Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 μg ml⁻¹ while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.     

The effect of sperm preparation and co-incubation time on in vitro fertilization of Bos indicus oocytes.

The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm-oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18h. After co-incubation oocytes were transferred to the culture medium and culture for 44h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P<0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm-oocyte co-incubation time was observed (P>0.05). However, sperm-oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P<0.05) progressively at 6h (63.3 and 54.4%) and 12h (77.6 and 67.6%). No differences (P>0.05) were observed between 12 and 18h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P>0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12h, and the prolongation of that time for up to 18h had no detrimental effect on fertilization.     

The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Mechanism of formation of the putative advanced glycosylation end product and protein cross-link 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole

2-(2-Furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) is a fluorescent molecule which was originally discovered in chloroform extract of ammoniacal solution of acid-hydrolyzed glycated proteins and proposed to represent a protein cross-link. The absence of a lysyl residue side chain and other observations promoted a detailed study of its mechanism of formation. Glycated alpha-t-Butoxycarbonyllysine was incubated for 29 days and periodically assayed for FFI and FFI-like fluorescence. Whereas fluorescence increased over time, FFI recovery was unexpectedly highest on day 0 and lowest on day 29, suggesting that FFI was directly derived from Amadori products. FFI was also recovered from hydrolysates of glycated neopentylamine, furosine, and browned poly-L-lysine but was virtually undetectable in similar solutions basified with NaOH, triethylamine, or pyridine instead of ammonia. Gas chromatography-mass spectrometry analysis of FFI from similar hydrolysates basified in the presence of 15N-enriched NH4Cl revealed for all precursors a parent ion peak at 230 instead of 228 m/e units, suggesting that the two imidazole nitrogen atoms had been incorporated from free ammonia into FFI. Spontaneous FFI synthesis occurred when furosine was reacted with aqueous ammonia at room temperature. These results do not support the proposition that FFI is an advanced glycosylation end product or a protein cross-link. They suggest that FFI is formed from ammonia and furosine which are by-products of acid-hydrolyzed glycated proteins.     

Membrane microheterogeneity: Förster resonance energy transfer characterization of lateral membrane domains

Lateral membrane heterogeneity, in the form of lipid rafts and microdomains, is currently implicated in cell processes including signal transduction, endocytosis, and cholesterol trafficking. Various biophysical techniques have been used to detect and characterize lateral membrane domains. Among these, Förster resonance energy transfer (FRET) has the crucial advantage of being sensitive to domain sizes smaller than 50-100 nm, below the resolution of optical microscopy but, apparently, similar to those of rafts in cell membranes. In the last decade, several formalisms for the analysis of FRET in heterogeneous membrane systems have been derived and applied to the study of microdomains. They are critically described and illustrated here.     

Phylogeography of the chestnut‐tailed antbird (Myrmeciza hemimelaena) clarifies the role of rivers in Amazonian biogeography

Aim We examined patterns of spatial and temporal diversification of the Amazonian endemic chestnut‐tailed antbird, Mymeciza hemimelaena (Thamnophilidae), to evaluate the diversification of a widespread avian taxon across rivers that potentially represent major natural barriers. Location Lowland Amazonia. Methods Sequences of the mitochondrial ND2 and cytochrome b genes were investigated from 65 individuals distributed throughout the entire range of M. hemimelaena, and including the two currently valid subspecies M. h. hemimelaena and M. h. pallens. Based on a combination of phylogeographic tools, molecular dating, and population genetic methods, we reconstructed a spatio‐temporal scenario of diversification of M. hemimelaena in the Amazon. Results The data revealed three genetically divergent and monophyletic groups in M. hemimelaena, which can also be distinguished by a combination of morphological and vocal characters. Two of these clades correspond to the previously described taxa M. h. hemimelaena and M. h. pallens, which are separated by the upper Madeira River, a main Amazonian tributary. The third clade is distributed between the middle reaches of the Madeira River and the much smaller tributaries Jiparaná and Aripuanã, and, although currently treated as M. h. pallens, clearly constitutes an independent evolutionary lineage probably deserving separate species status. Molecular clock and population genetic analyses indicate that diversification in this group occurred throughout the Pleistocene, with demographic fluctuations assumed for M. h. hemimelaena and M. h. pallens. Main conclusions The findings implicate rivers as barriers driving diversification in the M. hemimelaena complex. Levels of mitochondrial DNA divergence and associated morphological and vocal traits support its division into at least three separate species with comparatively small ranges. The existence of a previously unrecognized lineage in the M. hemimelaena complex, and the high degree of population structuring found in M. h. hemimelaena underscore the pervasiveness of cryptic endemism throughout Amazonia and the importance of DNA‐based taxonomic and phylogeographic studies in providing the accurate estimates of diversity that are essential for conservation planning.     

Genomic Characterization of Novel Circular ssDNA Viruses from Insectivorous Bats in Southern Brazil

Circoviruses are highly prevalent porcine and avian pathogens. In recent years, novel circular ssDNA genomes have recently been detected in a variety of fecal and environmental samples using deep sequencing approaches. In this study the identification of genomes of novel circoviruses and cycloviruses in feces of insectivorous bats is reported. Pan-reactive primers were used targeting the conserved rep region of circoviruses and cycloviruses to screen DNA bat fecal samples. Using this approach, partial rep sequences were detected which formed five phylogenetic groups distributed among the Circovirus and the recently proposed Cyclovirus genera of the Circoviridae. Further analysis using inverse PCR and Sanger sequencing led to the characterization of four new putative members of the family Circoviridae with genome size ranging from 1,608 to 1,790 nt, two inversely arranged ORFs, and canonical nonamer sequences atop a stem loop.