Title: Increased synthesis of poly(3-hydroxydodecanoate) by random mutagenesis of polyhydroxyalkanoate synthase Hiroe A, Watanabe S, Kobayashi M, Nomura CT, Tsuge T Ref: Applied Microbiology & Biotechnology, 102:7927, 2018 : PubMed
Poly(3-hydroxydodecanoate) [P(3HDD)], a medium-chain-length polyhydroxyalkanoate (PHA), is expected to be used as a novel type of bioplastic characterized by a soft and transparent nature. In this study, to achieve a high yield of P(3HDD), PHA synthase was modified through random mutagenesis of a region of the PHA synthase 1 gene from Pseudomonas putida KT2440 (phaC1(Pp)). Screening of the mutant library using a beta-oxidation-deficient Escherichia coli LSBJ was performed. As a result, four mutants, designated w10, w14, w309, and w311, were selected from 10,000 mutants. The w311 mutant had two amino acid replacements (E358G and N398S), and showed the highest production of P(3HDD) with increased polymer molecular weights when compared to the native enzyme. Saturation mutagenesis at the N398 position, which was found to be highly conserved among Pseudomonas PhaCs, revealed that amino acids with hydrophobic and smaller residues either retained or increased P(3HDD) production. This study demonstrates the benefit of using the PHA synthase mutants to enhance the production of P(3HDD).
Title: Characterization of a helminthosporic acid analog that is a selective agonist of gibberellin receptor Miyazaki S, Tomita K, Yamane H, Kobayashi M, Asami T, Nakajima M Ref: Bioorganic & Medicinal Chemistry Lett, 28:2465, 2018 : PubMed
Helminthosporol, a natural growth regulator isolated from a fungus, stimulates hypocotyl growth and seed germination, similar to gibberellin (GA). We recently reported that helminthosporic acid (H-acid), a synthetic analog of helminthosporol, acts as an agonist of GA receptor. In this study, we showed that a H-acid analog, in which the hydroxymethyl group at the C-8 position of H-acid was converted to a keto group, acts as a selective GA receptor agonist. 1) This analog shows higher hypocotyl elongation activity in Arabidopsis than H-acid does, and induces the degradation of DELLA protein and 2) leads to the formation of the GID1-DELLA complex and 3) regulates the expression of GA-related genes. In addition, 4) its hypocotyl elongation activity was not observed in a atgid1a single mutant, and 5) this analog could promote only the interaction between specific GA receptors and DELLA proteins in vitro. Taken together, our results strongly suggest that the selectivity of the reported H-acid analog depends on the specificity of its GA receptor binding activity.
OBJECTIVES: Drugs are mainly metabolized by hepatic enzymes, the activity of which can differ between individuals. Although it is ideal to measure the hepatic clearance of liver-targeted drugs in individualized medicine, blood enzyme tests typically measure metabolic drug clearance in the entire body, and not just in the liver. We investigated whether I-iomazenil imaging can directly assess and quantify the activity of hepatic drug-metabolizing enzymes. MATERIALS AND METHODS: Hepatic enzymes that metabolize I-iomazenil were identified by thin-layer chromatography in mouse liver homogenates with bis(4-nitrophenyl) phosphate (BNPP) inhibitor for carboxylesterase enzymes and nicotinamide adenine dinucleotide phosphate (NADPH) generator for cytochrome P450 enzymes. Whole-body images of mice were acquired using I-iomazenil with and without BNPP, and the distribution was also obtained. The metabolism of I-iomazenil in the blood, liver, gall bladder, and bladder was investigated by thin-layer chromatography. RESULTS: From the in-vitro metabolism of I-iomazenil using BNPP, the enzyme converting I-iomazenil to I-R-COOH was identified as carboxylesterase, and that converting I-iomazenil to M2 was identified as cytochrome P450 in experiments with and without an NADPH generator. The biological distribution and whole-body imaging showed increased accumulation in the liver of mice administered BNPP compared with normal mice, but decreased levels in the gall bladder and small intestine. The main fraction in bile and urine was I-R-COOH, with two unknown metabolites (M1 and M2), I, and I-iomazenil also being present. CONCLUSION: I-iomazenil whole-body imaging has good possibility of direct measurement of hepatic carboxylesterase activity as accumulation of I-R-COOH in the gall bladder through bile and in the bladder through urine.
Title: Effects of Silodosin, an alpha-Adrenoceptor Antagonist, and Distigmine, an Acetylcholinesterase Inhibitor, and Their Combined Effects on Impaired Voiding Function in Zucker Diabetic Fatty Rats Tatemichi S, Tsuchioka K, Yonekubo S, Maruyama K, Kobayashi M Ref: Pharmacology, 95:285, 2015 : PubMed
BACKGROUND/AIMS: To evaluate the effects of silodosin (alpha1A-adrenoceptor antagonist) and distigmine (acetylcholinesterase inhibitor), alone or in combination, on voiding dysfunction in Zucker diabetic fatty (ZDF) rats, a type 2 diabetes model, by pressure flow study. METHODS: Male ZDF rats were anesthetized with urethane and a catheter was implanted into the bladder through the dome. Saline was continuously infused into the bladder at 6 ml/h to induce the micturition reflex. Intravesical pressure and micturition volume were recorded continuously and various urodynamic parameters were calculated using a waveform analysis system. RESULTS: Increased bladder capacity, residual volume, and urethral resistance and decreased maximum detrusor contraction velocity and urine flow rate, considered to be detrusor underactivity-like symptoms, were observed in ZDF rats. Although both silodosin and distigmine improved impaired voiding function, administration of both drugs in combination was more effective than either drug alone. CONCLUSIONS: ZDF rats showed symptoms suggestive of detrusor underactivity, and silodosin tended to ameliorate these symptoms in ZDF rats. These results suggested that an alpha1A-adrenoceptor antagonists may be effective against the voiding disorder accompanying not only bladder outlet obstruction but also deficiency of bladder function. Moreover, combined administration of an alpha1A-adrenoceptor antagonist with an acetylcholinesterase inhibitor may have additive efficacy in clinical use. (c) 2015 S. Karger AG, Basel.
BACKGROUND: Thellungiella halophila (also known as T. salsuginea) is a model halophyte with a small size, short life cycle, and small genome. Thellungiella genes exhibit a high degree of sequence identity with Arabidopsis genes (90% at the cDNA level). We previously generated a full-length enriched cDNA library of T. halophila from various tissues and from whole plants treated with salinity, chilling, freezing stress, or ABA. We determined the DNA sequences of 20 000 cDNAs at both the 5'- and 3' ends, and identified 9569 distinct genes. RESULTS: Here, we completely sequenced 1047 Thellungiella full-length cDNAs representing abiotic-stress-related genes, transcription factor genes, and protein phosphatase 2C genes. The predicted coding sequences, 5'-UTRs, and 3'-UTRs were compared with those of orthologous genes from Arabidopsis for length, sequence similarity, and structure. The 5'-UTR sequences of Thellungiella and Arabidopsis orthologs shared a significant level of similarity, although the motifs were rearranged. While examining the stress-related Thellungiella coding sequences, we found a short splicing variant of T. halophila salt overly sensitive 1 (ThSOS1), designated ThSOS1S. ThSOS1S contains the transmembrane domain of ThSOS1 but lacks the C-terminal hydrophilic region. The expression level of ThSOS1S under normal growth conditions was higher than that of ThSOS1. We also compared the expression levels of Na+-transport-system genes between Thellungiella and Arabidopsis by using full-length cDNAs from each species as probes. Several genes that play essential roles in Na+ excretion, compartmentation, and diffusion (SOS1, SOS2, NHX1, and HKT1) were expressed at higher levels in Thellungiella than in Arabidopsis. CONCLUSIONS: The full-length cDNA sequences obtained in this study will be essential for the ongoing annotation of the Thellungiella genome, especially for further improvement of gene prediction. Moreover, they will enable us to find splicing variants such as ThSOS1S (AB562331).
Alternative splicing (AS) is a mechanism by which multiple types of mature mRNAs are generated from a single pre-mature mRNA. In this study, we completely sequenced 1800 full-length cDNAs from Arabidopsis thaliana, which had 5' and/or 3' sequences that were previously found to have AS events or alternative transcription start sites. Unexpectedly, these sequences gave us further evidence of AS, as 601 out of 1800 transcripts showed novel AS events. We focused on the combination patterns of multiple AS events within individual genes. Interestingly, some specific AS event combination patterns tended to appear more frequently than expected. The two most common patterns were: (i) alternative donor-0 approximately 12 times of exon skips-alternative acceptor and (ii) several times ( approximately 8) of retained introns. We also found that multiple AS events in a transcript tend to have the same effects concerning the length of the mature mRNA. Our current results are consistent with our previous observations, which showed changes in AS profiles under different conditions, and suggest the involvement of hypothetical cis- and trans-acting factors in the regulation of AS events.
Title: Dysideamine, a new sesquiterpene aminoquinone, protects hippocampal neuronal cells against iodoacetic acid-induced cell death Suna H, Arai M, Tsubotani Y, Hayashi A, Setiawan A, Kobayashi M Ref: Bioorganic & Medicinal Chemistry, 17:3968, 2009 : PubMed
In the course of our search for neuroprotective agents, dysideamine (1), a new sesquiterpene aminoquinone, was isolated along with bolinaquinone (2) from Indonesian marine sponge of Dysidea sp. Compounds 1 and 2 showed neuroprotective effect against iodoacetic acid (IAA)-induced cell death at 10 microM concentration in mouse HT22 hippocampal neuronal cells. Dysideamine (1) inhibited production of reactive oxygen species (ROS) by IAA treatment, whereas it exhibited no effect on depletion of intracellular ATP of the IAA-treated HT22 cells. Moreover, 1 induced neurite outgrowth against mouse neuroblastoma Neuro 2A cells with increase of acetylcholinesterase (AChE) activity, which is a marker of neuronal differentiation.
In Arabidopsis, three receptors exist for the phytohormone gibberellin. Of the three, only a double loss-of-function mutant (atgid1a atgid1c) shows a dwarf phenotype, while other double and all single mutants show no abnormality in height. In this study we show that the expression of AtGID1b-GUS mRNA, driven by the AtGID1b promoter, is low in inflorescence stems, but may be 10% of AtGID1a-GUS mRNA, driven by the AtGID1a promoter. However, AtGID1b-GUS enzymatic activity does not exist in them. This factor strongly suggests that atgid1a atgid1c lacks sufficient AtGID1b protein for normal stem growth. In the stamens of pAtGID1c::AtGID1c-GUS transformants, we detected clear AtGID1c-GUS activity, while another atgid1a atgid1b, which has short stamens in its flowers, causes the adhesion of little pollen to stigmas thus leading to its low fertility. We then evaluated the affinity of the AtGID1-DELLA interaction by a competitive yeast three-hybrid system and also by QCM apparatus. AtGID1c showed a quite lower affinity to RGL2, the major DELLA protein in floral buds, than AtGID1a or AtGID1b. The low affinity of the AtGID1c-RGL2 interaction is likely to be responsible for the failure of AtGID1c to hold RGL2, which is required for normal stamen development. Taken together with expressional information of DELLA genes, we propose that in a double loss-of-function mutant of gibberellin receptors, the emergence of any phenotype(s) depends on the abundance of the remaining receptor and its preference to DELLA proteins existing at a target site.
The Culex pipiens complex consists of vector mosquitoes that transmit important human pathogens. In this study we established a simplified method to distinguish three members of the Cx. pipiens complex, Cx. p. pallens Coquillet, Cx. p. form molestus Forskal, and Cx. quinquefasciatus Say, collected in Japan. Sequence analysis of the Drosophila Ace-orthologous acetylcholinesterase (Ace) gene (668 to 680 bp) revealed that a single polymorphic region characterizes each species. Based on this region, specific primers that distinguish Cx. p. form molestus (ACEpip2) and Cx. p. pallens (ACEpall2) were newly designed. Polymerase chain reactions were performed with the genomic DNA of Culex mosquitoes as the template, and these primers clearly distinguished two Culex spp. The accuracy of the designed primers was evaluated with 38 colonies of mosquito samples collected from 9 prefectures of Japan. The testing revealed that the distribution of anautogenous Cx. p. pipiens has not been confirmed in Japan. It also revealed that the male of Cx. p. pallens possesses an Ace gene haplotype that is highly similar to the sequence of Cx. quinquefasciatus. This improved method allows the evaluation of vector competence of Cx. p.molestus, which is the suspected vector of West Nile virus.
Arabidopsis carries three receptor genes for the phytohormone gibberellin (GA), AtGID1a, AtGID1b and AtGID1c. Expression of each gene in the rice gid1-1 mutant for GA receptors causes reversion of its severely dwarfed phenotype and GA insensitivity to a normal level, even though each loss-of-function mutant shows no clear phenotype in Arabidopsis (Nakajima et al., 2006). In this paper, we report the functional redundancy and specificity of each AtGID1 by analyzing the multiple mutants for loss of function. Seeds of the double knockout mutants atgid1a atgid1b, atgid1a atgid1c and atgid1b atgid1c germinated normally. The double knockout mutant atgid1a atgid1c showed a dwarf phenotype, while other double mutants were of normal height compared to the wild-type. The stamens of the double knockout mutant atgid1a atgid1b were significantly shorter than those of the wild-type, and this leads to low fertility. A severe disarrangement of the pattern on its seed surface was also observed. The triple knockout mutant atgid1a atgid1b atgid1c did not germinate voluntarily, and only started to grow when the seed coat was peeled off after soaking. Seedlings of the triple knockout mutants were severe dwarfs, only a few millimeters high after growing for 1 month. Moreover, the triple knockout seedlings completely lost their ability to respond to exogenously applied GA. These results show that all AtGID1s function as GA receptors in Arabidopsis, but have specific role(s) for growth and development.
Two cDNA sequences encoding Drosophila Ace-orthologous and -paralogous acetylcholinesterase precursors (AO- and AP-AChE precursors, respectively), were identified from the body louse, Pediculus humanus humanus L. In vitro inhibition studies with an insecticide-susceptible body louse strain exhibited a simplex inhibitory response of AChE. The I50 values of fenitroxon and carbaryl were estimated to be 2.2 and 1.9 microM for the susceptible lice, respectively. The mRNA level of AP-AChE gene was 3.1- and 9.3-fold higher than that of AO-AChE gene in the abdomen and the combined parts of the head and thorax, respectively, suggesting, due to its abundance, the potential significance of the AP-AChE isoform in Pediculus human lice in association with the efficacy of AChE-targeting pediculicides.
Three gibberellin (GA) receptor genes (AtGID1a, AtGID1b and AtGID1c), each an ortholog of the rice GA receptor gene (OsGID1), were cloned from Arabidopsis, and the characteristics of their recombinant proteins were examined. The GA-binding activities of the three recombinant proteins were confirmed by an in vitro assay. Biochemical analyses revealed similar ligand selectivity among the recombinants, and all recombinants showed higher affinity to GA(4) than to other GAs. AtGID1b was unique in its binding affinity to GA(4) and in its pH dependence when compared with the other two, by only showing binding in a narrow pH range (pH 6.4-7.5) with 10-fold higher affinity (apparent K(d) for GA(4) = 3 x 10(-8) m) than AtGID1a and AtGID1c. A two-hybrid yeast system only showed in vivo interaction in the presence of GA(4) between each AtGID1 and the Arabidopsis DELLA proteins (AtDELLAs), negative regulators of GA signaling. For this interaction with AtDELLAs, AtGID1b required only one-tenth of the amount of GA(4) that was necessary for interaction between the other AtGID1s and AtDELLAs, reflecting its lower K(d) value. AtDELLA boosted the GA-binding activity of AtGID1 in vitro, which suggests the formation of a complex between AtDELLA and AtGID1-GA that binds AtGID1 to GA more tightly. The expression of each AtGID1 clone in the rice gid1-1 mutant rescued the GA-insensitive dwarf phenotype. These results demonstrate that all three AtGID1s functioned as GA receptors in Arabidopsis.
Tea catechins, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG), have been shown to be epimerized to (-)-catechin (C), (-)-gallocatechin (GC), (-)-catechin gallate (CG), and (-)-gallocatechin gallate (GCG), respectively, during heat treatment. In this study, we examined the effect of tea catechins rich in ECG and EGCG and heat-treated tea catechins rich in CG and GCG on postprandial hypertriacylglycerolemia in rats. Both tea catechins and heat-treated tea catechins suppressed postprandial hypertriacylglycerolemia. Lymphatic recovery of (14)C-trioleoylglycerol in rats cannulated in the thoracic duct was delayed by the administration of tea catechins and heat-treated tea catechins. Tea catechins and heat-treated tea catechins had the same effect on all variables tested. These catechin preparations dose-dependently inhibited the activity of pancreatic lipase in vitro. When purified catechins were used, only those with a galloyl moiety inhibited the activity of pancreatic lipase. These results suggest that catechins with a galloyl moiety suppress postprandial hypertriacylglycerolemia by slowing down triacylglycerol absorption through the inhibition of pancreatic lipase. Because postprandial hypertriacylglycerolemia is a risk factor for coronary heart disease, our results suggest that catechins with a galloyl moiety may prevent this disease.
Gibberellins (GAs) are phytohormones that are essential for many developmental processes in plants. It has been postulated that plants have both membrane-bound and soluble GA receptors; however, no GA receptors have yet been identified. Here we report the isolation and characterization of a new GA-insensitive dwarf mutant of rice, gid1. The GID1 gene encodes an unknown protein with similarity to the hormone-sensitive lipases, and we observed preferential localization of a GID1-green fluorescent protein (GFP) signal in nuclei. Recombinant glutathione S-transferase (GST)-GID1 had a high affinity only for biologically active GAs, whereas mutated GST-GID1 corresponding to three gid1 alleles had no GA-binding affinity. The dissociation constant for GA4 was estimated to be around 10(-7) M, enough to account for the GA dependency of shoot elongation. Moreover, GID1 bound to SLR1, a rice DELLA protein, in a GA-dependent manner in yeast cells. GID1 overexpression resulted in a GA-hypersensitive phenotype. Together, our results indicate that GID1 is a soluble receptor mediating GA signalling in rice.
Title: Pyridoacridine Alkaloids Inducing Neuronal Differentiation in a Neuroblastoma Cell Line, from Marine Sponge Biemna fortis Aoki S, Wei H, Matsui K, Rachmat R, Kobayashi M Ref: Bioorganic & Medicinal Chemistry, 11:1969, 2003 : PubMed
A new and three known pyridoacridine alkaloids were isolated from the Indonesian marine sponge Biemna fortis as neuronal differentiation inducers against a murine neuroblastoma cell line, Neuro 2A. The chemical structure of the new compound, labuanine A (1), was determined by spectroscopic study and chemical conversion. These pyridoacridine alkaloids induced multipolar neuritogenesis in more than 50% of cells at 0.03-3 micro M concentration. Compound 3, which showed the strongest neuritogenic activity among them, also induced increase of acetylcholinesterase, a neuronal marker in Neuro 2A and arrested cell cycle at the G2/M phase.
We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.
Title: A novel lipase from Pseudomonas fluorescens HU380: gene cloning, overproduction, renaturation-activation, two-step purification, and characterization Kojima Y, Kobayashi M, Shimizu S Ref: J Biosci Bioeng, 96:242, 2003 : PubMed
The extracellular lipase gene (lipA) from Pseudomonas fluorescens HU380 was cloned from a genomic library constructed in pBluescript SK+. Nucleotide sequence analysis revealed an open reading frame of 1854 by encoding the lipase. Its deduced amino acid sequence included internal amino acid sequences of the lipase from this strain: The lipase showed significant sequence similarity to lipases of Serratia marcescens strains and P. fluorescens strains. In Escherichia coli, lipA was expressed in the form of inclusion bodies, which were subsequently solubilized by urea followed by dialysis. The refolded protein was soluble and biologically active. The lipase purified from the E. coli transformant by this denaturation-renaturation procedure followed by only two steps of column chromatographs exhibited the same electrophoretic mobility as did the enzyme purified from P. fluorescens HU380, and both enzymes were quite similar in physicochemical properties such as specific activity, suggesting that the recombinant lipase protein has an intrinsic folding capability in vitro. The function of its C-terminal region is also discussed.
The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.
Title: Lembehyne a, a spongean polyacetylene, induces neuronal differentiation in neuroblastoma cell Aoki S, Matsui K, Takata T, Hong W, Kobayashi M Ref: Biochemical & Biophysical Research Communications, 289:558, 2001 : PubMed
Lembehyne A (LB-A), a spongean polyacetylene, induced neuronal cell differentiation in a neuroblastoma cell line, Neuro 2A. The LB-A treatment of Neuro 2A cells predominantly resulted in a morphological change with bipolar neurites. The acetylcholinesterase activity of Neuro 2A was also increased by the treatment of LB-A. Furthermore, the cell cycle of Neuro 2A cells was found to be specifically blocked at the G1 phase by LB-A. The structure-activity relationship study using the LB-A analogues revealed the importance of the terminal 1-yn-3-ol and unsaturated long-chain alkyl moieties for the neuronal differentiation activity of LB-A.
Title: Expression and functional analysis of a gene cluster involved in the synthesis of decaprenoxanthin reveals the mechanisms for C50 carotenoid formation Krubasik P, Kobayashi M, Sandmann G Ref: European Journal of Biochemistry, 268:3702, 2001 : PubMed
Corynebacterium glutamicum accumulates the C50 carotenoid decaprenoxanthin. Rescued DNA from transposon color mutants of this Gram-positive bacterium was used to clone the carotenoid biosynthetic gene cluster. By sequence comparison and functional complementation, the genes involved in the synthesis of carotenoids with 50 carbon atoms were identified. The genes crtE, encoding a geranylgeranyl pyrophosphate synthase, crtB, encoding a phytoene synthase, and crtI, encoding a phytoene desaturase, are responsible for the formation of lycopene. The products of three novel genes, crtYe and crtYf, with sequence similarities to heterodimeric lycopene cyclase crtYc and crtYd, together with crtEb which exhibits a prenyl transferase motif, were involved in the conversion of C40 acyclic lycopene to cyclic C50 carotenoids. Using functional complementation in Escherichia coli, it could be shown that the elongation of lycopene to the acyclic C50 carotenoid flavuxanthin by the addition of C5 isoprenoid units at positions C-2 and C-2' is catalyzed by the crtEb gene product. Subsequently, the gene products of crtYe and crtYf in a concerted action convert the acyclic flavuxanthin into the cyclic C50 carotene, decaprenoxanthin, forming two epsilon-ionone groups. The mechanisms, involving two individual steps for the formation of cyclic C50 carotenoids from lycopene, are proposed on the basis of these results.
A 45-year-old woman was referred to our hospital because of hyperthyroidism complicated by atrial fibrillation and heart failure. Laboratory data revealed pancytopenia, with a white blood cell count of 2,600/microliter, red blood cell count of 330 x 10(4)/microliter, and platelet count of 6.2 x 10(4)/microliter. The patient had normal transaminase levels, but tests for hepaplastin and cholinesterase showed values of 34% and 1.4 U/ml, respectively, indicating liver dysfunction. There was also decreased excretion of indocyanine green. After initiation of treatment with 30 mg thiamazole and 20 mg propranolol daily, the patient's thyroid function normalized and the other abnormal laboratory findings such as pancytopenia and liver dysfunction also disappeared. Pancytopenia is a rare complication of hyperthyroidism. In this case, various laboratory abnormalities were normalized by antithyroid therapy alone, indicating that the hyperthyroidism itself was closely related to the pathogenesis of pancytopenia and liver dysfunction.
We report that there is a time-related change in the phospholipase C (PLC) activities of rat brain cytosol and membrane fractions after iv injection of a soman-like or a sarin-like organophosphorous agent (bis(isopropyl methyl)phosphonate [BIMP] and bis(pinacolyl methyl)phosphonate [BPMP]). PLCgamma was activated in the brain cytosol fraction from BPMP-injected rats. The phosphorylating activity of rat brain membrane fractions were enhanced by BPMP treatment. The brain membrane fractions from BPMP-treated rats phosphorylated several proteins, including supposedly PLCgamma in the brain cytosol fraction from control rats in vitro. These results suggest that soman and sarin may stimulate a membrane tyrosine kinase, including growth factor receptors, directly or indirectly.
Title: Altered cholinergic mechanisms and blood pressure regulation in the rostral ventrolateral medulla of DOCA-salt hypertensive rats Kubo T, Fukumori R, Kobayashi M, Yamaguchi H Ref: Brain Research Bulletin, 45:327, 1998 : PubMed
We examined whether cholinergic transmission in the rostral ventrolateral medulla (RVLM) of deoxycorticosterone acetate-salt hypertensive rats (DHR) is enhanced and the enhancement is involved in the maintenance of hypertension in DHR, and whether cholineacetyltransferase (ChAT) activities and ChAT mRNA expression are enhanced in neurons intrinsic to the RVLM of DHR. Rats were anesthetized, paralyzed, and artificially ventilated. Unilateral microinjection of cholinergic agents into the RVLM produced a pressor response. The pressor response to physostigmine was greater in DHR than in control rats, whereas the response to carbachol was the same in both sets of rats. Bilateral microinjection of scopolamine into the RVLM produced a decrease in blood pressure. The depressor response was greater in DHR than in control rats. The number of ChAT-activity-detected neurons in the RVLM was greater in DHR than in control rats. The number of ChAT mRNA-expressing neurons in the RVLM was also clearly greater in DHR than in control rats. These results demonstrate that cholinergic transmission in the RVLM is enhanced in DHR, and this enhancement may play a role in the maintenance of hypertension in DHR. It is probable that enhanced activity of cholinergic neurons intrinsic to the RVLM is at least in part, responsible for the enhanced cholinergic transmission in the RVLM of DHR.
One of the hydrolysis products of sarin (isopropyl methylphosphonofluoridate) was detected in formalin-fixed brain tissues of victims poisoned in the Tokyo subway terrorist attack. Part of this procedure, used for the detection of sarin hydrolysis products in erythrocytes of sarin victims, has been described previously. The test materials were four individual cerebellums, which had been stored in formalin fixative for about 2 years. Sarin-bound acetylcholinesterase (AChE) was solubilized from these cerebellums, purified by immunoaffinity chromatography, and digested with trypsin. Then the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion, subjected to trimethylsilyl derivatization (TMS), and detected by gas chromatography-mass spectrometry. Peaks at m/z 225 and m/z 240, which are indicative of TMS-methylphosphonic acid, were observed within the retention time range of authentic methylphosphonic acid. However, no isopropyl methylphosphonic acid was detected in the formalin-fixed cerebellums of these 4 sarin victims, probably because the isopropoxy group of isopropyl methylphosphonic acid underwent chemical hydrolysis during storage. This procedure will be useful for the forensic diagnosis of poisoning by protein-bound, highly toxic agents, such as sarin, which are easily hydrolysed. This appears to be the first time that intoxication by a nerve agent has been demonstrated by analyzing formalin-fixed brains obtained at autopsy.
Title: Working memory failure by combined blockade of muscarinic and beta- adrenergic transmission in the rat hippocampus Ohno M, Kobayashi M, Kishi A, Watanabe S Ref: Neuroreport, 8:1571, 1997 : PubMed
INTRAHIPPOCAMPAL administration of the muscarinic acetylcholine receptor antagonist scopolamine at a dose of 3.2 micrograms/side significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points) in the working memory task with a three-panel runway setup, whereas 0.32 microgram/side scopolamine did not affect working memory errors. The beta-adrenoceptor antagonist propranolol (10 mg/kg, i.p.) had no effect on working memory error, but it produced a significant increase in working memory errors when administered in combination with intrahippocampal scopolamine at the behaviourally ineffective dose (0.32 microgram/side). The increase in working memory errors induced by intrahippocampal administration of 0.32 microgram/side scopolamine to rats treated with 10 mg/kg propranolol was decreased by concurrent injection of the cholinesterase inhibitor physostigmine (3.2 micrograms/side). D-Cycloserine (the partial agonist at the glycine bindings site on the NMDA receptor/channel complex) at a dose of 10 micrograms/side reduced the increase in working memory errors induced by intrahippocampal 0.32 microgram/side scopolamine combined with 10 mg/kg propranolol. These results suggest that neural mechanisms regulated cooperatively by hippocampal muscarinic and beta-adrenergic transmission underlie working memory performance, and that modification of NMDA function contributes to such interactive regulation of working memory processes in the hippocampus.
Title: Screening of novel microbial enzymes for the production of biologically and chemically useful compounds. Shimizu S, Ogawa J, Kataoka M, Kobayashi M Ref: Advances in Biochemical Engineering Biotechnology, 58:45, 1997 : PubMed
Enzymes have been generally accepted as superior catalysts in organic synthesis. Micro-organisms in particular have been regarded as treasure sources of useful enzymes. The synthetic technology using microbial enzymes or micro-organisms themselves is called microbial transformation. In designing a microbial transformation process, one of the most important points is to find a suitable enzyme for the reaction of interest. Various kinds of novel enzymes for specific transformations have been discovered in micro-organisms and their potential characteristics revealed. This article reviews our current results on the discovery of novel enzymes for the production of biologically and chemically useful compounds, and emphasizes the importance of screening enzymes in a diverse microbial world.
Title: [Synthesis of estimated metabolites of 9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta[b]quinoline monohydrochloride monohydrate (NIK-247). II. Synthesis of dihydroxylated metabolites] Komatsu T, Yano M, Iwamoto M, Kobayashi M, Suzuki K Ref: Yakugaku Zasshi, 115:1022, 1995 : PubMed
The two dihydroxylated metabolites of 9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta[b]quinoline monohydrochloride monohydrate (NIK-247), which is a new drug for the treatment of dementia, were synthesized to determine their chemical structures. Reduction of the tricyclic diketone, 9-amino-2,3,6,7-tetrahydro-1H-cyclopenta[b]quinoline-1,8(5H)-dione, with equivalent molar of NaBH4, afforded the racemic two alcohols, (+/-)-9-amino-2,3,5,6,7,8-hexahydro-8-hydroxy-1H-cyclopenta[b]quinoli n-1-on e and (+/-)-9-amino-2,3,5,6,7,8-hexahydro-1-hydroxy-1H-cyclopenta[b]quinoli n-8- one. (+)-9-Amino-2,3,5,6,7,8-hexahydro-8-hydroxy-1H-cyclopenta[b]quinolin+ ++-1-one was obtained by optical resolution of the corresponding racemic hydroxyketone using (-)-di-p-toluoyl-L-tartaric acid. The optically active dihydroxylated metabolites were obtained by reduction of the (+)-8-hydroxy-1-one with NaBH4.
The precise localization of an endothelin (ET) receptor subtype, the ETB receptor, in porcine lung was elucidated by in vitro microautoradiography using a novel ETB-selective radioligand, [125I]BQ-3020 ([125I-Tyr]-N-acetyl-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp -Ile-Ile-Trp). Of the labeled native ET isopeptides, [125I]ET-3 is selective for ETB receptors. However, [125I]ET-3 was not suitable for autoradiography due to its high degree of non-specific binding. On the other hand, [125I]BQ-3020 showed extremely low non-specific binding on autoradiography. The distribution of [125I]BQ-3020 binding in porcine lung was clearly different from that of [125I]ET-1, which showed more widespread binding than [125I]BQ-3020 due to a high affinity to both ETA and ETB receptors. [125I]BQ-3020 was found to bind to parenchyma, parasympathetic ganglia, pulmonary and submucosal plexuses, but bound only slightly to circular smooth muscle layers and the epithelium of airway tracts. Although [125I]ET-1 bound to the smooth muscle layer of all blood vessels, the binding of [125I]BQ-3020 differed among blood vessels. [125I]BQ-3020 binding in blood vessels paralleled acetylcholinesterase activity, suggesting that ETB receptors in blood vessels are located on parasympathetic nerves. Thus, the radioligand [125I]BQ-3020 is very useful for studying the precise localization of ETB receptors.
