Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

Thiazolidinediones upregulate impaired fatty acid uptake in skeletal muscle of type 2 diabetic subjects

We examined the regulation of free fatty acid (FFA, palmitate) uptake into skeletal muscle cells of nondiabetic and type 2 diabetic subjects. Palmitate uptake included a protein-mediated component that was inhibited by phloretin. The protein-mediated component of uptake in muscle cells from type 2 diabetic subjects (78 +/- 13 nmol. mg protein-1. min-1) was reduced compared with that in nondiabetic muscle (150 +/- 17, P < 0.01). Acute insulin exposure caused a modest (16 +/- 5%, P < 0.025) but significant increase in protein-mediated uptake in nondiabetic muscle. There was no significant insulin effect in diabetic muscle (+19 +/- 19%, P = not significant). Chronic (4 day) treatment with a series of thiazolidinediones, troglitazone (Tgz), rosiglitazone (Rgz), and pioglitazone (Pio) increased FFA uptake. Only the phloretin-inhibitable component was increased by treatment, which normalized this activity in diabetic muscle cells. Under the same conditions, FFA oxidation was also increased by thiazolidinedione treatment. Increases in FFA uptake and oxidation were associated with upregulation of fatty acid translocase (FAT/CD36) expression. FAT/CD36 protein was increased by Tgz (90 +/- 22% over control), Rgz (146 +/- 42%), and Pio (111 +/- 37%, P < 0.05 for all 3) treatment. Tgz treatment had no effect on fatty acid transporter protein-1 and membrane-associated plasmalemmal fatty acid-binding protein mRNA expression. We conclude that FFA uptake into cultured muscle cells is, in part, protein mediated and acutely insulin responsive. The basal activity of FFA uptake is impaired in type 2 diabetes. In addition, chronic thiazolidinedione treatment increased FFA uptake and oxidation into cultured human skeletal muscle cells in concert with upregulation of FAT/CD36 expression. Increased FFA uptake and oxidation may contribute to lower circulating FFA levels and reduced insulin resistance in skeletal muscle of individuals with type 2 diabetes following thiazolidinedione treatment.     

Development of Sustained Release Capsules Containing “Coated Matrix Granules of Metoprolol Tartrate”

The objective of this investigation was to prepare sustained release capsule containing coated matrix granules of metoprolol tartrate and to study its in vitro release and in vivo absorption. The design of dosage form was performed by choosing hydrophilic hydroxypropyl methyl cellulose (HPMC K100M) and hydrophobic ethyl cellulose (EC) polymers as matrix builders and Eudragit® RL/RS as coating polymers. Granules were prepared by composing drug with HPMC K100M, EC, dicalcium phosphate by wet granulation method with subsequent coating. Optimized formulation of metoprolol tartrate was formed by using 30% HPMC K100M, 20% EC, and ratio of Eudragit® RS/RL as 97.5:2.5 at 25% coating level. Capsules were filled with free flowing optimized granules of uniform drug content. This extended the release period upto 12 h in vitro study. Similarity factor and mean dissolution time were also reported to compare various dissolution profiles. The network formed by HPMC and EC had been coupled satisfactorily with the controlled resistance offered by Eudragit® RS. The release mechanism of capsules followed Korsemeyer–Peppas model that indicated significant contribution of erosion effect of hydrophilic polymer. Biopharmaceutical study of this optimized dosage form in rabbit model showed 10 h prolonged drug release in vivo. A close correlation (R² = 0.9434) was established between the in vitro release and the in vivo absorption of drug. The results suggested that wet granulation with subsequent coating by fluidized bed technique, is a suitable method to formulate sustained release capsules of metoprolol tartrate and it can perform therapeutically better than conventional immediate release dosage form.Key words: biopharmaceutical evaluation, coated granules, metoprolol tartrate, sustained release     

An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes

Background  

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN) viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype.     

A role for the mouse 12/15-lipoxygenase pathway in promoting epithelial wound healing and host defense

The surface of the eye actively suppresses inflammation while maintaining a remarkable capacity for epithelial wound repair. Our understanding of mechanisms that balance inflammatory/reparative responses to provide effective host defense while preserving tissue function is limited, in particular, in the cornea. Lipoxin A(4) (LXA(4)) and docosahexaenoic acid-derived neuroprotectin D1 (NPD1) are lipid autacoids formed by 12/15-lipoxygenase (LOX) pathways that exhibit anti-inflammatory and neuroprotective properties. Here, we demonstrate that mouse corneas generate endogenous LXA(4) and NPD1. 12/15-LOX (Alox15) and LXA(4) receptor mRNA expression as well as LXA(4) formation were abrogated by epithelial removal and restored during wound healing. Amplification of these pathways by topical treatment with LXA(4) or NPD1 (1 microg) increased the rate of re-epithelialization (65-90%, n = 6-10, p < 0.03) and attenuated the sequelae of thermal injury. In contrast, the proinflammatory eicosanoids, LTB(4) and 12R-hydroxyeicosatrienoic acid, had no impact on corneal re-epithelialization. Epithelial removal induced a temporally defined influx of neutrophils into the stroma as well as formation of the proinflammatory chemokine KC. Topical treatment with LXA(4) and NPD1 significantly increased PMNs in the cornea while abrogating KC formation by 60%. More importantly, Alox15-deficient mice exhibited a defect in both corneal re-epithelialization and neutrophil recruitment that correlated with a 43% reduction in endogenous LXA(4) formation. Collectively, these results identify a novel action for the mouse 12/15-LOX (Alox15) and its products, LXA(4) and NPD1, in wound healing that is distinct from their well established anti-inflammatory properties.     

M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase

The sliding β-clamp, an important component of the DNA replication and repair machinery, is drawing increasing attention as a therapeutic target. We report the crystal structure of the M. tuberculosis β-clamp (Mtbβ-clamp) to 3.0 Å resolution. The protein crystallized in the space group C222₁ with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 Å respectively. Mtbβ-clamp is a dimer, and exhibits head-to-tail association similar to other bacterial clamps. Each monomer folds into three domains with similar structures respectively and associates with its dimeric partner through 6 salt-bridges and about 21 polar interactions. Affinity experiments involving a blunt DNA duplex, primed-DNA and nicked DNA respectively show that Mtbβ-clamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent K_d of 300 nM. In bacteria like E. coli, the β-clamp is known to interact with subunits of the clamp loader, NAD⁺ -dependent DNA ligase (LigA) and other partners. We tested the interactions of the Mtbβ-clamp with MtbLigA and the γ-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtbβ-clamp interacts in vitro with the γ-clamp loader, it does not interact with MtbLigA unlike in bacteria like E. coli where it does. Modeling studies involving earlier peptide complexes reveal that the peptide-binding site is largely conserved despite lower sequence identity between bacterial clamps. Overall the results suggest that other as-yet-unidentified factors may mediate interactions between the clamp, LigA and DNA in mycobacteria.     

Mechanisms of resistance in the rice cultivar Manikpukha to the rice stem nematode Ditylenchus angustus

The incompatible interaction between the rice cultivar Manikpukha and the rice stem nematode Ditylenchus angustus has been reported recently. This research focuses on the underlying mechanisms of resistance in Manikpukha. Invasion, post‐infection development and reproduction of D. angustus were compared in compatible and incompatible interactions to identify the stage in which resistance occurs. The results indicate that resistance in Manikpukha is associated with reduced development and reproduction, implying that resistance acts post‐invasion. We studied the possible involvement of three classical defence hormones, salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), in response to infection in a compatible interaction using biosynthesis/signalling‐deficient transgenic rice lines. All three hormones appear to have an influence on the basal defence of Nipponbare against the stem nematode. Although hormone application increases basal defences, expression studies and hormone analyses after nematode infection in Manikpukha did not show a clear involvement of the hormone defense pathways for SA, ET and JA. However, it seems that OsPAL1 plays a pivotal role in resistance, indicating that the phenylpropanoid pathway and its products might be key players in the incompatible interaction. Lignin measurement showed that, although basal levels are similar, Manikpukha had a significantly higher lignin content on nematode infection, whereas it was decreased in the susceptible cultivar. The results presented here show that SA, ET and JA are involved in basal defences, but the resistance of Manikpukha against D. angustus probably relies on products of the phenylpropanoid pathway.     

Effect of 28-homobrassinolide on the drought stress-induced changes in photosynthesis and antioxidant system of <Emphasis Type="Italic">Brassica juncea</Emphasis> L.

To investigate the effect of exogenously applied 28-homobrassinolide (HBL) on drought-stressed plants, photosynthesis and antioxidant systems were examined in Indian mustard (Brassica juncea L.). Seedlings of Indian mustard were subjected to drought stress for 7 days at the 8–14 (DS1)/15–21 (DS2) days’ stage of growth and then returned to normal conditions of growth. These seedlings were sprayed with HBL (0.01 μM) at the 30-day stage and were sampled at 60 days to assess the changes in growth, photosynthesis and antioxidant enzymes. Plants exposed to stress at either of the stages of growth exhibited a significant decrease in growth and photosynthesis. The exposure of plants to stress at an earlier stage (DS1) was more inhibitory than that at a later stage (DS2). However, the follow-up treatment with HBL significantly improved the values of these parameters and also overcame the inhibitory effect of water stress. The activity of antioxidant enzymes [catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7) and superoxide dismutase (E.C. 1.15.1.1)] and proline content in leaves exhibited an increase in response to both the treatment factors, where their interaction had an additive effect. It was, therefore, concluded that the elevated antioxidant system, at least in part, was responsible for amelioration of the drought stress.