Adenosine deaminase affects ligand-induced signalling by interacting with cell surface adenosine receptors

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto-enzyme. At the plasma membrane, an adenosine deaminase binding protein (CD26, also known as dipeptidylpeptidase IV) has been identified but the functional role of this ADA/CD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A₁ adenosine receptor (A₁R) is a second ecto-ADA binding protein. Binding of ADA to A₁R increased its affinity for the ligand thus suggesting that ADA was needed for an effective coupling between A₁R and heterotrimeric G proteins. This was confirmed by the fact that ASA, independently of its catalytic behaviour, enhanced the ligand-induced second messenger production via A₁R. These findings demonstrate that, apart from the cleavage of adenosine, a further role of ecto-adenosine deaminase on the cell surface is to facilitate the signal transduction via A₁R.     

Alu-splice PCR: a simple method to isolate exon-containing fragments from cloned human genomic DNA

We have developed a simple, straightforward procedure to isolate exons from cloned human genomic DNA. The method is PCR based and relies upon the conservation of splice-site sequences and the frequency of Alu repeat elements in the genome to capture coding sequences. We designed two different sets of primers: a primer from each end of the Alu element and primers with the 5′ or 3′ splice-site consensus sequences. Putative exons were amplified by PCR using YAC DNA as starting material. We applied Alu-splice PCR to two overlapping YACs, 72H9 and 860G11, from human chromosome 21. Sequence and northern analysis of 37 initial clones resulted in the identification of five novel exons. Received: 17 July 1997 / Accepted: 28 August 1997     

A new breeding population of Mediterranean Gulls Larus melanocephalus in the species' main wintering area maintains independent spatial dynamics

We studied a newly established breeding population of the range‐expanding Mediterranean Gull Larus melanocephalus in eastern Spain, situated in close proximity to the species' main wintering area. By investigating the origin, population composition and wintering area of the new breeders, we found that recruitment from locally wintering birds was unlikely and that the emerging colonies were probably attracting birds from populations wintering 700–1200 km away in Portugal and southern Spain. Our findings reveal that expanding populations may follow their own dynamics, independently of other populations of the same species, and may consist of different individuals altogether.     

Soil drying procedure affects the DNA quantification of <Emphasis Type="Italic">Lactarius vinosus</Emphasis> but does not change the fungal community composition

Drying soil samples before DNA extraction is commonly used for specific fungal DNA quantification and metabarcoding studies, but the impact of different drying procedures on both the specific fungal DNA quantity and the fungal community composition has not been analyzed. We tested three different drying procedures (freeze-drying, oven-drying, and room temperature) on 12 different soil samples to determine (a) the soil mycelium biomass of the ectomycorrhizal species Lactarius vinosus using qPCR with a specifically designed TaqMan® probe and (b) the fungal community composition and diversity using the PacBio® RS II sequencing platform. Mycelium biomass of L. vinosus was significantly greater in the freeze-dried soil samples than in samples dried at oven and room temperature. However, drying procedures had no effect on fungal community composition or on fungal diversity. In addition, there were no significant differences in the proportions of fungi according to their functional roles (moulds vs. mycorrhizal species) in response to drying procedures. Only six out of 1139 operational taxonomic units (OTUs) had increased their relative proportions after soil drying at room temperature, with five of these OTUs classified as mould or yeast species. However, the magnitude of these changes was small, with an overall increase in relative abundance of these OTUs of approximately 2 %. These results suggest that DNA degradation may occur especially after drying soil samples at room temperature, but affecting equally nearly all fungi and therefore causing no significant differences in diversity and community composition. Despite the minimal effects caused by the drying procedures at the fungal community composition, freeze-drying resulted in higher concentrations of L. vinosus DNA and prevented potential colonization from opportunistic species.     

Multiple Paternity in a Reintroduced Population of the Orinoco Crocodile (Crocodylus intermedius) at the El Frío Biological Station,Venezuela

The success of a reintroduction program is determined by the ability of individuals to reproduce and thrive. Hence, an understanding of the mating system and breeding strategies of reintroduced species can be critical to the success, evaluation and effective management of reintroduction programs. As one of the most threatened crocodile species in the world, the Orinoco crocodile (Crocodylus intermedius) has been reduced to only a few wild populations in the Llanos of Venezuela and Colombia. One of these populations was founded by reintroduction at Caño Macanillal and La Ramera lagoon within the El Frío Biological Station, Venezuela. Twenty egg clutches of C. intermedius were collected at the El Frío Biological Station for incubation in the lab and release of juveniles after one year. Analyzing 17 polymorphic microsatellite loci from 335 hatchlings we found multiple paternity in C. intermedius, with half of the 20 clutches fathered by two or three males. Sixteen mothers and 14 fathers were inferred by reconstruction of multilocus parental genotypes. Our findings showed skewed paternal contributions to multiple-sired clutches in four of the clutches (40%), leading to an overall unequal contribution of offspring among fathers with six of the 14 inferred males fathering 90% of the total offspring, and three of those six males fathering more than 70% of the total offspring. Our results provide the first evidence of multiple paternity occurring in the Orinoco crocodile and confirm the success of reintroduction efforts of this critically endangered species in the El Frío Biological Station, Venezuela.     

Failure of an enriched nitrite-DPAO population to use nitrate as an electron acceptor

The metabolism of denitrifying polyphosphate accumulating organisms (DPAO) is not completely known. Recent reports suggest the existence of two types of DPAOs: those that can use nitrate and nitrite as electron acceptors (nitrate-DPAO) and those that can only use nitrite (nitrite-DPAO). Then, the survival of nitrite-DPAO in nitrate reducing environments is due to the existence of flanking denitrifying species, which reduce nitrate to nitrite. This works aims at a better understanding of the nitrite-DPAO population. For this aim, a nitrite-DPAO population was previously selected in a SBR using nitrite as electron acceptor. Then, nitrate utilisation by nitrite-DPAO was studied within a short-term period (4 days) and within a long-term period (50 days) with simultaneous nitrite and nitrate additions. The results obtained clearly indicate that nitrite-DPAO fail to use nitrate as electron acceptor even after 50 days of periodic dosing of nitrate and agree with the dual DPAO theory. Moreover, this failure casts doubts on the feasibility of nitrite based EBPR systems (i.e. partial nitrification + nitrite-DPAO) because these systems will not be able to denitrify an occasional nitrate inlet, which will remain in the effluent.     

Phylogeography and phylogeny of the epineritic cosmopolitan bonitos of the genus Sarda (Cuvier): inferred patterns of intra‐ and inter‐oceanic connectivity derived from nuclear and mitochondrial DNA data

Aim To reconstruct the phylogenetic relationships of the four species of the genus Sarda (Sarda sarda, Sarda orientalis, Sarda australis and Sarda chilensis) and their phylogeographic history in the context of historical and ecological biogeography. Also, to reconstruct within‐species phylogenetic relationships to test whether the North Atlantic and Mediterranean populations of Atlantic bonito (S. sarda) warrant subspecies status, and the validity of the allopatric northern and southern populations of eastern Pacific bonito (S. chiliensis), recognized as S. chiliensis lineolata and S. chiliensis chiliensis. Location Representative samples of all four Sarda species collected world‐wide were analysed. Methods Phylogenetic inference was carried out with neighbour‐joining, maximum parsimony and maximum likelihood, employing nucleotide sequences of the mitochondrial DNA (mtDNA) control region I (CR‐I) and of the single‐copy nuclear DNA (nDNA) Tmo‐4c4 gene. Analysis of molecular variance was used on the mtDNA data to estimate the extent of geographic population structuring. Results Gene trees derived from mtDNA and nDNA data yielded concordant phylogenies that support the monophyly of the genus Sarda. The following sibling pairs received strong statistical support: striped bonito (S. orientalis) with Australian bonito (S. australis), and Atlantic bonito (S. sarda) with eastern Pacific bonito (S. chiliensis). Furthermore, the origin of S. sarda mtDNA is paraphyletic with respect to S. chiliensis, and these results are indicative of introgression. The analysis of Tmo‐4c4 sequences corroborates the ancestral hybridization between these allopatric species. Comparisons of north‐west Atlantic and Mediterranean populations of S. sarda using mtDNA CR‐I data revealed substantial genetic differentiation. By contrast, no differences between the putative northern and southern allopatric subspecies of S. chiliensis were detected. Main conclusions The monophyly of the genus Sarda as indicated by morphology is corroborated using both molecular markers. However, molecular phylogenies depicted a paraphyletic relationship between S. sarda and S. chiliensis. This phylogeographical relationship is better explained by an ancestral introgression facilitated by trans‐Arctic contact during the Pleistocene. The pronounced genetic differentiation between S. sarda samples from the north‐west Atlantic and the Mediterranean is consistent with the differentiation of these two regions, but not with the amphi‐Atlantic speciation hypothesis. Finally, the S. chiliensis lineolata and S. chiliensis chiliensis subspecies status is not supported by the molecular data.     

Phorbol Esters Induce Neurotransmitter Release in Cholinergic Synaptosomes from Torpedo Electric Organ

The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity. This drug showed no effect on potassium-evoked ACh release. Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.