Noninvasive Method of DNA Isolation From Fecal Epithelial Tissue of Dairy Animals

A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.     

Length–weight relationships of three freshwater fish species from Rupnaryan and Kangsabati River,West Bengal,India

The length–weight relationships (LWRs) of three freshwater fish species from the Kangsabati and Rupnaryan river in West Bengal, India are presented, namely as Amblypharyngodon microlepis (Bleeker, 1853), Parambassis lala (Hamilton, 1822) and Macrognathus aculeatus (Bloch, 1786). Gill‐nets (mesh sizes with 0.5 cm–4 cm), cast‐nets (up to 1 × 1 cm mesh size with up to 4.0 m² area) and scoop‐nets (0.3 × 0.3 cm and 0.5 × 0.5 cm mesh size) were used from January, 2017 to April, 2018. Sampling was done every 15 days during this period. The value of parameter “b” ranged from 2.751 to 2.895 with highly significant correlations (r² > 0.95).     

Comparative Genomic Analysis of Buffalo (Bubalus bubalis) NOD1 and NOD2 Receptors and Their Functional Role in In-Vitro Cellular Immune Response

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo—a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.     

Synthesis and evaluation of 1,2,4-triazolo[1,5-c]pyrimidine derivatives as A2A receptor-selective antagonists

Movement disorders such as Parkinson’s disease and Huntington’s disease are serious life-limiting and debilitating movement disorders. Their onset typically occurs from mid-life to late in life, and effective diagnostic techniques for detecting and following the disease course are lacking. Our goal is to develop receptor imaging agents for positron emission tomography (PET) that selectively target the most relevant subtype of adenosine receptors (AR) that are highly expressed in the striatum, that is, the A_2A AR. To further this goal, we have synthesized and characterized pharmacologically a family of high affinity A_2A AR ligands, based on the known antagonist, SCH 442416 (R = –Me), which have structural variability on the terminus (R = –Et, –i-Pr, –allyl, and others). A O-fluoroethyl analogue suitable for use as a PET tracer had a K_i value of 12.4 nM and was highly selective for the A_2A AR in comparison to the A₁ and A₃ ARs.     

Molecular probes for the A2A adenosine receptor based on a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine scaffold

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A_2A adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for ¹⁸F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, K_i 15 nM). The potent and A_2AAR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A_2AAR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A_2AAR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A_2AAR.     

Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas’ disease. We have undertaken a detailed structure–activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme–ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60–70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.     

Functional organization of TRPC-Ca2+ channels and regulation of calcium microdomains

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1-TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca(2+) influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP(2)) hydrolysis, generation of IP(3) and DAG, and IP(3)-induced Ca(2+) release from the intracellular Ca(2+) store via inositol trisphosphate receptor (IP(3)R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca(2+) entry mechanisms. The former is regulated by the emptying/refilling of internal Ca(2+) store(s) while the latter depends on PIP(2) hydrolysis (due to changes in PIP(2) per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca(2+) entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca(2+)](i) signals which are critical for precise control of downstream cellular functions.     

Structural insights into the MDP binding and CARD–CARD interaction in zebrafish (Danio rerio) NOD2: a molecular dynamics approach

Nucleotide binding and oligomerization domain (NOD2) is a key component of innate immunity that is highly specific for muramyl dipeptide (MDP)—a peptidoglycan component of bacterial cell wall. MDP recognition by NOD2–leucine rich repeat (LRR) domain activates NF‐κB signaling through a protein–protein interaction between caspase activating and recruitment domains (CARDs) of NOD2 and downstream receptor interacting and activating protein kinase 2 (RIP2). Due to the lack of crystal/NMR structures, MDP recognition and CARD–CARD interaction are poorly understood. Herein, we have predicted the probable MDP and CARD–CARD binding surfaces in zebrafish NOD2 (zNOD2) using various in silico methodologies. The results show that the conserved residues Phe819, Phe871, Trp875, Trp929, Trp899, and Arg845 located at the concave face of zNOD2–LRR confer MDP recognition by hydrophobic and hydrogen bond (H‐bond) interactions. Molecular dynamics simulations reveal a stable association between the electropositive surface on zNOD2–CARDa and the electronegative surface on zRIP2–CARD reinforced mostly by H‐bonds and electrostatic interactions. Importantly, a 3.5 Å salt bridge is observed between Arg60 of zNOD2–CARDa and Asp494 of zRIP2–CARD. Arg11 and Lys53 of zNOD2–CARDa and Ser498 and Glu508 of zRIP2–CARD are critical residues for CARD–CARD interaction and NOD2 signaling. The 2.7 Å H‐bond between Lys104 of the linker and Glu508 of zRIP2–CARD suggests a possible role of the linker for shaping CARD–CARD interaction. These findings are consistent with existing mutagenesis data. We provide first insight into MDP recognition and CARD–CARD interaction in the zebrafish that will be useful to understand the molecular basis of NOD signaling in a broader perspective. Copyright © 2014 John Wiley & Sons, Ltd.     

Threats to Fish and Fisheries of Kangsabati Reservoir,West Bengal,India

Fishes belonging to the Orders Beloniformes, Cypriniformes, Perciformes, Siluriformes and Synbranchiformes are usually found in the Kangsabati reservoir of West Bengal, India. In recent years among the trapped fishes fishermen failed to get certain fish species which were available to them in the last decade. This prompted us to conduct a survey of ichthyofauna of the said reservoir in respect to the water parameters, keeping in view the anthropogenic activity-induced pollution scenario. It is revealed that the fishes belonging to the species Xenentodon cancila, Nemacheilus savona, Sillaginopsis panijus, Pangasius sutchi, Colisa sota, Mystus cavasius, Mystus seenghala and Mastacembelus armatus are completely absent in the survey area. It is most likely that the eutrophication-induced causes especially, variations in composition and density of plankton as well as the undesirable changes in physical and chemical properties of the water have forced these fishes to migrate elsewhere.