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1.
Methods Mol Biol ; 2201: 109-116, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975793

RESUMO

The opioid receptors have been an interesting target for the drug industry for decades. These receptors were pharmacologically characterized in the 1970s and several drugs and peptides have emerged over the years. In 2012, the crystal structures were also demonstrated, with new data on the receptor sites, and thus new possibilities will appear. The role of opioids in the brain has attracted considerable interest in several diseases, especially pain and drug dependence. The opioid receptors are G-protein-coupled receptors (GPCR ) that are Gi coupled which make them suitable for studying the receptor functionality. The [35S]GTP γS autoradiography assay is a good option that has the benefit of generating both anatomical and functional data in the area of interest. It is based on the first step of the signaling mechanism of GPCRs. When a ligand binds to the receptor GTP will replace GDP on the a-subunit of the G-protein, leading to a dissociation of the ßγ-subunit. These subunits will start a cascade of second messengers and subsequently a physiological response.


Assuntos
Autorradiografia/métodos , Guanosina 5'-O-(3-Tiotrifosfato)/química , Receptores Opioides mu/metabolismo , Analgésicos Opioides/metabolismo , Animais , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Receptores Opioides/metabolismo , Transdução de Sinais , Radioisótopos de Enxofre/química , Radioisótopos de Enxofre/metabolismo
2.
Biomolecules ; 10(12)2020 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-33297334

RESUMO

The 26S proteasome is the endpoint of the ubiquitin- and ATP-dependent degradation pathway. Over the years, ATP was regarded as completely essential for 26S proteasome function due to its role in ubiquitin-signaling, substrate unfolding and ensuring its structural integrity. We have previously reported that physiological concentrations of NADH are efficient in replacing ATP to maintain the integrity of an enzymatically functional 26S PC. However, the substrate specificity of the NADH-stabilized 26S proteasome complex (26S PC) was never assessed. Here, we show that the binding of NADH to the 26S PC inhibits the ATP-dependent and ubiquitin-independent degradation of the structured ODC enzyme. Moreover, the NADH-stabilized 26S PC is efficient in degrading intrinsically disordered protein (IDP) substrates that might not require ATP-dependent unfolding, such as p27, Tau, c-Fos and more. In some cases, NADH-26S proteasomes were more efficient in processing IDPs than the ATP-26S PC. These results indicate that in vitro, physiological concentrations of NADH can alter the processivity of ATP-dependent 26S PC substrates such as ODC and, more importantly, the NADH-stabilized 26S PCs promote the efficient degradation of many IDPs. Thus, ATP-independent, NADH-dependent 26S proteasome activity exemplifies a new principle of how mitochondria might directly regulate 26S proteasome substrate specificity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Trifosfato de Adenosina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , NAD/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Trifosfato de Adenosina/análogos & derivados , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Intrinsicamente Desordenadas/genética , Camundongos , Células NIH 3T3 , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Biossíntese de Proteínas , Proteólise , Proteínas Proto-Oncogênicas c-fos/genética , Coelhos , Radioisótopos de Enxofre , Fatores de Transcrição/genética , Ubiquitina/genética , Ubiquitina/metabolismo
3.
Methods Mol Biol ; 2116: 99-108, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32221916

RESUMO

The amount of a protein that is made in a cell is determined not only by the corresponding mRNA level but also by the efficiency with which the mRNA is translated. Very powerful transcriptome-wide methods are available to analyze both the density of ribosomes on each mRNA and the rate at which polypeptides are elongated. However, for many research questions, simpler, less expensive methods are more suitable. Here we describe two methods to assess the general translation status of cells: polysome profiling by sucrose density gradient centrifugation and metabolic labeling using radioactive amino acids. Both methods can also be used to examine translation of individual mRNAs.


Assuntos
Marcação por Isótopo/métodos , Polirribossomos/metabolismo , Biossíntese de Proteínas , Trypanosoma brucei brucei/genética , Aminoácidos/química , Aminoácidos/metabolismo , Centrifugação com Gradiente de Concentração/métodos , Metabolômica/métodos , Parasitologia/métodos , Polirribossomos/química , RNA Mensageiro/química , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , RNA de Protozoário/química , RNA de Protozoário/isolamento & purificação , RNA de Protozoário/metabolismo , Radioisótopos de Enxofre/química , Trypanosoma brucei brucei/metabolismo
4.
Neurotox Res ; 37(2): 366-379, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31292883

RESUMO

Attractive due to an alleged high biocompatibility, silica nanoparticles have been widely used in the field of nanomedicine; however, their proven capacity to induce the synthesis and release of pro-inflammatory cytokines in several cellular models has raised concern about their safety. Glutamate, the main excitatory amino acid transmitter triggers a wide variety of signal transduction cascades that regulate protein synthesis at transcriptional and translational levels. A stimulus-dependent dynamic change in the protein repertoire in neurons and glia cells is the molecular framework of higher brain functions. Within the cerebellum, Bergmann glia cells are the most abundant non-neuronal cells and span the entire molecular layer of the cerebellar cortex, wrapping the synapses in this structure. Taking into consideration the functional role of Bergmann glia in terms of the recycling of glutamate, lactate supply to neurons, and prevention of neurotoxic insults, we decided to investigate the possibility that silica nanoparticles affect Bergmann glia and by these means alter the major excitatory neurotransmitter system in the brain. To this end, we exposed cultured chick cerebellar Bergmann glia cells to silica nanoparticles and measured [35S]-methionine incorporation into newly synthesized polypeptides. Our results demonstrate that exposure of the cultured cells to silica nanoparticles exerts a time- and dose-dependent modulation of protein synthesis. Furthermore, altered patterns of eukaryotic initiation factor 2 alpha and eukaryotic elongation factor 2 phosphorylation were present upon nanoparticle exposure. These results demonstrate that glia cells respond to the presence of this nanomaterial modifying their proteome, presumably in an effort to overcome any plausible neurotoxic effect.


Assuntos
Nanopartículas/efeitos adversos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Dióxido de Silício/efeitos adversos , Animais , Embrião de Galinha , Relação Dose-Resposta a Droga , Quinase do Fator 2 de Elongação/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Metionina/metabolismo , Fosforilação , Cultura Primária de Células , Radioisótopos de Enxofre/metabolismo , Fatores de Tempo
5.
ACS Chem Neurosci ; 10(11): 4476-4491, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31618004

RESUMO

G-protein-coupled receptors (GPCRs), also known as 7-transmembrane receptors, are the single largest class of drug targets. Consequently, a large amount of preclinical assays having GPCRs as molecular targets has been released to public sources like the Chemical European Molecular Biology Laboratory (ChEMBL) database. These data are also very complex covering changes in drug chemical structure and assay conditions like c0 = activity parameter (Ki, IC50, etc.), c1 = target protein, c2 = cell line, c3 = assay organism, etc., making difficult the analysis of these databases that are placed in the borders of a Big Data challenge. One of the aims of this work is to develop a computational model able to predict new GPCRs targeting drugs taking into consideration multiple conditions of assay. Another objective is to perform new predictive and experimental studies of selective 5-HTA2 receptor agonist, antagonist, or inverse agonist in human comparing the results with those from the literature. In this work, we combined Perturbation Theory (PT) and Machine Learning (ML) to seek a general PTML model for this data set. We analyzed 343 738 unique compounds with 812 072 end points (assay outcomes), with 185 different experimental parameters, 592 protein targets, 51 cell lines, and/or 55 organisms (species). The best PTML linear model found has three input variables only and predicted 56 202/58 653 positive outcomes (sensitivity = 95.8%) and 470 230/550 401 control cases (specificity = 85.4%) in training series. The model also predicted correctly 18 732/19 549 (95.8%) of positive outcomes and 156 739/183 469 (85.4%) of cases in external validation series. To illustrate its practical use, we used the model to predict the outcomes of six different 5-HT2A receptor drugs, namely, TCB-2, DOI, DOB, altanserin, pimavanserin, and nelotanserin, in a very large number of different pharmacological assays. 5-HT2A receptors are altered in schizophrenia and represent drug target for antipsychotic therapeutic activity. The model correctly predicted 93.83% (76 of 86) experimental results for these compounds reported in ChEMBL. Moreover, [35S]GTPγS binding assays were performed experimentally with the same six drugs with the aim of determining their potency and efficacy in the modulation of G-proteins in human brain tissue. The antagonist ketanserin was included as inactive drug with demonstrated affinity for 5-HT2A/C receptors. Our results demonstrate that some of these drugs, previously described as serotonin 5-HT2A receptor agonists, antagonists, or inverse agonists, are not so specific and show different intrinsic activity to that previously reported. Overall, this work opens a new gate for the prediction of GPCRs targeting compounds.


Assuntos
Big Data , Bases de Dados de Compostos Químicos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Aprendizado de Máquina , Receptores Acoplados a Proteínas G/metabolismo , Radioisótopos de Enxofre/metabolismo , Adulto , Idoso , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Serotoninérgicos/metabolismo , Serotoninérgicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
6.
BMC Cancer ; 19(1): 956, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615471

RESUMO

BACKGROUND: 99mTc-Rituximab is a new specific radiopharmaceutical that binds to the CD20 receptor which is highly expressed on the surface of B cells. We conducted a study in which 99mTc-Rituximab was compared with filtered 99mTc-sulfur colloid (fTcSC) for sentinel lymph node (SLN) detection in patients with breast cancer. METHOD: The study is divided into three parts. 1. Initially, 25 patients were selected for an internal controlled trial to received both 99mTc-Rituximab and fTcSC, the interval time is separated by ≥2 days. 2. Then, 91 patients were selected for a randomized controlled trial (41 and 50 patients in the 99mTc-Rituximab and fTcSC groups, respectively). All patients were administered either agent at the 6- and 12-o' clock positions by subareolar injection technique. SLN mapping was then performed 2 h after injection. 3. Serial dynamic images were further acquired for 2 h in 31 patients (22 and 9 patients from 99mTc-Rituximab and fTcSC cohorts, respectively). RESULTS: The identification rate of lymphoscintigraphy and SLNB in all and axilla regions for 99mTc-Rituximab and 99mTc-SC were 98.5% vs 98.7, 100% vs 98.4%, respectively. The mean number of SLNs identified by 99mTc-Rituximab and fTcSC was respectively 2.72 and 3.28, with a significant difference of P = 0.013 (paired sample t-test). The difference exists in the internal mammary and clavicular area, not in the axillary. The mean number of axillary sentinel lymph node biopsy (SLNB) for 99mTc-Rituximab and fTcSC was 2.95 vs 3.14, respectively, and no significant difference existed. 99mTc-Rituximab also exhibited a significantly faster injection site clearance rate when compared with fTcSC (0.193 ± 0.057 h- 1 vs 0.021 ± 0.007 h- 1, respectively). CONCLUSION: No significant difference was observed in identification rate and number of axillary SLN imaging and SLNB, between the two tracers. Compared to fTcSC, 99mTc-Rituximab based imaging demonstrated a fewer number of secondary lymph nodes and had faster injection site clearance rate. TRIAL REGISTRATION: www.chictr.org.cn, ChiCTR1900024990 (retrospectively registered August 6, 2019).


Assuntos
Neoplasias da Mama/patologia , Coloides/química , Compostos de Organotecnécio/administração & dosagem , Rituximab/química , Linfonodo Sentinela/diagnóstico por imagem , Radioisótopos de Enxofre/química , Tecnécio/química , Adulto , Idoso , Axila , Estudos de Coortes , Feminino , Meia-Vida , Humanos , Linfocintigrafia/métodos , Pessoa de Meia-Idade , Compostos Radiofarmacêuticos , Linfonodo Sentinela/patologia , Biópsia de Linfonodo Sentinela
7.
Chem Res Toxicol ; 32(10): 1955-1964, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31496220

RESUMO

Acyl glucuronides (AGs) are reactive metabolites of carboxylic acid-containing drugs, which are associated with idiosyncratic toxicity (IDT) such as anaphylaxis, drug-induced liver injury, and so on. In this study, we developed a new in vitro approach for the quantitative assessment of the reactivity of AGs and their toxicity risk. Thirteen test drugs were incubated with human liver microsomes and uridine 5'-diphospho-glucuronic acid in the presence of cysteine (Cys) as a trapping agent. Both acylation and glycation Cys adducts formed from the AGs of the test drugs and were analyzed by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry. Acylation Cys adduct formation can closely reflect the reactivity of AGs to predict their IDT risk. Subsequently, we performed a quantitative trapping assay using radiometric analysis, with [35S]-labeled Cys ([35S]Cys) as the trapping agent, and the results showed that the test drugs associated with IDT resulted in a high product formation of [35S]Cys adducts. In conclusion, this approach can be used for the easy and quantitative evaluation of the reactivity of AGs without the need for authentic AG standards and to screen the potential IDT of new chemical entities during the early drug discovery phase.


Assuntos
Cisteína/química , Glucuronídeos/análise , Glucuronídeos/toxicidade , Glucuronídeos/química , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Radioisótopos de Enxofre
8.
Neuropharmacology ; 158: 107731, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31376424

RESUMO

Disruption of the hypothalamic-pituitary-adrenal axis is an established finding in patients with anxiety and/or depression. Chronic corticosterone administration in animals has been proposed as a model for the study of these stress-related disorders and the antidepressant action. Alterations of the central noradrenergic system and specifically of inhibitory α2-adrenoceptors seem to be part of the pathophysiology of depression and contribute to the antidepressant activity. The present study evaluates in male rats the effect of chronic corticosterone treatment during 35 days (16-20 mg kg-1 day-1) on the sensitivity of α2-adrenoceptors expressed in the somatodendritic and terminal noradrenergic areas locus coeruleus (LC) and prefrontal cortex (PFC), respectively. Further, the effect of chronic fluoxetine treatment (5 mg kg-1, i.p., since the 15th day) on the sensitivity of α2-adrenoceptors was examined under control conditions and in corticosterone-treated rats. The α2-adrenoceptor functionality was analysed in vitro by agonist-mediated [35S]GTPγS binding stimulation and in vivo through the modulation of noradrenaline (NA) release evaluated by dual-probe microdialysis. The concentration-effect curves of the [35S]GTPγS binding stimulation by the agonist UK14304 (5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) demonstrated a desensitization of cortical α2-adrenoceptors induced by corticosterone (-logEC50 = 6.7 ±â€¯0.2 vs 8.2 ±â€¯0.3 in controls) that was reverted by fluoxetine treatment (-logEC50 = 7.5 ±â€¯0.3). Local administration of the α2-adrenoceptor antagonist RS79948 ((8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine) (0.1-100 µmol L-1) into the LC induced a concentration-dependent NA increase in the PFC of the control group (Emax = 191 ±â€¯30%) but non-significant effect was observed in corticosterone-treated rats (Emax = 133 ±â€¯46%), reflecting a desensitization of α2-adrenoceptors that control the firing of noradrenergic neurons. Fluoxetine treatment did not alter the corticosterone-induced desensitization in this area (Emax = 136 ±â€¯19%). No effect of fluoxetine on α2-adrenoceptor functionality was observed in control animals (Emax = 223 ±â€¯30%). In PFC, the local administration of RS79948 increased NA in controls (Emax = 226 ±â€¯27%) without effect in the corticosterone group (Emax = 115 ±â€¯26%), suggesting a corticosterone-induced desensitization of terminal α2-adrenoceptors. Fluoxetine administration prevented the desensitization induced by corticosterone in the PFC (Emax = 233 ±â€¯33%) whereas desensitized α2-adrenoceptors in control animals (Emax = -24 ±â€¯10%). These data indicate that chronic corticosterone increases noradrenergic activity by acting at different α2-adrenoceptor subpopulations. Treatment with the antidepressant fluoxetine seems to counteract these changes by acting mainly on presynaptic α2-adrenoceptors expressed in terminal areas.


Assuntos
Neurônios Adrenérgicos/efeitos dos fármacos , Antidepressivos de Segunda Geração/farmacologia , Corticosterona/farmacologia , Fluoxetina/farmacologia , Locus Cerúleo/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Neurônios Adrenérgicos/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Animais , Tartarato de Brimonidina/farmacologia , Corpo Celular/efeitos dos fármacos , Corpo Celular/metabolismo , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Modelos Animais de Doenças , Guanosina 5'-O-(3-Tiotrifosfato) , Sistema Hipotálamo-Hipofisário/metabolismo , Técnicas In Vitro , Isoquinolinas/farmacologia , Locus Cerúleo/metabolismo , Masculino , Microdiálise , Naftiridinas/farmacologia , Norepinefrina/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Córtex Pré-Frontal/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Ratos , Receptores Adrenérgicos alfa 2/metabolismo , Estresse Psicológico/metabolismo , Radioisótopos de Enxofre
9.
J Environ Radioact ; 208-209: 106022, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31351333

RESUMO

The knowledge of groundwater residence times in (vulnerable) aquifers is essential for the sustainable management of the associated groundwater resources. A powerful tool for related investigations is the application of naturally occurring radioisotopes as water age indicators. However, due to the limited number of suitable (i.e. omnipresent, short-lived and easily detectable) radionuclides only few studies focus on groundwater ages below one year. A natural radionuclide that does have the potential to cover this time range is 35S (87.4 day half-life). 35S is continually produced in the upper atmosphere and transferred with the rain to the groundwater. Since no natural sources of 35S exist in the subsurface the decrease of the 35S activity concentration in such young groundwater can be used for the determination of its age. Still, 35S activities in precipitation (and hence even more in groundwater) are very low and necessitate appropriate analytical protocols based on liquid scintillation counting (LSC). This turns out to be challenging due to the required large sample volumes and due to potentially high SO42- loads of the samples, both limiting the range of possible applications of 35S as indicator for short groundwater residence times. In the paper we present an improved straightforward LSC based approach for the detection of 35S in natural water samples. We recommend using Insta-Gel Plus as scintillation cocktail for allowing a homogeneous suspension of 35S-containing BaSO4 in the cocktail. The recommended improvements in instrument setting concern the LSC (TriCarb 3170 Tr/SL) counting window, the pulse decay discriminator setting and the delay before burst setting. The settings allow measuring low activity concentrations of 35S, which was previously pre-concentrated from natural water samples, containing SO42- loads of up to 1500 mg with a reasonably high statistical reliability.


Assuntos
Água Subterrânea/química , Monitoramento de Radiação/métodos , Poluentes Radioativos da Água/análise , Contagem de Cintilação , Radioisótopos de Enxofre/análise
10.
Methods Mol Biol ; 2009: 111-127, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31152399

RESUMO

Proteins can be radiolabeled either during synthesis, typically using 35S-cysteine/methionine (35S-Cys/Met), or after synthesis, by adding a radiolabeled posttranslational modification. Here we describe how protein S-palmitoylation, and its dynamics, can be monitored by 3H-palmitate labeling and how the importance of S-palmitoylation in protein biogenesis and turnover can be investigated using 35S-Cys/Met pulse-chase metabolic labeling. Proteins frequently have multiple palmitoylation sites. The importance thereof on the design and interpretation of metabolic labeling experiments is discussed.


Assuntos
Marcação por Isótopo/métodos , Lipoilação , Ácido Palmítico/metabolismo , Processamento de Proteína Pós-Traducional , Radioisótopos de Enxofre , Linhagem Celular Transformada , Humanos
12.
Sci Rep ; 9(1): 4433, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30872714

RESUMO

Correlating cultural, technological and ecological aspects of both Upper Pleistocene modern humans (UPMHs) and Neandertals provides a useful approach for achieving robust predictions about what makes us human. Here we present ecological information for a period of special relevance in human evolution, the time of replacement of Neandertals by modern humans during the Late Pleistocene in Europe. Using the stable isotopic approach, we shed light on aspects of diet and mobility of the late Neandertals and UPMHs from the cave sites of the Troisième caverne of Goyet and Spy in Belgium. We demonstrate that their diet was essentially similar, relying on the same terrestrial herbivores, whereas mobility strategies indicate considerable differences between Neandertal groups, as well as in comparison to UPMHs. Our results indicate that UPMHs exploited their environment to a greater extent than Neandertals and support the hypothesis that UPMHs had a substantial impact not only on the population dynamics of large mammals but also on the whole structure of the ecosystem since their initial arrival in Europe.


Assuntos
Evolução Biológica , Radioisótopos de Carbono/análise , Dieta/tendências , Ecossistema , Emigração e Imigração/estatística & dados numéricos , Radioisótopos de Nitrogênio/análise , Radioisótopos de Enxofre/análise , Animais , Antropologia Física , Dieta/estatística & dados numéricos , Proteínas na Dieta/análise , Fósseis , Hominidae , Humanos , Homem de Neandertal
13.
Eur Neuropsychopharmacol ; 29(4): 566-576, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30773388

RESUMO

Cigarette (CIG) smoking often precedes the use of illegal drugs. Electronic-cigarettes (e-CIGs) have been promoted as a means of stopping smoking and reducing the harmful effects of CIGs on the population. However, although e-CIGs eliminate some of the morbidity associated with combustible tobacco, they are still nicotine-delivery devices. In order to study whether the nicotine delivered via e-CIG acts as "a gateway drug" to the use of cannabis, we analysed the behavioural and molecular effects of 7 weeks' pre-exposure to air (AIR), e-CIGs or CIGs on addiction-related conditioned place preference (CPP) in mice using a sub-threshold (0.01 mg/kg) dose of delta-9-tetrahydrocannabinol (Δ9-THC), the principal psychoactive constituent of cannabis. After 8 and 66 days of withdrawal, this Δ9-THC dose was ineffective in inducing CPP in mice pre-exposed to pump-driven AIR, but very effective in mice pre-exposed to e-CIGs or CIGs. Exposure to e-CIGs or CIGs increases the expression of ΔFosB in the nucleus accumbens (NAc), which remains high during short-term e-CIG or CIG withdrawal and long-term CIG withdrawal and is not influenced by treatment with Δ9-THC. At the end of e-CIG or CIG exposure and during withdrawal, the mice also had a higher AMPA receptors GluA1/GluA2-3 ratio in the NAc. Chronic nicotine exposure increases sensitivity to rewarding effects of Δ9-THC in mice and produces long-lasting neurobiological changes regardless of the delivery method (CIG vs. e-CIG). The exposure to passive tobacco smoke or e-CIG vapours can similarly increase vulnerability to the effects of cannabis and possibly other drugs of abuse.


Assuntos
Condicionamento Psicológico/efeitos dos fármacos , Dronabinol/farmacologia , Sistemas Eletrônicos de Liberação de Nicotina , Produtos do Tabaco/efeitos adversos , Animais , Cicloexanóis/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Masculino , Camundongos , Núcleo Accumbens/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ensaio Radioligante , Receptores de AMPA/metabolismo , Radioisótopos de Enxofre/metabolismo , Dispositivos para o Abandono do Uso de Tabaco/efeitos adversos , Trítio/metabolismo
14.
Cell Tissue Res ; 375(2): 507-529, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30259139

RESUMO

The subcommissural organ (SCO) is an ancient and conserved brain gland secreting into cerebrospinal fluid (CSF) glycoproteins that form the Reissner fiber (RF). The present investigation was designed to further investigate the dynamic of the biosynthetic process of RF glycoproteins prior and after their release into the CSF, to identify the RF proteome and N-glycome and to clarify the mechanism of assembly of RF glycoproteins. Various methodological approaches were used: biosynthetic labelling injecting 35S-cysteine and 3H-galactose into the CSF, injection of antibodies against galectin-1 into the cerebrospinal fluid, light and electron microscopical methods; isolated bovine RF was used for proteome analyses by mass spectrometry and glycome analysis by xCGE-LIF. The biosynthetic labelling study further supported that a small pool of SCO-spondin molecules rapidly enter the secretory pathways after its synthesis, while most of the SCO-spondin molecules are stored in the rough endoplasmic reticulum for hours or days before entering the secretory pathway and being released to assemble into RF. The proteomic analysis of RF revealed clusterin and galectin-1 as partners of SCO-spondin; the in vivo use of anti-galectin-1 showed that this lectin is essential for the assembly of RF. Galectin-1 is not secreted by the SCO but evidence was obtained that it would be secreted by multiciliated ependymal cells lying close to the SCO. Further, a surprising variety and complexity of glycan structures were identified in the RF N-glycome that further expands the potential functions of RF to a level not previously envisaged. A model of the macromolecular organization of Reissner fiber is proposed.


Assuntos
Glicoproteínas/metabolismo , Órgão Subcomissural/fisiologia , Animais , Bovinos , Cisteína/metabolismo , Citoplasma/metabolismo , Epêndima/citologia , Epêndima/metabolismo , Galactose/metabolismo , Galectina 1/metabolismo , Glicoproteínas/ultraestrutura , Glicosilação , Masculino , Polissacarídeos/química , Polissacarídeos/metabolismo , Ratos Sprague-Dawley , Via Secretória , Coloração e Rotulagem , Órgão Subcomissural/ultraestrutura , Radioisótopos de Enxofre/metabolismo , Trítio/metabolismo
15.
Methods Mol Biol ; 1855: 417-436, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30426436

RESUMO

This report discusses recent methods of sample preparation and gel electrophoresis for 35S immunoprecipitation (IP) and IP western blotting. In both methods, IP is used to obtain purified proteins, and sodium dodecyl sulfate polyacrylamide gel electrophoresis is used to separate the proteins on a gel. In 35S IP, the proteins are radiolabeled and visualized on film by fluorography; in IP blotting, proteins are transferred onto nitrocellulose paper, and antibodies are used to detect specific proteins. A similar IP and SDS-PAGE method can be used for both procedures, but IP blotting has the potential advantages of improvement in sensitivity for low-abundance proteins and enhanced specificity for identification of proteins from a mixture. Some of the technical adaptations discussed here to facilitate IP blotting and avoid loss of beads or purified proteins may also be useful for 35S IP.


Assuntos
Western Blotting/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Imunoprecipitação/métodos , Proteínas/análise , Animais , Colódio/química , Humanos , Proteínas/isolamento & purificação , Radioisótopos de Enxofre/análise
16.
Cold Spring Harb Protoc ; 2018(9)2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30181224

RESUMO

Pulse-chase labeling of antigens with [35S]methionine is used to determine the relative half-life of a protein. In this protocol, intracellular unlabeled methionine levels are reduced by starvation of cells for 0.5-1 h, and then the cells are briefly labeled with [35S]methionine to create the pulse of newly synthesized proteins. Upon completion of cell labeling, the addition of Chasing medium containing an excess of unlabeled methionine is used to create the chase, reducing the likelihood that any remaining [35S]methionine will be incorporated into newly synthesized proteins. Labeling and chasing reactions of adherent cells can be directly performed in cell culture dishes in an incubator, whereas suspension cells are labeled and chased in a polypropylene tube kept in a water bath set at 37°C. At intervals after the pulse, aliquots of chased labeled cells are collected and pelleted with the option of immediately preparing cell lysates or freezing and storing the cell pellets at -80°C. Upon cell lysis and antigen purification by immunoprecipitation, SDS-PAGE-resolved proteins can be fixed on the gel and enhanced with fluorography or can be transferred to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane followed by autoradiography or exposure in a phosphorimager. Membrane blotting has the advantage of allowing for detection of the target of interest by probing with an antigen-specific antibody to confirm that equal amounts of steady-state levels of the target protein were immunoprecipitated at each interval.


Assuntos
Antígenos/metabolismo , Metionina/metabolismo , Proteínas/metabolismo , Radioisótopos de Enxofre/metabolismo , Autorradiografia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Meia-Vida
17.
Methods Mol Biol ; 1841: 113-130, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30259483

RESUMO

Despite all its apparent limitations proteome analysis based on two-dimensional protein gels combined with mass spectrometry is still the method of choice to study global protein synthesis activity in bacterial cells. Alterations in global protein synthesis play an important role during adaptation of bacteria to changing environmental conditions which are rather the role than the exception in their natural habitats. The protein synthesis pattern in response to a certain stimulus is highly specific and reflects the new challenges the bacterium has to meet. Here we present the techniques to analyze global protein synthesis in bacteria as exemplified by Staphylococcus aureus which is an important human pathogen and one main cause of nosocomial infections with severe outcome.


Assuntos
Proteínas de Bactérias/metabolismo , Proteoma , Proteômica , Staphylococcus aureus/metabolismo , Mineração de Dados , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional/métodos , Humanos , Marcação por Isótopo , Espectrometria de Massas , Proteômica/métodos , Staphylococcus aureus/genética , Radioisótopos de Enxofre , Transcriptoma , Fluxo de Trabalho
18.
Methods Mol Biol ; 1841: 131-140, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30259484

RESUMO

The half-life of a particular protein is highly variable, reaching from minutes to hours, over days and weeks to years or even a whole life time of an organism (e.g., α-crystalline of the mammalian eye). Thus, controlling protein activity by proteolysis is the most dramatic and unambiguous decision by any organism, because reconstitution of the destroyed protein activity requires an "expensive" new synthesis. To distinguish degradation from protein synthesis and accumulation only one method comes into consideration-pulse-chase labeling. In our hands, the most accurate method to track the fate of a single protein is radioactive pulse-chase labeling combined with immunoprecipitation. Besides a detailed description of the standard protocol, the general applicability as well as certain improvements of the method will be discussed here.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Bioensaio , Estabilidade Proteica , Trifosfato de Adenosina/metabolismo , Bioensaio/métodos , Imunoprecipitação/métodos , Marcação por Isótopo , Proteólise , Radioisótopos de Enxofre
19.
Methods Mol Biol ; 1841: 163-174, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30259486

RESUMO

Quantitative proteome profiling of microorganisms by isotopic labeling of amino acids is still a challenge, because only microorganisms with auxotrophic character are able to embed amino acids into their biomass in a quantitatively correct manner. Here, we describe an isotopic labeling technique (sulfur stable isotope labeling of amino acids for quantification, SULAQ) for the sulfur-containing amino acids cysteine and methionine in a broad range of organisms. The metabolic labeling approach is suitable for gel-based and gel-free protein analysis.


Assuntos
Aminoácidos , Marcação por Isótopo , Proteoma , Proteômica , Isótopos de Enxofre , Radioisótopos de Enxofre , Aminoácidos/química , Proteínas de Bactérias , Cromatografia Líquida , Biologia Computacional , Espectrometria de Massas , Proteólise , Proteômica/métodos
20.
Purinergic Signal ; 14(2): 177-190, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29492786

RESUMO

Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A1 receptor and G-protein was assessed by means of two [35S]GTPγS binding assays, i.e., conventional filtration method and [35S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A1 receptor-mediated Gαi-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [35S]GTPγS binding determined with conventional assay derives from functional activation of Gαi/o proteins (not restricted only to Gαi-3) coupled to adenosine A1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [35S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %Emax values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A1 receptor/G-protein interaction in postmortem human brain tissue.


Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Imunoprecipitação/métodos , Receptor A1 de Adenosina/metabolismo , Animais , Ligação Competitiva , Feminino , Humanos , Antagonistas de Receptores Purinérgicos P1 , Ratos , Radioisótopos de Enxofre/metabolismo
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