Pro-melanin-concentrating hormone gene (PMCH) is localized on human chromosome 12q and rat chromosome 7

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that may be involved in regulation of the stress response and food intake behavior in mammals. MCH and two other putative neuropeptides, NEI and NGE, are encoded by the same precursor, designated pro-melanin-concentrating hormone (PMCH). A panel of somatic cell hybrids segregating either human or rat chromosomes was used to determine the chromosomal localization of the PMCH locus. It was assigned to human chromosome 12q and to rat chromosome 7. This is the first neuropeptide-encoding gene found in this new synteny group conserved in rat and human.     

High-affinity selective inhibitor against phospholipase A2 (PLA2): a computational study

Phospholipase A₂ (PLA₂) is the most abundant protein found in snake venom. PLA₂ induces a variety of pharmacological effects such as neurotoxicity, myotoxicity and cardiotoxicity as well as anticoagulant, hemolytic, anti-platelet, hypertensive, hemorrhagic and edema inducing effects. In this study, the three dimensional structure of PLA₂ of Naja sputatrix (Malayan spitting cobra) was modeled by I-TASSER, SWISS-MODEL, PRIME and MODELLER programs. The best model was selected based on overall stereo-chemical quality. Further, molecular dynamics simulation was performed to know the stability of the modeled protein using Gromacs software. Average structure was generated during the simulation period of 10?ns. High throughput virtual screening was employed through different databases (Asinex, Hit finder, Maybridge, TOSLab and ZINC databases) against PLA₂. The top seven compounds were selected based on the docking score and free energy binding calculations. These compounds were analyzed by quantum polarized ligand docking, induced fit docking and density functional theory calculation. Furthermore, the stability of lead molecules in the active site of PLA₂ was employed by MD simulation. The results show that selected lead molecules were highly stable in the active site of PLA₂.     

Studies on the measurement of phospholipase A2 (PLA2) and PLA2 inhibitor activities in ram semen.

Extraction with Tris-citrate or Tris-NaCl-EGTA improved the yield of phospholipase A2 (PLA2) from ram semen by 40-50 fold over the previously recommended method of extraction by dilute (0.18 N) sulphuric acid. The enzyme activity in the citrate extract deteriorated more rapidly than in Tris-NaCl-EGTA. The semen PLA2 activity was optimum at pH 8.0, heat sensitive at 70 degrees C for 30 min, activated by Ca2+ (although approximately 60% activity was also found in the absence of calcium) and did not exist as a pro-enzyme. The semen PLA2 activity was equally distributed among the sperm and seminal plasma (SP) components of ram semen. However, the low levels of PLA2 activity in the SP of vasectomised rams tend to suggest that PLA2 in the SP fraction may have originated from testicular or epididymal secretions or leakage, from sperm. PLA, in sperm exists as a large molecular weight aggregate, whereas in SP it is present as a smaller aggregate. In addition to PLA2, semen also contained PLA2 inhibitor activities. Inhibition was observed against PLA2s from bee venom, pig pancreas and oviductal extracts. The inhibitory activity is presumed to be due to a large molecular weight protein as the inhibitor activity was not extracted in a chloroform:methanol (2:1; v/v) mixture, it was non-dialysable, precipitated by 10% trichloroacetic acid and destroyed by proteases. The inhibitor activity was distributed in various molecular weight fractions of sperm, SP and SP from vasectomised rams.     

Molecular characterization of murine pancreatic phospholipase A(2)

The pancreatic secretory phospholipase A(2) (sPLA(2)IB) is considered to be a digestive enzyme, although it has several important receptor-mediated functions. In this study, using the newly isolated murine sPLA(2)IB cDNA clone as a probe, we demonstrate that in addition to the pancreas, the sPLA(2)IB mRNA was expressed in extrapancreatic organs such as the liver, spleen, duodenum, colon, and lungs. We also demonstrate that sPLA(2)IB mRNA expression was detectable from the 17(th) day of gestation in the developing mouse fetus, coinciding with the time of completion of differentiation of the pancreas. Furthermore, the mRNA expression pattern of sPLA(2)IB was distinct from those of sPLA(2)IIA and cPLA(2) in various tissues examined. The murine sPLA(2)IB gene structure is well conserved, consistent with findings in other mammalian species, and this gene mapped to the region of mouse chromosome 5F1-G1.1. Taken together, our results suggest that sPLA(2)IB plays important roles both in the pancreas and in extrapancreatic tissues and that in the mouse, its expression is developmentally regulated.     

Cloning of the human phospholipase A2 activating protein (hPLAP) gene on the chromosome 9p21 melanoma deleted region

Cutaneous malignant melanoma (CMM) is a common skin cancer. About 50% of CMM sporadic tumours have lost one copy of the chromosome 9p21 region. To identify genes involved in the initiation and/or progression of CMM we have characterised the 9p21 melanoma deleted region and screened the human expressed sequence tag (EST) databases (dbEST) to search for expressed genes. We have identified the gene that encodes the human orthologue of the rat phospholipase A2 activating protein (PLAP). PLAP was considered a potential candidate to be involved in malignant melanoma because it maps to the critical region for CMM and because the PLA2 gene has been identified as a modifier of the APC gene, responsible for the adenomatous polyposis phenotype in the mouse. PLAP encodes a protein of 738 amino acids and has a high DNA (90%) and protein (97%) sequence similarity with the rat and mouse PLAP protein. PLAP has a region of WD40 repeats in the amino-terminus, which allows us to include this protein in the superfamily of beta-transducin proteins. Northern blot hybridisation gave a fragment of 4.5 kb, with higher expression in heart compared to other tissues. PLAP was localised at chromosome 9p21, between marker AFM218xg11 and TEK. SSCP analysis of the coding region of PLAP revealed no variants in the studied samples, but one of six CMM samples analysed by RT-PCR showed specific inactivation of PLAP. Despite PLAP's important role in mediating several cellular responses and its localisation to the chromosome 9p21 region deleted in CMM, it is unlikely that point mutations or deletions in the coding region of PLAP are responsible for the initiation or progression of CMM. Further studies on PLAP inactivation should be performed to clarify its potential involvement in CMM.     

Mechanism of human group V phospholipase A2 (PLA2)-induced leukotriene biosynthesis in human neutrophils. A potential role of heparan sulfate binding in PLA2 internalization and degradation

Human group V phospholipase A(2) (hVPLA(2)) has been shown to have high activity to elicit leukotriene production in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To determine the mechanism by which hVPLA(2) interacts with cell membranes to induce leukotriene formation, we mutated surface cationic residues and a catalytic residue of hVPLA(2) and measured the interactions of mutants with model membranes, immobilized heparin, and human neutrophils. These studies showed that cationic residues, Lys(7), Lys(11), and Arg(34), constitute a part of the interfacial binding surface of hVPLA(2), which accounts for its moderate preference for anionic membranes. Additionally, hVPLA(2) binds heparin with high affinity and has a well defined heparin-binding site. The site is composed of Arg(100), Lys(101), Lys(107), Arg(108), and Arg(111), and is spatially distinct from its interfacial binding surface. Importantly, the activities of the mutants to hydrolyze cell membrane phospholipids and induce leukotriene biosynthesis, when enzymes were added exogenously to neutrophils, correlated with their activities on phosphatidylcholine membranes but not with their affinities for anionic membranes and heparin. These results indicate that hVPLA(2) acts directly on the outer plasma membranes of neutrophils to release fatty acids and lysophospholipids. Further studies suggest that products of hVPLA(2) hydrolysis trigger the cellular leukotriene production by activating cellular enzymes involved in leukotriene formation. Finally, the temporal and spatial resolution of exogenously added hVPLA(2) and mutants suggests that binding to cell surface heparan sulfate proteoglycans is important for the internalization and clearance of cell surface-bound hVPLA(2).     

Localization of a human brain sodium channel gene (SCN2A) to chromosome 2

A DNA probe derived from a human genomic library has been used to localize on human chromosomes a gene coding for the alpha-subunit of the brain type II sodium channel (SCN2A). Hybridization of the probe to Southern blots made with DNAs from a rodent-human somatic cell hybrid panel indicates localization to the long arm of human chromosome 2. In situ hybridization to metaphase chromosomes confirms this assignment and indicates regional localization to 2q21-q33. The probe also reveals a frequent two-allele HaeIII RFLP.