Analysis and accurate quantification of CpG methylation by MALDI mass spectrometry

As the DNA sequence of the human genome is now nearly finished, the main task of genome research is to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the development of cancer. Even minor changes in the degree of methylation can have severe consequences. An accurate quantification of the methylation status at any given position of the genome is a powerful diagnostic indicator. Here we present the first assay for the analysis and precise quantification of methylation on CpG positions in simplex and multiplex reactions based on matrix-assisted laser desorption/ ionisation mass spectrometry detection. Calibration curves for CpGs in two genes were established and an algorithm was developed to account for systematic fluctuations. Regression analysis gave R² ≥ 0.99 and standard deviation around 2% for the different positions. The limit of detection was ~5% for the minor isomer. Calibrations showed no significant differences when carried out as simplex or multiplex analyses. All variable parameters were thoroughly investigated, several paraffin-embedded tissue biopsies were analysed and results were verified by established methods like analysis of cloned material. Mass spectrometric results were also compared to chip hybridisation.     

Eclipse-synchrony relationship in Escherichia coli strains with mutations affecting sequestration, initiation of replication and superhelicity of the bacterial chromosome

Initiation of replication from oriC on the Escherichia coli chromosomes occurs once and only once per generation at the same cell mass per origin. During rapid growth there are overlapping replication cycles, and initiation occurs synchronously at two or more copies of oriC. Since the bacterial growth can vary over a wide range (from three divisions per hour to 2.5 hours or more per division) the frequency of initiation should change in coordination with bacterial growth. Prevention of reinitiation from a newly replicated origin by temporary sequestration of the hemi-methylated GATC-sites in the origin region provides the molecular/genetic basis for the maintenance of the eclipse period between two successive rounds of replication. Sequestration is also believed to be responsible for initiation synchrony, since inactivation of either the seqA or the dam gene abolishes synchrony while drastically reducing the eclipse. In this work, we attempted to examine the functional relationship(s) between the eclipse period and the synchrony of initiation in E.coli strains by direct measurements of these parameters by density-shift centrifugation and flow-cytometric analyses, respectively. The eclipse period, measured as a fraction of DNA-duplication times, varied continuously from 0.6 for the wild-type E.coli K12 to 0.1 for strains with mutations in seqA, dam, dnaA, topA and gyr genes (all of which have been shown to cause asynchrony) and their various combinations. The asynchrony index, a quantitative indicator for the loss of synchrony of initiation, changed from low (synchronous) to high (asynchronous) values in a step-function-like relationship with the eclipse. An eclipse period of approximately 0.5 generation time appeared to be the critical value for the switch from synchronous to asynchronous initiation.     

HIV-1 gag expression is quantitatively dependent on the ratio of native and optimized codons

There is a significant variation of codon usage bias among different species and even among genes within the same organisms. Codon optimization, this is, gene redesigning with the use of codons preferred for the specific expression system, results in improved expression of heterologous genes in bacteria, plants, yeast, mammalian cells, and transgenic animals. The mechanisms preventing expression of genes with rare or low-usage codons at adequate levels are not completely elucidated. Human immunodeficiency virus (HIV) represents an interesting model for studying how differences in codon usage affect gene expression in heterologous systems. Construction of synthetic genes with optimized codons demonstrated that the codon-usage effects might be a major impediment to the efficient expression of HIV gag/pol and env gene products in mammalian cells. According to another hypothesis, the poor expression of HIV structural proteins even without HIV context is attributed to the so-called cis-acting inhibitory elements (INS), which are located within the protein-coding region. They consist of AU-rich sequences and may be inactivated through the introduction of multiple mutations over the large regions of gag gene. In our work, we evaluated expression of hybrid HIV-1 gag mRNAs where wild-type (A-rich) gag sequences were combined with artificial sequences. In such "humanized" gag fragments with adapted codon usage, AT-content was significantly reduced in favor of G and C nucleotides without any changes in protein sequence. We show that wild-type gag sequences negatively influence expression of gag-reporter, and the addition of fragments with optimized codons to gag mRNA partially rescues its expression. The results demonstrate that the expression of HIV-1 gag is determined by the ratio of optimized and rare codons within mRNA. Our data also indicates that some wtgag fragments counteract the influence of the other wtgag sequences, which cause the inhibition of gag expression. The presented data do not contradict the concept of INS; yet, it makes the definition of INS more complex. This supports the idea of a broader role of the selected codon usage in influencing the expression of HIV proteins in mammalian cells.     

Internal and external mycoflora of the American dog tick,Dermacentor variabilis (Acari: Ixodidae), and its ecological implications

Scopulariopsis brevicaulis, the anamorph of Microascus brevicaulis (Microascaceae, Ascomycota), has been identified in the body contents of the tick Dermacentor variabilis. After topical application of the fungal inoculum, tick mortality was marked. This is the first account describing the internal mycoflora of D. variabilis with a novel technique used to recover potential biological control agents.     

A robust subspace algorithm for principal component analysis

We present a noise robust PCA algorithm which is an extension of the Oja subspace algorithm and allows tuning the noise sensitivity. We derive a loss function which is minimized by this algorithm and interpret it in a noisy PCA setting. Results on the local stability analysis of this algorithm are given and it is shown that the locally stable equilibria are those which minimize the loss function.     

Insecticidal pyrido[1,2-a]azepine alkaloids and related derivatives from Stemona species

Eight new alkaloids, the pyrido[1,2-a]azepines stemokerrin, methoxystemokerrin-N-oxide, oxystemokerrin, oxystemokerrin-N-oxide, and pyridostemin, along with the pyrrolo[1,2-a]azepines dehydroprotostemonine, oxyprotostemonine, and stemocochinin were isolated from four Stemona species together with the known compounds protostemonine, stemofoline, 2'-hydroxystemofoline, and parvistemonine. Their structures were elucidated by 1H and 13C NMR including 2D methods and two key compounds additionally by X-ray diffraction. Besides the formation of a six membered piperidine ring, additional oxygen bridges and N-oxides contributed to structural diversity. The co-occurrence of pyrrolo- and pyridoazepines suggested biosynthetic connections starting from more widespread protostemonine type precursors. Bioassays with lipophilic crude extracts against Spodoptera littoralis displayed very strong insecticidal activity for the roots of S. curtisii and S. cochinchinensis, moderate activity for S. kerrii, but only weak effects for the unidentified species HG 915. The insect toxicity was mainly caused by the accumulation of stemofoline, oxystemokerrin, and dehydroprotostemonine displaying two different modes of action. Based on the various insecticidal activities of 13 derivatives structure-activity relationships became apparent.     

Basic limnology of fifty-one lakes in Costa Rica

We visited 51 lakes in Costa Rica as part of a broad-based survey to document their physical and chemical characteristics and how these relate to the mode of formation and geographical distribution of the lakes. The four oxbow lakes were low in elevation and tended to be turbid, high in conductivity and CO2, but low in dissolved O2; one of these, L. Gandoca, had a hypolimnion essentially composed of sea water. These were similar to the four wetland lakes, but the latter instead had low conductivities and pH, and turbidity was often due to tannins rather than suspended sediments. The thirteen artificial lakes formed a very heterogenous group, whose features varied depending on local factors. The thirteen lakes dammed by landslides, lava flows, or lahars occurred in areas with steep slopes, and were more likely to be stratified than most other types of lakes. The eight lakes that occupy volcanic craters tended to be deep, stratified, clear, and cool; two of these, L. Hule and L. Río Cuarto, appeared to be oligomictic (tending toward meromictic). The nine glacial lakes, all located above 3440 m elevation near Cerro Chirripó, were clear, cold, dilute, and are probably polymictic. Cluster analysis resulted in three significant groups of lakes. Cluster 1 included four calcium-rich lakes (average 48 mg l-1), Cluster 2 included fourteen lakes with more Si than Ca+2 and higher Cl- than the other clusters, and Cluster 3 included the remaining thirty-three lakes that were generally less concentrated. Each cluster included lakes of various origins located in different geographical regions; these data indicate that, apart from the high-altitude glacial lakes and lakes in the Miravalles area, similarity in lake chemistry is independent of lake distribution.     

Antifungal diterpenes from Hypoestes serpens (Acanthaceae)

Two new diterpenes, fusicoserpenol A and dolabeserpenoic acid A, with antifungal activity, were isolated from leaves of Hypoestes serpens (Acanthaceae). Their structures were elucidated by means of spectrometric methods including 1D and 2D NMR experiments and MS analysis. X-ray crystallographic analysis confirmed the structure of fusicoserpenol A and established the relative configuration.     

Yaf9, a novel NuA4 histone acetyltransferase subunit,is required for the cellular response to spindle stress in yeast

Yaf9 is one of three proteins in budding yeast containing a YEATS domain. We show that Yaf9 is part of a large complex and that it coprecipitates with three known subunits of the NuA4 histone acetyltransferase. Although Esa1, the catalytic subunit of NuA4, is essential for viability, we found that yaf9 Delta mutants are viable but hypersensitive to microtubule depolymerizing agents and synthetically lethal with two different mutants of the mitotic apparatus. Microtubules depolymerized more readily in the yaf9Delta mutant compared to the wild type in the presence of nocodazole, and recovery of microtubule polymerization and cell division from limiting concentrations of nocodazole was inhibited. Two other NuA4 mutants (esa1-1851 and yng2 Delta) and nonacetylatable histone H4 mutants were also sensitive to benomyl. Furthermore, wild-type budding yeast were more resistant to benomyl when grown in the presence of trichostatin A, a histone deacetylase inhibitor. These results strongly suggest that acetylation of histone H4 by NuA4 is required for the cellular resistance to spindle stress.