bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of the interaction between protein phosphatase 1 glycogen-targeting subunit and glycogen phosphorylase increases glycogen synthesis in primary rat hepatocytes
Authors: Zibrova Darya , Grempler Rolf , Streicher Rüdiger , Kauschke Stefan G.
Published: May 2008

In Type 2 diabetes, increased glycogenolysis contributes to the hyperglycaemic state, therefore the inhibition of GP (glycogen phosphorylase), a key glycogenolytic enzyme, is one of the possibilities to lower plasma glucose levels. Following this strategy, a number of GPis (GP inhibitors) have been described. However, certain critical issues are associated with their mode of action, e.g. an impairment of muscle function. The interaction between GP and the liver glycogen targeting subunit (termed GL) of PP1 (protein phosphatase 1) has emerged as a new potential anti-diabetic target, as the disruption of this interaction should increase glycogen synthesis, potentially providing an alternative approach to counteract the enhanced glycogenolysis without inhibiting GP activity. We identified an inhibitor of the GL–GP interaction (termed GL–GPi) and characterized its mechanism of action in comparison with direct GPis. In primary rat hepatocytes, at elevated glucose levels, the GL–GPi increased glycogen synthesis similarly to direct GPis. Direct GPis significantly reduced the cellular GP activity, caused a dephosphorylation of the enzyme and decreased the amounts of GP in the glycogen-enriched fraction; the GL–GPi did not influence any of these parameters. Both mechanisms increased glycogen accumulation at elevated glucose levels. However, at low glucose levels, only direct GPis led to increased glycogen amounts, whereas the GL–GPi allowed the mobilization of glycogen because it did not block the activity of GP. Due to this characteristic, GL–GPi in comparison with GPis could offer an advantageous risk/benefit profile circumventing the potential downsides of a complete prevention of glycogen breakdown while retaining glucose- lowering efficacy, suggesting that inhibition of the GL–GP interaction may provide an attractive novel approach for rebalancing the disturbed glycogen metabolism in diabetic patients.
Analysis of carboxyl tail function in the skeletal muscle Cl− channel hClC-1
Authors: Ma Linlin , Rychkov Grigori Y. , Hughes Bernard P. , Bretag Allan H.
Published: May 2008

Human ClC-1 (skeletal muscle Cl− channel) has a long cytoplasmic C-tail (carboxyl tail), containing two CBS (cystathionine β-synthase) domains, which is very important for channel function. We have now investigated its significance further, using deletion and alanine-scanning mutagenesis, split channels, GST (glutathione transferase)-pull-down and whole-cell patch-clamping. In tagged split-channel experiments, we have demonstrated strong binding between an N-terminal membrane-resident fragment (terminating mid-C-tail at Ser720 and containing CBS1) and its complement (containing CBS2). This interaction is not affected by deletion of some sequences, suggested previously to be important, particularly in channel gating. Contact between CBS1 and CBS2, however, may make a major contribution to assembly of functional channels from such co-expressed complements, although the possibility that C-tail fragments could, in addition, bind to other parts of the membrane-resident component has not been eliminated. We now show such an interaction between a membrane-resident component terminating at Ser720 (but with CBS1 deleted) and a complete C-tail beginning at Leu598. Channel function is rescued in patch-clamped HEK-293T (human embryonic kidney) cells co-expressing these same fragments. From our own results and those of others, we conclude that the CBS1–CBS2 interaction is not sufficient, in itself, for channel assembly, but rather that this might normally assist in bringing some part of the CBS2/C-tail region into appropriate proximity with the membrane-resident portion of the protein. Previously conflicting and anomalous results can now be explained by an hypothesis that, for split channels to be functional, at least one membrane-resident component must include a plasma membrane trafficking signal between Leu665 and Lys680.
Cep57, a multidomain protein with unique microtubule and centrosomal localization domains
Authors: Momotani Ko , Khromov Alexander S. , Miyake Tsuyoshi , Stukenberg P. Todd , Somlyo Avril V.
Published: May 2008

The present study demonstrates different functional domains of a recently described centrosomal protein, Cep57 (centrosomal protein 57). Endogenous Cep57 protein and ectopic expression of full-length protein or the N-terminal coiled-coil domain localize to the centrosome internal to γ-tubulin, suggesting that it is either on both centrioles or on a centromatrix component. The N-terminus can also multimerize with the N-terminus of other Cep57 molecules. The C-terminus contains a second coiled-coil domain that directly binds to MTs (microtubules). This domain both nucleates and bundles MTs in vitro. This activity was also seen in vivo, as overexpression of full-length Cep57 or the C-terminus generates nocodazole-resistant MT cables in cells. Based on the present findings, we propose that Cep57 serves as a link with its N-terminus anchored to the centriole or centromatrix and its C-terminus to MTs.
Analysis of endogenous Bax complexes during apoptosis using blue native PAGE: implications for Bax activation and oligomerization
Authors: Valentijn Anthony J. , Upton John-Paul , Gilmore Andrew P.
Published: May 2008

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.
Regulation of Ca2+/calmodulin kinase II by a small C-terminal domain phosphatase
Authors: Gangopadhyay Samudra S. , Gallant Cynthia , Sundberg Eric J. , Lane William S. , Morgan Kathleen G.
Published: May 2008

We present here the identification and characterization of an SCP3 (small C-terminal domain phosphatase-3) homologue in smooth muscle and show, for the first time, that it dephosphorylates CaMKII [Ca2+/CaM (calmodulin)-dependent protein kinase II]. SCP3 is a PP2C (protein phosphatase 2C)-type phosphatase that is primarily expressed in vascular smooth muscle tissues and specifically binds to the association domain of the CaMKIIγ G-2 variant. The dephosphorylation is site-specific, excluding the Thr287 associated with Ca2+/CaM-independent activation of the kinase. As a result, the autonomous activity of CaMKIIγ G-2 is not affected by the phosphatase activity of SCP3. SCP3 co-localizes with CaMKIIγ G-2 on cytoskeletal filaments, but is excluded from the nucleus in differentiated vascular smooth muscle cells. Upon depolarization-induced Ca2+ influx, CaMKIIγ G-2 is activated and dissociates from SCP3. Subsequently, CaMKIIγ G-2 is targeted to cortical adhesion plaques. We show here that SCP3 regulates phosphorylation sites in the catalytic domain, but not those involved in regulation of kinase activation. This selective dephosphorylation by SCP3 creates a constitutively active kinase that can then be differentially regulated by other phosphorylation-dependent regulatory mechanisms.
The role of double covalent flavin binding in chito-oligosaccharide oxidase from Fusarium graminearum
Authors: Heuts Dominic P. H. M. , Winter Remko T. , Damsma Gerke E. , Janssen Dick B. , Fraaije Marco W.
Published: May 2008

ChitO (chito-oligosaccharide oxidase) from Fusarium graminearum catalyses the regioselective oxidation of N-acetylated oligosaccharides. The enzyme harbours an FAD cofactor that is covalently attached to His94 and Cys154. The functional role of this unusual bi-covalent flavin–protein linkage was studied by site-directed mutagenesis. The double mutant (H94A/C154A) was not expressed, which suggests that a covalent flavin–protein bond is needed for protein stability. The single mutants H94A and C154A were expressed as FAD-containing enzymes in which one of the covalent FAD–protein bonds was disrupted relative to the wild-type enzyme. Both mutants were poorly active, as the kcat decreased (8.3- and 3-fold respectively) and the Km increased drastically (34- and 75-fold respectively) when using GlcNac as the substrate. Pre-steady-state analysis revealed that the rate of reduction in the mutant enzymes is decreased by 3 orders of magnitude when compared with wild-type ChitO (kred=750 s−1) and thereby limits the turnover rate. Spectroelectrochemical titrations revealed that wild-type ChitO exhibits a relatively high redox potential (+131 mV) and the C154A mutant displays a lower potential (+70 mV), while the H94A mutant displays a relatively high potential of approximately +164 mV. The results show that a high redox potential is not the only prerequisite to ensure efficient catalysis and that removal of either of the covalent bonds may perturb the geometry of the Michaelis complex. Besides tuning the redox properties, the bi-covalent binding of the FAD cofactor in ChitO is essential for a catalytically competent conformation of the active site.
Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applications in agriculture
Authors: Dirk Lynnette M. A. , Schmidt Jack J. , Cai Yiying , Barnes Jonathan C. , Hanger Katherine M. , Nayak Nihar R. , Williams Mark A. , Grossman Robert B. , Houtz Robert L. , Rodgers David W.
Published: May 2008

The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 Å (1 Å=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr178 as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr2 in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr178 to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr178 can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.
Hydrogen peroxide mediates the radiation-induced mutator phenotype in mammalian cells
Authors: Dayal Disha , Martin Sean M. , Limoli Charles L. , Spitz Douglas R.
Published: May 2008

Chronic oxidative stress has been associated with genomic instability following exposure to ionizing radiation. However, results showing direct causal linkages between specific ROS (reactive oxygen species) and the ionizing radiation-induced mutator phenotype are lacking. The present study demonstrates that ionizing radiation-induced genomically unstable cells (characterized by chromosomal instability and an increase in mutation and gene amplification frequencies) show a 3-fold increase in steady-state levels of hydrogen peroxide, but not superoxide. Furthermore, stable clones isolated from parallel studies showed significant increases in catalase and GPx (glutathione peroxidase) activity. Treatment of unstable cells with PEG-CAT (polyethylene glycol-conjugated catalase) reduced the mutation frequency and mutation rate in a dose-dependent fashion. In addition, inhibiting catalase activity in the stable clones using AT (3-aminotriazole) increased mutation frequency and rate. These results clearly demonstrate the causal relationship between chronic oxidative stress mediated by hydrogen peroxide and the mutator phenotype that persists for many generations following exposure of mammalian cells to ionizing radiation.
A cyclic peptidylic inhibitor of murine urokinase-type plasminogen activator: changing species specificity by substitution of a single residue
Authors: Andersen Lisbeth M. , Wind Troels , Hansen Hanne D. , Andreasen Peter A.
Published: May 2008

uPA (urokinase-type plasminogen activator) is a potential therapeutic target in a variety of pathological conditions, including cancer. In order to find new principles for inhibiting uPA in murine cancer models, we screened a phage-displayed peptide library with murine uPA as bait. We thereby isolated several murine uPA-binding peptide sequences, the predominant of which was the disulfide-bridged constrained sequence CPAYSRYLDC, which we will refer to as mupain-1. A chemically synthesized peptide corresponding to this sequence was found to be a competitive inhibitor of murine uPA, inhibiting its activity towards a low-molecular-mass chromogenic substrate as well as towards its natural substrate plasminogen. The Ki value for inhibition as well as the KD value for binding were approx. 400 nM. Among a variety of other murine and human serine proteases, including trypsin, mupain-1 was found to be highly selective for murine uPA and did not even measurably inhibit human uPA. The cyclic structure of mupain-1 was indispensable for binding. Alanine scanning mutagenesis identified Arg6 of mupain-1 as the P1 residue and indicated an extended binding interaction including the P5, P3, P2, P1 and P1′ residues of mupain-1 and the specificity pocket, the catalytic triad and amino acids 41, 99 and 192 located in and around the active site of murine uPA. Exchanging His99 of human uPA by a tyrosine residue, the corresponding residue in murine uPA, conferred mupain-1 susceptibility on to the latter. Peptide-derived inhibitors, such as mupain-1, may provide novel mechanistic information about enzyme–inhibitor interactions, provide alternative methodologies for designing effective protease inhibitors, and be used for target validation in murine model systems.
Critical role for cross-linking of trimeric lectin domains of surfactant protein D in antiviral activity against influenza A virus
Authors: Tecle Tesfaldet , White Mitchell R. , Sorensen Grith , Gantz Donald , Kacak Nilgun , Holmskov Uffe , Smith Kelly , Crouch Erika C. , Hartshorn Kevan L.
Published: May 2008

Collectins are multimeric host defence lectins with trimeric CRDs (carbohydrate-recognition domains) and collagen and N-terminal domains that form higher-order structures composed of four or more trimers. Recombinant trimers composed of only the CRD and adjacent neck domain (termed NCRD) retain binding activity for some ligands and mediate some functional activities. The lung collectin SP-D (surfactant protein D) has strong neutralizing activity for IAVs (influenza A viruses) in vitro and in vivo, however, the NCRD derived from SP-D has weak viral-binding ability and lacks neutralizing activity. Using a panel of mAbs (monoclonal antibodies) directed against the NCRD in the present study we show that mAbs binding near the lectin site inhibit antiviral activity of full-length SP-D, but mAbs which bind other sites on the CRD do not. Two of the non-blocking mAbs significantly increased binding and antiviral activity of NCRDs as assessed by haemagglutination and neuraminidase inhibition and by viral neutralization. mAb-mediated cross-linking also enabled NCRDs to induce viral aggregation and to increase viral uptake by neutrophils and virus-induced respiratory burst responses by these cells. These results show that antiviral activities of SP-D can be reproduced without the N-terminal and collagen domains and that cross-linking of NCRDs is essential for antiviral activity of SP-D with respect to IAV.
Conserved amphiphilic feature is essential for periplasmic chaperone HdeA to support acid resistance in enteric bacteria
Authors: Wu Ye E. , Hong Weizhe , Liu Chong , Zhang Lingqing , Chang Zengyi
Published: May 2008

The extremely acidic environment of the mammalian stomach (pH 1–3) represents a stressful challenge for enteric pathogenic bacteria, including Escherichia coli, Shigella and Brucella. The hdeA (hns-dependent expression A) gene was found to be crucial for the survival of these enteric bacteria under extremely low pH conditions. We recently demonstrated that HdeA is able to exhibit chaperone-like activity exclusively within the stomach pH range by transforming from a well-folded conformation at higher pH values (above pH 3) into an unfolded conformation at extremely low pH values (below pH 3). This study was performed to characterize the action mechanisms and underlying specific structural features for HdeA to function in this unfolded conformation. In the present study, we demonstrate that the conserved ‘amphiphilic’ feature of HdeA, i.e. the exposure of the conserved hydrophobic region and highly charged terminal regions, is essential for exhibiting chaperone-like activity under extremely low pH conditions. Mutations that disrupt this amphiphilic feature markedly reduced the chaperone-like activity of HdeA. The results also strongly suggest that this acid-induced chaperone-like activity of HdeA is crucial for acid resistance of the enteric bacteria. Moreover, our new understanding of this amphiphilic structural feature of HdeA helps to better interpret how this unfolded (disordered) conformation could be functionally active.
The intact CFTR protein mediates ATPase rather than adenylate kinase activity
Authors: Ramjeesingh Mohabir , Ugwu Francisca , Stratford Fiona L. L. , Huan Ling-Jun , Li Canhui , Bear Christine E.
Published: May 2008

The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain–domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.
Diverse roles for chloroplast stromal and thylakoid-bound ascorbate peroxidases in plant stress responses
Authors: Kangasjärvi Saijaliisa , Lepistö Anna , Hännikäinen Kati , Piippo Mirva , Luomala Eeva-Maria , Aro Eva-Mari , Rintamäki Eevi
Published: May 2008

Photosynthetic light reactions comprise a significant source of hydrogen peroxide (H2O2) in illuminated leaves. APXs (ascorbate peroxidases) reduce H2O2 to water and play an important role in the antioxidant system of plants. In the present study we addressed the significance of chloroplast APXs in stress tolerance and signalling in Arabidopsis thaliana. To this end, T-DNA (transfer DNA) insertion mutants tapx, sapx and tapx sapx, lacking the tAPX (thylakoid-bound APX), sAPX (stromal APX) or both respectively, were characterized. Photo-oxidative stress during germination led to bleaching of chloroplasts in sapx single-mutant and particularly in the tapx sapx double-mutant plants, whereas the greening process of wild-type and tapx plants was only partially impaired. Mature leaves of tapx sapx double mutants were also susceptible to short-term photo-oxidative stress induced by high light or methyl viologen treatments. After a 2-week acclimation period under high light or under low temperature, none of the mutants exhibited enhanced stress symptoms. Immunoblot analysis revealed that high-light-stress-acclimated tapx sapx double mutants compensated for the absence of tAPX and sAPX by increasing the level of 2-cysteine peroxiredoxin. Furthermore, the absence of tAPX and sAPX induced alterations in the transcriptomic profile of tapx sapx double-mutant plants already under quite optimal growth conditions. We conclude that sAPX is particularly important for photoprotection during the early greening process. In mature leaves, tAPX and sAPX are functionally redundant, and crucial upon sudden onset of oxidative stress. Moreover, chloroplast APXs contribute to chloroplast retrograde signalling pathways upon slight fluctuations in the accumulation of H2O2 in chloroplasts.
Proteomic analysis of polymeric salivary mucins: no evidence for MUC19 in human saliva
Authors: Rousseau Karine , Kirkham Sara , Johnson Lindsay , Fitzpatrick Brian , Howard Marj , Adams Emily J. , Rogers Duncan F. , Knight David , Clegg Peter , Thornton David J.
Published: May 2008

MUC5B is the predominant polymeric mucin in human saliva [Thornton, Khan, Mehrotra, Howard, Veerman, Packer and Sheehan (1999) Glycobiology 9, 293–302], where it contributes to oral cavity hydration and protection. More recently, the gene for another putative polymeric mucin, MUC19, has been shown to be expressed in human salivary glands [Chen, Zhao, Kalaslavadi, Hamati, Nehrke, Le, Ann and Wu (2004) Am. J. Respir. Cell Mol. Biol. 30, 155–165]. However, to date, the MUC19 mucin has not been isolated from human saliva. Our aim was therefore to purify and characterize the MUC19 glycoprotein from human saliva. Saliva was solubilized in 4 M guanidinium chloride and the high-density mucins were purified by density-gradient centrifugation. The presence of MUC19 was investigated using tandem MS of tryptic peptides derived from this mucin preparation. Using this approach, we found multiple MUC5B-derived tryptic peptides, but were unable to detect any putative MUC19 peptides. These results suggest that MUC19 is not a major component in human saliva. In contrast, using the same experimental approach, we identified Muc19 and Muc5b glycoproteins in horse saliva. Moreover, we also identified Muc19 from pig, cow and rat saliva; the saliva of cow and rat also contained Muc5b; however, due to the lack of pig Muc5b genomic sequence data, we were unable to identify Muc5b in pig saliva. Our results suggest that unlike human saliva, which contains MUC5B, cow, horse and rat saliva are a heterogeneous mixture of Muc5b and Muc19. The functional consequence of these species differences remains to be elucidated.
A functional link between housekeeping selenoproteins and phase II enzymes
Authors: Sengupta Aniruddha , Carlson Bradley A. , Weaver James A. , Novoselov Sergey V. , Fomenko Dmitri E. , Gladyshev Vadim N. , Hatfield Dolph L.
Published: May 2008

Sec (selenocysteine) is biosynthesized on its tRNA and incorporated into selenium-containing proteins (selenoproteins) as the 21st amino acid residue. Selenoprotein synthesis is dependent on Sec tRNA and the expression of this class of proteins can be modulated by altering Sec tRNA expression. The gene encoding Sec tRNA (Trsp) is a single-copy gene and its targeted removal in liver demonstrated that selenoproteins are essential for proper function wherein their absence leads to necrosis and hepatocellular degeneration. In the present study, we found that the complete loss of selenoproteins in liver was compensated for by an enhanced expression of several phase II response genes and their corresponding gene products. The replacement of selenoprotein synthesis in mice carrying mutant Trsp transgenes, wherein housekeeping, but not stress-related selenoproteins are expressed, led to normal expression of phase II response genes. Thus the present study provides evidence for a functional link between housekeeping selenoproteins and phase II enzymes.
Activation of the insulin receptor (IR) by insulin and a synthetic peptide has different effects on gene expression in IR-transfected L6 myoblasts
Authors: Jensen Maja , Palsgaard Jane , Borup Rehannah , de Meyts Pierre , Schäffer Lauge
Published: May 2008

Single-chain peptides have been recently produced that display either mimetic or antagonistic properties against the insulin and IGF-1 (insulin-like growth factor 1) receptors. We have shown previously that the insulin mimetic peptide S597 leads to significant differences in receptor activation and initiation of downstream signalling cascades despite similar binding affinity and in vivo hypoglycaemic potency. It is still unclear how two ligands can initiate different signalling responses through the IR (insulin receptor). To investigate further how the activation of the IR by insulin and S597 differentially activates post-receptor signalling, we studied the gene expression profile in response to IR activation by either insulin or S597 using microarray technology. We found striking differences between the patterns induced by these two ligands. Most remarkable was that almost half of the genes differentially regulated by insulin and S597 were involved in cell proliferation and growth. Insulin either selectively regulated the expression of these genes or was a more potent regulator. Furthermore, we found that half of the differentially regulated genes interact with the genes involved with the MAPK (mitogen-activated protein kinase) pathway. These findings support our signalling results obtained previously and confirm that the main difference between S597 and insulin stimulation resides in the activation of the MAPK pathway. In conclusion, we show that insulin and S597 acting via the same receptor differentially affect gene expression in cells, resulting in a different mitogenicity of the two ligands, a finding which has critical therapeutic implications.
Phosphorylation of eIF2α in response to 26S proteasome inhibition is mediated by the haem-regulated inhibitor (HRI) kinase
Authors: Yerlikaya Azmi , Kimball Scot R. , Stanley Bruce A.
Published: May 2008

The present study demonstrates that even brief inhibition of degradation by the 26S proteasome inhibits global protein synthesis, mediated through increased phosphorylation of eIF2α (eukaryotic translational initiation factor 2α) by the HRI (haem-regulated inhibitor) kinase. Exposure of COS-7 cells to the proteasome inhibitor MG-132 (the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-leucinal) for 4 h resulted in a 55–60% decrease in protein synthesis rate compared with control cells. This repression of protein synthesis after treatment with MG-132 is not due to induction of apoptosis, which is known to occur after longer periods of 26S inhibition. Instead, we observed a significantly increased phosphorylation of eIF2α, which is known to repress global protein synthesis. In three MEF (mouse embryonic fibroblast) knockout cell lines lacking one of the four kinases known to phosphorylate eIF2α, increased phosphorylation of eIF2α still occurred after inhibition of the 26S proteasome. These three cell lines included a deletion of the PKR (double-stranded-RNA-dependent protein kinase); a deletion of the PERK (PKR-like endoplasmic reticulum resident kinase); or a deletion of the GCN2 (positive general control of transcription-2) kinase, indicating that none of these kinases was primarily responsible for the observed phosphorylation of eIF2α. In contrast, in a fourth MEF knockout cell line, HRI−/− cells lacking the HRI kinase failed to increase eIF2α phosphorylation upon proteasome inhibitor treatment (MG-132 or various doses of Bortezomib), indicating that the HRI kinase is the primary kinase activated by brief treatment of MEFs with 26S proteasome inhibitors.
Redox state of quinone affects sensitivity of Acanthamoeba castellanii mitochondrial uncoupling protein to purine nucleotides
Authors: Swida Aleksandra , Woyda-Ploszczyca Andrzej , Jarmuszkiewicz Wieslawa
Published: May 2008

We studied FFA (free fatty acid)-induced uncoupling activity in Acanthamoeba castellanii mitochondria in the non-phosphorylating state. Either succinate or external NADH was used as a respiratory substrate to determine the proton conductance curves and the relationships between respiratory rate and the quinone reduction level. Our determinations of the membranous quinone reduction level in non-phosphorylating mitochondria show that activation of UCP (uncoupling protein) activity leads to a PN (purine nucleotide)-sensitive decrease in the quinone redox state. The gradual decrease in the rate of quinone-reducing pathways (using titration of dehydrogenase activities) progressively leads to a full inhibitory effect of GDP on LA (linoleic acid) induced proton conductance. This inhibition cannot be attributed to changes in the membrane potential. Indeed, the lack of GDP inhibitory effect observed when the decrease in respiratory rate is accompanied by an increase in the quinone reduction level (using titration of the quinol-oxidizing pathway) proves that the inhibition by nucleotides can be revealed only for a low quinone redox state. It must be underlined that, in A. castellanii non-phosphorylating mitochondria, the transition of the inhibitory effect of GDP on LA-induced UCP-mediated uncoupling is observed for the same range of quinone reduction levels (between 50% and 40%) as that observed previously for phosphorylating conditions. This observation, drawn from the two different metabolic states of mitochondria, indicates that quinone could affect UCP activity through sensitivity to PNs.
Important role of the LKB1–AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice
Authors: Huang Xu , Wullschleger Stephan , Shpiro Natalia , McGuire Victoria A. , Sakamoto Kei , Woods Yvonne L. , Mcburnie Wendy , Fleming Stewart , Alessi Dario R.
Published: May 2008

The LKB1 tumour suppressor phosphorylates and activates AMPK (AMP-activated protein kinase) when cellular energy levels are low, thereby suppressing growth through multiple pathways, including inhibiting the mTORC1 (mammalian target of rapamycin complex 1) kinase that is activated in the majority of human cancers. Blood glucose-lowering Type 2 diabetes drugs also induce LKB1 to activate AMPK, indicating that these compounds could be used to suppress growth of tumour cells. In the present study, we investigated the importance of the LKB1–AMPK pathway in regulating tumorigenesis in mice resulting from deficiency of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor, which drives cell growth through overactivation of the Akt and mTOR (mammalian target of rapamycin) kinases. We demonstrate that inhibition of AMPK resulting from a hypomorphic mutation that decreases LKB1 expression does not lead to tumorigenesis on its own, but markedly accelerates tumour development in PTEN+/− mice. In contrast, activating the AMPK pathway by administration of metformin, phenformin or A-769662 to PTEN+/− mice significantly delayed tumour onset. We demonstrate that LKB1 is required for activators of AMPK to inhibit mTORC1 signalling as well as cell growth in PTEN-deficient cells. Our findings highlight, using an animal model relevant to understanding human cancer, the vital role that the LKB1–AMPK pathway plays in suppressing tumorigenesis resulting from loss of the PTEN tumour suppressor. They also suggest that pharmacological inhibition of LKB1 and/or AMPK would be undesirable, at least for the treatment of cancers in which the mTORC1 pathway is activated. Most importantly, our results demonstrate the potential of AMPK activators, such as clinically approved metformin, as anticancer agents, which will suppress tumour development by triggering a physiological signalling pathway that potently inhibits cell growth.
Adult-type hypolactasia is not a predisposing factor for the early functional and structural changes of atherosclerosis: the Cardiovascular Risk in Young Finns Study
Authors: Lehtimäki Terho , Hutri-Kähönen Nina , Kähönen Mika , Hemminki Jukka , Mikkilä Vera , Laaksonen Marika , Räsänen Leena , Mononen Nina , Juonala Markus , Marniemi Jukka , Viikari Jorma , Raitakari Olli
Published: May 2008

Individuals suffering from ATH (adult-type hypolactasia), defined by the LCT (gene encoding lactase-phlorizin hydrolase) C/C−13910 genotype (rs4988235), use less milk and dairy products and may have higher plasma HDL (high-density lipoprotein) and lower triacylglycerol (triglyceride) concentrations than their counterparts without ATH. To investigate the effects of ATH status on the early markers of atherosclerosis, we examined its association with CIMT (carotid intima-media thickness), CAC (carotid artery compliance) and brachial artery FMD (flow-mediated dilation) in a young population-based cohort of otherwise healthy individuals. As part of the Cardiovascular Risk in Young Finns Study, we performed CIMT, CAC and FMD analyses, LCT C/T−13910 genotyping and risk factor determination in 2109 young subjects 24–39 years of age (45% males) at the time of the examination. The consumption of both milk and dairy products was lowest and the consumption of alcohol highest in subjects with the C/C−13910 genotype (P<0.001 for all) in comparison with subjects without ATH (TT+CT). In multivariate analysis, no significant association between ATH status and CIMT, CAC or brachial artery FMD was found after adjustment for the use of alcohol, dairy products and all other major risk factors of coronary artery disease. In otherwise similar statistical analysis, the results remained non-significant when females and males were analysed in their own groups. In conclusion, the finding does not support the involvement of ATH in the pathogenesis of early atherosclerosis.
Higher levels of collagen and facilitated healing protect against ventricular rupture following myocardial infarction
Authors: Fang Lu , Gao Xiao-Ming , Samuel Chrishan S. , Su Yidan , Lim Yean Leng , Dart Anthony M. , Du Xiao-Jun
Published: May 2008

The mechanism of cardiac rupture after MI (myocardial infarction) is not fully understood. Rupture has not been reported in most laboratory species, including the rat, but does occur in mice. We have reported previously that β2-TG mice (transgenic mice with cardiac-restricted overexpression of β2-adrenergic receptors) had a lower incidence of rupture compared with NTG (non-transgenic) littermates. We hypothesized that the difference in the incidence of rupture between rodents and specific mouse strains is due to the difference in collagen content following MI. In the present study, we compared the difference in matrix remodelling post-MI between β2-TG and NTG mice and between mice and rats. MI was induced by ligation of the left main coronary artery. Following MI, tensile strength, insoluble and soluble collagen content and gelatinase expression were determined in the infarcted and non-infarcted myocardium. Better preserved tensile strength measured as TTR [tension-to-rupture; 88±14 and 58±3% of the respective sham group values for β2-TG compared with NTG mice (P<0.05); 108±7 and 32±4% of the respective sham group values for rats compared with 129sv mice (P<0.01)] and less severe acute infarct expansion after MI were found in rats compared with mice or in β2-TG compared with NTG mice. These differences were associated with a higher content of pre-existing fibril collagen in the normal myocardium of β2-TG compared with NTG mice (1.6-fold) or rats compared with 129sv mice (2-fold) and an accelerated fibrotic healing in the infarcted myocardium. Additionally, a less pronounced increase in MMP-9 (matrix metalloproteinase-9) activity was observed in the infarcted myocardium of rats compared with 129sv mice. We conclude that a higher collagen level is associated with facilitated fibrotic healing of an infarct and preserves the tensile strength of infarcted myocardium, thereby preventing cardiac rupture and acute ventricular remodelling.
Local expression of Toll-like receptor 4 at the site of ruptured plaques in patients with acute myocardial infarction
Authors: Ishikawa Yuh , Satoh Mamoru , Itoh Tomonori , Minami Yoshitaka , Takahashi Yuji , Akamura Motoyuki
Published: May 2008

Several reports suggest that a chronic inflammatory process plays a key role in coronary artery plaque instability and subsequent occlusive thrombosis. In a previous study, we found that TLR4 (Toll-like receptor 4) mediates the synthesis of cytokines in circulating monocytes of patients with AMI (acute myocardial infarction); however, it remains unclear whether TLRs are expressed at the site of the ruptured plaque in these patients. The aim of the present study was to determine whether TLR2 and TLR4 are expressed at the site of ruptured plaques in patients with AMI and to compare this with systemic levels. The study included 62 patients with AMI, 20 patients with SA (stable angina) and 32 subjects with a normal coronary angiogram (control). Local samples from the site of the ruptured plaque were taken from patients with AMI using aspiration catheterization. Systemic blood samples from the aorta were taken from patients with AMI and SA and controls. Systemic levels of TLR2 and TLR4 were higher in patients with AMI than in patients with SA and controls. In patients with AMI, local TLR4 levels were higher than systemic levels. There was no significant difference in TLR2 levels between local and systemic samples. TLR4 immunostaining was positive in infiltrating macrophages in ruptured plaque material. Cardiac events were observed in 16 patients with AMI at the time of the 6-month follow-up study. Local and systemic levels of TLR4 were higher in patients with AMI with cardiac events than in those without. These results indicate an increase in monocytic TLR4 expression not only in the systemic circulation, but also at the site of plaque rupture. In conclusion, expression of both systemic and local plaque TLR4 may be one of the mechanisms responsible for the pathogenesis of AMI.
Circulating vascular endothelial growth factor and systemic inflammatory markers in patients with stable and exacerbated chronic obstructive pulmonary disease
Authors: Valipour Arschang , Schreder Martin , Wolzt Michael , Saliba Sleman , Kapiotis Sonja , Eickhoff Philipp , Burghuber Otto Chris
Published: May 2008

The aim of the present study was to assess circulating levels of VEGF (vascular endothelial growth factor), a biomarker with prognostic significance in cardiovascular disease, and markers of systemic inflammation in patients with stable and exacerbated COPD (chronic obstructive pulmonary disease). Lung function parameters, arterial blood gas analysis and circulating levels of VEGF, IL-6 (interleukin-6), TNF-α (tumour necrosis factor-α), CRP (C-reactive protein), fibrinogen and the peripheral blood neutrophil cell count were assessed in 30 patients on admission to the hospital for acute exacerbation of COPD, in 30 age-, gender- and BMI (body mass index)-matched patients with stable COPD, and 30 matched controls with normal lung function. Patients with acute exacerbated COPD had higher circulating concentrations of VEGF (P<0.001), IL-6 (P<0.05) and CRP (P<0.01) and an increased blood neutrophil cell count (P<0.05) compared with patients with stable COPD and healthy controls. VEGF levels in exacerbated COPD correlated with systemic inflammatory markers, such as CRP (r=0.61, P<0.005), IL-6 (r=0.46; P<0.01) and fibrinogen (r=0.39, P<0.05). In patients with stable COPD, there was a significant relationship between circulating VEGF levels and the percentage of the predicted FEV1 (forced expiratory volume in 1 s) (r=0.47, P<0.01). Recovery from the exacerbation resulted in a significant decrease in both circulating VEGF levels and markers of systemic inflammation. In conclusion, circulating levels of VEGF and markers of systemic inflammation are up-regulated in patients with acute exacerbated COPD and decrease after recovery from the exacerbation.
Gender-specific regulation of pancreatic islet blood flow, insulin levels and glycaemia in spontaneously diabetic Goto–Kakizaki rats
Authors: Huang Zhen , Jansson Leif , Sjöholm Åke
Published: May 2008

Patients with diabetes are often treated with a statin for hyperlipidaemia and an ACE (angiotensin-converting enzyme) inhibitor or angiotensin receptor antagonist for hypertension or albuminuria. These drugs may also exert beneficial metabolic effects, causing improved glucose tolerance in patients. Gender-related differences have also been observed in the clinical responsiveness to these drugs, but the mechanism behind this is unclear. In the present study, we have investigated whether these drugs and the fatty acid palmitate influence the pancreatic microcirculation, thereby having an impact on insulin secretion and glycaemia in vivo, in spontaneously diabetic male and female Goto–Kakizaki rats. In male rats, pancreatic IBF (islet blood flow) and total PBF (pancreatic blood flow) were increased significantly by pravastatin, captopril and irbesartan. Serum insulin levels were increased by pravastatin and captopril. Palmitate suppressed pancreatic IBF and increased blood glucose. In female animals, pancreatic IBF was stimulated by captopril, candesartan and irbesartan. Total PBF was increased by captopril, candesartan and irbesartan, and by pravastatin. Palmitate suppressed pancreatic IBF and serum insulin secretion. In conclusion, the present study lends support to the view that a local pancreatic RAS (renin–angiotensin system) and pravastatin may be selectively influencing the pancreatic microcirculation and therefore affecting insulin secretion and glycaemia. NEFAs (non-esterified fatty acids) impaired pancreatic IBF, suppressed insulin secretion and increased blood glucose. Substantial gender-related differences in the vascular and metabolic responses to these drugs prevail in this animal model of diabetes.
Importance of the high-molecular-mass isoform of adiponectin in improved insulin sensitivity with rosiglitazone treatment in HIV disease
Authors: Qurashi Saima , Mynarcik Dennis C. , McNurlan Margaret A. , Ahn Hongshik , Ferris Robert , Gelato Marie C.
Published: May 2008

The present study was designed to investigate the relationship of isoforms of adiponectin to insulin sensitivity in subjects with HIV-associated insulin resistance in response to treatment with the thiazolidinedione, rosiglitazone. The two isoforms of adiponectin, HMW (high-molecular-mass) and LMW (low-molecular-mass), were separated by sucrose-gradient-density centrifugation. The amount of adiponectin in gradient fractions was determined by ELISA. Peripheral insulin sensitivity (Rd) was determined with hyperinsulinaemic–euglycaemic clamp, whereas hepatic sensitivity [HOMA (Homoeostasis Model Assessment) %S] was based on basal glucose and insulin values. Treatment with rosiglitazone for 3 months resulted in a significant improvement in the index of hepatic insulin sensitivity (86.4±15% compared with 139±23; P=0.007) as well as peripheral insulin sensitivity (4.04±0.23 compared with 6.17±0.66 mg of glucose/kg of lean body mass per min; P<0.001). Improvement in HOMA was associated with increased levels of HMW adiponectin (r=0.541, P=0.045), but not LMW adiponectin. The present study suggests that the HMW isoform of adiponectin is important in the regulation of rosiglitazone-mediated improvement in insulin sensitivity in individuals with HIV-associated insulin resistance, particularly in the liver.
Impairment of arginine metabolism in rats after massive intestinal resection: effect of parenteral nutrition supplemented with citrulline compared with arginine
Authors: Osowska Sylwia , Neveux Nathalie , Nakib Samir , Lasserre Virginie , Cynober Luc , Moinard Christophe
Published: May 2008

Arginine homoeostasis is impaired in short bowel syndrome, but its supplementation in short bowel syndrome patients remains controversial. Recently, we demonstrated the benefits of citrulline supplementation by the enteral route in resected rats. Since the first step in managing short bowel syndrome is to initiate total parenteral nutrition, we hypothesized that parenteral citrulline supplementation would be more appropriate in this situation than arginine supplementation. In the present study, 24 rats were assigned to four groups. The sham group underwent transection whereas the three other groups underwent resection (R) of 80% of the small intestine. All rats were then fed exclusively by total parenteral nutrition as follows: supplementation with citrulline (R+CIT), with arginine (R+ARG) or no supplementation (R). All of the rats received isocaloric and isonitrogenous nutrition for 4 days. Nitrogen balance was measured daily. Rats were then killed and the blood was collected and the intestinal mucosa and extensor digitorum longus muscle were removed for amino acid and protein analysis. Citrulline and arginine increased mucosal protein content in the ileum (compared with sham and R, P<0.05). However, only citrulline prevented extensor digitorum longus atrophy (R+CIT, 130±3 mg compared with R, 100±6 mg and R+ARG, 110±2 mg, P<0.05). In addition, arginine worsened nitrogen balance (R+ARG, 104±46 mg/72 h compared with R, 249±69 mg/72 h, P<0.05). Only citrulline was able to prevent muscle atrophy and it was achieved independently from any noticeable effect on the gut in particular because citrulline and arginine share the same effect on mucosal ileal protein content. These results suggest that citrulline should be considered as a potential supplement for total parenteral nutrition of short bowel syndrome patients.