Proteomic analysis of kidneys from selenoprotein M transgenic rats in response to increased bioability of selenium

Background

To characterize changes in global protein expression in kidneys of transgenic rats overexpressing human selenoprotein M (SelM) in response to increased bioabivility of selenium (Sel), total proteins extracted from kidneys of 10-week-old CMV/hSelM Tg and wild-type rats were separated by 2-dimensional gel electrophoresis and measured for changes in expression.

Results

Ten and three proteins showing high antioxidant enzymatic activity were up- and down-regulated, respectively, in SelM-overexpressing CMV/hSelM Tg rats compared to controls based on an arbitrary 2-fold difference. Up-regulated proteins included LAP3, BAIAP2L1, CRP2, CD73 antigen, PDGF D, KIAA143 homolog, PRPPS-AP2, ZFP313, HSP-60, and N-WASP, whereas down-regulated proteins included ALKDH3, rMCP-3, and STC-1. After Sel treatment, five of the up-regulated proteins were significantly increased in expression in wild-type rats, whereas there were no changes in CMV/hSelM Tg rats. Only two of the down-regulated proteins showed reduced expression in wild-type and Tg rats after Sel treatment.

Conclusions

These results show the primary novel biological evidences that new functional protein groups and individual proteins in kidneys of Tg rats relate to Sel biology including the response to Sel treatment and SelM expression.     

IN VITRO ANTIOXIDANT ACTIVITIES OF THE FERMENTED MARINE MICROALGA PAVLOVA LUTHERI (HAPTOPHYTA) WITH THE YEAST HANSENULA POLYMORPHA1

Microalgae are major primary producers of organic matter in aquatic environments through their photosynthetic activities. Fermented microalga (Pavlova lutheri Butcher) preparation (FMP) is the product of yeast fermentation by Hansenula polymorpha. It was tested for the antioxidant activities including lipid peroxidation inhibitory activity, free‐radical‐scavenging activity, inhibition of reactive oxygen species (ROS) on mouse macrophages (RAW264.7 cell), and inhibited myeloperoxidase (MPO) activity in human myeloid cells (HL60). FMP exhibited the highest antioxidant activity on free‐radical scavenging, inhibitory intracellular ROS, and inhibited MPO activity. MTT [3‐(4,5‐dimethyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay showed no cytotoxicity in mouse macrophages (RAW264.7 cell), human myeloid cells (HL60), and human fetal lung fibroblast cell line (MRC‐5). Furthermore, the antioxidative mechanism of FMP was evaluated by protein expression levels of antioxidant enzyme (superoxide dismutase [SOD] and glutathione [GSH]) using Western blot. The results obtained in the present study indicated that FMP is a potential source of natural antioxidant.     

Klebsiella pneumoniae secretes outer membrane vesicles that induce the innate immune response

Outer membrane vesicles (OMVs) derived from pathogenic Gram-negative bacteria are an important vehicle for delivery of effector molecules to host cells, but the production of OMVs from Klebsiella pneumoniae, an opportunistic pathogen of both nosocomial and community-acquired infections, and their role in bacterial pathogenesis have not yet been determined. In the present study, we examined the production of OMVs from K. pneumoniae and determined the induction of the innate immune response against K. pneumoniae OMVs. Klebsiella pneumoniae ATCC 13883 produced and secreted OMVs during in vitro culture. Proteomic analysis revealed that 159 different proteins were associated with K. pneumoniae OMVs. Klebsiella pneumoniae OMVs did not inhibit cell growth or induce cell death. However, these vesicles induced expression of proinflammatory cytokine genes such as interleukin (IL)-1β and IL-8 in epithelial cells. An intratracheal challenge of K. pneumoniae OMVs in neutropenic mice resulted in severe lung pathology similar to K. pneumoniae infection. In conclusion, K. pneumoniae produces OMVs like other pathogenic Gram-negative bacteria and K. pneumoniae OMVs are a molecular complex that induces the innate immune response.     

Development of a Plasmid Display System using GAL4 DNA Binding Domain for the <Emphasis Type="Italic">in Vitro</Emphasis> Screening of Functional Proteins

A plasmid display system using GAL4 DNA binding domain (GAL4 DBD) was constructed to enrich the molecular diversity and in vitro selection of functional proteins. Model proteins used were enhanced green fluorescent protein (EGFP) and glutathione S-transferase (GST). The feasibility of this display system was examined using enrichment experiments of target protein from a model protein mixture and identifying the encoding genes by PCR, in which the model protein mixture includes GAL4 DBD/GST fusion protein, GAL4 DBD/EGFP fusion protein, and xylanase. Target proteins of GAL4 DBD/GST and GAL4 DBD/EGFP from the model protein mixture were efficiently isolated by the plasmid display, respectively. The results show that the display system is sufficiently sensitive to select a target protein from a protein mixture, and that it is possible to discover the functional proteins from large libraries using relatively simple approaches.     

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.     

Immunoevasive property of a polydnaviral product, CpBV-lectin, protects the parasitoid egg from hemocytic encapsulation of Plutella xylostella (Lepidoptera: Yponomeutidae)

Immunosuppression is the main pathological symptom of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae), parasitized by an endoparasitoid wasp, Cotesia plutellae (vestalis, Hymenoptera: Braconidae). C. plutellae bracovirus (CpBV), which is a symbiotic virus of C. plutellae, has been known to be the main parasitic factor in the host-parasitoid interaction. CpBV-lectin, encoded in the viral genome and expressed in P. xylostella during early parasitization stage, was suspected to play a role in immunoevasion of defense response. Here we expressed CpBV-lectin in Sf9 cells using a recombinant baculovirus for subsequent functional assays. The recombinant CpBV-lectin exhibited hemagglutination against vertebrate erythrocytes. Its hemagglutinating activity increased with calcium, but inhibited by adding EDTA, indicating its C-type lectin property. CpBV-lectin showed specific carbohydrate-binding affinity against N-acetyl glucosamine and N-acetyl neuraminic acid. The role of this CpBV-lectin in immunosuppression was analyzed by exposing hemocytes of nonparasitized P. xylostella to rat erythrocytes or FITC-labeled bacteria pretreated with recombinant CpBV-lectin, which resulted in significant reduction in adhesion or phagocytosis, respectively. The immunosuppressive activity of CpBV-lectin was further analyzed under in vitro encapsulation response of hemocytes against parasitoid eggs collected at 1- or 24-h post-parasitization. Hemocytic encapsulation was observed against 1-h eggs but not against 24-h eggs. When the 1-h eggs were pretreated with the recombinant CpBV-lectin, encapsulation response was completely inhibited, where CpBV-lectin bound to the parasitoid eggs, but not to hemocytes. These results suggest that CpBV-lectin interferes with hemocyte recognition by masking hemocyte-binding sites on the parasitoid eggs.     

Fecal microbiota and diets of muskox female adults and calves

In mammals, the gut microbiome is vertically transmitted during maternal lactation at birth. In this study, we investigated the gut microbiome and diets of muskox, a large herbivore inhabiting in the high Arctic. We compared the microbiota composition using bacterial 16S rRNA gene sequencing and diets using stable isotope analysis of muskox feces of six female adults and four calves on Ella Island, East Greenland. Firmicutes were the most abundant bacterial phylum in both the adults and calves, comprising 94.36% and 94.03%, respectively. Significant differences were observed in the relative abundance of the two Firmicutes families. The adults were primarily dominated by Ruminococcaceae (73.90%), and the calves were dominated by both Ruminococcaceae (56.25%) and Lachnospiraceae (24.00%). Stable isotope analysis of the feces in the study area revealed that both adults and calves had similar ranges of ¹³C and ¹⁵N, likely derived from the dominant diet plants. Despite their similar diets, the different gut microbiome compositions in muskox adults and calves indicate that the gut microbiome of the calves may not be fully colonized to the extent of that of the adults.     

First Report of Soil‐Borne Wheat Mosaic Virus (SBWMV)‐Infecting Triticale in Poland

The virus in naturally infected, stunted triticale plants was identified as soil‐borne wheat mosaic virus (SBWMV). The infected plants were collected in the Southern Wielkopolska region (Western Poland). Molecular analysis including RT‐PCR, and sequencing of the complete coding sequence of coat protein gene, was performed. The sequence of the Polish isolate of SBWMV (SBWMV‐Pol1) shared 100, 99 and 98% identities with the corresponding regions of De1 (AF519799), OKL‐1 (X81639) and US‐Nebraska (L07938) isolates of SBWMV, respectively. Phylogenetic analyses showed that the Polish isolate, SBWMV‐Pol1, clustered together with other SBWMV isolates. This is the first report of the occurrence of SBWMV in Poland and the second of its presence in Europe.