Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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A deoxyribonuclease (DNase) was isolated from viscera of the cold-adapted marine bivalve Icelandic scallop. The 42 kDa DNase was shown to be a single polypeptide which catalyses DNA hydrolysis in the absence of divalent cations. The isolated enzyme showed maximal activity at pH 6 and no activity above pH 7.2 against native DNA. The scallop DNase was slightly more susceptible to heat denaturation than porcine DNase II and makes double-strand breaks in circular DNA substrate as the porcine enzyme. The N-terminal sequence of the scallop DNase was shown to be closely similar to DNase II (EC 3.1.22.1) proteins from other organisms. The scallop DNase is in addition to plancitoxin I from A. planci, the only DNase II enzyme isolated from marine invertebrates.
Comp Biochem Physiol B Biochem Mol Biol 2006 Mar
PMID:Deoxyribonuclease II from the Icelandic scallop (Chlamys islandica): isolation and partial characterization. 1642 84

Xenopus oocytes have been utilized in a number of laboratories as an experimental system to study a variety of biological processes. Here, we describe its application to functional studies of spliceosomal small nuclear RNAs (snRNAs) in pre-messenger RNA (pre-mRNA) splicing, a process that occurs extremely efficiently in Xenopus oocytes. A DNA oligonucleotide complementary to an snRNA of interest is injected into the oocyte cytoplasm. The oligonucleotide subsequently diffuses into the nucleus and hybridizes to the target snRNA, thereby triggering snRNA degradation via endogenous RNase H activity. By the time the endogenous snRNA is depleted, the DNA oligonucleotide itself is degraded by endogenous deoxyribonuclease (DNase) activity. In principle, this procedure enables one to quantitatively deplete any snRNA of choice. Subsequently, a rescuing snRNA that is constructed in vitro may be injected into the snRNA-depleted oocytes to restore the splicing function. After reconstitution, a radiolabeled splicing substrate is injected into the nuclei of the oocytes. These oocyte nuclei are then manually isolated and used to prepare both nuclear RNA for splicing assays and nuclear extract for spliceosome assembly assays. The ability of an injected rescuing snRNA to reconstitute splicing can therefore be tested. Because all types of rescuing snRNAs (e.g., mutant snRNAs, snRNAs with or without modified nucleotides) can be constructed readily, the results obtained from this procedure provide valuable information on the function of a particular snRNA of interest in pre-mRNA splicing.
Methods Mol Biol 2006
PMID:Pre-mRNA splicing in the nuclei of Xenopus oocytes. 1673 22

Failure of ligamentous support of the genital tract to resist intra-abdominal pressure is a plausible underlying mechanism for the development of pelvic organ prolapse, but the nature of the molecular response of pelvic tissue support remains unknown. We hypothesized that the expression of genes coding for proteins involved in maintaining the cellular and extracellular integrity would be altered as a result of mechanical stretch. Therefore, cDNA microarrays were used to examine the difference in transcriptional profile in RNA of primary culture fibroblasts subjected to mechanical stretch and those that remained static. Out of 34 mechano-responsive genes identified (P < 0.05), four were coding for regulation of actin cytoskeleton remodelling, and its interaction with the extracellular matrix proteins; these are phosphatidyl inositol-4-phosphate 5-kinase (PIP5K1C), the human signal-induced proliferation associated gene-1 (SIPA-1), TNFRSF1A-associated via death domain (TRADD) and deoxyribonuclease 1-like 1 (DNase 1-L1). The transcriptosomal changes led us to investigate the phenotypic consequences of stretch, levormeloxifene and estradiol (E(2)) on the cytoskeleton of cultured fibroblasts. The percentage of cells with abnormal F-actin configuration was significantly higher in fibroblasts subjected to stretch compared with the static model (P < 0.0001). Levormeloxifene caused similar significant alterations in actin morphology of the static fibroblasts. The use of E(2) did not reverse the process or protect the cells from the effect of stretch, but significantly increased the rate of fibroblast proliferation, suggestive of a role in healing process. Mechanical stretch and/or levormeloxifene disturb the fibroblasts ability to maintain the cytoskeleton architecture and we speculate that they may disrupt ligamentous integrity and result in clinical prolapse.
Mol Hum Reprod 2008 Feb
PMID:Changes in transcription profile and cytoskeleton morphology in pelvic ligament fibroblasts in response to stretch: the effects of estradiol and levormeloxifene. 1818 56

The protocol described in this unit is a fast and streamlined procedure for preparing total cytoplasmic RNA from many cultures simultaneously for nuclease protection analysis. It is scaled for small cultures--1 to 2 dishes of adherent cells or 10 to 20 ml of a suspension culture. The procedure works well for many cell types. If full-length RNA is required, ribonuclease inhibitors should be added to the lysis buffer (as described in this unit) or the guanidinium isothiocyanate method should be used. Finally, if RNA is isolated from transiently transfected cells, a is provided for the treatment of RNA with deoxyribonuclease to remove transfected DNA. This modification is especially critical if the RNA is to be assayed by nuclease protection using uniformly labeled probes.
Curr Protoc Mol Biol 2002 May
PMID:Preparation of cytoplasmic RNA from tissue culture cells. 1826 14

Enzymes immobilized on solid-phase matrices have found various applications in biotechnology, molecular biology and molecular diagnostics and can serve as industrial catalysts and as specific reagents for analytical procedures. A wide range of supports have been utilized for immobilization among which particle-based supports are the most commonly implemented. Type of support used for immobilization is one of the key considerations in practical application due to different immobilization efficiency, ligand utilization and the mass transfer regime. The mass transfer between the mobile and the particulate stationary phase is often a bottleneck for the entire process due to slow pore diffusion of large molecules. In contrast, monoliths due to their structure enable almost flow-independent properties. Consequently, the overall behavior of the immobilized ligand reflects its intrinsic reaction kinetics. Therefore, such an immobilized system is expected to allow higher throughput because of higher enzyme efficiency, especially pronounced for macromolecular substrates having low mobility. In this work, different methods for immobilization of enzymes on Convective Interaction Media monolithic supports are presented. In particular, enzymes acting on macromolecular substrates, such as trypsin, deoxyribonuclease and ribonuclease, are described in detail. Immobilized efficiency is evaluated for different immobilization procedures in terms of biologic activity and long-term stability. Finally, their performance on real samples is demonstrated.
Methods Mol Biol 2008
PMID:Monolithic bioreactors for macromolecules. 1882 60

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair that allows for the enhanced repair of the transcribed strand of active genes. A classical method to study DNA repair in vivo consists in the molecular analysis of UV-induced DNA damages at specific loci. Cells are irradiated with a defined dose of UV light leading to the formation of DNA lesions and incubated in the dark to allow repair. About 90% of the photoproducts consist of cyclobutane pyrimidine dimers, which can be cleaved by the DNA nicking activity of the T4 endonuclease V (T4endoV) repair enzyme. Strand-specific repair in a suitable restriction fragment is determined by alkaline gel electrophoresis followed by Southern blot transfer and indirect end-labeling using a single-stranded probe. Recent approaches have assessed the role of transcription factors in TCR by analyzing RNA polymerase II occupancy on a damaged template by chromatin immunoprecipitation (ChIP). Cells are treated with formaldehyde in vivo to cross-link proteins to DNA and enrichment of a protein of interest is done by subsequent immunoprecipitation. Upon reversal of the protein-DNA cross-links, the amount of coprecipitated DNA fragments can be detected by quantitative PCR. To perform ChIP on UV-damaged templates, we included an in vitro photoreactivation step prior to PCR analysis to ensure that all precipitated DNA fragments serve as substrates for the PCR reaction. Here, we provide a detailed protocol for both the DNA repair analysis and the ChIP approaches to study TCR in chromatin.
Methods Mol Biol 2009
PMID:Methods to study transcription-coupled repair in chromatin. 1938 41

PTH regulates transcription of a number of genes involved in bone remodeling and calcium homeostasis. We have previously shown that the matrix metalloproteinase-13 (MMP-13) gene is induced by PTH in osteoblastic cells as a secondary response through the protein kinase A pathway requiring the runt domain and activator protein 1 binding sites of the proximal promoter. Here, we investigated the changes PTH causes in histone acetylation in this region (which contains the only deoxyribonuclease-hypersensitive sites in the promoter) leading to MMP-13 gene activation in these cells. Chromatin immunoprecipitation experiments revealed that PTH rapidly increased histone H4 acetylation followed by histone H3 acetylation associated with the different regions of the MMP-13 proximal promoter. The hormone also stimulated p300 histone acetyl transferase activity and increased p300 bound to the MMP-13 proximal promoter, and this required protein synthesis. Upon PTH treatment, Runx2, already bound to the runt domain site of the MMP-13 promoter, interacted with p300, which then acetylated histones H4 and H3. The knockdown of either Runx2 or p300 by RNA interference reduced PTH-induced acetylation of histones H3 and H4, association of p300 with the MMP-13 promoter, and resultant MMP-13 gene transcription. Overall, our studies suggest that without altering the gross chromatin structure, PTH stimulates acetylation of histones H3 and H4 via recruitment of p300 to Runx2 bound to the MMP-13 promoter, resulting in gene activation. This work establishes the molecular basis of transcriptional regulation in osteoblasts by PTH, a hormone acting through a G-protein coupled receptor.
Mol Endocrinol 2009 Aug
PMID:Runx2 recruits p300 to mediate parathyroid hormone's effects on histone acetylation and transcriptional activation of the matrix metalloproteinase-13 gene. 1942 55

This review describes the nature and applications of ribosome inactivating proteins (RIPs) from Momordica charantia (bitter melon). RIPs from the plant kingdom have received much attention in biomedical research because they target conserved host protein synthesis machinery and show specificity towards human and animal cell targets. Recent studies aimed at unravelling the enzymatic activities of the M charantia RIPs provide a structural basis for their activities. It has been reported that RIPs are member of the single chain ribosome inactivating protein (SCRIP) family which act irreversibly on ribosome by removing adenine residue from eukaryotic ribosomal RNA. Various activities of RIPs include anti-tumor, broad anti-viral, ribonuclease and deoxyribonuclease. MAP30 (Momordica Anti-HIV Protein), alpha- and beta-momorcharins inhibit HIV replication in acutely and chronically infected cells and thus are considered potential therapeutic agent in HIV infection and AIDS. Further, MAP30 improved the efficacy of anti-HIV therapy when used in combination with other anti-viral drugs. MAP30 holds therapeutic promise over other RIPs because not only it is active against infection and replication of both HSV and HIV but is non toxic to normal cells. Here we review the nature, action, structure function relationship and applications of RIPs from Momordica charantia and evaluate their potential for anti-cancer and anti-viral therapy.
Curr Mol Med 2009 Dec
PMID:Ribosome inactivating proteins (RIPs) from Momordica charantia for anti viral therapy. 2038 79

Nick translation is the name given to a reaction that is used to replace cold nucleoside triphosphates in a double-stranded DNA molecule with radioactive ones (1,2). Free 3'-hydroxyl groups are created within the unlabeled DNA (nicks) by deoxyribonuclease 1 (DNAse 1). DNA polymerase 1 from E. coli will then catalyze the addition of a nucleotide residue to the 3'-hydroxyl terminus of the nick. At the same time, the 5'- to 3'-exonuclease activity of this enzyme will eliminate the nucleotide unit from the 5'-phosphoryl terminus of the nick. Thus a new nucleotide with a free 3'-OH group will have been incorporated at the position where the original nucleotide was excised, and the nick will have been shifted along by one nucleotide unit in a 3' direction. This 3' shift, or translation, of the nick will result in the sequential addition of new nucleotides to the DNA while the pre-existing nucleotides will be removed. If radioactively labeled deoxyribonucleoside triphosphates are used as substrates, up to 50% of the residues in the DNA can be labeled.Furthermore, Rigby et al. have shown (2) that the DNA is labeled throughout at a uniform specific activity, which is an important requirement if the DNA is to be used as a probe in molecular hybridization experiments.
Methods Mol Biol 1985
PMID:Radiolabeling of DNA by nick translation. 2137 2

Intramuscular injection of naked plasmid DNA is known (1-3) to elicit humoral and cell-mediated immune responses against the encoded antigen. It is thought (2,3) that immunity follows DNA uptake by muscle cells, leading to the expression and extracellular release of the antigen which is then taken up by antigen presenting cells (APC). In addition, it is feasible that some of the injected DNA is taken up directly by APC. Disadvantages (1-3) of naked DNA vaccination include: uptake of DNA by only a minor fraction of muscle cells, exposure of DNA to deoxyribonuclease in the interstitial fluid thus necessitating the use of relatively large quantities of DNA, and, in some cases, injection into regenerating muscle in order to enhance immunity. We have recently proposed (1,4) that DNA immunization via liposomes (phospholipid vesicles) could circumvent the need of muscle involvement and instead facilitate (5) uptake of DNA by APC infiltrating the site of injection or in the lymphatics, at the same time protecting DNA from nuclease attack (6). Moreover, transfection of APC with liposomal DNA could be promoted by the judicial choice of vesicle surface charge, size and lipid composition, or by the co-entrapment, together with DNA, of plasmids expressing appropriate cytokines (e.g., interleukin 2), or immunostimulatory sequences.
Methods Mol Med 2000
PMID:Entrapment of plasmid DNA vaccines into liposomes by dehydration/rehydration. 2137 30


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