Efficient shoot organogenesis in petioles of yam (Dioscorea spp)

Shoot organogenesis was successfully achieved in petiole explants excised from 6 to 8-week old in vitro plantlets of yam Dioscorea rotundata P. cv. Kponan fissa, Dioscorea cayenensis L. cv. Krengle IB14 and cv. Krengle IB35, and Dioscorea alata L. cv. Bete bete. Only the basipetal portion of the petiole acquired competence, and plants regenerated within 21?days on MS medium supplemented with 2?% sucrose, 100?mg/l myo-inositol, 10???M kinetin and 1.5?mM putrescine referred to as yam regeneration medium (YRM). Plant regeneration was significantly (p?<?0.01) higher (42?%) with 2?% sucrose compared to 1, 3 and 4?% sucrose which produced 4, 25 and 15?% regeneration respectively. The age of the donor yam plantlet was critical to regeneration, and the highest regeneration efficiency was obtained with explants from 8-week old plantlets. Addition of the antioxidants lipoic acid (19.4???M), and l-cysteine (28.5???M) to the culture medium stimulated axillary branching in regenerated shoots. Among the four yam cultivars tested, cv. Kponan fissa, cv. Krengle IB14 and cv. Bete bete had similar response at 10???M kinetin, while the cv. Krengle IB35 regenerated best at a lower concentration of kinetin (0.5???M).     

Detection of disordered regions in globular proteins using 13C‐detected NMR

Characterization of disordered regions in globular proteins constitutes a significant challenge. Here, we report an approach based on ¹³C‐detected nuclear magnetic resonance experiments for the identification and assignment of disordered regions in large proteins. Using this method, we demonstrate that disordered fragments can be accurately identified in two homologs of menin, a globular protein with a molecular weight over 50 kDa. Our work provides an efficient way to characterize disordered fragments in globular proteins for structural biology applications.     

Empathy manipulation impacts music-induced emotions: a psychophysiological study on opera

This study investigated the effects of voluntarily empathizing with a musical performer (i.e., cognitive empathy) on music-induced emotions and their underlying physiological activity. N = 56 participants watched video-clips of two operatic compositions performed in concerts, with low or high empathy instructions. Heart rate and heart rate variability, skin conductance level (SCL), and respiration rate (RR) were measured during music listening, and music-induced emotions were quantified using the Geneva Emotional Music Scale immediately after music listening. Listening to the aria with sad content in a high empathy condition facilitated the emotion of nostalgia and decreased SCL, in comparison to the low empathy condition. Listening to the song with happy content in a high empathy condition also facilitated the emotion of power and increased RR, in comparison to the low empathy condition. To our knowledge, this study offers the first experimental evidence that cognitive empathy influences emotion psychophysiology during music listening.     

Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis

Most of the glycosyltransferases (GTs) that catalyze the formation of plant cell wall carbohydrates remain to be biochemically characterized. This can be achieved only if specific assays are available for these enzymes. Here we present a protocol for in vitro assays of processive and nonprocessive membrane-bound GTs. The assays are either based on the use of radioactive nucleotide sugars (NDP sugars; e.g., UDP-[U-(14)C]glucose) and the quantification of the radiolabeled monosaccharides incorporated into soluble or insoluble carbohydrates, or on the coupling of the GT reaction with that of pyruvate kinase (PK) and the oxidation of NADH by lactate dehydrogenase (LDH). The radiometric assays are more suitable for exploratory work on poorly characterized enzymes, whereas the spectrophotometric assays require the availability of highly enriched GTs. Both assays can be performed within 1 d, depending on the number of fractions to be assayed or reaction mixtures to be tested.     

The P450-4 Gene of Gibberella fujikuroi Encodes ent-Kaurene Oxidase in the Gibberellin Biosynthesis Pathway

At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a P450-4 knockout mutant identified ent-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized efficiently to GA₄. The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic acid after transformation with P450-4. The B1-41a mutant, described as blocked between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P450-4. There is a single nucleotide difference between the sequence of the B1-41a and wild-type P450-4 alleles at the 3′ consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that P450-4 encodes a multifunctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.     

Isolation of high quality RNA from seeds and tubers of the Mexican yam bean (Pachyrhizus erosus)

Pachyrhizus erosus is a tuberous legume native to Central America that has great potential for development as a food crop. It produces both protein rich grain and starch filled tubers. There are two major limitations to its dietary use, the high levels of rotenone found in the grains and the low starch content of the tubers, both of which must be addressed, for development of the crop. The low variability of the existing gene pool of the genus limits the use of conventional plant breeding to address these problems. Genetic engineering technology is, therefore, being adopted. For this purpose, an efficient means of RNA isolation from yam bean tissues was developed. The quality of RNA obtained by this method was tested byin vitro translation and was sufficient for use in RT-PCR.     

Matrix metalloproteinase-12 inhibitors: synthesis,structure-activity relationships and intestinal absorption of novel sugar-based biphenylsulfonamide carboxylates

MMP-12 is a validated target in pulmonary and cardiovascular diseases. The principal obstacles to clinical development of MMP-12 inhibitors are an inadequate selectivity for the target enzyme and a poor water solubility, with consequent poor oral bioavailability. We recently reported a new class of sugar-based arylsulfonamide carboxylates with a nanomolar activity for MMP-12, a good selectivity and an improved water solubility. In this study, we designed and synthesized new derivatives to characterize the structure-activity relationship (SAR) within this class of glycoconjugate inhibitors. All the new derivatives were tested on human recombinant MMP-12 and MMP-9 in order to evaluate their affinity and the selectivity for the target enzyme. Among them, the four most promising compounds were selected to assess their intestinal permeability using an ex vivo everted gut sac model. Given the high polarity and structural similarity to glucose, compound 3 was demonstrated to cross the intestinal membrane by using the facilitative GLUT2 transport.