Atomic determinants of state-dependent block of sodium channels by charged local anesthetics and benzocaine

Molecular modeling predicts that a local anesthetic (LA) lidocaine binds to the resting and open Na(v)1.5 in different modes, interacting with LA-sensing residues known from experiments. Besides the major pathway via the open activation gate, LAs can reach the inner pore via a "sidewalk" between D3S6, D4S6, and D3P. The ammonium group of a cationic LA binds in the focus of the pore-helices macrodipoles, which also stabilize a Na(+) ion chelated by two benzocaine molecules. The LA's cationic group and a Na(+) ion in the selectivity filter repel each other suggesting that the Na(+) depletion upon slow inactivation would stabilize a LA, while a LA would stabilize slow-inactivated states.     

Effects of bovine spermatozoa preparation on embryonic development in vitro

The aim of our research was to examine the ability of density gradient preparation BoviPure^? and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/thawed semen from six Simmental bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity and acrosomal status were evaluated and compared before and after sperm processing using BoviPure^? and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were significant differences (P < 0.05) between the sperm characteristics before and after BoviPure^?, but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods. A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly higher using BoviPure^? method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of Day 7 morulas and blastocysts showed that BoviPure^? treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure^? method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So, we concluded that BoviPure^? could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for IVP of bovine embryos.     

unc-93(e1500): A Behavioral Mutant of CAENORHABDITIS ELEGANS That Defines a Gene with a Wild-Type Null Phenotype

The uncoordinated, egg-laying-defective mutation, unc-93(e1500) III, of the nematode Caenorhabditis elegans spontaneously reverts to a wild-type phenotype. We describe 102 spontaneous and mutagen-induced revertants that define three loci, two extragenic (sup-9 II and sup-10 X) and one intragenic. Genetic analysis suggests that e1500 is a rare visible allele that generates a toxic product and that intragenic reversion, resulting from the generation of null alleles of the unc-93 gene, eliminates the toxic product. We propose that the genetic properties of the unc-93 locus, including the spontaneous reversion of the e1500 mutation, indicate that unc-93 may be a member of a multigene family. The extragenic suppressors also appear to arise as the result of elimination of gene activity; these genes may encode regulatory functions or products that interact with the unc-93 gene product. Genes such as unc-93, sup-9 and sup-10 may be useful for genetic manipulations, including the generation of deficiencies and mutagen testing.     

Refining the genetic portrait of Portuguese Roma through X-chromosomal markers

Due to differences in transmission between X-chromosomal and autosomal DNA, the comparison of data derived from both markers allows deeper insight into the forces that shape the patterns of genetic diversity in populations. In this study, we applied this comparative approach to a sample of Portuguese Roma (Gypsies) by analyzing 43 X-chromosomal markers and 53 autosomal markers. Portuguese individuals of non-Gypsy ancestry were also studied. Compared with the host population, reduced levels of diversity on the X chromosome and autosomes were detected in Gypsies; this result was in line with known patterns of genetic diversity typical of Roma groups. As a consequence of the complex demographic past of the Roma, during which admixture and genetic drift played major roles, the amount of linkage disequilibrium (LD) on the X chromosome in Gypsies was considerably higher than that observed in non-Gypsies. When the pattern of differentiation on the X chromosome was compared with that of autosomes, there was evidence for asymmetries in female and male effective population sizes during the admixture between Roma and non-Roma. This result supplements previous data provided by mtDNA and the Y chromosome, underlining the importance of using combined information from the X chromosome and autosomes to dissect patterns of genetic diversity. Following the out-of-India dispersion, the Roma acquired a complex genetic pattern that was influenced by drift and introgression with surrounding populations, with important contributions from both males and females. We provide evidence that a sex-biased admixture with Europeans is probably associated with the founding of the Portuguese Gypsies.     

The effect of PKC phosphorylation on the “architectural” properties of HMGB1 protein

High mobility group box (HMGB)1 protein acts as an architectural element, promoting the assembly of active nucleoprotein complexes due to its ability to bend DNA and to bind preferentially to distorted DNA structures. The behavior of HMGB1 as an "architect" of chromatin defines it as an important factor in many cellular processes such as repair, replication and remodeling. It was shown that the post-synthetic acetylation of HMGB1 at Lys2 modulated its essential properties as a structure-specific nuclear protein. We studied the role of PKC phosphorylation on the "architectural" properties of HMGB1, (i) the effect for the formation of a stable complex with DNA damaged by the anti-tumour drug cis-platinum and (ii) the influence on the ability of HMGB1 protein to bend short DNA fragments. PKC-phosphorylated recombinant HMGB1 increased about an order of magnitude its affinity to cis-platinated DNA, a finding that has already been reported for in vivo acetylated protein. Regarding the effect on the protein's DNA bending ability, it was enhanced upon phosphorylation as demonstrated by the stimulation of DNA circularization. We showed also that PKC phosphorylated the recombinant protein in vitro simultaneously at two target sites. Our results demonstrate that the PKC phosphorylation of HMGB1 has a considerable effect on the fundamental properties of the protein; therefore this post-synthetic modification may serve as a modulator of the HMGB1 participation in different nuclear processes.     

Identification and regulation of a molecular module for bleb-based cell motility

Single-cell migration is a key process in development, homeostasis, and disease. Nevertheless, the control over basic cellular mechanisms directing cells into motile behavior in?vivo is largely unknown. Here, we report on the identification of a minimal set of parameters the regulation of which confers proper morphology and cell motility. Zebrafish primordial germ cells rendered immotile by knockdown of Dead end, a negative regulator of miRNA function, were used as a platform for identifying processes restoring motility. We have defined myosin contractility, cell adhesion, and cortex properties as factors whose proper regulation is sufficient for restoring cell migration of this cell type. Tight control over the level of these cellular features, achieved through a balance between miRNA-430 function and the action of the RNA-binding protein Dead end, effectively transforms immotile primordial germ cells into polarized cells that actively migrate relative to cells in their environment.     

Circular dichroism and guanine quadruplexes

Circular dichroism (CD) is remarkably sensitive to the conformational states of nucleic acids; therefore, CD spectroscopy has been used to study most features of DNA and RNA structures. Quadruplexes are among the significant noncanonical nucleic acids architectures that have received special attentions recently. This article presents examples on the contribution of CD spectroscopy to our knowledge of quadruplex structures and their polymorphism. The examples were selected to demonstrate the potential of this simple method in the quadruplex field. As CD spectroscopy detects only the global feature of a macromolecule, it should preferably be used in combination with other techniques. On the other hand, CD spectroscopy, often as a pioneering approach, can reveal the formation of particular structural arrangements, to search for the conditions stabilizing the structures, to follow the transitions between various structural states, to explore kinetics of their appearance, to determine thermodynamic parameters and also detect formation of higher order structures. This article aims to show that CD spectroscopy is an important complementary technique to NMR spectroscopy and X-ray diffraction in quadruplex studies.     

Proteomic analysis of wheat proteins recognized by IgE antibodies of allergic patients

Wheat belongs to six major food allergens inducing IgE-mediated hypersensitivity reaction manifesting as cutaneous, gastrointestinal, and respiratory symptoms. Although cereals are a staple food item in most diets, only a few wheat proteins causing hypersensitivity have been identified. To characterize wheat allergens, salt-soluble wheat extracts were separated by 1-DE and 2-DE and IgE-binding proteins were detected by immunoblotting using sera of patients with allergy to ingested wheat. Proteins, frequently recognized by IgE on 2-DE were analyzed by MALDI-TOF and QTOF and their spectrum was completed by 1-DE and LCQ(DECA) nLC-MS/MS IT technique. Using all three techniques we identified 19 potential wheat allergens such as alpha-amylase inhibitors, beta-amylase, profilin, serpin, beta-D-glucan exohydrolase, and 27K protein. Employing newly developed ELISA, levels of IgE Abs against Sulamit wheat extract and alpha-amylase inhibitors type 1 and 3 were quantified and shown to be significantly elevated in sera of allergic patients compared to those of healthy controls. The level of IgE Abs against alpha-amylase inhibitor type 3 was lower, slightly above the cut-off value in the majority of patients' sera. Our findings contribute to the identification of wheat allergens aimed to increase the specificity of serum IgE and cell activation diagnostic assays.     

Occurrence of bifidobacteria in faeces of calves fed milk or a combined diet

The development of faecal bacteria composition in calves fed milk or a combined diet was investigated from 4 to 21 days of age. On day 7, bifidobacteria in faeces of milk-fed calves already increased from about 7.6 to 9.2 log CFU/g and did not change until the end of the study, whereas in calves fed the combined diet bifidobacteria only moderately increased to 7.9 log CFU/g and decreased slowly until day 21. The counts of bifidobacteria in calves on a combined diet were significantly (p < 0.01) lower compared to those in milk-fed calves. Bifidobacterial counts determined by cultivation or by fluorescence in situ hybridisation (FISH) did not differ significantly. Our results showed that the occurrence of bifidobacteria in calf faeces is highly dependent on the diet composition. Faecal bacteria flora of calves fed exclusively by milk is rich in bifidobacteria, but in calves on a combined diet coliforms dominated.     

Neoparamoeba perurans is a cosmopolitan aetiological agent of amoebic gill disease

Previously we described a new member of the Neoparamoeba genus, N. perurans, and showed that it is an agent of amoebic gill disease (AGD) of Atlantic salmon Salmo salar cultured in southeast Tasmania, Australia. Given the broad distribution of cases of AGD, we were interested in extending our studies to epizootics in farmed fish from other sites around the world. Oligonucleotide probes that hybridise with the 18S rRNA of N. perurans, N. branchiphila or N. pemaquidensis were used to examine archival samples of AGD in Tasmania as well as samples obtained from 4 host fish species cultured across 6 countries. In archival samples, N. perurans was the only detectable amoeba, confirming that it has been the predominant aetiological agent of AGD in Tasmania since epizootics were first reported. N. perurans was also the exclusive agent of AGD in 4 host species across 6 countries. Together, these data show that N. perurans is a cosmopolitan agent of AGD and, therefore, of significance to the global mariculture industry.