Antibodies to the ciliary membrane of Paramecium tetraurelia alter membrane excitability.

Immobilization of Paramecium followed the binding of antibodies to the major proteins of the ciliary membrane (the immobilization antigens, i-antigens, approximately 250,000 mol wt). Immunoelectron microscopy showed this binding to be serotype-specific and to occur over the entire cell surface. Antibody binding also reduced the current through the Ca-channel of the excitable ciliary membrane as monitored using a voltage-clamp. The residual Ca-current appeared normal in its voltage sensitivity and kinetics. As a secondary consequence of antibody binding, the Ca-induced K-current was also reduced. The resting membrane characteristics and other activatable currents, however, were not significantly altered by the antibody treatment. Since monovalent fragments of the antibodies also reduced the current but did not immobilize the cell, the electrophysiological effects were not the secondary consequences of immobilization. Antibodies against the second most abundant family of proteins (42,000-45,000 mol wt) had similar electrophysiological effects as revealed by experiments in which the Paramecia and the serum were heterologous with respect to the i-antigen but homologous with respect to the 42,000-45,000-mol-wt proteins. Protease treatment, shown to remove the surface antigen, also caused a reduction of the Ca-inward current. The loss of the inward Ca-current does not seem to be due to a drop in the driving force for Ca++ entry since increasing the external Ca++ or reducing the internal Ca++ (through EGTA injection) did not restore the current. Here we discuss the possibilities that (a) the major proteins define the functional environment of the Ca-channel and that (b) the Ca-channel is more susceptible to certain general changes in the membrane.

Phosphoenolpyruvate carboxylase from soybean nodule cytosol. Evidence for isoenzymes and kinetics of the most active component.

Phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylase (phosphorylating), EC 4.1.1.31) from plant cells of soybean nodules was studied to assess its role in providing carbon skeletons for aspartate and asparagine synthesis. The enzyme was purified 119-fold by (NH4)2SO4 fractionation and DEAE-cellulose, BioGel A-1.5m, and hydroxyapatite chromatography. Five activity bands were resolved with discontinuous polyacrylamide gel electrophoresis. A small quantity of enzyme from the most active band was separated from the others by preparative electrophoresis. The apparent Michaelis constants of this enzyme for phosphoenolpyruvate and HCO3- were 9.4.10(-2) and 4.1.10(-1) mM, respectively. A series of metabolite tested at 1 mM had no significant effect on enzyme activity. These experiments indicate that the major factors directly controlling phosphoenolpyruvate carboxylase activity in vivo are phosphoenolpypyruvate and HCO3- concentrations.

Reliability and validity of alcohol-induced heart rate increase as a measure of sensitivity to the stimulant properties of alcohol.

RATIONALE: Alcohol-induced heart rate (HR) stimulation during the rising limb of the blood alcohol curve reliably discriminates between individuals at differential risk for alcoholism, and appears to be a potential psychophysiological index of psychomotor stimulation from alcohol. OBJECTIVES: Three studies are presented which explore the reliability and convergent and discriminant validity of this alcohol response index. METHODS: Young men with and without a multigenerational family history of alcoholism were administered a 1.0 ml/kg dose of 95% USP alcohol. Resting baseline cardiac and subjective measures were assessed before and after alcohol consumption. RESULTS: Study 1 demonstrated that alcohol-induced HR stimulation was significantly and positively related to alcohol-induced changes in mood. Study 2 demonstrated that alcohol-induced HR stimulation was reliable across two alcohol administration sessions (r=0.33-0.66, P<0.01). Study 3 explored the relationship between the proposed index and measures of sensitivity to alcohol previously linked to genetic predisposition to alcoholism. Multiple regression analysis indicated that alcohol-induced HR increase and reduced subjective intoxication (measured using the Subjective High Assessment Scale) were both positively associated with alcohol-induced changes in mood states that have previously been shown to be sensitive to the effects of stimulant drugs and the reinforcing effects of alcohol. CONCLUSIONS: Sensitivity to alcohol-induced heart-rate stimulation during the ascending limb of the blood alcohol curve may be a useful and informative marker for understanding susceptibility to alcoholism.

Provocation, acute alcohol intoxication, cognitive performance, and aggression.

This study investigated the relationships between provocation, acute alcohol intoxication, impaired frontal-lobe function, and aggressive behavior. The authors ranked 114 men according to their performance on two neuropsychological tests associated with frontal-lobe function. Forty-eight men (24 with scores in the upper and 24 with scores in the lower performance quartiles) participated in the full study. Half completed and aggression task while intoxicated, the remainder while sober. Aggression was defined as shock intensity delivered to a sham opponent. Shock intensity significantly increased as a main effect of provocation, alcohol intoxication, and lower cognitive performance. Furthermore, provocation interacted significantly with test performance such that individuals in the lower cognitive performance quartile responded to increased provocation with heightened aggression.

A biosocial model of the alcohol-aggression relationship.

Four of alcohol's dose- and rate-dependent pharmacological properties may increase the likelihood of human aggression. As an anxiolytic, alcohol is capable of reducing the inhibitory effect of fear on manifestation of aggressive behavior. As a psychomotor stimulant, alcohol can potentiate aggressive behavior, once evoked, or lower the threshold for such evocation. Alcohol-related disruption of certain higher order cognitive functions may reduce the inhibitory control generally exercised by previously established knowledge and decrease ability to plan in the face of threat or punishment. Finally, alcohol's ability to increase pain sensitivity may increase the likelihood of defensive aggression. Discussion of the nature and relevance of these pharmacological properties is structured according to a heuristic and synthetic schema, predicated upon consideration of an inhibitory neuropsychological structure--the individually and culturally determined general expectancy set.

Acute alcohol intoxication and cognitive functioning.

Acute alcohol intoxication produces changes in the cognitive functioning of normal individuals. These changes appear similar prima facie to those exhibited by individuals who sustain prefrontal lobe damage during adulthood. In order to test the validity of this observation, and to control for the confounding effects of expectancy, 72 male subjects were administered a battery of neuropsychological tests, within the context of a balanced-placebo design. Each subject received one of three widely different doses of alcohol. Analysis of the results of the cognitive test battery demonstrated that a high dose of alcohol detrimentally affects a number of functions associated with the prefrontal and temporal lobes, including planning, verbal fluency, memory and complex motor control. Expectancy does not appear to play a significant role in determining this effect. The implications of this pattern of impairment are analyzed and discussed.

Cognitive dysfunction and the inherited predisposition to alcoholism.

A battery of neuropsychological tests was administered to 22 nonalcoholic sons of male alcoholics (SOMAs) from families with extensive histories of male alcoholism and to 22 nonalcoholic controls with no history of familial alcoholism. In each group 11 subjects were tested while sober and 11 were tested while alcohol-intoxicated. Analyses of the results of this battery suggested (1) that SOMAs may be characterized by comparative decrements in those cognitive functions associated with the organization of novel information, dependent in theory upon the prefrontal cortex; and (2) that alcohol detrimentally affects delayed memory, associated with the temporal cortex, equally across groups. Of these SOMAs 20 had previously participated in one of two studies that demonstrated their cardiovascular hyper-reactivity to threat/stress and their increased sensitivity to the reactivity-dampening effects of alcohol intoxication. Correlational analyses of the results of the present and previous studies demonstrated the existence of a highly significant relationship between cognitive impairment, cardiovascular hyper-reactivity and susceptibility to the reactivity-dampening effects of alcohol.

Factor structure and correlates of the Tridimensional Personality Questionnaire.

Cloninger has proposed a model linking personality characteristics to patterns of responses to various stimuli, including alcohol. The model also uses personality characteristics to divide alcoholics into two types. In order to assess the relevant aspects of personality, the Tridimensional Personality Questionnaire (TPQ) was developed. This study examined the factor structure and correlates of the questionnaire to shed light on its validity. A confirmatory factor analysis of the TPQ failed to replicate the three proposed factors of novelty seeking, harm avoidance and reward dependence. Alternative models also failed to fit the data. The three factors were correlated with other personality measures theoretically linked to drinking (MacAndrew, Socialization and Sensation-Seeking scales), quantity and frequency measures of alcohol use and a measure of alcohol abuse (the Michigan Alcoholism Screening Test). The pattern of correlations provides evidence for the convergent validity of the TPQ. Nevertheless, the failure to replicate the factor structure suggests that the scale requires further revision before the model can be adequately tested.

The Tridimensional Personality Questionnaire and the inherited risk for alcoholism.

The Tridimensional Personality Questionnaire (TPQ) (Cloninger, 1987c) was administered to four groups of young men. The first group was composed of nonalcoholic sons of male alcoholics with extensive multigenerational family histories of male alcohol abuse. The second was made up of nonalcoholic men with alcoholic fathers. The third group was composed of nonalcoholic men with no family history of alcoholism, taken from the general population. The fourth group contained male undergraduates with no family history of alcoholism. There were no significant differences between the mean scores obtained by members of all four groups on the three major subscales of the TPQ.

Enzymatic hydrolysis of ammonia-treated sugar beet pulp.

Sugar beet pulp is a carbohydrate-rich coproduct generated by the table sugar industry. Beet pulp has shown promise as a feedstock for ethanol production using enzymes to hydrolyze polymeric carbohydrates and engineered bacteria to ferment sugars to ethanol. In this study, sugar beet pulp underwent an ammonia pressurization depressurization (APD) pretreatment in which the pulp was exploded by the sudden evaporation of ammonia in a reactor vessel. APD was found to substantially increase hydrolysis efficiency of the cellulose component, but when hemicellulose- and pectin-degrading enzymes were added, treated pulp hydrolysis was no better than the untreated control.

Polymyxin E production by P. amylolyticus.

AIMS: To investigate antibiotic production by bacteria isolated from the hindgut of Tipula abdominalis, the aquatic crane fly. METHODS AND RESULTS: A group of five isolates with 99.1% 16S rRNA sequence similarity to Paenibacillus amylolyticus were identified as antibacterial producers using the cross-streak method against both Gram-positive and Gram-negative bacteria. For one isolate, P. amylolyticus C27, biochemical tests were performed to confirm 16S rRNA identification and the antibacterials were purified using chromatographic methods. Postsource decay (PSD) mass spectroscopy (MS) was used to identify the antimicrobials, which were found to be polymyxins E(1) and E(2). Investigation of the remaining four isolates using PSD MS revealed they all produce polymyxins E(1) and E(2) as well. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Although variants of the polymyxin antibiotics are known to be produced by several species within the Paenibacillus genus, this first investigation of antibacterial production by bacteria isolated from the hindgut of T. abdominalis describes a novel source for polymyxin E production as well as the first report of antibiotic production by P. amylolyticus.

Dependence of oxygen-tolerant nitrogenase activity on divalent cations in Azotobacter vinelandii.

Nitrogenase activity of washed Azotobacter vinelandii cells was enhanced by the addition of Ca2+ and Mg2+, and the enhancement increased with the O2 concentration. In assays provided with a level of O2 that was initially supraoptimal and inhibitory to nitrogenase activity, the addition of Ca2+ or Mg2+ affected both the maximum respiration rate (Vmax) of the cells and the apparent affinity [KS(O2)] of cell respiration for O2. Changes in these parameters correlated with changes in nitrogenase activity. Aeration-dependent increases in Vmax and KS(O2) were inhibited by rifampin and chloramphenicol and were also observed in ammonium-grown cultures.

Fluorescence monitoring of flavins during preparation of respiratory membranes.

Azotobacter vinelandii large and small membrane particles were examined by fluorescence spectroscopy through purification to qualitatively monitor contamination by non-respiratory flavin. Flavin was analyzed by observing the effects of reduction by dithionite or NAD(P)H and subsequent oxidation. Flavin of the large particles did not change significantly with purification on a sucrose gradient. The small particle or R3 fraction contained relatively large amounts of non-respiratory flavin. Small particles eluted from a Sepharose CL-6B column with a fluorescence peak but still contained contaminating flavin. After centrifugation on a sucrose gradient, the flavin of these particles was essentially the same as the large particles. This method is an improvement over just observation of fluorescence intensity for monitoring flavoprotein purity of membrane particle preparations.

Small GTP-binding proteins associated with secretory vesicles of Paramecium.

GTP-binding proteins act as molecular switches in a variety of membrane-associated processes, including secretion. One group of GTP-binding proteins, 20-30 kDa, is related to the product of the ras proto-oncogene. In Saccharomyces cerevisiae, ras-like GTP-binding proteins regulate vesicular traffic in secretion. The ciliate protist Paramecium tetraurelia contains secretory vesicles (trichocysts) whose protein contents are released by regulated exocytosis. Using [alpha-32P]GTP and an on-blot assay for GTP-binding, we detected at least seven GTP-binding proteins of low molecular mass (22-31 kDa) in extracts of Paramecium tetraurelia. Subcellular fractions contained characteristic subsets of these seven; cilia were enriched for the smallest (22 kDa). The pattern of GTP-binding proteins was altered in two mutants defective in the formation or discharge of trichocysts. Trichocysts isolated with their surrounding membranes intact contained two minor GTP-binding proteins (23.5 and 29 kDa) and one major GTP-binding protein (23 kDa) that were absent from demembranated trichocysts. This differential localization of GTP-binding proteins suggests functional specialization of specific GTP-binding proteins in ciliary motility and exocytosis.

Cytokinins in Vinca rosea L. Crown Gall Tumor Tissue as Influenced by Compounds Containing Reduced Nitrogen.

Several compounds containing reduced nitrogen markedly increased the yields of cell-division compounds extractable from an A6 Vinca rosea L. crown gall tumor tissue. Casein hydrolysate, several amino acids, and ammonium salts were effective. Both trans-zeatin and ribosyl-trans-zeatin were substantially increased in total amount per culture and in concentration. These two compounds have been identified by several criteria including mass spectra. The reduced nitrogen treatments also caused the appearance of a cytokinin not previously detected in this tissue; it has not yet been identified. The tumor tissue rapidly absorbed [8-(14)C]adenine from a liquid medium. Within 1 hour, the tissue converted some of the adenine to zeatin and ribosylzeatin, and greater degrees of conversion occurred in 2-, 4-, and 8-hour periods. The tissue grown on a medium containing ammonium chloride accumulated considerably greater quantities of the two cytokinins made from the labeled adenine during each incubation period.

Properties of pyruvate kinase from soybean nodule cytosol.

The properties of pyruvate kinase from soybean (Glycine max L.) nodule cytosol were examined to determine what influence the N(2) fixation process might have on this supposed key control enzyme. A crude enzyme preparation was prepared by chromatography of cytosol extract on a diethylaminoethyl-cellulose column. ATP and citrate at 5 mm concentrations inhibited pyruvate kinase 27 and 34%, respectively. Enzyme activation was hyperbolic with respect to both K(+) and NH(4) (+) concentrations. In the presence of physiological concentrations of K(+) and high phosphoenolpyruvate (PEP) concentrations, NH(4) (+) inhibited enzyme activity. Comparisons of kinetic parameters (V(max) and apparent K(a)) for NH(4) (+) and K(+) with inhibition curves indicated that inhibition was very likely a result of competition of the ions for activation site(s) on the pyruvate kinase. In addition, apparent K(a) (monovalent cation) and K(m) (PEP) were influenced by PEP and monovalent cation concentrations, respectively. This effect may reflect a fundamental difference between plant and animal pyruvate kinases. It is concluded that control of cytosol pyruvate kinase may be closely related to reactions involved in the assimilation of NH(4) (+).

Glucosyl Zeatin and Glucosyl Ribosylzeatin from Vinca rosea L. Crown Gall Tumor Tissue.

Recently detected but unidentified cytokinin activity in crown gall tumor tissue from Vinca rosea L. grown on media containing sources of reduced nitrogen has now been attributed to two adenine-type cytokinins. These compounds are glucopyranosyl derivatives of zeatin and ribosylzeatin. The substitution in each case is on the isopentenyl chain of the parent compound. Neither of these compounds had activity in the soybean callus bioassay at concentrations lower than 1 nm whereas zeatin had activity at 0.1 nm.

Information processing, neuropsychological function, and the inherited predisposition to alcoholism.

Sons of male alcoholics are at particularly heightened risk for the development of alcoholism. This heightened risk frequently appears in association with increased incidence of conduct disorder or hyperactivity, with deficits in abstract thinking and poor school performance, with abnormalities in cued psychophysiological response, and with increased sensitivity to the putatively stress-response-dampening effects of alcohol intoxication. This risk and its associated features are discussed within the context of a neuropsychological theory, predicted on the notions (1) that deficits in cognitive functions theoretically dependent upon the intact functioning of the prefrontal cortex could underlie manifestation of the idiosyncracies commonly attributed to sons of male alcoholics, and (2) that acute alcohol intoxication could relieve the subjective discomfort associated with the consequences of such deficits.