Developmental and hormone-induced changes of mitochondrial electron transport chain enzyme activities during the last instar larval development of maize stem borer, Chilo partellus (Lepidoptera: Crambidae).

The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F1F0ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development.

20-hydroxyecdysone mediates fat body arylphorin regulation during development of rice moth, Corcyra cephalonica.

Arylphorin hexamerins are one of the major insect storage proteins involved in diverse functions during metamorphosis. However, their regulation during development is not elucidated so far. In the present study, we documented 20-hydroxyecdysone (20E)-mediated regulation of arylphorin expression in the fat body of the stored grain pest, Corcyra cephalonica. Based on the differential developmental expression and 20E-induced transcriptional as well as translational level alterations of arylphorin, we isolated the 5' upstream region of the gene to analyze regulatory motifs. Promoter motif analysis revealed the presence of ecdysone response element (ERE). Transient transfection studies showed the functionality of the ERE. Enzyme mobility shift experiments with radiolabelled, cold and mutated probes indicate ERE-nuclear factor binding. This study is the first to report transcriptional regulation of arylphorins by 20E in lepdopteran insect species.

Early exposure of 17α-ethynylestradiol and diethylstilbestrol induces morphological changes and alters ovarian steroidogenic pathway enzyme gene expression in catfish, Clarias gariepinus.

Environmental estrogens are major cause of endocrine disruption in vertebrates, including aquatic organisms. Teleosts are valuable and popular models for studying the effects of endocrine disrupting chemicals (EDCs) in the environment. In the present study, we investigated the changes caused by exposure to the synthetic estrogens 17α-ethynylestradiol (EE2 ) and diethylstilbesterol (DES) during early stages of growth and sex differentiation of air-breathing catfish, Clarias gariepinus, at the morphological, histological, and molecular levels. Catfish hatchlings, 0 day post hatch (dph) were exposed continuously to sublethal doses of EE2 (50 ng/L) and DES (10 ng/L) until 50 dph and subsequently monitored for ovarian structural changes and alteration in the gene expression of steroidogenic enzymes till adulthood. Treated fish exhibited morphological deformities such as spinal curvature, stunted growth, and yolk-sac fluid retention. In addition to ovarian atrophy, DES-treated fish showed either rudimentary or malformed ovaries. Detailed histological studies revealed precocious oocyte development as well as follicular atresia. Further, transcript levels of various steroidogenic enzyme and transcription factor genes were altered in response to EE2 and DES. Activity of the rate-limiting enzyme of estrogen biosynthesis, aromatase, in the ovary as well as the brain of treated fish was in accordance with transcript level changes. These developmental and molecular effects imparted by EE2 and DES during early life stages of catfish could demonstrate the deleterious effects of estrogen exposure and provide reliable markers for estrogenic EDCs exposure in the environment.

Two-dimensional proteomic analysis of gonads of air-breathing catfish, Clarias batrachus after the exposure of endosulfan and malathion.

Endocrine disrupting chemicals have raised public concern, since their effects have been found to interfere with the physiological systems of various organisms, especially during critical stage of development and reproduction. Endosulfan and malathion, pesticides widely used for agricultural purposes, have been known to disrupt physiological functions in aquatic organisms. The current work analyzes the effects of endosulfan (2.5 parts per billion [ppb]) and malathion (10 ppb) on the reproductive physiology of catfish (Clarias batrachus) by evaluating protein expression profiles after 21 days of exposure. The proteomic profile of testis and ovary after exposure to endosulfan showed downregulation of proteins such as ubiquitin and Esco2, and upregulation in melanocortin-receptor-2 respectively. Malathion exposed ovary showed upregulated prolactin levels. Identification of proteins differentially expressed in gonads due to the exposure to these pesticides may serve as crucial indications to denote their disruptive effects at the level of proteins.

Gene expression analysis in gonads and brain of catfish Clarias batrachus after the exposure of malathion.

Pesticides like malathion have the potential to disrupt development and reproduction of aquatic organisms including fishes. To investigate the likely consequences of malathion exposure at low doses in juvenile catfish, Clarias batrachus, we studied the expression pattern of genes encoding certain transcription factors, activin A, sex steroid or orphan nuclear receptors and steroidogenic enzymes which are known to be involved in gonadal development along with histological changes. To compare further, we also analyzed certain brain specific genes related to gonadal axis. Fifty days post hatch catfish fingerlings were exposed continuously to 1 and 10 µg/L of malathion for 21 days. Results from these experiments indicated that transcript levels of various genes were altered by the treatments, which may further affect the gonadal development either directly or indirectly through brain. Histological analysis revealed slow progression of spermatogenesis in testis, while in ovary, the oil droplet oocytes were found to be higher after treatment (10 µg/L). Our findings revealed that the exposure of malathion, even at low doses, hinder or modulate early gonadal development differentially by targeting gene expression pattern of transcription factors, activin A, sex steroid or orphan nuclear receptors and steroidogenic enzymes with an evidence on histological changes. Further, some of the genes showed differential expression at the level of brain in male and female sex after the exposure of malathion.

Endosulfan and flutamide impair testicular development in the juvenile Asian catfish, Clarias batrachus.

Endosulfan and flutamide, a widely used pesticide and a prostate cancer/infertility drug, respectively, have an increased risk of causing endocrine disruption if they reach water bodies. Though many studies are available on neurotoxicity/bioaccumulation of endosulfan and receptor antagonism of flutamide, only little is known about their impact on testicular steroidogenesis at molecular level. Sex steroids play an important role in sex differentiation of lower vertebrates including fishes. Hence, a small change in their levels caused by endocrine disruptors affects the gonadal development of aquatic vertebrates significantly. The aim of this study was to evaluate the effects of endosulfan and flutamide on testis-related transcription factor and steroidogenic enzyme genes with a comparison on the levels of androgens during critical period of catfish testicular development. We also analyzed the correlation between the above-mentioned genes and catfish gonadotropin-releasing hormone (cfGnRH)-tryptophan hydroxylase2 (tph2). The Asian catfish, Clarias batrachus males at 50 days post hatch (dph) were exposed to very low dose of endosulfan (2.5 µg/L) and flutamide (33 µg/L), alone and in combination for 50 days. The doses used in this study were far less than those used in the previous studies of flutamide and reported levels of endosulfan in surface water and sediments. Sampling was done at end of the treatments (100 dph) to perform testicular germ cell count (histology), measurements of testosterone (T) and 11-ketotestosterone (11-KT) by enzyme immunoassay and transcript quantification by quantitative real-time PCR. In general, treatments decreased the expression of several genes including testis-related transcription factors (dmrt1, sox9a and wt1), steroidogenic enzymes (11ß-hsd2, 17ß-hsd12 and P450c17), steroidogenic acute regulatory protein and orphan nuclear receptors (nr2c1 and Ad4BP/SF-1). In contrast, the transcripts of cfGnRH and tph2 were elevated in the brain of all treated groups with maximum elevation in the endosulfan group. However, combination of endosulfan and flutamide (E+F) treatment showed minor antagonism in a few results of transcript quantification. Levels of T and 11-KT were elevated after flutamide and E+F treatments while no change was seen in the endosulfan group signifying the effect of flutamide as an androgen receptor antagonist. All the treatments modulated testis growth by decreasing the progression of differentiation of spermatogonia to spermatocytes. Based on these results, we suggest that the exposure to endosulfan and flutamide, even at low doses, impairs testicular development either directly or indirectly at the level of brain.

Endosulfan and flutamide, alone and in combination, target ovarian growth in juvenile catfish, Clarias batrachus.

Juvenile Catfish(es), Clarias batrachus of 50 days post hatch (dph) were exposed to endosulfan (2.5 parts per billion [ppb]) and flutamide (33 ppb), alone and in combination for 50 days to access their impact on ovarian development. The doses used in this study were nominal considering pervious reports. Sampling was done at 100 dph to perform histology and measurement of various transcripts, estradiol-17ß and aromatase activity. In general, treatments enhanced expression of ovary-specific transcription factors, steroidogenic enzymes steroidogenic acute regulatory protein and aromatases while transcripts of tryptophan hydroxylase2 (tph2) and catfish gonadotropin-releasing hormone declined in the brain of all treated groups with maximum reduction in the endosulfan group. Significant reduction of tph2 immunoreactivity in the forebrain/telencephalon-preoptic area endorsed our results. Increased number of pre-vitellogenic and less immature oocytes in the treated groups indicated hastened ovarian growth. Elevated ovarian aromatase activity and plasma estradiol-17ß levels were noticed in the treated groups with maximum being in the endosulfan group. These data together demonstrate that the exposure of endosulfan causes synchronous precocious ovarian development better than flutamide, alone or in combination. Our results suggest that both endosulfan and flutamide alter ovarian growth by triggering precocious development in catfish.

Gender differences in tryptophan hydroxylase-2 mRNA, serotonin, and 5-hydroxytryptophan levels in the brain of catfish, Clarias gariepinus, during sex differentiation.

Tryptophan hydroxylase (tph) is the key regulator in serotonin (5-HT) biosynthesis that stimulates the release of GnRH and gonadotropins by acting at the level of hypothalamo-hypophyseal axis. In brain, 5-HT is expressed predominantly in preoptic area-hypothalamus (POA-HYP) region in teleosts. Therefore, in the present study we isolated tph2 from catfish brain to evaluate its expression pattern in male and female brains during early development. Tph2 cloned from catfish brain is 2.768 Kb in length which encodes predicted protein of 488 amino acid residues. The characterization of recombinant tph2 was done by transient transfection in CHO cells. Tissue distribution of tph2 revealed ubiquitous expression except ovary. Real time PCR analysis in discrete regions of adult male brain revealed that tph2 mRNA was abundant in the POA-HYP and optic tectum+cerebellum+thalamus (OCT) regions. Differential expression of tph2 was observed at mRNA and protein levels in the POA-HYP and OCT regions of male and female brains during development that further correlate with the 5-hydroxytryptophan (5-HTP) and 5-HT levels measured using HPLC method in these regions of male and female brains. Tph2 immunoreactive neurons were observed in different regions of brain at 50 days post hatch using catfish specific tph2 antibody. Changes in tph2 mRNA expression, 5-HTP, and 5-HT levels in the POA-HYP+OCT region of brains of methyltestosterone and para-chlorophenylalanine treated fishes during development further endorse our results. Based on our results, we propose that the serotonergic system is involved in brain sex differentiation in teleosts.

Cloning, expression and enzyme activity analysis of testicular 11beta-hydroxysteroid dehydrogenase during seasonal cycle and after hCG induction in air-breathing catfish Clarias gariepinus.

A full-length cDNA encoding 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) was cloned from testis of air-breathing catfish, Clarias gariepinus which showed high sequence homology to zebrafish and eel. The open reading frame of 11beta-HSD2 was then transfected to COS-7 cells, which converted 11beta-hydroxytestosterone (11-OHT) to 11-ketotestosterone (11-KT). Using NAD(+), 11beta-HSD2 from testicular microsomes oxidized 11-OHT with apparent K(m) 56+/-4nM and V(max) 55+/-6pmol/h/mgprotein values. Tissue distribution analysis revealed prominent expression in testis, anterior kidney, liver and gills. Expression of 11beta-HSD2 in testis and serum levels of 11-KT were high in the prespawning phase. Administration of human chorionic gonadotropin (hCG) during prespawning and resting phases revealed initial rise in 11beta-HSD2 transcript at 4h followed by gradual increase at 8h, 12h and peaking at 24h, only in testis of prespawning phase. Rate of conversion of 11-OHT to 11-KT by testicular microsomes during different testicular phases and after hCG administration corroborated well with the expression of 11beta-HSD2. Ontogeny study indicated that this enzyme is expressed during testicular development. Thus the spatio-temporal expression supported with putative dehydrogenase activity and circulating 11-KT levels clearly suggest a major role for 11beta-HSD2 during testicular differentiation and seasonal testicular cycle in catfish.

Bowman-Birk proteinase inhibitor from Cajanus cajan seeds: purification, characterization, and insecticidal properties.

A red gram proteinase inhibitor (RgPI) was purified from red gram ( Cajanus cajan ) seeds by using ammonium sulfate precipitation and ion-exchange, affinity, and gel filtration chromatography. SDS-PAGE under nonreducing condition revealed two protein bands with molecular masses of approximately 8.5 and approximately 16.5 kDa corresponding to monomeric and dimeric forms of RgPI, respectively. Similarly, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry also confirmed the presence of dimer as well as other oligomeric forms: trimer, tetramer, and pentamer. Reduction of RgPI with dithiothreitol (DTT) led to the dissociation of the dimeric and oligomeric forms. Native-PAGE and two-dimensional gel electrophoresis indicated the existence of isoinhibitors with pI values of 5.95, 6.25, 6.50, 6.90, and 7.15, respectively. The MALDI-TOF-TOF mass spectrum and N-terminal sequence 'DQHHSSKACC' suggested that the isolated RgPI is a member of the Bowman-Birk inhibitor family. RgPI exhibited noncompetitive type inhibitory activity against bovine pancreatic trypsin and chymotrypsin, with inhibition constants of 292 and 2265 nM, respectively. It was stable up to a temperature of 80 degrees C and was active over a wide pH range between 2 and 12. However, reduction with DTT or 2-mercaptoethanol resulted in loss of inhibitory activity against trypsin and chymotrypsin. It also decreased the activity of larval midgut trypsin-like proteinases in Manduca sexta . Its insecticidal property was further confirmed by reduction in the growth and development of these larvae, when supplemented in the diet.

Dimorphic expression of tryptophan hydroxylase in the brain of XX and XY Nile tilapia during early development.

Serotonin (5-HT) is well known for modulating the release of GnRH and gonadotropin in teleosts. Reports on increased female:male ratio after the blockade of 5-HT biosynthesis proposed a role for 5-HT in brain sex differentiation. Two types of tryptophan hydroxylase (Tph), rate-limiting enzyme in the biosynthesis of 5-HT were cloned from vertebrates. In the present study, we cloned Tph from brain and evaluated its importance during early development of XX and XY Nile tilapia. Tph cloned from tilapia brain is 1888 bp in length and it encodes predicted protein of 462 amino acid residues. Tph activity of tilapia was confirmed by demonstrating the conversion of L-tryptophan to 5-hydroxy tryptophan by the recombinant protein after transient transfection of this cDNA clone in COS-7 cells. Northern blot identified single transcript around 2kb in male brain. Tissue distribution of Tph revealed high abundance in brain, kidney, liver and testis. Semi-quantitative RT-PCR revealed exclusive expression of Tph in the male brain from 5 to 20 days post hatch (dph) while in the female brain, it was from 25 dph. These results were authenticated by localization of Tph transcripts in olfactory bulb-telencephalon region of 11 dph male brain using in situ hybridization. Tph immunoreactivity (-ir) was also evident in the nucleus preopticus-periventricularis area of male brain as early as 12 dph. However, Tph-ir was observed in several regions of both male and female brain without any distinction from 30 dph. Dimorphic expression pattern of Tph during early brain development around the critical period (7-21 dph) of gonadal sex determination and differentiation may implicate a role for Tph in brain sex differentiation of tilapia.

Purification and characterization of a Bowman-Birk proteinase inhibitor from the seeds of black gram (Vigna mungo).

A proteinase inhibitor (BgPI) was purified from black gram, Vigna mungo (cv. TAU-1) seeds by using ammonium sulfate fractionation, followed by ion-exchange, affinity and gel-filtration chromatography. BgPI showed a single band in SDS-PAGE under non-reducing condition with an apparent molecular mass of approximately 8kDa correlating to the peak 8041.5Da in matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum. BgPI existed in different isoinhibitor forms with pI values ranging from 4.3 to 6.0. The internal sequence "SIPPQCHCADIR" of a peak 1453.7 m/z, obtained from MALDI-TOF-TOF showed 100% similarity with Bowman-Birk inhibitor (BBI) family. BgPI exhibited non-competitive-type inhibitory activity against both bovine pancreatic trypsin (K(i) of 309.8nM) and chymotrypsin (K(i) of 10.7muM), however, with a molar ratio of 1:2 with trypsin. BgPI was stable up to a temperature of 80 degrees C and active over a wide pH range between 2 and 12. The temperature-induced conformational changes in secondary structure are reversed when BgPI was cooled from 90 to 25 degrees C. Further, upon reduction with dithiothreitol, BgPI lost both its inhibitory activity as well as secondary structural conformation. Lysine residue(s) present in the reactive site of BgPI play an important role in inhibiting the bovine trypsin activity. The present study provides detailed biochemical characteristic features of a BBI type serine proteinase inhibitor isolated from V. mungo.

Cloning and expression of StAR during gonadal cycle and hCG-induced oocyte maturation of air-breathing catfish, Clarias gariepinus.

Complementary DNAs encoding steroidogenic acute regulatory protein (StAR) have been isolated from different fish species, yet the relevance of StAR during gonadal cycle and more importantly in final oocyte maturation has not been assessed so far. A cDNA encoding StAR was isolated from the ovarian follicles of air-breathing catfish, Clarias gariepinus. Catfish StAR exhibited 55 to 72% identity at nucleotide level with other vertebrate orthologs. RT-PCR analysis of tissue distribution pattern demonstrated the presence of StAR mRNA in various tissues including gonads, kidney, liver, brain and intestine of catfish. Real-time RT-PCR analysis revealed high expression of StAR mRNA in the pre-spawning phase of ovary while it was low in preparatory, spawning and regressed phases. In testis, maximum expression was noticed during the preparatory phase. During human chorionic gonadotropin (hCG)-induced oocyte maturation, both in vitro and in vivo, StAR mRNA levels were augmented by 2 h and then declined gradually to reach basal levels by 12 h as that of saline-treated controls. Taken together, high level of expression during hCG-induced oocyte maturation vis-à-vis in spawning suggests a role for StAR, in addition to the steroidogenic enzyme genes in final oocyte maturation.

Inhibitors from pigeonpea active against lepidopteran gut proteinases.

The proteinase inhibitors (PIs) active against bovine pancreatic trypsin, chymotrypsin, and insect midgut trypsin-like proteinases were found in the seeds of 14 cultivars and eight wild types of pigeonpea, Cajanus cajan L.. The inhibitory activity of PIs against trypsin and chymotrypsin, as well as their activity profile on gelatin-polyacrylamide gel electrophoresis (PAGE) were identical among the various cultivars. In contrast to cultivars, the wild types showed differences in inhibitory activity of PIs and their activity profile on gelatin-PAGE. The PIs from all cultivars and few wild types showed 10- to 50-fold higher activity against midgut trypsin-like proteinases of Achaea janata (L.) (Lepidoptera: Noctuidae), compared with bovine pancreatic trypsin. However, the PIs from both cultivars and wild types showed three- to nine-fold less activity against Spodoptera litura (F.) (Lepidoptera: Noctuidae) midgut trypsin-like proteinases, compared with bovine pancreatic trypsin. This inhibitory potential of PIs from cultivars and wild types, toward midgut trypsin-like proteinases from A. janata was further evident by the strong activity profile visualized on gelatin-PAGE. These results further suggest that the inhibitory potential of PIs from pigeonpea cultivars and wild types could be exploited in management of nonhost insects.

Fulminant hepatic failure in rats induces oxidative stress differentially in cerebral cortex, cerebellum and pons medulla.

Hepatic Encephalopathy (HE) is one of the most common complications of acute liver diseases and is known to have profound influence on the brain. Most of the studies, available from the literature are pertaining to whole brain homogenates or mitochondria. Since brain is highly heterogeneous with functions localized in specific areas, the present study was aimed to assess the oxidative stress in different regions of brain-cerebral cortex, cerebellum and pons medulla during acute HE. Acute liver failure was induced in 3-month old adult male Wistar rats by intraperitoneal injection of thioacetamide (300 mg/kg body weight for two days), a well known hepatotoxin. Oxidative stress conditions were assessed by free radical production, lipid peroxidation, nitric oxide levels, GSH/GSSG ratio and antioxidant enzyme machinery in three distinct structures of rat braincerebral cortex, cerebellum and pons medulla. Results of the present study indicate a significant increase in malondialdehyde (MDA) levels, reactive oxygen species (ROS), total nitric oxide levels [(NO) estimated by measuring (nitrites + nitrates)] and a decrease in GSH/GSSG ratio in all the regions of brain. There was also a marked decrease in the activity of the antioxidant enzymes-glutathione peroxidase, glutathione reductase and catalase while the super oxide dismutase activity (SOD) increased. However, the present study also revealed that pons medulla and cerebral cortex were more susceptible to oxidative stress than cerebellum. The increased vulnerability to oxidative stress in pons medulla could be due to the increased NO levels and increased activity of SOD and decreased glutathione peroxidase and glutathione reductase activities. In summary, the present study revealed that oxidative stress prevails in different cerebral regions analyzed during thioacetamide-induced acute liver failure with more pronounced effects on pons medulla and cerebral cortex.

Co-administration of C-Phycocyanin ameliorates thioacetamide-induced hepatic encephalopathy in Wistar rats.

Fulminant hepatic failure (FHF) is a condition with a sudden onset of necrosis followed by degeneration of hepatocytes, without any previously established liver disease, generally occurring within hours or days. FHF is associated with a wide spectrum of neuropsychiatric alterations ranging from stupor to coma, culminating in death. In the present study FHF was induced in rats by the administration of thioacetamide (TAA). Oxidative stress is thought to play a prominent role in the pathophysiology of cerebral changes during FHF leading to the assumption that antioxidants might offer protection. Hence, in the present study the protective effect of C-Phycocyanin (C-PC), a natural antioxidant, was evaluated on TAA-induced tissue damage. C-Phycocyanin was administered intraperitoneally twice at 24 h interval (50 mg/kg body weight) along with the hepatotoxin TAA (300 mg/kg body weight). The animals were sacrificed 18 h after the second injection of TAA treatment and various biochemical parameters were analysed in liver, serum and brain tissues. These studies revealed significant prevention of TAA-induced liver damage by C-PC, as evidenced by a) increase in survival rate; b) the prevention of leakage of liver enzymes (AAT and AST) and ammonia into serum; c) increase in prothrombin time and d) liver histopathology. Ultrastructural studies of astrocytes of different regions of brain clearly showed a decrease in edema after C-PC treatment. TAA-induced histopathological lesions in different regions of the brain namely cerebral cortex, cerebellum and pons medulla were significantly reduced by the co-administration of C-PC with TAA. Further C-PC treatment resulted in a) decrease in the levels of tryptophan and markers of lipid peroxidation and b) elevation in the activity levels of catalase, glutathione peroxidase in different regions of brain. These studies reveal the potential of C-PC in ameliorating TAA-induced hepatic encephalopathy by improving antioxidant defenses.

Changes in cerebral membrane lipid composition and fluidity during thioacetamide-induced hepatic encephalopathy.

Lipids are an essential structural and functional component of cellular membranes. Changes in membrane lipid composition are known to affect the activities of many membrane-associated enzymes, endocytosis, exocytosis, membrane fusion and neurotransmitter uptake, and have been implicated in the pathophysiology of many neurodegenerative disorders. In the present study, we investigated changes in the lipid composition of membranes isolated from the cerebral cortex of rats treated with thioacetamide (TAA), a hepatotoxin that induces fulminant hepatic failure (FHF) and thereon hepatic encephalopathy (HE). HE refers to acute neuropsychiatric changes accompanying FHF. The estimation of membrane phospholipids, cholesterol and fatty acid content in cerebral cortex membranes from TAA-treated rats revealed a decrease in cholesterol, phosphatidylserine, sphingomyelin, a monounsaturated fatty acid, namely oleic acid, and the polyunsaturated fatty acids gamma-linolenic acid, decosa hexanoic acid and arachidonic acid compared with controls. Assessment of membrane fluidity with pyrene, 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene revealed a decrease in the annular membrane fluidity, whereas the global fluidity was unaffected. The level of the thiobarbituric acid reactive species marker for lipid peroxidation also increased in membranes from TAA-treated rats, thereby indicating the prevalence of oxidative stress. Results from the present study demonstrate gross alterations in cerebral cortical membrane lipid composition and fluidity during TAA-induced HE, and their possible implications in the pathogenesis of this condition are also discussed.

Thiourea-induced thyroid hormone depletion impairs testicular recrudescence in the air-breathing catfish, Clarias gariepinus.

We used thiourea-induced thyroid hormone depletion as a strategy to understand the influence of thyroid hormones on testicular recrudescence of the air-breathing catfish, Clarias gariepinus. Treatment with 0.03% thiourea via immersion for 21 days induced hypothyroidism (thyroid hormone depletion) as evidenced by significantly reduced serum T(3) levels. Thiourea-treated males had narrowed seminiferous lobules with fewer spermatozoa in testis, very little or no secretory fluid, reduced protein and sialic acid levels in seminal vesicles when compared to controls. The histological changes were accompanied by reduction in serum and tissue levels of testosterone (T) and 11-ketotestosterone (11-KT), a potent male specific androgen in fish. Qualitative changes in the localization of catfish gonadotropin-releasing hormone (cfGnRH) and luteinizing hormone (LH, heterologous system) revealed a reduction in the distribution of immunoreactive neuronal cells and fibers in thyroid depleted fish. Interestingly, thiourea-withdrawal group showed physiological and histological signs of recovery after 21 days such as reappearance of spermatozoa and partial restoration of 11-KT and T levels. These data demonstrate that thyroid hormones play a significant role in testicular function of catfish. The mechanism of action includes modulating sex steroids either directly or through the hypothalamo (GnRH)-hypophyseal (LH) axis.

Complete sequence, expression and evolution of two members of the hexamerin protein family during the larval development of the rice moth, Corcyra cephalonica.

Three distinct types of storage hexamerins are expressed in the "last-instar" larvae of the rice moth, Corcyra cephalonica. A cDNA expression library was constructed from fat body-RNA and screened with a polyclonal antibody raised against purified hexamerin (SP2) of Corcyra cephalonica. Two slightly different "full-length" hexamerin cDNA clones (Hex2a and Hex2b) were isolated and sequenced. Both include open reading frames of 2109 bp which are translated into polypeptides of 703 amino acids with 92.5% identity. Signal peptides of 19 amino acids are present at the N-termini. The 684 amino acids native proteins have a high content of aryl groups (17.6%). According to both the criteria for amino acid composition and the phylogenetic analysis, Hex2a and Hex2b belong to the lepidopteran arylphorins. Northern blot studies revealed that the Hex2 genes are species- and tissue-specifically expressed in fat body cells of "last-instar" (= 5th) larvae.

Identification, characterization, immunocytochemical localization, and developmental changes in the activity of calcium/calmodulin-dependent protein kinase II in the CNS of Bombyx mori during postembryonic development.

In the present investigation, in vitro phosphorylation of CNS proteins of the silkworm Bombyx mori during the postembryonic development have been studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of phosphorylated proteins revealed the presence of major phosphoproteins of 59/60 kDa. Based on molecular mass, calcium/calmodulin-dependent autophosphorylation, substrate specificity, KN-62 inhibition, apparent Km for ATP and syntide-2, these proteins were identified as calcium/calmodulin-dependent protein kinase II (CaM kinase II). Anti-rat CaM kinase II monoclonal antibody showed immunoreactivity with Bombyx CaM kinase II isoforms. This kinase showed a high degree of autophosphorylation in neural tissue. During postembryonic development of Bombyx, two distinct peaks of enzyme activity could be noticed, one at the late-larval and another at the late-pupal stage, which were associated with an increase in amount of the enzyme. These results suggested that the expression of CaM kinase II in the CNS of Bombyx was developmentally regulated.