Human midbrain precursors activate the expected developmental genetic program and differentiate long-term to functional A9 dopamine neurons in vitro. Enhancement by Bcl-X(L)

Understanding the molecular programs of the generation of human dopaminergic neurons (DAn) from their ventral mesencephalic (VM) precursors is of key importance for basic studies, progress in cell therapy, drug screening and pharmacology in the context of Parkinson's disease. The nature of human DAn precursors in vitro is poorly understood, their properties unstable, and their availability highly limited. Here we present positive evidence that human VM precursors retaining their genuine properties and long-term capacity to generate A9 type Substantia nigra human DAn (hVM1 model cell line) can be propagated in culture. During a one month differentiation, these cells activate all key genes needed to progress from pro-neural and pro-dopaminergic precursors to mature and functional DAn. For the first time, we demonstrate that gene cascades are correctly activated during differentiation, resulting in the generation of mature DAn. These DAn have morphological and functional properties undistinguishable from those generated by VM primary neuronal cultures. In addition, we have found that the forced expression of Bcl-X(L) induces an increase in the expression of key developmental genes (MSX1, NGN2), maintenance of PITX3 expression temporal profile, and also enhances genes involved in DAn long-term function, maintenance and survival (EN1, LMX1B, NURR1 and PITX3). As a result, Bcl-X(L) anticipates and enhances DAn generation.     

Imidazo[1,2-a]pyrazines as novel PI3K inhibitors

Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of its signaling pathway in a wide variety of human cancers. We describe herein a novel series of imidazo[1,2-a]pyrazines as PI3K inhibitors.     

Aryl-alcohol Oxidase Involved in Lignin Degradation: A MECHANISTIC STUDY BASED ON STEADY AND PRE-STEADY STATE KINETICS AND PRIMARY AND SOLVENT ISOTOPE EFFECTS WITH TWO ALCOHOL SUBSTRATES*

Aryl-alcohol oxidase (AAO) is a FAD-containing enzyme in the GMC (glucose-methanol-choline oxidase) family of oxidoreductases. AAO participates in fungal degradation of lignin, a process of high ecological and biotechnological relevance, by providing the hydrogen peroxide required by ligninolytic peroxidases. In the Pleurotus species, this peroxide is generated in the redox cycling of p-anisaldehyde, an extracellular fungal metabolite. In addition to p-anisyl alcohol, the enzyme also oxidizes other polyunsaturated primary alcohols. Its reaction mechanism was investigated here using p-anisyl alcohol and 2,4-hexadien-1-ol as two AAO model substrates. Steady state kinetic parameters and enzyme-monitored turnover were consistent with a sequential mechanism in which O₂ reacts with reduced AAO before release of the aldehyde product. Pre-steady state analysis revealed that the AAO reductive half-reaction is essentially irreversible and rate limiting during catalysis. Substrate and solvent kinetic isotope effects under steady and pre-steady state conditions (the latter showing ∼9-fold slower enzyme reduction when α-bideuterated substrates were used, and ∼13-fold slower reduction when both substrate and solvent effects were simultaneously evaluated) revealed a synchronous mechanism in which hydride transfer from substrate α-carbon to FAD and proton abstraction from hydroxyl occur simultaneously. This significantly differs from the general mechanism proposed for other members of the GMC oxidoreductase family that implies hydride transfer from a previously stabilized substrate alkoxide.Wood and other lignocellulosic materials are the main source of renewable materials in earth. White-rot basidiomycetes are essential contributors to carbon cycling in forest and other land ecosystems because of their ability to degrade lignocellulose to carbon dioxide and water. This ability confers to these fungi and their ligninolytic enzymes high interest in industrial processes, such as bioethanol production and paper pulp manufacturing, where the removal of lignin is a previous and essential step to use the cellulose present in plant biomass as a source for renewable fuels, chemicals, and materials (1).Aryl-alcohol oxidase (AAO)⁵ is an extracellular FAD-containing enzyme (2) that, in collaboration with myceliar aryl-alcohol dehydrogenases, participates in lignin degradation by some white-rot fungi, such as Pleurotus (and Bjerkandera) species, by generating hydrogen peroxide in the redox cycling of aromatic fungal metabolites, such as p-anisaldehyde (3, 4). Fungal high redox-potential peroxidases catalyze the oxidative degradation of lignin by this extracellular peroxide (5).AAO was cloned for the first time in Pleurotus eryngii (6), a fungus of biotechnological interest because of its ability to degrade lignin selectively (7). The AAO amino acid sequence revealed moderate homology with glucose oxidase from Aspergillus niger (8), a flavoenzyme in the glucose-methanol-choline oxidases (GMC) oxidoreductase family. The reported molecular model of AAO (9), based on the glucose oxidase crystal structure (10), showed common features with the overall structural topology of bacterial choline oxidase and almond hydroxynitrile lyase (a lyase with oxidoreductase structure), as well as with other members of the GMC family; such as the extracellular flavoenzymes pyranose-2-oxidase and cellobiose dehydrogenase from white-rot basidiomycetes, and bacterial cholesterol oxidase (11–15). In particular, P. eryngii AAO conserves two histidine residues, His-502 and His-546 (supplemental Fig. S1), involved in catalysis in different members of this family (the second residue is an asparagine in some of them) (9).Non-glycosylated P. eryngii AAO expressed in Escherichia coli (16) is used for further characterization studies. The enzyme catalyzes the oxidative dehydrogenation of unsaturated alcohols with a primary hydroxyl at Cα, exhibiting broad substrate specificity. In addition to benzyl alcohols, its active site also binds and oxidizes aliphatic polyunsaturated primary alcohols (such as 2,4-hexadien-1-ol), naphthyl, and cinnamyl alcohols, and shows low activity on some aromatic aldehydes (17). Methanol and other saturated alcohols are not AAO substrates, and the monounsaturated allyl alcohol is very slowly oxidized (2).It is suggested that the AAO catalytic mechanism proceeds via electrophilic attack and direct transfer of a hydride to the flavin (17). A recent mutational study confirmed the strict requirement for catalysis of His-502 and His-546 located near the isoalloxazine ring of FAD (supplemental Fig. S1), as well as the involvement of two aromatic residues (18). Here we present the first study on the reaction mechanism of AAO in which substrate and solvent kinetic isotope effect (KIE), in combination with bisubstrate steady state and pre-steady state kinetic approaches, have been used to investigate the mechanism of polyunsaturated primary alcohol oxidation by AAO. Its natural substrate, p-anisyl alcohol, as well as a structurally different (non-aromatic) AAO substrate, 2,4-hexadien-1-ol, were chosen as two models for the different AAO alcohol substrates.     

Coenzyme binding and hydride transfer in Rhodobacter capsulatus ferredoxin/flavodoxin NADP(H) oxidoreductase

Ferredoxin-NADP(H) reductases catalyse the reversible hydride/electron exchange between NADP(H) and ferredoxin/flavodoxin, comprising a structurally defined family of flavoenzymes with two distinct subclasses. Those present in Gram-negative bacteria (FPRs) display turnover numbers of 1-5 s(-1) while the homologues of cyanobacteria and plants (FNRs) developed a 100-fold activity increase. We investigated nucleotide interactions and hydride transfer in Rhodobacter capsulatus FPR comparing them to those reported for FNRs. NADP(H) binding proceeds as in FNRs with stacking of the nicotinamide on the flavin, which resulted in formation of charge-transfer complexes prior to hydride exchange. The affinity of FPR for both NADP(H) and 2'-P-AMP was 100-fold lower than that of FNRs. The crystal structure of FPR in complex with 2'-P-AMP and NADP(+) allowed modelling of the adenosine ring system bound to the protein, whereas the nicotinamide portion was either not visible or protruding toward solvent in different obtained crystals. Stabilising contacts with the active site residues are different in the two reductase classes. We conclude that evolution to higher activities in FNRs was partially favoured by modification of NADP(H) binding in the initial complexes through changes in the active site residues involved in stabilisation of the adenosine portion of the nucleotide and in the mobile C-terminus of FPR.     

Rhizobia from Lanzarote,the Canary Islands,That Nodulate Phaseolus vulgaris Have Characteristics in Common with Sinorhizobium meliloti Isolates from Mainland Spain

The stable, low-molecular-weight (LMW) RNA fractions of several rhizobial isolates of Phaseolus vulgaris grown in the soil of Lanzarote, an island of the Canary Islands, were identical to a less-common pattern found within Sinorhizobium meliloti (assigned to group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northern Spain. The P. vulgaris isolates and the group II LMW RNA S. meliloti isolates also were distinguishable in that both had two conserved inserts of 20 and 46 bp in the 16S-23S internal transcribed spacer region that were not present in other strains of S. meliloti. The isolates from P. vulgaris nodulated bean but not Medicago sativa, while those recovered from Medicago, Melilotus, and Trigonella spp. nodulated both host legumes. The bean isolates also were distinguished from those of Medicago, Melilotus, and Trigonella spp. by nodC sequence analysis. The nodC sequences of the bean isolates were most similar to those reported for S. meliloti bv. mediterranense and Sinorhizobium fredii bv. mediterranense (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"DQ333891","term_id":"84872507","term_text":"DQ333891"}}DQ333891 and {"type":"entrez-nucleotide","attrs":{"text":"AF217267","term_id":"13810627","term_text":"AF217267"}}AF217267, respectively). None of the evidence placed the bean isolates from Lanzarote in the genus Rhizobium, which perhaps is inconsistent with seed-borne transmission of Rhizobium etli from the Americas to the Canaries as an explanation for the presence of bean-nodulating rhizobia in soils of Lanzarote.A remarkable attribute of Phaseolus vulgaris (common bean) is its ability to nodulate with rhizobia from at least 20 different legume genera (summarized in reference 1). Of particular relevance is the report by Ishizawa (16), who described P. vulgaris nodulation ranging from doubtful to good by 14 strains recovered from Medicago sativa, Medicago denticulata, and Melilotus alba, while nodulation of the latter three legumes by four bean strains was negative.At the time of the host range experiments, such as those described by Ishizawa (16), rhizobial nomenclature depended on the legume host of origin; the taxonomy of the strains was based on cross-inoculation groups. Consequently, no information was available about the genetic relationships among the rhizobial strains that originated from the different host legume genera and formed nodules on P. vulgaris. Eventually, rhizobial nomenclature based on the cross-inoculation groups was abandoned because of the many unexplainable and incongruous nodulation data (44). The cross-inoculation groups consisted of different rhizobial species within the single genus Rhizobium. Eventually, rhizobial taxonomy was expanded to several different genera based on estimates of their phylogeny (38). Phylogenies of bean-nodulating rhizobia were estimated from variations in the 16S rRNA gene sequence (39), even though subsequently it became clear that this method is significantly limited by histories of genetic exchange and recombination (6, 40). Most reported phylogenies of rhizobia nodulating P. vulgaris have placed them in the genus Rhizobium (3, 39), but several surveys with isolates from North Africa and Spain have demonstrated that rhizobia in the genus Sinorhizobium also nodulate this legume species (12, 23, 24, 25, 41), supporting the nodulation data originally published by Ishizawa (16). The number of isolates described as originating from nodules of P. vulgaris in the genus Sinorhizobium is small, and for the most part, from the published evidence, it has been suggested that they are affiliated with Sinorhizobium fredii. However, nodules of P. vulgaris growing in a single Tunisian soil where beans are cultivated yielded four isolates that, according to the data, appeared to support an affiliation with Sinorhizobium meliloti rather than S. fredii (25). Whether these four cultures were of the same rhizobial genotype constituting a single example of S. meliloti isolated from P. vulgaris is unknown.P. vulgaris was introduced into Europe as a crop plant as early as the 16th century (31) but never became a very important part of agriculture in Lanzarote, one of the Canary Islands that lie in the Atlantic Ocean to the west of the North African coast. Since there is no record of any nodulation studies with P. vulgaris cultivated on Lanzarote Island, the first objective of this study was to examine bean plants that had grown in Lanzarote soil for nodulation. Considering that the diversity of rhizobia able to nodulate bean plants is extremely wide, the second objective was to characterize the isolates originating from the nodules of plants grown in Lanzarote soil.(Part of this work was presented as a poster at the First International Meeting on Microbial Phosphate Solubilization, Salamanca, Spain, July 2002.)     

Population genetic structure of the Antarctic ascidian Aplidium falklandicum from Scotia Arc and South Shetland Islands

In sessile marine organisms, gene flow between populations depends mainly on free-living reproductive stages (such as larvae and gametes), and usually the strength of genetic structure is related to the time spent in the plankton and physical factors as oceanographic conditions. In Antarctica, abyssal depths that surround the continent and the Polar Front are considered strong barriers for benthic marine fauna, keeping the continent isolated from other shelves. The only available shallow water habitats between South America and the Antarctic continent are those around the Scotia Arc Islands; there are no shallow water habitats between the other southern continents and Antarctica. In this work, ISSRs-PCR markers were used to study the genetic structure of populations of Aplidium falklandicum, a compound ascidian with short-lived lecitotrophic larvae. A highly significant genetic differentiation (Φ_ST = 0.405; P < 0.05) and a pattern of isolation by distance were found. A genetic landscape approach identified a discontinuity in genetic diversity, coincident with the southernmost registered position of the Polar Front. For A. falklandicum, a species with presumably low capacity of long distance dispersal, the abyssal depths together with the large geographic distances create a barrier for gene flow.     

Strains nodulating Lupinus albus on different continents belong to several new chromosomal and symbiotic lineages within Bradyrhizobium

In this work we analysed different chromosomal and symbiotic markers in rhizobial strains nodulating Lupinus albus (white lupin) in several continents. Collectively the analysis of their rrs and atpD genes, and 16S-23S intergenic spacers (ITS), showed that they belong to at least four chromosomal lineages within the genus Bradyrhizobium. Most isolates from the Canary Islands (near to the African continent) grouped with some strains isolated on mainland Spain and were identified as Bradyrhizobium canariense. These strains are divided into two ITS subgroups coincident with those previously described from isolates nodulating Ornithopus. The remaining strains isolated on mainland Spain grouped with most isolates from Chile (American continent) forming a new lineage related to Bradyrhizobium japonicum. The strains BLUT2 and ISLU207 isolated from the Canary Islands and Chile, respectively, formed two new lineages phylogenetically close to different species of Bradyrhizobium depending on the marker analyzed. The analysis of the nodC gene showed that all strains nodulating L. albus belong to the biovar genistearum; nevertheless they form four different nodC lineages of which lineage C is at present exclusively formed by L. albus endosymbionts isolated from different continents.