Mh Haron

Ethanol-induced attenuation of oxidative stress is unable to alter mRNA expression pattern of catalase, glutathione reductase, glutathione-S-transferase (GST1A), and superoxide dismutase (SOD3) enzymes in Japanese rice fish (Oryzias latipes) embryogenesis

Although the mechanism of ethanol toxicity during embryogenesis is unknown, our earlier studies on Japanese rice fish (Oryzias latipes) embryos indicated that the effects might be mediated through oxidative stress. In this study we have determined the oxidative stress and the mRNA content of four antioxidant enzymes (catalase, glutathione reductase, glutathione-S-transferase, and superoxide dismutase) during Japanese rice fish embryogenesis (from 0 day post-fertilization to hatching) and after exposing the embryos to ethanol (100 and 300 mM) for 48 h at three stages (0-2, 1-3 and 4-6 days post-fertilization, dpf) of organogenesis. We observed that oxidative stress was minimal in blastula, gastrula or neurula stages, increased gradually with the advancement of morphogenesis and reached its maximum level in hatchlings. The antioxidant enzyme mRNAs were constitutively expressed throughout development; however, the expression pattern was not identical among the enzymes. Catalase and superoxide dismutase (SOD) mRNAs were minimal in the fertilized eggs, but increased significantly in 1 dpf and then either sharply dropped (SOD) or maintained a steady-state (catalase). Glutathione-S-transferase (GST) was very high in fertilized eggs and sharply dropped 1 dpf and then gradually increased thereafter. Glutathione reductase (GR) maintained a steady-state throughout the development. Ethanol was able to attenuate oxidative stress in embryos exposed only to 300 mM 1-3 dpf; no significant difference with controls was observed in other ethanol-treated groups. The antioxidant enzyme mRNAs also remained unaltered after ethanol treatment. From these data we conclude that the attenuation of oxidative stress by ethanol is probably due to the inhibition of normal growth of the embryos rather than by inhibiting catalase, GST, GR or SOD-dependent activities.

Oral administration of a Spirulina extract enriched for Braun-type lipoproteins protects mice against influenza A (H1N1) virus infection

A growing body of research indicates that oral administration of bacteria (such as probiotics) can exhibit a protective effect against influenza A (H1N1) viral infection in mice. In the present study, we used a mouse model to examine whether oral administration of Immulina(®), a commercial extract from the cyanobacteria Arthrospira (Spirulina) platensis, can reduce the severity of illness resulting from influenza A (H1N1) viral infection. The main active compounds within Immulina(®) are bacterial Braun-type lipoproteins that activate innate immune cells through a toll-like receptor (TLR) 2-dependent pathway. Mice that were fed Immulina(®) for 30 days before and 21 days after infection with influenza A (H1N1) virus exhibited a statistically significant reduction in the severity of infection. Compared to the control group, Immulina(®)-fed mice exhibited less weight loss, increased appetite, decreased clinical signs of disease, and lower lung histopathology scores. The results from the present study adds to the increasing evidence that oral administration of bacterial components that activate innate immune cells, whether derived from a bacterial preparation (probiotics or cyanobacteria) or from plant material containing endophytic bacteria, can exhibit a protective effect against influenza A (H1N1) viral infection.

Feasibility of Medaka (Oryzias latipes) as an Animal Model to Study Fetal Alcohol Spectrum Disorder

Abstract Alcohol is recognized as a classic teratogen capable of inducing a wide range of developmental abnormalities. Alcohol abuse during pregnancy produces permanent brain damage in the fetus and is associated with the development of a life-long behavioral, social, and cognitive disorder now known as fetal alcohol spectrum disorder (FASD). The most clinically recognizable form of FASD is fetal alcohol syndrome (FAS) which is characterized by a set of characteristics including facial dysmorphogenesis, mental dysfunction, growth retardation, and cardiovascular and limb defects. Due to ethical constraints, human studies of FASD are very limited; however, our current understanding of FASD is mainly based on studies on several model vertebrate and invertebrate organisms. The fish embryo, especially zebrafish ( Danio rerio ) and Japanese medaka ( Oryzias latipes ), have proven utility for studying ethanols damaging effects during morphogenesis. These two fish are long-established models for research in developmental biology and have been used to explore ethanols effect on neurogenesis, cardiogenesis, intracellular signaling, neurobehavioral aspects, and apoptosis. Zebrafish are the center of attraction as a fish model of FASD; however, we have demonstrated that medaka embryos exposed to ethanol during development have several phenotypic features in craniofacial, cardiovascular structures and many biochemical parameters which are comparable to FASD phenotypes observed in human. In this chapter, we reviewed our findings and propose that medaka embryogenesis, like zebrafish, may be a very useful model for investigating the molecular endpoints of FASD.

Modulation of ethanol toxicity by Asian ginseng (Panax ginseng) in Japanese ricefish (Oryzias latipes) embryogenesis

Alcohol consumption by women during pregnancy often induces fetal alcohol spectrum disorder (FASD) in children who have serious central nervous system (CNS), cardiovascular, and craniofacial defects. Prevention of FASD, other than women abstaining from alcohol drinking during pregnancy, is not known. A limitation of the use of synthetic anti-alcoholic drugs during pregnancy led us to investigate herbal products. In particular, many plants including Asian ginseng (Panax ginseng) have therapeutic potential for the treatment of alcoholism. We used Japanese ricefish (medaka) (Oryzias latipes), an animal model of FASD, for identifying herbal medicines that can attenuate ethanol toxicity. Fertilized eggs in standard laboratory conditions were exposed to ginseng (PG) root extract (0-2 mg/mL) either 0-2 (group A) or 1-3 (group B) day post fertilization (dpf) followed by maintenance in a clean hatching solution. The calculated IC50 as determined 10 dpf in A and B groups were 355.3±1.12 and 679.7±1.6 μg/mL, respectively. Simultaneous exposure of embryos in sub-lethal concentrations of PG (50-200 μg/mL) and ethanol (300 mM) for 48 h disrupted vessel circulation and enhanced mortality. However, PG (100 μg/mL) may partially protect trabecular cartilage (TC) deformities in the neurocranium in B group embryos induced by ethanol (300 mM). To understand the mechanism, embryonic ethanol concentration was measured at 2 dpf and adh5, adh8, aldh2, aldh9a, catalase, GST, and GR mRNAs were analyzed at 6 dpf. It was observed that although ethanol is able to reduce adh8 and GST mRNA contents, the simultaneous addition of PG was unable to alter ethanol level as well as mRNA contents in these embryos. Therefore, antagonistic effects of PG on ethanol toxicity are mediated by a mechanism which is different from those regulating ethanol metabolism and oxidative stress.

Activities and Prevalence of Proteobacteria Members Colonizing Echinacea purpurea Fully Account for Macrophage Activation Exhibited by Extracts of This Botanical

Evidence supports the theory that bacterial communities colonizing Echinacea purpurea contribute to the innate immune enhancing activity of this botanical. Previously, we reported that only about half of the variation in in vitro monocyte stimulating activity exhibited by E. purpurea extracts could be accounted for by total bacterial load within the plant material. In the current study, we test the hypothesis that the type of bacteria, in addition to bacterial load, is necessary to fully account for extract activity. Bacterial community composition within commercial and freshly harvested (wild and cultivated) E. purpurea aerial samples was determined using high-throughput 16S rRNA gene pyrosequencing. Bacterial isolates representing 38 different taxa identified to be present within E. purpurea were acquired, and the activity exhibited by the extracts of these isolates varied by over 8000-fold. Members of the Proteobacteria exhibited the highest potency for in vitro macrophage activation and were the most predominant taxa. Furthermore, the mean activity exhibited by the Echinacea extracts could be solely accounted for by the activities and prevalence of Proteobacteria members comprising the plant-associated bacterial community. The efficacy of E. purpurea material for use against respiratory infections may be determined by the Proteobacterial community composition of this plant, since ingestion of bacteria (probiotics) is reported to have a protective effect against this health condition.

Developmental regulation of neuroligin genes in Japanese ricefish (Oryzias latipes) embryogenesis maintains the rhythm during ethanol-induced fetal alcohol spectrum disorder.

Although prenatal alcohol exposure is the potential cause of fetal alcohol spectrum disorder (FASD) in humans, the molecular mechanism(s) of FASD is yet unknown. We have used Japanese ricefish (Oryzias latipes) embryogenesis as an animal model of FASD and reported that this model has effectively generated several phenotypic features in the cardiovasculature and neurocranial cartilages by developmental ethanol exposure which is analogous to human FASD phenotypes. As FASD is a neurobehavioral disorder, we are searching for a molecular target of ethanol that alters neurological functions. In this communication, we have focused on neuroligin genes (nlgn) which are known to be active at the postsynaptic side of both excitatory and inhibitory synapses of the central nervous system. There are six human NLGN homologs of Japanese ricefish reported in public data bases. We have partially cloned these genes and analyzed their expression pattern during normal development and also after exposing the embryos to ethanol. Our data indicate that the expression of all six nlgn genes in Japanese ricefish embryos is developmentally regulated. Although ethanol is able to induce developmental abnormalities in Japanese ricefish embryogenesis comparable to the FASD phenotypes, quantitative real-time PCR (qPCR) analysis of nlgn mRNAs indicate unresponsiveness of these genes to ethanol. We conclude that the disruption of the developmental rhythm of Japanese ricefish embryogenesis by ethanol that leads to FASD may not affect the nlgn gene expression at the message level.

Bacterial components are the major contributors to the macrophage stimulating activity exhibited by extracts of common edible mushrooms

Recent studies have indicated that a major contributor to the innate immune enhancing properties of some medicinal plants is derived from the cell wall components of bacteria colonizing these plants. The purpose of the current study was to assess if the bacteria present within edible and medicinal mushrooms substantially contribute to the innate immune stimulating potential of these mushrooms. Whole mushrooms from thirteen types of edible fungi and individual parts from Agaricus bisporus were analyzed for in vitro macrophage activation as well as bacterial lipopolysaccharides (LPS) content, cell load, and community composition. Substantial variation between samples was observed in macrophage activation (over 500-fold), total bacterial load (over 200-fold), and LPS content (over 10 million-fold). Both LPS content (ρ = 0.832, p < 0.0001) and total bacterial load (ρ = 0.701, p < 0.0001) correlated significantly with macrophage activation in the whole mushroom extracts. Extract activity was negated by treatment with NaOH, conditions that inactivate LPS and other bacterial components. Significant correlations between macrophage activation and total bacterial load (ρ = 0.723, p = 0.0001) and LPS content (ρ = 0.951, p < 0.0001) were also observed between different tissues of Agaricus bisporus. Pseudomonas and Flavobacterium were the most prevalent genera identified in the different tissue parts and these taxa were significantly correlated with in vitro macrophage activation (ρ = 0.697, p < 0.0001 and ρ = 0.659, p = 0.0001, respectively). These results indicate that components derived from mushroom associated bacteria contribute substantially to the innate immune enhancing activity exhibited by mushrooms and may result in similar therapeutic actions as reported for ingestion of bacterial preparations such as probiotics.

Asian Ginseng (Panax ginseng) Attenuates Ethanol-induced Neurocranial Cartilage Deformities in a Fish Model of Fetal Alcohol Spectrum Disorder

Fetal Alcohol spectrum disorder (FASD) is an important clinical problem resulting from prenatal alcohol exposure. It has serious central nervous system (CNS), cardiovascular, and craniofacial defects. Prevention of FASD, other than women abstaining from drinking alcohol during pregnancy, is not known. The synthetic drugs recommended for the treatment of alcoholism cannot be used by women during pregnancy which led us to investigate on natural products. Many herbs including Asian ginseng (Panax ginseng) have therapeutic potencies on alcoholism. Recently, ginseng has been shown to inhibit ethanol-induced teratogenesis in mouse embryos in vitro. We therefore tested the potency of Asian ginseng (PG) as an antialcoholic agent using medaka (Oryzias latipes) as an animal model. Previously we have demonstrated that medaka embryos exposed to ethanol developed neurocranial cartilage deformities which are analogous to the craniofacial abnormalities observed in human FASD phenotypes [1]. Moreover, the chondrification in cartilages, specifically trabecular cartilages (TC), was inhibited by ethanol [2]. In the present experiments we have tested whether PG can attenuate ethanol-induced TC deformities in medaka. Fertilized medaka eggs in standard laboratory conditions (16L: 8D, 25 0C) were exposed to PG root extract (0–2mg/ml) for 0–2 day post fertilization (dpf). The calculated IC50 of PG as determined on 10 dpf is 355±1g/ml (n=3, r2=0.9001). In separate experiments embryos were exposed to sub-lethal concentrations of PG (50 and 200µg/ml) with or without ethanol (100 and 300 mM) either for 0–2 dpf or 1–3 dpf and the developmental abnormalities were assessed in embryos and hatchlings. It was observed that PG dose-dependently prevent TC deformity in hatchlings and reduce thrombus formation in embryos; however, embryo mortality were enhanced. It is therefore concluded that PG is able to attenuate some of the ethanol- induced birth defects in medaka even though the embryo mortality was aggravated. Acknowledgements: This research is supported in part by the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No 586408–2-0009. The project described was supported by grant number 5P20RR021929 from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. References: [1] Wang X, et al. (2006) Birth Def Res, 77B: 29–39. [2] Hu Y, et al. (2009). Comp Biochem Physiol, 150C: 495–502.