Rice OsRAD21-2 is Expressed in Actively Dividing Tissues and its Ectopic Expression in Yeast Results in Aberrant Cell Division and Growth

Rad21 and its meiotic counterpart Rec8,the key components of the cohesin complex,are essential for sister chromatid cohesion and chromosome segregation in mitosis and meiosis,respectively.In contrast to yeast and vertebrates,which have only two RAD21/REC8 genes,the rice genome encodes four Rad21/Rec8 proteins.Here,we report on the cloning and characterization of OsRAD21-2 from rice(Oryza sativa L.).Phylogenetic analysis of the full-length amino acids showed that OsRad21-2 was grouped into the plant-specific...     

Directed neural differentiation of duck embryonic germ cells

Although the avian primordial germ cells (PGCs) have been used to produce transgenic birds, their characteristics largely remain unknown. The isolation, culture, biological characterization, and directed neural differentiation of duck EG cells were assayed in this study. The Results showed that the EG cells were got by isolating embryonic gonad and surrounding tissue from 7-day-old duck embryo. The PGCs co-cultured with their gonadal somatic cells were well grown. After passaging, the EG cells were incubated in medium with cytokines and Mitomycin C on inactivated duck embryonic fibroblasts (DEFs) feeder layers. After several passages, alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) resulted positive, cellular markers detection positive for SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. Karyotype analysis showed the EG cells kept diploid condition and the hereditary feature was stable in accordance with varietal characteristics of duck. These cells grew continuously for 11 passages on DEFs. Under induction of medium with BME, RA, and IBMX, the EG cells lost undifferentiated state, large amount of neural cells appeared with the formation of neural cells networks. Special Nissl body was found by toluidine blue stain after induced for 7 days. Immunofluorescence staining results indicated that differentiated EG cells expressed Nestin, NSE, and GFAP positive. The expression of Nestin, NSE, and GFAP mRNA were positive by RT-PCR. The results revealed that RA can obviously promote the directed differentiation of duck EG cells into neural lineage. The duck EG cells will be useful for the production of transgenic birds, for cell replacement therapy and for studies of germ cell differentiation.     

Poly(ADP-ribose) polymerase 1 is involved in glucose toxicity through SIRT1 modulation in HepG2 hepatocytes

Accelerated glucose metabolism leads to oxidative stress and DNA damage in cells; these effects are related to glucose toxicity. The precise mechanisms of glucose toxicity are still unclear. The aim of this work was to investigate the mechanism of poly(ADP‐ribose) polymerase 1 (PARP1), which is a DNA repair enzyme activated by high‐glucose‐induced oxidative stress, and its effect on glucose toxicity in HepG2 hepatocytes. HepG2 cells were cultured under normal (5.5 mM) or high (30 mM) glucose conditions for 4 days. PJ34, which is an inhibitor of PARP1, was used to determine the downstream effects of PARP1 activation. PARP1 activity in 30 mM‐glucose‐treated cells was more than that in 5.5 mM‐glucose‐treated cells, and the activity correlated with the increase in ROS generation and DNA damage. PJ34 suppressed PARP1 activation and prevented the high‐glucose‐induced suppression of SIRT1 and AMP‐activated protein kinase (AMPK) activity, which was similar to its effect on the restoration of intracellular nicotinamide adenine dinucleotide (NAD) content. Further, the phosphorylation of insulin receptor was attenuated in response to insulin stimulation under high glucose conditions, and PJ34 could reverse this effect. The results of transfection of HepG2 cells with PARP1 small interfering RNA were similar to those obtained by treatment of the cells with PARP1 inhibitor PJ34. These data suggest that high‐glucose‐induced PARP1 activation might play a role in glucose toxicity by down‐regulating SIRT1 and AMPK activity through NAD depletion and resulting in insulin insensitivity. J. Cell. Biochem. 112: 299–306, 2011. © 2010 Wiley‐Liss, Inc.     

Molecular fossils illuminate the evolution of retroviruses following a macroevolutionary transition from land to water

The ancestor of cetaceans underwent a macroevolutionary transition from land to water early in the Eocene Period >50 million years ago. However, little is known about how diverse retroviruses evolved during this shift from terrestrial to aquatic environments. Did retroviruses transition into water accompanying their hosts? Did retroviruses infect cetaceans through cross-species transmission after cetaceans invaded the aquatic environments? Endogenous retroviruses (ERVs) provide important molecular fossils for tracing the evolution of retroviruses during this macroevolutionary transition. Here, we use a phylogenomic approach to study the origin and evolution of ERVs in cetaceans. We identify a total of 8,724 ERVs within the genomes of 25 cetaceans, and phylogenetic analyses suggest these ERVs cluster into 315 independent lineages, each of which represents one or more independent endogenization events. We find that cetacean ERVs originated through two possible routes. 298 ERV lineages may derive from retrovirus endogenization that occurred before or during the transition from land to water of cetaceans, and most of these cetacean ERVs were reaching evolutionary dead-ends. 17 ERV lineages are likely to arise from independent retrovirus endogenization events that occurred after the split of mysticetes and odontocetes, indicating that diverse retroviruses infected cetaceans through cross-species transmission from non-cetacean mammals after the transition to aquatic life of cetaceans. Both integration time and synteny analyses support the recent or ongoing activity of multiple retroviral lineages in cetaceans, some of which proliferated into hundreds of copies within the host genomes. Although ERVs only recorded a proportion of past retroviral infections, our findings illuminate the complex evolution of retroviruses during one of the most marked macroevolutionary transitions in vertebrate history.     

Comparative effects of NaCl and NaHCO3 stresses on respiratory metabolism,antioxidant system,nutritional status,and organic acid metabolism in tomato roots

Physiological responses of tomato roots to NaCl and NaHCO₃ stresses were investigated in a hydroponic setting. The relative growth rate of tomato plants was significantly reduced in both NaCl and NaHCO₃ treatments, especially under NaHCO₃ stress. Tomato root respiration increased under low concentrations of NaCl and NaHCO₃ stresses. However, high concentrations of both NaCl and NaHCO₃ significantly inhibited respiration, especially in the NaHCO₃ treatment. With increasing concentration of NaCl and NaHCO₃ treatment, root Na accumulation increased, while accumulation of N, P, K, Fe, and Mg was significantly lower. Compared to NaCl, NaHCO₃ treatment resulted in more dramatic changes in these nutrients. All organic acids investigated were increased by NaHCO₃ after 5 days of treatment, but only oxalate, tartrate and malate were induced by NaCl. This implies that global regulation of organic acids might play an important role in tomato’s alkali stress tolerance. Compared to NaCl treatments, NaHCO₃ treatments induced much higher levels of reactive oxygen species (ROS) and lipid peroxidation after 5 days of treatment, which was accompanied by higher activities of antioxidant enzymes and higher concentrations of ascorbate–glutathione. However, after 10 days of treatment, 100 mM NaHCO₃ stress led to lower accumulation of ROS, antioxidant enzyme activities, and ascorbate–glutathione content. This may have been because root metabolism had almost completely stopped, as indicated by lower root respiration and activity.     

Biochemical and kinetic characterization of the multifunctional β-glucosidase/β-xylosidase/α-arabinosidase,Bgxa1

Functional screening of a metagenomic library constructed with DNA extracted from the rumen contents of a grass/hay-fed dairy cow identified a protein, β-glucosidase/β-xylosidase/α-arabinosidase gene (Bgxa1), with high levels of β-glucosidase activity. Purified Bgxa1 was highly active against p-nitrophenyl-β-d-glucopyranoside (pNPG), cellobiose, p-nitrophenyl-β-d-xylopyranoside (pNPX) and p-nitrophenyl-α-d-arabinofuranoside (pNPAf), suggesting it is a multifunctional β-glucosidase/β-xylosidase/α-arabinosidase. Kinetic analysis of the protein indicated that Bgxa1 has the greatest catalytic activity against pNPG followed by pNPAf and pNPX, respectively. The catalytic efficiency of β-glucosidase activity was 100× greater than β-xylosidase or α-arabinosidase. The pH and temperature optima for the hydrolysis of selected substrates also differed considerably with optima of pH 6.0/45 °C and pH 8.5/40 °C for pNPG and pNPX, respectively. The pH dependence of pNPAf hydrolysis displayed a bimodal distribution with maxima at both pH 6.5 and pH 8.5. The enzyme exhibited substrate-dependent responses to changes in ionic strength. Bgxa1 was highly stable over a broad pH range retaining at least 70 % of its relative catalytic activity from pH 5.0–10.0 with pNPG as a substrate. Homology modelling was employed to probe the structural basis of the unique specificity of Bgxa1 and revealed the deletion of the PA14 domain and insertions in loops adjacent to the active site. This domain has been found to be an important determinant in the substrate specificity of proteins related to Bgxa1. It is postulated that these indels are, in part, responsible for the multifunctional activity of Bgxa1. Bgxa1 acted synergistically with endoxylanase (Xyn10N18) when incubated with birchwood xylan, increasing the release of reducing sugars by 168 % as compared to Xyn10N18 alone. Examination of Bgxa1 and Xyn10N18 synergy with a cellulase for the saccharification of alkali-treated straw revealed that synergism among the three enzymes enhanced sugar release by 180 % as compared to cellulase alone. Our results suggest that Bgxa1 has a number of properties that make it an interesting candidate for the saccharification of lignocellulosic material.