Expression,refolding and purification of a human interleukin-17A variant

A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T₇ promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris–HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2–8 °C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.     

Eukaryotic-like Ser/Thr Protein Kinases SpkC/F/K Are Involved in Phosphorylation of GroES in the Cyanobacterium Synechocystis

Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.     

16S rRNA phylogenetic analysis and quantification of <Emphasis Type="Italic">Korarchaeota</Emphasis> indigenous to the hot springs of Kamchatka,Russia

The candidate archaeal division Korarchaeota is known primarily from deeply branching sequences of 16S rRNA genes PCR-amplified from hydrothermal springs. Parallels between the phylogeny of these genes and the geographic locations where they were identified suggested that Korarchaeota exhibit a high level of endemism. In this study, the influence of geographic isolation and select environmental factors on the diversification of the Korarchaeota was investigated. Fourteen hot springs from three different regions of Kamchatka, Russia were screened by PCR using Korarchaeota-specific and general Archaea 16S rRNA gene-targeting primers, cloning, and sequencing. Phylogenetic analyses of these sequences with Korarchaeota 16S rRNA sequences previously identified from around the world suggested that all Kamchatka sequences cluster together in a unique clade that subdivides by region within the peninsula. Consistent with endemism, 16S rRNA gene group-specific quantitative PCR of all Kamchatka samples detected only the single clade of Korarchaeota that was found by the non-quantitative PCR screening. In addition, their genes were measured in only low numbers; small Korarchaeota populations would present fewer chances for dispersal to and colonization of other sites. Across the entire division of Korarchaeota, common geographic locations, temperatures, or salinities of identification sites united sequence clusters at different phylogenetic levels, suggesting varied roles of these factors in the diversification of Korarchaeota.     

Aldose reductase deficiency in mice protects from ragweed pollen extract (RWE)-induced allergic asthma

Background

Childhood hospitalization related to asthma remains at historically high levels, and its incidence is on the rise world-wide. Previously, we have demonstrated that aldose reductase (AR), a regulatory enzyme of polyol pathway, is a major mediator of allergen-induced asthma pathogenesis in mouse models. Here, using AR null (AR^-/-) mice we have investigated the effect of AR deficiency on the pathogenesis of ragweed pollen extract (RWE)-induced allergic asthma in mice and also examined the efficacy of enteral administration of highly specific AR inhibitor, fidarestat.

Methods

The wild type (WT) and AR^-/- mice were sensitized and challenged with RWE to induce allergic asthma. AR inhibitor, fidarestat was administered orally. Airway hyper-responsiveness was measured in unrestrained animals using whole body plethysmography. Mucin levels and Th2 cytokine in broncho-alveolar lavage (BAL) were determined using mouse anti-Muc5A/C ELISA kit and multiplex cytokine array, respectively. Eosinophils infiltration and goblet cells were assessed by H&E and periodic acid Schiff (PAS)-staining of formalin-fixed, paraffin-embedded lung sections. T regulatory cells were assessed in spleen derived CD4⁺CD25⁺ T cells population.

Results

Deficiency of AR in mice led to significantly decreased PENH, a marker of airway hyper-responsiveness, metaplasia of airway epithelial cells and mucus hyper-secretion following RWE-challenge. This was accompanied by a dramatic decrease in infiltration of eosinophils into sub-epithelium of lung as well as in BAL and release of Th2 cytokines in response to RWE-challenge of AR^-/- mice. Further, enteral administration of fidarestat significantly prevented eosinophils infiltration, airway hyper-responsiveness and also markedly increased population of T regulatory (CD4⁺CD25⁺FoxP3⁺) cells as compared to RWE-sensitized and challenged mice not treated with fidarestat.

Conclusion

Our results using AR^-/- mice strongly suggest the role of AR in allergic asthma pathogenesis and effectiveness of oral administration of AR inhibitor in RWE-induced asthma in mice supports the use of AR inhibitors in the treatment of allergic asthma.     

Effects of short-term glucocorticoid treatment on changes in cartilage matrix degradation and chondrocyte gene expression induced by mechanical injury and inflammatory cytokines

Introduction  

Traumatic joint injury damages cartilage and causes adjacent joint tissues to release inflammatory cytokines, increasing the risk of developing osteoarthritis. The main objective of this study was to determine whether the combined catabolic effects of mechanical injury, tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R) on cartilage could be abolished by short-term treatment with glucocorticoids such as dexamethasone.     

Effects of low concentration of endosulfan on proliferation, ERK1/2 pathway, apoptosis and senescence in Nile tilapia (Oreochromis niloticus) splenocytes

Endosulfan is a potent organochlorinated pesticide that is known to induce side effects in aquatic organisms, including Oreochromis niloticus (Nile tilapia). It has been previously shown that endosulfan induces oxidative stress and non-specific activation of splenic macrophages and exacerbated serum interleukin-2 synthesis in Nile tilapia. Endosulfan may promote proliferation of T cells through MAP kinase (MAPK) activated signal transductions. The ERK family of MAPKs includes ERK1 and ERK2. Phosphorylated ERK1/2 (pERK1/2) molecules are involved in many aspects of cellular survival, and are important for apoptosis or oxidative stress-induced senescence. In order to study the mechanisms by which endosulfan affects fish health, the present study was aimed at evaluating the in vitro effects of this insecticide on proliferation, the ERK1/2 pathway, apoptosis and cell senescence in splenocytes from Nile tilapia. Lymphoproliferation was evaluated by colorimetric method using the WST-1 assay. Flow cytometry was used to assess pERK1/2, apoptosis and senescence, using Annexin V-FITC and β-galactosidase respectively. Experimental data showed that exposure to 7 μg mL(-1) of endosulfan per se increased cellular proliferation, but decreased the lymphoproliferative response to mitogenic stimulus with PMA + ionomycin. Splenocytes exposed to endosulfan for 15-180 min showed significantly higher levels of pERK1/2 than the non-exposed control. Endosulfan mediated a decrease in etoposide-induced apoptosis and provoked cell senescence. In conclusion, exposure of immune cells to a low concentration of endosulfan deregulates their function and may facilitate the development of multiple diseases.     

Immunolocalization of an alternative respiratory chain in Antonospora (Paranosema) locustae spores: mitosomes retain their role in microsporidial energy metabolism

Microsporidia are a group of fungus-related intracellular parasites with severely reduced metabolic machinery. They lack canonical mitochondria, a Krebs cycle, and a respiratory chain but possess genes encoding glycolysis enzymes, a glycerol phosphate shuttle, and ATP/ADP carriers to import host ATP. The recent finding of alternative oxidase genes in two clades suggests that microsporidial mitosomes may retain an alternative respiratory pathway. We expressed the fragments of mitochondrial chaperone Hsp70 (mitHsp70), mitochondrial glycerol-3-phosphate dehydrogenase (mitG3PDH), and alternative oxidase (AOX) from the microsporidium Antonospora (Paranosema) locustae in Escherichia coli. Immunoblotting with antibodies against recombinant polypeptides demonstrated specific accumulation of both metabolic enzymes in A. locustae spores. At the same time comparable amounts of mitochondrial Hsp70 were found in spores and in stages of intracellular development as well. Immunoelectron microscopy of ultrathin cryosections of spores confirmed mitosomal localization of the studied proteins. Small amounts of enzymes of an alternative respiratory chain in merogonial and early sporogonial stages, alongside their accumulation in mature spores, suggest conspicuous changes in components and functions of mitosomes during the life cycle of microsporidia and the important role of these organelles in parasite energy metabolism, at least at the final stages of sporogenesis.     

Lipid motif of a bacterial antigen mediates immune responses via TLR2 signaling

The cross-talk between the innate and the adaptive immune system is facilitated by the initial interaction of antigen with dendritic cells. As DCs express a large array of TLRs, evidence has accumulated that engagement of these molecules contributes to the activation of adaptive immunity. We have evaluated the immunostimulatory role of the highly-conserved outer membrane lipoprotein P6 from non-typeable Haemophilus influenzae (NTHI) to determine whether the presence of the lipid motif plays a critical role on its immunogenicity. We undertook a systematic analysis of the role that the lipid motif plays in the activation of DCs and the subsequent stimulation of antigen-specific T and B cells. To facilitate our studies, recombinant P6 protein that lacked the lipid motif was generated. Mice immunized with non-lipidated rP6 were unable to elicit high titers of anti-P6 Ig. Expression of the lipid motif on P6 was also required for proliferation and cytokine secretion by antigen-specific T cells. Upregulation of T cell costimulatory molecules was abrogated in DCs exposed to non-lipidated rP6 and in TLR2(-/-) DCs exposed to native P6, thereby resulting in diminished adaptive immune responses. Absence of either the lipid motif on the antigen or TLR2 expression resulted in diminished cytokine production from stimulated DCs. Collectively, our data suggest that the lipid motif of the lipoprotein antigen is essential for triggering TLR2 signaling and effective stimulation of APCs. Our studies establish the pivotal role of a bacterial lipid motif on activating both innate and adaptive immune responses to an otherwise poorly immunogenic protein antigen.     

Energy metabolism in H460 lung cancer cells: effects of histone deacetylase inhibitors

Background

Tumor cells are characterized by accelerated growth usually accompanied by up-regulated pathways that ultimately increase the rate of ATP production. These cells can suffer metabolic reprogramming, resulting in distinct bioenergetic phenotypes, generally enhancing glycolysis channeled to lactate production. In the present work we showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin. This treatment was able to shift energy metabolism by activating mitochondrial systems such as the respiratory chain and oxidative phosphorylation that were largely repressed in the untreated controls.

Methodology/Principal Findings

Various cellular and biochemical parameters were evaluated in lung cancer H460 cells treated with the histone deacetylase inhibitors (HDACis), sodium butyrate (NaB) and trichostatin A (TSA). NaB and TSA reduced glycolytic flux, assayed by lactate release by H460 cells in a concentration dependent manner. NaB inhibited the expression of glucose transporter type 1 (GLUT 1), but substantially increased mitochondria bound hexokinase (HK) activity. NaB induced increase in HK activity was associated to isoform HK I and was accompanied by 1.5 fold increase in HK I mRNA expression and cognate protein biosynthesis. Lactate dehydrogenase (LDH) and pyruvate kinase (PYK) activities were unchanged by HDACis suggesting that the increase in the HK activity was not coupled to glycolytic flux. High resolution respirometry of H460 cells revealed NaB-dependent increased rates of oxygen consumption coupled to ATP synthesis. Metabolomic analysis showed that NaB altered the glycolytic metabolite profile of intact H460 cells. Concomitantly we detected an activation of the pentose phosphate pathway (PPP). The high O₂ consumption in NaB-treated cells was shown to be unrelated to mitochondrial biogenesis since citrate synthase (CS) activity and the amount of mitochondrial DNA remained unchanged.

Conclusion

NaB and TSA induced an increase in mitochondrial function and oxidative metabolism in H460 lung tumor cells concomitant with a less proliferative cellular phenotype.