Preparation of (2S, 3R)-methyl-3-phenylglycidate using whole cells of Pseudomonas putida

Preparation of (2S, 3R)-methyl 3-phenylglycidate via enantioselective hydrolysis of racemic phenylglycidate was carried out using whole cells of Pseudomonas putida. Under optimal conditions (2S, 3R)-methyl-3-phenylglycidate could be got with ee value 99 and 48% chemical yield.     

Genomic organization and evolution of the 5S ribosomal DNA in Tilapiini fishes

Summary 5S rDNA sequences present an intense dynamism and have proved to be valuable as genetic markers to distinguish closed related species and also in the understanding of the evolutionary dynamic of repetitive sequences in the genomes. In order to identify patterns of 5S rDNA organization and their evolution in the genome of fish species, such genomic segment was investigated in the tilapias Oreochromis niloticus and Tilapia rendalli, and in the hybrid O. urolepis hornorum × O. mossambicus. A dual 5S rDNA system was identified in the three analyzed tilapia samples. Although each 5S rDNA class was conserved among the three samples, a distinct 5S rDNA genome organization pattern could be evidenced for each sample. The presence of a dual 5S rDNA system seems to be a general trait among non-related teleost fish orders, suggesting that evolutionary events of duplication have occurred before the divergence of the main groups of teleost fishes.     

A molecular phylogenetic investigation of zoothamnium (ciliophora, peritrichia, sessilida)

The gene coding for 18S small subunit ribosomal RNA (ssu rRNA) was sequenced in seven free-living, marine species of the sessiline peritrich genus Zoothamnium. These were Zoothamnium niveum, Zoothamnium alternans, Zoothamnium pelagicum, and four unidentified species. The ssu rRNA gene also was sequenced in Vorticella convallaria, Vorticella microstoma, and in an unidentified, freshwater species of Vorticella. Phylogenetic trees were constructed using these new sequences to test a previously published phylogenetic association between Zoothamnium arbuscula, currently in the family Zoothamniidae, and peritrichs in the family Vorticellidae. Trees constructed by means of neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian inference methods all had similar topologies. The seven new sequences of Zoothamnium species grouped into three well-supported clades, each of which contained a diversity of morphological types. The three clades formed a poorly supported, larger clade that was deeply divergent from Z. arbuscula, which remained more closely associated with vorticellid peritrichs. It is apparent that Zoothamnium is a richly diverse genus and that a much more intensive investigation, involving both morphological and molecular data and a wider selection of species, will be necessary to resolve its phylogeny. A greater amount of molecular diversity than is predicted by morphological data exists within all major clades of sessiline peritrichs that have been included in molecular phylogenies, indicating that characteristics of stalk and peristomial structure traditionally used to differentiate taxa at the generic level and above may not be uniformly reliable.     

Isolation and characterization of a bacterial strain of the genus Ochrobactrum with methyl parathion mineralizing activity

AIMS: To isolate and characterize a methyl parathion (MP)-mineralizing bacterium, and to elucidate the degradative pathway of MP and localize the responsible degrading genes. METHODS AND RESULTS: A bacterial strain, designated B2, capable of mineralizing MP was isolated from the MP-polluted soil. Analysis of the 16S rRNA gene sequence and phenotypic analysis suggested that strain B2 had a close relationship with Ochrobactrum anthropi. B2 could totally degrade MP and four metabolites [p-nitrophenol (PNP), 4-nitrocatechol (4-NC), 1,2,4-benzenetriol (BT) and hydroquinone (HQ)] were identified by HPLC and gas chromatography-mass spectrometry analyses. Plasmid curing of strain B2 resulted in the loss of ability of B2 to degrade PNP, but not the ability to hydrolyse MP. CONCLUSIONS: Ochrobactrum sp. B2 can mineralize MP rapidly via PNP, 4-NC, BT and HQ pathway. B2 harbours a plasmid encoding the ability to degrade PNP, while MP-hydrolysing activity is encoded on the bacterial chromosome. SIGNIFICANCE AND IMPACT OF THE STUDY: This new bacterial strain (B2) capable of mineralizing MP will be useful in a pure-culture remediation process of organophosphate pesticides and their metabolites such as nitroaromatics.     

Pasteurella] caballi infection not limited to horses - a closer look at taxon 42 of Bisgaard

AIM: To investigate if taxon 42 of Bisgaard isolated from pigs represents genuine [Pasteurella] caballi, which was previously only isolated from horses. METHODS AND RESULTS: A total of 15 field isolates from horses and pigs from five different countries representing three continents were subjected to extended phenotypical characterization. Although minor differences were observed between taxon 42 and [P.] caballi, these differences did not allow phenotypic separation. Ribotyping based on HindIII digestion showed five profiles based on nine band positions. One [P.] caballi strain and two taxon 42 strains shared the same profile. Ribotyping using HpaII gave a higher diversity with nine profiles based on ten band positions. While no profiles were shared between the taxon 42 and [P.] caballi strains, pattern analysis showed that two of the taxon 42 isolates were most similar (91% similarity) with a [P.] caballi isolate. The 16S rRNA gene sequencing of one strain of taxon 42 and one strain of [P.] caballi was performed and compared with the published sequence for the type strain of [P.] caballi. The three strains showed nearly identical sequences with at least 99.8% similarity. DNA re-associations measured by the micro-well method were 79 and 77%, respectively between the type strain of [P.] caballi and two strains of taxon 42 representing distinct ribotypes and confirmed that taxon 42 belongs to [P.] caballi. CONCLUSION: The present investigation documents that [P.] caballi can be isolated from clinical respiratory specimens from pigs and the recognized association with respiratory infections in horses and horse bite infection in humans. Strains classified as taxon 42 are [P.] caballi isolated from pigs and for both pigs and horses, lesions mainly include the respiratory tract. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will improve the diagnostics and progress studies of virulence and epidemiology of [P.] caballi.     

Reevaluating the classification of Halobacteroides and Haloanaerobacter species based on sequence comparisons of the 16S ribosomal RNA gene

Abstract The 16S rRNA gene (rDNA) sequence analysis of four halophilic anaerobes: Halobacteroides halobius, H. lacunaris, Haloanaerobacter (Hb.) chitinovorans and H. acetoethylicus confirmed that they were all members of the family Haloanaerobiaceae. H. lacunaris and H. halobius were found to be more closely related to each other and were distantly related to Sporohalobacter lortetti and the members of the genera Haloanaerobium and Halothermothrix . These data are in agreement with their assignment to the genus Halobacteroides . Further analysis indicated that Hb. chitinovorans was closely affiliated to members of the genus Halobacteroides , and therefore we propose to transfer it to the genus Halobacteroides as H. chitinovorans comb. nov. This transfer would invalidate the genus Haloanaerobacter , as Hb. chitinovorans is the only member of this genus. The 16S rDNA sequence analysis of H. acetoethylicum indicated that it was very closely related to members of the genus Haloanaerobium , viz. Haloanaerobium (Ha.) praevalens, Ha. salsugo , and Ha. alcaliphilum , and hence we propose to transfer it to the genus Haloanaerobium as Ha. acetoethylicus comb. nov.     

Optimizing conditions for cryopreservation of an insect cell line

A cell line (UM-BGE-2) derived from embryos of the cockroach Blattella germanica was frozen to ?196 °C under a variety of conditions and cell viability was assayed after warming. It was found that cell viability was affected by the cooling rate, the warming rate, the controlled cooling endpoint temperature, and the type and concentration of cryoprotectant. The best survival for cells suspended in Grace's tissue culture medium containing 1 M Me₂SO was obtained when cells were cooled at 1 °C/ min to at least ?90 °C before being placed in liquid nitrogen and warmed at more than 900 °C/min. Cultures initiated from these frozen cells produce typical growth curves and appear normal after several passages.     

Influence of phosphorus and sulfur nutrition on composition ofClitoria ternatea L.

Summary A greenhouse experiment on a silt loam surface soil (Typic Hapludult) was done to investigate effects of P and S on yield, quality aspects and sugar reserves in the tropical forage legumeClitoria ternatea L. Four levels of P and two N treatments (NH₄NO₃ vs symbiotic) were arranged in a factorial design with four replications. After the first cutting two levels of S were imposed on this design.Phosphorus enhanced dry matter yield in the first cutting. Its effect was smaller in the second cutting. Amount of P required to produce maximum plant yield dropped from 200 mg/kg soil (or more) at the first cutting to 50–100 mg/kg at the second. Added S improved growth at suboptimal levels of P. At optimal P and S, symbiotic and +N plants yielded alike.Phosphorus and S fertilization caused several changes in plant composition. Nitrogen concentration was raised by S treatment and lowered by P. Combined addition of P and S lowered plant nitrate content. In symbiotic plants, soluble sugar concentrations were higher than in N-treated plants, and were increased by P and S treatment. In N-treated plants, neither P nor S increased reducing sugar concentration, but they increased total sugar.     

Chromosomal diversification of reef fishes from genus <Emphasis Type="Italic">Centropyge</Emphasis>(Perciformes,Pomacanthidae)

The genus Centropyge is remarkable for species richness, composing a highly specialized fish group amongst members from family Pomacanthidae. However, cytogenetical reports are nearly absent in these animals. New data are provided from karyotypical studies carried out on Centropyge aurantonotus from the Brazilian coast of the Atlantic Ocean and C. ferrugatus from the Philippines Sea of the Indo-Pacific Ocean. Both species present 2n=48 but karyotypes are differentiated by fundamental number. C. aurantonotus has a great number of biarmed chromosomes (4 m + 14 sm+16 st+4 a), while C. ferrugatus presents only acrocentric chromosomes. Single nucleolar organizer regions (NORs) are located at interstitial position of an acrocentric pair in C. ferrugatus and on short arms of a subtelocentric pair in C. aurantonotus, as confirmed by fluorescent in situ hybridization (FISH) with 18S rDNA probes. Heterochromatin is distributed over NOR and centromeric regions in both species, but additional GC-rich heterochromatic blocks on short arms of up to eight chromosomal pairs can be detected in C. aurantonotus. 5S rDNA segments were located interstitially on two chromosomal pairs in C. ferrugatus and on nine pairs in C. aurantonotus, mostly equivalent to heterochromatic blocks on short arms of biarmed chromosomes. C. ferrugatus can be considered a species in which basal chromosomal features proposed for modern Teleosteans were conserved. The derived karyotype pattern of C. aurantonotus seems to be determined by pericentric inversions and heterochromatin addition which probably determined the notorious dispersion of 5S rRNA (pseudo)genes. It is demonstrated that, even within a group generally characterized by cytogenetical homogeneity as the family Pomacanthidae, diversified karyotypes can be found.     

A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant proteins. Up to 20mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system we successfully produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion, the present tagging system offers a convenient general method for the robust expression and efficient purification of a variety of recombinant proteins.