Molecular cloning, expression, and characterization of a novel endo-alpha-N-acetylgalactosaminidase from Enterococcus faecalis

We report here the molecular cloning, expression and characterization of a novel endo-alpha-N-acetylgalactosaminidase, classified into the GH101 family, from Enterococcus faecalis (endo-EF). The recombinant endo-EF was found to catalyze the liberation of core1-disaccharides (Galbeta1-3GalNAc) from core1-pNP (Galbeta1-3GalNAcalpha-pNP) like other GH101 family enzymes. However, endo-EF seems to differ in specificity from the GH101 enzymes reported to date, because it was able to release trisaccharides from core2-pNP (Galbeta1-3[GlcNAcbeta1-6]GalNAcalpha-pNP) and tetrasaccharides from Gal-core2-pNP (Galbeta1-3[Galbeta1-3GlcNAcbeta1-6]GalNAcalpha-pNP). Interestingly, the enzyme could transfer not only core1-disaccharides but also core2-trisaccharides to alkanols generating alkyl-oligosaccharides. Endo-EF should facilitate O-glycoprotein research.     

Remodeling of synaptic actin induced by photoconductive stimulation.

Use-dependent synapse remodeling is thought to provide a cellular mechanism for encoding durable memories, yet whether activity triggers an actual structural change has remained controversial. We use photoconductive stimulation to demonstrate activity-dependent morphological synaptic plasticity by video imaging of GFP-actin at individual synapses. A single tetanus transiently moves presynaptic actin toward and postsynaptic actin away from the synaptic junction. Repetitive spaced tetani induce glutamate receptor-dependent stable restructuring of synapses. Presynaptic actin redistributes and forms new puncta that label for an active synapse marker FM5-95 within 2 hr. Postsynaptic actin sprouts projections toward the new presynaptic actin puncta, resembling the axon-dendrite interaction during synaptogenesis. Our results indicate that activity-dependent presynaptic structural plasticity facilitates the formation of new active presynaptic terminals.     

Cryopreservation of hairy root cultures of Vinca minor (L.) by encapsulation-dehydration

Hairy root cultures of Vinca minor and Ajuga reptans var. atropurpurea could be cryopreserved when the roots were precultured and encapsulated in 2% (w/v) alginate beads with 0.3 M sucrose and 0.5 M glycerol and dehydrated until the bead weight reached 25% of the initial weight before cooling in liquid nitrogen. Preculture and encapsulation of the roots with abscisic acid was effective in increasing the survival rates. For V. minor root tips moreover a sufficiently high survival rate of more than 70% was attained by eliminating glycerol from the preculture medium and dehydration of beads until 23% of the initial weight was reached instead of 25%.     

Chemical reactivity of labile sulfur of iron-sulfur proteins The reaction of triphenyl phosphine

The reaction of triphenyl phosphine to iron-sulfur proteins from adrenal cortex mitochondria, spinach chloroplasts, and Clostridium pasteurianum was investigated. As ethanol concentrations in the reaction mixture increased, the rate of the reaction decreased. In the simultaneous presence of 1 M KCl and 5 M urea, the reaction rate reached at maximum. Under these conditions the initial rates of the decolorization reaction by the phosphine were found to be 8.7, 0.88, and 1.8 nmol of ferrodoxin per min at 25°C for adrenal, spinach, and clostridial ferredoxins, respectively. The kinetic curves for the reaction of the phosphine sulfide formation, the loss of labile sulfur, and the deterioriation of visible absorption showed a similar pattern with a comparable rate. During this reaction, the complete reduction of ferric ions present in ferredoxin was observed with a fast rate under either aerobic or anaerobic conditions.These results suggest that the iron atoms in ferredoxin are first reduced by the intramolecular reductants in the presence of triphenyl phosphine with the concomitant formation of S₂^2?, which then reacts with triphenyl phosphine resulting in the formation of triphenyl phosphine sulfide.     

Quantitation of fumonisins in corn by HPLC with o-phthalaldehyde postcolumn derivatization and their identification by LC/MS

Fumonisins B₁(FB₁) and B₂(FB₂) were isocratically separated on a fluorocarbon column without using an ion pair reagent and nonvolatile buffer during the HPLC and were detected by an o-phthalaldehyde postcolumn derivatization system using a fluorescence detector. The minimum detectable concentrations of FB₁ and FB₂ in corn by this system were 0.01 μg/g and 0.01 μg/g, respectively. The separated fumonisins were further identified by a directly interfaced ion trap MS using electrospray ionization. FB₁ and FB₂ in naturally contaminated corn were identified in the selective ion monitoring mode at concentrations of 3.75μg/g and 1.44 μg/g, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic variation and population structure of a threatened timber tree <Emphasis Type="Italic">Dalbergia cochinchinensis</Emphasis> in Cambodia

Dalbergia cochinchinensis Pierre ex Laness. (Fabaceae) is a commercially important tree in Southeast Asia. Although this species is under legal protections, illegal logging and disorderly developments have reduced its populations, and the conservation of this species is currently of much concern. In this study, we determined nucleotide sequences at six chloroplasts and ten nuclear loci in four populations of D. cochinchinensis in Cambodia, followed by population genetic analyses. The average silent nucleotide diversity over the nuclear loci, excluding one with an exceptionally high value, was 0.0057 in the entire population, and the mean F _ST across the nuclear loci between each population pair was between 0.135 and 0.467. Thus, the nucleotide diversity in the studied populations was not low compared with that in other tree species, and the level of population differentiation was high. Neutrality test statistics indicated a recent reduction of population size and a subdivision of the population within this species. The divergence times and migration rates were estimated with a likelihood-based method assuming the isolation with migration model. Based on the results, the three populations split 68,000–138,000 years ago, possibly corresponding to the start of the last glacial period, and the level of gene flow among the populations was very low thereafter. Moreover, after the split, population sizes were reduced considerably. Notably, the nucleotide diversity in an insertion sequence in a noncoding region of nuclear C4H was much higher than the mean nucleotide diversity in silent sites across other nuclear genes, indicating that the region was affected by selection.     

Actin in action: the interplay between the actin cytoskeleton and synaptic efficacy

Synapse regulation exploits the capacity of actin to function as a stable structural component or as a dynamic filament. Beyond its well-appreciated role in eliciting visible morphological changes at the synapse, the emerging picture points to an active contribution of actin to the modulation of the efficacy of pre- and postsynaptic terminals. Moreover, by engaging distinct pools of actin and divergent signalling pathways, actin-dependent morphological plasticity could be uncoupled from modulation of synaptic strength. The aim of this Review is to highlight some of the recent progress in elucidating the role of the actin cytoskeleton in synaptic function.