Agrobacterium-mediated transformation of Guignardia citricarpa: An efficient tool to gene transfer and random mutagenesis

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.     

Sequence determinants for the reaction specificity of murine (12R)-lipoxygenase: targeted substrate modification and site-directed mutagenesis

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.     

Thioacetamide-Induced Fulminant Hepatic Failure Induces Cerebral Mitochondrial Dysfunction by Altering the Electron Transport Chain Complexes

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.     

Synchrotron ultraviolet microspectroscopy on rat cortical bone: involvement of tyrosine and tryptophan in the osteocyte and its environment

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.     

Selective inhibition of cyclooxygenase-2 (COX-2) by 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3, a less calcemic vitamin D analog

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.     

Stereocontrol of arachidonic acid oxygenation by vertebrate lipoxygenases: newly cloned zebrafish lipoxygenase 1 does not follow the Ala-versus-Gly concept

Animal lipoxygenases (LOXs) are classified according to their specificity of arachidonic acid oxygenation, and previous sequence alignments suggested that S-LOXs contain a conserved Ala at a critical position at the active site but R-LOXs carry a Gly instead. Here we cloned, expressed, and characterized a novel LOX isoform from the model vertebrate Danio rerio (zebrafish) that carries a Gly at this critical position, classifying this enzyme as putative arachidonic acid R-LOX. Surprisingly, the almost exclusive arachidonic acid oxygenation product was 12S-H(p)ETE (hydro(pero)xyeicosatetraenoic acid), and extensive mutation around Gly-410 failed to induce R-lipoxygenation. This finding prompted us to explore the importance of the corresponding amino acids in other vertebrate S-LOXs. We found that Ala-to-Gly exchange in human 15-LOX2 and human platelet 12-LOX induced major alterations in the reaction specificity with an increase of specific R-oxygenation products. For mouse 5-LOX and 12/15-LOX from rabbits, men, rhesus monkeys, orangutans, and mice, only minor alterations in the reaction specificity were observed. For these enzymes, S-HETE (hydroxyeicosatetraenoic acid) isomers remained the major oxygenation products, whereas chiral R-HETEs contributed only 10-30% to the total product mixture. Taken together these data indicate that the Ala-versus-Gly concept may not always predict the reaction specificity of vertebrate LOX isoforms.     

Betanin a betacyanin pigment purified from fruits of Opuntia ficus-indica induces apoptosis in human chronic myeloid leukemia Cell line-K562

Betalains are water-soluble nitrogenous vacuolar pigments present in flowers and fruits of many caryophyllales with potent antioxidant properties. In the present study the antiproliferative effects of betanin, a principle betacyanin pigment, isolated from the fruits of Opuntia ficus-indica, was evaluated on human chronic myeloid leukemia cell line (K562). The results show dose and time dependent decrease in the proliferation of K562 cells treated with betanin with an IC(50) of 40 microM. Further studies involving scanning and transmission electron microscopy revealed the apoptotic characteristics such as chromatin condensation, cell shrinkage and membrane blebbing. Agarose electrophoresis of genomic DNA of cells treated with betanin showed fragmentation pattern typical for apoptotic cells. Flow cytometric analysis of cells treated with 40 microM betanin showed 28.4% of cells in sub G0/G1 phase. Betanin treatment to the cells also induced the release of cytochrome c into the cytosol, poly (ADP) ribose polymerase (PARP) cleavage, down regulation Bcl-2, and reduction in the membrane potentials. Confocal microscopic studies on the cells treated with betanin suggest the entry of betanin into the cells. These studies thus demonstrate that betanin induces apoptosis in K562 cells through the intrinsic pathway and is mediated by the release of cytochrome c from mitochondria into the cytosol, and PARP cleavage. The antiproliferative effects of betanin add further value to the nutritional characteristics of the fruits of O. ficus-indica.     

Design,synthesis, and biological evaluation of prenylated chalcones as 5-LOX inhibitors

Ten novel mono- and di-O-prenylated chalcone derivatives were designed on the basis of a homology derived molecular model of 5-lipoxygenase (5-LOX). The compounds were docked into 5-LOX active site and the binding characteristics were quantified using LUDI. To verify our theoretical assumption, the molecules were synthesized and tested for their 5-LOX inhibitory activities. The synthesis was carried out by Claisen–Schmidt condensation reaction of mono- and di-O-prenylated acetophenones with appropriate aldehydes. 5-LOX in vitro inhibition assay showed higher potency of di-O-prenylated chalcones than their mono-O-prenylated chalcone analogs. Compound 5e exhibited good inhibition with an IC₅₀ at 4 μM. The overall trend for the binding energies calculated and LUDI score was in good qualitative agreement with the experimental data. Further, the compound 5e showed potent anti-proliferative effects (GI₅₀ at 9 μM) on breast cancer cell line, MCF-7.     

A cyclo peptide activates signaling events and promotes growth and the production of the bone matrix

The interaction of bone cells and their underlying extracellular matrix impacts biological processes such as maintenance of tissue integrity. The biological recognition of the extracellular matrix by attached cells is mediated by the activity of integrins that recognize adhesive-specific domains. The most widely recognized adhesive motif is the RGD sequence, common to many of the adhesive matrix molecules. Here, we show that cyclo DFKRG which was previously selected to increase cell adhesion of human bone marrow stromal cells (HBMSC), increases both cell differentiation and mineralization through activation of tyrosine kinases, focal adhesion kinase (p(125)FAK) and Mitogen Activated Protein (MAP) kinases.