SBTX, a new toxic protein distinct from soyatoxin and other toxic soybean [Glycine max] proteins, and its inhibitory effect on Cercospora sojina growth

SBTX, a novel toxin from soybean, was purified by ammonium sulfate fractionation followed by chromatographic steps DEAE-Cellulose, CM-Sepharose and Superdex 200 HR fast-protein liquid chromatography (FPLC). Lethality of SBTX to mice (LD50 5.6 mg/kg) was used as parameter in the purification steps. SBTX is a 44-kDa basic glycoprotein composed of two polypeptide chains (27 and 17 kDa) linked by a disulfide bond. The N-terminal sequences of the 44 and 27 kDa chains were identical (ADPTFGFTPLGLSEKANLQIMKAYD), differing from that of 17 kDa (PNPKVFFDMTIGGQSAGRIVMEEYA). SBTX contains high levels of Glx, Ala, Asx, Gly and Lys and showed maximum absorption at 280 nm, epsilon(1 cm) (1%) of 6.3, and fluorescence emission in the 290-450nm range upon excitation at 280nm. The secondary structure content was 35% alpha-helix, 13% beta-strand and beta-sheet, 27% beta-turn, 25% unordered, and 1% aromatic residues. Immunological assays showed that SBTX was related to other toxic proteins, such as soyatoxin and canatoxin, and cross-reacted weekly with soybean trypsin inhibitor and agglutinin, but it was devoid of protease-inhibitory and hemagglutinating activities. The inhibitory effect of SBTX on growth of Cercospora sojina, fungus causing frogeye leaf spot in soybeans, was observed at 50 mu g/ml, concentration 112 times lesser than that found to be lethal to mice. This effect on phytopathogenic fungus is a potential attribute for the development of transgenic plants with enhanced resistance to pathogens. (c) 2007 Elsevier Ltd. All rights reserved.

A ditryptophan cross-link is responsible for the covalent dimerization of human superoxide dismutase 1 during its bicarbonate-dependent peroxidase activity

Unlike intermolecular disulfide bonds, other protein cross-links arising from oxidative modifications cannot be reversed and are presumably more toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain poorly characterized. For instance, the antioxidant enzyme human superoxide dismutase 1 (hSod1) has been reported to undergo non-disulfide covalent dimerization and further oligomerization during its bicarbonate-dependent peroxidase activity. The dimerization was shown to be dependent on the oxidation of the single, solvent-exposed TrP(32) residue of hSod1, but the covalent dimer was not isolated nor was its structure determined. In this work, the hSod1 covalent dimer was isolated, digested with trypsin in H(2)O and H(2)(18)O, and analyzed by UV-Vis spectroscopy and mass spectrometry (MS). The results demonstrate that the covalent dimer consists of two hSod1 subunits cross-linked by a ditryptophan, which contains a bond between C3 and N1 of the respective Trp(32) residues. We further demonstrate that the cross-link cleaves under usual MS/MS conditions leading to apparently unmodified Trp(32), partially hinders proteolysis, and provides a mechanism to explain the formation of hSod1 covalent trimers and tetramers. This characterization of the covalent hSod1 dimer identifies a novel oxidative modification of protein Trp residues and provides clues for studying its occurrence in vivo. (C) 2010 Elsevier Inc. All rights reserved.

Between-strand disulfides: Forbidden disulfides linking adjacent ß-Strands

Between-strand disulfides (BSDs) connect cysteine (Cys) residues across adjacent strands of β-sheets. There are four BSD types which can be found in regular β-structure: CSDs, which link residues immediately opposite each other in the β-structure (residues i and j); ETDs, which connect Cys out of register by one residue (i and j ± 1); BDDs, which join Cys at positions i and j ± 2; and BFDs, which link residues i and j ± 3. Formation of these disulfides was initially predicted to be forbidden, producing too much local strain in the protein fold. However, BSDs do exist in nature. Significantly, their high levels of strain allow them to be involved in redox processes under physiological conditions. Here we characterise BSD motifs found in the Protein Data Bank (PDB), discussing important intrinsic factors, such as the disulfide conformation and torsional strain, and extrinsic factors, such as the influence of the β-sheet environment on the disulfide and vice versa. We also discuss the biological importance of BSDs, including the prevalence of non-homologous examples in the PDB, the conservation of BSD motifs amongst related proteins (BSD clusters) and experimental evidence for BSD redox activity. For clusters of homologous BSDs we present detailed data of the disulfide properties and the variations of these properties amongst the “redundant” structures. Identification of disulfides with the potential to be involved in biological redox processes via the analysis of these data will provide important insights into the function and mechanism of BSD-containing proteins. Characterisation of thiol-based redox signalling pathways will lead to significant breakthroughs in understanding the molecular basis of oxidative stress and associated pathways, such as ageing and neurodegenerative diseases.

Cross cultural exchange to support reasoning about socio-scientific sustainability issues

In this article, we describe a project on reasoning about socio-scientific issues (SSIs), involving French and Australian pre-service science teachers engaged in on-line discussion and development of a wiki. In the research, we developed frameworks for looking at the quality of reasoning about 'socially acute' sustainability questions. We found the level of reasoning was enhanced by the cross-cultural exchange, and identified the importance of context in framing reasoning quality. We argue that science teachers could effectively adapt this approach to develop students' scientific literacy and embed the 'science as a human endeavour' strand of the Australian Curriculum in their practice.

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. Structured digital abstract XfDsbC and XfDsbC bind by x ray scattering (View Interaction: 1, 2) XfDsbC and XfDsbC bind by molecular sieving (View interaction) XfDsbC and XfDsbC bind by comigration in non denaturing gel electrophoresis (View interaction) XfDsbC and XfDsbC bind by cross-linking study (View Interaction: 1, 2) XfDsbC and XfDsbC bind by dynamic light scattering (View Interaction: 1, 2)

Electron Interactions with Disulfide Bridges

Because of its electronic properties, sulfur plays a major role in a variety of metabolic processes and, more in general, in the chemistry of life. In particular, S-S bridges between cysteines are present in the amino acid backbone of proteins. Protein disulfur radical anions may decay following different paths through competing intra and intermolecular routes, including bond cleavage, disproportionation, protein-protein cross linking, and electron transfer. Indeed, mass spectrometry ECD (electron capture dissociation massspectroscopy) studies have shown that capture of low-energy (<0.2 eV) electrons by multiply protonated proteins is followed by dissociation of S-S bonds holding two peptide chains together. In view of the importance of organic sulfur chemistry, we report on electron interactions with disulphide bridges. To study these interactions we used as prototypes the molecules dimethyl sulfide [(CH3)2S] and dimethyl disulfide [(H3C)S2(CH3)]. We seek to better understand the electron-induced cleavage of the disulfide bond. To explore dissociative processes we performed electron scattering calculations with the Schwinger Multichannel Method with pseudopotentials (SMCPP), recently parallelized with OpenMP directives and optimized with subroutines for linear algebra (BLAS) and LAPACK routines. Elastic cross sections obtained for different S-S bond lengths indicate stabilization of the anion formed by electron attachment to a σ*SS antibonding orbital, such that dissociation would be expected.

Effects of Strand Geometry on Selected Properties of Long-Stand Structural Composite Lumber Made from Northeastern Hardwoods

Structural composite lumber (SCL) products often possess significantly higher design values than the top grades of solid lumber, making it a popular choice for both residential and commercial applications. The enhanced mechanical properties of SCL are mainly due to defect randomization and densification of the wood fiber, both largely functions of the size, shape and composition (species) of the wood element. Traditionally, SCL manufacturers have used thin, rectangular elements produced from either moderate density softwoods or low density hardwoods. Higher density hardwood species have been avoided, as they require higher pressures to adequately densify and consolidate the wood furnish. These higher pressures can lead to increased manufacturing costs, damage to the wood fiber and/or a product that is too dense, making it heavy and unreceptive to common mechanical fastening techniques. In the northeastern United States high density, diffuse-porous hardwoods (such as maple, beech and birch) are abundant. Use of these species as primary furnish for a SCL product may allow for a competitive advantage in terms of resource cost against products that rely on veneer grade logs. Proximity to this abundant and relatively inexpensive resource may facilitate entry of SCL production facilities in the northeastern United States, where currently none exist. However, modifications to current strand sizes, geometries or production techniques will likely be required to allow for use of these species. A new SCL product concept has been invented allowing for use of these high density hardwoods. The product, referred to as long-strand structural composite lumber (LSSCL), uses strands of significantly larger cross sectional areas and volumes than existing SCL products. In spite of the large strand size, satisfactory consolidation is achieved without excessive densification of the wood fiber through use of a symmetrical strand geometric cross-section. LSSCL density is similar to that of existing SCL products, but is due mainly to the inherent density of the species, rather than through densification. An experiment was designed and conducted producing LSSCL from both large (7/16”) and small (1/4”) strands, of both square and triangular geometric cross sections. Testing results indicate that the large, triangular strands produce LSSCL beams with projected design values of: Modulus of elasticity (MOEapp) – 1,750,000 psi; Allowable bending stress (Fb) – 2750 psi; Allowable shear stress (Fv) – 260 psi. Several modifications are recommended which may lead to improvement of these values, likely allowing for competition against existing SCL products.

Design, synthesis and cellular and molecular pharmacology of 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401): A novel non-cross-resistant anthracycline antibiotic with the intrinsic ability to circumvent P -glycoprotein-mediated drug resistance

We designed and synthesized a novel daunorubicin (DNR) analogue that effectively circumvents P-glycoprotein (P-gp)-mediated drug resistance. The fully protected carbohydrate intermediate 1,2-dibromoacosamine was prepared from acosamine and effectively coupled to daunomycinone in high yield. Deprotection under alkaline conditions yielded 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401). The in vitro cytotoxicity and cellular and molecular pharmacology of WP401 were compared with those of DNR in a panel of wild-type cell lines (KB-3-1, P388S, and HL60S) and their multidrug-resistant (MDR) counterparts (KB-V1, P388/DOX, and HL60/DOX). Fluorescent spectrophotometry, flow cytometry, and confocal laser scanning microscopy were used to measure intracellular accumulation, retention, and subcellular distribution of these agents. All MDR cell lines exhibited reduced DNR uptake that was restored, upon incubation with either verapamil (VER) or cyclosporin A (CSA), to the level found in sensitive cell lines. In contrast, the uptake of WP401 was essentially the same in the absence or presence of VER or CSA in all tested cell lines. The in vitro cytotoxicity of WP401 was similar to that of DNR in the sensitive cell lines but significantly higher in resistant cell lines (resistance index (RI) of 2-6 for WP401 vs 75-85 for DNR). To ascertain whether drug-mediated cytotoxicity and retention were accompanied by DNA strand breaks, DNA single- and double-strand breaks were assessed by alkaline elution. High levels of such breaks were obtained using 0.1-2 $\mu$g/mL of WP401 in both sensitive and resistant cells. In contrast, DNR caused strand breaks only in sensitive cells and not much in resistant cells. We also compared drug-induced DNA fragmentation similar to that induced by DNR. However, in P-gp-positive cells, WP401 induced 2- to 5-fold more DNA fragmentation than DNR. This increased DNA strand breakage by WP401 was correlated with its increased uptake and cytotoxicity in these cell lines. Overall these results indicate that WP401 is more cytotoxic than DNR in MDR cells and that this phenomenon might be related to the reduced basicity of the amino group and increased lipophilicity of WP401. ^

Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex.

Agrobacterium tumefaciens VirB proteins are essential for gene transfer from bacteria to plants. These proteins are postulated to form a transport pore to allow transfer of the T-strand DNA intermediate. To study the function of the VirB proteins in DNA transfer, we developed an expression system in A. tumefaciens. Analysis of one VirB protein, VirB9, by Western blot assays showed that under nonreducing conditions VirB9, when expressed alone, migrates as a approximately 31-kDa band but that it migrates as a approximately 36-kDa band when expressed with all other VirB proteins. The 36-kDa band is converted to the 31-kDa band by the reducing agent 2-mercaptoethanol. Using strains that contain a deletion in a defined virB gene and strains that express specific VirB proteins, we demonstrate that the 36-kDa band is composed of VirB9 and VirB7 that are linked to each other by a disulfide bond. Mutational studies demonstrate that cysteine residues at positions 24 of VirB7 and 262 of VirB9 participate in the formation of this complex.

Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus.

The Agrobacterium tumefaciens VirB7 lipoprotein contributes to the stabilization of VirB proteins during biogenesis of the putative T-complex transport apparatus. Here, we report that stabilization of VirB7 itself is correlated with its ability to form disulfide cross-linked homodimers via a reactive Cys-24 residue. Three types of beta-mercaptoethanol-dissociable complexes were visualized with VirB7 and/or a VirB7::PhoA41 fusion protein: (i) a 9-kDa complex corresponding in size to a VirB7 homodimer, (ii) a 54-kDa complex corresponding in size to a VirB7/VirB7::PhoA41 mixed dimer, and (iii) a 102-kDa complex corresponding to a VirB7::PhoA41 homodimer. A VirB7C24S mutant protein was immunologically undetectable, whereas the corresponding VirB7C24S::PhoA41 derivative accumulated to detectable levels but failed to form dissociable homodimers or mixed dimers with wild-type VirB7. We further report that VirB7-dependent stabilization of VirB9 is correlated with the ability of these two proteins to dimerize via formation of a disulfide bridge between reactive Cys-24 and Cys-262 residues, respectively. Two types of dissociable complexes were visualized: (i) a 36-kDa complex corresponding in size to a VirB7/VirB9 heterodimer and (ii) an 84-kDa complex corresponding in size to a VirB7/VirB9::PhoA293 heterodimer. A VirB9C262S mutant protein was immunologically undetectable, whereas the corresponding VirB9C262S::PhoA293 derivative accumulated to detectable levels but failed to form dissociable heterodimers with wild-type VirB7. Taken together, these results support a model in which the formation of disulfide cross-linked VirB7 dimers represent critical early steps in the biogenesis of the T-complex transport apparatus.

Locating a strand of the Seattle Fault Zone in southeast Seattle, Washington through topographic analysis, borehole analysis and ground penetrating radar

Contributing to the evaluation of seismic hazards, a previously unmapped strand of the Seattle Fault Zone (SFZ), cutting across the southwest side of Lake Washington and southeast Seattle, is located and characterized on the basis of bathymetry, borehole logs, and ground penetrating radar (GPR). Previous geologic mapping and geophysical analysis of the Seattle area have generally mapped the locations of some strands of the SFZ, though a complete and accurate understanding of locations of all individual strands of the fault system is still incomplete. A bathymetric scarp-like feature and co-linear aeromagnetic anomaly lineament defined the extent of the study area. A 2-dimensional lithology cross-section was constructed using six boreholes, chosen from suitable boreholes in the study area. In addition, two GPR transects, oblique to the proposed fault trend, served to identify physical differences in subsurface materials. The proposed fault trace follows the previously mapped contact between the Oligocene Blakeley Formation and Quaternary deposits, and topographic changes in slope. GPR profiles in Seward Park and across the proposed fault location show the contact between the Blakeley Formation and unconsolidated glacial deposits, but it does not constrain an offset. However, north-dipping beds in the Blakely Formation are consistent with previous interpretations of P-wave seismic profiles on Mercer Island and Bellevue, Washington. The profiles show the mapped location of the aeromagnetic lineament in Lake Washington and the inferred location of the steeply-dipping, high-amplitude bedrock reflector, representing a fault strand. This north-dipping reflector is likely the same feature identified in my analysis. I characterize the strand as a splay fault, antithetic to the frontal fault of the SFZ. This new fault may pose a geologic hazard to the region.

Knots in rings - The circular knotted protein momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys(1)-Cys(18) disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.

Cross-resistance to elvitegravir and dolutegravir in 502 patients failing on raltegravir: a French national study of raltegravir-experienced HIV-1-infected patients

International audience

Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response

Tese de Doutoramento em Ciências Veterinárias na especialidade de Sanidade Animal