falciparum and designated PfPDE1. PfPDE1 cDNA encodes an 884-amino-acid protein, including six putative transmembrane domains in the N-terminus followed by a catalytic domain. The PfPDE1 gene is a single-copy gene consisting of two exons and a 170 bp intron. PfPDE1 transcripts were abundant in the ring form of the asexual blood stages of the parasite. The C-terminal catalytic domain of PfPDE1, produced in Escherichia coli, specifically hydrolysed cGMP with a K(m) value of 0.65 microM. Among the PDE inhibitors tested, a PDE5 inhibitor, zaprinast, was the most effective, having an IC50 value of 3.8 microM. The non-specific PDE inhibitors IBMX (3-isobutyl-1-methylxanthine), theophylline and the antimalarial chloroquine had IC50 values of over 100 microM. Membrane fractions prepared from P. falciparum at mixed asexual blood stages showed potent cGMP hydrolytic activity compared with cytosolic fractions. This hydrolytic activity was sensitive to zaprinast with an IC50 value of 4.1 microM, but insensitive to IBMX and theophylline. Furthermore, an in vitro antimalarial activity assay demonstrated that zaprinast inhibited the growth of the asexual blood parasites, with an ED50 value of 35 microM. The impact of cyclic nucleotide signalling on the cellular development of this parasite has previously been discussed. Thus this enzyme is suggested to be a novel potential target for the treatment of the disease malaria.

Animals↗
PubMed2004

Transcriptional activation of phosphodiesterase 7B1 by dopamine D1 receptor stimulation through the cyclic AMP/cyclic AMP-dependent protein kinase/cyclic AMP-response element binding protein pathway in primary striatal neurons.

Phosphodiesterase (PDE) 7B, a cAMP-specific PDE which is dominantly expressed in striatum, is expected to be involved in dopaminergic signaling in striatal neurons. Here we show, for the first time, the involvement of the dopaminergic signaling pathway in transcriptional activation of rat PDE7B in primary striatal culture. RT-PCR analysis revealed that dopamine, D1 agonist, forskolin and 8-Br-cAMP stimulation potentiated PDE7B transcription in striatal neurons, while D2 agonist failed to activate the PDE7B transcription. Pre-treatment with D1 antagonist abolished the dopamine- or D1 agonist-induced transcriptional activation of PDE7B. The activation of PDE7B transcription by these stimulators was completely ablated by pre-treatment of the cells with a cAMP-dependent protein kinase inhibitor, H-89. RT-PCR using splice variant-specific primers revealed that transcription of PDE7B1, but not of other splice variants, was activated by D1 agonist. We determined the putative transcription start site of PDE7B1, a brain-specific splice variant of PDE7B, by 5'-RACE and identified a promoter region of PDE7B1. Sequence analysis of the PDE7B1 promoter revealed the presence of a canonical cAMP-response element at 166 bp upstream of the putative transcription start site. The cAMP-responsiveness of the PDE7B1 promoter was demonstrated by functional promoter analysis using the luciferase reporter system. Deletion and mutation of the cAMP-response element site in the PDE7B1 promoter abolished the forskolin-induced activation of the PDE7B1 promoter activity. Electrophoretic mobility shift assay showed the binding of cAMP-response element binding protein to the PDE7B1 promoter. These data demonstrate the dopamine D1 receptor-mediated transcriptional activation of PDE7B through the cAMP/cAMP-dependent protein kinase/cAMP-response element binding protein pathway in striatal neurons.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

mRNA expression patterns of the cGMP-hydrolyzing phosphodiesterases types 2, 5, and 9 during development of the rat brain.

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP-hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP-hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions.

Animals↗
PubMed2003

Molecular comparison of rat cyclic nucleotide phosphodiesterase 8 family: unique expression of PDE8B in rat brain.

Cyclic nucleotide phosphodiestease (PDE) type 8 is categorized into a family of 3-isobutyl-1-methylxanthine-insensitive PDE hydrolyzing cAMP with high affinity. We have isolated cDNAs encoding rat PDE8A and PDE8B from brain and testis, respectively. The sequence analysis demonstrated that rat PDE8A was a protein of 823 amino acid residues. Rat PDE8B protein was predicted as an N-terminal truncated form of 760 amino acid residues. Both of rat PDE8 proteins include REC, PAS and catalytic PDE domains. Tissue-specific expression patterns of rat PDE8A and PDE8B transcripts were demonstrated by Northern blot analysis. Rat PDE8A transcripts were rich in the liver and testis, and those of rat PDE8B were particularly abundant in the brain and were not expressed in the thyroid gland, while human thyroid gland contains PDE8B transcripts at a high level. Rat PDE8B transcripts were found in all brain regions other than cerebellum and shown to exist in the neuronal cells in in situ hybridization. Mouse PDE8B1 sequence was also identified by a database search and sequence alignment, revealing a protein of 885 amino acid residues, which is 99% and 96% identical to rat and human PDE8B1, respectively. As well as rat PDE8B, expression of mouse PDE8B transcripts was not confined to the thyroid gland. Species-dependent tissue expression pattern was quite unique features of PDE8B.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

Subcellular localization of cyclic nucleotide phosphodiesterase type 10A variants, and alteration of the localization by cAMP-dependent protein kinase-dependent phosphorylation.

Our previous studies have suggested that two phosphodiesterase type 10A (PDE10A) variants, PDE10A1 and PDE10A2 transcripts, are mainly expressed in humans and that PDE10A2 and PDE10A3 transcripts are major variants in rats. In the present study, immunoblot analysis demonstrated that PDE10A proteins, especially PDE10A2, are more abundant in membrane fractions than in cytosolic fractions of rat striatum. Recombinant PDE10A1 and PDE10A3 were produced only in cytosolic fractions of transfected PC12h cells. By contrast, recombinant PDE10A2 was present mainly in membrane fractions. This finding agreed well with the result of subcellular fractionation of PDE10A in rat striatum. Immunocytochemical analysis showed that PDE10A2 was localized in the Golgi apparatus of transfected PC12h cells. PDE10A2 was phosphorylated by cAMP-dependent protein kinase (PKA) at Thr16. Interestingly, recombinant protein of wild-type PDE10A2, but not PDE10A2 mutant with an Ala replacement at Thr16, was distributed to cytosolic fractions by co-transfection with a plasmid encoding the catalytic subunit of PKA. A PDE10A2 mutant with Glu substitution at Thr16, which can be a mimic of phosphorylation, was localized in the cytosolic fractions of transfected PC12h cells. These observations implied that phosphorylation of PDE10A2 at Thr16 by PKA caused alteration of subcellular localization of PDE10A2 from the Golgi apparatus to cytosol. It is hypothesized that cAMP signaling in the Golgi area and the cytosol in neurons is controlled through alteration of subcellular localization of PDE10A brought by activation of PKA in response to intracellular elevations of cAMP.

Alternative Splicing↗
PubMed2003

Novel membrane protein containing glycerophosphodiester phosphodiesterase motif is transiently expressed during osteoblast differentiation.

Osteoblast maturation is a multistep series of events characterized by an integrated cascade of gene expression that are accompanied by specific phenotypic alterations. To find new osteoblast-related genes we cloned differentially expressed cDNAs characteristic of specific differentiation stages in the mouse osteoblast-like MC3T3-E1 cells by a differential display method. We identified a novel cDNA encoding a putative glycerophosphodiester phosphodiesterase, GDE3, which specifically was expressed at the stage of matrix maturation. Interestingly, the deduced amino acid sequence contains 539 amino acids including seven putative transmembrane domains and a glycerophosphodiester phosphodiesterase region in one of the extracellular loops. Northern blot analysis revealed that GDE3 was also expressed in spleen as well as primary calvarial osteoblasts and femur. We next transfected HEK293T cells with GDE3 with green fluorescent protein fused to the C terminus. The green fluorescent protein-fused protein accumulated at the cell periphery, and the transfected cells overexpressing the protein changed from a spread form to rounded form with disappearance of actin filaments. Immunofluorescence staining with GDE3 antibody and phalloidin in MC3T3-E1 cells indicated that endogenous GDE3 might be co-localized with the actin cytoskeleton. To identify a role for GDE3 in osteoblast differentiation, MC3T3-E1 cells stably expressing the full-length protein were constructed. Expression of GDE3 showed morphological changes, resulting in dramatic increases in alkaline phosphatase activity and calcium deposit. These results suggest that GDE3 might be a novel seven-transmembrane protein with a GP-PDE-like extracellular motif expressed during the osteoblast differentiation that dramatically accelerates the program of osteoblast differentiation and is involved in the morphological change of cells.

3T3 Cells↗
PubMed2003

1,7- and 2,7-naphthyridine derivatives as potent and highly specific PDE5 inhibitors.

Novel 1,7- and 2,7-naphthyridine derivatives, designed by the introduction of nitrogen atom into the phenyl ring of previously reported 4-aryl-1-isoquinolinone derivatives, were disclosed as a new structural class of potent and specific PDE5 inhibitors. Among them, 2,7-naphthyridine 4c showed potent PDE5 inhibition (IC(50)=0.23 nM) and one of the best PDE5 specificities against PDEs1-4,6 (>100,000-fold selective versus PDE1-4, 240-fold selective vs PDE6). This compound showed more potent relaxant effects on isolated rabbit corpus cavernosum (EC(30)=5.0 nM) than Sildenafil (EC(30)=8.7 nM). The compound 4c (T-0156) was selected for further biological and pharmacological evaluation of erectile dysfunction.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2003

The Serratia marcescens bioH gene encodes an esterase.

The 3.9 kb chromosomal DNA was cloned from Serratia marcescens Sr41, which confers on Escherichia coli cells a phenotype of clear halo formation on tributyrin agar plates. Three complete open reading frames (ORFs) were identified in the inserted DNA, and one ORF was demonstrated to encode a 28 kDa protein of 255 amino acids related to esterase activity. Interestingly, the ORF was 70% identical to a product of the E. coli bioH gene, which lies at a locus separated from the bioABFCD operon and acts in the early steps of the biotin synthetic pathway before pimeloyl-CoA synthesis. This gene complemented a bioH-deficient mutation of E. coli. From the sequence analysis, BioH is presumed to be a serine hydrolase, which belongs to the alpha/beta hydrolase-fold family comprising a wide variety of hydrolases including esterases. A catalytic triad composed of a nucleophilic residue (Ser80), an acidic residue (Asp206), and histidine (His234) was conserved in BioH, and the nucleophilic residue Ser, a catalytic center, was situated in the consensus sequence of G-X-S-X-G-G, a nucleophile elbow. Although the enzymatic function of BioH is not yet elucidated, the bioH gene products from S. marcescens and E. coli show esterase activity, which may imply the hydrolysis of a precursor leading to pimeloyl-CoA ester. The esterase activity of BioH and its CoA binding activity recently reported agree with a current hypothesis of pimeloyl-CoA ester synthesis from CoA and acylester derivatives including an acyl-carrier protein.

Amino Acid Sequence↗
PubMed2003

Comparison of enzymatic characterization and gene organization of cyclic nucleotide phosphodiesterase 8 family in humans.

Full-length cDNAs of human cyclic nucleotide phosphodiesterase 8B (PDE8B) were isolated. Enzymatic characteristics of a dominant variant encoding a protein of 885 residues (PDE8B1) were compared with those of PDE8A1. The recombinant PDE8A1 and PDE8B1 proteins of an entire form were produced in both cytosolic and membrane fractions of the transfected COS cells. The human PDE8B1 was a high-affinity cAMP-PDE with K(m) value of 101+/-12 nM for cAMP, which is greater than that of PDE8A1 (40+/-1 nM). Relative V(max) value of PDE8A1 was 57+/-8% compared with that of PDE8B1 (100+/-12%). Although PDE8A1 was moderately inhibited by dipyridamole with IC(50) value of 8+/-2 microM, the compound antagonized the PDE8B1 activity at three-fold higher concentration (IC(50)=23+/-2 microM). The human PDE8B gene was composed of 22 exons, spanning over 217 kb. Although overall sequence identity between PDE8A1 and PDE8B1 was 68%, positions of junctions of each exon between the PDE8A1 and PDE8B1 sequences were well matched, indicating evolutionary relatedness of both genes.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

Gram-negative bacterial ATP-binding cassette protein exporter family and diverse secretory proteins.

Protein translocation to the extracellular space is essential for the invasion, colonization, and survival of pathogenic gram-negative bacteria within a host organism. In addition to the N-terminal signal sequence-dependent secretion system, which is specific for protein transport to the periplasmic space, there are five major systems (type I, II, III, IV, and V) that are known to be involved in protein secretion into the extracellular space. Of the systems, the type I pathway, which is composed of three membrane components including an ATP-binding cassette (ABC) protein, translocates proteins into the extracellular space from the cytosol by directly using the energy generated from ATP hydrolysis, and therefore, the system is a member of the ABC transporter family and is also known as the ABC exporter. To date, ABC exporters have been discovered to be involved in the secretion of a wide variety of exoproteins including RTX (repeats-in-toxin) toxins, cell surface layer proteins, proteases, lipases, bacteriocins, heme-acquisition proteins, and nodulation-related proteins such as the exoglucanases of gram-negative bacteria. A secretory protein and its associated specific ABC exporter are encoded in the same gene cluster in most cases, and ABC exporters show substrate specificity for secretion. Consequently, ABC exporters are present based primarily on the number of secretory protein genes. A secretion signal is situated in the C-terminal region of secretory proteins, however, the characteristics of the secretion signal are not fully understood. Secretory substrates and their linked ABC exporters are reviewed in the following paper.

Journal Article↗
PubMed2003

cGMP-phosphodiesterase activity is up-regulated in response to pressure overload of rat ventricles.

Although expression of natriuretic peptides in cardiac tissues is up-regulated in response to pressure overload, no significant change in cGMP level in hypertrophied ventricles was observed. Activities of two cyclic nucleotide phosphodiesterase (PDE) isoforms, Ca2+/calmodulin-stimulated PDE (PDE1) and cGMP-stimulated PDE (PDE2), were significantly higher in rat left ventricles 14 days after aortic banding. The absence of significant changes in PDE1A and PDE2A mRNA levels indicated that the two PDE activities were post-transcriptionally up-regulated. These results suggested that the increased cGMP-PDE activity in response to pressure overload plays an important role in neutralizing cGMP action in cardiac tissue.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2002

Enzymological and pharmacological profile of T-0156, a potent and selective phosphodiesterase type 5 inhibitor.

The enzymological and pharmacological properties of 2-(2-Methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), a new phosphodiesterase type 5 inhibitor, were studied in vitro and in vivo. The inhibitory effects of T-0156 on six phosphodiesterase isozymes isolated from canine tissues were investigated. T-0156 specifically inhibited the hydrolysis of cyclic guanosine monophosphate (cGMP) by phosphodiesterase type 5, at low concentration (IC(50)=0.23 nM), in a competitive manner. T-0156 also inhibited phosphodiesterase type 6 with IC(50) value of 56 nM, which was 240-fold higher than that for inhibition of phosphodiesterase type 5. T-0156 had low potencies against phosphodiesterase types 1, 2, 3, and 4 (IC(50)>10 microM). In the isolated rabbit corpus cavernosum, T-0156 at 10 and 100 nM increased cGMP levels (100 nM T-0156-treated: 6.0+/-1.5 pmol/mg protein, vehicle-treated: 1.1+/-0.4 pmol/mg protein, P<0.05), causing relaxation of the tissue. T-0156 at 1 to 100 nM potentiated the electrical field stimulation-induced relaxation in the isolated rabbit corpus cavernosum in a concentration-dependent manner (100 nM T-0156-treated: 76.9+/-19.8%, vehicle-treated: 12.3+/-10.1%, P<0.05). Intraduodenal administration of T-0156 at 100 to 1000 microg/kg potentiated the pelvic nerve stimulation-induced tumescence in anesthetized dogs (1000 microg/kg T-0156-treated: 279.0+/-38.4%, vehicle-treated: 9.8+/-4.5%, P<0.05). These results suggested that T-0156 enhanced the nitric oxide (NO)/cGMP pathway, probably through blockade of phosphodiesterase type 5 in vitro and in vivo experimental conditions. The present study clearly showed that T-0156 is a potent and highly selective phosphodiesterase type 5 inhibitor, which is a useful tool for pharmacological studies in vitro and in vivo.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2002

Novel alternative splice variants of rat phosphodiesterase 7B showing unique tissue-specific expression and phosphorylation.

cDNA species coding for novel variants of cyclic-AMP-specific phosphodiesterases (PDEs), namely the PDE7B family, were isolated from rats and characterized. Rat PDE7B1 (RNPDE7B1) was composed of 446 amino acid residues. Rat PDE7B2 (RNPDE7B2) and PDE7B3 (RNPDE7B3), which possessed unique N-terminal sequences, consisted of 359 and 459 residues respectively. Northern hybridization analysis showed that rat PDE7B transcripts were particularly abundant in the striatum and testis. PCR analyses revealed that rat PDE7B2 transcripts were restricted to the testis and that low levels of PDE7B3 transcripts were expressed in the heart, lung and skeletal muscle. In situ hybridization analysis demonstrated that rat PDE7B transcripts were expressed in striatal neurons and spermatocytes. In spermatocytes, rat PDE7B transcripts were expressed in a stage-specific manner during spermatogenesis. The K(m) values of recombinant rat PDE7B1, PDE7B2 and PDE7B3 for cAMP were 0.05, 0.07 and 0.05 microM respectively. Each rat PDE7B variant was the most sensitive to 3-isobutyl-1-methylxanthine (IC(50) 1.5-2.1 microM). Two phosphorylation sites for cAMP-dependent protein kinase (PKA) were found in rat PDE7B1 and PDE7B3, whereas rat PDE7B2 possessed one site. PKA-dependent phosphorylation was observed in C-terminal phosphorylation sites of three rat PDE7B variants, in addition to unique N-terminal regions of rat PDE7B1 and PDE7B3. Unique tissue distribution and PKA-dependent phosphorylation of PDE7B variants suggested that each variant has a specific role for cellular functions via cAMP signalling in various tissues.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2002

Role of repetitive nine-residue sequence motifs in secretion, enzymatic activity, and protein conformation of a family I.3 lipase.

A family I.3 lipase from Pseudomonas sp. MIS38 (PML) contains 12 repeats of a nine-residue sequence motif in the C-terminal region. To elucidate the role of these repetitive sequences, mutant proteins PML5, PML4, PML1, and PML0, in which 7, 8, 11, and all 12 of the repetitive sequences are deleted, and PMLdelta19, in which 19 C-terminal residues are truncated, were constructed. Escherichia coli DH5 cells carrying the Serratia marcescens Lip system permitted the secretion of the wild-type and all of the mutant proteins except for PMLdelta19, although they were partially accumulated in the cells in an insoluble form as well. Both the secretion level and cellular content of the proteins decreased in the order PML > PML5 > PML4 > PML1 > PML0, indicating that repetitive sequences are not required for secretion of PML but are important for its stability in the cells. All the mutant proteins were purified in a refolded form and their biochemical properties were characterized. CD spectra, the Ca2+ contents, and susceptibility to chymotryptic digestion strongly suggested that the five repetitive sequences remaining in PML5 are sufficient to form a beta-roll structure, whereas the four in PML4 are not. PML5 and PMLdelta19 showed both lipase and esterase activities, whereas PML4, PML1, and PML0 were inactive. These results suggest that the enzymatic activity of PML is not seriously affected by a deletion or truncation at the C-terminal region as long as a succession of repetitive sequences can build a beta-roll structure.

Journal Article↗