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R A Bennett

Publications and source records attributed to R A Bennett.

At least 37 records · Page 2Linked to original sources
PubMed1999

Single-turnover analysis of mutant human apurinic/apyrimidinic endonuclease.

Apurinic/apyrimidinic endonuclease (AP endo) is a key enzyme in the repair of oxidatively damaged DNA. Using single-turnover conditions, we recently described substrate binding parameters for wild type human AP endo. In this study, we utilized four enzyme mutants, D283A, D308A, D283A/D308A, and H309N, and assayed them under steady state and single-turnover conditions. The turnover number of the single aspartate mutants was decreased 10-30-fold in comparison to that of the wild type. The decrease in the turnover number was accompanied by a 17- and 50-fold decrease in the forward rate constant (kon) for substrate binding by D308A and D283A, respectively. The dissociation rate constant for substrate (koff) was unchanged for the D308A mutant but was 10 times faster for the D283A mutant than for the wild type. The apparent Km values for both of the single aspartate mutants were about equal to their respective KD values. To account for the kinetic behavior of the D308A mutant, it was necessary to insert a conformational change into the kinetic scheme. In contrast to the single aspartate mutants, the turnover number for the double mutant was 500-fold lower than that of the wild type, its apparent Km was 2.5-fold higher, and binding to substrate was weak. Mutation of His309 caused the greatest decrease in activity, resulting in a turnover number that was more than 30000-fold lower than that of the wild type and an apparent Km that was 13-fold higher, supporting the notion that His309 is intimately involved in catalysis. Molecular dynamics simulation techniques suggested that conversion of either aspartate to alanine resulted in major shifts in the spatial localization of key amino acids. Despite the fact that the two aspartates flank His309, the movement they engendered was distinct, consistent with the differences in catalytic behavior. We suggest that the conformation of the active site is largely maintained by the two aspartates, which enable efficient binding and cleavage of abasic site-containing DNA.

Alanine↗
PubMed1999

Peripheral neuropathy in a turkey vulture with lead toxicosis.

Clinical, electromyographic, and pathologic findings characteristic of lead toxicosis were detected in a turkey vulture (Cathartes aura). The bird had generalized lower motor neuron dysfunction that progressed over 5 days. Electromyography revealed diffuse denervation potentials and a presumed decrement in the sciatic-tibial nerve conduction velocity. Histologic examination of peripheral nerves obtained at necropsy revealed changes that could be compatible with lead-induced neuropathy. Lead toxicosis was confirmed by determination of blood lead concentrations. Lead toxicosis causing neurologic disorders in birds has been described. However, this report emphasizes the effects of lead on the peripheral nervous system and demonstrates the use of electromyography for diagnosis of peripheral neuropathy in birds.

Animals↗
PubMed1999

Surgical management of bifid sternum in two African grey parrots.

Transposition of the pectoral muscles for treatment of caudal bifid (cleft) sternum in 2 unrelated African Grey Parrots is described. The birds did not have clinical signs of ventilatory compromise prior to surgery; however, both had cutaneous ulcers over the defects. The pectoral muscles provided a thick pad over the heart, minimizing the risk of trauma to the heart.

Animals↗
PubMed1999

The Saccharomyces cerevisiae ETH1 gene, an inducible homolog of exonuclease III that provides resistance to DNA-damaging agents and limits spontaneous mutagenesis.

The recently sequenced Saccharomyces cerevisiae genome was searched for a gene with homology to the gene encoding the major human AP endonuclease, a component of the highly conserved DNA base excision repair pathway. An open reading frame was found to encode a putative protein (34% identical to the Schizosaccharomyces pombe eth1(+) [open reading frame SPBC3D6.10] gene product) with a 347-residue segment homologous to the exonuclease III family of AP endonucleases. Synthesis of mRNA from ETH1 in wild-type cells was induced sixfold relative to that in untreated cells after exposure to the alkylating agent methyl methanesulfonate (MMS). To investigate the function of ETH1, deletions of the open reading frame were made in a wild-type strain and a strain deficient in the known yeast AP endonuclease encoded by APN1. eth1 strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were approximately 3-fold more sensitive to MMS and approximately 10-fold more sensitive to hydrogen peroxide than was the wild type. Double-mutant strains (apn1 eth1) were approximately 15-fold more sensitive to MMS and approximately 2- to 3-fold more sensitive to hydrogen peroxide and phleomycin D1 than were apn1 strains. Elimination of ETH1 in apn1 strains also increased spontaneous mutation rates 9- or 31-fold compared to the wild type as determined by reversion to adenine or lysine prototrophy, respectively. Transformation of apn1 eth1 cells with an expression vector containing ETH1 reversed the hypersensitivity to MMS and limited the rate of spontaneous mutagenesis. Expression of ETH1 in a dut-1 xthA3 Escherichia coli strain demonstrated that the gene product functionally complements the missing AP endonuclease activity. Thus, in apn1 cells where the major AP endonuclease activity is missing, ETH1 offers an alternate capacity for repair of spontaneous or induced damage to DNA that is normally repaired by Apn1 protein.

Alkylating Agents↗
PubMed1998

Dynamics of the interaction of human apurinic endonuclease (Ape1) with its substrate and product.

We investigated the interaction dynamics of human abasic endonuclease, the Ape1 protein (also called Ref1, Hap1, or Apex), with its DNA substrate and incised product using electrophoretic assays and site-specific amino acid substitutions. Changing aspartate 283 to alanine (D283A) left 10% residual activity, contrary to a previous report, but complementation of repair-deficient bacteria by the D283A Ape1 protein was consistent with its activity in vitro. The D308A, D283/D308A double mutant, and histidine 309 to asparagine proteins had 22, 1, and approximately 0. 02% of wild-type Ape1 activity, respectively. Despite this range of enzymatic activities, all the mutant proteins had near-wild-type binding affinity specific for DNA containing a synthetic abasic site. Thus, substrate recognition and cleavage are genetically separable steps. Both the wild-type and mutant Ape1 proteins bound strongly to the enzyme incision product, an incised abasic site, which suggested that Ape1 might exhibit product inhibition. The use of human DNA polymerase beta to increase Ape1 activity by eliminating the incision product supports this conclusion. Notably, the complexes of the D283A, D308A, and D283A/D308A double mutant proteins with both intact and incised abasic DNA were significantly more stable than complexes containing wild-type Ape1, which may contribute to the lower turnover numbers of the mutant enzymes. Wild-type Ape1 protein bound tightly to DNA containing a one-nucleotide gap but not to DNA with a nick, consistent with the proposal that substrate recognition by Ape1 involves a space bracketed by duplex DNA, rather than mere flexibility of the DNA.

Amino Acid Substitution↗
PubMed1998

Rapid dissociation of human apurinic endonuclease (Ape1) from incised DNA induced by magnesium.

Repair of apurinic/apyrimidinic (AP) sites is initiated by AP endonucleases, such as the human Ape1 protein (also called Hap1, Apex, and Ref1). This and related enzymes show strong dependence on divalent cations, particularly magnesium. Here we explore the role of this metal in different stages of the Ape1 reaction: substrate binding, cleavage, and product release. We examined DNA binding using an electrophoretic approach and DNA cleavage in single-turnover and steady-state reactions. Magnesium at low to moderate concentrations accelerated both substrate and product release by wild-type Ape1 protein. For a mutant Ape1 protein with an aspartate to alanine substitution at residue 308, substrate in preformed protein-DNA complexes was more efficiently cleaved before release in contrast to wild-type Ape1, whereas product release was accelerated dramatically. The magnesium dependence of steady-state AP endonuclease reactions was sigmoidal for both wild-type and the aspartate 308 to alanine protein but was not sigmoidal for an aspartate 283 to alanine derivative of Ape1. These results show that magnesium affects both DNA interactions with and phosphodiester cleavage by Ape1 and can change the rate-limiting step of the reaction. Structural studies will need to be interpreted in the context of these diverse effects of the metal.

Carbon-Oxygen Lyases↗
PubMed1998

Removal of a thymoma via median sternotomy in a rabbit with recurrent appendicular neurofibrosarcoma.

A 9-year-old rabbit was evaluated for a mass on the right elbow. As a result of preoperative radiography, another mass was identified in the cranial portion of the mediastinum. Ultrasound guided fine needle aspiration was used for cytologic evaluation of the mass in the mediastinum and resulted in a presumptive diagnosis of thymoma. A median sternotomy approach was chosen to allow wide exposure for meticulous dissection in the cranial portion of the thoracic region. Histologic examination and immunoperoxidase labeling of lymphocytes with CD3 (a T cell marker) confirmed the diagnosis. The mass on the right elbow was subsequently removed and identified as a neurofibrosarcoma. Multiple appendicular masses that developed at separate locations and distinct times were excised and classified as locally invasive primary neurofibrosarcomas. Postmortem examination 9 months after initial evaluation confirmed recurrence of the neurofibrosarcoma at previous surgical sites. Gross or histologic evidence of thymoma was not evident. Prognosis is dismal for incompletely excised soft tissue sarcomas, because they are locally invasive and often recur.

Animals↗
PubMed1998

Cardiopulmonary and anesthetic effects of propofol administered intraosseously to green iguanas.

OBJECTIVE: To determine cardiopulmonary effects of intraosseous administration of propofol in green iguanas (Iguana iguana). DESIGN: Prospective study. ANIMALS: 14 green iguanas. PROCEDURE: Anesthesia was induced in 4 iguanas with propofol (10 mg/kg [4.5 mg/lb] of body weight, intraosseously). Heart and respiratory rates, functional hemoglobin oxygen saturation (SpO2), end-tidal CO2 concentration, and cloacal temperature were recorded. Ten additional iguanas were given propofol intraosseously for induction (5 mg/kg [2.3 mg/lb] and maintenance (0.5 mg/kg/min [0.23 mg/lb/min], q 30 min) of anesthesia. Heart and respiratory rates, cloacal temperature, and SpO2 were recorded. RESULTS: Mean induction time for the first 4 iguanas was 1.2 minutes. A significant decrease in heart rate was seen 1 minute after induction of anesthesia. All iguanas were apneic, but spontaneous ventilation resumed within 5 minutes. End-tidal CO2 concentration decreased from 46 mm of Hg 4 minutes after induction of anesthesia to 32 mm of Hg 30 minutes after induction of anesthesia. Mean duration of anesthesia was 27 minutes. Mean induction time for the other 10 iguanas was 3 minutes. A significant decrease in heart rate was detected 35 minutes after induction of anesthesia and persisted until 120 minutes. Mean SpO2 value decreased from 79% 5 minutes after induction of anesthesia to 64% 30 minutes after induction of anesthesia. Mean recovery time was 57 minutes. CLINICAL IMPLICATIONS: Propofol is an effective anesthetic agent for use in green iguanas. It is recommended that iguanas be intubated, provided oxygen, and given assisted ventilation after administration of propofol to prevent hypoxemia and hypercapnia.

Anesthesia↗
PubMed1997

Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway.

Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase beta (Polbeta) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein-protein contact between Polbeta and human AP endonuclease (Ape protein). In vitro, binding of Ape protein to uncleaved AP sites loads Polbeta into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Polbeta exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5'-terminal deoxyribose 5-phosphate by Polbeta. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.

DNA Polymerase I↗
PubMed1997

Incidence of contralateral versus ipsilateral neurological signs associated with lateralised Hansen type I disc extrusion.

Asymmetrical neurological signs were noted in 50 dogs presenting with Hansen type I thoracolumbar disc extrusion. Thoracolumbar myelograms and surgical decompression were performed in all cases. Dogs were divided into two groups (acute and chronic) based on the duration of clinical signs prior to presentation to the University of Georgia. Lateralising extradural cord compressive lesions were noted on all myelograms. In the acute group, 35 per cent of the dogs had asymmetrical neurological signs contralateral to the myelographic and surgical lesion, while in the chronic group only 11 per cent had neurological signs contralateral to the lesion. There was found to be no significant difference in frequency of contralateral asymmetrical clinical signs between the two groups (Fischer's exact test; P = 0.095). The high frequency of contralateral signs documents the importance of thoracolumbar myelography for accurate localisation of the disc material before decompressive surgery.

Acute Disease↗
PubMed1997

Regulation of eukaryotic abasic endonucleases and their role in genetic stability.

Abasic (AP) sites in DNA arise from spontaneous reactions or the action of DNA glycosylases and represent a loss of genetic information. The AP sites can be mutagenic or cytotoxic, and their repair is initiated by class II AP endonucleases, which incise immediately 5' to AP sites. The main enzyme of S. cerevisiae. Apn1, provides cellular resistance to oxidants (e.g., H2O2) or alkylating agents, and limits the spontaneous mutation rate. AP endonucleases from other species can replace Apn1 function in yeast to different extents. We studied the main human enzyme, Ape, with respect to its incision specificity in vitro and the expression of the APE gene in vivo. The results suggest that Ape evolved to act preferentially on AP sites compared to deoxyribose fragments located at oxidative strand breaks and that the incision modes of Ape and Apn1 may be fundamentally different. We also defined the functional APE promoter, and showed that APE expression is transiently downregulated during the regeneration of epidermis after wounding. This latter effect may lead to a window of vulnerability for DNA damage and perhaps mutagenesis during the healing of epidermal and other wounds. Such unexpected effects on the expression of DNA repair enzymes need to be taken into account in analyzing the susceptibility of different tissues to carcinogens.

DNA Damage↗
PubMed1996

End-joining of free radical-mediated DNA double-strand breaks in vitro is blocked by the kinase inhibitor wortmannin at a step preceding removal of damaged 3' termini.

Both mammalian cells and Xenopus eggs possess activities for the joining of nonhomologous DNA ends, and such activities may play a major role in double-strand break repair. In order to dissect the biochemical processing of breaks with oxidatively modified ends, vectors containing various site-specific double-strand breaks with 3'-phosphoglycolate termini were constructed and treated with Xenopus egg extracts. These vectors were rejoined by the extracts at rates 30-100 times slower than comparable 3'-hydroxyl vectors. Vectors with blunt or cohesive 3'-phosphoglycolate ends yielded single repair products corresponding to simple phosphoglycolate removal followed by ligation, while a vector with mismatched ends was also rejoined but yielded a mixture of products. Addition of the kinase inhibitors wortmannin and dimethylaminopurine not only blocked rejoining, but also suppressed phosphoglycolate removal, implying an early, essential, kinase-dependent restriction point in the pathway. The results suggest that double-strand breaks with oxidatively modified ends are repaired in Xenopus eggs by a highly conservative and stringently regulated end-joining pathway, in which all biochemical processing of the breaks is contingent on both end alignment and a specific phosphorylation event. Several lines of indirect evidence suggest DNA-dependent protein kinase as a likely candidate for effecting this phosphorylation.

Androstadienes↗
PubMed1996

Construction of a vector containing a site-specific DNA double-strand break with 3'-phosphoglycolate termini and analysis of the products of end-joining in CV-1 cells.

Previous studies have shown that linearized SV40-based shuttle vectors transfected into mammalian cells are efficiently recircularized by an error-prone end-joining pathway. To determine whether and with what specificity free radical-mediated double-strand breaks are rejoined by this pathway, a structural mimic of such a break was introduced at a specific site in an SV40-based shuttle vector, by ligating purified 3'-phosphoglycolate-terminated oligonucleotides into 3' recessed ends generated in the linearized vector. These terminally blocked linear vectors were efficiently repaired and replicated when transfected into simian CV-1 cells. Sequencing across the repair joints in progeny plasmid indicated that, for a blunt-ended vector, the most frequent mechanism of rejoining was splicing at a terminal 4-base homology; however, a significant fraction of the joints retained all bases from both ends of the break, consistent with a mechanism involving simple 3'-phosphoglycolate removal, followed by blunt-end ligation. For the analogous 3'-hydroxyl terminated break, the fraction of simple blunt-end ligations was considerably higher. For a phosphoglycolate-terminated vector with cohesive ends the most frequent repair mechanism was simple ligation of the annealed cohesive ends, presumably preceded by phosphoglycolate removal. For all these substrates, the remaining repair joints showed small or large deletions from one or both of the ends, usually with apparent annealing at short (1-4-base) homologies. The results suggest that while breaks with 3'-phosphoglycolates can be repaired, these blocked termini represent a significant barrier to DNA end-joining, and can significantly alter its specificity. The presence of cohesive ends appears to improve markedly the fidelity of rejoining for terminally blocked double-strand breaks.

Animals↗
PubMed1996

Immunohistochemical localization of transforming growth factor-alpha, epidermal growth factor (EGF), and EGF receptor in the human fetal ovary.

Peptide growth factors are thought to be involved in adult ovarian regulatory functions. However, little is known about the role of growth factors in human fetal ovarian development. This study is an attempt to identify and localize transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), and EGF receptor (EGF-R) in human fetal ovaries. Ovaries were obtained from first and second trimester elective abortuses. Immunohistochemistry was performed on paraffin sections of these specimens after fixation. We examined the sections microscopically using the specific antibodies against TGF alpha, EGF, and EGF-R. Phosphate-buffered saline and preimmune IgG were used as negative controls. First and second trimester ovaries stained positively for all three proteins. Staining was significantly more intense in the oocytes than in the stroma. Negative controls did not stain. These results combined with our previous demonstration of messenger ribonucleic acid for these growth factors suggest roles for TGF alpha, EGF, and EGF-R in human fetal ovarian development. The strong staining in the oocytes suggests a possible autocrine or paracrine role of these growth factors in human oocyte growth in utero.

Epidermal Growth Factor↗
PubMed1995

Trans-complementation by human apurinic endonuclease (Ape) of hypersensitivity to DNA damage and spontaneous mutator phenotype in apn1-yeast.

Abasic (AP) sites in DNA are potentially lethal and mutagenic. 'Class II' AP endonucleases initiate the repair of these and other DNA lesions. In yeast, the predominant enzyme of this type is Apn1, and its elimination sensitizes the cells to killing by simple alkylating agents or oxidants, and raises the rate of spontaneous mutation. We investigated the ability of the major human class II AP endonuclease, Ape, which is structurally unrelated to Apn1, to replace the yeast enzyme in vivo. Confocal immunomicroscopy studies indicate that approximately 25% of the Ape expressed in yeast is present in the nucleus. High-level Ape expression corresponding to approximately 7000 molecules per nucleus, equal to the normal Apn1 copy number, restored resistance to methyl methanesulfonate to near wild-type levels in Apn1-deficient (apn1-) yeast. Ape expression in apn1- yeast provided little protection against H2O2 challenges, consistent with the weak 3'-repair diesterase activity of the human enzyme. Ape expression at approximately 2000 molecules per nucleus reduced the spontaneous mutation rate of apn1- yeast to that seen for wild-type cells. Because Ape has a powerful AP endonuclease but weak 3'-diesterase activity, these findings indicate that endogenously generated AP sites can drive spontaneous mutagenesis.

DNA Damage↗
PubMed1994

Single base-pair deletions induced by bleomycin at potential double-strand cleavage sites in the aprt gene of stationary phase Chinese hamster ovary D422 cells.

One possible mechanism for the generation of deletion mutations is inaccurate repair of DNA double-strand breaks. In an attempt to detect such aberrant repair events in intact cells, confluent stationary phase cultures of chinese hamster ovary D422 cells, which are hemizygous for aprt, were treated for two days with low concentrations of bleomycin, and aprt mutant clones were selected and analyzed by polymerase chain reaction and DNA sequencing. Bleomycin was quite mutagenic in stationary phase cells, increasing the mutant frequency by five to 40-fold at 5 to 50% survival. While spontaneous mutations generated under these conditions were predominantly base substitutions, the majority of the bleomycin-induced mutations were very small deletions, with lesser numbers of large deletions/rearrangements and base substitutions. Although the small deletions tended to be clustered in several short segments of the gene, nucleosome positioning studies indicated that there was no consistent phasing of nucleosomes in aprt, suggesting that the clustering was due to sequence specificity rather than chromatin structure. About half of the bleomycin-induced mutations were single-base-pair (-1) deletions, and the majority of these involved deletion of one C in a G-Cn sequence (n > or = 2). At such sites, bleomycin is known to induce double-strand breaks by fragmentation of deoxyribose moieties at the same sequence position in both strands, resulting in a blunt-ended double-strand break with 5'-phosphate and 3'-phosphoglycolate termini. Thus, this sequence specificity is consistent with a model in which bleomycin-induced -1 deletions are generated by a double-strand break rejoining process involving removal of phosphoglycolate moieties from both 3' ends, followed by blunt-end ligation. The results support the view that repair of free radical-mediated double-strand breaks in mammalian cells in G1/G0 phase can be effected by such simple end-joining mechanisms, without the need for homologous recombination.

Adenine Phosphoribosyltransferase↗