Biomedical subjects
Publications and source records attributed to G L Carter.
We have increased the production of bovine chymosin in Aspergillus niger var. awamori to more than one gram per liter of secreted authentic enzyme by combining a mutagenesis protocol with a novel robotic screening program. Analysis of the superior chymosin producing strains indicated that they have enhanced capabilities to secrete extracellular proteins.
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L1210 cell lines were generated which were resistant to specific ribonucleotide reductase inhibitors. Hydroxyurea-resistant L1210 cells (HU-7) were cross-resistant to IMPY but sensitive to deoxyadenosine and deoxyguanosine. Deoxyadenosine-resistant L1210 cells (Y-8) were cross-resistant to 2-fluorodeoxyadenosine and showed only a small increase in resistance to hydroxyurea or IMPY. L1210 cells which were generated in the presence of deoxyadenosine/EHNA/IMPY/Desferal were markedly resistant to deoxyadenosine, deoxyguanosine and 2-fluorodeoxyadenosine with moderate increases in resistance to IMPY. The HU-7, Y-8 and ED2 cell lines were sensitive to the inhibitory effects of MAIQ and HAG-IQ. The HU-7 L1210 cell line had elevated levels of ribonucleotide reductase activity and this activity showed normal inhibition by hydroxyurea, IMPY, dATP, dGTP and dTTP. The Y-8 L1210 cell line did not have elevated levels of ribonucleotide reductase activity, but had altered allosteric properties relative to dATP. The ED2 L1210 cell line had elevated levels of ribonucleotide reductase activity and had altered allosteric properties relative to dATP. These data show that resistance to ribonucleotide reductase inhibitors is specifically generated in response to the particular drug. The biochemical basis can be related to either increased levels of ribonucleotide reductase activity or loss of feedback control by dATP or both.
Injuries and fatalities from recreational hunting accidents have been studied much less than firearm accidents occurring in urban populations. The available data indicate that hunting accidents may account for a significant number of unintentional firearm accidents in areas outside commonly studied urban settings. Legislative efforts to control handgun availability can be expected to have little impact on hunting accident statistics. The development of automatic firearm safety devices, promotion of hunter safety programs, and greater participation by the medical community in preventive measures may impact the problem. Similar efforts have already been influential in reducing other forms of accidental injury through promotion of seat-belt use, local motorcycle helmet laws, use of infant car seats, and, most recently, regulations regarding all-terrain vehicles.
Ribonucleotide reductase consists of two non-identical subunits, non-heme iron subunit (NHI) and effector-binding subunit (EB), that are encoded by different genes and that can be specifically and independently inhibited. L1210 cell lines were selected for resistance to hydroxyurea (HU-7), deoxyadenosine (Y-8), or the combination of pyrazoloimidazole and deoxyadenosine (ED2). The gene for the NHI was amplified in the HU-7, Y-8, and ED2 cell lines; there was no amplification of the gene for the EB. The mRNA for the NHI was increased in the HU-7, Y-8, and ED2 cells, but there was no change in the mRNA levels for the EB. Reductase activity was increased in the HU-7, ED2 cells, but not in the Y-8 cells. The reductase activities in the Y-8 and the ED2 cells were not subject to feedback inhibition by dATP. These data show that the mechanisms of resistance to inhibitors directed at this reductase are varied and do not require increased enzyme activity. Further, gene amplification or increased mRNA levels did not necessarily result in increased levels of cellular enzyme.
Ribonucleotide reductase consists of two non-identical protein subunits that are required for enzyme activity. These subunits are encoded by different genes and are not expressed coordinately as the cells pass through the cell cycle. Using specific cDNAs for the non-heme iron (NHI) and the effector-binding (EB) subunits the levels of the mRNAs for these two subunits were determined in leukemia L1210 cells during the transition from the G0/G1 phase to the S and G2/M phases of the cell cycle. Synchronized populations of L1210 cells were obtained either by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) treatment or by enrichment by elutriation centrifugation. The changes in the levels of the mRNAs for NHI and EB subunits were compared with the changes in the levels of the mRNAs for actin, p53, c-myc, thymidine incorporation into DNA, and DNA content by flow cytometric measurements. Synchronization of the cells by the two methods resulted in quantitative differences in the responses. The EGTA synchronized L1210 cells showed maximal increases of 9.3- and 5.7-fold in the mRNAs for the NHI and EB subunits, respectively. The peak level of the NHI mRNA was observed at 12 hr after the addition of calcium ions. The peak increase in the level of the mRNA for the EB subunit was observed between 12 and 15 hr after the addition of calcium ions. The rate of increase for the mRNA for c-myc was greater than the increase in the mRNA for the NHI subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
Hydroxyurea is an inhibitor of ribonucleotide reductase and is specifically directed at the non-heme iron subunit (which contains the free radical) of this enzyme. Leukemia L1210 cells, grown in the presence of increasing concentrations of hydroxyurea, developed resistance to hydroxyurea. For hydroxyurea, the wild-type L1210 cells required a drug concentration of 85 microM to inhibit cell growth by 50%, and the hydroxyurea-resistant (HU-7-S7) cells required a concentration of approximately 2000 microM. The resistant L1210 cells were cross-resistant to 2,3-dihydro-1H-pyrazolo[2,3-a]imidazole/Desferal. However, these HU-7-S7 cells remained sensitive to 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone and 1-isoquinolylmethylene-N-hydroxy-N'-amino-guanidine tosylate (inhibitors directed at the same subunit as hydroxyurea). The HU-7-S7 cells retained their sensitivity to deoxyadenosine/erythro-9-(2-hydroxy-3-nonyl)adenine and deoxyguanosine/8-amino-guanosine (inhibitors directed at the effector-binding subunit of ribonucleotide reductase). The L1210 cells developed for resistance to hydroxyurea were sensitive to the non-ribonucleotide reductase inhibitors, methotrexate and 1-beta-D-arabinofuranosylcytosine. Ribonucleotide reductase activity was elevated in the HU-7-S7 cells (CDP reductase, 5.5-fold increase; ADP reductase, 13.2-fold increase). The addition of exogenous effector-binding subunit caused much greater stimulation of reductase activities in the extracts from the resistant cells than from the wild-type cells. The reductase activity in cell-free extracts from the resistant cells was inhibited by hydroxyurea, 2,3-dihydro-1H-pyrazolo[2,3-a]imidazole and dATP to the same extent as the activity from the wild-type L1210 cells. These data indicate that resistance to hydroxyurea in these L1210 cells is to some extent related to increased reductase activity. However, the specificity of resistance of these L1210 cells to inhibitors of ribonucleotide reductase depends on the nature of the inhibitor and the subunit at which the inhibitor is directed.
Leukemia L1210 cell lines, ED1 and ED2, were generated which were resistant to the cytotoxic effects of deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine and deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine plus 2,3-dihydro-1H-pyrazole[2,3a]imidazole/Desferal, respectively. The ED1 and ED2 were characterized to show that these cell lines had increased levels of ribonucleotide reductase as measured by CDP reduction. The reductase activity in crude cell-free extracts from the ED1 and ED2 cells was not inhibited by dATP. For CDP reductase, the activation by adenylylimido diphosphate and inhibition by dGTP and dTTP in these extracts from the ED1 and ED2 cells were the same as for the wild-type L1210 cells. The ED1 and ED2 cells were highly cross-resistant, as measured by growth inhibition, to deoxyguanosine/8-aminoguanosine, 2-fluorodeoxyadenosine, and 2-fluoroadenine arabinoside. While the ED2 cells showed resistance to 2,3-dihydro-1H-pyrazole-[2,3a]-imidazole/Desferal (6-fold), the ED1 and ED2 cell lines showed less resistance to hydroxyurea, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone, and the dialdehyde of inosine. These data indicate that the mechanisms of resistance to the ribonucleotide reductase inhibitors are related to the increased level of ribonucleotide reductase activity and to the decreased sensitivity of the effector-binding subunit to dATP.
Violence in the emergency department is a common concern. However, most aspects of this problem remain unstudied because no organization or government agency tracks such data and no regulatory or administrative guidelines adequately address its management. We surveyed 170 US teaching hospital ED medical directors with respect to violence and security issues and received responses from 127 (74.7%). Among other findings, 41 institutions report at least one verbal threat each day, and 23 report at least one threat with a weapon each month. Four-point physical restraint is used by 125 of the 127 facilities. Personnel in 32 of these facilities restrain at least one patient each day. Seventeen institutions report having significantly injured a patient during restraint in the last five years, resulting in one death. Twenty institutions report involvement with litigation with respect to restraint. Only 51 institutions provide ED nurses with formal training in recognition and management of aggression and violence, and only 79 institutions have security personnel present in the ED 24 hours a day. A sizable number of facilities receiving frequent threats and batteries are not among those with 24-hour-a-day security personnel. A preventative, risk-management approach that addresses environmental factors, training policies, restraint, security arrangements, and legal precedents is suggested.
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Maximum growth of L1210 cells in culture required the presence of free extracellular calcium ions. Reducing the free extracellular calcium ion concentration with EGTA served to decrease the growth rate of the cells. The decrease in cell growth was not due to cell death but rather due to the "pile-up" of the L1210 cells in the GO/Gl phase of the cell cycle. With the readdition of excess calcium ions, there was a lag period of 3 to 6 hours before the L1210 cells initiated DNA synthesis or transited from the G0/G1 phase to S-phase. Cells enriched for S and G2/M phase by elutriation and which were incubated in EGTA-containing culture medium, continued through the cell cycle and were blocked in GO/Gl. These data indicate that the proliferation of L1210 cells in culture requires a calcium ion-dependent process to allow movement from the G0/G1 to S-phase of the cell cycle.
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It was found that nucleoside 5'-diphosphates could serve as effectors of ribonucleotide reductase. ADP was an activator of CDP reduction; ADP reduction was activated by dGDP; GDP reduction was activated by dTDP. Conversely, dADP inhibited the reduction of CDP, UDP, GDP, and ADP; dGDP inhibited UDP and GDP reductions; and dTDP inhibited UDP reduction. The inhibition of UDP reduction by dADP, dTDP, and dGDP was at least equal to that observed for dATP, dTTP, and dGTP, respectively. In these experiments with the nucleoside diphosphates as effectors, high-pressure liquid chromatography analysis of the reaction mixtures showed that no nucleoside 5'-triphosphates were found during the reaction period which could account for the effects seen with the nucleoside diphosphates as effectors. Further experiments were carried out in which adenyl-5'-yl imidodiphosphate was used as the positive effector of CDP and UDP reductions in place of ATP. Under these conditions, CDP and UDP reductions were inhibited by dADP, dTDP, and dGDP to the same extent observed in the presence of ATP. ADP served not only as a substrate for ribonucleotide reductase but also as an activator of CDP and UDP reductions. The direct products (dNDPs) also served as positive and negative effectors. Dixon plots indicated that the dNDPs were acting as noncompetitive inhibitors with respect to the substrate. ADP increased the sedimentation velocity of the ribonucleotide reductase in a manner similar to ATP. These data are consistent with the allosteric effects seen with the nucleoside 5'-triphosphates. Additionally, from the thorough study of the role of effectors on UDP reduction, it is clear that UDP reduction was most sensitive to the negative effectors dATP, dADP, dTTP, dTDP, dGTP, and dGDP.
A series of N-hydroxy-N'-aminoguanidine derivatives was studied for their effects on L1210 cell growth and ribonucleotide reductase activity. With the twelve compounds studied, there was a good correlation between the inhibition of L1210 cell growth and the inhibition of ribonucleotide reductase activity. The most potent compound required concentrations of only 1.4 and 2 microM for 50% inhibition of L1210 cell growth and ribonucleotide reductase activity respectively. These guanidine analogs specifically inhibited the conversion of [14C]cytidine and deoxycytidine nucleotides in the nucleotide pool and the incorporation of [14C]cytidine into DNA without altering the incorporation of [14C]cytidine into RNA. Ribonucleotide reductase activity in drug-treated cells was reduced markedly. Iron-chelating agents did not either increase or decrease the inhibition caused by the N-hydroxy-N'-aminoguanidine derivatives. No evidence was obtained that these derivatives selectively inactivated one of the subunits of ribonucleotide reductase. These compounds appear to inhibit ribonucleotide reductase by a mechanism different from hydroxyurea or the thiosemicarbazone derivatives.
Ribonucleotide reductase is a key enzyme in DNA replication and, as such, has been a target for antitumor agents. This enzyme is composed of two nonidentical protein subunits which can be specifically and independently inhibited. Combinations of drugs directed at the effector-binding and non-heme iron subunits of ribonucleotide reductase resulted in the synergistic inhibition of L1210 cell growth and synergistic L1210 cell kill. These combinations included dAdo/EHNA/IMPY/Desferal; dAdo/EHNA/hydroxyurea/Desferal (the EHNA was required to protect dAdo from deamination while Desferal modulated the effects of IMPY or hydroxyurea); 2-F-araA/IMPY/Desferal and 2-F-2'-dAdo/IMPY/Desferal (EHNA was not required to protect 2-F-araA or 2-F-2'-dAdo from deamination); and dGuo/8-AGuo/IMPY/Desferal (8-AGuo was required to protect dGuo from phosphorolysis). Although thymidine alone inhibited L1210 cell growth, it was not possible to potentiate the effects of thymidine with the pyrimidine nucleoside phosphorylase inhibitors, acyclothymidine, 5-chlorouracil and 2,6-dihydroxypyridine. Combinations of drugs directed at the ribonucleotide reductase and DNA polymerase sites were studied for their effects on L1210 cell growth. With these combinations, no synergistic inhibition of L1210 cell growth was observed. The combinations of aphidicolin and IMPY/Desferal and aphidicolin and dAdo/EHNA inhibited L1210 cell growth in an additive manner; the combinations of IMPY/Desferal and BuAU or IMPY/Desferal and BuPdG resulted in antagonistic inhibition of L1210 cell growth. From these results it is clear that combination chemotherapy directed at independent sites of the same key target enzyme can result in strong synergistic inhibition of cell growth and cytotoxicity offering a clear therapeutic advantage. In contrast, the combinations directed at sequential key enzymes (e.g. ribonucleotide reductase and DNA polymerase) did not result in synergistic inhibition of cell growth. The utility of combinations of drugs directed at specific but independent sites of the target enzyme (e.g. ribonucleotide reductase) has been demonstrated in tumor cell systems in culture and now must be demonstrated in vivo.
Ribonucleotide reductase catalyzes the rate-limiting step in DNA synthesis. It represents a key metabolic site at which specific inhibitors have been directed as potential antitumor agents. Several different classes of ribonucleotide reductase inhibitors have been generated and studied. Because of the nature of the DNA polymerase reaction in which all four dNTPs are required, the initial velocity vs dNTP concentration curve gives sigmoidal rather than hyperbolic kinetics. As a result, a 50 per cent decrease in ribonucleotide reductase activity causes a decrease in DNA polymerase activity of 75 per cent or greater depending on the ratio of [dNTP] to its Km. This has been demonstrated with theoretical calculations, actual DNA polymerase determinations and precursor studies in intact tumor cells. The structural requirements for a compound to serve as a specific inhibitor of ribonucleotide reductase, either as the non-heme iron or effector-binding subunit, are stringent. Each protein subunit comprising the active enzyme can be specifically and independently inhibited. When combinations of agents, each directed at one of the subunits of ribonucleotide reductase, are used, strong synergistic inhibition of L1210 cell growth and synergistic cytotoxicity result.
Ribonucleotide reductase from tumor cells consists of two non-identical components which can be specifically and independently inhibited. Combinations of agents directed at the individual components gave synergistic inhibition of L1210 cell growth in culture. Utilizing hydroxyurea and deoxyadenosine or IMPY and deoxyadenosine as the parent combinations, modulators were used to potentiate the effects of each of these drugs. EHNA was used to prevent the deamination of deoxyadenosine while Desferal was utilized to increase the effects of hydroxyurea and IMPY. Combinations consisting of deoxyadenosine/EHNA plus IMPY/Desferal and deoxyadenosine/EHNA plus hydroxyurea/Desferal gave synergistic inhibition of L1210 cell growth. Utilizing these combination chemotherapies, the concentrations of each of the agents could be kept to minimal, essentially non-inhibitory levels and yet still achieve complete inhibition of L1210 cell growth with the specifically generated four-drug combinations.