Role of Ngamma-acetyldiaminobutyrate as an enzyme stabilizer and an intermediate in the biosynthesis of hydroxyectoine.

Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC). This strain accumulates large amounts of Ngamma-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794-25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A (1)H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated that H. elongata can synthesize hydroxyectoine by two different pathways-directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.     

Cytotoxicity and genotoxicity of chitooligosaccharides upon lymphocytes

Two COS mixtures and a low molecular weight chitosan (LMWC) were tested for potential cytotoxicity and genotoxicity upon human lymphocytes. Genotoxicity was evaluated in vitro by cytokinesis-blocked micronucleus and alkaline comet assays, while cytotoxicity was assessed by flow cytometry analysis. Our results suggest that COS do not exhibit any genotoxicity upon human lymphocytes, independently of MW or concentration. However, above 0.07 mg/mL COS induced strong cytotoxic effects. According to the concentration used, such cytotoxicity will induce cell death, essentially by necrosis (>0.10 mg/mL) and/or apoptosis (<0.10 mg/mL). The level of necrosis/apoptosis induced by high COS concentrations, suggests a promising use as apoptosis inducers in specific cancer situations.     

Diversity of ndo genes in mangrove sediments exposed to different sources of polycyclic aromatic hydrocarbon pollution

Polycyclic aromatic hydrocarbon (PAH) pollutants originating from oil spills and wood and fuel combustion are pollutants which are among the major threats to mangrove ecosystems. In this study, the composition and relative abundance in the sediment bacterial communities of naphthalene dioxygenase (ndo) genes which are important for bacterial adaptation to environmental PAH contamination were investigated. Three urban mangrove sites which had characteristic compositions and levels of PAH compounds in the sediments were selected. The diversity and relative abundance of ndo genes in total community DNA were assessed by a newly developed ndo denaturing gradient gel electrophoresis (DGGE) approach and by PCR amplification with primers targeting ndo genes with subsequent Southern blot hybridization analyses. Bacterial populations inhabiting sediments of urban mangroves under the impact of different sources of PAH contamination harbor distinct ndo genotypes. Sequencing of cloned ndo amplicons comigrating with dominant DGGE bands revealed new ndo genotypes. PCR-Southern blot analysis and ndo DGGE showed that the frequently studied nah and phn genotypes were not detected as dominant ndo types in the mangrove sediments. However, ndo genotypes related to nagAc-like genes were detected, but only in oil-contaminated mangrove sediments. The long-term impact of PAH contamination, together with the specific environmental conditions at each site, may have affected the abundance and diversity of ndo genes in sediments of urban mangroves.     

A rapid exocytosis mode in chromaffin cells with a neuronal phenotype

We have used astrocyte-conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM-promoted neuronal phenotype, secretory vesicles and intracellular Ca2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre-spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7-9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM-treated cells correspond to the exocytosis of large dense-core vesicles (LDCV). On the other hand, experiments performed in EGTA-loaded cells suggest that ACM treatment promotes a better coupling between voltage-gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC.     

Absence of microspore polarity, symmetric divisions and pollen cell fate in Brachiaria decumbens (Gramineae).

Meiotic division and male gametophyte development were analyzed in one tetraploid (2n = 4x = 36) accession of Brachiaria decumbens cv. Basilisk that showed some pollen sterility. Meiotic process was typical of polyploids in that it consisted of multiple chromosome associations. Precocious chromosome migration to the poles, laggards, and micronucleus formation were abundant in both meiosis I and II and resulted in tetrads with micronuclei. After callose dissolution, microspores were released into the anther locule and had the semblance of being normal. Although each microspore initiated its differentiation by pollen mitosis, in 43.24% of the microspores, nuclear polarization was not observed and the typical hemispherical cell plate was not detected. Division was symmetric and microspores lacked differentiation between the vegetative and the generative cell. Both nuclei were of equal size, presented equal chromatin condensation, and had a spherical shape. After the first pollen mitosis and cytokinesis, each cell underwent a new symmetric mitosis without nuclear polarization. At the end of the second pollen mitosis, four equal nuclei were observed in each pollen grain. After the second cytokinesis, the cells gave rise to four equal-sized pollen grains with a similar tetrad configuration that initially remained together. Sterile pollen grains resulted from abnormal pollen mitosis. This anomaly may be explained by a mutation, probably affecting microtubule cytoskeleton formation. The importance of this male-sterile mutation for Brachiaria breeding programs is discussed.