Structural stability and reversible unfolding of recombinant porcine S100A12

Porcine S100A12 is a member of the S100 proteins, family of small acidic calcium-binding proteins characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events such as the regulation of protein phosphorylation, enzymatic activity, protein-protein interaction, Ca(2+) homeostasis, inflammatory processes and intermediate filament polymerization. In addition, members of this family bind Zn(2+) or Ca(2+) with cooperative effect on binding. In this study, the gene sequence encoding porcine S100A12 was obtained by the synthetic gene approach using E. coli codon bias. Additionally, we report a thermodynamic study of the recombinant S100A12 using circular dichroism, fluorescence and isothermal titration calorimetry. The results of urea and temperature induced unfolding and refolding processes indicated a reversible two-state process. Also, the ANS fluorescence studies showed that in presence of divalent ions the protein exposes hydrophobic sites which could facilitate the interaction with other proteins and trigger the physiological responses. (c) 2008 Elsevier B.V. All rights reserved.

The stability and aggregation properties of the GTPase domain from human SEPT4

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTR At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease. (C) 2008 Elsevier B.V. All rights reserved.

Skipping of exon 30 in C5 gene results in complete human C5 deficiency and demonstrates the importance of C5d and CUB domains for stability

The deficiency of complement C5 is rare and frequently associated with severe and recurrent infections, especially caused by Neisseria spp. We observed the absence of component C5 in the serum of 3 siblings from a Brazilian family with history of consanguinity. The patients had suffered from recurrent episodes of meningitis and other less severe infections. Sera from these patients were unable to mediate hemolytic activity either by the classical or alternative pathways and presented extremely low levels of C5 protein (13, 0.9 and 1.0 mu g/ml-normal range: 45-190 mu g/ml). Hemolytic activity could be restored by the addition of purified C5 to deficient serum. Sequencing of sibling C5 cDNA revealed a homozygous 153 bp deletion that corresponds precisely to exon 30. The parents carried the same deletion but only in one allele. Sequencing of the corresponding region in the genomic DNA revealed a C to C substitution within intron 30 and, most significantly, the substitution of GAG(4028) for GAA(4028) at the 3` end of exon 30 which is most likely responsible for skipping of exon 30. The resulting in-frame deletion in the C5 mRNA codes for a mutant C5 protein lacking residues 1289-1339. These residues map to the CUB and C5d domains of the C5 alpha chain. This deletion is expected to produce a non-functional and unstable C5 protein which is more susceptible to degradation. (C) 2009 Published by Elsevier Ltd.

Decisions with Impact on Energy Reduction Opportunities : Swedish manufacturers and buyers of new single-family houses

This article discusses some of the complexities of human decision-making. It aims, in particular, at relating the nature of decision-making to the illusory dichotomies of change and stability, individual actions and cultural sharing. Serving as an illustration to the discussion of the article is ongoing fieldwork in contexts of buying, selling and constructing pre-fabricated detached houses in the central Sweden, and the very specific question of how decisions to install one kind of heating-system rather than another come about. A common reductionism is to narrow down the understanding of decisions about heating systems and energy consumption to conscious choices made by individual householders. I have asked myself whether, on the contrary, anyone actually makes such decisions at all. Perhaps some of these decisions are merely outcomes of interaction between different individuals with their respective responsibilities and focuses of interest.

Genetic heterogeneity of within-family variance of body weight in Atlantic salmon (Salmo salar)

BACKGROUND: Canalization is defined as the stability of a genotype against minor variations in both environment and genetics. Genetic variation in degree of canalization causes heterogeneity of within-family variance. The aims of this study are twofold: (1) quantify genetic heterogeneity of (within-family) residual variance in Atlantic salmon and (2) test whether the observed heterogeneity of (within-family) residual variance can be explained by simple scaling effects. RESULTS: Analysis of body weight in Atlantic salmon using a double hierarchical generalized linear model (DHGLM) revealed substantial heterogeneity of within-family variance. The 95% prediction interval for within-family variance ranged from ~0.4 to 1.2 kg2, implying that the within-family variance of the most extreme high families is expected to be approximately three times larger than the extreme low families. For cross-sectional data, DHGLM with an animal mean sub-model resulted in severe bias, while a corresponding sire-dam model was appropriate. Heterogeneity of variance was not sensitive to Box-Cox transformations of phenotypes, which implies that heterogeneity of variance exists beyond what would be expected from simple scaling effects. CONCLUSIONS: Substantial heterogeneity of within-family variance was found for body weight in Atlantic salmon. A tendency towards higher variance with higher means (scaling effects) was observed, but heterogeneity of within-family variance existed beyond what could be explained by simple scaling effects. For cross-sectional data, using the animal mean sub-model in the DHGLM resulted in biased estimates of variance components, which differed substantially both from a standard linear mean animal model and a sire-dam DHGLM model. Although genetic differences in canalization were observed, selection for increased canalization is difficult, because there is limited individual information for the variance sub-model, especially when based on cross-sectional data. Furthermore, potential macro-environmental changes (diet, climatic region, etc.) may make genetic heterogeneity of variance a less stable trait over time and space.

Dynamical erosion of asteroid groups in the region of the Phocaea family

In a previous paper, the current state of knowledge of the region containing the Phocaea dynamical family was revised. Here, the dynamical evolution and possible origin of the Phocaea dynamical family and asteroid groups in the region are investigated. First, I study the case of asteroids at high eccentricity (e > 0.31). I find that these objects are unstable because of encounters with Mars on time-scales of up to 270 Myr. The minimum time needed by members of the Phocaea classical family to reach the orbital locations of these objects, 370 Myr, can be used to set a lower limit on the age of the Phocaea family.Next, attention is focused on the chaotic layer previously identified near the nu(6) secular resonance border. Using analytical and numerical tools, I find that the presence of the nu(6) secular resonance forces asteroids with vertical bar g-g(6)vertical bar < 2.55 arcsec yr(-1) to reach eccentricities high enough to allow them to experience deep, close encounters with Mars. Results of the analytical model of Yoshikawa and of my numerical simulations fully explain the low-inclination chaotic region found by Carruba.Finally, I investigate the long-term stability of the minor families and clumps identified in the previous paper, with particular emphasis on a clump only identifiable in the domain of proper frequencies (n, g, g - s) around (6246) Komurotoru. I find that while the clumps identified in the space of proper elements quickly disperse when the Yarkovsky effect is considered, the family around (19536) is still observable for time-scales of more than 50 Myr. The (6246) clump, characterized by its interaction with the nu(5) + nu(16) and 2 nu(6) - nu(16) secular resonances, is robust on time-scales of 50 Myr. I confirm that this group may be the first clump ever detected in the frequency domain that can be associated with a real collisional event.

Dynamical erosion of asteroid groups in the region of the Pallas family

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An analysis of the region of the Phocaea dynamical family

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Stability results for impulsive functional differential equations with infinite delay

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

HETEROCHROMATIN AND KARYOTYPE REORGANIZATION IN FISH OF THE FAMILY ANOSTOMIDAE (CHARACIFORMES)

Mitotic chromosomes of four fish species of the family Anostomidae, belonging to the genera Leporinus, Leporellus, and Schizodon, were studied. With 2n = 54 meta- and submetacentric chromosomes, this family appears to be characterized by marked karyotypic stability. Although perceptible differences exist, mainly in the amount of constitutive heterochromatin present in the chromosomes of these species, these differences do not affect the structure and/or size of these chromosomes. Chromatin substitutions and/or modifications may have led, in one direction, to an increase in heterochromatin in some species and, in the opposite direction, to heterochromatin reduction in others. Whether these changes are accompanied by changes in the amount of euchromatin in the chromosomes is an open question. The nucleolar organizer regions, which may be located on different chromosomes in the various species, may also be indicators of reorganization of these karyotypes.

Development of a topical formulation containing S. Lutea extract: Stability, in vitro studies and cutaneous permeation

Flavonoids have been widely incorporated into cosmetic and dermatological formulations, affording benefits such as antioxidant action, improved skin tone and fewer lines and wrinkles. Brazil has a huge biodiversity, with one of the richest flora in the world, and existing studies justify the quest for greater research efforts in this area. The cajazeira (Spondias lutea L.), a plant of the Anacardiaceae family from tropical America, is widely disseminated in Brazil. This plant was chosen because of the presence of flavonoids that exhibit antioxidant activity. The purpose of this research was to develop a stable topical formulation containing Spondias lutea extract with the aim of preventing skin diseases caused by UV radiation. Hydro ethanolic extract of Spondias lutea fruit was prepared and assayed for its the flavonoids content. The antioxidant activity was estimated by DPPH and superoxide assay. The physicochemical stability and skin permeation of the cream containing 8% (w/w) of extract were assessed. The results showed that the S. lutea extract possessed antioxidant activity, and that it is possible to obtain a stable cosmetic containing the extract, which is able to penetrate the skin. Thus, it is possible to use this extract to produce an anti-aging cosmetic.

Comparative analysis of three hyperthermophilic GH1 and GH3 family members with industrial potential

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Septin C-Terminal Domain Interactions: Implications for Filament Stability and Assembly

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high alpha-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K (D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K (D) of 4 mu M, and a moderate alpha-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.

Improvement of fungal arabinofuranosidase thermal stability by reversible immobilization

A gene encoding a-L-arabinofuranosidase (abfA) from Aspergillus niveus was identified, cloned, and successfully expressed in Aspergillus nidulans. Based on amino acid sequence comparison, the 88.6 kDa enzyme could be assigned to the GH family 51. The characterization of the purified recombinant AbfA revealed that the enzyme was active at a limited pH range (pH 4.0-5.0) and an optimum temperature of 70 degrees C. The AbfA was able to hydrolyze arabinoxylan, xylan from birchwood, debranched arabinan, and 4-nitrophenyl arabinofuranoside. Synergistic reactions using both AbfA and endoxylanase were also assessed. The highest degree of synergy was obtained after the sequential treatment of the substrate with endoxylanase, followed by AbfA, which was observed to release noticeably more reducing sugars than that of either enzyme acting individually. The immobilization of AbfA was performed via ionic adsorption onto various supports: agarose activated by polyethyleneimine polymers, cyanogen bromide activated Sepharose, DEAE-Sepharose, and Sepharose-Q The Sepharose-Q derivative remained fully active at pH 5 after 360 min at 60 degrees C, whereas the free AbfA was inactivated after 60 min. A synergistic effect of arabinoxylan hydrolysis by AbfA immobilized in Sepharose-Q and endoxylanase immobilized in glyoxyl agarose was also observed. The stabilization of arabinofuranosidases using immobilization tools is a novel and interesting topic. (C) 2012 Elsevier Ltd. All rights reserved.

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCe15A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.