Biomedical subjects
Publications and source records attributed to M S Miller.
The first step in reducing the cost of poor quality is to identify the defects controlled by materiel management. This article discusses the defects the authors have identified and measured in their efforts to reduce quality costs.
A series of novel N-methyl-D-aspartate antagonists acting at the phencyclidine site has been identified. Compound 2 has a Ki = 8 +/- 1 nM (vs [3H]thienylcyclidine, [3H]TCP) as a mixture of enantiomers. Resolution and further testing indicate that (-)-2, Ki = 4 +/- 0.7 nM, is a potent and selective TCP site ligand with neuroprotective activity in cultured neurons in the presence of excitotoxic concentrations of NMDA (IC50 = 26 nM). Compound (-)-2 is > 1000-fold selective for the TCP site vs a panel of receptor types including opiate, adrenergic, serotonergic, dopamine, adenosine, dihydropyridine, and benzodiazepine and displays increased selectivity for the activated (open) NMDA receptor-ion channel complex vs PCP and MK801 as measured by patch recordings in cultured, voltage-clamped neurons. Highly enhanced "open-channel" selectivity leads to tentative classification of these ligands as uncompetitive vs NMDA. Ligands with these characteristics may enable deconvolution of the pharmacologic effects associated with typical noncompetitive NMDA antagonists. We report here on the identification, synthesis, and activity of compounds of this structural class.
Testicular toxicity and interstitial cell tumours induced by cadmium are prevented by zinc or by low dose cadmium pretreatments. The mechanism of this tolerance is unknown, though metallothionein (MT) is thought to play a role in tissue resistance to cadmium toxicity. Thus, the possible involvement of the testicular MT gene in metal-induced tolerance to cadmium toxicity was studied. Rats were pretreated with zinc (1.0 mmol kg-1, s.c.). Histological examination of the testes indicated such pretreatments prevented the necrotizing effects of subsequent doses of cadmium (20 mumol kg-1, s.c.) administered 24 h later. RNA was extracted from testes or liver 24 h after zinc pretreatment, and analysed by the slot blot technique using the p2A10 cDNA probe to the MT gene. Zinc pretreatment had little effect on MT RNA in the testes, and such pretreatments did not alter testicular cadmium-binding protein capacity. In contrast, RNAs derived from livers of zinc pretreated rats showed marked increases in MT RNA and MT protein. Hence, the testicular MT gene does not appear to play a major role in the induced tolerance to cadmium toxicity and carcinogenesis generated by zinc.
We examined how reinforcer rate, quality, delay, and response effort combined to influence the choices of 6 youths with learning and behavior difficulties, and the viability of an assessment methodology derived from matching theory for determining differential responsiveness to those reinforcer and response dimensions. The students were given two concurrent sets of math problems that were equal on two dimensions but competed on two other dimensions (e.g., one set yielded higher rate and lower quality reinforcement than the other). Competing dimensions were counterbalanced across the six conditions of the initial assessment phase, permitting assessment of each dimension on time allocation. The conditions resulting in the most and least time allocated to one problem set alternative relative to the other were then replicated. Time allocated to each of the problems within sets was differentially affected by the reinforcer and/or response dimensions, with allocation patterns varying across students. The results are discussed in the context of implications for the design of treatments and extrapolations from basic research on matching and behavioral economics.
Sprague-Dawley rats were pretreated with a single i.p. injection of either 2.25 mL/kg of phosphate-buffered saline (PBS) or 22.5 mg/kg of dimethylnitrosamine (DMN) followed 2 hr later by a single i.p. injection of either 1.35 mg/kg of dexamethasone (DEX) or the vehicle, a 50% ethanol solution, both delivered in a volume of 3 mL/kg. RNA levels of the hormone-inducible, specialized liver function genes, tyrosine aminotransferase (TAT) and glutamine synthetase (GS), were monitored 4, 5, 6, 7, 8, and 10 hr after the DEX injection. Maximal induction of both the TAT (26-fold) and GS (6-fold) RNAs occurred 6 hr after DEX administration in PBS-pretreated animals. Pretreatment with DMN caused at least a 42% inhibition of DEX-induced RNA accumulation at every time point examined, with greater than 90% inhibition occurring when the genes were maximally induced at 6 hr. This inhibition was not due to any alterations of the glucocorticoid receptors as DMN had no effect on the binding affinity or amounts of glucocorticoid receptors present in rat hepatic cytosols. These results suggest that chemical carcinogens such as DMN may affect normal gene function in vivo by inhibiting the cellular response to hormone receptors mediating differentiation-associated, specialized cell functions.
Generation of free radicals during reperfusion after organ ischemia has been implicated in the pathogenesis of ischemic injury. We have previously shown that a combination of intravenous polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) and catalase (PEG-CAT), at a dose of 10,000 U/kg each, is effective in reducing infarct size in a focal cerebral ischemia model in the rat. It is not clear whether PEG-SOD alone is sufficient to reduce ischemic brain injury. In this study we determined the therapeutic efficacy of PEG-SOD and its dose-response curve. In a range of 1,000-30,000 U/kg, PEG-SOD exhibited a U-shaped dose-response curve. Only 10,000 U/kg significantly reduced infarct size [control 121 +/- 12 mm3 (mean +/- SE), n = 35; PEG-SOD 95 +/- 10 mm3, n = 36, P < 0.05]. PEG-SOD at the doses tested did not have significant acute hemodynamic effects but had a tendency to improve postischemic hypotension. This beneficial effect of PEG-SOD on blood pressure did not appear to fully account for the treatment effect of PEG-SOD on infarct size. The narrow therapeutic dose range of PEG-SOD in this study and similar findings of SOD in other investigations may contribute to the inconsistent protective effects of SOD preparations in ischemia-reperfusion injury in the literature.
Members of the cytochrome P4502E and 2B gene families have been implicated in the activation of nitrosamines to reactive species capable of binding to cellular macromolecules and initiating tumor formation in various rodent species. This study was initiated to determine the relative prevalence of these isozymes and their response to ethanol during pregnancy and late gestation. Nonpregnant and pregnant hamsters were given a 10% ethanol solution in their drinking water for 10 days (gestation days 5-15) prior to being killed. RNA blot analysis of liver and lung tissue from nonpregnant, pregnant, and fetal hamsters demonstrated tissue-specific expression of CYP2E and 2B in adult and fetal animals. The levels of RNA expression of both P450s in fetal hamsters were less than 30% of nonpregnant adult values. In pregnant hamsters, the hepatic levels of CYP2E and 2B RNAs were decreased compared to nonpregnant animals. In contrast, the pulmonary levels of CYP2B RNA were increased in pregnant versus non-pregnant hamsters, while no effect of pregnancy on the levels of CYP2E RNA was seen. Although rats contain a single CYP2E1 gene transcript. Northern analysis demonstrated the presence of 1.8 and 2.8 kb bands in both liver and lung tissue of the hamster. Pretreatment with ethanol had little effect on the levels of either P450 RNA species in the lungs or livers of nonpregnant, pregnant, and fetal hamsters. These results demonstrate differences in the levels of expression of members of the CYP2E and 2B gene families during pregnancy and late gestation compared to nonpregnant adult hamsters. Fetal animals, like the adults, apparently respond to ethanol treatment by altering the levels of these P450 isozymes at the post-transcriptional level.
The effects of a number of phenobarbital-type inducers on selected drug-metabolizing enzymes in male F344/NCr rats were determined by measuring specific catalytic activities and/or by measuring the levels of RNA which hybridize with specific probes for the corresponding genes. The effects on hepatic CYP2B1 were assessed by measuring the levels of CYP2B1-specific RNA and benzyloxyresorufin O-dealkylase and testosterone 16 beta-hydroxylase activities. Levels of CYP3A were monitored by measuring the rate of hydroxylation of testosterone at the 6 beta-position. Microsomal epoxide hydrolase activity was determined by measurement of cellular RNA specific for this form and by assaying the hydrolysis of benzo[a]pyrene-4,5-oxide. UDP-glucuronyltransferase activity was assayed by measuring the glucuronidation of 3-hydroxybenz[a]anthracene. Levels of glutathione S-transferase Ya/Yc were measured by quantifying total cellular RNA coding for the proteins. When male F344/NCr rats were administered various doses of phenobarbital or dichlorodiphenyltrichloroethane (DDT), strong correlations between the induction of CYP2B1 and the induction of epoxide hydrolase or UDP-glucuronyltransferase activities were observed. Treatment of rats with barbiturates, hydantoins, halogenated pesticides such as DDT or alpha-hexachlorocyclohexane, 2,4,5,2',4',5'-hexachlorobiphenyl, CYP2B1 inhibitors such as clotrimazole or clonazepam, or such structurally-diverse compounds as 2-hexanone or diallyl sulfide resulted in induction of CYP2B1-mediated enzyme activity and induction of certain other forms of cytochrome P450, microsomal epoxide hydrolase, at least one form of UDP-glucuronyltransferase, and multiple forms of glutathione S-transferase. This suggests that, as a class, compounds which induce CYP2B1 also induce a coordinate hepatic pleiotropic response which includes induction of these other phase I and phase II drug-metabolizing enzymes.
Phenobarbital (PB) and certain structurally-related compounds induce a variety of hepatic drug-metabolizing enzymes in many strains of rats. Thus, following administration of PB (300, 500 ppm), barbital (BB, 1500 ppm) or 5-ethyl-5-phenylhydantoin (EPH, 500 ppm), CYP2B1-mediated benzyloxyresorufin O-dealkylase activity and epoxide hydrolase activity were profoundly induced in female DA and F344/NCr rats. In contrast, outbred female lean and obese Zucker rats showed markedly reduced CYP2B1 responses (less than 15% and less than 5% of those observed in the female DA or F344/NCr rat) to PB (doses less than or equal to 300 ppm), BB (1500 ppm) or EPH (500 ppm). In parallel studies, profound increases in RNA levels coding for CYP2B1, glutathione S-transferases Ya/Yc (alpha subclass), or epoxide hydrolase were detected in the female F344/NCr rat following treatment with PB (300 ppm), BB (1500 ppm) or EPH (500 ppm). In contrast, lean Zucker rats showed a strong response only to the highest dose of PB (500 ppm), implying that the diminished response in the Zucker rats may occur at some pretranslational level. Similar studies with lower doses of PB, EPH or BB in male lean Zucker rats showed a decreased response, relative to that in male F344/NCr rats. However, this insensitivity was not as profound as that observed in the female Zucker rats. In fact, the response to PB-type inducers in male or female Zucker rats is probably most clearly explained as a shift of the dose-response curve sharply to the right (decreased responsiveness, compared to F344/NCr or DA rats of the same sex). This decreased responsiveness of female lean Zucker rats to induction of CYP2B1, relative to that of F344/NCr rats, was also observed with the structurally-diverse PB-type inducers clonazepam, clotrimazole and 2-hexanone. In contrast, the female Zucker rat (obese or lean) displayed a pronounced response to induction of CYP1A-mediated ethoxyresorufin O-deethylase activity by beta-naphthoflavone, a prototype inducer of CYP1A1 and CYP1A2. The Zucker rat would thus appear to represent a potentially exploitable genetic model for examining the mechanism of enzyme induction by the myriad xenobiotics which induce a PB-type response.
Pregnant Sprague-Dawley rats were treated with a single ip dose of either olive oil or 40 mg/kg of 3-methylcholanthrene on gestation day 20 and sacrificed at various times after injection. Determination of aryl hydrocarbon hydroxylase activity 24 hr after injection revealed that treatment with 3-methylcholanthrene resulted in a 10.5-fold stimulation of enzymatic activity in liver 800 x g supernatants. Western blot analysis with monoclonal antibody 1-7-1 confirmed these results and demonstrated the presence of a 3-methylcholanthrene-inducible P-450 isozyme. Using Northern and slot blot techniques, the induction of steady-state levels of CYPIA1 RNA was shown to occur as early as 4 hr following 3-methylcholanthrene injection. CYPIA1 RNA levels were induced 31.6-fold over values obtained from oil-treated tissues at this time. This appears to be the optimal time to study changes in the levels of CYPIA1 RNA gene expression in the fetus following transplacental exposure to polycyclic aromatic hydrocarbons. By 12 to 24 hr postinjection, the induction of CYPIA1 RNA levels declined to 3.5- to 8.5-fold above control values. These results demonstrate that the kinetics of induction of the CYPIA1 gene during the fetal period differed from that seen in adults.
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The ontogeny and transplacental inducibility of UDP-glucuronosyl transferase (UDPGT) activities potentially relevant to detoxification of polycyclic aromatic hydrocarbons were studied in (C57BL/6 x DBA/2) F1 or (DBA/2 x C57BL/6) F1 fetal mouse liver, with p-nitrophenol (PNP) and 3-hydroxybenzo[a]pyrene (3-OH-BP) as substrates. Both UDPGT activities developed during the late fetal period and reached almost 60% of the adult activity at term; PNP, but not 3-OH-BP UDPGT decreased significantly on postnatal day 1 before rising to adult levels. A single exposure to beta-naphthoflavone (beta NF; 150 mg/kg) on day 17 of gestation induced the PNP-UDPGT activity significantly (1.5-fold) by day 19 in the B6D2 F1s but not D2B6 F1s. A single dose of 3-methylcholanthrene (MC; 100 mg/kg) or 2,3,7,8,-tetrachlorodibenzo-p-dioxin (10 micrograms/kg) did not induce, but three injections of MC also resulted in significant induction in the fetuses of C57BL/6 mothers. 3-OH-BP-UDPGT was not significantly induced by any of the chemicals in either genetic cross. In a parallel study, a gene for an inducible mouse UDPGT, designated UDPGTm-1, was shown by Northern blotting to be expressed in fetal liver by day 18 of gestation at low levels relative to adults, but was not induced transplacentally by MC, beta NF or phenobarbital (PB). These results show that (1) at least two functionally defined UDPGT activities toward phenolic substrates are present in the late fetal mouse liver; (2) one of these is transplacentally inducible by beta NF and MC, but only in fetuses of C57BL/6 mothers, (3) induction where achieved was relatively small in magnitude, and (4) a gene of a PB-inducible UDPGT was expressed at low levels in the fetuses but was not induced transplacentally.
The administration of 3-methylcholanthrene (MC) to pregnant mice results in the formation of lung tumors in the offspring. Previous work has shown that fetuses demonstrating inducibility of aryl hydrocarbon metabolism develop two to five times more lung tumors than induction-nonresponsive littermates. In this study, the effects of fetal versus adult MC exposure were compared with regard to both induction of aryl hydrocarbon hydroxylase activity (AHH) in lung and dependence of lung tumorigenesis on the Ah genotype. In inducible (C57BL/6 X DBA/2)F1 fetal lung supernatants, a single ip injection of 100 mg/kg of MC to the mothers resulted in a maximal 50-fold induction of AHH activity by 8 hr, which persisted for 48 hr. The enzyme data agreed well with RNA blot analysis, as MC caused maximal induction of P450IA1 RNA by 4 hr. For comparison, adult (F1 X DBA/2) mice were given three weekly injections of 100 mg/kg MC and tumor incidences were determined after 16 weeks. No differences were observed between responsive and nonresponsive mice of either sex in the number of mice bearing lung tumors, nor did the tumor multiplicity differ between responsive and nonresponsive males. However, noninducible female mice had a significantly higher tumor multiplicity than their inducible counterparts (p less than 0.025). Single ip injections of MC to adult F1 mice revealed that lung AHH activity was increased only 4- to 7-fold in the adult animal compared to the large fetal induction ratio. The difference in the magnitude of induction was due to the higher constitutive levels of AHH activity seen in adult tissue (4- to 14-fold greater than maximal basal fetal levels), as fetal and adult supernatants showed similar levels of induced activity following MC treatment. These results suggest that the correlation between susceptibility to MC-initiated lung tumors and induction of cytochrome P450IA1 is a unique property of the fetus and may be due, in part, to the low basal levels of fetal activating enzymes and their high induction ratio during the fetal period.
Previous studies have shown that treatment of rat hepatoma cells (the Fao clone of Reuber H-35 cells) with 500 ng/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) causes a 78% decrease in dexamethasone (DEX)-induced tyrosine aminotransferase (TAT) enzyme activity and a concurrent 75% decline in total steroid-induced TAT steady-state RNA levels. To determine if this inhibition was a specific or more general effect on inducible-gene expression, the effects of MNNG on other genes were examined. MNNG had little effect on total DEX or Cd-induced metallothionein (MT) RNA levels when the cells were treated with 1 microM DEX or 3 microM CdCl2 for 4 h. In addition, the carcinogen had no effect on the basal level of MT-specific total RNA, nor did it alter the total RNA levels of the alpha-tubulin gene. Although attempts were made to measure the levels of the glucocorticoid receptor by both biochemical and molecular methods, receptor levels were too low to quantitate accurately. However, the lack of effect of MNNG on steroid-induced MT RNA levels suggests that the inhibitory effect of the carcinogen was not mediated through alterations in glucocorticoid receptor function. MNNG had no effect on cell number or viability, nor did the carcinogen alter the methylation pattern of the TAT gene as determined from MspI/HpaII digests. The results suggest that MNNG mediates its inhibitory effects by a specific interaction with either the TAT gene itself or some other regulatory factor(s) involved in TAT RNA transcription or stability. This effect was relatively gene specific, since expression of the inducible, specialized liver function TAT gene was inhibited by MNNG but expression of the more ubiquitous and inducible MT gene and the constitutively expressed alpha-tubulin gene were not.
Treatment of pregnant mice with 3-methylcholanthrene (MC) causes lung and liver tumors in the offspring, the incidences of which are greatly influenced by the Ah locus regulated induction phenotype for aryl hydrocarbon hydroxylase activity (AHH) in both the mother and fetuses. In order to examine the biochemical and molecular mechanisms responsible for the modulating effect of maternal environment on tumor susceptibility, reciprocal crosses between responsive C57BL/6 and non-responsive DBA/2 mice were made and the pregnant mothers were treated i.p. on the 17th day of gestation with either olive oil alone, 30 mg/kg of MC, or 30 mg/kg of beta-naphthoflavone (beta NF). At various times after injection, the mothers were killed and the fetuses removed for enzymatic and molecular blot analysis. In fetal lung tissues, the absolute levels and relative induction ratios of AHH activity from D2B6F1 fetuses were very similar to those obtained in B6D2F1 fetuses during the first 24 h following a transplacental exposure to either inducing agent. This was also the case 48 h after an injection of beta NF. However, 48 h after exposure to MC, the AHH activity in fetal lungs from B6 mothers had declined to practically control values, whereas fetal lungs from D2 mothers still exhibited a high level of AHH activity. Similar induction kinetics for the CYPIA1 gene were obtained in fetal livers. These results were confirmed at the RNA level by quantitative slot-blot analysis of fetal RNA preparations. In both organs, treatment with inducing agents for the P450IA1 gene resulted in a rapid and early induction of CYPIA1 RNA by 4 h. Fetuses from D2 mothers, however, showed a more sustained induction of CYPIA1 RNA following exposure to MC than did fetuses from B6 mothers. These results suggest that the observed increase in tumor susceptibility observed in the offspring of D2 mothers compared to the offspring of B6 mothers was due, at least in part, to the differences in the persistence of induction of the CYPIA1 gene locus, and may be the result of differences in the clearance rates of MC from the fetal and maternal compartments or its pharmacokinetic distribution in the two types of maternal environments.
An elastinolytic proteinase of Aspergillus flavus has been isolated to homogeneity, and its physical and biochemical properties have been characterized. Two purification protocols were compared; an initial step of ion-exchange chromatography was found to be equivalent to ammonium sulfate precipitation at neutral pH. A combination of gel filtration and adsorption chromatographies on the resultant crude enzyme produced highly purified elastase with yields of 5 to 10%. The enzyme is a 23-kilodalton protein with a pI of 7.6. The enzyme activity is markedly inhibited by numerous metal ions. Aspergillus elastase appears to be a metalloproteinase EC 3.4.24.X), as determined by its sensitivity to 1,10-phenanthroline.
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The incidence of cardiopulmonary disease increases with age. Degenerative valvular disease, chronic obstructive pulmonary disease, and arrhythmias are common in the geriatric dog. Chronic bronchial disease, pulmonary neoplasia, and arrhythmias occur in the geriatric cat. Systemic diseases in both species often show cardiopulmonary manifestations. Medical management to treat the underlying disease and to control clinical signs is complicated by altered absorption, metabolism, and elimination of drugs.