On the cyclotron resonance mechanism for magnetic field effects on transmembrane ion conductivity

The cyclotron resonance model, recently proposed to account for physiological response to weak environmental magnetic fields, is shown to violate the laws of classical mechanics. Further, it is argued that the ubiquitous presence of dynamic friction in fluid media precludes significant magnetic effects on membrane ion transport.     

Crystal structure of the leadzyme at 1.8 A resolution: metal ion binding and the implications for catalytic mechanism and allo site ion regulation

The leadzyme is a small ribozyme, derived from in vitro selection, which catalyzes site specific, Pb(2+)-dependent RNA cleavage. Pb(2+) is required for activity; Mg(2+) inhibits activity, while many divalent and trivalent ions enhance it. The leadzyme structure consists of an RNA duplex interrupted by a trinucleotide bulge. Here, crystal structures determined to 1.8 A resolution, both with Mg(2+) as the sole divalent counterion and with Mg(2+) and Sr(2+) (which mimics Pb(2+) with respect to binding but not catalysis), reveal the metal ion interactions with both the ground state and precatalytic conformations of the leadzyme. Mg(H(2)O)(6)(2+) ions bridge complementary strands of the duplex at multiple locations by binding tandem purines of one RNA strand in the major groove. At one site, Mg(H(2)O)(6)(2+) ligates the phosphodiester backbone of the trinucleotide bulge in the ground state conformation, but not in the precatalytic conformation, suggesting (a) Mg(2+) may inhibit leadzyme activity by stabilizing the ground state and (b) metal ions which displace Mg(2+) from this site may activate the leadzyme. Binding of Sr(2+) to the presumed catalytic Pb(2+) site in the precatalytic leadzyme induces local structural changes in a manner that would facilitate alignment of the catalytic ribose 2'-hydroxyl with the scissile bond for cleavage. These data support a model wherein binding of a catalytic ion to a precatalytic conformation of the leadzyme, in conjunction with the flexibility of the trinucleotide bulge, may facilitate structural rearrangements around the scissle phosphodiester bond favoring configurations that allow bond cleavage.     

A channel mechanism for electrogenic ion pumps

A model of active ion transport is analyzed in which an essential part of the pump molecule is an ion channel. Ion translocation in the channel is described as a series of jumps between binding sites which are separated by energy barriers. Pumping action results from a transient energy-dependent modification of the barrier structure of the channel and requires only minor conformational changes of the pump molecule. This model is applied to the lightdriven proton pump of Halobacterium and to redox-coupled proton pumps in the mitochondrial respiratory chain. Similar considerations may be used to describe ATP-dependent ion transport.     

A possible mechanism of ammonium ion regulation of photosynthetic carbon flow in higher plants

Addition of NH₄⁺ to the photosynthesizing leaf cells of Dolichos lab lab L. var. Lignosis Prain and leaf discs of Vigna sinensis L. savi ex Hassk caused a significant increase in the flow of photosynthetic carbon toward amino acids with a concomitant decrease toward sugars without affecting the over-all photosynthetic rate. Similar diversion of photosynthetic carbon away from sugars was also observed in the photosynthesizing isolated chloroplasts of V. sinensis, but the latter differed in that they accumulated organic acids rather than amino acids. In an effort to understand the mechanism of NH₄⁺-mediated regulation, the specific and total activities of NAD(P)-glutamate dehydrogenase, glutamine synthetase, pyruvate kinase, alkaline fructose 1,6-bisphosphatase, and NAD(P)-glyceraldehyde-3-phosphate dehydrogenase of the cells of D. lab lab were checked but none was affected by the added ammonium salts even after prolonged incubation. At certain concentrations, ammonium ions abolished the light activation of NADP-glyceraldehyde-3-phosphate dehydrogenase and alkaline fructose 1,6-bisphosphatase in isolated chloroplasts from dark-adapted Vigna leaves without interfering with the basal dark activity of these enzymes. Based on these observations, a possible mechanism of action of NH₄⁺ in regulating the photosynthetic carbon flow is postulated.     

The regulation of extramitochondrial free calcium ion concentration by rat liver mitochondria

The mechanism whereby rat liver mitochondria regulate the extramitochondrial concentration of free Ca(2+) was investigated. At 30 degrees C and pH7.0, mitochondria can maintain a steady-state pCa(2+) (0) (the negative logarithm of the free extramitochondrial Ca(2+) concentration) of 6.1 (0.8mum). This represents a true steady state, as slight displacements in pCa(2+) (0) away from 6.1 result in net Ca(2+) uptake or efflux in order to restore pCa(2+) (0) to its original value. In the absence of added permeant weak acid, the steady-state pCa(2+) (0) is virtually independent of the Ca(2+) accumulated in the matrix until 60nmol of Ca(2+)/mg of protein has been taken up. The steady-state pCa(2+) (0) is also independent of the membrane potential, as long as the latter parameter is above a critical value. When the membrane potential is below this value, pCa(2+) (0) is variable and appears to be governed by thermodynamic equilibration of Ca(2+) across a Ca(2+) uniport. Permeant weak acids increase, and N-ethylmaleimide decreases, the capacity of mitochondria to buffer pCa(2+) (0) in the region of 6 (1mum-free Ca(2+)) while accumulating Ca(2+). Permeant acids delay the build-up of the transmembrane pH gradient as Ca(2+) is accumulated, and consequently delay the fall in membrane potential to values insufficient to maintain a pCa(2+) (0) of 6. The steady-state pCa(2+) (0) is affected by temperature, incubation pH and Mg(2+). The activity of the Ca(2+) uniport, rather than that of the respiratory chain, is rate-limiting when pCa(2+) (0) is greater than 5.3 (free Ca(2+) less than 5mum). When the Ca(2+) electrochemical gradient is in excess, the activity of the uniport decreases by 2-fold for every 0.12 increase in pCa(2+) (0) (fall in free Ca(2+)). At pCa(2+) (0) 6.1, the activity of the Ca(2+) uniport is kinetically limited to 5nmol of Ca(2+)/min per mg of protein, even when the Ca(2+) electrochemical gradient is large. A steady-state cycling of Ca(2+) through independent influx and efflux pathways provides a model which is kinetically and thermodynamically consistent with the present observations, and which predicts an extremely precise regulation of pCa(2+) (0) by liver mitochondria in vivo.     

A channel mechanism for electrogenic ion pumps.

A model of active ion transport is analyzed in which an essential part of the pumps molecule is an ion channel. Ion translocation in the channel is described as a series of jumps between binding sites which are separated by energy barriers. Pumping action results from a transient energy-dependent modification of the barrier structure of the channel and requires only minor conformational changes of the pump molecule. This model is applied to the light-driven proton pump of Halobacterium and to redox-coupled proton pumps in the mitochondrial respiratory chain. Similar considerations may be used to describe ATP-dependent ion transport.     

A physical analysis of the ion parametric resonance model

We show, in elementary terms, using for the most part only elementary mathematics, the physical bases for the ion parametric resonance model so as to clarify the assumptions and consequences of the model. The analysis shows why, contrary to earlier conclusions, no combination of weak DC and AC magnetic fields can modify the transition rate to the ground state of excited ions. Although reinterpretations of the biological consequences of the motion of the excited ions circumvent that particular objection to the model, those changes introduce other difficulties. Also, other objections to the mechanism still stand; hence the model cannot account for any purported biological effects of weak extremely low frequency magnetic fields. Bioelectromagnetics 19:181–191, 1998. © 1998 Wiley-Liss, Inc.     

The regulation of the intracellular sodium ion concentration in cultured chick embryo heart cells.

Using digital imaging microscopy with the fluorescent indicator sodium-binding benzofuran isophtalate, we examined the cytosolic Na+ concentration ([Na+]i) in individual chick embryo heart cells. Inhibition of the Na(+)-H+ exchanger using Na(+)-free (Li+ substituted) medium and inhibition of the Na(+)-efflux through the Na(+)-Ca2+ exchanger using Ca(2+)-free medium didn't change the [Na+]i. The opening of voltage-dependent Na+ channels with veratridine (150 micrograms/ml) and inhibition of the Na(+)-K(+)-Cl(-)-cotransporter with bumetanide (10 microM) led to an increase in [Na+]i by 107% and 86%, respectively, suggesting that the Na+ channels and the Na(+)-K(+)-Cl- cotransporter predominantly regulate the [Na+]i in cultured chick embryo heart cells.     

A generic mechanism for adaptive growth rate regulation

How can a microorganism adapt to a variety of environmental conditions despite the existence of a limited number of signal transduction mechanisms? We show that for any growing cells whose gene expression fluctuate stochastically, the adaptive cellular state is inevitably selected by noise, even without a specific signal transduction network for it. In general, changes in protein concentration in a cell are given by its synthesis minus dilution and degradation, both of which are proportional to the rate of cell growth. In an adaptive state with a higher growth speed, both terms are large and balanced. Under the presence of noise in gene expression, the adaptive state is less affected by stochasticity since both the synthesis and dilution terms are large, while for a nonadaptive state both the terms are smaller so that cells are easily kicked out of the original state by noise. Hence, escape time from a cellular state and the cellular growth rate are negatively correlated. This leads to a selection of adaptive states with higher growth rates, and model simulations confirm this selection to take place in general. The results suggest a general form of adaptation that has never been brought to light—a process that requires no specific mechanisms for sensory adaptation. The present scheme may help explain a wide range of cellular adaptive responses including the metabolic flux optimization for maximal cell growth.     

A three-tiered mechanism for regulation of planar cell polarity

Some epithelial cells are polarized along an axis orthogonal to their apical-basal axes. Recent studies in Drosophila lead to the view that three classes of signaling molecules govern the planar cell polarity (PCP) pathway. The first class, or module, functions across whole tissues, providing directional information to individual cells. The second module, apparently shared by all planar polarized tissues, and related to the canonical Wnt signaling pathway, interprets the directional signal to produce subcellular asymmetries. The third modules are tissue specific, acting to translate subcellular asymmetry into the appropriate morphological manifestations in the different cell types.     

A novel mechanism for regulation of vacuolar acidification.

We have recently demonstrated that Cys-254 of the 73-kDa A subunit of the clathrin-coated vesicle (H+)-ATPase is responsible for sensitivity of the enzyme to sulfhydryl reagents (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 5817-5822). In the present study we observe that for the purified enzyme, disulfide bond formation causes inactivation of proton transport which is reversed by dithiothreitol (DTT). DTT also restores activity of the oxidized enzyme following treatment with N-ethylmaleimide (NEM). These results indicate that disulfide bond formation between the NEM-reactive cysteine (Cys-254) and a closely proximal cysteine residue leads to inactivation of the (H+)-ATPase. To test whether sulfhydryl-disulfide bond interchange may play a role in regulating vacuolar acidification in vivo, we have determined what fraction of the (H+)-ATPase is disulfide-bonded in native clathrin-coated vesicles. Vesicles were isolated under conditions that prevent any change in the oxidation state of the sulfhydryl groups. NEM treatment of vesicles causes nearly complete loss of activity while subsequent treatment with DTT restores 50% of the activity of the fully reduced vesicles. By contrast, treatment of fully reduced vesicles with NEM leads to inactivation which is not reversed by DTT. These results indicate that a significant fraction of the clathrin-coated vesicle (H+)-ATPase exists in an inactive, disulfide-bonded state and suggest that sulfhydryl-disulfide bond interconversion may play a role in controlling vacuolar (H+)-ATPase (V-ATPase) activity in vivo.     

A mechanism for rapid neurosteroidal regulation of parenting behaviour

While systemic steroid hormones are known to regulate reproductive behaviour, the actual mechanisms of steroidal regulation remain largely unknown. Steroidogenic enzyme activity can rapidly modulate social behaviour by influencing neurosteroid production. In fish, the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) synthesizes 11-ketotestosterone (KT, a potent teleost androgen) and deactivates cortisol (the primary teleost glucocorticoid), and both of these steroid hormones can regulate behaviour. Here, we investigated the role of neurosteroidogenesis in regulating parenting in a haremic bidirectionally hermaphroditic fish, Lythrypnus dalli, where males provide all requisite parental care. Using an in vitro assay, we found that an 11β-HSD inhibitor, carbenoxolone (CBX), reduced brain and testicular KT synthesis by 90% or more. We modulated neurosteroid levels in parenting males via intracerebroventricular injection of CBX. Within only 20 min, CBX transiently eliminated parenting behaviour, but not other social behaviour, suggesting an enzymatic mechanism for rapid neurosteroidal regulation of parenting. Consistent with our proposed mechanism, elevating KT levels rescued parenting when paired with CBX, while cortisol alone did not affect parenting. Females paired with the experimental males opportunistically consumed unattended eggs, which reduced male reproductive success by 15%, but some females also exhibited parenting behaviour and these females had elevated brain KT. Brain KT levels appear to regulate the expression of parenting behaviour as a result of changes in neural 11β-HSD activity.     

Weighing on autophagy: A novel mechanism for the CNS regulation of energy balance

Comment on: Coupé B, et al. Cell Metab 2012; 15:247–55, Kaushik S, et al. EMBO Rep 2012; 13:258-65 and Quan W, et al. Endocrinology 2012; 153: In pressAutophagy has received considerable attention over the past decade owing to the fact that alteration of this cellular process, which degrades cytoplasmic materials, including organelles and misfolded proteins, contributes to a variety of diseases, such as cancer, muscular disorders and neurodegeneration.¹ Recent studies using conditional, cell-specific gene-targeting methods have also revealed the importance of autophagy in the regulation of energy balance. For example, the deletion of essential autophagy genes, such as the autophagy-related gene (Atg) 7, in the liver, pancreas or adipose tissue produces alterations in body weight, adiposity and glucose homeostasis.^2-6 Appetite, energy expenditure and metabolism are also carefully regulated by the central nervous system (CNS), particularly the pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus. These neurons act as major negative regulators of energy balance by reducing food intake and increasing energy expenditure. However, the role of CNS autophagy in the regulation of energy balance is largely unknown.Three recent studies, including one from our laboratory, have implicated CNS autophagy in the pathogenesis of obesity (Fig. 1).^7-9 These studies used a conditional cre-loxP approach to specifically delete Atg7 from POMC neurons. Pomc-Cre; Atg7^loxploxp mice display higher body weights, hyperphagia and impaired glucose tolerance.^7-9 These mice also exhibit an increased adiposity that is associated with leptin resistance.^7-9 Mice lacking autophagy in POMC neurons develop an increased sensitivity to weight gain when they are fed a high-fat/high-energy diet.^8,9Open in a separate windowFigure 1. Schematic diagram illustrating the metabolic and structural effects of autophagy deletion in hypothalamic POMC neurons. POMC, pro-opiomelanocortin; Atg, autophagy-related gene.Autophagy plays a particularly important role in biological processes that involve massive cellular elimination, such as neural development.¹⁰ Autophagy is constitutively present in the hypothalamus during important periods of development, and the loss of Atg7 in POMC neurons produces marked structural alterations during the first weeks of postnatal life and prior to the development of obesity(Fig. 1).⁷ Pomc-Cre; Atg7^loxPloxP mice exhibit a reduced density of POMC-containing projections to each of their target nuclei, including the paraventricular nucleus of the hypothalamus, as early as postnatal day 14. These abnormalities in POMC neural projections persist throughout adult life and appear to be the result of diminished capacity of POMC neurons to extend axons.⁷ However, all developmental processes are not affected by autophagy deficiency. No changes in POMC cell numbers between Pomc-Cre; Atg7^loxploxp and control mice are observed,^7-9 which suggests that autophagy does not influence neurogenesis or programmed cell death, but that it specifically affects axonal growth. However, autophagy may be involved in hypothalamic neurodegeneration, because aging is associated with a decline in hypothalamic autophagy and the accumulation of p62 (a polyubiquitin-binding protein that is normally degraded by autophagy) specifically in POMC neurons.⁸ In addition, axonal swelling, which is a hallmark of neurodegeneration, is observed in the hypothalamus of mice that lack autophagy in POMC neurons (Coupé and Bouret, unpublished data).Together, these recent studies suggest that autophagy is required for the proper development and function of POMC neurons, and that autophagy deficiency in POMC neurons causes marked metabolic and structural alterations. Autophagy is highly regulated by nutrient availability,¹¹ including during perinatal life, and further studies may provide novel mechanistic insights on the influence of perinatal dietary changes on the susceptibility to metabolic diseases.     

A role for p53 in the frequency and mechanism of mutation

The tumor suppressor protein, p53, is often referred to as the guardian of the genome. When p53 function is impaired, its ability to preserve genomic integrity is compromised. This may result in an increase in mutation on both a molecular and chromosomal level and contribute to the progression to a malignant phenotype. In order to study the effect of p53 function on the acquisition of mutation, in vitro and in vivo models have been developed in which both the frequency and mechanism of mutation can be analyzed. In human lymphoblastoid cells in which p53 function was impaired, both the spontaneous and induced mutant frequency increased at the autosomal thymidine kinase (TK) locus. The mutant frequency increased to a greater extent in cell lines in which p53 harbored a point mutation than in those lines in which a "null" mutation had been introduced by molecular targeting or by viral degradation indicating a possible "gain-of-function" associated with the mutant protein. Further, molecular analysis revealed that the loss of p53 function was associated with a greater tendency towards loss-of-heterozygosity (LOH) within the TK gene that was due to non-homologous recombination than that found in wild-type cells. Most data obtained from the in vivo models uses the LacI reporter gene that does not efficiently detect mutation that results in LOH. However, studies that have examined the effect of p53 status on mutation in the adenine phosphoribosyl transferase (APRT) gene in transgenic mice also suggest that loss of p53 function results in an increase in mutation resulting from non-homologous recombination. The results of these studies provide clear and convincing evidence that p53 plays a role in modulating the mutant frequency and the mechanism of mutation. In addition, the types of mutation that occur within the p53 gene are also of importance in determining the mutant frequency and the pathways leading to mutation.     

Matrix regulation of skeletal cell apoptosis III: mechanism of ion pair-induced apoptosis

Our previous work has demonstrated that while the Ca(2+) and Pi ions acting in concert function as a potent osteoblast apoptogen, the underlying mechanisms by which it activates cell death is not known. We hypothesize that the ion pair causes release of Ca(2+) from intracellular stores ([Ca(2+)]i); the increase in intracellular calcium prompts the mitochondria to uptake more calcium. This accumulation of calcium eventually results in the loss of mitochondrial membrane potential (MMP) and, subsequently, apoptosis. To test this hypothesis, we evaluated apoptosome formation in MC3T3-E1 osteoblast-like cells treated with the ion pair. Western blot analysis indicated migration of cytochrome-c and Smac/DIABLO from mitochondria to the cytoplasm. Inhibition of either the electron transfer chain (with antimycin a and rotenone), or the activation of a MMP transition (with bongkrekic acid) inhibited apoptosis in a dose-dependent manner. Pre-treating osteoblasts with ruthenium red, a Ca(2+) uniporter inhibitor of both mitochondria and the endoplasmic reticulum (ER), also completely abolished Ca(2+.)Pi-induced apoptosis. Moreover, we showed that an increase in [Ca(2+)]i preceded the increase in MMP over the first 45 min of treatment; a mitochondrial membrane permeability transition was evident at 75 min. To determine the role of ER, Ca(2+) stores in the generation of the apoptotic signal by the ion pair, cells were treated with several inhibitors. Apoptosis was inhibited when cells were treated with dantrolene, an inhibitor of ER ryanodine receptors, and 2-aminodiphenylborate, an IP3 Ca(2+) channel inhibitor, but not cyclopiazonic acid, an ER Ca(2)-ATPase inhibitor. Together, these data demonstrate that Ca(2+) Pi-induced osteoblast apoptosis is characterized by the generation of an apoptosome and that Ca(2+) release from ER stores may promote ion pair-dependent cell death.