Signal-peptide-peptidase-like 2a (SPPL2a) is targeted to lysosomes/late endosomes by a tyrosine motif in its C-terminal tail

Signal-peptide-peptidase-like 2A (SPPL2a), an aspartyl intramembrane protease, has been implicated in the proteolysis of TNF-alpha, Fas Ligand and Bri2. Here, we show that endogenous SPPL2a - in agreement with overexpression studies - is localised in membranes of lysosomes/late endosomes. Furthermore, we have analysed the molecular determinants for lysosomal sorting of SPPL2a by creating chimaeric constructs between SPPL2a and its plasma membrane localised homologue SPPL2b. Lysosomal transport of SPPL2a critically depends on its cytosolic carboxyterminal tail. A canonical tyrosine-based sorting motif of the YXX? type at position 498 is sufficient to direct SPPL2a to lysosomal/late endosomal compartments. This motif accounts for the differential localisation of the homologous proteases SPPL2a and SPPL2b and thereby influences the access to substrates and biological function of SPPL2a.     

TLR3 but not TLR7/8 ligand induces allergic sensitization to inhaled allergen

Epidemiological studies suggest that viral infections during childhood are a risk factor for the development of asthma. However, the role of virus-specific pattern recognition receptors in this process is not well defined. In the current study, we compare the effects of the inhaled viral TLR ligands polyinosinic-polycytidylic acid (TLR3) and resiquimod (TLR7/8) on sensitization to a model allergen (OVA) in a murine model. Both compounds enhance the migration, activation, and Ag-processing of myeloid dendritic cells from the lung to the draining lymph nodes comparable to the effects of LPS. Application of polyinosinic-polycytidylic acid [poly(I:C)] or LPS induces production of allergen-specific IgE and IgG1, whereas resiquimod (R848) had no effect. In addition, rechallenge of mice with OVA resulted in airway inflammation and mucus production in animals that received either poly(I:C) or LPS but not after application of R848. In summary, these results show that activation of TLR3 in combination with inhaled allergen results in induction of dendritic cell activation and migration similar to the effects of LPS. This leads to the development of allergic airway disease after allergen rechallenge, whereas mice treated with R848 did not develop allergic airway disease. These findings give further insight into the effects of stimulation of different TLRs on the development of asthma.     

Inactivation of NF1 in CNS causes increased glial progenitor proliferation and optic glioma formation

The gene responsible for neurofibromatosis type 1 (NF1) encodes a tumor suppressor that functions as a negative regulator of the Ras proto-oncogene. Individuals with germline mutations in NF1 are predisposed to the development of benign and malignant tumors of the peripheral and central nervous system (CNS). Children with this disease suffer a high incidence of optic gliomas, a benign but potentially debilitating tumor of the optic nerve; and an increased incidence of malignant astrocytoma, reactive astrogliosis and intellectual deficits. In the present study, we have sought insight into the molecular and cellular basis of NF1-associated CNS pathologies. We show that mice genetically engineered to lack NF1 in CNS exhibit a variety of defects in glial cells. Primary among these is a developmental defect resulting in global reactive astrogliosis in the adult brain and increased proliferation of glial progenitor cells leading to enlarged optic nerves. As a consequence, all of the mutant optic nerves develop hyperplastic lesions, some of which progress to optic pathway gliomas. These data point to hyperproliferative glial progenitors as the source of the optic tumors and provide a genetic model for NF1-associated astrogliosis and optic glioma.     

Analysing the yeast complexome—the Complex Portal rising to the challenge

The EMBL-EBI Complex Portal is a knowledgebase of macromolecular complexes providing persistent stable identifiers. Entries are linked to literature evidence and provide details of complex membership, function, structure and complex-specific Gene Ontology annotations. Data are freely available and downloadable in HUPO-PSI community standards and missing entries can be requested for curation. In collaboration with Saccharomyces Genome Database and UniProt, the yeast complexome, a compendium of all known heteromeric assemblies from the model organism Saccharomyces cerevisiae, was curated. This expansion of knowledge and scope has led to a 50% increase in curated complexes compared to the previously published dataset, CYC2008. The yeast complexome is used as a reference resource for the analysis of complexes from large-scale experiments. Our analysis showed that genes coding for proteins in complexes tend to have more genetic interactions, are co-expressed with more genes, are more multifunctional, localize more often in the nucleus, and are more often involved in nucleic acid-related metabolic processes and processes where large machineries are the predominant functional drivers. A comparison to genetic interactions showed that about 40% of expanded co-complex pairs also have genetic interactions, suggesting strong functional links between complex members.     

Purification,characterization and partial sequence of a pro‐inflammatory lectin from seeds of Canavalia oxyphylla Standl. & L. O. Williams

Recent studies have shown that lectins are promising tools for use in various biotechnological processes, as well as studies of various pathological mechanisms, isolation, and characterization of glycoconjugates and understanding the mechanisms underlying pathological mechanisms conditions, including the inflammatory response. This study aimed to purify, characterize physicochemically, and predict the biological activity of Canavalia oxyphylla lectin (CoxyL) in vitro and in vivo. CoxyL was purified by a single‐step affinity chromatography in Sephadex® G‐50 column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the pure lectin consists of a major band of 30 kDa (α‐chain) and two minor components (β‐chain and γ‐chain) of 16 and 13 kDa, respectively. These data were further confirmed by electrospray ionization mass spectrometry, suggesting that CoxyL is a typical ConA‐like lectin. In comparison with the average molecular mass of α‐chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. CoxyL presented hemagglutinating activity that was specifically inhibited by monosaccharides (D‐glucose, D‐mannose, and α‐methyl‐D‐mannoside) and glycoproteins (ovalbumin and fetuin). Moreover, CoxyL was shown to be thermostable, exhibiting full hemagglutinating activity up to 60°C, and it was pH‐sensitive for 1 h, exhibiting maximal activity at pH 7.0. CoxyL caused toxicity to Artemia nauplii and induced paw edema in rats. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses. Copyright © 2014 John Wiley & Sons, Ltd.     

Total low-molecular-weight antioxidants as a summary parameter, quantified in biological samples by a chemiluminescence inhibition assay

Aerobic metabolism requires a complex antioxidative system to balance reactive oxygen species (ROS). When in excess, ROS disrupt cellular activities and molecular structures. Owing to the variety of ROS, there are different antioxidative activities that require various tests for their detection. The so-called 'total antioxidative capacity' inhibition assay presented in this paper can be used to quantify the overall activity of low-molecular-weight antioxidants (AOs) in biological samples. The assay is based on enhanced horseradish peroxidase-catalyzed luminol chemiluminescence. It can be fine-tuned so that the biological samples meet the requirements of the light detector. A detailed protocol describing all relevant parameters is provided. The procedure is quick, inexpensive and reproducible. The assay can be used with diverse biological materials such as plant extracts and blood plasma. Hence, it is applicable to fields as diverse as crop breeding, medical diagnostics or food sciences. The time needed for the assay depends on the number of samples and their AO content. The protocol takes one working day to complete when five samples with five replicates are measured sequentially.     

Denaturing high-performance liquid chromatography in the detection of ABCA1 gene mutations in familial HDL deficiency

Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Because these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons. The aim of this study was to validate denaturing high-performance liquid chromatography (DHPLC) in mutation detection as an alternative to systematic sequencing. Exons of the ABCA1 gene were amplified using primers employed for sequencing. Temperatures for DHPLC were deducted from a software and empirically defined for each amplicon. To assess DHPLC reliability, we tested 30 sequence variants found in FHD patients and controls. Combined DHPLC and sequencing was applied to the genotyping of new FHD patients. Most of the amplicons required from two to five temperature conditions to obtain partially denatured DNA over the entire amplicon length. Twenty-nine of the variants found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and amplification conditions. DHPLC and sequencing analysis of new FHD patients revealed that all amplicons showing a heteroduplex DHPLC profile contained sequence variants. No variants were detected in amplicons with a homoduplex profile. DHPLC is a sensitive and reliable method for the detection of ABCA1 gene mutations.     

Hesperidin attenuates inflammation and oxidative damage in pleural exudates and liver of rat model of pleurisy

Objectives: This study investigated the potential anti-inflammatory effect of hesperidin against carrageenan induced pleurisy in rat model.

Methods: Twenty-four adult female Wistar rats (350 - 450g) were grouped as follows: Group I: rats were administered saline solution only (Normal control group); Group II: rats were administered saline solution (NaCl 0.9%) orally and injected with carrageenan (Inflammation control group); Group III: rats were administered hesperidin only (Hesperidin group); Group IV: rats were administered hesperidin orally and intrapleurally injected with 2% carrageenan (Inflammation treated with hesperidin group). The exudate volume, total leukocyte count, reactive oxygen species (ROS), myeloperoxidase (MPO),δ–aminolevulinate dehydratase (δ-ALA-D), catalase (CAT), superoxide dismutase (SOD), activities as well as non-protein thiol group (NPSH) and thiobarbituric acid reactive substances (TBARS) levels were determined.

Results: Pretreatment with hesperidin at a dose of 80 mg/kg orally per day for 21 days, minimized the increase in pleural exudate volume and leucocyte count and modulated the activities of MPO, SOD and CAT, as well as the levels of ROS, NPSH and TBARS in carrageenan-induced rats.

Conclusion: Our results suggest that hesperidin can elicit its anti-inflammatory action by blocking exudate and leukocyte influx into pleural cavity, inhibiting MPO activity and preventing oxidative damage.     

Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo.     

Reactive oxygen and nitrogen species are involved in sorbitol-induced apoptosis of human erithroleukaemia cells K562

In this study, we found that production of both reactive oxygen (ROS) and nitrogen (RNS) species is a very early event related to treatment with hyperosmotic concentration of sorbitol. The production of nitric oxide (NO) was paralleled by the increase of the mRNA and protein level of the inducible form of the nitric oxide synthase (iNOS). ROS and RNS enhancement, process concomitant to the failure of mitochondrial trans-membrane potential (ΔΨ), was necessary for the induction of apoptosis as demonstrated by the protection against sorbitol-mediated toxicity observed after treatment with ROS scavengers or NOS inhibitors. The synergistic action of ROS and RNS was finally demonstrated by pre-treatment with rosmarinic acid that, by powerfully buffering both these species, prevents impairment of ΔΨ and cell death. Overall results suggest that the occurrence of apoptosis upon sorbitol treatment is an event mediated by oxidative/nitrosative stress rather than a canonical hyperosmotic shock.