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1.
J Small Anim Pract ; 63(6): 486-491, 2022 06.
Article in English | MEDLINE | ID: mdl-34913482

ABSTRACT

Malignant pulmonary neoplasia associated with cystic airspaces is a well-recognised disease entity in humans. Two elderly dogs, previously diagnosed with a solitary emphysematous bulla, presented with non-specific clinical signs. At presentation, pulmonary auscultation was unremarkable. In both cases, thoracic CT demonstrated the transformation of the cystic airspace lesions characterised by a progressive increase of the solid component and reduction of the air component. Cytological evaluation and subsequent surgical excision followed by histopathology confirmed pulmonary carcinoma in both cases. These two cases represent the first demonstration of possible malignant transformation of pulmonary cystic airspace in dogs. Veterinarians should consider neoplastic transformation as a differential diagnosis in cases of cystic airspaces, particularly cases with features including thickening or irregularity of the wall, associated soft-tissue nodules or solid and non-solid tissue intermixed within clusters of multiple cystic airspaces. Ongoing monitoring of cystic airspace lesions through diagnostic imaging is recommended.


Subject(s)
Carcinoma , Cysts , Dog Diseases , Lung Neoplasms , Animals , Carcinoma/complications , Carcinoma/diagnosis , Carcinoma/surgery , Carcinoma/veterinary , Cysts/complications , Cysts/diagnostic imaging , Cysts/surgery , Cysts/veterinary , Dog Diseases/diagnostic imaging , Dog Diseases/surgery , Dogs , Lung , Lung Neoplasms/diagnosis , Lung Neoplasms/veterinary , Tomography, X-Ray Computed/methods , Tomography, X-Ray Computed/veterinary
2.
Soc Work ; 47(4): 461-70, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12450016

ABSTRACT

This article examines families' perceptions about involvement in residential treatment from the viewpoints of African American and non-African American family members. Focus group interviews found that all family members shared some common positive and negative experiences. However, unique issues remained for African American caregivers. The costs to children of being separated from their families and communities, fears regarding the use of medications, cultural dissimilarities of staff and clients, staff stereotyping, and a commitment to advocating for children other than their own were themes frequently expressed by African American family members. Implications for social services professionals serving African American families are highlighted.


Subject(s)
Black or African American/psychology , Caregivers/psychology , Family Relations/ethnology , Residential Treatment , Social Work, Psychiatric , Child , Community Participation , Cultural Diversity , Focus Groups , Humans , Interviews as Topic , Professional Competence , Qualitative Research
3.
J Biol Chem ; 276(42): 38555-62, 2001 Oct 19.
Article in English | MEDLINE | ID: mdl-11504725

ABSTRACT

Mutations in human mitochondrial DNA influence aging, induce severe neuromuscular pathologies, cause maternally inherited metabolic diseases, and suppress apoptosis. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we investigated the fidelity of DNA synthesis by human pol gamma. Comparison of the wild-type 140-kDa catalytic subunit to its exonuclease-deficient derivative indicates pol gamma has high base substitution fidelity that results from high nucleotide selectivity and exonucleolytic proofreading. pol gamma is also relatively accurate for single-base additions and deletions in non-iterated and short repetitive sequences. However, when copying homopolymeric sequences longer than four nucleotides, pol gamma has low frameshift fidelity and also generates base substitutions inferred to result from a primer dislocation mechanism. The ability of pol gamma both to make and to proofread dislocation intermediates is the first such evidence for a family A polymerase. Including the p55 accessory subunit, which confers processivity to the pol gamma catalytic subunit, decreases frameshift and base substitution fidelity. Kinetic analyses indicate that p55 promotes extension of mismatched termini to lower the fidelity. These data suggest that homopolymeric runs in mitochondrial DNA may be particularly prone to frameshift mutation in vivo due to replication errors by pol gamma.


Subject(s)
DNA-Directed DNA Polymerase/biosynthesis , DNA-Directed DNA Polymerase/chemistry , Base Pair Mismatch , Catalysis , DNA Polymerase gamma , DNA Repair , DNA, Mitochondrial/metabolism , DNA-Directed DNA Polymerase/metabolism , Frameshift Mutation , Gene Deletion , Humans , Kinetics , Mutagenesis , Mutation , Phenotype , Recombinant Proteins/metabolism , Repetitive Sequences, Nucleic Acid
4.
J Biol Chem ; 274(53): 38197-203, 1999 Dec 31.
Article in English | MEDLINE | ID: mdl-10608893

ABSTRACT

Human DNA polymerase gamma is composed of a 140-kDa catalytic subunit and a smaller accessory protein variously reported to be 43-54 kDa. Immunoblot analysis of the purified, heterodimeric native human polymerase gamma complex identified the accessory subunit as 55 kDa. We isolated the full-length cDNA encoding a 55-kDa polypeptide, expressed the cDNA in Escherichia coli and purified the 55-kDa protein to homogeneity. Recombinant Hp55 forms a high affinity, salt-stable complex with Hp140 during protein affinity chromatography. Immunoprecipitation, gel filtration, and sedimentation analyses revealed a 190-kDa complex indicative of a native heterodimer. Reconstitution of Hp140.Hp55 raises the salt optimum of Hp140, stimulates the polymerase and exonuclease activities, and increases the processivity of the enzyme by several 100-fold. Similar to Hp140, isolated Hp55 binds DNA with moderate strength and was a specificity for double-stranded primer-template DNA. However, Hp140.Hp55 has a surprisingly high affinity for DNA, and kinetic analyses indicate Hp55 enhances the affinity of Hp140 for primer termini by 2 orders of magnitude. Thus the enhanced DNA binding caused by Hp55 is the basis for the salt tolerance and high processivity characteristic of DNA polymerase gamma. Observation of native DNA polymerase gamma both as an Hp140 monomer and as a heterodimer with Hp55 supports the notion that the two forms act in mitochondrial DNA repair and replication. Additionally, association of Hp55 with Hp140 protects the polymerase from inhibition by N-ethylmaleimide.


Subject(s)
DNA Replication , DNA-Directed DNA Polymerase/metabolism , DNA/metabolism , Ethylmaleimide/pharmacology , Mitochondria/enzymology , Base Sequence , Catalytic Domain , DNA Polymerase gamma , DNA Primers , DNA, Complementary , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Dimerization , Escherichia coli/genetics , Humans , Molecular Sequence Data , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sodium Chloride
5.
Proc Natl Acad Sci U S A ; 95(21): 12244-8, 1998 Oct 13.
Article in English | MEDLINE | ID: mdl-9770471

ABSTRACT

Mitochondria have been proposed to possess base excision repair processes to correct oxidative damage to the mitochondrial genome. As the only DNA polymerase (pol) present in mitochondria, pol gamma is necessarily implicated in such processes. Therefore, we tested the ability of the catalytic subunit of human pol gamma to participate in uracil-provoked base excision repair reconstituted in vitro with purified components. Subsequent to actions of uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease, human pol gamma was able to fill a single nucleotide gap in the presence of a 5' terminal deoxyribose phosphate (dRP) flap. We report here that the catalytic subunit of human pol gamma catalyzes release of the dRP residue from incised apurinic/apyrimidinic sites to produce a substrate for DNA ligase. The heat sensitivity of this activity suggests the dRP lyase function requires a three-dimensional protein structure. The dRP lyase activity does not require divalent metal ions, and the ability to trap covalent enzyme-DNA complexes with NaBH4 strongly implicates a Schiff base intermediate in a beta-elimination reaction mechanism.


Subject(s)
DNA Repair , Mitochondria/metabolism , Base Sequence , Catalysis , DNA Polymerase gamma , DNA Primers , DNA-Directed DNA Polymerase/metabolism , Humans , Molecular Sequence Data
6.
Biochemistry ; 37(29): 10529-39, 1998 Jul 21.
Article in English | MEDLINE | ID: mdl-9671525

ABSTRACT

The human DNA polymerase gamma catalytic subunit was overexpressed in recombinant baculovirus-infected insect cells, and the 136 000 Da protein was purified to homogeneity. Application of the same purification protocol to HeLa mitochondrial lysates permitted isolation of native DNA polymerase gamma as a single subunit, allowing direct comparison of the native and recombinant enzymes without interference of other polypeptides. Both forms exhibited identical properties, and the DNA polymerase and 3' --> 5' exonuclease activities were shown unambiguously to reside in the catalytic polypeptide. The salt sensitivity and moderate processivity of the isolated catalytic subunit suggest other factors could be required to restore the salt tolerance and highly processive DNA synthesis typical of gamma polymerases. To facilitate our understanding of mitochondrial DNA replication and mutagenesis as well as cytotoxicity mediated by antiviral nucleotide analogues, we also constructed two site-directed mutant proteins of the human DNA polymerase gamma. Substituting alanine for two essential acidic residues in the exonuclease motif selectively eliminated the 3' --> 5' exonucleolytic function of the purified mutant polymerase gamma. Replacement of a tyrosine residue critical for sugar recognition with phenylalanine in polymerase motif B reduced dideoxynucleotide inhibition by a factor of 5000 with only minor effects on overall polymerase function.


Subject(s)
Amino Acids/metabolism , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Deoxyribonucleotides/metabolism , Exonucleases/metabolism , Recombinant Proteins/chemistry , Amino Acid Sequence , Catalysis , DNA Polymerase gamma , DNA-Directed DNA Polymerase/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Humans , Mutagenesis, Site-Directed , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Substrate Specificity/genetics
7.
J Biol Chem ; 272(16): 10917-21, 1997 Apr 18.
Article in English | MEDLINE | ID: mdl-9099749

ABSTRACT

HeLa nuclear extract was resolved into a depleted fraction incapable of supporting mismatch repair in vitro, and repair activity was restored upon the addition of a purified fraction isolated from HeLa cells by in vitro complementation assay. The highly enriched complementing activity copurified with a DNA polymerase, and the most pure fraction contained DNA polymerase delta but was free of detectable DNA polymerases alpha and epsilon. Calf thymus DNA polymerase delta also fully restored mismatch repair to the depleted extract, indicating DNA polymerase delta is required for mismatch repair in human cells. However, due to the presence of DNA polymerases alpha and epsilon in the depleted extract, potential involvement of one or both of these activities in the reaction cannot be excluded.


Subject(s)
Cell Nucleus/metabolism , DNA Repair , DNA-Directed DNA Polymerase/metabolism , Amino Acid Sequence , Animals , Antibodies , Base Composition , Cattle , Cell Fractionation , Cell Nucleus/ultrastructure , Chromatography, Affinity , Chromatography, Ion Exchange , DNA/chemistry , DNA Polymerase II/metabolism , DNA Polymerase III , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/isolation & purification , Electrophoresis, Polyacrylamide Gel , HeLa Cells , Humans , Immunoblotting , KB Cells , Molecular Sequence Data , Nucleic Acid Heteroduplexes , Peptide Fragments/chemistry , Peptide Fragments/immunology , Proliferating Cell Nuclear Antigen/analysis
8.
Science ; 268(5219): 1909-12, 1995 Jun 30.
Article in English | MEDLINE | ID: mdl-7604264

ABSTRACT

A mismatch-binding heterodimer of hMSH2 and a 160-kilodalton polypeptide has been isolated from HeLa cells by virtue of its ability to restore mismatch repair to nuclear extracts of hMSH2-deficient LoVo colorectal tumor cells. This heterodimer, designated hMutS alpha, also restores mismatch repair to extracts of alkylation-tolerant MT1 lymphoblastoid cells and HCT-15 colorectal tumor cells, which are selectively defective in the repair of base-base and single-nucleotide insertion-deletion mismatches. Because HOT-15 cells appear to be free of hMSH2 mutations, this selective repair defect is likely a result of a deficiency of the hMutS alpha 160-kilodalton subunit, and mutations in the corresponding gene may confer hypermutability and cancer predisposition.


Subject(s)
Colorectal Neoplasms/genetics , DNA Repair/genetics , DNA, Neoplasm/metabolism , DNA-Binding Proteins/isolation & purification , Amino Acid Sequence , Base Composition , Base Sequence , Colorectal Neoplasms/chemistry , DNA Damage , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/physiology , HeLa Cells , Humans , Molecular Sequence Data , Molecular Weight , Mutation , Nucleic Acid Heteroduplexes/metabolism , Sequence Deletion , Tumor Cells, Cultured
9.
Cell ; 75(6): 1227-36, 1993 Dec 17.
Article in English | MEDLINE | ID: mdl-8261516

ABSTRACT

A subset of sporadic colorectal tumors and most tumors developing in hereditary nonpolyposis colorectal cancer patients display frequent alterations in microsatellite sequences. Such tumors have been thought to manifest replication errors (RER+), but the basis for the alterations has remained conjectural. We demonstrate that the mutation rate of (CA)n repeats in RER+ tumor cells is at least 100-fold that in RER- tumor cells and show by in vitro assay that increased mutability of RER+ cells is associated with a profound defect in strand-specific mismatch repair. This deficiency was observed with microsatellite heteroduplexes as well as with heteroduplexes containing single base-base mismatches and affected an early step in the repair pathway. Thus, a true mutator phenotype exists in a subset of tumor cells, the responsible defect is likely to cause transitions and transversions in addition to microsatellite alterations, and a biochemical basis for this phenotype has been identified.


Subject(s)
DNA Repair/genetics , DNA Replication/genetics , Mutagenesis, Insertional , Mutation , Base Sequence , Cell Line , Cell Nucleus/metabolism , DNA, Neoplasm/metabolism , Genetic Vectors , HeLa Cells , Humans , Molecular Sequence Data , Nucleic Acid Heteroduplexes/metabolism , Oligodeoxyribonucleotides , Polymerase Chain Reaction , Repetitive Sequences, Nucleic Acid , Restriction Mapping , Tumor Cells, Cultured
10.
Proc Natl Acad Sci U S A ; 90(14): 6424-8, 1993 Jul 15.
Article in English | MEDLINE | ID: mdl-8341649

ABSTRACT

The human lymphoblastoid MT1 B-cell line was previously isolated as one of a series of mutant cells able to survive the cytotoxic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MT1 cells nevertheless remain sensitive to mutagenesis by MNNG and display a mutator phenotype. These phenotypes have been attributed to a single genetic alteration postulated to confer a defect in strand-specific mismatch repair, a proposal that attributes the cytotoxic effect of DNA alkylation in wild-type cells to futile attempts to correct mispairs that arise during replication of alkylated template strands. Our results support this view. MNNG-induced mutations in the HPRT gene of MT1 cells are almost exclusively G.C-->A.T transitions, while spontaneous mutations observed in this mutator cell line are single-nucleotide insertions, transversions, and A.T-->G.C transitions. In vitro assay has demonstrated that the MT1 line is in fact deficient in strand-specific correction of all eight base-base mispairs. This defect, which is manifest at or prior to the excision stage of the reaction, is due to simple deficiency of a required activity because MT1 nuclear extracts can be complemented by a partially purified HeLa fraction to restore in vitro repair. These findings substantiate the idea that strand-specific mismatch repair contributes to alkylation-induced cytotoxicity and imply that this process serves as a barrier to spontaneous transition, transversion, and insertion/deletion mutations in mammalian cells.


Subject(s)
B-Lymphocytes/physiology , DNA Repair/genetics , Hypoxanthine Phosphoribosyltransferase/genetics , Mutagenesis/genetics , Alkylation , Cell Line , Cell Nucleus , HeLa Cells , Humans , Mutation
11.
Methods Enzymol ; 218: 587-609, 1993.
Article in English | MEDLINE | ID: mdl-8510549

ABSTRACT

We have presented several protocols for producing an in situ activity gel that allows detection of various DNA-metabolizing enzymes. Both nondenaturing polyacrylamide and SDS-polyacrylamide activity gel electrophoresis procedures were detailed. Combining the use of defined [32P]DNA substrates with product analysis, these procedures detected a wide spectrum of enzymatic activities. The ability to detect 7 different catalytic activities of 15 different enzymes provides encouragement for expanded applications. It is hoped that others will find this technique applicable for detecting these enzymes and other activities in different biological systems. The modification of DNA in situ and the creation of intermediate substrates within activity gels should prove extremely useful for dissecting the enzymatic steps of DNA replication, repair, recombination, and restriction, as well as the metabolic pathways of other nucleic acids.


Subject(s)
DNA Polymerase I/analysis , DNA, Viral/metabolism , DNA/metabolism , Escherichia coli/enzymology , Exodeoxyribonucleases/analysis , Bacteriophage M13/genetics , Base Sequence , DNA Polymerase I/isolation & purification , DNA Polymerase I/metabolism , Electrophoresis, Polyacrylamide Gel/methods , Exodeoxyribonuclease V , Exodeoxyribonucleases/isolation & purification , Exodeoxyribonucleases/metabolism , Indicators and Reagents , Molecular Sequence Data , Molecular Weight , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/isolation & purification , Oligodeoxyribonucleotides/metabolism , Phosphorus Radioisotopes , Substrate Specificity
12.
J Biol Chem ; 266(36): 24702-11, 1991 Dec 25.
Article in English | MEDLINE | ID: mdl-1662214

ABSTRACT

Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product.


Subject(s)
DNA Polymerase III/metabolism , Exonucleases/metabolism , Liver/enzymology , Animals , Base Sequence , Catalysis , DNA/metabolism , DNA Polymerase III/antagonists & inhibitors , Electrophoresis, Polyacrylamide Gel , Exonucleases/antagonists & inhibitors , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Protein Denaturation , Substrate Specificity , Swine
13.
Biochemistry ; 30(10): 2655-64, 1991 Mar 12.
Article in English | MEDLINE | ID: mdl-2001353

ABSTRACT

We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps. Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition. Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E. coli DNA ligase activity, respectively. Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.


Subject(s)
DNA Ligases/metabolism , DNA-Directed DNA Polymerase/metabolism , N-Glycosyl Hydrolases/metabolism , Base Sequence , DNA Glycosylases , DNA, Bacterial/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Escherichia coli/metabolism , Hot Temperature , Molecular Sequence Data , Substrate Specificity
14.
Nucleic Acids Res ; 18(24): 7317-22, 1990 Dec 25.
Article in English | MEDLINE | ID: mdl-2175431

ABSTRACT

Thermus aquaticus DNA polymerase was shown to contain an associated 5' to 3' exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novel in situ activity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2 or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5'-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5'-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5'-hydroxylated residue. Hydrolysis of a 5'-terminal residue from a nick was preferred threefold over the same 5'-end of duplex DNA. The 5' to 3' exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction.


Subject(s)
DNA-Directed DNA Polymerase/metabolism , Exonucleases/metabolism , Thermus/enzymology , Base Sequence , Centrifugation, Density Gradient , DNA, Bacterial/metabolism , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Molecular Weight , Substrate Specificity , Taq Polymerase
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