Genetic basis of starvation survival in nondifferentiating bacteria.
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Bacteria↗
Biomedical subjects
J E Schultz
Publications and source records attributed to J E Schultz.
Paramecium: a model system for the study of excitable cells.
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Action Potentials↗
Cloning of the adenylate cyclase genetic determinant of Bordetella pertussis and its expression in Escherichia coli and B. pertussis.
A recombinant plasmid, pRMB1, identified from a gene library of B. pertussis, restored adenylate cyclase (AC) and haemolysin (HLY) activities to B. pertussis BP348 (a Tn5-insertion mutant deficient in both these activities). B. pertussis BP348 was considerably less virulent than wild type strains of B. pertussis when 3-week-old mice were challenged intranasally; possession of pRMB1 restored virulence. Neither AC nor HLY activities were expressed in E. coli harbouring pRMB1. However, expression of calmodulin-responsive AC was obtained in E. coli when restriction fragments of pRMB1 were subcloned into other vectors; expression depended on the lac and tac promoters of these vectors. The enzyme was not readily solubilized from urea extracts of E. coli and required sonication for efficient release. One plasmid conferred a specific AC enzymic activity to E. coli which was greater than that for wild type B. pertussis strains. Unlike extracts of B. pertussis, extracts from E. coli expressing enzymic AC activity, did not elevate cAMP levels in S49 lymphoma cells. A second plasmid, pRMB2, was identified from the gene library, which contained a trans-acting regulatory determinant required for expression of AC, HLY and other virulence-associated factors in B. pertussis.
Adenylyl Cyclases↗
Unusual stereospecificity of the potential antidepressant rolipram on the cyclic AMP generating system from rat brain cortex.
Adrenoceptor sensitivity of central synapses is thought to be involved in regulation of mood. Persistent changes exceeding normal fluctuations conceivably could lead to disease states like manic-depressive illness. Several treatment regimens which affect mood in man reversibly alter the density of beta-adrenoceptors in animals in a time- and dose-dependent manner. Most prominently, many antidepressants which inhibit neurotransmitter uptake downregulate postsynaptic adrenoceptors, and thus diminish intracellular cyclic AMP formation elicited by noradrenaline. In addition, other modulations of presynaptic nerve activity, e.g. electroconvulsive shock or treatment with thyroid hormones, also cause adaptational responses at the postsynapse. In general, functional integrity of the pre/postsynaptic unit had to be maintained strictly to observe postsynaptic downregulation. Using a highly potent and stereospecific inhibitor of cyclic AMP hydrolysis we demonstrate that downregulation can also be elicited by postsynaptic mechanisms. Treatment of rats with rolipram, a phenylpyrrolidinone derivative, decreases adrenoceptor density in a time- and dose-dependent fashion. The ED50 for the racemate is 0.35 mg/kg, for the (-)-isomer 0.18 mg/kg. The (+)-isomer is inactive. This effect correlates with the drug's potency to inhibit a calmodulin/calcium-insensitive phosphodiesterase from brain cortex (ED50 racemate: 1.2 microM; (-)-isomer: 0.4 microM). Using methods of computer-assisted drug design to identify common stereochemical features of rolipram and the unrelated tricyclic antidepressants, we found that calculated low energy conformations fit onto a four-point model which specifies four hypothetical binding sites between a drug and its presumptive receptor site. These findings may indicate that all antidepressants share a common, hitherto unknown site of action irrespective of their known biochemical activities.
Animals↗
Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli.
Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).
Bacterial Proteins↗
Starvation-induced cross protection against heat or H2O2 challenge in Escherichia coli.
Glucose- or nitrogen-starved cultures of Escherichia coli exhibited enhanced resistance to heat (57 degrees C) or H2O2 (15 mM) challenge, compared with their exponentially growing counterparts. The degree of resistance increased with the time for which the cells were starved prior to the challenge, with 4 h of starvation providing the maximal protection. Protein synthesis during starvation was essential for these cross protections, since chloramphenicol addition at the onset of starvation prevented the development of thermal or oxidative resistance. Starved cultures also demonstrated stronger thermal and oxidative resistance than did growing cultures adapted to heat, H2O2, or ethanol prior to the heat or H2O2 challenge. Two-dimensional gel electrophoresis of 35S-pulse-labeled proteins showed that subsets of the 30 glucose starvation proteins were also synthesized during heat or H2O2 adaptation; three proteins were common to all three stresses. Most of the common proteins were among the previously identified Pex proteins (J.E. Schultz, G. I. Latter, and A. Matin, J. Bacteriol. 170:3903-3909, 1988), which are independent of cyclic AMP positive control for their induction during starvation. Induction of starvation proteins dependent on cyclic AMP was not important in these cross protections, since a delta cya strain of E. coli K-12 exhibited the same degree of resistance to heat or H2O2 as the wild-type parent did during both growth and starvation.
Autoradiography↗
Ionic regulation of adenylate cyclase from the cilia of Paramecium tetraurelia.
The kinetics of the ionic regulation of an adenylate cyclase associated with the excitable ciliary membrane from Paramecium tetraurelia was examined. Glycerol (30%, v/v) stabilized the enzyme, and activated by an increase in Vmax. (3-fold) and a decrease in the apparent Km for MgATP (6-fold). Kinetic analysis of Mg2+ effects showed a stimulation via a single metal-binding site separate from the substrate site, with a dissociation constant, Ks, of 0.27 mM. Analysis of Ca2+ effects showed (i) an uncompetitive inhibition with respect to substrate MgATP, and (ii) dependence of the extent of inhibition on the free Mg2+ concentration. Ki values ranged from 4 to 130 microM-Ca2+ in the presence of 0.55-2 mM-Mg2+ respectively. This indicates competition between Mg2+ and Ca2+ at the metal-binding site. The Ca2+ effect was specific; Sr2+ and Ba2+ were almost without effect, and 100 microM-Ba2+ did not interfere with the Ca2+ inhibition. The actions of Ca2+ were readily reversible after addition of EGTA. K+ activated the adenylate cyclase at concentrations around 20 mM. The stimulatory potency of K+ was dependent on the free Mg2+ concentration. At 1 mM free Mg2+, 20 mM-K+ doubled the adenylate cyclase activity. The inhibitory Ca2+ and stimulatory K+ inputs were independent of each other.
Adenosine Triphosphate↗
Effect of tryptic calmodulin fragments on guanylate cyclase activity from Paramecium tetraurelia.
Tryptic bovine brain calmodulin fragments 1-77 or 1-106 reactivated La-inactivated ciliary guanylate cyclase from Paramecium dose-dependently up to 60%. They were 20-fold less potent compared to bovine brain calmodulin. Fragment 78-148 was even less active. Concomitant addition of fragments 1-77 and 78-148 had no additive effect. Genetically engineered calmodulin lacking a blocked amino terminus and trimethyllysine at position 115 reactivated La-treated guanylate cyclase as good as bovine brain calmodulin. After detergent solubilization of La-inactivated guanylate cyclase intact bovine brain calmodulin and calmodulin fragments 1-77 and 78-148 were equipotent. 80% Reactivation was obtained with 40 microM of either fragment.
Animals↗
Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type 1.
The binding of Shiga toxin isolated from the bacterium Shigella dysenteriae type 1 to a series of glycolipids and to cells or cell homogenates has been studied. Bound toxin was detected using either 125I-labeled toxin or specific monoclonal antibody and 125I-labeled anti-antibody. Overlay of toxin on thin-layer chromatograms with separated glycolipids and binding to glycolipids coated in microtiter wells established that the toxin specifically bound to Gal alpha 1-4Gal beta (galabiose) placed terminally or internally in the oligosaccharide chain. No glycolipid shown to lack this sequence binds the toxin. Most of the glycolipids with internally placed galabiose were not active, indicating a sterical hindrance for toxin access to the binding epitope. Binding of toxin to HeLa cells in monolayers could be inhibited by preincubation of the toxin with galabiose covalently linked to bovine serum albumin (BSA), but not with free oligosaccharides containing galabiose or with lactose coupled to BSA. This demonstrated that the inhibition is specifically dependent on galabiose and requires multivalency of the disaccharide to be efficient. The inhibitory effect was successively enhanced by increasing the substitution on BSA (7, 18, and 25 mol of galabiose/mol of BSA). The BSA-coupled galabiose could also prevent the cytotoxic effect on HeLa cells (detachment of killed cells). There are cell lines with a dense number of receptor sites, but which are resistant to toxin action (uptake and inhibition of protein synthesis) which may suggest two types of receptor substances which are functionally different and unevenly expressed. In analogy with the mechanism earlier formulated for cholera toxin, we propose glycolipid-bound, bilayer-close galabiose as the functional receptor for membrane penetration of the toxin, while galabiose bound in glycoproteins affords binding sites but is not able to mediate penetration.
Bacterial Toxins↗
Amino acid sequence of a novel calmodulin from Paramecium tetraurelia that contains dimethyllysine in the first domain.
A class of Paramecium behavioral mutants called pantophobiacs have a deficiency in calcium-dependent potassium efflux, and this deficiency can be corrected by the microinjection of wild-type Paramecium calmodulin (Hinrichsen, R. D., Burgess-Cassler, A., Soltvelt, B. C., Hennessey, T., and Kung, C. (1986) Science 232, 503-506). As a starting point in investigations of which features allow wild-type Paramecium calmodulin to fully restore this behavior while other calmodulins are inactive or poorly effective, we elucidated the amino acid sequence of the wild-type calmodulin. We utilized an approach that combined Edman chemistry with mass spectrometry. This approach resulted in the identification of a new post-translational modification in calmodulin: N epsilon,N epsilon-dimethyllysine at residue 13. This particular modification has not been described for calmodulins studied previously. The only other first-domain modification that has been described for any calmodulin is acetylation of the amino terminus (Watterson, D. M., Sharief, F., and Vanaman, T. C. (1980) J. Biol. Chem. 255, 962-975). These results along with analyses of pantophobiac calmodulin and calmodulin binding proteins will provide insight into calmodulin's role in a well-defined behavioral mutant.
Acetylation↗
The potential antidepressant tiflucarbine down-regulates beta-adrenoceptors in rat brain.
Subchronic treatment of rats with the potential antidepressant tiflucarbine down-regulated the noradrenaline (NA) responses of the cAMP system in cerebral cortex. A concomitant 25% decrease in dihydroalprenolol binding sites in cerebral cortical membranes was observed. The effect was dose-dependent (ED50 = 6 mg/kg), required a 9 days' treatment period, and was reversible 5 days after discontinuation of treatment. Tiflucarbine treatment increased the specific activity of soluble calmodulin (CaM)-dependent phosphodiesterase in rat brain. Tiflucarbine bound to CaM and inhibited its interaction with the phosphodiesterase. Adrenergic denervation by 6-hydroxydopamine (6-OHDA) injection prevented both the beta-adrenoceptor down-regulation and the increase in specific activity of the phosphodiesterase. We suggest that a synergistic interaction between a presynaptic phosphodiesterase inhibition and the NA reuptake blockade was responsible for the down-regulation induced by tiflucarbine. The data are compatible with the reported antidepressant properties of this drug.
Adenylyl Cyclase Inhibitors↗
Rolipram, a stereospecific inhibitor of calmodulin-independent phosphodiesterase, causes beta-adrenoceptor subsensitivity in rat cerebral cortex.
Prolonged pretreatment of rats with the atypical antidepressant rolipram attenuates noradrenaline (NA) sensitivity of the cerebral cortical cAMP generating system. The development of this down-regulation is time (7 d treatment required) and dose dependent (EC50 = 0.35 mg/kg). Density of beta-adrenoceptor as measured by (-)-3H-dihydroalprenolol [(-)-3H-DHA] binding is also reduced by rolipram pretreatment. The effect of rolipram is absolutely stereospecific for the (-)-enantiomer (ED50 = 0.18 mg/kg). In addition, only with this isomer, a reduction in daily weight gain was found compared to sham treated controls. Presynaptic denervation using intracerebroventricular (i.c.v.) injections of 6-hydroxydopamine (6-OHDA) prior to or during rolipram treatment did not completely block the effect of a rolipram treatment on down-regulation of cerebral cortical beta-adrenoceptors. The data favor a pre- and postsynaptic action of rolipram different from all other antidepressants studied so far in this experimental setting. Rolipram is known as inhibitor of brain phosphodiesterase. Using partially purified calmodulin-independent phosphodiesterase from brain it is shown that exclusively the (-)-enantiomer of rolipram inhibits phosphodiesterase with an IC50 of 1.25 mumol/l whereas the (+)-isomer possesses little potency. Since a marked stereospecificity for the (-)-isomer of rolipram was displayed in all pharmacological parameters tested so far with (+)- and (-)-rolipram, it is suggested that stereospecific and isozyme specific inhibition of cAMP-phosphodiesterase is, at least in part, related to the mechanism of action of the potential antidepressant drug rolipram and possibly of other antidepressants as well.
Adenylyl Cyclases↗
Starvation proteins in Escherichia coli: kinetics of synthesis and role in starvation survival.
Starvation proteins synthesized by Escherichia coli at the onset of carbon starvation (R. G. Groat and A. Matin, J. Indust. Microbiol. 1:69-73, 1986) exhibited four temporal classes of synthesis in response to glucose or succinate starvation, indicating sequential expression of carbon starvation response (cst) genes. A cst mutant of E. coli showed greatly impaired carbon starvation survival. Thus, it appears that E. coli undergoes a significant molecular realignment in response to starvation, which increases its resistance to this stress. New polypeptides were also synthesized by E. coli in response to phosphate or nitrogen starvation. Some of these polypeptides were unique to a given starvation regimen, but at least 13 appeared to be synthesized regardless of the nutrient deprivation causing the starvation.
Bacterial Proteins↗
Effects of lithium and antidepressants on electrophysiological and biochemical processes in the CNS.
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Animals↗
Regulation of nitrogen fixation in Rhodospirillum rubrum grown under dark, fermentative conditions.
Rhodospirillum rubrum was shown to grow fermentatively on fructose with N2 as a nitrogen source. The nitrogenase activity of these cells was regulated by the NH4+ switch-off/switch-on mechanism in a manner identical to that for photosynthetically grown cells. In vitro, the inactive nitrogenase Fe protein from fermenting cells was reactivated by an endogenous membrane-bound, Mn2+-dependent activating enzyme that was interchangeable with the activating enzyme isolated from photosynthetic membranes.
Darkness↗
Chronic thyroxine treatment of rats down-regulates the noradrenergic cyclic AMP generating system in cerebral cortex.
Treatment of Sprague-Dawley rats for 9 days with 15 micrograms/kg of tri-iodo-thyronine, 50 micrograms/kg of thyroxine (T4) or 500 micrograms/kg of thyrotropin-releasing hormone decreased the number of beta receptors in cerebral cortex as measured by dihydroalprenolol binding. The dissociation constants of dihydroalprenolol (3.5 nM) were not altered by the treatment. Only tri-iodo-thyronine and T4 administration resulted in a concomitant reduction of norepinephrine (NE) elicited cyclic AMP formation in cerebral cortical slices. This process required at least a 7-day treatment period and was dose-dependent. Application of 1 microgram of T4 per kg for 9 days significantly diminished the NE responsivity of the cyclic AMP synthesizing system. Dose-response curves with NE indicate a reduction of the maximal response after T4 treatment with no change in ED50. An almost additive interaction between the effects of T4 and a low dose (3 mg/kg) of the tricyclic antidepressant imipramine was observed. Striking differences in the response of the adrenoceptor coupled adenylate cyclase to a 9-day T4 treatment were found when different rat strains, i.e., Fischer F-344, Long-Evans, Wistar and Sprague-Dawley were used. The hyperthyroid state of the animals was ascertained by measurements of plasma levels of tri-iodo-thyronine and T4. Down-regulation of NE sensitive adenylate cyclase by T4 treatment required intact synaptic structures because denervation by i.c.v. injection of 6-hydroxydopamine abolished the effect of T4 treatment. This is indicative of a postsynaptic localization of the down-regulated cyclic AMP generating system. The data stress the importance of the neuroendocrine system for adrenoceptor regulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Ionic regulation of cyclic AMP levels in Paramecium tetraurelia in vivo.
cAMP levels in Paramecium increased dose dependently after a step increase of [Ca] or [Sr] in the incubation, provided K was present. Two mM Ca or Sr tripled cAMP concentrations within 3 s and induced an increase in forward swimming speed. The increase in cAMP formation was strictly dependent on the Donnan ratio [K]: square root [Ca]. Na, Li, or tetraethylammonium could not replace K. The data provide evidence for regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the K: Ca ratio.
Animals↗
Effect of temperature on membrane fluidity and calcium conductance of the excitable ciliary membrane from Paramecium.
Fluorescence anisotropy and average fluorescence lifetime of diphenylhexatriene were measured in artificial lipid membrane vesicles. Within the temperature range investigated (15-52 degrees C) both parameters correlate and can be used interchangeably to measure membrane fluidity. Fluorescence anisotropy of DPH in membrane vesicles of cilia from the protozoan Paramecium tetraurelia decreased slightly from 5 to 37 degrees C, yet, no phase transition was observed. An estimated flow activation energy of approx. 2 kcal/mol indicated that the ciliary membrane is very rigid and not readily susceptible to environmental stimuli. The ciliary membrane contains two domains of different membrane fluidity as indicated by two distinct fluorescence lifetimes of diphenylhexatriene of 7.9 and 12.4 ns, respectively. Ca2+ flux into ciliary membrane vesicles of Paramecium as measured with the Ca2+ indicator dye arsenazo III showed a nonlinear temperature dependency from 5 to 35 degrees C with a minimum around 15 degrees C and increasing flux rates at higher and lower temperatures. The fraction of vesicles permeable for Ca2+ remained unaffected by temperature. The differences in temperature dependency of Ca2+ conductance and membrane fluidity indicate that the Ca2+ permeability of the ciliary membrane is a membrane property which is not directly affected by the fluidity of its lipid environment.
Animals↗