Obstructive sleep apnea: Plasma endothelin-1 precursor but not endothelin-1 levels are elevated and decline with nasal continuous positive airway pressure

Assessment of plasma endothelin-1 (ET-1) reveals conflicting results in cerebral and noncerebral conditions. Obstructive sleep apnea (OSA) syndrome has been used as a definite challenge for the investigation of endothelin measurements. Despite marked sleep-related breathing disturbances in untreated patients peripherally measurable ET-1 concentrations remained within the normal range and did not change after an appropriate therapy with continuous positive airway pressure (CPAP). In contrast, its precursor, big ET-1, was considerably elevated in untreated patients and dropped to normal values after long-term CPAP depending on compliance. Relatively stable big ET-1 elevations in untreated patients, during sleep and wakefulness, suggest that a general endothelial alteration beyond that explained by a direct impact of nocturnal breathing disturbances on the vascular system occurs. CPAP-therapy effectively lowered plasma big ET-1 in compliant patients and thus possibly their related risk for vascular diseases. Big ET-1 has been demonstrated to be a more appropriate marker of endothelial alteration than ET-1 because of its longer half-life. Simultaneous measurements are to be recommended.     

Ipangulines and minalobines, chemotaxonomic markers of the infrageneric Ipomoea taxon subgenus Quamoclit, section Mina

A comprehensive GC-MS analysis of 8 Ipomoea species belonging to the subgenus Quamoclit, section Mina revealed that the members of this taxon form combinations of two necine bases with rare necic acids resulting in unique pyrrolizidine alkaloids. The occurrence and diversity of these metabolites show remarkable variations: Some species, especially Ipomoea hederifolia and Ipomoea lobata, are able to synthesize a large number of alkaloids whereas others, especially Ipomoea coccinea and Ipomoea quamoclit, are poor synthesizers with only a few compounds. However, these metabolites are apparently chemotaxonomic markers of this infrageneric taxon in general. They represent either esters of (-)-platynecine (altogether 48 ipangulines and 4 further esters including results of a previous study) or esters of (-)-trachelanthamidine, an additional novel structural type called minalobines (altogether 21 alkaloids). Both types are characterized by section-specific rare necic acids, e.g., ipangulinic/isoipangulinic acid, phenylacetic acid. The alkaloids of Ipomoea cholulensis, I. coccinea, I. hederifolia, Ipomoea neei, and Ipomoea quamoclit were mono and diesters of platynecine. Minalobines turned out to be metabolites of I. lobata (Cerv.) Thell. (syn.: Mina lobata Cerv.) lacking ipangulines. The major alkaloid of this species, minalobine R, has been isolated and identified as 9-O-(threo-2-hydroxy-2-methyl-3-phenylacetoxy-butyryl)-(-)-trachelanthamidine on the basis of spectral data. Apparently only two of the species included in this study, Ipomoea cristulata and Ipomoea sloteri, are able to synthesize both, ipangulines as well as minalobines. Minalobine O could be isolated as a major alkaloid of I. cristulata, its structure has been established as 9-O-(erythro-2-hydroxy-2-methyl-3-tigloyloxy-butyryl)-(-)-trachelanthamidine on the basis of spectral data.     

Organelle proteomics of rat synaptic proteins: correlation-profiling by isotope-coded affinity tagging in conjunction with liquid chromatography-tandem mass spectrometry to reveal post-synaptic density specific proteins

Organelle proteomics is the method of choice for global analysis of cellular proteins. However, it is difficult to isolate organelles to homogeneity. Recently, correlation-profiling has been used to filter off the contaminants ad hoc and to disclose the genuine organelle-specific proteins. In the present study, we further extend the method to include subcellular compartments that contain proteins shared by multiple distinct subcellular domains. We performed correlation profiling of proteins contained in synaptic membrane and postsynaptic density (PSD) fractions isolated from rat brain. Proteins were labeled with isotope-coded affinity-tag reagents, digested with trypsin, and resulting peptides were resolved by cation exchange chromatography followed by reversed phase chromatography. Peptides were then subjected to mass spectrometry for quantification and identification. We confirm that the core PSD proteins were enriched in the PSD preparation. Other functional protein groups such as cytoskeleton-associated proteins, protein kinases and phosphatases, signaling components and regulators, as well as proteins involved in energy production partitioned to multiple organelles. When analyzed as groups, they were shown to accumulate to a lesser extent. Mitochondrial proteins and transporters were generally strongly depleted from the PSD fraction confirming that they were contaminants of the PSD preparation. Finally, immunoelectron microscopy was performed on selected proteins to validate the proteomics results, and confirm that synaptophysin that was highly depleted in the PSD preparation is localized in the presynaptic compartment, whereas LASP-1 that was slightly enriched in the PSD preparation is present in the PSD as well as other subdomains within the synapse.     

Potent inhibitory activity of chimeric oligonucleotides targeting two different sites of human telomerase

Suppression of telomerase activity in tumor cells has been considered as a new anticancer strategy. Here, we present chimeric oligonucleotides (chimeric ODNs) as a new type of telomerase inhibitor that contains differently modified oligomers to address two different sites of telomerase: the RNA template and a suggested protein motif. We have shown previously that phosphorothioate-modified oligonucleotides (PS ODNs) interact in a length-dependent rather than in a sequence-dependent manner, presumably with the protein part of the primer-binding site of telomerase, causing strong inhibition of telomerase. In the present study, we demonstrate that extensions of these PS ODNs at their 3'-ends with an antisense oligomer partial sequence covering 11 bases of the RNA template cause significantly increased inhibitory activity, with IC(50) values between 0.60 and 0.95 nM in a Telomeric Repeat Amplification Protocol (TRAP) assay based on U-87 cell lysates. The enhanced inhibitory activity is observed regardless of whether the antisense part is modified (phosphodiester, PO; 2'-O-methylribosyl, 2'-OMe/PO; phosphoramidate, PAM). However, inside intact U-87 cells, these modifications of the antisense part proved to be essential for efficient telomerase inhibition 20 hours after transfection. In particular, the chimeric ODNs containing PAM or 2'-OMe/PO modifications, when complexed with lipofectin, were most efficient telomerase inhibitors (ID(50) = 0.04 and 0.06 microM, respectively). In conclusion, ODNs of this new type emerged as powerful inhibitors of human telomerase and are, therefore, promising candidates for further investigations of the anticancer strategy of telomerase inhibition.     

DNA variation at the invertase locus invGE/GF is associated with tuber quality traits in populations of potato breeding clones

Starch and sugar content of potato tubers are quantitative traits, which are models for the candidate gene approach for identifying the molecular basis of quantitative trait loci (QTL) in noninbred plants. Starch and sugar content are also important for the quality of processed products such as potato chips and French fries. A high content of the reducing sugars glucose and fructose results in inferior chip quality. Tuber starch content affects nutritional quality. Functional and genetic models suggest that genes encoding invertases control, among other things, tuber sugar content. The invGE/GF locus on potato chromosome IX consists of duplicated invertase genes invGE and invGF and colocalizes with cold-sweetening QTL Sug9. DNA variation at invGE/GF was analyzed in 188 tetraploid potato cultivars, which have been assessed for chip quality and tuber starch content. Two closely correlated invertase alleles, invGE-f and invGF-d, were associated with better chip quality in three breeding populations. Allele invGF-b was associated with lower tuber starch content. The potato invertase gene invGE is orthologous to the tomato invertase gene Lin5, which is causal for the fruit-sugar-yield QTL Brix9-2-5, suggesting that natural variation of sugar yield in tomato fruits and sugar content of potato tubers is controlled by functional variants of orthologous invertase genes.     

A systematic comparison and evaluation of biclustering methods for gene expression data

MOTIVATION: In recent years, there have been various efforts to overcome the limitations of standard clustering approaches for the analysis of gene expression data by grouping genes and samples simultaneously. The underlying concept, which is often referred to as biclustering, allows to identify sets of genes sharing compatible expression patterns across subsets of samples, and its usefulness has been demonstrated for different organisms and datasets. Several biclustering methods have been proposed in the literature; however, it is not clear how the different techniques compare with each other with respect to the biological relevance of the clusters as well as with other characteristics such as robustness and sensitivity to noise. Accordingly, no guidelines concerning the choice of the biclustering method are currently available. RESULTS: First, this paper provides a methodology for comparing and validating biclustering methods that includes a simple binary reference model. Although this model captures the essential features of most biclustering approaches, it is still simple enough to exactly determine all optimal groupings; to this end, we propose a fast divide-and-conquer algorithm (Bimax). Second, we evaluate the performance of five salient biclustering algorithms together with the reference model and a hierarchical clustering method on various synthetic and real datasets for Saccharomyces cerevisiae and Arabidopsis thaliana. The comparison reveals that (1) biclustering in general has advantages over a conventional hierarchical clustering approach, (2) there are considerable performance differences between the tested methods and (3) already the simple reference model delivers relevant patterns within all considered settings.     

Neuronal Ca2+ signaling via caldendrin and calneurons

The calcium sensor protein caldendrin is abundantly expressed in neurons and is thought to play an important role in different aspects of synapto-dendritic Ca2+ signaling. Caldendrin is highly abundant in the postsynaptic density of a subset of excitatory synapses in brain and its distinct localization raises several decisive questions about its function. Previous work suggests that caldendrin is tightly associated with Ca2+ - and Ca2+ release channels and might be involved in different aspects of the organization of the postsynaptic scaffold as well as with synapse-to-nucleus communication. In this report we introduce two new EF-hand calcium sensor proteins termed calneurons that apart from calmodulin represent the closest homologues of caldendrin in brain. Calneurons have a different EF-hand organization than other calcium sensor proteins, are prominently expressed in neurons and will presumably bind Ca2+ with higher affinity than caldendrin. Despite some significant structural differences it is conceivable that they are involved in similar Ca2+ regulated processes like caldendrin and neuronal calcium sensor proteins.     

Laminating the hippocampus

Lamination of neurons and fibre projections is a fundamental organizational principle of the mammalian cerebral cortex. A laminated organization is likely to be essential for cortical function, as studies in mutant mice have revealed causal relationships between lamination defects and functional deficits. Unveiling the determinants of the laminated cortical architecture will contribute to our understanding of how cortical functions have evolved in phylogenetic and ontogenetic development. Recently, the hippocampus, with its clearly segregated cell and fibre layers, has become a major subject of studies on cortical lamination.     

Detection of activity of telomerase in tumor cells using fiber optical biosensors

Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 degrees C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.