TargetMine, an integrated data warehouse for candidate gene prioritisation and target discovery

Prioritising candidate genes for further experimental characterisation is a non-trivial challenge in drug discovery and biomedical research in general. An integrated approach that combines results from multiple data types is best suited for optimal target selection. We developed TargetMine, a data warehouse for efficient target prioritisation. TargetMine utilises the InterMine framework, with new data models such as protein-DNA interactions integrated in a novel way. It enables complicated searches that are difficult to perform with existing tools and it also offers integration of custom annotations and in-house experimental data. We proposed an objective protocol for target prioritisation using TargetMine and set up a benchmarking procedure to evaluate its performance. The results show that the protocol can identify known disease-associated genes with high precision and coverage. A demonstration version of TargetMine is available at http://targetmine.nibio.go.jp/.     

Spice phenolics inhibit human PMNL 5-lipoxygenase

A wide variety of phenolic compounds and flavonoids present in spices possess potent antioxidant, antimutagenic and anticarcinogenic activities. We examined whether 5-lipoxygenase (5-LO), the key enzyme involved in biosynthesis of leukotrienes is a possible target for the spices. Effect of aqueous extracts of turmeric, cloves, pepper, chili, cinnamon, onion and also their respective active principles viz., curcumin, eugenol, piperine, capsaicin, cinnamaldehyde, quercetin, and allyl sulfide were tested on human PMNL 5-LO activity by spectrophotomeric and HPLC methods. The formation of 5-LO product 5-HETE was significantly inhibited in a concentration-dependent manner with IC(50) values of 0.122-1.44 mg for aqueous extracts of spices and 25-83 microM for active principles, respectively. The order of inhibitory activity was of quercetin>eugenol>curcumin>cinnamaldehyde>piperine>capsaicin>allyl sulfide. Quercetin, eugenol and curcumin with one or more phenolic ring and methoxy groups in their structure showed high inhibitory effect, while the non-phenolic spice principle allyl sulfide showed least inhibitory effect on 5-LO. The inhibitory effect of quercetin, curcumin and eugenol was similar to that of synthetic 5-LO inhibitors-phenidone and NDGA. Moreover, the inhibitory potency of aqueous extracts of spice correlated with the active principles of their respective spices. The synergistic or antagonistic effect of mixtures of spice active principles and spice extracts were investigated and all the combinations of spice active principles/extracts exerted synergistic effect in inhibiting 5-LO activity. These findings clearly suggest that phenolic compounds present in spices might have physiological role in modulating 5-LO pathway.     

Immunotoxicity of carbaryl in chicken

Effect of methyl carbonate pesticide, carbonyl, was studied on macrophage functions, lymphocyte proliferation and delayed type hypersensitivity response. Sixteen adult chicken, vaccinated against Newcaslte disease, were procured and randomly divided in two experimental groups. Chicken of group I served as control, while group II birds were given carbaryl at 20 ppm (No observable effect level, NOEL) in feed for 3 months. To measure the functional activity of phagocytic cells, nitroblue tetrazolium (NBT) reduction test was performed on peripheral blood leucocytes. Concanvalin A (Con-A) and lipopolysaccharide stimulated proliferation of T and B lymphocytes was assessed using MTT dye method. At the end of experiment, the phagocytic capacities of macrophages were significantly reduced in carbaryl treated group. Lymphocyte proliferation responses to Con-A and LPS were (23 and 28%, respectively lower) in chicken fed with carbaryl. Delayed hypersensitivity reaction to tuberculin was reduced to 77% of control values indicating inhibition of cell mediated immune response. The present study suggested of immunosuppressive effect of (NOEL dose carbaryl) in chicken.     

Ischemic preconditioning in isolated perfused mouse heart: Reduction in infarct size without improvement of post-ischemic ventricular function

Genetically engineered mice provide an excellent tool to study the role of a particular gene in biological systems and will be increasingly used as models to understand the signal transduction mechanisms involved in ischemic preconditioning (IP). However, the phenomenon of IP has not been well characterized in this species. We therefore attempted to examine whether IP could protect isolated mouse heart against global ischemia/reperfusion (GI/R) injury. Thirty adult mice hearts were perfused at constant pressure of 55 mmHg in Langendorff mode. Following 20 min equilibration, the hearts were randomized into three groups (n = 10/each): (1) Control Group; (2) IP2.5 Group: IP with two cycles of 2.5 min GI + 2.5 min R; (3) IP5 Group: IP with 5 min GI + 5 min R. All hearts were then subjected to 20 min of GI and 30 min R (37°C). Ventricular developed force was measured by a force transducer attached to the apex. Leakage of CK and LDH was measured in coronary efflux. Infarct size was determined by tetrazolium staining. Following sustained GI/R, infarct size was significantly reduced in IP2.5 (13.8 ± 2.3%), but not in IP5 (20.1 ± 4.0%), when compared with non-preconditioned control (23.6 ± 3.8%) hearts. CK and LDH release was also reduced in both IP2.5 and IP5 groups. No significant improvement in post-ischemic ventricular contractile function was observed in either IP groups. We conclude that IP with repetitive cycles of brief GI/R is able to reduce myocardial infarct size and intracellular enzyme leakage caused by a sustained GI/R in the isolated perfused mouse heart. This anti-necrosis cardioprotection induced by IP was not associated with the amelioration of post-ischemic ventricular dysfunction.     

Molecular constitution of breast but not other reproductive tissues is rich in growth promoting molecules: A possible link to highest incidence of tumor growths

In the current study we tested if highest incidence of benign as well as cancer growths in breast tissue is due to constitutive molecular composition of this tissue. To delineate the molecular basis, we compared the expression of nine functional gene modules (total 578 genes) that regulate major positive growth and negative inhibitory signals in normal breast with two other reproductive tissues, ovary and uterus. We present data to demonstrate that breast tissues constitutively have very highly elevated levels of several growth promoting molecules and diminished levels of inhibitory molecules which may, in part, contribute for highest incidence of tumor growths in this tissue.     

Oxygen radical-mediated lipid peroxidation and inhibition of Ca2+-ATPase activity of cardiac sarcoplasmic reticulum

Oxygen radicals have been implicated as important mediators of myocardial ischemic and reperfusion injury. A major product of oxygen radical formation is the highly reactive hydroxyl radical via a biological Fenton reaction. The sarcoplasmic reticulum is one of the major target organelles injured by this process. Using a oxygen radical generating system consisting of dihydroxyfumarate and Fe3+-ADP, we studied lipid peroxidation and Ca2+-ATPase of cardiac sarcoplasmic reticulum. Incubation of sarcoplasmic reticulum with dihydroxyfumarate plus Fe3+-ADP significantly inhibited enzyme activity. Addition of superoxide dismutase, superoxide dismutase plus catalase (15 micrograms/ml) or iron chelator, deferoxamine (1.25-1000 microM) protected Ca2+-ATPase activity. Time course studies showed that this system inhibited enzyme activity in 7.5 to 10 min. Similar exposure of sarcoplasmic reticulum to dihydroxyfumarate plus Fe3+-ADP stimulated malondialdehyde formation. This effect was inhibited by superoxide dismutase, catalase, singlet oxygen, and hydroxyl radical scavengers. EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide verified production of the hydroxyl radical. The combination of dihydroxyfumarate and Fe3+-ADP resulted in a spectrum of hydroxyl radical spin trap adduct, which was abolished by ethanol, catalase, mannitol, and superoxide dismutase. The results demonstrate the role of oxygen radicals in causing inactivation of Ca2+-ATPase and inhibition of lipid peroxidation of the sarcoplasmic reticulum which could possibly be one of the important mechanisms of oxygen radical-mediated myocardial injury.     

Interaction of Aryl Hydrocarbon Receptor-interacting Protein-like 1 with the Farnesyl Moiety

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor specific chaperone of the visual effector enzyme phosphodiesterase-6 (PDE6). AIPL1 has been shown to bind the farnesylated PDE6A subunit. Mutations in AIPL1 are thought to destabilize PDE6 and thereby cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Here, we examined the solution structure of AIPL1 by small angle x-ray scattering. A structural model of AIPL1 with the best fit to the scattering data features two independent FK506-binding protein (FKBP)-like and tetratricopeptide repeat domains. Guided by the model, we tested the hypothesis that AIPL1 directly binds the farnesyl moiety. Our studies revealed high affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thus uncovering a novel function of this domain. Mutational analysis of the potential farnesyl-binding sites on AIPL1 identified two critical residues, Cys-89 and Leu-147, located in close proximity in the structure model. The L147A mutation and the LCA-linked C89R mutation prevented the binding of the farnesyl-Cys probe to AIPL1. Furthermore, Cys-89 and Leu-147 flank the unique insert region of AIPL1, deletion of which also abolished the farnesyl interaction. Our results suggest that the binding of PDE6A farnesyl is essential to normal function of AIPL1 and its disruption is one of the mechanisms underlying LCA.     

Integrating information from existing databases for enhanced function annotation of genes, genomes and networks

Uncovering functional associations for genes and gene products remains one of the most significant challenges in biology. The classical approaches, such as homology detection, are mainly suited for predicting approximate molecular function of a protein and should be used in context with other methods. Several studies have emerged that employ knowledge-based procedures to extract functional data for genes from a variety of biological sources. However, data derived from a single biological resource often provides only a limited perspective on their functional associations largely due to systematic bias in the underlying data. The post-genomic era has witnessed the emergence of knowledge-based studies that aim to decipher functional associations by combining several biological evidence types. These are expected to provide better insights into the functional aspects of diverse genes, genomes and networks.