Release of Endogenous Amino Acids from Striatal Neurons in Primary Culture

Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl-cellulose, ATP-agarose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442-fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory subunit from the type II cyclic AMP-dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G-200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 microM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity.     

Glutamate Dehydrogenase in Human Brain: Regional Distribution and Properties

Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25 degrees C and it was 10-fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5-fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With alpha-ketoglutarate (alpha-KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Km alpha-KG = 1.9 X 10(-3) M, KmNADH = 0.21 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M, and in leukocytes they were Km alpha-KG = 1.7 X 10(-3) M, KmNADH = 0.24 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.     

Comparison of health problems related to work and environmental measurements in two office buildings with different ventilation systems.

A cross sectional survey investigating "building sickness" was carried out in two buildings with similar populations of office workers but differing ventilation systems, one being fully air conditioned with humidification and the other naturally ventilated. The prevalence of symptoms related to work was assessed by a questionnaire administered by a doctor. A stratified, randomly selected sample of workers was seen (84% response). Building sickness includes several distinct syndromes related to work, most of which were significantly more common in the air conditioned building than the naturally ventilated building--namely, rhinitis (28% v 5%), nasal blockage and dry throat (35% v 9%), lethargy (36% v 13%), and headache (31% v 15%). The prevalence of work related asthma and humidifier fever was low and did not differ significantly between the two buildings. An environmental assessment of the offices was performed to attempt to identify possible factors responsible for the differences in the prevalence of disease. Globe temperature, dry bulb temperature, relative humidity, moisture content, air velocity, positive and negative ions, and carbon monoxide, ozone, and formaldehyde concentrations were all measured. None of these factors differed between the buildings, suggesting that building sickness is caused by other factors.     

Unusual properties of two branched RNA''s with circular and linear components.

Irradiation with ultraviolet light was used to create two nonlinear RNA molecules. Circular potato spindle tuber viroid (PSTV) RNA was crosslinked at a single site to generate a figure eight-shaped molecule; 5S rRNA from HeLa cells was transformed into an alpha-shaped molecule with a small circular element and two arms (1). Crosslinked RNA's could be separated from their untreated counterparts by electrophoresis in polyacrylamide gels containing urea. The gel mobility of crosslinked PSTV was not altered by boiling, treatment with E. coli RNase III or glyoxalation. However, mild nuclease digestion ("nicking") produced derivatives which migrated more slowly than the starting material in gels of certain polyacrylamide concentrations, but not in others. Limited nuclease digestion of crosslinked 5S rRNA did not generate any detectable products with reduced mobility in the gels tested. Thus, the ability of the "nicking assay" to reveal circular elements within nonlinear RNA's can vary depending upon the composition of the gel chosen for analysis and on the size of the circular element relative to the rest of the molecule.     

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.     

tRNA binding sites of ribosomes from Escherichia coli

70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites. The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding. It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM. A third binding site in addition to P and A sites was observed for deacylated tRNAPhe. At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low. Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites. These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome. Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely. Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one. This difference possibly indicates some specificity of the E. coli ribosome for tRNAs from the same organism.     

Epitopes on foot-and-mouth disease virus outer capsid protein VP1 involved in neutralization and cell attachment.

Foot-and-mouth disease virus structural protein VP1 elicits neutralizing and protective antibody and is probably the viral attachment protein which interacts with cellular receptor sites on cultured cells. To study the relationships between epitopes on the molecule related to neutralization and cell attachment, we tested monoclonal antibodies prepared against type A12 virus, isolated A12 VP1, and a CNBr-generated A12 VP1 fragment for neutralization and effect on viral absorption. The antibodies selected for analysis neutralized viral infectivity with varying efficiencies. One group of antibodies caused a high degree of viral aggregation and inhibited the adsorption of virus to cells by 50 to 70%. A second group of antibodies caused little or no viral aggregation but inhibited the adsorption of virus to cells by 80 to 90%. One antibody, which is specific for the intact virion, caused little viral aggregation and had no effect on the binding of virus to specific cellular receptor sites. Thus, at least three antigenic areas on the surface of foot-and-mouth disease virus which were involved in neutralization were demonstrated. One of the antigenic sites appears to have been responsible for interaction with the cellular receptor sites on the surface of susceptible cells.     

Cortisol production by dispersed guinea-pig adrenal cells; a specific, sensitive and reproducible response to ACTH....and its fragments

Naturally occurring steroids and peptide hormones, tested at supraphysiological concentrations, were without effect on basal and human (h) 1-39 ACTH (NIBSC code 74/555, 25 ng/l (5.5 X 10(-12) mol/l] stimulated cortisol production. Further, low concentrations of angiotensin II, N-pro-opiocortin (N terminal fragment 1-76) and gamma-MSH all of which have been reported to synergise with ACTH with regard to cortisol production, were without significant effect alone or in combination with ACTH over the range 2.2 X 10(-13) to 5.5 X 10(-12) mol/l. The activity of h 1-39 was compared with that of the ACTH related peptides 1-24, 1-18, 1-17, 1-16, 1-13-NH2 (alpha MSH), 1-10 and 4-10. The dose responses were parallel and the same maximal cortisol output was observed with all the peptides except the 1-10 fragment. Half maximal stimulation occurred at 3.1 X 10(-12) (1-24), 4.4 X 10(-12) (h 1-39), 1.5 X 10(-11) (1-39), 3.3 X 10(-10) (1-18), 5 X 10(-9) (1-13-NH2), 8 X 10(-9) (1-17), 2 X 10(-7) (1-16) and 1 X 10(-5) (4-10) mol/l respectively. Interference by the above ACTH-derived peptides in cortisol secretion by the cells in response to 5.5 X 10(-12) mol/l h 1-39 ACTH was minimal over the range 5.2 X 10(-12)-2.2 X 10(-6) mol/l. The sensitivity of the adrenal cells to h 1-39 ACTH was such that 2 ng/l (4.4 X 10(-13) mol/l) provoked cortisol secretion over the control (P less than 0.05, n = 17). The coefficient of variation within assay for each dose on the full standard curve (2.2 X 10(-13)-1.1 X 10(-10) mol/l) was less than 10% (n = 6). Half maximal stimulation was given by 14.5 ng/l (3.2 X 10(-12) mol/l). Between control and 1.1 X 10(-10) mol/l ACTH there was a 32 +/- 8 (mean +/- SD, n = 9) fold change in cortisol production.     

Detection of noncontingent feedback in EMG biofeedback

Noncontingent feedback is frequently used as a placebo control procedure in biofeedback research. Researchers, however, have criticized this procedure for lacking credibility because of easy detection. The present study examined detection of false feedback in biofeedback with EMG. Contingent feedback (CF), truly random false feedback (FF), and controlled false feedback (CFF) groups were compared for changes in EMG levels, report of inaccurate feedback, and report of learning muscle activity reduction. The results indicated that FF procedures are easily detected; therefore, differences found between the FF and CF groups may be influenced by extraneous variables. The CFF group did not detect false feedback, but subjects reported some suspicions in later trials. With more trials, CFF may have also been detected. These results indicate a need for more attention to appropriate placebo control procedures in evaluating the parameters and efficacy of biofeedback.