Millimeter wave-induced vibrational modes in DNA as a possible alternative to animal tests to probe for carcinogenic mutations.

Developing methods for alternative testing is increasingly important due to dwindling funding resources and increasing costs associated with animal testing and legislation. We propose to test the feasibility of a new and novel method for detecting DNA mutagenesis using millimeter wave spectroscopy. Although millimeter wave spectroscopy has been known since the 1950s, the cost was prohibitive and studies did not extend to large biological proteins such as DNA. Recent advances have made this technology feasible for developing laboratory and field equipment. We present preliminary findings for lesion-induced vibrational modes in DNA observed from 80 to 1000 gigahertz (GHz). These findings suggest that there are vibrational modes that can be used as identification resonances. These modes are associated with localized defects of the DNA polymers. They are unique for each defect/lesion, and should be easy to detect. We described a field-detecting detector based on the local modes.

Predicting the presence of choledocholithiasis in patients with symptomatic cholelithiasis.

OBJECTIVES: Accurate preoperative prediction of choledocholithiasis is essential in order to minimize patient risk and curtail health care expenditures. This study was designed to identify independent risk factors for choledocholithiasis in patients who had undergone cholecystectomy for symptomatic cholelithiasis and to develop a predictive model based on those factors. METHODS: The charts of 1264 consecutive patients who had undergone cholecystectomy at one of three North Carolina hospitals between January 1, 1989 and December 31, 1991 were reviewed; 465 of these patients had confirmed presence or absence of choledocholithiasis by cholangiography and/or common bile duct exploration and were eligible for analysis. Candidate predictor variables included age and maximum preoperative values for each of the following: temperature, alkaline phosphatase, bilirubin, AST, amylase, white blood cell count, and common bile duct diameter. Model development and validation were conducted using standard data-splitting (60% "training," 40% "test") and logistic regression techniques. RESULTS: Choledocholithiasis was confirmed in 115 (25%) of the 465 eligible patients. Univariate analysis identified bilirubin, common bile duct diameter, AST, temperature, alkaline phosphatase, and age as predictors. Multivariable analysis subsequently identified bilirubin, common bile duct diameter, AST, alkaline phosphatase, and age as independent predictors of choledocholithiasis. A final model containing these variables (except age, whose contribution to the model was small) accurately predicted choledocholithiasis (c-index = 0.76). CONCLUSIONS: Accurate estimates of choledocholithiasis risk can be made using maximum preoperative bilirubin, common bile duct diameter, AST, and alkaline phosphatase values. Use of the model may help physicians select those patients with symptomatic cholelithiasis who would most likely benefit from further investigation to exclude choledocholithiasis.

Orienting and defense responses to punishment: effects on learning.

Two groups of students attempted to learn sequences of letter-number pairs. For both groups, a tone signalled each error. However, for aversive punishment subjects, a mildly painful electric shock followed the tone 20% of the time, whereas the informational punishment subjects received only the tone. Skin conductance responses (SCRs) and cardiac interbeat intervals indicated the presence of an orienting response to the tone in informational punishment subjects and a defense response to the tone in aversive punishment subjects. Accompanying these were significant differences in behavior: aversive punishment subjects completed fewer sequences and had higher error rates. The two groups did not differ in measures of tonic arousal. Session trends suggested that the cardiac orienting response developed in both groups as subjects learned to use the information in the punishment contingency. Defense responses to aversive punishers may complete with orienting responses necessary to the efficient learning of complex tasks.

Changes in erythrocyte enzyme activities during erythrocyte aging in alcoholism.

Aldehyde dehydrogenase, glucose-6-phosphate dehydrogenase, and pyruvate kinase activities were determined in erythrocytes of various ages, separated by Percoll gradient centrifugation, in 13 alcoholic patients and eight control subjects. The total erythrocyte activities of all three enzymes were not affected by alcoholism, however, the youngest cells of alcoholics had a decreased aldehyde dehydrogenase activity, while both glucose-6-phosphate dehydrogenase and pyruvate kinase activities were increased. The depression of aldehyde dehydrogenase activity not only persisted, but became more marked after 2 weeks of abstinence, while the enhanced activities of the two other enzymes returned to normal. These observations suggest that chronic alcohol ingestion suppresses aldehyde dehydrogenase in the bone marrow, while it enhances other erythrocytic enzymes.

Characterization of human erythrocyte aldehyde dehydrogenase.

Human erythrocyte aldehyde dehydrogenase was purified to homogeneity. The enzyme exhibited a single band of activity on starch gel electrophoresis and on isoelectric focusing. It was a tetramer with an estimated molecular weight of 230,000 daltons and an isoelectric point of 5.0. Its pH optimum of 8.5, Michaelis-Menten constant for acetaldehyde of 46 microM, and high sensitivity to noncompetitive inhibition by disulfiram resembled human liver cytosolic aldehyde dehydrogenase. Low concentrations of magnesium (5-10 microM) resulted in enhancement of erythrocyte aldehyde dehydrogenase activity, whereas higher physiological concentrations of magnesium resulted in uncompetitive inhibition of enzyme activity. Magnesium inhibited the enzyme activity by increasing the binding of NADH to the enzyme as had been found to be the case for the inhibitory effect of magnesium on the human liver cytosolic enzyme. Erythrocyte aldehyde dehydrogenase may metabolize small amounts of acetaldehyde escaping the liver during ethanol metabolism and protect extrahepatic tissues from acetaldehyde toxicity.

Effect of growth hormone on alcohol dehydrogenase activity in hepatocyte culture.

The effect of growth hormone on the activity of alcohol dehydrogenase was determined in hepatocyte culture from normal and hypophysectomized male rats. Alcohol dehydrogenase activity was highest in hepatocytes harvested from hypophysectomized rats. The enzyme activity remained stable in hepatocytes harvested from normal rats during 2 to 6 days of culture but declined steadily in hepatocytes cultured from hypophysectomized rats. The combination of growth hormone (1 microgram per ml) and corticosterone (1 microM) increased alcohol dehydrogenase activity in hepatocytes from normal rats, while neither hormone alone had an effect. Corticosterone (1 microM) prevented the decline of the enzyme activity in hepatocytes from hypophysectomized rats, and the combination of growth hormone (1 microgram per ml) and corticosterone (1 microM) resulted in a further increase in enzyme activity. The increases in alcohol dehydrogenase, due to the exposure of the hepatocytes to the combination of growth hormone and corticosterone, were associated with increases in the rate of ethanol elimination. These observations indicate that growth hormone enhances liver alcohol dehydrogenase activity and ethanol elimination, and that this effect is dependent on the permissive influence of corticosterone.

Effect of glucagon on alcohol dehydrogenase activity in rat hepatocyte culture.

The effect of glucagon on the activity of alcohol dehydrogenase in rat hepatocyte culture was determined. Glucagon concentrations of 0.1 nM enhanced, whereas concentrations greater than 1 nM decreased, alcohol dehydrogenase. These effects became apparent after exposure of the cultures to glucagon for 4 or more days. The presence of corticosterone (1 microM) prevented the enhancing effect of 0.1 nM glucagon on alcohol dehydrogenase activity. The changes in alcohol dehydrogenase caused by glucagon were associated with parallel changes in the rate of ethanol elimination. Alcohol dehydrogenase appears to be rate-limiting for ethanol oxidation, as uncoupling of oxidative phosphorylation did not modify the rate of ethanol elimination. These studies suggest a physiologic role of glucagon in enhancing liver alcohol dehydrogenase activity, whereas higher pharmacologic concentrations of glucagon have an opposite, depressant effect.

Behavioral test of tolerance for aversive mechanical stimuli in sympathectomized cats.

Cats were studied behaviorally to determine their suitability as an animal model for the post-sympathectomy hyperalgesia reported to occur in humans. For this study a device and methodology were developed which allow humane testing of tolerance for intense mechanical stimulation of the hindlegs. Behavioral tolerance was measured quantitatively before and after unilateral sympathectomy. The results from this preliminary study of 6 cats are remarkably similar to those reported for humans; 1 of the 6 cats showed a decreased tolerance on the sympathectomized side which was delayed in onset and of limited duration. The new methodology appears to provide relatively stable, quantitative measures of tolerance for aversive stimulation, and the cat shows promise as an animal model for post-sympathectomy hyperalgesia.

Analgesia from rostral brain stem stimulation in the rat.

Rats implanted with bipolar stimulating electrodes in the rostral medial brain stem were tested for brain stimulation-produced analgesia using tail-flick, pinch and hot-plate tests. Potent analgesia across all three tests was obtained from stimulation of sites in the gray matter surrounding the aqueduct and the caudal portion of the third ventricle, the posterior hypothalamus, the midline area of the caudal thalamus and the pretectal region of the meso-diencephalic junction. The analgesia obtained from these sites was comparable to that produced by stimulation of the previously studied caudal periaqueductal gray matter: it outlasted the period of brain stimulation, was not due to a generalized motor debilitation of the animal, and was not correlated with changes in electrographic activity. Stimulation of sites in the caudal thalamus and pretectal area yielded analgesia without stimulation-induced aversive reactions, confirming the potential of these sites for use in the relief of clinical pain in man.