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1.
Maria Beatriz Calderan Rodrigues Léia Cecília de Lima Fávaro Ana Paula de Souza Pallu Anderson Ferreira Fernanda de Souza Sebastianes Maria Juliana Calderan Rodrigues Marcel Bellato Spósito Welington Luiz de Araújo Aline Aparecida Pizzirani-Kleiner 《Fungal biology》2013,117(7-8):556-568
Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone – AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant–pathogen interaction. 相似文献
2.
C-Phycocyanin,a selective cyclooxygenase-2 inhibitor,induces apoptosis in lipopolysaccharide-stimulated RAW 264.7 macrophages 总被引:3,自引:0,他引:3
Reddy MC Subhashini J Mahipal SV Bhat VB Srinivas Reddy P Kiranmai G Madyastha KM Reddanna P 《Biochemical and biophysical research communications》2003,304(2):385-392
C-Phycocyanin (C-PC) is one of the major biliproteins of Spirulina platensis, a blue green algae, with antioxidant and radical scavenging properties. It is also known to exhibit anti-inflammatory and anti-cancer properties. However, the mechanism of action of C-PC is not clearly understood. Previously, we have shown that C-PC selectively inhibits cyclooxygenase-2 (COX-2), an inducible isoform that is upregulated during inflammation and cancer. In view of the reported induction of apoptosis in cancer cells by cyclooxygenase-2 inhibitors, the present study is undertaken to test the effect of C-PC on LPS stimulated RAW 264.7 mouse macrophage cell line. These studies have shown a dose dependent reduction in the growth and multiplication of macrophage cell line by C-PC. This decrease in cell number appears to be mediated by C-PC induced apoptosis as evidenced by flow cytometric and confocal microscopic studies. Cells treated with 20 micro M C-PC showed typical nuclear condensation and 16.6% of cells in sub-G(o)/G(1) phase. These cells also showed DNA fragmentation in a dose dependent manner. The studies on poly(ADP ribose) polymerase (PARP) cleavage showed typical fragmentation pattern in C-PC treated cells. This C-PC induced apoptosis in RAW 264.7 cells appears to be mediated by the release of cytochrome c from mitochondria and independent of Bcl-2 expression. These effects of C-PC on RAW 264.7 cells may be due to reduced PGE(2) levels as a result of COX-2 inhibition. 相似文献
3.
Enhanced production of antimicrobial sesquiterpenes and lipoxygenase metabolites in elicitor-treated hairy root cultures of Solanum tuberosum 总被引:2,自引:0,他引:2
Komaraiah P Reddy GV Reddy PS Raghavendra AS Ramakrishna SV Reddanna P 《Biotechnology letters》2003,25(8):593-597
Potato (Solanum tuberosum) hairy root cultures, established by infecting potato tuber discs with Agrobacterium rhizogenes, were used as a model system for the production of antimicrobial sesquiterpenes and lipoxygenase (LOX) metabolites. Of the four sesquiterpene phytoalexins (rishitin, lubimin, phytuberin and phytuberol) detected in elicitor-treated hairy root cultures, rishitin (213 g g–1 dry wt) was the most predominant followed by lubimin (171 g g–1 dry wt). The elicitors also induced LOX activity (25-fold increase) and LOX metabolites, mainly 9-hydroxyoctadecadienoic acid and 9-hydroxyoctadecatrienoic acid, in potato hairy root cultures. The combination of fungal elicitor plus cyclodextrin was the most effective elicitor treatment, followed by methyl jasmonate plus cyclodextrin in inducing sesquiterpenes and LOX metabolites. 相似文献
4.
Glutathione S-transferases (GSTs, EC 2.5.1.18) are multifunctional and multigene products. They are versatile enzymes and participate in the nucleophilic attack of the sulphur atom of glutathione on the electrophilic centers of various endogenous and xenobiotic compounds. Out of the five, , and , major classes of GSTs, GST- has significance in the diagnosis of cancers as it is expressed abundantly in tumor cells. This protein is a single gene product, coded by seven exons, that is having 24 kDa mass and pI value of 7.0. Four upstream elements such as two enhancers, and one of each of AP-1 site and GC box regulate gene. During chemical carcinogenesis because of jun/fos oncogenes (AP-1) regulatory elements, specifically GST- is expressed in liver. Therefore this gene product could be used as marker protein for the detection of chemical toxicity and carcinogenesis. 相似文献
5.
Meruvu S Walther M Ivanov I Hammarström S Fürstenberger G Krieg P Reddanna P Kuhn H 《The Journal of biological chemistry》2005,280(44):36633-36641
Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe(390), Gly(441), Ala(455), and Val(631) align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe(390) and Ala(455) did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly(441) to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val(631), which aligns with the positional determinant Ile(593) of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val(631) for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val(631) as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products. 相似文献
6.
Fulminant hepatic failure (FHF) is an acute form of hepatic encephalopathy resulting from severe inflammatory or necrotic
liver damage without any previously established liver damage. This develops as a complication due to viral infections, and
drug abuse. FHF also occurs in acute disorders like Reye’s syndrome. Although the exact mechanisms in the etiology of FHF
are not understood, elevated levels of brain ammonia have been consistently reported. Such increased ammonia levels are suggested
to alter neurotransmission signals and impair cerebral energy metabolism due to mitochondrial dysfunctions. In the present
study we have examined the role of cerebral electron transport chain complexes, including complex I, II, III IV, and pyruvate
dehydrogenase in the non-synaptic mitochondria isolated from the cortex of the thioacetamide-induced FHF rats. Further, we
have examined if the structure of mitochondria is altered. The results of the current study demonstrated a decrease in the
activity of the complex I by 31 and 48% at 18 and 24 h respectively after the thioacetamide injection. Similarly, the activity
of electron transport chain complex III was inhibited by 35 and 52% respectively, at 18 and 24 h, respectively. The complex
II and complex IV, on the other hand, revealed unaltered activity. Further the activity of pyruvate dehydrogenase at 18 and
24 h after the induction of FHF was inhibited by 29 and 43%, respectively. Our results also suggest mitochondrial swelling
in FHF induced rats. The inhibition of the respiratory complexes III and I and pyruvate dehydrogenase might lead to the increased
production of free radical resulting in oxidative stress and cerebral energy disturbances thereby leading to mitochondrial
swelling and further contributing to the pathogenesis of FHF. 相似文献
7.
S Pallu GY Rochefort C Jaffre M Refregiers DB Maurel D Benaitreau E Lespessailles F Jamme C Chappard CL Benhamou 《PloS one》2012,7(8):e43930
Alcohol induced osteoporosis is characterized by a bone mass decrease and microarchitecture alterations. Having observed an excess in osteocyte apoptosis, we aimed to assess the bone tissue biochemistry, particularly in the osteocyte and its environment. For this purpose, we used a model of alcohol induced osteoporosis in rats. Bone sections of cortical bone were investigated using synchrotron UV-microspectrofluorescence at subcellular resolution. We show that bone present three fluorescence peaks at 305, 333 and 385 nm, respectively corresponding to tyrosine, tryptophan and collagen. We have determined that tyrosine/collagen and tryptophan/collagen ratios were higher in the strong alcohol consumption group. Tryptophan is related to the serotonin metabolism involved in bone formation, while tyrosine is involved in the activity of tyrosine kinases and phosphatases in osteocytes. Our experiment represents the first combined synchrotron UV microspectroscopy analysis of bone tissue with a quantitative biochemical characterization in the osteocyte and surrounding matrix performed separately. 相似文献
8.
9.
Glutathione-S-transferase (GST), superoxide dismutase (SOD), Xanthine oxidase, selenium-dependent glutathione peroxidase (GPxI), catalase activities and malondialdehyde (MDA) content were determined in liver of three groups of exercised rats (E) viz., one day (E1), 10 days (E10) and 60 days (E60). GST, SOD and xanthine oxidase activities increased significantly with the increase in exercise period. Lipid peroxidation, expressed in terms of MDA formation, also increased in the liver of all the three groups. But catalase activity decreased significantly during exercise. Further, GPxI did not show any significant change in its activity in response to exercise. Our findings indicate that: 1) The significant increase in GST activity suggests their induction aimed at counteracting the oxidant stress induced during exercise; 2) The significant increase in xanthine oxidase and SOD activities indicates the generation of more superoxide anion radicals and their removal, respectively. 3) The significant reduction in catalase activity denotes the decreased formation of hydrogenperoxides during exercise; and 4) The pattern of changes in the activity level of GPxI indicate its least participation during exercise. However, in another way it is giving a scope for the involvement of GPxII associated with GST in the reduction of organic hydroperoxides. Further more, the relative increase in MDA is considered as the indicator of the rate of lipid peroxidation in the wake of exhaustive exercise. 相似文献
10.
Aparna R Subhashini J Roy KR Reddy GS Robinson M Uskokovic MR Venkateswara Reddy G Reddanna P 《Journal of cellular biochemistry》2008,104(5):1832-1842
Inducible cyclooxygenase-2 (COX-2) has been implicated to play a role in inflammation and carcinogenesis and selective COX-2 inhibitors have been considered as anti-inflammatory and cancer chemopreventive agents. 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), the active hormonal form of vitamin D3 also has been considered to be a cancer chemopreventive agent in addition to its important role in maintaining calcium homeostasis. Based on these observations, we studied the direct effect of 1alpha,25(OH)2D3 and one of its less calcemic synthetic analogs, 1alpha,25(OH)2-16-ene-23-yne-D3 on the activity of both COX-1 and COX-2 in an in vitro enzyme assay. Preliminary data indicated that both 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited selectively the activity of COX-2 with no effect on the activity of COX-1. Out of the two compounds, 1alpha,25(OH)2-16-ene-23-yne-D3 was found to be more effective with an IC50 of 5.8 nM. Therefore, the rest of the experiments were performed using 1alpha,25(OH)2-16-ene-23-yne-D3 only. 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited the proliferation of lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) with a reduction in the expression of COX-2 along with other inflammatory mediators like inducible nitric oxide synthase (iNOS) and interleukin-2 (IL-2). Furthermore, 1alpha,25(OH)2-16-ene-23-yne-D3 also inhibited carrageenan induced inflammation in an air pouch of a rat and effectively reduced the expression of COX-2, iNOS, and IL-2 in the tissues of the same air pouch. In both cases, 1alpha,25(OH)2-16-ene-23-yne-D3 did not show any effect on the expression of COX-1. In summary, our results indicate that 1alpha,25(OH)2-16-ene-23-yne-D3, a less calcemic vitamin D analog, exhibits potent anti-inflammatory effects and is a selective COX-2 inhibitor. 相似文献