Cys mutants in functional regions of Sticholysin I clarify the participation of these residues in pore formation

Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg(52) in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu(2), Phe(15)) did not modify the toxin`s permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation. (C) 2011 Elsevier Ltd. All rights reserved.

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64 kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22 kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation. (C) 2007 Elsevier Ltd. All rights reserved.

PILZ Protein Structure and Interactions with PILB and the FIMX EAL Domain: Implications for Control of Type IV Pilus Biogenesis

The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3`-> 5`)cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv Citri (PilZ(XAC1133), this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ(XAC1133) shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which NZ proteins regulate specific processes is not clear. In this study, we show that PilZ(XAC1133) binds to PilB, an ATPase required for TV polymerization, and to the EAL domain of FiMX(XAC2398), which regulates TV biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimXX(AC2398) in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ(XAC1133). Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ(XAC1133) interactions with FimX(XAC2398) and PilB(XAC3239) are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of ""degenerate"" GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery. (C) 2009 Elsevier Ltd. All rights reserved.

Formation of out of plane oxime metallacycles in [Cu(2)] and [Cu(4)] complexes

The reaction Of Cu(ClO(4))(2)center dot 6H(2)O with dimethylglyoxime (H(2)dmg) in a 1:1 mole ratio in aqueous methanol at room temperature affords the dinuclear complex [Cu(2)(mu-Hdmg)(4)] (1). Reaction of 1 with [Cu(bpy)(H(2)O)(2)](ClO(4))(2) (bpy = 2,2`-bipyridine) in a 1:1 mole ratio in aqueous methanol at room temperature yields the tetranuclear complex [Cu(2)(mu-HdMg)(2)(mu-dMg)(2)(bpy)(2)(H(2)O)(2)](ClO(4))(2) (2). The direct reaction of Cu(ClO(4))(2)center dot 6H(2)O with H(2)dmg and bpy in a 2:21 mole ratio in aqueous methanol at room temperature also yields 2 quantitatively. The complexes 1 and 2 were structurally characterized by X-ray crystallography. Unlike the binding in Ni/Co-dmg, two different types of N-O bridging modes during the oxime based metallacycle formation and stacking of square planar units have been identified in these complexes. The neutral dinuclear complex 1 has CuN(4)O coordination spheres and complex 2 consists of a dicationic [Cu(2)(mu-HdMg)(2)(mu-dMg)(2)(bpy)(2)(H(2)O)(2)](2+) unit and two uncoordinated ClO(4)(-) anions having CuN(4)O and CuN(2)O(3) coordination spheres. The two copper(II) ions are at a distance of 3.846(8) angstrom in 1 for the trans out of plane link and at 3.419(10) and 3.684(10) angstrom in 2 for the trans out of plane and cis in plane arrangements, respectively. The average Cu-N(oxime) distances are 1.953 and 1.935 angstrom, respectively. The average basal and apical Cu-N(oxime) distances are 1.945, 2.295 and 2.429 angstrom. The UV-Vis spectra of 2 is similar to the spectrum of the reaction mixture of 1 and [Cu(bpy)(H(2)O)(2)](2+). Variable temperature magnetic properties measurement shows that the interaction between the paramagnetic copper centers in complex I is antiferromagnetic in nature. The EPR spectra of frozen solution of the complexes at 77 K consist of axially symmetric fine-structure transitions (Delta M(S) = 1) and half-field signals (Delta M(S) = 2) at ca. 1600 G, suggesting the presence of appreciable Cu-Cu interactions. (C) 2009 Elsevier Ltd. All rights reserved.

Exploring new molecular species on the (1)[H, Se, F] singlet potential energy surface: Energetics, structures, IR spectra, and heats of formation

Structural, energetic, and vibrational properties of new molecular species, HSeF and HFSe, the associated transition state, and dissociation fragments are investigated using a state-of-the-art theoretical approach, CCSD(T)/CBS. HSeF is a normal covalently bonded molecule 38.98 kcal mol (1) more stable than the complex HF-Se, which shows an unusual structure with a central fluorine atom and a bond angle of 101.8 degrees.A barrier (Delta G(#)) of 49.01 kcal mol (1) separates the two species. Vibrational frequencies are also quite distinct. Heats of formation are evaluated for the diatomic fragments and HSeF. Final Delta(f)H values depend on the experimental accuracy of those of Se(g) and H(2)Se. (c) 2009 Elsevier B.V. All rights reserved.

Structure of polyaniline formed in different inorganic porous materials: A spectroscopic study

In this work, the electronic and structural characterization of polyaniline (PANI) formed in cavities of zeolites Y (ZY) and Mordenite (MOR) and montmorillonite (MMT) clay having Cu(II) as oxidant agent are presented. The formation of PANI and its structure is analyzed by Resonance Raman, UV-Vis-NIR, FT-IR and N K XANES techniques. In all cases the structure of PANT formed is different from the ""free"" polymer. The presence of azo bonds linked to phenazine-like rings are observed only for PANI-MMT composites, independent of the kind of oxidant agent employed in the synthesis. The presence of Cu(II) ions leads to the formation of Phenosafranine-like rings. The presence of these phenazine-like rings in the structure of confined PANT chains can also contribute to the enhancement of the thermal stability observed for all composites. (C) 2008 Elsevier Ltd. All rights reserved.

Effect of the chain length in the structure of imidazolic ionic liquids and dimethylformamide solutions probed by Raman spectroscopy

The Raman band assigned to the nu(C=O)mode in N,N-dimethylformamide (at ca. 1660 cm(-1)) was used as a probe to study a group of ionic liquids 1-alkyl-3-methylimidazolium bromide ([C(n)Mlm]Br) with different alkyl groups (n = 2, 4, 6, 8 and 10 carbons) in binary equimolar binary mixtures with dimethylformamide. Due to the high electric dipole moment of the group C=O, there is a substantial coupling between adjacent molecules in the solution, and the corresponding Raman band involves both vibrational and reorientational modes. Different chain lengths of the ILs lead to different extents of the uncoupling of adjacent molecules of dimethylformamide, resulting in different shifts for this band in the mixtures. Information about the organization of ionic liquids in solution was obtained and a model of aggregation for these systems is proposed. (C) 2010 Elsevier B.V. All rights reserved.

Flavoring agents present in a dentifrice can modify volatile sulphur compounds (VSCs) formation in morning bad breath

This study aimed to evaluate the effects of a flavor-containing dentifrice on the formation of volatile sulphur compounds (VSCs) in morning bad breath. A two-step, blinded, crossover, randomized study was carried out in 50 dental students with a healthy periodontium divided into two experimental groups: flavor-containing dentifrice (test) and non-flavor-containing dentifrice (control). The volunteers received the designated dentifrice and a new toothbrush for a 3 X/day brushing regimen for 2 periods of 30 days. A seven-day washout interval was used between the periods. The assessed parameters were: plaque index (PI), gingival index (GI), organoleptic breath scores (ORG), VSC levels (as measured by a portable sulphide monitor) before (H1) and after (H2) cleaning of the tongue, tongue coating (TC) wet weight and BANA test from TC samples. The intra-group analysis showed a decrease in ORG, from 3 to 2, after 30 days for the test group (p < 0.05). The inter-group analysis showed lower values in ORG, H1 and H2 for the test group (p < 0.05). There was no difference between the amount of TC between groups and the presence of flavor also did not interfere in the BANA results between groups (p > 0.05). These findings suggest that a flavor-containing dentifrice seems to prevent VSCs formation in morning bad breath regardless of the amount of TC in periodontally healthy subjects.

Endodontic treatment of dental formation anomalies

PURPOSE: Dental fusion is defined as the union of two dental germs at some stage of their development. The aim of this article is to report the endodontic treatment of two clinical cases of dental fusion. CASE DESCRIPTION: In the first case, the patient was referred by an orthodontist for endodontic treatment of tooth 12, which was fused to 13. Surgical separation and later replacement of the involved elements in the dental arch was indicated. In the second case, the patient sought dental attendance due to spontaneous pain. In the radiographic exam, gemination in tooth 11 and fusion of 21 with a supernumerary tooth was observed. The fused teeth were endodontically treated, and patients were referred to other dental specialties to reestablish esthetics and function. CONCLUSION: The dentist must be able to diagnose, differentiate and treat these dental anomalies adequately, with the goal of maintaining patients' oral health.

Evaluation of the dental structure loss produced during maintenance and replacement of occlusal amalgam restorations

The aim of this in vitro study was to evaluate four different approaches to the decision of changing or not defective amalgam restorations in first primary molar teeth concerning the loss of dental structure. Ditched amalgam restorations (n = 11) were submitted to four different treatments, as follows: Control group - polishing and finishing of the restorations were carried out; Amalgam group - the ditched amalgam restorations were replaced by new amalgam restorations; Composite resin group - the initial amalgam restorations were replaced by composite resin restorations; Flowable resin group - the ditching around the amalgam restorations was filled with flowable resin. Images of the sectioned teeth were made and the area of the cavities before and after the procedures was determined by image analysis software to assess structural loss. The data were submitted to ANOVA complemented by the Student Newman Keuls test (p < 0.05). The cavities in all the groups presented significantly greater areas after the procedures. However, the amalgam group showed more substantial dental loss. The other three groups presented no statistically significant difference in dental structure loss after the re-treatments. Thus, replacing ditched amalgam restorations by other similar restorations resulted in a significant dental structure loss while maintaining them or replacing them by resin restorations did not result in significant loss.

Characterization of oil shale residue and rejects from irati formation by electron paramagnetic resonance

In this study, sedimentary organic matter of oil shale rejects, calschist, shale fine and the so called retorted shale from Irati formation was characterized. EPR was used to analyse the samples regarding loss of signal in g = 2.003 associated to the organic free radical with the calcined samples and washing with hydrogen peroxide. The radical signal was detected in all samples, however, for the calschist and shale fine samples another signal was identified at g = 2.000 which disappeared when the sample was heated at 400 ºC. Hydrogen peroxide washing was also performed and it was noted that after washing the signal appeared around g = 2.000 for all samples, including retorted shale, which might be due to the quartz E1 defect.

Transpressive dextral shear in the Italva-Itaperuna section, Northern State of Rio de Janeiro, Brazil

Structural analysis carried out on a segment of the Neoproterozoic Ribeira Belt, southeastern Brazil, show that it represents part of the transpressive dextral orogen related to the Central Mantiqueira Province. NNE-trending and steeply dipping regional mylonitic belts form anastomosed geometry, and describe a map-scale, S-C-like structure that is characterized by their deflection towards NE near the Além Paraíba Lineament. Lithological and structural control related to deformation partition were responsible for the formation of felsic mylonitic granulites with S-type granites lenses developed in ductile shear zones, alternated with less deformed intermediate to basic granulites associated with charnockites. The dextral shear sense indicators are consistent with transpressive deformation in the region and are common especially at the border of the main shear zones. The presence of S-type leucogranite may lead to variations of linear and planar relationships, which result in local extension zones. These elements are consistent with oblique continental collision considering the São Francisco Craton as a stable block.

Marine influence in the Barreiras Formation, State of Alagoas, northeastern Brazil

Tidal processes were important for deposition of the Barreiras Formation located in northern Brazil, while correlatable deposits in northeastern Brazil have been traditionally related to continental environments. Facies analysis in southern Alagoas revealed that the Barreiras Formation consists of cross-stratified conglomerates and sandstones (facies Sx and Cgx), compound cross-stratified sandstones (facies Cx), and heterolithic beddings (facies H). A significant portion of these deposits occurs within channel morphologies displaying fining and thinning upward successions. An abundance of sedimentary features is comparable to those from the northern Brazilian counterpart. These include: tidal bundles; herringbone cross-stratification; heterolithic beddings with sandstone and mudstone beds in sharp contacts; and ichnofossils mostly consisting of Ophiomorpha nodosa, Skolithos and Planolites. Altogether, these features point to a marginal marine depositional setting dominated by tidal processes, which are related to an estuarine system, an interpretation also provided for the Barreiras Formation in northern Brazil. The widespread occurrence of deposits with unambiguous evidence of tidal processes in the Barreiras Formation of northern Brazil, and now in the State of Alagoas, leads to argue that the early/middle Miocene worldwide marine transgression might have left a much more widespread sedimentary record along the Brazilian coast than currently regarded.

Benthic macrofauna structure in the northeast area of Todos os Santos Bay, Bahia State, Brazil: patterns of spatial and seasonal distribution

Structure of intertidal and subtidal benthic macrofauna in the northeastern region of Todos os Santos Bay (TSB), northeast Brazil, was investigated during a period of two years. Relationships with environmental parameters were studied through uni-and multivariate statistical analyses, and the main distributional patterns shown to be especially related to sediment type and content of organic fractions (Carbon, Nitrogen, Phosphorus), on both temporal and spatial scales. Polychaete annelids accounted for more than 70% of the total fauna and showed low densities, species richness and diversity, except for the area situated on the reef banks. These banks constitute a peculiar environment in relation to the rest of the region by having coarse sediments poor in organic matter and rich in biodetritic carbonates besides an abundant and diverse fauna. The intertidal region and the shallower area nearer to the oil refinery RLAM, with sediments composed mainly of fine sand, seem to constitute an unstable system with few highly dominant species, such as Armandia polyophthalma and Laeonereis acuta. In the other regions of TSB, where muddy bottoms predominated, densities and diversity were low, especially in the stations near the refinery. Here the lowest values of the biological indicators occurred together with the highest organic compound content. In addition, the nearest sites (stations 4 and 7) were sometimes azoic. The adjacent Caboto, considered as a control area at first, presented low density but intermediate values of species diversity, which indicates a less disturbed environment in relation to the pelitic infralittoral in front of the refinery. The results of the ordination analyses evidenced five homogeneous groups of stations (intertidal; reef banks; pelitic infralittoral; mixed sediments; Caboto) with different specific patterns, a fact which seems to be mainly related to granulometry and chemical sediment characteristics.

Population structure of the lancelet Branchiostoma caribaeum (Cephalochordata: Branchiostomidae) in the Baía de Guanabara, Rio de Janeiro, southeastern Brazil

Population structure of the lancelet Branchiostoma caribaeum Sandevall, 1853 was studied in four surveys, corresponding to austral seasons, in a tropical bay, southeast of Brazil. Abundance was higher in the spring and was positively correlated to coarse sediments, limiting its occurrence to some sectors of the sampling area. Body length and biomass differed seasonally but not between sexes. Sexually mature individuals occurred in all seasons, suggesting continuous breeding that is typical of tropical species. Variation in the frequency of small specimens indicates temporal differences in the intensity of breeding. The body length of recruits differed from other population of lancelets and the small length which B. caribaeum attained sexual maturity in Guanabara Bay may be related to local environmental stress or the great availability of food.