Structural insights into the interaction between prion protein and nucleic acid

The infectious agent of transmissible spongiform encephalopathies (TSE) is believed to comprise, at least in part, the prion protein (PrP). Other molecules can modulate the conversion of the normal PrP(C) into the pathological conformer (PrP(Sc)), but the identity and mechanisms of action of the key physiological factors remain unclear. PrP can bind to nucleic acids with relatively high affinity. Here, we report small-angle X-ray scattering (SAXS) and nuclear magnetic resonance spectroscopy measurements of the tight complex of PrP with an 18 bp DNA sequence. This double-stranded DNA sequence (E2DBS) binds with nanomolar affinity to the full-length recombinant mouse PrP. The SAXS data show that formation of the rPrP-DNA complex leads to larger values of the maximum dimension and radius of gyration. In addition, the SAXS studies reveal that the globular domain of PrP participates importantly in the formation of the complex. The changes in NMR HSQC spectra were clustered in two major regions: one in the disordered portion of the PrP and the other in the globular domain. Although interaction is mediated mainly through the PrP globular domain, the unstructured region is also recruited to the complex. This visualization of the complex provides insight into how oligonucleotides bind to PrP and opens new avenues to the design of compounds against prion diseases.     

Simple and rapid method determination for metformin in human plasma using high performance liquid chromatography tandem mass spectrometry: application to pharmacokinetic studies

A rapid and simple method for quantitation of metformin (MET) in human plasma by HPLC-MS/MS was developed and validated. The sample preparation consists of plasma deproteinization using acetonitrile. The mobile phase consisted of water-acetonitrile and formic acid (55/45/0.048, v/v/%) and the run time was 3 min. A pursuit C(18) (100 mm x 2.0 mm i.d., 3 microm) column connected to a guard column MS-pursuit (0.20 mm x 0.20 mm i.d., 5 microm) was used. The range of the calibration curve was from 20 to 5000 ng/mL, the limit of quantitation being 20 ng/mL. The detection was performed on a mass spectrometer (ESI+), using metoprolol as internal standard. The calibration curves have r(2) values of 0.995 (CV=0.24%, n=10). The accuracy and precision were between 90.74 and 106.7% and coefficients of variations (CV) of 1.10 and 4.35%, respectively. The method was applied to determine the pharmacokinetic parameters: C(max) (1667.25 ng/mL) and T(max) (3.89 h).     

Germination of Geonoma brevispatha (Arecaceae) in laboratory and its relation to the palm spatial distribution in a swamp forest

Geonoma brevispatha is a clonal palm species commonly found as tussocks in swamp and riparian forests in SE Brazil, occurring in the very transition between flooded pits/channels and dry mounds. Aiming to verify whether the germination responses could explain the spatial pattern of the palm tussocks in swamp forests, we investigated the germination in relation to the main environmental factors to which the seeds are subjected in their natural habitat. The seeds are recalcitrant since germination percentage is reduced with loss of moisture content. Germination did not differ on moist filter paper, semi-immersion (water up to half of the seed diameter) or complete immersion (water up to double of the seed diameter). Seeds did not germinate when stored in a closed bottle with water (that is, under low oxygen concentration), but remained viable for up to 8 weeks under this condition. The capacity to germinate in water with minimum oxygen availability may be considered ecologically advantageous in a swamp habitat. On the other hand, the capacity to germinate in moist filter paper indicates that, in natural conditions, the seed can germinate, provided there is a critical minimum of water availability. We concluded that the germination responses per se are not enough to explain the palm tussocks spatial pattern, but they are very well tuned with the conditions of the transitional zone between channels/pits (ever flooded micro-sites) and mounds (well-drained micro-sites), in which the species occurs in the swamp forest.     

Diversity distribution and floristic differentiation of the coastal lowland vegetation: implications for the conservation of the Brazilian Atlantic Forest

Floristic differentiation and vegetation definition is an important step to recognize biome distribution and for biodiversity conservation. Here, we aim to verify if the distribution of the costal lowland vegetation in Brazilian littoral is congruent with climatic gradient and the previous vegetation definitions. Additionally we discussed the importance of terms for the Atlantic Forest conservation. Our study was based on floristic and geo-climatic data from 58 published surveys. We generate a checklist of 1088 woody species and verified species distribution according to environmental gradient using a Detrended Correspondence Analysis (DCA). We compared DCA??s groups with the a priori vegetation definition and generate an a posteriori classification using TWINSPAN. DCA and TWINSPAN resulted in groups determined mainly by rainfall (r = ?0.65) and soil sandiness (r = ?0.71). Those groups were not congruent with both the previous vegetation definitions. The coastal lowland vegetation comprises two distinctive floristic groups representing forests and scrubs that occur in wetter climates (Ombrophilous lowland forests) in the Brazilian states of Santa Catarina, Paraná and São Paulo and in drier climates of Espírito Santo, Rio de Janeiro (Restinga-Northern group) and Rio Grande do Sul (Restinga-Southern group) states. The floristic and historical relationships between Ombrophylous lowland forests and Restingas suggest that conservation initiatives should be more conservative and treat collectively all coastal lowland vegetation as a biodiversity hotspot.     

A DNA Vaccine against Yellow Fever Virus: Development and Evaluation

Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies.     

Ubiquitin-mediated internalization of connexin43 is independent of the canonical endocytic tyrosine-sorting signal

Gap junctions are specialized cell-cell contacts that provide direct intercellular communication between eukaryotic cells. The tyrosine-sorting signal (YXX?), present at amino acids 286-289 of Cx43 (connexin43), has been implicated in the internalization of the protein. In recent years, ubiquitination of Cx43 has also been proposed to regulate gap junction intercellular communication; however, the underlying mechanism and molecular players involved remain elusive. In the present study, we demonstrate that ubiquitinated Cx43 is internalized through a mechanism that is independent of the YXX? signal. Indeed, expression of a Cx43-Ub (ubiquitin) chimaera was shown to drive the internalization of a mutant Cx43 in which the YXX? motif was eliminated. Immunofluorescence, cycloheximide-chase and cell-surface-protein biotinylation experiments demonstrate that oligomerization of Cx43-Ub into hemichannels containing wild-type Cx43 or mutant Cx43Y286A is sufficient to drive the internalization of the protein. Furthermore, the internalization of Cx43 induced by Cx43-Ub was shown to depend on its interaction with epidermal growth factor receptor substrate 15.     

Immunohistochemical expression of types I and III collagen antibodies in the temporomandibular joint disc of human foetuses

The objective was to study the morphology of the articular disc and analyse the immunohistochemical expression of types I and III collagen markers in the temporomandibular joint (TMJ) disc of human foetuses of different gestational ages. Twenty TMJ from human foetuses supplied by Universidade Federal de Uberaba with gestational ages from 17 to 24 weeks were studied. The gestational age of the foetuses was determined by measuring the crown-rump (CR) length. Macroscopically, the foetuses were fixed in 10% formalin solution and dissected by removing the skin and subcutaneous tissue and exposing the deep structures. Immunohistochemical markers of type I and III were used to characterize the existence of collagen fibres. Analysis of the immunohistochemical markers of types I and III collagen revealed the presence of heterotypical fibril networks.     

Use of sodC versus ctrA for real-time polymerase chain reaction-based detection of Neisseria meningitidis in sterile body fluids

We evaluated the use of a newly described sodC-based real-time-polymerase chain reaction (RT-PCR) assay for detecting Neisseria meningitidis in normally sterile sites, such as cerebrospinal fluid and serum. The sodC-based RT-PCR assay has an advantage over ctrA for detecting nongroupable N. meningitidis isolates, which are commonly present in asymptomatic pharyngeal carriage. However, in our study, sodC-based RT-PCR was 7.5% less sensitive than ctrA. Given the public health impact of possible false-negative results due to the use of the sodC target gene alone, sodC-based RT-PCR for the diagnosis of meningococcal meningitis should be used with caution.     

Otters and fish farms in the Sado estuary: ecological and socio-economic basis of a conflict

The degree of conflict between otter conservation and fish farming was assessed at Sado estuary (SW Portugal), using ecological (otter visiting rates to fish farms and consumption of commercial fish) and socio-economic (past and current instruments and policies addressing the conflict and a social impact assessment, including a discourse analysis of relevant stakeholders) parameters. The study concerned 14 fish farms producing Sparus aurata, Dicentrarchus labrax, Solea senegalensis and Solea solea. Results indicate high visiting rates in most fish farms (average: 76%), although in only 29%, species stocked were the most consumed prey. Other marine species and freshwater prey were the basis of otter diet in the remaining fish farms. The conflict is quite consensual among most fish farmers, and the results indicate that the perceived conflict by fish farmers has an ecological basis, although there are large gaps between effective and perceived predation. No specific instruments exist in Portugal to address the conflict, but some not specifically targeted can have an effect (e.g. species protection legislation and aquaculture licensing), although with limitations to effectively contribute to its mitigation (e.g. lack of enforcement and supervision). Formulating and assessing solutions is the following step, using a participatory approach to the development and evaluation of mitigation/compensation strategies, capable of providing an effective reconciliation of the conflict.