bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ohtani Receives 2007 N. L. Bowen Award
Authors: Karato Shun‐Ichiro , Ohtani Eiji 
Published: May 2008

It is my pleasure to present Eiji Ohtani, one of the recipients of the N. L. Bowen Award of the American Geophysical Union.Eiji Ohtani is indisputably the most prominent leader in the experimental studies of properties of Earth materials, particularly the melting relationships and the properties of melts under high pressures and temperatures. Eiji started his brilliant career in Mineo Kumazawa's lab at Nagoya University in the early 1970s. Mineo Kumazawa is one of the pioneers of large‐volume high‐pressure devices (others include Naoto Kawai and Syun‐iti Akimoto), and Eiji played a major role as a young student in establishing new techniques of high‐pressure and high‐temperature experiments using a large‐volume apparatus. His reputation was established already in the early 1980s based on his seminal papers on the melting of fayalite and forsterite under high pressures (to 20 gigapascals).
In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex
Authors: Mcandrew Rosemary S. , Olson Bradley J. S. C. , Kadirjan-Kalbach Deena K. , Chi-Ham Cecilia L. , Vitha Stanislav , Froehlich John E. , Osteryoung Katherine W. 
Published: May 2008

FtsZ1 and FtsZ2 are phylogenetically distinct homologues of the tubulin-like bacterial cell division protein FtsZ that play major roles in the initiation and progression of plastid division in plant cells. Both proteins are components of a mid-plastid ring, the Z-ring, which functions as a contractile ring on the stromal surface of the chloroplast IEM (inner envelope membrane). FtsZ1 and FtsZ2 have been shown to interact, but their in vivo biochemical properties are largely unknown. To gain insight into the in vivo biochemical relationship between FtsZ1 and FtsZ2, in the present study we investigated their molecular levels in wild-type Arabidopsis thaliana plants and endogenous interactions in Arabidopsis and pea. Quantitative immunoblotting and morphometric analysis showed that the average total FtsZ concentration in chloroplasts of 3-week-old Arabidopsis plants is comparable with that in Escherichia coli. FtsZ levels declined as plants matured, but the molar ratio between FtsZ1 and FtsZ2 remained constant at approx. 1:2, suggesting that this stoichiometry is regulated and functionally important. Density-gradient centrifugation, native gel electrophoresis, gel filtration and co-immunoprecipitation experiments showed that a portion of the FtsZ1 and FtsZ2 in Arabidopsis and pea chloroplasts is stably associated in a complex of ∼200–245 kDa. This complex also contains the FtsZ2-interacting protein ARC6 (accumulation and replicatioin of chloroplasts 6), an IEM protein, and analysis of density-gradient fractions suggests the presence of the FtsZ1-interacting protein ARC3. Based on the mid-plastid localization of ARC6 and ARC3 and their postulated roles in promoting and inhibiting chloroplast FtsZ polymer formation respectively, we hypothesize that the FtsZ1–FtsZ2–ARC3–ARC6 complex represents an unpolymerized IEM-associated pool of FtsZ that contributes to the dynamic regulation of Z-ring assembly and remodelling at the plastid division site in vivo.
Kinetic, thermodynamic and X-ray structural insights into the interaction of melatonin and analogues with quinone reductase 2
Authors: Calamini Barbara , Santarsiero Bernard D. , Boutin Jean A. , Mesecar Andrew D. 
Published: May 2008

Melatonin exerts its biological effects through at least two transmembrane G-protein-coupled receptors, MT1 and MT2, and a lower-affinity cytosolic binding site, designated MT3. MT3 has recently been identified as QR2 (quinone reductase 2) (EC 1.10.99.2) which is of significance since it links the antioxidant effects of melatonin to a mechanism of action. Initially, QR2 was believed to function analogously to QR1 in protecting cells from highly reactive quinones. However, recent studies indicate that QR2 may actually transform certain quinone substrates into more highly reactive compounds capable of causing cellular damage. Therefore it is hypothesized that inhibition of QR2 in certain cases may lead to protection of cells against these highly reactive species. Since melatonin is known to inhibit QR2 activity, but its binding site and mode of inhibition are not known, we determined the mechanism of inhibition of QR2 by melatonin and a series of melatonin and 5-hydroxytryptamine (serotonin) analogues, and we determined the X-ray structures of melatonin and 2-iodomelatonin in complex with QR2 to between 1.5 and 1.8 Å (1 Å=0.1 nm) resolution. Finally, the thermodynamic binding constants for melatonin and 2-iodomelatonin were determined by ITC (isothermal titration calorimetry). The kinetic results indicate that melatonin is a competitive inhibitor against N-methyldihydronicotinamide (Ki=7.2 μM) and uncompetitive against menadione (Ki=92 μM), and the X-ray structures shows that melatonin binds in multiple orientations within the active sites of the QR2 dimer as opposed to an allosteric site. These results provide new insights into the binding mechanisms of melatonin and analogues to QR2.
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 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.
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.
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.
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.
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.
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.
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&lt;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.