Xyloglucan-cellulose interaction depends on the sidechains and molecular weight of xyloglucan.

Recent papers have brought evidence against the hypothesis that the fucosyl branching of primary wall xyloglucans (Xg) are responsible for their higher capacity of binding to cellulose. Reinforcement of this questioning has been obtained in this work where we show that the binding capacity was improved when the molecular weight (MW) of the Xg polymers is decreased by enzymatic hydrolysis. Moreover, the enthalpy changes associated with the adsorption process between Xg and cellulose is similar for Xgs with similar MW (but differing in the fine structure such as the presence/absence of fucose). On the basis of these results, we suggest that the fine structure and MW of Xg determines the energy and amount of binding to cellulose, respectively. Thus, the occurrence of different fine structural domains of Xg (e.g. the presence of fucose and the distribution of galactoses) might have several different functions in the wall. Besides the structural function in primary wall, these results might have impact on the packing features of storage Xg in seed cotyledons, since the MW and absence of fucose could also be associated with the self-association capacity.     

Development and neuromodulation of spinal locomotor networks in the metamorphosing frog

Metamorphosis in the anuran frog, Xenopus laevis, involves profound structural and functional transformations in most of the organism's physiological systems as it encounters a complete alteration in body plan, habitat, mode of respiration and diet. The metamorphic process also involves a transition in locomotory strategy from axial-based undulatory swimming using alternating contractions of left and right trunk muscles, to bilaterally-synchronous kicking of the newly developed hindlimbs in the young adult. At critical stages during this behavioural switch, functional larval and adult locomotor systems co-exist in the same animal, implying a progressive and dynamic reconfiguration of underlying spinal circuitry and neuronal properties as limbs are added and the tail regresses. To elucidate the neurobiological basis of this developmental process, we use electrophysiological, pharmacological and neuroanatomical approaches to study isolated in vitro brain stem/spinal cord preparations at different metamorphic stages. Our data show that the emergence of secondary limb motor circuitry, as it supersedes the primary larval network, spans a developmental period when limb circuitry is present but not functional, functional but co-opted into the axial network, functionally separable from the axial network, and ultimately alone after axial circuitry disappears with tail resorption. Furthermore, recent experiments on spontaneously active in vitro preparations from intermediate metamorphic stage animals have revealed that the biogenic amines serotonin (5-HT) and noradrenaline (NA) exert short-term adaptive control over circuit activity and inter-network coordination: whereas bath-applied 5-HT couples axial and appendicular rhythms into a single unified pattern, NA has an opposite decoupling effect. Moreover, the progressive and region-specific appearance of spinal cord neurons that contain another neuromodulator, nitric oxide (NO), suggests it plays a role in the maturation of limb locomotor circuitry. In summary, during Xenopus metamorphosis the network responsible for limb movements is progressively segregated from an axial precursor, and supra- and intra-spinal modulatory inputs are likely to play crucial roles in both its functional flexibility and maturation.     

Dorso-ventral limb polarity and origin of the ridge: On the fringe of independence?

Molecular and developmental studies of limb pattern formation have recently gained widespread attention. The fact that vertebrate limbs are amenable to both genetic and embryological manipulations has established this model system as a valuable paradigm for studying vertebrate development. Limb buds are polarised along all three major axes and the establishment of the dorso-ventral (DV) polarity is dependent upon cues localised in the trunk, where a DV ectodermal interface is produced by confrontation of dorsal and ventral identities. By analogy to Drosophila imaginal disc development, this interface has been proposed to determine and position an ectodermal organising centre, the Apical Ectodermal Ridge (AER), controlling limb bud outgrowth. Recent fate mapping studies⁽¹⁾ and studies of genes regulating DV limb polarity^(2-6), AER formation^(7,8) and differentiation⁽⁹⁾ suggest, however, that DV patterning and AER induction, though coordinately regulated during limb bud outgrowth, may early on be more dissociated than expected.     

Bioproduction of lauryl lactone and 4-vinyl guaiacol as value-added chemicals in two-phase biotransformation systems

Recombinant Escherichia coli whole-cell biocatalysts harboring either a Baeyer–Villiger monooxygenase or ferulic acid decarboxylase were employed in organic-aqueous two-phase bioreactor systems. The feasibility of the bioproduction of water-insoluble products, viz., lauryl lactone from cyclododecanone and 4-vinyl guaiacol from ferulic acid were examined. Using hexadecane as the organic phase, 10∼16 g of lauryl lactone were produced in a 3-l bioreactor that operated in a semicontinuous mode compared to 2.4 g of product in a batch mode. For the decarboxylation of ferulic acid, a new recombinant biocatalyst, ferulic acid decarboxylase derived from Bacillus pumilus, was constructed. Selected solvents as well as other parameters for in situ recovery of vinyl guaiacol were investigated. Up to 13.8 g vinyl guaiacol (purity of 98.4%) were obtained from 25 g of ferulic acid in a 2-l working volume bioreactor by using octane as organic phase. These selected examples highlight the superiority of the two-phase biotransformations systems over the conventional batch mode.     

Development of a novel expression,ZIMAX/KZI,for determination of the counter-anion effect on the antimicrobial activity of tetrabutylammonium salts

Due to the increasing number of strains of drug-resistant bacteria, the development of new antibiotics has become increasingly important. The antibacterial properties of quaternary amines and their derivatives on both Gram-positive and Gram-negative bacteria are well known. However, an encompassing study with specific emphasis on the role of the counter-anion has not been reported in the literature. By monitoring the Zone of Inhibition of various concentrations of tetrabutylammonium (TBA) salts, we observed that the counter anion plays a significant role in activity. We developed a novel method of reporting activity using zone of inhibition tests (ZI_MAX/K_ZI) and found it to be strongly correlated with the minimum inhibitory concentration (MIC).     

Molecular characterization of a second copy of holocarboxylase synthetase gene (hcs2) in Arabidopsis thaliana.

Holocarboxylase synthetase (HCS), catalyzing the covalent attachment of biotin, is ubiquitously represented in living organisms. Indeed, the biotinylation is a post-translational modification that allows the transformation of inactive biotin-dependent carboxylases, which are committed in fundamental metabolisms such as fatty acid synthesis, into their active holo form. Among other living organisms, plants present a peculiarly complex situation. In pea, HCS activity has been detected in three subcellular compartments and the systematic sequencing of the Arabidopsis genome revealed the occurrence of two hcs genes (hcs1 and hcs2). Hcs1 gene product had been previously characterized at molecular and biochemical levels. Here, by PCR amplification, we cloned an hcs2 cDNA from Arabidopsis thaliana (Ws ecotype) mRNA. We observed the occurrence of multiple cDNA forms which resulted from the alternative splicing of hcs2 mRNA. Furthermore, we evidenced a nucleotide polymorphism at the hcs2 gene within the Ws ecotype, which affected splicing of hcs2 mRNA. This contrasted sharply with the situation at hcs1 locus. However, this polymorphism had no apparent effect on total HCS activity in planta. Finally, hcs2 mRNAs were found 4-fold less abundant than hcs1 mRNA and the most abundant hcs2 mRNA spliced variant should code for a truncated protein. We discuss the possible role of such a multiplicity of putative HCS proteins in plants and discuss the involvement of each of hcs genes in the correct realization of biotinylation.     

Use of the LA 640 for a simple method of measuring the concentration of muscle lactate

A simple technique was elaborated for the measurement of muscle lactate concentration. It was tested on samples of muscle vastus lateralis taken by the Bergstr?m (1962) needle biopsy technique at the end of 20 min exercise bouts corresponding to 60-70% of Vo2 max. The biopsies were freshly frozen in liquid nitrogen, powdered and weighed in a cryostat at -20 degrees C. The extraction were made by saponine and the lactate measured in the saponine solution by an electrochemical-enzymatic method (LA 640). The results concern: the time of taking the biopsies and the freezing time (27 +/- 11 s and 34 +/- 9 s respectively); the accuracy of weighing (inducing a 1% uncertainty in the final result); a comparison of the saponine extraction with the perchloric acid extraction and a checking of the extraction capacity of the former; the accuracy of the whole measurement (the mean relative confidence limits are +/- 8-10%; the reproducibility of the technique through measurement of the variation coefficient (18%) calculated on measurements performed at a 15 days interval in 6 trained subjects. The discussion of the results and their comparison with those of the literature lead to the conclusion that the described method is suitable for muscle lactate measurements.     

Two-step purification and N-terminal amino acid sequence analysis of the rat Mr 90,000 heat shock protein

A fast and efficient method for the isolation of the rat Mr approximately 90,000 heat shock protein is presented, comprising a two-step high-performance anion-exchange and gel-permeation column chromatography. The Mr approximately 90,000 protein was purified to electrophoretic homogeneity in high yield (up to 2 mg per 10g of normal rat liver) in 4-5 h. The purified protein was recognized on protein immunoblots by monospecific rabbit antibodies raised against the rat glucocorticoid receptor-associated Mr approximately 90,000 non-ligand-binding protein. The N-terminal sequence of the first 25 amino acids of the purified protein showed a high degree of similarity with Mr approximately 90,000 heat shock proteins from calf, human, Drosophila, and yeast.