Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Psychrotrophic Bacillus cereus is a limiting factor for the shelf-life of pasteurized milk, particularly during the grazing season. Potential sources of contamination and factors that might affect the spore content of milk were studied in detail for a group of eight cows during three 2-wk study periods from June to September over 2 yr. The spore content of milk was strongly associated with the degree of contamination of the teats with soil. High water content of soil, low evaporation of water and dirty access alloys were the most important factors correlating with high spore concentrations. The spore content of soil varied from < 50 to 380,000/g, depending on time and sampling site. The milking equipment did not contribute significantly to the contamination. The spore contents in air during milking (< 100 cfu/m3) and in feed (silage, hay, fresh grass, and concentrates) were too low to be of importance for contamination. The spore content in dung was also low. Further support that soil was the major contamination source was found by comparison of genetic fingerprints by random amplified polymorphic DNA polymerase chain reaction of isolates of B. cereus from soil and milk and by teat cleansing experiments, which resulted in reduced contamination levels in milk.
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PMID:Bacillus cereus spores in raw milk: factors affecting the contamination of milk during the grazing period. 1006 52

Rhodium(II) carboxylates and their derivatives constitute a promising class of second-generation transition metal compounds with anticancer properties. While most transition metal anticancer compounds chelate DNA and cause extensive chromosomal damage, rhodium(II) carboxylates act on the enzyme DNA polymerase alpha and hence cause minimal chromosomal damage. Rhodium(II) citrate, a recent member of the rhodium(II) carboxylate family is highly promising as an antitumor agent. However, due to its high water solubility, a high systemic dose is necessary to achieve efficacy. In this paper, we have explored the complexation of rhodium(II) citrate with hydroxypropyl-beta-cyclodextrin as a means to improve encapsulation and release kinetics from poly(dl-lactic-co-glycolic) acid (PLGA) and poly(anhydride) microspheres. We observed that complexation of rhodium(II) citrate with hydroxypropyl-beta-cyclodextrin significantly increased both the encapsulation efficiency and duration of release in both polymer systems.
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PMID:Encapsulation and release of rhodium(II) citrate and its association complex with hydroxypropyl-beta-cyclodextrin from biodegradable polymer microspheres. 1022 52

The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.
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PMID:Mutational and pH studies of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase. 1045 97

DNA prepared from soil usually contains a brown-tinted inhibitor of the polymerase chain reaction (PCR) which limits the sensitivity of this technique for specific detection of microorganisms. To localize the inhibitor, soil fractions were tested for their inhibitory effect on the PCR reaction. A highly inhibitory activity, sufficient to account for the inhibition typically exhibited by soil DNA, was found to be tightly associated with the soil microorganism fraction. After cell breakage, the inhibitory material became soluble, and was not separable from DNA by standard purification procedures. A method was derived by which most of the inhibitory material could be selectively solubilized from the microorganism fraction without cell breakage, using successive washes with buffers differing in EDTA concentration. This technique was used to isolate a substance with characteristics suggesting that it is the major PCR inhibitor contaminating DNA purified from soil. It was found to be an organic, water-soluble compound of high molecular weight, and was present in a variety of soil types from different locations. It was found to be distinctly different in its solubility properties from humic and fulvic acids, and also in its FT-IR and NMR spectra. It forms a complex with protein and may inhibit the PCR reaction by an interaction with Taq DNA polymerase.
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PMID:Purification and characterization of a common soil component which inhibits the polymerase chain reaction. 1093 57

Continuous administration in the drinking water of hepatocarcinogen N-nitrosodiethylamine (NDEA) to male rats (200 mg/L) for 60 days resulted in DNA damage in the form of single strand breaks. The damage, which is measured as a shift in the sedimentation of DNA in alkaline sucrose density gradients, was found to be maximum at the fourth week of treatment, and the sedimentation pattern of DNA was found to return to near normal size by the seventh week of NDEA treatment. Simultaneously, there were perturbations in the nuclear enzymes involved in DNA replication and repair. Activities of DNA polymerase beta, DNA ligase, and topoisomerase were found to increase in as early as the first week of NDEA treatment and reached the maximum at the fourth week, and thereafter declined to normal level by the eighth week of treatment. Concomitantly, the activities of DNA polymerase alpha, DNA primase, and RNA polymerase which were unaltered in the initial period of carcinogen treatment recorded a marked increase after sixth week of NDEA treatment. Results suggest that administration of NDEA inflicts DNA damage, which is manifested as increase in DNA repair enzymes in the initial period and activated DNA replicative enzymes at a later period, indicating the active proliferation of transformed cells.
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PMID:Damage to DNA and activity of nuclear DNA repair and replicative enzymes following N-nitrosodiethylamine treatment to rats. 1096 99

(-)-beta-D-2,6-Diaminopurine dioxolane (DAPD), is a nucleoside reverse transcriptase (RT) inhibitor with activity against human immunodeficiency virus type 1 (HIV-1). DAPD, which was designed as a water-soluble prodrug, is deaminated by adenosine deaminase to give (-)-beta-D-dioxolane guanine (DXG). By using calf adenosine deaminase a K(m) value of 15 +/- 0.7 microM was determined for DAPD, which was similar to the K(m) value for adenosine. However, the k(cat) for DAPD was 540-fold slower than the k(cat) for adenosine. In CEM cells and peripheral blood mononuclear cells exposed to DAPD or DXG, only the 5'-triphosphate of DXG (DXG-TP) was detected. DXG-TP is a potent alternative substrate inhibitor of HIV-1 RT. Rapid transient kinetic studies show the efficiency of incorporation for DXG-TP to be lower than that measured for the natural substrate, 2'-deoxyguanosine 5'-triphosphate. DXG-TP is a weak inhibitor of human DNA polymerases alpha and beta. Against the large subunit of human DNA polymerase gamma a K(i) value of 4.3 +/- 0.4 microM was determined for DXG-TP. DXG showed little or no cytotoxicity and no mitochondrial toxicity at the concentrations tested.
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PMID:Mechanism of action of 1-beta-D-2,6-diaminopurine dioxolane, a prodrug of the human immunodeficiency virus type 1 inhibitor 1-beta-D-dioxolane guanosine. 1112 Sep 59

We have estimated pre-steady-state kinetic parameters for the addition of a single nucleotide residue by a set of RB69 DNA polymerase mutants in which four highly conserved residues in the fingers domain have been replaced by Ala. The relationship between the kinetic constants exhibited by the mutants and the structure of the ternary complex [Franklin, M., Wang, J., and Steitz T. (2001) Cell 105, 657-667] was consistent with the following sets of interactions between the conserved residues and oxygen atoms in the triphosphate portion of the incoming dNTP: (i) the epsilon-amino group of K560 contacts oxygen atoms of the alpha- and gamma-phosphates, (ii) the amide side chain of Asn 564 forms a hydrogen bond via a water molecule with the nonbridging oxygen of the beta-phosphate, and (iii) the epsilon-amino and delta-guanidino groups of K486 and R482, respectively, contact the nonbridging oxygens of the gamma-phosphate. We have also determined the pre-steady-state kinetic parameters for the addition of both dCTP and dCDP onto a 13/20mer primer/template with an exo(-) derivative of RB69 DNA polymerase and have shown that the deoxynucleoside diphosphate can be incorporated, in contrast to the behavior of the Klenow fragment which cannot use dCDP as a substrate. We have shown that, with RB69 DNA polymerase, in contrast to the Klenow fragment, there is no inhibition of the primer-extension reaction by incoming NTPs having either noncomplementary bases or ribo- instead of a deoxyribose moieties. This implies that the mode of recognition of incoming dNTPs and triggering of the conformational change, which is thought to occur prior to the chemical step, differs between these two enzymes.
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PMID:Correlation of the kinetics of finger domain mutants in RB69 DNA polymerase with its structure. 1185 99

The epsilon subunit of the Escherichia coli replicative DNA polymerase III is the proofreading 3'-5' exonuclease. Structures of its catalytic N-terminal domain (epsilon186) were determined at two pH values (5.8 and 8.5) at resolutions of 1.7-1.8 A, in complex with two Mn(II) ions and a nucleotide product of its reaction, thymidine 5'-monophosphate. The protein structure is built around a core five-stranded beta sheet that is a common feature of members of the DnaQ superfamily. The structures were identical, except for differences in the way TMP and water molecules are coordinated to the binuclear metal center in the active site. These data are used to develop a mechanism for epsilon and to produce a plausible model of the complex of epsilon186 with DNA.
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PMID:Structural basis for proofreading during replication of the Escherichia coli chromosome. 1193 58

High throughput screening identified 2-acetamido-thiazolylthio acetic ester 1 as an inhibitor of cyclin-dependent kinase 2 (CDK2). Because this compound is inactive in cells and unstable in plasma, we have stabilized it to metabolic hydrolysis by replacing the ester moiety with a 5-ethyl-substituted oxazole as in compound 14. Combinatorial and parallel synthesis provided a rapid analysis of the structure-activity relationship (SAR) for these inhibitors of CDK2, and over 100 analogues with IC(50) values in the 1-10 nM range were rapidly prepared. The X-ray crystallographic data of the inhibitors bound to the active site of CDK2 protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues displayed potent and broad spectrum antiproliferative activity across a panel of tumor cell lines in vitro. In addition, A2780 ovarian carcinoma cells undergo rapid apoptosis following exposure to CDK2 inhibitors of this class. Mechanism of action studies have confirmed that the phosphorylation of CDK2 substrates such as RB, histone H1, and DNA polymerase alpha (p70 subunit) is reduced in the presence of compound 14. Further optimization led to compounds such as water soluble 45, which possesses a favorable pharmacokinetic profile in mice and demonstrates significant antitumor activity in vivo in several murine and human models, including an engineered murine mammary tumor that overexpresses cyclin E, the coactivator of CDK2.
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PMID:Discovery of aminothiazole inhibitors of cyclin-dependent kinase 2: synthesis, X-ray crystallographic analysis, and biological activities. 1219 Mar 13

Intracerebral infusion of lysed erythrocytes causes brain edema without inducing ischemic cerebral blood flow. Reports have indicated that oxidative damage contributes to secondary brain injury in stroke. In the present study, we investigated whether erythrocyte lysis after intracerebral hemorrhage (ICH) might result in oxidative brain damage. This study had four parts. Male Sprague-Dawley rats received an infusion of autologous lysed erythrocytes into the right striatum. Control rats only had a needle insertion. Neurological deficits, brain water and ion contents were determined in the first part. In the second part, hemoxygenase-1 (HO-1), manganese superoxide dismutase (Mn-SOD), copper/zinc SOD (CuZn-SOD) and protein carbonyl levels were determined by Western blot analysis. In the third part, immunohistochemistry was performed for HO-1. DNA damage was examined using DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) in the fourth part. Infusion of lysed RBCs induced marked edema in the ipsilateral striatum and profound neurological deficits. Western blot analysis and immunohistochemistry indicated that HO-1 was upregulated 24 h after infusion of lysed red blood cells. Both Mn-SOD and CuZn-SOD contents decreased, protein carbonyl levels increased in the ipsilateral striatum, and there was the appearance of PANT- and TUNEL-positive cells suggesting oxidative mechanisms in the erythrocyte-induced brain injury. In conclusion, oxidative stress caused by components of the lysed erythrocytes contributes to the brain injury after ICH.
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PMID:Oxidative brain injury from extravasated erythrocytes after intracerebral hemorrhage. 1238 37


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