Probiotic Potential of a Lactobacillus Bacterium of Canine Faecal-Origin and Its Impact on Select Gut Health Indices and Immune Response of Dogs

The objective of the present study was to develop a probiotic of canine-origin for its potential application in pet nutrition. Accordingly, 32 lactic acid bacteria (LAB) strains were isolated from faeces of dogs, out of which 9 strains were short-listed for further in vitro testing based on the aggregation time and cell surface hydrophobicity. The results of acid-, bile- and phenol-tolerance tests indicated that out of the nine, isolate cPRO23 was having better resistance to these adverse conditions likely to be encountered in the gastrointestinal tract. The isolate also showed optimal enzymatic activities for amylase, lipase and protease. Further assessments also indicated its superiority in terms of co-aggregation and antagonistic activity against pathogenic strains of Salmonella typhimurium and Salmonella enteritidis. Subsequently, the isolate was identified through 16S rRNA sequencing and sequence homology, and designated as Lactobacillus johnsonii CPN23. The candidate probiotic was then evaluated in vivo using 15 adult Labrador dogs, divided into 3 groups, viz. CON (with no probiotics), dPRO (with Lactobacillus acidophilus NCDC 15 as a conventional dairy-origin probiotic) and cPRO (with L. johnsonii CPN23 as a canine-origin probiotic). Results of the 9-week study indicated that supplementation of cPRO improved (P < 0.05) the faecal concentration of acetate and butyrate with a concomitant reduction (P < 0.05) in faecal ammonia. The cell-mediated immune response, assessed as delayed-type hypersensitivity reaction to phytohaemagglutinin-P, was better (P < 0.05) in dogs fed cPRO as compared to the CON dogs. There were, however, no variations evident in the antibody response to sheep-erythrocytes among the three groups. It is concluded that the canine-origin L. johnsonii CPN23, in addition to possessing all the in vitro functional attributes of a candidate probiotic, also has the potential to be used as a probiotic in pet nutrition programs.     

Analysis of reported SCO2 gene mutations affecting cytochrome c oxidase activity in various diseases

A large number of mutations have been reported in SCO2 (synthesis of cytochrome c oxidase) gene in association with COXdeficiency reported in different diseases such as cardioencephalomyopathy, cardiomyopathy and Leigh syndrome. However, veryfew of these mutations have been functionally analyzed.SCO2 gene encodes for an essential assembly factor for the formation ofcytochrome c oxidase (COX). It is a nuclear encoded protein that helps in transfer of copper ions to COX. This study is an attemptto understand the possible effect of these mutations on the structure and function of SCO2 protein, by using different in silico tools.As per Human Gene Mutation Database, total 11 non synonymous variations have been reported in SCO2 gene. Among these 11variations, only E140K and R171W are functionally proven to cause COX deficiency. They have been used as controls in this study.The remaining variations were further analyzed using ClustalW, SIFT, PolyPhen-2, GOR4, MuPro and Panther softwares. Ascompared to the results of the controls, most of these variations were predicted to affect the structure of SCO2 protein and hence,may cause COX dysfunction. Thus, we hypothesize that these variations have the potential to result in a disease phenotype andshould be investigated by subsequent functional analyses. This will help in an appropriate diagnosis and management of the widespectrum of COX deficiency diseases.     

Molecular modelling study on human histamine H1 receptor and its applications in virtual lead identification for designing novel inverse agonists

Human histamine H1 receptor (HHR1) is one of the G protein-coupled receptors (GPCRs) known for their constitutive activation in the absence of agonist binding. Inverse agonists are the compounds that inhibit this constitutive activity of GPCRs. HHR1 is involved in allergic reactions and is also known to be constitutively active. An updated quantitative pharmacophore model, Hypo1, has been developed using a diverse set of known HHR1 inverse agonists employing the HypoGen algorithm as implemented in Accelrys Discovery Studio 2.1. Hypo1 comprised four pharmacophore features (each one of hydrogen bond acceptor, hydrophobic, ring aromatic and positive ionisable group) along with a high correlation value of 0.944. This pharmacophore model was validated using an external test set containing 25 diverse inverse agonists and CatScramble method. Three chemical databases were screened for novel chemical scaffolds using Hypo1 as a query, to be utilised in drug design. The 3D structure of HHR1 has been constructed using human β2 adrenergic receptor. Molecular docking studies were performed with the database hit compounds using GOLD 4.1 program. The combination of all results led us to identify novel compounds to be deployed in designing new generation HHR1 inverse agonists.     

BCR/ABL Directly Inhibits Expression of SHIP, an SH2-Containing Polyinositol-5-Phosphatase Involved in the Regulation of Hematopoiesis

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML), a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and granulocyte lineage cells. The SH2-containing inositol-5-phosphatase SHIP is a 145-kDa protein which has been shown to regulate hematopoiesis in mice. Targeted disruption of the murine SHIP gene results in a myeloproliferative syndrome characterized by a dramatic increase in numbers of granulocyte-macrophage progenitor cells in the marrow and spleen. Also, hematopoietic progenitor cells from SHIP(-/-) mice are hyperresponsive to certain hematopoietic growth factors, a phenotype very similar to the effects of BCR/ABL in murine cells. In a series of BCR/ABL-transformed hematopoietic cell lines, Philadelphia chromosome (Ph)-positive cell lines, and primary cells from patients with CML, the expression of SHIP was found to be absent or substantially reduced compared to untransformed cell lines or leukemia cells lacking BCR/ABL. Ba/F3 cells in which expression of BCR/ABL was under the control of a tetracycline-inducible promoter showed rapid loss of p145 SHIP, coincident with induction of BCR/ABL expression. Also, an ABL-specific tyrosine kinase inhibitor, CGP57148B (STI571), rapidly caused reexpression of SHIP, indicating that BCR/ABL directly, but reversibly, regulates the expression of SHIP protein. The estimated half-life of SHIP protein was reduced from 18 h to less than 3 h. However, SHIP mRNA also decreased in response to BCR/ABL, suggesting that SHIP protein levels could be affected by more than one mechanism. Reexpression of SHIP in BCR/ABL-transformed Ba/F3 cells altered the biological behavior of cells in culture. The reduction of SHIP due to BCR/ABL is likely to directly contribute to the pathogenesis of CML.     

Effect of Rice Straw Incorporation on Phytotoxicity of Isoxaflutole to an Exotic Weed Phalaris minor Retz.

Phalaris minorRetz. is a major exotic annual weed in the wheat (Triticum aestivum L.) crop. Unharvested rice (Oryza sativa L.) straw, unburned and burned, is often incorporated in the field prior to cultivating wheat. Isoxaflutole (Balance), a pre-emergent systemic soil applied herbicide, has potential to control P. minor. Glasshouse experiments were conducted to determine the phytotoxicity of isoxaflutole defined by reductions in relation to shoot length of P. minor when grown in unamended soil or soil amended with unburned or burned rice straw. A 120 g soil was amended with 0, 1, 2 and 4 g of unburned or burned rice straw, and placed in 150 mL styrofoam pots. Appropriate amount of isoxaflutole (75% active ingredient, ai) was added to pots to get final concentration of 0, 7.5, 30, 60 and 120 μg ai/pot. Unamended soil and soil amended with unburned or burned rice straw were analyzed for pH and organic matter; two important determinants of isoxaflutole activity. Results indicate a significant reduction in shoot length of P. minor when grown in soil treated with isoxaflutole at 30, 60 or 120 μg ai/pot. Inhibition in the shoot length of P. minor was observed when soil amended with unburned straw was treated with isoxaflutole at 7.5 and 30 μg ai/pot compared with unamended soil treated with similar amounts of isoxaflutole. No significant change in isoxaflutole toxicity was observed when soil amended with unburned straw was treated with isoxaflutole at 60 and 120 lg ai/pot compared with unamended soil treated with similar amounts of isoxaflutole. Isoxaflutole phytotoxicity to P. minor shoot length was eliminated when soil amended with burned straw was treated with isoxaflutole at 7.5 and 30 μg ai/pot. P. minor shoot length was greater when soil amended with burned straw was treated with isoxaflutole at 60 and 120 μg ai/pot relative to herbicide-treated unamended soils. We conclude that incorporation of burned rice straw greatly reduces the phytotoxicity of isoxaflutole toP. minor.