Microcystin-LR (MCLR) induces a compensation of PP2A activity mediated by α4 protein in HEK293 cells

Protein phosphatase 2A (PP2A) is a major protein phosphatase with important cell functions. Known and utilized as a potent inhibitor of PP2A, microcystin-LR (MCLR) targets PP2A as a core element that affects numerous cellular mechanisms. But apart from direct inhibition, the exact effect of MCLR on PP2A in cell is largely unknown, specifically with regard to cellular response and autoregulation. Here, we show that a low concentration of MCLR stimulates, rather than inhibits, PP2A activity in HEK293 cells. Immunoprecipitation and immunofluorescence assays reveal that the catalytic subunit and a regulatory subunit of PP2A, termed α4, dissociate from inactive complex upon MCLR exposure, suggesting that the released catalytic subunit regains activity and thereby compensates the activity loss. At high concentrations of MCLR, PP2A activity decreases along with dissociation of the core enzyme and altered post-translational modification of its catalytic subunit. In addition, the dissociation of α4 and PP2A may contribute to destabilization of HEK293 cells cytoskeleton architecture, detachment to extracellular matrix and further anoikis. Our data provide a novel PP2A upregulation mechanism and challenge the recognition of MCLR only as a PP2A inhibitor in cells.     

Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors

GABA_A receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α₅ has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α₅ subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α₅ subunit-containing GABA_A receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α_4/6 subunit-containing GABA_A receptors, but is sensitive to L-655708, an antagonist of the GABA_A receptors containing α₅ subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α₅ subunit in the turtle spinal cord. Together, these results suggest that GABA_A receptors containing the α₅ subunit mediate the tonic inhibitory currents observed in VHIs.     

A gene (Bmn) controllingβ-mannosidase activity in the mouse is located in the distal part of chromosome 3

A gene (Bmn) with a major effect on -mannosidase activity in kidney and liver of the house mouse was revealed by assay with the synthetic substratep-nitrophenyl--d-mannoside. Activity is low in DBA/2J and CSB mice and high in C57BL/6J mice. By the use of the BXD series of recombinant inbred strains and by crosses between C57BL and CSB, it was possible to map the gene to the distal part of chromosome 3 by demonstration of linkage to a gene for cadmium resistance,cdm, as well as to theAdh-3 locus.This work was supported by Swedish Natural Science Research Council Project B-BU 2992-108.     

Mechanisms of α1-adrenoceptor mediated QT prolongation in the diabetic rat heart

AimsDiabetes mellitus is associated with changes of α₁-adrenoceptor (α₁-AR) on heart electrical function and expression. In this study, we investigated the ionic basis underlying abnormal α₁-AR mediated QT prolongation in the diabetic rat hearts.Main methodsElectrophysiological and biochemical techniques were used in Streptozotocin (STZ)-induced diabetic and control rat hearts.Key findingsIn both control and diabetic rats, the α₁-AR agonist, phenylephrine (PE, 10–100 µM) prolonged the rate-corrected QT intervals (QTc) and action potential durations at 30% (APD₃₀) and 90% (APD₉₀) repolarization levels with the increased QTc and APD₉₀ significantly greater in diabetic rats. PE significantly decreased the transient outward K⁺ current (I_to) and the steady-state K⁺ current (I_ss) in both control and diabetic rats but had no effects on the delayed rectifier K⁺ current (I_k). However, PE induced a greater reduction mainly in the I_ss, but not I_to, in diabetic rats. Furthermore, using RT–PCR and Western blot analyses, we found that α_1A-ARs were over-expressed in the left ventricular tissues of the diabetic rat hearts at both the mRNA and the protein levels.SignificanceThese data suggested that in diabetic hearts, a greater sensitivity of the α_1A-AR mediated the larger suppression of I_ss and resulted in a more prolonged APD₉₀ and QTc. Thus, higher α_1A-AR expression levels in diabetic heart may underlie this type of diabetic cardiomyopathy and suggests that α_1A-AR may serve as a therapeutic target.     

The antitumour activity of the interferon inducer bropirimine is partially mediated by endogenous tumour necrosis factor α

Summary Pyrimidinones, like 2-amino-5-bromo-6-phenyl-4-pyrimidinone (bropirimine), are potent immunomodulators. Natural killer cell activity and macrophage cytotoxicity are increased after bropirimine treatment, an effect exerted through induction of cytokines. Up to now, the interferons have been supposed to be the main mediators but we have found that tumour necrosis factor (TNF) can also be an important mediator of the bropirimine antitumour effects. Increased serum levels of TNF were seen in rats after intraperitoneal administration of 200 mg/kg bropirimine on 2 consecutive days. We also found that the tumour-growth-inhibiting effect of the drug on a colon carcinoma in rats could be reduced about 40% by giving the rats rabbit anti-TNF serum just prior to drug treatment. These results indicate that bropirimine can induce the release of TNF in vivo and that this endogenous TNF may be important as far as the antitumour effect of the drug is concerned.     

The slow-onset nature of allosteric inhibition in α-isopropylmalate synthase from Mycobacterium tuberculosis is mediated by a flexible loop

The identification of structure-function relationships in allosteric enzymes is essential to describing a molecular mechanism for allosteric processes. The enzyme α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) is subject to slow-onset, allosteric inhibition by l-leucine. Here we report that alternate amino acids act as rapid equilibrium noncompetitive inhibitors of MtIPMS failing to display biphasic inhibition kinetics. Amino acid substitutions on a flexible loop covering the regulatory binding pocket generate enzyme variants that have significant affinity for l-leucine but lack biphasic inhibition kinetics. Taken together, these results are consistent with the flexible loop mediating the slow-onset step of allosteric inhibition.     

Site specific inhibition by α-benzyl-α-bromomalodinitrile (BBMD) of electron transport in spinach chloroplasts

The addition of α-benzyl-α-bromomalodinitrile to different controlled states (non-phosphorylating [2]. phosphorylating [3], ATP-inhibited [4] and uncoupled) of photosynthetic electron transport to ferricyanide or benzoquinone demonstrate a significant inhibition in isolated spinach chloroplasts. α-Benzyl-α-bromomalodinitrile pretreatement of isolated chloroplasts or addition of α-benzyl-α-bromomalodinitrile at the onset of illumination completely abolished the O₂ evolving reaction. The level of the steady state fluorescence in intact chloroplasts showed a α-benzyl-α-bromomalodinitrile concentration-dependent increase. The gradual decrease in the reoxidation capacity of the reduced quencher, Q with increasing α-benzyl-α-bromomalodinitrile concentrations provides evidence for an additional inhibitory site for α-benzyl-α-bromomalodinitrile between the two photosystems.     

Ammonium-induced increase in NADH-glutamate dehydrogenase activity is caused by de-novo synthesis of the α-subunit

When callus derived from shoot segments of Vitis vinifera L. was transferred to ammonium-containing medium the aminating activity of NAD(H)-glutamate dehydrogenase (GDH, EC 1.4.1.2) increased significantly. This increase in enzyme activity closely paralleled an increase in the protein of the GDH -subunit (43.0 kDa), as detected by sodium dodecyl sulfate (SDS) gel electrophoresis and Western-blotting. A similar correlation was observed between the deaminating activity and the -subunit (42.5 kDa) which both decreased during this treatment. Using [³⁵S]methionine and immunochemical detection it was shown that the rate of synthesis of the -subunit increased considerably in the ammonium-containing medium while there was no detectable synthesis of the -subunit. At the isoenzyme level, ammonium caused an increase in the de-novo synthesis and hence the activity staining of the more anodic isoenzymes, which are hexameric and consist mainly of -subunits. The results indicate that the increase in NADH-GDH specific activity was due to de-novo synthesis of the -subunit of GDH and the assembly of only the more anodic isoenzymes.Abbreviations GDH glutamate dehydrogenase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Modulation of the activity of cytosolic phospholipase A2�� (cPLA2��) by cellular sphingolipids and inhibition of cPLA2�� by sphingomyelin

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2α (cPLA2α) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2α inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2α. Phosphatidylcholine vesicles containing SM reduced cPLA2α activity. Furthermore, SM disturbed the binding of cPLA2α to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2α-dependent release of AA by inhibiting the binding of cPLA2α to glycerophospholipids.     

Effective inhibition of melanoma by BI-69A11 is mediated by dual targeting of the AKT and NF-κB pathways

In melanoma, the activation of pro-survival signaling pathways, such as the AKT and NF-κB pathways, is critical for tumor growth. We have recently reported that the AKT inhibitor BI-69A11 causes efficient inhibition of melanoma growth. Here, we show that in addition to its AKT inhibitory activity, BI-69A11 also targets the NF-κB pathway. In melanoma cell lines, BI-69A11 inhibited TNF-α-stimulated IKKα/β and IκB phosphorylation as well as NF-κB reporter gene expression. Furthermore, the effective inhibition of melanoma growth by BI-69A11 was attenuated upon NF-κB activation. Mechanistically, reduced NF-κB signaling by BI-69-A11 is mediated by the inhibition of sphingosine kinase 1, identified in a screen of 315 kinases. Significantly, we demonstrate that BI-69A11 is well tolerated and orally active against UACC 903 and SW1 melanoma xenografts. Our results demonstrate that BI-69A11 inhibits both the AKT and the NF-κB pathways and that the dual targeting of these pathways may be efficacious as a therapeutic strategy in melanoma.     

Inhibition of activity of the ethylene-forming enzyme by α(p-chlorophenoxy)isobutyric acid

(p-Chlorophenoxy)isobutyric acid (PCIB) inhibited indole-3-acetic acid (IAA)-induced ethylene production in etiolated mung bean hypocotyl sections. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC) was not significantly affected by PCIB, indicating that PCIB exerted its effect primarily by inhibiting the activity of the ethylene-forming enzyme (EFE). This conclusion was supported by the observations that PCIB inhibited the conversion of exogenously applied ACC to ethylene. The inhibitory effect of PCIB was already evident with 0.05 mM PCIB, and it increased with time after application of the inhibitor. PCIB also significantly inhibited ethylene production in apple fruit tissues, but it only slightly reduced the level of endogenous ACC. Similar to mung bean, EFE activity in apple tissue was significantly inhibited by PCIB. The possibility that PCIB also inhibits auxin-induced ACC synthase activity is discussed.     

Serotoninergic agonist and antagonist are modulators of the α2-adrenoceptor activity in rat brain cortex membranes

The influence of activation and inhibition of serotonin receptors by serotonin (5HT) and miancerin on binding of specific nonselective α₂-antagonist [³H]RX821002 in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction for α₂-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of the [³H]RX821002 binding to α₂-adrenoceptors were as follows: K _d = 1.57 ± 0.276 nM, B _max = 7.24 ± 1.63 fmol/mg protein, n = 2. In the case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters K _d1 = 0.82 ± 0.06; K _d2 = 2.65 ± 0.22 nM; B _m1 = 1.65 ± 0.23; B _m2 = 4.20 ± 0.11 fmol/mg protein; n = 2. The affinity of high-affinity receptors increased twofold and the affininty of low-affinity receptors decreased by 69% as compared to control values. The concentration of high-affinity receptors decreased 4.4-fold, and of low-affinity, 1.7-fold. The value of maximal reaction (B _max) decreased by 20%. In the case of miancerin-induced inhibition of 5HT-receptors the character of ligand binding also changed; two pools of receptors were detected with the following parameters: K _d1 = 0.48 ± 0.09; K _d2 = 3.79 ± 0.71 nM; B ₁ = 0.63 ± 0.17; B ₂ = 4.75 ± 0.21 fmol/mg protein; n = 2. The affinity of high-affinity receptors pool increased by 70% and that of low-affinity receptors decreased by 76% as compared to control values. The concentration of active high-affinity and low-affinity α₂-adrenoceptors decreased by 70% and 141%, respectively. The total amount of the receptors (B _max) decreased by 26%. The data suggest that α₂-adrenoceptors in rat cerebral cortex exist as dimers. Modulatory effects of serotonin and miancerin on specific antagonist binding to α₂-adrenoceptors may be accomplished by altering the character and binding parameters of the nonselective α₂-antagonist [³H]RX821002.     

Microlocating lithium in the mouse embryo by use of a (n, α) nuclear reaction

Summary The quantitative imaging of lithium distribution, in histological sections of 15-days old mouse embryos (whose mother had been submitted to Li-treatment), was performed using⁶Li isotope as tracer,⁶Li(n,)³H nuclear reaction for detection, and dielectric track detectors. Despite the particular difficulties of cryosectioning the embryos without disturbing the lithium distribution, the Li regionalization appeared to be very clear-cut. The ectomesodermic tissues were significantly more loaded with lithium than the endodermic ones. This is probably related to the ectomesodermic tissues being also those most sensitive to the teratogenic effect of lithium. The Li-distribution in the embryo brain was almost homogeneous, instead of being heterogeneous as in adult brain. The mean Li-concentration in the embryo brain was not much below the Li concentration in the grey matter of the mother brain, but it was significantly larger than that in the white matter of the mother brain. Our results are discussed in the context of teratogenic effects observed in situ during mammalian development.     

GABA(A) receptors containing the α2 subunit are critical for direction-selective inhibition in the retina

Far from being a simple sensor, the retina actively participates in processing visual signals. One of the best understood aspects of this processing is the detection of motion direction. Direction-selective (DS) retinal circuits include several subtypes of ganglion cells (GCs) and inhibitory interneurons, such as starburst amacrine cells (SACs). Recent studies demonstrated a surprising complexity in the arrangement of synapses in the DS circuit, i.e. between SACs and DS ganglion cells. Thus, to fully understand retinal DS mechanisms, detailed knowledge of all synaptic elements involved, particularly the nature and localization of neurotransmitter receptors, is needed. Since inhibition from SACs onto DSGCs is crucial for generating retinal direction selectivity, we investigate here the nature of the GABA receptors mediating this interaction. We found that in the inner plexiform layer (IPL) of mouse and rabbit retina, GABA(A) receptor subunit α2 (GABA(A)R α2) aggregated in synaptic clusters along two bands overlapping the dendritic plexuses of both ON and OFF SACs. On distal dendrites of individually labeled SACs in rabbit, GABA(A)R α2 was aligned with the majority of varicosities, the cell's output structures, and found postsynaptically on DSGC dendrites, both in the ON and OFF portion of the IPL. In GABA(A)R α2 knock-out (KO) mice, light responses of retinal GCs recorded with two-photon calcium imaging revealed a significant impairment of DS responses compared to their wild-type littermates. We observed a dramatic drop in the proportion of cells exhibiting DS phenotype in both the ON and ON-OFF populations, which strongly supports our anatomical findings that α2-containing GABA(A)Rs are critical for mediating retinal DS inhibition. Our study reveals for the first time, to the best of our knowledge, the precise functional localization of a specific receptor subunit in the retinal DS circuit.     

The α1d-adrenoceptor subtype is involved in the noradrenaline-induced contractions of rat aorta

The pA2 value of several a1-adrenoceptor antagonists on noradrenaline-induced contractions of rat aorta and their affinity for the cloned α1a-, α1b- and α1d-adrenoceptor subtypes were evaluated. Selective or moderately selective α1d-, partially selective α1b-, selective α1a- and non subtype-selective α1-adrenoceptors antagonists were included in the study. The potency of these compounds on rat aorta was well correlated with the affinity observed for the aid-adrenoceptor subtype. A poor correlation was found for the α1b- and α1a-subtypes. These results suggest that, the α1d-subtype plays a determining role in rat aorta contractions induced by noradrenaline.     

Inhibition of adenylate cyclase activity in the goldfish melanophore is mediated by α 2-adrenoceptors and a pertussis toxin-sensitive GTP-binding protein

The signal-transduction system that mediates the melanosome-aggregating response in melanophores of the black-moor goldfish, Carassius auratus, was investigated by examining the inhibition of adenylate cyclase activity mediated by -adrenoceptors in cultured cells. When the melanophores were incubated with 1 mmol·l^-1 3-isobutyl-1-methylxanthine for 5 min, the intracellular level of cyclic adenosine-3,5-monophosphate increased two- to three-fold. Norepinephrine at 100 nmol·l^-1 and naphazoline at 1 mol·l^-1 inhibited the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3,5-monophosphate in the cells in both the presence and the absence of isoproterenol, a -adrenergic agonist. Methoxamine and phenylephrine also reduced the extent of accumulation of cyclic adenosine-3,5-monophosphate, but only when they were present at relatively high concentrations (above 100 mol·l^-1). The range of concentrations at which norepinephrine inhibited the accumulation of cyclic adenosine-3,5-monophosphate was consistent with the range at which it induced the aggregation of melanosomes. Pretreatment of the cells with pertussis toxin (1 g·ml^-1) for 15 h or treatment with 100 nmol·l^-1 yohimbine (an ₂-adrenergic antagonist) inhibited the effects of the -adrenergic agonists on both the aggregation of melanosomes and the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3,5-monophosphate, but prazosin (an ₁adrenergic antagonist) at 100 nmol·l^-1 was not inhibitory. These results indicate that the melanosome-aggregating response of the goldfish melanophore is induced mainly via inhibition of the activity of adenylate cyclase, which occurs as result of stimulation of a pathway that involves ₁adrenergic and a inhibitory GTP-binding protein.Abbreviations A-kinase cAMP-dependent protein kinase - BSS balanced salts solution - CaM calmodulin - cAMP cyclic adenosine-3,5-monophosphate - Clo clonidine - EDTA ethylenediaminetetra-acetic acid - G-protein GTP-binding protein - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - IBMX 3-isobutyl-1-methylxanthine - IP₃ inositol 1,4,5-trisphosphate Mex, methoxamine - MSH melanocyte-stimulating hormone - Nap naphazoline - NE norepinephrine - Oxy oxymetazoline - Phe phenylephrine - PTX pertussis toxin     

CaV2 channel subtype expression in rat sympathetic neurons is selectively regulated by α2δ subunits

Type two voltage gated calcium (Ca_V2) channels are the primary mediators of neurotransmission at neuronal presynapses, but their function at neural soma is also important in regulating excitability.¹ Catterall WA. Voltage-gated calcium channels. Cold Spring Harb Perspect Biol. 2011;3:a003947. doi:10.1101/cshperspect.a003947. PMID:21746798[Crossref], [PubMed], [Web of Science ®] [Google Scholar] Mechanisms that regulate Ca_V2 channel expression at synapses have been studied extensively, which motivated us to perform similar studies in the soma. Rat sympathetic neurons from the superior cervical ganglion (SCG) natively express Ca_V2.2 and Ca_V2.3.² Zhu Y, Ikeda SR. Adenosine modulates voltage-gated Ca2+ channels in adult rat sympathetic neurons. J Neurophysiol. 1993;70:610-20. PMID:8410161[PubMed], [Web of Science ®] [Google Scholar] We noted previously that heterologous expression of Ca_V2.1 but not Ca_V2.2 results in increased calcium current in SCG neurons.³ Beqollari D, Kammermeier PJ. The interaction between mGluR1 and the calcium channel Cav(2).(1) preserves coupling in the presence of long Homer proteins. Neuropharmacology. 2013;66:302-10. doi:10.1016/j.neuropharm.2012.05.038. PMID:22659088[Crossref], [PubMed], [Web of Science ®] [Google Scholar] In the present study, we extended these observations to show that both Ca_V2.1 and Ca_V2.3 expression resulted in increased calcium currents while Ca_V2.2 expression did not. Further, Ca_V2.1 could displace native Ca_V2.2 channels, but Ca_V2.3 expression could not. Heterologous expression of the individual accessory subunits α₂δ-1, α₂δ-2, α₂δ-3, or β4 alone failed to increase current density, suggesting that the calcium current ceiling when Ca_V2.2 was over-expressed was not due to lack of these subunits. Interestingly, introduction of recombinant α2δ subunits produced surprising effects on displacement of native Ca_V2.2 by recombinant channels. Both α₂δ-1 and α₂δ-2 seemed to promote Ca_V2.2 displacement by recombinant channel expression, while α₂δ-3 appeared to protect Ca_V2.2 from displacement. Thus, we observe a selective prioritization of Ca_V channel functional expression in neurons by specific α₂δ subunits. These data highlight a new function for α₂δ subtypes that could shed light on subtype selectivity of Ca_V2 membrane expression.     

Tetramerization of human guanylate-binding protein 1 is mediated by coiled-coil formation of the C-terminal α-helices

The human guanylate-binding protein 1 (hGBP1) is a large GTP-binding protein belonging to the dynamin family, a common feature of which is nucleotide-dependent assembly to homotypic oligomers. Assembly leads to stimulation of GTPase activity, which, in the case of dynamin, is responsible for scission of vesicles from membranes. By yeast two-hybrid and biochemical experiments we addressed intermolecular interactions between all subdomains of hGBP1 and identified the C-terminal subdomain, α12/13, as a new interaction site for self-assembly. α12/13 represents a stable subdomain of hGBP1, as shown by CD spectroscopy. In addition to contacts between GTPase domains leading to dimer formation, the interaction between two α12/13 subdomains, in the course of GTP hydrolysis, results in tetramer formation of the protein. With the help of CD spectroscopy we showed coiled-coil formation of two α12/13 subdomains and concentration-dependent measurements allow estimating a value for the dissociation constant of 7.3 μM. We suggest GTP hydrolysis-driven release of the α12/13 subdomain, making it available for coiled-coil formation. Furthermore, we can demonstrate the biological relevance of hGBP1 tetramer formation in living cells by chemical cross-link experiments.