Isolation and characterization of kinetoplast DNA and RNA of Phytomonas davidi

Kinetoplast DNA networks were isolated from stationary-phase culture forms of Phytomonas davidi. The networks banded in CsCl at a density of 1.699 g/ml and consisted of covalently closed circular molecules. The networks were sensitive to shear forces and exhibited several discrete sedimenting components in neutral and alkaline sucrose. Closed monomeric minicircles were isolated from sonicated networks by alkaline band sedimentation. Closed monomers showed a heterogeneous banding pattern on electrophoresis in acrylamide-agarose gels and had sedimentation coefficients of 20.5 S in alkaline sucrose and 11 S in neutral sucrose. The mean minicircle molecular weight as measured by cospreading with φXRF II was 0.70 × 10⁶ or 1064 nucleotide pairs. Minicircles exhibited a sequence microheterogeneity as evidenced by restriction enzyme analysis, melting analysis, and renaturation kinetics. Network maxicircles were evidenced by the appearance of high molecular weight fragments after restriction with several enzymes and by the existence of supertwisted “edge loops” extending out from the periphery of networks. The maxicircle molecular weight was estimated to be approximately 24 × 10⁶. A purified kinetoplast-mitochondrion fraction was found to contain 9 and 12 S RNA species that comigrated with L. tarentolae 9 and 12 S kinetoplast RNAs.     

SIDD: A Semantically Integrated Database towards a Global View of Human Disease

Background

A number of databases have been developed to collect disease-related molecular, phenotypic and environmental features (DR-MPEs), such as genes, non-coding RNAs, genetic variations, drugs, phenotypes and environmental factors. However, each of current databases focused on only one or two DR-MPEs. There is an urgent demand to develop an integrated database, which can establish semantic associations among disease-related databases and link them to provide a global view of human disease at the biological level. This database, once developed, will facilitate researchers to query various DR-MPEs through disease, and investigate disease mechanisms from different types of data.

Methodology

To establish an integrated disease-associated database, disease vocabularies used in different databases are mapped to Disease Ontology (DO) through semantic match. 4,284 and 4,186 disease terms from Medical Subject Headings (MeSH) and Online Mendelian Inheritance in Man (OMIM) respectively are mapped to DO. Then, the relationships between DR-MPEs and diseases are extracted and merged from different source databases for reducing the data redundancy.

Conclusions

A semantically integrated disease-associated database (SIDD) is developed, which integrates 18 disease-associated databases, for researchers to browse multiple types of DR-MPEs in a view. A web interface allows easy navigation for querying information through browsing a disease ontology tree or searching a disease term. Furthermore, a network visualization tool using Cytoscape Web plugin has been implemented in SIDD. It enhances the SIDD usage when viewing the relationships between diseases and DR-MPEs. The current version of SIDD (Jul 2013) documents 4,465,131 entries relating to 139,365 DR-MPEs, and to 3,824 human diseases. The database can be freely accessed from: http://mlg.hit.edu.cn/SIDD.     

Dual targeting of organellar seryl-tRNA synthetase to maize mitochondria and chloroplasts

Aminoacyl-tRNA synthetases (AARSs) play a critical role in translation and are thus required in three plant protein-synthesizing compartments: cytosol, mitochondria and plastids. A systematic study had previously shown extensive sharing of organellar AARSs from Arabidopsis thaliana, mostly between mitochondria and chloroplasts. However, distribution of AARSs from monocot species, such as maize, has never been experimentally investigated. Here we demonstrate dual targeting of maize seryl-tRNA synthetase, SerZMo, into both mitochondria and chloroplasts using combination of complementary methods, including in vitro import assay, transient expression analysis of green fluorescent protein (GFP) fusions and immunodetection. We also show that SerZMo dual localization is established by the virtue of an ambiguous targeting peptide. Full-length SerZMo protein fused to GFP is targeted to chloroplast stromules, indicating that SerZMo protein performs its function in plastid stroma. The deletion mutant lacking N-terminal region of the ambiguous SerZMo targeting peptide was neither targeted into mitochondria nor chloroplasts, indicating the importance of this region in both mitochondrial and chloroplastic import.     

Up-regulation of vimentin expression in low-density malignant glioma cells as immediate and late effects under irradiation and temozolomide treatment

Nervous system tumors are one of the leading causes of cancer related death. Specific mechanisms facilitating the invasive behavior of gliomas remain obscure. Advanced simulation models of the in vivo response to therapy conditions should potentially improve malignant glioma treatment. Expressional profiling of vimentin––one of reliable pro-invasive tumor makers––in those simulation models was the goal of this study, in order to estimate a pro-invasive response of surviving malignant glioma cells under clinically relevant therapeutic conditions. Human U87-MG malignant glioma cells were used. These cells are characterized by the wild p53-phenotype, which is relevant for the majority of primary malignant glioblastomas. Experimental design foresaw the cells to undergo either irradiation or chemo-treatment with temozolomide alone, or combined treatment. Expression profiling of vimentin was performed by quantitative “Real-Time”-PCR under all treatment conditions simulating diverse tumor regions. Here we demonstrated that vimentin expression patterns in human malignant glioma cells strongly depend on cellular density, algorithms of drug delivery and chemo/radio treatment. Substantial differences were recognized between immediate and late therapy effects. Significant increase in vimentin expression levels was detected particularly in low-density cell cultures under durable treatment with constant concentration levels of temezolomide. Simulation of variable intratumoral regional conditions (central intratumoral regions vs. disseminated malignant cells in peripheral regions) demonstrated differential response of vimentin expression in malignant glioma cell cultures treated under clinically relevant conditions. Slight ebbing of expression levels as late effects of the treatment in confluent cultures may correspond to necrotic processes clinically observed in central intratumoral regions. Contrary, in disseminated malignant cells of peripheral regions therapy resulted in vimentin-inducing effects. This is in agreement with the clinical observations of an increased aggressiveness and malignancy grade of post-operatively chemo/radio-treated malignant gliomas.     

Change in microbial communities in acetate- and glucose-fed microbial fuel cells in the presence of light

Power densities produced by microbial fuel cells (MFCs) in natural systems are changed by exposure to light through the enrichment of photosynthetic microorganisms. When MFCs with brush anodes were exposed to light (4000 lx), power densities increased by 8–10% for glucose-fed reactors, and 34% for acetate-fed reactors. Denaturing gradient gel electrophoresis (DGGE) profiles based on the 16S rRNA gene showed that exposure to high light levels changed the microbial communities on the anodes. Based on 16S rRNA gene clone libraries of light-exposed systems the anode communities using glucose were also significantly different than those fed acetate. Dominant bacteria that are known exoelectrogens were identified in the anode biofilm, including a purple nonsulfur (PNS) photosynthetic bacterium, Rhodopseudomonas palustris, and a dissimilatory iron-reducing bacterium, Geobacter sulfurreducens. Pure culture tests confirmed that PNS photosynthetic bacteria increased power production when exposed to high light intensities (4000 lx). These results demonstrate that power production and community composition are affected by light conditions as well as electron donors in single-chamber air-cathode MFCs.     

High level soluble expression and one-step purification of IBDV VP2 protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To improve the expression of soluble IBDV VP2 protein by using different tagged vectors in Escherichia coli.

Results

Fusion tags, Grifin, MBP, SUMO, thioredoxin, γ-crystallin, ArsC and PpiB, enhanced the expression and solubility of VP2 protein. The fusion proteins were purified by Ni–NTA chromatography, MBP-VP2 showed the highest purity about 90 %. After removing the MBP tag, VP2 self-assembled into virus-like particles, ~25 nm diam. Results from AGP suggested the recombinant IBDV VP2 protein identified by reference serum like IBDV.

Conclusion

All the seven tags enhanced the expression and solubility of IBDV VP2 protein. The recombinant protein self-assembly into virus like particles and possess antigenicity as reference IBDV.

     

Protective effects of activated vitamin D receptor on radiation‐induced intestinal injury

Radiation‐induced intestinal injury (RIII) is a common complication after radiation therapy in patients with pelvic, abdominal, or retroperitoneal tumours. Recently, in the model of DSS (Dextran Sulfate Sodium Salt) ‐induced intestinal inflammatory injury, it has been found in the study that transgenic mice expressing hVDR in IEC (Intestinal Epithelial Cell) manifest highly anti‐injury properties in colitis, suggesting that activated VDR in the epithelial cells of intestine may inhibit colitis by protecting the mucosal epithelial barrier. In this study, we investigated the effect of the expression and regulation of VDR on the protection of RIII, and the radiosensitivity in vitro experiments, and explored the initial mechanism of VDR in regulating radiosensitivity of IEC. As a result, we found that the expression of VDR in intestinal tissues and cells in mice can be induced by ionizing radiation. VDR agonists are able to prolong the average survival time of mice after radiation and reduce the radiation‐induced intestinal injury. For lack of vitamin D, the radiosensitivity of intestinal epithelial cells in mice increased, which can be reduced by VDR activation. Ensuing VDR activation, the radiation‐induced intestinal stem cells damage is decreased, and the regeneration and differentiation of intestinal stem cells is promoted as well. Finally, on the basis of sequencing analysis, we validated and found that VDR may target the HIF/PDK1 pathway to mitigate RIII. We concluded that agonism or upregulation of VDR expression attenuates radiation‐induced intestinal damage in mice and promotes the repair of epithelial damage in intestinal stem cells.     

Mutations in methylenetetrahydrofolate reductase and in cysthationine beta synthase: is there a link to homocysteine levels in peripheral arterial disease?

Peripheral arterial disease (PAD) is an atherosclerotic disturbance characterized by a progressive obstruction of lower limb arteries. Many risk factors associated with PAD development have being reported in the literature. The present study aimed to investigate whether mutations in the methylenetetrahydrofolate reductase (MTHFR) or in the cystathionine beta synthase (CBS) genes are associated with higher levels of homocysteine and the risk of PAD in patients from Brazil. This study analyzed 39 patients with PAD and 32 without PAD in whom risk factors and C677T mutations in the MTHFR gene and both 844ins68 and T833C mutations in the CBS gene were investigated. Although higher levels of homocysteine could be observed in patients with PAD compared to controls, no association between the increase of homocysteine and the frequency of C677T, 844ins68, and T833C mutations could be observed. The results suggest that these mutations do not appear to be related to either homocysteine levels or the development of the disease. However, hyperhomocysteinemia and smoking are important factors in PAD development.