Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes

The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.     

Production of a chimeric enzyme tool associating the Trichoderma reesei swollenin with the Aspergillus niger feruloyl esterase A for release of ferulic acid

The main goals of this work were to produce the fusion protein of the Trichoderma reesei swollenin I (SWOI) and Aspergillus niger feruloyl esterase A (FAEA) and to study the effect of the physical association of the fusion partners on the efficiency of the enzyme. The fusion protein was produced up to 25 mg l⁻¹ in the T. reesei strains Rut-C30 and CL847. In parallel, FAEA alone was produced for use as a control protein in application tests. Recombinant FAEA and SWOI–FAEA were purified to homogeneity and characterized. The biochemical and kinetic characteristics of the two recombinant proteins were found to be similar to those of native FAEA, except for the temperature stability and specific activity of the SWOI–FAEA. Finally, the SWOI–FAEA protein was tested for release of ferulic acid from wheat bran. A period of 24 h of enzymatic hydrolysis with the SWOI–FAEA improved the efficiency of ferulic acid release by 50% compared with the results obtained using the free FAEA and SWOI. Ferulic acid is used as an antioxidant and flavor precursor in the food and pharmaceutical industries. This is the first report of a potential application of the SWOI protein fused with an enzyme of industrial interest.     

Functional Characterization of the Role of the N-terminal Domain of the c/Nip1 Subunit of Eukaryotic Initiation Factor 3 (eIF3) in AUG Recognition

In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5'-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1-45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60-137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site.     

Multidomain Human Peroxidasin 1 Is a Highly Glycosylated and Stable Homotrimeric High Spin Ferric Peroxidase

Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Here, hsPxd01 and various truncated variants lacking nonenzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure, and unfolding pathway are reported. The homotrimeric iron protein contains a covalently bound ferric high spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of −233 ± 5 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (k_cat/K_M^app) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking.     

Is diversification history of maize influencing selection of soil bacteria by roots?

A wide range of plant lines has been propagated by farmers during crop selection and dissemination, but consequences of this crop diversification on plant-microbe interactions have been neglected. Our hypothesis was that crop evolutionary history shaped the way the resulting lines interact with soil bacteria in their rhizospheres. Here, the significance of maize diversification as a factor influencing selection of soil bacteria by seedling roots was assessed by comparing rhizobacterial community composition of inbred lines representing the five main genetic groups of maize, cultivated in a same European soil. Rhizobacterial community composition of 21-day-old seedlings was analysed using a 16S rRNA taxonomic microarray targeting 19 bacterial phyla. Rhizobacterial community composition of inbred lines depended on the maize genetic group. Differences were largely due to the prevalence of certain Betaproteobacteria and especially Burkholderia, as confirmed by quantitative PCR and cloning/sequencing. However, these differences in bacterial root colonization did not correlate with plant microsatellite genetic distances between maize genetic groups or individual lines. Therefore, the genetic structure of maize that arose during crop diversification (resulting in five main groups), but not the extent of maize diversification itself (as determined by maize genetic distances), was a significant factor shaping rhizobacterial community composition of seedlings.     

Construction, database integration, and application of an Oenothera EST library

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.     

Genetics of host response to Leishmania tropica in mice - different control of skin pathology, chemokine reaction, and invasion into spleen and liver

Background

Leishmaniasis is a disease caused by protozoan parasites of genus Leishmania. The frequent involvement of Leishmania tropica in human leishmaniasis has been recognized only recently. Similarly as L. major, L. tropica causes cutaneous leishmaniasis in humans, but can also visceralize and cause systemic illness. The relationship between the host genotype and disease manifestations is poorly understood because there were no suitable animal models.

Methods

We studied susceptibility to L. tropica, using BALB/c-c-STS/A (CcS/Dem) recombinant congenic (RC) strains, which differ greatly in susceptibility to L. major. Mice were infected with L. tropica and skin lesions, cytokine and chemokine levels in serum, and parasite numbers in organs were measured.

Principal Findings

Females of BALB/c and several RC strains developed skin lesions. In some strains parasites visceralized and were detected in spleen and liver. Importantly, the strain distribution pattern of symptoms caused by L. tropica was different from that observed after L. major infection. Moreover, sex differently influenced infection with L. tropica and L. major. L. major-infected males exhibited either higher or similar skin pathology as females, whereas L. tropica-infected females were more susceptible than males. The majority of L. tropica-infected strains exhibited increased levels of chemokines CCL2, CCL3 and CCL5. CcS-16 females, which developed the largest lesions, exhibited a unique systemic chemokine reaction, characterized by additional transient early peaks of CCL3 and CCL5, which were not present in CcS-16 males nor in any other strain.

Conclusion

Comparison of L. tropica and L. major infections indicates that the strain patterns of response are species-specific, with different sex effects and largely different host susceptibility genes.     

Preparation and pharmacological profile of 7-(alpha-azolylbenzyl)-1H-indoles and indolines as new aromatase inhibitors

Aromatase (P450 arom) is a target of pharmacological interest for the treatment of breast cancer. New series of 7-(alpha-azolylbenzyl)-1H-indoles and indolines were synthesized as non-steroidal inhibitors of P450 arom. Selectivity was studied towards P450 17alpha enzyme. The most active compound, 1-ethyl-7-[(imidazol-1-yl)(4-chlorophenyl)methyl]-1H-indole 12c exhibited promising relative potency (rp) of 336 (rp of aminoglutethimide=1) and most of the described azoles were active and selective towards P450 arom.     

High Fat Diet Accelerates Pathogenesis of Murine Crohn’s Disease-Like Ileitis Independently of Obesity

Background

Obesity has been associated with a more severe disease course in inflammatory bowel disease (IBD) and epidemiological data identified dietary fats but not obesity as risk factors for the development of IBD. Crohn’s disease is one of the two major IBD phenotypes and mostly affects the terminal ileum. Despite recent observations that high fat diets (HFD) impair intestinal barrier functions and drive pathobiont selection relevant for chronic inflammation in the colon, mechanisms of high fat diets in the pathogenesis of Crohn’s disease are not known. The aim of this study was to characterize the effect of HFD on the development of chronic ileal inflammation in a murine model of Crohn’s disease-like ileitis.

Methods

TNF^ΔARE/WT mice and wildtype C57BL/6 littermates were fed a HFD compared to control diet for different durations. Intestinal pathology and metabolic parameters (glucose tolerance, mesenteric tissue characteristics) were assessed. Intestinal barrier integrity was characterized at different levels including polyethylene glycol (PEG) translocation, endotoxin in portal vein plasma and cellular markers of barrier function. Inflammatory activation of epithelial cells as well as immune cell infiltration into ileal tissue were determined and related to luminal factors.

Results

HFD aggravated ileal inflammation but did not induce significant overweight or typical metabolic disorders in TNF^ΔARE/WT. Expression of the tight junction protein Occludin was markedly reduced in the ileal epithelium of HFD mice independently of inflammation, and translocation of endotoxin was increased. Epithelial cells showed enhanced expression of inflammation-related activation markers, along with enhanced luminal factors-driven recruitment of dendritic cells and Th17-biased lymphocyte infiltration into the lamina propria.

Conclusions

HFD feeding, independently of obesity, accelerated disease onset of small intestinal inflammation in Crohn’s disease-relevant mouse model through mechanisms that involve increased intestinal permeability and altered luminal factors, leading to enhanced dendritic cell recruitment and promoted Th17 immune responses.