Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants

By using barley seeds, developmental changes of ADPglucose (ADPG)-producing sucrose synthase (SS) and ADPG pyrophosphorylase (AGPase) have been compared with those of UDPglucose (UDPG), ADPG, sucrose (Suc) and starch contents. Both ADPG-synthesizing SS and AGPase activity patterns were found to correlate well with those of ADPG and starch contents. Remarkably, however, maximal activities of ADPG-synthesizing SS were found to be several fold higher than those of AGPase throughout seed development, the highest rate of starch accumulation being well accounted for by SS. Kinetic analyses of SS from barley endosperms and potato tubers in the Suc cleavage direction showed similar K(m) values for ADP and UDP, whereas apparent affinity for Suc was shown to be higher in the presence of UDP than with ADP. Moreover, measurements of transglucosylation activities in starch granules incubated with purified SS, ADP and [U-(14)C]Suc revealed a low inhibitory effect of UDP. The ADPG and UDPG contents in the transgenic S-112 SS and starch deficient potato mutant [Zrenner et al. (1995) Plant J. 7: 97] were found to be 35% and 30% of those measured in wild-type plants, whereas both glucose-1-phosphate and glucose-6-phosphate contents were found to be normal as compared with those of wild-type plants. The overall results thus strongly support a novel gluconeogenic mechanism reported previously [Pozueta-Romero et al. (1999) CRIT: Rev. Plant Sci. 18: 489] wherein SS catalyses directly the de novo production of ADPG linked to starch biosynthesis in heterotrophic tissues of plants.     

Oligomeric State in the Crystal Structure of Modular FAD Synthetase Provides Insights into Its Sequential Catalysis in Prokaryotes

The crystal structure of the modular flavin adenine dinucleotide (FAD) synthetase from Corynebacterium ammoniagenes has been solved at 1.95 Å resolution. The structure of C. ammoniagenes FAD synthetase presents two catalytic modules—a C-terminus with ATP-riboflavin kinase activity and an N-terminus with ATP-flavin mononucleotide (FMN) adenylyltransferase activity—that are responsible for the synthesis of FAD from riboflavin in two sequential steps. In the monomeric structure, the active sites from both modules are placed 40 Å away, preventing the direct transfer of the product from the first reaction (FMN) to the second catalytic site, where it acts as substrate. Crystallographic and biophysical studies revealed a hexameric assembly formed by the interaction of two trimers. Each trimer presents a head-tail configuration, with FMN adenylyltransferase and riboflavin kinase modules from different protomers approaching the active sites and allowing the direct transfer of FMN. Experimental results provide molecular-level evidences of the mechanism of the synthesis of FMN and FAD in prokaryotes in which the oligomeric state could be involved in the regulation of the catalytic efficiency of the modular enzyme.     

The C-terminal extension of bacterial flavodoxin-reductases: Involvement in the hydride transfer mechanism from the coenzyme

To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD:NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP⁺ than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the C-terminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding.     

Engineers have more sons, nurses have more daughters: an evolutionary psychological extension of Baron-Cohen's extreme male brain theory of autism

In his extreme male brain theory of autism, Baron-Cohen postulates that having a typically male brain was adaptive for ancestral men and having a typically female brain was adaptive for ancestral women. He also suggests that brain types are substantially heritable. These postulates, combined with the insight from the Trivers-Willard hypothesis regarding parental ability to vary offspring sex ratio, lead to the prediction that people who have strong male brains should have more sons than daughters, and people who have strong female brains should have more daughters than sons. The analysis of the 1994 US General Social Survey data provides support for this prediction. Our results suggest potentially fruitful extensions of both Baron-Cohen's theory and the Trivers-Willard hypothesis.     

Structural backgrounds for the formation of a catalytically competent complex with NADP(H) during hydride transfer in ferredoxin–NADP+ reductases

The role of the highly conserved C266 and L268 of pea ferredoxin–NADP⁺ reductase (FNR) in formation of the catalytically competent complex of the enzyme with NADP(H) was investigated. Previous studies suggest that the volume of these side-chains, situated facing the side of the C-terminal Y308 catalytic residue not stacking the flavin isoalloxazine ring, may be directly involved in the fine-tuning of the catalytic efficiency of the enzyme. Wild-type pea FNR as well as single and double mutants of C266 and L268 residues were analysed by fast transient-kinetic techniques and their midpoint reduction potentials were determined. For the C266A, C266M and C266A/L268A mutants a significant reduction in the overall hydride transfer (HT) rates was observed along with the absence of charge-transfer complex formation. The HT rate constants for NADPH oxidation were lower than those for NADP⁺ reduction, reaching a 30-fold decrease in the double mutant. In agreement, these variants exhibited more negative midpoint potentials with respect to the wild-type enzyme. The three-dimensional structures of C266M and L268V variants were solved. The C266M mutant shows a displacement of E306 away from the relevant residue S90 to accommodate the bulky methionine introduced. The overall findings indicate that in FNR the volume of the residue at position 266 is essential to attain the catalytic architecture between the nicotinamide and isoalloxazine rings at the active site and, therefore, for an efficient HT process. In addition, flexibility of the 268–270 loop appears to be critical for FNR to achieve catalytically competent complexes with NADP(H).     

Antimicrobial effects of terpinen-4-ol against oral pathogens and its capacity for the modulation of gene expression

Abstract

This study evaluated the antibacterial activity of terpinen-4-ol against Streptococcus mutans and Lactobacillus acidophilus and its influence on gbpA (S. mutans) and slpA (L. acidophilus) gene expression. As measured by XTT assay, the concentrations of terpinen-4-ol that effectively inhibited the biofilm were 0.24% and 0.95% for S. mutans and L. acidophilus, respectively. Confocal microscopy revealed the presence of a biofilm attached to the enamel and dentin block surfaces with significant terpinen-4-ol effects against these microorganisms. The expression of the gbpA and slpA genes involved in adherence and biofilm formation was investigated using RT-PCR. Expression of these genes decreased after 15?min with 0.24% and 0.95% terpinen-4-ol in S. mutans and L. acidophilus, respectively. These findings demonstrate the antimicrobial activity of terpinen-4-ol and its ability to modulate the expression of gbpA and slpA genes, emphasizing the therapeutic capacity of terpinen-4-ol as an alternative to inhibit adherence in biofilm.     

Proceedings of the symposium on brain mechanisms of taste recognition memory and neural plasticity held at the ninth European congress of psychology on 6 July 2005

Research on the neurobiological substrates of taste-relatedlearning and memory is a broad and rapidly evolving multidisciplinaryfield. Historically, research on taste aversion learning hascontributed critically both to the behavioral and the neurobiologicalassessment of learning systems. Since the discovery by Garciaet al. (1955) of conditioned taste aversion and its peculiarfeatures, such as one-trial and long-delay learning, taste aversionlearning represented a challenge for learning theorists andcontributed decisively to the development of modern learningtheories. Also, the search