RESUMO
Immunometabolism has emerged as a key focus for immunologists, with metabolic change in immune cells becoming as important a determinant for specific immune effector responses as discrete signaling pathways. A key output for these changes involves post-translational modification (PTM) of proteins by metabolites. Products of glycolysis and Krebs cycle pathways can mediate these events, as can lipids, amino acids, and polyamines. A rich and diverse set of PTMs in macrophages and T cells has been uncovered, altering phenotype and modulating immunity and inflammation in different contexts. We review the recent findings in this area and speculate whether they could be of use in the effort to develop therapeutics for immune-related diseases.
Assuntos
Doenças do Sistema Imunitário/metabolismo , Imunoterapia/tendências , Inflamação/metabolismo , Macrófagos/metabolismo , Linfócitos T/metabolismo , Animais , Ciclo do Ácido Cítrico , Glicólise , Humanos , Doenças do Sistema Imunitário/terapia , Imunidade , Processamento de Proteína Pós-Traducional , Transdução de Sinais/imunologiaRESUMO
Toxicity tests evaluated chronic and sublethal effects of fog oil (FO) on a freshwater endangered fish. FO is released during military training as an obscurant smoke that can drift into aquatic habitats. Fountain darters, Etheostoma fonticola, of four distinct life stages were exposed under laboratory conditions to three forms of FO. FO was vaporized into smoke and allowed to settle onto water, violently agitated with water, and dosed onto water followed by photo-oxidization by ultraviolet irradiation. Single smoke exposures of spawning adult fish did not affect egg production, egg viability, or adult fish survival in 21-day tests. Multiple daily smoke exposures induced mortality after 5 days for larvae fish. Larvae and juvenile fish were more sensitive than eggs in 96-h lethal concentration (LC50) tests with FOwater mixtures and photo-oxidized FO. Water-soluble FO components photo-modified by ultraviolet radiation were the most toxic, thus indicating the value of examining weathering and aging of chemicals for the best determination of environmental impact.
Assuntos
Óleos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Espécies em Perigo de Extinção , Militares/educação , Percas , Medição de Risco , Tempo (Meteorologia)RESUMO
Following the fusion of synaptic vesicles with the presynaptic plasma membrane of nerve terminals by the process of exocytosis, synaptic-vesicle components are recycled to replenish the vesicle pool. Here we use a pH-sensitive green fluorescent protein to measure the residence time of VAMP, a vesicle-associated SNARE protein important for membrane fusion, on the surfaces of synaptic terminals of hippocampal neurons following exocytosis. The time course of VAMP retrieval depends linearly on the amount of VAMP that is added to the plasma membrane, with retrieval occurring between about 4 seconds and 90 seconds after exocytosis, and newly internalized vesicles are rapidly acidified. These data are well described by a model in which endocytosis appears to be saturable, but proceeds with an initial maximum velocity of about one vesicle per second. We also find that, following exocytosis, a portion of the newly inserted VAMP appears on the surface of the axon.
Assuntos
Hipocampo/citologia , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Potenciais de Ação/fisiologia , Animais , Membrana Celular/metabolismo , Células Cultivadas , Endocitose/fisiologia , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Cinética , Fusão de Membrana/fisiologia , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Terminações Pré-Sinápticas/metabolismo , Proteínas R-SNARE , Ratos , Ratos Sprague-Dawley , Proteínas SNARERESUMO
The nature of synaptic vesicle recycling at nerve terminals has been a subject of considerable debate for >35 years. Here, we report the use of an optical strategy that allows the exocytosis and retrieval of synaptic components to be tracked in real time at single-molecule sensitivity in living nerve terminals. This approach has allowed us to examine the recycling of synaptic vesicles in response to single action potentials. Our results show that, after exocytosis, individual synaptic vesicles are retrieved by a stochastic process with an exponential distribution of delay times, with a mean time of approximately 14 s. We propose that evidence for fast endocytosis, such as that proposed to support the presence of kiss-and-run, is likely explained by the stochastic nature of a slower process.
Assuntos
Endocitose , Exocitose , Microscopia de Fluorescência/métodos , Vesículas Sinápticas/fisiologia , Animais , Células Cultivadas , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Fotodegradação , Ratos , Ratos Sprague-DawleyRESUMO
Cell-cell adhesion is at the top of a molecular cascade of protein interactions that leads to the remodeling of epithelial cell structure and function. The earliest events that initiate this cascade are poorly understood. Using high resolution differential interference contrast microscopy and retrospective immunohistochemistry, we observed that cell-cell contact in MDCK epithelial cells consists of distinct stages that correlate with specific changes in the interaction of E-cadherin with the cytoskeleton. We show that formation of a stable contact is preceded by numerous, transient contacts. During this time and immediately following formation of a stable contact, there are no detectable changes in the distribution, relative amount, or Triton X-100 insolubility of E-cadherin at the contact. After a lag period of approximately 10 min, there is a rapid acquisition of Triton X-100 insolubility of E-cadherin localized to the stable contact. Significantly, the total amount of E-cadherin at the contact remains unchanged during this time. The increase in the Triton X-100 insoluble pool of E-cadherin does not correlate with changes in the distribution of actin or fodrin, suggesting that the acquisition of the Triton X-100 insolubility is due to changes in E-cadherin itself, or closely associated proteins such as the catenins. The 10 minute lag period, and subsequent prompt and localized nature of E-cadherin reorganization indicate a form of signaling is occurring.
Assuntos
Caderinas/fisiologia , Adesão Celular , Actinas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Citoesqueleto/ultraestrutura , Cães , Células Epiteliais , Imunofluorescência , Técnicas In Vitro , Proteínas dos Microfilamentos/metabolismo , Polietilenoglicóis/farmacologia , Fatores de Tempo , Gravação em VídeoRESUMO
The synapsins are a family of four neuron-specific phosphoproteins that have been implicated in the regulation of neurotransmitter release. Nevertheless, knock-out mice lacking synapsin Ia and Ib, family members that are major substrates for cAMP and Ca2+/ Calmodulin (CaM)-dependent protein kinases, show limited phenotypic changes when analyzed electrophysiologically (Rosahl, T.W., D. Spillane, M. Missler, J. Herz, D.K. Selig, J.R. Wolff, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1995. Nature (Lond.). 375: 488-493; Rosahl, T.W., M. Geppert, D. Spillane, D., J. Herz, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1993. Cell. 75:661-670; Li, L., L.S. Chin, O. Shupliakov, L. Brodin, T.S. Sihra, O. Hvalby, V. Jensen, D. Zheng, J.O. McNamara, P. Greengard, and P. Andersen. 1995. Proc. Natl. Acad. Sci. USA. 92:9235-9239; see also Pieribone, V.A., O. Shupliakov, L. Brodin, S. Hilfiker-Rothenfluh, A.J. Czernik, and P. Greengard. 1995. Nature (Lond.). 375:493-497). Here, using the optical tracer FM 1-43, we characterize the details of synaptic vesicle recycling at individual synaptic boutons in hippocampal cell cultures derived from mice lacking synapsin I or wild-type equivalents. These studies show that both the number of vesicles exocytosed during brief action potential trains and the total recycling vesicle pool are significantly reduced in the synapsin I-deficient mice, while the kinetics of endocytosis and synaptic vesicle repriming appear normal.
Assuntos
Sinapsinas/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Células Cultivadas , Endocitose , Exocitose , Corantes Fluorescentes/química , Hipocampo/citologia , Cinética , Camundongos , Camundongos Knockout , Compostos de Piridínio/química , Compostos de Amônio Quaternário/química , Sinapsinas/genéticaRESUMO
Strong steric interactions among proteins on crowded living cell surfaces were revealed by measurements of the equilibrium spatial distributions of proteins in applied potential gradients. The fraction of accessible surface occupied by mobile surface proteins can be accurately represented by including steric exclusion in the statistical thermodynamic analysis of the data. The analyses revealed enhanced, concentration-dependent activity coefficients, implying unanticipated thermodynamic activity even at typical cell surface receptor concentrations.
Assuntos
Membrana Celular/fisiologia , Fluidez de Membrana , Proteínas de Membrana/fisiologia , Animais , Ratos , Receptores Fc/fisiologia , Receptores de IgE , Termodinâmica , Células Tumorais CultivadasRESUMO
Restriction of sodium, potassium adenosine triphosphatase (Na+,K(+)-ATPase) to either the apical or basal-lateral membrane domain of polarized epithelial cells is fundamental to vectorial ion and solute transport in many tissues and organs. A restricted membrane distribution of Na+,K(+)-ATPase in Madin-Darby canine kidney (MDCK) epithelial cells was found experimentally to be generated by preferential retention of active enzyme in the basal-lateral membrane domain and selective inactivation and loss from the apical membrane domain, rather than by vectorial targeting of newly synthesized protein from the Golgi complex to the basal-lateral membrane domain. These results show how different distributions of the same subunits of Na+,K(+)-ATPase may be generated in normal polarized epithelial and in disease states.
Assuntos
Membrana Celular/enzimologia , Polaridade Celular , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Sítios de Ligação , Comunicação Celular , Linhagem Celular , Membrana Celular/fisiologia , Cães , Epitélio/enzimologia , Epitélio/fisiologia , Cinética , Ouabaína/metabolismoRESUMO
Careful functional dissection of mouse neuromuscular junctions (NMJs) formed in the absence of NCAM reveal that in spite of relatively normal morphology and ultrastructure, the presynaptic terminals have profound alterations in the mechanisms of recycling of synaptic vesicles. These alterations, including the appearance of a brefeldin-sensitive pathway, leave synaptic transmission impaired for certain types of activity. NCAMs thus appear to play a critical role in the molecular organization and function of synaptic terminals.
Assuntos
Moléculas de Adesão de Célula Nervosa/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Humanos , Transmissão Sináptica/fisiologiaRESUMO
Using the fluorescent membrane label FM 1-43, we have measured the release, reuptake, and repriming of synaptic vesicles in response to action potential stimulation of cultured hippocampal neurons. We find that approximately 90% of a recycling vesicle pool is released during 60 s of 10 Hz action potential firing, and that a single action potential releases approximately 0.5% of that pool. Our data also indicate that endocytic reuptake of vesicle membrane externalized by 10 Hz action potentials lags exocytosis, with a half-time on the order of 20 s, and that the minimum time for repriming of an endocytosed vesicle is on the order of 15 s. Finally, we find that once vesicles have undergone this repriming period, they become functionally mixed in the vesicle pool within a few minutes; the probability of release for recently recycled vesicles is indistinguishable from that of vesicles that have resided within the bouton for much longer periods.
Assuntos
Hipocampo/fisiologia , Sinapses/fisiologia , Vesículas Sinápticas/fisiologia , Potenciais de Ação , Animais , Células Cultivadas , Estimulação Elétrica , Endocitose , Exocitose , Cinética , Microscopia de Fluorescência , Ratos , Ratos Sprague-DawleyRESUMO
We have studied synaptic plasticity in hippocampal cell cultures using a new imaging approach that allows unambiguous discrimination of presynaptic function at the level of single synaptic boutons. Employing a protocol designed to test for use-dependent plasticity resembling N-methyl-D-aspartate receptor-dependent long-term potentiation (NMDA-type LTP), we find that brief tetanic stimuli induce a potentiation of evoked synaptic vesicle turnover that lasts for at least 1 hr. Induction of this clearly presynaptic potentiation is blocked by putative postsynaptic glutamate receptor antagonists, suggesting that a retrograde induction signal might be involved. Potentiation appears to occur approximately equally at boutons of low and high initial release probabilities, and evidently does not involve an increase in the size of the total recycling synaptic vesicle pool.
Assuntos
Sinapses/fisiologia , Vesículas Sinápticas/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Potenciais de Ação , Animais , Estimulação Elétrica , Eletrofisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/citologia , Plasticidade Neuronal , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
We used the fluorescent membrane probe FM 1-43 to label recycling synaptic vesicles within the presynaptic boutons of dissociated hippocampal neurons in culture. Quantitative time-lapse fluorescence imaging was employed in combination with rapid superfusion techniques to study the dynamics of synaptic vesicles within single boutons. This approach enabled us to measure exocytosis and to analyze the kinetics of endocytosis and the preparation of endocytosed vesicles for re-release (repriming). Our measurements indicate that under sustained membrane depolarization, endocytosis persists much longer than exocytosis, with a t1/2 approximately 60 s (approximately 24 degrees C); once internalized, vesicles become reavailable for exocytosis in approximately 30 s. Furthermore, we have shown that endocytosis is not dependent on membrane potential and, unlike exocytosis, that it is independent of extracellular Ca2+.
Assuntos
Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Compostos de Piridínio , Compostos de Amônio Quaternário , Vesículas Sinápticas/fisiologia , Vesículas Sinápticas/ultraestrutura , Animais , Células Cultivadas , Endocitose , Exocitose , Imunofluorescência , Corantes Fluorescentes , Hipocampo/citologia , Hipocampo/fisiologia , Hipocampo/ultraestrutura , Cinética , Potenciais da Membrana , Neurônios/ultraestrutura , Tratos Piramidais/citologia , Tratos Piramidais/fisiologia , Tratos Piramidais/ultraestrutura , Ratos , Ratos Sprague-Dawley , Sinapsinas/análiseRESUMO
A pH-sensitive form of green-fluorescent protein (GFP) fused to the lumenal domain of VAMP (synapto-pHluorin) provides a sensitive optical probe to track the net balance between exocytosis and endocytosis of this protein at small synaptic terminals of the central nervous system. Here we used a reversible proton-pump blocker that prevents vesicle re-acidification upon endocytosis to trap vesicles in the alkaline state during recycling. In combination with optical measurements of synapto-pHluorin, we used alkaline trapping to examine the kinetic components of exocytosis and endocytosis separately at synaptic terminals. Using this approach, we show that, in addition to controlling exocytosis, intracellular calcium levels tightly regulate the speed of endocytosis, increasing it to a maximal speed of approximately one vesicle per second.
Assuntos
Cálcio/fisiologia , Endocitose/fisiologia , Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Potenciais de Ação/fisiologia , Álcalis/metabolismo , Animais , Estimulação Elétrica , Exocitose/fisiologia , Hipocampo/fisiologia , Membranas Intracelulares/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas SNARE , Sinapses/fisiologiaRESUMO
Presynaptic modulation of synaptic transmission provides an important basis for control of synaptic function. The synapsins, a family of highly conserved proteins associated with synaptic vesicles, have long been implicated in the regulation of neurotransmitter release. However, direct physiological measurements of the molecular mechanisms have been lacking. Here we show that in living hippocampal terminals, green fluorescent protein (GFP)-labeled synapsin Ia dissociates from synaptic vesicles, disperses into axons during action potential (AP) firing, and reclusters to synapses after the cessation of synaptic activity. Using various mutated forms of synapsin Ia that prevent phosphorylation at specific sites, we performed simultaneous FM 4-64 measurements of vesicle pool mobilization along with synapsin dispersion kinetics. These studies indicate that the rate of synapsin dispersion is controlled by phosphorylation, which in turn controls the kinetics of vesicle pool turnover. Thus synapsin acts as a phosphorylation-state-dependent regulator of synaptic vesicle mobilization, and hence, neurotransmitter release.
Assuntos
Potenciais de Ação/fisiologia , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Sinapsinas/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Vetores Genéticos , Proteínas de Fluorescência Verde , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Imuno-Histoquímica , Indicadores e Reagentes/metabolismo , Cinética , Proteínas Luminescentes/genética , Camundongos , Camundongos Knockout , Fosforilação , Terminações Pré-Sinápticas/ultraestrutura , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Sinapsinas/genética , Vesículas Sinápticas/ultraestrutura , Sinaptofisina/metabolismo , Fatores de TempoRESUMO
Understanding the detailed molecular events that support chemical synaptic transmission requires high-resolution methods that provide quantitative information combined with molecular specificity. In recent years, many new technological approaches, including genetically encoded fluorescent indicators, ultra-thin sectioning, and live-cell imaging have been brought to bear on understanding the cell biology and physiology of presynaptic terminals.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Terminações Pré-Sinápticas/química , Animais , Corantes Fluorescentes/análise , Humanos , Neurônios/química , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Compostos de Piridínio/análise , Compostos de Amônio Quaternário/análise , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/química , Vesículas Sinápticas/fisiologiaRESUMO
The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central event in many signal transduction processes in cellular physiology. Recent advances in optical techniques for measuring [Ca2+]i as well as developments in quantitative low light level fluorescence microscopy have led to the application of these methods to the study of subcellular [Ca2+]i in many biological systems. In the following paper we describe some techniques in our laboratory to provide quantitative high spatio-temporal resolution measurements of [Ca2+]i in individual living cells during the signal transduction of cell surface receptor ligand interactions. In particular, we are studying the changes in [Ca2+]i induced by the micro-aggregation of immunoglobulin E (IgE) receptor complexes on the surface of rat basophilic leukemia (RBL) cells (a tumor mast cell line) by multivalent antigen. We seek to understand the mechanisms which are involved in the detection of these cell surface events which lead to changes in [Ca2+]i as well as the interactions between the various subcellular components which impart the delicate control of [Ca2+]i during cellular stimulation. The limitations and properties of the technology used for these studies will be discussed, and some illustrative examples of the type of [Ca2+]i changes found in this biological system will be given.
Assuntos
Cálcio/metabolismo , Transdução de Sinais , Animais , Antígenos , Antígenos de Diferenciação de Linfócitos B/metabolismo , Benzofuranos , Fura-2 , Processamento de Imagem Assistida por Computador , Imunoglobulina E/metabolismo , Leucemia Basofílica Aguda , Ratos , Agregação de Receptores , Receptores Fc/metabolismo , Receptores de IgE , Células Tumorais CultivadasRESUMO
Memory functioning was contrasted in 40 schizophrenic patients with and without tardive dyskinesia (TD). Visual and verbal memory tests were used to investigate specific types of impairments. The presence of TD was ascertained using the Abnormal Involuntary Movement Scale (AIMS). TD patients scored significantly lower than non-TD patients on two measures of visual learning, though no differences were found for verbal learning or immediate recall. These results are consistent with previous reports that schizophrenic patients with TD demonstrate impaired cognitive functioning. They also raise the possibility that the neurochemical and structural changes underlying TD may produce specific deficits in memory for visual materials. In addition, a significant relationship was found between total score on the Brief Psychiatric Rating Scale (BPRS) and performance on all of the test measures included in the cognitive test battery. This demonstrates the importance of attending to the overall level of schizophrenic symptomatology when evaluating results from experimental learning tasks.
Assuntos
Discinesia Induzida por Medicamentos/psicologia , Transtornos da Memória/complicações , Esquizofrenia/complicações , Psicologia do Esquizofrênico , Humanos , MasculinoRESUMO
OBJECTIVE: To determine the predictors of outcome in cardiac surgical patients with prolonged ICU stay. DESIGN: Inception cohort with retrospective chart review. SETTING: Adult cardiovascular ICU. PATIENTS: All patients admitted after cardiac surgery who stayed in ICU for at least 14 consecutive days. INTERVENTIONS: Collection of data, including preoperative demographics, comorbidity, routine laboratory testing, surgical procedure, duration of cardiopulmonary bypass and aortic cross-clamping, postoperative requirement for transfusion and intra-aortic balloon counterpulsation, and postoperative indexes of organ dysfunction 14 and 28 days after surgery. An organ failure score (OFS) was calculated for days 1, 14, and 28. OUTCOME MEASURES: Hospital mortality. RESULTS: One hundred forty-one of 324 (43.5%) ICU admissions lasting at least 14 days resulted in hospital mortality. Seventy-four of 166 (45%) ICU admissions lasting at least 28 days resulted in hospital mortality. Preoperative demographics, morbidity, and indexes of organ failure in the first 24 h after surgery were not predictive of hospital mortality. Indexes of organ failure predictive of hospital death at 14 days included requirement for epinephrine infusion, diminished Glasgow coma scale, requirement for dialysis, greater value of BUN, lower value of creatinine, greater value of bilirubin, greater value of arterial PCO2, lower platelet count, and lower value of serum albumin. After a 28-day stay in ICU, the indexes of organ failure predictive of hospital mortality included requirement for dopamine or norepinephrine infusions, diminished Glasgow coma score, greater value of bilirubin, greater value of arterial PCO2, lower value of serum albumin, and advanced age. The area under the receiver operating characteristic curve for the OFS on day 1 was 0.55+/-0.04 (p=0.12), on day 14 it was 0.75+/-0.03 (p<0.0001), and on day 28 it was 0.76+/-0.04 (p<0.0001). CONCLUSION: Preoperative health status and early organ failure were not predictive of late hospital mortality. The pattern of late organ failure associated with hospital mortality changed with time.