Rhizobium etli taxonomy revised with novel genomic data and analyses

The taxonomic position of Phaseolus vulgaris rhizobial strains with available sequenced genomes was examined. Phylogenetic analyses with concatenated conserved genomic fragments accounting for over half of each genome showed that Rhizobium strains CIAT 652, Ch24-10 (newly reported genome) and CNPAF 512 constituted a well-supported group independent from Rhizobium etli CFN 42(T). DNA-DNA hybridization results indicated that CIAT 652, Ch24-10 and CNPAF 512 could correspond to R. etli, although the hybridization values were at the borderline that distinguishes different species. In contrast, experimental hybridization results were higher (over 80%) with Rhizobium phaseoli type strain ATCC 14482(T) in congruence to phylogenetic and ANIm analyses. The latter criterion allowed the reclassification of R. etli strains 8C-3 and Brasil5 as R. phaseoli. It was therefore concluded, based on all the evidence, that the CIAT 652, Ch24-10, and CNPAF 512 strains should be reclassified as R. phaseoli in spite of several common features linking them to R. etli. The R. phaseoli and R. etli speciation process seems to be a more recent event than the speciation that has occurred among other sister species, such as R. leguminosarum-R. etli or R. rhizogenes-R. tropici.     

Bradyrhizobium rifense sp. nov. isolated from effective nodules of Cytisus villosus grown in the Moroccan Rif

Two bradyrhizobial strains, CTAW71(T) and CTAW69, previously isolated from root nodules of Cytisus villosus, have been analysed using a polyphasic approach. These strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium cytisi, whose type strain CTAW11(T) presented 99.8% identity with respect to strain CTAW71(T). Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII harboured by strain CTAW71(T) were divergent to those from B. cytisi CTAW11(T), with identity values of 93%, 95% and 97%, respectively. These differences were congruent with DNA-DNA hybridization analysis that revealed an average of 37% relatedness between strain CTAW71(T) and B. cytisi CTAW11(T). Phenotypic characteristics were identical for strains CTAW71(T) and CTAW69, but differed from those of the described species from genus Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose that strains CTAW71(T) and CTAW69 should be classified into a new species for which the name Bradyrhizobium rifense sp. nov. is proposed (type strain CTAW71(T)=LMG 26781(T)=CECT 8066(T)).     

Effect of acepromazine and haloperidol in male Iberian Ibex (Capra pyrenaica) captured by box-trap

Short-acting neuroleptic drugs are used to prevent adverse effects of stress in wildlife. We compared the effect of acepromazine and haloperidol in Iberian ibex (Capra pyrenaica) captured with box-traps. We captured 23 male Iberian ibex at the National Game Reserve of Ports de Tortosa i Beseit, northeastern Spain, March 2003-June 2005. Seven animals received 0.1 mg/kg of acepromazine maleate, eight received 0.33 mg/kg of haloperidol and eight animals acted as controls. Clinical, hematologic, and serum biochemical parameters were analyzed. Both treatments decreased rectal temperature, white blood cells, lymphocytes, and concentrations of creatinine, creatine kinase, and lactate dehydrogenase. Acepromazine also decreased red blood cells, packed cell volume, hemoglobin concentration, neutrophils, and concentrations of glucose and cholesterol. Haloperidol also decreased heart rate and concentrations of urea and potassium. Our results demonstrate the suitability of using acepromazine and haloperidol in capture operations to reduce stress and prevent its adverse effects.     

Roost characteristics as indicators for heterothermic behavior of forest-dwelling bats

Many forest-dwelling bats spend their diurnal inactivity period in tree cavities. During this time bats can save energy through heterothermy. A heterothermic response (torpor) is characterized by a lowered body temperature, reduced metabolic rate, and reduction of other physiological processes, and can be influenced by the microclimatic conditions of roost cavities. The thermal and physical characteristics of roosts used by the sympatric, ecologically, and morphologically similar bat species Myotis bechsteinii, M. nattereri, and Plecotus auritus were compared. These three species differ in their heterothermic behavior, with the lowest skin temperatures observed for P. auritus. Therefore, we hypothesized that roosts occupied by the three species should differ in roost characteristics and microclimatic conditions, whereby P. auritus should select colder and thermally less stable roosts. The results showed that horizontal depth of the cavity, diameter of the roost tree, and microclimatic conditions within roosts differed among species. Roosts of P. auritus had the lowest horizontal depth, lowest thermal stability, and lowest mean minimum roost temperatures. Height of the roost, diameter of the roost tree, and vertical depth were also shown to influence microclimatic conditions. With increasing diameter of the tree and increasing horizontal depth, mean minimum roost temperature increased and thermal stability improved. Furthermore, with ascending height above ground insulation and mean roost temperatures increased. Our results imply that species such as P. auritus, which use pronounced torpor as a primary energy saving strategy, prefer colder cavities that support their heterothermic strategy.     

Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites

Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to beta-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of beta-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.     

Recovery of neuropathy target esterase activity after inhibition with mipafox and O-hexyl O-2,5-dichlorophenyl phosphoramidate in bovine chromaffin cell cultures

Neuropathy target esterase (NTE) is a membrane protein present in various tissues whose physiological function has been recently suggested to be the maintenance of phosphatidylcholine homeostasis. Inhibition and further modification of NTE by certain organophosphorus compounds (OPs) were related to the induction of the "organophosphorus induced delayed neuropathy". Bovine chromaffin cells were cultured at 75,000cells/well in 96-well plates and exposed to 25microM mipafox or 3microM O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) for 60min. Inhibitors were removed by washing cells three times with Krebs solution. Then NTE activity was assayed at 0, 24, 48 and 120h after exposure using the Biomek 1000 workstation. Immediately after mipafox treatment NTE activity represented 3% of the control (6.7+/-1.9mU/10(6) cells). At 24, 48 and 120h after removing inhibitor, recorded activities were 33%, 42% and 111% of their respective controls (5.7+/-3.1; 5.7+/-1.9; 5.4+/-0.0mU/10(6) cells, respectively). Treatment with HDCP also displayed a time-dependent pattern of NTE recovery. As NTE inhibited by phosphoramidates is not reactivated in homogenized tissues, these results confirm a time-dependent regeneration of NTE after inhibition by neuropathic OPs.     

Synthesis and biological activities of high affinity taxane-based fluorescent probes

Three fluorescent probes 3a, 3b, and 4 have been synthesized through conjugation of fluorescein and difluorescein groups to the 7-OH of C-2 modified paclitaxel and cephalomannine derivatives with very high affinity to microtubules. All these probes exhibited potent tubulin assembly promotion and tumor cell killing activities, thus may be useful as tools for the determination of thermodynamic parameters and exploration of ligand–microtubule interactions.     

The analysis of core and symbiotic genes of rhizobia nodulating Vicia from different continents reveals their common phylogenetic origin and suggests the distribution of Rhizobium leguminosarum strains together with Vicia seeds

In this work, we analysed the core and symbiotic genes of rhizobial strains isolated from Vicia sativa in three soils from the Northwest of Spain, and compared them with other Vicia endosymbionts isolated in other geographical locations. The analysis of rrs, recA and atpD genes and 16S–23S rRNA intergenic spacer showed that the Spanish strains nodulating V. sativa are phylogenetically close to those isolated from V. sativa and V. faba in different European, American and Asian countries forming a group related to Rhizobium leguminosarum. The analysis of the nodC gene of strains nodulating V. sativa and V. faba in different continents showed they belong to a phylogenetically compact group indicating that these legumes are restrictive hosts. The results of the nodC gene analysis allow the delineation of the biovar viciae showing a common phylogenetic origin of V. sativa and V. faba endosymbionts in several continents. Since these two legume species are indigenous from Europe, our results suggest a world distribution of strains from R. leguminosarum together with the V. sativa and V. faba seeds and a close coevolution among chromosome, symbiotic genes and legume host in this Rhizobium–Vicia symbiosis.     

Ticks Parasitizing Wild Birds in Portugal: Detection of Rickettsia aeschlimannii, R. helvetica and R. massiliae

From January 2002 to December 2004, 152 ticks were collected from 40 wild birds recovered in Santo André Natural Reserve and Monsanto Forestal Park, Portugal mainland. Five ticks species were identified from 22 species of birds, and new host record were provided for some species. In addition, 32 (21%) ticks were screened by PCR to detect infections with agents belonging to order Rickettsiales: Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Rickettsia spp. PCR amplicons were obtained in 5 (15.6%) tick samples. Rickettsia DNA exhibiting gltA sequences similar to those of Rickettsia aeschilimannii, R. helvetica and R. massiliae were identified in Hyalomma marginatum, Ixodes ventalloi and in Rhipicephalus turanicus, respectively. This is the first report of rickettsiae infections in ticks collected from wild birds in Portugal. Giving the results presented above wild birds play an important role in the maintenance and dissemination of several tick species and associated rickettsiae.