Potent and selective xanthine-based inhibitors of phosphodiesterase 5

Inhibitors of PDE5 are useful therapeutic agents for treatment of erectile dysfunction. A series of novel xanthine derivatives has been identified as potent inhibitors of PDE5, with good levels of selectivity against other PDE isoforms, including PDE6. Studies in the dog indicate excellent oral bioavailability for compound 21.     

The suitability of different DHB isomers as matrices for the MALDI-TOF MS analysis of phospholipids: which isomer for what purpose?

Although the analysis of large biomolecules is the prime application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), there is also increasing interest in lipid analysis. Since lipids possess relatively small molecular weights, matrix signals should be as small as possible to avoid overlap with lipid peaks. Although 2,5-dihydroxybenzoic acid (DHB) is an established MALDI matrix, the question whether just this isomer is ideal for lipid analysis was not yet addressed. UV absorptions of all six DHB isomers were determined and their laser desorption spectra recorded. In addition, all isomers were used as matrices to record positive and negative ion mass spectra of selected phospholipids (phosphatidylcholine and -serine): In the order 2,5-, 2,6-, 2,3- and 2,4-DHB, the quality of the positive ion lipid spectra decreases. This correlates well with the decreasing acidity of the applied DHB isomers. The 3,4- and 3,5- isomers give only very weak positive ion signals especially of acidic lipids. In contrast, the most suitable matrices in the negative ion mode are 2,5-, 2,4- and 3,5-DHB. 2,6-DHB does not provide any signal in the negative ion mode due to its marked acidity. Finally, differences in the crystallization behavior of the pure matrix and the matrix/lipid co-crystals were also monitored by atomic force microscopy (AFM): 2,5-DHB gave the smallest crystals and the skinniest layer. It is concluded that basically all DHB isomers can be used as MALDI matrices but the 2,5-isomer represents the most versatile compound. Dedicated to Prof. Dr. Klaus Arnold on the occasion of his 65th birthday.     

In vivo TRPC functions in the cardiopulmonary vasculature

Cardiovascular diseases are the leading cause of death in the industrialized countries. The cardiovascular system includes the systemic blood circulation, the heart and the pulmonary circulation providing sufficient blood flow and oxygen to peripheral tissues and organs according to their metabolic demand. This review focuses on three major cell types of the cardiovascular system: myocytes of the heart as well as smooth muscle cells and endothelial cells from the systemic and pulmonary circulation. Ion channels initiate and regulate contraction in all three cell types, and the identification of their genes has significantly improved our knowledge of signal transduction pathways in these cells. Among the ion channels expressed in smooth muscle cells, cation channels of the TRPC family allow for the entry of Na(+) and Ca(2+). Physiological functions of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 in the cardiovascular system, dissected by down-regulating channel activity in isolated tissues or by the analysis of gene-deficient mouse models, are reviewed. Possible functional roles and physiological regulation of TRPCs as homomeric or heteromeric channels in these cell types are discussed. Moreover, TRP channels may also be responsible for pathophysiological processes of the cardiovascular system like hypertension as well as cardiac hypertrophy and increased endothelial permeability.     

Basolateral Cl channels in primary airway epithelial cultures

Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance (G(Cl)) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral G(Cl) was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl(2) = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl(2) > tamoxifen > 4,4'-dinitro-2,2'-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of G(Cl) were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive G(Cl); 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive G(Cl); and 3) a forskolin-activated, linear G(Cl), which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl(-) secretion or support transcellular Cl(-) absorption.     

GAPDH is not regulated in human glioblastoma under hypoxic conditions

Background

Gene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions in vitro in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and β-actin expression was comparatively examined in vivo in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1α protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.

Results

We observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied in vitro. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation in vivo.

Conclusion

GAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.     

Small molecule enhancers of rapamycin-induced TOR inhibition promote autophagy, reduce toxicity in Huntington's disease models and enhance killing of mycobacteria by macrophages

Upregulation of autophagy may have therapeutic benefit in a range of diseases that includes neurodegenerative conditions caused by intracytosolic aggregate-prone proteins, such as Huntington's disease, and certain infectious diseases, such as tuberculosis. The best-characterized drug that enhances autophagy is rapamycin, an inhibitor of the TOR (target of rapamycin) proteins, which are widely conserved from yeast to man. Unfortunately, the side effects of rapamycin, especially immunosuppression, preclude its use in treating certain diseases including tuberculosis, which accounts for approximately 2 million deaths worldwide each year, spurring interest in finding novel drugs that selectively enhance autophagy. We have recently reported a novel two-step screening process for the discovery of such compounds. We first identified compounds that enhance the growth-inhibitory effects of rapamycin in the budding yeast Saccharomyces cerevisiae, which we termed small molecule enhancers of rapamycin (SMERs). Next we showed that three SMERs induced autophagy independently, or downstream of mTOR, in mammalian cells, and furthermore enhanced the clearance of a mutant huntingtin fragment in Huntington's disease cell models. These SMERs also protected against mutant huntingtin fragment toxicity in Drosophila. We have subsequently tested two of the SMERs in models of tuberculosis and both enhance the killing of mycobacteria by primary human macrophages.     

A hypothesis on the identification of the editing enzyme in plant organelles

RNA editing in plant organelles is an enigmatic process leading to conversion of cytidines into uridines. Editing specificity is determined by proteins; both those known so far are pentatricopeptide repeat (PPR) proteins. The enzyme catalysing RNA editing in plants is still totally unknown. We propose that the DYW domain found in many higher plant PPR proteins is the missing catalytic domain. This hypothesis is based on two compelling observations: (i) the DYW domain contains invariant residues that match the active site of cytidine deaminases; (ii) the phylogenetic distribution of the DYW domain is strictly correlated with RNA editing.     

15N magnetic relaxation study of backbone dynamics of the ribosome-associated cold shock response protein Yfia of Escherichia coli

In the solution structure of the ribosome-associated cold shock response protein Yfia of Escherichia coli in the free state two structural segments can be distinguished: a well structured, rigid N-terminal part displaying a betaalphabetabetabetaalpha topology and a flexible C-terminal tail comprising last 20 amino-acid residues. The backbone dynamics of Yfia protein was studied by (15)N nuclear magnetic relaxation at three magnetic fields and analyzed using model-free approach. The overall diffusional tumbling of the N-terminal part is strongly anisotropic with a number of short stretches showing increased mobility either on a subnanosecond time scale, or a micro- to millisecond time scale, or both. In contrast, the unstructured polypeptide chain of the C-terminal part, which cannot be regarded as a rigid structure, shows the predominance of fast local motions over slower ones, both becoming faster closer to the C-terminus.     

Multivariate Analysis of Complex DNA Sequence Electropherograms for High-Throughput Quantitative Analysis of Mixed Microbial Populations

High-throughput quantification of genetically coherent units (GCUs) is essential for deciphering population dynamics and species interactions within a community of microbes. Current techniques for microbial community analyses are, however, not suitable for this kind of high-throughput application. Here, we demonstrate the use of multivariate statistical analysis of complex DNA sequence electropherograms for the effective and accurate estimation of relative genotype abundance in cell samples from mixed microbial populations. The procedure is no more labor-intensive than standard automated DNA sequencing and provides a very effective means of quantitative data acquisition from experimental microbial communities. We present results with the Campylobacter jejuni strain-specific marker gene gltA, as well as the 16S rRNA gene, which is a universal marker across bacterial assemblages. The statistical models computed for these genes are applied to genetic data from two different experimental settings, namely, a chicken infection model and a multispecies anaerobic fermentation model, demonstrating collection of time series data from model bacterial communities. The method presented here is, however, applicable to any experimental scenario where the interest is quantification of GCUs in genetically heterogeneous DNA samples.