Modulation of Endogenous Antioxidant Enzymes by Nitric Oxide in Rat C6 Glial Cells

Abstract: To understand the possible mechanism of nitric oxide (NO)-mediated cytotoxicity, we investigated the effect of NO on the endogenous antioxidant enzymes (AOEs) catalase, glutathione peroxidase (GPX), and CuZn- and Mn-superoxide dismutases (SODs) in rat C₆ glial cells under conditions in which these cells expressed oligodendrocyte-like properties as evidenced by the expression of 2′,3′-cyclic-nucleotide 3′-phosphohydrolase. The 24-h treatment with S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, decreased the activities and the protein levels of catalase, GPX, and Mn-SOD in a dose-dependent manner. Alternatively, the activity and the protein level of CuZn-SOD were increased. 2-Phenyl-4,4, 5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a NO scavenger, blocked the effect of SNAP. Moreover, the treatment of C₆ cells with sodium nitroprusside, another NO donor, or with a combination of lipopolysaccharide (LPS) and interferon-γ (IFN-γ), which induce excessive production of NO, also significantly modulated the AOE activities in a manner similar to that seen with SNAP treatment. The compounds/enzymes that inhibit the production of NO (e.g., N-nitro-l -arginine methyl ester hydrochloride, arginase, and PTIO) blocked the effects of LPS and IFN-γ on the activities of AOEs. Treatment with SNAP and a combination of LPS and IFN-γ also modulated the mRNA levels of AOEs, parallel to the changes in their protein levels and activities, except for Mn-SOD where the combination of LPS and IFN-γ markedly stimulated the mRNA expression. In spite of the stimulation of mRNA level, LPS and IFN-γ significantly inhibited the activity of Mn-SOD within the first 24 h of incubation; however, Mn-SOD activity gradually increased with the increase in time of incubation. These results suggest that alterations in the status of AOEs by NO may be the basis of NO-induced cytotoxicity in disease states associated with excessive NO production.     

Overreliance on the hexon gene, leading to misclassification of human adenoviruses

The genome of human adenovirus (HAdV) D30 was sequenced in depth. Sequence assembly and analysis revealed two distinct viral sequences with identical hexon genes, which were the same as the one previously reported for HAdV-D30. However, one of the two viruses was found to be a recombinant of HAdV-D29. Exclusive reliance on serum neutralization can lead to mischaracterization of adenoviruses and miss coinfections. Whole-genome sequencing remains the gold standard for proper classification of HAdVs.     

A case-by-case evolutionary analysis of four imprinted retrogenes

Retroposition is a widespread phenomenon resulting in the generation of new genes that are initially related to a parent gene via very high coding sequence similarity. We examine the evolutionary fate of four retrogenes generated by such an event; mouse Inpp5f_v2, Mcts2, Nap1l5, and U2af1-rs1. These genes are all subject to the epigenetic phenomenon of parental imprinting. We first provide new data on the age of these retrogene insertions. Using codon-based models of sequence evolution, we show these retrogenes have diverse evolutionary trajectories, including divergence from the parent coding sequence under positive selection pressure, purifying selection pressure maintaining parent-retrogene similarity, and neutral evolution. Examination of the expression pattern of retrogenes shows an atypical, broad pattern across multiple tissues. Protein 3D structure modeling reveals that a positively selected residue in U2af1-rs1, not shared by its parent, may influence protein conformation. Our case-by-case analysis of the evolution of four imprinted retrogenes reveals that this interesting class of imprinted genes, while similar in regulation and sequence characteristics, follow very varied evolutionary paths.     

Dehydrocostuslactone and plant growth activity of derived guaianolides

The stereostructures of two new guaianolides, isodehydrocostuslactone and isozaluzanin C, isolated previously from Saussurea lappa, have been confirmed by their correlation with dehydrocostuslactone. Twenty new derivatives have been synthesized from these guaianolides and these have been tested as plant growth regulators. The conjugated lactones which have an exocyclic methylene group at C-4 conjugated with a C-3 ketone, show distinct enhancement in their root-forming potential, as compared with their 3-deoxy derivatives. Of further significance is the fact that these ketones display maximum activity only at lower concentrations. Other compounds show the expected structure-biological activity relationships displayed in general by guaianolides. However, the presence of an epoxide at the C-3, C-4 position does not affect the biological activity, which is indeed the case when the epoxide group occupies the C-4, C-14 position in guaianolides. The major biological parameter studied was rooting in-stem cuttings of Phaseolus aureus.     

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.     

Improving piezoelectric cell printing accuracy and reliability through neutral buoyancy of suspensions

The sedimentation and aggregation of cells within inkjet printing systems has been hypothesized to negatively impact printer performance. The purpose of this study was to investigate this influence through the use of neutral buoyancy. Ficoll PM400 was used to create neutrally buoyant MCF‐7 breast cancer cell suspensions, which were ejected using a piezoelectric drop‐on‐demand inkjet printing system. It was found that using a neutrally buoyant suspension greatly increased the reproducibility of consistent cell counts, and eliminated nozzle clogging. Moreover, the use of Ficoll PM400 was shown to not affect cellular viability. This is the first demonstration of such scale and accuracy achieved using a piezoelectric inkjet printing system for cellular dispensing. Biotechnol. Bioeng. 2012; 109: 2932–2940. © 2012 Wiley Periodicals, Inc.     

A comparative study of oocyte development in cotton stainers (Dysdercus spp., pyrrhocoridae) and the factors that control egg production

The relative importance of three factors-mating, nutrition, and crowding-in regulating the reproductive biology of Dysdercus fasciatus Signoret, D. nigrofasciatus Stål and D. cardinalis Gerst. was studied under standard laboratory conditions. Observations on mated and virgin females of these three species of Dysdercus showed that there were no significant differences in pre-oviposition period and inter-oviposition period. In D. fasciatus and D. nigrofasciatus there was, further, no significant difference in the rate of oocyte development; but D. cardinalis differed from these two species in that mating had a profound effect on the rate of oocyte development and consequently on the first oviposition. However, in all three species of Dysdercus mating had a significant effect on the number of eggs produced in each batch, the egg batches of mated females being significantly larger than those produced by virgin females. Further, in both D. fasciatus and D. nigrofasciatus virgin females lived longer than mated females but in D. nigrofasciatus mating had no significant effect on adult longevity. Mating activity was drastically reduced in starved adult bugs. But where it occurred under these conditions, mating did not have any effect on the development of the oocytes, even in D. cardinalis. Under starvation conditions, growth proceeded up to the stage where yolk deposition would normally occur, then stopped; the oocytes showed no signs of degeneration or oosorption. Crowding of female adults led to the acceleration of oocyte development and the onset of oviposition in all three species. These findings are discussed in relation to the known field biology of the three Dysdercus species.

---

Résumé La biologie de la reproduction de 3 espèces de Dysdercus du Kenya a été étudiée au laboratoire. Des 3 espèces, D. fasciatus Signoret présente les périodes les plus courtes de pré-copulation et de pré-oviposition. Puisque cette espèce est inféodée à un relativement petit nombre de plantes-hôtes, qui ont une courte saison de fructification procurant alors une ample source de nourriture, elle tire un avantage maximum de la brièveté de cette période favorable, grâce à la réduction des phases de pré-copulation et de pré-oviposition.Par contre D. nigrofasciatus Stål est adaptée à un très grand nombre de plantes hôtes et pour elle il n'est pas indispensable d'avoir une très grande capacité de reproduction. Cela s'applique encore mieux à D. cardinalis Gerst. qui a une plus longue période de pré-copulation et de pré-oviposition. Cette espèce préfère les plantes qui sont dispersées sur une grande superficie et cela exige une longue période de vol exploratoire avant de s'alimenter et de se reproduire.Chacune de ces 3 espèces s'accouple de façon presque continue, mais les observations faites au laboratoire montrent que l'accouplement n'a aucun effet notable sur la longévité des adultes, sur la durée des phases de pré-oviposition ou d'inter-oviposition et sur le nombre total d'ufs pondus chez D. fasciatus et D. nigrofasciatus. Chez D. cardinalis, au contraire, l'accouplement a un effet important sur la vitesse de croissance des ovocytes, mais non sur les autres aspects de la reproduction évoqués ci-dessus.Le rassemblement en masse des femelles adultes accélère le développement des ovocytes et déclenche la ponte dans chacune des 3 espèces de Dysdercus.L'alimentation a un effet sur le développement des ovocytes. De jeunes femelles adultes doivent être alimentées pendant au moins 3–4 jours avant de déposer leur première ponte. Après chaque ponte une alimentation plus abondante est encore nécessaire pour assurer le maintien de la production d'ufs. Les femelles de chacune des 3 espèces peuvent survivre avec de l'eau pure mais leurs ovocytes arrêtent leur développement au stade où normalement se dépose le vitellus, mais ils ne sont pas résorbés.

     

Adaptive associations between total body color dimorphism and climatic stress‐related traits in a stenothermal circumtropical Drosophila species

Abstract Low desiccation resistance of Drosophila ananassae reflects its rarity outside the humid tropics. However, the ability of this sensitive species to evolve under seasonally varying subtropical areas is largely unknown. D. ananassae flies are mostly lighter during the rainy season but darker and lighter flies occur in the autumn season in northern India. We tested the hypothesis whether seasonally varying alternative body color phenotypes of D. ananassae vary in their levels of environmental stress tolerances and mating behavior. Thus, we investigated D. ananassae flies collected during rainy and autumn seasons for changes in body melanization and their genetic basis, desiccation‐related traits, cold tolerance and mating propensity. On the basis of genetic crosses, we found total body color dimorphism consistent with a single gene model in both sexes of D. ananassae. A significant increase in the frequency of the dark morph was observed during the drier autumn season, and body color phenotypes showed significant deviations from Hardy‐Weinberg equilibrium, which suggests climatic selection plays a role. Resistance to desiccation as well as cold stress were two‐ to three‐fold higher in the dark body color strain as compared with the light strain. On the basis of no‐choice mating experiments, we observed significantly higher assortative matings between dark morphs under desiccation or cold stress, and between light morphs under hot or higher humidity conditions. To the best of our knowledge, this is the first report on the ecological significance of seasonally varying total body color dimorphism in a tropical species, D. ananassae.