Acacia senegal gum: continuum of molecular species differing by their protein to sugar ratio, molecular weight, and charges

The main chemical and physical features of the Acacia senegal exudate gum and its molecular fractions isolated by chromatographies were determined using a wide variety of methods. Three main molecular fractions were isolated after hydrophobic interaction chromatography (HIC) and biochemical analyses confirmed the presence of an arabinogalactan-peptide (FI), an arabinogalactan-protein (FII), and a glycoprotein (FIII) fraction as described commonly in the literature. Further purification of FIII using size exclusion chromatography revealed three distinct populations. A wide molecular weight distribution within each population with the presence of at least two distinct molecular species per population was identified by high performance size exclusion chromatography coupled to on line multi-angle laser light scattering (HPSEC-MALLS). In addition, both sugars content (neutral and uronic acids) and UV profiles revealed that FIII was composed of a continuum of molecular species differing both by their protein-to-sugar ratio and molecular weight. FI and FII had average molecular weight M(w) of 2.86 x 10(5) and 1.86 x 10(6) g.mol(-1), respectively, and a low polydispersity index (M(w)()/M(n) approximately 1.3). The three populations identified in FIII after HIC separation had M(w) of 2.67 x 10(6), 7.76 x 10(5), and 2.95 x 10(5) g.mol(-1) and very low polydispersity indexes (1.13, 1.04, and 1.01). Estimation of the polypeptide backbone length in the three fractions gave 43, 2253, and 4443 amino acid residues, respectively, hydroxyproline (Hyp) and serine being the most prominent residues within FI and FII, Hyp and Asx (asparagine + aspartic acid) within FIII. Secondary structure prediction from circular dichroism data resulted in polyproline II, beta-sheet, and random coil structures for FII and FIII, whereas no secondary structure was identified in FI. The existence of exposed tryptophanyl residues to the solvent was noticed by fluorescence in FII and FIII, tryptophan residues being absent from FI. In addition, 8-5' non cyclic diferulic acid was identified to be covalently linked to carbohydrate moieties of FII. Infrared spectroscopy identified the different vibrations of saccharidic and peptidic bonds with absorbance amplitudes in agreement with sugar and protein elementary analyses. Titration measurements in order to evaluate the number of charges on total Acacia gum and its molecular fractions revealed that 100% of charges came from polysaccharidic moieties (i.e., glucuronic acids) in FI. Charges coming from polysaccharidic moieties were of 91.3% and 37.9% for FII and FIII, respectively, the remaining 8.7% and 62.1% charges in FII and FIII molecular fractions coming from the polypeptidic backbone.     

Tissue-type plasminogen activator rescues neurones from serum deprivation-induced apoptosis through a mechanism independent of its proteolytic activity

Although the mechanism of action of tissue-type plasminogen activator (tPA) in excitotoxic necrosis is well documented, whether this serine protease can influence the apoptotic cascade remains a subject of debate. Here, we report that tPA protects cultured cortical neurones against apoptotic cell death induced by serum deprivation, an effect associated with a reduction of caspase-3 activation. Interestingly, blocking tPA proteolytic activity by either tPA stop or neuroserpin did not prevent this neuroprotection. Similarly, prevention of the interaction between tPA and its receptor low-density lipoprotein receptor-related protein (LRP) could not alter tPA anti-apoptotic activity. Interestingly, the survival-promoting effect of tPA was blocked by the phosphatidylinositol-3 (PI-3) kinase inhibitor, LY294002, but not by the mitogen-activated protein (MAP) kinase inhibitor, U0126. In conclusion, the present demonstration of an anti-apoptotic effect of tPA, independent of its enzymatic activity, reveals an additional level of complexity in our understanding of this critical mediator of brain physiology and pathology.     

Human mast cell-derived gelatinase B (matrix metalloproteinase-9) is regulated by inflammatory cytokines: role in cell migration

Mast cells are key effectors in the pathogenesis of inflammatory and tissue destructive diseases such as rheumatoid arthritis (RA). These cells contain specialized secretory granules loaded with bioactive molecules including cytokines, growth factors, and proteases that are released upon activation. This study investigated the regulation of matrix metalloproteinase MMP-9 (gelatinase B) in human mast cells by cytokines that are known to be involved in the pathogenesis of RA. Immunohistochemical staining of synovial tissue showed abundant expression of MMP-9 by synovial tissue mast cells in patients with RA but not in normal controls. The expression, activity, and production of MMP-9 in mast cells was confirmed by RT-PCR, zymography, and Western blotting using cord blood-derived human mast cells (CB-HMC). Treatment of CB-HMC with TNF-alpha significantly increased the expression of MMP-9 mRNA and up-regulated the activity of MMP-9 in a time- and dose-dependent manner. By contrast, IFN-gamma inhibited MMP-9 mRNA and protein expression. The cytokine-mediated regulation of MMP-9 was also apparent in the human mast cell line (HMC-1) and in mouse bone marrow-derived mast cells. Furthermore, TNF-alpha significantly increased the invasiveness of CB-HMC across Matrigel-coated membranes while the addition of IFN-gamma, rTIMP-1, or pharmacological MMP inhibitors significantly reduced this process. These observations suggest that MMP-9 is not a stored product in mast cells but these cells are capable of producing this enzyme under inflammatory conditions that may facilitate the migration of mast cell progenitors to sites of inflammation and may also contribute to local tissue damage.     

Actinide speciation in relation to biological processes

In case of accidental release of radionuclides into the environment, actinides represent a severe health risk to human beings following internal contamination (inhalation, ingestion or wound). For a better understanding of the actinide behaviour in man (in term of metabolism, retention, excretion) and in specific biological systems (organs, cells or biochemical pathways), it is of prime importance to have a good knowledge of the relevant actinide solution chemistry and biochemistry, in particular of the thermodynamic constants needed for computing actinide speciation. To a large extent, speciation governs bioavailability and toxicity of elements and has a significant impact on the mechanisms by which toxics accumulate in cell compartments and organs and by which elements are transferred and transported from cell to cell. From another viewpoint, speciation is the prerequisite for the design and success of potential decorporation therapies. The purpose of this review is to present the state of the art of actinide knowledge within biological media. It is also to discuss how actinide speciation can be determined or predicted and to highlight the areas where information is lacking with the aim to encourage new research efforts.     

SLiMDisc: short, linear motif discovery, correcting for common evolutionary descent

Many important interactions of proteins are facilitated by short, linear motifs (SLiMs) within a protein's primary sequence. Our aim was to establish robust methods for discovering putative functional motifs. The strongest evidence for such motifs is obtained when the same motifs occur in unrelated proteins, evolving by convergence. In practise, searches for such motifs are often swamped by motifs shared in related proteins that are identical by descent. Prediction of motifs among sets of biologically related proteins, including those both with and without detectable similarity, were made using the TEIRESIAS algorithm. The number of motif occurrences arising through common evolutionary descent were normalized based on treatment of BLAST local alignments. Motifs were ranked according to a score derived from the product of the normalized number of occurrences and the information content. The method was shown to significantly outperform methods that do not discount evolutionary relatedness, when applied to known SLiMs from a subset of the eukaryotic linear motif (ELM) database. An implementation of Multiple Spanning Tree weighting outperformed two other weighting schemes, in a variety of settings.     

Regulation of ventilation in the caiman (Caiman latirostris): effects of inspired CO2 on pulmonary and upper airway chemoreceptors

In order to study the relative roles of receptors in the upper airways, lungs and systemic circulation in modulating the ventilatory response of caiman (Caiman latirostris) to inhaled CO₂, gas mixtures of varying concentrations of CO₂ were administered to animals breathing through an intact respiratory system, via a tracheal cannula by-passing the upper airways (before and after vagotomy), or via a cannula delivering gas to the upper airways alone. While increasing levels of hypercarbia led to a progressive increase in tidal volume in animals with intact respiratory systems (Series I), breathing frequency did not change until the CO₂ level reached 7%, at which time it decreased. Despite this, at the higher levels of hypercarbia, the net effect was a large and progressive increase in total ventilation. There were no associated changes in heart rate or arterial blood pressure. On return to air, there was an immediate change in breathing pattern; breathing frequency increased above air-breathing values, roughly to the same maximum level regardless of the level of CO₂ the animal had been previously breathing, and tidal volume returned rapidly toward resting (baseline) values. Total ventilation slowly returned to air breathing values. Administration of CO₂ via different routes indicated that inhaled CO₂ acted at both upper airway and pulmonary CO₂-sensitive receptors to modify breathing pattern without inhibiting breathing overall. Our data suggest that in caiman, high levels of inspired CO₂ promote slow, deep breathing. This will decrease dead-space ventilation and may reduce stratification in the saccular portions of the lung.