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Biomedical subjects

K W Miller

Publications and source records attributed to K W Miller.

At least 109 records · Page 6Linked to original sources
PubMed1987

Cotranscription of the Escherichia coli isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) genes. Fine-structure mapping of the lsp internal promoter.

We have applied the technique of Northern blotting hybridization analysis to examine the transcription of the Escherichia coli isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) genes. RNA samples from the wild-type strain CS412 and CS412 containing the plasmids pMT521 and pSYC890 were examined with ileS- and lsp-specific 32P-labeled probes. pMT521 is a pBR322 derivative into which a chromosomal DNA fragment containing the ileS and lsp genes has been cloned downstream of the pBR322 tet promoter. pSYC890 is a derivative of pMT521 which lacks the tet promoter, but nevertheless is active in lsp mRNA transcription due to the presence of a weak lsp internal promoter located in the distal portion of the upstream ileS DNA, RNA from CS412(pMT521) exhibited a high level of a 5500-nucleotide ileS-lsp cotranscript that hybridized to both probes. RNA from CS412(pSYC890) contained a high level of low molecular weight lsp-specific transcripts from the lsp internal promoter. However, only high molecular weight (5000-6500 nucleotides) cotranscripts were detected in RNA from CS412 by Northern blotting analysis. A trace level of lsp-specific mRNA was detected in CS412 by S1 nuclease mapping. The 5'-end of this transcript was mapped using RNA from CS412(pSYC890). The mRNA begins 192/193 base pairs upstream of the lsp translation initiation codon at a site within ileS DNA that codes for the aspartate residue located 61 amino acids from the carboxyl-terminal of ILES. In conclusion, most of the transcription of lsp in wild-type E. coli originates from upstream of ileS and yields ileS-lsp cotranscripts.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Anesthetic potencies of secondary alcohol enantiomers.

The Meyer-Overton rule has been interpreted to mean that general anesthetics act at a nonpolar site, either in a lipid bilayer or a protein. Optical isomers, also called enantiomers, are pairs of compounds with the same molecular formula and functional groups, but which differ in the arrangement of the groups around an "asymmetric" carbon atom and in the direction they rotate plane-polarized light. By definition, enantiomers that are anesthetics can distinguish between stereoselective and nonselective sites of anesthetic action. We used such enantiomers to determine whether anesthetics are stereoselective in their actions on animals by measuring the potencies of a homologous series of secondary aliphatic alcohols from 2-butanol through 2-octanol in tadpoles, using reversible loss of righting reflex as the endpoint. None of the isomeric pairs exhibited significant differences in potency. Anesthetic potency increased logarithmically with the number of carbon atoms in the hydrocarbon chain of the alcohol. The ED50 +/- SE (mM) for the (+) and (-) forms of the alcohols, respectively, were as follows: 2-butanol 17 +/- 1.2, 17 +/- 1.1; 2-pentanol 4.7 +/- 0.28, 4.8 +/- 0.27; 2-hexanol 1.33 +/- 0.068, 1.42 +/- 0.079; 2-heptanol 0.32 +/- 0.011, 0.33 +/- 0.020; and 2-octanol 0.063 +/- 0.0042, 0.061 +/- 0.0032. These data demonstrate a lack of stereoselectivity in the interactions between the anesthetic secondary alcohols and their site of action in animals.

1-Octanol↗
PubMed1987

Barbiturates bind to an allosteric regulatory site on nicotinic acetylcholine receptor-rich membranes.

The ability of barbiturates to bind to acetylcholine receptor-rich membranes purified from the electroplaques of Torpedo nobiliana was examined by centrifugation assay. [14C]Amobarbital both partitioned into the membrane and bound displaceably to a site with an equilibrium dissociation constant of 12 microM. This low affinity made the stoichiometry difficult to obtain despite the high specific activity of acetylcholine receptors in this membrane preparation. However, the data are not inconsistent with a stoichiometry of one barbiturate-binding site per acetylcholine-binding site. Displaceable [14C]amobarbital binding was completely inhibited by barbiturates (IC50: amobarbital, 28 microM; secobarbital, 110 microM; pentobarbital, 400 microM; phenobarbital, 690 microM; butabarbital, 690 microM; and barbital, 5.1 mM. alpha-Bungarotoxin had no effect, but cholinergic ligands that convert the acetylcholine receptor to the desensitized state (acetylcholine, carbamylcholine, and, to a lesser extent, d-tubocurarine) partially inhibited displaceable [14C]amobarbital binding. This cholinergic inhibition was prevented by preincubation with alpha-bungarotoxin, implying an allosteric mediation through the classical cholinergic site. This negative interaction between the cholinergic and the barbiturate sites was mutual with barbiturates partially decreasing equilibrium [3H]acetylcholine binding in a saturable fashion with relative affinities that parallel those for inhibiting [14C]amobarbital binding (IC50). These data establish a mutual negative heterotropic interaction between barbiturate-binding sites and cholinergic binding sites on the nicotinic acetylcholine receptor from Torpedo.

Acetylcholine↗
PubMed1987

Estimating shelf-life of drugs in solution.

The use of pseudo-first-order chemical reaction kinetics and Arrhenius thermodynamic principles in estimating the effect of temperature on shelf-life of drug solutions is described. The degradation of many drugs in aqueous solutions occurs by hydrolysis. Equations using activation energy of the hydrolysis reaction (Ea) derived from Arrhenius principles, rate constants, and temperature are used to illustrate the exponential decrease in drug shelf-life proportional to an arithmetic increase in temperature. Use of the Q value, a factor based on Ea, rate constants, and temperature change, is described. The range of Ea for pharmaceutically important degradation reactions is relatively limited; thus, near room temperature, Q values of 2-5 can be used to estimate the effect of temperature change on shelf-lives of drugs in solution. To conservatively estimate effect on shelf-life when Ea is not known, a Q value of 2 is used to predict the increase in shelf-life obtained by decreasing storage temperature, and a Q of 5 is used to predict decrease in shelf-life from increasing the temperature. Sample calculations are used to show the application of equations based on these principles to practical temperature-dependent problems regarding shelf-life. Given information about shelf-life at a certain temperature, pharmacists can estimate safe shelf-life under other temperature conditions by using these equations.

Chemistry, Pharmaceutical↗
PubMed1986

Actions of general anesthetics on acetylcholine receptor-rich membranes from Torpedo californica.

The molecular mechanisms by which general anesthetics act on postsynaptic membranes can only be worked out in a highly purified, homogeneous system. The nicotinic acetylcholine receptor-rich membranes from the electric tissue of Torpedo californica are currently the only postsynaptic membranes that fulfill this condition. Is this peripheral synapse acted on with a pharmacologic specificity similar to that for general anesthesia, and how much less sensitive is it to anesthetic action than the unknown central site? To answer these questions, the authors studied the effects of 13 anesthetic compounds, including volatile general anesthetics, alkanols, and urethane, on the equilibrium binding of 3H-acetylcholine to these nicotinic receptors. As the anesthetic concentration was raised, all the agents first increased acetylcholine binding steeply and then, with few exceptions, decreased it again at higher concentrations. Anesthetics increased acetylcholine binding by decreasing acetylcholine's dissociation constant without changing the Hill coefficient or the number of sites. To a first approximation, the relative ability of these agents to increase 3H-acetylcholine binding parallels that of anesthesia in vivo as predicted by the Meyer-Overton lipid solubility rule. On average, they produced half maximal increases in acetylcholine binding (EC50) at about four times the concentration that causes loss of righting reflex in one-half of a group of animals (ED50). However, a few agents deviated from this relationship. They were the agents with greatest general anesthetic potency in both the volatile anesthetic series (thiomethoxyflurane) and the normal alcohol series (octanol), and required up to 17 times their ED50s to achieve a half effect on acetylcholine binding. Although the concentrations required were high, these effects were reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Octanol↗
PubMed1985

An isocratic high-performance liquid chromatography method for the simultaneous analysis of plasma retinol, alpha-tocopherol, and various carotenoids.

An isocratic high-performance liquid chromatography method for the simultaneous determination of various fat-soluble vitamins and carotenoids is reported. The method utilizes a Radial-Pak C-18, 5-microns column and an elution solvent composed of methanol:acetonitrile:chloroform (25:60:15). Only 100 microliters of plasma sample is required for one determination. Retinol, alpha-tocopherol, alpha-carotene, beta-carotene, lycopene, zeaxanthin, and two other unidentified carotenoids can be clearly separated and quantified in one HPLC run using alpha-tocopheryl acetate or tocol as the internal standard. The eluted peaks are quantified by either a photodiode-array detector at preprogrammed wavelengths at the absorption maxima of the compounds or a dual-wavelength detector at 280 and 436 nm. The total run time is 16 min. With an automatic injector and a programmable detector, the system allows unattended operation. The within-run and day-to-day coefficients of variation range from 1 to 8%. The lower limits of determination are 2, 40, and 2 ng for retinol, alpha-tocopherol, and carotenes, respectively. In addition, the system monitors the absorption spectra of the eluant during the HPLC run; this allows the spectral identification of various compounds separated in the same run.

Carotenoids↗
PubMed1985

Evidence for a dual mechanism in the anesthetic action of an opioid peptide.

Loss of righting reflex (LRR) produced by various concentrations of the leucine-enkephalin analog BW831c (TYR.D-ALA.GLY.PHE.D-LEU.NHEt.HCI) was determined in amphibia at 1 atm and 120 atm of helium. EC50 for LRR was 22.1 +/- 1.6 microM and 44 +/- 6.9 microM, respectively. The octanol/water partition coefficient (P) was 26 +/- 3.6, suggesting that this peptide is sufficiently lipid soluble for a classic Meyer-Overton type of anesthetic action. The ratio (EC50 at 120 atm)/(EC50 at 1 atm) for the peptide (2.0 +/- 0.31) was essentially the same as that for the long-chain alcohol, octanol (1.8 +/- 0.08), and similar to those reported for phenobarbital and the gaseous anesthetics. Thus, peptide-induced LRR was reversible by pressure. Peptide-induced LRR also was completely reversible by naloxone, whereas octanol-induced LRR was unaffected by up to 100 microM naloxone. These findings are consistent with a dual mechanism of anesthetic action for this peptide: one, an opiate receptor-specific mechanism, reversible with the specific opiate antagonist, naloxone; the other, a nonspecific mechanism, related to lipid solubility and reversible with the application of the physical agent, pressure.

1-Octanol↗
PubMed1985

The physiological effects of hydrostatic pressure are not equivalent to those of helium pressure on Rana pipiens.

The effects of helium pressure and hydrostatic pressure on Rana pipiens were compared. Both agents caused paralysis at pressures greater than 135 atmospheres (1 atm = 101.325 kPa), but the median pressure for hydrostatic-pressure-induced paralysis was 35 atm less than that for helium pressure. When the ability of both pressurizing agents to reverse urethane-induced anaesthesia was compared, it was found that hydrostatic pressure raised the median dose for anaesthesia 2.2-fold more per atmosphere than did helium pressure. Animals that were lightly anaesthetized by urethane at 110 atm hydrostatic pressure became more deeply anaesthetized when helium was admitted isobarically into the pressure chamber. This difference in depth of anaesthesia between hydrostatic pressure and helium pressure is consistent with helium possessing an inherent anaesthetic effect. The abilities of other gases to pressure-reverse urethane anaesthesia were also determined. The degree of attenuation of the full pressure reversal effect observed with hydrostatic pressure was proportional to the lipid solubility of the gases, increasing in the order helium, neon, hydrogen, nitrogen and argon. Our data on the difference between hydrostatic and helium pressure are consistent with the critical volume hypothesis.

Anesthesia↗
PubMed1985

Nutritional status of the high esophageal cancer risk population in Linxian, People's Republic of China: effects of vitamin supplementation.

Plasma levels of nutrients in 196 individuals were assayed as part of a study of the feasibility of a nutrition intervention trial in Linxian, a county in North Central China with exceptionally high rates of esophageal cancer. High-performance liquid chromatography analyses of samples collected in April 1983 showed low (relative to United States standards) base-line levels for retinol, alpha-tocopherol, and beta- and alpha-carotene. Repeat sampling in August 1983 revealed significantly increased plasma levels of retinol and alpha-tocopherol among those who had in the interim received daily supplementation with multivitamin pills containing the Recommended Dietary Allowance levels of those nutrients but not among those without supplementation. Levels of carotenes, which were not included in the pills, tended to increase regardless of supplementation, consistent with seasonal variations in availability of carotenoid-containing foods. Atomic absorption spectrophotometry analyses showed essentially normal levels of copper and zinc in plasma. Neither was affected by vitamin supplementation (the pills did not contain minerals) nor seasonal variation. The data are generally consistent with prior biochemical surveys in indicating marginal or low status of several nutrients in Linxian and in showing that supplementation with vitamins can effectively raise blood nutrient levels.

Adult↗
PubMed1985

Effects of riboflavin deficiency on metabolism of nitrosamines by rat liver microsomes.

The effects of riboflavin deficiency on the metabolism of N-nitrosodimethylamine [(DMN) CAS: 62-75-9] and other nitrosamines were examined in rats. After weanling rats were put on a riboflavin-deficient diet, the development of the deficiency was monitored by the growth rate and the erythrocyte glutathione reductase activation coefficient. In the riboflavin-deficient rats, the liver microsomal NADPH-cytochrome c reductase activity was lower but the cytochrome P450 content was higher than that of the control. The metabolism of DMN was dependent on the severity of the deficiency. During mild deficiency, which was observed mainly with Sprague-Dawley rats, the microsomal DMN demethylase (DMNd) activity was elevated 30-80%, but the metabolism of N-nitrosomethylbenzylamine (CAS: 937-40-6) and three other nitrosamines was slightly decreased. Dietary restriction in the pair-fed group also caused an elevation of DMNd activity above that of the ad libitum control group due to a partial fasting effect. During severe deficiency, which was observed mainly with Wistar rats, however, the metabolism of DMN, as well as the oxidation of benzo[a]pyrene, was decreased. Preincubation with flavin adenine dinucleotide and flavin mononucleotide enhanced the DMNd activity of the microsomes from riboflavin-deficient rats but not that from control rats. The results suggest that, depending on the alterations of the monooxygenase enzyme system during the development of the deficiency, riboflavin deficiencies may have different effects on the metabolism of DMN and some other carcinogens.

Animals↗
PubMed1984

Estradiol-induced alteration of very-low-density lipoprotein assembly. Possible competition among apoproteins for incorporation into nascent very-low-density lipoprotein.

The hypothesis that the apoprotein composition of nascent very-low-density lipoprotein (VLDL) secreted by the hepatocyte is determined by the relative rates of apoprotein synthesis and their affinities of binding to VLDL was tested using chick hepatocytes in monolayer culture. Chick cells were chosen for the study of lipoprotein assembly since estradiol treatment can be used to alter the composition of the apoprotein mixture synthesized by these cells. The secretion of apoprotein (apo) B by estradiol-treated hepatocytes was elevated 4.2-fold above the basal level measured in control cells. Furthermore, estradiol-treated cells secreted apo-II, a major VLDL apoprotein not synthesized prior to estradiol treatment, at a level equivalent to that of apo-B. However, no difference in the secretion of apo-A-I and other newly identified nascent VLDL apoproteins was detected. These changes in relative rates of apoprotein synthesis altered the composition of nascent VLDL secreted by control versus estradiol-induced cells from: apo-B, 22 to 40%; apo-II, 0 to 32%; apo-37 kDa, 14 to 6%; apo-A-I, 31 to 12%; apo-17 kDa, 10 to 4%; apo-9 kDa, 15 to less than 10%; and apo-6 kDa, 8 to less than 2%. To investigate the basis for the preferential incorporation of apo-B and apo-II into nascent VLDL, the relative affinities of the apoproteins for VLDL were compared by measuring their capacities to transfer from VLDL into other lipoprotein or nonlipoprotein density classes. Culture medium containing [3H]leucine-labeled VLDL was incubated with plasma deficient in lipoproteins of rho less than 1.006 g/ml. Within 30 min of incubation at 37 degrees C, 3H-labeled apo-A-I and apo-9 kDa exchanged between VLDL and high-density lipoprotein, whereas apo-37 kDa exchanged between VLDL and the rho greater than 1.21 g/ml fraction. Neither apo-B nor apo-II underwent transfer from nascent VLDL. These results suggest that the relative rates of input of apoproteins into the secretory pathway and their affinities of binding to the nascent VLDL particle determine their extent of incorporation into, and, thus, the apoprotein composition of secreted VLDL.

Animals↗
PubMed1984

Simultaneous determination of plasma retinol, alpha-tocopherol, lycopene, alpha-carotene, and beta-carotene by high-performance liquid chromatography.

Reverse-phase high-performance liquid chromatography was used for the simultaneous determination of retinol, alpha-tocopherol, lycopene, alpha-carotene, and beta-carotene in human plasma. A multiple solvent system of methanol followed by a mixture of methanol:acetonitrile:chloroform (47:42:11) provided clear separation of these compounds. With retinyl acetate as an internal standard, standard curves were developed for each compound on the basis of peak area ratios. The coefficient of variation was less than 10% in all cases within a run. Between-run reproducibility was within +/- 2 standard deviations. The method required 100 to 200 microliter of plasma and 16 min of elution time per sample. Stability studies showed plasma samples could be stored at -10 degrees C with occasional freeze-thaw cycles for 3 to 5 weeks. This method should prove useful in clinical and epidemiological work.

Carotenoids↗
PubMed1984

The interactions between pressure and anaesthetics.

Compression of animals causes excitation, which has recently posed a barrier to deeper diving. The broad question addressed here is how far the inert gas breathed modifies the excitatory effects of hydrostatic pressure. By using aquatic animals we first show that helium postpones the onset of pressure-induced paralysis by some 35 atm. Next we show that in mammals compressed with helium, five anaesthetic gases (nitrogen, argon, nitrous oxide, carbon tetrafluoride, sulphur hexafluoride) all elevated dose-dependently the median pressure of four distinct phases of the high pressure neurological syndrome (h.p.n.s.) (complete spasms, clonic convulsions, tonic convulsions and non-tonic death). All the gases were equally efficacious relative to their anaesthetic potency. However, the sensitivity of each phase of the h.p.n.s. to anaesthetic gases differed. Most notably, the median pressure for tonic convulsions was elevated about three times more by a given partial pressure of anaesthetic gas than were the median pressures for complete spasms or non-tonic death. These observations can be fitted remarkably well by the hypothesis that a given phase of the h.p.n.s. is activated when some hydrophobic region is compressed beyond a certain critical amount by the application of pressure. Absorption of an inert gas in this region will cause it to expand, tending to elevate the median pressure for that phase of the h.p.n.s. Our data and analysis allow the following conclusions relevant to diving practice. All gases protect against the h.p.n.s. but some phases of this complex syndrome are more effectively controlled than others. Although addition of a second inert gas to helium allows substantial increases in the pressure at which h.p.n.s. occurs, the onset of anaesthesia (or inert gas narcosis) will limit the ultimate gain. The composition of therapeutic gas mixtures becomes more narrowly defined as the pressure increases. The optimum mixture may be different for each phase of the h.p.n.s., and the order of presentation of the h.p.n.s. symptoms may be changed by the second inert gas. We may also predict that physiological sites may exist where helium acts like an anaesthetic. If such sites resulted in physiological dysfunction, addition of a second gas would exacerbate the situation.

Anesthetics↗
PubMed1984

Synthesis, processing, and secretion of hepatic very low density lipoprotein.

Very low density lipoprotein (VLDL) is the major vehicle in the plasma which carries triacylglycerol synthesized in the liver to peripheral tissues for utilization. Estrogen-induced chick parenchymal liver cells (hepatocytes) synthesize and secrete large amounts of VLDL. These cells, in a primary monolayer culture system developed in this laboratory, have been employed to study the operative and regulatory aspects of VLDL synthesis, assembly, and secretion. Some 10 min are required for the translation of the principle VLDL protein constituent, apolipoprotein B, and 30-35 min are required for the two newly translated chick VLDL apolipoproteins, apolipoprotein B and apolipoprotein II, to be secreted. Apolipoprotein B is synthesized on membrane-bound polysomes as a contiguous polypeptide chain of 350K molecular weight (MW) and is not assembled posttranslationally from smaller-peptide precursors. Translocation of puromycin-discharged apolipoprotein B nascent chains into the endoplasmic reticulum lumen and their subsequent secretion are independent of both ongoing protein synthesis and the attachment of the nascent peptides to ribosomes. Apolipoprotein B nascent chains discharged by puromycin assemble with glycerolipid (mainly triacylglycerol) and are secreted as immunoprecipitable VLDL. Core oligosaccharides are added to the apolipoprotein B nascent chain co-translationally in at least two stages, at molecular weights of approximately 120K and approximately 280K . Inhibition of N-linked glycosylation of apolipoprotein B with tunicamycin affects neither the assembly of glycerolipids into VLDL nor the secretion of the VLDL particle, indicating that aglyco -apolipoprotein B can serve as a functional component for VLDL assembly and secretion. Active synthesis of the VLDL apolipoproteins is required, however, for glycerolipid assembly into VLDL and secretion from the hepatocyte. The differential kinetics with which newly synthesized apolipoproteins and glycerolipids are secreted as VLDL and the timing of the effects of protein-synthesis inhibitors on their secretion indicate that VLDL constituents are assembled sequentially in the intact liver cell. The bulk of the VLDL triacylglycerol and some VLDL phosphoglyceride is introduced early in the secretory pathway proximal, yet subsequent to apopeptide synthesis, while a significant fraction of VLDL phosphoglyceride associates with the resulting triacylglycerol-rich lipid-protein complexes just prior to their secretion as mature VLDL. Within the context of current models for VLDL structure, the late assembly of phosphoglyceride into VLDL is taken to represent a surface maturation of the nascent VLDL particle.

Animals↗
PubMed1984

Studies on the mechanisms of induction of N-nitrosodimethylamine demethylase by fasting, acetone, and ethanol.

Previous work has shown that induction of a high-affinity NADPH-dependent nitrosodimethylamine demethylase (NDMAd) in liver microsomes occurs in rats due to fasting, ethanol consumption, and streptozotocin-induced diabetes. Several lines of observations suggest that this is due to the induction of specific cytochrome P-450 isozymes. Induction of P-450 species by ethanol has also been observed by other investigators. Since each of the above altered metabolic states has in common elevated levels of ketone bodies, the possible role of acetone, a known inducer of NDMAd, in the induction of the demethylase activity was investigated. Levels of endogenous acetone in fasted rats correlated (r = 0.72) with a three- to fourfold increase in NDMAd activity. However, a dose-response experiment showed endogenous levels of acetone to be capable of causing at most 40% of the induction in fasted rats. This suggests that other ketone bodies or factors may have contributed to the induction. The induction of NDMAd by ethanol was enhanced by alcohol dehydrogenase inhibitors pyrazole and acetaldehyde oxime, suggesting that ethanol, rather than its metabolites, was responsible for the induction.

Acetone↗
PubMed1984

Potentiation of the hepatotoxicity of N-nitrosodimethylamine by fasting, diabetes, acetone, and isopropanol.

Previous studies indicate that pretreatment with acetone or isopropanol, fasting, and streptozotocin-induced diabetes enhance hepatic microsomal nitroso-dimethylamine (NDMA) demethylase in rats. This study demonstrates that these same treatments also potentiate the hepatotoxicity of NDMA as indicated by plasma glutamic pyruvate transaminase (GPT) levels and histologic data. Pretreatment with acetone or isopropanol (2.5 ml/kg) and 2 days of fasting caused a 2-fold potentiation of NDMA-induced plasma GPT elevation, whereas streptozotocin-induced diabetes caused a 4.6-fold potentiation. The centrilobular necrosis produced by NDMA was more severe after pretreatment with the inducers. NDMA treatment also decreased hepatic microsomal demethylase activity. These results lend support to the concept that a NDMA demethylase is responsible for the activation of NDMA in vivo to a toxic intermediate, and induction of this enzyme activity potentiates NDMA hepatotoxicity.

1-Propanol↗