Stereochemical control over Mn(II)-thio versus Mn(II)-oxy coordination in adenosine 5'-O-(1-thiodiphosphate) complexes at the active site of creatine kinase

The stereochemical configurations of the Mn(II) complexes with the resolved epimers of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S), bound at the active site of creatine kinase, have been determined in order to assess the relative strengths of enzymic stereoselectivity versus Lewis acid/base preferences in metal-ligand binding. Electron paramagnetic resonance (EPR) data have been obtained for Mn(II) in anion-stabilized, dead-end (transition-state analogue) complexes, in ternary enzyme-MnIIADP alpha S complexes, and in the central complexes of the equilibrium mixture. The modes of coordination of Mn(II) at P alpha in the nitrate-stabilized, dead-end complexes with each epimer of ADP alpha S were ascertained by EPR measurements with (Rp)-[alpha-17O]ADP alpha S and (Sp)-[alpha-17O]ADP alpha S. The EPR spectrum for the complex with (Rp)-[alpha-17O]ADP alpha S showed inhomogeneous broadening due to unresolved superhyperfine coupling from coordinated 17O at P alpha. By contrast, the EPR spectrum for Mn(II) in complex with (Sp)-[alpha-17O]ADP alpha S is indistinguishable from that obtained for a matched sample with unlabeled (Sp)-ADP alpha S. A reduction in the magnitude of the 55Mn hyperfine coupling constant in the spectrum for the complex containing (Sp)-ADP alpha S is indicative of Mn(II)-thio coordination at P alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Olfactory cytochrome P-450. Studies with suicide substrates of the haemoprotein.

1. The olfactory epithelium of male hamsters has been found to be extremely active in the cumene hydroperoxide-supported oxidation of tetramethylphenylenediamine, and this peroxidase activity has been shown to be cytochrome P-450-dependent. 2. The interaction of a series of suicide substrates of cytochrome P-450 with the hepatic and olfactory mono-oxygenase systems has been assessed by determination of peroxidase, 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) activities after treatment in vivo with these compounds. Chloramphenicol, OOS-trimethylphosphorothiolate and two dihydropyridines [DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) and 4-ethyl DDC (3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine)] all caused similar percentage inhibitions of hepatic and olfactory activities, but the absolute amounts of enzymic activity lost were considerably greater in the latter tissue. In contrast, halothane had little effect upon hepatic cytochrome P-450-dependent reactions, whereas it severely inhibited those of the olfactory epithelium. 3. The time course of loss and recovery of hepatic and olfactory peroxidase, ECOD and EROD activities after a single dose of 4-ethyl DDC was studied. The rates of loss of activity observed were very similar, irrespective of tissue or reaction examined. In the olfactory epithelium, all three activities recovered concurrently and at a rate similar to that of the hepatic peroxidase activity. In contrast, the hepatic de-ethylation of 7-ethoxycoumarin and 7-ethoxy-resorufin recovered significantly more rapidly. 4. It is suggested that this behaviour is due to 4-ethyl DDC acting not only as a suicidal inhibitor but also as an inducer of certain forms of cytochrome P-450 in the liver; in the olfactory epithelium, however, inactivation, but not induction, occurs. Classical inducing agents were reported to have no effect upon olfactory cytochrome P-450, and in the present study neither phenobarbitone nor beta-naphthoflavone treatment had any effect upon olfactory cytochrome P-450-dependent reactions, although it induced those of the liver.     

Effect of microspore stage and media on anther culture of peanut (Arachis hypogaea L.)

This study was designed to study the effects of stage of microspore development and culture medium on androgenic response in peanut (Arachis hypogaea L.). Anthers of various developmental stages were cultured for 7 days, then fixed and observed cytologically. Three sets of media, involving different basal media, growth regulators, sucrose levels and glutamine concentrations, were tested. In all experiments, the stage of development of the microspores at the time of culture was highly significant. The early uninucleate microspores stage was identified as producing the highest anther response rating. The effect of media was nonsignificant in all experiments. However, the stepwise modification of the media through the course of the study resulted in an almost 8 x increase in anther response rating. Numerically, the best media tested was N₆ basal medium with 1 mg 1^-1 NAA, 0.1 mg 1^-1 BA, 5.5% sucrose, and 3.5 g 1^-1 glutamine. While no haploids were obtained, four-nucleate cells were observed, indicating the potential in peanuts for an androgenic reponse.     

Rhizobium meliloti exopolysaccharides: genetic analyses and symbiotic importance.

Genetic experiments have indicated that succinoglycan (EPS I), the acidic Calcofluor-binding exopolysaccharide, of the nitrogen-fixing bacterium Rhizobium meliloti strain Rm1021 is required for nodule invasion and possibly for later events in nodule development on alfalfa and other hosts. Fourteen exo loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. We have identified two loci, exoR and exoS, that are involved in the regulation of EPS I synthesis in the free-living state. Certain exo mutations which completely abolish EPS I production are lethal in an exoR95 or exoS96 background. Histochemical analyses of the expression of exo genes during nodulation using exo::TnphoA fusions have indicated that the exo genes are expressed most strongly in the invasion zone. In addition, we have discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for exopolysaccharides in symbiosis are discussed.     

Retention of oxidized glutathione by isolated rat liver mitochondria during hydroperoxide treatment

The addition of tert-butyl hydroperoxide (t-BuOOH) to isolated mitochondria resulted in oxidation of approximately 80% of the mitochondrial reduced glutathione (GSH) independently of the dose of t-BuOOH (1-5 mM). Concomitant with the oxidation of GSH inside the mitochondria was the formation of GSH-protein mixed disulfides (protein-SSG), with approximately 1% of the mitochondrial protein thiols involved. A dose-dependent rate of GSH recovery was observed, via the reduction of oxidized GSH (GSSG) and a slower reduction of protein-SSG. Although t-BuOOH administration affected the respiratory control ratio, the mitochondria remained coupled and loss of the matrix enzyme, citrate synthase, was not increased over the control and was less than 3% over 60 min. A slow loss of GSH out of the coupled non-treated mitochondria was not increased by t-BuOOH treatment, in fact, a dose-dependent drop of GSH levels occurred in the medium. However, no GSSG was found outside the mitochondria, indicating the necessary involvement of enzymes in the t-BuOOH-induced conversion of GSH to GSSG. The absence of GSSG in the medium also suggests that, unlike the plasma membrane, the mitochondrial membranes do not have the ability to export GSSG as a response to oxidative stress. Our results demonstrate the inability of mitochondria to export GSSG during oxidative stress and may explain the protective role of mitochondrial GSH in cytotoxicity.     

Miconia zanonii (Melastomataceae: Miconieae), a new species from Hispaniola

Miconia zanonii, which occurs in broad-leaved cloud forests at four localities in the Cordillera Central of the Dominican Republic, is described and illustrated. It is compared withM. krugii andM. samanensis, two probably close relatives withinMiconia sect.Chaenopleura.     

The effect of glucocorticoids on the in vivo conversion of androstenedione to oestrone

In vitro studies have previously shown that the activity of the aromatase enzyme system, which is responsible for the conversion of androstenedione to oestrone, can be stimulated by natural and synthetic glucocorticoids and also by ACTH. In view of the potential physiological importance of such a regulatory mechanism we have examined the effect of administration of dexamethasone, and of ACTH on the conversion of androstenedione to oestrone in vivo. The transfer constants for the conversion of androstenedione to oestrone [( rho]AEBU) measured in two women before administration of dexamethasone were 1.0% and 1.1% and after were 0.9% and 1.2%. Similarly no increase in conversion of androstenedione to oestrone [( rho]AE1BB) was detected after ACTH stimulation (pre = 0.74%, post = 0.77%). It is concluded from this study that glucocorticoids and ACTH do not have a role in regulating aromatase activity in vivo.     

Amiodarone inhibits T4 to T3 conversion and alpha-glycerophosphate dehydrogenase and malic enzyme levels in rat liver

Amiodarone has been found to decrease serum T3 by blocking peripheral T4 5'-deiodinase. This reduction in T3 levels may contribute to the effectiveness of this drug in moderating cardiac arrhythmias. To further characterize the effect of amiodarone on thyroid hormone metabolism and biological action, male Sprague-Dawley rats were thyroidectomized and then fed 500 ug T4 or 50 ug T3 and 500 mg amiodarone/kg of powdered diet for 6 to 8 weeks. Hepatic and cardiac levels of T4, T3, alpha-glycerophosphate dehydrogenase (GPD) and malic enzyme (ME) were used as indicators of thyroid hormone availability and action at the cellular level. Conversion of T4 to T3 was measured in liver homogenates. Serum TSH, T4 and T3 were also measured. Amiodarone reduced hepatic GPD and ME in thyroidectomized rats receiving dietary T4. Liver T4 levels were significantly increased by amiodarone and the T3/T4 ratio was reduced (P less than .05). Amiodarone inhibited hepatic T4 to T3 conversion and decreased serum T3. The decreased T3 action at the cellular level, indicated by the reduction in hepatic GPD and ME, is not due to pharmacologic effects of amiodarone since these enzyme levels were not affected by amiodarone in thyroidectomized rats replaced with T3.     

Observation of manganese(II)-ligand superhyperfine couplings in complexes with proteins by electron spin-echo spectroscopy

Pulsed electron paramagnetic resonance spectroscopy has been used to detect Mn(II)-ligand superhyperfine couplings in complexes with creatine kinase and in the Mn(II) metalloprotein concanavalin A. Electron spin-echo envelopes from Mn(II), bound in these complexes, are modulated by superhyperfine interactions between Mn(II) and nearby, weakly coupled nuclear spins. The characteristic frequencies of the modulations were obtained by Fourier transformation of the three-pulse, spin-echo envelopes. In transition-state analogue complexes of creatine kinase (enzyme-MnIIADP-anion-creatine), superhyperfine interactions from the directly coordinated nitrogen of the thiocyanate ligand give envelope modulations. The source of the modulations was confirmed by measurements with the 14N and 15N forms of thiocyanate. On the other hand, the nitrogen of coordinated nitrate, which is two bonds removed from the paramagnetic center, does not produce detectable modulations. In spectra for Mn(II) concanavalin A, envelope modulations are detected due to the nitrogen of the coordinated histidine residue. Complexes prepared in 2H2O give strong signals due to weakly coupled 2H. For Mn(II)-doped single crystals of sodium pyrophosphate, signals are observed in the frequency domain spectra that are due to coupling from 31P. Phosphorus signals from the ADP ligand in complexes with creatine kinase show approximately the same coupling constant but have a much broader line width.