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Droplet microfluidics offers exquisite control over the flows of multiple fluids in micro-scale, enabling fabrication of advanced microspheres with precisely tuneable structures. The main goal of this work was to design monodispersed carboxylated polystyrene microspheres with a developed pore structure (a specific surface area more than 200 m2/g) using microfluidic technology. We investigated the influence of the composition of monomer phase for the stable formation of droplets. Under the stable region, the resulting microspheres (with diameter 50 µm) showed narrow size distribution having a coefficient of variation of below 2%. The obtained microspheres are characterised by morphology and surface structure by means of electron microscopy. The structure of cross-linked microspheres is investigated by solid-state 1H NMR spectroscopy. Finally, these microspheres have great potential for the effective sorption of biologically active substances (bovine serum albumin).
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Microfluídica/métodos , Microesferas , Poliestirenos/química , Albúmina Sérica Bovina/química , PorosidadRESUMEN
Blast overpressure (OB) injury in rodents has been employed for modeling the traumatic brain injury (TBI) induced by an improvised explosive device (IED) in military service personnel. IED's can cause respiratory arrest if directed at the thorax due to the fluid-tissue interface of the lungs but it is unclear what respiratory changes occur in a head-directed OB injury. The diaphragm is the primary muscle of inspiration and electromyographic (EMG) recordings from this muscle are used for recording breathing in anesthetized and conscious rats. The breathing pattern of the rodents will be recorded during the OB injury. Our results indicate that a dorsal directed closed-head OB injury results in a neurally mediated apnea followed by respiratory timing changes.
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Traumatismos por Explosión/fisiopatología , Lesiones Encefálicas/fisiopatología , Respiración , Animales , Apnea/fisiopatología , Diafragma/fisiopatología , Modelos Animales de Enfermedad , Electrodos Implantados , Electromiografía , Masculino , Presión/efectos adversos , Distribución Aleatoria , Ratas Sprague-DawleyRESUMEN
CONTEXT: New biomarkers are needed in acetaminophen (APAP) hepatotoxicity. Plasma argininosuccinate synthetase (ASS) is a promising candidate. OBJECTIVE: Characterize ASS in APAP hepatotoxicity. METHODS: ASS was measured in plasma from rodents and humans with APAP hepatotoxicity. RESULTS: In mice, ASS increased before injury, peaked before alanine aminotransferase (ALT) and decreased rapidly. Fischer rats had a greater increase in ASS relative to ALT. Patients with abnormal liver test results had very high ASS compared to controls. ASS appeared to increase early in some patients, and declined rapidly in all. CONCLUSIONS: ASS may be a useful biomarker of acute cell death in APAP hepatotoxicity.
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Acetaminofén/envenenamiento , Argininosuccinato Sintasa/sangre , Sobredosis de Droga/sangre , Animales , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
Liver and kidney damage associated with polytrauma, endotoxic shock/sepsis, and organ transplantation, are among the leading causes of the multiple organ failure. Development of novel sensitive biomarkers that detect early stages of liver and kidney injury is vital for the effective diagnostics and treatment of these life-threatening conditions. Previously, we identified several hepatic proteins, including Argininosuccinate Synthase (ASS) and sulfotransferases which were degraded in the liver and rapidly released into circulation during Ischemia/Reperfusion (I/R) injury. Here we compared sensitivity and specificity of the newly developed sandwich ELISA assays for ASS and the sulfotransferase isoform SULT2A1 with the standard clinical liver and kidney tests Alanine Aminotransferase (ALT) and Aspartate Transaminase (AST) in various pre-clinical models of acute injury. Our data suggest that ASS and SULT2A1 have superior characteristics for liver and kidney health assessment in endotoxemia, Ischemia/Reperfusion (I/R), chemical and drug-induced liver injury and may be of high potential value for clinical applications.
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Severe blast exposures are frequently complicated with fatal intracranial hemorrhages. However, many more sustain low level blasts without tissue damage detectable by brain imaging. To investigate effects of nonlethal blast on thrombin-related biomarkers, rats were subjected to two different types of head-directed blast: 1) moderate "composite" blast with strong head acceleration or 2) moderate primary blast, without head acceleration. Thrombin generation (TG) ex vivo after blast was studied by calibrated automated thrombography (CAT). In the same blood samples, we assessed maximal concentration of TG (TGmax), start time, peak time, mean time, and concentrations of protein markers for vascular/hemostatic dysfunctions: integrin α/ß, soluble endothelial selectin (sE-selectin), soluble intercellular cell adhesion molecule-1 (sICAM-1), and matrix metalloproteinases (MMP)-2, MMP-8, and MMP-13. Blast remarkably affected all TG indices. In animals exposed to "composite" blast, TGmax peaked at 6 h (â¼4.5-fold vs. control), sustained at day 1 (â¼3.8-fold increase), and declined to a 2-fold increase over control at day 7 post-blast. After primary blast, TGmax also rose to â¼4.2-fold of control at 6 h, dropped to â¼1.7-fold of control at day 1, and then exhibited a slight secondary increase at 2-fold of control at day 7. Other TG indices did not differ significantly between two types of blast exposure. The changes were also observed in other microvascular/inflammatory/hemostatic biomarkers. Integrin α/ß and sICAM-1 levels were elevated after both "composite" and primary blast at 6 h, 1 day, and 7 days. sE-selectin exhibited near normal levels after "composite" blast, but increased significantly at 7 days after primary blast; MMP-2, MMP-8, and MMP-13 slightly rose after "composite" blast and significantly increased (â¼2-4-fold) after primary blast. In summary, CAT may have a clinical diagnostic utility in combination with selected set of microvascular/inflammatory biomarkers in patients subjected to low/moderate level blast exposures.
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Traumatismos por Explosión/sangre , Lesiones Encefálicas/sangre , Trombina/metabolismo , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Traumatismos por Explosión/etiología , Lesiones Encefálicas/etiología , Calibración , Modelos Animales de Enfermedad , Fluorometría/métodos , Ratas , Trombina/análisis , Factores de TiempoRESUMEN
Explosive overpressure brain injury (OBI) impacts the lives of both military and civilian population. We hypothesize that a single exposure to OBI results in increased hypothalamic expression of oxidative stress and activation of the sympatho-adrenal medullary axis. Since a key component of blast-induced organ injury is the primary overpressure wave, we assessed selective biochemical markers of autonomic function and oxidative stress in male Sprague Dawley rats subjected to head-directed overpressure insult. Rats were subjected to single head-directed OBI with a 358kPa peak overpressure at the target. Control rats were exposed to just noise signal being placed at ~2m distance from the shock tube nozzle. Sympathetic nervous system activation of the adrenal medullae (AM) was evaluated at 6h following blast injury by assessing the expression of catecholamine biosynthesizing enzymes, tyrosine hydroxylase (TH), dopamine-ß hydroxylase (DßH), neuropeptide Y (NPY) along with plasma norepinephrine (NE). TH, DßH and NPY expression increased 20%, 25%, and 91% respectively, following OBI (P<0.05). Plasma NE was also significantly elevated by 23% (P<0.05) following OBI. OBI significantly elevated TH (49%, P<0.05) in the nucleus tractus solitarius (NTS) of the brain stem while AT1 receptor expression and NADPH oxidase activity, a marker of oxidative stress, was elevated in the hypothalamus following OBI. Collectively, the increased levels of TH, DßH and NPY expression in the rat AM, elevated TH in NTS along with increased plasma NE suggest that single OBI exposure results in increased sympathoexcitation. The mechanism may involve the elevated AT1 receptor expression and NADPH oxidase levels in the hypothalamus. Taken together, such effects may be important factors contributing to pathology of brain injury and autonomic dysfunction associated with the clinical profile of patients following OBI.
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Médula Suprarrenal/lesiones , Médula Suprarrenal/metabolismo , Traumatismos por Explosión/metabolismo , Catecolaminas/biosíntesis , Hipotálamo/lesiones , Hipotálamo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Masculino , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
BACKGROUND: The heterogeneity of liver cancer, in particular hepatocellular carcinoma (HCC), portrays the requirement of multiple targets for both its treatment and prevention. Multifaceted agents, minimally or non-toxic for normal hepatocytes, are required to address the molecular diversity of HCC, including the resistance of putative liver cancer stem cells to chemotherapy. METHODS: We designed and synthesized two fatty acid ethers of isopropylamino propanol, C16:0-AIP-1 and C18:1-AIP-2 (jointly named AIPs), and evaluated their anti-proliferative effects on the human HCC cell line Huh7 and the murine hepatoma cell line BNL 1MEA.7R.1, both in vitro and in an in vivo allograft mouse model. RESULTS: We found that AIP-1 and AIP-2 inhibited proliferation and caused cell death in both Huh7 and BNL 1MEA.7R.1 cells. Importantly, AIP-1 and AIP-2 were found to block the activation of putative liver cancer stem cells as manifested by suppression of clonal 'carcinosphere' development in growth factor-free and anchorage-free medium. The AIPs exhibited a relatively low toxicity against normal human or rat hepatocytes in primary cultures. In addition, we found that the AIPs utilized multifaceted pathways that mediate both autophagy and apoptosis in HCC, including the inhibition of AKTs and CAMK-1. In immune-competent mice, the AIPs significantly reduced BNL 1MEA.7R.1 cell-driven tumor allograft development, with a higher efficiency than sorafenib. A combination of AIP-1 + AIP-2 was most effective in reducing the tumor allograft incidence. CONCLUSIONS: AIPs represent a novel class of simple fatty acid derivatives that are effective against liver tumors via diverse pathways. They show a low toxicity towards normal hepatocytes. The addition of AIPs may represent a new avenue towards the management of chronic liver injury and, ultimately, the prevention and treatment of HCC.
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Amidas/farmacología , Éter/farmacología , Ácidos Grasos/farmacología , Neoplasias Hepáticas/patología , Trasplante de Neoplasias , Amidas/síntesis química , Amidas/química , Amidas/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sistema Libre de Células , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Éter/síntesis química , Éter/química , Éter/uso terapéutico , Ácidos Grasos/uso terapéutico , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis/efectos de los fármacos , Espectrina/metabolismo , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologíaRESUMEN
Argininosuccinate synthase (ASS) plays an important role in regulating metabolic functions in mammals. We previously reported that hepatic ASS is released into circulation at very high concentrations in response to endotoxin and acute liver injury. We propose that ASS may serve as a novel biomarker for various inflammatory conditions. Our data showed that ASS accumulated in serum and urine of septic, obese or tumor mice in a condition-dependent fashion. Moreover, ASS significantly increased in urine within the first week after tumor cell implantation in mice which subsequently develop tumors. These results suggest that ASS is a novel biomarker increased upon diverse inflammatory conditions.
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Argininosuccinatoliasa/sangre , Argininosuccinatoliasa/orina , Carcinoma Hepatocelular/orina , Neoplasias Hepáticas Experimentales/orina , Obesidad/orina , Sepsis/orina , Animales , Bilis/química , Biomarcadores/sangre , Biomarcadores/orina , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patología , Inflamación , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas Experimentales/sangre , Neoplasias Hepáticas Experimentales/diagnóstico , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/sangre , Obesidad/diagnóstico , Obesidad/patología , Sepsis/sangre , Sepsis/diagnóstico , Sepsis/patologíaRESUMEN
A number of experimental models of blast brain injury have been implemented in rodents and larger animals. However, the variety of blast sources and the complexity of blast wave biophysics have made data on injury mechanisms and biomarkers difficult to analyze and compare. Recently, we showed the importance of rat position toward blast generated by an external shock tube. In this study, we further characterized blast producing moderate traumatic brain injury and defined "composite" blast and primary blast exposure set-ups. Schlieren optics visualized interaction between the head and a shock wave generated by external shock tube, revealing strong head acceleration upon positioning the rat on-axis with the shock tube (composite blast), but negligible skull movement upon peak overpressure exposure off-axis (primary blast). Brain injury signatures of a primary blast hitting the frontal head were assessed and compared to damage produced by composite blast. Low to negligible levels of neurodegeneration were found following primary blast compared to composite blast by silver staining. However, persistent gliosis in hippocampus and accumulation of GFAP/CNPase in circulation was detected after both primary and composite blast. Also, markers of vascular/endothelial inflammation integrin alpha/beta, soluble intercellular adhesion molecule-1, and L-selectin along with neurotrophic factor nerve growth factor-beta were increased in serum within 6 h post-blasts and persisted for 7 days thereafter. In contrast, systemic IL-1, IL-10, fractalkine, neuroendocrine peptide Orexin A, and VEGF receptor Neuropilin-2 (NRP-2) were raised predominantly after primary blast exposure. In conclusion, biomarkers of major pathological pathways were elevated at all blast set-ups. The most significant and persistent changes in neuro-glial markers were found after composite blast, while primary blast instigated prominent systemic cytokine/chemokine, Orexin A, and Neuropilin-2 release, particularly when primary blast impacted rats with unprotected body.
RESUMEN
Lipopolysaccharide (LPS), a structural component of Gram-negative bacteria, is implicated in the pathogenesis of endotoxemia/sepsis and multi-organ injury, including liver damage. We have shown that argininosuccinate synthase (ASS), a hepatic enzyme of the urea cycle, accumulates in circulation within 1h after treatment with both LPS alone and hepatotoxic combination of LPS and D-Galactosamine. These findings indicate ASS as a sensitive biomarker of liver responses to bacterial endotoxin. Furthermore, we suggest that the ASS release represents a potential counteracting liver reaction to LPS, and demonstrates anti-LPS activity of recombinant ASS (rASS) in vitro and in rodent models of endotoxemia in vivo. rASS physically bound to LPS, as indicated by a gel shift assay, and suppressed Escherichia coli growth in cultures consistent with direct antimicrobial properties of ASS. rASS reduced LPS cytotoxicity, TNF-α production, and increased cell viability in cultured mouse macrophages, even when added one hour following LPS challenge. Intraperitoneal injection of rASS (5 mg/kg) after treatment with a high dose of LPS remarkably increased survival of rodents, with a concomitant decrease of sepsis markers TNF-α, C-reactive protein (CRP), and lactate dehydrogenase (LDH) levels in blood. These results suggest that the endogenous ASS constitutes a novel liver-derived component of the innate immune response to bacterial LPS, and that recombinant ASS could mitigate the lethal effects of bacterial endotoxins during sepsis.
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Argininosuccinato Sintasa/inmunología , Infecciones Bacterianas/inmunología , Inmunidad Innata/inmunología , Lipopolisacáridos/antagonistas & inhibidores , Hígado/inmunología , Animales , Argininosuccinato Sintasa/sangre , Argininosuccinato Sintasa/metabolismo , Infecciones Bacterianas/metabolismo , Proteína C-Reactiva/antagonistas & inhibidores , Supervivencia Celular/inmunología , Células Cultivadas , Endotoxemia/sangre , Endotoxemia/inmunología , Endotoxemia/metabolismo , Endotoxinas/inmunología , Endotoxinas/metabolismo , Escherichia coli/inmunología , Escherichia coli/metabolismo , Galactosamina/inmunología , Humanos , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/sangre , Lipopolisacáridos/inmunología , Lipopolisacáridos/toxicidad , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Neuronal cell death after traumatic brain injury, Alzheimer's disease and ischemic stroke may in part be mediated through endoplasmic reticulum (ER) stress and unfolded protein response (UPR). UPR results in induction of molecular chaperone GRP78 and the ER-resident caspase-12, whose activation has been proposed to be mediated by calpain and caspase processing, although their relative contribution remains unclear. In this study we induced ER stress with thapsigargin (TG), and determined the activation profile of calpain-2, caspase-3, caspase-7, and caspase-12 by analyses of protein levels, corresponding substrates and breakdown products (BDP). Specific calpain and caspase activity was assessed by analysis of αII-spectrin BDP of 145 kDa (SBDP145), BDP of 150 kDa (SBDP150) and BDP of 120 kDa (SBDP120). Decrease in pro-calpain-2 protein and increased SBDP145 levels by 3 h after TG treatment indicated early calpain activity. Active caspase-7 (p20) increase occurred after 8 h, followed by concomitant up-regulation of active caspase-3 and SBDP120 after 24 h. In vitro digestion experiments supported that SBDP120 was exclusively generated by active caspase-3 and validated that kinectin and co-chaperone p23 were calpain and caspase-7 substrates, respectively. Pro-caspase-12 protein processing by the specific action of calpain and caspase-3/7 was observed in a time-dependent manner. N-terminal pro-domain processing of pro-caspase-12 by calpain generated a 38 kDa fragment, while caspase-3/7 generated a 35 kDa fragment. Antibody developed specifically against the caspase-3/7 C-terminal cleavage site D(341) detected the presence of large subunit (p20) containing 23 kDa fragment that increased after 24 h of TG treatment. Significant caspase-12 enzyme activity was only detected after 24 h of TG treatment and was completely inhibited by caspase 3/7 inhibitor DEVD-fmk and partially by calpain inhibitor SNJ-1945. ER-stress-induced cell death pathway in TG-treated PC12 cells was characterized by up-regulation of GRP-78 and processing and activation of caspase-12 by the orchestrated proteolytic activity of calpain-2 and caspase-3/7.
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Calpaína , Caspasa 12/metabolismo , Caspasas , Muerte Celular/fisiología , Retículo Endoplásmico/enzimología , Activación Enzimática/fisiología , Proteínas de Choque Térmico/metabolismo , Proteínas de Microfilamentos/análisis , Estrés Oxidativo/fisiología , Fragmentos de Péptidos/análisis , Respuesta de Proteína Desplegada/fisiología , Proteínas de Transporte Vesicular/análisis , Secuencia de Aminoácidos , Animales , Calpaína/metabolismo , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Diferenciación Celular , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas de Microfilamentos/química , Datos de Secuencia Molecular , Neuronas/química , Neuronas/fisiología , Estrés Oxidativo/efectos de los fármacos , Fragmentos de Péptidos/química , Ratas , Especificidad por Sustrato , Tapsigargina/farmacología , Factores de Tiempo , Respuesta de Proteína Desplegada/efectos de los fármacos , Proteínas de Transporte Vesicular/químicaRESUMEN
OBJECTIVES: Existing experimental approaches for studies of blast impact in small animals are insufficient and lacking consistency. Here, we present a comprehensive model, with repeatable blast signatures of controlled duration, peak pressure, and transmitted impulse, accurately reproducing blast impact in laboratory animals. MATERIALS: Rat survival, brain pathomorphology, and levels of putative biomarkers of brain injury glial fibrillary acid protein (GFAP), neuron-specific enolase, and ubiquitin C-terminal hydrolase (UCH)-L1 were examined in brain, cerebrospinal fluid (CSF), and blood after 10 msec of 358 kPa peak overpressure blast exposure. RESULTS: The high-speed imaging demonstrated a strong head acceleration/jolting accompanied by typical intracranial hematomas and brain swelling. Microscopic injury was revealed by prominent silver staining in deep brain areas, including the nucleus subthalamicus zone, suggesting both diffused and focal neurodegeneration. GFAP and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), markers of astroglia and oligodendroglia, accumulated substantially in the hippocampus 24 hours after blast and persisted for 30 days postblast. However, GFAP content in the blood significantly increased 24 hours after injury, followed by a decline and subsequent accumulation in CSF in a time-dependent fashion. A similar profile is shown for UCH-L1 increase in blood, whereas increased CSF levels of UCH-L1 persisted throughout 14 days after blast and varied significantly in individual rats. Neuron-specific enolase levels in blood were significantly elevated within 24 hours and 48 hours postblast. CONCLUSIONS: The proposed model of controlled nonpenetrating blast in rats demonstrates the critical pathologic and biochemical signatures of blast brain injury that may be triggered by cerebrovascular responses, including blood-brain barrier disruption, glia responses, and neuroglial alterations.
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Traumatismos por Explosión/patología , Conmoción Encefálica/patología , Lesiones Encefálicas/patología , Lesión Axonal Difusa/patología , Modelos Animales de Enfermedad , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Presión del Aire , Animales , Biomarcadores/metabolismo , Barrera Hematoencefálica/fisiología , Western Blotting , Corteza Cerebral/lesiones , Corteza Cerebral/patología , Ensayo de Inmunoadsorción Enzimática , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/patología , Hipocampo/lesiones , Hipocampo/patología , Fosfopiruvato Hidratasa/metabolismo , Ratas , Proteínas S100/metabolismo , Ubiquitina Tiolesterasa/metabolismoRESUMEN
The nature of warfare in the 21st century has led to a significant increase in primary blast or over-pressurization injuries to the whole body and head, which manifest as a complex of neuro-somatic damage, including traumatic brain injury (TBI). Identifying relevant pathogenic pathways in reproducible experimental models of primary blast wave exposure is therefore vital to the development of biomarkers for diagnostics of blast brain injury. Comparative analysis of mechanisms and putative biomarkers of blast brain injury is complicated by a deficiency of experimental studies. In this article, we present an overview of current TBI biomarkers, as well as outline experimental strategies to investigate molecular signatures of blast neurotrauma and to develop a pathway network map for novel biomarker discovery. These biomarkers will be effective for triaging and managing both combat and civilian casualities.
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Traumatismos por Explosión/fisiopatología , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/fisiopatología , Encéfalo/metabolismo , Encéfalo/fisiopatología , Modelos Biológicos , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Traumatismos por Explosión/complicaciones , Encéfalo/patología , Lesiones Encefálicas/etiología , Arterias Cerebrales/metabolismo , Arterias Cerebrales/patología , Arterias Cerebrales/fisiopatología , Humanos , Mediadores de Inflamación/análisis , Mediadores de Inflamación/metabolismo , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/fisiologíaRESUMEN
DNA fragmentation factors (DFF) form protein complexes consisting of nuclease DFF40/CAD and inhibitory chaperon DFF45/ICAD. Although activated caspase-3 has been shown to cleave DFF complexes with the release of active DFF40 and DNA fragmentation, the organ-specific mechanisms of DFF turnover during liver injury accompanied by massive apoptosis are unclear. In this study, we investigated hepatic profile of DFF40-immunopositive proteins in two models of liver injury in rats: acute ischemia/reperfusion (I/R) and chronic alcohol administration. We show that DFF40-like proteins occur in intact rat liver mainly as a 52kDa protein. Hepatic I/R-induced caspase-3 activation and a time-dependent accumulation of DFF40-positive protein fragments (40 and 20kDa), most likely via specific caspase-3 cleavage as evidenced by in vitro digestion of intact liver tissue with recombinant caspase-3. In addition, immunoprecipitation with DFF40 followed by Western blot with active caspase-3 antibody revealed the presence of active caspase-3 in DFF40-immunopositive 20kDa proteins. Chronic alcohol administration in rats also resulted in a dose-dependent fragmentation of DFF40 proteins similar to I/R injury. Collectively, these data demonstrate that DFF40 immunopositive proteins exist in the liver as distinct, tissue-specific molecular forms that may be processed by caspase-3 during both acute and chronic liver injury.
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Caspasa 3/metabolismo , Desoxirribonucleasas/metabolismo , Hepatopatías/enzimología , Hígado/enzimología , Enfermedad Aguda , Animales , Enfermedad Crónica , Desoxirribonucleasas/análisis , Activación Enzimática , Hígado/irrigación sanguínea , Hepatopatías Alcohólicas/enzimología , Proteínas de Unión a Poli-ADP-Ribosa , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/enzimologíaRESUMEN
Survivin attenuates apoptosis by inhibiting cleavage of some cell proteins by activated caspase-3. We recently discovered strong up-regulation of survivin, primarily in astrocytes and a sub-set of neurons, after traumatic brain injury (TBI) in rats. In this study we characterized co-expression of survivin with activated caspase-3 and downstream DNA fragmentation (TUNEL) in astrocytes and neurons after TBI. Western blot analysis revealed significant time-dependent increases in active caspase-3 between 5 and 14 days post-injury. No difference was observed between the proportion of survivin-positive and survivin-negative cells labeled with active caspase-3 at 5 or 7 days post-injury, as indicated by dual fluorescent immunostaining. Labeling of survivin-negative cells with TUNEL was, however, significantly greater than for survivin-positive cells, suggesting that expression of survivin may attenuate DNA cleavage and progression of apoptosis. A higher proportion of astrocytes than neurons accumulated active caspase-3. In contrast, co-localization with TUNEL was significantly higher for neurons than for astrocytes. These data suggest that survivin expression may attenuate DNA cleavage and cell death, and that this mechanism operates in a cell type-specific manner after TBI.
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Lesiones Encefálicas/metabolismo , Caspasas/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Apoptosis/fisiología , Astrocitos/metabolismo , Western Blotting/métodos , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Caspasa 3 , Recuento de Células/métodos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Activación Enzimática/fisiología , Lateralidad Funcional/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Etiquetado Corte-Fin in Situ/métodos , Masculino , Modelos Biológicos , Neuronas/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
In this study, we examined the expression and cellular localization of survivin and proliferating cell nuclear antigen (PCNA) after controlled cortical impact traumatic brain injury (TBI) in rats. There was a remarkable and sustained induction of survivin mRNA and protein in the ipsilateral cortex and hippocampus of rats after TBI, peaking at five days post injury. In contrast, both survivin mRNA and protein were virtually undetectable in craniotomy control animals. Concomitantly, expression of PCNA was also significantly enhanced in the ipsilateral cortex and hippocampus of these rats with similar temporal and spatial patterns. Immunohistochemistry revealed that survivin and PCNA were co-expressed in the same cells and had a focal distribution within the injured brain. Further analysis revealed a frequent co-localization of survivin and GFAP, an astrocytic marker, in both the ipsilateral cortex and hippocampus, while a much smaller subset of cells showed co-localization of survivin and NeuN, a mature neuronal marker. Neuronal localization of survivin was observed predominantly in the ipsilateral cortex and contralateral hippocampus after TBI. PCNA protein expression was detected in both astrocytes and neurons of the ipsilateral cortex and hippocampus after TBI. Collectively these data demonstrate that the anti-apoptotic protein survivin, previously characterized in cancer cells, is abundantly expressed in brain tissues of adult rats subjected to TBI. We found survivin expression in both astrocytes and a sub-set of neurons. In addition, the expression of survivin was co-incident with PCNA, a cell cycle protein. This suggests that survivin may be involved in regulation of neural cell proliferative responses after traumatic brain injury.
Asunto(s)
Astrocitos/metabolismo , Lesiones Encefálicas/metabolismo , Proteínas Asociadas a Microtúbulos/biosíntesis , Neuronas/metabolismo , Regulación hacia Arriba/fisiología , Animales , Astrocitos/química , Proteínas Inhibidoras de la Apoptosis , Masculino , Proteínas Asociadas a Microtúbulos/análisis , Proteínas de Neoplasias , Neuronas/química , Antígeno Nuclear de Célula en Proliferación/análisis , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Ratas , Ratas Sprague-Dawley , SurvivinRESUMEN
Neural stem/progenitor cells are clonogenic in vitro and produce neurospheres in serum-free medium containing epidermal growth factor (EGF) and fibroblast growth factor (FGF2). Here, we demonstrate that lysophosphatidic acid (LPA) instigated the clonal generation of neurospheres from dissociated mouse postnatal forebrain in the absence of EGF and FGF2. LPA induced proliferation of cells which co-expressed Sca-1 antigen and AC133, markers of primitive hematopoietic and neural stem/progenitor cells. Clonal expansion of these cells induced by LPA was inhibited by diacylglycerol- pyrophosphate (DGPP), an antagonist of the LPA receptor subtypes LPA1 and LPA3. Moreover, Sca-1- and AC133-positive cells of these neurospheres expressed LPA1, LPA2, and LPA3, suggesting important roles for these LPA receptors in proliferation of neural progenitors. LPA induced neurospheres to differentiate on an adherent laminin/poly-L-ornithine matrix. In differentiating neurospheres, LPA receptors co-localized with betaIII-tubulin, nestin, and CNPase, but not with glial fibrillary acidic protein (GFAP), a marker of astrocyte lineage. Our results demonstrate for the first time that lysophosphatidic acid induces clonal neurosphere development via proliferation of AC133/Sca-1-positive stem cells by a receptor-dependent mechanism. This differentiation was characterized by the initial co-localization of neural specific antigens at sites of LPA receptor expression upon their interaction with the inducing agonist.
Asunto(s)
Glicerol/análogos & derivados , Glicoproteínas/biosíntesis , Lisofosfolípidos/farmacología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas Nucleares/biosíntesis , Receptores del Ácido Lisofosfatídico/fisiología , Antígeno AC133 , Animales , Antígenos CD , Astrocitos/citología , Ataxina-1 , Ataxinas , Encéfalo/metabolismo , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Difosfatos/farmacología , Factor de Crecimiento Epidérmico/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteína Ácida Fibrilar de la Glía/química , Glicerol/farmacología , Inmunohistoquímica , Lisofosfolípidos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Oligodendroglía/metabolismo , Péptidos , Prosencéfalo/metabolismo , Células Madre/citologíaRESUMEN
Growth factor lysophosphatidic acid (LPA) regulates cell proliferation and differentiation and increases motility and survival in several cell types, mostly via G-protein-coupled receptors encoded by endothelial differentiation genes (EDG). We show herein that hepatic oval (stem) cell proliferation, induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in a mouse model of chronic liver injury, was associated with the expression of LPA1, LPA2, and LPA3 receptor subtypes; only LPA1 receptor protein was detectable in normal liver by western blot. In the injured liver, enhanced LPA1 receptor was identified predominantly in oval cells along the portal tract, proliferating ductular epithelial cells, and small cells, which were located in the nearby parenchyma and formed clusters. Interestingly, the LPA1 receptor was co-expressed in DDC-treated livers with the stem cell antigen SCA-1, suggesting that this receptor may be associated with bone marrow-derived progenitors. All three receptors for LPA were detected mostly in small cells in the vicinity of the portal tract, and co-localized with the A6 antigen, a marker of ductular oval cells. In addition, hepatic levels of endogenous LPA were significantly higher in DDC-fed mice compared to normal animals. We propose that the expression of diverse LPA receptors may be a necessary part of the mechanism responsible for activation of oval cells during liver injury. As a result, LPA and its analogs may represent critical endogenous mediators, which regulate survival, increase motility, and modulate proliferation and differentiation of hepatocyte progenitors in regenerating liver.
Asunto(s)
Diferenciación Celular/genética , Hígado/patología , Lisofosfolípidos/toxicidad , Animales , Western Blotting , Endotelio/patología , Endotelio/fisiología , Inmunohistoquímica/métodos , Hígado/efectos de los fármacos , Hígado/lesiones , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
The biological roles of phospholipid growth factors lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been broadly investigated. The cellular effects of LPA and S1P are mediated predominantly via endothelial differentiation gene (EDG) receptors. Yet, the biological significance of LPA, S1P and their EDG receptors in cells of the liver remains unclear. Recent data demonstrate the presence of EDG2 and EDG4 mRNA for LPA receptor in a murine hepatocyte cell line transformed with human TGF-alpha, and in primary mouse hepatocytes. EDG2 receptor protein is expressed in mouse liver, where it appears to be located in nonparenchymal cells. Moreover, we have obtained data suggesting that proliferation of small hepatocyte-progenitors and stem (oval) cells during liver injury is associated with the expression of EDG2 and EDG4 receptors. LPA, and possibly S1P, appear to be essential factors that control proliferation and motility of hepatic stellate cells (HSC) and hepatoma cells. It is proposed that LPA, S1P and their respective EDG receptors play important roles in pathophysiology of chronic liver injury and fibrogenesis. The underlying mechanisms recruited by LPA and S1P in pathogenesis of liver injury remain to be investigated.