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His
Terms modified by His Selected AbstractsGenetic polymorphism of sulfotransferase 1A1, cigarette smoking, hazardous chemical exposure and urothelial cancer risk in a Taiwanese populationINTERNATIONAL JOURNAL OF UROLOGY, Issue 12 2008Yuan-Hung Wang Objectives: To investigate the association between genetic polymorphism of sulfotransferase1A1 (SULT1A1), cigarette smoking, hazardous chemical exposure and urothelial cancer risk in a Taiwanese population. Methods: In a hospital-based case,control study, a total of 300 urothelial cancer (UC) cases and 300 cancer-free controls frequency-matched by age and gender were recruited from September 1998 to December 2005. The SULT1A1 arginine213histidine (Arg213His) polymorphism was genotyped using a polymerase chain reaction,restriction fragment length polymorphism method. Results: We found that the significantly increased UC risks of ever smokers and heavy smokers (,28 pack-years) were 2.1 (95% confidence interval [CI] = 1.4,3.3) and 2.2 (95% CI = 1.3,3.6), respectively. An increased UC risk of 1.8 (95% CI = 0.8,3.8) was observed among individuals with more than one item of hazardous chemical exposure, but it was not statistically significant. Compared with study subjects carrying the SULT1A1 Arg/Arg genotype, those with SULT1A1 Arg/His or His/His genotypes have a significantly decreased UC risk (Odds ratio [OR] = 0.5, 95% CI = 0.3,0.8). Heavy smokers carrying the SULT1A1 Arg/Arg genotype have a significantly increased UC risk (OR = 5.2, 95% CI = 2.3,11.6). Individuals who had been exposed to more than one item of hazardous chemicals and who carried the SULT1A1 Arg/Arg genotype have a significantly increased UC risk (OR = 3.7, 95% CI = 1.4,9.7). The highest significant increased UC risk (OR = 16.1, 95% CI = 2.9,87.2) was observed among ever smokers with hazardous chemical exposure and the SULT1A1 Arg/Arg genotype. Conclusions: SULT1A1 Arg213His polymorphism is associated with the development of UC, especially among cigarette smokers exposed to hazardous chemicals. [source] Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutaseFEBS JOURNAL, Issue 24 2000Elucidation of the roles of amino acids involved in substrate binding, catalysis The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis. The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site-directed mutagenesis. All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm::HIS) in good yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. cerevisiae. The S62A mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate (3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques. [source] Analysis of 3D problems using a new enhanced strain hexahedral elementINTERNATIONAL JOURNAL FOR NUMERICAL METHODS IN ENGINEERING, Issue 11 2003P. M. A. Areias Abstract The now classical enhanced strain technique, employed with success for more than 10 years in solid, both 2D and 3D and shell finite elements, is here explored in a versatile 3D low-order element which is identified as HIS. The quest for accurate results in a wide range of problems, from solid analysis including near-incompressibility to the analysis of locking-prone beam and shell bending problems leads to a general 3D element. This element, put here to test in various contexts, is found to be suitable in the analysis of both linear problems and general non-linear problems including finite strain plasticity. The formulation is based on the enrichment of the deformation gradient and approximations to the shape function material derivatives. Both the equilibrium equations and their variation are completely exposed and deduced, from which internal forces and consistent tangent stiffness follow. A stabilizing term is included, in a simple and natural form. Two sets of examples are detailed: the accuracy tests in the linear elastic regime and several finite strain tests. Some examples involve finite strain plasticity. In both sets the element behaves very well, as is illustrated in numerous examples. Copyright © 2003 John Wiley & Sons, Ltd. [source] Usefulness of Interatrial Conduction Time to Distinguish Between Focal Atrial Tachyarrhythmias Originating from the Superior Vena Cava and the Right Superior Pulmonary VeinJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 12 2008KUAN-CHENG CHANG M.D. Objective: Differentiation of the tachycardia originating from the superior vena cava (SVC) or the right superior pulmonary vein (RSPV) is limited by the similar surface P-wave morphology and intraatrial activation pattern during tachycardia. We sought to find a simple method to distinguish between the two tachycardias by analyzing the interatrial conduction time. Methods: Sixteen consecutive patients consisting of 8 with SVC tachycardia and the other 8 with RSPV tachycardia were studied. The interatrial conduction time from the high right atrium (HRA) to the distal coronary sinus (DCS) and the intraatrial conduction time from the HRA to the atrial electrogram at the His bundle region (HIS) were measured during the sinus beat (SR) and during the tachycardia-triggering ectopic atrial premature beat (APB). The differences of interatrial (,[HRA-DCS]SR-APB) and intraatrial (,[HRA-HIS]SR-APB) conduction time between SR and APB were then obtained. Results: The mean ,[HRA-DCS]SR-APB was 1.0 ± 5.2 ms (95% confident interval [CI],3.3,5.3 ms) in SVC tachycardia and 38.5 ± 8.8 ms (95% CI 31.1,45.9 ms) in RSPV tachycardia. The mean ,[HRA-HIS]SR-APB was 1.5 ± 5.3 ms (95% CI ,2.9,5.9 ms) in SVC tachycardia and 19.9 ± 12.0 ms (95% CI 9.9,29.9 ms) in RSPV tachycardia. The difference of ,[HRA-DCS]SR-APB between SVC and RSPV tachycardias was wider than that of ,[HRA-HIS]SR-APB (37.5 ± 9.3 ms vs. 18.4 ± 15.4 ms, P < 0.01). Conclusions: The wide difference of the interatrial conduction time ,[HRA-DCS]SR-APB between SVC and RSPV tachycardias is a useful parameter to distinguish the two tachycardias and may avoid unnecessary atrial transseptal puncture. [source] History and Trends in Clinical Information Systems in the United StatesJOURNAL OF NURSING SCHOLARSHIP, Issue 1 2001Nancy Staggers Purpose: To provide a synopsis of issues about clinical information systems for nurses not schooled in nursing informatics. Organizing construct: The past, present, and future of clinical computing, including major factors resulting in the early hospital information systems (HIS) and decision support systems (DSS) in the United States, current advances and issues in managing clinical information, and future trends and issues. Methods: Literature review and analysis. Findings and Conclusions: The first HIS and DSS were used in the late 1960s and were focused on applications for acute care. The change from fee-for-service to managed care required a change in the design of clinical information systems toward more patient-centered systems that span the care continuum, such as the computer-based patient record (CPR). Current difficulties with CPR systems include lack of systems integration, data standardization, and implementation. Increased advances in information and technology integration and increased use of the Internet for health information will shape the future of clinical information systems. [source] Dietary strategies to improve the growth and feed utilization of barramundi, Lates calcarifer under high water temperature conditionsAQUACULTURE NUTRITION, Issue 4 2010B. GLENCROSS Abstract Several dietary strategies to ameliorate poorer growth observed to occur at temperatures above the upper thermal optima were examined with juvenile barramundi (Lates calcarifer). A reference (REF) and three experimental diets, one with an increased protein to energy ratio (PRO), another with an increased level of the amino acid histidine (HIS) and a third with supplementation of dietary nucleotides (NUC), were each fed to fish at either 30 °C or 37 °C for a 28-day period. Growth was affected by both temperature and diet. Fish fed the PRO diet at 30 °C grew fastest, but not faster than those fed the NUC diet at the same temperature. The addition of the amino acid histidine to the diet did not improve growth rates at either temperature. At water temperatures of 37 °C, only the fish fed the PRO diet had growth rates equivalent to those of fish at the 30 °C temperatures. Other key factors including feed intake, feed conversion rate, nutrient and energy retention and plasma enzymology were also all affected by temperature and diet. This study shows that the use of a diet with an increased protein to energy ratio provides significant benefits in terms of reducing the impact of growth retardation at higher temperatures. [source] Improved Voltammetric Response of L -Tyrosine on Multiwalled Carbon Nanotubes-Ionic Liquid Composite Coated Glassy Electrodes in the Presence of Cupric IonELECTROANALYSIS, Issue 19 2008Liqin Liu Abstract L -Tyrosine can exhibit a small anodic peak on multiwalled carbon nanotubes (MWCNTs) coated glassy carbon electrodes (GCE). At pH,5.5 its peak potential is 0.70,V (vs. SCE). When an ionic liquid (i.e., 1-octyl-3-methylimidazolium hexafluorophosphate, [omim][PF6]) is introduced on the MWCNT coat, the peak becomes bigger. Furthermore, in the presence of Cu2+ ion the anodic peak of L -tyrosine increases further due to the formation of Cu2+ - L -tyrosine complex, while the peak potential keeps unchanged. Therefore, a sensitive voltammetry based on the oxidation of Cu2+ - L -tyrosine complex on MWCNTs-[omim][PF6] composite coated electrode is developed for L -tyrosine. Under the optimized conditions, the anodic peak current is linear to L -tyrosine concentration in the range of 1×10,8,5×10,6 M, and the detection limit is 8×10,9 M. The modified electrode shows good reproducibility and stability. In addition, the voltammetric behavior of other amino acids is explored. It is found that among them tryptophan (Trp) and histidine (His) can also produce sensitive anodic peak under same experimental conditions, and their detection limits are 4×10,9 M and 4×10,6 M, respectively. [source] Determination of 1-methylhistidine and 3-methylhistidine by capillary and chip electrophoresis with contactless conductivity detectionELECTROPHORESIS, Issue 13 2007Petr T, ma Dr. Abstract CE with capacitively coupled contactless detection (C4D) was used to determine 3-methylhistidine (3-MH) and 1-methylhistidine (1-MH). The C4D response to 3-MH was studied in a BGE consisting of 500,mM acetic acid and ammonia at varying concentration and the results were compared with the theory. Complete separation of a model mixture of 3-MH, 1-MH, and histidine (His) was attained in two optimized BGEs, one containing 500,mM HAc, 20,mM NH4OH, and 0.1 % m/v hydroxyethylcellulose (HEC), pH,3.4 (I) and the other consisting of 100,mM morpholinoethanesulfonic acid (MES), 25,mM LiOH, and 0.1 % m/v HEC, pH,5.5 (II). These optimized BGEs were tested in CE/C4D analyses of urine. Promising results were obtained for separation and determination of 3-MH, 1-MH, and His on a silicon microchip, using aluminum strips as the C4D electrodes; the three analytes were baseline-separated within less than 30,s with a separation channel effective length of 38,mm. The LOD were satisfactory and amounted to 26.4,,M for 3-MH and 18.3,,M for 1-MH. [source] ,And the beat goes on' The cardiac conduction system: the wiring system of the heartEXPERIMENTAL PHYSIOLOGY, Issue 10 2009Mark R. Boyett The cardiac conduction system (CCS), consisting of the sino-atrial node, atrioventricular node and His,Purkinje system, is responsible for the initiation and co-ordination of the heart beat. In the last decade, our understanding of the CCS has been transformed. Immunohistochemistry, used in conjunction with anatomical techniques, has transformed our understanding of its anatomy; arguably, we now understand the position of the sino-atrial node (not the same as in medical textbooks), and our new understanding of the atrioventricular node anatomy means that we can compute its physiological and pathophysiological behaviour. Ion channel expression in the CCS has been shown to be fundamentally different from that in the working myocardium. Dysfunction of the CCS has previously been attributed to fibrosis, but it is now clear that remodelling of ion channels plays an important role in dysfunction during ageing, heart failure and atrial fibrillation. Differences in ion channel expression may even be responsible for the bradycardia in the athlete and differences in heart rate among different species (such as humans and mice). Recent work has highlighted less well-known components of the CCS, including tricuspid, mitral and aortic rings and even a third (retro-aortic) node. These additional tissues do not participate in the initiation and co-ordination of the heart beat and instead they are likely to be the source of various life-threatening arrhythmias. During embryological development, all parts of the CCS have been shown to develop from the primary myocardium of the linear heart tube, partly under the influence of the transcription factor, Tbx3. [source] Multifunctional host defense peptides: intracellular-targeting antimicrobial peptidesFEBS JOURNAL, Issue 22 2009Pierre Nicolas There is widespread acceptance that cationic antimicrobial peptides, apart from their membrane-permeabilizing/disrupting properties, also operate through interactions with intracellular targets, or disruption of key cellular processes. Examples of intracellular activity include inhibition of DNA and protein synthesis, inhibition of chaperone-assisted protein folding and enzymatic activity, and inhibition of cytoplasmic membrane septum formation and cell wall synthesis. The purpose of this minireview is to question some widely held views about intracellular-targeting antimicrobial peptides. In particular, I focus on the relative contributions of intracellular targeting and membrane disruption to the overall killing strategy of antimicrobial peptides, as well as on mechanisms whereby some peptides are able to translocate spontaneously across the plasma membrane. Currently, there are no more than three peptides that have been convincingly demonstrated to enter microbial cells without the involvement of stereospecific interactions with a receptor/docking molecule and, once in the cell, to interfere with cellular functions. From the limited data currently available, it seems unlikely that this property, which is isolated in particular peptide families, is also shared by the hundreds of naturally occurring antimicrobial peptides that differ in length, amino acid composition, sequence, hydrophobicity, amphipathicity, and membrane-bound conformation. Microbial cell entry and/or membrane damage associated with membrane phase/transient pore or long-lived transitions could be a feature common to intracellular-targeting antimicrobial peptides and mammalian cell-penetrating peptides that have an overrepresentation of one or two amino acids, i.e. Trp and Pro, His, or Arg. Differences in membrane lipid composition, as well as differential lipid recruitment by peptides, may provide a basis for microbial cell killing on one hand, and mammalian cell passage on the other. [source] Spectroscopic and DNA-binding characterization of the isolated heme-bound basic helix,loop,helix-PAS-A domain of neuronal PAS protein 2 (NPAS2), a transcription activator protein associated with circadian rhythmsFEBS JOURNAL, Issue 11 2006Yuji Mukaiyama Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm-associated transcription factor with two heme-binding sites on two PAS domains. In the present study, we compared the optical absorption spectra, resonance Raman spectra, heme-binding kinetics and DNA-binding characteristics of the isolated fragment containing the N-terminal basic helix,loop,helix (bHLH) of the first PAS (PAS-A) domain of NPAS2 with those of the PAS-A domain alone. We found that the heme-bound bHLH-PAS-A domain mainly exists as a dimer in solution. The Soret absorption peak of the Fe(III) complex for bHLH-PAS-A (421 nm) was located at a wavelength 9 nm higher than for isolated PAS-A (412 nm). The axial ligand trans to CO in bHLH-PAS-A appears to be His, based on the resonance Raman spectra. In addition, the rate constant for heme association with apo-bHLH-PAS (3.3 × 107 mol,1·s,1) was more than two orders of magnitude higher than for association with apo-PAS-A (< 105 mol,1·s,1). These results suggest that the bHLH domain assists in stable heme binding to NPAS2. Both optical and resonance Raman spectra indicated that the Fe(II),NO heme complex is five-coordinated. Using the quartz-crystal microbalance method, we found that the bHLH-PAS-A domain binds specifically to the E-box DNA sequence in the presence, but not in the absence, of heme. On the basis of these results, we discuss the mode of heme binding by bHLH-PAS-A and its potential role in regulating DNA binding. [source] Disruption of transport activity in a D93H mutant thiamine transporter 1, from a Rogers Syndrome familyFEBS JOURNAL, Issue 22 2003Dana Baron Rogers syndrome is an autosomal recessive disorder resulting in megaloblastic anemia, diabetes mellitus, and sensorineural deafness. The gene associated with this disease encodes for thiamine transporter 1 (THTR1), a member of the SLC19 solute carrier family including THTR2 and the reduced folate carrier (RFC). Using transient transfections into NIH3T3 cells of a D93H mutant THTR1derived from a Rogers syndrome family, we determined the expression, post-translational modification, plasma membrane targeting and thiamine transport activity. We also explored the impact on methotrexate (MTX) transport activity of a homologous missense D88H mutation in the human RFC, a close homologue of THTR1. Western blot analysis revealed that the D93H mutant THTR1 was normally expressed and underwent a complete N -glycosylation. However, while this mutant THTR1 was targeted to the plasma membrane, it was completely devoid of thiamine transport activity. Consistently, introduction into MTX transport null cells of a homologous D88H mutation in the hRFC did not result in restoration of MTX transport activity, thereby suggesting that D88 is an essential residue for MTX transport activity. These results suggest that the D93H mutation does not interfere with transporter expression, glycosylation and plasma membrane targeting. However, the substitution of this negatively charged amino acid (Asp93) by a positively charged residue (His) in an extremely conserved region (the border of transmembrane domain 2/intracellular loop 2) in the SLC19 family, presumably inflicts deleterious structural alterations that abolish thiamine binding and/or translocation. Hence, this functional characterization of the D93H mutation provides a molecular basis for Rogers syndrome. [source] Resonance Raman study of multihemic c -type cytochromes from Desulfuromonas acetoxidansFEBS JOURNAL, Issue 4 2000Genevičve Chottard Two multihemic cytochromes c from the sulfur reducing bacteria Desulfuromonas acetoxidans have been studied by optical and resonance Raman spectroscopy: cytochrome c551.5, a trihemic cytochrome and cytochrome c Mr 50 000, a recently isolated high molecular mass cytochrome. The redox and Raman characteristics of cytochrome c551.5 are compared to those of the tetrahemic cytochromes c3 from Desulfovibrio. While the redox behavior, followed by spectroelectrochemistry, is similar to that of cytochrome c3, showing the same conformational change after reduction of the highest potential heme, the Raman data show a contribution from a His, form of the axial ligands and lead to the assignment of a band at 218 cm,1 to the Fe(III),(His)2 stretching vibration. The Raman data on cytochrome c Mr 50 000 are in favor of an entirely low spin species with two different sets of axial ligands. A partially reduced state is easily accessible by ascorbate addition. [source] Characterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatusFEBS JOURNAL, Issue 3 20002S] ferredoxin, Redox properties, molecular interaction with a [2Fe A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6 -tag fusion from an Escherichia coli clone over-expressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48-kDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 , Em1 of > 80 mV. The reduction of SQ by dithionite was significantly accelerated when the [2Fe,2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be ,275 ± 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of , 60 000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Fe,2S] ferredoxins, including FdV and FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprA -deleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wild-type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions. [source] Bacillus subtilis contains a cyclodextrin-binding protein which is part of a putative ABC-transporterFEMS MICROBIOLOGY LETTERS, Issue 1 2001Annette Kamionka Abstract Bacillus subtilis is able to grow on ,-, ,- and ,-cyclodextrins as a carbon source via a yet unknown metabolizing system. Sequence analysis of the B. subtilis genome reveals that the putative yvfK-yvfO operon seems to be involved in cyclodextrin utilization, containing the open reading frame yvfK, now termed cycB. The amino acid sequence derived from the DNA sequence bears high similarities to solute-binding proteins from B. subtilis, as well as to cymE from Klebsiella oxytoca and malE from Escherichia coli, both encoding solute-binding proteins able to interact with cyclodextrins. A [His]6 -tagged variant of CycB from B. subtilis was constructed, overproduced in E. coli and purified. The modified protein has been used to study its substrate specificity by surface plasmon resonance and fluorescence spectroscopy. From these data, CycB can be classified as a cyclodextrin-binding protein which interacts with all three natural cyclodextrins: ,, , and ,, thereby showing the highest affinity to ,-cyclodextrin. [source] Pendred's syndrome with goiter and enlarged vestibular aqueducts diagnosed by PDS gene mutation,HEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 7 2002Hajime Ishinaga MD Abstract Background Pendred's syndrome (PS) is an autosomal recessive disorder characterized by goiter and congenital sensorineural hearing loss. Recent advances in molecular biology revealed the gene responsible for PS (PDS) and provided an important aid for the diagnosis of this condition. Methods A case of PS with huge goiter and congenital hearing impairment was diagnosed by mutational analysis of the PDS gene. Results Physical examination and computer tomography CT revealed a diffuse swelling of the thyroid gland. Thyroid function tests were normal, and the perchlorate discharge test was negative. Audiologic examination confirmed sensorineural hearing loss, and temporal bone CT revealed bilateral enlarged vestibular aqueducts. The mutational analysis revealed that the patient was homozygous for His 723 Arg (2168A,G) in exon 19, a missense mutation. Conclusions The results of thyroid function tests in PS patients are usually normal, and the positive perchlorate discharge test has been used for the diagnosis. However, this is a nonspecific test and is not sensitive enough for PS. In our case, despite a negative perchlorate test, the patient was diagnosed by mutational analysis and received total thyroidectomy to relieve respiratory distress caused by thyroid enlargement. This is the first report of a mutation detected in the thyroid tissue and clearly shows that the mutation caused histopathologic change in that gland. © 2002 Wiley Periodicals, Inc. [source] Functional Hydrogel Surfaces: Binding Kinesin-Based Molecular Motor Proteins to Selected Patterned Sites,ADVANCED FUNCTIONAL MATERIALS, Issue 8 2005T. Yu Abstract Hydrogel microstructures with micrometer-scale topography and controllable functionality have great potential for numerous nanobiotechnology applications including, for example, three-dimensional structures that exhibit controlled interactions with proteins and cells. Taking advantage of the strong affinity of histidine (His) residues for metal-ion,nitrilotriacetic acid (NTA) complexes, we have chemically modified hydrogels to enable protein immobilization with retention of activity by incorporating 2-methacrylamidobutyl nitrilotriacetic acid, an NTA-containing monomer that can be copolymerized with a series of monomers to form NTA-containing hydrogels. By varying the NTA-monomer composition in the hydrogels, it is possible to control the amount of protein bound to the hydrogel surface. The retention of biological activity was demonstrated by microtubule gliding assays. Normally, hydrogels are resistant to protein binding, but we have selected these materials because of their porous nature. Bringing together hydrogel functionalization and soft-lithography patterning techniques, it was possible to create a hybrid hydrogel superstructure that possesses binding specificity to His-tagged protein in selected sites. This type of surface and microstructure is not only advantageous for motor protein integration, but it can also be generally applied to the formation of His-tagged molecules for sensors and biochip applications. [source] Sequence variation in trypsin- and chymotrypsin-like cDNAs from the midgut of Ostrinia nubilalis: methods for allelic differentiation of candidate Bacillus thuringiensis resistance genesINSECT MOLECULAR BIOLOGY, Issue 1 2006B. S. Coates Abstract Midgut expressed alkaline serine proteases of Lepidoptera function in conversion of Bacillus thuringiensis (Bt) protoxin to active toxin, and reduced level of transcript T23 is associated with Ostrinia nubilalis resistance to Dipel® Bt formulations. Three groups of trypsin- (OnT25, OnT23, and OnT3) and two chymotrypsin-like (OnC1 and OnC2) cDNAs were isolated from O. nubilalis midgut tissue. Intraspecific groupings are based on cDNA similarity and peptide phylogeny. Derived serine proteases showed a catalytic triad (His, Asp, and Ser; except transcript OnT23a), three substrate specificity-determining residues, and three paired disulphide bonds. RT-PCR indicated all transcripts are expressed in the midgut. Mendelian-inherited genomic markers for loci OnT23, OnT3 and OnC1 will be useful for association of alleles with bioassayed Bt toxin resistance phenotypes. [source] Disease-associated casein kinase I , mutation may promote adenomatous polyps formation via a Wnt/,-catenin independent mechanismINTERNATIONAL JOURNAL OF CANCER, Issue 5 2007I-Chun Tsai Abstract The Wnt signaling pathway is critical for embryonic development and is dysregulated in multiple cancers. Two closely related isoforms of casein kinase I (CKI, and ,) are positive regulators of this pathway. We speculated that mutations in the autoinhibitory domain of CKI,/, might upregulate CKI,/, activity and hence Wnt signaling and increase the risk of adenomatous polyps and colon cancer. Exons encoding the CKI, and CKI, regulatory domains were sequenced from DNA obtained from individuals with adenomatous polyps and a family history of colon cancer unaffected by familial adenomatous polyposis or hereditary nonpolyposis colorectal cancer (HNPCC). A CKI, missense mutation, changing a highly conserved residue, Arg324, to His (R324H), was found in an individual with large and multiple polyps diagnosed at a relatively young age. Two findings indicate that this mutation is biologically active. First, ectopic ventral expression of CKI,(R324H) in Xenopus embryos results in secondary axis formation with an additional distinctive phenotype (altered morphological movements) similar to that seen with unregulated CKI,. Second, CKI,(R324H) is more potent than wildtype CKI, in transformation of RKO colon cancer cells. Although the R324H mutation does not significantly change CKI, kinase activity in an in vitro kinase assay or Wnt/,-catenin signal transduction as assessed by a ,-catenin reporter assay, it alters morphogenetic movements via a ,-catenin-independent mechanism in early Xenopus development. This novel human CKI, mutation may alter the physiological role and enhance the transforming ability of CKI, through a Wnt/,-catenin independent mechanism and thereby influence colonic adenoma development. © 2006 Wiley-Liss, Inc. [source] Single nucleotide polymorphisms in the XPG gene: Determination of role in DNA repair and breast cancer riskINTERNATIONAL JOURNAL OF CANCER, Issue 5 2003Rajiv Kumar Abstract In this study we determined the effect of single nucleotide polymorphisms in the XPG gene on DNA repair and breast cancer susceptibility. Ninety individuals, with previously studied DNA repair rate at 24 hr of 2 types of UV-specific cyclobutane pyrimidines dimers (CPDs) in skin were genotyped for XPG polymorphism at codon 1104 (exon 15 G>C; Asp > His). The repair rate of TT=C dimer was similar in both wild-type GG homozygotes and GC heterozygotes, whereas, for TT=T, dimer repair was non-significantly (Student's t -test, p = 0.34) lower in GC heterozygotes than wild-type GG homozygotes. Genotyping of 220 breast cancer cases and 308 controls for the same single nucleotide polymorphism in exon 15 of the XPG gene exhibited marginally significant increased frequency of the variant allele (,2 3.84, p = 0.05; OR 1.33, 95% CI 1.0,1.8) in cases (C-allele 0.29) compared to controls (C-allele 0.24). Combined heterozygote and variant homozygote genotype frequency was also higher in cases than controls (,2 4.79, p = 0.03; OR 1.50, 95%CI 1.04,2.16). © 2002 Wiley-Liss, Inc. [source] pKa optimized catalysis in serine proteinases, an ab initio study on the catalaytic HisINTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 11 2007Péter Hudáky Abstract First principle models of catalytic apparatus of enzymes can be used for studying stability as well as the atomic details of a catalytic mechanism. For example, the catalytic triad of chymotrypsin was recently investigated by using an ab initio geometry optimized (Hudáky and Perczel, Proteins: Struct Funct Genet, 2006, 62, 749) self-stabilizing molecule ensemble without the presence of the complete enzyme and substrate. Several parameters of the above catalytic reaction turned out to be the same within the model and the in vitro enzymatic reaction. Among the numerous parameters of the catalytic process geometrical changes of the catalytic histidine was investigated here and the variation of its pKa value was determined. A relatively large range, 3.5 unit, was determined as the variation of pKa as function of the conformational subspace available in serine proteases. Comparing PDB structures of the free and the complex enzymes it was shown, that histidine, after accepting the proton from the OH group of the catalytic serine, undergoes a minor conformational change accompanied by a 2.5 unit decrease in pKa. We conclude that the changes of pKa during catalysis are predominantly determined by the geometrical arrangement of the histidine moiety and this change serves as a significant driving force in the catalytic process. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2007 [source] Density functional study of the heme moiety of cytochrome c,,INTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 5 2005Anil Kumar Abstract A model of cytochrome c (Cyt-c) including the porphyrin ring, a methionine residue (Met), and the imidazole ring of histidine (His), the latter two being situated above and below the iron (Fe) atom of the porphyrin ring, was studied using Density Functional Theory (DFT). The geometries of the model Cyt-c complex with the Fe atom in two different charge states were fully optimized, i.e., in singlet and triplet states for Fe and in doublet and quartet states for Fe3+. The B3LYP method of DFT along with the 3-21G* basis set for C, H, N, and O atoms and the Lanl2dz basis set for the Fe atom was used. We found that with Fe3+, the doublet spin state is the ground state and the quartet state lies slightly above it. The geometry of the singlet spin state is similar to that of the doublet and quartet states. However, methionine has different conformations when Fe has zero charge (singlet, triplet states) relative to the situation when Fe has +3 charges (doublet, quartet states). The Met chain is folded instead of remaining extended in going from the singlet or triplet spin state to the doublet or quartet state and the folding is stabilized by an intramolecular CH..O hydrogen bond. The optimized geometrical parameters of the model of Cyt-c are usually in satisfactory agreement with those observed experimentally. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005 [source] Catalytic mechanism and substrate selectivity of aldo-keto reductases: Insights from structure-function studies of Candida tenuis xylose reductaseIUBMB LIFE, Issue 9 2006Regina Kratzer Abstract Aldo-keto reductases (AKRs) constitute a large protein superfamily of mainly NAD(P)-dependent oxidoreductases involved in carbonyl metabolism. Catalysis is promoted by a conserved tetrad of active site residues (Tyr, Lys, Asp and His). Recent results of structure-function relationship studies for xylose reductase (AKR2B5) require an update of the proposed catalytic mechanism. Electrostatic stabilization by the ,-NH3+ group of Lys is a key source of catalytic power of xylose reductase. A molecular-level analysis of the substrate binding pocket of xylose reductase provides a case of how a very broadly specific AKR achieves the requisite selectivity for its physiological substrate and could serve as the basis for the design of novel reductases with improved specificities for biocatalytic applications. iubmb Life, 58: 499-507, 2006 [source] HATODAS II , heavy-atom database system with potentiality scoringJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 3 2009Michihiro Sugahara HATODAS II is the second version of HATODAS (the Heavy-Atom Database System), which suggests potential heavy-atom reagents for the derivatization of protein crystals. The present expanded database contains 3103 heavy-atom binding sites, which is four times more than the previous version. HATODAS II has three new criteria to evaluate the feasibility of the search results: (1) potentiality scoring for the predicted heavy-atom reagents, (2) exclusion of the disordered amino acid residues based on the secondary structure prediction and (3) consideration of the solvent accessibility of amino acid residues from a homology model. In the point mutation option, HATODAS II suggests possible mutation sites into reactive amino acid residues such as Met, Cys and His, on the basis of multiple sequence alignments of homologous proteins. These new features allow the user to make a well informed decision as to the possible heavy-atom derivatization experiments of protein crystals. [source] Characterization and heterologous expression of a novel lysophospholipase gene from Antrodia cinnamomeaJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2010K.-H. Hsu Abstract Aims:, A novel lysophospholipase (LysoPL) from the basidiomycetous fungi Antrodia cinnamomea named ACLysoPL was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results:, The gene encoding ACLysoPL was obtained from expressed sequence tags from A. cinnamomea. The full length of this gene has a 945 -bp open reading frame encoding 314 amino acids with a molecular weight of 35·5 kDa. ACLysoPL contains a lipase consensus sequence (GXSXG) motif and a Ser,His,Asp catalytic triad. A putative peroxisomal targeting signal type 1 was found in the C-terminal. Heterologous expression of ACLysoPL in E. coli showed that the enzyme preferentially hydrolyses long-chain acyl esterases at pH 7 and 30°C. ACLysoPL is a psychrophilic enzyme about 40% of whose maximum activity remained at 4°C. The LysoPL activities with lysophospholipids as substrate were analysed by gas chromatography/mass spectrometry. Conclusion:, We have identified and characterized a gene named ACLysoPL encoding a protein performing LysoPL and esterase activities. Significance and Impact of the Study:, This is the first LysoPL of A. cinnamomea identified and characterized at the molecular level. [source] Negative effects of the amino acids Lys, His, and Thr on S6K1 phosphorylation in mammary epithelial cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2008Rotem Ladovsky Prizant Abstract The role of essential amino acids (AA) on protein synthesis via the mTOR pathway was studied in murine mammary epithelial cells cultured under lactogenic conditions. Leu, Ile, and Val increased S6K1 phosphorylation compared to that measured in AA-deprived cells. Trp, Phe, and Met had no effect. Surprisingly, Lys, His, and Thr inhibited S6K1 phosphorylation in both murine and bovine mammary cells. Thr exhibited the most potent inhibition, being the only amino acid that competed with Leu's positive role. In non-deprived cells, there was no observable effect of Lys, His, or Thr on S6K1 phosphorylation at concentrations up to five times those in the medium. However, their addition as a mix revealed a synergistic negative effect. Supplementation of Lys, His, and Thr abrogated mTOR Ser 2448 phosphorylation, with no effect on Akt Ser 473,an mTORC2 target. This confirms specific mTORC1 regulation of S6K1 phosphorylation. The individual supplementation of Lys, His, and Thr maintained a low level of IRS-1 phosphorylation, which was dose-dependently increased by their combined addition. Thus, in parallel to inhibiting S6K1 activity, these AA may act synergistically to activate an additional kinase, phosphorylating IRS-1 via an S6K1-independent pathway. In cultures supplemented by Lys, His, and Thr, cellular protein synthesis decreased by up to 65%. A more pronounced effect was observed on ,-casein synthesis. These findings indicate that positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediate the synthesis rates of total and specific milk proteins in mammary epithelial cells. J. Cell. Biochem. 105: 1038,1047, 2008. © 2008 Wiley-Liss, Inc. [source] Simultaneous inhibition of anti-coagulation and inflammation: crystal structure of phospholipase A2 complexed with indomethacin at 1.4,Ĺ resolution reveals the presence of the new common ligand-binding siteJOURNAL OF MOLECULAR RECOGNITION, Issue 6 2009Nagendra Singh Abstract A novel ligand-binding site with functional implications has been identified in phospholipase A2 (PLA2). The binding of non-steroidal anti-inflammatory agent indomethacin at this site blocks both catalytic and anti-coagulant actions of PLA2. A group IIA PLA2 has been isolated from Daboia russelli pulchella (Russell's viper) which is enzymatically active as well as induces a strong anti-coagulant action. The binding studies have shown that indomethacin reduces the effects of both anti-coagulant and pro-inflammatory actions of PLA2. A group IIA PLA2 was co-crystallized with indomethacin and the structure of the complex has been determined at 1.4,Ĺ resolution. The structure determination has revealed the presence of an indomethacin molecule in the structure of PLA2 at a site which is distinct from the conventional substrate-binding site. One of the carboxylic group oxygen atoms of indomethacin interacts with Asp 49 and His 48 through the catalytically important water molecule OW 18 while the second carboxylic oxygen atom forms an ionic interaction with the side chain of Lys 69. It is well known that the residues, His 48 and Asp 49 are essential for catalysis while Lys 69 is a part of the anti-coagulant loop (residues, 54,77). Indomethacin binds in such a manner that it blocks the access to both, it works as a dual inhibitor for catalytic and anti-coagulant actions of PLA2. This new binding site in PLA2 has been observed for the first time and indomethacin is the first compound that has been shown to bind at this novel site resulting in the prevention of anti-coagulation and inflammation. Copyright © 2009 John Wiley & Sons, Ltd. [source] Oligohis-tags: mechanisms of binding to Ni2+ -NTA surfacesJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2009Steven Knecht Abstract Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni2+ immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C - or N -terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His-tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His-tags to Ni2+ -NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid-phase peptide synthesis (SPPS). Binding to Ni2+ -NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (KD) of 14,±,1,nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding. Copyright © 2009 John Wiley & Sons, Ltd. [source] Binding of synthetic peptides by a human monoclonal IgM with an unusual combining site structureJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2001Allen B. Edmundson Abstract Using X-ray crystallography, a human monoclonal IgM cryoglobulin (Mez) was found to have an unusual combining site topography. Analysis of the unliganded Fv at 2.6,Ĺ resolution revealed that the HCDR3 had partitioned the active site into two compartments [Ramsland PA et al. 2000. Mol. Immunol. 37: 295,310]. The two cavities had dimensions and chemical properties that were compatible with the binding of peptides. In this study, libraries of peptides were prepared using solid-phase synthesis. Binding of the intact Mez IgM to these peptides was tested by enzyme-linked immunoassays. Screening of 400 dipeptides revealed that binding was markedly skewed toward amino acids with aromatic side-chains (Phe and Trp), especially when located in the second position. Preferential recognition of aromatic side-chains by Mez IgM was confirmed with larger peptides of three to five residues, but C-terminal positioning was not favored in these peptides. Mez IgM also showed binding propensities for acidic residues (Asp and Glu) as well as several other side-chains with different chemical properties, including His, Pro, Asn and Gln. Mez IgM recognized sets of overlapping octapeptides representing the sequences of the constant domains of human IgG1 heavy chains. These peptides represented similar stretches of polypeptide on the three-dimensional structures of all three constant domains (CH1, CH2 and CH3). Thus, Mez IgM may recognize structurally homologous regions of immunoglobulin domains, which were conserved during the evolution of the immune system. Copyright © 2001 John Wiley & Sons, Ltd. [source] Full-length bovine spp24 [spp24 (24-203)] inhibits BMP-2 induced bone formation,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2008Chananit Sintuu Abstract Secreted phosphoprotein 24 kDa (spp24) is a bone matrix protein. It contains a TGF-, receptor II homology 1 (TRH1) domain. A cyclic, synthetic 19 amino acid peptide (bone morphogenetic protein binding peptide or BBP) based on the sequence of the TRH1 domain enhances BMP-2 induced osteogenesis. Many observations suggest that different size forms of this protein have very different effects (inhibiting or enhancing) on BMP-2 induced osteogenesis. Using the stable recombinant Met(His)6 -tagged secretory form of full-length (fl) bovine spp24 [Met(His)6 -spp24 (residues 24,203)] and transgenic (TG) mice expressing fl bovine spp24 (residues 1,203), we have demonstrated that spp24 inhibits BMP-2 induced bone formation. The effects of Met(His)6 -spp24 (24,203) were determined in the ectopic bone-forming bioassay in male mice. Implantation of 5 µg of BMP-2 stimulated bone formation, assessed densitometrically as bone area and mineral content. When Met(His)6 -spp24 (24,203) was implanted with BMP-2, it elicited a dose-dependent decrease in BMP-2-medicated ectopic bone formation. When added at a 50-fold excess (w/w), Met(His)6 -spp24 (24,203) completely ablated the effects of BMP-2, while addition of a 10-fold excess had no effect. Constitutive expression of fl bovine spp24 (1,203) under the control of the osteocalcin promoter in TG female mice reduced femoral and vertebral bone mineral density at 3 months of age and reduced femoral BMD at 8 months of age, but had no effects in male mice, which can exhibit less osteocalcin-promoter driven gene transcription than females. Histomorphometric analysis demonstrated that bone volume and trabecular thickness were lower in TG female mice at 3 months of age than in sex- and age-matched wild type (WT) controls. Thus, fl spp24 and its secretory isoform (Met(His)6 -spp24 [24,203]), which contain a BMP-binding or TRH1 motif, inhibit ectopic bone formation in male mice and adversely affects BMD and histological parameters related to bone mass and formation in female mice expressing the human transgene. Under these conditions, fl spp24 acts as a BMP antagonist in vivo. © 2008 Orthopaedic Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:753,758, 2008 [source] | ||||||||||||||||||||||||||||||||||||||||