Root parasitic plants such as Striga, Orobanche, and Phelipanche spp. cause serious damage to crop production world-wide. Deletion of the Low Germination Stimulant 1 (LGS1) gene gives a Striga-resistance trait in sorghum (Sorghum bicolor). The LGS1 gene encodes a sulfotransferase-like protein, but its function has not been elucidated. Since the profile of strigolactones (SLs) that induce seed germination in root parasitic plants is altered in the lgs1 mutant, LGS1 is thought to be an SL biosynthetic enzyme. In order to clarify the enzymatic function of LGS1, we looked for candidate SL substrates that accumulate in the lgs1 mutants and performed in vivo and in vitro metabolism experiments. We found the SL precursor 18-hydroxycarlactonoic acid (18-OH-CLA) is a substrate for LGS1. CYP711A cytochrome P450 enzymes (SbMAX1 proteins) in sorghum produce 18-OH-CLA. When LGS1 and SbMAX1 coding sequences were co-expressed in Nicotiana benthamiana with the upstream SL biosynthesis genes from sorghum, the canonical SLs 5-deoxystrigol and 4-deoxyorobanchol were produced. This finding showed that LGS1 in sorghum uses a sulfo group to catalyze leaving of a hydroxyl group and cyclization of 18-OH-CLA. A similar SL biosynthetic pathway has not been found in other plant species.
Title: Chemical identification of 18-hydroxycarlactonoic acid as an LjMAX1 product and in planta conversion of its methyl ester to canonical and non-canonical strigolactones in Lotus japonicus Mori N, Sado A, Xie X, Yoneyama K, Asami K, Seto Y, Nomura T, Yamaguchi S, Akiyama K Ref: Phytochemistry, 174:112349, 2020 : PubMed
Strigolactones (SLs) are a group of plant apocarotenoids that act as rhizosphere signaling molecules for both arbuscular mycorrhizal fungi and root parasitic plants. They also regulate plant architecture as phytohormones. The model legume Lotus japonicus (synonym of Lotus corniculatus) produces canonical 5-deoxystrigol (5DS) and non-canonical lotuslactone (LL). The biosynthesis pathways of the two SLs remain elusive. In this study, we characterized the L. japonicus MAX1 homolog, LjMAX1, found in the Lotus japonicus genome assembly build 2.5. The L. japonicus max1 LORE1 insertion mutant was deficient in 5DS and LL production. A recombinant LjMAX1 protein expressed in yeast microsomes converted carlactone (CL) to 18-hydroxycarlactonoic acid (18-OH-CLA) via carlactonoic acid (CLA). Identity of 18-OH-CLA was confirmed by comparison of the methyl ester derivative of the MAX1 product with chemically synthesized methyl 18-hydroycarlactonoate (18-OH-MeCLA) using LC-MS/MS. (11R)-CL was detected as an endogenous compound in the root of L. japonicus.(13)C-labeled CL, CLA, and 18-OH-MeCLA were converted to [(13)C]-5DS and LL in plant feeding experiments using L. japonicus WT. These results showed that LjMAX1 is the crucial enzyme in the biosynthesis of Lotus SLs and that 18-hydroxylated carlactonoates are possible precursors for SL biosynthesis in L. japonicus.
Title: Identification of two oxygenase genes involved in the respective biosynthetic pathways of canonical and non-canonical strigolactones in Lotus japonicus Mori N, Nomura T, Akiyama K Ref: Planta, 251:40, 2020 : PubMed
A cytochrome P450 and a 2-oxoglutarate-dependent dioxygenase genes responsible, respectively, for the biosyntheses of canonical and non-canonical strigolactones in Lotus japonicus were identified by transcriptome profiling and mutant screening. Strigolactones (SLs) are a group of apocarotenoids with diverse structures that act as phytohormones and rhizosphere signals. The model legume Lotus japonicus produces both canonical and non-canonical SLs, 5-deoxystrigol (5DS) and lotuslactone (LL), respectively, through oxidation of a common intermediate carlactone by the cytochrome P450 (CYP) enzyme MAX1. However, the pathways downstream of MAX1 and the branching point in the biosyntheses of 5DS and LL have not been elucidated. Here, we identified a CYP and a 2-oxoglutarate-dependent dioxygenase (2OGD) genes responsible, respectively, for the formation of Lotus SLs by transcriptome profiling using RNA-seq and screening of SL-deficient mutants from the Lotus retrotransposon 1 (LORE1) insertion mutant resource. The CYP and 2OGD genes were named DSD and LLD, respectively, after 5DS or LL defective phenotype of the mutants. The involvements of the genes in Lotus SL biosyntheses were confirmed by restoration of the mutant phenotype using Agrobacterium rhizogenes-mediated transformation to generate transgenic roots expressing the coding sequence. The transcript levels of DSD and LLD in roots as well as the levels of 5DS and LL in root exudates were reduced by phosphate fertilization and gibberellin treatment. This study can provide the opportunity to investigate how and why plants produce the two classes of SLs.
Title: High cholinesterase predicts tolerance to sorafenib treatment and improved prognosis in patients with transarterial chemoembolization refractory intermediate stage hepatocellular carcinoma Takaki S, Fukuhara T, Mori N, Tsuji K Ref: Mol Clin Oncol, 12:60, 2020 : PubMed
Although sorafenib is the standard treatment for patients with advanced hepatocellular carcinoma (HCC), the predictive factors sorafenib tolerance in intermediate-stage HCC cannot be accurately determined. The aim of the current study was to identify the predictive characteristics for the continuation of sorafenib treatment (>/=400 mg) in patients with transarterial chemoembolization (TACE)-refractory intermediate HCC and to identify candidates for second-line sorafenib treatment. A total of 33 TACE-refractory intermediate patients with HCC that were treated with sorafenib, and who had reached progressive disease (PD), were analyzed in the present retrospective study. Of 33 patients, 6 patients (18.1%) were able to continue sorafenib treatment (>/=400 mg) until PD, however, a total of 27 patients (71.9%) were unable to continue treatment (<400 mg). The current study compared the baseline characteristics parameters to sorafenib >/=400 mg and <400 mg using a logistic regression model. The overall survival (OS) of patients receiving sorafenib >/=400 mg treatment was significantly increased compared with patients receiving sorafenib treatment <400 mg [554.5 days (228-674) vs. 219 days (134-369); P=0.0315). A univariate analysis was performed and indicated that Age (<75 years; P=0.021), total cholesterol (>180 mg/dl; P=0.026) and cholinesterase (ChE; >/=220 U/l; P=0.024) were significant factors, and a multivariate analysis indicated that ChE (>/=220 U/l) was a significant prognostic factor (HR: 11.9; 95% CI: 1.19-118.0; P=0.004). Both progression-free survival [279 (204-403) vs. 117.5 (63-197) days; P=0.0136] and OS [470 (277-679) vs. 171.5 (80-236) days; P=0.0004] were significantly increased in patients with ChE levels >/=220 U/l compared with patients exhibiting ChE levels <220 U/l. Baseline high value of ChE in intermediate-stage HCC predicts the ability to continue sorafenib treatment at >/=400 mg.
Strigolactones (SLs) regulate important aspects of plant growth and stress responses. Many diverse types of SL occur in plants, but a complete picture of biosynthesis remains unclear. In Arabidopsis thaliana, we have demonstrated that MAX1, a cytochrome P450 monooxygenase, converts carlactone (CL) into carlactonoic acid (CLA) and that LBO, a 2-oxoglutarate-dependent dioxygenase, can convert methyl carlactonoate (MeCLA) into a metabolite called [MeCLA + 16 Da]. In the present study, feeding experiments with deuterated MeCLAs revealed that [MeCLA + 16 Da] is hydroxymethyl carlactonoate (1'-HO-MeCLA). Importantly, this LBO metabolite was detected in plants. Interestingly, other related compounds, methyl 4-hydroxycarlactonoate (4-HO-MeCLA) and methyl 16-hydroxycarlactonoate (16-HO-MeCLA), were also found to accumulate in lbo mutants. 3-HO-, 4-HO-, and 16-HO-CL were detected in plants, but their expected corresponding metabolites, HO-CLAs, were absent in max1 mutants. These results suggest that HO-CL derivatives may be predominant SLs in Arabidopsis, produced through MAX1 and LBO.
Root exudates from Lotus japonicus were found to contain at least three different hyphal branching-inducing compounds for the arbuscular mycorrhizal (AM) fungus Gigaspora margarita, one of which had been previously identified as (+)-5-deoxystrigol (5DS), a canonical strigolactone (SL). One of the two remaining unknown hyphal branching inducers was purified and named lotuslactone. Its structure was determined as methyl (E)-2-(3-acetoxy-2-hydroxy-7-methyl-1-oxo-1,2,3,4,5,6-hexahydroazulen-2-yl)-3-(((R)-4-methyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)acrylate, by 1D and 2D NMR spectroscopy, and HR-ESI- and EI-MS. Although lotuslactone, a non-canonical SL, contains the AB-ring and the enol ether-bridged D-ring, it lacks the C-ring and has a seven-membered cycloheptadiene in the A-ring part as in medicaol, a major SL of Medicago truncatula. Lotuslactone was much less active than 5DS, but showed comparable activity to methyl carlactonoate (MeCLA) in inducing hyphal branching of G. margarita. Other natural non-canonical SLs including avenaol, heliolactone, and zealactone (methyl zealactonoate) were also found to be moderate to weak inducers of hyphal branching in the AM fungus. Lotuslactone strongly elicited seed germination in Phelipanche ramosa and Orobanche minor, but Striga hermonthica seeds were 100-fold less sensitive to this stimulant.
Strigolactones (SLs) are carotenoid-derived plant secondary metabolites that play important roles in various aspects of plant growth and development as plant hormones, and in rhizosphere communications with symbiotic microbes and also root parasitic weeds. Therefore, sophisticated regulation of the biosynthesis, perception and functions of SLs is expected to promote symbiosis of beneficial microbes including arbuscular mycorrhizal (AM) fungi and also to retard parasitism by devastating root parasitic weeds. We have developed SL mimics with different skeletons, SL biosynthesis inhibitors acting at different biosynthetic steps, SL perception inhibitors that covalently bind to the SL receptor D14, and SL function inhibitors that bind to the serine residue at the catalytic site. In greenhouse pot tests, TIS108, an azole-type SL biosynthesis inhibitor effectively reduced numbers of attached root parasites Orobanche minor and Striga hermonthica without affecting their host plants; tomato and rice, respectively. AM colonization resulted in weak but distinctly enhanced plant resistance to pathogens. SL mimics can be used to promote AM symbiosis and to reduce the application rate of systemic-acquired resistance inducers which are generally phytotoxic to horticultural crops. (c) 2019 Society of Chemical Industry.
The parasitic plant Striga hermonthica has been causing devastating damage to the crop production in Africa. Because Striga requires host-generated strigolactones to germinate, the identification of selective and potent strigolactone agonists could help control these noxious weeds. We developed a selective agonist, sphynolactone-7, a hybrid molecule originated from chemical screening, that contains two functional modules derived from a synthetic scaffold and a core component of strigolactones. Cooperative action of these modules in the activation of a high-affinity strigolactone receptor ShHTL7 allows sphynolactone-7 to provoke Striga germination with potency in the femtomolar range. We demonstrate that sphynolactone-7 is effective for reducing Striga parasitism without impinging on host strigolactone-related processes.
Strigolactones (SLs) are a class of plant hormones which regulate shoot branching and function as host recognition signals for symbionts and parasites in the rhizosphere. However, steps in SL biosynthesis after carlactone (CL) formation remain elusive. This study elucidated the common and diverse functions of MAX1 homologs which catalyze CL oxidation. We have reported previously that ArabidopsisMAX1 converts CL to carlactonoic acid (CLA), whereas a rice MAX1 homolog has been shown to catalyze the conversion of CL to 4-deoxyorobanchol (4DO). To determine which reaction is conserved in the plant kingdom, we investigated the enzymatic function of MAX1 homologs in Arabidopsis, rice, maize, tomato, poplar and Selaginella moellendorffii. The conversion of CL to CLA was found to be a common reaction catalyzed by MAX1 homologs, and MAX1s can be classified into three types: A1-type, converting CL to CLA; A2-type, converting CL to 4DO via CLA; and A3-type, converting CL to CLA and 4DO to orobanchol. CLA was detected in root exudates from poplar and Selaginella, but not ubiquitously in other plants examined in this study, suggesting its role as a species-specific signal in the rhizosphere. This study provides new insights into the roles of MAX1 in endogenous and rhizosphere signaling.
Title: Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in arbuscular mycorrhizal fungi Mori N, Nishiuma K, Sugiyama T, Hayashi H, Akiyama K Ref: Phytochemistry, 130:90, 2016 : PubMed
Hyphal branching in the vicinity of host roots is a host recognition response of arbuscular mycorrhizal fungi. This morphological event is elicited by strigolactones. Strigolactones are carotenoid-derived terpenoids that are synthesized from carlactone and its oxidized derivatives. To test the possibility that carlactone and its oxidized derivatives might act as host-derived precolonization signals in arbuscular mycorrhizal symbiosis, carlactone, carlactonoic acid, and methyl carlactonoate as well as monohydroxycarlactones, 4-, 18-, and 19-hydroxycarlactones, were synthesized chemically and evaluated for hyphal branching-inducing activity in germinating spores of the arbuscular mycorrhizal fungus Gigaspora margarita. Hyphal branching activity was found to correlate with the degree of oxidation at C-19 methyl. Carlactone was only weakly active (100 ng/disc), whereas carlactonoic acid showed comparable activity to the natural canonical strigolactones such as strigol and sorgomol (100 pg/disc). Hydroxylation at either C-4 or C-18 did not significantly affect the activity. A series of carlactone analogues, named AD ester and AA'D diester, was synthesized by reacting formyl Meldrum's acid with benzyl, cyclohexylmethyl, and cyclogeranyl alcohols (the A-ring part), followed by coupling of the potassium enolates of the resulting formylacetic esters with the D-ring butenolide. AD ester analogues exhibited moderate activity (1 ng-100 pg/disc), while AA'D diester analogues having cyclohexylmethyl and cyclogeranyl groups were highly active on the AM fungus (10 pg/disc). These results indicate that the oxidation of methyl to carboxyl at C-19 in carlactone is a prerequisite but BC-ring formation is not essential to show hyphal branching activity comparable to that of canonical strigolactones.
CONTEXT: Both neuropsychological and functional magnetic resonance imaging studies have shown deficiencies in face perception in subjects with autism spectrum disorders (ASD). The fusiform gyrus has been regarded as the key structure in face perception. The cholinergic system is known to regulate the function of the visual pathway, including the fusiform gyrus. OBJECTIVES: To determine whether central acetylcholinesterase activity, a marker for the cholinergic system, is altered in ASD and whether the alteration in acetylcholinesterase activity, if any, is correlated with their social functioning. DESIGN: Using positron emission tomography and a radiotracer, N-[(11)C]methyl-4-piperidyl acetate ([(11)C]MP4A), regional cerebrocortical acetylcholinesterase activities were estimated by reference tissue-based linear least-squares analysis and expressed in terms of the rate constant k(3). Current and childhood autism symptoms in the adult subjects with ASD were assessed by the Autism Diagnostic Observation Schedule and the Autism Diagnostic Interview-Revised, respectively. Voxel-based analyses as well as region of interest-based methods were used for between-subject analysis and within-subject correlation analysis with respect to clinical variables. SETTING: Participants recruited from the community. PARTICIPANTS: Twenty adult subjects with ASD (14 male and 6 female; age range, 18-33 years; mean [SD] intelligence quotient, 91.6 [4.3]) and 20 age-, sex-, and intelligence quotient-matched healthy controls. RESULTS: Both voxel- and region of interest-based analyses revealed significantly lower [(11)C]MP4A k(3) values in the bilateral fusiform gyri of subjects with ASD than in those of controls (P < .05, corrected). The fusiform k(3) values in subjects with ASD were negatively correlated with their social disabilities as assessed by Autism Diagnostic Observation Schedule as well as Autism Diagnostic Interview-Revised. CONCLUSIONS: The results suggest that a deficit in cholinergic innervations of the fusiform gyrus, which can be observed in adults with ASD, may be related to not only current but also childhood impairment of social functioning.
Title: Donepezil and concurrent sertraline treatment is associated with increased hippocampal volume in a patient with depression Suda S, Sugihara G, Suyama R, Mori N, Takei N Ref: J Clin Psychiatry, 71:806, 2010 : PubMed
Title: Partial cloning of the rat choline acetyltransferase gene and in situ localization of its transcripts in the cell body of cholinergic neurons in the brain stem and spinal cord Mori N, Tajima Y, Sakaguchi H, Vandenbergh DJ, Nawa H, Salvaterra PM Ref: Brain Research Mol Brain Res, 17:101, 1993 : PubMed
We have isolated recombinant lambda (lambda) phages which contain a part of the rat choline acetyltransferase (ChAT) gene. Restriction and Southern blot analyses using synthetic oligonucleotides indicate that these clones overlap one another and contain at least four exons which reside in 16.4 kb of sequence encoding from the middle to the 3' end, but not the 5'-region, of the rat ChAT gene. Partial sequence analyses revealed that the clones contain an exon whose nucleotide sequence corresponds to a highly conserved region of ChAT during evolution. RNase protection mapping experiments show that sequences represented by this exon are expressed at high levels in the spinal cord of adult rats and at low but detectable levels in PC12 cells. By using the genomic sequences, including the exon, as a hybridization probe, we have detected ChAT mRNAs in situ in rat tissues. In situ hybridization experiments using radioactive and non-radioactive probes revealed that cholinergic motoneurons in the spinal cord, the laterodorsal tegmental nucleus as well as the hypoglossal nucleus in the brain stem were labeled, suggesting that the genomic sequence can be used as a probe to measure the ChAT mRNA levels in those cholinergic neurons. The results also indicate that the non-radioactive method gives a better resolution in localizing the expression of ChAT transcripts in the cytoplasm of cholinergic neurons.
In a Japanese patient with familial LPL deficiency, a new null allelic mutation, one base pair deletion at nucleotide position 916 was identified in exon 5 of one allele. In exon 3 of the other allele, we found the same nonsense mutation as we described previously in other Japanese kindreds. For the deletional mutant allele, we developed a simple detection method and constructed the DNA haplotype.
The DNA sequences were determined for the lipoprotein lipase (LPL) gene from five unrelated Japanese patients with familial LPL deficiency. The results demonstrated that all five patients are homozygotes for distinct point mutations dispersed throughout the LPL gene. Patient 1 has a G-to-A transition at the first nucleotide of intron 2, which abolishes normal splicing. Patient 2 has a nonsense mutation in exon 3 (Tyr61----Stop) and patient 3 in exon 8 (Trp382----Stop). The latter mutation emphasizes the importance of the carboxy-terminal portion of the enzyme in the expression of LPL activity. Missense mutations were identified in patient 4 (Asp204----Glu) and patient 5 (Arg243----His) in the strictly conserved amino acids. Expression study of both mutant genes in COS-1 cells produced inactive enzymes, establishing the functional significance of the two mis-sense mutations. In these patients, postheparin plasma LPL mass was either virtually absent (patients 1 and 2) or significantly decreased (patients 3-5). To detect these mutations more easily, we developed a rapid diagnostic test for each mutation. We also determined the DNA haplotypes for patients and confirmed the occurrence of multiple mutations on the chromosomes with an identical haplotype. These results demonstrate that familial LPL deficiency is a heterogeneous genetic disease caused by a wide variety of gene mutations.
Title: Evolutionary origin of cholinergic macromolecules and thyroglobulin Mori N, Itoh N, Salvaterra PM Ref: Proc Natl Acad Sci U S A, 84:2813, 1987 : PubMed
We have compared the amino acid sequences of proteins that are involved in acetylcholine (AcCho) metabolism and cholinergic neurotransmission: choline acetyltransferase (ChoAcTase), acetylcholinesterase (AcChoEase), and a neuronal alpha subunit of nicotinic AcCho receptor (AcChoR). A comparison of Drosophila ChoAcTase and rat neuronal alpha subunit of AcChoR shows a limited segmental type homology, which may suggest a similar acetylcholine binding site in the two proteins evolving by convergence. We note a global homology of 21-44% identity between Drosophila ChoAcTase and Torpedo AcChoEase. Six homologous segments of 40-60 amino acids cover 38% and 54% of the sequences, raising the possibility of a common evolutionary origin. We also note that mammalian thyroglobulin (TG), the precursor for thyroid hormones, contains an AcChoEase-like sequence at its carboxyl end. This homology raises the possibility that the gene for TG has evolved by gene fusion or condensation (i.e., recruiting a preexisting redundant copy of a gene for AcChoEase during vertebrate evolution). Our results demonstrate that the record of evolutionary history for nervous system proteins can be read across the boundaries of separation between vertebrates and invertebrates. They also provide molecular evidence for the common evolutionary origins of the nervous and endocrine systems in vertebrates--both evolving to make intercellular communication possible.
Title: A butyrylcholinesterase inhibitor produced by Penicillium sp. no. C-81 and its identity with mycelianamide Nagasawa T, Mori N, Tani Y, Ogata K Ref: J Antibiot (Tokyo), 29:526, 1976 : PubMed
A screening aimed at obtaining a cholinesterase inhibitor of microbial origin was carried out using Pseudomonas butyrylcholinesterase. A mycelium-extract of a fungus strain, belonging to the genus Penicillium, was found to produce such an enzyme inhibitor. The inhibitor was purified and crystallized as colorless leaflets. From physical and chemical studies, the inhibitor was identified as being identical with an antibiotic, mycelianamide, though this compound was not known to have enzyme inhibitor activity. The kinetics of the inhibition of Pseudomonas butyrylcholinesterase were also studied. Horse serum cholinesterase and hog liver carboxylesterase were also inhibited by the isolated Penicillium C-81 inhibitor, but lipase and acetylcholinesterase were not.
