Purification and characterization of two alpha-amylases from Toxoplasma gondii.

Two distinct alpha-amylases have been identified in Toxoplasma gondii. They were purified close to homogeneity from cytoplasmic and membrane fractions. The apparent molecular weight of the cytoplasmic amylase was 22,300 Da and that of the membrane enzyme was 39,600 Da by gel filtration, and 25,000 and 41,000 Da by SDS gel electrophoresis, respectively. The physicochemical and catalytic properties of both enzymes showed them to be very different. Cytoplasmic alpha-amylase had an acid isoelectric point and its optimum pH was pH 5.0; its activity was unaffected by NaCl, Ca2+, or EDTA. The membrane alpha-amylase had an isoelectric point of 7.7 and an optimum pH of 8.0. It was affected by Ca2+, inhibited by EDTA, and activated eight-fold by NaCl. Both amylases were inactivated by temperatures above 65 degrees C, but cytoplasmic amylase was more resistant to thermal denaturation.     

Chicken cystatin stimulates nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages via cytokine synthesis.

Cystatins are natural tight-binding, reversible inhibitors of cysteine proteases. We have shown that cystatins also stimulate nitric oxide (NO) production by interferon-gamma-activated mouse peritoneal macrophages [Verdot, L., Lalmanach, G., Vercruysse, V., Hartman, S., Lucius, R., Hoebeke, J., Gauthier F. & Vray, B. (1996) J. Biol. Chem. 271, 28077-28081]. The present study was undertaken to further document this new function. Macrophages activated with interferon-gamma and then stimulated with interferon-gamma plus chicken cystatin generated increased amounts of NO in comparison with macrophages only activated with interferon-gamma. Interferon-gamma-activated macrophages must be incubated with chicken cystatin for at least 8 h to upregulate NO production. NO induction was due to increased inducible nitric oxide synthase protein synthesis. Macrophages incubated with chicken cystatin alone or with interferon-gamma plus chicken cystatin produced increased amounts of both tumor necrosis factor alpha and interleukin 10. The addition of recombinant murine tumor necrosis factor alpha alone or in combination with recombinant murine interleukin-10 mimicked the effect of chicken cystatin. The addition of neutralizing anti-(tumor necrosis factor alpha) antibodies reduced sharply NO production by chicken cystatin/interferon-gamma-activated mouse peritoneal macrophages. Taken together, these data suggest that chicken cystatin induces the synthesis of tumor necrosis factor alpha and interleukin 10. In turn, these two cytokines stimulate the production of NO by interferon-gamma-activated macrophages. The findings point to a new relationship between cystatins, cytokines, inflammation and the immune response.     

Monoclonal antibodies against the native or denatured forms of muscarinic acetylcholine receptors.

BALB/c mice were immunized with affinity-purified muscarinic acetylcholine receptors from calf brain and their splenocytes fused with NS1 myeloma cells. Hybrid cultures were grown and selected for production of antibodies on the basis of enzyme immunoassays on calf and rat forebrain membrane preparations. Thirty-four clones were retained and six of them further subcloned. Two of these subclones produced antibodies that selectively recognized muscarinic acetylcholine receptor-bearing membranes. The M-35b antibodies interacted only with native digitonin-solubilized receptors, and not with denatured receptors. The M-23c antibodies did not react with active digitonin-solubilized receptors but recognized the denatured form. The M-23c antibodies should thus be useful in the purification of the receptor and its precursor translation products, while the M-35b antibodies could be used for the immunocytochemical localization of the receptor in cells and tissues of different species.     

A blood group A specific lectin from the seeds of Crotalaria striata

A lectin, monospecific for human blood group A red blood cells was extracted from seeds of Crotalaria striata and purified by molecular sieving on Sephadex G-100 and ion-exchange on DEAE-cellulose. A molecular mass of 30 kDa was determined by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions. Molecular sieving on a Superose 12 column indicated a molecular mass of 110 kDa, suggesting the tetrameric nature of the native protein. Amino-acid composition showed the presence of aminated carbohydrate residues on the lectin. N-terminal amino-acid sequencing showed a striking similarity with the N-terminal sequence of the lectin from Crotalaria juncea, which is blood-group non-specific. The potency order of agglutination inhibition with galactose containing monosaccharides was N-acetyl-D-galactosamine greater than D-galactose greater than D-galactosamine as found for blood-group-A-specific lectins from other species.     

Interaction of oncodazole (R 17934), a new antitumoral drug, with rat brain tubulin.

Oncodazole (R 17934), methyl [5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl] carbamate (I), a new synthetic drug with anti-tumoral activity, inhibits the polymerization of rat brain tubulin $\text{in vitro}$. It has no depolymerizing effect on preformed microtubules $\text{in vitro}$. Binding studies by means of molecular sieving and equilibrium dialysis indicates that the drug binds to purified rat brain tubulin in a mole to mole ratio. Finally the drug competitively inhibits colchicine binding to purified rat brain tubulin. From these results the conclusion may be drawn that oncodazole is a true microtubule inhibitor.     

Sequence analysis of peptide fragments from the intrinsic membrane protein of calf lens fibers MP26 and its natural maturation product MP22

Calf lens fiber plasma membranes, containing only the intrinsic membrane protein MP26 and its maturation product MP22 were treated with proteolytic enzymes such as trypsin, protease V8 from S. aureus or with chemical agents as CNBr in formic acid. The cleavage products, purified by electrophoresis, were analysed for their amino acid composition and N-terminal sequences. Proteolysis gave rise to peptides which were mainly shortened at the C-terminal end of the molecules. While the V8 protease produced a fragment with a similar N-terminal sequence as the maturation product MP22, trypsin yielded another cleavage product. Chemical hydrolysis yielded large fragments (11-15 kDa) with hydrophobic N-terminal sequences. Our results suggest that MP26 is characterised by an N-terminal signal sequence and possesses other hydrophobic domains which could function as untranslocated insertion sequences.     

Insyght: navigating amongst abundant homologues,syntenies and gene functional annotations in bacteria,it's that symbol!

High-throughput techniques have considerably increased the potential of comparative genomics whilst simultaneously posing many new challenges. One of those challenges involves efficiently mining the large amount of data produced and exploring the landscape of both conserved and idiosyncratic genomic regions across multiple genomes. Domains of application of these analyses are diverse: identification of evolutionary events, inference of gene functions, detection of niche-specific genes or phylogenetic profiling. Insyght is a comparative genomic visualization tool that combines three complementary displays: (i) a table for thoroughly browsing amongst homologues, (ii) a comparator of orthologue functional annotations and (iii) a genomic organization view designed to improve the legibility of rearrangements and distinctive loci. The latter display combines symbolic and proportional graphical paradigms. Synchronized navigation across multiple species and interoperability between the views are core features of Insyght. A gene filter mechanism is provided that helps the user to build a biologically relevant gene set according to multiple criteria such as presence/absence of homologues and/or various annotations. We illustrate the use of Insyght with scenarios. Currently, only Bacteria and Archaea are supported. A public instance is available at http://genome.jouy.inra.fr/Insyght. The tool is freely downloadable for private data set analysis.     

Evidence of oxidative stress and mitochondrial respiratory chain dysfunction in an in vitro model of sepsis-induced kidney injury

To investigate the role of oxidative stress and/or mitochondrial impairment in the occurrence of acute kidney injury (AKI) during sepsis, we developed a sepsis-induced in vitro model using proximal tubular epithelial cells exposed to a bacterial endotoxin (lipopolysaccharide, LPS). This investigation has provided key features on the relationship between oxidative stress and mitochondrial respiratory chain activity defects.     

Catalytic IgG from patients with hemophilia A inactivate therapeutic factor VIII

Factor VIII (FVIII) inhibitors are anti-FVIII IgG that arise in up to 50% of the patients with hemophilia A, upon therapeutic administration of exogenous FVIII. Factor VIII inhibitors neutralize the activity of the administered FVIII by sterically hindering its interaction with molecules of the coagulation cascade, or by forming immune complexes with FVIII and accelerating its clearance from the circulation. We have shown previously that a subset of anti-factor VIII IgG hydrolyzes FVIII. FVIII-hydrolyzing IgG are detected in over 50% of inhibitor-positive patients with severe hemophilia A, and are not found in inhibitor-negative patients. Although human proficient catalytic Abs have been described in a number of inflammatory and autoimmune disorders, their pathological relevance remains elusive. We demonstrate here that the kinetics of FVIII degradation by FVIII-hydrolyzing IgG are compatible with a pathogenic role for IgG catalysts. We also report that FVIII-hydrolyzing IgG from each patient exhibit multiple cleavage sites on FVIII and that, while the specificity of cleavage varies from one patient to another, catalytic IgG preferentially hydrolyze peptide bonds containing basic amino acids.