Characterization of Neurospora crassa mutant stratins deficient in adenylate cyclase activity.

Mycelial extracts from Neurospora crassa strains having any one of five different mutations of the cr-1 allele (“crisp”) exhibited an adenylate cyclase specific activity 2 to 3% of that found in wild-type strains. The enzyme deficiency seemed to be specific for the cr-1 mutation but not for the “crisp” morphology and recessive in heterocaryons carrying mutated and wild-type cr-1 alleles. The reduced adenylate cyclase activity detected in extracts from cr-1 mutants was not due to an impairment in the extraction of membranes, to a preferential inactivation of the enzyme after extraction, or to the presence of inhibitors.     

Refractoriness of ovarian adenylate cyclase to continued hormonal stimulation.

Culture of preovulatory rat follicles with luteinizing hormone, follicle-stimulating hormone or prostaglandin E2 for 24 h reduced the subsequent response of adenylate cyclase to the homologous by 80, 50 and 90%, respectively; yet follicles refractory to luteinizing hormone fully responded to follicle-stimulating hormone responded to luteinizing hormone and prostaglandin E2, and those refractory to prostaglandin E2 could be stimulated by either gonadotropin. Desensitization of the adenylate cyclase system by luteinizing hormone was achieved by hormone concentrations of 0.8--2.0 mug/ml in the medium; a lower dose of luteinizing hormone (0.4 mug/ml), though effective in stimulating adenylate cyclase, did not induce refractoriness. Prostaglandin E2 caused partial refractoriness at dose levels of 0.1--0.25 mug/ml; higher dose levels were more effective. These findings suggest that continued exposure to the preovulatory follicle to elevated levels of hormones may cause perturbations in either the interaction between the hormone and its specific receptor or in a subsequent step essential for activation of adenylate cyclase.     

Agonist-specific refractoriness induced by isoproterenol. Studies with mutant cells.

The beta-adrenergic catecholamine isoproterenol produces a large, rapid, but often a transient, elevation in cellular content of cyclic AMP. We have used the S49 mouse lymphoma cell line, in which genetic variants with specific defects in the pathway of cyclic AMP generation and function have been isolated, to study the increase and subsequent decrease in cyclic AMP levels (termed refractoriness) following incubation of cells with isoproterenol. In wild type S49 cells, isoproterenol produces a peak response in the cellular content of cyclic AMP within 30 min, but the cyclic AMP level falls rapidly thereafter, approaching basal levels by 6 h. Neither inactivation of the drug nor secretion of a nonspecific inhibitor of adenylate cyclase appears to account for the refractoriness. Because isoproterenol refractory cells can still be stimulated by cholera toxin, refractoriness to isoproterenol does not represent a generalized decrease in cellular cyclic AMP response. Particulate preparations from refractory cells have a selective loss of isoproterenol-responsive adenylate cyclase activity, but their activation constants and stereoselectivity for (-)- and (+)-isoproterenol are unaltered. In addition, refractory cells have decreased specific binding of the beta-adrenergic antagonist [125I]iodohydroxybenzylpindolol. This decrease appears to represent a reduction in the number, but not the affinity, of beta-adrenergic receptor sites. Similar studies in an S49 clone that lacks the enzyme cyclic AMP-dependent protein kinase yield essentially identical findings. Because kinase-deficient cells do not induce the cyclic AMP-degrading enzyme phosphodiesterase after the cellular content of cyclic AMP is increased, induced of phosphodiesterase cannot account for refractoriness to isoproterenol. Cyclic AMP-dependent protein kinase does not appear to be required for either the decrease in beta-adrenergic receptors and isoproterenol-responsive adenylate cyclase, nor does it appear to be required for the development of refractoriness to isoproterenol. In contrast, an S49 clone lacking hormone-responsive adenylate cyclase activity but retaining beta-adrenergic receptors does not appear to lose receptors after being incubated with isoproterenol, either alone or together with dibutyryl cyclic AMP. Therefore, in this clone, receptor occupancy alone or in combination with elevated cyclic AMP levels is insufficient to cause refractoriness. Refractoriness thus appears to require intact adenylate cyclase. This suggests that adenylate cyclase may exert regulatory controls on beta-adrenergic receptors in addition to generation of cyclic AMP.     

In vitro studies with "transfer factor". II. Transfer of the cell-migration inhibition correlate of delayed hypersensitivity in humans with nondialyzable components of leucocyte lysates from humans sensitized to histoplasmin and coccidioidin

“Nonsensitive” cells from skin-test-negative individuals were incubated with whole cell lysates, nondialyzable and dialyzable fractions of leucocyte lysates obtained from strongly skin test sensitive histoplasmin, and/or coccidioidin sensitive donors and specific antigen. “Nonsensitive” cells incubated with whole cell lysate or nondialyzable fractions of lysate in the presence of specific antigen released a substance (MIF) which specifically inhibited the migration of guinea pig macrophages. Dialyzable fractions from active cell lysates incubated with “nonsensitive” cells and specific antigen did not liberate MIF from “nonsensitive” cells. Cell lysates, nondialyzable fractions, or dialyzable fractions alone, or in combination with nonspecific antigens in the presence of “nonsensitive” cells failed to inhibit the migration of guinea pig macrophages.     

Refractoriness of ovarian adenylate cyclase to continued hormonal stimulation

Culture of preovulatory rat follicles with luteinizing hormone, folliclestimulating hormone or prostaglandin E₂ for 24 h reduced the subsequent response of adenylate cyclase to the homologous hormone by 80, 50 and 90%, respectively; yet follicles refractory to luteinizing hormone fully responded to follicle-stimulating hormone or prostaglandin E₂, those refractory to follicle-stimulating hormone responded to luteinizing hormone and prostaglandin E₂, and those refractory to prostaglandin E₂ could be stimulated by either gonadotropin. Desensitization of the adenylate cyclase system by luteinizing hormone was achieved by hormone concentrations of 0.8−2.0 μg/ml in the mediem; a lower dose of luteinizing hormone (0.4 μg/ml), though effective in stimulating adenylate cyclase, did not induce refractoriness. Prostaglandin E₂ caused partial refractoriness at dose levels of 0.1–0.25 μg/ml; higher dose levels were more effective. These findings suggest that continued exposure of the preovulatory follicle to elevated levels of hormones may cause perturbations in either the interaction between the hormone and its specific receptor or in a subsequent step essential for activation of adenylate cyclase.     

Functional integrity of desensitized beta-adrenergic receptors

The adenylate cyclase-coupled beta 2-adrenergic receptor of the frog erythrocyte has served as a useful model system for elucidating the mechanisms of catecholamine-induced densensitization. In this system, it has been previously demonstrated that agonist-induced refractoriness is associated with sequestration of the beta-adrenergic receptors in vesicles away from the cell surface and from their effector unit, the adenylate cyclase system (Stadel, J.M., Strulovici, B., Nambi, P., Lavin, T.N., Briggs, M.M., Caron, M.G., and Lefkowitz, R.J. (1983) J. Biol. Chem. 258, 3032-3038). These internalized beta-adrenergic receptors appear to be structurally intact as assessed by photoaffinity labeling, but their functional status has previously been unknown. In the present studies, we sought to assess the functionality of the sequestered vesicular receptors by fusing them to Xenopus laevis erythrocytes. This cell is suitable for such studies, since it has almost no detectable beta-adrenergic receptor or catecholamine-sensitive adenylate cyclase, but contains prostaglandin E1-stimulable adenylate cyclase. Fusion of beta-adrenergic receptor-containing vesicles from desensitized frog erythrocytes with X. laevis erythrocytes results in a 30-fold stimulation of the hybrid adenylate cyclase by the beta-adrenergic agonist isoproterenol. This effect was entirely blocked by the beta-antagonist propranolol. The catecholamine-sensitive adenylate cyclase activity established in the vesicle-Xenopus hybrids showed the characteristic agonist potency series of the donor frog erythrocyte beta 2-adrenergic receptor. Fusion of vesicles from desensitized frog erythrocytes in which the beta-adrenergic receptors had been inactivated with the group specific reagent dicyclohexylcarbodiimide, or of vesicles derived from control frog erythrocytes, which contain low amounts of beta-adrenergic receptor, did not establish catecholamine-sensitive adenylate cyclase activity in the hybrids. These data demonstrate that beta-adrenergic receptors internalized during desensitization retain their functionality when recoupled to an adenylate cyclase system from a different source. The functional uncoupling of these receptors during desensitization is thus more likely due to their sequestration away from the other components of the adenylate cyclase than to any alterations in the receptors themselves.     

Cellular immunity in gnotobiotic primates induced by transfer factor.

Human dialyzable transfer factor (TF) was found capable of inducing in vivo (skin test) and in vitro blastogenesis) cellular immunity in gnotobiotic nonhuman primates. Because the animals were gnotobiotic (germ-free) and had not been skin tested previously, these data support the hypothesis that induction of cell-mediated immunity in recipients of TF does not require a “priming” exposure to specific and/or cross-reacting antigens, and that this induction may be due to an antigen-specific informational effect of TF.In addition, these results support the antigenic specificity of TF, in that recipient primates developed cellular reactivity only against donor “positive” but not against donor “negative” antigens.     

Induction of refractoriness to thyrotropin stimulation in cultured thyroid cells. Dependence on new protein synthesis.

Cultured dog thyroid cells were used to investigate the mechanism by which previous exposure to thyrotropin (TSH) induces refractoriness to further TSH stimulation of cellular adenosine 3'-5'-monophosphate (cAMP). Refractoriness of the cAMP response to TSH could not be overcome by exposure of the cells to supramaximal stimulatory concentrations of TSH. Although an unknown factor present in human and fetal calf serum was found to inhibit the thyroid cell cAMP response to TSH, this factor could not account for refractoriness because refractoriness could be induced in the absence of serum. Induction of thyroid refractoriness did not appear to be related to cellular concentrations of cyclic AMP, because equal refractoriness was produced by TSH alone or TSH plus the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine. In addition, preincubation of thyroid cells in 10(-4) M cAMP did not result in subsequent refractoriness. Recovery from the refractory process required almost 24 h. Short term (15 min) stimulation with TSH did not produce thyroid cell refractoriness, and reversal of the stimulation was obtained by thorough washing of the cells. Long term TSH stimulation (16 h), however, resulted in both supramaximal cAMP response to TSH, and inclusion of TSH together with cycloheximide did not produce refractoriness. Cyclic AMP phosphodiesterase activity in thyroid cell homogenate was unaltered by TSH or dibutyryl cyclic AMP pretreatment of the cells for up to 24 h, or cycloheximide for up to 4 h. In contrast, TSH-stimulated, but not F--stimulated, adenylate cyclase activity was reduced in thyroid cell homogenates after preincubation of the cells in TSH. Refractoriness to TSH stimulation was not associated with an alteration in the binding of 125I-TSH to cultured thyroid cells. These studies suggest that the thyroid cAMP response to TSH is modulated by an inhibitory mechanism dependent upon new protein synthesis. TSH stimulation itself increases the degree of this inhibition through a mechanism not involving cAMP.     

Adenyl Cyclase Activity in Cultivated Human Skin Fibroblasts

CYCLIC 3′,5′-AMP (cyclic AMP) is an important regulator of cellular processes^{1, 2}. In target cells, hormones stimulate adenyl cyclase, an enzyme which catalyses the conversion of ATP to cyclic AMP, which acts as a “second messenger” on either a membrane or an enzyme system and produces a specific physiological response. Hormonal stimulation of adenyl cyclase in several tissues has been documented².     

Activation of rat lung particulate guanylate cyclase due to filipin induced fluidity change

Involvement of cholesterol in the regulation of rat lung particulate guanylate cyclase was studied with filipin. The enzyme was not activated to a great extent by sodium nitroprusside alone; however, in presence of filipin nitroprusside activated the enzyme about 12–16 fold over the basal. Filipin did not affect the soluble enzyme significantly. The changes induced by filipin did not cause solubilization of proteins or enzyme. The Arrhenius plot of filipin-treated particulate enzyme did not have a “break” which was evident with untreated enzyme. The results suggest that the sequestering of cholesterol by filipin can modulate the activity of membrane-associated guanylate cyclase probably by changing the membrane fluidity.     

The mechanism of action of soluble lymphocyte mediators. V. Mechanism of refractoriness to adenylate cyclase stimulators induced by migration inhibitory factor (MIF).

Exposure of purified guinea pig macrophages to lymphocyte culture supernatants, containing migration inhibitory factor (MIF), for a minimum of 1 hr results in reduced cyclic 3′,5′-adenosine monophosphate (cAMP) accumulation in response to adenylate cyclase stimulators, which persists for at least 6 hr. MIF-induced refractoriness is not due to: inactivation of the stimulating agents, destruction of membrane receptors, excessive leakage of cAMP from the cells, or activation of cAMP phosphodiesterase. Refractoriness develops normally in the absence of protein synthesis. It is concluded that MIF-induced refractoriness is an expression of diminished cAMP synthesis. However, MIF does not directly inhibit basal or stimulated adenylate cyclase, nor are prostaglandin synthesis intermediates responsible for the reduced cAMP production. Evidence is presented in support of the proposal that MIF interferes with the signal transfer from membrane receptors to the enzyme by an effect on cytoplasmic microtubules.     

Bursting synchronization dynamics of pancreatic β-cells with electrical and chemical coupling

Based on bifurcation analysis, the synchronization behaviors of two identical pancreatic β-cells connected by electrical and chemical coupling are investigated, respectively. Various firing patterns are produced in coupled cells when a single cell exhibits tonic spiking or square-wave bursting individually, irrespectively of what the cells are connected by electrical or chemical coupling. On the one hand, cells can burst synchronously for both weak electrical and chemical coupling when an isolated cell exhibits tonic spiking itself. In particular, for electrically coupled cells, under the variation of the coupling strength there exist complex transition processes of synchronous firing patterns such as “fold/limit cycle” type of bursting, then anti-phase continuous spiking, followed by the “fold/torus” type of bursting, and finally in-phase tonic spiking. On the other hand, it is shown that when the individual cell exhibits square-wave bursting, suitable coupling strength can make the electrically coupled system generate “fold/Hopf” bursting via “fold/fold” hysteresis loop; whereas, the chemically coupled cells generate “fold/subHopf” bursting. Especially, chemically coupled bursters can exhibit inverse period-adding bursting sequence. Fast–slow dynamics analysis is applied to explore the generation mechanism of these bursting oscillations. The above analysis of bursting types and the transition may provide us with better insight into understanding the role of coupling in the dynamic behaviors of pancreatic β-cells.     

Modulation of adenylate cyclase/cyclic AMP response by thyrotropin and prostaglandin E2 in cultured thyroid cells. 2. Positive regulation.

Two different independent processes are operating in cultured thyroid cells to regulate adenylate cyclase/cyclic AMP responsiveness to thyroid stimulators (thyrotropin and prostaglandin E2): firstly, refractoriness or negative regulation [preceding paper], which is specific for each thyroid stimulator, is not mediated by cyclic AMP and is not accompanied by alteration of adenylate cyclase activity; secondly, positive regulation which is characterized by an augmentation of the cyclic AMP response stimulated by thyrotropin and prostaglandin E2. This process is not specific for each thyroid stimulator and is a state of increased susceptibility of cyclic AMP synthesis to stimulation, accompanied by increased activity of the catalytic subunit of adenylate cyclase. Positive regulation is apparently mediated by increased intracellular cyclic AMP levels. It is a time-dependent and dose-dependent process. Very low concentrations (5-50 micronU/ml) of thyrotropin augmented cyclic AMP synthesis stimulated by thyrotropin and prostaglandin E2 whereas higher concentrations (above 0.1 mU/ml) augmented prostaglandin E2 stimulation but induced refractoriness to thyrotropin. Prostaglandin E2 (0.1 to 10 micronM) augmented thyrotropin stimulation and dibutyryl adenosine 3':5'-monophosphate (0.3 to 2 mM) augmented thyrotropin and prostaglandin E2 stimulation. Positive regulation is a slow process which develops within days and increases up to day 5 in culture. Experiments using inhibitors suggested that protein synthesis is required for the full expression of the increase in adenylate cyclase activity induced by the studied thyroid stimulators.     

Specific MIF release by rat lymphocytes following incubation with syngeneic anti-tumor “Immune” RNA

Other investigators have previously shown that normal nonimmune lymphoid cells, after incubation with “Immune” RNA, will release MIF when these cells are incubated with the specific antigen used to immunize the RNA donor. This conversion can be detected with the macrophage migration inhibition assay. These observations have been confirmed in a system involving the transfer of immune response to tumor associated antigens with syngeneic “Immune” RNA. Syngeneic “Immune” RNA was extracted from the spleens of Fischer 344/N rats bearing growing transplants of one or another of two syngeneic chemically induced sarcomas. Normal, nonimmune Fischer 344/N spleen cells were incubated with these RNA preparations. When these RNA-incubated spleen cells were exposed to solubilized antigens from that particular tumor used to immunize the RNA donor, MIF was released. RNAse treatment of the “Immune” RNA abrogated the response, while DNAse or pronase treatment did not.     

Rapid,gregarious settlement of the larvae of the marine echiuran Urechis caupo Fisher & MacGinitie 1928

Larvae of Urechis caupo Fisher & MacGinitie, reared in the laboratory, were exposed to potential settlement stimuli, including natural sediment from adult burrows, and “scent” obtained from the skin of adult animals. Competent larvae settled rapidly and specifically in response to adult burrow sediment when compared with their responses to other natural and abiotic sediments. Larvae also responded specifically to chemical “scent” from adult animals when the “scent” of another echiuran worm, Listriolobus pelodes Fisher served as a control. Larval responses to chemical “scent” were as great as their responses to natural burrow sediment. Hence, it is likely that larvae settle gregariously in nature in response to “scent” on sediment grains of adult burrows. The chemical “scent” had a molecular weight between ≈3500 and 14000 daltons, as determined by dialysis. It quickly lost its effectiveness in promoting settlement after it was heated to 80 °C, but was relatively stable at ambient ocean temperatures, retaining its effectiveness for several days. It was soluble in sea water. However, larvae did not respond to the chemical “scent”, unless it was adsorbed onto a surface. Purely tactile stimuli, such as the shape, texture, and size-distribution of particles, were not important settlement cues during these experiments.     

Minimal Hodgkin–Huxley type models for different classes of cortical and thalamic neurons

We review here the development of Hodgkin–Huxley (HH) type models of cerebral cortex and thalamic neurons for network simulations. The intrinsic electrophysiological properties of cortical neurons were analyzed from several preparations, and we selected the four most prominent electrophysiological classes of neurons. These four classes are “fast spiking” “regular spiking” “intrinsically bursting” and “low-threshold spike” cells. For each class, we fit “minimal” HH type models to experimental data. The models contain the minimal set of voltage-dependent currents to account for the data. To obtain models as generic as possible, we used data from different preparations in vivo and in vitro, such as rat somatosensory cortex and thalamus, guinea-pig visual and frontal cortex, ferret visual cortex, cat visual cortex and cat association cortex. For two cell classes, we used automatic fitting procedures applied to several cells, which revealed substantial cell-to-cell variability within each class. The selection of such cellular models constitutes a necessary step towards building network simulations of the thalamocortical system with realistic cellular dynamical properties.     

ACTH-induced refractoriness in cultured adrenal cell line (Y1).

Treatment of cultured mouse adrenal cells Y₁ with ACTH induced cell refractoriness to further hormonal stimulation. When ACTH was added to the cells every 2 hours the first addition increased the levels of 20αOH-progesterone and cAMP secreted into the medium. Upon the second and third additions of ACTH the levels of 20αOH-progesterone and cAMP secreted were greatly diminished and upon the fourth addition of ACTH were absent. Prolonged incubation (14 hours) with different concentrations of ACTH (5 × 10^?11 M to 10^?6 M) induced a dose-related steroidogenic refractoriness to further ACTH stimulation, 10^?8 M ACTH inducing complete refractoriness. The number of ACTH binding sites of cell particles prepared from desensitized cells was similar to that of the control but ACTH failed to stimulate the adenylate cyclase of desensitized cells, whereas the enzyme responded fully to NaF and Gpp(NH)p. The cAMP phosphodiesterase activity was similar in both desensitized and control cells. In addition the steroidogenic response to dibutyryl cAMP of desensitized cells was abolished. Thus, ACTH-induced adrenal cell desensitization seems to be related to at least two phenomena : a defect in the “coupling” between the hormone-receptor sites and the adenylate cyclase and an alteration of certain steps beyond cAMP formation.     

Modulation of adenylate cyclase/cyclic AMP response by thyrotropin and prostaglandin E2 in cultured thyroid cells. 1. Negative regulation.

Isolated porcine thyroid cells, cultured in the presence of thyrotropin (greater than or equal to 0.25 mU/ml) or prostaglandin E2 (greater than or equal to 0.1 micron), showed decreased adenosine 3':5'-monophosphate (cyclic AMP) response to further thyrotropin or prostaglandin E2 stimulation, respectively. Kinetics of the refractory process to thyrotropin and prostaglandin E2 are different: (a) maximal refractoriness to prostaglandin E2 was attained after 2--6 h exposure to prostaglandin E2 while refractoriness to thyrotropin was maximal only after 12--24 h; (b) the degree of refractoriness to prostaglandin E2 was much greater than that to thyrotropin. Refractoriness to thyrotropin or prostaglandin E2 is characterized: by specificity for each thyroid stimulator; by dependence upon the dose of thyrotropin or prostaglandin E2 in culture, e.g. induction of high degree of refractoriness with 0.5 mU/ml thyrotropin (or 1 micron prostaglandin E2), which elicits only a small cyclic AMP increase; by time requirement for induction; by partial effect; by changes of maximum activation of cyclic AMP response; by reversibility. This refractoriness of the cyclic AMP response was not induced by dibutyryl adenosine 3':5'-monophosphate. It was not attributed to increased cyclic AMP-phosphodiesterase activity, but to alterations in the receptor-adenylate cyclase system. Prevention of refractoriness to thyrotropin or prostaglandin E2 by incubation of cells in the presence of actinomycin D, puromycin and cycloheximide suggests that new RNA and protein syntheses are required for the development of the refractory state.     

Ecological Stability: Reality,Misconceptions, and Implications for Risk Assessment

Over a long time frame, an ecological system may not exhibit constancy due to successional and evolutionary changes in the species composing the system. However, over shorter time frames an ecological system exhibits a certain degree of constancy (i.e., varies within defined bounds). Traditionally, ecologists considered this short-term constancy to reflect a “balance of nature,” which was viewed akin to the simple homeostatic dynamics of physiological systems. This is an appealing perspective because the disruption of the system's “balance” (i.e., its ”health“) can be ascertained by comparing the system's current state after the imposition of a perturbation with the societally desired state (i.e., baseline). Recently, ecologists have started to develop a much more complex, and perhaps more realistic, perspective regarding ecosystem dynamics, which does not depend upon homeostasis with a single baseline state. This new view includes stochastic variation, nonlinear dynamics and alternative states, and poses a challenge for assessing environmental “health” and the risk of creating “unhealthy” ecological systems