Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8)

The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.     

STRUCTURE AND FUNCTION OF BENTHIC ALGAL COMMUNITIES IN AN EXTREMELY ACID RIVER1

The composition of algal species and pigments and the structural and functional characteristics of the algal community were investigated in an acid stream of southwestern Spain, the Río Tinto. The algal community had low diversity and showed few seasonal differences. It was mainly made up of Klebsormidium flaccidum Kütz. (Silva, Mattox & Blackwell) that produced long greenish or purplish filaments, Pinnularia acoricola Hust. (producing brown patches) and Euglena mutabilis Schmitz. The algal filaments made up a consistent biofilm that also included fungal hyphae, iron bacterial sheaths, diatoms, and mineral particles. HPLC analyses on Río Tinto samples showed that undegraded chl accounted for 67% of the total chl in the filamentous patches but were a minority in the brown patch (2.6%). The brown patch had a concentration of carotenoids eight times lower than that observed in the green patch. When chl concentrations were weighted for the proportion of the different patches on the streambed, undegraded chl a accounted for 89.2 mg chl a·m ^{? 2} of stream surface area (5.4 g C·m ^{? 2}). This high algal biomass was supported by relatively high nutrient concentrations and by a high phosphatase activity (V_max = 137.7 nmol methylumbelliferyl substrate·cm ^{? 2}·h ^{? 1} 1 Received 15 July 2002. Accepted 17 February 2003. , K_m = 0.0045 μM). The remarkable algal biomass in Río Tinto potentially contributed to the bacterial–fungal community and to the macroinvertebrate community and emphasizes the role that the algae may have in the organic matter cycling and energy flow in extreme systems dominated by heterotrophic microorganisms.     

Paraoxonase1-192 polymorphism modulates the effects of regular and acute exercise on paraoxonase1 activity

Regular exercise practise is a protective factor against coronary heart disease and enhances antioxidant systems, whereas acute exercise appears to be a major source of increased oxidative stress. Paraoxonase1 (PON1) is an antioxidant HDL-linked enzyme, whose activity toward paraoxon (PON1 activity) is strongly modulated by the PON1-192 polymorphism, comprising Q and R alleles for low and high PON1 activity, respectively. Another polymorphism at the PON1 locus, the PON1-55, modulates PON1 protein and activity levels. PON1 activity, lipid levels, and oxidized LDL concentration were determined in 17 healthy young volunteers before and after a 16-weeks aerobic exercise training period. Furthermore, PON1 activity was analyzed after a bout of exercise in both situations. We found that regular exercise was associated with a decrease in oxidized LDL levels, and an increase in PON1 activity in QQ subjects and with a decrease in PON1 activity in R carriers. A bout of exercise produced an increase in PON1 activity just after the bout of exercise, followed by a decrease in its activity. A recovery of the basal PON1 activity levels at 24 h was found in QQ subjects regardless of their training status and in trained R carriers, but not in untrained R carriers. These results suggest that the effects of regular and acute exercise on PON1 activity levels are modulated by PON1-192 polymorphism. Changes were less evident for the PON1-55 polymorphism.     

Can a Place of Origin of the Main Cystic Fibrosis Mutations Be Identified?

The genetic background of the mutations that most often cause cystic fibrosis (CF) is different from that of non-CF chromosomes in populations of European origin. It is not known whether these haplotype backgrounds could be found at high frequencies in populations in which CF is, at present, not common; such populations would be candidates for the place of origin of CF mutations. An analysis of haplotypes of CF transmembrane conductance regulator, together with their variation in specific CF chromosomes, in a worldwide survey of normal chromosomes shows (1) a very low frequency or absence of the most common CF haplotypes in all populations analyzed and (2) a strong genetic variability and divergence, among various populations, of the chromosomes that carry disease-causing mutations. The depth of the gene genealogy associated with disease-causing mutations may be greater than that of the evolutionary process that gave rise to present-day human populations. The concept of "population of origin" lacks either spatial or temporal meaning for mutations that are likely to have been present in Europeans before the ethnogenesis of present populations; subsequent population processes may have erased the traces of their geographic origin.     

Molecular phylogeny and evolution of the extinct bovid Myotragus balearicus

Myotragus balearicus was a dwarf artiodactyl endemic to the Eastern Balearic Islands, where it evolved in isolation for more than 5 million years before becoming extinct between 3640 and 2135 cal BC (calibrated years BC). Numerous unusual apomorphies obscure the relationship between Myotragus and the extant Caprinae. Therefore, genetic data for this species would significantly contribute to the clarification of its taxonomic position. In this study, we amplify, sequence, and clone a 338-base pair (bp) segment of the mitochondrial cytochrome b (cyt b) gene from a >9Kyr Myotragus subfossil from la Cova des Gorgs (Mallorca). Our results confirm the phylogenetic affinity of Myotragus with the sheep (Ovis) and the takin (Budorcas). In each tree, the Myotragus branch is long in comparison with the other taxa, which may be evidence of a local change in the rate of evolution in cyt b. This rate change may be due to in part to an early age of first reproduction and short generation time in Myotragus, factors that are potentially related to the extreme reduction in size of the adult Myotragus as compared to the other Caprinae.     

Molecular cloning and characterization of gilthead sea bream (Sparus aurata) growth hormone receptor (GHR). Assessment of alternative splicing

The full-length growth hormone receptor (GHR) of gilthead sea bream (Sparus aurata) was cloned and sequenced by RT-PCR and rapid amplification of 5'and 3'ends. The open reading frame codes for a mature 609 amino acid protein with a hydrophobic transmembrane region and all the characteristic motifs of GHRs. Sequence analysis revealed a 96 and 76% of amino acid identity with black sea bream (Acanthopagrus schlegeli) and turbot (Scophthalmus maximus) GHRs, respectively, but this amino acid identity decreases up to 52% for goldfish (Carassius auratus) GHR. By means of real-time PCR assays, concurrent changes in the hepatic expression of GHRs and insulin-like growth factor-I (IGF-I) was evidenced. Moreover, their regulation occurred in conjunction with the summer spurt of growth rates and circulating levels of GH and IGF-I. Search of alternative splicing was carried out exhaustively for gilthead sea bream GHR, but Northern blot and 3' RACE failed to demonstrate the occurrence of short alternative messengers. Besides, RT-PCR screening did not reveal deletions or insertions that could lead to alternative reading frames. In agreement with this, cross-linking assays only evidenced two protein bands that match well with the size of glycosylated and non-glycosylated forms of the full-length GHR. If so, it appears that alternative splicing at the 3'end does not occur in gilthead sea bream, although different messengers for truncated or longer GHR variants already exist in turbot and black sea bream, respectively. The physiological relevance of this finding remains unclear, but perhaps it points out large inter-species differences in the heterogeneity of the GHR population.     

Structure–activity studies on acetylcholinesterase inhibition in the lycorine series of Amaryllidaceae alkaloids

The synthesis of differentially functionalized analogs of the Amaryllidaceae alkaloid lycorine, accessed via a concise chemoselective silylation strategy, is described uncovering two of the most potent inhibitors of acetylcholinesterase (AChE) identified to date in this series. Important elements of this novel pharmacophore were elucidated through structure–activity relationship (SAR) studies.