Gq-coupled receptors as mechanosensors mediating myogenic vasoconstriction

Despite the central physiological function of the myogenic response, the underlying signalling pathways and the identity of mechanosensors in vascular smooth muscle (VSM) are still elusive. In contrast to present thinking, we show that membrane stretch does not primarily gate mechanosensitive transient receptor potential (TRP) ion channels, but leads to agonist-independent activation of G(q/11)-coupled receptors, which subsequently signal to TRPC channels in a G protein- and phospholipase C-dependent manner. Mechanically activated receptors adopt an active conformation, allowing for productive G protein coupling and recruitment of beta-arrestin. Agonist-independent receptor activation by mechanical stimuli is blocked by specific antagonists and inverse agonists. Increasing the AT(1) angiotensin II receptor density in mechanically unresponsive rat aortic A7r5 cells resulted in mechanosensitivity. Myogenic tone of cerebral and renal arteries is profoundly diminished by the inverse angiotensin II AT(1) receptor agonist losartan independently of angiotensin II (AII) secretion. This inhibitory effect is enhanced in blood vessels of mice deficient in the regulator of G-protein signalling-2. These findings suggest that G(q/11)-coupled receptors function as sensors of membrane stretch in VSM cells.     

Multimode selective detection of mercury by chiroptical fluorescent sensors based on methionine/cysteine

Two multimode Hg(II) sensors, L‐MethBQA and L‐CysBQA, were obtained by fusing methionine or S‐methyl cysteine, into a bis‐quinolyl amine‐based chiral podand scaffold. Quinolyl groups serve as the fluorophore and possess nitrogen lone pairs capable of chelating metal ions. On exposure to Hg²⁺ or Zn²⁺, these sensors show signal enhancement in fluorescence. However, Cu²⁺ quenches their fluorescence in 30:70 acetontrile/water. L‐CysBQA complexes with Hg²⁺, producing an exciton‐coupled circular dichroism spectrum with the opposite sign to the one that is produced by Cu²⁺ or Zn²⁺ complexation. L‐CysBQA binds Hg²⁺ more strongly than Zn²⁺ and is shown to differentiate Hg²⁺ from other metal ions, such as Zn²⁺, Cu²⁺, Ni²⁺, and Pb²⁺, exceptionally well. The synergistic use of relatively soft sulfur, quinoline‐based chiral ligands and chiroptically enhanced fluorescence detection results in high sensitivity and selectivity for Hg²⁺. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Sulfhydryl-reactive, cleavable, and radioiodinatable benzophenone photoprobes for study of protein-protein interaction

The major task in proteomics is to understand how proteins interact with their partners. The photo-cross-linking technique enables direct probing of protein-protein interaction. Here we report the development of three novel sulfhydryl-reactive benzophenone photoprobes of short "arm" length, each with a substitution of either amino, iodo, or nitro at the para-position, rendering the benzophenone moiety directly radioiodinatable. Their potential for study of protein-protein interaction was assessed using the inhibitory subunit of rod cGMP phosphodiesterase (PDEgamma) and the activated transducin alphasubunit (G alpha t-GTPgammaS) as a model system. These photoprobes proved to be stable at neutral pH and dithiothreitol-cleavable in addition. The PDEgamma constructs derivatized at the C-terminal positions with these probes could be readily purified, had unaltered PDEgamma functional activity, and were shown to photo-cross-link to G alpha t-GTPgammaS with an efficiency as high as 40%. Additionally, the amino benzophenone probe was radioiodinated, facilitating sensitive detection of label transfer. The uniquely combined features of these benzophenone photoprobes promise robust and flexible methods for characterization of protein-protein interaction, either by mass spectrometry when a nonradioactive label is available or by autoradiography when using radioiodinated derivatives.     

When secondary comes first – The importance of non-canonical DNA structures

Secondary structure-forming DNA motifs have achieved infamy because of their association with a variety of human diseases and cancer. The 3rd FASEB summer conference on dynamic DNA structures focused on the mechanisms responsible for the instabilities inherent to repetitive DNA and presented many exciting and novel aspects related to the metabolism of secondary structures. In addition, the meeting encompassed talks and posters on the dynamic structures that are generated during DNA metabolism including nicked DNA, Holliday junctions and RNA:DNA hybrids. New approaches for analysis and sequencing technologies put forth secondary structures and other DNA intermediates as vital regulators of a variety of cellular processes that contribute to evolution, polymorphisms and diseases.     

Vertebrate evolution reflected in the evolution of nuclear ribosomal internal transcribed spacer 2

In eukaryotes, mature rRNA sequences are produced from single large (45S) precursor (pre-rRNA) as the result of successive removal of spacers through a series of rapid and intricate actions of endo- and exonucleases. The excision of internal transcribed spacer (ITS2), a eukaryotic-specific insertion, remains the most elusive processing step. ITS2 is the element mandatory for all eukaryotic pre-rRNAs that contain at least three processing cleavage sites for precise 5.8S and 28S formation. Conserved core sequences (cis-elements) binding to trans-factors provide for precise rRNA processing, whereas rapidly diverging regions between the core sequences preserve internal complementarity, which guarantees the spatial integrity of ITS2. Characteristic differences in the formation of such insertions during evolution should reflect the relationships between taxa. The phylogeny of the reptiles and the relationships between taxa proposed by scientists are controversial. To delineate the structural and functional features preserved among reptilian ITS2s, we cloned and sequenced 58 ITS2s belonging to four reptile orders: Squamata, Crocodilians, Aves, and Testudines. We studied the subsequent alignment and folding of variable regions. The sizes and packing of the loop–stems between conserved consensus segments in reptiles vary considerably between taxa. Our phylogenetic trees constructed on the basis of the reptile ITS2s primary structural alignments revealed a split between Iguania clade and all other taxa. True lizards (suborder Scleroglossa) and snakes (suborder Serpentes) show sister relationships, as well as the two other reptilian orders, Crocodilia + Aves and Testudines. In summary, our phylogenetic trees exhibit a mix of specific features deduced or, to the contrary, rejected earlier by other authors.     

Mitochondrial aberrations in mucolipidosis Type IV

Mucolipidosis type IV is a genetic lysosomal storage disease associated with degenerative processes in the brain, eye, and other tissues. Mucolipidosis type IV results from mutations in the gene MCOLN1, which codes for the TRP family ion channel, mucolipin 1. The connection between lysosomal dysfunction and degenerative processes in mucolipidosis type IV is unclear. Here we report that mucolipidosis type IV and several unrelated lysosomal storage diseases are associated with significant mitochondrial fragmentation and decreased mitochondrial Ca2+ buffering efficiency. The mitochondrial alterations observed in these lysosomal storage diseases are reproduced in control cells by treatment with lysosomal inhibitors and with the autophagy inhibitor 3-methyladenine. This suggests that inefficient autophagolysosomal recycling of mitochondria generates fragmented, effete mitochondria in mucolipidosis. Mitochondria accumulate that cannot properly buffer calcium fluxes in the cell. A decrease in mitochondrial Ca2+ buffering capacity in cells affected by these lysosomal storage diseases is associated with increased sensitivity to apoptosis induced by Ca2+-mobilizing agonists and executed via a caspase-8-dependent pathway. Deficient Ca2+ homeostasis may represent a common mechanism of degenerative cell death in several lysosomal storage diseases.     

Characterization of Avian-like Influenza A (H4N6) Virus Isolated from Caspian Seal in 2012

正Dear Editor Marine mammals are widely distributed and can be found almost in all coastal waters and coastlines around the world. The interface areas between marine and terrestrial environments provide natural habitats for aquatic and semiaquatic mammals as well as for reservoir species of avian influenza viruses (AIV)(Runstadler et al. 2013). Previous     

In vitro assembly,purification, and crystallization of the rab geranylgeranyl transferase:substrate complex

Posttranslational modification with the geranygeranyl moiety is essential for the ability of Rab GTPases to control processes of membrane docking and fusion. This modification is conferred by Rab geranylgeranyltransferase (RabGGTase), which catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab proteins. The enzyme consists of a catalytic alpha/beta heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane. In order to understand the structural basis of Rab prenylation, we have investigated in vitro assembly and crystallization of the RabGGTase:REP-1:Rab complex. In order to ensure maximal stability of the ternary complex, we generated its monoprenylated form, which corresponds to a reaction intermediate and displays the highest affinity between the components. This was achieved by expressing the individual components in baculovirus and Escherichia coli systems with subsequent purification followed by in vitro monoprenylation of Rab7 with immobilized recombinant RabGGTase. Purified monoprenylated REP-1:Rab7 was complexed with recombinant RabGGTase and crystallized in hanging drops. The crystals obtained initially diffract to 8 A on an in-house X-ray source.