Preextinction viral RNA can interfere with infectivity

When the error rate during the copying of genetic material exceeds a threshold value, the genetic information cannot be maintained. This concept is the basis of a new antiviral strategy termed lethal mutagenesis or virus entry into error catastrophe. Critical for its success is preventing survival of residual infectious virus or virus mutants that escape the transition into error catastrophe. Here we document that mutated, preextinction foot-and-mouth disease virus (FMDV) RNA can interfere with and delay viral production up to 30 h when cotransfected in BHK-21 cells with standard RNA. Interference depended on the physical integrity of preextinction RNA and was not observed with unrelated RNAs or with nonmutated, defective FMDV RNA. These results suggest that this type of interference requires large size, preextinction FMDV RNA and is mediated neither by small interfering RNAs nor by RNAs that can compete with infectious RNA for host cell factors. A model based on the aberrant expression of mutated RNA as it is expected to occur in the initial stages of the transition into error catastrophe is proposed. Interference mediated by preextinction RNA indicates an advantage of mutagenesis versus inhibition in preventing the survival of virus escape mutants during antiviral treatments.     

Characterization of rotavirus cell entry

While recently we have learned much about the viral and cellular proteins involved in the initial attachment of rotaviruses to MA104 cells, the mechanism by which these viruses reach the interior of the cell is poorly understood. For this study, we observed the effects of drugs and of dominant-negative mutants, known to impair clathrin-mediated endocytosis and endocytosis mediated by caveolae, on rotavirus cell infection. Rotaviruses were able to enter cells in the presence of compounds that inhibit clathrin-mediated endocytosis as well as cells overexpressing a dominant-negative form of Eps15, a protein crucial for the assembly of clathrin coats. We also found that rotaviruses infected cells in which caveolar uptake was blocked; treatment with the cholesterol binding agents nystatin and filipin, as well as transfection of cells with dominant-negative caveolin-1 and caveolin-3 mutants, had no effect on rotavirus infection. Interestingly, cells treated with methyl-beta-cyclodextrin, a drug that sequesters cholesterol from membranes, and cells expressing a dominant-negative mutant of the large GTPase dynamin, which is known to function in several membrane scission events, were not infected by rotaviruses, indicating that cholesterol and dynamin play a role in the entry of rotaviruses.     

Antibacterial activity of ethanolic and aqueous extracts of Acacia aroma Gill. ex Hook et Arn

The purpose of the present study was to investigate the antibacterial activity of seven ethanolic extracts and three aqueous extracts from various parts (leaves, stems and flowers) of A. aroma against 163 strains of antibiotic multi-resistant bacteria. The disc diffusion assay was performed to evaluate antibacterial activity of the A. aroma crude extracts, against several Gram-positive bacteria (E. faecalis, S. aureus, coagulase-negative stahylococci, S. pyogenes, S. agalactiae, S. aureus ATCC 29213, E. faecalis ATCC 29212) and Gram-negative bacteria (E. coli., K. pneumoniae, P. mirabilis, E. cloacae, S. marcescens, M morganii, A. baumannii, P. aeruginosa, S. maltophilia, E. coli ATCC 35218, P. aeruginosa ATCC 27853, E. coli ATCC 25922). All ethanolic extracts showed activity against gram-positive bacteria. Among all obtained extracts, only leaf and flower fluid extracts showed activity against Gram-negative bacteria. Based on this bioassay, leaf fluid extracts tended to be the most potent, followed by flower fluid extracts. Minimal inhibitory concentration (MIC) values of extracts and antibiotics were comparatively determined by agar and broth dilution methods. Both extracts were active against S. aureus, coagulase-negative stahylococci, E. faecalis and E. faecium and all tested Gram-negative bacteria with MIC values from 0.067 to 0.308 mg/ml. In this study the minimal bactericidal concentration (MBC) values were identical or twice as high than the corresponding MIC for leaf extracts and four or eight times higher than MIC values for flower extracts. This may indicate a bactericidal effect. Stored extracts have similar antibacterial activity as recently obtained extracts. The A. aroma extracts of leaves and flowers may be useful as antibacterial agents against Gram- negative and Gram-positive antibiotic multi-resistant microorganisms.     

Divide and conquer: development and cell cycle genes in plant transformation

Genetic transformation and regeneration of transgenic plants remains unfeasible for the majority of plant species. We propose that inducible expression and/or suppression of the genes that control the cell cycle and development, by altering chromatin structure and exerting epigenetic control of gene expression, might substantially improve competence for transformation and/or regeneration. Transformation efficiency was higher in cells with nuclei at the S and G2 phases, and manipulating the genes whose activation or silencing promote the G1-S transition has increased both transient and stable transformation. Controlling the cell cycle directly, using RBR and VIP1, or indirectly, through hormone regulation using IPT and ESR1, has improved rates of stable transformation. Other target genes that might promote incorporation of DNA and/or pluripotency of cells include HP1, CycD3 and CycD1. The availability of large EST databanks, complete plant-genome sequences and/or inducible gene expression systems create opportunities for testing homologous genes to increase competence of transformation and regeneration.     

Microbial degradation of palm (Elaeis guineensis) biodiesel

The kinetics of biodegradation of palm-derived fatty methyl and ethyl esters (Elaeis guineensis biodiesel) by a wild-type aerobic bacterial population was measured at 20 degrees C, as the rate of oxygen uptake by a manometric technique. The methyl and ethyl biodiesels were obtained by potassium-hydroxide catalysed trans-esterification of palm oil, respectively. The bacterial flora included the genera Bacillus, Proteus, Pseudomonas, Citrobacter and Enterobacter. The rate of oxygen uptake for palm biodiesel is similar to the quantity observed in the biodegradation of 1.0 mM solutions of simple substrates such as carbohydrates or amino acids. Palm methyl or ethyl biodiesel is subjected to facile aerobic biodegradation by wild-type bacteria commonly present in natural open environments. This result should lessen any environmental concern for its use as alternative fuel, solvent or lubricant.     

The induction of NOS2 expression by the hybrid cecropin A-melittin antibiotic peptide CA(1-8)M(1-18) in the monocytic line RAW 264.7 is triggered by a temporary and reversible plasma membrane permeation

There is an increasing awareness of immune cell modulation by antimicrobial peptides. While this process often requires specific receptors for the peptides involved, several reports point out to a receptor-independent process. The cecropin A-melittin hybrid peptide CA(1-8)M(1-18) (KWKLFKKIGIGAVLKVLTTGLPALIS-amide) modifies gene expression in the macrophage line RAW 264.7 in the absence of any previous macrophage priming, suggesting a membrane permeation process. To further analyze the initial steps of this mechanism, we have studied the interaction of the peptide with these cells. Below 2 microM, CA(1-8)M(1-18) causes a concentration-dependent membrane depolarization partially reversible with time. At 2 microM, the accumulation of the SYTOX green vital dye is one half of that achieved with 0.05% Triton X-100. The binding level, as assessed by fluorescein-labeled CA(1-8)M(1-18), varies from 7.7+/-1.2 to 37.4+/-3.9 x 10(6) molecules/cell over a 0.5-4.0 microM concentration range. Electrophysiological experiments with 0.5 microM CA(1-8)M(1-18), a concentration that triggers maximal NOS2 expression and minimal toxicity, show a reversible current induction in the RAW 264.7 plasma membrane that is maintained as far as peptide is present. This activation of the macrophage involves the production of nitric oxide, a metabolite lethal for many pathogens that results from unspecific membrane permeation by antimicrobial peptides, and represents a new mode of action that may open new therapeutic possibilities for these compounds against intracellular pathogens.     

Molecular mechanisms and binding site location for the noncompetitive antagonist crystal violet on nicotinic acetylcholine receptors

We investigated the molecular mechanisms and the binding site location for the fluorophor crystal violet (CrV), a noncompetitive antagonist of the nicotinic acetylcholine receptor (AChR). To this end, radiolabeled competition binding, fluorescence spectroscopy, Schild-type analysis, patch-clamp recordings, and molecular dynamics approaches were used. The results indicate that (i) CrV interacts with the desensitized Torpedo AChR with higher affinity than with the resting state at several temperatures (5-37 degrees C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [(3)H]thienylcyclohexylpiperidine binding to the desensitized or resting AChR is mediated by a steric mechanism; (iii) tetracaine inhibits CrV binding to the resting AChR, probably by a steric mechanism; (iv) barbiturates modulate CrV binding to the resting AChR by an allosteric mechanism; (v) CrV itself induces AChR desensitization; (vi) CrV decreases the peak of macroscopic currents by acting on the resting AChR but without affecting the desensitization rate from the open state; and (vii) two tertiary amino groups from CrV may bind to the alpha1-Glu(262) residues (located at position 20') in the resting state. We conclude that the CrV binding site overlaps the PCP locus in the resting and desensitized state. The noncompetitive action of CrV may be explained by an allosteric mechanism in which the binding of CrV to the extracellular mouth of the resting receptor leads to an inhibition of channel opening. Binding of CrV probably increases desensitization of the resting channel and stabilizes the desensitized state.     

Anti-Trypanosoma cruzi and cytotoxic activities of Eugenia uniflora L

Chagas disease is caused by Trypanosoma cruzi, being considered a public health problem. An alternative to combat this pathogen is the use of natural products isolated from fruits such as Eugenia uniflora, a plant used by traditional communities as food and medicine due to its antimicrobial and biological activities. Ethanolic extract from E. uniflora was used to evaluate in vitro anti-epimastigote and cytotoxic activity. This is the first record of anti-Trypanosoma activity of E. uniflora, demonstrating that a concentration presenting 50% of activity (EC(50)) was 62.76 μg/mL. Minimum inhibitory concentration (MIC) was ≤ 1024 μg/mL. Our results indicate that E. uniflora could be a source of plant-derived natural products with anti-epimastigote activity with low toxicity.