Oxidation of alkyl nitronates catalyzed by 2-nitropropane dioxygenase from Hansenula mrakii

2-Nitropropane dioxygenase from Hansenula mrakii was expressed in Escherichia coli cells and purified in active and stable form using 60% saturation of ammonium sulfate and a single chromatographic step onto a DEAE column. MALDI-TOF mass spectrometric and spectrophotometric analyses of the flavin extracted by heat or acid denaturation of the enzyme indicated that FMN, and not FAD as erroneously reported previously, is present in a 1:1 stoichiometry with the protein. Inductively coupled plasma mass spectrometric analysis of the enzyme established that H. mrakii 2-nitropropane dioxygenase contains negligible amounts of iron, manganese, zinc, and copper ions, which are not catalytically relevant. Anaerobic substrate reduction and kinetic data using a Clark oxygen electrode to measure rates of oxygen consumption indicated that the enzyme is active on a broad range of alkyl nitronates, with a marked preference for unbranched substrates over propyl-2-nitronate. Interestingly, the enzyme reacts poorly, if at all, with nitroalkanes, as suggested by lack of both anaerobic reduction of the enzyme-bound flavin and consumption of oxygen with nitroethane, nitrobutane, and 2-nitropropane. Finally, both the tight binding of sulfite (K_d = 90 μM, at pH 8 and 15 °C) to the enzyme and the formation of the anionic flavosemiquinone upon anaerobic incubation with alkyl nitronates are consistent with the presence of a positively charged group in proximity of the N(1)C(2)O atoms of the FMN cofactor.     

Impaired Temperature Stress Response of a Streptococcus thermophilus deoD Mutant

An insertional deoD mutant of Streptococcus thermophilus strain SFi39 had a reduced growth rate at 20°C and an enhanced survival capacity to heat shock compared to the wild type, indicating that the deoD product is involved in temperature shock adaptation. We report evidence that ppGpp is implicated in this dual response.     

The influence on coagulation of transdermal estrogen hormone replacement therapy as a preoperative preparation of the tissue before vaginal hysterectomy

In 32 postmenopausal patients who underwent vaginal hysterectomy due to the presence of uterine prolapse at the Department of Uro-gynaecology and Pelvic Floor Disorders in the Clinic of Gynaecology and Obstetrics, Medical School, Skopje in the period from 1st January 2002 to 1st January 2003, and who were preoperatively treated with transdermal estradiol 50 microg/day during 14 days the following parameters of the coagulating status were estimated: prothrombin time (PT) that is expressed in: absolute value, percentage and INR; activated partial thromboplastin time (aPTT Pathrombin SL); thrombin time and platelets number before and after hormone replacement therapy. After 14-day transdermal estrogen therapy, the parameters: PT, PT%, PT INR, aPTT Pathrombin SL didn't expressed significant changes, the thrombin time expressed significant extension, and the platelets expressed a significant decrease. According to our results, the transdermal estrogens might not have any influence on the hepatic synthesis of coagulating factors till the step of prothrombin formation. They might have an essential influence on the step of prothrombin transformation into thrombin, as well as on the process of megacaryocytes segregation into platelets.     

Birch pollen-associated peanut allergies in children

Panallergens show structural similarities, and they are responsible for many cross-reactions between pollen and plant food sources. The aim of the present study was to investigate IgE reactivity to peanut allergen components in children with birch pollen allergy. Patients experienced symptoms of allergic asthma, allergic rhinitis, and urticaria, and they underwent a complete diagnostic evaluation, including skin prick test (SPT), specific IgE (sIgE) to birch pollen allergen (t3), peanut allergen (f13). In addition, measurement of sIgE to the major birch allergen components, Betula verrucosa (Bet v1, Bet v2), and to peanut allergen components, Arachis hypogaea (genuine componens: Ara h1, Ara h2, Ara h3, and cross-reactive Ara h8) was performed, by using a microarray technique (component resolved diagnosis, CRD). SPT to birch extract was positive in all children, and SPT to peanut extract was positive in 51 % of them. sIgE to both allergens was increased in 39 % of children, 55 % of them had increased sIgE (t3), and one child had increased sIgE (f13). CRD results confirmed that some children were sensitized to Bet v1 only, and some children to genuine Ara h only. Bet v1/Ara h8 cross-reactivity was found in 16 % of children. Results of the present study reveal that SPT, sIgE, and CRD may detect sensitization and co-sensitization with birch and peanut allergens/allergen components, and CRD may help to differentiate sensitization to genuine peanut components from sensitization to peanut cross-reactive component in birch-sensitive children. Diagnostic approach has to be individualized for each patient.     

A proteinaceous elicitor Sm1 from the beneficial fungus Trichoderma virens is required for induced systemic resistance in maize

We have previously shown that the beneficial filamentous fungus Trichoderma virens secretes the highly effective hydrophobin-like elicitor Sm1 that induces systemic disease resistance in the dicot cotton (Gossypium hirsutum). In this study we tested whether colonization of roots by T. virens can induce systemic protection against a foliar pathogen in the monocot maize (Zea mays), and we further demonstrated the importance of Sm1 during maize-fungal interactions using a functional genomics approach. Maize seedlings were inoculated with T. virens Gv29-8 wild type and transformants in which SM1 was disrupted or constitutively overexpressed in a hydroponic system or in soil-grown maize seedlings challenged with the pathogen Colletotrichum graminicola. We show that similar to dicot plants, colonization of maize roots by T. virens induces systemic protection of the leaves inoculated with C. graminicola. This protection was associated with notable induction of jasmonic acid- and green leaf volatile-biosynthetic genes. Neither deletion nor overexpression of SM1 affected normal growth or development of T. virens, conidial germination, production of gliotoxin, hyphal coiling, hydrophobicity, or the ability to colonize maize roots. Plant bioassays showed that maize grown with SM1-deletion strains exhibited the same levels of systemic protection as non-Trichoderma-treated plants. Moreover, deletion and overexpression of SM1 resulted in significantly reduced and enhanced levels of disease protection, respectively, compared to the wild type. These data together indicate that T. virens is able to effectively activate systemic disease protection in maize and that the functional Sm1 elicitor is required for this activity.     

Beneficial effect of agmatine in the acute phase of experimental autoimmune encephalomyelitis in iNOS-/- knockout mice

The aim of the study was to investigate the hypothesis that agmatine (AGM) provides protection against oxidative stress in experimental autoimmune encephalomyelitis (EAE).     

Dimerization controls the activity of fungal elicitors that trigger systemic resistance in plants

The soilborne fungus Trichoderma virens secretes a small protein (Sm1) that induces local and systemic defenses in plants. This protein belongs to the ceratoplatanin protein family and is mainly present as a monomer in culture filtrates. However, Hypocrea atroviride (the telomorph form of Trichoderma atroviride) secretes an Sm1-homologous protein, Epl1, with high levels of dimerization. Nonetheless, the molecular mechanisms involved in recognition and the signaling pathways involved in the induction of systemic resistance in plants are still unclear. In this report, we demonstrate that Sm1 and Epl1 are mainly produced as monomer and a dimer, respectively, in the presence of maize seedlings. The results presented show that the ability to induce plant defenses reside only in the monomeric form of both Sm1 and Epl1, and we demonstrate for the first time that the monomeric form of Epl1, likewise Sm1, induces defenses in maize plants. Biochemical analyses indicate that monomeric Sm1 is produced as a glycoprotein, but the glycosyl moiety is missing from its dimeric form, and Epl1 is produced as a nonglycosylated protein. Moreover, for Sm1 homologues in various fungal strains, there is a negative correlation between the presence of the glycosylation site and their ability to aggregate. We propose a subdivision in the ceratoplatanin protein family according to the presence of the glycosylation site and the ability of the proteins to aggregate. The data presented suggest that the elicitor's aggregation may control the Trichoderma-plant molecular dialogue and block the activation of induced systemic resistance in plants.     

Physiological and cell ultrastructure disturbances in wheat seedlings generated by <Emphasis Type="Italic">Chenopodium murale</Emphasis> hairy root exudate

Chenopodium murale L. is an invasive weed species significantly interfering with wheat crop. However, the complete nature of its allelopathic influence on crops is not yet fully understood. In the present study, the focus is made on establishing the relation between plant morphophysiological changes and oxidative stress, induced by allelopathic extract. Phytotoxic medium of C. murale hairy root clone R5 reduced the germination rate (24% less than control value) of wheat cv. Nata?a seeds, as well as seedling growth, diminishing shoot and root length significantly, decreased total chlorophyll content, and induced abnormal root gravitropism. The R5 treatment caused cellular structural abnormalities, reflecting on the root and leaf cell shape and organization. These abnormalities mostly included the increased number of mitochondria and reorganization of the vacuolar compartment, changes in nucleus shape, and chloroplast organization and distribution. The most significant structural changes were observed in cell wall in the form of amoeboid protrusions and folds leading to its irregular shape. These structural alterations were accompanied by an oxidative stress in tissues of treated wheat seedlings, reflected as increased level of H₂O₂ and other ROS molecules, an increase of radical scavenging capacity and total phenolic content. Accordingly, the retardation of wheat seedling growth by C. murale allelochemicals may represent a consequence of complex activity involving both cell structure alteration and physiological processes.     

TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance in multiple myeloma through NF-кβ inhibition

Multiple myeloma (MM) is a B-cell malignancy characterized by an accumulation of abnormal clonal plasma cells in the bone marrow. Introduction of the proteasome-inhibitor bortezomib has improved MM prognosis and survival; however hypoxia-induced or acquired bortezomib resistance remains a clinical problem. This study highlighted the role of thioredoxin reductase 1 (TrxR1) in the hypoxia-induced and acquired bortezomib resistance in MM. Higher TrxR1 gene expression correlated with high-risk disease, adverse overall survival, and poor prognosis in myeloma patients. We demonstrated that hypoxia induced bortezomib resistance in myeloma cells and increased TrxR1 protein levels. Inhibition of TrxR1 using auranofin overcame hypoxia-induced bortezomib resistance and restored the sensitivity of hypoxic-myeloma cells to bortezomib. Hypoxia increased NF-кβ subunit p65 nuclear protein levels and TrxR1 inhibition decreased hypoxia-induced NF-кβ p65 protein levels in the nucleus and reduced the expression of NF-кβ-regulated genes. In addition, higher TrxR1 protein levels were observed in bortezomib-resistant myeloma cells compared to the naïve cells, and its inhibition using either auranofin or TrxR1-specific siRNAs reversed bortezomib resistance. TrxR1 inhibition reduced p65 mRNA and protein expression in bortezomib-resistant myeloma cells, and also decreased the expression of NF-кβ-regulated anti-apoptotic and proliferative genes. Thus, TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance by inhibiting the NF-кβ signaling pathway. Our findings demonstrate that elevated TrxR1 levels correlate with the acquisition of bortezomib resistance in MM. We propose considering TrxR1-inhibiting drugs, such as auranofin, either for single agent or combination therapy to circumvent bortezomib-resistance and improve survival outcomes of MM patients.