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High temperature is one of the main environmental factors causing economic losses to the poultry industry, as it reduces growth and production performance of chickens. The heat shock proteins (HSPs) play a key role in cellular defense mechanisms during exposure in a hot environment. The aim of this study was to evaluate the expression level of the candidate genes in the liver of Khorasan native chickens under acute heat stress. Sixteen 42 days old chickens were divided into two groups; the control (25 °C and 50% humidity) and heat-treated (42 °C and 50% humidity), and then the liver was sampled. The level of gene expression of HSPB1, HSPB9, SERPINH1, HSPA2 and HSP110 were evaluated using the reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) method. The results of the analysis of variance revealed that the expression of HSPA2 and HSP110 was significantly increased. In the biological processes of gene ontology, three processes had FDR < 0.01. HSPA2 and HSPB1 involved in the processes that stimulated cells against increasing temperature. The results indicated that Khorasan native chickens have suitable resistance to acute heat stress. Furthermore, HSPA2 has the ability to express under high ambient temperature in order to protect the structure of cellular proteins. [ABSTRACT FROM AUTHOR]

Abstract Polymerases represent an attractive molecular target for antibacterial drug development, antiviral intervention and cancer therapy. Over the past decade, academic groups and scientists from pharmaceutical industry have developed a large plethora of different functional assays to monitor the enzymatic reaction catalyzed by polymerases. These assays were used to enable high-throughput screening (HTS) for lead discovery purposes, as well as hit-to-lead (H2L) drug profiling activities. In both cases the choice of the assay technology is critical and to the best of our knowledge, there is no review available to help scientists to choose the most suitable assay. This review summarizes the most common functional assays developed to monitor the enzymatic activity of polymerases and discusses the advantages and disadvantages of each assay. Assays are presented and evaluated in term of cost, ease of use, high-throughput screening compatibility and liability towards delivering false positives and false negatives. Highlights • Overview on functional assays to monitor the enzymatic reaction of polymerases. • Application of assays in high-throughput screening (HTS) for lead discovery. • Recommendation for choosing the appropriate assay technology. • Evaluation of assays in term of cost, ease of use and HTS compatibility. • Application of assays for antibacterial, antiviral anticancer drug development. [ABSTRACT FROM AUTHOR]

Background: Previous studies showed the role of vitamin D (Vit D) on the progression of chronic urticaria. To the best of our knowledge, there are no other results regarding the contribution of single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) and vitamin D binding protein (VDBP) genes in chronic urticaria (CU). Aim: In the present study, we investigated the Vit pathway and the association between VDR and VDBP gene polymorphisms and CU risk in Iranian population. Methods: All participating individuals in the present study were evaluated for serumVit D and VDBP concentration VDR rs1544410 and rs2228570 and VDBP rs7041using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The associations of studied analytes and three SNPs with clinical and laboratory outcomes were investigated in CU patients. Results: Patients with CU showed lower Vit D compared to controls (19.26 ± 1.26 vs. 31.72 ± 7.14ng/ml, P-value=0.006). There was a significant correlation between Vit D levels and urticaria activity score. Serum VDBP was significantly higher in CU patients than controls (1317.3 ± 183.71 vs. 395.77 ± 12.96 µg/ml, P-value <0.0001) and had a positive correlation to progression of CU. The A allele of this polymorphism might be a potential risk factor for progression of CU [odds ratio 4.3434, 95% confidence interval (1.7331-10.8852), Z-statistic=3.133, P-value=0.0017]. Conclusion: In summary, this study demonstrated that change in Vit D pathway in the level of gene or protein may be a risk factor for progression of CU. [ABSTRACT FROM AUTHOR]

Keap1 binds to the transcription factor Nrf2 and negatively modulates the expression of genes involved in cellular protection against oxidative stress. Small molecules have been discovered to inhibit the Nrf2:Keap1 interactions and act as antagonists of Keap1. The affinities of these small molecules are not very high and need further improvement in follow up hit-to-lead programs. In addition to the affinity parameters K i , K d , and IC 50 thermodynamic parameters provide useful information for the selection and optimization of these hit molecules at the early stage of the lead discovery process. In this letter a tracer displacement assay was used to determine the thermodynamic signature of some of the known inhibitors of the Nrf2:Keap1 interaction. An optimized assay protocol is presented, which can be applied to other small molecules in hit-to-lead programs in a medium throughput manner. [ABSTRACT FROM AUTHOR]

Parathyroid hormone-related protein (PTHrP) is widely expressed in the fibrous outer layer of the periosteum (PO), and the PTH/PTHrP type I receptor (PTHR1) is expressed in the inner PO cambial layer. The cambial layer gives rise to the PO osteoblasts (OBs) and osteoclasts (OCs) that model/remodel the cortical bone surface during development as well as during fracture healing. PTHrP has been implicated in the regulation of PO modeling during development, but nothing is known as regards a role of PTHrP in this location during fracture healing. We propose that PTHrP in the fibrous layer of the PO may be a key regulatory factor in remodeling bone formation during fracture repair. We first assessed whether PTHrP expression in the fibrous PO is associated with PO osteoblast induction in the subjacent cambial PO using a tibial fracture model in PTHrP-lacZ mice. Our results revealed that both PTHrP expression and osteoblast induction in PO were induced 3 days post-fracture. We then investigated a potential functional role of PO PTHrP during fracture repair by performing tibial fracture surgery in 10-week-old CD1 control and PTHrP conditional knockout (PTHrP cKO) mice that lack PO PTHrP. We found that callus size and formation as well as woven bone mineralization in PTHrP cKO mice were impaired compared to that in CD1 mice. Concordant with these findings, functional enzyme staining revealed impaired OB formation and OC activity in the cKO mice. We conclude that deleting PO PTHrP impairs cartilaginous callus formation, maturation and ossification as well as remodeling during fracture healing. These data are the initial genetic evidence suggesting that PO PTHrP may induce osteoblastic activity and regulate fracture healing on the cortical bone surface. [ABSTRACT FROM AUTHOR]

Highlights: [•] U–Pb detrital zircon ages from Neoproterozoic sediments of the ANS are presented. [•] The Wadi Igla Formation in the Eastern Desert of Egypt was deposited at ≤628±6Ma. [•] The Thalbah Group sediments span a depositional period between ca. 635 and 596Ma. [•] The Thalbah Group has a multi-phase Cryogenian-early Ediacaran tectonic evolution. [•] Detrital zircon age distributions may reflect the onset of collision at ca. 596Ma. [ABSTRACT FROM AUTHOR]

We report here on the screening of a fragment library against a G-quadruplex element in the human c-MYC promoter. The ten fragment hits had significant concordance between a biophysical assay, in silico modelling and c-MYC expression inhibition, highlighting the feasibility of applying a fragment-based approach to the targeting of a quadruplex nucleic acid. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondrial complex I (proton pumping NADH:ubiquinone oxidoreductase) is the largest and most complicated component of the respiratory electron transfer chain. Despite its central role in biological energy conversion the structure and function of this membrane integral multiprotein complex is still poorly understood. Recent insights into the structure of complex I by X-ray crystallography have shown that iron–sulfur cluster N2, the immediate electron donor for ubiquinone, resides about 30Å above the membrane domain and mutagenesis studies suggested that the active site for the hydrophobic substrate is located next to this redox-center. To trace the path for the hydrophobic tail of ubiquinone when it enters the peripheral arm of complex I, we performed an extensive structure/function analysis of complex I from Yarrowia lipolytica monitoring the interaction of site-directed mutants with five ubiquinone derivatives carrying different tails. The catalytic activity of a subset of mutants was strictly dependent on the presence of intact isoprenoid moieties in the tail. Overall a consistent picture emerged suggesting that the tail of ubiquinone enters through a narrow path at the interface between the 49-kDa and PSST subunits. Most notably we identified a set of methionines that seems to form a hydrophobic gate to the active site reminiscent to the M-domains involved in the interaction with hydrophobic targeting sequences with the signal recognition particle of the endoplasmic reticulum. Interestingly, two of the amino acids critical for the interaction with the ubiquinone tail are different in bovine complex I and we could show that one of these exchanges is responsible for the lower sensitivity of Y. lipolytica complex I towards the inhibitor rotenone. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). [Copyright &y& Elsevier]

A homogenous fluorescence polarization (FP) assay was developed to monitor the enzymatic activity of polymerases. Under the optimized conditions established in this study, the assay provides highly robust and reproducible data. Miniaturization of the assay for high-throughput screening and compound testing was also performed. The sensitivity of the newly developed assay was confirmed using 2′,3′-dideoxyadenosine-5′-triphosphate (ddATP), a chain-elongating inhibitor of the polymerase reaction. Side-by-side comparison of the presented fluorescence polarization assay with already well established PicoGreen® fluorescence intensity assay revealed that the performance of both formats is comparable with good assay sensitivity. However, the direct ratiometric readout of the presented FP assay makes it superior over existing colorimetric and fluorescence intensity based assays in terms of susceptibility to false positives. Moreover, due to its generic nature the presented FP assay can be applied to other polymerases and is compatible with identification of inhibitors and requirements of hit-to-lead programs. [ABSTRACT FROM AUTHOR]

In Gram-positive bacteria and other prokaryotes containing succinate:menaquinone reductases, it has previously been shown that the succinate oxidase and succinate:menaquinone reductase activities are lost when the transmembrane electrochemical proton potential, Δp, is abolished by the rupture of the bacteria or by the addition of a protonophore. It has been proposed that the endergonic reduction of menaquinone by succinate is driven by the electrochemical proton potential. Opposite sides of the cytoplasmic membrane were envisaged to be separately involved in the binding of protons upon the reduction of menaquinone and their release upon succinate oxidation, with the two reactions linked by the transfer of two electrons through the enzyme. However, it has previously been argued that the observed Δp dependence is not associated specifically with the succinate:menaquinone reductase. Definitive insight into the mechanism of catalysis of this reaction requires a corresponding functional characterization of an isolated, membrane-bound succinate:menaquinone reductase from a Gram-positive bacterium. Here, we describe the purification, reconstitution into proteoliposomes, and functional characterization of the diheme-containing succinate:menaquinone reductase from the Gram-positive bacterium Bacillus licheniformis and, with the help of the design, synthesis, and characterization of quinones with finely tuned oxidation/reduction potentials, provide unequivocal evidence for Δp-dependent catalysis of succinate oxidation by quinone as well as for Δp generation upon catalysis of fumarate reduction by quinol. [ABSTRACT FROM AUTHOR]

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential (‘proton-motive force’), either by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by transmembrane proton transfer. Here we provide the first evidence that both of these mechanisms are combined in the case of a specific respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes, so as to facilitate transmembrane electron transfer by transmembrane proton transfer. We also demonstrate the non-functionality of this novel transmembrane proton transfer pathway (‘E-pathway’) in a variant QFR where a key glutamate residue has been replaced. The ‘E-pathway’, discussed on the basis of the 1.78-Å-resolution crystal structure of QFR, can be concluded to be essential also for the viability of pathogenic ɛ-proteobacteria such as Helicobacter pylori and is possibly relevant to proton transfer in other dihaem-containing membrane proteins, performing very different physiological functions. [ABSTRACT FROM AUTHOR]

A short, efficient one-step synthesis of primin and its isomer is devised. The synthesis is based on the introduction of an alkyl side chain via radical alkylation of 2-methoxy-benzoquinone. The required alkyl radical was either generated from the corresponding alkyl acid or alkyl boronic acid in the presence of persulfate under silver catalysis. The alkylation of 2-methoxy-benzoquinone occurs at the C-6 and C-5 position, generating the desired primin and iso-primin. The formation of the natural product primin was confirmed by NMR spectroscopy determining carbon–carbon constitution at 13 C natural abundance in an 1,1-ADEQUATE experiment. [ABSTRACT FROM AUTHOR]

Bacterial energy metabolism has become a promising target for next-generation tuberculosis chemotherapy. One strategy to hamper ATP production is to inhibit the respiratory oxidases. The respiratory chain of Mycobacterium tuberculosis comprises a cytochrome bcc:aa3 and a cytochrome bd ubiquinol oxidase that require a combined approach to block their activity. A quinazoline-type compound called ND-011992 has previously been reported to ineffectively inhibit bd oxidases, but to act bactericidal in combination with inhibitors of cytochrome bcc:aa3 oxidase. Due to the structural similarity of ND-011992 to quinazoline-type inhibitors of respiratory complex I, we suspected that this compound is also capable of blocking other respiratory chain complexes. Here, we synthesized ND-011992 and a bromine derivative to study their effect on the respiratory chain complexes of Escherichia coli. And indeed, ND-011992 was found to inhibit respiratory complex I and bo3 oxidase in addition to bd-I and bd-II oxidases. The IC50 values are all in the low micromolar range, with inhibition of complex I providing the lowest value with an IC50 of 0.12 µM. Thus, ND-011992 acts on both, quinone reductases and quinol oxidases and could be very well suited to regulate the activity of the entire respiratory chain. [ABSTRACT FROM AUTHOR]

Cytochrome bd oxidases are membrane proteins expressed by bacteria including a number of pathogens, which make them an attractive target for the discovery of new antibiotics. An electrochemical assay is developed to study the activity of these proteins and inhibition by quinone binding site tool compounds. The setup relies on their immobilization at electrodes specifically modified with gold nanoparticles, which allows achieving a direct electron transfer to/from the heme cofactors of this large enzyme. After optimization of the protein coverages, the assay shows at pH 7 a good reproducibility and readout stability over time, and it is thus suitable for further screening of small molecule collections. [ABSTRACT FROM AUTHOR]

The modeling of long bone surfaces during linear growth is a key developmental process, but its regulation is poorly understood. We report here that parathyroid hormone-related peptide ( PTHr P) expressed in the fibrous layer of the periosteum ( PO) drives the osteoclastic ( OC) resorption that models the metaphyseal-diaphyseal junction ( MDJ) in the proximal tibia and fibula during linear growth. PTHr P was conditionally deleted (c KO) in the PO via Scleraxis gene targeting ( Scx- Cre). In the lateral tibia, c KO of PTHr P led to a failure of modeling, such that the normal concave MDJ was replaced by a mound-like deformity. This was accompanied by a failure to induce receptor activator of NF-kB ligand ( RANKL) and a 75% reduction in OC number ( P ≤ 0.001) on the cortical surface. The MDJ also displayed a curious threefold increase in endocortical osteoblast mineral apposition rate ( P ≤ 0.001) and a thickened cortex, suggesting some form of coupling of endocortical bone formation to events on the PO surface. Because it fuses distally, the fibula is modeled only proximally and does so at an extraordinary rate, with an anteromedial cortex in CD-1 mice that was so moth-eaten that a clear PO surface could not be identified. The c KO fibula displayed a remarkable phenotype, with a misshapen club-like metaphysis and an enlargement in the 3 D size of the entire bone, manifest as a 40-45% increase in the PO circumference at the MDJ ( P ≤ 0.001) as well as the mid-diaphysis ( P ≤ 0.001). These tibial and fibular phenotypes were reproduced in a Scx- Cre-driven RANKL c KO mouse. We conclude that PTHr P in the fibrous PO mediates the modeling of the MDJ of long bones during linear growth, and that in a highly susceptible system such as the fibula this surface modeling defines the size and shape of the entire bone. [ABSTRACT FROM AUTHOR]

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria and may have evolved to protect cells against the production of damaging reactive oxygen species. Therefore, compounds that enhance mitochondrial uncoupling are potentially attractive anti-aging therapies; however, chronic ingestion is associated with a number of unwanted side effects. We have previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-directed and promotes oxidation of hepatic triglycerides by causing a subtle sustained increase in hepatic mitochondrial inefficiency. Here, we sought to leverage the higher therapeutic index of CRMP to test whether mild mitochondrial uncoupling in a liver-directed fashion could reduce oxidative damage and improve age-related metabolic disease and lifespan in diet-induced obese mice. Oral administration of CRMP (20 mg/[kg-day] × 4 weeks) reduced hepatic lipid content, protein kinase C epsilon activation, and hepatic insulin resistance in aged (74-week-old) high-fat diet (HFD)-fed C57BL/6J male mice, independently of changes in body weight, whole-body energy expenditure, food intake, or markers of hepatic mitochondrial biogenesis. CRMP treatment was also associated with a significant reduction in hepatic lipid peroxidation, protein carbonylation, and inflammation. Importantly, long-term (49 weeks) hepatic mitochondrial uncoupling initiated late in life (94-104 weeks), in conjugation with HFD feeding, protected mice against neoplastic disorders, including hepatocellular carcinoma (HCC), in a strain and sex-specific manner. Taken together, these studies illustrate the complex variation of aging and provide important proof-of-concept data to support further studies investigating the use of liver-directed mitochondrial uncouplers to promote healthy aging in humans. [ABSTRACT FROM AUTHOR]

Both the genomes of the epsilonproteobacteria Wolinella succinogenes and Campylobacter jejuni contain operons ( sdhABE) that encode for so far uncharacterized enzyme complexes annotated as ‘non-classical’ succinate:quinone reductases (SQRs). However, the role of such an enzyme ostensibly involved in aerobic respiration in an anaerobic organism such as W. succinogenes has hitherto been unknown. We have established the first genetic system for the manipulation and production of a member of the non-classical succinate:quinone oxidoreductase family. Biochemical characterization of the W. succinogenes enzyme reveals that the putative SQR is in fact a novel methylmenaquinol:fumarate reductase (MFR) with no detectable succinate oxidation activity, clearly indicative of its involvement in anaerobic metabolism. We demonstrate that the hydrophilic subunits of the MFR complex are, in contrast to all other previously characterized members of the superfamily, exported into the periplasm via the twin-arginine translocation (tat)-pathway. Furthermore we show that a single amino acid exchange (Ala86→His) in the flavoprotein of that enzyme complex is the only additional requirement for the covalent binding of the otherwise non-covalently bound FAD. Our results provide an explanation for the previously published puzzling observation that the C. jejuni sdhABE operon is upregulated in an oxygen-limited environment as compared with microaerophilic laboratory conditions. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet-induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance. [ABSTRACT FROM AUTHOR]

[Display omitted] • A collection of natural products based on noscapine has been synthesized. • Components tested for breast cancer cells MDA-MB-231 in the cell proliferation assay. • Two novel noscapine derivatives have been identified as having antiproliferative activity. • The growth inhibition of novel noscapine derivatives was superior to the lead compound. Noscapine is a natural product first isolated from the opium poppy (Papaver somniferum L.) with anticancer properties. In this work, we report the synthesis and cellular screening of a noscapine-based library. A library of novel noscapine derivatives was synthesized with modifications in the isoquinoline and phthalide scaffolds. The so generated library, consisting of fifty-seven derivatives of the natural product noscapine, was tested against MDA-MB-231 breast cancer cells in a cellular proliferation assay (with a Z' > 0.7). The screening resulted in the identification of two novel noscapine derivatives as inhibitors of MDA cell growth with IC 50 values of 5 µM and 1.5 µM, respectively. Both hit molecules have a five-fold and seventeen-fold higher potency, compared with that of lead compound noscapine (IC 50 26 µM). The identified active derivatives retain the tubulin-binding ability of noscapine. Further testing of both hit molecules, alongside the natural product against additional cancer cell lines (HepG2, HeLa and PC3 cells) confirmed our initial findings. Both molecules have improved anti-proliferative properties when compared to the initial natural product, noscapine. [ABSTRACT FROM AUTHOR]

Riboswitches are highly structured RNA elements that control the expression of many bacterial genes by binding directly to small metabolite molecules with high specificity and affinity. In Bacillus subtilis, two classes of riboswitches have been described that discriminate between guanine and adenine despite an extremely high degree of homology both in their primary and secondary structure. We have identified intermolecular base triples between both purine ligands and their respective riboswitch RNAs by NMR spectroscopy. Here, specificity is mediated by the formation of a Watson-Crick base pair between the guanine ligand and a C residue. or the adenine ligand and a U residue of the cognate riboswitch RNA, respectively. In addition, a second base-pairing interaction common to both riboswitch purine complexes involves a uridine residue of the RNA and the N3/N9 edge of the purine ligands. This base pairing is mediated by a previously undescribed hydrogen-bonding scheme that contributes to the affinity of the RNA-ligand interaction. The observed intermolecular hydrogen bonds between the purine ligands and the RNA rationalize the previously observed change in specificity upon a C to U mutation in the core of the purine riboswitch RNAs and the differences in the binding affinities for a number of purine analogs. [ABSTRACT FROM AUTHOR]

Objective: Spermatogonia are male germ line progenitors of sperm cells. In bovine, rodents and primates, undifferentiated spermatogonia and spermatogonial stem cells (SSCs) express the surface marker THY1. Since cells can be enriched from mixed populations by positive or negative selection on physical or functional characteristics such as cell surface markers, present study was aimed to compare the efficiency of two different enrichment methods: Magnetic activated cell sorting (MACS) and differential plating (DP) in pre-pubertal goat testes cell population. To comparison of two enrichment methods efficiency, real time PCR analysis, protein expression, and transplantation into recipient mice data were analyzed. For control group, the intact testis cells population were used. Materials and Methods: After 3 times enzymatic digestion of pre-pubertal goat testicular tissues, cells were cultured for 16 h in laminin-coated flasks and DP carried out. For MACS method, cell suspension were sorted using THY1 surface marker. Results: Real time PCR analysis revealed that expression of THY1, PLZF, VASA, BCL6B, UCHL1 as SCCs characteristic genes in THY1 positive cells were significantly higher than in the DP isolation cells and unselected total cells. Transplantation of THY1positive, DP isolated and intact population of testis cells (control) into the mice recipient seminiferous tubules revealed that THY1 positive cells had higher capacity for colony formation were compared to DP isolated and intact cells. Conclusion: The results indicated that isolation of undifferentiated testis cells using MACS method can be more efficient than differential plating [ABSTRACT FROM AUTHOR]