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.     

Presence and spatio-temporal habitat characterization of Dermatemys mawii (Testudines: Dermatemydidae) in the Grijalva-Usumacinta watershed, Tabasco, Mexico

The Central American River Turtle (Dermatemys mawii) is an endangered species that has been poorly studied. There are no reports on their population status, habitat condition, and the species distribution area is still unknown. This study analyzes the seasonal and spatial variations of their habitat and the presence/absence of D. mawii in three rivers within the Pantanos de Centla Biosphere Reserve (Tabasco, Mexico). For habitat characterization, natural segmentation of rivers was used and three sites per segment were identified, 9 in each rivers (Grijalva and Usumacinta) and 6 in Tabasquillo. Additionally, the evaluation of 11 environmental variables such as water hydrological, physicochemical characteristics and riparian and hydrophytic vegetation were carried out during two different seasons (dry and rainy). The presence/absence of species was assessed with eight fike nets that were set per segment, with a capture effort of 384 hours per trap. The capture per unit effort (CPUE) was used as an indicator of relative abundance. The results indicated spatio-temporal variations in habitat characteristics and the presence of environmental gradients. The principal components analysis (PCA) applied allowed us to determine that the first three components explained 67.8% of the environmental variability. The species presence was confirmed in all rivers, however significant differences exists in their relative abundance: the highest was registered in the Tabasquillo River where the species was present in both seasons and in all segments. Of the 11 environmental variables analyzed, the gradient, shelter and depth were the most indicative of species presence. The obtained results evidenced the importance of riparian vegetation as habitat for Dermatemys. This represents the first approach towards an action plan for a species and its habitat protection within the Pantanos de Centla Biosphere Reserve.     

Historical demography and phylogeography of a specialist bark beetle, Dendroctonus pseudotsugae Hopkins (Curculionidae: Scolytinae)

Contemporary distribution of North American species has been shaped by past glaciation events during the Quaternary period. However, their effects were not as severe in the southern Rocky Mountains and Northern Mexico as elsewhere in North America. In this context, we test hypotheses about the historical demography of Dendroctonus pseudotsugae, based on 136 haplotypes of mitochondrial cytochrome oxidase I. The phylogenetic analysis yielded four haplogroups corresponding to northwestern United States and southwestern Canada (NUS), southwestern United States (Arizona, SUS), northwestern Mexico (Sierra Madre Occidental, SMOC), and northeastern Mexico (Sierra Madre Oriental, SMOR). Predictions of demographic expansion were examined through neutrality tests against population growth and mismatch distribution. Results showed that the NUS and SMOC haplogroups have experienced demographic expansion events, whereas the SUS and SMOR haplogroups have not. Divergence times between pairs of haplogroups were estimated from early to middle Pleistocene. The longer divergence time between NUS and all other haplogroups could be the result of refugia within the Pacific Northwest and northern Rocky Mountains and long-term isolation from southernmost populations in Mexico. The results obtained in this study are in agreement with the evolutionary history of the host Douglas-fir, as the warmer climates of interglacial periods pushed conifers northward of Colorado, New Mexico, and Arizona, whereas environmental changes reduced the population size of Douglas-fir and forced fragmentation of distribution range southward into northern Mexico.     

Confirmation of Anopheles (Anopheles) calderoni Wilkerson, 1991 (Diptera: Culicidae) in Colombia and Ecuador through molecular and morphological correlation with topotypic material

The morphologically similar taxa Anopheles calderoni, Anopheles punctimacula, Anopheles malefactor and Anopheles guarao are commonly misidentified. Isofamilies collected in Valle de Cauca, Colombia, showed morphological characters most similar to An. calderoni, a species which has never previously been reported in Colombia. Although discontinuity of the postsubcostal pale spots on the costa (C) and first radial (R1) wing veins is purportedly diagnostic for An. calderoni, the degree of overlap of the distal postsubcostal spot on C and R1 were variable in Colombian specimens (0.003-0.024). In addition, in 98.2% of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7% of specimens examined. Correlation of DNA sequences of the second internal transcribed spacer and mtDNA cytochrome c oxidase subunit I gene (COI) barcodes (658 bp of the COI gene) generated from Colombian progeny material and wild-caught mosquitoes from Ecuador with those from the Peruvian type series of An. calderoni confirmed new country records. DNA barcodes generated for the closely related taxa, An. malefactor and An. punctimacula are also presented for the first time. Examination of museum specimens at the University of the Valle, Colombia, revealed the presence of An. calderoni in inland localities across Colombia and at elevations up to 1113 m.     

Peptide synthesis catalysed by a haloalkaliphilic serine protease from the archaeon Natrialba magadii (Nep)

Aims: Haloarchaeal proteases function optimally in high salt (low water activity); thus, they offer an advantage over the nonhalophilic counterparts as biocatalysts for protease‐catalysed peptide synthesis. The haloalkaliphilic archaeon Natrialba magadii secretes a solvent‐tolerant protease, Nep (Natrialba magadii extracellular protease). In this work, the ability of Nep to catalyse peptide synthesis was examined. Methods and Results: The tripeptide Ac‐Phe‐Gly‐Phe‐NH₂ was synthesized using Ac‐Phe‐OEt and Gly‐Phe‐NH₂ substrates as building blocks in the presence of Nep, 30% (v/v) dimethyl sulfoxide (DMSO) and 1·5 or 0·5 mol l^?1 NaCl. Purification and identification of the peptide product was achieved by RP‐HPLC and ESI‐MS, respectively. The native as well as the recombinant enzyme produced in Haloferax volcanii (HvNep) was similarly effective as catalysts for the synthesis of this model tripeptide with yields of up to 60% and without secondary hydrolysis of the product. HvNep catalysed the synthesis of various tripeptides with preference for those having aromatic amino acids in the P1 site. Conclusion: Nep is able to catalyse peptide synthesis under different salt concentrations in the presence of DMSO. Significance and Impact of Study: The catalytic property of Nep in peptide synthesis combined with overproduction of this protease in Hfx. volcanii anticipates the potential applicability of this haloarchaeal protease in biotechnology.     

Identification of novel α7 nAChR positive allosteric modulators with the use of pharmacophore in silico screening methods

The pharmacophore model of in house potent and selective α7 nAChR positive allosteric modulators is reported. The model was used to fish out commercially-available compounds from corporate 3D databases. As a result, novel α7 positive modulator chemotypes were identified. A rat full PK profile of a representative compound is also described.     

The structure of eukaryotic and prokaryotic complex I

The structures of the NADH dehydrogenases from Bos taurus and Aquifex aeolicus have been determined by 3D electron microscopy, and have been analyzed in comparison with the previously determined structure of Complex I from Yarrowia lipolytica. The results show a clearly preserved domain structure in the peripheral arm of complex I, which is similar in the bacterial and eukaryotic complex. The membrane arms of both eukaryotic complexes show a similar shape but also significant differences in distinctive domains. One of the major protuberances observed in Y. lipolytica complex I appears missing in the bovine complex, while a protuberance not found in Y. lipolytica connects in bovine complex I a domain of the peripheral arm to the membrane arm. The structural similarities of the peripheral arm agree with the common functional principle of all complex Is. The differences seen in the membrane arm may indicate differences in the regulatory mechanism of the enzyme in different species.