RESUMO
We reports a case of a newly formed thrombus in the left atrial appendage during cardiopulmonary bypass detected by transesophageal echocardiography in a patient with chronic atrial fibrillation and mitral stenosis. This case alerts the anesthesiologists of possible thrombus formation despite full heparinization during cardiac surgery and the importance of a comprehensive echocardiography examination.
RESUMO
When we treated rat bone marrow stromal cells (rBMSCs) with neuronal differentiation induction media, typical unfolded protein response (UPR) was observed. BIP/GRP78 protein expression was time-dependently increased, and three branches of UPR were all activated. ATF6 increased the transcription of XBP1 which was successfully spliced by IRE1. PERK was phosphorylated and it was followed by eIF2alpha phosphorylation. Transcription of two downstream targets of eIF2alpha, ATF4 and CHOP/GADD153, were transiently up-regulated with the peak level at 24 h. Immunocytochemical study showed clear coexpression of BIP and ATF4 with NeuN and Map2, respectively. UPR was also observed during the neuronal differentiation of mouse embryonic stem (mES) cells. Finally, chemical endoplasmic reticulum (ER) stress inducers, thapsigargin, tunicamycin, and brefeldin A, dose-dependently increased both mRNA and protein expressions of NF-L, and, its expression was specific to BIP-positive rBMSCs. Our results showing the induction of UPR during neuronal differentiations of rBMSCs and mES cells as well as NF-L expression by ER stress inducers strongly suggest the potential role of UPR in neuronal differentiation.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Embrionárias/citologia , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular , Meios de Cultura/farmacologia , Proteínas de Ligação a DNA , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dobramento de Proteína , Ratos , Células EstromaisRESUMO
Brain/kidney (B/K) protein is a novel double C2-like-domain protein that is highly expressed in rat brain and kidney, but its cellular localization and functional role in the kidney are still undetermined. We examined the cellular localization of B/K protein in the rat kidney under normal and ischemic conditions. Ischemia-reperfusion (I/R) injury was induced by clamping both renal arteries for 45 min, and animals were killed at 1 and 6 h and 1, 2, 3, 5, 7, 14, and 28 days after the reperfusion. Kidney tissues were processed for immunohistochemistry and immunoblot analyses using rabbit anti-B/K polyclonal antibodies. In control kidneys, B/K protein was expressed primarily in distal tubules including the thick ascending limb, distal convoluted and connecting tubules, and collecting duct. Notably, B/K protein was also expressed in the straight portion (S3 segment), but not in the S1 or S2, of proximal tubules, and podocytes of the glomerulus. In rat kidneys with I/R injury, expression of B/K protein was differentially regulated according to the anatomic location. In distal tubules, overall expression of B/K protein was markedly decreased. On the other hand, I/R injury significantly increased B/K protein expression in the S3 segment of the outer medulla as well as in the rat proximal tubular epithelial cell line NRK-52E in vitro. Furthermore, B/K protein was strongly expressed in many exfoliated cells in the lumen and urine. These findings suggest that B/K protein is closely associated with cell death in proximal tubules, which are vulnerable to I/R injury in the kidney.
Assuntos
Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Traumatismo por Reperfusão/metabolismo , Animais , Western Blotting , Morte Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Técnicas In Vitro , Córtex Renal/metabolismo , Medula Renal/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Sprague-Dawley , Sinaptotagminas , Urina/citologiaRESUMO
We have previously isolated a novel protein "B/K" that contains two C2-like domains. Here, we report the isolation and mRNA distribution of a human B/K isoform, and protein kinase A (PKA)-dependent phosphorylation of the B/K protein. The 1.5 kb human B/K cDNA clone exhibits 89% and 97% identities with rat B/K in the sequences of nucleotide and amino acid, respectively. Human B/K isoform encodes a 474 amino acid protein and shows structural features similar to the rat counterpart including two C2 domains, three consensus sequences for PKA, absence of a transmembrane region, and conservation of the N-terminal cysteine cluster. On Northern and dot blot analyses, a 3.0 kb B/K transcript was abundantly present in human brain, kidney, and prostate. Among the brain regions, strong signals were observed in the frontal and temporal lobes, the hippocampus, the hypothalamus, the amygdala, the substantia nigra, and the pituitary. Recombinant B/K proteins containing three consensus sites for PKA was very efficiently phosphorylated in vitro by PKA catalytic subunit. B/K protein which was overexpressed in LLC-PK1 cells was also strongly phosphorylated in vivo by vasopressin analog DDAVP, and PKA-specific inhibitor H 89 as well as type 2 vasopressin receptor antagonist specifically suppressed DDAVP-induced B/K phosphorylation. These results suggest that B/K proteins play a role as potential substrates for PKA in the area where they are expressed.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Fosfoproteínas/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/genética , Fosforilação , Isoformas de Proteínas/genética , Ratos , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , SinaptotagminasRESUMO
B/K protein is a newly identified member of double C2-like domain protein family. We examined the expression of B/K protein in the hippocampus of kainate-induced rat seizure model. Intraperitoneal injection of kainate increased the immunoreactivity to B/K protein in the CA1 to CA3 of the hippocampus. B/K protein expression began to increase at 6 h, reached the maximum at 12 h, and then returned nearly to the normal level at 72 h after the injection of kainate (12 mg/kg), and it was also dependent on the dose of kainate between 4 and 16 mg/kg. In electron microscopic and subcellular fractionation studies, B/K protein was localized in the endoplasmic reticulum (ER) of the hippocampus. Kainate also induced the expression of BiP, a typical ER stress marker protein, in the hippocampus and the cortex, and it was coexpressed with B/K protein. Moreover, thapsigargin-induced ER stress caused upregulation of B/K protein expression in PC12 cells. In conclusion, our data showing the induction of both B/K protein expression and ER stress response in the hippocampus of kainate seizure model, and ER-specific expression and ER stress-induced expression of B/K strongly suggest the possible role of B/K protein in epileptogenesis or epilepsy-induced neuronal damage.
