Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.     

Fractionated irradiation improves the mating performance of the West Indian sweet potato weevil Euscepes postfasciatus

• 1 The sterile insect technique (SIT) is widely used to suppress or eradicate target pest insect populations.
• 2 The effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females. The use of gamma radiation to induce sterility, however, negatively affects both somatic cells as well as reproductive cells. Consequently, mating performance of sterilized individuals decreases drastically over time. The mating propensity of sterilized Euscepes postfasciatus (Fairmaire) males irradiated with a single dose of 150 Gy (the current standard of the Okinawa Prefecture SIT programme) is equal to that of non‐irradiated weevils for the first 6 days.
• 3 Fractionated irradiation, in which a sterilizing dose is delivered over time in a series of smaller irradiations, reduces the damage of irradiation in insects. In the present study, we evaluated the effect of fractionated irradiation on male fertilization ability, longevity and mating propensity of E. postfasciatus for a period of 16 days after irradiation.
• 4 Although fractionated irradiation totalling 150 Gy was found to induce full sterility regardless of the number of individual doses, the mating propensity of male weevils sterilized by fractionated irradiation was maintained for the first 12 days. These results demonstrate that fractionated irradiation can be highly advantageous in programmes aimed at eradication of E. postfasciatus.

     

Cellulase applied to the leaves of sweet pepper (Capsicum annuum L. var. grossum) upregulates the production of salicylic and azelaic acids

Treating the leaves of sweet pepper (Capsicum annuum L. var. grossum) with an aqueous solution of cellulase resulted in a four-fold increase in the salicylic acid level compared to a control plant. The level of endogenous azelaic acid was also elevated by the cellulase treatment. Azelaic acid has recently been reported to act as a mobile "priming" agent to arm plants against pathogenic attack. Our results are consistent with this and that the cellulase treatment enhanced the ability of sweet pepper to withstand viral attack.     

Cell fate polarization in ascidian mesenchyme/muscle precursors by directed FGF signaling and role for an additional ectodermal FGF antagonizing signal in notochord/nerve cord precursors

Asymmetric cell division plays a fundamental role in generating various types of embryonic cell. In ascidian embryos, asymmetric cell divisions occur in the vegetal hemisphere in a manner similar to those found in Caenorhabditis elegans. Early divisions in embryos of both species involve inductive events on a single mother cell that result in production of daughters with different cell fates. Here we show in the ascidian Halocynthia roretzi that polarity of muscle/mesenchyme mother precursors is determined solely by the direction from which the FGF9/16/20 signal is presented, a role similar to that of Wnt signaling in the EMS and T cell divisions in C. elegans. However, polarity of nerve cord/notochord mother precursors is determined by possible antagonistic action between the FGF signal and a signal from anterior ectoderm, providing a new mechanism underlying asymmetric cell division. The ectoderm signal suppresses MAPK activation and expression of Hr-FoxA, which encodes an intrinsic competence factor for notochord induction, in the nerve cord lineage.     

Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities

NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon-carbon-based and carbon-oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism.     

Functional analysis of mutations in the PIS gene, which encodes Saccharomyces cerevisiae phosphatidylinositol synthase

Abstract The PIS gene for an enzyme phosphatidylinositol synthase having an increased K _m for myo-inositol, was isolated from Saccharomyces cerevisiae . The mutant PIS gene contained a CAA codon at position 114 instead of the CAC codon observed in the wild-type gene, resulting in alteration of the amino acid from His to Gln. Oligonucleotide mediated site-directed mutagenesis of PIS at codon 114 revealed that mutant genes with codons for Ala, Thr and Leu could support yeast cell growth in vivo, but those for Asp, Lys and Tyr could not. All mutant enzymes when expressed in Escherichia coli showed greatly reduced in vitro activity.     

Treatment of a depressive disorder patient with EEG-driven photic stimulation

This study examined the effects of electroencephalographic-(EEG-) driven photic stimulation on a case of depressive disorder, as measured by a psychometric test of mood states, EEG parameters, and several autonomic indices. The EEG-driven photic stimulation enhances the alpha rhythm of brain waves using photic signals, the brightness of which is modulated by a subject's own alpha rhythm. The patient was a 37-year-old businessman, who was treated for depression with medication during the 13 months prior to his first visit to our hospital. He underwent two sets of inpatient treatment sessions, comprising first 16 and then 18 treatment sessions. The treatments brought about the following changes: an improvement in general mood state, alpha rhythm increase, cardiac parasympathetic suppression, and increased skin conductance level. In addition, significant correlations between alpha rhythm increase and cardiac parasympathetic suppression or cardiac sympathetic predominance were observed with each inpatient treatment. Significant correlations between alpha rhythm increase, cardiac parasympathetic suppression, or cardiac sympathetic predominance and the improvement of general mood state were also observed. Thus, from these observations, it was concluded that the alpha enhancement induced by EEG-driven photic stimulation produced an improvement in the patient's depressive symptomatology connected with cardiac parasympathetic suppression and sympathetic predominance.     

BIOGEOGRAPHY AND TAXONOMY OF BATRACHOSPERMUM HELMINTHOSUM (BATRACHOSPERMALES,RHODOPHYTA) IN JAPAN INFERRED FROM RBCL GENE SEQUENCES1

Twenty‐seven individuals of Batrachospermum helminthosum Bory were collected from various sites in Japan, from temperate Iwate Prefecture to subtropical Okinawa Prefecture. The chloroplast‐encoded rbcL gene was sequenced from each sample. Phylogenetic analyses were performed using these and previously published sequences of 12 samples from North America. There were five haplotypes among the Japanese samples. Haplotype 1 was collected throughout a large geographical area corresponding to most of the sampling range in this study and was represented by most individuals (21 individuals). The other haplotypes were represented by one to three individuals, respectively. These distributions of haplotypes suggest the occurrence of one large metapopulation and/or recent colonization over a wide geographical area in Japan. Phylogenetic analysis showed two well‐supported clades within B. helminthosum, one clade containing four haplotypes (1–4) from Japan and the other clade comprising five North American haplotypes. Such differences between Japanese and North American haplotypes might be a result of continental drift and subsequent isolation in the Mesozoic era. The position of haplotype 5 from Okinawa in Japan is poorly resolved, but it is not closely related to the other four Japanese haplotypes. It is suggested that several taxonomic characteristics (sexuality, point of origin of carpogonium‐bearing branches, and trichogynes with or without basal knobs or branching) is not fixed genetically but is affected by environmental conditions. Based on the results of the present study, B. coerulescens Sirodot and B. elegans Sirodot should be placed in synonymy with B. helminthosum.     

Histochemical quantification of dopa-oxidase (tyrosinase) activity in epidermal melanocytes.

The graphical method of Lineweaver-Burk was applied to the histochemical system to obtain quantitative information on the dopa-oxidase activity in human epidermal melanocytes. In vitro experiment showed that the incubation time necessary for yielding a certain amount of dopa-melanin is related to the amount of enzyme present. In the case of histochemical experiment, the time required for the first appearance of dopa-melanin deposition in melanocytes at various substrate concentrations was plotted against the reciprocal of the substrate concentration. This technique made it possible to compare the dopa-oxidase activity at various sites of the tissue.