bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteasomal dysfunction activates the transcription factor SKN-1 and produces a selective oxidative-stress response in Caenorhabditis elegans
Authors: Kahn Nate W. , Rea Shane L. , Moyle Sarah , Kell Alison , Johnson Thomas E.
Published: December 2007

SKN-1 in the nematode worm Caenorhabditis elegans is functionally orthologous to mammalian NRF2 [NF-E2 (nuclear factor-E2)-related factor 2], a protein regulating response to oxidative stress. We have examined both the expression and activity of SKN-1 in response to a variety of oxidative stressors and to down-regulation of specific gene targets by RNAi (RNA interference). We used an SKN-1–GFP (green fluorescent protein) translational fusion to record changes in both skn-1 expression and SKN-1 nuclear localization, and a gst-4–GFP transcriptional fusion to measure SKN-1 transcriptional activity. GST-4 (glutathione transferase-4) is involved in the Phase II oxidative stress response and its expression is lost in an skn-1(zu67) mutant. In the present study, we show that the regulation of skn-1 is tied to the protein-degradation machinery of the cell. RNAi-targeted removal of most proteasome subunits in C. elegans caused nuclear localization of SKN-1 and, in some cases, induced transcription of gst-4. Most intriguingly, RNAi knockdown of proteasome core subunits caused nuclear localization of SKN-1 and induced gst-4, whereas RNAi knockdown of proteasome regulatory subunits resulted in nuclear localization of SKN-1 but did not induce gst-4. RNAi knockdown of ubiquitin-specific hydrolases and chaperonin components also caused nuclear localization of SKN-1 and, in some cases, also induced gst-4 transcription. skn-1 activation by proteasome dysfunction could be occurring by one or several mechanisms: (i) the reduced processivity of dysfunctional proteasomes may allow oxidatively damaged by-products to build up, which, in turn, activate the skn-1 stress response; (ii) dysfunctional proteasomes may activate the skn-1 stress response by blocking the constitutive turnover of SKN-1; and (iii) dysfunctional proteasomes may activate an unidentified signalling pathway that feeds back to control the skn-1 stress response.
MAPK-induced Ser727 phosphorylation promotes SUMOylation of STAT1
Authors: Vanhatupa Sari , Ungureanu Daniela , Paakkunainen Maija , Silvennoinen Olli
Published: December 2007

STAT1 (signal transducer and activator of transcription 1) is a critical mediator of IFN-γ (interferon-γ)-induced gene responses, and its function is regulated through phosphorylation of Tyr701 and Ser727. MAPK (mitogen-activated protein kinase) pathways mediate phosphorylation of Ser727 in response to microbial infections, stress stimuli and growth factors. Recently, STAT1 was found to become modified by PIAS (protein inhibitor of activated STAT)-mediated SUMO-1 (small ubiquitin-related modifier-1) conjugation at Lys703, but the regulation of this modification is largely unknown. Here, we have investigated the role of MAPK-induced Ser727 phosphorylation in regulation of STAT1 SUMOylation. Activation of the p38MAPK pathway by upstream activating kinase, MKK6 (MAPK kinase-6) or osmotic stress enhanced the SUMOylation of STAT1, which was counteracted by the p38MAPK inhibitor SB202190 or by dominant-negative p38MAPK. Activation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway by Raf-1 also enhanced Ser727 phosphorylation and SUMOylation of STAT1, and this induction was counteracted by PD98059 inhibitor. Mutation of Ser727 to alanine abolished the p38MAPK-induced SUMOylation. Furthermore, S727D and S727E mutations, which mimic the phosphorylation of Ser727, enhanced the basal SUMOylation of STAT1 and interaction between PIAS1 and STAT1. Taken together, these results identify Ser727 phosphorylation as a regulator of STAT1 SUMOylation and highlight the central role of Ser727 in co-ordination of STAT1 functions in cellular responses.
Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor
Authors: Ecke Denise , Hanck Theodor , Tulapurkar Mohan E. , Schäfer Rainer , Kassack Matthias , Stricker Rolf , Reiser Georg
Published: December 2007

Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP–P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.
Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells
Authors: Molinari Paola , Casella Ida , Costa Tommaso
Published: December 2007

Green bioluminescence in Renilla species is generated by a ∼100% efficient RET (resonance energy transfer) process that is caused by the direct association of a blue-emitting luciferase [Rluc (Renilla luciferase)] and an RGFP (Renilla green fluorescent protein). Despite the high efficiency, such a system has never been evaluated as a potential reporter of protein–protein interactions. To address the question, we compared and analysed in mammalian cells the bioluminescence of Rluc and RGFP co-expressed as free native proteins, or as fused single-chain polypeptides and tethered partners of self-assembling coiled coils. Here, we show that: (i) no spontaneous interactions generating detectable BRET (bioluminescence RET) signals occur between the free native proteins; (ii) high-efficiency BRET similar to that observed in Renilla occurs in both fusion proteins and self-interacting chimaeras, but only if the N-terminal of RGFP is free; (iii) the high-efficiency BRET interaction is associated with a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogues. Here, we propose a new functional complementation assay based on the detection of the high-efficiency BRET signal that is generated when the reporters Rluc and RGFP are brought into close proximity by a pair of interacting proteins to which they are linked. To demonstrate its performance, we implemented the assay to measure the interaction between GPCRs (G-protein-coupled receptors) and β-arrestins. We show that complementation-induced BRET allows detection of the GPCR–β-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response.
The N-terminal region of ABC50 interacts with eukaryotic initiation factor eIF2 and is a target for regulatory phosphorylation by CK2
Authors: Paytubi Sonia , Morrice Nicholas A. , Boudeau Jerome , Proud Christopher G.
Published: December 2007

ABC50 is an ABC (ATP-binding cassette) protein which, unlike most ABC proteins, lacks membrane-spanning domains. ABC50 interacts with eIF2 (eukaryotic initiation factor 2), a protein that plays a key role in translation initiation and in its control, and in regulation of ribosomes. Here, we establish that the interaction of ABC50 with eIF2 involves features in the N-terminal domain of ABC50, the region of ABC50 that differs most markedly from other ABC proteins. This region also shows no apparent similarity to the eIF2-binding domains of other partners of eIF2. In contrast, the N-terminus of ABC50 cannot bind to ribosomes by itself, but it can in conjunction with one of the nucleotide-binding domains. We demonstrate that ABC50 is a phosphoprotein and is phosphorylated at two sites by CK2. These sites, Ser-109 and Ser-140, lie in the N-terminal part of ABC50 but are not required for the binding of ABC50 to eIF2. Expression of a mutant of ABC50 in which both sites are mutated to alanine markedly decreased the association of eIF2 with 80S ribosomal and polysomal fractions.
Phorbol ester and hydrogen peroxide synergistically induce the interaction of diacylglycerol kinase γ with the Src homology 2 and C1 domains of β2-chimaerin
Authors: Yasuda Satoshi , Kai Masahiro , Imai Shin-ichi , Kanoh Hideo , Sakane Fumio
Published: December 2007

DGKγ (diacylglycerol kinase γ) was reported to interact with β2-chimaerin, a GAP (GTPase-activating protein) for Rac, in response to epidermal growth factor. Here we found that PMA and H2O2 also induced the interaction of DGKγ with β2-chimaerin. It is noteworthy that simultaneous addition of PMA and H2O2 synergistically enhanced the interaction. In this case, PMA was replaceable by DAG (diacylglycerol). The β2-chimaerin translocation from the cytoplasm to the plasma membrane caused by PMA plus H2O2 was further enhanced by the expression of DGKγ. Moreover, DGKγ apparently enhanced the β2-chimaerin GAP activity upon cell stimulation with PMA. PMA was found to be mainly required for a conversion of β2-chimaerin into an active form. On the other hand, H2O2 was suggested to induce a release of Zn2+ from the C1 domain of β2-chimaerin. By stepwise deletion analysis, we demonstrated that the SH2 (Src homology 2) and C1 domains of β2-chimaerin interacted with the N-terminal half of catalytic region of DGKγ. Unexpectedly, the SH2 domain of β2-chimaerin contributes to the interaction independently of phosphotyrosine. Taken together, these results suggest that the functional link between DGKγ and β2-chimaerin has a broad significance in response to a wide range of cell stimuli. Our work offers a novel mechanism of protein–protein interaction, that is, the phosphotyrosine-independent interaction of the SH2 domain acting in co-operation with the C1 domain.
Leber hereditary optic neuropathy mutations in the ND6 subunit of mitochondrial complex I affect ubiquinone reduction kinetics in a bacterial model of the enzyme
Authors: Pätsi Jukka , Kervinen Marko , Finel Moshe , Hassinen Ilmo E.
Published: December 2007

LHON (Leber hereditary optic neuropathy) is a maternally inherited disease that leads to sudden loss of central vision at a young age. There are three common primary LHON mutations, occurring at positions 3460, 11778 and 14484 in the human mtDNA (mitochondrial DNA), leading to amino acid substitutions in mitochondrial complex I subunits ND1, ND4 and ND6 respectively. We have now examined the effects of ND6 mutations on the function of complex I using the homologous NuoJ subunit of Escherichia coli NDH-1 (NADH:quinone oxidoreductase) as a model system. The assembly level of the NDH-1 mutants was assessed using electron transfer from deamino-NADH to the ‘shortcut’ electron acceptor HAR (hexammine ruthenium), whereas ubiquinone reductase activity was determined using DB (decylubiquinone) as a substrate. Mutant growth in minimal medium with malate as the main carbon source was used for initial screening of the efficiency of energy conservation by NDH-1. The results indicated that NuoJ-M64V, the equivalent of the common LHON mutation in ND6, had a mild effect on E. coli NDH-1 activity, while nearby mutations, particularly NuoJ-Y59F, NuoJ-V65G and NuoJ-M72V, severely impaired the DB reduction rate and cell growth on malate. NuoJ-Met64 and NuoJ-Met72 position mutants lowered the affinity of NDH-1 for DB and explicit C-type inhibitors, whereas NuoJ-Y59C displayed substrate inhibition by oxidized DB. The results are compatible with the notion that the ND6 subunit delineates the binding cavity of ubiquinone substrate, but does not directly take part in the catalytic reaction. How these changes in the enzyme's catalytic properties contribute to LHON pathogenesis is discussed.
Characterization of the interaction between the human DNA topoisomerase IIβ-binding protein 1 (TopBP1) and the cell division cycle 45 (Cdc45) protein
Authors: Schmidt Uta , Wollmann Yvonne , Franke Claudia , Grosse Frank , Saluz Hans-Peter , Hänel Frank
Published: December 2007

TopBP1 (topoisomerase IIβ-binding protein 1) is a BRCT [BRCA1 (breast-cancer susceptibility gene 1) C-terminal]-domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and DNA damage signalling. Experiments with fission yeast and Xenopus revealed that the TopBP1 homologues of these organisms are required for chromatin loading of the replication protein Cdc45 (cell division cycle 45). To improve our understanding of the physiological functions of human TopBP1, we investigated the interplay between human TopBP1 and Cdc45 proteins in synchronized HeLa-S3 cells. Using GST (glutathione transferase) pull-down and co-immunoprecipitation techniques, we showed a direct interaction between TopBP1 and Cdc45 in vitro and in vivo. The use of deletion mutants in GST pull-down assays identified the first and second as well as the sixth BRCT domains of TopBP1 to be responsible for the functional interaction with Cdc45. Moreover, the interaction between Cdc45 and the first and second BRCT domains of TopBP1 inhibited their transcriptional activation both in yeast and mammalian one-hybrid systems. Both proteins interacted exclusively at the G1/S boundary of cell cycle; only weak interaction could be found at the G2/M boundary. The overexpression of the sixth BRCT domain led to diminished loading of Cdc45 on to chromatin. These results suggest that human TopBP1 is involved in the formation of the initiation complex of replication in human cells and is required for the recruitment of Cdc45 to origins of DNA replication.
Lactococcus lactisas expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins
Authors: El Khattabi Mohamed , van Roosmalen Maarten L. , Jager Dennis , Metselaar Heidi , Permentier Hjalmar , Leenhouts Kees , Broos Jaap
Published: December 2007

Incorporation of Trp (tryptophan) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorpor-ation of such analogues into proteins is of considerable importance. The Gram-negative Escherichia coli is the only prokaryotic expression host regularly used for the incorporation of Trp analogues into recombinant proteins. Here, we present the use of the versatile Gram-positive expression host Lactococcus lactis for the incorporation of Trp analogues. The availability of a tightly regulated expression system for this organism, the potential to secrete modified proteins into the growth medium and the construction of the trp-synthetase deletion strain PA1002 of L. lactis rendered this organism potentially an efficient tool for the incorporation of Trp analogues into recombinant proteins. The Trp analogues 7-azatryptophan, 5-fluorotryptophan and 5-hydroxytryptophan were incorporated with efficiencies of >97, >97 and 89% respectively. Interestingly, 5-methylTrp (5-methyltryptophan) could be incorporated with 92% efficiency. Successful biosynthetical incorporation of 5-methylTrp into recombinant proteins has not been reported previously.
Uncoupling protein-2 contributes significantly to high mitochondrial proton leak in INS-1E insulinoma cells and attenuates glucose-stimulated insulin secretion
Authors: Affourtit Charles , Brand Martin D.
Published: December 2007

Proton leak exerts stronger control over ATP/ADP in mitochondria from clonal pancreatic β-cells (INS-1E) than in those from rat skeletal muscle, due to the higher proton conductance of INS-1E mitochondria [Affourtit and Brand (2006) Biochem. J. 393, 151–159]. In the present study, we demonstrate that high proton leak manifests itself at the cellular level too: the leak rate (measured as myxothiazol-sensitive, oligomycin-resistant respiration) was nearly four times higher in INS-1E cells than in myoblasts. This relatively high leak activity was decreased more than 30% upon knock-down of UCP2 (uncoupling protein-2) by RNAi (RNA interference). The high contribution of UCP2 to leak suggests that proton conductance through UCP2 accounts for approx. 20% of INS-1E respiration. UCP2 knock-down enhanced GSIS (glucose-stimulated insulin secretion), consistent with a role for UCP2 in β-cell physiology. We propose that the high mitochondrial proton leak in β-cells is a mechanism which amplifies the effect of physiological UCP2 regulators on cytoplasmic ATP/ADP and hence on insulin secretion.
Selective inhibition of binding of Bacillus thuringiensis Cry1Ab toxin to cadherin-like and aminopeptidase proteins in brush-border membranes and dissociated epithelial cells from Bombyx mori
Authors: Ibiza-Palacios M. Sales , Ferré Juan , Higurashi Satoshi , Miyamoto Kazuhisa , Sato Ryoichi , Escriche Baltasar
Published: December 2007

Binding analyses with denatured epithelial membrane proteins from Bt (Bacillus thuringiensis) demonstrated at least two kinds of proteins, APNs (aminopeptidases N) and cadherin-like proteins, as possible receptors for the Cry1A class of Bt toxins. Two alternative models have been proposed, both based on initial toxin binding to a cadherin-like protein, but one involving APN and the other not. We have used two Bombyx mori strains (J65 and Kin), which are highly susceptible to Cry1Ab, to study the role of these two types of receptors on Cry1Ab toxin binding and cytotoxicity by means of the inhibitory effect of antibodies. BBMVs (brush-border membrane vesicles) of strain J65 incubated with labelled 125I-Cry1Ab revealed a marked reduction in reversible and irreversible binding when anti-BtR175 (a cadherin-like protein) was used for BBMV pre-treatment. By contrast, the anti-APN1 antibody specifically affected the irreversible binding, while the reversible binding component was not affected. This is the first time that binding of Cry1Ab to APN1 and to a cadherin-like protein from BBMVs in solution has been shown. Dissociated epithelial cells from the Kin strain were used to test the inhibitory effect of the antibodies on the cytotoxicity of Cry1Ab. Pre-incubation of the cells with the anti-BtR175 antibody conferred protection against Cry1Ab, but not the anti-APN1 antibody. Therefore our results seem to support the two models of the mode of action of Cry1Ab in Lepidoptera, depending on whether BBMVs or intact dissociated cells are used, suggesting that both pathways may co-operate for the toxicity of Cry1A toxins in vivo.
Characterization of a new family of protein kinases from Arabidopsis containing phosphoinositide 3/4-kinase and ubiquitin-like domains
Authors: Galvão Rafaelo M. , Kota Uma , Soderblom Erik J. , Goshe Michael B. , Boss Wendy F.
Published: December 2007

At least two of the genes predicted to encode type II PI4K (phosphoinositide 4-kinase) in Arabidopsis thaliana (thale cress), namely AtPI4Kγ4 and AtPI4Kγ7, encode enzymes with catalytic properties similar to those of members of the PIKK (phosphoinositide kinase-related kinase) family. AtPI4Kγ4 and AtPI4Kγ7 undergo autophosphorylation and phosphorylate serine/threonine residues of protein substrates, but have no detectable lipid kinase activity. AtPI4Kγ4 and AtPI4Kγ7 are members of a subset of five putative AtPI4Ks that contain N-terminal UBL (ubiquitin-like) domains. In vitro analysis of AtPI4Kγ4 indicates that it interacts directly with, and phosphorylates, two proteins involved in the ubiquitin–proteasome system, namely UFD1 (ubiquitin fusion degradation 1) and RPN10 (regulatory particle non-ATPase 10). On the basis of the present results, we propose that AtPI4Kγ4 and AtPI4Kγ7 should be designated UbDKγ4 and UbDKγ7 (ubiquitin-like domain kinases γ4 and γ7). These UBL-domain-containing AtPI4Ks correspond to a new PIKK subfamily of protein kinases. Furthermore, UFD1 and RPN10 phosphorylation represents an additional mechanism by which their function can be regulated.
Kinetic and biochemical characterization ofPlasmodium falciparumGMP synthetase
Authors: Bhat Javaid Yousuf , Shastri Brahmanaspati Ganapathi , Balaram Hemalatha
Published: December 2007

Plasmodium falciparum, the causative agent of the fatal form of malaria, synthesizes GMP primarily from IMP and, hence, needs active GMPS (GMP synthetase) for its survival. GMPS, a G-type amidotransferase, catalyses the amination of XMP to GMP with the reaction occurring in two domains, the GAT (glutamine amidotransferase) and ATPPase (ATP pyrophosphatase). The GAT domain hydrolyses glutamine to glutamate and ammonia, while the ATPPase domain catalyses the formation of the intermediate AMP-XMP from ATP and XMP. Co-ordination of activity across the two domains, achieved through channelling of ammonia from GAT to the effector domain, is the hallmark of amidotransferases. Our studies aimed at understanding the kinetic mechanism of PfGMPS (Plasmodium falciparum GMPS) indicated steady-state ordered binding of ATP followed by XMP to the ATPPase domain with glutamine binding in a random manner to the GAT domain. We attribute the irreversible, Ping Pong step seen in initial velocity kinetics to the release of glutamate before the attack of the adenyl-XMP intermediate by ammonia. Specific aspects of the overall kinetic mechanism of PfGMPS are different from that reported for the human and Escherichia coli enzymes. Unlike human GMPS, absence of tight co-ordination of activity across the two domains was evident in the parasite enzyme. Variations seen in the inhibition by nucleosides and nucleotide analogues between human GMPS and PfGMPS highlighted differences in ligand specificity that could serve as a basis for the design of specific inhibitors. The present study represents the first report on recombinant His-tagged GMPS from parasitic protozoa.
Voltammetric characterization of the aerobic energy-dissipating nitrate reductase of Paracoccus pantotrophus: exploring the activity of a redox-balancing enzyme as a function of electrochemical potential
Authors: Gates Andrew J. , Richardson David J. , Butt Julea N.
Published: December 2007

Paracoccus pantotrophus expresses two nitrate reductases associated with respiratory electron transport, termed NapABC and NarGHI. Both enzymes derive electrons from ubiquinol to reduce nitrate to nitrite. However, while NarGHI harnesses the energy of the quinol/nitrate couple to generate a transmembrane proton gradient, NapABC dissipates the energy associated with these reducing equivalents. In the present paper we explore the nitrate reductase activity of purified NapAB as a function of electrochemical potential, substrate concentration and pH using protein film voltammetry. Nitrate reduction by NapAB is shown to occur at potentials below approx. 0.1 V at pH 7. These are lower potentials than required for NarGH nitrate reduction. The potentials required for Nap nitrate reduction are also likely to require ubiquinol/ubiquinone ratios higher than are needed to activate the H+-pumping oxidases expressed during aerobic growth where Nap levels are maximal. Thus the operational potentials of P. pantotrophus NapAB are consistent with a productive role in redox balancing. A Michaelis constant (KM) of approx. 45 μM was determined for NapAB nitrate reduction at pH 7. This is in line with studies on intact cells where nitrate reduction by Nap was described by a Monod constant (KS) of less than 15 μM. The voltammetric studies also disclosed maximal NapAB activity in a narrow window of potential. This behaviour is resistant to change of pH, nitrate concentration and inhibitor concentration and its possible mechanistic origins are discussed.
Mitochondria and cellular oxygen sensing in the HIF pathway
Authors: Taylor Cormac T.
Published: December 2007

Mitochondrial respiration is responsible for more than 90% of oxygen consumption in humans. Cells utilize oxygen as the final electron acceptor in the aerobic metabolism of glucose to generate ATP which fuels most active cellular processes. Consequently, a drop in tissue oxygen levels to the point where oxygen demand exceeds supply (termed hypoxia) leads rapidly to metabolic crisis and represents a severe threat to ongoing physiological function and ultimately, viability. Because of the central role of oxygen in metabolism, it is perhaps not surprising that we have evolved an efficient and rapid molecular response system which senses hypoxia in cells, leading to the induction of an array of adaptive genes which facilitate increased oxygen supply and support anaerobic ATP generation. This response is governed by HIF (hypoxia-inducible factor). The oxygen sensitivity of this pathway is conferred by a family of hydroxylases which repress HIF activity in normoxia allowing its rapid activation in hypoxia. Because of its importance in a diverse range of disease states, the mechanism by which cells sense hypoxia and transduce a signal to the HIF pathway is an area of intense investigation. Inhibition of mitochondrial function reverses hypoxia-induced HIF leading to speculation of a role for mitochondria in cellular oxygen sensing. However, the nature of the signal between mitochondria and oxygen-sensing hydroxylase enzymes has remained controversial. In the present review, two models of the role for mitochondria in oxygen sensing will be discussed and recent evidence will be presented which raises the possibility that these two models which implicate ROS (reactive oxygen species) and oxygen redistribution respectively may complement each other and facilitate rapid and dynamic activation of the HIF pathway in hypoxia.
Atrial natriuretic peptide gene promoter polymorphism is associated with left ventricular hypertrophy in hypertension
Authors: Xue Hao , Wang Shuxia , Wang Hu , Sun Kai , Song Xiaodong , Zhang Weili , Fu Chunyan , Han Yunfeng , Hui Rutai
Published: December 2007

Recent studies suggest that the ANP (atrial natriuretic peptide)/NPRA (type A natriuretic peptide receptor) system modulates ventricular remodelling and cardiac hypertrophy in hypertension in Western populations. In the present study, we tested for any association between two SNPs (single nucleotide polymorphisms) in the ANP gene (one in the promoter and one exonic) with cardiac hypertrophy. We tested the hypothesis in 2118 hypertensive patients, including 945 with LVH [LV (left ventricular) hypertrophy] and 1173 without LVH, as well as 816 healthy control subjects. All subjects were genotyped for the −A2843G and A188G polymorphisms. We found that the GG genotype at position −2843 conferred a 2.2-fold risk for LVH compared with the AA or AG genotypes, including septal wall thickness (11.8±1.4 mm for GG compared with 10.9±1.4 and 10.7±1.3 mm for AA and AG respectively; P<0.01), posterior wall thickness (11.8±2.8 mm for GG compared with 10.6±1.2 and 10.6±1.4 mm for AA and AG respectively; P<0.01), LV mass index (62.7±13.6 g/m2.7 for GG compared with 57.9±8.6 and 57.8±8.4 g/m2.7 for AA and AG respectively; P<0.05) and relative wall thickness (50.7±10.8% for GG compared with 44.3±7.3 and 43.5±6.8% for AA and AG respectively; P<0.05). Plasma levels of ANP were significantly lower in the hypertensive patients with LVH carrying the GG genotypes compared with those carrying the AA or AG genotypes (P<0.01). No association of GG genotype with echocardiographic variables and plasma ANP levels was identified in hypertensive patients without LVH and in control subjects (P>0.05). No significant association between the A188G genotype and echocardiographic variables was found in either hypertensive patients or controls (P>0.05). In conclusion, our findings indicate that the −A2843G polymorphism in the ANP gene promoter might be a genetic risk factor for the development of LVH in patients with hypertension.
Arterial stiffness and haemodynamic response to vasoactive medication in subjects with insulin-resistance syndrome
Authors: Brillante Divina G. , O'Sullivan Anthony J. , Johnstone Martina T. , Howes Laurence G.
Published: December 2007

INSR (insulin-resistance syndrome) affects 25% of the Australian population and is associated with increased cardiovascular risk. In the present study, we postulated that early cardiovascular changes in these individuals may be associated with an activated RAS (renin–angiotensin system). We studied 26 subjects: 13 with INSR [waist circumference, 99±6 cm; HOMA (homoeostasis model assessment) score, 2.5±0.3] and 13 NCs (normals controls; waist circumference, 77±2 cm; HOMA score, 1.4±0.2). All received intravenous GTN (glyceryl trinitrate; 10, 20 and 40 μg/min), L-NMMA (NG-monomethyl-L-arginine; 3 mg/kg of body weight), AngII (angiotensin II; 8 and 16 ng/min), the selective AT2R (AngII type 2 receptor) inhibitor PD123319 (10 and 20 μg/min) and AngII (16 ng/min)+PD123319 (20 μg/min). At the end of each infusion, arterial stiffness indices [SI (stiffness index) and RI (reflection index)] and haemodynamic parameters were measured. There was a significantly higher RI response to AngII (P=0.0004 for both 8 and 16 ng/min doses) and to PD123319 (P=0.004 and P=0.03 for 10 and 20 μg/min doses respectively) in subjects with INSR compared with NCs. Co-infusion of AngII and PD123319 did not lead to additive changes in RI. RI responses to L-NMMA and GTN were not significantly different in both groups. No significant differences in SI and haemodynamic responses were detected. In conclusion, AT1R (AngII type 1 receptor) and AT2R activity produce arterial stiffness changes in subjects with INSR. Evidence of increased AT1R- and AT2R-mediated responses in small-to-medium-sized arteries in INSR was found, and may play an early role in the pathogenesis of vascular changes in INSR before haemodynamic changes become apparent.
Endothelin, sex and hypertension
Authors: Tostes Rita C. , Fortes Zuleica B. , Callera Glaucia E. , Montezano Augusto C. , Touyz Rhian M. , Webb R. Clinton , Carvalho Maria Helena C.
Published: December 2007

The ETs (endothelins) comprise a family of three 21-amino-acid peptides (ET-1, ET-2 and ET-3) and 31-amino-acid ETs (ET-11–31, ET-21–31 and ET-31–31). ET-1 is synthesized from a biologically inactive precursor, big ET-1, by ECEs (ET-converting enzymes). The actions of ET-1 are mediated through activation of the G-protein-coupled ETA and ETB receptors, which are found in a variety of cells in the cardiovascular and renal systems. ET-1 has potent vasoconstrictor, mitogenic, pro-inflammatory and antinatriuretic properties, which have been implicated in the pathophysiology of a number of cardiovascular diseases. Overexpression of ET-1 has been consistently described in salt-sensitive models of hypertension and in models of renal failure, and has been associated with disease progression. Sex differences are observed in many aspects of mammalian cardiovascular function and pathology. Hypertension, as well as other cardiovascular diseases, is more common in men than in women of similar age. In experimental models of hypertension, males develop an earlier and more severe form of hypertension than do females. Although the reasons for these differences are not well established, the effects of gonadal hormones on arterial, neural and renal mechanisms that control blood pressure are considered contributing factors. Sex differences in the ET-1 pathway, with males displaying higher ET-1 levels, greater ET-1-mediated vasoconstrictor and enhanced pressor responses in comparison with females, are addressed in the present review. Sex-associated differences in the number and function of ETB receptors appear to be particularly important in the specific characteristics of hypertension between females and males. Although the gonadal hormones modulate some of the differences in the ET pathway in the cardiovascular system, a better understanding of the exact mechanisms involved in sex-related differences in this peptidergic system is needed. With further insights into these differences, we may learn that men and women could require different antihypertensive regimens.
Use of multiple fluorophores for evaluating microvascular permeability in control rats and rats with sepsis
Authors: Assaly Ragheb A. , Habib Robert H. , Azizi Mustafa , Shapiro Joseph I. , Dignam J. David
Published: December 2007

Capillary leak accompanying systemic inflammatory response conditions is a significant clinical problem. In the present study, we describe and verify a method for studying capillary leak that is based on the injection of proteins that differ significantly in size and have spectrally distinguishable fluorophores. Control (n=11) and post-CLP (caecal ligation and puncture; n=14) Sprague–Dawley rats were injected with tracer amounts of albumin and PEG–Alb [albumin covalently linked to methoxy-poly(ethylene glycol)] labelled with fluorescein and Texas Red. Blood samples were withdrawn between 5 min and 144 h, and the fluorescence of the labelled proteins was determined. The relative retention of the PEG–Alb and albumin was assessed via measurement of the TER (transcapillary escape rate; in %/h) and the t50% estimate, defined as the time when the actual concentration reached 50% of its baseline. The concentration–time trends for both albumin and PEG–Alb tracers exhibited two-compartmental behaviour and were analysed using bi-exponential modelling. Retention times were significantly greater for PEG–Alb in both control and CLP rats. TERPEG-Alb was significantly lower than TERalbumin for both control (8.1±5.6 compared with 14.8±7.1 %/h respectively; P<0.01) and CLP (14.8±6.6 compared with 22.5±7.3 %/h respectively; P<0.001) rats. The t50%[PEG–Alb] was substantially greater than the corresponding t50%[albumin] for both control (29.8±9.8 compared with 7.2±2.0 h respectively; P<0.001) and CLP (12.9±5.6 compared with 5.1±1.6 h respectively; P<0.001) rats. The result was similar irrespective of the fluorophore–protein combination, validating the multifluorophore technique. In conclusion, the double-fluorophore approach described in the present study may provide the future basis for a method to quantify capillary leak in disease.
Impairment of respiratory muscle function in pulmonary hypertension
Authors: Kabitz Hans-Joachim , Schwoerer Anja , Bremer Hinrich-Cordt , Sonntag Florian , Walterspacher Stephan , Walker David , Schaefer Vanessa , Ehlken Nicola , Staehler Gerd , Halank Michael , Klose Hans , Ghofrani Hossein A. , Hoeper Marius M. , Gruenig Ekkehard , Windisch Wolfram
Published: December 2007

It has been suggested that impaired respiratory muscle function occurs in patients with PH (pulmonary hypertension); however, comprehensive investigations of respiratory muscle function, including the application of non-volitional tests, needed to verify impairment of respiratory muscle strength in patients with PH have not yet been performed. In the present study, respiratory muscle function was assessed in 31 patients with PH (20 females and 11 males; mean pulmonary artery pressure, 51±20 mmHg; median World Health Organization class 3.0±0.5; 25 patients with pulmonary arterial hypertension and six patients with chronic thromboembolic PH) and in 31 control subjects (20 females and 11 males) well-matched for gender, age and BMI (body mass index). A 6-min walking test was performed to determine exercise capacity. Volitionally assessed maximal inspiratory (7.5±2.1 compared with 6.2±2.8 kPa; P=0.04) and expiratory (13.3±4.2 compared with 9.9±3.4 kPa; P<0.001) mouth pressures, sniff nasal (8.3±1.9 compared with 6.6±2.2 kPa; P=0.002) and transdiaphragmatic (11.3±2.5 compared with 8.7±2.5 kPa; P<0.001) pressures, non-volitionally assessed twitch mouth (1.46±0.43 compared with 0.97±0.41 kPa; P<0.001) and transdiaphragmatic (2.08±0.55 compared with 1.47±0.72 kPa; P=0.001) pressures during bilateral anterior magnetic phrenic nerve stimulation were markedly lower in patients with PH compared with control subjects. Maximal inspiratory mouth (r=0.58, P<0.001) and sniff transdiaphragmatic (r=0.43, P=0.02) pressures were correlated with the 6-min walking distance in patients with PH. In conclusion, the present study provides strong evidence that respiratory muscle strength is reduced in patients with PH compared with well-matched control subjects. Furthermore, the 6-min walking distance is significantly linked to parameters assessing inspiratory muscle strength.
Non-HDL cholesterol and apoB in dyslipidaemia
Authors: Sniderman Allan D. , Hogue Jean-Charles , Bergeron Jean , Gagné Claude , Couture Patrick
Published: December 2007

On the basis of a high correlation, non-HDL-C (non-high-density lipoprotein cholesterol) and apoB (apolipoprotein B) have been suggested to be of equivalent value for clinical practice; however, the strength of this relationship has not been examined in detail in patients with dyslipidaemia. The present study examines the variance of non-HDL-C compared with apoB in 1771 consecutive patients evaluated in a lipid clinic. These patients were divided into normolipidaemic subjects (n=407), type I hyperlipoproteinaemia (n=16), type IIa (n=736) and IIb (n=231) hyperlipoproteinaemia, type III hyperlipoproteinaemia (n=38), type IV hyperlipoproteinaemia (n=509) and type V hyperlipoproteinaemia (n=101). The relationship between non-HDL-C and apoB was examined both in terms of correlation and concordance. Correlation was high, but concordance was only moderate in the normolipidaemic subjects and in those with type IIa and type IIb hyperlipoproteinaemia. Correlation and concordance were both low in the subgroups with type III and type V hyperlipoproteinaemia. In those with type IV hyper-lipoproteinaemia, correlation was moderately high (r=0.74), but concordance was only fair. In conclusion, our results indicate that there is substantial variance of apoB for given values of non-HDL-C in many dyslipidaemic subjects. It follows that correlation is not adequate as a sole judge of equivalence of laboratory parameters.
Study on the interaction between tetracene and Cu(110) surface
Authors: Dou Weidong , Li Nana , Guan Dandan , Song Fei , Huang Han , Zhang Hanjie , Li Haiyang , He Pimo , Bao Shining , Chen Qiao , Zhou Wuzong
Published: December 2007

The electronic structure of tetracene on Cu (110) surface has been studied by using ultraviolet photoemission spectroscopy (UPS). The emission features from the organic molecule are located from 1to10eV below the Fermi level, and they shift in binding energy with increasing the coverage of the organic material. For the surface with multilayer of tetracene, six well-resolved features were found at 1.90, 3.40, 4.70, 5.95, 6.95, and 9.15eV below the Fermi level, respectively. On the surface with a lower coverage of tetracene, angle-resolved UPS measurements suggest that the molecular plane is parallel to the substrate. Density functional theory calculation confirms the flat-lying adsorption mode and shows that the tetracene molecule prefers to be adsorbed on the long bridge site with its long axis in the [11¯0] azimuth.
Simple model for transport phenomena: Microscopic construction of Maxwell demonlike engine
Authors: Chaudhuri Jyotipratim Ray , Chattopadhyay Sudip , Banik Suman Kumar
Published: December 2007

We present a microscopic Hamiltonian framework to develop Maxwell demonlike engine. Our model consists of an equilibrium thermal bath and a nonequilibrium bath, latter generated by driving with an external stationary, Gaussian noise. The engine we develop can be considered as a device to extract work by modifying internal fluctuations. Our theoretical analysis focuses on finding the essential ingredients necessary for generating fluctuation induced transport under nonequilibrium condition. An important outcome of our model is that the net motion occurs when the nonlinear bath is modulated by the external noise, creating the nonzero effective temperature even when the temperature of both the baths are the same.
The radiative lifetime of metastable CO (aΠ3, v=)
Authors: Gilijamse Joop J. , Hoekstra Steven , Meek Samuel A. , Metsälä Markus , van de Meerakker Sebastiaan Y. T. , Meijer Gerard , Groenenboom Gerrit C.
Published: December 2007

We present a combined experimental and theoretical study on the radiative lifetime of CO in the aΠ1,23, v=0 state. CO molecules in a beam are prepared in selected rotational levels of this metastable state, Stark-decelerated, and electrostatically trapped. From the phosphorescence decay in the trap, the radiative lifetime is measured to be 2.63±0.03ms for the aΠ13, v=0, J=1 level. From the spin-orbit coupling between the aΠ3 and the AΠ1 states a 20% longer radiative lifetime of 3.16ms is calculated for this level. It is concluded that coupling to other Π1 states contributes to the observed phosphorescence rate of metastable CO.
High-pressure phase equilibria with compressed gases
Authors: Ren Wei , Scurto Aaron M.
Published: December 2007

An apparatus is described that is capable of determining high-pressure vapor-liquid equilibrium, liquid-liquid equilibrium, solid-liquid-vapor equilibrium, vapor-liquid-liquid equilibrium, and mixture critical points and transitions. The device is capable of temperatures to 150°C and pressures to 300bars (higher with slight modifications). The construction and operation are described in detail and do not require the use of mercury. This method requires very low sample volumes and no analytical equipment nor system-specific calibration. The apparatus was verified by comparison with literature data for the decane-CO2 mixture and CO2-ionic liquid [1-hexyl-3-methyl-imidazolium bis(trifyl)imide)] systems. The experimental data have excellent agreement with the literature data that used different experimental methods. A rigorous error analysis of the system is also presented.
Structure, energetics, and bonding of amorphous Au–Si alloys
Authors: Lee Soo-Hwan , Hwang Gyeong S.
Published: December 2007

First principles periodic calculations based on gradient-corrected density functional theory have been performed to examine the structure, energetics, and bonding of amorphous Au–Si alloys with varying Au:Si composition ratios. Our results predict that the Au–Si alloy forms the most stable structure when the Si content is around 40–50at.%, with an energy gain of about 0.15eV/atom. In addition, the volume change per atom in the alloy exhibits a distinctive nonlinear trend, with the minimum value around 60at.% Si. The occurrence of the minimum in the Au–Si mixing energy and volume is attributed to strong hybridization of the Au 5d–Si 3p states. We also present variations in the radial distribution function and atomic coordination number as a function of Au:Si composition ratio, with discussion of the nature of local packing and chemical bonding in the Au–Si alloy system.
The dependence of the Tauc and Cody optical gaps associated with hydrogenated amorphous silicon on the film thickness: αl Experimental limitations and the impact of curvature in the Tauc and Cody plots
Authors: Mok Tat M. , O’Leary Stephen K.
Published: December 2007

Using a model for the optical spectrum associated with hydrogenated amorphous silicon, explicitly taking into account fundamental experimental limitations encountered, we theoretically determine the dependence of the Tauc and Cody optical gaps associated with hydrogenated amorphous silicon on the thickness of the film. We compare these results with that obtained from experiment. We find that the curvature in the Tauc plot plays a significant role in influencing the determination of the Tauc optical gap associated with hydrogenated amorphous silicon, thus affirming an earlier hypothesis of Cody et al. We also find that the spectral dependence of the refractive index plays an important role in influencing the determination of the Cody optical gap. It is thus clear that care must be exercised when drawing conclusions from the dependence of the Tauc and Cody optical gaps associated with hydrogenated amorphous silicon on the thickness of the film.
Oxidation of diamond films by atomic oxygen: High resolution electron energy loss spectroscopy studies
Authors: Shpilman Z. , Gouzman I. , Grossman E. , Akhvlediani R. , Hoffman A.
Published: December 2007

Diamond surface oxidation by atomic oxygen, annealing up to ∼700°C, and in situ exposure to thermally activated hydrogen were studied by high resolution electron energy loss spectroscopy (HREELS). After atomic oxygen (AO) exposure, HREELS revealed peaks associated with CHx groups, carbonyl, ether, and peroxide-type species and strong quenching of the diamond optical phonon and its overtones. Upon annealing of the oxidized surfaces, the diamond optical phonon overtones at 300 and 450meV emerge and carbonyl and peroxide species gradually desorb. The diamond surface was not completely regenerated after annealing to ∼700°C and in situ exposure to thermally activated hydrogen, probably due to the irreversible deterioration of the surface by AO.
A coherent nonlinear optical signal induced by electron correlations
Authors: Mukamel Shaul , Oszwałdowski Rafał , Yang Lijun
Published: December 2007

The correlated behavior of electrons determines the structure and optical properties of molecules, semiconductors, and other systems. Valuable information on these correlations is provided by measuring the response to femtosecond laser pulses, which probe the very short time period during which the excited particles remain correlated. The interpretation of four-wave-mixing techniques, commonly used to study the energy levels and dynamics of many-electron systems, is complicated by many competing effects and overlapping resonances. Here we propose a coherent optical technique, specifically designed to provide a background-free probe for electronic correlations in many-electron systems. The proposed signal pulse is generated only when the electrons are correlated, which gives rise to an extraordinary sensitivity. The peak pattern in two-dimensional plots, obtained by displaying the signal versus two frequencies conjugated to two pulse delays, provides a direct visualization and specific signatures of the many-electron wave functions.
Simultaneous correction of flat field and nonlinearity response of intensified charge-coupled devices
Authors: Williams Timothy C. , Shaddix Christopher R.
Published: December 2007

Intensified charge-coupled devices (ICCDs) are used extensively in many scientific and engineering environments to image weak or temporally short optical events. Care has to be taken in interpreting the images from ICCDs if quantitative results are required. In particular, nonuniform gain (flat field) and nonlinear response effects must be properly accounted for. Traditional flat-field corrections can only be applied in the linear regime of the ICCD camera, which limits the usable dynamic range. This paper reports a more general approach to image correction whereby the nonlinear gain response of each pixel of the ICCD is characterized over the full dynamic range of the camera. Image data can then be corrected for the combined effects of nonuniform gain and nonlinearity. The results from a two-color pyrometry measurement of soot field temperature are used to illustrate the capabilities of the new correction approach.