Prokaryotic Community Characterization in a Mesothermic and Water- Flooded Oil Reservoir in Colombia

The prokaryotic community at the “La Cira-Infantas” oil field, located in Colombia's Middle Magdalena Valley Basin, was characterized using 16S rRNA gene sequence analysis. This characterization is a first-step in assessing the dynamics of microbial degradation and defining strategies that may increase oil recovery and quality at the site. Two 16S rRNA gene libraries were generated from the total community DNA extracted from production water using both Eubacterial and Archaea universal primers. Sequence analysis of the libraries indicated that a large percentage of Eubacteria clones were affiliated with class α-, β- and δ-Proteobacteria, Clostridia and Bacteroidetes. Archaea clones were dominated by Methanobacteria and Methanococci. Annotations at these generic levels indicate that the prokaryotic community has the following metabolic capacities: i) reduction of sulfur-compounds and fermentation, ii) nitrate reduction and sulfide oxidation, iii) decomposition of aromatic compounds, and iv) methane production. These results are discussed in the context of the importance of the characterized metabolic capacities for oil biodegradation in the mesothermic and water-flooded environment of this reservoir.     

Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica

Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC₅₀ was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca²⁺ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis.     

The time-course of cyclic AMP signaling is critical for leukemia U-937 cell differentiation

The regulation of the cAMP signaling is intimately involved in several cellular processes, including cell differentiation. Here, we provide strong evidence supporting that the time-course of cAMP signal is critical for leukemia U-937 cell differentiation. Three stimulating-cAMP agents were used to analyze the correlation between cAMP time-course and cell differentiation. All three agents denoted similar cAMP maximal responses in dose-response experiments. The kinetic of desensitization showed differential characteristics, while H2 receptor desensitized homologously without affecting PGE2 or forskolin effect, PGE2 response showed mixed desensitization characterized by a homologous initial phase followed by a heterologous phase. Regarding forskolin, long-term stimuli attenuated PGE2 and H2 agonist response without affecting adenylyl cyclase activity. In the absence of phosphodiesterase inhibitors, the three agents induced similar maximal cAMP levels after 5 min, but only that induced by the H2 agonist returned to basal levels. Consistent with this observation, H2 agonist was not able to induce U-937 cell maturation in contrast to PGE2 and forskolin, supporting the importance of time-course signaling in the determination of cell behavior.     

Characterization of a cry2A Gene Cloned from an Isolate of Bacillus thuringiensis serovar sotto

A cry2A-type gene, designated as cry2(SKW), was cloned from Bacillus thuringiensis serovar sotto SKW01-10.2-06, and some unique features of the gene were revealed. The cry2(SKW) gene encoded a polypeptide of 635 residues with a predicted molecular mass of 71,137 Da. Cry2(SKW) had 95.4% identity with Cry2Aa in amino acid sequence and was two residues longer than Cry2Aa. Two open reading frames (ORFs), designated as orf1 and orf2, were present upstream of the cry2(SKW) and showed high homology with the corresponding ORFs in the cry2Aa operon. The Orf2 from SKW01-10.2-06 contained a region of repeated sequences. However, unlike Cry2Aa, Cry2(SKW) formed the cuboidal crystalline inclusions when the cry2(SKW) gene was expressed in an acrystalliferous B. thuringiensis strain in the absence of the upstream ORFs. Furthermore, Cry2(SKW) was less toxic to a lepidopteran species, Bombyx mori, than Cry2Aa in spite of high homology between the two proteins. Received: 17 July 1996 / Accepted: 5 December 1996     

Catalytic properties of endo-1,3-β-D-glucanase from the Vietnamese edible mussel Perna viridis

A β-1,3-glucanase with a molecular mass of 33 kDa was isolated in the homogeneous state from a crystalline stalk of the commercially available Vietnamese edible mussel Perna viridis. It hydrolyzes β-1,3-bonds in glucans and is capable of catalyzing the transglycosylation reaction. The β-1,3-glucanase has a K _m value of 0.3 mg/ml for the hydrolysis of laminaran and shows a maximum activity in the pH range from 4 to 6.5 and at 45°C. Its half-inactivation time is 180 min at 45°C and 20 min at 50°C. The enzyme was ascribed to glucan-endo-(1 → 3)-β-D-glucosidases (EC 3.2.1.39). The enzyme could be used in the structure determination of β-1,3-glucans and enzymatic synthesis of new carbohydrate-containing compounds.     

Study of the Degradation Activity and the Strategies to Promote the Bioavailability of Phenanthrene by Sphingomonas paucimobilis Strain 20006FA

The present study describes the phenanthrene-degrading activity of Sphingomonas paucimobilis 20006FA and its ability to promote the bioavailability of phenanthrene. S. paucimobilis 20006FA was isolated from a phenanthrene-contaminated soil microcosm. The strain was able to grow in liquid mineral medium saturated with phenanthrene as the sole carbon source, showing high phenanthrene elimination (52.9% of the supplied phenanthrene within 20 days). The accumulation of 1-hydroxy-2-naphthoic acid and salicylic acid as major phenanthrene metabolites and the capacity of the strain to grow with sodium salicylate as the sole source of carbon and energy indicated that the S. paucimobilis 20006FA possesses a complete phenanthrene degradation pathway. However, under the studied conditions, the strain was able to mineralize only the 10% of the consumed phenanthrene. Investigations on the cell ability to promote bioavailability of phenanthrene showed that the S. paucimobilis strain 20006FA exhibited low cell hydrophobicity (0.13), a pronounced chemotaxis toward phenanthrene, and it was able to reduce the surface tension of mineral liquid medium supplemented with phenanthrene as sole carbon source. Scanning electron micrographs revealed that: (1) in suspension cultures, cells formed flocks and showed small vesicles on the cell surface and (2) cells were also able to adhere to phenanthrene crystals and to produce biofilms. Clearly, the strain seems to exhibit two different mechanisms to enhance phenanthrene bioavailability: biosurfactant production and adhesion to the phenanthrene crystals.     

Rational design of new binding specificity by simultaneous mutagenesis of calmodulin and a target peptide

Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of target proteins in most cells. This promiscuity underlies the role of CaM as a shared participant in calcium-dependent signal transduction pathways but imposes a handicap on popular CaM-based calcium biosensors, which display an undesired tendency to cross-react with cellular proteins. Designed CaM/target pairs that retain high affinity for one another but lack affinity for wild-type CaM and its natural interaction partners would therefore be useful as sensor components and possibly also as elements of "synthetic" cellular-signaling networks. Here, we have adopted a rational approach to creating suitably modified CaM/target complexes by using computational design methods to guide parallel site-directed mutagenesis of both binding partners. A hierarchical design procedure was applied to suggest a small number of complementary mutations on CaM and on a peptide ligand derived from skeletal-muscle light-chain kinase (M13). Experimental analysis showed that the procedure was successful in identifying CaM and M13 mutants with novel specificity for one another. Importantly, the designed complexes retained an affinity comparable to the wild-type CaM/M13 complex. These results represent a step toward the creation of CaM and M13 derivatives with specificity fully orthogonal to the wild-type proteins and show that qualitatively accurate predictions may be obtained from computational methods applied simultaneously to two proteins involved in multiple-linked binding equilibria.     

Entamoeba histolytica: ultrastructure of the chromosomes and the mitotic spindle

We have analyzed by transmission electron microscopy the mitotic process of Entamoeba histolytica trophozoites in an asynchronous population of axenically cultured parasites. Our observations showed that nuclear microtubules, initially located at random in the karyosome during prophase, formed in subsequent stages a mitotic spindle closely related to the nuclear membrane at the polar regions of dividing nuclei. In late prophase and in anaphase, chromosomes appeared as dense bodies 0.1-0.5 microm. At least 15 chromosomes appeared in favorable planes of section, arranged as an incomplete elliptical circle, in close contact with microtubules. There was no morphological evidence of structures resembling the kinetochores of higher eukaryotes. When cut in cross-section, the mitotic spindle was made of 28-35 microtubular rosette assemblies. The latter probably correspond to a similar number of chromosomes, as has been shown by others with pulse-field electrophoresis and fluorescence microscopy of trophozoite spreads. In turn, each microtubular rosette was constituted by 7-12 parallel microtubules. In later stages of the metaphase, two sets of chromosomes were disposed forming a pair of elliptical circles. An additional finding in the dividing nuclei of E. histolytica trophozoites was the presence of compact conglomerates of numerous particles 50 nm in diameter, of similar electron density, shape, and size, probably corresponding to RNA episomes.