RESUMO
Psilocybin has recently attracted a great deal of attention as a clinical research and therapeutic tool. The aim of this paper is to bridge two major knowledge gaps regarding its behavioural pharmacology - sex differences and the underlying receptor mechanisms. We used psilocin (0.25, 1 and 4 mg/kg), an active metabolite of psilocybin, in two behavioural paradigms - the open-field test and prepulse inhibition (PPI) of the acoustic startle reaction. Sex differences were evaluated with respect to the phase of the female cycle. The contribution of serotonin receptors in the behavioural action was tested in male rats with selective serotonin receptor antagonists: 5-HT1A receptor antagonist (WAY100635 1 mg/kg), 5-HT2A receptor antagonist (MDL100907 0.5 mg/kg), 5-HT2B receptor antagonist (SB215505 1 mg/kg) and 5-HT2C receptor antagonist (SB242084 1 mg/kg). Psilocin induced dose-dependent inhibition of locomotion and suppression of normal behaviour in rats (behavioural serotonin syndrome, impaired PPI). The effects were more pronounced in male rats than in females. The inhibition of locomotion was normalized by 5-HT1A and 5-HT2B/C antagonists; however, PPI was not affected significantly by these antagonists. Our findings highlight an important issue of sex-specific reactions to psilocin and that apart from 5-HT2A-mediated effects 5-HT1A and 5-HT2C/B receptors also play an important role. These findings have implications for recent clinical trials.
Assuntos
Alucinógenos/farmacologia , Psilocibina/análogos & derivados , Receptores de Serotonina/efeitos dos fármacos , Serotonina/metabolismo , Animais , Relação Dose-Resposta a Droga , Ciclo Estral/fisiologia , Feminino , Alucinógenos/administração & dosagem , Locomoção/efeitos dos fármacos , Masculino , Psilocibina/administração & dosagem , Psilocibina/farmacologia , Ratos , Ratos Wistar , Receptores de Serotonina/metabolismo , Reflexo de Sobressalto/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Fatores SexuaisRESUMO
Dickeya and Pectobacterium species represent an important group of broad-host-range phytopathogens responsible for blackleg and soft rot diseases on numerous plants including many economically important plants. Although these species are commonly detected using cultural, serological, and molecular methods, these methods are sometimes insufficient to classify the bacteria correctly. On that account, this study was undertaken to investigate the feasibility of three individual analytical techniques, capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), for reliable classification of Dickeya and Pectobacterium species. Forty-three strains, representing different Dickeya and Pectobacterium species, namely Dickeya dianthicola, Dickeya dadantii, Dickeya dieffenbachiae, Dickeya chrysanthemi, Dickeya zeae, Dickeya paradisiaca, Dickeya solani, Pectobacterium carotovorum, and Pectobacterium atrosepticum, were selected for this purpose. Furthermore, the selected bacteria included one strain which could not be classified using traditional microbiological methods. Characterization of the bacteria was based on different pI values (CIEF), migration velocities (CZE), or specific mass fingerprints (MALDI-TOF MS) of intact cells. All the examined strains, including the undetermined bacterium, were characterized and classified correctly into respective species. MALDI-TOF MS provided the most reliable results in this respect.
Assuntos
Eletroforese Capilar/métodos , Enterobacteriaceae/química , Enterobacteriaceae/classificação , Pectobacterium/química , Pectobacterium/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Enterobacteriaceae/isolamento & purificação , Pectobacterium/isolamento & purificaçãoRESUMO
The effect of antibiotics on the microbial cells and concentration of antibiotics in the human body is essential for the effective use of antimicrobial therapy. The capillary isoelectric focusing is a suitable technique for the separation and the detection of bacteria, and amphoteric substances from nature. However, the determination of isoelectric points of ampholytic antibiotics by conventional techniques is time consuming. For this reason, capillary isoelectric focusing seems to be appropriate as a simple and reliable way for establishing them. The separation conditions for the capillary isoelectric focusing of selected ampholytic antibiotics with known isoelectric points and pK as, ampicillin (pI 4.9), ciprofloxacin (pI 7.4), ofloxacin (pI 7.1), tetracycline (pI 5.4), tigecycline (pI 9.7), and vancomycin (pI 8.1), were found and optimized in the suitable pH ranges pH 2.0-5.3, 2.0-9.6, and 9.0-10.4. The established values of isoelectric points correspond with those found in the literature except tigecycline. Its pI was not found in the literature. As an example of a possible procedure for direct detection of both ampholytic antibiotics and bacteria, Staphylococcus epidermidis, in the presence of culture media or whole human blood, was found. The changes of the bacterial cells after their treatment with tetracycline were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Capillary isoelectric focusing allows the fast and simple determination of isoelectric points of relevant antibiotics, their quantification from the environment, as well as studying their effectiveness on microorganisms in biological samples.
Assuntos
Antibacterianos/química , Sangue/microbiologia , Focalização Isoelétrica/métodos , Staphylococcus epidermidis/química , Antibacterianos/farmacologia , Meios de Cultura/química , Humanos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/instrumentação , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/isolamento & purificaçãoRESUMO
This study was undertaken to investigate feasibility of a combination of capillary isoelectric focusing (CIEF) in a tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and reliable identification of bacteria taken from plant-tissue-containing samples. Eight strains representing different species of the genus Dickeya were selected on the basis of close proximity of their isoelectric points: D. chrysanthemi, D. chrysanthemi bv. parthenii, D. chrysanthemi bv. chrysanthemi, D. dadantii, D. paradisiaca, D. solani, D. diffenbachiae, and D. dianthicola. Because the Dickeya species (spp.) cannot be easily discriminated from each other when CIEF is performed in a cylindrical FS capillary (commonly used in CIEF) even if a narrow pH gradient is used, a tapered FS capillary was employed instead, which enabled satisfactory discrimination of the examined bacteria due to enhanced separation efficiency of CIEF in the tapered FS capillary. CIEF in the tapered FS capillary was also successfully used for the detection and characterization of Dickeya spp. in a plant-tissue-containing sample. Then an off-line combination of CIEF with MALDI-TOF MS was employed for rapid and reliable identification of Dickeya spp. in the plant-tissue-containing sample. It was found that the presence of plant tissue did not affect the results, making the proposed procedure very promising with respect to the fast and reliable detection and identification of bacteria in plant-tissue-containing samples.
Assuntos
Enterobacteriaceae/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Eletroforese Capilar/métodos , Enterobacteriaceae/química , Focalização Isoelétrica/métodos , Extratos Vegetais/análise , Extratos Vegetais/isolamento & purificação , Proteínas de Plantas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normas , Fatores de TempoRESUMO
In this study electrophoretic and mass spectrometric analysis of three types of bacterial sample (intact cells, cell lysates, and "washed pellets") were used to develop an effective procedure for the characterization of bacteria. The samples were prepared from specific bacterial strains. Five strains representing different species of the family Rhizobiaceae were selected as model microorganisms: Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, R. galegae, R. loti, and Sinorhizobium meliloti. Samples of bacteria were subjected to analysis by four techniques: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), gel IEF, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These methods are potential alternatives to DNA-based methods for rapid and reliable characterization of bacteria. Capillary electrophoretic (CZE and CIEF) analysis of intact cells was suitable for characterization of different bacterial species. CIEF fingerprints of "washed pellets" and gel IEF of cell lysates helped to distinguish between closely related bacterial species that were not sufficiently differentiated by capillary electrophoretic analysis of intact cells. MALDI-TOF MS of "washed pellets" enabled more reliable characterization of bacteria than analysis of intact cells or cell lysates. Electrophoretic techniques and MALDI-TOF MS can both be successfully used to complement standard methods for rapid characterization of bacteria.
Assuntos
Eletroforese Capilar/métodos , Focalização Isoelétrica/métodos , Rhizobium/química , Sinorhizobium meliloti/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Viabilidade Microbiana , Rhizobium/classificação , Rhizobium/isolamento & purificação , Sinorhizobium meliloti/classificação , Sinorhizobium meliloti/isolamento & purificaçãoRESUMO
In modern medical diagnostics, where analytical chemistry plays a key role, fast and accurate identification of pathogens is becoming increasingly important. Infectious diseases pose a growing threat to public health due to population growth, international air travel, bacterial resistance to antibiotics, and other factors. For instance, the detection of SARS-CoV-2 in patient samples is a key tool to monitor the spread of the disease. While there are several techniques for identifying pathogens by their genetic code, most of these methods are too expensive or slow to effectively analyze clinical and environmental samples that may contain hundreds or even thousands of different microbes. Standard approaches (e.g., culture media and biochemical assays) are known to be very time- and labor-intensive. The purpose of this review paper is to highlight the problems associated with the analysis and identification of pathogens that cause many serious infections. Special attention was paid to the description of mechanisms and the explanation of the phenomena and processes occurring on the surface of pathogens as biocolloids (charge distribution). This review also highlights the importance of electromigration techniques and demonstrates their potential for pathogen pre-separation and fractionation and demonstrates the use of spectrometric methods, such as MALDI-TOF MS, for their detection and identification.
RESUMO
In recent years, characterization and identification of microorganisms has become very important in different fields of human activity. Conventional laboratory methods are time consuming, laborious, and they may provide both false positive or negative results, especially for closely related microorganisms. On that account, new methods for fast and reliable microbial characterization are of great interest. In particular, capillary electrophoretic techniques have a great potential for characterization of microorganisms due to their unique surface properties. Cell surface proteins play a key role in this respect. Since CIEF represents one of the most efficient techniques for protein separation, it was consequently applied to the analysis of microbial cells. This review describes, after a brief introduction to CIEF of proteins, recent developments in CIEF of diverse microorganisms (viruses, bacteria, yeasts, and fungi). Possible application schemes in human and veterinary medicine as well as in plant protection and in biosecurity are outlined.
Assuntos
Bactérias/química , Fungos/química , Focalização Isoelétrica/métodos , Proteínas/química , Vírus/química , Animais , Bactérias/isolamento & purificação , Fungos/isolamento & purificação , Humanos , Proteínas/análise , Proteínas/isolamento & purificação , Vírus/isolamento & purificaçãoRESUMO
Biofilm-positive strains of Candida parapsilosis are the second most common yeasts responsible for bloodstream infections. This pathogen is difficult to identify by standard methods from other phenotypically indistinguishable species, biofilm-negative Candida orthopsilosis and biofilm-positive Candida metapsilosis. From a medical point of view, important information is especially whether the strains form biofilm. The biofilm formation enables yeast to colonize artificial surfaces thereby protecting the yeast cell against antifungal agents. The commonly used genotypic methods including different modifications of the polymerase chain reaction have some disadvantages. Therefore, a rapid and reliable method able to identify phenotypically indistinguishable C. "psilosis" species is still of great interest. In this study, the four well-established analytical techniques: gel isoelectric focusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, were applied in order to discriminate C. "psilosis" species. The ability of these techniques to differentiate between biofilm-positive and biofilm-negative strains was further investigated. Our results have revealed that the proposed methods, especially matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact yeast cells, can be used as the efficient tools for discrimination and identification of the phenotypically indistinguishable microorganisms.
Assuntos
Candida/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Biofilmes , Candida/classificação , Candida/patogenicidade , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Reação em Cadeia da Polimerase , Especificidade da EspécieRESUMO
Influenza A is viral disease, which is a cause of yearly epidemics and, potentially, pandemics. The conventional techniques used today are equipment-demanding, time-consuming and laborious. Recently, we have confirmed that the capillary isoelectric focusing is a suitable fast alternative for the verifying of virus purity. In the wide pH gradient of pH range 2.0-7.5 the isoelectric points for subtypes of equine (H3N8) and swine (H1N2) influenza A viruses were determined approximately as 6.6 and 6.5, respectively. In this contribution we have verified these findings using different isolates of different viral subtypes of swine influenza, H1N1, H1N2, and of equine influenza, H3N8, H7N7, which were separated by capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) in the narrow pH gradient pH range from 6.0 to 7.0. It was found that the isoelectric points of different isolates and subtypes of equine and swine influenza are almost independent of their origin. The electromigration velocities of subtypes of equine or swine influenza viruses were dependent on the antigenic subtypes of their surface glycoproteins. The detection sensitivity of the influenza viruses labeled by the fluorescent non-ionogenic tenside based on poly(ethylene glycol)pyrenebutanoate for fluorometric detection was increased and down to ten labeled viruses were detected. The isoelectric points of the native and labeled equine and swine influenza A viruses and their subtypes do not differ. According to our experiments these methods appear to be useful for the fast preliminary differentiation of influenza viruses in future.
Assuntos
Eletroforese Capilar/métodos , Vírus da Influenza A/classificação , Focalização Isoelétrica/métodos , Animais , Éteres/química , Corantes Fluorescentes/química , Fluorometria , Cavalos , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Vírus da Influenza A Subtipo H7N7/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Polivinil/química , Espectrofotometria Ultravioleta , SuínosRESUMO
Trace analysis of microorganisms in real biological samples needs very sensitive methods for their detection. Most procedures for detecting and quantifying pathogens require a sample preparation step including concentrating microorganisms from large sample volumes with high and reproducible efficiency. Electromigration techniques have great potential to include the preconcentration, separation, and detection of whole cells and therefore they can rapidly indicate the presence of pathogens. The preconcentration and separation of microorganisms from real suspensions utilising a combination of filtration and capillary isoelectric focusing was developed and the possibility for its application to real samples was verified. For our experiments, spores of Monilinia species and of Penicillium expansum were selected as model bioparticles, as they cause major losses in agrosystems. The isoelectric points of the spores of M. laxa, M. fructigena, M. fruticola, and P. expansum were determined and the method was verified using real samples taken directly from infected apples. The coupling of a filtration cartridge with a separation capillary can improve the detection limit of isoelectric focusing with UV detection by at least 4 orders of magnitude. Spores of M. fructigena and of M. laxa in numbers of hundreds of particles per milliliter were detected on a visually noninfected apple surface which was cross-contaminated during handling and storage. The efficiency of preconcentration and a preliminary identification was verified by the phenotyping technique after cultivation of the spores sampled from the apple surface.
Assuntos
Ascomicetos/isolamento & purificação , Filtração/métodos , Focalização Isoelétrica/métodos , Malus/microbiologia , Penicillium/isolamento & purificação , Esporos Fúngicos/isolamento & purificação , Eletroforese Capilar/instrumentação , Eletroforese Capilar/métodos , Desenho de Equipamento , Filtração/instrumentação , Focalização Isoelétrica/instrumentação , Sensibilidade e EspecificidadeRESUMO
The detection and identification of pathogens currently relies upon a very diverse range of techniques and skills, from traditional cultivation and taxonomic procedures to modern rapid and sensitive diagnostic methods. Real-time PCR is now exploited as a front line diagnostic screening tool in human, animal and plant health as well as bio-security. Nevertheless, new techniques for pathogen identification, particularly of unknown samples, are needed. In this study we propose the combination of electrophoresis-based procedures for the fast differentiation of microorganisms. The method feasibility is proved on the model of seven similar strains of Pseudomonas syringae pathovars from 37 sources, identified by gas chromatography of fatty acid methyl esters. The results from the routine laboratory were compared with results of the combination of the developed capillary and gel electrophoresis as well as mass spectrometry. According to our experiments appropriate combination of electromigration techniques appears to be useful for the fast and economical differentiation of unknown microorganisms.
Assuntos
Eletroforese Capilar/métodos , Pseudomonas syringae/classificação , Cromatografia Gasosa/métodos , Ácidos Graxos/análise , Pseudomonas syringae/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
In immunocompromised people fungal agents are able to cause serious infections with high mortality rate. An early diagnosis can increase the chances of survival of the affected patients. Simultaneously, the fungi produce toxins and they are frequent cause of allergy. Currently, various methods are used for detection and identification of these pathogens. They use microscopic examination and growth characteristic of the fungi. New methods are based on the analysis of structural elements of the target microorganisms such as proteins, polysaccharides, glycoproteins, nucleic acids, etc. for the construction of antibodies, probes, and primers for detection. The above-mentioned methods are time-consuming and elaborate. Here hydrophobic conidia from the cultures of different strains of the filamentous fungi were focused and separated by capillary zone electrophoresis and capillary isoelectric focusing. The detection was optimized by dynamic modifying of conidia by the nonionogenic tenside on the basis of pyrenebutanoate. Down to 10 labeled conidia of the fungal strains were fluorometrically detected, and isoelectric points of conidia were determined. The observed isoelectric points were compared with those obtained from the separation of the cultured clinical samples, and they were found to be not host-specific.
Assuntos
Eletroforese Capilar/métodos , Focalização Isoelétrica/métodos , Esporos Fúngicos/química , Fungos/química , Humanos , Espectrofotometria Ultravioleta , Esporos Fúngicos/isolamento & purificaçãoRESUMO
Excessive stimulation of NMDA receptors with glutamate or other potent agonists such as NMDA leads to excitotoxicity and neural injury. In this study, we aimed to provide insight into an animal model of brain excitotoxic damage; single unilateral infusion of NMDA at mild dose into the hippocampal formation. NMDA infusion induced chronic, focal neurodegeneration in the proximity of the injection site. The lesion was accompanied by severe and progressive neuroinflammation and affected preferentially principal neurons while sparing GABAergic interneurons. Furthermore, the unilateral lesion did not cause significant impairment of spatial learning abilities. Finally, GluN1 and GluN2B subunits of NMDA receptor were significantly upregulated up to 3 days after the NMDA infusion, while GABAA α5 subunit was downregulated at 30 days after the lesion. Taken together, a single infusion of NMDA into the hippocampal formation represents an animal model of excitotoxicity-induced chronic neurodegeneration of principal neurons accompanied by severe neuroinflammation and subunit specific changes in NMDA and GABAA receptors.
Assuntos
Hipocampo/metabolismo , N-Metilaspartato/toxicidade , Doenças Neurodegenerativas/metabolismo , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Modelos Animais de Doenças , Lateralidade Funcional , Hipocampo/diagnóstico por imagem , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , N-Metilaspartato/administração & dosagem , Degeneração Neural/diagnóstico por imagem , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/patologia , Neuroimunomodulação/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Ratos Long-EvansRESUMO
Perinatal immune challenge leads to neurodevelopmental dysfunction, permanent immune dysregulation and abnormal behaviour, which have been shown to have translational validity to findings in human neuropsychiatric disorders (e.g. schizophrenia, mood and anxiety disorders, autism, Parkinson's disease and Alzheimer's disease). The aim of this animal study was to elucidate the influence of early immune stimulation triggered by systemic postnatal lipopolysaccharide administration on biochemical, histopathological and morphological measures, which may be relevant to the neurobiology of human psychopathology. In the present study of adult male Wistar rats we examined the brain and plasma levels of monoamines (dopamine, serotonin), their metabolites, the levels of the main excitatory and inhibitory neurotransmitters glutamate and γ-aminobutyric acid and the levels of tryptophan and its metabolites from the kynurenine catabolic pathway. Further, we focused on histopathological and morphological markers related to pathogenesis of brain diseases--glial cell activation, neurodegeneration, hippocampal volume reduction and dopaminergic synthesis in the substantia nigra. Our results show that early immune stimulation in adult animals alters the levels of neurotransmitters and their metabolites, activates the kynurenine pathway of tryptophan metabolism and leads to astrogliosis, hippocampal volume reduction and a decrease of tyrosine hydroxylase immunoreactivity in the substantia nigra. These findings support the crucial pathophysiological role of early immune stimulation in the above mentioned neuropsychiatric disorders.
Assuntos
Transtornos Mentais/imunologia , Transtornos Mentais/metabolismo , Neuroimunomodulação , Animais , Astrócitos/metabolismo , Monoaminas Biogênicas/sangue , Monoaminas Biogênicas/metabolismo , Peso Corporal , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Cinurenina/metabolismo , Masculino , Transtornos Mentais/patologia , Transtornos Mentais/psicologia , Redes e Vias Metabólicas , Metaboloma , Microglia/metabolismo , Neurotransmissores/metabolismo , Psicopatologia , Ratos , Triptofano/metabolismoRESUMO
In the present study we investigated the potential antipsychotic effects of the mGlu2/3 agonist LY379268 on changes in EEG power spectra and coherence in the ketamine model of psychosis. In order to use behaviorally active drug doses, experiments detecting changes in locomotor activity and sensorimotor gating were also conducted. In EEG experiments, adult male Wistar rats were injected with ketamine 30 mg/kg i.p. and LY379268 3 mg/kg i.p. Cortical EEG was recorded from twelve (2 × 6) electrodes placed homolaterally on each hemisphere. To avoid interference with the behavioral hyperactivity of ketamine challenge, the behavioral activity of animals was simultaneously registered at the time of recording. Subsequent power spectral and coherence analyses were assessed in epochs corresponding to behavioral inactivity. Analysis of segments with behavioral activity compared to inactivity was also performed. The effects of LY379268 3 mg/kg i.p. on the deficits in sensorimotor processing and on hyperlocomotion induced by ketamine were evaluated in the test of prepulse inhibition of acoustic startle reaction (PPI ASR) and in the open field. LY379268 reversed the ketamine-induced hyperlocomotion but had no effect on ketamine-induced PPI deficits. In EEG epochs corresponding to behavioral inactivity ketamine decreased the power in the delta band, induced a power increase in the high frequency bands and globally decreased EEG coherence. Pretreatment with the LY379268 completely reversed the ketamine-induced power increase in high frequency bands and had a partial effect on EEG coherence. LY379268 alone induced a decrease of beta, high beta and low-gamma power, and an increase in coherence in high frequency bands. Additional analysis revealed that behavioral activity increases power as well as coherence in most frequency bands. In conclusion, agonism of mGlu2/3 receptors was effective in reversing most of the changes induced by ketamine, however due to the lack of effectiveness on PPI deficits its potential antipsychotic properties remain disputable.
Assuntos
Aminoácidos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Modelos Animais de Doenças , Eletroencefalografia/efeitos dos fármacos , Transtornos Psicóticos/tratamento farmacológico , Receptores de Glutamato Metabotrópico/agonistas , Aminoácidos/farmacologia , Animais , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Eletroencefalografia/métodos , Ketamina/toxicidade , Masculino , Transtornos Psicóticos/fisiopatologia , Ratos , Ratos Wistar , Resultado do TratamentoRESUMO
In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cow's milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2×10(6) mL(-1). Finally, cow's milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample.
Assuntos
Análise de Alimentos/métodos , Focalização Isoelétrica/métodos , Lactobacillus/isolamento & purificação , Leite/microbiologia , Probióticos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Calibragem , Bovinos , Eletroforese Capilar/métodos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/instrumentação , PasteurizaçãoRESUMO
RATIONALE AND OBJECTIVES: Behavioral, neurochemical and pharmaco-EEG profiles of a new synthetic drug 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in rats were examined. MATERIALS AND METHODS: Locomotor effects, prepulse inhibition (PPI) of acoustic startle reaction (ASR), dopamine and its metabolite levels in nucleus accumbens (NAc), EEG power spectra and coherence in freely moving rats were analysed. Amphetamine was used as a reference compound. RESULTS: 2C-B had a biphasic effect on locomotion with initial inhibitory followed by excitatory effect; amphetamine induced only hyperlocomotion. Both drugs induced deficits in the PPI; however they had opposite effects on ASR. 2C-B increased dopamine but decreased 3,4-dihydroxyphenylacetic acid (DOPAC) in the NAc. Low doses of 2C-B induced a decrease in EEG power spectra and coherence. On the contrary, high dose of 2C-B 50 mg/kg had a temporally biphasic effect with an initial decrease followed by an increase in EEG power; decrease as well as increase in EEG coherence was observed. Amphetamine mainly induced an increase in EEG power and coherence in theta and alpha bands. Increases in the theta and alpha power and coherence in 2C-B and amphetamine were temporally linked to an increase in locomotor activity and DA levels in NAc. CONCLUSIONS: 2C-B is a centrally active compound similar to other hallucinogens, entactogens and stimulants. Increased dopamine and decreased DOPAC in the NAc may reflect its psychotomimetic and addictive potential and monoaminoxidase inhibition. Alterations in brain functional connectivity reflected the behavioral and neurochemical changes produced by the drug; a correlation between EEG changes and locomotor behavior was observed.
Assuntos
Comportamento Animal/efeitos dos fármacos , Dimetoxifeniletilamina/análogos & derivados , Alucinógenos/farmacologia , Atividade Motora/efeitos dos fármacos , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Anfetamina/farmacologia , Animais , Dimetoxifeniletilamina/administração & dosagem , Dimetoxifeniletilamina/farmacologia , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Eletroencefalografia , Alucinógenos/administração & dosagem , Masculino , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Ratos , Ratos Wistar , Reflexo de Sobressalto/efeitos dos fármacosRESUMO
During bacterial infections of the central nervous system the number of microorganisms in the cerebrospinal fluid is often ranging from few up to hundreds of cells per milliliter. The electrophoretic techniques with the UV-detection reach a detection limit for whole cells of approximately 10(7)cells per milliliter. The coupling of the filtration cartridge with capillary isoelectric focusing can improve the detection limit by four orders of magnitude. In order to improve the detection limit the red non-ionogenic surfactant 1-[[4-(phenylazo)phenyl]azo]-2-hydroxy-3-naphthoic acid polyethylene glycol ester, PAPAN 1000, has been prepared and used for the dynamic labeling of analytes before filtration of the sample with a concentration modulation in the analysis of proteins or microorganisms. Values of isoelectric points of labeled analytes have been calculated using pI markers detectable at 515 nm and have been found comparable with pI of the native compounds. Minimum detectable amounts of proteins and microorganisms were lower than nanograms and a hundred labeled cells, respectively. The introduced method, coupling of the filtration cerebrospinal fluid spiked with microorganisms and labeled by PAPAN, facilitates their rapid CIEF separation in the pH gradient pH range of 2-5 at their clinically important level 10(1) to 10(2) cells per milliliter.
Assuntos
Infecções Bacterianas/líquido cefalorraquidiano , Candida/isolamento & purificação , Eletroforese Capilar/métodos , Escherichia coli/isolamento & purificação , Micoses/líquido cefalorraquidiano , Saccharomyces cerevisiae/isolamento & purificação , Staphylococcus/isolamento & purificação , Infecções Bacterianas/diagnóstico , Humanos , Focalização Isoelétrica/métodos , Limite de Detecção , Micoses/diagnóstico , Naftóis/química , Polietilenoglicóis/química , Tensoativos/químicaRESUMO
This review focuses on the relationship between psychotropic drugs and adult hippocampal neurogenesis. Adult neurogenesis is important for learning and memory, as well as for depression and anxiety. There is some evidence that chronic treatment with opiates, stimulants and entactogens decreases neurogenesis and consequently impairs cognitive function, as well as inducing depressive-like behaviour in animals during drug withdrawal. On the other hand, NMDA receptor antagonists increase neurogenesis, but negatively affect cognitive function and have an antidepressant-like profile. We suggest that drug-induced changes in neurogenesis have a greater and more concise effect on emotive state reflecting the direction of influencing new cells proliferation than the performance of cognitive tasks. In this review we provide some evidence for this assumption.
Assuntos
Envelhecimento/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Psicotrópicos/farmacologia , Envelhecimento/fisiologia , Animais , Cognição/efeitos dos fármacos , Cognição/fisiologia , Emoções/efeitos dos fármacos , Emoções/fisiologia , Hipocampo/fisiologia , Humanos , Neurogênese/fisiologiaRESUMO
Microbial strains are now spreading out of their original geographical areas of incidence and previously adequate morphological identification methods often must be accompanied by a phenotypic characterization for the successful microbial identification. The fungal genus Monilinia represents a suitable example. Monilinia species represent important fruit pathogens responsible for major losses in fruit production. Four closely related spp. of Monilinia: Monilinia laxa, Monilinia fructigena, Monilinia fructicola and Monilia polystroma have been yet identified. However, the classical characterization methods are not sufficient for current requirements, especially for phytosanitary purposes. In this study, rapid and reproducible methods have been developed for the characterization of Monilinia spp. based on the utilization of five well-established analytical techniques: CZE, CIEF, gel IEF, SDS-PAGE and MALDI-TOF MS, respectively. The applicability of these techniques for the identification of unknown spores of Monilinia spp. collected from infected fruits was also evaluated. It was found that isoelectric points, migration velocities or the protein patterns can be used as the identification markers in the case of cultivated filamentous fungi. Moreover, the results obtained by capillary electromigration techniques are independent on the host origin of the spores. On the other hand, the host origin of the fungi can play an important role in the precise fungi identification by the other techniques.