Expression of truncated tobacco osmotin in Escherichia coli: purification and antifungal activity

PURPOSE OF WORK: Tobacco osmotin is a functional homolog of mammalian adiponectin, and has antifungal activity. This work was undertaken to produce recombinant osmotin that has previously been unsuccessful because of its toxicity. Expression of recombinant tobacco osmotin (rOSM) in Escherichia coli inclusion bodies has been achieved. The optimal pH for rOSM expression in ZYM 505 medium is 7.0 at OD(650) of 1.5 of culture growth. The rOSM from the inclusion body was extracted with 8 M urea, and purified using CM-cellulose and cobalt-agarose bead affinity chromatography to a high purity. Approximately 80% of the rOSM remained bound to CM-cellulose and Cobalt-agarose beads after initial elution. The yield of purified rOSM was between 40 and 50 mg from 2 l of culture. Repeated elution of protein from CM-cellulose and Co-agarose increased the yield of rOSM to 200 mg from 2 l culture. The purified rOSM showed variable antifungal activities against two pathogenic yeast strains; Cryptococcus neoformans, Candida albicans, and non-pathogenic strains; Saccharomyces cerevisiae and Pichia methanolica.     

Identification of a second pyridoxine (pyridoxamine) 5′-phosphate oxidase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Pyridoxine (pyridoxamine) 5′-phosphate oxidase (PPOX) is involved in the biosynthetic pathway of vitamin B₆, converting pyridoxine 5′-phosphate (PNP) or pyridoxamine 5′-phosphate (PMP) into pyridoxal 5′-phosphate (PLP). PLP is a well-known cofactor of numerous enzymes including transamination and decarboxylation reactions. We have previously identified a PPOX (AtPPOX-1) protein encoded by At5g49970 in Arabidopsis thaliana. Here, we report a second PPOX in Arabidopsis, which was named as AtPPOX-2 encoded by At2g46580. The RT-PCR amplified cDNA of AtPPOX-2 was cloned into an Escherichia coli expression vector and a yeast shuttle vector. Both PPOX enzyme assay and complementation of the oxidative stress sensitivity phenotype of a yeast PDX3 deletion mutant demonstrated that At2g46580 encodes a PPOX protein (AtPPOX-2). The catalytic efficiency of AtPPOX-1 is approximately 300-fold higher than that of AtPPOX-2 for PNP. Based on bioinformatic analysis, AtPPOX-2 has a putative mitochondrial transit peptide at the N-terminus. The truncated AtPPOX-2 without 18 amino acids at the N-terminal end lost PPOX activity, suggesting that the N-terminal 18 amino acids are necessary for the enzyme activity of AtPPOX-2. Phylogenetic analysis of AtPPOX-2 homologs from all domains of life suggests that AtPPOX-2 homologs in plants are the product of lateral gene transfer from the cyanobacterial endosymbionts from which plastids are derived.     

Kidins220/ARMS mediates the integration of the neurotrophin and VEGF pathways in the vascular and nervous systems

Signaling downstream of receptor tyrosine kinases controls cell differentiation and survival. How signals from different receptors are integrated is, however, still poorly understood. In this work, we have identified Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin repeat-rich membrane spanning) as a main player in the modulation of neurotrophin and vascular endothelial growth factor (VEGF) signaling in vivo, and a primary determinant for neuronal and cardiovascular development. Kidins220(-/-) embryos die at late stages of gestation, and show extensive cell death in the central and peripheral nervous systems. Primary neurons from Kidins220(-/-) mice exhibit reduced responsiveness to brain-derived neurotrophic factor, in terms of activation of mitogen-activated protein kinase signaling, neurite outgrowth and potentiation of excitatory postsynaptic currents. In addition, mice lacking Kidins220 display striking cardiovascular abnormalities, possibly due to impaired VEGF signaling. In support of this hypothesis, we demonstrate that Kidins220 constitutively interacts with VEGFR2. These findings, together with the data presented in the accompanying paper, indicate that Kidins220 mediates the integration of several growth factor receptor pathways during development, and mediates the activation of distinct downstream cascades according to the location and timing of stimulation.     

Folate and folate-PEG-PAMAM dendrimers: synthesis, characterization, and targeted anticancer drug delivery potential in tumor bearing mice

Ligand-mediated targeting of drugs especially in anticancer drug delivery is an effective approach. Dendrimers, due to unique surface topologies, can be a choice in this context. In the present study, PAMAM (polyamidoamine) dendrimers up to fourth generation were synthesized and characterized through infrared (IR), nuclear magnetic resonance (NMR), electrospray ionization (ESI) mass spectrometric, and transmission electron microscopic (TEM) techniques. Primary amines present on the dendritic surface were conjugated through folic acid and folic acid-PEG (poly(ethylene glycol))-NHS (N-hydroxysuccinimide) conjugates. Tumor in mice was induced through the use of KB cell culture. Prepared dendritic conjugates were evaluated for the anticancer drug delivery potential using 5-FU (5-fluorouracil) in tumor-bearing mice. Approximately 31% of 5-FU was loaded in folate-PEG-dendritic conjugates. Results indicated that folate-PEG-dendrimer conjugate was significantly safe and effective in tumor targeting compared to a non-PEGylated formulation. Tailoring of dendrimers via PEG-folic acid reduced hemolytic toxicity, which led to a sustained drug release pattern as well as highest accumulation in the tumor area.     

Y418C: a novel mutation in exon 9 of the glucocerebrosidase gene of a patient with Gaucher disease creates a new Bgl I site

We report a novel mutation in exon 9 of the glucocerebrosidase gene of a patient with Gaucher disease and of Sardinian origin.     

Auxin signaling and transport promote susceptibility to the root-infecting fungal pathogen Fusarium oxysporum in Arabidopsis

Fusarium oxysporum is a root-infecting fungal pathogen that causes wilt disease on a broad range of plant species, including the model plant Arabidopsis thaliana. Currently, very little is known about the molecular or physiological processes that are activated in the host during infection and the roles these processes play in resistance and susceptibility to F. oxysporum. In this study, we analyzed global gene expression profiles of F. oxysporum-infected Arabidopsis plants. Genes involved in jasmonate biosynthesis as well as jasmonate-dependent defense were coordinately induced by F. oxysporum. Similarly, tryptophan pathway genes, including those involved in both indole-glucosinolate and auxin biosynthesis, were upregulated in both the leaves and the roots of inoculated plants. Analysis of plants expressing the DR5:GUS construct suggested that root auxin homeostasis was altered during F. oxysporum infection. However, Arabidopsis mutants with altered auxin and tryptophan-derived metabolites such as indole-glucosinolates and camalexin did not show an altered resistance to this pathogen. In contrast, several auxin-signaling mutants were more resistant to F. oxysporum. Chemical or genetic alteration of polar auxin transport also conferred increased pathogen resistance. Our results suggest that, similarly to many other pathogenic and nonpathogenic or beneficial soil organisms, F. oxysporum requires components of auxin signaling and transport to colonize the plant more effectively. Potential mechanisms of auxin signaling and transport-mediated F. oxysporum susceptibility are discussed.     

Skin regeneration in adult axolotls: a blueprint for scar-free healing in vertebrates

While considerable progress has been made towards understanding the complex processes and pathways that regulate human wound healing, regenerative medicine has been unable to develop therapies that coax the natural wound environment to heal scar-free. The inability to induce perfect skin regeneration stems partly from our limited understanding of how scar-free healing occurs in a natural setting. Here we have investigated the wound repair process in adult axolotls and demonstrate that they are capable of perfectly repairing full thickness excisional wounds made on the flank. In the context of mammalian wound repair, our findings reveal a substantial reduction in hemostasis, reduced neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM) during scar-free healing. Additionally, we test the hypothesis that metamorphosis leads to scarring and instead show that terrestrial axolotls also heal scar-free, albeit at a slower rate. Analysis of newly forming dermal ECM suggests that low levels of fibronectin and high levels of tenascin-C promote regeneration in lieu of scarring. Lastly, a genetic analysis during wound healing comparing epidermis between aquatic and terrestrial axolotls suggests that matrix metalloproteinases may regulate the fibrotic response. Our findings outline a blueprint to understand the cellular and molecular mechanisms coordinating scar-free healing that will be useful towards elucidating new regenerative therapies targeting fibrosis and wound repair.