Phosphodiesterase isoform‐specific expression induced by traumatic brain injury

Traumatic brain injury (TBI) results in significant inflammation which contributes to the evolving pathology. Previously, we have demonstrated that cyclic AMP (cAMP), a molecule involved in inflammation, is down‐regulated after TBI. To determine the mechanism by which cAMP is down‐regulated after TBI, we determined whether TBI induces changes in phosphodiesterase (PDE) expression. Adult male Sprague Dawley rats received moderate parasagittal fluid‐percussion brain injury (FPI) or sham injury, and the ipsilateral, parietal cortex was analyzed by western blotting. In the ipsilateral parietal cortex, expression of PDE1A, PDE4B2, and PDE4D2, significantly increased from 30 min to 24 h post‐injury. PDE10A significantly increased at 6 and 24 h after TBI. Phosphorylation of PDE4A significantly increased from 6 h to 7 days post‐injury. In contrast, PDE1B, PD4A5, and PDE4A8 significantly decreased after TBI. No changes were observed with PDE1C, PDE3A, PDE4B1/3, PDE4B4, PDE4D3, PDE4D4, PDE8A, or PDE8B. Co‐localization studies showed that PDE1A, PDE4B2, and phospho‐PDE4A were neuronally expressed, whereas PDE4D2 was expressed in neither neurons nor glia. These findings suggest that therapies to reduce inflammation after TBI could be facilitated with targeted therapies, in particular for PDE1A, PDE4B2, PDE4D2, or PDE10A.     

Purification,primary structure and potential functions of a novel lectin from Bauhinia forficata seeds

A new lectin, BfL, was purified from Bauhinia forficata seeds by ammonium sulfate fractionation, DEAE-Sephadex ion exchange chromatography, Sepharose-4B and chitin affinity chromatographies and Superdex 75 size exclusion chromatography. The molecular homogeneity and purity of BfL were assessed by reversed-phase HPLC. BfL appeared as a single band of approximately 27.0 kDa on SDS-PAGE under non-reducing and reducing conditions, and its molecular weight was determined to be 27,850 Da by LC/ESI-MS. BfL is a glycoprotein with a carbohydrate content of 6.24% determined by the phenol–sulfuric acid method. Fetuin, asialofetuin, thyroglobulin and azocasein inhibited the hemagglutinating activity of BfL, whereas saccharides did not. BfL hemagglutinating activity was stable at 100 °C for 30 min, pH-dependent, with the highest activity at pH 6.0, and metal-independent. The primary structure of BfL shows similarity with other lectins from the genus Bauhinia. Deconvolution of the BfL circular dichroism (CD) spectrum indicated the presence of α-helix and β structures. BfL increases coagulation time, but this effect is not related to human plasma kallikrein or human factor Xa inhibition. BfL also inhibits ADP- and epinephrine-induced platelet aggregation in a dose-dependent manner and is the only currently described lectin from Bauhinia that exhibits anticoagulant and antiplatelet aggregating properties.     

Evaluation of lead toxicity in Erica andevalensis as an alternative species for revegetation of contaminated soils

Although revegetation using native flora is a low cost way to stabilize soil and restore the landscape contaminated with metals, little is known regarding the Pb-tolerance of many of these species. For this purpose, we evaluated the tolerance of Erica andevalensis to Pb by growing plants in nutrient solutions with increasing concentrations of Pb (up to 100 microM). Plant growth and different physiological parameters were determined to ascertain tolerance to metal stress. Additionally, an electron microscopy study coupled with EDX-analysis was performed to get clues on the Pb uptake and translocation from roots into stem and leaves. The LOEC (the lowest observed effect concentration) of Pb was 40 microM while the IC50 (inhibition concentration) was 80 microM Pb. Chemical analysis revealed a root > stem > leaf accumulation pattern. There was a severe reduction in fresh biomass and chlorophyll concentration at the highest Pb dose. The SEM-EDX study indicated that Pb was mostly located in root epidermal tissues. The blockage of Pb on the root probably avoided its toxic effects by limiting Pb transport to other tissues.     

Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 Å resolution were obtained using hanging drop method by vapor diffusion at 18 °C. The refined structure shows the conservative β-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function.     

Purification of a lectin with antibacterial activity from Bothrops leucurus snake venom

A novel lectin was isolated from Bothrops leucurus snake venom using a combination of affinity and gel filtration chromatographies. The lectin (BlL) agglutinated glutaraldehyde-treated rabbit and human erythrocytes with preference for rabbit erythrocytes. Galactose, raffinose, lactose, fetal bovine serum and casein inhibited lectin-induced rabbit erythrocyte agglutination. BlL, with a molecular mass of 30 kDa and composed of two subunits of 15 kDa, showed dependence on calcium. BlL is an acidic protein with highest activity over the pH range of 4.0-7.0 and stable under heating to 70°C. Fluorescence emission spectra showed tryptophan residues partially buried within the lectin structure. The percentages of secondary structure revealed by circular dichroism were 1% α-helix, 44% β-sheet, 24% β-turn and 31% unordered. BlL showed effective antibacterial activity against Gram-positive bacteria Staphylococcus aureus, Enterococcus faecalis and Bacillus subtilis with minimal inhibitory concentrations of 31.25, 62.25 and 125 μg/mL, respectively. In conclusion, B. leucurus snake venom contains a galactoside-binding lectin with antibacterial activity.     

Metazoan parasites of Macruronus magellanicus from southern Chile as biological tags

Examination of 450 specimens of Macruronus magellanicus from two fishing grounds in southern Chile (Talcahuano: 36°40'S, Punta Arenas: 54° S) revealed 24 205 metazoan parasites, belonging to 15 taxa with low specificity: Chondracanthus australis and Neobrachiella sp. (Copepoda); Elytrophalloides oatesi, Gonocerca phycidis, Derogenes varicus and Brachyphallus parvus , (Digenea); Hepatoxylon trichiuri, Scolex pleuronectis, Clestobothrium crassiceps, Grillotia dollfusi , Pseudophyllidea gen. sp. (Cestoda); Anisakidae, Cucullanus sp. (Nematoda), Corynosoma australe and Acanthocephala gen. sp. (Acanthocephala). The list includes parasites that are common to other merluccids from the zone. The parasitological evidence does not suggest the existence of two discrete stocks, but the existence of a migratory process from south to north.     

Expression of functional human glutaminase in baculovirus system: affinity purification, kinetic and molecular characterization

Glutaminase catalyzes the hydrolysis of glutamine yielding stoichiometric amounts of glutamate plus ammonium ions. In mammals, there are two different genes encoding for glutaminase, known as liver (L) and kidney (K) types. The human L-type isoform expressed in baculovirus yielded functional recombinant enzyme in Sf9 insect cells. A novel affinity chromatography method, based on its specific interaction with a PDZ protein, was developed for purification. Kinetic constants were determined for the purified human isozyme, which showed an allosteric behaviour for glutamine, with a Hill index of 2.7 and S(0.5) values of 32 and 64 mM for high and low P(i) concentrations, respectively. Whereas the protein showed a low P(i) dependence typical for L-type glutaminases, the enzyme was unexpectedly inhibited by glutamate, a kinetic characteristic exclusive of K-type isozymes, and was slightly activated by ammonia, unlike the classical liver enzymes which show an absolute dependence on ammonia. Subcellular fractionation demonstrates that recombinant human glutaminase was targeted to both mitochondria and nucleus, and in both locations the protein was catalytically active. This is the first report of the expression of a functional L-type mammalian glutaminase enzyme. The study also provides a simple and efficient method for affinity purification of the recombinant enzyme. Moreover, the data imply that this human enzyme may represent a new isoform different from classical kidney and liver isozymes.     

Removal of pesticides from white wine by the use of fining agents and filtration

The effects of four clarification agents (bentonite, charcoal, PVPP and potassium caseinate) on the removal of residues of three fungicides (famoxadone, fluquinconazole and trifloxystrobin) applied directly to a racked white wine, elaborated from Airen variety grapes from the D.O. Region of Jumilla (Murcia, Spain) are studied. The clarified wines were filtered with 0.45 microm nylon filters to determine the influence of this winemaking process in the disappearance of fungicide residues. Hydro-alcoholic solutions with the three fungicides at concentrations of 1 and 2 ppm were then added through intense stirring to each of the containers. Two hours later, the corresponding clarifying agent was added with intense stirring for some minutes. The containers were then sealed and left to settle for five days. Once the clean wines had been racked, they were filtered through nylon 0.45 microm pore filters. All assays were performed three times. Analytical determination of fluquinconazole and trifloxystrobin was performed by gas chromatography with an electron captor detector (ECD), while that of famoxadone was made using an HPLC-DAD. For the three fungicides, the highest elimination is produced with the clarification by charcoal, reaching Levels of removal of 100% in all cases. For the four clarifying agents, the highest elimination is produced for the fluquinconazole residues. The fungicide which is retained most in the lees is famoxadone, since it has the lowest solubility of the three pesticides studied. The highest percentage of residues in the lees is obtained for the assay with charcoal. The filtration process of the clarified wines using the four agents studied is not effective, since the elimination percentage is generally lower than 10% of the initial residues in the non-clarified wines.     

Arginine Citrullination at the C-Terminal Domain Controls RNA Polymerase II Transcription

1. Download high-res image (125KB)
2. Download full-size image