Regioselective glucosidation of <Emphasis Type="Italic">trans</Emphasis>-resveratrol in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing glucosyltransferase from <Emphasis Type="Italic">Phytolacca americana</Emphasis>

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.     

Host plant use for oviposition by Trigonotylus caelestialium (Hemiptera: Miridae) and Stenotus rubrovittatus (Hemiptera: Miridae)

The main hosts and sites of oviposition for the two bugs, Trigonotylus caelestialium (Kirkaldy) (Hemiptera: Miridae) and Stenotus rubrovittatus (Matsumura) (Hemiptera: Miridae), that cause pecky rice were investigated in 24 poaceous plants. Nymphs of T. caelestialium emerged from both spikelets and leaf sheaths, while nymphs of S. rubrovittatus emerged almost exclusively from spikelets. Suitable plants for oviposition by T. caelestialium are Lolium multiflorum, Digitaria violascens and Hordeum murinum, while Poa annua, Anthoxanthum odoratum, Alopecurus aequalis and D. violascens were preferentially used by S. rubrovittatus. There was a greater difference in the number of nymphs emerging from different plants for S. rubrovittatus than for T. caelestialium. This difference may be because T. caelestialium can oviposit on leaf sheaths if the spikelets are not suitable for oviposition, whereas S. rubrovittatus only oviposits on spikelets. Although both bugs oviposited on spikelets, the internal oviposition sites were different. In D. ciliaris, T. caelestialium laid all eggs between the lemma of the first floret and the second floret, whereas S. rubrovittatus laid eggs almost exclusively inside the second floret. In contrast, in P. annua, T. caelestialium laid all eggs inside the florets, whereas S. rubrovittatus laid eggs both between and inside the florets.     

Distribution of pink-pigmented facultative methylotrophs on leaves of vegetables

The distribution of pink-pigmented facultative methylotrophs (PPFMs) on the leaves of various vegetables was studied. All kinds of vegetable leaves tested gave pink-pigmented colonies on agar plates containing methanol as sole carbon source. The numbers of PPFMs on the leaves, colony-forming units (CFU)/g of fresh leaves, differed among the plants, although they were planted and grown at the same farm. Commercial green perilla, Perilla frutescens viridis (Makino) Makino, gave the highest counts of PPFMs (2.0-4.1×10(7) CFU/g) of all the commercial vegetable leaves tested, amounting to 15% of total microbes on the leaves. The PPFMs isolated from seeds of two varieties of perilla, the red and green varieties, exhibited high sequence similarity as to the 16S rRNA gene to two different Methylobacterium species, M. fujisawaense DSM5686(T) and M. radiotolerans JCM2831(T) respectively, suggesting that there is specific interaction between perilla and the PPFMs.     

Msn5p is involved in formaldehyde resistance but not in oxidative stress response in the methylotrophic yeast Candida boidinii

Methylotrophic yeasts, which can utilize methanol as sole carbon and energy source, are exposed to two toxic metabolic intermediates, formaldehyde and hydrogen peroxide, during growth on methanol. Here we report that Msn5p, an importin-β family nuclear exporter, participated in the formaldehyde resistance mechanism but not in the hydrogen peroxide resistance mechanism in Candida boidinii. Disruption of the MSN5 gene in this yeast caused retardation of growth on formaldehyde-generating growth substrates such as methanol and methylamine, but the expression levels of the methanol-metabolizing enzymes did not fall. The Msn5p-depleted strain was sensitive to formaldehyde but not to hydrogen peroxide. Furthermore, a yellow fluorescent protein-tagged Msn5p was diffuse in the cytoplasm of C. boidinii when the cells were treated with high concentrations of formaldehyde or ethanol, but was predominantly associated with the nuclei following treatment with hydrogen peroxide.     

Glycosylation of trans-Resveratrol by Plant-Cultured Cells

Plant-cultured cells of Catharanthus roseus converted trans-resveratrol into its 3-O-β-D-glucopyranoside, 4'-O-β-D-glucopyranoside, 3-O-(6-O-β-D-xylopyranosyl)-β-D-glucopyranoside, and 3-O-(6-O-α-L-arabinopyranosyl)-β-D-glucopyranoside. The 3-O-(6-O-β-D-xylopyranosyl)-β-D-glucopyranoside and 3-O-(6-O-α-L-arabinopyranosyl)-β-D-glucopyranoside compounds of trans-resveratrol are both new. Incubation of plant-cultured cells of Ipomoea batatas and Strophanthus gratus with trans-resveratrol gave trans-resveratrol 3-O-β-D-glucopyranoside and trans-resveratrol 4'-O-β-D-glucopyranoside.     

Secreted Nucleobindin-2 Inhibits 3T3-L1 Adipocyte Differentiation

Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARγ, aP2, and adipsin). When Nucleobindin- 2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.     

Effect of mycelial morphology on ergothioneine production during liquid fermentation of Lentinula edodes

The morphological type of a microorganism generally influences its metabolite production. In the present study, we investigated the effects of the mycelial morphology of shiitake (Lentinula edodes) on the production of 2-mercaptohistidine trimethylbetaine (ergothioneine, ESH) during liquid fermentation. Analyses of the distribution of ESH in mycelial cells of different morphological types revealed that the ESH content of pellets obtained from the liquid fermentation media was much greater than the content in the free mycelia and clumps. The concentration of ESH in pellets on day 15 of liquid fermentation reached 0.79 mg/g dry weight (DW), which is approximately three times the concentration found in mycelia clumps (0.28 mg/g DW) and free mycelia (0.31 mg/g DW). Macroscopic image analysis of the development and morphological changes of the pellets during a liquid fermentation period of up to 25 days indicated that pellet growth showed a highly positive correlation with the increase in ESH concentration (r ² = 0.9851). A reduced agitation rate of 50 rpm for the culture medium was suitable for pellet formation and size enlargement. The results obtained in this work would be helpful in guiding the intentional manipulation of the distribution and enrichment of ESH in L. edodes through changes in liquid fermentation conditions.