Syntheses and crystal structures of cyano-bridged cluster-metal layered coordination polymers [Cu(dien)]3[W4Te4(CN)12] · 9H2O and [Ni(en)(NH3)]3[W4Se4(CN)12] · 7.5H2O

Two new tetrahedral tungsten cyanide cluster compounds, [Cu(dien)]₃[W₄Te₄(CN)₁₂] · 9H₂O (1) (dien=diethylenetriamine) and [Ni(en)(NH₃)]₃[W₄Se₄(CN)₁₂] · 7.5H₂O (2) (en=ethylenediamine), were synthesized by treating aqueous solutions of the saltlike cluster compound K₆[W₄Te₄(CN)₁₂] · 5H₂O/K₆[W₄Se₄(CN)₁₂] · 6H₂O with copper(II)/nickel(II) chloride in aqueous ammonia containing dien/en. The cyano-bridged layered coordination polymeric compounds were characterized by single-crystal X-ray diffraction analysis: monoclinic, space group P2₁ for 1; trigonal, space group for 2. Structures of 1 and 2 consist of infinite neutral layers of cluster components {W₄Te₄(CN)₁₂}/{W₄Se₄(CN)₁₂} connected, one another by {Cu(dien)} or {Ni(en)(NH₃)} fragments, respectively.     

Role of protein-tyrosine phosphatase SHP2 in focal adhesion kinase down-regulation during neutrophil cathepsin G-induced cardiomyocytes anoikis

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.     

The domain of the Bacillus subtilis DEAD-box helicase YxiN that is responsible for specific binding of 23S rRNA has an RNA recognition motif fold

The YxiN protein of Bacillus subtilis is a member of the DbpA subfamily of prokaryotic DEAD-box RNA helicases. Like DbpA, it binds with high affinity and specificity to segments of 23S ribosomal RNA as short as 32 nucleotides (nt) that include hairpin 92. Several experiments have shown that the 76-residue carboxy-terminal domain of YxiN is responsible for the high-affinity RNA binding. The domain has been crystallized and its structure has been solved to 1.7 Angstroms resolution. The structure reveals an RNA recognition motif (RRM) fold that is found in many eukaryotic RNA binding proteins; the RRM fold was not apparent from the amino acid sequence. The domain has two solvent exposed aromatic residues at sites that correspond to the aromatic residues of the ribonucleoprotein (RNP) motifs RNP1 and RNP2 that are essential for RNA binding in many RRMs. However, mutagenesis of these residues (Tyr404 and Tyr447) to alanine has little effect on RNA affinity, suggesting that the YxiN domain binds target RNAs in a manner that differs from the binding mode commonly found in many eukaryotic RRMs.     

Expression of an expanded polyglutamine domain in yeast causes death with apoptotic markers

Huntington's disease is caused by specific mutations in huntingtin protein. Expansion of a polyglutamine (polyQ) repeat of huntingtin leads to protein aggregation in neurons followed by cell death with apoptotic markers. The connection between the aggregation and the degeneration of neurons is poorly understood. Here, we show that the physiological consequences of expanded polyQ domain expression in yeast are similar to those in neurons. In particular, expression of expanded polyQ in yeast causes apoptotic changes in mitochondria, caspase activation, nuclear DNA fragmentation and death. Similar to neurons, at the late stages of expression the expanded polyQ accumulates in the nuclei and seems to affect the cell cycle of yeast. Interestingly, nuclear localization of the aggregates is dependent on functional caspase Yca1. We speculate that the aggregates in the nuclei disturb the cell cycle and thus contribute to the development of the cell death process in both systems. Our data show that expression of the polyQ construct in yeast can be used to model patho-physiological effects of polyQ expansion in neurons.     

Elucidation of (-)-epicatechin metabolites after ingestion of chocolate by healthy humans

After absorption in the gastrointestinal tract, (-)-epicatechin is extensively transformed into various conjugated metabolites. These metabolites, chemically different from the aglycone forms found in foods, are the compounds that reach the circulatory system and the target organs. Therefore, it is imperative to identify and quantify these circulating metabolites to investigate their roles in the biological effects associated with (-)-epicatechin intake. Using authentic synthetic standards of (-)-epicatechin sulfates, glucuronides, and O-methyl sulfates, a novel LC-MS/MS-MRM analytical methodology to quantify (-)-epicatechin metabolites in biological matrices was developed and validated. The optimized method was subsequently applied to the analysis of plasma and urine metabolites after consumption of dark chocolate, an (-)-epicatechin-rich food, by humans. (-)-Epicatechin-3'-β-d-glucuronide (C(max) 290±49nM), (-)-epicatechin 3'-sulfate (C(max) 233±60nM), and 3'-O-methyl epicatechin sulfates substituted in the 4', 5, and 7 positions were the most relevant (-)-epicatechin metabolites in plasma. When plasmatic metabolites were divided into their substituent groups, it was revealed that (-)-epicatechin glucuronides, sulfates, and O-methyl sulfates represented 33±4, 28±5, and 33±4% of total metabolites (AUC(0-24)(h)), respectively, after dark chocolate consumption. Similar metabolites were found in urine samples collected over 24h. The total urine excretion of (-)-epicatechin was 20±2% of the amount ingested. In conclusion, we describe the entire metabolite profile and its degree of elimination after administration of (-)-epicatechin-containing food. These results will help us understand more precisely the mechanisms and the main metabolites involved in the beneficial physiological effects of flavanols.     

Human galectin 3 binding protein interacts with recombinant adeno-associated virus type 6

Recombinant adeno-associated viruses (rAAVs) hold enormous potential for human gene therapy. Despite the well-established safety and efficacy of rAAVs for in vivo gene transfer, there is still little information concerning the fate of vectors in blood following systemic delivery. We screened for serum proteins interacting with different AAV serotypes in humans, macaques, dogs, and mice. We report that serotypes rAAV-1, -5, and -6 but not serotypes rAAV-2, -7, -8, -9, and -10 interact in human sera with galectin 3 binding protein (hu-G3BP), a soluble scavenger receptor. Among the three serotypes, rAAV-6 has the most important capacities for binding to G3BP. rAAV-6 also bound G3BP in dog sera but not in macaque and mouse sera. In mice, rAAV-6 interacted with another protein of the innate immune system, C-reactive protein (CRP). Furthermore, interaction of hu-G3BP with rAAV-6 led to the formation of aggregates and hampered transduction when the two were codelivered into the mouse. Based on these data, we propose that species-specific interactions of AAVs with blood proteins may differentially impact vector distribution and efficacy in different animal models.     

Hit clustering can improve virtual fragment screening: CDK2 and PARP1 case studies

Virtual fragment screening could be a promising alternative to existing experimental screening techniques. However, reliable methods of in silico fragment screening are yet to be established and validated. In order to develop such an approach we first checked how successful the existing molecular docking methods can be in predicting fragment binding affinities and poses. Using our Lead Finder docking software the RMSD of the binding energy prediction was observed to be 1.35 kcal/mol(-1) on a set of 26 experimentally characterized fragment inhibitors, and the RMSD of the predicted binding pose from the experimental one was <1.5 ?. Then, we explored docking of 68 fragments obtained from 39 drug molecules for which co-crystal structures were available from the PDB. It appeared that fragments that participate in oriented non-covalent interactions, such as hydrogen bonds and metal coordination, could be correctly docked in 70-80% of cases suggesting the potential success of rediscovering of corresponding drugs by in silico fragment approach. Based on these findings we've developed a virtual fragment screening technique which involved structural filtration of protein-ligand complexes for specific interactions and subsequent clustering in order to minimize the number of preferable starting fragment candidates. Application of this method led to 2 millimolar-scale fragment PARP1 inhibitors with a new scaffold.     

Natural causes of programmed death of yeast Saccharomyces cerevisiae

The existence of cell death program in unicellular organisms has been reported for a number of species. Nevertheless, the question why the ability to commit suicide has been maintained throughout evolution is far from being solved. While it is believed that altruistic death of individual yeast cells could be beneficial for the population, it is generally not known (i) what is wrong with the individuals destined for elimination, (ii) what is the critical value of the parameter that makes a cell unfit and (iii) how the cell monitors this parameter. Studies performed on yeast Saccharomyces cerevisiae allow us to hypothesize on ways of possible solutions of these problems. Here we argue that (a) the main parameter for life-or-death decision measured by the cell is the degree of damage to the genetic material, (b) its critical value is dictated by quorum sensing machinery, and (c) it is measured by monitoring delays in cell division.     

Thrips (Thysanoptera) identification using artificial neural networks

We studied the use of a supervised artificial neural network (ANN) model for semi-automated identification of 18 common European species of Thysanoptera from four genera: Aeolothrips Haliday (Aeolothripidae), Chirothrips Haliday, Dendrothrips Uzel, and Limothrips Haliday (all Thripidae). As input data, we entered 17 continuous morphometric and two qualitative two-state characters measured or determined on different parts of the thrips body (head, pronotum, forewing and ovipositor) and the sex. Our experimental data set included 498 thrips specimens. A relatively simple ANN architecture (multilayer perceptrons with a single hidden layer) enabled a 97% correct simultaneous identification of both males and females of all the 18 species in an independent test. This high reliability of classification is promising for a wider application of ANN in the practice of Thysanoptera identification.