Effects of EEG of the osmotic opening of the blood-brain barrier in rats

EEG activity was recorded in rats submitted to osmotic opening of the BBB by intracarotid mannitol infusion.This procedure produced an immediate short-lasting depression of the EEG and a tardive paroxysmal EEG activity. Both these phenomena were more relevant on the ipsilateral hemisphere. In some instances a tonico-clonic seizure was recorded.Pre-treatment with diazepam abolished the occurrence of the tardive EEG and behavioral modifications.In accord with previous findings, focal seizure activity is likely to be responsible for the metabolic abnormalities associated with osmotic opening of the BBB. This preparation therefore produces in the brain unphysiological states in respect to local metabolism and electrical function.     

Contribution to catalysis and stability of the five cysteines in Escherichia coli aspartate aminotransferase. Preparation and properties of a cysteine-free enzyme.

The five cysteines, at positions 82, 191, 192, 270, and 401, of Escherichia coli aspartate aminotransferase (AATase) were, individually and in some combinations, converted to alanine by site-directed mutagenesis (C82A, C191A, C192A, C270A, C401A). Cys-191, which is conserved in all AATase isozymes, was mutated to serine as well (C191S). A quintuple mutant, with all cysteines converted to alanines (Quint), was also constructed. The effects of these single and multiple mutations were examined by steady-state kinetics and urea denaturation. The thermal stabilities of Quint and of the wild-type enzyme (WT) were determined by differential scanning calorimetry. The mutants had kcat values up to 50% greater than that of WT and KMAsp and KM alpha-KG values up to 1.5- and 3.3-fold higher than that of WT. The mutants C82A and C191A exhibit nearly the same CM in urea denaturation experiments as WT, while the other single mutants and Quint are less stable, with CM differences of up to 0.7 M urea. Quint is also less thermostable than WT, with a delta TM of 3.3-4.4 degrees C. Thus the five cysteine replacements yield small, but significant, changes in catalytic and denaturation parameters, but none of the cysteines was found to be essential. The changes manifested in the mutation of the conserved Cys-191 to alanine are no greater than those observed with the four nonconserved cysteines. We consider the evolutionary implications of these findings.     

Antagonism by theophylline of respiratory inhibition induced by adenosine

The effects on respiration of an analogue of adenosine, L-2-N6-(phenylisopropyl)adenosine (PIA), and of the methylxanthine, theophylline, were determined in 19 vagotomized glomectomized cats whose end-tidal PCO2 was kept constant by means of a servo-controlled ventilator. Integrated phrenic nerve activity was used to represent respiratory output. Our results show that PIA, whether given systemically or into the third cerebral ventricle, depressed respiration. Systemically administered theophylline stimulated respiration. Theophylline given intravenously, or into the third ventricle not only reversed the depressive effects of previously administered PIA but caused further increases of respiration above the control level. Prior systemic administration of theophylline blocked both respiratory and hypotensive effects of subsequently administered PIA. Effects of either agent on medullary extracellular fluid pH did not explain the results. We conclude that the adenosine analogue PIA, acts to inhibit neurons in the brain that are involved in the control of respiration and that its effects are blocked by theophylline. We suggest that adenosine acts as a tonic modulator of respiration and that theophylline stimulates breathing by competitive antagonism of adenosine at neuronal receptor sites.     

Effect of bromocriptine on prostacyclin release and cyclic nucleotides on rat aortic and uterine tissues

The effect of bromocriptine mesylate on cyclic nucleotides and PGI₂ release by rat aortic and uterine tissues was investigated. Treatment of rats with bromocriptine (10 mg kg⁻¹ I.P. daily for 14 days) increased PGI₂ release by the thoracic aorta from 0.67 ± 0.02 to 1.4 ± 0.03 ng/mg wet tissue (P < 0.001; n = 6). This increase was antagonized by treatment with sulpiride (15 mg kg⁻¹). Incubation of the arterial tissue with bromocriptive (50 ug ml⁻) in vitro also stimulated PGI₂ release. Mepacrine (160 μg ml⁻) significantly decreased both basal and stimulated PGI₂ release. Incubation of myometrial tissue from pregnant rats with bromocriptine (50 μg ml⁻¹) in vitro significantly decreased PGI₂ release from 1.25 ± 0.07 to 0.60 ± 0.08 ng/mg wet tissue (P < 0.05, n = 6).It also elevated uterine cAMP from 40 ± 2 to 64 ± 3 pmoles/100 mg wet tissue. Both effects were antagonized by sulpiride. Bromocriptine did not affect uterine cGMP or the cyclic nucleotides in the aorta. It is concluded that the increase in aortic PGI₂ was mediated via activation of dopamine D-2 receptors that stimulate phospholipase A₂ enzyme. The decrease in myometrial PGI₂ release may be related to the increase in uterine cAMP resulting from activation of dopamine D-1 receptors. Previous studies suggested a role for PGI₂ in implantation in the rat. The results suggest that the inhibitory effèct on uterine PGI₂ may underlie the reported inhibition of bromocriptine on implantation. On broad basis, the decrease in uterine PGI₂ together with the reported luteolytic effect of bromocriptine point to a potential role for the compound in postcoital contraception.     

Thiostrepton resistance mutations in the gene for 23S ribosomal RNA of halobacteria

Mutants of Halobacterium (H.) halobium and H. cutirubrum were isolated which are resistant to the 70S ribosome inhibitor thiostrepton. Using primer extension analysis, resistance was shown to correlate with base changes at position 1159, which corresponds to position 1067 of the E. coli 23S rRNA. In four mutants, A1159 was replaced by U, in one mutant by G. The results show that not only methylation (Cundliffe & Thompson (1979) Nature 278, 859-861) of A1067 (E. coli nomenclature), but also base changes at this position cause high-level resistance to thiostrepton.     

Application of halophilic nuclease H of Micrococcus varians subsp. halophilus to commercial production of flavoring agent 5'-GMP

RNA was degraded at 60 degrees C for 24 h by halophilic nuclease H in supernatants from broth cultures of Micrococcus varians subsp. halophilus containing 12% NaCl. Since contaminating 5'-nucleotidase exhibited almost no activity under these conditions, the 5'-GMP formed could be recovered from the reaction mixture, and the yield was 805 mg from 5 g of RNA.