RESUMO
Six years ago, a study performed in our department reported that the incidence of postoperative residual curarisation (PORC) was 39%. The reassessment of neuromuscular monitoring and reversal of neuromuscular block in routine anaesthetic practice is relevant now that sugammadex has become available. The incidence of PORC, defined by a train-of-four (TOF) <90%, was evaluated at post-anaesthesia care unit (PACU) arrival in patients whose neuromuscular block had been reversed with neostigmine or sugammadex and those in whom reversal was felt unnecessary (adequate spontaneous recovery). During the PACU stay we recorded the oxygen saturation (SpO(2)) at arrival, episodes of SpO(2) <90%, airway manoeuvres and/or stimulation required to maintain SpO(2) >90%, and the need for re-intubation. In total, 624 patients were studied. Fifteen percent (66/441) of the patients who were not reversed, 15% (21/139) of those who were reversed with neostigmine and 2% (1/44) of those who received sugammadex exhibited PORC (P=0.06). No patient required reintubation in the PACU. The absence of neuromuscular monitoring and pharmacological reversal before extubation were not associated with PORC. A TOF <90% at PACU arrival was not associated with SpO(2) <90% during the PACU stay. Body mass index was the only independent predictor of SpO(2) <90% during the stay in the PACU. These findings indicate that in recent years, the incidence of PORC, defined by a TOF <90%, has dramatically decreased in our institution. The differences in PORC were not statistically significant between patients who received sugammadex for reversal and patients with spontaneous recovery or neostigmine reversal.
Assuntos
Neostigmina/farmacologia , Bloqueio Neuromuscular/métodos , Oxigênio/metabolismo , gama-Ciclodextrinas/farmacologia , Adulto , Idoso , Período de Recuperação da Anestesia , Índice de Massa Corporal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Parassimpatomiméticos/farmacologia , Estudos Prospectivos , Transtornos Respiratórios/etiologia , Sugammadex , Fatores de TempoRESUMO
The past decade there has been an enormous advance in imaging technology, most obvious in the field of magnetic resonance imaging (MRI) and computed tomography (CT). Today nearly every radiology department has a multislice CT (MSCT) available for routine imaging, many of which are increasingly being replaced by last generation 16- and 64-slice CT scanners. However, the use of fast CT scanners requires a better insight in acquisition and contrast media injection protocols in order to achieve the best possible result. It is the aim of this article to give a review of the basic principles of CT protocol design for the chest and the kinetics of contrast media injection.
Assuntos
Meios de Contraste/administração & dosagem , Angiografia Coronária/métodos , Radiografia Torácica , Tomografia Computadorizada por Raios X/métodos , Débito Cardíaco/efeitos dos fármacos , Humanos , Injeções , Fatores de TempoRESUMO
This paper presents our experience on the characterization of cystic pancreatic lesions using CT and MRI. First of all, true cystic pancreatic neoplasms should be differentiated from pseudocysts. Noninvasive characterization of cystic pancreatic neoplasms continues to rely principally on CT and MRI. Despite the presence of classic radiological characteristics of various cystic pancreatic neoplasms, these lesions continue to be problematic for the radiologist. They are most frequently incidental findings that are not related to the reason for imaging the patients in whom they are discovered. The radiological and clinical challenges are to determine the benign or malignant nature of the lesion and its potential resectability. The heterogeneity among cystic lesions and overlap in imaging characteristics should cause radiologists to approach the specific characterization of cystic pancreatic masses with a substantial degree of humility. Recommending an appropriate management approach based on imaging findings, in conjunction with clinical information and clinical consultation, may therefore be more important than attempting to assign a specific diagnosis to a cystic pancreatic lesion.
Assuntos
Imageamento por Ressonância Magnética/métodos , Cisto Pancreático/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Tomografia Computadorizada por Raios X/métodos , Meios de Contraste , Diagnóstico Diferencial , Humanos , Sensibilidade e EspecificidadeAssuntos
Asfixia Neonatal/diagnóstico por imagem , Clavícula/anormalidades , Osteocondrodisplasias/diagnóstico por imagem , Costelas/anormalidades , Síndrome de Costela Curta e Polidactilia/diagnóstico por imagem , Asfixia Neonatal/genética , Aberrações Cromossômicas , Clavícula/diagnóstico por imagem , Diagnóstico Diferencial , Nanismo/diagnóstico por imagem , Nanismo/genética , Genes Recessivos , Humanos , Recém-Nascido , Osteocondrodisplasias/genética , Radiografia , Costelas/diagnóstico por imagem , Síndrome de Costela Curta e Polidactilia/genéticaRESUMO
Our earlier investigations have demonstrated a critical difference in the efficacy of orally administered porcine compared to human or mouse insulin (no effect) in preventing type I diabetes in two distinct experimental models. Based on these findings one has to assume that certain insulins might not be suitable for the induction of oral 'tolerance'/bystander suppression, which might be one cause for recent failures in human oral antigen trials. Here we demonstrate that coupling to the non-toxic subunit of cholera toxin (CTB) can abolish these differences in efficacy between human and porcine insulin. As expected, an added benefit was the much smaller oral antigen dose required to induce CD4+ insulin-B specific regulatory cells that bystander-suppress autoaggressive responses. Mechanistically we found that uptake or transport of insulin-CTB conjugates in the gut occurs at least partially via binding to GM-1, which would explain the enhanced clinical efficacy. Both B chains bound well to major histocompatibility complex (MHC) class II, indicating comparable immunological potential once uptake and processing has occurred. Thus, our findings delineate a pathway to overcome issues in oral antigen choice for prevention of type I diabetes.
Assuntos
Autoantígenos/administração & dosagem , Toxina da Cólera/administração & dosagem , Diabetes Mellitus Tipo 1/prevenção & controle , Imunização/métodos , Insulina/administração & dosagem , Vacinas Conjugadas/administração & dosagem , Administração Oral , Animais , Autoantígenos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Toxina da Cólera/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Esquema de Medicação , Feminino , Humanos , Insulina/metabolismo , Mucosa Intestinal/metabolismo , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Modelos Animais , Suínos , Vacinas Conjugadas/metabolismoRESUMO
The ABRF-98SEQ sample was the 11th in a series of amino acid sequencing studies performed by the Protein Sequence Research Group of the Association of Biomolecular Resource Facilities. This study was designed to aid participants' laboratories in determining their abilities to analyze the amino acid sequence of a peptide at high sensitivity using Edman degradation, mass spectrometry (MS), or both. ABRF-98SEQ is a 17-amino acid synthetic peptide (IFDDEIEEVQALYPTER) that resembles a typical tryptic peptide. It was distributed at the 2.8-pmol level. The sample was sent dried in a microfuge tube accompanied by instructions on solubilizing the sample and by a survey form. Including tentative calls, the correct sequence was obtained by 16% of the responding participants, compared with only 6% in the 1997 study when the low-level peptide was a minor component of a mixture. This increase probably reflects the purity of ABRF-98SEQ. A secondary factor in the increase in correct calls may be the larger number of respondents this year reporting that they perform sequence analysis at the 1- to 10-pmol level. Most respondents who obtained the correct sequence used a combination of Edman sequencing and molecular weight determination by MS. Overall, the accuracy and sensitivity of peptide sequencing by Edman degradation continue to improve and are clearly aided by the use of MS for molecular weight determination. Although peptide sequencing by MS is not yet routinely practiced by the participating laboratories, results of this study indicate that MS-derived sequence data, when properly interpreted, are valuable for correcting, completing, or corroborating sequence assignments derived by Edman.
RESUMO
Stanniocalcin (STC) is a hormone that was originally identified in fish, where it inhibits calcium uptake by the gills and gut and stimulates phosphate adsorption by the kidney. Recently, two mammalian homologues of stanniocalcin were identified. The first (STC1) shows 61% identity to the fish stanniocalcins and appears to have a function similar to that of the fish stanniocalcins. The second homologue (STC2) is 30-38% identical to the fish stanniocalcins, and is characterized by unique cysteine and histidine motifs that are not found in the other stanniocalcins. We purified both the native hamster and recombinant human STC2 proteins and obtained a partial amino acid sequence of the hamster protein. Both proteins behave as a disulfide bonded homodimer, which undergoes post-translational modification(s). The STC2 gene was localized to human chromosome 5q35. Northern blot analysis revealed that the primary site of human STC2 production is the pancreas, and immunostaining localized the STC2 protein to a subpopulation of cells in the islet. Double immunostaining for STC2 and either insulin or glucagon revealed that STC2 protein is found in the alpha cells, but not the beta cells. We speculate that STC2 may play a role in glucose homeostasis.
Assuntos
Glicoproteínas/análise , Pâncreas/química , Sequência de Aminoácidos , Animais , Northern Blotting , Células CHO , Mapeamento Cromossômico , Cricetinae , Glicoproteínas/química , Glicoproteínas/imunologia , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Proteínas Recombinantes/imunologiaAssuntos
Biotecnologia , Política Pública , Pesquisa , Laboratórios/economia , Pesquisadores/economiaRESUMO
We describe a two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-based approach for detecting and quantifying secreted recombinant proteins in media conditioned by baby hamster kidney (BHK) cells. Seven secreted proteins were analyzed in this system: leptin, thrombopoietin, thrombin, glycoprotein 130, soluble interleukin-2 receptor, and two novel sequences obtained from sequence database searches. BHK cells transfected with plasmids encoding each of these proteins, and cells transfected with empty plasmids (control cells), were metabolically labeled and the resulting conditioned media were analyzed by 2-D PAGE. Gel images derived from cells expressing recombinant proteins were compared with images from control cells in order to identify spots corresponding to the expressed proteins. All seven of the test proteins were successfully detected using this method. The sensitivity of the system was evaluated by diluting samples derived from high-expressing clones with conditioned media from control cells. The sensitivity of detection was protein-dependent, but recombinant proteins expressed at levels as low as 10 ng/mL could be detected. Quantification of recombinant protein levels was achieved by measuring spot intensities using phosphorimager analysis. The intensity of spots corresponding to recombinant proteins were compared with the spot intensity of an endogenous BHK protein which had been calibrated to a known standard. Estimates of the levels of expressed protein determined using this technique correlated with the levels determined using standard affinity assays. We conclude that this system provides a reliable method for quantifying levels of protein expression when specific assays are unavailable.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas Recombinantes/metabolismo , Animais , Linhagem Celular , Cricetinae , Leptina , Glicoproteínas de Membrana , Biossíntese de Proteínas , Receptores de Interleucina-2 , Proteínas Recombinantes/análise , Sensibilidade e Especificidade , Trombina/biossíntese , Trombopoetina/biossínteseRESUMO
Voltage-gated L-type Ca2+ channels mediate Ca2+ entry into cells in response to membrane depolarization. Ca2+ entry through the cardiac Ca2+ channel determines the rate and force of contraction, and modulation of Ca2+ channel activity by beta-adrenergic agents acting through adenosine 3',5'-cyclic monophosphate-(cAMP)-dependent protein phosphorylation contributes to physiological regulation of cardiac function by the sympathetic nervous system. Immunoblotting experiments using site-directed anti-peptide antibodies against different peptide segments indicate that the alpha 1 subunit of the cardiac L-type Ca2+ channel exists in two size forms with apparent molecular masses of 240 and 210 kDa, which we call alpha 1(242) and alpha 1(210), Alpha 1(242) corresponds to the full-length cardiac alpha 1 subunit predicted from its cDNA sequence, while alpha 1(210) is truncated at its COOH terminus. Only alpha 1(242) is phosphorylated in vitro by cAMP-dependent protein kinase. Protein microsequencing and peptide mapping of wild-type and mutant fusion proteins show that this phosphorylation occurs at serine 1928 near the COOH terminus. Phosphorylation of this residue can be detected by phosphospecific antibodies raised against the corresponding phosphopeptide. Experiments with these antibodies show that alpha 1(242) is phosphorylated in intact cells expressing the cardiac alpha 1 subunit in response to increased intracellular levels of cAMP. These results identify serine 1928 on the alpha 1 subunit as a possible site of regulation by cAMP-dependent phosphorylation.
Assuntos
Canais de Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miocárdio/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Canais de Cálcio/genética , Bovinos , Dados de Sequência Molecular , Mutagênese , Mapeamento de Peptídeos , Peptídeos/metabolismo , Fosforilação , Testes de Precipitina , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Voltage-gated sodium channels in brain neurons are complexes of a pore-forming alpha subunit with smaller beta 1 and beta 2 subunits. cDNA cloning and sequencing showed that the beta 2 subunit is a 186 residue glycoprotein with an extracellular NH2-terminal domain containing an immunoglobulin-like fold with similarity to the neural cell adhesion molecule (CAM) contactin, a single transmembrane segment, and a small intracellular domain. Coexpression of beta 2 with alpha subunits in Xenopus oocytes increases functional expression, modulates gating, and causes up to a 4-fold increase in the capacitance of the oocyte, which results from an increase in the surface area of the plasma membrane microvilli. beta 2 subunits are unique among the auxiliary subunits of ion channels in combining channel modulation with a CAM motif and the ability to expand the cell membrane surface area. They may be important regulators of sodium channel expression and localization in neurons.
Assuntos
Química Encefálica/fisiologia , Moléculas de Adesão Celular Neuronais , Moléculas de Adesão Celular/fisiologia , Canais de Sódio/fisiologia , Sequência de Aminoácidos , Animais , Encéfalo/embriologia , Membrana Celular/fisiologia , Clonagem Molecular , Contactinas , DNA Complementar/genética , Expressão Gênica/fisiologia , Imunoglobulinas/genética , Glicoproteínas de Membrana/fisiologia , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Oócitos/fisiologia , Ratos , Canais de Sódio/ultraestrutura , Medula Espinal/química , Medula Espinal/embriologia , XenopusRESUMO
Previous saxitoxin binding studies indicated two forms of the sodium channel in the fetal rat brain; a low-affinity precursor located in an internal membrane compartment, present exclusively in growth cones and a high-affinity mature form present in the plasmalemma of growth cones and characteristic of synapses. This raises the questions (1) of the presence or absence of the beta2 subunit in these channel forms and (2) of the developmental regulation of the the beta2 subunit. Antibodies against the alpha and beta2 channel subunits were used to probe Western blots of subcellular fractions from rat brains at embryonic day 18 (E18), pups at postnatal (P) days 7-25, and adults, as well as purified sodium channels from adult brain. In both synaptosomes and the purified sodium channel the beta2 antibody recognized the expected band at 38 kDa under reducing conditions. However, in contrast to the alpha subunit, this band was absent at E18 and became apparent only from P7 onwards. At the earlier time intervals a very prominent immunoreactive band of unknown identity was evident at 260--300kDa, which declined in intensity concomitant with the appearance of the 38 kDa beta2 band. These data indicate that beta2 subunits are regulated independently from alpha subunits, are absent in differentiating neurons, and hence are not necessary for insertion of the sodium channel into the plasmalemma, at least during early development of the neuron.
Assuntos
Encéfalo/metabolismo , Terminações Nervosas/metabolismo , Neurônios/metabolismo , Canais de Sódio/metabolismo , Frações Subcelulares/metabolismo , Animais , Western Blotting , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Dissulfetos/metabolismo , Desenvolvimento Embrionário e Fetal/fisiologia , Neurônios/citologia , Oxirredução , Fragmentos de Peptídeos/metabolismo , Ratos , Saxitoxina/metabolismoRESUMO
This study examines the proteolysis of the carboxy terminal domain of the full-length (alpha 1(212)) and truncated (alpha 1(190)) forms of the rabbit skeletal muscle L-type calcium channel alpha 1 subunit by calpain I and calpain II. Although both forms of the alpha 1 subunit show little sensitivity to proteolysis by calpain II, alpha 1(212) is relatively more sensitive than alpha 1(190) to digestion by calpain I, the form of the enzyme regulated by micromolar concentrations of calcium. Calpain I cleaves a 37-kDa fragment from the C-terminus of alpha 1(212) in a time- and concentration-dependent manner and proteolysis is independent of the alpha 1(212) phosphorylation state. This proteolytic cleavage removes the major site of cyclic AMP-dependent phosphorylation from alpha 1(212) and may provide a mechanism for modifying the cyclic AMP-dependent regulation of L-type calcium channels in skeletal muscle.
Assuntos
Canais de Cálcio/química , Calpaína/sangue , Animais , Canais de Cálcio/isolamento & purificação , Canais de Cálcio/metabolismo , Calpaína/isolamento & purificação , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Hidrólise , Cinética , Microssomos/metabolismo , Músculos/metabolismo , Fragmentos de Peptídeos/isolamento & purificação , Fosfopeptídeos/isolamento & purificação , Fosforilação , CoelhosAssuntos
Canais Iônicos/química , Animais , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Humanos , Canais Iônicos/metabolismo , Substâncias Macromoleculares , Modelos Estruturais , Canais de Potássio/química , Canais de Potássio/metabolismo , Estrutura Secundária de Proteína , Canais de Sódio/química , Canais de Sódio/metabolismoRESUMO
Among the various voltage-sensitive Ca2+ channels present in PC12 cells are the dihydropyridine (DHP)-sensitive L-channel, the omega-conotoxin (omega-CgTx)-sensitive N-channel, and an atypical omega-CgTx/DHP-insensitive Ca2+ channel. Depolarization-evoked Ca2+ entry and [3H]dopamine release is mediated by L-type Ca2+ channels determined by the use of Ca2+ channel antagonists, and a single protein of 250 kDa is recognized by L-type-specific antibodies. Screening of a PC12 cDNA library revealed two types of Ca2+ channels which were identified by partial sequencing. A pc12-L clone displayed virtually identical sequence homology to the cardiac L-type channel. The identical sequence homology of the single alternative splicing region confirmed clone pc12-L as the rbC-I transcript, a cardiac-neuronal alpha 1 subunit expressed in rat brain. Clone pc12-N displayed identical sequence homology to rbB-I, a neuronal alpha 1 subunit of the N-type Ca2+ channel expressed in rat brain; Northern blot analysis identified RNA of a size similar to that previously described for rat brain.
Assuntos
Canais de Cálcio/metabolismo , Dopamina/metabolismo , Miocárdio/metabolismo , Animais , Sequência de Bases , Canais de Cálcio/classificação , Canais de Cálcio/genética , Canais de Cálcio Tipo L , Clonagem Molecular , DNA Complementar/genética , Dados de Sequência Molecular , Proteínas Musculares/metabolismo , Células PC12 , Peptídeos/farmacologia , Ratos , Homologia de Sequência do Ácido Nucleico , Venenos de Aranha/farmacologia , ômega-Agatoxina IVA , ômega-Conotoxina GVIARESUMO
Membrane vesicles enriched in both ryanodine receptor and dihydropyridine receptor were obtained from rabbit skeletal muscle and solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Analysis of the sedimentation behavior of the solubilized proteins showed the existence of a population of alpha 1 subunits of the dihydropyridine receptor which cosedimented with the ryanodine receptor. Solubilized proteins were immunoprecipitated with antibodies directed against either the ryanodine receptor or the alpha 1, alpha 2, or beta subunits of the dihydropyridine receptor. Immunoprecipitated proteins were identified by Western blot analysis and by specific labeling with [3H]ryanodine or [3H]PN200-110. Immunoprecipitation of the solubilized proteins with antibodies directed against the dihydropyridine receptor led to the coimmunoprecipitation of the ryanodine receptor. Conversely, immunoprecipitation with antibodies directed against the ryanodine receptor led to an immune complex containing both receptors, but these antibodies were unable to precipitate purified dihydropyridine receptor. These results demonstrate that ryanodine receptor and dihydropyridine receptor are present in the triad membrane preparation in a complex which may play an important role in excitation-contraction coupling.
Assuntos
Canais de Cálcio/metabolismo , Proteínas Musculares/metabolismo , Músculos/metabolismo , Animais , Western Blotting , Canais de Cálcio Tipo L , Contração Muscular , Testes de Precipitina , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina , SolubilidadeRESUMO
Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent protein kinase (cA-PK) and protein kinase C. Using synthetic peptides and protein microsequencing, we have determined that the alpha subunit of rat brain Na+ channel is selectively phosphorylated by cA-PK in vitro and in intact cells on 4 serine residues in the intracellular loop connecting homologous domains I and II. Ser-623 was most rapidly and extensively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was most extensively phosphorylated in mammalian cells transfected with the alpha subunit of type IIA Na+ channel in response to an increase in intracellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylated Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to lesser extents. Phosphatase 2A selectively dephosphorylated Ser-610. Together these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent phosphorylation results from selective phosphorylation and dephosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.