Trichostatin A improves histone acetylation in bovine somatic cell nuclear transfer early embryos

Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming following somatic cell nuclear transfer (SCNT), and may underlie the observed reduced viability of cloned embryos. In the present study, we tested the effects of the histone deacetylase inhibitor (HDACi), trichostatin A (TSA), on development and histone acetylation of cloned bovine preimplantation embryos. Our results indicated that treating activated reconstructed SCNT embryos with 50 nM TSA for 13 h produced eight-cell embryos with levels of acetylation of histone H4 at lysine 5 (AcH4K5) similar to fertilized counterparts and significantly greater than in control NT embryos (p < 0.005). Further, TSA treatment resulted in SCNT embryos with preimplantation developmental potential similar to fertilized counterparts, as no difference was observed in cleavage and blastocyst rates or in blastocyst total cell number (p > 0.05). Measurement of eight selected developmentally important genes in single blastocysts showed a similar expression profile among the three treatment groups, with the exception of Nanog, Cdx2, and DNMT3b, whose expression levels were higher in TSA-treated NT than in in vitro fertilized (IVF) embryos. Data presented herein demonstrate that TSA can improve at least one epigenetic mark in early cloned bovine embryos. However, evaluation of development to full-term is necessary to ascertain whether this effect reflects a true increase in developmental potential.     

Ghrelin alleviates biliary obstruction-induced chronic hepatic injury in rats

BACKGROUND: Reactive oxygen species and oxidative stress are implicated in hepatic stellate cell activation and liver fibrosis, which are initiated by recruitment of inflammatory cells and by activation of cytokines. OBJECTIVE: The possible anti-oxidant and anti-inflammatory effects of ghrelin were evaluated in a hepatic fibrosis model in rats with bile duct ligation (BDL). METHODS: Under anesthesia, bile ducts of Sprague Dawley rats were ligated, and half of the rats were subcutaneously administered with ghrelin (10 ng/kg/day) and the rest with saline for 28 days. Sham-operated control groups were administered saline or ghrelin. On the 28th day of the study, rats were decapitated and malondialdehyde (MDA) content--an index of lipid peroxidation, and myeloperoxidase (MPO) activity--an index of neutrophil infiltration--were determined in the liver tissues. Oxidant-induced tissue fibrosis was determined by collagen contents, while the hepatic injury was analyzed microscopically. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels and lactate dehydrogenase (LDH) levels were determined to assess liver function and tissue damage, respectively. Pro-inflammatory cytokines; TNF-alpha, IL-1beta and IL-6 were also assayed in plasma samples. RESULTS: In the saline-treated BDL group, hepatic MDA levels, MPO activity and collagen content were increased (p<0.001), suggesting oxidative organ damage, as confirmed histologically. In the ghrelin-treated BDL group, however, all of the oxidant responses were reversed significantly (p<0.05-p<0.001). Serum AST, ALT, LDH levels, and cytokines were elevated in the BDL group as compared to the control group, while this increase was significantly decreased by ghrelin treatment. CONCLUSION: Owing to the anti-inflammatory and anti-oxidant effect as demonstrated in our study, it is possible to speculate that exogenously administered ghrelin may possess an antifibrotic effect against biliary obstruction-induced liver fibrosis. Thus, it seems likely that ghrelin may be of potential therapeutic value in protecting the liver fibrosis and oxidative injury due to biliary obstruction.     

Interspecies nuclear transfer: implications for embryonic stem cell biology

Accessibility of human oocytes for research poses a serious ethical challenge to society. This fact categorically holds true when pursuing some of the most promising areas of research, such as somatic cell nuclear transfer and embryonic stem cell studies. One approach to overcoming this limitation is to use an oocyte from one species and a somatic cell from another. Recently, several attempts to capture the promises of this approach have met with varying success, ranging from establishing human embryonic stem cells to obtaining live offspring in animals. This review focuses on the challenges and opportunities presented by the formidable task of overcoming biological differences among species.     

Characterization of hemocyanin from the peacock mantis shrimp Odontodactylus scyllarus (Malacostraca:Hoplocarida)

Hemocyanin is the blue respiratory protein of many arthropod species. While its structure, evolution, and physiological function have been studied in detail in Decapoda, there is little information on hemocyanins from other crustacean taxa. Here, we have investigated the hemocyanin of the peacock mantis shrimp Odontodactylus scyllarus, which belongs to the Stomatopoda (Hoplocarida). O. scyllarus hemocyanin forms a dodecamer (2 × 6-mer), which is composed of at least four distinct subunit types. We obtained the full-length cDNA sequences of three hemocyanin subunits, while a fourth cDNA was incomplete at its 5′ end. The complete full-length cDNAs of O. scyllarus hemocyanin translate into polypeptides of 650–662 amino acids, which include signal peptides of 16 or 17 amino acids. The predicted molecular masses of 73.1–75.1 kDa correspond well with the main hemolymph proteins detected by SDS-PAGE and Western blotting using various anti-hemocyanin antibodies. Phylogenetic analyses show that O. scyllarus hemocyanins belong to the β-type of malacostracan hemocyanin subunits, which diverged from the other subunits before the radiation of the malacostracan subclasses around 520 million years ago. Molecular clock analysis revealed an ancient and complex pattern of hemocyanin subunit evolution in Malacostraca and also allowed dating divergence times of malacostracan taxa.     

Metformin does not prevent DNA damage in lymphocytes despite its antioxidant properties against cumene hydroperoxide-induced oxidative stress

Metformin (1-(diaminomethylidene)-3,3-dimethyl-guanidine), which is the most commonly prescribed oral antihyperglycaemic drug in the world, was reported to have several antioxidant properties such as the inhibition of advanced glycation end-products. In addition to its use in the treatment of diabetes, it has been suggested that metformin may be a promising anti-aging agent. The present work was aimed at assessing the possible protective effects of metformin against DNA-damage induction by oxidative stress in vitro. The effects of metformin were compared with those of N-acetylcysteine (NAC). For this purpose, peripheral blood lymphocytes from aged (n = 10) and young (n = 10) individuals were pre-incubated with various concentrations of metformin (10–50 μM), followed by incubation with 15 μM cumene hydroperoxide (CumOOH) for 48 h, under conditions of low oxidant level, which do not induce cell death. Protection against oxidative DNA damage was evaluated by use of the Comet assay and the cytokinesis-block micronucleus technique. Changes in the levels of malondialdehyde + 4-hydroxy-alkenals, an index of oxidative stress, were also measured in lymphocytes. At concentrations ranging from 10 μM to 50 μM, metformin did not protect the lymphocytes from DNA damage, while 50 μM NAC possessed an effective protective effect against CumOOH-induced DNA damage. Furthermore, NAC, but not metformin, inhibited DNA fragmentation induced by CumOOH. In contrast to the lack of protection against oxidative damage in lymphocyte cultures, metformin significantly protected the cells from lipid peroxidation in both age groups, although not as effective as NAC in preventing the peroxidative damage at the highest doses. Within the limitations of this study, the results indicate that pharmacological concentrations of metformin are unable to protect against DNA damage induced by a pro-oxidant stimulus in cultured human lymphocytes, despite its antioxidant properties.     

Cyclic-diGMP signal transduction systems in Vibrio cholerae: modulation of rugosity and biofilm formation

Cyclic di-guanylic acid (c-diGMP) is a second messenger that modulates the cell surface properties of several microorganisms. Concentrations of c-diGMP in the cell are controlled by the opposing activities of diguanylate cyclases and phosphodiesterases, which are carried out by proteins harbouring GGDEF and EAL domains respectively. In this study, we report that the cellular levels of c-diGMP are higher in the Vibrio cholerae rugose variant compared with the smooth variant. Modulation of cellular c-diGMP levels by overexpressing proteins with GGDEF or EAL domains increased or decreased colony rugosity respectively. Several genes encoding proteins with either GGDEF or EAL domains are differentially expressed between the two V. cholerae variants. The generation and characterization of null mutants of these genes (cdgA-E, rocS and mbaA) revealed that rugose colony formation, exopolysaccharide production, motility and biofilm formation are controlled by their action. Furthermore, epistasis analysis suggested that cdgC, rocS and mbaA act in convergent pathways to regulate the phenotypic properties of the rugose and smooth variants, and are part of the VpsR, VpsT and HapR signal transduction pathway.     

Identification and characterization of cyclic diguanylate signaling systems controlling rugosity in Vibrio cholerae

Vibrio cholerae, the causative agent of the disease cholera, can generate rugose variants that have an increased capacity to form biofilms. Rugosity and biofilm formation are critical for the environmental survival and transmission of the pathogen, and these processes are controlled by cyclic diguanylate (c-di-GMP) signaling systems. c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). Proteins that contain GGDEF domains act as DGCs, whereas proteins that contain EAL or HD-GYP domains act as PDEs. In the V. cholerae genome there are 62 genes that are predicted to encode proteins capable of modulating the cellular c-di-GMP concentration. We previously identified two DGCs, VpvC and CdgA, that can control the switch between smooth and rugose. To identify other c-di-GMP signaling proteins involved in rugosity, we generated in-frame deletion mutants of all genes predicted to encode proteins with GGDEF and EAL domains and then searched for mutants with altered rugosity. In this study, we identified two new genes, cdgG and cdgH, involved in rugosity control. We determined that CdgH acts as a DGC and positively regulates rugosity, whereas CdgG does not have DGC activity and negatively regulates rugosity. In addition, epistasis analysis with CdgG, CdgH, and other DGCs and PDEs controlling rugosity revealed that CdgG and CdgH act in parallel with previously identified c-di-GMP signaling proteins to control rugosity in V. cholerae. We also determined that PilZ domain-containing c-di-GMP binding proteins contribute minimally to rugosity, indicating that there are additional c-di-GMP binding proteins controlling rugosity in V. cholerae.     

Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis in Vibrio cholerae

The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.Vibrio cholerae, a rod-shaped, highly motile, gram-negative bacterium, is the causative agent of the life threatening diarrheal disease cholera (59). The type II secretion (T2S) system plays an important role in the pathogenesis of V. cholerae by secreting cholera toxin (63), which is largely responsible for the symptoms of the disease (33). The T2S system is widespread and well conserved in gram-negative bacteria inhabiting a variety of ecological niches and likely contributes to environmental survival as well as to virulence (11, 21). In V. cholerae, secretion via the T2S machinery is supported by a transenvelope complex of 12 Eps proteins (EpsC to EpsN) and the type 4 prepilin peptidase PilD (VcpD) (25, 44, 63). Transport of exoproteins by the T2S system occurs via a two-step process. The first step, which is either Sec or Tat dependent, requires recognition of the N-terminal signal peptide of the exoproteins and translocation through the inner membrane to the periplasm. Then the folded proteins engage the T2S machinery and are subsequently exported across the outer membrane to the extracellular milieu (23, 29).Besides periplasmic accumulation of exoproteins, additional phenotypes of T2S mutants are reported for an increasing number of species, possibly indicating involvement of the T2S system in other important cellular processes. For example, alterations in outer membrane protein composition have been described for T2S mutants of V. cholerae, Aeromonas hydrophila, marine Vibrio sp. strain 60, and Shewanella oneidensis (30, 32, 63, 64). The levels of outer membrane porins OmpU, OmpT, and OmpS are decreased in T2S mutants of V. cholerae (63, 65), and likewise, disruption of T2S genes in A. hydrophila leads to diminished quantities of OmpF and OmpS (30). Similarly, the amounts of the c-type cytochromes MtrC and OmcA in the outer membranes of S. oneidensis T2S mutants are reduced (64). Furthermore, we have shown that inactivation of the T2S system in V. cholerae results in a reduced growth rate, compromised outer membrane integrity, and, as a consequence, induction of RpoE activity. In addition, our studies showed that V. cholerae T2S mutants are unable to survive the passage through the infant mouse gastrointestinal tract (65). Growth defects at low temperatures under laboratory conditions as well as in tap water and amoebae were also observed for T2S mutants of Legionella pneumophila (68).Interestingly, differential abundance of proteins involved in phosphate metabolism and iron uptake has been revealed by proteomic analysis of culture supernatants isolated from wild-type and T2S mutant strains of Pseudoaltermonas tunicata (22). Based on these results, it has been suggested that the T2S system might be involved in iron acquisition. Similarly, certain T2S mutants of Erwinia chrysanthemi exhibit defects indicative of changes in iron homeostasis (17). It has also been noted that the level of aconitate hydratase, an iron-sulfur cluster-containing enzyme, is reduced in L. pneumophila T2S mutants (16).In this study, in an attempt to explain the phenotypes associated with loss of T2S, we performed microarray gene expression profiling of wild-type and T2S-deficient strains. Our data revealed that inactivation of the T2S machinery results in a metabolic feedback loop leading to oxidative stress and changes in iron metabolism. By analyzing another V. cholerae mutant that shares a similar cell envelope phenotype while remaining competent for T2S, we show that the changes in iron homeostasis and oxidative stress are linked to cell envelope damage and extracytoplasmic stress.