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Considering the importance of efficiently obtaining somatic resource banks as an ex-situ conservation strategy for wild mammals, we evaluated two techniques (slow freezing - SF and solid-surface vitrification - SSV) for the cryopreservation of ear cartilage and skin from six-banded armadillos. Additionally, we analyzed the effects of two combinations of intracellular cryoprotectants (3.0 M or 6.0 M ethylene glycol - EG and dimethyl sulfoxide - Me 2 SO) on SSV. Tissues not subjected to cryopreservation were used as controls. All samples were evaluated for morphological analysis and cell ability during culture. The thickness of the basal layer was similar to the control only for tissues derived from SF, while SSV ensured the preservation of the cartilage thickness. Moreover, fragments derived from SF and SSV, especially in the 3.0 M EG-Me 2 SO group, resulted in dermis thickness and total skin similar to the control. All cryopreserved techniques maintained normal patterns of the fibroblasts, epidermal cells, and melanocytes. While only SF-derived fragments maintained the number of degenerated chondrocytes similar to the control, no difference was observed between groups for normal chondrocytes and lacunae. Moreover, SSV maintained the collagen fibers percentage of the tissues even after warming. After culture, SF and SSV techniques were efficient for the recovery of the somatic cells in all parameters evaluated, except the day of subconfluence, which was greater for the SSV group with 6.0 M EG-Me 2 SO. In summary, SSV, especially with 3 M EG-Me 2 SO, was as efficient as SF in preserving ear skin and cartilage derived from six-banded armadillos. • Solid-surface vitrification with 3 M EG-Me 2 SO was as efficient as slow freezing in preserving skin of six-banded armadillos. • Solid-surface vitrification is suggested as the most convenient technique than slow freezing for six-banded armadillos • The efficiency of the vitrification depends on the use of lower concentrations of permeating cryoprotectants at 3 M • Our data provide an important basis for the formation of tissue bank for six-banded armadillos. [ABSTRACT FROM AUTHOR]

Aim: Aim of the present study was to carry out the partial purification and biochemical characterization of glutathione S-transferase (GST) from the somatic tissue of ruminal amphistome parasite, Gastrothylax crumenifer (Gc) infecting Indian water buffalo (Bubalus bubalis). Materials and Methods: The crude somatic homogenate of Gc was subjected to progressive ammonium sulfate precipitation followed by size exclusion chromatography in a Sephacryl S 100-HR column. The partially purified GST was assayed spectrophotometrically, and the corresponding enzyme activity was also recorded in polyacrylamide gel. GST isolated from the amphistome parasite was also exposed to variable changes in temperature and the pH gradient of the assay mixture. Results: The precipitated amphistome GST molecules showed maximum activity in the sixth elution fraction. The GST subunit appeared as a single band in the reducing polyacrylamide gel electrophoresis with an apparent molecular weight of 26 kDa. The GST proteins were found to be fairly stable up to 37°C, beyond this the activity got heavily impaired. Further, the GST obtained showed a pH optima of 7.5. Conclusion: Present findings showed that GST from Gc could be conveniently purified using gel filtration chromatography. The purified enzyme showed maximum stability and activity at 4°C. [ABSTRACT FROM AUTHOR]

Background: As a major epigenetic component, DNA methylation plays important functions in individual development and various diseases. DNA methylation has been well studied in human and model organisms, but only limited data exist in economically important animals like cattle. Results: Using reduced representation bisulphite sequencing (RRBS), we obtained single-base-resolution maps of bovine DNA methylation from ten somatic tissues. In total, we evaluated 1,868,049 cytosines in CG-enriched regions. While we found slightly low methylation levels (29.87 to 38.06 %) in cattle, the methylation contexts (CGs and non-CGs) of cattle showed similar methylation patterns to other species. Non-CG methylation was detected but methylation levels in somatic tissues were significantly lower than in pluripotent cells. To study the potential function of the methylation, we detected 10,794 differentially methylated cytosines (DMCs) and 836 differentially methylated CG islands (DMIs). Further analyses in the same tissues revealed many DMCs (including non-CGs) and DMIs, which were highly correlated with the expression of genes involved in tissue development. Conclusions: In summary, our study provides a baseline dataset and essential information for DNA methylation profiles of cattle. [ABSTRACT FROM AUTHOR]

Ancestral TCDD exposure could induce epigenetic transgenerational phenotypes, which may be mediated in part by imprinted gene inheritance. The aim of our study was to evaluate the transgenerational effects of ancestral TCDD exposure on the imprinted gene insulin-like growth factor-2 (Igf2) in rat somatic tissue. TCDD was administered daily by oral gavage to groups of F0 pregnant SD rats at dose levels of 0 (control), 200 or 800 ng/kg bw during gestation day 8–14. Animal transgenerational model of ancestral exposure to TCDD was carefully built, avoiding sibling inbreeding. Hepatic Igf2 expression of the TCDD male progeny was decreased concomitantly with hepatic damage and increased activities of serum hepatic enzymes both in the F1 and F3 generation. Imprinted Control Region (ICR) of Igf2 manifested a hypermethylated pattern, whereas methylation status in the Differentially Methylated Region 2 (DMR2) showed a hypomethylated manner in the F1 generation. These epigenetic alterations in these two regions maintained similar trends in the F3 generation. Meanwhile, the expressions of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) changed in a non-monotonic manner both in the F1 and F3 generation. This study provides evidence that ancestral TCDD exposure may promote epigenetic transgenerational alterations of imprinted gene Igf2 in adult somatic tissue. [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome system is central to the regulation of cellular proteostasis. Nevertheless, the impact of in vivo proteasome dysfunction on the proteostasis networks and the aging processes remains poorly understood. We found that RNAi-mediated knockdown of 20 S proteasome subunits in Drosophila melanogaster resulted in larval lethality. We therefore studied the molecular effects of proteasome dysfunction in adult flies by developing a model of dose-dependent pharmacological proteasome inhibition. Impaired proteasome function promoted several 'old-age' phenotypes and markedly reduced flies' lifespan. In young somatic tissues and in gonads of all ages, loss of proteasome activity induced higher expression levels and assembly rates of proteasome subunits. Proteasome dysfunction was signaled to the proteostasis network by reactive oxygen species that originated from malfunctioning mitochondria and triggered an Nrf2-dependent upregulation of the proteasome subunits. RNAi-mediated Nrf2 knockdown reduced proteasome activities, flies' resistance to stress, as well as longevity. Conversely, inducible activation of Nrf2 in transgenic flies upregulated basal proteasome expression and activity independently of age and conferred resistance to proteotoxic stress. Interestingly, prolonged Nrf2 overexpression reduced longevity, indicating that excessive activation of the proteostasis pathways can be detrimental. Our in vivo studies add new knowledge on the proteotoxic stress-related regulation of the proteostasis networks in higher metazoans. Proteasome dysfunction triggers the activation of an Nrf2-dependent tissue- and age-specific regulatory circuit aiming to adjust the cellular proteasome activity according to temporal and/or spatial proteolytic demands. Prolonged deregulation of this proteostasis circuit accelerates aging. [ABSTRACT FROM AUTHOR]

Abstract: The developmental timing of all types of cells must be synchronized and spatially coordinated to achieve the organized development of a multicellular organism. Previously, we found RNAi of asb-1, encoding a germline-specific isoform of mitochondrial ATP synthase b subunit, caused 100% penetrant sterility in Caenorhabditis elegans. ATP synthase is one of the five complexes of the mitochondrial respiratory chain, and defects in some of the components of the chain are known to slow the growth and extend the lifespan of worms. We found that development of asb-1 mutant germ line was not arrested at any stage, but did slow to half the rate of wild type, whereas the rate of somatic development was the same in asb-1 mutants as that of wild type, indicating that asb-1 is required to maintain the rate of germline development but has no effect on somatic development. Among ATP synthase subunit genes, RNAi of asg-1, encoding a germline-specific isoform of the g subunit, also caused asb-1-like sterility, indicating that some other germline-specific components are also required to maintain the rate of germline development. Both asb-1 and asg-1 are located on autosomes while they possess counterparts, asb-2 and asg-2, respectively, on X chromosome, which are both required for somatic development. Chromosomal locations of the genes may be the basis of the segregation of germline/somatic functions of each gene, as were demonstrated for other autosomal/X-linked duplicated gene pairs. [Copyright &y& Elsevier]

SummaryTsetse flies Glossina spp. (Diptera: Glossinidae) harbor three different symbiotic microorganisms, one being an intracellular Rickettsia of the genus Wolbachia. This bacterium infects a wide range of arthropods, where it causes a variety of reproductive abnormalities, one of which is termed cytoplasmic incompatibility (CI) that, when expressed, results in embryonic death due to disruptions in fertilization events. We report here that in colonized flies, Wolbachia infections can be detected in 100% of sampled individuals, while infections vary significantly in field populations. Based on Wolbachia Surface Protein (wsp) gene sequence analysis, the infections associated with different fly species are all unique within the A group of the Wolbachia pipientis clade. In addition to being present in germ-line tissues, Wolbachia infections have been found in somatic tissues of several insects. Using a Wolbachia-specific PCR-based assay, the tissue tropism of infections in Glossina morsitans morsitans Westwood, Glossina brevipalpis Newstead and Glossina austeni Newstead were analysed. While infections in G. m. morsitans and G. brevipalpis were limited to reproductive tissues, in G. austeni, Wolbachia could be detected in various somatic tissues. [ABSTRACT FROM AUTHOR]