Distinct signaling functions for Shc isoforms in the heart

Thrombin activates protease-activated receptor-1 (PAR-1) and engages signaling pathways that influence the growth and survival of cardiomyocytes as well as extracellular matrix remodeling by cardiac fibroblasts. This study examines the role of Shc proteins in PAR-1-dependent signaling pathways that influence ventricular remodeling. We show that thrombin increases p46Shc/p52Shc phosphorylation at Tyr(239)/Tyr(240) and Tyr(317) (and p66Shc-Ser(36) phosphorylation) via a pertussis toxin-insensitive epidermal growth factor receptor (EGFR) transactivation pathway in cardiac fibroblasts; p66Shc-Ser(36) phosphorylation is via a MEK-dependent mechanism. In contrast, cardiac fibroblasts express beta(2)-adrenergic receptors that activate ERK through a pertussis toxin-sensitive EGFR transactivation pathway that does not involve Shc isoforms or lead to p66Shc-Ser(36) phosphorylation. In cardiomyocytes, thrombin triggers MEK-dependent p66Shc-Ser(36) phosphorylation, but this is not via EGFR transactivation (or associated with Shc-Tyr(239)/Tyr(240) and/or Tyr(317) phosphorylation). Importantly, p66Shc protein expression is detected in neonatal, but not adult, cardiomyocytes; p66Shc expression is induced (via a mechanism that requires protein kinase C and MEK activity) by Pasteurella multocida toxin, a Galpha(q) agonist that promotes cardiomyocyte hypertrophy. These results identify novel regulation of individual Shc isoforms in receptor-dependent pathways leading to cardiac hypertrophy and the transition to heart failure. The observations that p66Shc expression is induced by a Galpha(q) agonist and that PAR-1 activation leads to p66Shc-Ser(36) phosphorylation identifies p66Shc as a novel candidate hypertrophy-induced mediator of cardiomyocyte apoptosis and heart failure.     

Antagonistic interactions of kleisins and DNA with bacterial Condensin MukB

MukBEF is a bacterial SMC (structural maintenance of chromosome) complex required for faithful chromosome segregation in Escherichia coli. The SMC subunit of the complex, MukB, promotes DNA condensation in vitro and in vivo; however, all three subunits are required for the function of MukBEF. We report here that MukEF disrupts MukB x DNA complex. Preassembled MukBEF was inert in DNA binding or reshaping. Similarly, the association of MukEF with DNA-bound MukB served to displace MukB from DNA. When purified from cells, MukBEF existed as a mixture of MukEF-saturated and unsaturated complexes. The holoenzyme was unstable and could only bind DNA upon dissociation of MukEF. The DNA reshaping properties of unsaturated MukBEF were identical to those of MukB. Furthermore, the unsaturated MukBEF was stable and proficient in DNA binding. These results support the view that kleisins are not directly involved in DNA binding but rather bridge distant DNA-bound MukBs.     

Mass spectral characterization of organophosphate-labeled lysine in peptides

Organophosphate (OP) esters bind covalently to the active site serine of enzymes in the serine hydrolase family. Recently, mass spectrometry identified covalent binding of OPs to tyrosine in a wide variety of proteins when purified proteins were incubated with OPs. In the current work, manual inspection of tandem mass spectrometry (MS/MS) data led to the realization that lysines also make a covalent bond with OPs. OP-labeled lysine residues were found in seven proteins that had been treated with either chlorpyrifos oxon (CPO) or diisopropylfluorophosphate (DFP): human serum albumin (K212, K414, K199, and K351), human keratin 1 (K211 and K355), human keratin 10 (K163), bovine tubulin alpha (K60, K336, K163, K394, and K401), bovine tubulin beta (K58), bovine actin (K113, K291, K326, K315, and K328), and mouse transferrin (K296 and K626). These results suggest that OP binding to lysine is a general phenomenon. Characteristic fragments specific for CPO-labeled lysine appeared at 237.1, 220.0, 192.0, 163.9, 128.9, and 83.9 amu. Characteristic fragments specific for DFP-labeled lysine appeared at 164.0, 181.2, and 83.8 amu. This new OP-binding motif to lysine suggests new directions to search for mechanisms of long-term effects of OP exposure and in the search for biomarkers of OP exposure.     

Replication Independent Formation of Extrachromosomal Circular DNA in Mammalian Cell-Free System

Extrachromosomal circular DNA (eccDNA) is a pool of circular double stranded DNA molecules found in all eukaryotic cells and composed of repeated chromosomal sequences. It was proposed to be involved in genomic instability, aging and alternative telomere lengthening. Our study presents novel mammalian cell-free system for eccDNA generation. Using purified protein extract we show that eccDNA formation does not involve de-novo DNA synthesis suggesting that eccDNA is generated through excision of chromosomal sequences. This process is carried out by sequence- independent enzymes as human protein extract can produce mouse- specific eccDNA from high molecular weight mouse DNA, and vice versa. EccDNA production does not depend on ATP, requires residual amounts of Mg²⁺ and is enhanced by double strand DNA breaks.     

Incorporation of N‐amidino‐pyroglutamic acid into peptides using intramolecular cyclization of α‐guanidinoglutaric acid

N‐terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N‐amidino‐amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block—N‐amidino‐pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N‐amidino‐proline using RuO₄ did not produce positive results, N‐amidino‐Glp‐Phe‐OH was synthesized on Wang polymer by cyclization of α‐guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N‐amidino‐Glp‐Phe‐OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N‐amidino‐Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The retinal pigment epithelial cells of the adult newt and rat under conditions of in vitro organotypic culture

To understand why the retinal pigment epithelium (RPE) has different potentials for neural differentiation in lower and higher vertebrates, the RPEs of adult newts and rats were compared under similar in vitro cultivation conditions. The RPEs of both animal species were organotypically cultivated within the posterior eye wall under constant rotation in the serum medium free of growth factors. Comparison of the cell morphology, proliferation, and expression of pan-neural markers demonstrated that the RPE cells of adult newts and rats under similar in vitro conditions displayed both similarities and differemces. They were able to synthesize DNA but rarely divided mitotically. In addition, part of the RPE cells of both the newt and the rat were dislodged from the layer, migrated, and acquired a macrophage phenotype. However, the majority of the cells retained the initial morphology and remained within the layer. In several cases, these cells displayed the initial characteristics of neural differentiation, namely, expression of pan-neural proteins. The difference between the newt and rat RPE cells was in the ability of the former to generate in vitro an additional row of dedifferentiated NF-200-positive cells, characteristic of in vivo newt retinal regeneration. These data demonstrate that the RPE cells of the adult newt and rat retain the potential of manifesting neural cell traits; however, more advanced changes towards differentiation are characteristic of only the newt RPE.     

A review of Turkish Diglyphus Walker (Hymenoptera: Eulophidae), with description of a new species

Two species Diglyphus begini Ashmead and D. sabulosus Erdös are new records for Turkey; one new species is described Diglyphus sensilis sp. nov., three species have new host record Liriomyza sativae and Chromatomyia horticola; 10 species of Diglyphus from Turkey are keyed.     

LH2 Complex from Sulfur Bacteria Allochromatium vinosum – Natural Singlet Oxygen Sensor

Biochemistry (Moscow) - It was established that in a heterogeneous model system, which consisted of two types of complexes: reaction center or core complex of photosystem 2 of higher plants and LH2...     

Hidden population size estimation and diagnostics using two respondent-driven samples with applications in Armenia

Estimating the size of hidden populations is essential to understand the magnitude of social and healthcare needs, risk behaviors, and disease burden. However, due to the hidden nature of these populations, they are difficult to survey, and there are no gold standard size estimation methods. Many different methods and variations exist, and diagnostic tools are needed to help researchers assess method-specific assumptions as well as compare between methods. Further, because many necessary mathematical assumptions are unrealistic for real survey implementation, assessment of how robust methods are to deviations from the stated assumptions is essential. We describe diagnostics and assess the performance of a new population size estimation method, capture–recapture with successive sampling population size estimation (CR-SS-PSE), which we apply to data from 3 years of studies from three cities and three hidden populations in Armenia. CR-SS-PSE relies on data from two sequential respondent-driven sampling surveys and extends the successive sampling population size estimation (SS-PSE) framework by using the number of individuals in the overlap between the two surveys and a model for the successive sampling process to estimate population size. We demonstrate that CR-SS-PSE is more robust to violations of successive sampling assumptions than SS-PSE. Further, we compare the CR-SS-PSE estimates to population size estimations using other common methods, including unique object and service multipliers, wisdom of the crowd, and two-source capture–recapture to illustrate volatility across estimation methods.     

Influence of the spacer region between the Shine–Dalgarno box and the start codon for fine-tuning of the translation efficiency in Escherichia coli

Translation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine–Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next-generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine–Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100-fold range.