Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:BIOD00001 (DNase I)
 8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Initial experiments in our laboratory have successfully reassembled infectious polyoma virions from dissociated virion products. Virions treated with ethyleneglycol-bis-N,N'-tetraacetic acid and the reducing agent beta-mercaptoethanol at pH 7.5 were dissociated to a 48S DNA-protein complex and capsomere subunits. The virion dissociation products were not infectious by plaque assay and lacked hemagglutination activity. These virion dissociation products were reassembled to intact virions by overnight dialysis against a reassembly buffer containing CaCl2, dimethyl sulfoxide, and Triton X-100 in phosphate-buffered saline at pH 7.4. The biophysical characteristics of the reassembled virions were identical to those of untreated virions in that the reassembled virions had a sedimentation value of 240S in sucrose gradients and a buoyant density of 1.315 g/cm3 in CsCl isopycnic gradients. The reassembled virions were intact as determined by electron microscopy and were found to be 60% resistant to DNase I treatment. Biologically, the reassembled purified virions were found to partially regain both hemagglutinating activity and plaque-forming ability.
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PMID:In vitro reassembly of infectious polyoma virions. 22 81

The mouse sperm nucleus, after the removal of protamines and DNA, consisted of a skeletal structure that conformed to the original nuclear shape. Sperm were extracted with 1% SDS, and the isolated nuclei, along with the enveloping perinuclear theca, were incubated in 25 mM dithiothreitol, and exposed to different reagents in an effort to displace the protamines, P1 and P2. Protamines, labeled with [3H]arginine, were displaced from the nucleus by CaCl2.MgCl2, but only partially by anionic detergents, monovalent cations, and polyvalent anions. Displacement of P1 and P2 was achieved by digesting the nuclei with DNase I and simultaneously extracting with CaCl2.MgCl2 (3:2; mol:mol) in stepwise increments of 125, 150, 175, 200, and 250 mM. Protamine displacement was concentration-dependent, occurring with an EC50 of approximately 205 mM and with maximal displacement at approximately 250 mM CaCl2.MgCl2. The nucleus was reduced to a skeletal structure consisting of the perinuclear theca and an internal network of transverse fibers. The evidence was consistent with the former being derived from the perforatorium and postacrosomal nuclear sheath (both cytoplasmic structures), whereas the fibers were most likely of nuclear origin. By SDS-PAGE and isoelectric focusing (IEF), perinuclear matrices consisted of greater than or equal to 230 protein spots, with M(r)s in the range of 70,000 to 8000 and pIs of greater than or equal to 7.5 to approximately 4.7, respectively. Monoclonal antibodies prepared against perinuclear matrices bound to specific proteins on IEF immunoblots and, based on light and electron microscopic observations, to discrete domains of the sperm perinuclear theca and nucleus.
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PMID:The perinuclear matrix as a structural element of the mouse sperm nucleus. 151 Oct 98

The natural transformation of Acinetobacter calcoaceticus BD413 (trpE27) was characterized with respect to features that might be important for a possible gene transfer by extracellular DNA in natural environments. Transformation of competent cells with chromosomal DNA (marker trp+) occurred in aqueous solutions of single divalent cations. Uptake of DNA into the DNase I-resistant state but not the binding of DNA to cells was strongly stimulated by divalent cations. An increase of transformation of nearly 3 orders of magnitude was obtained as a response to the presence of 0.25 mM Ca2+. With CaCl2 solutions the transformation frequencies approached the highest values obtained under standard broth conditions, followed by MnCl2 and MgCl2. It is concluded that transformation requires divalent cations. DNA competition experiments showed that A. calcoaceticus does not discriminate between homologous and heterologous DNA. Furthermore, circular plasmid DNA competed with chromosomal DNA fragments and vice versa. The equally efficient transformation with plasmid pKT210 isolated from A. calcoaceticus or Escherichia coli indicated absence of DNA restriction in transformation. High efficiency plasmid transformation was obtained in samples of non-sterile natural groundwater and in non-sterile extracts of fresh and air-dried soil. Heat-treatment (10 min, 80 degrees C) of the non-sterile liquid samples increased transformation only in the dried soil extract, probably by inactivation of DNases. The results presented suggest that competent cells of A. calcoaceticus can take up free high molecular weight DNA including plasmids of any source in natural environments such as soil, sediment or groundwater.
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PMID:Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater. 159 Jul 8

Previous studies of chromatin melting had been done at low salt concentrations to maintain optically clear solutions permitting observation of hyperchromic effects that accompany the unstacking of bases in DNA. Scanning calorimetry does not require clear solutions and so allowed more nearly physiological levels of salt to be used in the present work. HeLa chromatin went through two major structural transitions of 73 and 85 degrees C in 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2. Proteinase K treatment eliminated the 73 degrees C transition without affecting the other one. DNase I digestion eliminated the 85 degrees C transition and shifted the other from 73 to 58 degrees C. Supported by previous reports that salt concentrations like these would largely eliminate the effect of histones on the Tm of DNA, the present observations indicate that the collapse of the core histone complex occurs at 73-76 degrees C (transition II) and the nucleosomal DNA unstacks at 85-87 degrees C (transition III). They also show that the thermal stability of the histone octamer is enhanced by the DNA folded around it in the nucleosome; although the histone core raises the Tm of the DNA in low salt, in physiological salt conditions it is the DNA that stabilizes the protein complex.
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PMID:In physiological salt conditions the core proteins of the nucleosomes in large chromatin fragments denature at 73 degrees C and the DNA unstacks at 85 degrees C. 270 3

Depolarization of whole brain synaptosomes, which stimulates transmitter release, also affects regulation of the assembly of actin microfilaments. Lysates of depolarized synaptosomes contain 20% less cytoskeletal actin than lysates of unstimulated synaptosomes. Parameters affecting the assembly of actin are modified before lysis, but release of actin from the Triton-insoluble cytoskeleton does not occur until after lysis. Actin released from the cytoskeleton is not precipitated with myosin, indicating that it consists of monomers and/or short oligomers. Synaptosomes were incubated for 12 sec in one of three solutions of identical ionic strength but of different salt mixtures: 75 mM KCl-2 mM CaCl2, 5 mM KCl-2mM CaCl2, or 75 mM KCl-0.1 mM EGTA. Synaptosomes were then lysed in an F-actin stabilizing buffer containing 1% Triton X-100. Control synaptosomes (no incubation) were lysed directly into the same lysis buffer containing one of the three different salt mixtures. The cytoskeletal and noncytoskeletal actin pools were separated 25 sec after lysis by centrifugation at 10(4) X g for 1 min, and the actin in each pool was quantitated by the DNase I inhibition assay. The drop in cytoskeletal actin induced by depolarization is maximized by including Ca2+ in the depolarizing buffer, and it is blocked completely by adding a neutral thiol protease inhibitor, leupeptin, to either the pre- or post-lysis buffer. The drop is also completely reversed by repolarizing the synaptosomes.
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PMID:Reorganization of actin in depolarized synaptosomes. 286 5

Using a gel overlay technique we have previously described a 90,000-mol wt actin-binding protein in a number of hormone-secreting tissues and tentatively identified this protein as gelsolin. Gelsolin is a protein that cuts or solates cross-linked actin filaments and can also serve as a nucleating site for actin polymerization. The objective of this study was to isolate this protein from a hamster insulin-secreting (HIT) cell line and compare the immunologic properties and peptide maps of purified rabbit macrophage gelsolin, human platelet gelsolin, and the HIT cell 90,000-mol wt protein. DNase I-Sepharose retained the HIT cell actin-binding proteins in 1 mM CaCl2; some of the 90,000-mol wt protein could then be eluted with 1 mM EGTA. The remaining actin-binding proteins were eluted using a buffer containing SDS. The EGTA peak fractions contained two major protein bands of Mr = 90,000 and 42,000, which suggested that a 90,000-mol wt-actin complex was eluted from the DNase I-Sepharose column. Specific antibodies to the human platelet and rabbit macrophage gelsolins bound to the 90,000-mol wt bands in the eluates, but did not crossreact with other actin-binding proteins. Indirect immunofluorescence using an anti-human platelet gelsolin antibody localized the 90,000-mol wt protein to stress fibers that were also stained with phalloidin, which suggested that gelsolin is associated with actin in vivo. Tryptic peptide maps of all three radioiodinated gelsolins were virtually indistinguishable. Thus, gelsolin is a highly conserved gene product found in at least three diverse cell types, an insulin-secreting beta cell line, macrophages, and platelets, and may link a transient increase in Ca2+ cellular levels with changes in actin polymerization and/or the gel-sol state of these cells.
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PMID:Gelsolin, a Ca2+-dependent actin-binding protein in a hamster insulin-secreting cell line. 298 50

Cytoplasmic actin from cultured fibroblasts has been purified to homogeneity and characterized with respect to its polymerization and structure. It was found to be qualitatively similar to muscle actin in all respects, but significant quantitative differences in its properties were demonstrated. Although BHK actin did not polymerize in unfractionated cytoplasmic extracts, the purified BHK actin polymerized into filaments both in magnesium and calcium. The critical concentration, measured by the DNase I inhibition assay and by fluorimetry, was the same as that of muscle actin both in magnesium and calcium. Polymerization of pyrene-labelled BHK and muscle actin was followed by fluorimetry. Significant differences in kinetics were found under both ionic conditions tested. In the absence of Mg2+ ions (0.2 mM CaCl2, 85 mM KCl), BHK actin polymerized at a much slower rate than muscle actin. In the presence of magnesium and EGTA, the nucleation phase for BHK actin polymerization was shorter than that for muscle actin and the kinetics of polymerization was different. The structure of BHK actin filaments in the electron micrographs was very similar to that of muscle actin. In high concentrations of magnesium, BHK actin formed paracrystals which had the same appearance as muscle actin paracrystals. However, calcium-induced formation of actin paracrystals required higher concentration of Ca2+ ions for BHK actin than for muscle actin (12 mM and 8 mM respectively). These results suggest differences in divalent cation binding to both high- and low-affinity sites of the two actins.
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PMID:Isolation and characterization of actin from cultured BHK cells. 342 41

Nuclear envelopes were prepared from purified rat liver nuclei by lysis with heparin, digestion with deoxyribonuclease I (DNase I), or sonication. The envelopes were fractionated by centrifugation on sucrose density gradients and analyzed for protein kinase activity using endogenous and exogenous protein substrates and [gamma-32 P]ATP. The protein kinase activity toward endogenous proteins was markedly affected by the method used to isolate the envelopes, with sonication producing a preparation with very low activity. At least 12 phosphoproteins in nuclear envelopes isolated by the heparin or DNase I method were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A 32P-labeled material migrating with an apparent Mr = 3000 was extracted with chloroform:methanol:HCl and was identified as a mixture of phospholipids. Total 32P incorporation into nuclear envelopes peaked at 5 min of incubation, followed by a decrease in labeled products. This decrease was due to both phosphoprotein phosphatase activity and degradation of the lipid products. The highest protein kinase activity toward endogenous proteins was expressed with [gamma-32P]ATP in the presence of MgCl2; however, some phosphorylation also occurred with MnCl2, CoCl2, NiCl2, and [gamma-32P]GTP in the presence of MgCl2. Nuclear envelope protein phosphorylation was unaffected by cyclic nucleotides and calmodulin, slightly inhibited by CaCl2, MnCl2, CoCl2, disulfides, and sulfhydryl alkylating agents, and strongly inhibited by LaCl3 and phosphatidylglycerol. Nuclear porelamina complexes isolated from phosphorylated envelopes contained phosphoproteins of 7, 20, 51, 59, and 70 kDa. Incubation of pore-lamina complexes isolated from unlabeled envelopes with [gamma-32P]ATP resulted in 32P incorporation into the 20-, 51-, and 50-kDa proteins.
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PMID:Phosphorylation of rat liver nuclear envelopes. I. Characterization of in vitro protein phosphorylation. 630 4

An endodeoxyribonuclease has been purified to near homogeneity from rat small intestinal mucosa by a procedure involving Con A-Sepharose affinity chromatography. During the initial steps of purification, the presence of 5 mM CaCl2 was essential for stability of the enzyme activity. The enzyme has a molecular weight of 32 000 and an isoelectric point of 4.7. NaCl, sulfhydryl reagents, and iodoacetate strongly inhibited the reaction, but tRNA did not. The enzyme required divalent cations for activity and had a pH optimum of pH 6.2 with Co2+ and pH 7.7 with Mn2+. In both optimum conditions, the enzyme hydrolyzed native DNA more rapidly than denatured DNA, and the average chain lengths of limit digestion products of native and denatured DNA were 8 and 10, respectively, at pH 6.2 and 9 and 11, respectively, at pH 7.7. The enzyme activity to produce acid-soluble fractions from linear DNA substrate was similar in the two optimum conditions, but the activity to nick double-stranded, superhelical circular DNA substrate was significantly higher at pH 6.2 than at pH 7.7. The endonuclease formed single-strand breaks making 5'-phosphoryl and 3'-hydroxyl termini, and deoxythymidine was present at the 5' termini with a frequency of about 50% in both optimum conditions. Bovine pancreatic DNase I antibody and G-action inhibited the enzyme activity. Thus this endonuclease is classified as a DNase I.
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PMID:Purification and properties of a neutral endodeoxyribonuclease from rat small intestinal mucosa. 707 91

A thermodynamic analysis of the binding of the TATA binding protein (TBP) from Saccharomyces cerevisiae to the adenovirus E4 promoter was conducted using quantitative DNase I "footprint" titration techniques. These studies were conducted to provide a foundation for studies of TBP structure-function relations and its assembly into transcription preinitiation complexes. The binding of TBP to the E4 promoter is well described by the Langmuir binding polynomial, suggesting that no linked equilibria contribute to the binding reaction under the conditions examined. Van't Hoff analysis yielded a nonlinear dependence on temperature with the TBP-E4 promoter interaction displaying maximal affinity at 30 degrees C. An unusually negative value of the apparent standard heat capacity change, delta Cp degrees = -3.5 +/- 0.5 kcal/mol.K, was determined from these data. The dependence of the TBP-E4 promoter interaction on [KCl] indicates that 3.6 +/- 0.3 K+ ions are displaced upon complex formation. Within experimental error, no linkage of proton binding with the TBP-E4 promoter interaction is detectable between pH 5.9 and 8.7. Rates of association of TBP for the E4 promoter were obtained using a novel implementation of a quench-flow device and DNase I "footprinting" techniques. The value determined for the second-order rate constant at pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 30 degrees C (ka = 5.2 +/- 0.5) x 10(5) M-1 s-1) confirms the results obtained by Hawley and co-workers [Hoopes, B.C., LeBlanc, J.F., & Hawley, D.K. (1992) J. Biol. Chem. 267, 11539-11547] and extends them through TBP concentrations of 636 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thermodynamic and kinetic characterization of the binding of the TATA binding protein to the adenovirus E4 promoter. 763 96


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