Metal-free artificial nucleases based on simple oxime and hydroxylamine scaffolds

Hydrolysis of DNA is of increasing importance in biotechnology and medicine. In this Letter, we present the DNA-cleavage potential of metal-free hydroxylamines and oximes as new members of nucleic acid cleavage agents.     

Preferential location of lidocaine and etidocaine in lecithin bilayers as determined by EPR, fluorescence and 2H NMR

We have examined the effect of the uncharged species of lidocaine (LDC) and etidocaine (EDC) on the acyl chain moiety of egg phosphatidylcholine liposomes. Changes in membrane organization caused by both anesthetics were detected through the use of EPR spin labels (5, 7 and 12 doxyl stearic acid methyl ester) or fluorescence probes (4, 6, 10, 16 pyrene-fatty acids). The disturbance caused by the LA was greater when the probes were inserted in more external positions of the acyl chain and decreased towards the hydrophobic core of the membrane. The results indicate a preferential insertion of LDC at the polar interface of the bilayer and in the first half of the acyl chain, for EDC. Additionally, (2)H NMR spectra of multilamellar liposomes composed by acyl chain-perdeutero DMPC and EPC (1:4 mol%) allowed the determination of the segmental order (S(mol)) and dynamics (T(1)) of the acyl chain region. In accordance to the fluorescence and EPR results, changes in molecular orientation and dynamics are more prominent if the LA preferential location is more superficial, as for LDC while EDC seems to organize the acyl chain region between carbons 2-8, which is indicative of its positioning. We propose that the preferential location of LDC and EDC inside the bilayers creates a "transient site", which is related to the anesthetic potency since it could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel.     

Effects of phenol in antioxidant metabolism in matrinxã, Brycon amazonicus (Teleostei; Characidae)

Parameters of the antioxidant defense systems of Brycon amazonicus (matrinx?--a neotropical fish) exposed to phenol for 96 h plus the recovery over 1 and 2 weeks were studied in erythrocytes and liver. Hematocrit increase was observed during phenol exposure and recovery for 1 week. Total superoxide dismutases (SOD), glutathione peroxidase (GPx) and reduced glutathione (GSH) did not change during phenol exposure. Erythrocyte glucose-6-phosphate dehydrogenase (G6PDH) increased during that period while catalase (CAT) activity decreased during phenol exposure and recovery for 2 weeks. In the liver, SOD and CAT did not change, whereas GPx increased in the first week of recovery and decreased after 2 weeks. A late response was observed for G6PDH activity which increased only at the second week. Ascorbate concentration in the brain decreased during phenol exposure and increased over recovery. From our results it appears that the oxidative stress was limited in matrinx? exposed to phenol, but seemed to occur during the recovery period.     

Species-specific mitochondrial DNA markers for identification of non-invasive samples from sympatric carnivores in the Iberian Peninsula

Genetic species identification of non-invasively collected samples has become an important tool in ecological research, management and conservation and wildlife forensics. This is especially true for carnivores, due to their elusive nature, and is crucial when several ecologically and phylogenetically close species, with similar faeces, hairs, bones and/or pelts, occur in sympatry. This is the case of the Iberian Peninsula, a region with a carnivore community of 16 species—about two-thirds of the European carnivore fauna. Here we present a simple, efficient and reliable PCR-based protocol, using a novel set of species-specific primers, for the unambiguous identification to species of non-invasively collected samples or forensic materials from Iberian carnivores. For each species, from the consensus of all cytochrome b haplotypes, found here and previously reported, we designed species-specific primer pairs for short fragments, the most likely to persist in low-quantity and degraded DNA samples. The predicted specificity of each primer pair was assessed through PCR of positive DNA extracts from the carnivore species, from an exhaustive array of potential prey and from humans. The robustness of PCR amplification for non-invasively sampled DNA was tested with scat samples. The primers did not produce false positives and correctly identified all carnivore samples to the species level. In comparison with sequencing and PCR-RFLP assays, our method is, respectively, cost- and time-effective, and is especially suited for monitoring surveys targeting multiple populations/species. It also introduces an approach that works for a whole community of carnivores living sympatrically over a large geographic area.     

Antimutagenicity of rosmarinic acid in Swiss mice evaluated by the micronucleus assay

Rosmarinic acid (RA) is a natural phenolic compound which presents different biological activities such as antitumor, antibacterial, anti-inflammatory, hepatoprotective and cardioprotective properties. In view of its important biological activities, the study of the effects of RA on genetic material becomes relevant. Thus, the objective of the present study was to evaluate the mutagenic and/or antimutagenic potential of RA on peripheral blood cells of Swiss mice using the micronucleus assay. Three doses of RA (50, 100 and 200 mg/kg body weight, b.w.) were used for the evaluation of its mutagenic potential. In the antimutagenicity assays, the different concentrations of RA were combined with the chemotherapeutic agent doxorubicin (DXR, 15 mg/kg b.w.). Peripheral blood samples were collected 24, 48 and 72 h after treatment for the evaluation of micronucleated polychromatic erythrocytes (MNPCEs). The results of the mutagenicity assays showed no increase in the frequency of micronuclei in animals treated with different concentrations of RA when compared to the negative controls. Treatment with different concentrations of RA combined with DXR revealed a significant reduction in the frequency of micronuclei compared to animals treated with DXR only. Although the mechanisms underlying the protective effect of RA are not completely understood, the putative antioxidant activity of RA might explain its effect on DXR mutagenicity.     

Seasonal variations in the biochemical composition of the crayfish Parastacus defossus (Crustacea, Decapoda) in its natural environment

The crayfish Parastacus defossus occurs in Uruguay and the state of Rio Grande do Sul in Brazil. It lives in swamps and lakes, where it digs sloped subterranean tunnels that are used as burrows. Because there is little information about the biology, physiology and ecology of this species, the aim of this study was to identify the seasonal variations of its carbohydrate and lipid metabolism. Crayfish were collected monthly (from November 2002 to February 2004) in the Lami region, Porto Alegre municipality (30 degrees 11'41'S-51 degrees 06'00'W). Haemolymph samples, used for determination of glucose, total proteins, triglycerides, total cholesterol and total lipids, were collected in the field using potassium oxalate as an anti-clotting agent. The animals and haemolymph samples were immediately frozen in the field. In the laboratory, the hepatopancreas, gills and abdominal muscles were removed for determination of glycogen, triglycerides, total cholesterol and total lipids. The findings suggest that in P. defossus, lipids are an important reserve of energy used during reproduction in both males and females; whereas glycogen may be used during periods of intense activity or environmental stress.     

Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability

The objectives of this study were to assess the effects of induced testicular degeneration in Bos taurus indicus (Nellore) bulls on changes in seminal characteristics and fertilizing ability of sperm. Four Nellore bulls (30–36-month-old, 500–550 kg) with good seminal quality (>80% motile and morphologically normal sperm) had scrotal insulation applied for 5 d. Semen was collected by electroejaculation and cryopreserved at the pre-insulation moment, and 7, 14, and 21 d after insulation was removed. Gross motility, vigor of sperm movement (1–5), acrosome integrity, sperm morphology (phase-contrast microscopy), nuclear vacuoles and abnormal chromatin (Feulgen-stain) were determined after sperm preparations for in vitro fertilization (IVF). Prior to IVF, sperm were separated using a Percoll gradient (45% and 90%). Normal sperm decreased (P < 0.05) 14 and 21 d after insulation was removed. On 14 and 21 d, the incidence of head defects (9.7 ± 0.6 and 17.0 ± 0.8, respectively; mean ± S.E.M.) was higher (P < 0.05) in agreement with the incidence of nuclear vacuoles (14.0 ± 5.0 and 12.3 ± 2.3) and abnormal chromatin (24.4 ± 7.2 and 30.8 ± 2.8). Although the frequency of cleaved oocytes decreased only on 21 d (P < 0.05), blastocyst rates were lower (P < 0.05) than pre-insulation on 14 and 21 d. In regression analyses, only nuclear vacuoles, head defects and intact acrosome accounted for differences in cleavage (R² = 0.38, 0.48, and 0.30, respectively) and blastocyst rates (R² = 0.35, 0.37, and 0.44). Abnormal chromatin was associated only with blastocyst rates (R² = 0.35). In conclusion, blastocyst rate was more sensitive than cleavage rate and the assessment of nuclear integrity is recommended to predict the fertilizing ability of bull sperm.     

Wild Manihot species: botanical aspects, geographic distribution and economic value

A total of 98 Manihot species have been recognized in the genus. All of them are native to the tropics of the New World, particularly Brazil and Mexico. The cultigen, Manihot esculenta Crantz (cassava), grows throughout the lowland tropics. Wild species vary in growth habit from acaulescent or short shrubs to tree-like. Because of their adaptations to different conditions, they are gene reservoirs for tackling many abiotic and biotic stresses such as improving root quality and resistance to diseases. They have been used successfully by the first author for improving protein content, seed-fertility, apomixis, resistance to mealy bug, and tolerance to drought. A table of the most important species from an economic viewpoint is presented.     

Virulence in murine model shows the existence of two distinct populations of Brazilian Vaccinia virus strains

Brazilian Vaccinia virus had been isolated from sentinel mice, rodents and recently from humans, cows and calves during outbreaks on dairy farms in several rural areas in Brazil, leading to high economic and social impact. Some phylogenetic studies have demonstrated the existence of two different populations of Brazilian Vaccinia virus strains circulating in nature, but little is known about their biological characteristics. Therefore, our goal was to study the virulence pattern of seven Brazilian Vaccinia virus strains. Infected BALB/c mice were monitored for morbidity, mortality and viral replication in organs as trachea, lungs, heart, kidneys, liver, brain and spleen. Based on the virulence potential, the Brazilian Vaccinia virus strains were grouped into two groups. One group contained GP1V, VBH, SAV and BAV which caused disease and death in infected mice and the second one included ARAV, GP2V and PSTV which did not cause any clinical signals or death in infected BALB/c mice. The subdivision of Brazilian Vaccinia virus strains into two groups is in agreement with previous genetic studies. Those data reinforce the existence of different populations circulating in Brazil regarding the genetic and virulence characteristics.