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Single Amino Acid Substitution (single + amino_acid_substitution)
Selected AbstractsIdentification of key residues involved in mediating the in vivo anti-tumor/anti-endothelial activity of AlphastatinJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2007C. A. STATON Summary., Background :,We have recently shown that Alphastatin, a 24-amino-acid peptide (ADSGEGDFLAEGGGVRGPRVVERH) derived from human fibrinogen has anti-endothelial properties in vitro and in vivo. Objectives:, The aim of this study was to determine the activity of a terminally modified (stabilized) form of Alphastatin in vitro and in vivo and to identify the key residues required for this activity. Methods:, The in vitro activity of modified Alphastatin, truncates and mutants was determined by endothelial cell (HuDMEC) tubule formation and migration. Active peptides were then assessed in vivo using syngeneic murine subcutaneous 4T1 mammary carcinomas. Results:, Modified Alphastatin-inhibited HuDMEC migration and tubule formation in response to multiple growth factors and caused a 45% inhibition in tumor growth when administered intravenously at 0.25 mg kg,1 (three times per week). Intravenous (i.v.) administration proved non-toxic at all doses investigated, whereas oral and intraperitoneal (i.p.) administration demonstrated neither anti-tumor activity nor toxicity. Truncations of Alphastatin revealed an 11-amino-acid peptide (DFLAEGGGVRG), termed AHN419, which inhibited endothelial cell activity in vitro; however, intravenous AHN419 caused a non-significant growth inhibition in vivo. Single amino acid substitutions to alanine along the entire length of Alphastatin indicated that additional residues outside the AHN419 sequence were required for full activity. Conclusions:, Terminal modification of Alphastatin altered the in vivo efficacy and these studies suggest that a hydrophobic cluster (Phe8, Leu9, Ala10 and Val15) is essential for the biological activity, but additional residues, including Ser3-Gly14, Pro18-Val20 and Arg23 are required for full inhibitory activity of Alphastatin. [source] Interdomain side-chain interactions in human ,D crystallin influencing folding and stabilityPROTEIN SCIENCE, Issue 8 2005Shannon L. Flaugh Abstract Human ,D crystallin (H,D-Crys) is a two domain, ,-sheet eye lens protein that must remain soluble throughout life for lens transparency. Single amino acid substitutions of H,D-Crys are associated with juvenile-onset cataracts. Features of the interface between the two domains conserved among ,-crystallins are a central six-residue hydrophobic cluster, and two pairs of interacting residues flanking the cluster. In H,D-Crys these pairs are Gln54/Gln143 and Arg79/Met147. We previously reported contributions of the hydrophobic cluster residues to protein stability. In this study alanine substitutions of the flanking residue pairs were constructed and analyzed. Equilibrium unfolding/refolding experiments at 37°C revealed a plateau in the unfolding/refolding transitions, suggesting population of a partially folded intermediate with a folded C-terminal domain (C-td) and unfolded N-terminal domain (N-td). The N-td was destabilized by substituting residues from both domains. In contrast, the C-td was not significantly affected by substitutions of either domain. Refolding rates of the N-td were significantly decreased for mutants of either domain. In contrast, refolding rates of the C-td were similar to wild type for mutants of either domain. Therefore, domain interface residues of the folded C-td probably nucleate refolding of the N-td. We suggest that these residues stabilize the native state by shielding the central hydrophobic cluster from solvent. Glutamine and methionine side chains are among the residues covalently damaged in aged and cataractous lenses. Such damage may generate partially unfolded, aggregation- prone conformations of H,D-Crys that could be significant in cataract. [source] Growth hormone excess and the development of growth hormone receptor antagonistsEXPERIMENTAL PHYSIOLOGY, Issue 11 2008C. E. Higham In 1990, a single amino acid substitution in the growth hormone (GH) gene at position 119 was found to transform the consequent protein from an agonist to an antagonist at the growth hormone receptor (GHR). Further amino acid substitutions plus prolongation of the half-life of the protein by pegylation resulted in the first clinically effective GHR antagonist, pegvisomant. Following extensive clinical trials, this medication has emerged as the most efficacious therapy for treatment-resistant acromegaly. Subsequent advances in our understanding of GH,GHR interactions and downstream GH signalling pathways suggest that pegvisomant binds to preformed GHR dimers and prevents rotational changes within the receptor,GH complex necessary for intracellular signalling to occur. This article reviews the discovery of pegvisomant, from initial experimental data to successful licensing of the drug for treatment-resistant acromegaly, and discusses its other potential therapeutic uses in diseases with abnormalities in the GH,IGF-I axis. [source] Membrane localization itself but not binding to IICBGlc is directly responsible for the inactivation of the global repressor Mlc in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 3 2004Yuya Tanaka Summary Mlc is a global transcriptional repressor involved in the regulation of genes linked to glucose metabolism. The activity of Mlc is modulated through the interaction with a major glucose transporter, IICBGlc, in response to external glucose. To understand how IICBGlc,Mlc interaction controls the repressor activity of Mlc, we attempted to isolate Mlc mutants that retain the ability to repress target genes even in the presence of glucose. The Mlc mutants were tested for their ability to interact with IICBGlc. Mutants in which a single amino acid substitution occurs in the N-terminal portion were no longer able to bind to IICBGlc, suggesting that the N-terminal region of Mlc is primarily responsible for the interaction with IICBGlc. To examine whether the Mlc,IICBGlc interaction and/or the membrane localization of Mlc per se are essential for the inactivation of Mlc, the properties of several hybrid proteins in which either IIBGlc or Mlc is fused to membrane proteins were analysed. The cytoplasmic IIBGlc domain failed to inhibit the Mlc action although it retains the ability to bind Mlc in cells. However, it gained the ability to inhibit the Mlc activity when it was fused to a membrane protein LacY. In addition, we showed that Mlc is inactivated when fused to membrane proteins but not when fused to cytoplasmic proteins. We conclude that the IICBGlc,Mlc interaction is dispensable for the inactivation of Mlc, and that membrane localization is directly responsible for the inactivation of Mlc. [source] Familial amyloidotic polyneuropathy (ATTR Val30Met) with widespread cerebral amyloid angiopathy and lethal cerebral hemorrhagePATHOLOGY INTERNATIONAL, Issue 6 2001Naomi Sakashita We report an autopsy case of familial amyloidotic polyneuropathy (FAP) with cerebral hemorrhage. A 38-year-old woman with a typical FAP pedigree started developing severe diarrhea and sensori-motor polyneuropathy at the age of 28 years; autonomic nervous system, heart and renal dysfunction manifested themselves in the following years. Genetic analysis revealed a single amino acid substitution at codon 30 of transthyretin (ATTR Val30Met). Ten years after her initial symptoms, the patient died of a sudden convulsive attack and respiratory failure. Autopsy revealed lethal cerebral hemorrhages and uremic lungs. Histochemical and immunohistochemical analyses revealed TTR-derived amyloid protein in every tissue examined, particularly in glomeruli and peripheral vessels. Severe meningo-cerebrovascular amyloidosis was also detected. Because uremia causes oxidative damage to the vascular system and amyloid formation is closely associated with oxidative stress, it is possible that uremic endothelial damage facilitated an unusual cerebral amyloid deposition. In typical FAP (ATTR Val30Met), cerebral amyloid angiopathy does not usually have clinical manifestations. However, cerebral amyloid angiopathy should be considered to explain FAP symptoms when some risk factors such as uremic vascular damage are accompanying features. [source] Perioperative care of a patient with Beare,Stevenson syndromePEDIATRIC ANESTHESIA, Issue 12 2005SARA UPMEYER DO Summary Beare,Stevenson syndrome is a craniofacial syndrome consisting of a specific pattern of craniosynostosis resulting in a cloverleaf skull deformity and hydrocephalus, down-slanting palpebral fissures, proptosis, hypertelorism, strabismus, dysmorphic ears, choanal atresia, cleft palate, cutis gyratum, acanthosis nigricans, and abnormal genitalia. Its primary cause has been identified as a single amino acid substitution in fibroblast growth factor receptor 2. Of primary importance to the anesthesiologist are issues related to airway management resulting from midface hypoplasia, choanal atresia, and airway abnormalities (tracheal stenosis). Additional issues affecting airway management include associated cervical spine and foramen magnum abnormalities. The authors present their experience caring for a patient with Beare,Stevenson syndrome and discuss the anesthesia care of these patients. [source] Conformational changes induced by a single amino acid substitution in the trans -membrane domain of Vpu: Implications for HIV-1 susceptibility to channel blocking drugsPROTEIN SCIENCE, Issue 10 2007Sang Ho Park Abstract The channel-forming trans -membrane domain of Vpu (Vpu TM) from HIV-1 is known to enhance virion release from the infected cells and is a potential target for ion-channel blockers. The substitution of alanine at position 18 by a histidine (A18H) has been shown to render HIV-1 infections susceptible to rimantadine, a channel blocker of M2 protein from the influenza virus. In order to describe the influence of the mutation on the structure and rimantadine susceptibility of Vpu, we determined the structure of A18H Vpu TM, and compared it to those of wild-type Vpu TM and M2 TM. Both isotropic and orientationally dependent NMR frequencies of the backbone amide resonance of His18 were perturbed by rimantadine, and those of Ile15 and Trp22 were also affected, suggesting that His18 is the key residue for rimantadine binding and that residues located on the same face of the TM helix are also involved. A18H Vpu TM has an ideal, straight ,-helix spanning residues 6,27 with an average tilt angle of 41° in C14 phospholipid bicelles, indicating that the tilt angle is increased by 11° compared to that of wild-type Vpu TM. The longer helix formed by the A18H mutation has a larger tilt angle to compensate for the hydrophobic mismatch with the length of the phospholipids in the bilayer. These results demonstrate that the local change of the primary structure plays an important role in secondary and tertiary structures of Vpu TM in lipid bilayers and affects its ability to interact with channel blockers. [source] Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3PROTEIN SCIENCE, Issue 9 2006Mingyue He Abstract The crystal structures of the Fab, fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 104 -fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position. [source] Genetic analysis of response regulator activation in bacterial chemotaxis suggests an intermolecular mechanismPROTEIN SCIENCE, Issue 11 2002Sandra Da Re Abstract Response regulator proteins of two-component systems are usually activated by phosphorylation. The phosphorylated response regulator protein CheY,P mediates the chemotaxis response in Escherichia coli. We performed random mutagenesis and selected CheY mutants that are constitutively active in the absence of phosphorylation. Although a single amino acid substitution can lead to constitutive activation, no single DNA base change can effect such a transition. Numerous different sets of mutations that activate in synergy were selected in several different combinations. These mutations were all located on the side of CheY defined by ,4, ,5, ,5, and ,1. Our findings argue against the two-state hypothesis for response regulator activation. We propose an alternative intermolecular mechanism that involves a dynamic interplay between response regulators and their effector targets. [source] The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activityPROTEIN SCIENCE, Issue 12 2001Anne Chapman-Smith BCCP, biotin carboxyl carrier protein; IPTG, isopropyl-1-thio-,-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; S.D., standard deviation Abstract Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5,-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain. [source] C3H/HeJ Mouse Model for Spontaneous Chronic Otitis Media,THE LARYNGOSCOPE, Issue 7 2006Carol J. MacArthur MD Abstract Objectives/Hypothesis: Chronic otitis media is a significant clinical problem. Understanding the mechanisms of chronic otitis media is critical for its control. However, little is known of these processes as a result of lack of animal models of spontaneous otitis media. The C3H/HeJ mouse has a single amino acid substitution in its toll-like receptor 4 (TLR4), making it insensitive to endotoxin. As a result, these mice cannot clear Gram-negative bacteria. The chronically inflamed middle ear in this animal provides us the opportunity to study spontaneous chronic otitis media. Study Design and Methods: Otoscopy and auditory brain response (ABR) evaluation of C3H/HeJ mice at 3, 5, 7, 9, and 11 months were carried out under sedation. At 12 months of age, mice were killed and histologic analysis of the middle ear, inner ear, and eustachian tube was carried out. Results: Tympanic membrane visualization and ABR thresholds in 7- to 8-month-old C3H/HeJ mice showed that approximately half developed middle and inner ear disease spontaneously. The significant elevation of thresholds suggested a sensorineural component in addition to the conductive loss. Middle and inner ear histology showed some degree of middle and inner ear inflammation in half the mice, paralleling the ABR data. Conclusions: The histopathologic changes reported here in the C3H/HeJ mouse model of chronic otitis media have been reported in human chronic otitis media. This spontaneous model of chronic otitis media will be valuable for the characterization of middle and inner ear inflammatory disease processes that are induced by middle ear infections. [source] Sporadic onset of erythermalgia: A gain-of-function mutation in Nav1.7ANNALS OF NEUROLOGY, Issue 3 2006Chongyang Han BS Objective Inherited erythermalgia (erythromelalgia) is an autosomal dominant disorder in which patients experience severe burning pain in the extremities, in response to mild thermal stimuli and exercise. Although mutations in sodium channel Nav1.7 have been shown to underlie erythermalgia in several multigeneration families with the disease that have been investigated to date, the molecular basis of erythermalgia in sporadic cases is enigmatic. We investigated the role of Nav1.7 in a sporadic case of erythermalgia in a Chinese family. Methods Genomic DNA from patients and their asymptomatic family members were sequenced to identify mutations in Nav1.7. Whole-cell patch clamp analysis was used to characterize biophysical properties of wild-type and mutant Nav1.7 channels in mammalian cells. Results A single amino acid substitution in the DIIS4-S5 linker of Nav1.7 was present in two children whose parents were asymptomatic. The asymptomatic father was genetically mosaic for the mutation. This mutation produces a hyperpolarizing shift in channel activation and an increase in amplitude of the response to slow, small depolarizations. Interpretation Founder mutations in Nav1.7, which can confer hyperexcitability on peripheral sensory neurons, can underlie sporadic erythermalgia. Ann Neurol 2006 [source] Fine antigenic variation within H5N1 influenza virus hemagglutinin's antigenic sites defined by yeast cell surface displayEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2009Jian Li Abstract Fifteen strains of mAb specific for HA of the A/Hong Kong/482/97 (H5N1) influenza virus were generated. The HA antigenic sites of the human A/Hong Kong/482/97 (H5N1) influenza virus were defined by using yeast cell surface-displaying system and anti-H5 HA mAb. Evolution analysis of H5 HA identified residues that exhibit diversifying selection in the antigenic sites and demonstrated surprising differences between residue variation of H5 HA and H3 HA. A conserved neutralizing epitope in the H5 HA protein recognized by mAb H5M9 was found using viruses isolated from 1997,2006. Seven single amino acid substitutions were introduced into the HA antigenic sites, respectively, and the alteration of antigenicity was assessed. The structure obtained by homology-modeling and molecular dynamic methods showed that a subtle substitution at residue 124 propagates throughout its nearby loop (152,159). We discuss how the structural changes caused by point mutation might explain the altered antigenicity of the HA protein. The results demonstrate the existence of immunodominant positions in the H5 HA protein, alteration of these residues might improve the immunogenicity of vaccine strains. [source] Interaction of ribosome recycling factor and elongation factor EF-G with E. coli ribosomes studied by the surface plasmon resonance techniqueGENES TO CELLS, Issue 12 2000Tetsuya Ishino Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the post-termination complex of a ribosome after the release of polypeptides. How RRF dissociates the complex has long been puzzling. Crystal structures of RRF molecules have been solved recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does. The formation of ribosome complexes on the surface-coupled RRF and elongation factor EF-G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique. RRF interacted with 70S ribosomes as well as 50S and 30S subunits, although it interacted preferentially with 50S subunits, which was clearly seen under high but physiological ionic conditions. This 50S interaction was diminished by a single amino acid substitutions for Arg132 of RRF, which did not appreciably affect the protein folding but nullified the activity in vivo and in vitro. Moreover, a set of antibiotics that inhibited the RRF,50S interaction were also inhibitory to the polysome breakdown activity of RRF in vitro. The BIACORE technique also worked very well in demonstrating the action of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre- and post-translocation intermediates catalysed by EF-G. These results suggest that the preferential interplay of RRF with the 50S subunit may be of biological significance, probably reflecting the mode of RRF action. The BIACORE technique proved useful for real-time monitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling factor. [source] Isolated sulfite oxidase deficiency: identification of 12 novel SUOX mutations in 10 patientsHUMAN MUTATION, Issue 1 2002Jean L. Johnson Abstract We report twelve novel mutations in patients with isolated sulfite oxidase deficiency. The mutations are in SUOX, the gene that encodes the molybdohemoprotein sulfite oxidase. These include two frameshift mutations, a four-basepair deletion (562del4) and a single-basepair insertion (113insC), both resulting in premature termination. Nonsense mutations predicting Y343X and Q364X substitutions were identified in a homozygous state in three patients, the latter in two sibs. The remaining eight are missense mutations generating single amino acid substitutions. From the position of the substituted residues, seven of these mutations are considered to be causative of the enzyme deficiency: I201L, R211Q, G305S, R309H, K322R, Q339R, and W393R. The eighth, a C>T transition, predicts an R319C substitution, which could affect the binding of the molybdenum cofactor and thus severely reduce sulfite oxidase activity. This mutation, however, is downstream of a frameshift mutation and is therefore not the causative mutation in this individual. © 2002 Wiley-Liss, Inc. [source] Rhesus macaque antibody molecules: sequences and heterogeneity of alpha and gamma constant regionsIMMUNOLOGY, Issue 1 2004Franco Scinicariello Summary Rhesus macaques (Macaca mulatta) are extensively used in vaccine development. Macaques infected with simian immunodeficiency viruses (SIV) or simian-human immunodeficiency viruses (SHIV) are the best animal model currently available for acquired-immune-deficiency-syndrome-related studies. Recent results emphasize the importance of antibody responses in controlling HIV and SIV infection. Despite the increasing attention placed on humoral immunity in these models, very limited information is available on rhesus macaque antibody molecules. Therefore, we sequenced, cloned and characterized immunoglobulin gamma (IGHG) and alpha (IGHA) chain constant region genes from rhesus macaques of Indian and Chinese origin. Although it is currently thought that rhesus macaques express three IgG subclasses, we identified four IGHG genes, which were designated IGHG1, IGHG2, IGHG3 and IGHG4 on the basis of sequence similarities with the four human genes encoding the IgG1, IgG2, IgG3 and IgG4 subclasses. The four genes were expressed at least at the messenger RNA level, as demonstrated by real-time reverse transcription polymerase chain reaction (RT-PCR). The level of intraspecies heterogeneity was very high for IGHA genes, whereas IGHG genes were remarkably similar in all animals examined. However, single amino acid substitutions were present in IGHG2 and IGHG4 genes, indicating the presence of IgG polymorphism possibly resulting in the expression of different allotypes. Two IgA alleles were identified in several animals and RT-PCR showed that both alleles may be expressed. Presence of immunoglobulin gene polymorphism appears to reflect the unusually high levels of intraspecies heterogeneity already demonstrated for major histocompatibility complex genes in this non-human primate species. [source] Structural alterations in a type IV pilus subunit protein result in concurrent defects in multicellular behaviour and adherence to host tissueMOLECULAR MICROBIOLOGY, Issue 2 2001Hae-Sun Moon Park The ability of bacteria to establish complex communities on surfaces is believed to require both bacterial,substratum and bacterial,bacterial interactions, and type IV pili appear to play a critical but incompletely defined role in both these processes. Using the human pathogen Neisseria gonorrhoeae, spontaneous mutants defective in bacterial self-aggregative behaviour but quantitatively unaltered in pilus fibre expression were isolated by a unique selective scheme. The mutants, carrying single amino acid substitutions within the conserved amino-terminal domain of the pilus fibre subunit, were reduced in the ability to adhere to a human epithelial cell line. Co-expression of the altered alleles in the context of a wild-type pilE gene confirmed that they were dominant negative with respect to aggregation and human cell adherence. Strains expressing two copies of the altered alleles produced twice as much purifiable pili but retained the aggregative and adherence defects. Finally, the defects in aggregative behaviour and adherence of each of the mutants were suppressed by a loss-of-function mutation in the twitching motility gene pilT. The correlations between self-aggregation and the net capacity of the microbial population to adhere efficiently demonstrates the potential significance of bacterial cell,cell interactions to colonization. [source] | |||||||||||||