Structural modeling and gene expression analysis of phosvitinless vitellogenin (vgc) in the Indian freshwater murrel, Channa punctatus (Bloch, 1793).

Vitellogenin (Vg) is a female-specific egg-yolk precursor protein, synthesized in the liver of fish in response to estrogens. In the present study, complete gene of phosvitinless vitellogenin (vgc) was sequenced, its 3D structure was predicted and validated by web-based softwares. The complete nucleotide sequence of vgc was 4126 bp which encodes for 1272 amino acids and showed the presence of three conserved domains viz. LPD_N, DUF1943 and DUF1944. The retrieved amino acid sequence of VgC protein was subjected to in silico analysis for understanding the structural and functional properties of protein. mRNA levels of multiple vg genes have also been quantified during annual reproductive cycle employing qPCR. A correlation has been observed between seasonal changes in gonadosomatic index with estradiol levels and hepatic expression of three types of vg genes (vga, vgb, vgc) during ovarian cycle of murrel. During preparatory phase, when photoperiod and temperature are low; low titre of E2 in blood induces expression of vgc gene. A rapid increase in the levels of E2 favours induction of vgb and vga genes in liver of murrel during early pre-spawning phase when photoperiod is long and temperature is high in nature. These results suggest that among three vitellogenin proteins, VgC is synthesized earlier than VgA and VgB during oogenesis.

Molecular cloning and bioinformatic characterization of Gonadotropin Inhibitory Hormone (GnIH) and its receptors in the freshwater murrel, Channa punctatus (Bloch, 1793).

Gonadotropin inhibitory hormone belonging to the RFamide peptide family, a hypothalamic neuropeptide, regulates Hypothalamus-pituitary-gonadal (HPG) axis and inhibits gonadal development. GnIH polypeptide precursor has an Arg-Phe-NH2 (RFamide) motif at the C-terminal, which has LPXRF (X = Q or L) domain. The actions of GnIH are mediated through G-protein coupled receptors and upto three receptors have been characterized in many teleosts. GnIH exerts its inhibitory effect on the HPG axis through direct interaction with GnRH and Kisspeptin neurons in the brain and acts directly on the pituitary gonadotrophs. To decipher the role of GnIH in Indian freshwater murrel, Channa punctatus, we sequenced the cDNA encoding GnIH and its two receptors. The identified GnIH mRNA encodes three RFamide peptides having -MPMRF, -MPQRF, and -LPQRFamide motifs. In silico analysis of the amino acid sequence of GnIH exhibits its molecular and functional properties and the protein-protein interaction with significant factors regulating the HPG axis. The 3-D structure of GnIH and its receptors, provides more relevant information about the active residues of these proteins which might be involved in their functioning and interaction with other proteins. Molecular dynamic simulation of GnIH protein has provided more insight into its dynamic behavior. The expression of GnIH and its receptors, shows an inverse correlation with gonadal development during the annual reproductive cycle.

Role of cathepsins B and D in proteolysis of yolk in the catfish Clarias gariepinus.

Yolk processing pathways vary in the oocytes of benthophil and pelagophil teleosts. The present study investigated the yolk processing pattern in the oocytes of the fresh water catfish Clarias gariepinus at vitellogenic, maturation, and ovulated stages. This study concludes that during maturation stage, an electrophoretic shift in the major peptide band on Polyacrylamide gel electrophoresis (PAGE) occurs due to a decrease in the size of the yolk protein. The PMF spectrum of corresponding peptides from vitellogenic and ovulated oocytes revealed a difference in the minor ions. A minor difference in the molecular weight of the corresponding peptides occurs due to a difference in their amino acid composition. Maximal activity of the proteases cathepsin D and cathepsin B was observed in the vitellogenic oocytes, thus confirming their role in the processing of yolk. A significant transient increase in the activity of cathepsin B in the mature oocytes also suggests its role in oocyte maturation.

In silico analysis of kiss2, expression studies and protein-protein interaction with gonadotropin-releasing hormone 2 (GnRH2) and luteinizing hormone beta (LHß) in Heteropneustes fossilis.

Kisspeptins, encoded by the kiss genes, are neuropeptides that regulate the onset of puberty, maturation of gonads, and fertility in higher vertebrates including fishes. The gene ontology suggests that kisspeptin plays an important role not only in reproduction but also in cell signaling, immune response and metabolic processes, and to decipher protein-protein interactions, computational approach has been favored. The present investigation focuses on the detailed structural analysis and molecular docking of kiss2 gene using in silico tools. A putative kiss2 protein of 113 amino acids was encoded by an open reading frame of 342 bp kiss2 gene. The protein is of 13.12 kDa with isoelectric point of 9.45. The secondary structure of the protein indicates more than 50% random coils, followed by 34% of alpha helix and 13% extended strand. The protein was found to be extracellular and secretory in nature. Since, protein-protein interactions play a very crucial role in every cellular process and due to unavailability of crystal structure of our protein of interest in fishes computational approach has been employed. The 3D PDB modeling and the molecular docking of kiss2, Gonadotropin-releasing hormone 2 (GnRH2) and luteinizing hormone beta (LHß) proteins in fishes have been demonstrated applying protein-docking approach. Molecular interactions of kiss2 protein were the highest with kisspeptin receptor 2 and lowest for the neuropeptide FF-amide peptide precursor protein. Expression of kiss2 transcripts, mainly in the brain and ovary of H. fossilis, supports its hypothalamic-pituitary-gonadal axis signaling and reproductive function. Further, changes in expression patterns of kiss2 mRNA during different developmental stages, indicate its potential role in embryonic development also. The present study conclusively reveals interaction of kiss2 with other neuropeptides. Prediction of binding structures and identification of key residues in protein-protein interaction illustrate direct interaction among these proteins, playing a cardinal role in neuroendocrine regulation of reproduction in catfish. Communicated by Ramaswamy H. Sarma.

Amelioration of immune and digestive system through weed supplemented feed against Aeromonas hydrophila in Clarias gariepinus.

Aquaculture is one of the important globally growing industries. It serves as an important food source of protein for human beings. With the expanding demand for the fish and their products it has become extremely important to improve the aquaculture practices. Aquaculture in India has witnessed huge mortalities caused by bacteria, viruses, fungi, nematodes etc. Aquatic weeds plants are harmful for aquaculture in many ways. Present study is aimed to overcome the disease caused by Aeromonas hydrophila (fish pathogenic bacteria) through feed supplementation of two aquatic weed plants (Azolla pinnata and Ceratophyllum demersum). The fish were divided into 6 groups: experimental groups (fish fed on supplementary feed at 5% and 2.5% concentration for individual plant and challenged with bacteria), positive control (fish fed on non-supplemented feed and challenged with bacteria) and negative control (fish fed on non-supplementary feed and not challenged with bacteria). It was observed that supplemented feed enhanced both cell mediated and humoral immunity in fish. Therefore, we advocate that feed formulated with incorporation of Azolla pinnata and Ceratophyllum demersum leaf powder at 5% and 2.5% could be used to prevent disease caused by A. hydrophila or can be used to enhance fish health by boosting its immune system. The results of this study also showed an improved digestibility in fish fed on supplemented feed.

Structural analysis and characterization of egg-envelope in the Indian freshwater murrel, Channa punctatus.

Egg-envelope, an acellular coat, surrounds the egg and is essential for vitellogenin incorporation. It also plays a pivotal role during fertilization and provides protection to the developing embryo. In the present study, scanning electron microscopy was used to elucidate the structural details of isolated egg-envelopes from the Indian freshwater murrel, Channa punctatus. Several pores and single micropyle were observed on outer surface, whereas inner layer indicated deposition of proteinaceous material. The constituent proteins of egg-envelope were further characterized by Fourier transform infrared (FT-IR) spectroscopy, and electrophoresis and mass-spectrometry (MALDI-TOF-MS/MS). The secondary structure of egg-envelope proteins showed the presence of antiparallel ß-pleated sheets and aromatic amino acids. These proteins resolved into two peptides (130 kDa and 68 kDa) under denaturing conditions, which exhibited glycoprotein nature. The peptide band with low molecular mass showed significant similarity with transmembrane protein, whereas peptide band with high molecular mass matched with choriogenin protein of other fishes. These results confirm that chorion is derived from precursor protein, Choriogenin, in murrel. Chemical composition of egg-envelope supports that chorion is responsible exchange material and chemical defence during embryogenesis.

Ligand and Structure Based Models for the Identification of Beta 2 Adrenergic Receptor Antagonists.

Ligand bound beta 2 adrenergic receptor (ß2AR) crystal structures are in use for screening of compound libraries for identifying inducers and blockers. However, in case of G protein coupled receptors (GPCR), docking and binding energy (BE) calculations are not enough to discriminate agonist and antagonists. Absence of a reliable model for discriminating ß2AR antagonist is still a major hurdle. Docking of known antagonists and agonists into activated and ground state ß2AR revealed several key intermolecular interactions and H-bonding patterns, which in combination, emerged as a model for prediction of antagonists. Present study identifies an alternative binding orientation, within the binding pocket, for blockers and a minimum grid size to lessen the false positive predictions. Cluster analysis revealed structural variability among the antagonists and a conserved pattern in case of agonists. A combination of docking and structure activity relationship (SAR) model reliably discriminated antagonists. Based on key intermolecular interactions, a set of agonists and antagonists useful to SAR, quantitative structure activity relationship (QSAR) and pharmacophore modeling, has also been proposed for identifying antagonists.

Cross Talk between KGF and KITLG Proteins Implicated with Ovarian Folliculogenesis in Buffalo Bubalus bubalis.

Molecular interactions between mesenchymal-derived Keratinocyte growth factor (KGF) and Kit ligand (KITLG) are essential for follicular development. These factors are expressed by theca and granulosa cells. We determined full length coding sequence of buffalo KGF and KITLG proteins having 194 and 274 amino acids, respectively. The recombinant KGF and KITLG proteins were solubilized in 10 mM Tris, pH 7.5 and 50 mM Tris, pH 7.4 and purified using Ni-NTA column and GST affinity chromatography, respectively. The purity and molecular weight of His-KGF (~23 kDa) and GST-KITLG (~57 kDa) proteins were confirmed by SDS-PAGE and western blotting. The co-immunoprecipitation assay accompanied with computational analysis demonstrated the interaction between KGF and KITLG proteins. We deduced 3D structures of the candidate proteins and assessed their binding based on protein docking. In the process, KGF specific residues, Lys123, Glu135, Lys140, Lys155 and Trp156 and KITLG specific ones, Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 involved in the formation of KGF-KITLG complex were detected. The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface. Additionally, in-silico site directed mutagenesis enabled the assessment of changes that occurred in the binding energies of mutated KGF-KITLG protein complex. Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex. This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.

Transcriptional dynamics of homeobox C11 gene in water buffalo bubalus bubalis.

The Hox complex contains 39 genes clustered into four groups involved in cell differentiation and development. We cloned full-length sequence of Hoxc11 gene from water buffalo Bubalus bubalis, assessed its copy number, localized the same onto the chromosome 5, and studied its evolutionary conservation across the species. Northern hybridization of Hoxc11 showed a 2.2 kb band in the tissues analyzed. Real-Time PCR showed highest expression of Hoxc11 gene in lung followed by spleen, spermatozoa, and testis. Six interacting partners of this gene showed higher expression in spleen, lung, testis, and spermatozoa. During the early stages of development, Hoxc11 and its interacting partners both showed lower expression, which then became prominent during the age of 1-3 years, regressed drastically thereafter, and remained so until the animal's life time (∼ 20 years). The high expression of Hoxc11 and its interacting partners in spermatozoa and testis during the onset of puberty suggests its likely role in the differentiation of gonads and subsequent reproductive activities. Additional work on Hoxc11 especially, in the context of respiratory, immunological, and in/fertility in other species, including humans would be useful for establishing its broader biological significance towards the enrichment of functional and comparative genomics.

Morphology, morphogenesis, and molecular phylogeny of Sterkiella tetracirrata n. sp. (Ciliophora, Oxytrichidae), from the Silent Valley National Park, India.

The morphology and morphogenesis during cell division of Sterkiella tetracirrata n. sp., isolated from a soil sample collected from the Silent Valley National Park, Kerala, India, were investigated using live observation, protargol staining and scanning electron microscopy. The new species differs from its congeners by the following combination of features: cell size in vivo 85-110×35-50µm, on average 84×37µm in protargol preparations; four ellipsoidal macronuclear nodules; 31 adoral membranelles; 17 frontal-ventral-transverse cirri consisting of three frontal, four frontoventral, one buccal, three ventral, two pretransverse and invariably four transverse cirri; resting cyst with separate macronuclear nodules. Sterkiella tetracirrata differs from the similar species S. terricola in the number of transverse cirri (invariably 4 vs. 3) and in the number of adoral membranelles (24-35 vs. 22 or 23). Morphogenesis resembles that of its congeners S. nova and S. histriomuscorum. Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to either S. cavicola or S. histriomuscorum. The analyses support the morphological evidence (e.g., similarity in the oral apparatus and the dorsal kinety pattern) that Gastrostyla and Pattersoniella evolved from a Sterkiella-like ancestor.

Todralazine protects zebrafish from lethal effects of ionizing radiation: role of hematopoietic cell expansion.

The Johns Hopkins Clinical Compound Library (JHCCL), a collection of Food and Drug Administration (FDA)-approved small molecules (1400), was screened in silico for identification of novel ß2AR blockers and tested for hematopoietic stem cell (HSC) expansion and radioprotection in zebrafish embryos. Docking studies, followed by the capacity to hasten erythropoiesis, identified todralazine (Binding energy, -8.4 kcal/mol) as a potential HSC-modulating agent. Todralazine (5 µM) significantly increased erythropoiesis in caudal hematopoietic tissue (CHT) in wild-type and anemic zebrafish embryos (2.33- and 1.44-folds, respectively) when compared with untreated and anemic control groups. Todralazine (5 µM) treatment also led to an increased number of erythroid progenitors, as revealed from the increased expression of erythroid progenitor-specific genes in the CHT region. Consistent with these effects, zebrafish embryos, Tg(cmyb:gfp), treated with 5 µM todralazine from 24 to 36 hours post fertilization (hpf) showed increased (approximately two-folds) number of HSCs at the aorta-gonad-mesonephros region (AGM). Similarly, expression of HSC marker genes, runx1 (3.3-folds), and cMyb (1.41-folds) also increased in case of todralazine-treated embryos, further supporting its HSC expansion potential. Metoprolol, a known beta blocker, also induced HSC expansion (1.36- and 1.48-fold increase in runx1 and cMyb, respectively). Todralazine (5 µM) when added 30 min before 20 Gy gamma radiation, protected zebrafish from radiation-induced organ toxicity, apoptosis, and improved survival (80% survival advantage over 6 days). The 2-deoxyribose degradation test further suggested hydroxyl (OH) radical scavenging potential of todralazine, and the same is recapitulated in vivo. These results suggest that todralazine is a potential HSC expanding agent, which might be acting along with important functions, such as antioxidant and free radical scavenging, in manifesting radioprotection.

Differential expression of Homeobox C11 protein in water buffalo Bubalus bubalis and its putative 3D structure.

BACKGROUND: The Homeobox (Hox) family complex contains 39 genes, clustered into four groups (A-D) all expressing in sequential manner. The HOX proteins are transcriptional factors involved in regulation of pattern formation of the anterio-posterior body axis across the species. Most of the Hox family genes have been studied with respect to their organization and expression during the embryonic stages. However, expression pattern of Homeobox C11 (Hoxc11) gene in the 5' region, particularly in higher mammals remains largely unexplored. RESULTS: We cloned and expressed Homeobox C11 (Hoxc11) gene from water buffalo Bubalus bubalis. The recombinant HOXC11 protein expressed as inclusion bodies was solubilized in Tris buffer (10 mM, pH-6.5) and purified using Ni-NTA affinity column. The purity and molecular weight of HOXC11 protein (~33 kDa) were confirmed by SDS-PAGE and western blot analysis. Employing immunohistochemistry approach, we localized HOXC11 protein in the nuclei across the tissues of buffalo. Western blot analysis showed highest expression of HOXC11 protein in kidney and lung although its possible renal and respiratory roles are not yet established. Electrophoretic mobility shift assay (EMSA) demonstrated the specific binding of HOXC11 protein with the promoter element, CE-LPH1 of lactase-phlorizin hydrolase (LPH) gene showing reduced mobility of the protein-DNA complex, corroborating with earlier report on the possible role of this protein in intestinal functions. In silico analysis of HOXC11 showed predominance of α helices and presence of six conserved domains. We deduced the putative 3D structure of HOXC11 protein and fifteen possible DNA interacting residues within the homeodomain. CONCLUSIONS: Present study augments our understanding on the specific expression of HOXC11 protein in kidney and lung in water buffalo. The fifteen DNA interacting residues reported herein provide an opportunity to establish much broader structural and functional perspectives of HOXC11 protein in the context of genome analysis in general and animal biotechnology in particular.

Aromatase activity in brain and ovary: seasonal variations correlated with circannual gonadal cycle in the catfish, Heteropneustes fossilis.

Seasonal variations in the aromatase activity in H. fossilis estimated by a microassay were correlated with the sex steroids, vitellogenin in and ovarian weight during circannual reproductive cycle. In the female catfish, aromatase activity was detectable in the hypothalamus throughout the year whereas in ovary only during active vitellogenesis. In the catfish, hypothalamic aromatase levels increased two times during annual gonadal cycle, once in a fully gravid fish and then in a reproductively quiescent fish. On the other hand, increase in the ovarian aromatase activity was observed only during vitellogenesis, which showed a direct correlation with plasma levels of sex steroids. Further, plasma levels of testosterone and estradiol suggested a precursor-product relationship. At the completion of vitellogenesis, ovarian aromatase activity declined sharply resulting in elevation of plasma testosterone levels, which in turn could be utilized as substrate by the hypothalamic aromatase whose activity was the highest in the postvitellogenic catfish. At least two isoforms of gene, cyp19a and cyp19b, coding for aromatase in ovary and brain respectively were expressed in the catfish. Aromatase activity was more concentrated in those areas of catfish brain, which have been implicated in the control of reproduction.

Immunostimulatory effect of artificial feed supplemented with indigenous plants on Clarias gariepinus against Aeromonas hydrophila.

The antibacterial activity of methanol extracts of Ficus benghalensis (prop-root) and Leucaena leucocephala (pod seed) was evaluated by measurement of zone of inhibition against pathogenic bacteria, Escherichia coli and Aeromonas hydrophila. Control artificial feed and artificial feed supplemented with 5% powder of F. benghalensis and L. leucocephala were prepared. Juvenile Clarias gariepinus were divided into four groups, acclimatized to laboratory conditions and fed with respective feeds for 20 days prior to the experiment. Immunomodulatory response of supplementary feed was studied by challenging the fish intraperitoneally at weekly intervals, with A. hydrophila. One set of fish, not challenged with A. hydrophila was used as a negative control, to analyze any detrimental effect of supplementary feed, while positive control, comprised of challenged fish fed with non-supplemented feed. Other two groups of fish were challenged with A. hydrophila and fed with respective supplementary feeds. Blood was collected on weekly intervals for four weeks and serum samples were analyzed to evaluate the damage of fish by A. hydrophila through liver function tests. The increase in the levels of Serum glutamic oxaloacetic transaminase (SGOT) and Serum Glutamic pyruvate transaminase (SGPT) in positive control group indicated the damage of liver & kidney. However the levels did not change significantly in fish fed with supplementary feeds when compared to negative control group. Nitric oxide, SOD, ALP and lipid peroxidase indicated lower stress levels in these fish compared to positive control. Fish fed with supplementary feed showed increased lysozyme activity and phagocytic index indicating an increase in non-specific immune response. The immunoglobulin levels of in serum were analyzed by homologous sandwich ELISA, which showed higher antibody production in fish fed with supplementary feed. The current study suggests conclusively, immunostimulatory role of F. benghalensis (prop-roots) and L. leucocephala (pod seed) in C. gariepinus when supplemented in artificial feed.

Middle-redox potential laccase from Ganoderma sp.: its application in improvement of feed for monogastric animals.

The variables influencing laccase production by white-rot fungus Ganoderma sp. rckk-02 were optimized employing response surface methodology. Malt extract (6.0% w/v), lignin (0.5% w/v) and pH (5.5) were found to be the most significant factors for enhanced laccase production by 7 fold (226.0 U/ml) as compared to unoptimized growth conditions (32.0 U/ml). The N-terminal sequence of laccase revealed its distinct amino acid profile (S- I- R- N- S- G), which suggested it as a novel enzyme. The Far-UV CD spectrum of the laccase showed single broad negative trough at around 213 nm, a typical signature of all ß proteins. The laccase was found to fall in the range of middle redox potential laccases. Purified laccase at dosage of 2.5 Ug(-1) body weight when supplemented with pelleted diet of rats, a significant improvement (p < 0.05) in nutrients digestibility without causing any elevation of blood stress enzymes was observed.

Immunostimulatory response induced by supplementation of Ficus benghalensis root powder, in the artificial feed the Indian freshwater murrel, Channa punctatus.

Methanol extract from the dried aerial root of Ficus benghalensis, was used to evaluate antibacterial activity on the bacterial strains of Aeromonas hydrophila and Escherichia coli, by disc diffusion method. In order to study, if there is any immunostimulatory response of F. benghalensis, immunized fish were fed with supplementary artificial feed containing 5% F. benghalensis dried root powder. There was no marked difference in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in control and treated fish, suggesting that the supplementary feed had no adverse effect on liver or kidney. Serum lysozyme, tissue super oxide dismutase (SOD), percentage phagocytosis, phagocytotic index, nitric oxide (NO), total serum protein and immunoglobulin increased significantly in the treated fish compared to control fish. Serum immunoglobulin levels were estimated by development of a sandwich ELISA, and levels were found to increase with successive immunizations of BSA.

Design and docking studies of [diethylenetriaminepentaacetic acid-(amino acid)2] with acetylcholine receptor as a molecular imaging agent for single-photon emission computed tomographic application.

The acetylcholine receptor is an essential link between the brain and the muscles, so it is a sensitive location for attack. In this study, some reversible [diethylenetriaminepentaacetic acid-(amino acid)2] have been docked computationally to the active site of the acetylcholine receptor. The induced fit method was employed to perform the automolecular docking for these systems. The result of docking studies generated thermodynamic properties, such as free energy of bindings (Glide score) and their weak electrostatic interactions. On the basis of these results, scintigraphic imaging studies were performed in mice. Among the radiotracers evaluated in this study, compound derived from 5-hydroxytryptophan/tryptophan exhibited remarkable localization in the brain, whereas radiotracer derived from l-histidine shows moderate accumulation in the brain. Preliminary studies with these amino acid-based ligands are encouraging to carrying out further in vivo experiments for targeted imaging.

Differential modulation of apoptotic gene expression by N-acetyl-L-cysteine in Leydig cells stimulated persistently with hCG in vivo.

The present study was designed to investigate the molecular mechanisms of NAC (150 mg/kg bw twice/week) action in vivo under repeated hCG (100 IU/rat/day) stimulation to adult rats. Leydig cell refractoriness led to a significant decline in serum testosterone and intracellular cAMP by day 30 of chronic hCG intervention which improved significantly following NAC co-administration. It inhibited the rise in lipid peroxidation, improved the activity of antioxidant enzymes along with intracellular glutathione and total antioxidant capacity in the target cells. Leydig cell apoptosis declined significantly (P<0.001) with down-regulation of upstream, Fas, FasL, caspase-8, Bax and caspase-9, JNK/pJNK and downstream caspase-3 and PARP. On the other hand, anti-apoptotic Bcl2, NF-kß, and Akt were up-regulated. Taken together, the above findings indicate that the specificity of NAC action was not restricted to regulating marker proteins in the extrinsic and JNK pathways as seen in vitro but extended to include intrinsic pathway of metazoan apoptosis as well.

Nutritional and toxicological assessment of white-rot fermented animal feed.

The fungal fermented wheat straws as animal feeds have been evaluated for its toxicological and nutritional status in male rats (Holtzman strain). Digestibility of dry matter and other nutrients as well as fiber fractions were found significantly higher (P < 0.05) in straw fermented with either Ganoderma sp. rckk02 (T3) or Crinipellis sp. RCK-1 (T4) than unfermented straw (T1) or straw fermented with Pycnoporus cinnabarinus (T2). The aflatoxin B1, B2, G1 and G2 were either absent or present in permissive levels in T3 and T4 diets and exhibited normal stress enzyme activity in case of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) enzymes whereas, rats fed on T2 diet showed elevated levels of stress enzymes (ALT, AST and LDH activity), 100% high morbidity and 8.3% mortality. This study suggests that Ganoderma sp. rckk02 and Crinipellis sp. RCK-1 are efficient in improving the nutritive value of poor quality straw and do not posses any threat for their subsequent use as ruminant feed.

AR versus ER (α) expression in the testis and pituitary following chronic estrogen administration in adult rat.

Modulation of the testis-pituitary axis has direct relevance to the expression of androgen and estrogen receptors. Androgen receptor (AR) and estrogen receptor (ERα) expression during hypospermatogenesis after chronic estrogen administration to rats was studied in the adult testis and pituitary utilizing immunohistochemistry, western blotting, and RT-PCR. Both organs demonstrated higher AR transcriptional activity gradually increasing from 15 days (d) to 30 d of estrogen treatment. However, the AR protein as measured by either immunostaining or western blotting demonstrated a significant decline. A distinct break down of the AR protein in the pituitary into two specific bands was seen. In contrast, higher ERα transcriptional activity coincided well with the rise in protein and immunoexpression in both organs. FSH and testosterone (serum, intra-testicular testosterone) were found significantly (p < 0.001) lowered compared with raised estradiol levels. Spermatogenesis was adversely affected and was associated with a significant increase in cell apoptosis in both organs. The pituitary demonstrated a higher rate of apoptosis at the end of 30 d of estrogen treatment. Taken together, the above data indicate that chronic estrogenization to adult rats up-regulates ERα but down-regulates AR protein expression in testis and pituitary which probably has a direct association to the marked rise in cell apoptosis in these organs.