Purification and amino acid sequence of fructose-1,6-bisphosphate aldolase from the electric organ of Electrophorus electricus (L.)

A soluble fructose-1,6-bisphosphate aldolase enzyme has been purified 50.2-fold (2.36%) at the homogeneity from the electric organ of Electrophorus electricus by one step of DEAE-52 anion exchange chromatography followed by Superose-12 gel filtration-FPLC. Like other aldolase enzymes the E. electricus protein is a dimer with two identical subunits of 45 kDa. The N-terminal (20 residues) revealed a high homology with S. aurata (75%, goldfish), R. ratus and M. musculus (mouse, 80%) enzymes.     

Benzodiazepine calcitonin gene-related peptide (CGRP) receptor antagonists: optimization of the 4-substituted piperidine

In our continuing effort to identify CGRP receptor antagonists for the acute treatment of migraine, we have undertaken a study to evaluate alternative 4-substituted piperidines to the lead dihydroquinazolinone 1. In this regard, we have identified the piperidinyl-azabenzimidazolone and phenylimidazolinone structures which, when incorporated into the benzodiazepine core, afford potent CGRP receptor antagonists (e.g., 18 and 29). These studies produced a potent analog (18) which overcomes the instability issues associated with the lead structure 1. A general pharmacophore for the 4-substituted piperidine component of these CGRP receptor antagonists is also presented.     

Synthesis, labeling, and biological evaluation of halogenated 2-quinolinecarboxamides as potential radioligands for the visualization of peripheral benzodiazepine receptors

The previous exploration of the structure-affinity relationships concerning 4-phenyl-2-quinolinecarboxamide peripheral benzodiazepine receptor (PBR) ligands 6 showed as an interesting result the importance of the presence of a chlorine atom in the methylene carbon at position 3 of the quinoline nucleus. The subnanomolar PBR affinity shown by N-benzyl-3-chloromethyl-N-methyl-4-phenyl-2-quinolinecarboxamide (6b) suggested its chlorine atom to be replaced with other halogens in order to optimize the interaction of the quinolinecarboxamide derivatives with PBR and to develop suitable candidates for positron emission tomography (PET) or single photon emission computed tomography (SPECT) studies. The binding studies led to the discovery of fluoromethyl derivative 6a, which showed an IC50 value of 0.11 nM and is, therefore, one of the most potent PBR ligands so far described. Fluoromethyl derivative 6a has been labeled with 11C (t1/2=20.4 min, beta+=99.8%) starting from the corresponding des-methyl precursor (14) using [11C]CH3I in the presence of tetrabutylammonium hydroxide in DMF with a 35-40% radiochemical yield (corrected for decay) and 1.5 Ci/micromol of specific radioactivity. Ex vivo rat biodistribution and inhibition (following intravenous pre-administration of PK11195) studies showed that [11C]6a rapidly and specifically accumulated in PBR-rich tissues such as heart, lung, kidney, spleen, and adrenal, and at a lower level in other peripheral organs and in the brain. The images obtained in mouse with small animal YAP-(S)PET essentially confirmed the result of the ex vivo biodistribution experiments. The biological data suggest that [11C]6a is a promising radioligand for peripheral benzodiazepine receptor PET imaging in vivo.     

Forest Management and Plant Species Diversity in Chestnut Stands of Three Mediterranean Areas

Over many centuries, chestnut fruits had an important role as food, while chestnut wood was used for local purposes. Today sweet chestnut stands are very common around the western Mediterranean Basin, and it is necessary to analyze the dynamic of plant species diversity in different chestnut stand types (groves and coppices) to guide management strategies that will allow the conservation of biodiversity. Our objective was to analyze consequences on plant species diversity of various management strategies in chestnut stands of three Mediterranean areas, Salamanca (Spain), the Cévennes (France), and Etna volcano (Italy). We found that plant species diversity is different according to management types; it is higher in groves than in coppice stands. We also demonstrated that Castanea sativa cultivated groves were characterized by small heliophillous therophytes. C. sativa abandoned groves, mixed C. sativa–Quercus pyrenaica coppice stands, Q. pyrenaica coppice stands, and young C. sativa coppice stands were characterized by hemicryptophytes with anemochorous dispersal mode and chamaephytes. Medium and old C. sativa coppice stands (that differ by the shoot age) were characterized by phanerophytes with zoochorous dispersal mode. Human perturbations maintain a quite high level of species diversity. In contrast, the abandonment of chestnut stands leads to homogeneous vegetation with decreasing diversity. One solution could be to maintain a landscape mosaic constituted of diverse chestnut stands modified by human activities (groves, cultivated or abandoned, and coppice stands). This could enhance regional plant diversity.     

In vitro inhibitory activity of boropinic acid against Helicobacter pylori

In this study, we assessed in vitro minimum inhibitory concentration (MIC) values of some natural geranyloxycoumarins, geranyloxy- and isopentenyloxy acids against growth of Helicobacter pylori. Boropinic acid, active principle isolated from Boronia pinnata (Fam. Rutaceae), was seen to be the most effective compound with a MIC value of 1.62 microg/mL.     

Navajo neurohepatopathy is caused by a mutation in the MPV17 gene

Navajo neurohepatopathy (NNH) is an autosomal recessive disease that is prevalent among Navajo children in the southwestern United States. The major clinical features are hepatopathy, peripheral neuropathy, corneal anesthesia and scarring, acral mutilation, cerebral leukoencephalopathy, failure to thrive, and recurrent metabolic acidosis with intercurrent infections. Infantile, childhood, and classic forms of NNH have been described. Mitochondrial DNA (mtDNA) depletion was detected in the livers of two patients, suggesting a primary defect in mtDNA maintenance. Homozygosity mapping of two families with NNH suggested linkage to chromosome 2p24. This locus includes the MPV17 gene, which, when mutated, causes a hepatocerebral form of mtDNA depletion. Sequencing of the MPV17 gene in six patients with NNH from five families revealed the homozygous R50Q mutation described elsewhere. Identification of a single missense mutation in patients with NNH confirms that the disease is probably due to a founder effect and extends the phenotypic spectrum associated with MPV17 mutations.     

Mutations in structural genes of complex I associated with neurological diseases

This paper summarizes observations on the genetic and biochemical basis of hereditary defects of complex I (NADH-ubiquinone oxidoreductase) of the respiratory chain in human neurological patients. Two different types of functional defects of the complex are described. In one type mutations in the NDUFS1 and NDUFS4 nuclear structural genes of the complex were identified in two unrelated families. Both NDUFS1 and NDUFS4 neurological disorders were transmitted by autosomic recessive inheritance. The two mutations resulted in different impact on cellular metabolism. The NDUFS4 mutation, giving a more severe, fatal pathological pattern, resulted in a defective assembly of the complex and complete suppression of the enzymatic activity. The NDUFS1 mutation, with less severe progressive pathology, caused only partial inhibition of the complex but enhanced production of oxygen free radicals. In the second type of deficiencies extensive mutational analysis did not reveal pathogenic mutations in complex I genes but a decline in the level and activity of complex I, III, and IV were found, apparently associated with alteration in the cardiolipin membrane distribution.     

MÉNAGE À TROIS—TWO ENDEMIC SPECIES OF DECEPTIVE ORCHIDS AND ONE POLLINATOR SPECIES

In the sexually deceptive orchid genus Ophrys , reproductive isolation is based on the specific attraction of males of a single pollinator species by mimicking the female species-specific sex pheromone. Changes in the odor composition can lead to hybridization and speciation by the attraction of a new pollinator that acts as an isolation barrier toward other sympatrically occurring Ophrys species. On Sardinia, we investigated the evolutionary origin of two sympatrically occurring endemic species, Ophrys chestermanii and O. normanii , which are both pollinated by males of the cuckoo bumblebee Bombus vestalis . Chemical and electrophysiological analyses of floral scent and genetic analyses with amplified fragment length polymorphisms and plastid-markers clearly showed that O. normanii is neither a hybrid nor a hybrid species. The two species evolved from different ancestors, viz. O. normanii from O. tenthredinifera and O. chestermanii from O. annae , and converged to the same pollinator attracted by the same bouquet of polar compounds. In spite of sympatry, pollinator sharing and overlapping blooming periods, no evidence has been obtained for gene flow between O. chestermanii and O. normanii indicating an unusual case among sexually deceptive orchids in which postmating rather than premating reproductive isolation mechanisms strongly prevent interspecific gene flow.     

Trypanosoma cruzi: Isolation and characterization of aspartyl proteases

Two aspartyl proteases activities were identified and isolated from Trypanosoma cruzi epimastigotes: cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II). One was isolated from a soluble fraction (CZP-II) and the other was solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CZP-I). The molecular mass of both proteases was estimated to be 120 kDa by HPLC gel filtration and the activity of the enzymes was detected in a doublet of bands (56 and 48 kDa) by substrate-sodium dodecyl sulphate-polyacrylamide-gelatin gel electrophoresis. Substrate specificity studies indicated that the enzymes consistently hydrolyze the cathepsin D substrate Phe-Ala-Ala-Phe (4-NO₂)-Phe-Val-Leu-O₄MP but failed to hydrolyze serine and other protease substrates. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (?68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (?80%). These findings show that both proteases are novel T. cruzi acidic proteases. The physiological function of these enzymes in T. cruzi has under investigation.