Functional expression, localization, and biochemical characterization of thioredoxin glutathione reductase from air-breathing magur catfish, Clarias magur.

The glutathione (GSH) and thioredoxin (Trx) systems regulate cellular redox homeostasis and maintain antioxidant defense in most eukaryotes. We earlier reported the absence of gene coding for the glutathione reductase (GR) enzyme of the GSH system in the facultative air-breathing catfish, Clarias magur. Here, we identified three thioredoxin reductase (TrxR) genes, one of which was later confirmed as a thioredoxin glutathione reductase (TGR). We then characterized the novel recombinant TGR enzyme of C. magur (CmTGR). The tissue-specific expression of the txnrd genes and the tissue-specific activity of the TrxR enzyme were analyzed. The recombinant CmTGR is a dimer of ~133 kDa. The protein showed TrxR activity with 5,5'-diothiobis (2-nitrobenzoic acid) reduction assay with a Km of 304.40 µM and GR activity with a Km of 58.91 µM. Phylogenetic analysis showed that the CmTGR was related to the TrxRs of fishes and distantly related to the TGRs of platyhelminth parasites. The structural analysis revealed the conserved glutaredoxin active site and FAD- and NADPH-binding sites. To our knowledge, this is the first report of the presence of a TGR in any fish. This unusual presence of TGR in C. magur is crucial as it helps maintain redox homeostasis under environmental stressors-induced oxidative stress.

Deciphering the role of aquaporin 1 in the adaptation of the stinging catfish Heteropneustes fossilis to environmental hypertonicity by molecular dynamics simulation studies.

A thorough investigation of the water permeability of H. fossilis aquaporin 1 (hfAQP1) in a hypertonic environment can provide a useful insight into the understanding of the underlying molecular mechanism of its high tolerance to salinity. Here, we constructed a 3 D homology model of hfAQP1 by taking Bos taurus AQP1, AQP0, and human AQP2 as templates using I-TASSER. The model obtained has similar structural organizations with mammalian AQP1s in all aspects. We investigated the water permeability of the modeled hfAQP1 in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane under neutral and 100 mM hypersalinity by subjecting each system to a 100 ns molecular dynamics simulation. Our results show that hypersalinity hinders water permeation across the membrane through the hfAQP1 channel. A change in the intermolecular distance between key residues of the ar/R selectivity filter along with charge redistribution resulted in the accommodation of only 2-6 water molecules inside the channel at once under hypersaline conditions. We investigated the mRNA expression pattern of hfaqp1 in osmoregulatory organs of H. fossilis in response to 100 mM hypertonicity by using qPCR analysis. The transcript was downregulated in kidney and GI tract, but upregulated in the Gills. Thus, the catfish survive in a hypertonic environment by reducing the transport of water in its cellular systems and downregulating the expression of the hfaqp1 gene. The results observed in our study can shed more light on the functionality of AQP1 in catfishes under salinity stress and aid in future researches on solving more gating mechanisms involved in its regulation.Communicated by Ramaswamy H. Sarma.

Transcriptome analysis of gills reveals novel insights into the molecular response of stinging catfish (Heteropneustes fossilis) to environmental hypertonicity.

The stinging catfish Heteropneustes fossilis is a champion survivor under hypertonic stress and is suggested to be a profitable candidate for culture in slightly saline water in coastal regions. Fish gills are an essential site of osmoregulation and other physiological processes. To investigate the stress responses and mechanisms of salinity tolerance in stinging catfish, we sampled gills tissues from control and hypertonicity (100 mM NaCl solution) treated adult catfish and assessed for transcriptomic profiling by high throughput sequencing. The raw data generated was filtered and assembled for de novo transcriptome assembly. The final contig assembly produced a total of 1,71,478 unigene transcripts with an average transcript length of 898 bp and a GC content of 45%. A total of 22,231 transcripts matched with Chordata with BLAST search and were functionally annotated, out of which 21,814 were best-hit transcripts aligned with the UniProt database. Comparative transcriptomic analysis revealed that a total of 1951 genes were differentially expressed in the gills of NaCl-treated fish compared to the control. Functional and enrichment analysis of the Differentially expressed genes demonstrated that several GO pathway terms were significantly over-represented, such as 'catalytic activity', 'hydrolase activity' in molecular function category, 'membrane', 'integral component of membrane' in cellular component category and 'metabolic process', 'regulation of transcription' in biological process category. The functional analysis study of DEGs demonstrated that tolerance to hypertonic stress by stinging catfish is associated with a few pathways related to stress response, immune response, biosynthesis, metabolism, molecular transport, cytoskeleton remodeling, apoptosis, cell signaling, transcriptional regulation, etc. The present study provides a novel insight into the molecular responses of the air-breathing stinging catfish against salinity stress, which could elucidate the underlying mechanisms of adaptation of this stenohaline species under various environmental constraints.

Differential expression of aquaporin genes and the influence of environmental hypertonicity on their expression in juveniles of air-breathing stinging catfish (Heteropneustes fossilis).

Aquaporins (AQPs) are a superfamily of transmembrane channel proteins that are responsible for the transport of water and some other molecules to and from the cell, mainly for osmoregulation under anisotonicity. We investigated here the expression patterns of different AQP isoforms and also during exposure to hypertonicity (300 mOsmol/L) for 48 h in juvenile stages of air-breathing stinging catfish (Heteropneustes fossilis). A total of 8 mRNA transcripts for different isoforms of AQPs and their translated proteins could be detected in the anterior and posterior regions of S1, S2, and S3 stages of juveniles of stinging catfish at variable levels. In general, more expression of mRNAs for different aqp genes was seen in the S2 and S3 juveniles than in the S1 juveniles. Most interestingly, exposure to hypertonicity of S2 juveniles for a period of 48 h led to increased expression of most of the aqp genes both at transcriptional and translational levels, except for aqp3 in the anterior and posterior regions and aqp1 in the anterior region, showing maximum expression at later stages of hypertonic exposure. Thus, it is evident that AQPs play crucial roles in maintaining the water and ionic balances under anisotonic conditions even at the early developmental stages of stinging catfish as a biochemical adaptational strategy to survive and grow in anisotonic environment.

Identifying miRNAs in the modulation of gene regulation associated with ammonia toxicity in catfish, Clarias magur (Linnaeus, 1758).

BACKGROUND: The small non-coding microRNAs play a vital role in post-transcriptional gene regulation associated with different physiological events such as metabolism, stress, etc. The freshwater catfish, Clarias magur, can grow within hyper ammonia containing stagnant water bodies and/or muddy substratum. We intended to identify organ-specific miRNAs associated with ammonia stress management. METHODS AND RESULTS: The miRNA-libraries were generated from QC passed total RNA extracted from liver, muscle, and kidney of ammonia-treated (exposed to 25 mM NH4Cl for 14 days) and untreated catfish. The libraries were validated using High sensitivity D1000 Screen tape. The trimmed quality-filtered reads for control and treated samples of kidney were 19,406,210; 14,904,423; for liver 15,467,727; 18,582,072; and for muscle 25,081,345; 19,782,182 respectively. Total 120 known and 150 novel differentially expressed miRNAs were identified, out of which miR-200, miR-217, miR-122, miR-133, miR-145, miR-221, miR-19, miR-138, miR-34, and miR-184 were predicted to be involved in the metabolism of nitrogen. The key miRNAs targeted several genes associated with urea synthesis like Glutaminase 2, Argininosuccinate lyase, Glutamate dehydrogenase 1, Alanine aminotransferase 2-like, Aspartate aminotransferase, cytoplasmic-like, Glutamate ionotropic receptor NMDA type subunit 2A, etc. CONCLUSIONS: This is the first report of miRNAs, which serve as a vital resource for regulating nitrogen metabolism in freshwater catfish, C. magur. The data will be resourceful for further evaluating the regulatory role of miRNAs in fishes, which grow and reproduce very well in hazardous ammonia-contaminated water bodies.

Molecular characterization of superoxide dismutase and catalase genes, and the induction of antioxidant genes under the zinc oxide nanoparticle-induced oxidative stress in air-breathing magur catfish (Clarias magur).

The deduced amino acid sequences from the complete cDNA coding sequences of three antioxidant enzyme genes (sod1, sod2, and cat) demonstrated that phylogenetically the magur catfish (Clarias magur) is very much close to other bony fishes with complete conservation of active site residues among piscine, amphibian, and mammalian species. The three-dimensional structures of three antioxidant enzyme proteins are very much similar to mammalian counterparts, thereby suggesting the functional similarities of these enzymes. Exposure to ZnO NPs resulted in an oxidative stress as evidenced by an initial sharp rise of intracellular concentrations of hydrogen peroxide (H2O2) and malondialdehyde (MDA) but decreased gradually at later stages. The level of glutathione (GSH) also increased gradually in all the tissues examined after an initial decrease. Biochemical and gene expression analyses indicated that the magur catfish has the ability to defend the ZnO NP-induced oxidative stress by inducing the SOD/CAT enzyme system and also the GSH-related enzymes that are mediated through the activation of various antioxidant-related genes both at the transcriptional and translational levels in various tissues. Furthermore, it appeared that the stimulation of NO, as a consequence of induction nos2 gene, under NP-induced oxidative stress serves as a modulator to induce the SOD/CAT system in various tissues of magur catfish as an antioxidant strategy. Thus, it can be contemplated that the magur catfish possesses a very efficient antioxidant defensive mechanisms to defend against the oxidative stress and also from related cellular damages during exposure to ZnO NPs into their natural environment.

Increase in PPARγ inhibitory phosphorylation by Fetuin-A through the activation of Ras-MEK-ERK pathway causes insulin resistance.

Obesity induced insulin resistance is primarily regulated by the inhibitory phosphorylation of peroxisome proliferator-activated receptor γ at serine 273 (PPARγS273) which has been shown to be regulated by MEK and ERK. An upstream regulatory molecule of this pathway could be a therapeutic option. Here we analyzed the involvement of Fetuin-A (FetA), a key hepato-adipokine implicated in insulin resistance, as an upstream regulator molecule for the regulation of PPARγ inhibitory phosphorylation. Mice fed with standard diet (SD), high fat diet (HFD) and HFD with FetA knockdown (HFD-FetAKD) were used to examine the role of FetA on PPARγS273 phosphorylation in adipocytes. The mechanism of regulation and its effect on skeletal muscle were studied using primary adipocytes, 3T3-L1 (preadipocyte) and C2C12 (myotube) cell lines. Increased FetA in HFD mice strongly correlated with augmentation of PPARγS273 phosphorylation in inflamed adipocytes while knockdown of FetA suppressed it. This effect of FetA was mediated through the activation of Ras which in turn activated MEK and ERK. On addressing how FetA could stimulate activation of Ras, we found that FetA triggered TNFα in inflamed adipocytes which induced Ras activation. The ensuing sharp fall in adiponectin level attenuated AMPK activation in skeletal muscle cells affecting mitochondrial ATP production. Our data reveal the essential role of FetA induced activation of Ras in regulating PPARγ inhibitory phosphorylation through Ras-MEK-ERK pathway which downregulates adiponectin disrupting skeletal muscle mitochondrial bioenergetics. Thus, FetA mediated PPARγ inactivation has adverse consequences upon adipocyte-myocyte crosstalk leading to disruption of energy homeostasis and loss of insulin sensitivity.

Genome-wide identification of novel long non-coding RNAs and their possible roles in hypoxic zebrafish brain.

Long non-coding RNAs (lncRNAs) are the master regulators of numerous biological processes. Hypoxia causes oxidative stress with severe and detrimental effects on brain function and acts as a critical initiating factor in the pathogenesis of Alzheimer's disease (AD). From the RNA-Seq in the forebrain (Fb), midbrain (Mb), and hindbrain (Hb) regions of hypoxic and normoxic zebrafish, we identified novel lncRNAs, whose potential cis targets showed involvement in neuronal development and differentiation pathways. Under hypoxia, several lncRNAs and mRNAs were differentially expressed. Co-expression studies indicated that the Fb and Hb regions' potential lncRNA target genes were involved in the AD pathogenesis. In contrast, those in Mb (cry1b, per1a, cipca) was responsible for regulating circadian rhythm. We identified specific lncRNAs present in the syntenic regions between zebrafish and humans, possibly functionally conserved. We thus identified several conserved lncRNAs as the probable regulators of AD genes (adrb3b, cav1, stat3, bace2, apoeb, psen1, s100b).

Induction of nitric oxide synthesis: a strategy to defend against high environmental ammonia-induced oxidative stress in primary hepatocytes of air-breathing catfish, Clarias magur.

Air-breathing magur catfish (Clarias magur) regularly face the problem of exposure to high environmental ammonia (HEA) as one of the major pollutants in their natural habitats that causes considerable toxic effects at the cellular level, including that of oxidative stress. The major objective of the present study was to demonstrate the antioxidant activity of endogenously produced nitric oxide (NO) to defend against ammonia-induced oxidative stress in primary hepatocytes of magur catfish during exposure to HEA. Exposure to NH4Cl (5 mmol l-1) led to a significant increase in intracellular ammonia concentration with a sharp rise of hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations within 3 h in primary hepatocytes, which decreased gradually at later stages of treatment. This phenomenon was accompanied by a significant increase in superoxide dismutase (SOD) and catalase (CAT) activity as a consequence of induction of corresponding genes. HEA exposure also led to the stimulation of NO production due to induction of inducible nitric oxide synthase (iNOS) activity, as a consequence of up-regulation of the nos2 gene. Most interestingly, when NO production by hepatocytes under ammonia stress was blocked by adding certain inhibitors [aminoguanidine and 3-(4-methylphenylsulfonyl)-2-propenenitrile] to the culture medium, there was a further rise of H2O2 and MDA concentrations in hepatocytes. These were accompanied by the lowering of SOD and CAT activity with less expression of corresponding genes. Thus, it can be contemplated that magur catfish use the strategy of stimulation of NO production, which ultimately induces the SOD-CAT enzyme system to defend against ammonia-induced oxidative stress.

Molecular characterization and ornithine-urea cycle genes expression in air-breathing magur catfish (Clarias magur) during exposure to high external ammonia.

The air-breathing magur catfish (Clarias magur) is a potential ureogenic teleost because of its functional ornithine-urea cycle (OUC), unlike typical freshwater teleosts. The ability to convert ammonia waste to urea was a significant step towards land-based life forms from aquatic predecessors. Here we investigated the molecular characterization of some OUC genes and the molecular basis of stimulation of ureogenesis via the OUC in magur catfish. The deduced amino acid sequences from the complete cDNA coding sequences of ornithine transcarbamyolase, argininosuccinate synthase, and argininosuccinate lyase indicated that phylogenetically magur catfish is very close to other ureogenic catfishes. Ammonia exposure led to a significant induction of major OUC genes and the gene products in hepatic and in certain non-hepatic tissues of magur catfish. Hence, it is reasonable to assume that the induction of ureogenesis in magur catfish under hyper-ammonia stress is mediated through the activation of OUC genes as an adaptational strategy.

Orthopantomography and Cone-Beam Computed Tomography for the Relation of Inferior Alveolar Nerve to the Impacted Mandibular Third Molars.

CONTEXT: Relation of inferior alveolar nerve to the impacted mandibular third molars (IMTMs). AIMS: The aim of this study was to assess the reliability of seven specific radiographic signs of mandibular third molar root that are observed on orthopantomography (OPG) and to predict the proximity and the absence of corticalization between the mandibular canal and IMTM root on cone-beam computed tomography (CBCT) images. SETTINGS AND DESIGN: The present study was conducted in the Department of Oral and Maxillofacial Surgery, Raja Rajeswari Dental College and Hospital. SUBJECTS AND METHODS: Data set of 30 panoramic images was retrieved between the year of 2015 and 2016 indicated for extraction of lower third molars. The sample consisted of 30 individuals, who underwent preoperative radiographic evaluation before the extraction of impacted mandibular third molars (IMTM). Patients aged above 18 years with any of the seven specific signs observed on the panoramic radiograph which includes darkening, deflection, narrowing of roots, bifid root apex, diversion, narrowing of canal and interruption in the white line of the canal were included in the study. If any of the above mentioned seven specific sign were present, the patient was subjected to CBCT. On the CBCT images, the canal was traced in three planes. The acquired images were assessed for the presence or absence of corticalization. STATISTICAL ANALYSIS USED: Descriptive and inferential statistical analyses were used. Proportions were compared using the Chi-square test and Student's t-test. RESULTS: Among the 4 subjects, diagnosed with an absence of corticalization, patients with isolated darkening of root P = 0.001 and patients with isolated interruption in white line P = 0.69. Patients with darkening of root in association with interruption in white lines on OPG showed the absence of corticalization on CBCT findings P = 0.001, respectively. CONCLUSIONS: This study showed the poor reliability of radiographic signs seen on OPG on predicting the proximity of third mandibular root with mandibular canal related to CBCT finding. Four were diagnosed with the absence of corticalization in CBCT findings.

Highly Selective Detection of Hypochlorous Acid by a Bis-heteroleptic Ru(II) Complex of Pyridyl-1,2,3-triazole Ligand via C(sp2)-H Hydroxylation.

A Ru(II) complex (Ru-1) of a substituted pyridyl-1,2,3-triazole ligand (BtPT) for highly selective "light-up" detection of hypochlorous acid is presented. An unusual anti-Markovnikov HOCl addition to the C═C bond of 1,2,3-triazole and a highly specific C(sp2)-H hydroxylation over epoxidation made Ru-1 a highly selective luminescent HOCl probe. The abnormal regio- and stereoselective HOCl addition and subsequent hydroxylation mechanism in detail is supported by the combination of ESI-MS, 1H/13C NMR spectroscopy, and 1H NMR titration. The hydroxylation at the C5 center in 1,2,3-triazole increases the electron density and makes BtPT a better σ-donor as well as π-donor, which in turn increases the 3MC-3MLCT energy gap and inhibits the nonradiative decay from the excited state of Ru-1 and is the key reason for luminescence light-up. Most importantly, the exogenous and endogenous HOCl imaging in the living HEK293T cells is also demonstrated. The probe showed low cytotoxicity and efficiently permeated the cell membrane. The cell-imaging experiments revealed rapid staining of the extranuclear region of HEK293T cells which clearly indicates the presence of cytoplasmic HOCl. The endogenous HOCl generation and imaging, stimulated by lipopolysaccharides (LPS) and paraquat in the HEK293T cells, is also demonstrated.

Transcriptome analysis reveals novel insights in air-breathing magur catfish (Clarias magur) in response to high environmental ammonia.

The facultative air-breathing magur catfish (Clarias magur) frequently face different environmental challenges, such as hyper-ammonia, and desiccation stresses in their natural habitats. All these stresses lead to higher accumulation of body ammonia, thereby causing various harmful effects to the fish due to its toxicity. Nonetheless, the mechanisms underlying ammonia-induced toxicity is yet not clear. In the present study, we used RNA sequencing and utilized a modified method for de novo assembly of the transcriptome to provide an exhaustive study on the transcriptomic alterations of magur catfish in response to high environmental ammonia (HEA; 25 mM NH4Cl). The final contig assembly produced a total of 311,076 unique transcripts (termed as unigenes) with a GC content of 48.3% and the average length of 599 bp. A considerable number of SSR marker associated with these unigenes were also detected. A total of 279,156 transcripts were successfully annotated by using various databases. Comparative transcriptomic analysis revealed a total of 3453 and 19,455 genes were differentially expressed in the liver and brain tissues, respectively, in ammonia-treated fish compared to the control. Enrichment analysis of the differentially expressed genes (DEGs) showed that several GO and KEGG pathway terms were significantly over-represented. Functional analysis of significantly elevated DEGs demonstrated that ammonia stress tolerance of the magur catfish was associated with quite a few pathways related to immune response, oxidative stress, and apoptosis, as well as few transporter proteins involved with ammonia and urea transport. Both liver and brain tissues showed HEA-mediated oxidative damage with consequent activation of antioxidant machinery. However, elevated ROS levels led to an activation of inflammatory cytokines and thus innate immune response in the liver. Conversely, in the brain ROS-mediated irreversible cell damages activated apoptosis via both p53-Bax-Bcl2 and caspase-mediated pathways. The present study provides a novel understanding of the molecular responses of this air-breathing catfish against the ammonia-induced stressors, which could elucidate the underlying mechanisms of adaptation of this facultative air-breather living under various environmental constraints.

Antioxidant activity of endogenously produced nitric oxide against the zinc oxide nanoparticle-induced oxidative stress in primary hepatocytes of air-breathing catfish, Clarias magur.

The facultative air-breathing magur catfish (Clarias magur) regularly encounter various environmental challenges including the exposure to nanomaterials discarded as industrial wastes in water bodies. The present investigation aimed at determining the possible ZnO NP-induced oxidative stress and also the antioxidant strategy of nitric oxide (NO), generated endogenously, in primary hepatocytes of magur catfish. Exposure of primary hepatocytes to different concentrations of ZnO NPs (5 and 10 µg/mL) led to a sharp rise of intracellular concentrations of hydrogen peroxide (H2O2) and malondialdehyde (MDA) within 6 h, which decreased gradually at later stages. This phenomenon was accompanied by an initial decrease of superoxide dismutase (SOD) and catalase (CAT) activities, the expression of their corresponding genes and the enzyme protein levels, with a subsequent significant increase of all these parameters at later stages. Most interestingly, exposure to ZnO NPs also stimulated the NO production by the primary hepatocytes as a consequence of induction of inducible nitric oxide synthase (iNOS) activity, higher expression of nos2 gene and iNOS protein. Furthermore, when the NO production by the hepatocytes was inhibited by either aminoguanidine (inhibitor for iNOS) or BAY (inhibitor for NFκB) in the presence of ZnO NPs, the intracellular concentrations of H2O2 and MDA was significantly elevated. This elevation was accompanied by a subsequent decrease of sod and cat genes expression, thereby suggesting that the inhibition of NO production leads to oxidative stress. Thus, it is believed that the magur catfish uses the strategy of stimulation of endogenous NO production by inducing the nos2 gene and simultaneous NO-mediated induction of sod and cat genes to defend against the NP-induced oxidative stress. It is the first report of such NO-mediated antioxidant strategy in any teleost fish to defend against the NP-induced oxidative stress and corresponding cellular damages.

Hyper-ammonia stress causes induction of inducible nitric oxide synthase gene and more production of nitric oxide in air-breathing magur catfish, Clarias magur (Hamilton).

Nitric oxide (NO) is an important signalling molecule that plays diverse physiological functions in several vertebrates including that of adaptation to various stressful stimuli. The air-breathing magur catfish (Clarias magur) is known to tolerate a very high external ammonia (HEA) stress in its natural habitats. We report here the possible induction of inducible nitric oxide (inos) gene and more generation of NO in magur catfish exposed to HEA. Exposure to HEA (25 mM NH4Cl) for 14 days led to the higher accumulation of NO in different tissues of magur catfish and also more efflux of NO from the perfused liver of NH4Cl-treated fish as a consequence of high build of toxic ammonia in body tissues. More synthesis and accumulation of NO in body tissues was associated with the induction of iNOS activity, which otherwise was not detectable in control fish. The stimulation of iNOS activity in HEA exposed fish was mainly due to induction of inos gene as evidenced by more expression of inos mRNA and also more abundance of iNOS protein in different tissues of magur catfish. Immunocytochemical analysis indicated the zonal specific expression of iNOS protein in different tissues of magur catfish. The augmentation of iNOS in the fish under HEA could be an adaptive strategy of the fish to defend against the ammonia stress through the generation of NO. Therefore, the present finding identifies the potential role of iNOS to enhance the adaptive capacity and survivability of catfish under various adverse environmental and pathological conditions that it faces in its natural habitats.

Differential expression of multiple glutamine synthetase genes in air-breathing magur catfish, Clarias magur and their induction under hyper-ammonia stress.

The present study demonstrates the unique presence of three different gs genes (cmgs01, cmgs02, and cmgs03) in air-breathing ureogenic magur catfish (Clarias magur), which is otherwise reported to be encoded by a single gene in higher vertebrates. Of these three genes, two (cmgs01and cmgs03) were identified as 'liver' form, predominantly expressed in liver cells, and the third one as 'brain' form (cmgs02), expressed chiefly in brain cells. Molecular characterization studies have revealed conservation of homologous active site residues in all the three gs genes. In silico analysis, accompanied by GS enzyme assay and Western blot analysis of different GS isoforms in different subcellular fractions indicated the mitochondrial localization of cmGS01 and cmGS03 in liver and kidney cells and cytosolic localization of cmGS02 in brain cells. Further, exposure of magur catfish to high external ammonia (HEA; 25 mM NH4Cl) led to a significant induction of multiple gs genes as evidenced by higher expression of different gs mRNAs at variable levels in different tissues. The cmgs01 and cmgs03 mRNA levels elevated significantly in liver, kidney, muscle, and gills, whereas the cmgs02 mRNA level increased considerably in the brain after 14 days of exposure to HEA. These increases in mRNA levels were associated with a significant rise in cmGS01 and cmGS03 proteins in liver, kidney, muscle, and gills, and the cmGS02 protein in the brain after 14 days of exposure to HEA. Therefore, it can be concluded that the unique differential expression of three gs genes and their induction under high ammonia level probably helps in detoxification of ammonia to glutamine and further to urea via the ornithine-urea cycle in ureogenic as well as non-ureogenic tissues of these magur catfish.

Lipopolysaccharide causes NFĸB-mediated induction of inducible nitric oxide synthase gene and more production of nitric oxide in air-breathing catfish, Clarias magur (Hamilton).

The magur catfish (Clarias magur) is a facultative air-breather and regularly encounters with various environmental changes along with exposure to various bacterial pathogens in its natural habitats. Occurrence of various biochemical adaptational strategies related to nitrogen metabolism in magur catfish is already known. The present investigation aimed at determining the possible induction of inducible nitric oxide synthase (inos) gene and stimulation of nitric oxide (NO) production in this catfish while challenging with lipopolysaccharide (LPS, a bacterial endotoxin) treatment, and also to determine the involvement of nuclear factor kappa B (NFkB) in induction of inos gene. Intra-peritoneal injection of LPS led to more production and accumulation of NO in different body tissues of magur catfish as a consequence of induction of iNOS activity. The induction of iNOS activity was associated with the induction of inos gene as evidenced by more expression of inos mRNA and more abundance of iNOS enzyme protein in different tissues of magur catfish with certain variations in zonal specific expression patterns. Similar observations related to more production of NO and induction of inos gene were also made when the isolated hepatocytes were treated with LPS in vitro condition. LPS treatment also led to activation of NFĸB in hepatic cells. However, in presence of a specific inhibitor of NFkB, the LPS-mediated induction of inos gene and extra production of NO were almost blocked, thereby suggesting that the induction of inos gene due to LPS treatment was mediated via the NFkB in magur catfish. It is hypothesized that the induction of iNOS activity, and more synthesis and accumulation of NO could serve as indicators to determine the pathophysiological conditions of the fish living in bacterial contaminated water bodies. Further, it can be contemplated that more synthesis of NO through iNOS enzyme probably serves as an important pharmacological tool against fish pathogen and also plays an important role in host defense mechanisms in this unique group of magur catfish.

Unique mitochondrial localization of arginase 1 and 2 in hepatocytes of air-breathing walking catfish, Clarias batrachus and their differential expression patterns under hyper-ammonia stress.

Arginase (ARG) catalyzes the final step of ornithine-urea cycle (OUC) leading to a conversion of L-arginine to L-ornithine and urea. Several isoforms of ARG have been reported in vertebrates, out of which the two predominant isoforms are the cytosolic ARG1 and the mitochondrial ARG2. The air-breathing walking catfish (Clarias batrachus) is frequently being challenged by different environmental insults such as hyper-ammonia, dehydration and osmotic stresses in their natural habitats throughout the year. The present study investigated the active presence of ARG1 and ARG2 isoforms in hepatocytes along with unique localization of both the isoforms inside the mitochondria, and also their specific expression patterns under hyper-ammonia stress (5mM NH4Cl) in isolated hepatocytes of walking catfish. Initially, full length sequences of both arg1 and arg2 genes were obtained by RACE-PCR. Studies on molecular characterization demonstrated the presence of all the conserved amino acids required for stability and activity of binuclear metal center in both the isoforms. Phylogenetic analysis of the amino acid sequences of ARG isoforms showed a differentiation of the ARG1 and ARG2 into two distinct clusters with their respective isoforms from other species. Most interestingly, both the isoforms of ARG in hepatocytes were found to be localized inside the mitochondria as evidenced by the presence of mitochondrial target peptide (mTP) in N-terminal of the derived amino acid sequences, and exclusive localization of ARG activity in the mitochondrial fraction. This was additionally confirmed by Western blot analysis of ARGs in mitochondrial and cytosolic fractions, and by immunocytochemical analysis in isolated hepatocytes. Although the possible reasons associated with the presence of both the isoforms of ARGs inside the mitochondria is not clearly understood, perhaps this mitochondrial localization of ARG is functionally advantageous in this catfish for the synthesis of N-acetyl-l-glutamate, the allosteric regulator for the first OUC enzyme, the carbamoyl phosphate synthetase III, and for supplying ornithine required for citrulline synthesis intramitochondrially. Furthermore, the ammonia stress, due to exposure to high external ammonia, led to greater synthesis of urea-N probably as a consequence of induction of ureogenesis, as evidenced by a larger accumulation of urea-N in hepatocytes and higher secretion in culture media parallel to the increased concentration of ammonia-N in hepatocytes. Ammonia stress also led to specific coordinated patterns of induction of both the arg genes in isolated hepatocytes of walking catfish.

Ammonia stress under high environmental ammonia induces Hsp70 and Hsp90 in the mud eel, Monopterus cuchia.

The obligatory air-breathing mud eel (Monopterus cuchia) is frequently being challenged with high environmental ammonia (HEA) exposure in its natural habitats. The present study investigated the possible induction of heat shock protein 70 and 90 (hsp70, hsc70, hsp90α and hsp90ß) genes and more expression of Hsp70 and Hsp90 proteins under ammonia stress in different tissues of the mud eel after exposure to HEA (50 mM NH4Cl) for 14 days. HEA resulted in significant accumulation of toxic ammonia in different body tissues and plasma, which was accompanied with the stimulation of oxidative stress in the mud eel as evidenced by more accumulation of malondialdehyde (MDA) and hydrogen peroxide (H2O2) during exposure to HEA. Further, hyper-ammonia stress led to significant increase in the levels of mRNA transcripts for inducible hsp70 and hsp90α genes and also their translated proteins in different tissues probably as a consequence of induction of hsp70 and hsp90α genes in the mud eel. However, hyper-ammonia stress was neither associated with any significant alterations in the levels of mRNA transcripts for constitutive hsc70 and hsp90ß genes nor their translated proteins in any of the tissues studied. More abundance of Hsp70 and Hsp90α proteins might be one of the strategies adopted by the mud eel to defend itself from the ammonia-induced cellular damages under ammonia stress. Further, this is the first report of ammonia-induced induction of hsp70 and hsp90α genes under hyper-ammonia stress in any freshwater air-breathing teleost.

Induction of Inducible Nitric Oxide Synthase by Lipopolysaccharide and the Influences of Cell Volume Changes, Stress Hormones and Oxidative Stress on Nitric Oxide Efflux from the Perfused Liver of Air-Breathing Catfish, Heteropneustes fossilis.

The air-breathing singhi catfish (Heteropneustes fossilis) is frequently being challenged by bacterial contaminants, and different environmental insults like osmotic, hyper-ammonia, dehydration and oxidative stresses in its natural habitats throughout the year. The main objectives of the present investigation were to determine (a) the possible induction of inducible nitric oxide synthase (iNOS) gene with enhanced production of nitric oxide (NO) by intra-peritoneal injection of lipopolysaccharide (LPS) (a bacterial endotoxin), and (b) to determine the effects of hepatic cell volume changes due to anisotonicity or by infusion of certain metabolites, stress hormones and by induction of oxidative stress on production of NO from the iNOS-induced perfused liver of singhi catfish. Intra-peritoneal injection of LPS led to induction of iNOS gene and localized tissue specific expression of iNOS enzyme with more production and accumulation of NO in different tissues of singhi catfish. Further, changes of hydration status/cell volume, caused either by anisotonicity or by infusion of certain metabolites such as glutamine plus glycine and adenosine, affected the NO production from the perfused liver of iNOS-induced singhi catfish. In general, increase of hydration status/cell swelling due to hypotonicity caused decrease, and decrease of hydration status/cell shrinkage due to hypertonicity caused increase of NO efflux from the perfused liver, thus suggesting that changes in hydration status/cell volume of hepatic cells serve as a potent modulator for regulating the NO production. Significant increase of NO efflux from the perfused liver was also observed while infusing the liver with stress hormones like epinephrine and norepinephrine, accompanied with decrease of hydration status/cell volume of hepatic cells. Further, oxidative stress, caused due to infusion of t-butyl hydroperoxide and hydrogen peroxide separately, in the perfused liver of singhi catfish, resulted in significant increase of NO efflux accompanied with decrease of hydration status/cell volume of hepatic cells. However, the reasons for these cell volume-sensitive changes of NO efflux from the liver of singhi catfish are not fully understood with the available data. Nonetheless, enhanced or decreased production of NO from the perfused liver under osmotic stress, in presence of stress hormones and oxidative stress reflected its potential role in cellular homeostasis and also for better adaptations under environmental challenges. This is the first report of osmosensitive and oxidative stress-induced changes of NO production and efflux from the liver of any teleosts. Further, the level of expression of iNOS in this singhi catfish could also serve as an important indicator to determine the pathological status of the external environment.