The polypeptides COX2A and COX2B are essential components of the mitochondrial cytochrome c oxidase of Toxoplasma gondii

Two genes encoding cytochrome c oxidase subunits, Cox2a and Cox2b, are present in the nuclear genomes of apicomplexan parasites and show sequence similarity to corresponding genes in chlorophycean algae. We explored the presence of COX2A and COX2B subunits in the cytochrome c oxidase of Toxoplasma gondii. Antibodies were raised against a synthetic peptide containing a 14-residue fragment of the COX2A polypeptide and against a hexa-histidine-tagged recombinant COX2B protein. Two distinct immunochemical stainings localized the COX2A and COX2B proteins in the parasite's mitochondria. A mitochondria-enriched fraction exhibited cyanide-sensitive oxygen uptake in the presence of succinate. T. gondii mitochondria were solubilized and subjected to Blue Native Electrophoresis followed by second dimension electrophoresis. Selected protein spots from the 2D gels were subjected to mass spectrometry analysis and polypeptides of mitochondrial complexes III, IV and V were identified. Subunits COX2A and COX2B were detected immunochemically and found to co-migrate with complex IV; therefore, they are subunits of the parasite's cytochrome c oxidase. The apparent molecular mass of the T. gondii mature COX2A subunit differs from that of the chlorophycean alga Polytomella sp. The data suggest that during its biogenesis, the mitochondrial targeting sequence of the apicomplexan COX2A precursor protein may be processed differently than the one from its algal counterpart.     

Structural requirements of chromokinesin Kif4A for its proper function in mitosis

Human Kif4A is a member of the Kinesin-4 family of kinesins. Kif4A is thought to be a bona fide chromokinesin because it possesses a motor domain and associates with condensed chromosomes during mitosis. Genetic deletion of Kif4A promotes tumorigenic phenotypes in mouse embryonic cells. Kif4A is critical for mitotic regulation including chromosome condensation, spindle organization and cytokinesis. However, the precise chromatin-binding domain of Kif4A has not been characterized. Herein, we report the identification of two conserved motifs critical for chromatin-binding: the first leucine Zip motif (Zip1) of a leucine Zip/Basic/leucine Zip region (ZBZ) previously thought to be a nuclear localization signal (NLS), and a cysteine-rich (CR) motif within the C-terminal region of Kif4A. Furthermore, by depleting endogenous Kif4A via RNAi and concurrently expressing RNAi-resistant Kif4A versions, we observed that wild type Kif4A, but not the mutants deficient in DNA-binding (Zip1 or CR deleted) or ATPase activity (K94A point mutant), was able to rescue the RNAi-elicited abnormal mitotic profile. Taken together, our results show that both the Zip1 and CR motifs are important for Kif4A chromatin-binding and its mitotic function.     

Ectopic expression of angiopoietin-1 promotes neuronal differentiation in neural progenitor cells through the Akt pathway

In regions of adult neurogenesis, neural progenitor cells (NPCs) are found in close proximity to blood vessels within a so-called ‘vascular niche’. Neurogenesis is linked to angiogenesis via certain growth factors. We propose that angiopoietin-1 (Ang1), which is similar to VEGF, has a unique role in neurogenesis independent of its role in angiogenesis. In this study, primary cultures of NPCs were transduced with recombinant adenoviruses expressing Ang1 and induced to differentiate with dibutyryl cyclic AMP (dbcAMP). Neuronal differentiation was evaluated by quantitative PCR, immunofluorescence microscopy and Western blot analysis. The results show that ectopic expression of Ang1 promotes neuronal differentiation and neurite outgrowth in NPCs, while this effect was blocked by the presence of anti-Tie2 receptor antibody or the PI3-K inhibitor, LY294002. Our results suggest that Ang1, identified originally as an angiogenic factor, can also stimulate in vitro neurogenesis in NPCs through the Akt pathway.     

Accelerated cellular senescence phenotype of GAPDH-depleted human lung carcinoma cells

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal glycolytic enzyme, and a signaling molecule which acts at the interface between stress factors and the cellular apoptotic machinery. Earlier, we found that knockdown of GAPDH in human carcinoma cell lines resulted in cell proliferation arrest and chemoresistance to S phase-specific cytotoxic agents. To elucidate the mechanism by which GAPDH depletion arrests cell proliferation, we examined the effect of GAPDH knockdown on human carcinoma cells A549. Our results show that GAPDH-depleted cells establish senescence phenotype, as revealed by proliferation arrest, changes in morphology, SA-β-galactosidase staining, and more than 2-fold up-regulation of senescence-associated genes DEC1 and GLB1. Accelerated senescence following GAPDH depletion results from compromised glycolysis and energy crisis leading to the sustained AMPK activation via phosphorylation of α subunit at Thr172. Our findings demonstrate that GAPDH depletion switches human tumor cells to senescent phenotype via AMPK network, in the absence of DNA damage. Rescue experiments using metabolic and genetic models confirmed that GAPDH has important regulatory functions linking the energy metabolism and the cell cycle networks. Induction of senescence in LKB1-deficient non-small cell lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

Effects of chromatin decondensation on alternative NHEJ

In cells of higher eukaryotes, repair of DNA double strand breaks (DSBs) utilizes different forms of potentially error-prone non-homologous end joining (NHEJ): canonical DNA-PK-dependent (C-NHEJ) and alternative backup pathways (A-NHEJ). In contrast to C-NHEJ, A-NHEJ shows pronounced efficiency fluctuations throughout the cell cycle and is severely compromised as cells cease proliferating and enter the plateau phase (Windhofer et al., 2007 [23]). The molecular mechanisms underpinning this response remain unknown but changes in chromatin structure are prime candidate-A-NHEJ-modulators. Since parameters beyond chromatin acetylation appear to determine A-NHEJ efficiency (Manova et al., 2012 0210 and 0380), we study here the role of chromatin decondensation mediated either by treatment with 5′-aza-2′-deoxycytidine (AzadC) or growth in hypotonic conditions, on A-NHEJ. We report that both treatments have no detectable effect on C-NHEJ but provoke, specifically for A-NHEJ, cell-growth-dependent effects. These results uncover for the first time a link between A-NHEJ and chromatin organization and provide means for understanding the regulatory mechanisms underpinning the growth-state dependency of A-NHEJ. A-NHEJ is implicated in the formation of chromosomal translocations and in chromosome fusions that underlie genomic instability and carcinogenesis. The observations reported here may therefore contribute to the development of drug-based A-NHEJ suppression-strategies aiming at optimizing cancer treatment outcomes and possibly also at suppressing carcinogenesis.     

Vibrio harveyi hemolysin induces ultrastructural changes and apoptosis in flounder (Paralichthys olivaceus) cells

Vibrio harveyi hemolysin (VHH) is considered a major pathogenic virulence factor to fish. However, the VHH active-site mutant has lost all hemolytic and phospholipase activities as well as pathogenicity. In this study, the effect of VHH on erythrocytes and a gill cell line from flounder was elucidated. Erythrocyte membranes formed thin tubular protrusions immediately after exposure to VHH, and membrane corrugations were evident after extended incubation. In contrast, the mutant VHH did not induce any gross morphological changes. With VHH-treated FG-9307 cells, a cell line derived from flounder gill, destruction of organelles and formation of features resembling apoptotic bodies were observed. Immunogold staining showed that a large amount of VHH was deposited on the membranes and membrane debris of erythrocytes and FG-9307 cells after treatment with VHH. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in VHH-treated FG-9307 cells using DAPI staining. Moreover, cell cycle analysis showed that VHH increased the proportion of cells in G1 phase. In addition, VHH significantly increased the percentage of apoptosis, the number of TUNEL positive apoptotic cells, and caspase-3 activity in FG-9307 cells when compared with the untreated controls. These data suggested that VHH killed the cells through apoptosis via the caspase activation pathway.     

Hypoxic preconditioning reinforces cellular functions of autologous peripheral blood-derived cells in rabbit hindlimb ischemia model

Peripheral blood mononuclear cell (PBMNC) is one of powerful tools for therapeutic angiogenesis in hindlimb ischemia. However, traditional approaches with transplanted PBMNCs show poor therapeutic effects in severe ischemia patients. In this study, we used autograft models to determine whether hypoxic pretreatment effectively enhances the cellular functions of PBMNCs and improves hindlimb ischemia. Rabbit PBMNCs were cultured in the hypoxic condition. After pretreatment, cell adhesion, stress resistance, and expression of angiogenic factor were evaluated in vitro. To examine in vivo effects, we autografted preconditioned PBMNCs into a rabbit hindlimb ischemia model on postoperative day (POD) 7. Preconditioned PBMNCs displayed significantly enhanced functional capacities in resistance to oxidative stress, cell viability, and production of vascular endothelial growth factor. In addition, autologous transplantation of preconditioned PBMNCs significantly induced new vessels and improved limb blood flow. Importantly, preconditioned PBMNCs can accelerate vessel formation despite transplantation on POD 7, whereas untreated PBMNCs showed poor vascularization. Our study demonstrated that hypoxic preconditioning of PBMNCs is a feasible approach for increasing the retention of transplanted cells and enhancing therapeutic angiogenesis in ischemic tissue.     

Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice

In this study, we identified and functionally characterized the mitochondrial heat shock protein 70 (mtHsp70). Over-expression of mtHsp70 suppressed heat- and H₂O₂-induced programmed cell death (PCD) in rice protoplasts, as reflected by higher cell viability, decreased DNA laddering and chromatin condensation. Mitochondrial membrane potential (Δψ_m) after heat shock was destroyed gradually in protoplasts, but mtHsp70 over-expression showed higher Δψ_m relative to the vector control cells, and partially inhibited cytochrome c release from mitochondria to cytosol. Heat treatment also significantly increased reactive oxygen species (ROS) generation, a phenomenon not observed in protoplasts over-expressing mtHsp70. Together, these results suggest that mtHsp70 may suppress PCD in rice protoplasts by maintaining mitochondrial Δψ_m and inhibiting the amplification of ROS.     

The chemotaxonomic significance of two bioactive caffeic acid esters,nepetoidins A and B,in the Lamiaceae

A survey of leaf surface constituents in the family Lamiaceae using HPLC with diode array detection revealed the presence of two characteristic phenolic compounds in many species. The distribution of these phenolics in the Lamiaceae was found to be of taxonomic significance, as they were present in the great majority of species investigated for the subfamily Nepetoideae, including representatives of the well-known genera of culinary herbs, mint, rosemary, sage, thyme and basil. In contrast, they were absent from species of the other subfamilies of Lamiaceae studied and from the related families Verbenaceae, Scrophulariaceae, Acanthaceae and Buddlejaceae. The compounds were isolated from Plectranthus crassus and identified by NMR spectroscopy as the known caffeic acid esters (Z,E)-[2-(3,5-dihydroxyphenyl)ethenyl] 3-(3,4-dihydroxyphenyl)-2-propenoate and (Z,E)-[2-(3,4-dihydroxyphenyl)ethenyl] 3-(3,4-dihydroxyphenyl)-2-propenoate, for which the trivial names nepetoidins A and B are proposed. The presence of this pair of caffeic acid esters adds another character to the chemical, palynological and embryological features distinguishing the Nepetoideae from the other subfamilies of Lamiaceae and related families, and supports the view that the Nepetoideae are a specialised and monophyletic group within the family. Nepetoidin B was shown to have a greater antioxidant activity than gallic, rosmarinic and caffeic acids, and showed activity as an insect phagostimulant. Both compounds were antifungal.     

Allosteric modulators of human A2B adenosine receptor

Background

Among adenosine receptors (ARs) the A_2B subtype exhibits low affinity for the endogenous agonist compared with the A₁, A_2A, and A₃ subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A_2B AR represents an important pharmacological target.

Methods

We evaluated seven 1-benzyl-3-ketoindole derivatives (7–9) for their ability to act as positive or negative allosteric modulators of human A_2B AR through binding and functional assays using CHO cells expressing human A₁, A_2A, A_2B, and A₃ ARs.

Results

The investigated compounds behaved as specific positive or negative allosteric modulators of human A_2B AR depending on small differences in their structures. The positive allosteric modulators 7a,b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b,c and 9a,b reduced agonist potency and efficacy.

Conclusions

A number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive (7a,b) or negative (8c, 9a,b) allosteric modulators of human A_2B AR.

General significance

The 1-benzyl-3-ketoindole derivatives 7–9 acting as positive or negative allosteric modulators of human A_2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A_2B AR.     

Pharmacological evidence that Ca²+ channels and, to a lesser extent, K+ channels mediate the relaxation of testosterone in the canine basilar artery

Testosterone induces vasorelaxation through non-genomic mechanisms in several isolated blood vessels, but no study has reported its effects on the canine basilar artery, an important artery implicated in cerebral vasospasm. Hence, this study has investigated the mechanisms involved in testosterone-induced relaxation of the canine basilar artery. For this purpose, the vasorelaxant effects of testosterone were evaluated in KCl- and/or PGF_2α-precontracted arterial rings in vitro in the absence or presence of several antagonists/inhibitors/blockers; the effect of testosterone on the contractile responses to CaCl₂ was also determined. Testosterone (10-180 μM) produced concentration-dependent relaxations of KCl- or PGF_2α-precontracted arterial rings which were: (i) unaffected by flutamide (10 μM), dl-aminoglutethimide (10 μM), actinomycin D (10 μM), cycloheximide (10 μM), SQ 22,536 (100 μM) or ODQ (30 μM); and (ii) significantly attenuated by the blockers 4-aminopyridine (K_V; 1 mM), BaCl₂ (K_IR; 30 μM), iberiotoxin (BKCa2+; 20 nM), but not by glybenclamide (K_ATP; 10 μM). In addition, testosterone (31, 56 and 180 μM) and nifedipine (0.01-1 μM) produced a concentration-dependent blockade of the contraction to CaCl₂ (10 μM to 10 mM) in arterial rings depolarized by 60 mM KCl. These results, taken together, show that testosterone relaxes the canine basilar artery mainly by blockade of voltage-dependent Ca²⁺ channels and, to a lesser extent, by activation of K⁺ channels (K_IR, K_V and BKCa2+). This effect does not involve genomic mechanisms, production of cAMP/cGMP or the conversion of testosterone to 17β-estradiol.     

Cholesterol modulates the antagonist-binding function of hippocampal serotonin1A receptors

The serotonin_1A receptor is the most extensively studied member of the family of seven transmembrane domain G-protein coupled serotonin receptors. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions such as sleep, mood, pain, addiction, locomotion, sexual activity, depression, anxiety, alcohol abuse, aggression and learning. Since a significant portion of the protein lies embedded in the membrane and the ligand-binding pocket is defined by the transmembrane stretches in such receptors, membrane composition and organization represent a crucial parameter in the structure-function analysis of G-protein coupled receptors. In this paper, we have monitored the role of membrane cholesterol in the ligand-binding function of the hippocampal serotonin_1A receptor. Our results demonstrate that the reduction of membrane cholesterol significantly attenuates the antagonist-binding function of the serotonin_1A receptor. Based on prior pharmacological knowledge regarding the requirements for the antagonist to bind the receptor, our results indicate that membrane cholesterol modulates receptor function independently of its ability to interact with G-proteins. These effects on ligand-binding function of the receptor are predominantly reversed upon cholesterol-replenishment of cholesterol-depleted membranes. When viewed in the light of our earlier results on the effect of cholesterol depletion on the serotonin_1A receptor/G-protein interaction, these results comprehensively demonstrate the importance of cholesterol in the serotonin_1A receptor function and form the basis for understanding lipid-protein interactions involving this important neuronal receptor.     

Biochemical characterization of missense mutations in the Arf/Arl-family small GTPase Arl6 causing Bardet-Biedl syndrome

Bardet-Biedl syndrome (BBS) is a pleiotropically genetic disorder, whose etiology is linked to cilia. Mutations in the Arf/Arl-family GTPase Arl6 have been recently shown to be responsible for BBS type 3. Here we show that BBS mutations alter the guanine nucleotide-binding properties of Arl6. Specifically, substitution of 31st Threonine to Arginine selectively abrogates the GTP-binding ability of Arl6 without affecting GDP-binding/dissociating properties. Furthermore, all the BBS mutations in Arl6 result in low expression of the mutant proteins, which can be restored by the inhibition of the proteasome. These findings implicate that Arl6 mutants are destabilized and eliminated by the proteasome in cells, probably due to the altered nucleotide-binding properties.     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.     

On the metabolic activation of the carcinogen N-hydroxy-N-2-acetylaminofluorene : III. Oxidation with horseradish peroxidase to yield 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene

Previous studies have shown that the carcinogen N-hydroxy-2-acetylaminofluorene is converted by one-electron oxidants to a free nitroxide radical which dismutates to N-acetoxy-2-acetylaminofluorene and 2-nitrosofluorene. The present study shows that the same oxidation can be achieved with horseradish peroxidase and H₂O₂. The free radical intermediate was detected by its ESR signal, and the yields of N-acetoxy-2-acetylaminofluorene and of 2-nitrosofluorene were determined under a number of conditions. Addition of tRNA to the reaction mixture containing N-acetoxy-N-2-acetyl[2′-³H]aminofluorene yielded tRNA-bound radioactivity; addition of guanosine yielded a reaction product which appears to be N-guanosin-8-yl)-2-acetylaminofluorene. The latter compound has previously been identified as a reaction product of N-acetoxy-2-acetylaminofluorene and guanosine. Preliminary attempts to demonstrate the formation of a nitroxide free radical or its dismutation products with rat liver mixed function oxidase systems were not successful.     

Quantitative assessment of peptide-lipid interactions.: Ubiquitous fluorescence methodologies

Peptide-membrane interactions have been gaining increased relevance, mainly in biomedical investigation, as the potential of the natural, nature-based and synthetic peptides as new drugs or drug candidates also expands. These peptides must face the cell membrane when they interfere with or participate in intracellular processes. Additionally, several peptide drugs and drug leads actions occur at the membrane level (e.g., antimicrobial peptides, cell-penetrating peptides and enveloped viruses membrane fusion inhibitors). Here we explore fluorescence spectroscopy methods that can be used to monitor such interactions. Two main approaches are considered, centered either on the peptide or on the membrane. On the first, we consider mainly the methodologies based on the intrinsic fluorescence of the aminoacid residues tryptophan and tyrosine. Regarding membrane-centric approaches, we review methods based on lipophilic probes sensitive to membrane potentials. The use of fluorescence constitutes a simple and sensitive method to measure these events. Unraveling the molecular mechanisms that govern these interactions can unlock the key to understand specific biological processes involving natural peptides or to optimize the action of a peptide drug.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.