Cloning, characterization and localization of three novel class III peroxidases in lignifying xylem of Norway spruce (Picea abies)

Plant class III peroxidases (POXs) take part in the formation of lignin and maturation of plant cell walls. However, only a few examples of such peroxidases from gymnosperm tree species with highly lignified xylem tracheids have been implicated so far. We report here cDNA cloning of three xylem-expressed class III peroxidase encoding genes from Norway spruce (Picea abies). The translated proteins, PX1, PX2 and PX3, contain the conserved amino acids required for heme-binding and peroxidase catalysis. They all begin with putative secretion signal propeptide sequences but diverge substantially at phylogenetic level, grouping to two subclusters when aligned with other class III plant peroxidases. In situ hybridization analysis on expression of the three POXs in Norway spruce seedlings showed that mRNA coding for PX1 and PX2 accumulated in the cytoplasm of young, developing tracheids within the current growth ring where lignification is occurring. Function of the putative N-terminal secretion signal peptides for PX1, PX2 and PX3 was confirmed by constructing chimeric fusions with EGFP (enhanced green fluorescent protein) and expressing them in tobacco protoplasts. Full-length coding region of px1 was also heterologously expressed in Catharanthus roseus hairy root cultures. Thus, at least the spruce PX1 peroxidase is processed via the endoplasmic reticulum (ER) most likely for secretion to the cell wall. Thereby, PX1 displays correct spatiotemporal localization for participation in the maturation of the spruce tracheid secondary cell wall.     

Responses of PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) in MCF-7 cells are culture condition dependent

To compare the effects of the food toxin 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and estradiol in hormone-responsive MCF-7 cells, the cells were exposed to different concentrations of either PhIP or estradiol. The effect of various culture conditions (e.g. phenol red, FBS, vehicle (DMSO/EtOH) and seeding density) on responses was studied. Cells were continuously grown with steroid-containing or -deprived medium, or switched from steroid-containing to -deprived medium for the experiments to minimize the effect of background estrogenicity. Effects of PhIP and estradiol on cell viability and proliferation were determined by ATP analysis and Ki-67 immunocytochemistry. Expression of estrogen receptor alpha, cell stress markers (p53 and ERK) and estrogen responsive proteins (c-myc and ERK) were immunoblotted. All concentrations of estradiol induced cell proliferation, viability and changes in protein expression, typical for estrogenic responses. PhIP, however, increased viability only at low concentrations and depending on culture conditions. No changes in protein expressions by PhIP were noted, not even when switching cells from steroid-containing to -deprived medium which down-regulated the expression of proteins at basal level. Vehicle affected significantly viability, especially after exposure to PhIP, but not protein expression while medium changes affected both. In conclusion, the effects of PhIP and estradiol in MCF-7 cells are dependent on culture conditions. The detected PhIP-induced changes are weaker compared to those induced by estradiol.     

Oxalate decarboxylase: biotechnological update and prevalence of the enzyme in filamentous fungi

Oxalate decarboxylase (ODC) is a manganese-containing, multimeric enzyme of the cupin protein superfamily. ODC is one of the three enzymes identified to decompose oxalic acid and oxalate, and within ODC catalysis, oxalate is split into formate and CO₂. This primarily intracellular enzyme is found in fungi and bacteria, and currently the best characterized enzyme is the Bacillus subtilis OxdC. Although the physiological role of ODC is yet unidentified, the feasibility of this enzyme in diverse biotechnological applications has been recognized for a long time. ODC could be exploited, e.g., in diagnostics, therapeutics, process industry, and agriculture. So far, the sources of ODC enzyme have been limited including only a few fungal and bacterial species. Thus, there is potential for identification and cloning of new ODC variants with diverse biochemical properties allowing e.g. more enzyme fitness to process applications. This review gives an insight to current knowledge on the biochemical characteristics of ODC, and the relevance of oxalate-converting enzymes in biotechnological applications. Particular emphasis is given to fungal enzymes and the inter-connection of ODC to fungal metabolism of oxalic acid.     

In vitro culture of Lippia dulcis (Trev.): light intensity and wavelength effects on growth,antioxidant defense,and volatile compound production

In Vitro Cellular & Developmental Biology - Plant - Plants of the Verbenaceae family are well known for having constituents with important bioactive properties. The objective of this study was...     

Identification of MAMDC1 as a Candidate Susceptibility Gene for Systemic Lupus Erythematosus (SLE)

Background

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with multiple susceptibility genes. We have previously reported suggestive linkage to the chromosomal region 14q21-q23 in Finnish SLE families.

Principal Findings

Genetic fine mapping of this region in the same family material, together with a large collection of parent affected trios from UK and two independent case-control cohorts from Finland and Sweden, indicated that a novel uncharacterized gene, MAMDC1 (MAM domain containing glycosylphosphatidylinositol anchor 2, also known as MDGA2, MIM 611128), represents a putative susceptibility gene for SLE. In a combined analysis of the whole dataset, significant evidence of association was detected for the MAMDC1 intronic single nucleotide polymorphisms (SNP) rs961616 (P –value = 0.001, Odds Ratio (OR) = 1.292, 95% CI 1.103–1.513) and rs2297926 (P –value = 0.003, OR = 1.349, 95% CI 1.109–1.640). By Northern blot, real-time PCR (qRT-PCR) and immunohistochemical (IHC) analyses, we show that MAMDC1 is expressed in several tissues and cell types, and that the corresponding mRNA is up-regulated by the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) in THP-1 monocytes. Based on its homology to known proteins with similar structure, MAMDC1 appears to be a novel member of the adhesion molecules of the immunoglobulin superfamily (IgCAM), which is involved in cell adhesion, migration, and recruitment to inflammatory sites. Remarkably, some IgCAMs have been shown to interact with ITGAM, the product of another SLE susceptibility gene recently discovered in two independent genome wide association (GWA) scans.

Significance

Further studies focused on MAMDC1 and other molecules involved in these pathways might thus provide new insight into the pathogenesis of SLE.     

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study

Background and Aims

Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD.

Methods

We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0–4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals.

Results

We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III.

Conclusions

This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.     

Magnetic field protects plants against high light by slowing down production of singlet oxygen

Recombination of the primary radical pair of photosystem II (PSII) of photosynthesis may produce the triplet state of the primary donor of PSII. Triplet formation is potentially harmful because chlorophyll triplets can react with molecular oxygen to produce the reactive singlet oxygen (1O?). The yield of 1O? is expected to be directly proportional to the triplet yield and the triplet yield of charge recombination can be lowered with a magnetic field of 100-300 mT. In this study, we illuminated intact pumpkin leaves with strong light in the presence and absence of a magnetic field and found that the magnetic field protects against photoinhibition of PSII. The result suggests that radical pair recombination is responsible for significant part of 1O? production in the chloroplast. The magnetic field effect vanished if leaves were illuminated in the presence of lincomycin, an inhibitor of chloroplast protein synthesis, or if isolated thylakoid membranes were exposed to light. These data, in turn, indicate that 1O? produced by the recombination of the primary charge pair is not directly involved in photoinactivation of PSII but instead damages PSII by inhibiting the repair of photoinhibited PSII. We also found that an Arabidopsis thaliana mutant lacking α-tocopherol, a scavenger of 1O?, is more sensitive to photoinhibition than the wild-type in the absence but not in the presence of lincomycin, confirming that the target of 1O? is the repair mechanism.     

Collagen XVIII,a basement membrane heparan sulfate proteoglycan,interacts with L-selectin and monocyte chemoattractant protein-1

Leukocyte infiltration during inflammation is mediated by the sequential actions of adhesion molecules and chemokines. By using a rat ureteral obstruction model, we showed previously that L-selectin plays an important role in leukocyte infiltration into the kidney. Here we report the purification, identification, and characterization of an L-selectin-binding heparan sulfate proteoglycan (HSPG) expressed in the rat kidney. Partial amino acid sequencing and Western blotting analyses showed that the L-selectin-binding HSPG is collagen XVIII, a basement membrane HSPG. The binding of L-selectin to isolated collagen XVIII was specifically inhibited by an anti-L-selectin monoclonal antibody, EDTA, treatment of the collagen XVIII with heparitinase or heparin but not by chemically desulfated heparin. A cell binding assay showed that the L-selectin-collagen XVIII interaction mediates cell adhesion. Interestingly, collagen XVIII also interacted with a chemokine, monocyte chemoattractant protein-1, and presented it to a monocytic cell line, THP-1, which enhanced the alpha(4)beta(1) integrin-mediated binding of the THP-1 cells to vascular cell adhesion molecule-1. Thus, collagen XVIII may provide a link between selectin-mediated cell adhesion and chemokine-induced cellular activation and accelerate the progression of leukocyte infiltration in renal inflammation.