Regulation of the penicillinase genes of Bacillus licheniformis: interaction of the pen repressor with its operators.

The synthesis of the inducible enzyme penicillinase of Bacillus licheniformis is negatively controlled by a repressor (D.A. Dubnau and M.R. Pollock, J. Gen. Microbiol. 41:7-21, 1965; D. J. Sherratt and J. F. Collins, J. Gen. Microbiol. 76:217-230,1973). The molecular organization of the genes coding for penicillinase (penP) and its repressor (penI) has recently been determined (T. Himeno, T. Imanaka, and S. Aiba, J. Bacteriol. 168:1128-1132, 1986). These two genes are transcribed divergently from within a 364-nucleotide region separating the coding sequences. We cloned and sequenced the repressor gene (penIc) from strain 749/C that constitutively produces penicillinase. The penIc and penI+ (wild-type) genes were expressed in Escherichia coli. Complementation analysis indicated that the repressor is the only trans-acting protein required to regulate the expression of the penI and penP genes. We purified the wild-type repressor protein, used it in gel retardation and DNase I protection experiments, and identified three operators positioned in the region between the penP and penI coding sequences. The spatial arrangement of the operators and the hierarchy in repressor binding seen in the protection experiments indicate that (i) the penI gene product represses the expression of the penP gene by physically blocking the RNA polymerase-binding site and (ii) the penI gene is autoregulated.     

Glutathione reductase: solvent equilibrium and kinetic isotope effects

Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D2O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456.     

Influence of ion gradients on the transbilayer distribution of dibucaine in large unilamellar vesicles

The uptake of dibucaine into large unilamellar vesicles in response to proton gradients (delta pH; inside acidic) or membrane potentials (delta psi; inside negative) has been investigated. Dibucaine uptake in response to delta pH proceeds rapidly in a manner consistent with permeation of the neutral (deprotonated) form of the drug, reaching a Henderson-Hasselbach equilibrium where [dibucaine]in/[dibucaine]out = [H+]in/[H+]out and where the absolute amount of drug accumulated is sensitive to the buffering capacity of the interior environment. Under appropriate conditions, high absolute interior concentrations of the drug can be achieved (approximately 120 mM) in combination with high trapping efficiencies (in excess of 90%). Dibucaine uptake in response to delta psi proceeds more than an order of magnitude more slowly and cannot be directly attributed to uptake in response to the delta pH induced by delta psi. This induced delta pH is too small (less than or equal to 1.5 pH units) to account for the transmembrane dibucaine concentration gradients achieved and does not come to electrochemical equilibrium with delta psi. Results supporting the possibility that the charged (protonated) form of dibucaine can be accumulated in response to delta psi were obtained by employing a permanently positively charged dibucaine analogue (N-methyldibucaine). Further, the results suggest that delta psi-dependent uptake may depend on formation of a precipitate of the drug in the vesicle interior. The uptake of dibucaine into vesicles in response to ion gradients is of direct utility in drug delivery and controlled release applications and is related to processes of drug sequestration by cells and organelles in vivo.     

Systolic pressure gradients between the wall of the left ventricle, the left ventricular chamber, and the aorta during positive inotropic states: implications for left ventricular efficiency

To study systolic pressure gradients developed between the left ventricular wall, its chamber, and the aortic root, in one group of dogs left ventricle ventral wall intramyocardial pressure, left ventricular outflow tract pressure, and aorta pressure were compared with aortic flow as well as left ventricular dimension changes during control conditions as well as during positive intropic states induced by isoproterenol, stellate ganglion stimulation, and noradrenaline. In another group of dogs systolic pressures in the ventral wall of the left ventricle, the main portion of the left ventricular chamber, and the aorta were compared with aortic flow during similar interventions, before and after the administration of phentolamine. Pressure gradients between the wall of the left ventricle and the outflow tract of the left ventricle were minimal during control states, but during the three positive inotropic states were increased significantly. In contrast, pressure gradients between the outflow tract of the left ventricle and the aortic root were insignificant during positive inotropic states; those between the wall and main portion of the chamber were only significantly different during left stellate ganglion stimulation. The data derived from these experiments indicate that useful peak power output of the left ventricle (systolic aortic pressure X flow) is unchanged following isoproterenol infusion, but is increased by stellate ganglion stimulation and noradrenaline. The useful peak power output index (an index of left ventricular efficiency derived by dividing useful peak power output by peak intramyocardial pressure) was reduced more by isoproterenol than the other two interventions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydralazine-dependent carbon dioxide free radical formation by metabolizing mitochondria

The addition of hydralazine (1-hydrazinophthalazine) to rat liver mitochondria metabolizing malate/glutamate causes formation of a carbon-centered free radical which was spin-trapped with phenyl-t-butylnitrone (PBN) or dimethylpyrrolidine-N-oxide (DMPO). The coupling constants of the spin-trapped free radical were AN = 16.1, AH beta = 4.6 G for PBN and AN = 15.9, AH beta = 18.9 G for DMPO-trapped radical in aqueous solution. The spin-trapped free radical was shown to be the carbon dioxide anion free radical by independent synthesis, high pressure liquid chromatography separation, and electron paramagnetic resonance characterization. The amount of carbon dioxide anion free radical produced was absolutely dependent upon the presence of hydralazine and varied depending on mitochondrial substrate, with by far the highest amount produced by pyruvate. Studies with 13C-labeled pyruvate demonstrated that the carbon dioxide free radical came from C-1 of this compound.     

The inhibition of commitment of mouse erythroleukemia cells by steroids involves a glucocorticoid-receptor mediated process(es) acting at the nuclear level

Dexamethasone has been shown to inhibit dimethylsulfoxide (DMSO)-induced differentiation of mouse erythroleukemia (or Friend) cells by blocking commitment to terminal erythroid maturation. In this study, we confirmed previous reports indicating the presence of glucocorticoid receptors in murine erythroleukemia cells and examined the mechanism(s) by which steroids block commitment. Untreated murine erythroleukemia cells contain dexamethasone receptors which decrease in number during DMSO-induced cell differentiation. When steroids of different classes (estrogenic, androgenic, glucocorticoid) were tested for inhibition of commitment and for displacement of [3H]dexamethasone from its receptors in DMSO-treated cells, we observed that the glucocorticoids dexamethasone, prednisolone and hydrocortisone, all blocked commitment and substantially displaced [3H]dexamethasone. In contrast, steroids other than glucocorticoids failed to inhibit commitment or displace [3H]dexamethasone. Analysis of kinetics of dexamethasone binding to chromatin revealed that dexamethasone binds to the nucleus via the receptor and preferentially interacts with active chromatin. Inhibition of commitment by dexamethasone persisted in cells released from this agent and reincubated with DMSO in the presence of another glucocorticoid of similar affinity to steroid receptors; inhibition of commitment, however, was not obtained when cells removed from dexamethasone were incubated in the presence of beta-estradiol, progesterone and testosterone. These data indicate that inhibition of commitment of mouse erythroleukemia cells by steroids is associated with binding to glucocorticoid receptors and may involve interactions of steroids and their receptors with regions of chromatin.     

Labelling of amoeboid microglial cells in rats of various ages following an intravenous injection of horseradish peroxidase

The macrophagic amoeboid microglial cells in the corpus callosum of postnatal rats were labelled following an intravenous injection of horseradish peroxidase (HRP). The earliest time when these cells were labelled was 3 h after the injection of HRP in postnatal (1-10 days) rats. Similar cells around the mesencephalic aqueduct and the fourth ventricle were also labelled. These cells, however, were weakly labelled in developing (11-20 days) and unlabelled in weaning (21-30 days) rats. The results suggest that in the postnatal rats, the HRP passed through the endothelial lining of the blood vessels and was then ingested by the amoeboid microglial cells. In the developing and older rats, the wall of blood vessels had developed fully thereby preventing the free passage of HRP into the brain tissues.     

Mass spectrometry and chromatography of t-butyldimethylsilyl derivatives of cytokinin bases

Di-(t-butyldimethylsilyl) derivatives of the cytokinin bases zeatin, cis-zeatin, and dihydrozeatin may be prepared quantitatively in the presence of dimethylaminopyridine. These derivatives have good gas chromatographic properties and are very suitable for gas chromatography-mass spectrometry analysis of cytokinin bases. The t-butyldimethylsilyl (tBuDMS) group at N-9 may be selectively hydrolyzed and the resulting mono-O-silyl derivatives are sufficiently stable to be subjected to thin-layer chromatography and high-performance liquid chromatography. The mass spectral fragmentation of the mono- and di-tBuDMS derivatives of adenine, zeatin, cis-zeatin, and dihydrozeatin and also of the mono-tBuDMS derivatives of N6-isopentenyladenine and 6-benzylaminopurine have been rationalized. The 9-tBuDMS moiety was characterized by an elimination of isobutene (M-56) and of isobutene plus a methyl radical (M-56-15).     

2',5'-Phosphodiesterase activity depends upon the presence of a 3'-hydroxyl moiety in the penultimate position of the oligonucleotide substrate

3'-Deoxyadenosine (3'dA, cordycepin)-substituted analogs of 2-5A core 5'-monophosphate (p5'A2'p5'A2'p5'A) were examined for their sensitivity toward degradation by the 2'-phosphodiesterase activity in cytoplasmic extracts of mouse L cells. The analogs, p5'(3'dA)-2'p5'A2'p5'A, p5'(3'dA)2'p5'A2'p5'(3'dA) and p5'A2'p5'A2'p5'(3'dA) were degraded at a rate comparable to p5'A2'p5'A2'p5'A itself. On the other hand, under the assay conditions examined p5'A2'p5'(3'dA)2'p5'A, like p5'(3'dA)2'p5'(3'dA)2'p5'(3'dA), was completely resistant to degradation. The data imply that sensitivity to the 2',5'-phosphodiesterase activity of mouse L cells requires the presence of 3'-hydroxyl moiety in the penultimate nucleotide.     

Assessment of mechanistic proposals for the binding of agonists to cardiac muscarinic receptors

N-[3H]Methylscopolamine has been used to characterize muscarinic receptors in crude homogenates prepared from hearts of Syrian golden hamsters. The Hill coefficient is one for specific binding of the radioligand itself and for its inhibition by muscarinic antagonists; markedly lower values are obtained for its inhibition by muscarinic agonists. The binding patterns of agonists have been analyzed in terms of a mixture of sites differing in affinity for the drug and reveal the following. All agonists discern at least two classes of receptor in atrial and ventricular homogenates. The number of classes and the relative size of each differ for different agonists in the same region and for the same agonist in different regions. Atrial and ventricular affinities are in good agreement for some agonists but differ for others. Guanylyl imidodiphosphate (GMP-PNP) is without effect on the specific binding of the radioligand but alters the binding of carbachol via an apparent redistribution of receptors from one class to another; the apparent affinity at either class remains unchanged. Carbachol reveals two classes of sites in ventricular preparations, and the nucleotide mediates an interconversion from higher to lower affinity; three classes are revealed in atrial preparations, and the nucleotide eliminates the sites of highest affinity with a concomitant increase in the number of sites of lowest affinity. Taken together, the data are incompatible with the notion of different, noninterconverting sites; rather, there appear to be several possible states of affinity such that the equilibrium distribution of receptors among the various states is determined by the tissue, by the agonist, and by neurohumoral modulators such as guanylyl nucleotides. The effects of agonists and GMP-PNP cannot be rationalized in terms of a ternary complex model in which the low Hill coefficients arise from a spontaneous equilibrium between receptor (R) and G protein (G) and in which agonists bind preferentially to the RG complex.