A switch from α‐helical to β‐strand conformation during co‐translational protein folding

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo‐EM structure determination to show that folding of a β‐barrel protein begins with formation of a dynamic α‐helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N‐terminal part of the nascent chain refolds to a β‐hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α‐helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl‐transferase center suggest that protein folding could modulate ribosome activity.     

High-performance liquid chromatographic determination of terguride in solid dosage forms and plasma

A simple and rapid HPLC method was developed to determine terguride in terguride hydrogenmaleate, coated tablets and plasma. The assay was carried out on a glass column of SGX CN (150 × 3.3 mm I.D.) using methanol and phosphate buffer solution (pH 7.0) as the mobile phase, with detection at 227 nm. Terguride was quantified using promethazine as an internal standards. The tablet matrix was extracted into methanol. Plasma samples were deproteinated with acetonitrile and the supernatant was injected into the HPLC system. The method is linear, quantitative and reproducible.     

Distribution of microcystin-producing and non-microcystin-producing Microcystis sp. in European freshwater bodies: detection of microcystins and microcystin genes in individual colonies

Microcystis is a well-known cyanobacterial genus frequently producing hepatotoxins named microcystins. Toxin production is encoded by microcystin genes (mcy). This study aims (i) to relate the mcy occurrence in individual colonies to the presence of microcystin, (ii) to assess whether morphological characteristics (morphospecies) are related to the occurrence of mcy genes, and (iii) to test whether there are geographical variations in morphospecies specificity and abundance of mcy genes. Individual colonies of nine different European countries were analysed by (1) morphological characteristics, (2) PCR to amplify a gene region within mcyA and mcyB indicative for microcystin biosynthesis, (3) matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to detect microcystins. Almost one hundred percent of the colonies predicted to produce microcystins by PCR analysis were found to contain microcystins. A high similarity in microcystin variants in the different colonies selected from lakes across Europe was demonstrated. The different morphospecies varied in the frequency with which they contained mcy genes. Most colonies (>75%) of M. aeruginosa and M. botrys contained the mcy genes, whereas < or = 20% of the colonies identified as M. ichthyoblabe and M. viridis gave a PCR product of the mcy genes. No colonies of M. wesenbergii gave a PCR product of either mcy gene. In addition, a positive relationship was found between the size of the colony and the frequency of those containing the mcy genes. It is concluded that the analysis of morphospecies is indicative for microcystin production, although the quantitative analysis of microcystin concentrations in water remains indispensable for hazard control.     

Occurrence of<Emphasis Type="Italic">penicillium expansum</Emphasis> and patulin in apples as raw materials for processing of foods — case study

The contamination of apples Gloster variety withPenicillium expansum was investigated in the case study. The apples serve as a raw material in thefood industry for processing of baby foodstuffs and other apple products. The core of 3 – 5 % apple samples has been contaminated with spores and mycelium of untested microfungi. The surface tissue of stored apples has not been damaged. Penicillium expansum was isolated in all tested apple samples. Patulin has been found at levels tens to hundreds of üg/kg in tested apple samples. Patulin was primarily associated with core and areas of decomposing tissue and can penetrate up to approx. 1 cm of the surrounding healthy tissue. The production of patulin after the experimental contamination of apples was tested after 14 days of storage (21 °C, dark). The maximum value of patulin was found 110 mg/kg of rotten part of apple sample.     

Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11-dependent genome fragmentation during meiosis

The Mre11/Rad50/Nbs1 complex is involved in many aspects of chromosome metabolism. Aberrant function of the complex is associated with defects in the DNA checkpoint, double-strand break repair, meiosis, and telomere maintenance. In this article, we report the consequences of Mre11 dysfunction for the stability of mitotic and meiotic chromosomes in Arabidopsis thaliana. Although plants homozygous for a T-DNA insertion in a conserved region of the MRE11 gene are viable, they exhibit growth defects and are infertile. Analysis of mitotic chromosomes prepared from the mutant plants revealed abundant dicentric chromosomes and chromosomal fragments. Fluorescence in situ hybridization showed that anaphase bridges are often formed by homologous chromosome arms. The frequency of chromosome fusions was not reduced in mre11 ku70 double mutants, suggesting that plants possess DNA end-joining activities independent of the Ku70/80 and Mre11 complexes. Cytogenetic examination of pollen mother cells revealed massive chromosome fragmentation and the absence of synapsis in the initial stages of meiosis. The fragmentation was substantially suppressed in mre11 spo11-1 double mutants, indicating that Mre11 is required for repair but not for the induction of Spo11-dependent meiotic DNA breaks in Arabidopsis.     

The signature of the<Emphasis Type="Italic"> Cestrum</Emphasis> genome suggests an evolutionary response to the loss of (TTTAGGG)<Subscript>n</Subscript> telomeres

The genus Cestrum in the Solanaceae family is unusual in lacking Arabidopsis-type telomeres (TTTAGGG)_n, although short interstitial telomeric sequences (ITSs) occur scattered throughout the genome in both orientations. To isolate candidate telomeric sequences in Cestrum we assumed that some of the ITSs were residues of the original telomeres and that they may still be located in the vicinity of present-day telomeres. Three sequence types associated with ITSs were cloned and characterized; these were termed NA3G, BR23 and A/T-rich minisatellite. These high copy number sequences are dispersed across the genome and clustered at a number of chromosomal loci. Their association with ITSs, which can act as recombination hotspots, might indicate past recombination and chromosomal fusion events, processes that may have contributed to the large size of Cestrum chromosomes. The sequences are frequently arranged as NA3G-ITS-BR23 blocks embedded in an A/T-rich minisatellite array. The A/T-rich minisatellite is of particular interest because the consensus 5-T_4–5AGCAG-3 might be a derivative of typical eukaryotic telomeric sequence motifs. The sequence is abundant at the end of some chromosomes in C. parqui and is found not only in Cestrum but also in the closely related genera Sessea and Vestia, which also lack Arabidopsis-type telomeric sequences. However, the sequence is absent from the Solanaceae genera investigated that are outside the group, including the closely related genus Streptosolen, which all have the Arabidopsis-type telomere. The data indicate that the A/T rich minisatellite might have evolved in response to the loss of Arabidopsis-type telomeres.Communicated by E.R. Schmidt     

The influence of the thymine C5 methyl group on spontaneous base pair breathing in DNA

Sequences of four or more AT base pairs without a 5'-TA-3' step, so-called A-tracts, influence the global properties of DNA by causing curvature of the helix axis if phased with the helical repeat and also influence nucleosome packaging. Hence it is interesting to understand this phenomenon on the molecular level, and numerous studies have been devoted to investigations of dynamical and structural features of A-tract DNA. It was early observed that anomalously slow base pair-opening kinetics were a striking physical property unique to DNA A-tracts (Leroy, J. L., Charretier, E., Kochoyan, M., and Gueron, M. (1988) Biochemistry 27, 8894-8898). Furthermore, a strong correlation between DNA curvature and anomalously slow base pair-opening dynamics was found. In the present work it is shown, using imino proton exchange measurements by NMR spectroscopy that the main contribution to the dampening of the base pair-opening fluctuations in A-tracts comes from the C5 methylation of the thymine base. Because the methyl group has been shown to have a very limited effect on the DNA curvature as well as the structure of the DNA helix, the thymine C5 methyl group stabilizes the helix directly. Empirical potential energy calculations show that methylation of the tract improves the stacking energy of a base pair with its neighbors in the tract by 3-4 kcal/mol.     

Stable variants of sperm aneuploidy among healthy men show associations between germinal and somatic aneuploidy

Repeated semen specimens from healthy men were analyzed by sperm fluorescence in situ hybridization (FISH), to identify men who consistently produced elevated frequencies of aneuploid sperm and to determine whether men who were identified as stable variants of sperm aneuploidy also exhibited higher frequencies of aneuploidy in their peripheral blood lymphocytes. Seven semen specimens were provided by each of 15 men over a 2-year period and were evaluated by the X-Y-8 multicolor sperm FISH method (i.e., approximately 1,050,000 sperm were analyzed from 105 specimens). Three men were identified as stable aneuploidy variants producing significantly higher frequencies of XY, disomy X, disomy Y, disomy 8, and/or diploid sperm over time. In addition, one man and three men were identified as sperm-morphology and sperm-motility variants, respectively. Strong correlations were found between the frequencies of sperm with autosomal and sex-chromosome aneuploidies and between the two types of meiosis II diploidy; but not between sperm aneuploidy and semen quality. A significant association was found between the frequencies of sex-chromosome aneuploidies in sperm and lymphocytes in a subset of 10 men (r2=0.67, P=.004), especially between XY sperm and sex-chromosome aneuploidy in lymphocytes (r2=0.70, P=.003). These findings suggest that certain apparently healthy men can produce significantly higher frequencies of both aneuploid sperm and lymphocytes. Serious long-term somatic and reproductive health consequences may include increased risks of aneuploidy-related somatic diseases and of having children with paternally transmitted aneuploidies, such as Klinefelter, Turner, triple-X, and XYY syndromes.