c-Jun N-terminal kinase activation by hydrogen peroxide in endothelial cells involves SRC-dependent epidermal growth factor receptor transactivation

The phenotypic properties of the endothelium are subject to modulation by oxidative stress, and the c-Jun N-terminal kinase (JNK) pathway is important in mediating cellular responses to stress, although activation of this pathway in endothelial cells has not been fully characterized. Therefore, we exposed endothelial cells to hydrogen peroxide (H(2)O(2)) and observed rapid activation of JNK within 15 min that involved phosphorylation of JNK and c-Jun and induction of AP-1 DNA binding activity. Inhibition of protein kinase C and phosphoinositide 3-kinase did not effect JNK activation. In contrast, the tyrosine kinase inhibitors, genistein, herbimycin A, and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) significantly attenuated H(2)O(2)-induced JNK activation as did endothelial cell adenoviral transfection with a dominant-negative form of Src, implicating Src as an upstream activator of JNK. Activation of JNK by H(2)O(2) was also inhibited by AG1478 and antisense oligonucleotides directed against the epidermal growth factor receptor (EGFR), implicating the EGFR in this process. Consistent with this observation, H(2)O(2) stimulated EGFR tyrosine phosphorylation and complex formation with Shc-Grb2 that was abolished by PP2, implicating Src in H(2)O(2)-induced EGFR activation. Tyrosine phosphorylation of the EGFR by H(2)O(2) did not involve receptor autophosphorylation at Tyr(1173) as assessed by an autophosphorylation-specific antibody. These data indicate that H(2)O(2)-induced JNK activation in endothelial cells involves the EGFR through an Src-dependent pathway that is distinct from EGFR ligand activation. These data represent one potential pathway for mediating oxidative stress-induced phenotypic changes in the endothelium.     

Polymorphisms in FTO and near TMEM18 associate with type 2 diabetes and predispose to younger age at diagnosis of diabetes

Variations in the FTO gene and near the TMEM18 gene are risk factors for common form of obesity, but have also been linked with type 2 diabetes (T2D). Our aim was to investigate the contribution of these variants to risk of T2D in a population in Latvia. Four single nucleotide polymorphisms (SNP) in the first and fourth intronic regions of FTO and one close to TMEM18 were genotyped in 987 patients with T2D and 1080 controls selected from the Latvian Genome Data Base (LGDB). We confirmed association of SNPs in the first intron (rs11642015, rs62048402 and rs9939609) of FTO and rs7561317 representing the TMEM18 locus with T2D. Association between SNP in FTO and T2D remained significant after correction for body mass index (BMI). The rs57103849 located in the fourth intron of FTO and rs7561317 in TMEM18 showed BMI independent association with younger age at diagnosis of T2D. Our results add to the evidence that BMI related variants in and near FTO and TMEM18 may increase the risk for T2D not only through secondary effects of obesity. The influence of variants in the fourth intron of the FTO gene on development of T2D may be mediated by mechanisms other than those manifested by SNPs in the first intron of the same gene.     

Cytogenetic mapping of a major locus for resistance to Fusarium head blight and crown rot of wheat on <Emphasis Type="Italic">Thinopyrum elongatum</Emphasis> 7EL and its pyramiding with valuable genes from a <Emphasis Type="Italic">Th. ponticum</Emphasis> homoeologous arm onto bread wheat 7DL

Key message

A major locus for resistance to different Fusarium diseases was mapped to the most distal end of Th. elongatum 7EL and pyramided with Th. ponticum beneficial genes onto wheat 7DL.

Abstract

Perennial Triticeae species of the Thinopyrum genus are among the richest sources of valuable genes/QTL for wheat improvement. One notable and yet unexploited attribute is the exceptionally effective resistance to a major wheat disease worldwide, Fusarium head blight, associated with the long arm of Thinopyrum elongatum chromosome 7E (7EL). We targeted the transfer of the temporarily designated Fhb-7EL locus into bread wheat, pyramiding it with a Th. ponticum 7el₁L segment stably inserted into the 7DL arm of wheat line T4. Desirable genes/QTL mapped along the T4 7el₁L segment determine resistance to wheat rusts (Lr19, Sr25) and enhancement of yield-related traits. Mapping of the Fhb-7EL QTL, prerequisite for successful pyramiding, was established here on the basis of a bioassay with Fusarium graminearum of different 7EL-7el₁L bread wheat recombinant lines. These were obtained without resorting to any genetic pairing promotion, but relying on the close 7EL-7el₁L homoeology, resulting in 20% pairing frequency between the two arms. Fhb-7EL resided in the telomeric portion and resistant recombinants could be isolated with useful combinations of more proximally located 7el₁L genes/QTL. The transferred Fhb-7EL locus was shown to reduce disease severity and fungal biomass in grains of infected recombinants by over 95%. The same Fhb-7EL was, for the first time, proved to be effective also against F. culmorum and F. pseudograminearum, predominant agents of crown rot. Prebreeding lines possessing a suitable 7EL-7el₁L gene/QTL assembly showed very promising yield performance in preliminary field tests.

     

Engineering new metal specificity in EF-hand peptides

Peptides (33-34 amino acids long) corresponding to the helix-turn-helix (EF-hand) motif of the calcium binding site I of Paramecium tetraurelia calmodulin have been synthesized. The linear sequence was unable to acquire a native-like conformation and calcium binding. However, incorporation of a well-positioned disulfide bond bridging the two putative helical regions greatly improved the ordered structure and binding properties. Analyzed by electrospray mass spectrometry, circular dichroism and time-resolved laser-induced fluorescence, such a disulfide-stabilized peptide is shown to acquire a calcium-dependent helical conformation and exhibits native-like affinity for calcium, terbium and europium ions with 30+/-1, 3.5+/-0.6 and 0.6+/-0.1 microM dissociation constants, respectively. Comparable affinities were calculated within the biological construct comprising the entire domain I of Arabidopsis taliana calmodulin. Single sequence mutation (Glu25Asp) in the binding loop of the peptide abolishes calcium affinity, but preserves lanthanide affinity, showing that metal selectivity can be modulated by specific mutations. Such disulfide-stabilized peptides represent useful models to engineer metal specificity in new calmodulin proteins, facilitating the development of new systems to monitor metal pollution in biosensors and to increase metal binding capability of bacterial and plant cells in bioremediation techniques.     

Adhesion to and biofilm formation on IB3-1 bronchial cells by <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> isolates from cystic fibrosis patients

Background  

Stenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains.     

Phenol hydroxylase from Acinetobacter radioresistens S13. Isolation and characterization of the regulatory component.

This paper reports the isolation and characterization of the regulatory moiety of the multicomponent enzyme phenol hydroxylase from Acinetobacter radioresistens S13 grown on phenol as the only carbon and energy source. The whole enzyme comprises an oxygenase moiety (PHO), a reductase moiety (PHR) and a regulatory moiety (PHI). PHR contains one FAD and one iron-sulfur cluster, whose function is electron transfer from NADH to the dinuclear iron centre of the oxygenase. PHI is required for catalysis of the conversion of phenol to catechol in vitro, but is not required for PHR activity towards alternative electron acceptors such as cytochrome c and Nitro Blue Tetrazolium. The molecular mass of PHI was determined to be 10 kDa by SDS/PAGE, 8.8 kDa by MALDI-TOF spectrometry and 18 kDa by gel-permeation. This finding suggests that the protein in its native state is a homodimer. The isoelectric point is 4.1. PHI does not contain any redox cofactor and does not bind ANS, a fluorescent probe for hydrophobic sites. The N-terminal sequence is similar to those of the regulatory proteins of phenol hydroxylase from A. calcoaceticus and Pseudomonas CF 600. In the reconstituted system, optimal reaction rate was achieved when the stoichiometry of the components was 2 PHR monomers: 1 PHI dimer: 1 PHO (alphabetagamma) dimer. PHI interacts specifically with PHR, promoting the enhancement of FAD fluorescence emission. This signal is diagnostic of a conformational change of PHR that might result in a better alignment with respect to PHO.     

Association of SOD2, GPX1, CAT, and TNF Genetic Polymorphisms with Oxidative Stress, Neurochemistry, Psychopathology, and Extrapyramidal Symptoms in Schizophrenia

There is a growing body of evidence confirming the involvement of oxidative stress and inflammation in pathogenesis of schizophrenia. Inter-individual variation in antioxidant capacity caused by different genetic profile could potentially influence patient’s susceptibility to oxidative damage. In this study we evaluated the polymorphisms of manganese superoxide dismutase SOD2Val16Ala, glutathione peroxidase GPX1Pro200Leu, catalase CAT-262C>T and CATc.66+78C>T, and tumour necrosis factor-alpha TNF-308G>A by assessing their association with biomarkers of oxidative stress, neurochemistry, psychopathology of schizophrenia and extrapyramidal symptoms in Caucasian schizophrenia patients treated with haloperidol depot. TNF-308G>A was associated with the increased risk of parkinsonism. No major role of polymorphism of SOD2Val16Ala, CAT-262C>T nor GPX1Pro200Leu in psychopathology of schizophrenia or extrapyramidal symptoms was observed. SOD2Val16Ala polymorphism was associated with dopamine plasma concentration and blood concentration ratio between reduced and oxidised form of glutathione, while GPX1Pro200Leu was related with concentration of reduced glutathione. CATc.66+78C>T was associated with noradrenaline plasma concentration and PANSS negative score. PANSS positive and general scores, were associated with the increased risk of tardive dyskinesia. PANSS positive, negative, and general scores, and GAF score were all associated with the increased risk of akathisia.     

Biological succession on silicone fouling‐release surfaces: Long‐term exposure tests in the harbour of ischia,Italy

A static test site was set up in the Harbour of Ischia (Gulf of Naples, Italy) to investigate the antifouling effectiveness of newly developed non‐polluting coatings. Two‐year exposure experiments were performed on sets of panels coated with silicone‐based coatings, and results were compared both to sets of panels coated with toxic agents, and non‐toxic epoxydic compounds. Abiotic factors, strength of adhesion of the temporal dynamics of succession of foulers were analyzed throughout the period of immersion. Brown algae constantly represented the “border point”; between the early community, dominated by sume, micro‐ and macroalgae, and the late community, mainly represented by bryozoans and molluscs, as well as polychaetes, sponges and tunicates. Brown algae, such as Ectocarpus siliculosus, tunicates (mainly Botryllus schlossen) and polychaetes (Hydroides elegans, Pileolaria pseudomilitaris) were demonstrated to be key species, triggering the community and influencing its development. Light was the main abiotic factor discriminating the community on the two sides of panels exposed to different irradiances. The best performing coatings (silicone easy release coatings without additives) substantially influenced community structure, shifting it to the earliest stages of colonization. Silicone coatings proved to be unsuitable for colonization by organisms typical of mature communities, due to their low energy surfaces. The results of the present paper demonstrate that silicone coatings technology represents an alternative to the use of biocidal antifouling paints.     

Transport and metabolism of L-lactate occur in mitochondria from cerebellar granule cells and are modified in cells undergoing low potassium dependent apoptosis

Having confirmed that externally added L-lactate can enter cerebellar granule cells, we investigated whether and how L-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions. (1) L-lactate enters mitochondria, perhaps via an L-lactate/H+ symporter, and is oxidized in a manner stimulated by ADP. The existence of an L-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the Km and Vmax values changed en route to apoptosis. (2) In both normal and apoptotic cell homogenates, externally added L-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirrored L-lactate uptake by mitochondria and occurred with a hyperbolic dependence on L-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition of L-lactate. The rate of the process depended on L-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellular L-lactate/pyruvate shuttle, whose activity was limited by the putative L-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier. (3) L-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putative L-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.