On the evolution of structure in aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. The evolution of the tRNA synthetases is of fundamental importance with respect to the nature of the biological cell and the transition from an RNA world to the modern world dominated by protein-enzymes. We present a structure-based phylogeny of the aminoacyl-tRNA synthetases. By using structural alignments of all of the aminoacyl-tRNA synthetases of known structure in combination with a new measure of structural homology, we have reconstructed the evolutionary history of these proteins. In order to derive unbiased statistics from the structural alignments, we introduce a multidimensional QR factorization which produces a nonredundant set of structures. Since protein structure is more highly conserved than protein sequence, this study has allowed us to glimpse the evolution of protein structure that predates the root of the universal phylogenetic tree. The extensive sequence-based phylogenetic analysis of the tRNA synthetases (Woese et al., Microbiol. Mol. Biol. Rev. 64:202-236, 2000) has further enabled us to reconstruct the complete evolutionary profile of these proteins and to make connections between major evolutionary events and the resulting changes in protein shape. We also discuss the effect of functional specificity on protein shape over the complex evolutionary course of the tRNA synthetases.     

Response of Daphnia to substances released from crowded congeners and conspecifics

The effects of chemicals released from crowded congeners andconspecifics on life history parameters of the freshwater zooplanktersDaphnia cucullata and Daphnia pulex were examined. Length andage at maturity of D. pulex were affected by crowding chemicals.Reproduction was lower in crowded medium, and ephippia wereproduced. Newborn D. pulex in crowded medium were significantlylonger than the controls. The intrinsic rate of population increaseof D. pulex was 14 and 25% lower than the control when exposedto crowded medium from D. cucullata and D. pulex, respectively.Neither urea nor ammonia (at 1 mg l^-1) seemed to be responsiblefor these effects in D. pulex. In D. cucullata, no significanteffect of crowding infochemicals on length and age at maturitywas found. However, crowding chemicals reduced reproduction.No ephippia were produced in crowded medium, but up to 83% non-developingeggs were observed in D. cucullata. Newborns were similarlysized in crowded and standard medium. The intrinsic rate ofpopulation increase of D. cucullata was 44 and 96% lower thanthe control when exposed to crowded medium from D. cucullataand D. pulex, respectively. Clearance rates of D. pulex weresignificantly reduced in crowded media compared with standardmedium, which could partly explain why the animals exposed tocrowding chemicals reacted as if they were food limited.     

Are UV-induced nonculturable Escherichia coli K-12 cells alive or dead?

Cells that have lost the ability to grow in culture could be defined operationally as either alive or dead depending on the method used to determine cell viability. As a consequence, the interpretation of the state of 'nonculturable' cells is often ambiguous. Escherichia coli K12 cells inactivated by UV-irradiation with a low (UV1) and a high (UV2) dose were used as a model of nonculturable cells. Cells inactivated by the UV1 dose lost 'culturability' but they were not lysed and maintained the capacity to respond to nutrient addition by protein synthesis and cell wall synthesis. The cells also retained both a high level of glucose transport and the capacity for metabolizing glucose. Moreover, during glucose incorporation, UV1-treated cells showed the capacity to respond to aeration conditions modifying their metabolic flux through the Embden-Meyerhof and pentose-phosphate pathways. However, nonculturable cells obtained by irradiation with the high UV2 dose showed several levels of metabolic imbalance and retained only residual metabolic activities. Nonculturable cells obtained by irradiation with UV1 and UV2 doses were diagnosed as active and inactive (dying) cells, respectively.     

A misfolded form of 5S rRNA is complexed with the Ro and La autoantigens.

In both vertebrate and invertebrate cells, the 60-kDa Ro autoantigen is bound to small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the 60-kDa Ro protein is also complexed with a class of 5S rRNA precursors that contain internal mutations. Because these 5S rRNA precursors are processed inefficiently and degraded eventually, the Ro protein may function in a quality control pathway for 5S rRNA biosynthesis. We have investigated the sequence and secondary structure determinants in the mutant 5S rRNAs that confer binding by the 60-kDa Ro protein. The mutant 5S rRNAs fold to form an alternative helix that is required for recognition by the 60-kDa Ro protein. Mutations that disrupt the alternative helix eliminate Ro protein binding, whereas compensatory changes that restore the helix are bound efficiently by the Ro protein. When the structure of the mutant RNA was probed using dimethylsulfate and oligonucleotide-directed RNase H cleavage, the results were consistent with the formation of the alternative structure. The La protein, which is also complexed with the mutant 5S rRNA precursors, protects similar sequences from nuclease digestion as does the 60-kDa Ro protein. Thus, the binding sites for these two proteins are either nearby on the RNA, or the two proteins may be complexed through protein-protein interactions. When the human Ro protein is expressed in the yeast Saccharomyces cerevisiae, the protein binds wild-type 5S rRNA precursors, suggesting that a population of wild-type precursors also folds into the alternative structure.     

Effects of temperature,salinity and agitation on byssus thread formation of zebra musselDreissena polymorpha

Byssus thread production ofD. polymorpha under different conditions of temperature, salinity and agitation were studied in the laboratory. The acclimation to salinity and temperature greatly affects the byssus production ofD. polymorpha. Byssus production of mussels was significantly reduced when temperature increased beyond 20°C and decreased below 10°C. Mussels with cut threads (for counting), produced a substantially increased number of threads. However, mussels with uncut byssus threads were comparatively more mobile. Byssus production of mussels did not vary significantly at salinities up to 3. Beyond this salinity byssus production was reduced significantly. Mussels increased their byssus production with increasing frequency of agitation.     

Simultaneous determination of azathioprine and 6-mercaptopurine by high-performance liquid chromatography

A specific, sensitive, single-step solid-phase extraction and reversed-phase high-performance liquid chromatographic method for the simultaneous determination of plasma 6-mercaptopurine and azathioprine concentrations is reported. Following solid-phase extraction, analytes are separated on a C₁₈ column with mobile phase consisting of 0.8% acetonitrile in 1 mM triethylamine, pH 3.2, run on a gradient system. Quantitation limits were 5 ng/ml and 2 ng/ml for azathioprine and 6-mercaptopurine, respectively. Peak heights correlated linearly to known extracted standards for 6-mercaptopurine and azathioprine (r = 0.999) over a range of 2–200 ng/ml. No chromatographic interferences were detected.     

Identification and quantitative determination of 3-chloro-2-hydroxypropylmercapturic acid and α-chlorohydrin in urine of rats treated with epichlorohydrin

Epichlorohydrin (ECH) is used in many industrial processes. Different toxic effects of ECH were found in rodents. The metabolism of ECH was investigated before in rats using [¹⁴C]ECH. The aim of this investigation was the development of non-radioactive quantitative analytical methods for measuring two urinary metabolites of ECH, namely 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) and α-chlorohydrin (α-CH). The identity of CHPMA and α-CH excreted in urine of rats treated with 5 to 35 mg/kg ECH was confirmed by GC-MS. The quantitative analysis of CHPMA, involving ethyl acetate extraction from acidified urine and subsequent methylation and analysis by gas chromatography-flame photometric detection (GC-FPD), showed a method limit of detection of 2 μg/ml. The analysis of α-CH, based on ethyl acetate extraction and subsequent analysis by GC-ECD, showed a method limit of detection of 2 μg/ml. CHPMA and α-CH derivatives could be determined quantitatively down to concentrations of 0.5 and 0.4 μg/ml urine, respectively, by selected-ion monitoring GC-MS under EI conditions. Cumulative urinary excretion of CHPMA and α-CH by rats treated with ECH were found to be 31 ± 10 and 1.4 ± 0.6% (n = 13) of the ECH dose, respectively. For CHPMA, the dose-excretion relationship suggested partially saturated ECH metabolism. For α-CH, the dose-excretion relationship was linear. With fractionated urine collection it was found that approximately 74 and 84% of the total cumulative excretion of CHPMA and α-CH, respectively, took place within the first 6 h after administration of ECH. From these investigations it is concluded that the GC-FPD and GC-ECD based methods developed are sufficiently sensitive to measure urinary excretion of CHPMA and α-CH in urine from rats administered 5 to 35 mg/kg ECH. It is anticipated that the analysis of CHPMA and α-CH based on GC-MS may be sufficiently sensitive to investigate urinary excretion from humans occupationally exposed to ECH.     

Cloning and characterization of theeapB andeapC genes ofCryphonectria parasitica encoding two new acid proteinases,and disruption ofeapC

Two new proteinases secreted byCryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that theeapB andeapC genes contain three and two introns, respectively. The products of theeapB andeapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. TheeapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of theeapC coding sequence in an endothiapepsin-deficient (EapA⁺)C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.