Glutathione (GSH) conjugates with dopamine (DA)-derived quinones to form reactive or non-reactive GSH-conjugates

In this study we demonstrate for the first time that GSH could rapidly conjugate with dopamine (DA)-derived DA-o-quinones without enzymatic catalysis to form short-lived intermediate GSH-conjugates (2-S-GSH-DA-o-quinone and 5-S-GSH-DA-o-quinone). These intermediate GSH-conjugates are unstable and would finally form reactive or non-reactive GSH-conjugates dependent on ambient reductive forces. Under insufficient reductive forces, the intermediate GSH-conjugates could cyclize spontaneously to form reactive 7-S-GSH-aminochrome (7-S-GSH-AM). The 7-S-GSH-AM is so reactive that it could further react with another GSH to form 4,7-bi-GSH-5,6-dihydroindole. Its reactivity could also abrogate tyrosinase activity in solutions. In addition, the 7-S-GSH-AM could further undergo internal rearrangement to form non-reactive 7-S-GSH-5,6-dihydroindole. From these novel findings, we propose two detrimental positive feedback loops involving accelerated DA oxidation, increased GSH consumption and impaired GSH detoxification efficiency, as the underlying chemical explanation for dopaminergic neuron degeneration in Parkinson's disease

A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

<p>Abstract</p> <p>Background</p> <p>Some plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy.</p> <p>Results</p> <p>Here, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm²of <it>Avicennia </it><it>officinalis </it>leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker^®Red, FM^®4-64, Texas Red^®).</p> <p>Conclusions</p> <p>The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic observations of salt glands could be achieved within a day. Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands. Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.</p

Systematic Identification of Factors for Provirus Silencing in Embryonic Stem Cells

Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs). Here, we systematically dissected the cellular factors involved in provirus repression in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen. Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing. RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs. ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs. Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28. Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome

Ultrastructure of Erythrophores and Xanthophores of the Siamese Fighting Fish, Betta splendens

The ultrastructural morphology of brightly colored pigment cells (chromatophores) of Siamese fighting fish, Betta splendens, was investigated by transmission electron microscopy. The major pigment cells in the epidermis and dermis of the red and golden strains were erythrophores and xanthophores, respectively. Specific combinations of these chromatophores formed the basis of pigmentation patterns in both strains. The ultrastructure of the erythrophores was characterized by ellipsoidal electron-lucent vesicles that had limiting membranes and inner lamellae. The latter appeared whorl-like due to a concentric arrangement of parallel membranes. The xanthophores contained small and large cytoplasmic vesicles that appeared hollow and electron-lucent, with some vesicles displaying slightly electron dense particles. Sections of some large vesicles also revealed a very thin membrane enveloping these droplet-like vesicles

Comparison of human alveolar osteoblasts cultured on polymer-ceramic composite scaffolds and tissue culture plates

The effects of medical grade polycaprolactone–tricalcium phosphate (mPCL–TCP) (80:20) scaffolds on primary human alveolar osteoblasts (AOs) were compared with standard tissue-culture plates. Of the seeded AOs, 70% adhered to and proliferated on the scaffold surface and within open and interconnected pores; they formed multi-layered sheets and collagen fibers with uniform distribution within 28 days. Elevation of alkaline phosphatase activity occurred in scaffold–cell constructs independent of osteogenic induction. AO proliferation rate increased and significant decrease in calcium concentration of the medium for both scaffolds and plates under induction conditions were seen. mPCL–TCP scaffolds significantly influenced the AO expression pattern of osterix and osteocalcin (OCN). Osteogenic induction down-regulated OCN at both RNA and protein level on scaffolds (3D) by day 7, and up-regulated OCN in cell-culture plates (2D) by day 14, but OCN levels on scaffolds were higher than on cell-culture plates. Immunocytochemical signals for type I collagen, osteopontin and osteocalcin were detected at the outer parts of scaffold–cell constructs. More mineral nodules were found in induced than in non-induced constructs. Only induced 2D cultures showed nodule formation. mPCL–TCP scaffolds appear to stimulate osteogenesis in vitro by activating a cellular response in AO's to form mineralized tissue. There is a fundamental difference between culturing AOs on 2D and 3D environments that should be considered when studying osteogenesis in vitro

Shallow multi-well plastic chip for thermal multiplexing

US8080411Granted Paten

p53, a novel inhibitor of apoptosis

p53 is a transcription factor known to induce apoptosis via transactivating the expression of pro-apoptotic proteins and by directly activating the mitochondria apoptotic pathway. p53 is also found to be mutated in 50% of human cancers with some of these tumours overexpressing both wild type (WT) and mutant p53. Overexpression of the p53 protein has been implicated with the more aggressive nature of these tumour cells, suggesting a possible gain-of-function of such mutants. In this study, the involvement of p53 in apoptosis via the caspase pathway was investigated. WT-p53 and three mutant p53 with mutations at the 1) N-terminus (D42Y), 2) central domain (R175H) and 3) C-terminus (R337H) were selected. The data collected demonstrated that p53 was able to inhibit the cleavage of caspases early in the apoptotic pathway. In vitro assays showed that addition of recombinant WT and the mutant p53 inhibited the cleavage of both caspase-9 and caspase-3 and subsequently PARP, while overexpression of p53 in mammalian cells yielded the same inhibition profile in vivo. Conversely, removal of p53 via siRNA and immunodepletetion showed accelerated caspase-9 activity and cleavage. Immunoprecipitation experiments and recombinant assay systems suggest that the inhibition by p53 is targeted at the active cleaved caspase-9. In addition, the presence of p53 (WT or mutant) in p53-null cells were able to confer a higher survival rate. These data therefore demonstrate that p53 may have an additional anti-apoptotic role via the inhibition of caspase-9, which is often masked by its pro-apoptotic functions. This anti-apoptotic role could manifest itself in a cancer background overexpressing mutant p53 which has lost its transcriptional activity and hence its ability to induce apoptosis via the induction of pro-apoptotic genes such as PUMA and NOXA. This newly discovered role of p53 could potentially explain the aggressive nature of cancers which overexpress p53.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Functional Genomics Approach to the Identification of Virulence Genes Involved in Edwardsiella tarda Pathogenesis

Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also of gastro- and extraintestinal infections in humans. Here, we report the identification of 14 virulence genes of pathogenic E. tarda that are essential for disseminated infection, via a genome-wide analysis. We screened 490 alkaline phosphatase fusion mutants from a library of 450,000 TnphoA transconjugants derived from strain PPD130/91, using fish as an infection model. Compared to the wild type, 15 mutants showed significant decreases in virulence. Six mutants had insertions in the known virulence-related genes, namely, fimA, gadB, katB, pstS, pstC, and ssrB. Some mutants corresponded to known genes (astA, isor, and ompS2) that had not been previously shown to be involved in pathogenesis, and three had insertions in two novel genes. In vivo infection kinetics experiments confirmed the inability of these attenuated mutants to proliferate and cause fatal infection in fish. Screening for the presence of the above-described virulence genes in six virulent and seven avirulent strains of E. tarda indicated that seven of the genes were specific to pathogenic E. tarda. The genes identified here may be used to develop vaccines and diagnostic kits as well as for further studying the pathogenesis of E. tarda and other pathogenic bacteria

Shallow multi-well plastic chip for thermal multiplexing

US7442542Granted Paten