RESUMEN
Clinical trials have shown the safety of mesenchymal stem/stromal cells (MSCs) transplantation, but the effectiveness of these treatments is limited. Since, transplanted MSCs will undergo metabolic disturbances in the bloodstream, we investigated the influence of blood plasmas of type 2 diabetes (T2D) patients on MSCs viability and examined whether apolipoprotein A-I (apoA-I) could protect cells from stressful conditions of serum deprivation (SD), hypoxia, and elevated concentrations of reactive oxygen species (ROS). ApoA-I exhibits anti-inflammatory, immune activities, improves glycemic control, and is suitable for T2D patients but its influence on MSCs remains unknown. For the first time we have shown that apoA-I decreases intracellular ROS and supports proliferative rate of MSCs, thereby increasing cell count in oxidation conditions. ApoA-I did not influence cell cycle when MSCs were predominantly in the G0/G1 phases under conditions of SD/hypoxia, activated proliferation rapidly, and reduced apoptosis during MSCs transition to the oxygenation or oxidation conditions. Finally, it was found that the blood plasma of T2D individuals had a cytotoxic effect on MSСs in 39% of cases and had a wide variability of antioxidant properties. ApoA-I protects cells under all adverse conditions and can increase the efficiency of MSCs transplantation in T2D patients.
Asunto(s)
Apolipoproteína A-I/metabolismo , Células Madre Mesenquimatosas/metabolismo , Estrés Fisiológico , Animales , Apolipoproteína A-I/química , Apolipoproteína A-I/farmacología , Apoptosis , Hipoxia de la Célula , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo , Conformación Proteica en Hélice alfa , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Nicho de Células Madre , Estrés Fisiológico/efectos de los fármacosRESUMEN
Traumatic brain injury (TBI) induces a cascade of primary and secondary events resulting in impairment of neuronal networks that eventually determines clinical outcome. The dynorphins, endogenous opioid peptides, have been implicated in secondary injury and neurodegeneration in rodent and human brain. To gain insight into the role of dynorphins in the brain's response to trauma, we analyzed short-term (1-day) and long-term (7-day) changes in dynorphin A (Dyn A) levels in the frontal cortex, hippocampus, and striatum, induced by unilateral left-side or right-side cortical TBI in mice. The effects of TBI were significantly different from those of sham surgery (Sham), while the sham surgery also produced noticeable effects. Both sham and TBI induced short-term changes and long-term changes in all three regions. Two types of responses were generally observed. In the hippocampus, Dyn A levels were predominantly altered ipsilateral to the injury. In the striatum and frontal cortex, injury to the right (R) hemisphere affected Dyn A levels to a greater extent than that seen in the left (L) hemisphere. The R-TBI but not L-TBI produced Dyn A changes in the striatum and frontal cortex at 7 days after injury. Effects of the R-side injury were similar in the two hemispheres. In naive animals, Dyn A was symmetrically distributed between the two hemispheres. Thus, trauma may reveal a lateralization in the mechanism mediating the response of Dyn A-expressing neuronal networks in the brain. These networks may differentially mediate effects of left and right brain injury on lateralized brain functions.
Asunto(s)
Lesiones Encefálicas/metabolismo , Dinorfinas/metabolismo , Análisis de Varianza , Animales , Cuerpo Estriado/metabolismo , Lateralidad Funcional/fisiología , Hipocampo/metabolismo , Masculino , Ratones , Corteza Prefrontal/metabolismo , RadioinmunoensayoRESUMEN
Malaria sporozoites must cross at least two cell barriers to reach their initial site of replication in the mammalian host. After transmission into the skin by an infected mosquito, they migrate towards small dermal capillaries, traverse the vascular endothelial layer, and rapidly home to the liver. To infect hepatocytes, the parasites must cross the sinusoidal cell layer, composed of specialized highly fenestrated sinusoidal endothelia and Kupffer cells, the resident macrophages of the liver (Fig. 1). The exact route Plasmodium sporozoites take to hepatocytes has been subject of controversial discussions for many years. Recent cell biological, microscopic, and genetic approaches have considerably enhanced our understanding of the initial events leading to the establishment of a malaria infection in the liver.
Asunto(s)
Hígado/parasitología , Plasmodium/patogenicidad , Esporozoítos/fisiología , Animales , Hepatocitos/inmunología , Hepatocitos/parasitología , Humanos , Macrófagos del Hígado/inmunología , Macrófagos del Hígado/parasitología , Malaria/inmunología , Malaria/parasitología , Modelos Biológicos , Plasmodium/inmunología , Plasmodium/fisiología , Esporozoítos/inmunologíaRESUMEN
Chronic pain is maintained in part by long-lasting neuroplastic changes in synapses and several proteins critical for synaptic plasticity are degraded by the ubiquitin-proteasome system (UPS). Here, we show that proteasome inhibitors administered intrathecally or subcutaneously prevented the development and reversed nerve injury-induced pain behavior. They also blocked pathological pain induced by sustained administration of morphine or spinal injection of dynorphin A, an endogenous mediator of chronic pain. Proteasome inhibitors blocked mechanical allodynia and thermal hyperalgesia in all three pain models although they did not modify responses to mechanical stimuli, but partially inhibited responses to thermal stimuli in control rats. In the spinal cord, these compounds abolished the enhanced capsaicin-evoked calcitonin gene-related peptide (CGRP) release and dynorphin A upregulation, both elicited by nerve injury. Model experiments demonstrated that the inhibitors may act directly on dynorphin-producing cells, blocking dynorphin secretion. Thus, the effects of proteasome inhibitors on chronic pain were apparently mediated through several cellular mechanisms indispensable for chronic pain, including those of dynorphin A release and postsynaptic actions, and of CGRP secretion. Levels of several UPS proteins were reduced in animals with neuropathic pain, suggesting that UPS downregulation, like effects of proteasome inhibitors, counteracts the development of chronic pain. The inhibitors did not produce marked or disabling motor disturbances at doses that were used to modify chronic pain. These results suggest that the UPS is a critical intracellular regulator of pathological pain, and that UPS-mediated protein degradation is required for maintenance of chronic pain and nociceptive, but not non-nociceptive responses in normal animals.
Asunto(s)
Dolor/enzimología , Dolor/fisiopatología , Complejo de la Endopetidasa Proteasomal/fisiología , Médula Espinal/enzimología , Ubiquitina/fisiología , Animales , Línea Celular Tumoral , Enfermedad Crónica , Masculino , Ratones , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Dolor/tratamiento farmacológico , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Inhibidores de Proteasoma , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatología , Ubiquitina/antagonistas & inhibidoresRESUMEN
After transmission by infected mosquitoes, malaria sporozoites rapidly travel to the liver. To infect hepatocytes, sporozoites traverse Kupffer cells, but surprisingly, the parasites are not killed by these resident macrophages of the liver. Here we show that Plasmodium sporozoites and recombinant circumsporozoite protein (CSP) suppress the respiratory burst in Kupffer cells. Sporozoites and CSP increased the intracellular concentration of cyclic adenosyl mono-phosphate (cAMP) and inositol 1,4,5-triphosphate in Kupffer cells, but not in hepatocytes or liver endothelia. Preincubation with cAMP analogues or inhibition of phosphodiesterase also inhibited the respiratory burst. By contrast, adenylyl cyclase inhibition abrogated the suppressive effect of sporozoites. Selective protein kinase A (PKA) inhibitors failed to reverse the CSP-mediated blockage and stimulation of the exchange protein directly activated by cAMP (EPAC), but not PKA inhibited the respiratory burst. Both blockage of the low-density lipoprotein receptor-related protein (LRP-1) with receptor-associated protein and elimination of cell surface proteoglycans inhibited the cAMP increase in Kupffer cells. We propose that by binding of CSP to LRP-1 and cell surface proteoglycans, malaria sporozoites induce a cAMP/EPAC-dependent, but PKA-independent signal transduction pathway that suppresses defence mechanisms in Kupffer cells. This allows the sporozoites to safely pass through these professional phagocytes and to develop inside neighbouring hepatocytes.
Asunto(s)
Macrófagos del Hígado/efectos de los fármacos , Plasmodium/metabolismo , Proteínas Protozoarias/farmacología , Estallido Respiratorio/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Hepatocitos/metabolismo , Hepatocitos/parasitología , Humanos , Macrófagos del Hígado/citología , Macrófagos del Hígado/metabolismo , Malaria/parasitología , Microscopía Fluorescente , Modelos Biológicos , Plasmodium/genética , Plasmodium/crecimiento & desarrollo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Esporozoítos/crecimiento & desarrollo , Esporozoítos/metabolismoRESUMEN
The initial site of replication for Plasmodium parasites in mammalian hosts are hepatocytes, cells that offer unique advantages for the extensive parasite replication occurring prior to the erythrocytic phase of the life cycle. The liver is the metabolic centre of the body and has an unusual relationship to the immune system. However, to reach hepatocytes, sporozoites must cross the sinusoidal barrier, composed of specialized endothelia and Kupffer cells, the resident macrophages of the liver. Mounting evidence suggests that, instead of taking what would seem a safer route through endothelia, the parasites traverse Kupffer cells yet suffer no harm. Kupffer cells have a broad range of responses towards incoming microorganisms, toxins and antigens which depend on the nature of the intruder, the experimental conditions and the environmental circumstances. Kupffer cells may become activated or remain anergic, produce pro- or anti-inflammatory mediators. Consequently, outcomes are diverse and include development of immunity or tolerance, parenchymal necrosis or regeneration, chronic cirrhotic transformation or acute liver failure. Here we review data concerning the unique structural and functional characteristics of Kupffer cells and their interactions with Plasmodium sporozoites in the context of a model in which these hepatic macrophages function as the sporozoite gate to the liver.
Asunto(s)
Macrófagos del Hígado/parasitología , Malaria/parasitología , Plasmodium/fisiología , Esporozoítos/fisiología , Animales , Humanos , Tolerancia Inmunológica , Macrófagos del Hígado/citología , Macrófagos del Hígado/inmunología , Estadios del Ciclo de Vida , Hígado/citología , Hígado/inmunología , Hígado/parasitología , Malaria/inmunología , Plasmodium/inmunología , Plasmodium/metabolismo , Vena Porta/inmunología , Vena Porta/parasitología , Esporozoítos/inmunología , Esporozoítos/metabolismoRESUMEN
The diversity of peptide ligands for a particular receptor may provide a greater dynamic range of functional responses, while maintaining selectivity in receptor activation. Dynorphin A (Dyn A), and dynorphin B (Dyn B) are endogenous opioid peptides that activate the kappa-opioid receptor (KOR). Here, we characterized interactions of big dynorphin (Big Dyn), a 32-amino acid prodynorphin-derived peptide consisting of Dyn A and Dyn B, with human KOR, mu- (hMOR) and delta- (hDOR) opioid receptors and opioid receptor-like receptor 1 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dyn A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dyn B. Dyn A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn, while Dyn B demonstrated low selectivity. In contrast, Big Dyn activated G proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR-mediated activation of G proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of G proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of radioimmunoassay (RIA) and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid (CSF) with the ratio of Big Dyn and Dyn B being approximately 1:3. The presence in the brain implies that Big Dyn, along with other dynorphins, is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR-mediated activation of G proteins.
Asunto(s)
Unión Competitiva/fisiología , Sistema Nervioso Central/metabolismo , Dinorfinas/líquido cefalorraquídeo , Receptores Opioides kappa/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Sistema Nervioso Central/efectos de los fármacos , Líquido Cefalorraquídeo/metabolismo , Dinorfinas/química , Dinorfinas/genética , Endorfinas/líquido cefalorraquídeo , Endorfinas/química , Endorfinas/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Ligandos , Ratones , Ratones Noqueados , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Dolor/genética , Dolor/metabolismo , Dolor/fisiopatología , Radioinmunoensayo , Ensayo de Unión Radioligante , Receptores Opioides/efectos de los fármacos , Receptores Opioides/metabolismo , Receptores Opioides delta/efectos de los fármacos , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/efectos de los fármacos , Receptores Opioides mu/efectos de los fármacos , Receptores Opioides mu/metabolismo , Receptor de NociceptinaRESUMEN
Dynorphins, endogenous kappa-opioid agonists widely expressed in the central nervous system, have been reported to increase following diverse pathophysiological processes, including excitotoxicity, chronic inflammation, and traumatic injury. These peptides have been implicated in cognitive impairment, especially that associated with aging. To determine whether absence of dynorphin confers any beneficial effect on spatial learning and memory, knockout mice lacking the coding exons of the gene encoding its precursor prodynorphin (Pdyn) were tested in a water maze task. Learning and memory assessment using a 3-day water maze protocol demonstrated that aged Pdyn knockout mice (13-17 months) perform comparatively better than similarly aged wild-type (WT) mice, based on acquisition and retention probe trial indices. There was no genotype effect on performance in the cued version of the swim task nor on average swim speed, suggesting the observed genotype effects are likely attributable to differences in cognitive rather than motor function. Young (3-6 months) mice performed significantly better than aged mice, but in young mice, no genotype difference was observed. To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus and frontal cortex of WT 129SvEv mice. Quantitative radioimmunoassay demonstrated that dynorphin A levels in frontal cortex, but not hippocampus, of 12- and 24-month mice were significantly elevated compared to 3-month mice. Although the underlying mechanisms have yet to be elucidated, the results suggest that chronic increases in endogenous dynorphin expression with age, especially in frontal cortex, may adversely affect learning and memory.
Asunto(s)
Envejecimiento/fisiología , Encefalinas/fisiología , Aprendizaje por Laberinto/fisiología , Precursores de Proteínas/fisiología , Percepción Espacial/fisiología , Conducta Espacial/fisiología , Factores de Edad , Análisis de Varianza , Animales , Conducta Animal/fisiología , Química Encefálica , Dinorfinas/metabolismo , Encefalinas/deficiencia , Lóbulo Frontal/metabolismo , Regulación de la Expresión Génica/fisiología , Genotipo , Hipocampo/metabolismo , Ratones , Ratones Noqueados , Precursores de Proteínas/deficiencia , Radioinmunoensayo/métodos , Tiempo de Reacción/genética , Retención en Psicología/fisiología , Natación , Factores de TiempoRESUMEN
Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.
Asunto(s)
Membrana Celular/metabolismo , Dinorfinas/química , Neuropéptidos/metabolismo , Animales , Células COS , Línea Celular , Núcleo Celular/metabolismo , Cerebelo/metabolismo , Dicroismo Circular , Clatrina/química , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Cinética , Microscopía Confocal , Neuronas/metabolismo , Células PC12 , Péptidos/química , Unión Proteica , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Espectrometría de Masa por Ionización de Electrospray , Factores de TiempoRESUMEN
A novel mechanism of protein biosynthesis regulation in liver under the action of reduced forms of steroid hormones (tetrahydrocortisol) and apolipoprotein A-I (apoA-I) is presented. Kupffer cells play an important role in uptake of the cortisol and high density lipoproteins (HDL) as well as in formation of the active complex, tetrahydrocortisol+apolipoprotein A-I (THC-apoA-I). If macrophages are stimulated by lipopolysaccharides (LPS), these processes enhance dramatically, thus causing parallel activation of nucleolar DNA expression and ribosome formation in hepatocytes. THC-apoA-I complex accelerates protein biosynthesis in primary cultures of hepatocytes, but not in macrophages and endotheliocytes.