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Acid Mutations (acid + mutation)
Kinds of Acid Mutations Selected AbstractsComparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epitheliumTHE JOURNAL OF GENE MEDICINE, Issue 12 2003Nils Loewen Abstract Background In neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2,5 days post-natal). In non-retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol -encoded functions intact. For HIV-1 vectors injected into brain, these have been used to simultaneously control for pseudotransduction and verify that long-term expression requires integration. Such experiments compare particles that differ only in a single amino acid within a single enzyme that forms a very small molar fraction of the virion. Class I integrase mutants have not been described for feline immunodeficiency virus (FIV) integrase, or tested in the eye for any lentiviral vector. Methods We compared subretinally and intravitreally injected FIV vectors and followed animals for up to 7 months, a duration that exceeds prior studies. We also compared the wild-type (WT) vector with one incorporating a single class I amino acid mutation in FIV integrase (D66V). A mock vector (packaging construct absent) was an alternative control. All vectors were vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped and were injected on day 7 of life. One group of animals received either subretinal or intravitreal injections of WT vector in the right eyes. Control left eyes were injected with mock vector. These animals were sacrificed at 2 or 7 days post-injection. A second group received subretinal injections of either WT vector or equivalent D66V vector (reverse transcriptase-normalized to WT), and were analyzed after 2, 3 and 7 months. All eyes were scored for marker gene (,-galactosidase) expression by an observer blinded to vector assignments. Results Subretinal FIV vector injections were much more effective than intravitreal injections. The RPE was the principal retinal layer transduced by the WT vector, and at least 50% of the area of the retina expressed the marker gene at 3 and 7 months. Occasional cells in inner retinal layers also expressed ,-galactosidase at these time points. The sustained retinal expression produced by subretinally injected vector was blocked by the D66V mutation. Conclusions These results show that class I integrase mutant FIV vectors are useful control vectors, and that VSV-G-pseudotyped FIV vectors produce extensive retinal expression for at least 215 days, the longest duration yet reported for lentiviral vectors in retina. Transgene expression is mostly restricted to RPE after post-natal day 7 in rats, suggesting that FIV vectors could be used to target RPE for gene therapy. Copyright © 2003 John Wiley & Sons, Ltd. [source] Characterization of selenocysteine methyltransferases from Astragalus species with contrasting selenium accumulation capacityTHE PLANT JOURNAL, Issue 1 2009Thomas G. Sors Summary A group of selenium (Se)-hyperaccumulating species belonging to the genus Astragalus are known for their capacity to accumulate up to 0.6% of their foliar dry weight as Se, with most of this Se being in the form of Se -methylselenocysteine (MeSeCys). Here, we report the isolation and molecular characterization of the gene that encodes a putative selenocysteine methyltransferase (SMT) enzyme from the non-accumulator Astragalus drummondii and biochemically compare it with an authentic SMT enzyme from the Se-hyperaccumulator Astragalus bisulcatus, a related species that lives within the same native habitat. The non-accumulator enzyme (AdSMT) shows a high degree of homology with the accumulator enzyme (AbSMT) but lacks the selenocysteine methyltransferase activity in vitro, explaining why little or no detectable levels of MeSeCys accumulation are observed in the non-accumulator plant. The insertion of mutations on the coding region of the non-accumulator AdSMT enzyme to better resemble enzymes that originate from Se accumulator species results in increased selenocysteine methyltransferase activity, but these mutations were not sufficient to fully gain the activity observed in the AbSMT accumulator enzyme. We demonstrate that SMT is localized predominantly within the chloroplast in Astragalus, the principal site of Se assimilation in plants. By using a site-directed mutagenesis approach, we show that an Ala to Thr amino acid mutation at the predicted active site of AbSMT results in a new enzymatic capacity to methylate homocysteine. The mutated AbSMT enzyme exhibited a sixfold higher capacity to methylate selenocysteine, thereby establishing the evolutionary relationship of SMT and homocysteine methyltransferase enzymes in plants. [source] A Single Point Mutation Reverses the Enantiopreference of Thermoanaerobacter ethanolicus Secondary Alcohol DehydrogenaseCHEMCATCHEM, Issue 1 2009The asymmetric reduction of benzylic and heteroaryl ketones to the corresponding (R)-alcohols using I86A Thermoanaerobacter ethanolicus alcohol dehydrogenase (I86A TeSADH) is described. This single amino acid mutation not only makes the active site of I86A TeSADH able to accommodate more sterically demanding substituents than those accommodated by wild-type TeSADH, but it also reverses the substrate stereospecificity of TeSADH. [source] Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee-infectious HC-J4 isolate to replicate efficiently in Huh-7 cellsJOURNAL OF VIRAL HEPATITIS, Issue 5 2004S. Maekawa Summary., Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh-7 cells. To extend the previous results to other isolated HCV clones, we constructed another HCV replicon from HC-J4, one of chimpanzee-infectious HCV clones. An HCV replicon derived from HC-J4 (RpJ4) consists of HCV-5, untranslated region, neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural region, NS3 to NS5B, and HCV-3, untranslated region. The adaptive mutations known to be required for HCV-Con1 replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC-J4) 2197 serine to proline, deletion of serine at aa.2201, and aa.2204 serine to isoleucine (RpJ4-S2197P, RpJ4-S22001del, and RpJ4-S2204I). RpJ4/ISDR mutant and RpJ4-S2201del/ISDR mutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). After transfection into Huh-7 cells and G418 selection, RpJ4 and RpJ4/ISDR mutants did not produce any colony. In contrast, G418-resistant cells were transduced efficiently by RpJ4-S2197P, RpJ4-S2204I, RpJ4-S2201del and RpJ4-S2201del/ISDR mutant, with the RpJ4-S2201del/ISDR mutant being most efficient. Hence the HCV replicon derived from HC-J4 can replicate efficiently following the introduction of adaptive mutations into the upstream region of ISDR. Moreover, additional introduction of mutations into ISDR further enhanced its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV replications. [source] NS5A mutations predict biochemical but not virological response to interferon-, treatment of sporadic hepatitis C virus infection in European patientsJOURNAL OF VIRAL HEPATITIS, Issue 4 2001I. Stratidaki The NS5A region of the hepatitis C virus (HCV) genome has been reported by Japanese but not European investigators to be a significant factor in predicting interferon (IFN) response patients with HCV of genotype 1. We correlated the NS5A region with treatment outcome in patients with sporadic HCV infection. Twenty-eight patients (10 men, 18 women, mean age 60 ± 2 years) with histologically proven HCV chronic hepatitis, genotype 1b, were treated with 6 MU IFN-, for 6 months. The 6954,7073 area of the NS5A region was directly sequenced for nucleotide and amino acids mutations and the results were related to biochemical and virological response. None of the patients had a strain with nucleotide sequence identical to the Japanese HCV-J. However, in five strains the nucleotide mutations led to synonymous amino acids and the amino acid sequences were identical to the prototype Japanese strain. Only 2/28 patients had four or more amino acid mutations (mutant strains) while 21 demonstrated an intermediate type and five belonged to the wild-type. The most frequent non-synonymous substitution was at position 6982 (A,G) corresponding to an amino acid change at codon 2218 (His,Arg). All patients with the wild-type were biochemical nonresponders while the two patients with the mutant strains had a sustained biochemical response. Twenty-three percent of the intermediate type had a sustained biochemical response. NS5A mutations predict the biochemical but not the virological response of patients. Virological response was poor and unrelated to the type of HCV strain. Biochemical responders had significantly lower amino acid mutations (1.14 ± 0.19) compared with nonresponders (2.57 ± 1.4, P < 0.003) as well as lower aminotransferase values (P < 0.01). Hence, mutational analysis of the NS5A region showed that our patients have a mutational profile similar to the European studies with a wild-type that is slightly different from the Japanese HCV-J sequence. The biochemical, but not the virological response to IFN-, is similar to the Japanese studies, with no response of the patients with wild-type sequence, a good response in the limited number of patients with mutant strains and 23% response rate in the patients with intermediate type sequences. [source] A pH-Based High-Throughput Screening of Sucrose-Utilizing Transglucosidases for the Development of Enzymatic Glucosylation ToolsCHEMCATCHEM, Issue 8 2010Elise Champion Dr. Abstract Sucrose-utilizing transglucosidases are valuable enzymatic tools for the diversification of carbohydrate-based molecules. Among them, recombinant amylosucrase from Neisseria polysaccharea is a glucansucrase that naturally catalyzes the synthesis of an amylose-like polymer as well as the transglucosylation of exogenous hydroxylated acceptors. A semirational engineering approach was recently undertaken to redesign the enzyme active site and adapt it to the glucosylation of a nonnatural acceptor, allyl 2- N -acetyl-2-deoxy-,- D -glucopyranoside (,- D -GlcpNAcOAll), to produce a key building block in the chemoenzymatic synthesis of Shigella flexneri 1b,serotype O-antigen repeating unit. This prior work shows the beneficial effect of single amino acid mutations at two positions (228 and 290) on the recognition of the acceptor by amylosucrase. On the basis of these first results, a library of about 8000 amylosucrase variants combining mutations at these two positions is constructed by saturation mutagenesis. The library is prescreened using a novel pH-sensitive colorimetric screening method for the detection of sucrose-utilizing amylosucrase variants, thereby reducing by about 95,% the size of the library to be subsequently screened for acceptor glucosylation. Active clones (5,% of the initial library) are then screened for acceptor recognition, leading to the isolation of 20 variants of potential interest for the production of the target disaccharide ,- D -Glcp-(1,4)-,- D -GlcpNAc. [source] Influence of the hydrophilic face on the folding ability and stability of ,-helix bundles: relevance to the peptide catalytic activityCHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2000S.E. Blondelle Although not the sole feature responsible, the packing of amino acid side chains in the interior of proteins is known to contribute to protein conformational specificity. While a number of amphipathic peptide sequences with optimized hydrophobic domains has been designed to fold into a desired aggregation state, the contribution of the amino acids located on the hydrophilic side of such peptides to the final packing has not been investigated thoroughly. A set of self-aggregating 18-mer peptides designed previously to adopt a high level of ,-helical conformation in benign buffer is used here to evaluate the effect of the nature of the amino acids located on the hydrophilic face on the packing of a four ,-helical bundle. These peptides differ from one another by only one to four amino acid mutations on the hydrophilic face of the helix and share the same hydrophobic core. The secondary and tertiary structures in the presence or absence of denaturants were determined by circular dichroism in the far- and near-UV regions, fluorescence and nuclear magnetic resonance spectroscopy. Significant differences in folding ability, as well as chemical and thermal stabilities, were found between the peptides studied. In particular, surface salt bridges may form which would increase both the stability and extent of the tertiary structure of the peptides. The structural behavior of the peptides may be related to their ability to catalyze the decarboxylation of oxaloacetate, with peptides that have a well-defined tertiary structure acting as true catalysts. [source] | ||||||||||