Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli : purification , biochemical and kinetic characterisation

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl-aminopeptidase I. The amino acid sequence, deduced from the nucleotide sequence, revealed that it consists of 209 amino acid residues and showed to have 98% homology with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary among the two sequences. The bovine enzyme was expressed in XL10-gold Esherichia coli cells. Immobilizied Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 3.3mg of PAP1 per litre culture. The purified enzyme had a specific activity of 1700 units/ml. SDS-PAGE produced a single band for bovine PAP1 with a molecular weight of ~23-24 kDa which is in good agreement with previously reported data on PAP1. Kinetic constants Km and Kcat were 59μΜ and 3.5s-1, respectively. It possessed an optimum pH between 9-9.5, a temperature of 37°C and showed an absolute requirement for a thiol-reducing agent (10mM DTT). EDTA didn’t prove to have an effect on enzyme activity. Competitive inhibition was seen with pyroglutamyl peptides pGlu-His-Pro-NH2 (TRH; Ki= 44.1 uM), pGlu-Ala- OH (Ki=141 uM) and pGlu-Val-OH (Ki=652.17)

Prediction of Protein Modification Sites of Pyrrolidone Carboxylic Acid Using mRMR Feature Selection and Analysis

Pyrrolidone carboxylic acid (PCA) is formed during a common post-translational modification (PTM) of extracellular and multi-pass membrane proteins. In this study, we developed a new predictor to predict the modification sites of PCA based on maximum relevance minimum redundancy (mRMR) and incremental feature selection (IFS). We incorporated 727 features that belonged to 7 kinds of protein properties to predict the modification sites, including sequence conservation, residual disorder, amino acid factor, secondary structure and solvent accessibility, gain/loss of amino acid during evolution, propensity of amino acid to be conserved at protein-protein interface and protein surface, and deviation of side chain carbon atom number. Among these 727 features, 244 features were selected by mRMR and IFS as the optimized features for the prediction, with which the prediction model achieved a maximum of MCC of 0.7812. Feature analysis showed that all feature types contributed to the modification process. Further site-specific feature analysis showed that the features derived from PCA's surrounding sites contributed more to the determination of PCA sites than other sites. The detailed feature analysis in this paper might provide important clues for understanding the mechanism of the PCA formation and guide relevant experimental validations

Breaking Functional Connectivity into Components: A Novel Approach Using an Individual-Based Model, and First Outcomes

Landscape connectivity is a key factor determining the viability of populations in fragmented landscapes. Predicting ‘functional connectivity’, namely whether a patch or a landscape functions as connected from the perspective of a focal species, poses various challenges. First, empirical data on the movement behaviour of species is often scarce. Second, animal-landscape interactions are bound to yield complex patterns. Lastly, functional connectivity involves various components that are rarely assessed separately. We introduce the spatially explicit, individual-based model FunCon as means to distinguish between components of functional connectivity and to assess how each of them affects the sensitivity of species and communities to landscape structures. We then present the results of exploratory simulations over six landscapes of different fragmentation levels and across a range of hypothetical bird species that differ in their response to habitat edges. i) Our results demonstrate that estimations of functional connectivity depend not only on the response of species to edges (avoidance versus penetration into the matrix), the movement mode investigated (home range movements versus dispersal), and the way in which the matrix is being crossed (random walk versus gap crossing), but also on the choice of connectivity measure (in this case, the model output examined). ii) We further show a strong effect of the mortality scenario applied, indicating that movement decisions that do not fully match the mortality risks are likely to reduce connectivity and enhance sensitivity to fragmentation. iii) Despite these complexities, some consistent patterns emerged. For instance, the ranking order of landscapes in terms of functional connectivity was mostly consistent across the entire range of hypothetical species, indicating that simple landscape indices can potentially serve as valuable surrogates for functional connectivity. Yet such simplifications must be carefully evaluated in terms of the components of functional connectivity they actually predict

Análise espacial de fragmentos florestais na Bacia do Rio Itapemirim, ES

Os objetivos deste trabalho foi mapear e analisar a estrutura da paisagem florestal em uma área representativa da Bacia do Rio Itapemirim, ES, por meio de índices de ecologia da paisagem. O mapeamento dos fragmentos florestais foi obtido utilizando técnicas de fotointerpretação na escala de 1:1500 do ortofotomosaico do ano 2007. Para o cálculo dos índices de ecologia, foi utilizada a extensão Patch Analyst dentro do aplicativo computacional ArcGIS 9.3. Foram encontrados 3.285 fragmentos florestais em toda a área, representando 17% de cobertura florestal. Os fragmentos mapeados foram divididos em classes de tamanho, sendo fragmentos pequenos aqueles menores que 5 ha; de tamanho médio aqueles entre 5 e 50 ha; e grandes os maiores que 50 ha. Os fragmentos pequenos encontravam-se em maior número (2.236), seguidos pelos fragmentos de tamanho médio (749) e, por último, pelos fragmentos grandes, que compreenderam apenas 100 manchas. O número de fragmentos de cada classe de tamanho possui relação inversa com a contribuição em área dessa classe dentro da fragmentação florestal. As análises quantitativas por meio de métricas da paisagem foram feitas com os grupos de índices de área; densidade e tamanho, forma; proximidade e área central, sendo este último obtido em diferentes simulações de efeito de borda (20,40, 60, 80, 100, 140 e 200 m). Para todos os índices houve diferenciações com relação às classes de tamanho dos fragmentos florestais. A maior parte dos fragmentos florestais da bacia, são pequenos, menores que 5 ha, sendo que a distância de borda de 100 metros elimina completamente a área central desses fragmentos. Apesar dos fragmentos grandes apresentarem os formatos mais irregulares, estes possuem maior índice de área central, mesmo sob o efeito da maior distância de efeito de borda

Vertical motion in the Indian summer monsoon

The vertical motion field in the Indian monsoon region has been studied for mean July, a strong monsoon day (7 July 1933) and a weak monsoon day (19 July 1963). We have used the quasi-geostrophic omega equation for this purpose, In addition to forcings due to thermal and differential voriticty advection, the vertical motion due to orography and friction have been included. The vertical motions due to the different forcing functions have been computed. The cumulative omega field due to all forcing functions and due to individual forcing functions together with their vertical profiles are delineated.   Conversion from potential to kinetic energy associated with the vertical circulations have been computed and presented in this paper, It is seen that the meridicnal circulation in the y-p plane is very marked on 7 July with ascent over north India and descent to the south. On 19th, however, uniform descent is seen everywhere. There is conversion from zonal available potential energy to zonal kinetic energy on the day of active monsoon. The conversion is much smaller on the day of weak monsoon. Other energy conversions are also discussed.</jats:p