Reactivity and stability of mycelium-bound carboxylesterase from Aspergillus oryzae.

The reactivity and thermostability of a novel mycelium-bound carboxylesterase from lyophilized cells of Aspergillus oryzae are explored in organic solvent. Ethanol acetylation was selected as reference esterification reaction. High carboxylesterase activity cells were used as biocatalyst in batch esterification tests at 12.5 < S(o) < 125 mmol L(-1), 5.0 < X(o) < 30 g L(-1), 0.49 < log P < 4.5 and 30 < T < 80 degrees C, as well as in residual activity tests after incubation at 40 < T < 90 degrees C. The starting rates of product formation were used to estimate with the Arrhenius model the apparent activation enthalpies of the enzymatic reaction (29-33 kJ mol(-1)), the reversible unfolding (56-63 kJ mol(-1)), and the irreversible denaturation (22 kJ mol(-1)) of the biocatalyst.     

Structure of human NMN adenylyltransferase. A key nuclear enzyme for NAD homeostasis.

Nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase alpha/beta-phosphodiesterases superfamily, catalyzes a universal step (NMN + ATP = NAD + PP(i)) in NAD biosynthesis. Localized within the nucleus, the activity of the human enzyme is greatly altered in tumor cells, rendering it a promising target for cancer chemotherapy. By using a combination of single isomorphous replacement and density modification techniques, the human NMNAT structure was solved by x-ray crystallography to a 2.5-A resolution, revealing a hexamer that is composed of alpha/beta-topology subunits. The active site topology of the enzyme, analyzed through homology modeling and structural comparison with other NMNATs, yielded convincing evidence for a substrate-induced conformational change. We also observed remarkable structural conservation in the ATP-recognition motifs GXXXPX(T/H)XXH and SXTXXR, which we take to be the universal signature for NMNATs. Structural comparison of human and prokaryotic NMNATs may also lead to the rational design of highly selective antimicrobial drugs.     

Ozone Quenching Properties of Isoprene and Its Antioxidant Role in Leaves

Isoprene is formed in and emitted by plants and the reason for this apparent carbon waste is still unclear. It has been proposed that isoprene stabilizes cell and particularly chloroplast thylakoid membranes. We tested if membrane stabilization or isoprene reactivity with ozone induces protection against acute ozone exposures. The reduction of visible, physiological, anatomical, and ultrastructural (chloroplast) damage shows that clones of plants sensitive to ozone and unable to emit isoprene become resistant to acute and short exposure to ozone if they are fumigated with exogenous isoprene, and that isoprene-emitting plants that are sensitive to ozone do not suffer damage when exposed to ozone. Isoprene-induced ozone resistance is associated with the maintenance of photochemical efficiency and with a low energy dissipation, as indicated by fluorescence quenching. This suggests that isoprene effectively stabilizes thylakoid membranes. However, when isoprene reacts with ozone within the leaves or in a humid atmosphere, it quenches the ozone concentration to levels that are less or non-toxic for plants. Thus, protection from ozone in plants fumigated with isoprene may be due to a direct ozone quenching rather than to an induced resistance at membrane level. Irrespective of the mechanism, isoprene is one of the most effective antioxidants in plants.     

Molecular Characterization and Identification of Bacillus clausii Strains Marketed for Use in Oral Bacteriotherapy

A substantial number of Bacillus species have been marketed for use in oral bacteriotherapy because of their purported ability to prevent or treat various gastrointestinal disorders. Recently, some of the Bacillus strains in Enterogermina, which is made up of aqueous suspensions of viable Bacillus spores, have been partially characterized and aligned with members of the Bacillus alcalophilus subgroup rather than with Bacillus subtilis, as previously reported. With a view toward verifying the original taxonomic position of the Enterogermina strains, we catalogued both phenotypic and genotypic traits exhibited by the four Bacillus strains isolated from the spore mixtures found in original commercial preparations dated 1975 and 1984 and commercial preparations now being propagated industrially. Analyses of physiological and biochemical traits, complete 16S rRNA gene sequences, DNA-DNA reassociation, tRNA intergenic spacer length polymorphism, single-strand conformation polymorphism of PCR-amplified spacer regions of tRNA genes, and randomly amplified polymorphic DNA led to the finding that all of the Enterogermina strains belong to a unique genospecies, which is unequivocally identified as the alkalitolerant species Bacillus clausii. Moreover, we provide evidence that in contrast to several reference strains of B. clausii, the strains constituting Enterogermina are characterized by a notable low level of intraspecific genome diversity and that each strain has remained the same for the last 25 years.     

Antibiotic-induced DNA methylation changes in calluses of Arabidopsis thaliana

This study was carried out to determine the involvement of the antibiotic kanamycin, commonly used as a selective agent in transformation protocols, in the phenomenon of somaclonal variation. Both genetic and epigenetic events were looked for. Two complementary approaches were used to evaluate global methylation changes in the genome of callus derived from the leaves of Arabidopsis thaliana L.; immunolabelling using monoclonal-antibodies raised against 5-methylcytosine in order to define the relative abundance of methylated cytosine; the analysis of methylation-sensitive polymorphism technique (MSAP) to assess methylation changes at CCGG sequences. In addition, the same samples were analysed using AFLPs in order to determine the extent of genomic change with respect to callus grown in the absence of kanamycin and plants grown from seed. The use of kanamycin as a selective agent caused extensive methylation changes in the genome with both hyper- and hypomethylation events seen. However, the net result was genome-wide hypomethylation: this effect was dosage dependent; the higher the dose, the greater the effect. At the same time, sequence mutation was detected.     

Adventitious shoot regeneration from leaf explants and stem nodes of Lilium

A method for the regeneration of lily plantlets (Lilium spp.) through different morphogenic pathways is described. Plant regeneration was obtained from in vitro cultured leaves of four lily hybrids, cultured on Murashige and Skoog's basal medium supplemented with cytokinins (TDZ and BA) and auxins (NAA and IBA) at different concentrations. Direct shoot regeneration occurred with all tested media for the Asiatic lilies `Elite' and `Pollyanna' and also for the Oriental hybrid `Star Gazer'. Callus developed on TDZ-enriched medium from leaf segments of L. longiflorum cv. `Snow Queen' regenerated by direct organogenesis. This occurred on a medium with auxin/ cytokinin balance which was lower than other genotypes. There were fewer problems of sterilization with leaves from sprouted bulbs than in vitro scale culture. This suggests that the leaf-segments obtained in this way could be an alternative to the scales as a source of material for propagation. A protocol for micropropagation based on bulblets from in vitro shoot-tip-derived stem nodes was also used. The development of pseudo-bulbets is particularly advantageous since it allows for structures characterised by absent or low dormancy. Regenerated shoots have been rooted and successfully acclimatized to greenhouse conditions where they flowered after the second year giving plants with true-to-type shape and colour.     

Ethiopians and Khoisan Share the Deepest Clades of the Human Y-Chromosome Phylogeny

The genetic structure of 126 Ethiopian and 139 Senegalese Y chromosomes was investigated by a hierarchical analysis of 30 diagnostic biallelic markers selected from the worldwide Y-chromosome genealogy. The present study reveals that (1) only the Ethiopians share with the Khoisan the deepest human Y-chromosome clades (the African-specific Groups I and II) but with a repertoire of very different haplotypes; (2) most of the Ethiopians and virtually all the Senegalese belong to Group III, whose precursor is believed to be involved in the first migration out of Africa; and (3) the Ethiopian Y chromosomes that fall into Groups VI, VIII, and IX may be explained by back migrations from Asia. The first observation confirms the ancestral affinity between the Ethiopians and the Khoisan, which has previously been suggested by both archaeological and genetic findings.     

The influence of canopy algae on vertical patterns of distribution of low-shore assemblages on rocky coasts in the northwest Mediterranean

Canopy-forming algae have been shown to play a fundamental role in the maintenance of understorey assemblages on rocky shores. In the Mediterranean, low-shore habitats are often monopolised by canopy algae of the genus Cystoseira and, in particular, by the species Cystoseira amentacea var. stricta. Alternatively, low-shore habitats are dominated by turf-forming algae and/or mussels. Previous studies showed that loss of Cystoseira, by natural or anthropogenic disturbances, resulted in the increase of turfing algae. Here, we propose that turf-forming algae may act as colonisers from nearby habitats, specifically from lower portions of the shore. The following hypotheses were tested to investigate this proposition: (1) in presence of Cystoseira, the assemblage living under its canopy will be distinct, in terms of composition and structure, from that found lower on the shore, (2) if the canopy of Cystoseira was removed, the differences between the two assemblages would decrease or disappear, (3) the effects of the canopy would be more important than other causes of variability at the spatial scale investigated (hundreds to thousands of metres), and (4) that effects of removal of the canopy would be consistent through time. These hypotheses were tested by means of a canopy removal experiment, involving several spatial and temporal repetitions of the manipulation.The assemblage underneath the canopy of Cystoseira was distinct from that found immediately lower on the shore, but when the canopy was removed there were no differences between the two. The effects of the canopy were consistent at the spatial and temporal scales investigated. Several species living under the canopy disappeared and were replaced by turf-forming species, resulting in a homogenisation of the two habitats and in a drastic loss of habitat diversity. This study shows that Cystoseira can be considered a habitat-forming species, responsible for the maintenance of two distinct low-shore assemblages. In this light, its importance should be taken into account by policies aiming to preserve biodiversity on rocky shores.