Knowledge-based design of reagentless fluorescent biosensors from recombinant antibodies

The possibility of obtaining from any antibody a fluorescent conjugate which responds to the binding of the antigen by a variation of its fluorescence, would be of great interest in the analytical sciences and for the construction of protein chips. This possibility was explored with antibody mAbD1.3 directed against hen egg white lysozyme. Rules of design were developed to identify the residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by mutagenesis. These rules were based on: the target residue belonging to a topological neighbourhood of the antigen in the structure of the complex between antibody and antigen; its absence of functional importance for the interaction with the antigen; and its solvent accessibility in the structure of the free antibody. Seventeen conjugates between the single-chain variable fragment scFv of mAbD1.3 and an environment-sensitive fluorophore were constructed. For six of the ten residues which fully satisfied the design rules, the relative variation of the fluorescence intensity between the free and bound states of the conjugate was comprised between 12 and 75% (in non-optimal buffer), and the affinity of the conjugate for lysozyme remained unchanged relative to the parental scFv. In contrast, such results were true for only one of the seven residues which failed to satisfy one of the rules and were used as controls. One of the conjugates was studied in more detail. Its fluorescence increased proportionally to the concentration of lysozyme in a nanomolar range, up to 90% in a defined buffer, and 40% in serum. This increase was specific for hen egg lysozyme and it was not observed with a closely related protein, turkey egg lysozyme. The residues which gave operational conjugates (six in V(L) and one in V(H)), were located in the immediate vicinity of residues which are functionally important, along the sequence of FvD1.3. The results suggest rules of design for constructing antigen-sensitive fluorescent conjugates from any antibody, in the absence of structural data.     

Apoptotic effect of sphingosine 1-phosphate and increased sphingosine 1-phosphate hydrolysis on mesangial cells cultured at low cell density

The lipid mediator sphingosine 1-phosphate (S1P) may alter the proliferation of mesangial cells during pathophysiological processes. Here, S1P stimulated proliferation of rat mesangial cells and phosphorylation of MAPKs at subconfluent cell density. Both effects were inhibited by pertussis toxin treatment. Mesangial cells expressed several S1P receptors of the endothelial differentiation gene family: EDG-1, -3, -5, and -8. Conversely, S1P induced apoptosis at low cell density (2 x 10(4) cells/cm(2)), which was demonstrated by flow cytometry and Hoechst staining. Apoptosis was observed also in quiescent or growing cells and was not reverted by lysophosphatidic acid or platelet-derived growth factor. S1P enhanced phosphorylation of SAPKs. Incubation with [(33)P]S1P, [(3)H]S1P, and [(3)H]sphingosine demonstrated increased S1P hydrolysis, resulting in enhanced intracellular sphingosine levels and decreased S1P levels. A rise in total ceramide levels was also observed; however, ceramide did not originate from [(3)H]sphingosine, and S1P-induced apoptosis was not inhibited by fumonisin B, precluding involvement of de novo ceramide synthesis in apoptosis. Therefore, we suggest that sphingosine accumulation and decreased S1P are primarily responsible for S1P-induced apoptosis. In conclusion, incubation of low-density mesangial cells with S1P results in apoptosis, presumably due to increased S1P hydrolysis.     

Channel openings are necessary but not sufficient for use-dependent block of cardiac Na(+) channels by flecainide: evidence from the analysis of disease-linked mutations

Na(+) channel blockers such as flecainide have found renewed usefulness in the diagnosis and treatment of two clinical syndromes arising from inherited mutations in SCN5A, the gene encoding the alpha subunit of the cardiac voltage-gated Na(+) channel. The Brugada syndrome (BrS) and the LQT-3 variant of the Long QT syndrome are caused by disease-linked SCN5A mutations that act to change functional and pharmacological properties of the channel. Here we have explored a set of SCN5A mutations linked both to BrS and LQT-3 to determine what disease-modified channel properties underlie distinct responses to the Na(+) channel blocker flecainide. We focused on flecainide block that develops with repetitive channel activity, so-called use-dependent block (UDB). Our results indicate that mutation-induced changes in the voltage-dependence of channel availability (inactivation) may act as determinants of flecainide block. The data further indicate that UDB by flecainide requires channel opening, but is not likely due to open channel block. Rather, flecainide appears to interact with inactivation states that follow depolarization-induced channel opening, and mutation-induced changes in channel inactivation will alter flecainide block independent of the disease to which the mutation is linked. Analysis of flecainide block of mutant channels linked to these rare disorders has provided novel insight into the molecular determinants of drug action.     

Phosphoinositide 3-kinases in lysophosphatidic acid signaling: regulation and cross-talk with the Ras/mitogen-activated protein kinase pathway

Recent reports have shown that phosphoinositide 3-kinases (PI3Ks) mediate various biological activities of lysophosphatidic acid (LPA), including cell proliferation or survival. In addition, these enzymes have been proposed to be early intermediates of mitogen-activated protein kinase (MAPK) activation. Here we summarize our current knowledge of the mechanisms underlying these observations. p110gamma is an isoform of PI3K that can be activated in vitro by Gbetagamma subunits and was therefore considered as the logical candidate to mediate responses induced by G protein-coupled receptor (GPCR) agonists. In agreement with this, p110gamma has been involved in different biochemical models linking Gbetagamma to MAPK activation. Nevertheless, its apparent tissue-specific distribution has raised questions regarding the physiological relevance of these models. In addition, LPA can activate p110beta, a member of the phosphotyrosine-dependent PI3K subfamily that participates in the mitogenic effect of LPA. Its activation is thought to involve a synergistic effect of Gbetagamma and phosphotyrosine motifs provided by a transactivated EGF receptor/Gab1 pathway. We are currently studying a possible role of p110beta upstream from Ras, suggesting that this protein could provide a novel connection between betagamma and the MAPK pathway.     

Truncated hemoglobins and nitric oxide action

Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they are scarcely related by sequence similarity to (non-)vertebrate hemoglobins, displaying amino acid sequences in the 115-130 residue range. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which hosts a unique hydrophobic cavity/tunnel system, traversing the protein matrix, from the molecular surface to the heme distal site. Such a protein matrix system may provide a path for diffusion of ligands to the heme. In Mycobacterium tuberculosis trHbN the heme-bound oxygen molecule is part of an extended hydrogen bond network including the heme distal residues TyrB10 and GlnE11. In vitro experiments have shown that M. tuberculosis trHbN supports efficiently nitric oxide dioxygenation, yielding nitrate. Such a reaction would provide a defense barrier against the nitrosative stress raised by host macrophages during lung infection. It is proposed that the whole protein architecture, the heme distal site hydrogen bonded network, and the unique protein matrix tunnel, are optimally designed to support the pseudo-catalytic role of trHbN in converting the reactive NO species into the harmless NO3-.     

Xyloglucan Octasaccharide XXLGol Derived from the Seeds of Hymenaea courbaril Acts as a Signaling Molecule

Treatment of the xyloglucan isolated from the seeds of Hymenaea courbaril with Humicola insolens endo-1,4-β-d-glucanase I produced xyloglucan oligosaccharides, which were then isolated and characterized. The two most abundant compounds were the heptasaccharide (XXXG) and the octasaccharide (XXLG), which were examined by reference to the biological activity of other structurally related xyloglucan compounds. The reduced oligomer (XXLGol) was shown to promote growth of wheat (Triticum aestivum) coleoptiles independently of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D). In the presence of 2,4-D, XXLGol at nanomolar concentrations increased the auxin-induced response. It was found that XXLGol is a signaling molecule, since it has the ability to induce, at nanomolar concentrations, a rapid increase in an α-l-fucosidase response in suspended cells or protoplasts of Rubus fruticosus L. and to modulate 2,4-D or gibberellic acid-induced α-l-fucosidase.     

Impacts of Climate Change on the Timing of the Production Season of Maple Syrup in Eastern Canada

Maple syrup production is an important economic activity in north-eastern North-America. The beginning and length of the production season is linked to daily variation in temperature. There are increasing concerns about the potential impact of climatic change on this industry. Here, we used weekly data of syrup yield for the 1999–2011 period from 121 maple stands in 11 regions of Québec (Canada) to predict how the period of production may be impacted by climate warming. The date at which the production begins is highly variable between years with an average range of 36 days among the regions. However, the average start date for a given region, which ranged from Julian day 65 to 83, was highly predictable (r² = 0.88) using the average temperature from January to April (T_J-A). A logistic model predicting the weekly presence or absence of production was also developed. Using the inputs of 77 future climate scenarios issued from global models, projections of future production timing were made based on average T_J-A and on the logistic model. The projections of both approaches were in very good agreement and suggest that the sap season will be displaced to occur 15–19 days earlier on average in the 2080–2100 period. The data also show that the displacement in time will not be accompanied by a greater between years variability in the beginning of the season. However, in the southern part of Québec, very short periods of syrup production due to unfavourable conditions in the spring will occur more frequently in the future although their absolute frequencies will remain low.     

Production,in Pichia pastoris,of a recombinant monomeric mapacalcine,a protein with anti-ischemic properties

Mapacalcine is a small homodimeric protein of 19 kDa with 9 disulfide bridges extracted from the Cliona vastifica sponge (Red Sea). It selectively blocks a calcium current insensitive to most calcium blockers. Specific receptors for mapacalcine have been described in a variety of tissues such as brain, smooth muscle, liver, and kidney. Previous works achieved on hepatocytes and nervous cells demonstrated that this protein selectively blocks a calcium influx triggered by an ischemia/reperfusion (I/R) shock and efficiently protects cells from death after I/R. The aim of this work was to produce the recombinant mapacalcine in the yeast Pichia pastoris. Mass spectrometry, light scattering analysis and biological characterization demonstrated that the recombinant mapacalcine obtained was a monomeric form with 4 disulfide bridges which retains the biological activity of the natural protein.