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Glutathione S-transferase from the Icelandic scallop (Chlamys islandica):: Isolation and partial characterization

COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY 2007年第4期144卷 403-407页

作者： Myrnes, Bjornar Nilsen, Inge W. Norwegian Inst Fisheries & Aquaculture N-9291 Tromso Norway

Glutathione S-transferase from the digestive gland of the cold-adapted marine bivalve Icelandic scallop was purified to apparent homogeneity by single GSTrap chromatography. The enzyme appeared to be a homodimer with subunit M-r 22,000 having an optimum catalytic activity at pH 6.5-7. Enzymatic analysis of scallop GST using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione resulted in apparent values K-m(GST) and K-m(DNB) for and KC of 0.3 mM and 0.4 mM, respectively. The scallop GST lost activity faster than porcine GST when exposed to increased temperatures, but both enzymes needed 10 min incubation at 60 degrees C for complete inactivation. A partial coding sequence was identified in cDNA synthesised from digestive gland mRNA. Comparison to known sequences indicates that the gene product is a glutathione S-transferase, and the predicted Icelandic scallop GST protein scores 40% sequence identity and 60% sequence similarity to mu-class proteins. (c) 2006 Elsevier Inc. All rights reserved.

关键词： invertebrate Icelandic scallop GST protein coding sequence purification

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Improved tropoelastin synthesis in the skin by codon optimization and nucleotide modification of tropoelastin-encoding synthetic mRNA

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MOLECULAR THERAPY-NUCLEIC ACIDS 2023年 33卷 642-654页

作者： Golombek, Sonia Hoffmann, Thomas Hann, Ludmilla Mandler, Markus Schmidhuber, Sabine Weber, Josefin Chang, Young-Tae Mehling, Roman Ladinig, Andrea Knecht, Christian Leyens, Johanna Schlensak, Christian Wendel, Hans Peter Schneeberger, Achim Avci-Adali, Meltem Univ Hosp Tubingen Dept Thorac & Cardiovasc Surg Calwerstr 7-1 D-72076 Tubingen Germany Accanis Biotech Karl Farkas Gasse 22 A-1030 Vienna Austria Pohang Univ Sci & Technol POSTECH Dept Chem Republ Koreea Pohang 37673 South Korea Eberhard Karls Univ Tubingen Werner Siemens Imaging Ctr Dept Preclin Imaging & Radiopharm Rontgenweg 13 D-72076 Tubingen Germany Univ Vet Med Univ Clin Swine Dept Farm Anim & Vet Publ Hlth Veterinarpl 1 A-1210 Vienna Austria Univ Hosp Tubingen Dept Thorac & Cardiovasc Surg Calwerstr 7-1 D-72076 Tubingen Germany

Loss of elastin due to aging, disease, or injury can lead to impaired tissue function. In this study, de novo tropoelastin (TE) synthesis is investigated in vitro and in vivo using different TE-encoding synthetic mRNA variants after codon optimization and nucleotide modification. Codon optimization shows a strong effect on protein synthesis without affecting cell viability in vitro, whereas nucleotide modifica-tions strongly modulate translation and reduce cell toxicity. Selected TE mRNA variants (3, 10, and 30 & mu;g) are then analyzed in vivo in porcine skin after intradermal application. Administration of 30 & mu;g of native TE mRNA with a me1 111 modification or 10 and 30 & mu;g of unmodified codon-optimized TE mRNA is required to increase TE protein expression in vivo. In contrast, just 3 & mu;g of a codon-optimized TE mRNA variant with the me1 111 modification is able to increase protein expression. Furthermore, skin toxicity is investigated in vitro by injecting 30 & mu;g of mRNA of selected TE mRNA variants into a human full-thickness skin model, and no toxic effects are observed. Thereby, for the first time, an increased dermal TE synthesis by exogenous administration of synthetic mRNA is demonstrated in vivo. Codon optimization of a synthetic mRNA can significantly increase protein expression and therapeutic outcome.

关键词： MT: Oligonucleotides: Therapies and Applications synthetic mRNA tropoelastin protein replacement therapy tissue regeneration mRNA optimization coding sequence nucleotide modification de novo synthesis

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sequence AND FUNCTIONAL-CHARACTERIZATION OF THE HUMAN PURINE NUCLEOSIDE PHOSPHORYLASE PROMOTER

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NUCLEIC ACIDS RESEARCH 1991年第18期19卷 5015-5020页

作者： JONSSON, JJ WILLIAMS, SR MCIVOR, RS UNIV MINNESOTA CTR HLTHINST HUMAN GENETBOX 206MINNEAPOLISMN 55455 UNIV MINNESOTA DEPT LAB MED & PATHOLMINNEAPOLISMN 55455 GENENTECH INC S SAN FRANCISCOCA 94080

Purine nucleoside phosphorylase (PNP) is a ubiquitously expressed enzyme which contributes to the catabolism and recycling of nucleotides. To characterize the promoter region of the human PNP gene, the nucleotide sequence from a BamHI site located in the 5' untranslated region extending 2237 bp upstream to an XbaI site was determined. The transcriptional start site as determined by primer extension was 119 bp upstream of the coding sequence and consisted of a 5'-CA-3' dimer with A at + 1. A TATA box was identified -24 to -29 bp upstream of the transcriptional start site. A CCAAT pentamer sequence in the inverted orientation was present at -51 to -55 bp and two GC rich regions were identified at -68 to -81 bp and -168 to -187 bp. Progressive 5' deletions of the 5' flanking region were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and transient expression measured after transfection of murine NIH/3T3 fibroblasts. A 91 bp promoter (the shortest tested) provided CAT activity at 60% the level of a 216 bp promoter, possibly due to removal of the GC rich region between -168 and -187 bp. Longer promoters resulted in CAT expression at similar or lower levels than the 216 bp promoter indicating that this region contained all of the 5' flanking sequences affecting transcription from the PNP promoter.

关键词： tr Plato's number PNP gene Upstream Anatomic Site promoter regions Transcription Initiation Site sequences TATA Box coding sequence GC Rich sequence A-91 Purine Nucleoside Phosphorylase Promoter transient expression

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Mutant fibrillin-1 monomers lacking EGF-like domains disrupt microfibril assembly and cause severe Marfan syndrome

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HUMAN MOLECULAR GENETICS 1996年第10期5卷 1581-1587页

作者： Liu, WG Qian, CP Comeau, K Brenn, T Furthmayr, H Francke, U STANFORD UNIV MED CTR BECKMAN CTR HOWARD HUGHES MED INST STANFORD CA 94305 USA STANFORD UNIV MED CTR DEPT PATHOL STANFORD CA 94305 USA STANFORD UNIV MED CTR DEPT GENET STANFORD CA 94305 USA

Marfan syndrome (MFS), a heritable connective tissue disorder, is caused by mutations in the gene coding for fibrillin-1 (FBN1), an extracellular matrix protein, One of the three major categories of FBN1 mutations involves exon-skipping, To rapidly detect such mutations, we developed a long RT-PCR method, Either three segments covering the entire FBN1 coding sequence or a single 8.9 kb FBN1 coding segment were amplified from reverse-transcribed total fibroblast RNA, Restriction fragment patterns of these RT-PCR products were compared and abnormal fragments were directly sequenced, Six exon-skipping mutations were identified in a panel of 60 MFS probands, All skipped exons encode calcium binding epidermal growth factor (EGF)-like domains and maintain the reading frame, In five probands, exon-skipping was due to point mutations in splice site sequences, and one had a 6 bp deletion in a donor splice site, Pulse-chase analysis of labelled fibrillin protein revealed normal levels of synthesis but significantly reduced matrix deposition, This dominant-negative effect of the mutant monomers is considered in the light of current models of fibrillin assembly, Probands with this type of FBN1 mutation include the most severe forms of MFS, such as neonatally lethal presentations.

关键词： .restriction monomers coding sequence probands Marfan syndrome EGF-like FBN mutations microfibril Mutant fibrillin long RT–PCR exon-skipping mutations

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Identification and functional characterization of a novel R621C mutation in the synphilin-1 gene in Parkinson's disease

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HUMAN MOLECULAR GENETICS 2003年第11期12卷 1223-1231页

作者： Marx, FP Holzmann, C Strauss, KM Li, L Eberhardt, O Gerhardt, E Cookson, MR Hernandez, D Farrer, MJ Kachergus, J Engelender, S Ross, CA Berger, K Schöls, L Schulz, JB Riess, O Krüger, R Univ Tubingen Dept Neurol Lab Neurodegenerat D-72076 Tubingen Germany Univ Rostock Dept Med Genet Rostock Germany Tongji Med Univ Union Hosp Dept Tradit Chinese Med Wuhan Peoples R China NIA Neurogenet Lab Bethesda MD 20892 USA Mayo Clin Neurogenet Lab Jacksonville FL USA Technion Israel Inst Technol Bruce Rappaport Fac Med Dept Pharmacol IL-31096 Haifa Israel Johns Hopkins Univ Sch Med Dept Psychiat Div Neurobiol Baltimore MD 21205 USA Univ Munster Dept Epidemiol & Social Med D-4400 Munster Germany Univ Bochum Bochum Germany Univ Tubingen Dept Med Genet Tubingen Germany

Synphilin-1 is linked to the pathogenesis of Parkinson's disease (PD) based on its identification as an alpha-synuclein (PARK1) and parkin (PARK2) interacting protein. Moreover, synphilin-1 is a component of Lewy bodies (LB) in brains of sporadic PD patients. Therefore, we performed a detailed mutation analysis of the synphilin-1 gene in 328 German familial and sporadic PD patients. In two apparently sporadic PD patients we deciphered a novel C to T transition in position 1861 of the coding sequence leading to an amino acid substitution from arginine to cysteine in position 621 (R621C). This mutation was absent in a total of 702 chromosomes of healthy German controls. To define a possible role of mutant synphilin-1 in the pathogenesis of PD we performed functional analyses in SH-SY5Y cells. We found synphilin-1 capable of producing cytoplasmic inclusions in transfected cells. Moreover we observed a significantly reduced number of inclusions in cells expressing C621 synphilin-1 compared with cells expressing wild-type (wt) synphilin-1, when subjected to proteasomal inhibition. C621 synphilin-1 transfected cells were more susceptible to staurosporine-induced cell death than cells expressing wt synphilin-1. Our findings argue in favour of a causative role of the R621C mutation in the synphilin-1 gene in PD and suggest that the formation of intracellular inclusions may be beneficial to cells and that a mutation in synphilin-1 that reduces this ability may sensitize neurons to cellular stress.

关键词： .Parkinson Disease sporadic interacting coding sequence Functional Characterization apparently arginine to cysteine synphilin PD patients

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Cloning and expression of neuron-specific enolase in the corpus luteum of dairy goats

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GENE 2012年第2期503卷 222-228页

作者： Meng, Xia Li, Feihu Chen, Shulin Tang, Caiyan Zhang, Wenhua Wang, Zhonghui Zhao, Shanting NW A&F Univ Coll Vet Med Yangling 712100 Shaanxi Peoples R China

Neuron-specific enolase (NSE) is the key molecular marker for diffuse neuroendocrine system (DNES) cells, its expression in the pregnant corpus luteum (CL) of dairy goats was studied by the immunofluorescence method and the ultra structural characteristics of luteal cells were detected by the electron microscopy to identify the existence of DNES cells in the pregnant CL of dairy goats. Besides, the coding sequence of dairy goats NSE gene was cloned and its biological information was analyzed. Results revealed that NSE immuno-positive cells exhibited widespread cytoplasmic staining throughout the whole pregnant CL In addition, these cells showed typical characteristics of DNES cells in the electron microscopy. These results suggested that many DNES cells exist in the pregnant CL of dairy goats. Meanwhile, we identified the coding sequence of dairy goats NSE (GenBank Accession No. JN887466). Its nucleotide sequence homology was found to be 97.9%, 89.3%, 90% and 92.6%, respectively, compared with that of Bos taurus, Rattus norvegicus, Mus musculus and Homo sapiens, while the amino acid sequence homology was 99.1%, 97%, 97.2% and 98.2% respectively. These results first showed that the functional amino acids coded by the NSE gene were highly conserved in Caprine, B. taurus, R. norvegicus, M. musculus and H. sapiens. It was implied that the gene NSE in dairy goats had close homology to that of NSE of other species. Our findings demonstrated the possible existence of DNES cells in pregnant CL, providing new clue for further understanding of interactions between the neuroendocrine and reproductive systems. Characterization of gene sequence of dairy goats NSE will enable us to synthesize interference RNA for further study on the role of NSE on the formation, function and apoptosis of pregnant CL in dairy goats. (C) 2012 Elsevier B.V. All rights reserved.

关键词： Neuron-specific enolase Corpus luteum Diffuse neuroendocrine system Immunofluorescence staining Molecular cloning coding sequence

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Classification studies based on a spectral representation of DNA

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JOURNAL OF THEORETICAL BIOLOGY 2010年第4期266卷 667-674页

作者： Bielinska-Waz, Dorota Subramaniam, Shankar Uniwersytet Mikolaja Kopernika Inst Fizyki PL-87100 Torun Poland Univ Calif San Diego Dept Bioengn San Diego CA 92093 USA Univ Calif San Diego Dept Chem San Diego CA 92103 USA Univ Calif San Diego Dept Biochem San Diego CA 92103 USA

The aim of this paper is to provide a new tool to classify the nucleic acid sequences. The profiles based on the values characterizing DNA sequences (descriptors derived from recently introduced graphical representation) are used as a basis of classification schemes related to different aspects of similarity of the sequences. New multicomponent similarity measures based on these descriptors have been defined. Each component of the new measures can be analyzed separately. The new measures are consistent with the standard ones but they contain more detailed information. In particular, it has been shown that within the new approach one can define a quantity which converges to the standard similarity measure. An application of the multicomponent similarity measure to families of histone sequences demonstrates the power and efficiency of the method. (c) 2010 Elsevier Ltd. All rights reserved.

关键词： Descriptors Similarity/dissimilarity analysis coding sequence Distribution moments

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Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco

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PLANT PHYSIOLOGY 1999年第4期119卷 1507-1516页

作者： Garcia, I Rodgers, M Pepin, R Hsieh, TF Matringe, M Rhone Poulenc CNRS UMR 41 F-69263 Lyon 09 France Rhone Poulenc Agrochim Dept Biotechnol F-69263 Lyon 09 France Texas A&M Univ Dept Biol College Stn TX 77843 USA

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We. performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

关键词： Recombinant Proteins Arabidopsis Enzymatic activity immunodetection COMPARTMENT 4-hydroxyphenylpyruvate 4-Hydroxyphenylpyruvate Dioxygenase Biochemical characteristics leaf extracts coding sequence Tobacco Products Dioxygen Dioxygenases Subcellular

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IDENTIFICATION OF PROTEIN-coding REGIONS IN GENOMIC DNA

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JOURNAL OF MOLECULAR BIOLOGY 1995年第1期248卷 1-18页

作者： SNYDER, EE STORMO, GD UNIV COLORADO DEPT MOLEC CELLULAR & DEV BIOL BOULDER CO 80309 USA

We have developed a computer program, GeneParser, which identifies and determines the fine structure of protein genes in genomic DNA sequences. The program scores all subintervals in a sequence for content statistics indicative of introns and exons, and for sites that identify their boundaries. This information is weighted by a neural network to approximate the log-likelihood that each subinterval exactly represents an intron or exon (first, internal or last). A dynamic programming algorithm is then applied to this data to find the combination of introns and exons that maximizes the likelihood function. Using this method, we can rapidly generate ranked suboptimal solutions, each of which is the optimum solution containing a given intron-exon junction. We have tested the system on a large collection of human genes. On sequences not used in training, we achieved a correlation coefficient for exon nucleotide prediction of 0.89. For a subset of G + C-rich genes, a correlation coefficient of 0.94 was achieved. We have also quantified the robustness of the method to substitution and frame-shift errors and show how the system can be optimized for performance on sequences with known levels of sequencing errors.

关键词： GENE IDENTIFICATION EXON STRUCTURE coding sequence ARTIFICIAL INTELLIGENCE DYNAMIC PROGRAMMING

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Wild-derived XY sex-reversal mutants in the medaka, Oryzias latipes

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GENETICS 2006年第4期173卷 2083-2090页

作者： Otake, Hiroyuki Shinomiya, Ai Matsuda, Masaru Hamaguchi, Satoshi Sakaizumi, Mitsuru Niigata Univ Grad Sch Sci & Technol Niigata 9502181 Japan Niigata Univ Dept Environm Sci Fac Sci Niigata 9502181 Japan Natl Inst Basic Biol Reprod Biol Lab Okazaki Aichi 4448585 Japan

The medaka, Oryzias latipes, has an XX/XY sex-determination mechanism. A Winked DM domain gene, DMY has been isolated by positional cloning as a sex-determining gene in this species. Previously, we found 23 XY sex-reversed females from 11 localities by examining the genotypic sex of wild-caught medaka. Genetic analyses revealed that all these females had Y-linked gene mutations. Here, we aimed to clarify the cause of this sex reversal. To achieve this, we screened for mutations in the amino acid coding sequence of DMY and examined DMY expression at 0 days after hatching (dah) using densitometric semiquantitative RT-PCR. We found that the mutants could be classified into two groups. One contained mutations in the amino acid coding sequence of DMY while the other had reduced DMY expression at 0 dab although the DMY coding sequence was normal. For the latter, histological analyses indicated that (YYwOur)-Y-wOur (Y-wOur, Y chromosome derived from an Oura XY female) individuals with the lowest DMY expression among the tested mutants were expected to develop into females at 0 dah. These results suggest that early testis development requires DMY expression above a threshold level. Mutants with reduced DMY expression may prove valuable for identifying DMY regulatory elements.

关键词： .localities coding sequence sex-determining latipes sex reversal wild-derived XY female medaka Oryzias DM domain XY Sex-Reversal Mutants DMY expression reduced DMY

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