Phenol degradation by a psychrotrophic strain of Pseudomonas putida

Summary Cell growth and phenol degradation kinetics were studied at 10°C for a psychrotrophic bacterium, Pseudomonas putida Q5. The batch studies were conducted for initial phenol concentrations, S_o, ranging from 14 to 1000 mg/1. The experimental data for 14<=S_o<=200 mg/1 were fitted by non-linear regression to the integrated Haldane substrate inhibition growth rate model. The values of the kinetic parameters were found to be: _m=0.119 h^–1, K _S=5.27 mg/1 and K _I=377 mg/1. The yield factor of dry biomass from substrate consumed was Y=0.55. Compared to mesophilic pseudomonads previously studied, the psychrotrophic strain grows on and degrades phenol at rates that are ca. 65–80% lower. However, use of the psychrotrophic microorganism may still be economically advantageous for waste-water treatment processes installed in cold climatic regions, and in cases where influent waste-water temperatures exhibit seasonal variation in the range 10–30°C.Nomenclature K _S saturation constant (mg/l) - K _I substrate inhibition constant (mg/l) - specific growth rate (h^–1) - _m maximum specific growth rate without substrate inhibition (h^–1) - _max maximum achievable specific growth rate with substrate inhibition (h^–1) - S substrate (phenol) concentration (mg/l) - S_o initial substrate concentration (mg/l) - S_max substrate concentration corresponding to _max (mg/l) - t time (h) - X cell concentration, dry basis (mg DW/l) - X_f final cell concentration, dry basis (mg DW/l) - X_o initial cell concentration, dry basis (mg DW/l) - Y yield factor (mg DW cell produced/mg substrate consumed)     

Identification of key structural determinants of the IntI1 integron integrase that influence attC x attI1 recombination efficiency

The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by several different IntI integrases. We have developed a genetic system to enrich and select mutants of IntI1 that provide a higher yield of recombination in order to identify key protein structural elements important for attC × attI1 recombination. We isolated mutants with higher activity on wild type and mutant attC sites. Interestingly, three out of four characterized IntI1 mutants selected on different substrates are mutants of the conserved aspartic acid in position 161. The IntI1 model we made based on the VchIntIA 3D structure suggests that substitution at this position, which plays a central role in multimer assembly, can increase or decrease the stability of the complex and accordingly influence the rate of attI × attC recombination versus attC × attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse.     

Initial Characterization of the Pf-Int Recombinase from the Malaria Parasite Plasmodium falciparum

Background

Genetic variation is an essential means of evolution and adaptation in many organisms in response to environmental change. Certain DNA alterations can be carried out by site-specific recombinases (SSRs) that fall into two families: the serine and the tyrosine recombinases. SSRs are seldom found in eukaryotes. A gene homologous to a tyrosine site-specific recombinase has been identified in the genome of Plasmodium falciparum. The sequence is highly conserved among five other members of Plasmodia.

Methodology/Principal Findings

The predicted open reading frame encodes for a ∼57 kDa protein containing a C-terminal domain including the putative tyrosine recombinase conserved active site residues R-H-R-(H/W)-Y. The N-terminus has the typical alpha-helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. Pf-Int mRNA is expressed differentially during the P. falciparum erythrocytic life stages, peaking in the schizont stage. Recombinant Pf-Int and affinity chromatography of DNA from genomic or synthetic origin were used to identify potential DNA targets after sequencing or micro-array hybridization. Interestingly, the sequences captured also included highly variable subtelomeric genes such as var, rif, and stevor sequences. Electrophoretic mobility shift assays with DNA were carried out to verify Pf-Int/DNA binding. Finally, Pf-Int knock-out parasites were created in order to investigate the biological role of Pf-Int.

Conclusions/Significance

Our data identify for the first time a malaria parasite gene with structural and functional features of recombinases. Pf-Int may bind to and alter DNA, either in a sequence specific or in a non-specific fashion, and may contribute to programmed or random DNA rearrangements. Pf-Int is the first molecular player identified with a potential role in genome plasticity in this pathogen. Finally, Pf-Int knock-out parasite is viable showing no detectable impact on blood stage development, which is compatible with such function.     

Patterns of DNA Barcode Variation in Canadian Marine Molluscs

Background

Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area.

Methodology/Principal Findings

This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0–26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%–46.5%), and showed a significant positive correlation with nearest neighbour distances.

Conclusions/Significance

DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.     

儿童颈髓区巨大动静脉畸形1例(英文)

脑动静脉畸形(AVM)是复杂的血管病变,是由于没有毛细血管床的脑动脉和静脉之间的异常连接引起的病灶[1]。脑动静脉畸形的临床表现有大出血、癫痫、神经功能障碍或头痛,但大部分患者无特异性症状,因此容易漏诊、误诊。手术切除、血管内治疗和放射治疗是动静脉畸形的选择疗法。血管内治疗可以作为其他难以治愈的脑动静脉畸形,或者作为手术切除或放射治疗前的辅助治疗,以减少脑动静脉畸形的血供或促进其收缩,进而促进手术切除或消融[2]。该病引起的颈部疼痛在儿科未见报道。我们收治1例颈髓区巨大动静脉畸形患儿,表现为持续性颈部疼痛并出现癫痫和呕吐症状,经采取分段外科胶栓塞术(第一次部分栓塞术后观察1年再行第二次栓塞),达到动静脉畸形的治愈。     

Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation

Background  

Gonadal differentiation in the mammalian fetus involves a complex dose-dependent genetic network. Initiation and progression of fetal ovarian and testicular pathways are accompanied by dynamic expression patterns of thousands of genes. We postulate these expression patterns are regulated by small non-coding RNAs called microRNAs (miRNAs). The aim of this study was to identify the expression of miRNAs in mammalian fetal gonads using sheep as a model.     

Refinement of the structure of Escherichia coli-derived rat intestinal fatty acid binding protein with bound oleate to 1.75-A resolution. Correlation with the structures of the apoprotein and the protein with bound palmitate.

The structure of rat intestinal fatty acid binding protein (I-FABP) with bound oleate (C18:1) has been refined with x-ray diffraction data to a resolution of 1.75 A. The protein contains 10 anti-parallel beta strands composed of 99 residues and 2 short helices of 14 residues. Oleate is located in the interior of the protein in a bent conformation with C1-C12 more ordered than C13-C18. Two of the eight ordered waters in I-FABP:oleate are part of a hydrogen bond network that includes the carboxylate of oleate, the guanidinium group of Arg106, the nitrogen of the indole group of Trp82, and the side chain of Gln115. Most of the methylenes of bound oleate reside in a crevice formed by hydrophobic and aromatic side chains. Tyr70 and Tyr117 envelop the acyl chain from C3 to C8 forming contacts with both the convex and concave faces of its van der Waals surface. The hydroxyls of each phenolic side chain hydrogen bond to ordered water molecules. Two ordered waters make van der Waals contact with the concave face of the bound fatty acid. The omega-terminal methyl of oleate is oriented so that it points toward the center of the benzene of Phe55 allowing it to form van der Waals interactions with its component methylenes. Comparison of the structure of I-FABP:oleate with a recently refined 1.19-A model of apoI-FABP and an earlier 2.0-A model of I-FABP:palmitate revealed a remarkable degree of similarity in the positions of their main chain and side chain atoms and in the conformations of the bound oleate and palmitate. The principal differences were confined to a few discrete regions of the protein. The helical domain, the type I turn between beta strands C and D, and the ring of Phe55 together form a solvent-accessible portal to the interior of the protein. They are repositioned in I-FABP:oleate (and I-FABP:palmitate) so that the binding cavity is even more accessible to solvent and its volume is increased. The side chain of Phe55 which shows discrete disorder in the apoprotein functions as an omega-terminal "sensing device": moving progressively outward toward the surface as the chain length of the bound fatty acid increases by 2 methylenes. Tyr70 and Tyr117 which also show discrete disorder in the apoprotein structure due to rotation around their C alpha-C beta bonds, are stabilized in a single, well ordered position in the holoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular evolution of mitochondrial 12S RNA and cytochrome b sequences in the pantherine lineage of Felidae

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among 17 Felidae species, notably 15 in the previously described pantherine lineage. The polymerase chain reaction (PCR) was used to generate sequences of 358 base pairs of the mitochondrial 12S RNA gene and 289 base pairs of the cytochrome b protein coding gene. DNA sequences were compared within and between 17 felid and five nonfelid carnivore species. Evolutionary trees were constructed using phenetic, cladistic, and maximum likelihood algorithms. The combined results suggested several phylogenetic relationships including (1) the recognition of a recently evolved monophyletic genus Panthera consisting of Panthera leo, P. pardus, P. onca, P. uncia, P. tigris, and Neofelis nebulosa; (2) the recent common ancestry of Acinonyx jubatus, the African cheetah, and Puma concolor, the American puma; and (3) two golden cat species, Profelis temmincki and Profelis aurata, are not sister species, and the latter is strongly associated with Caracal caracal. These data add to the growing database of vertebrate mtDNA sequences and, given the relatively recent divergence among the felids represented here (1-10 Myr), allow 12S and cytochrome b sequence evolution to be addressed over a time scale different from those addressed in most work on vertebrate mtDNA.      

Analysis of F2-isoprostanes as indicators of non-enzymatic lipid peroxidation in vivo by gas chromatography-mass spectrometry: development of a solid-phase extraction procedure

Recently, it has been reported that a series of prostaglanding F₂-like compounds (F₂-isoprostanes) are produced in vivo during peroxidation of arachidonic acid by a mechanism independent of the cyclooxygenase pathway. Of these, 8-epi-PGF_α² is shown to be a potent vasoconstrictor. We describe an improved method for analysing F₂-isoprostanes in biological fluids. The method involves solid-phase extraction on an octadecylsilane (C₁₈) and an aminopropyl (NH₂) cartridge. After conversion to pentafluorobenzyl ester and trimethylsilyl ether derivatives, F₂-isoprostanes are analysed by negative-ion chemical ionization mass spectrometry using tetradeuterated PGF_α² as the internal standard. The limit of detection of the assay was 10 pg/ml, with a coefficient of variation ranging from 9.4 to 15.1%. Analysis of plasma samples from healthy volunteers (n = 7) revealed no quantifiable levels of free (unesterified) 8-epi-PGF_α². However, the plasma samples contained 58 to 166 pg/ml of 8-epi-PGF_α² when analyzed for the total (sum of free and esterifield)_ F₂-isoprostances. The main advantages of the method lie in the improved recovery, gas chromatographic separation and speed compared to existing techniques.