Antibodies to nicotinic acetylcholine receptors used to probe the structural and functional relationships between brain α-bungarotoxin binding sites and nicotinic receptors

Antibodies against peripheral nicotinic acetylcholine receptors (nAChR) were used to determine the proportion of brain α-bungarotoxin binding sites that are immunologically related to the peripheral nAChR. The α-bungarotoxin binding component partially purified from rat brain was labelled with [¹²⁵I]α-bungarotoxin and reacted with increasing concentrations of rabbit anti(nAChR) antisera. At least 75% of the brain protein could be immunoprecipitated by rabbit anti(rat muscle junctional nAChR) antiserum (M) whereas an antiserum against Torpedo nAChR (J) was without effect and clearly failed to cross-react with the brain component. Both antisera precipitated 100% of [¹²⁵I]α-bungarotoxin-labelled nAChR from Torpedo marmorata. The lower precipitation of the brain protein was not a consequence of [¹²⁵I]α-bungarotoxin dissociating during the precipitation. We conclude that the majority of α-bungarotoxin binding sites in brain are clearly recognised by the crossreacting antiserum.Release of [³H]dopamine from striatal synaptosomes could be elicited by nicotine in a dose-dependent manner and the response was prevented by the ganglionic blocker mecamylamine, although antagonism by α-bungarotoxin was less clearcut. Preincubation of the synaptosomes with antiserum M resulted in a statistically significant decrease in the [³H]dopamine response to nicotine at all agonist concentrations tested. Antiserum J, however, had no consistent effect on the response. Thus the actions of the antisera parallel their ability to recognise the brain α-bungarotoxin binding component. We conclude that the cholinergic regulation of dopamine release is in part mediated through a nAChR that is immunologically related to the nAChR of the neuromuscular junction and to the α-bungarotoxin binding component that can be isolated from rat brain.     

Toward a better understanding of the interaction between TGF-β family members and their ALK receptors

Transforming growth factor-beta (TGF-β) proteins are a family of structurally related extracellular proteins that trigger their signaling functions through interaction with the extracellular domains of their cognate serine/threonine kinase receptors. The specificity of TGF-β/receptor binding is complex and gives rise to multiple functional roles. Additionally, it is not completely understood at the atomic level. Here, we use the most reliable computational methods currently available to study systems involving activin-like kinase (ALK) receptors ALK4 and ALK7 and their multiple TGF-β ligands. We built models for all these proteins and their complexes for which experimental structures are not available. By analyzing the surfaces of interaction in six different TGF-β/ALK complexes we could infer which are the structural distinctive features of the ligand-receptor binding mode. Furthermore, this study allowed us to rationalize why binding of the growth factors GDF3 and Nodal to the ALK4 receptor requires the Cripto co-factor, whilst binding to the ALK7 receptor does not.     

Quantitative correlation between the residual activity of β-hexosaminidase A and arylsulfatase A and the severity of the resulting lysosomal storage disease

Summary A previously suggested model for the correlation between residual activity of a lysosomal enzyme and the turnover rate of its substrate(s) has been extended to a discussion of substrate accumulation rates in individual cells and whole organs. With these considerations, much of the observed variability in age of onset and clinical phenotype, as well as the phenomenon of pseudodeficiency, can be understood as the consequences of small differences in the residual activity of the affected enzyme. In order to experimentally verify the basic assumptions on which this model rests, studies were performed in cell culture. The radiolabeled substrates ganglioside G_M2 and sulfatide were added to cultures of skin fibroblasts with different activities of -hexosaminidase A or arylsulfatase A, respectively, and their uptake and turnover measured. In both series of experiments, the correlation between residual enzyme activity and the turnover rate of the substrate was essentially as predicted: degradation increased steeply with residual activity, to reach the control level at a residual activity of approximately 10–15% of normal. All cells with an activity above this critical threshold had a normal turnover. Comparison of the results of these feeding studies with the clinical status of the donor of each cell line basically confirmed our notions but also revealed the limitations of the cell culture approach.     

A comparison of genetic diversity between the Galápagos Penguin and the Magellanic Penguin

The Galápagos Penguin (Spheniscusmendiculus) is a United States federallylisted endangered species with populations onthe Galápagos Islands of Fernandina andIsabela. Although the waters around theislands are normally productive, lowproductivity during El Niño years resultsin high adult penguin mortality and lowrecruitment in following years. We usedmicrosatellite markers developed for Spheniscus penguins to study the long termgenetic effects of serial bottleneck events inthe Galápagos Penguin, and compared thisvariation to that of its congener, theMagellanic penguin (Spheniscusmagellanicus). The observed heterozygosityfor the Galápagos Penguin was 3%,significantly lower than the 46%heterozygosity of the Magellanic Penguin. Thislow level of heterozygosity is directly relatedto its low effective population size. Whilethis population has survived long term,presumably without high levels of geneticvariation, we feel that the greater frequencyof El Niño events, coupled with increasedhuman impacts such as introduced disease, oildischarge, and competition with fisheries, mayput the species in particular danger ofextinction.     

A molecular modelling study of the interaction between β-cyclodextrin and the organophosphorothioate pesticide parathion

The interaction between parathion and -cyclodextrin was investigated by Molecular Dynamics. Several in vacuo trajectories were calculated for the system imposing a 1:1 stoichiometry. The influence of the solvent and temperature was considered. The results account for the formation of adducts which are stable at room temperature and involve mainly the nitrophenoxy group of the guest molecules which interacts with the hydrophobic cavity of the host by van der Waals forces.     

Interactions between inositol 1,4,5-trisphosphate receptors and ryanodine receptors in smooth muscle: one store or two?

This short review proposes a system of simplified functional models describing possible interactions between Ca(2+)-release channels associated with IP(3)Rs and RyRs in smooth muscle, and considers each of these models in the light of the available experimental evidence. Complete separation of IP(3)R- and RyR-gated stores seems to be unusual. Where both receptors release Ca(2+) from a common pool, simple interactions can occur since changes in the activation of one receptor type affects the availability of Ca(2+) for release through the other. Alterations in [Ca(2+)] within the sarcoplasmic reticulum can also affect the open probability of the release channels, and not just the Ca(2+)-flux through the channels when open, e.g., Ca(2+)-release through tonically active IP(3)Rs appears to limit SR Ca(2+)-content in some myocytes, and this modulates RyR activity, as indicated by changes in Ca(2+)-spark frequency. There is also evidence that intracellular release channels may co-operate, leading to positive feedback during activation. In particular, agonist-dependent activation of IP(3)Rs can promote activation of RyRs, amplifying and shaping the resulting Ca(2+)-signal. While there is little direct evidence as to the mechanism responsible for this interaction, some form of Ca(2+)-induced Ca(2+)-release in response to local increases in [Ca(2+)](c) seems likely.     

A global relationship between the heterotrophic and autotrophic components of soil respiration?

Soil surface CO₂ flux (R_S) is overwhelmingly the product of respiration by roots (autotrophic respiration, R_A) and soil organisms (heterotrophic respiration, R_H). Many studies have attempted to partition R_S into these two components, with highly variable results. This study analyzes published data encompassing 54 forest sites and shows that R_A and R_H are each strongly (R²>0.8) correlated to annual R_S across a wide range of forest ecosystems. Monte Carlo simulation showed that these correlations were significantly stronger than any correlation introduced as an artefact of measurement method. Biome type, measurement method, mean annual temperature, soil drainage, and leaf habit were not significant. For sites with available data, there was a significant (R²=0.56) correlation between total detritus input and R_H, while R_A was unrelated to net primary production. We discuss why R_A and R_H might be related to each other on large scales, as both ultimately depend on forest carbon balance and photosynthate supply. Limited data suggest that these or similar relationships have broad applicability in other ecosystem types. Site‐specific measurements are always more desirable than the application of inferred broad relationships, but belowground measurements are difficult and expensive, while measuring R_S is straightforward and commonly done. Thus the relationships presented here provide a useful method that can help constrain estimates of terrestrial carbon budgets.     

Alcohol effects on gamma-aminobutyric acid type A receptors: are extrasynaptic receptors the answer?

GABA(A) receptors have long been implicated in mediating at least part of the actions of ethanol in mammalian brain. However, until very recently, reports of the actions of EtOH on recombinant receptors have required very high doses of ethanol and animals lacking receptor subunits shown to be important for ethanol actions in vitro did not support the view that these subunits are crucial in ethanol actions. Recombinant alpha4beta3delta and alpha6beta3delta GABA(A) receptors are uniquely sensitive to ethanol, with a dose-response relationship mirroring the well known effects of alcohol consumption on the human brain. Receptors containing the delta subunit are thought to be located extrasynaptically and it will be important to determine if these extrasynaptic GABA(A) receptor subunit combinations mediate low dose alcohol effects in vivo.     

A functional link between Polo-like kinase 1 and the mammalian Target-Of-Rapamycin pathway?

Polo like kinase-1 is a key effector of cell division and its over-expression in several cancers is often linked with negative prognostic. We recently described that Plk1 is over-expressed in acute myeloid leukemia, and that its inhibition selectively reduces the proliferation of leukemic cells. Here, we report that Plk1 inhibition or depletion using pharmacological and siRNA approaches decreased the phosphorylation of two mTOR substrates in AML cells. In HCT116 cells, inducible expression of a constitutively active form of Plk1 leads to activation of mTOR, as shown by increased phosphorylation of its 4E-BP1 and RPS6 down-stream targets. In addition, HCT116 cells over-expressing the active form of Plk1 were characterized by abnormal growth that could be reversed by rapamycin, a specific inhibitor of the TORC1 complex. Altogether these data suggest the existence of a molecular and functional link between the Plk1 mitotic kinase and the mTOR pathway. Given the different established functions of Plk1 and mTOR during the cell cycle, we will discuss the possible meaning of this functional relationship.     

A possible relationship between the fatty acid composition of yeasts and the ‘petite’ mutation

A number of Crabtree-positive and Crabtree-negative yeasts were tested for their ability to yield respiratory-deficient (petite) mutants on treatment with acriflavine. Crabtree-positive species produced petite mutants but did not contain the polyunsaturated linoleic (C 18.2) and linolenic (C 18.3) fatty acids. Crabtree-negative species contained these fatty acids and were resistant to acriflavine. This work was supported in part by grant B/SR/5780 from the Science Research Council. We are grateful to the Brewer's Society for a Research Scholarship to Mr. B. Johnson. We thank Mr. A. Bradley for competent technical assistance.     

Cross-talk between allosteric and orthosteric binding sites of γ-amino butyric acid type A receptors (GABAA-Rs): A computational study revealing the structural basis of selectivity

The γ-amino butyric acid type A receptors (GABA_A-Rs) are GABA-gated chloride ion channels that mediate fast inhibitory neurotransmissions. Due to their essential role in normal brain function, neuromodulatory therapies are targeted at them for restoring GABA-mediated inhibition. The receptor modulation by benzodiazepine (BZD) shows therapeutically useful actions. The mechanisms, by which BZD-site performs selective transduction while modulating GABA_A-Rs, and its correlation with the occurrence of sedation is not fully known. In pursuance, we performed a computational study starting from modeling of α₂-subtype GABA_A-R, docking of α_1/2-selective ligands followed by molecular dynamics simulations of the obtained complexes. The results show that during early stages of activation, a) allosteric binding initiate structural changes through BZD-site for GABA-elicited activation; b) selective BZD-binders positively modulate orthosteric GABA-bound site with fin-like C- and F-loop movements, which supports twisting of inner and outer β-barrel; c) modulation by α_1/2-selective ligands was only evident at site 1, mimicking mandatory doubly bound state; d) strength of allosteric communication was prominent for α₂-modulators, however, the basic nature of allosteric-orthosteric site cross-talk remains same for both α_1/2-modulators; and e) ratio of hydrophobic:hydrophilic ligand contact surface decides α₂-selectivity, less value of ratio favors it. These insights would enable us to design better α₂-selective modulator/s. Altogether our computational study reveals early stages of allosteric modulation, highlighting subtype selective activation and pathways recommending GABA binding sites during selective modulation.

Communicated by Ramaswamy H. Sarma     

Characterization of postsynaptic β-adrenergic receptors and presynaptic muscarinic cholinergic receptors in the rat spleen

The β-adrenergic and muscarinic cholinergic receptors in the splenic homogenates of control and 6-hydroxydopamine (6-OHDA) treated rats were characterized. The specific binding of [³H]dihydroalprenolol (DHA) and [³H]quinuclidinyl benzilate (QNB) in the rat spleen were saturable and of high affinity and showed pharmacological specificity of splenic β-adrenergic and muscarinic cholinergic receptors. Following 6-OHDA treatment, the Bmax value for specific [³H](-)DHA binding to the rat spleen was significantly increased by 26 percent and 22 percent compared to control at 2 and 3 weeks without a change in the Kd. In contrast, there was a 38 percent decrease in the Bmax for [³H](-)QNB in the 6-OHDA treated rat spleen at 2 and 3 weeks respectively without a change in the Kd. The Bmax value at 5 weeks was significantly greater than that at 2 or 3 weeks. The splenic norepinephrine (NE) concentration was markedly reduced by the 6-OHDA treatment at 1 to 3 weeks, while there was a significant recovery in the splenic NE concentration at 5 weeks. Thus, our results strongly suggest that we are biochemically localizing muscarinic cholinergic receptors on the sympathetic nerves of the rat spleen and that the β-adrenergic receptors of the spleen are localized postsynaptically.     

Crosstalk between adenosine A1 and β1-adrenergic receptors regulates translocation of PKCε in isolated rat cardiomyocytes

Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and β(1)-adrenergic agonists on function are complex in the heart.