Molecular cloning and heterologous expression of novel glucosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxin.

Scopoletin is one of the phytoalexins in tobacco. Cells of the T-13 cell line (Nicotiana tabacum L. Bright Yellow) accumulate a large amount of scopoletin, also known as 7-hydroxy-6-methoxycoumarin, as a glucoconjugate, scopolin, in vacuoles. We report here the molecular cloning of glucosyltransferases that can catalyze the glucosylation of many kinds of secondary metabolites including scopoletin. Two cDNAs encoding glucosyltransferase (NtGT1a and NtGT1b) were isolated from a cDNA library derived from the tobacco T-13 cell line by screening with heterologous cDNAs as a probe. The deduced amino-acid sequences of NtGT1a and NtGT1b exhibited 92% identity with each other, approximately 20-50% identities with other reported glucosyltransferases. Heterologous expression of these genes in Escherichia coli showed that the recombinant enzymes had glucosylation activity against both flavonoids and coumarins. They also strongly reacted with 2-naphthol as a substrate. These recombinant enzymes can utilize UDP-glucose as the sugar donor, but they can also utilize UDP-xylose as a weak donor. RNA blot analysis showed that these genes are induced by salicylic acid and auxin, but the time course of the expression was different. This result is similar to the changes in scopoletin glucosylation activity in these tobacco cells after addition of these plant growth regulators. These results might suggest that one of the roles of the products of these genes is scopoletin glucosylation, in response to salicylic acid and/or auxin, together with the other glucosyltransferases in tobacco cells.     

A cold-adapted protease engineered by experimental evolution system.

A new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which we developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K(m) value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V. The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51. In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.     

Predictive and Prognostic Value of Metabolic Tumor Volume (MTV) in Patients with Laryngeal Carcinoma Treated by Radiotherapy (RT) / Concurrent Chemoradiotherapy (CCRT)

PurposeTo evaluate the predictive and prognostic value of pretreatment metabolic tumor volume (MTV) in patients with treated by radiotherapy (RT) or concurrent chemoradiotherapy (CCRT).MethodsWe reviewed the records of 118 patients with newly diagnosed laryngeal carcinoma, who had been treated by RT or CCRT. Pretreatment positron emission tomography (PET) was performed, and MTV values were obtained by contouring margins of standardized uptake value. Clinical factors and MTV were analyzed for their association with survival.ResultsPatients with residual disease showed a significantly higher MTV than those with a complete response (CR) after primary treatment. Univariate analysis showed that the patients with a high MTV had a significantly lower disease-free survival (DFS) (p < 0.001). Subsite (p = 0.010), T-stage (p < 0.001), nodal metastasis (p < 0.001) and clinical stage (p < 0.001) also correlated significantly with DFS. In the multivariate analysis, MTV and clinical stage were both found to be independent prognostic factors for DFS (p = 0.001, p = 0.034, respectively). The 3-year DFS for patients with a high MTV were significantly poorer than those with a low MTV (p < 0.001).ConclusionsMTV of the primary tumor is a significant prognostic factor for DFS in patients with laryngeal carcinoma treated by RT or CCRT. The results imply that MTV could be an important factor when planning treatment and follow-up for patients with laryngeal carcinoma.     

Dynamic Modulation of Thymidylate Synthase Gene Expression and Fluorouracil Sensitivity in Human Colorectal Cancer Cells

Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS) expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU) treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.     

Trigonelline Content in Coffee Beans and the Thermal Conversion of Trigonelline into Nicotinic Acid during the Roasting of Coffee Beans

The trigonelline content in coffee was determined by the microbiological assay method after demethylating the compound. The content was proved to be extremely high (up to 1 % on the wet basis). When trigonelline was heated to above 180°C, it was converted into nicotinic acid. Although the conversion rate was low, a nutritionally significant amount of nicotinic acid was formed during roasting coffee beans because of the high content of trigonelline in coffee beans. The optimum heating condition for nicotinic acid formation was found at 220°C for 20 min.     

Combined antiallodynic effect of Neurotropin® and pregabalin in rats with L5-spinal nerve ligation

AimsIn this study, we investigated the combined effect of Neurotropin® and pregabalin for L5-spinal nerve ligation (L5-SNL) model in rats and thiopental-induced sleep in mice.Main methodsThe left fifth lumbar nerve of rats was tightly ligated with silk sutures under pentobarbital anesthesia. The hindpaw withdrawal threshold was measured by application of von Frey filaments. Thiopental sodium was intravenously administered in mice and sleeping time was measured. In L5-SNL rats, an isobolographic analysis was performed to clarify the combined antiallodynic effect of Neurotropin and pregabalin 14 days after ligation in rats. In isobolographic analysis and thiopental-induced sleep test, Neurotropin and pregabalin were orally administered to coincide with the timing of the peak effect of each drug.Key findingsNeurotropin (50–200 NU/kg) and pregabalin (2.5–10 mg/kg) showed a dose-dependent antiallodynic action in L5-SNL rats. The antiallodynic effect of pregabalin was reversed by intrathecal injection of yohimbine or ondansetron. Isobolographic analysis suggested that the combined antiallodynic effect of Neurotropin and pregabalin in L5-SNL rats may have been more than a mere additive effect. Neurotropin (50–400 NU/kg) had no effect on thiopental-induced sleeping time whereas pregabalin (30–100 mg/kg) significantly prolonged it. When the dose of pregabalin was 30 mg/kg, Neurotropin (50–400 NU/kg) did not further exacerbate the prolongation effect of pregabalin on thiopental-induced sleep.SignificanceIt was suggested that when Neurotropin was administered in combination with pregabalin, it might provide more effective pain relief than that obtained with each agent alone in neuropathic pain without aggravating adverse effects of pregabalin.     

Effects of Mutations in the Substrate-Binding Domain of Poly[(R)-3-Hydroxybutyrate] (PHB) Depolymerase from Ralstonia pickettii T1 on PHB Degradation

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZ_RpiT1) adsorbs to denatured PHB (dPHB) via its substrate-binding domain (SBD) to enhance dPHB degradation. To evaluate the amino acid residues participating in dPHB adsorption, PhaZ_RpiT1 was subjected to a high-throughput screening system consisting of PCR-mediated random mutagenesis targeted to the SBD gene and a plate assay to estimate the effects of mutations in the SBD on dPHB degradation by PhaZ_RpiT1. Genetic analysis of the isolated mutants with lowered activity showed that Ser, Tyr, Val, Ala, and Leu residues in the SBD were replaced by other residues at high frequency. Some of the mutant enzymes, which contained the residues replaced at high frequency, were applied to assays of dPHB degradation and adsorption, revealing that those residues are essential for full activity of both dPHB degradation and adsorption. These results suggested that PhaZ_RpiT1 adsorbs on the surface of dPHB not only via hydrogen bonds between hydroxyl groups of Ser in the enzyme and carbonyl groups in the PHB polymer but also via hydrophobic interaction between hydrophobic residues in the enzyme and methyl groups in the PHB polymer. The L441H enzyme, which displayed lower dPHB degradation and adsorption abilities, was purified and applied to a dPHB degradation assay to compare it with the wild-type enzyme. The kinetic analysis of the dPHB degradation suggested that lowering the affinity of the SBD towards dPHB causes a decrease in the dPHB degradation rate without the loss of its hydrolytic activity for the polymer chain.     

Identification of cDNAs encoding pterocarpan reductase involved in isoflavan phytoalexin biosynthesis in Lotus japonicus by EST mining

Isoflavans and pterocarpans are the major biosynthetically connected phytoalexins in legumes. A search of the expressed sequence tag library of a model legume Lotus japonicus, which produces an (-)-isoflavan, for homologs of phenylcoumaran benzylic ether reductase catalyzing the reductive cleavage of dihydrofurans, yielded seven full-length cDNAs, and the encoded proteins were analyzed in vitro. Four of them cleaved the dihydrofuran of a pterocarpan medicarpin to yield an isoflavan (-)-vestitol and were designated pterocarpan reductase (PTR). Two PTRs displayed enantiospecificity to (-)-medicarpin, representing genuine L. japonicus PTRs, while the other two lacked enantiospecificity and were presumed to be evolutionarily primitive types.