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1.
Alterations to melanocortinergic, GABAergic and cannabinoid neurotransmission associated with olanzapine-induced weight gain 总被引:1,自引:0,他引:1
Background/Aim
Second generation antipsychotics (SGAs) are used to treat schizophrenia but can cause serious metabolic side-effects, such as obesity and diabetes. This study examined the effects of low to high doses of olanzapine on appetite/metabolic regulatory signals in the hypothalamus and brainstem to elucidate the mechanisms underlying olanzapine-induced obesity.Methodology/Results
Levels of pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and glutamic acid decarboxylase (GAD65, enzyme for GABA synthesis) mRNA expression, and cannabinoid CB1 receptor (CB1R) binding density (using [3H]SR-141716A) were examined in the arcuate nucleus (Arc) and dorsal vagal complex (DVC) of female Sprague Dawley rats following 0.25, 0.5, 1.0 or 2.0 mg/kg olanzapine or vehicle (3×/day, 14-days). Consistent with its weight gain liability, olanzapine significantly decreased anorexigenic POMC and increased orexigenic NPY mRNA expression in a dose-sensitive manner in the Arc. GAD65 mRNA expression increased and CB1R binding density decreased in the Arc and DVC. Alterations to neurotransmission signals in the brain significantly correlated with body weight and adiposity. The minimum dosage threshold required to induce weight gain in the rat was 0.5 mg/kg olanzapine.Conclusions
Olanzapine-induced weight gain is associated with reduced appetite-inhibiting POMC and increased NPY. This study also supports a role for the CB1R and GABA in the mechanisms underlying weight gain side-effects, possibly by altering POMC transmission. Metabolic dysfunction can be modelled in the female rat using low, clinically-comparable olanzapine doses when administered in-line with the half-life of the drug. 相似文献2.
Sejima E Yamauchi A Nishioku T Koga M Nakagama K Dohgu S Futagami K Kataoka Y 《Cellular and molecular neurobiology》2011,31(7):985-989
Olanzapine is known to be advantageous with respect to outcome and drug compliance in patients with schizophrenia. However,
olanzapine has adverse effects, including a higher incidence of weight gain and metabolic disturbances, when compared with
those of other antipsychotic agents. The mechanisms underlying these adverse events remain obscure. Female rats were orally
administered olanzapine (2 mg/kg) or vehicle once a day for 2 weeks to ascertain if hypothalamic AMP-activated protein kinase
(AMPK) mediates olanzapine-induced weight gain and hyperphagia. Body weight and food intake in each rat were evaluated every
day and every two days, respectively. After the termination of drug treatment, we measured the protein levels of AMPK and
phosphorylated AMPK in the hypothalamus using western blot analyses. Olanzapine significantly increased body weight and food
intake. The phosphorylation levels of AMPK were significantly elevated by olanzapine. These results suggest that activation
of hypothalamic AMPK may mediate hyperphagia and weight gain induced by chronic treatment with olanzapine. 相似文献
3.
Background
GABAA receptors (GABAAR) are composed of several subunits that determine sensitivity to drugs, synaptic localisation and function. Recent studies suggest that agonists targeting selective GABAAR subunits may have therapeutic value against the cognitive impairments observed in schizophrenia. In this study, we determined whether GABAAR binding deficits exist in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia and tested if changes in GABAAR binding are related to the changes in subunit mRNAs. The GABA orthosteric and the benzodiazepine allosteric binding sites were assessed autoradiographically using [3H]Muscimol and [3H]Flumazenil, respectively, in a large cohort of individuals with schizophrenia (n = 37) and their matched controls (n = 37). We measured, using qPCR, mRNA of β (β1, β2, β3), γ (γ1, γ2, γ2S for short and γ2L for long isoform, γ3) and δ subunits and used our previous measurements of GABAAR α subunit mRNAs in order to relate mRNAs and binding through correlation and regression analysis.Results
Significant increases in both [3H]Muscimol (p = 0.016) and [3H]Flumazenil (p = 0.012) binding were found in the DLPFC of schizophrenia patients. Expression levels of mRNA subunits measured did not show any significant difference in schizophrenia compared to controls. Regression analysis revealed that in schizophrenia, the [3H]Muscimol binding variance was most related to α4 mRNA levels and the [3H]Flumazenil binding variance was most related to γ2S subunit mRNA levels. [3H]Muscimol and [3H]Flumazenil binding were not affected by the lifetime anti-psychotics dose (chlorpromazine equivalent).Conclusions
We report parallel increases in orthosteric and allosteric GABAAR binding sites in the DLPFC in schizophrenia that may be related to a “shift” in subunit composition towards α4 and γ2S respectively, which may compromise normal GABAergic modulation and function. Our results may have implications for the development of treatment strategies that target specific GABAAR receptor subunits. 相似文献4.
Before a cell enters mitosis, the Golgi apparatus undergoes extensive fragmentation. This is required for the correct partitioning of the Golgi apparatus into daughter cells, and inhibition of this process leads to cell cycle arrest in G2 phase. AMP-activated protein kinase (AMPK) plays critical roles in regulating growth and reprogramming metabolism. Recent studies have suggested that AMPK promotes mitotic progression and Golgi disassembly, and that this seems independent of the cellular energy status. However, the molecular mechanism underlying these events is not well understood. Here, we show that both treatment with compound C and depletion of AMPKα2 (but not AMPKα1) delays the G2/M transition in synchronized HeLa cells, as evidenced by flow cytometry and mitotic index analysis. Furthermore, knockdown of AMPKα2 specifically delays further fragmentation of isolated Golgi stacks. Interestingly, pAMPKαThr172 signals transiently appear in the perinuclear region of late G2/early prophase cells, partially co-localizing with the Golgi matrix protein, GM-130. These Golgi pAMPKαThr172 signals were also specifically abolished by AMPKα2 knockdown, indicating specific spatio-temporal activation of AMPKα2 at Golgi complex during late G2/early prophases. We also found that the specific CaMKKβ inhibitor, STO-609, reduces the pAMPKα Thr172 signals in the perinuclear region of G2 phase cells and delays mitotic Golgi fragmentation. Taken together, these data suggest that AMPKα2 is the major catalytic subunit of AMPKα which regulates Golgi fragmentation and G2/M transition, and that the CaMKKβ activates AMPKα2 during late G2 phase. 相似文献
5.
6.
Aiysha Thompson Jeffrey W. Stephens Stephen C. Bain Venkateswarlu Kanamarlapudi 《PloS one》2016,11(4)
The glucagon-like peptide receptor (GLP-1R), which is a G-protein coupled receptor (GPCR), signals through both Gαs and Gαq coupled pathways and ERK phosphorylation to stimulate insulin secretion. The aim of this study was to determine molecular details of the effect of small molecule agonists, compounds 2 and B, on GLP-1R mediated cAMP production, intracellular Ca2+ accumulation, ERK phosphorylation and its internalisation. In human GLP-1R (hGLP-1R) expressing cells, compounds 2 and B induced cAMP production but caused no intracellular Ca2+ accumulation, ERK phosphorylation or hGLP-1R internalisation. GLP-1 antagonists Ex(9–39) and JANT-4 and the orthosteric binding site mutation (V36A) in hGLP-1R failed to inhibit compounds 2 and B induced cAMP production, confirming that their binding site distinct from the GLP-1 binding site on GLP-1R. However, K334A mutation of hGLP-1R, which affects Gαs coupling, inhibited GLP-1 as well as compounds 2 and B induced cAMP production, indicating that GLP-1, compounds 2 and B binding induce similar conformational changes in the GLP-1R for Gαs coupling. Additionally, compound 2 or B binding to the hGLP-1R had significantly reduced GLP-1 induced intracellular Ca2+ accumulation, ERK phosphorylation and hGLP-1R internalisation. This study illustrates pharmacology of differential activation of GLP-1R by GLP-1 and compounds 2 and B. 相似文献
7.
The TH2 cytokines, IL-4 and IL-13, play critical roles in inducing allergic lung inflammation and drive the alternative activation of macrophages (AAM). Although both cytokines share receptor subunits, IL-4 and IL-13 have differential roles in asthma pathogenesis: IL-4 regulates TH2 cell differentiation, while IL-13 regulates airway hyperreactivity and mucus production. Aside from controlling TH2 differentiation, the unique contribution of IL-4 signaling via the Type I receptor in airway inflammation remains unclear. Therefore, we analyzed responses in mice deficient in gamma c (γc) to elucidate the role of the Type I IL-4 receptor. OVA primed CD4+ OT-II T cells were adoptively transferred into RAG2−/− and γc
−/− mice and allergic lung disease was induced. Both γc
−/− and γcxRAG2−/− mice developed increased pulmonary inflammation and eosinophilia upon OVA challenge, compared to RAG2−/− mice. Characteristic AAM proteins FIZZ1 and YM1 were expressed in lung epithelial cells in both mouse strains, but greater numbers of FIZZ1+ or YM1+ airways were present in γc
−/− mice. Absence of γc in macrophages, however, resulted in reduced YM1 expression. We observed higher TH2 cytokine levels in the BAL and an altered DC phenotype in the γc
−/− recipient mice suggesting the potential for dysregulated T cell and dendritic cell (DC) activation in the γc-deficient environment. These results demonstrate that in absence of the Type I IL-4R, the Type II R can mediate allergic responses in the presence of TH2 effectors. However, the Type I R regulates AAM protein expression in macrophages. 相似文献
8.
β-cells in the pancreatic islet respond to elevated plasma glucose by secreting insulin to maintain glucose homeostasis. In addition to glucose stimulation, insulin secretion is modulated by numerous G-protein coupled receptors (GPCRs). The GPCR ligands Kisspeptin-10 (KP) and glucagon-like peptide-1 (GLP-1) potentiate insulin secretion through Gq and Gs-coupled receptors, respectively. Despite many studies, the signaling mechanisms by which KP and GLP-1 potentiate insulin release are not thoroughly understood. We investigated the downstream signaling pathways of these ligands and their affects on cellular redox potential, intracellular calcium activity ([Ca2+]i), and insulin secretion from β-cells within intact murine islets. In contrast to previous studies performed on single β-cells, neither KP nor GLP-1 affect [Ca2+]i upon stimulation with glucose. KP significantly increases the cellular redox potential, while no effect is observed with GLP-1, suggesting that KP and GLP-1 potentiate insulin secretion through different mechanisms. Co-treatment with KP and the Gβγ-subunit inhibitor gallein inhibits insulin secretion similar to that observed with gallein alone, while co-treatment with gallein and GLP-1 does not differ from GLP-1 alone. In contrast, co-treatment with the Gβγ activator mSIRK and either KP or GLP-1 stimulates insulin release similar to mSIRK alone. Neither gallein nor mSIRK alter [Ca2+]i activity in the presence of KP or GLP-1. These data suggest that KP likely alters insulin secretion through a Gβγ-dependent process that stimulates glucose metabolism without altering Ca2+ activity, while GLP-1 does so, at least partly, through a Gα-dependent pathway that is independent of both metabolism and Ca2+. 相似文献
9.
N. Arias M. T. Macarulla L. Aguirre M. G. Martínez-Casta?o M. P. Portillo 《Genes & nutrition》2014,9(1)
Quercetin exhibits a wide range of biological functions. The first aim of the present work was to analyze the effects of quercetin on fat accumulation in adipose tissue and glycemic control in rats. Any potential involvement of muscle fatty acid oxidation in its effect on glycemic control was also assessed. Animals were fed a high-fat high-sucrose diet either supplemented with quercetin (30 mg/kg body weight/day), or not supplemented, for 6 weeks. One week before killing, a glucose tolerance test was carried out. Muscle triacylglycerol content, serum glucose, insulin, fructosamine and free fatty acids were measured, and homeostatic model assessment for insulin resistance (HOMA-IR) was calculated. The activities of lipogenic enzymes and lipoprotein lipase in adipose tissue, carnitine palmitoyl transferase-1b (CPT-1b) and citrate synthase in skeletal muscle, and the expression of several genes, ACO, CD36, CPT-1b, PPAR-α, PGC-1α, UCP3, TFAM and COX-2 in skeletal muscle were analyzed. Quercetin caused no significant reduction in body weight or adipose tissue sizes. However, fructosamine, basal glucose and insulin, and consequently HOMA-IR, were significantly reduced by quercetin. No changes were observed in the activity of lipogenic enzymes and lipoprotein lipase. Muscle triacylglycerol content was similar in both experimental groups. The expression of ACO, CD36, CPT-1b, PPAR-α, PGC-1α, UCP3, TFAM and COX-2 remained unchanged. It can be concluded that quercetin is more effective as an anti-diabetic than as an anti-obesity biomolecule. The improvement in insulin resistance induced by this flavonoid is not mediated by a delipidating effect in skeletal muscle. 相似文献
10.
Gerasimos Anagnostopoulos Omar Motio Sijing Li Vincent Carbonnier Hui Chen Valentina Sica Sylvre Durand Mlanie Bourgin Fanny Aprahamian Nitharsshini Nirmalathasan Romain Donne Chantal Desdouets Marcelo Simon Sola Konstantina Kotta La Montgut Flavia Lambertucci Didier Surdez Grossetête Sandrine Olivier Delattre Maria Chiara Maiuri Jos Manuel Bravo-San Pedro Isabelle Martins Guido Kroemer 《Cell death & disease》2022,13(4)
11.
Olga A. Sergeeva Olaf Kletke Andrea Kragler Anja Poppek Wiebke Fleischer Stephan R. Schubring Boris G?rg Helmut L. Haas Xin-Ran Zhu Hermann Lübbert Günter Gisselmann Hanns Hatt 《The Journal of biological chemistry》2010,285(31):23985-23993
Nineteen GABAA receptor (GABAAR) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of β1-subunit-containing GABAARs is unknown. Here we report the discovery of a new structural class of GABAAR positive modulators with unique β1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed α1βxγ2L (x-for 1,2,3) GABAAR FDD were 6 times more potent at β1- versus β2- and β3-containing receptors. Serine at position 265 was essential for the high sensitivity of the β1-subunit to FDD and the β1N286W mutation nearly abolished modulation; vice versa the mutation β3N265S shifted FDD sensitivity toward the β1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to β1-negative cerebellar Purkinje neurons. Immunostaining for the β1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by β1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of β1-containing GABAARs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABAARs. 相似文献
12.
Inez Wens Ulrik Dalgas Frank Vandenabeele Lotte Grevendonk Kenneth Verboven Dominique Hansen Bert O. Eijnde 《PloS one》2015,10(9)
Introduction
Low-to-moderate intensity exercise improves muscle contractile properties and endurance capacity in multiple sclerosis (MS). The impact of high intensity exercise remains unknown.Methods
Thirty-four MS patients were randomized into a sedentary control group (SED, n = 11) and 2 exercise groups that performed 12 weeks of a high intensity interval (HITR, n = 12) or high intensity continuous cardiovascular training (HCTR, n = 11), both in combination with resistance training. M.vastus lateralis fiber cross sectional area (CSA) and proportion, knee-flexor/extensor strength, body composition, maximal endurance capacity and self-reported physical activity levels were assessed before and after 12 weeks.Results
Compared to SED, 12 weeks of high intensity exercise increased mean fiber CSA (HITR: +21±7%, HCTR: +23±5%). Furthermore, fiber type I CSA increased in HCTR (+29±6%), whereas type II (+23±7%) and IIa (+23±6%,) CSA increased in HITR. Muscle strength improved in HITR and HCTR (between +13±7% and +45±20%) and body fat percentage tended to decrease (HITR: -3.9±2.0% and HCTR: -2.5±1.2%). Furthermore, endurance capacity (Wmax +21±4%, time to exhaustion +24±5%, VO2max +17±5%) and lean tissue mass (+1.4±0.5%) only increased in HITR. Finally self-reported physical activity levels increased 73±19% and 86±27% in HCTR and HITR, respectively.Conclusion
High intensity cardiovascular exercise combined with resistance training was safe, well tolerated and improved muscle contractile characteristics and endurance capacity in MS.Trial Registration
ClinicalTrials.gov NCT01845896 相似文献13.
Mansi R. Khanna Floyd J. Mattie Kristen C. Browder Megan D. Radyk Stephanie E. Crilly Katelyn J. Bakerink Sandra L. Harper David W. Speicher Graham H. Thomas 《The Journal of biological chemistry》2015,290(2):706-715
The dominant paradigm for spectrin function is that (αβ)2-spectrin tetramers or higher order oligomers form membrane-associated two-dimensional networks in association with F-actin to reinforce the plasma membrane. Tetramerization is an essential event in such structures. We characterize the tetramerization interaction between α-spectrin and β-spectrins in Drosophila. Wild-type α-spectrin binds to both β- and βH-chains with high affinity, resembling other non-erythroid spectrins. However, α-specR22S, a tetramerization site mutant homologous to the pathological α-specR28S allele in humans, eliminates detectable binding to β-spectrin and reduces binding to βH-spectrin ∼1000-fold. Even though spectrins are essential proteins, α-spectrinR22S rescues α-spectrin mutants to adulthood with only minor phenotypes indicating that tetramerization, and thus conventional network formation, is not the essential function of non-erythroid spectrin. Our data provide the first rigorous test for the general requirement for tetramer-based non-erythroid spectrin networks throughout an organism and find that they have very limited roles, in direct contrast to the current paradigm. 相似文献
14.
Dane D. Jensen Michelle L. Halls Jane E. Murphy Meritxell Canals Fiore Cattaruzza Daniel P. Poole TinaMarie Lieu Hon-Wai Koon Charalabos Pothoulakis Nigel W. Bunnett 《The Journal of biological chemistry》2014,289(29):20283-20294
Although the intracellular trafficking of G protein-coupled receptors controls specific signaling events, it is unclear how the spatiotemporal control of signaling contributes to complex pathophysiological processes such as inflammation. By using bioluminescence resonance energy transfer and superresolution microscopy, we found that substance P (SP) induces the association of the neurokinin 1 receptor (NK1R) with two classes of proteins that regulate SP signaling from plasma and endosomal membranes: the scaffolding proteins β-arrestin (βARRs) 1 and 2 and the transmembrane metallopeptidases ECE-1c and ECE-1d. In HEK293 cells and non-transformed human colonocytes, we observed that G protein-coupled receptor kinase 2 and βARR1/2 terminate plasma membrane Ca2+ signaling and initiate receptor trafficking to endosomes that is necessary for sustained activation of ERKs in the nucleus. βARRs deliver the SP-NK1R endosomes, where ECE-1 associates with the complex, degrades SP, and allows the NK1R, freed from βARRs, to recycle. Thus, both ECE-1 and βARRs mediate the resensitization of NK1R Ca2+ signaling at the plasma membrane. Sustained exposure of colonocytes to SP activates NF-κB and stimulates IL-8 secretion. This proinflammatory signaling is unaffected by inhibition of the endosomal ERK pathway but is suppressed by ECE-1 inhibition or βARR2 knockdown. Inhibition of protein phosphatase 2A, which also contributes to sustained NK1R signaling at the plasma membrane, similarly attenuates IL-8 secretion. Thus, the primary function of βARRs and ECE-1 in SP-dependent inflammatory signaling is to promote resensitization, which allows the sustained NK1R signaling from the plasma membrane that drives inflammation. 相似文献
15.
Many atypical antipsychotic drugs cause weight gain, but the mechanism of this weight gain is unclear. To dissect the role of the dopamine D2 receptor (D2R), an important receptor in the pharmacology of antipsychotic drugs, we analyzed the effect of olanzapine, risperidone, and ziprasidone on changes in body weight and food intake in male wild-type (WT) and D2R knockout (D2R−/−) mice. The oral delivery of atypical antipsychotics, olanzapine (5 and 10 mg/kg), risperidone (0.1 and 1.0 mg/kg) and ziprasidone (10 and 20 mg/kg) in both strains mice for 2 weeks suppressed body weight gain, except for olanzapine treatment in D2R−/− mice. Olanzapine treatment suppressed body weight gain and decreased food intake in WT mice, but also reduced fat body mass and locomotor activity, whereas D2R−/− mice did not show these changes. Ziprasidone and risperidone treatment produced similar responses in WT and D2R−/− mice. These data suggest the involvement of D2R in the effect of olanzapine on metabolic regulation. Further studies are required to explore the implications of D2R activity in antipsychotic-mediated metabolic complications. 相似文献
16.
Taurine is a semi-essential sulfonic acid found at high concentrations in plasma and mammalian tissues which regulates osmolarity, ion channel activity and glucose homeostasis. The structural requirements of GABAA-receptors (GABAAR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABAAR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α1/2β1/3 receptors taurine was as efficient as GABA, whereas incorporation of the γ1/2 subunit reduced taurine efficacy to 60–90% of GABA. The mutation γ2F77I, which abolishes zolpidem potentiation, significantly reduced taurine efficacy at recombinant and native receptors compared to the wild type controls. As taurine was a full- or super- agonist at recombinant αxβ1δ-GABAAR, we generated a chimeric γ2 subunit carrying the δ subunit motif around F77 (MTVFLH). At α1/2β1γ2(MTVFLH) receptors taurine became a super-agonist, similar to δ-containing ternary receptors, but remained a partial agonist at β3-containing receptors. In conclusion, using site-directed mutagenesis we found structural determinants of taurine’s partial agonism at γ-containing GABAA receptors. Our study sheds new light on the β1 subunit conferring the widest range of taurine-efficacies modifying GABAAR function under (patho)physiological conditions. 相似文献
17.
Hua Zhang Wei Dou Cheng-Xi Jiang Zhao-Jun Wei Jian Liu Russell L Jones 《Plant signaling & behavior》2010,5(8):1031-1033
We recently reported that H2S could significantly promote the germination of wheat grains subjected to aluminum (Al3+) stress.1 In these experiments seeds were pretreated with the H2S donor NaHS for 12 h prior to Al3+ stress. During this pre-incubation period we observed that H2S increased the activity of grain amylase in the absence of Al3+. Using embryoless half grains of wheat we now show that H2S preferentially affects the activity of endosperm β-amylase and that α-amylase synthesis and activity is unaffected by this treatment.Key words: α-amylase, β-amylase, hydrogen sulfide, reactive sulfur species, seed germination, wheat (triticum)Cereal grains contain many acid hydrolases, some synthesized de novo by the scutellum and aleurone layer and others are found preformed in the starchy endosperm. The amylases are the best known of these types of enzymes. α-Amylases are synthesized and secreted by the scutellum and aleurone layer, and in the case of aleurone isoforms their synthesis is regulated by GAs and ABA.2,3 Whereas GAs stimulate the synthesis of α-amylases and many other secreted hydrolases, ABA inhibits these processes. β-Amylases, on the other hand, are preformed enzymes whose synthesis is not affected by ABA and GAs. Two forms of β-amylase are found in wheat grains, one is a soluble form present in ungerminated grains which can form high-molecular-weight homopolymers or heteropolymers; the other is bound in an inactive form via S-S linkages to proteins at the periphery of starch grains.4,5 The activation of β-amylases is thought to result at least in part from their release from endosperm proteins by the action of GA-induced proteases secreted from the aleurone layer.6To examine in more detail the effect of H2S on wheat grain amylases we used de-embryontated wheat grains where only the aleurone layer and starchy endosperm were the possible sources of amylase activity. Embryoless half grains of wheat (Triticum aestivum L., c.v Yangmai 158) were incubated in water, the H2S donor NaHS, GA, or combinations of these treatments for up to 12 h. After incubation soluble proteins were isolated from grains by homogenizing in buffer and amylase activity was determined colorimetrically using starch-I2KI. Figure 1A shows that during the first 8 h of incubation there was no increase in amylase activity from half grains incubated in H2O or GA but there was a small but significant increase in activity after 10 h and 12 h of incubation in these two treatments. By contrast, incubation in NaHS brought about a three-fold increase in amylase activity by six h and this increased to about five-fold above the initial activity by 12 h of incubation.Open in a separate windowFigure 1Amylolytic activity from wheat half grains measured colorimetrically (A), by diffusion into agar-starch (B) and following native gel electrophoresis (C and D). (A) Half grains were incubated in water (CK), 0.5 mM NaHS, 10 µM GA3 and 0.5 mM NaHS plus 10 µM GA3 for up to 12 h. Amylase activity was measured colorimetrically on grain homogenates by the starch I-KI method. (B) Half grains pre-incubated as (A) then transferred to agar containing 0.2% starch and 2 mM EDT A for a further 12 h. Amylolytic activity was determined by the diameter of the starch-free halo after flooding the agar-starch plate with I-KI solution. Seeds treated with CK, NaHS, GA3 and NaHS plus GA3 were lined from left to right in each plate, respectively. (C and D) Wheat grains incubated for 12 h in H2O, NaHS, GA3 and Hb (0.1 g/L) and combinations of these treatments, were homogenized and aliquots of extracts were electrophoresed by non-denaturing PAGE. After electrophoesis gels were soaked in starch, washed and stained with I-KI. Amylolytic activity is shown by cleared areas in the gel. For the gel in (C), homogentes were incubated with 25 mM EDT A to inactivate α-amylase and for (D), they were heated at 70°C to inactivate β-amylase.We confirmed these results by incubating wheat half gains on 0.2% agar containing 0.2% soluble starch and 2 mM EDTA (Fig. 1B). In this experiment, half grains were first incubated in H2O, GA or NAHS for up to 12 h as for the experiment shown in Figure 1A and half grains were transferred to agar to estimate amylase activity. Because α-amylases are Ca2+-containing metalloenzymes whose activities are dependent on Ca2+ binding we incorporated EDTA into the agar to favor the appearance of β-amylase activity. The data in Figure 1B confirm what we observed when we measured amylase activity colorimetrically, namely that starch degrading activity was high in half grains exposed to NaHS but low in those incubated in H2O or GA. From this experiment we also concluded that the starch degrading activity was likely a result of the activity of β-amylase as α-amylase activity would have been reduced or eliminated by the presence of EDTA.We established that the amylolytic activity produced by wheat grains in response to the NaHS donor was largely β-amylase by selectively inhibiting amylase activities following native gel electrophoresis. α-Amylases are heat stable but are sensitive to metal chelators, whereas β-amylases are denatured by heating but are not inhibited by chelators such as EDTA.4 Figure 1C and D show the activities of amylase isoforms measured by incubating non-denaturing polyacrylamide gels in 1% soluble starch followed by staining in I-KI. Figure 1C shows amylolytic activity after incubation of the gel in starch containing 25 mM EDTA. Two distinct sets of bands are seen together with minor bands of activity. Whereas group I amylases do not change significantly following treatment incubation in the presence of NaHS or GA, group II amylases show high activity in the presence of NaHS, but show no activity with GA (Fig. 1C). When the enzyme preparations were heated to 70°C for 15 min before electrophoresis almost all activity was lost showing that the activity seen in the absence of heating but in the presence of EDTA was likely that of β-amylase. We also used hemoglobin (Hb) a nonspecific H2S scavenger to show that the effects of NaHS were indeed via the production of H2S. Figure 1C shows that Hb almost completely abolished the inductive effect of NaHS on β-amylase activity.Although at present we have no direct evidence that H2S acts as an endogenous regulator of endosperm function in cereal grains, it is tempting to speculate that this is indeed the case. Our previous work showed an increase in the synthesis of H2S by wheat grains that is detected as early as 12 h following the start of imbibition in H2O.1 Although we have no information on the route of H2S synthesis in wheat, there is abundant evidence that plants synthesize H2S as its emission has been observed in many species.7–13 In plants, H2S is thought to be released from cysteine via a reversible O-acetylserine (thiol) lyase (OASTL) reaction, and recently several L- and D-cysteine-specific desulfhydrase candidates have been isolated and partially characterized from Arabidopsis thaliana, confirming H2S release by the action of desulfhydrases in various cellular compartments.14–17 The induction of L-cysteine desulfhydrase upon pathogen attack,18 freezing tolerance by H2S fumigation,19 emission of H2S from plants exposed to SO2 injury,10,20 and abiotic stresses tolerance in plants treated with H2S donor,1,21–24 all infer that H2S is involved in these responses.It is also tempting to speculate that H2S may work in cereal grains by influencing redox status.15,25 It has been proposed that H2S can bring about an increase in synthesis of glutathione from cysteine and an overall improvement of plant performance under stress. The release of β-amylase from starchy endosperm proteins or the dissociation of free β-amylase from small homopolymers or heteropolymers has been shown to be enhanced by reducing agents such as dithiothreitol and by S-H proteinases. We propose that one plausible action of H2S in cereal endosperm is to enhance reactive sulfur species that can lead to reduction of S-S bonds between β-amylase and its binding partners in the endosperm. Release of activated β-amylase would then be free to participate in starch degradation providing sugar units for seedling growth and development prior to the induction of α-amylases by GAs. 相似文献
18.
Olanzapine (Olz) is one of the most effective antipsychotic drugs commonly used for treating schizophrenia. Unfortunately, Olz administration is associated with severe weight gain and metabolic disturbances. Both patients and clinicians are highly interested in the development of new antipsychotics which are as effective as atypical antipsychotics but which have a lower propensity to induce metabolic side effects. In the present study, we examined two new derivatives of Olz; OlzEt (2-ethyl-4-(4′-methylpiperazin-1′-yl)-10Hbenzo[b]thieno[2,3-e][1,4]diazepine), and OlzHomo (2-ethyl-4-(4′-methyl-1′,4′-diazepan-1′-yl)-10H-benzo[b]thieno[2,3-e] [1,4]diazepine), for their tendency to induce weight gain in rats. Weight gain and metabolic changes were measured in female Sprague Dawley rats. Animals were treated orally with Olz, OlzEt, OlzHomo (3 or 6 mg/kg/day), or vehicle (n = 8), three times daily at eight-hour intervals for 5 weeks. Furthermore, a phencyclidine (PCP)-treated rat model was used to examine the prevention of PCP-induced hyperlocomotor activity relevant for schizophrenia therapy. Male Sprague Dawley rats were pre-treated with a single dose (3 mg/kg/day) of Olz, OlzEt, OlzHomo, or vehicle (n = 12), for 2 weeks. Locomotor activity was recorded following a subcutaneous injection with either saline or PCP (10 mg/kg). Olz was found to induce weight gain, hyperphagia, visceral fat accumulation, and metabolic changes associated with reduced histamatergic H1 receptor density in the hypothalamus of treated rats. In contrast, OlzEt and OlzHomo presented promising antipsychotic effects, which did not induce weight gain or fat deposition in the treated animals. Behavioural analysis showed OlzEt to attenuate PCP-induced hyperactivity to a level similar to that of Olz; however, OlzHomo showed a lower propensity to inhibit these stereotyped behaviours. Our data suggest that the therapeutic effectiveness of OlzHomo may be delivered at a higher dose than that of Olz and OlzEt. Overall, OlzEt and OlzHomo may offer a better pharmacological profile than Olz for treating patients with schizophrenia. Clinical trials are needed to test this hypothesis. 相似文献
19.
Kai B. Kaufmann Nafisah Al-Rifai Felix Ulbrich Nils Schallner Hannelore Rücker Monika Enzinger Hermina Petkes Sebastian Pitzl Ulrich Goebel Sabine Amslinger 《PloS one》2015,10(11)
Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1), is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2’,3,4,4’-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH) had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS) production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2’-hydroxychalcone, calythropsin and 2’-hydroxy-3,4,4’-trimethoxychalcone) prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2’,3,4,4''-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C6H4-TMC displayed no significant activity. Also, E-α-p-OMe-C6H4-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C6H4-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C6H4-TMC treatment. Overall, E-α-p-OMe-C6H4-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264.7 macrophages. The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway. 相似文献
20.
Regulation of the synthesis of barley aleurone alpha-amylase by gibberellic Acid and calcium ions
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The effects of gibberellic acid (GA3) and calcium ions on the production of α-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA3 or Ca2+ show qualitative and quantitative changes in hydrolase production following incubation in either GA3 or Ca2+ or both. Incubation in H2O or Ca2+ results in the production of low levels of α-amylase or acid phosphatase. The addition of GA3 to the incubation medium causes a 10- to 20-fold increase in the amounts of these enzymes released from the tissue, and addition of Ca2+ at 10 millimolar causes a further 8- to 9-fold increase in α-amylase release and a 75% increase in phosphatase release. Production of α-amylase isoenzymes is also modified by the levels of GA3 and Ca2+ in the incubation medium. α-Amylase 2 is produced under all conditions of incubation, while α-amylase 1 appears only when layers are incubated in GA3 or GA3 plus Ca2+. The synthesis of α-amylases 3 and 4 requires the presence of both GA3 and Ca2+ in the incubation medium. Laurell rocket immuno-electrophoresis shows that two distinct groups of α-amylase antigens are present in incubation media of aleurone layers incubated with both GA3 and Ca2+, while only one group of antigens is found in media of layers incubated in GA3 alone. Strontium ions can be substituted for Ca2+ in increasing hydrolase production, although higher concentrations of Sr2+ are required for maximal response. We conclude that GA3 is required for the production of α-amylase 1 and that both GA3 and either Ca2+ or Sr2+ are required for the production of isoenzymes 3 and 4 of barley aleurone α-amylase. 相似文献