Assuntos
Ciprofloxacina/administração & dosagem , Quimioterapia Combinada/administração & dosagem , Diálise Peritoneal/efeitos adversos , Peritonite/tratamento farmacológico , Administração Oral , Gentamicinas/administração & dosagem , Humanos , Injeções Intravenosas , Peritonite/etiologia , Peritonite/microbiologia , Vancomicina/administração & dosagemRESUMO
Recent years have seen an increase in the number of new vaccines available on the Canadian market, and increasing divergence in provincial and territorial immunization programs as jurisdictions must choose among available health interventions with limited funding. We present an analytical framework, which we have developed to assist in the analysis and comparison of potential immunization programs. The framework includes 58 criteria classified into 13 categories, including the burden of disease, vaccine characteristics and immunization strategy, cost-effectiveness, acceptability, feasibility, and evaluability of program, research questions, equity, ethical, legal and political considerations. To date this framework has been utilized in a variety of different contexts, such as to structure expert presentations and reports and to examine the degree of consensus and divergence among experts, and to establish priorities. It can be transformed for a variety of other uses such as educating health professionals and the general public about immunization.
Assuntos
Pesquisa sobre Serviços de Saúde/métodos , Programas de Imunização , Canadá , Política de Saúde , HumanosRESUMO
Early postoperative feeding (EPOF) practices among North American institutions were investigated using a survey questionnaire to obtain descriptive information regarding the overall utilization and criteria used to identify candidates for EPOF. EPOF was defined as the initiation of enteral nutrition support two to 48 hours postoperatively in major abdominal and thoracic surgical patients. Two hundred and ninety-seven questionnaires were mailed; 170 were completed. Sixty-nine (41%) institutions reported using EPOF. Feeding was initiated less than 12 hours postoperatively in 16% of centres; 84% reported EPOF 13-48 hours postoperatively. The majority (88%) of institutions did not have a specific nutritional guideline for determining which patients should receive EPOF. Objective and subjective nutritional indices, degree of preoperative malnutrition and type of surgery were considered by 23% of respondents when determining the need for EPOF. Percent weight loss, albumin and the anticipated postoperative NPO were considered the most reliable objective indices while decreased dietary intake, cachexic appearance and anorexia were considered the most reliable subjective indices. The results reveal that less than 50% of institutions surveyed use EPOF in major abdominal and thoracic surgical patients and the criteria used to identify candidates for EPOF were found to be variable.
Assuntos
Dietética/estatística & dados numéricos , Nutrição Enteral/estatística & dados numéricos , Cuidados Pós-Operatórios/normas , Abdome/cirurgia , Canadá , Protocolos Clínicos , Número de Leitos em Hospital , Hospitalização , Humanos , Cuidados Pós-Operatórios/estatística & dados numéricos , Inquéritos e Questionários , Cirurgia Torácica/reabilitação , Cirurgia Torácica/estatística & dados numéricos , Estados UnidosRESUMO
A retrospective study was conducted to provide a description of the risk, complications, fatality, and sequelae associated with invasive meningococcal disease in college students admitted in the Allegheny county (Pennsylvania) hospital system from January 1990 to May 1999.
Assuntos
Amputação Cirúrgica , Infecções Meningocócicas/complicações , Qualidade de Vida , Estudantes/estatística & dados numéricos , Adulto , Amputação Cirúrgica/psicologia , Feminino , Humanos , Masculino , Infecções Meningocócicas/mortalidade , Necrose , Neisseria meningitidis/isolamento & purificação , Pennsylvania/epidemiologia , Qualidade de Vida/psicologia , Estudos Retrospectivos , Fatores de Risco , SorotipagemRESUMO
To determine whether the previously demonstrated ability of delta-opioid receptors to interact simultaneously with multiple G-proteins was a function of high receptor levels, this interaction was investigated in Chinese hamster ovary cells stably expressing 10 different levels of cloned delta-opioid receptors, ranging from 18,000 to 1.6 x 10(6) receptors/cell. The opioid agonist D-Ala2,D-Leu5-enkephalin (DADLE) inhibited forskolin-stimulated adenylyl cyclase activity in all 10 clones with variable maximal inhibitory levels. Furthermore, opioid agonists altered incorporation of [alpha-32P]azidoanilido-GTP into at least four G-protein alpha-subunits in all 10 clones, three of which were determined to be Gi3 alpha, Gi2 alpha and Go2 alpha. This effect was concentration-dependent, naloxone-reversible, and delta-opioid agonist-specific and was blocked by pretreatment with pertussis toxin. Although DADLE induced an increase in the incorporation of [alpha-32P]azidoanilido-GTP into three of the four G alpha proteins that was independent of receptor density, the magnitude of this response was greater as receptor density increased. In addition, concentrations of DADLE required to promote 50% maximal labeling were similar for all four G alpha proteins within each clone and did not appear to be affected by receptor density. Therefore, the ability of delta-opioid receptors to interact with multiple G-proteins is independent of receptor density and there is also no apparent correlation between the amount of G-protein activated and the maximal effect of an agonist.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Opioides delta/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Leucina Encefalina-2-Alanina/farmacologia , Dados de Sequência Molecular , Naloxona/farmacologia , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides delta/genéticaRESUMO
Agonist-induced internalization of G protein-coupled receptors is influenced by many structural determinants including the carboxyl tail. To investigate the role of serine and threonine residues within the carboxyl tail, several mutants were constructed by truncating the carboxyl tail of the hemagglutinin-tagged mu-opioid receptor, thereby removing serines and threonines systematically. Neuro2A cells stably expressing the truncated receptors did not exhibit a significant alteration in the affinity of [3H]diprenorphine or etorphine for the receptor or the potency of etorphine to inhibit forskolin-stimulated adenylyl cyclase activity. Chronic etorphine treatment resulted in a time-dependent down-regulation of all the truncated receptors, except MOR1TAG355D, thus revealing the importance of the four amino acids between Ser355 and Glu359 (STIE). Surprisingly, deletion of the STIE sequence resulted in a receptor that down-regulated the same as the wild-type receptor. The involvement of multiple amino acids within the carboxyl tail was demonstrated by combining alanine substitutions of several putative G-protein-coupled receptor kinase phosphorylation sites. Systematic analysis of these receptors indicated that mutation of Ser356 and Ser363 to alanine attenuated agonist-mediated down-regulation. The magnitude of etorphine-induced phosphorylation of this mutant receptor, however, was similar to that of the wild-type mu-opioid receptor. Thus, phosphorylation of the carboxyl tail of the mu-opioid receptor is not an obligatory event for etorphine-induced down-regulation of the receptor.
Assuntos
Regulação para Baixo , Etorfina/farmacologia , Estrutura Secundária de Proteína , Receptores Opioides mu/metabolismo , Serina , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , Diprenorfina/metabolismo , Etorfina/metabolismo , Ácido Glutâmico , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores Opioides mu/biossíntese , Receptores Opioides mu/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , TransfecçãoRESUMO
Treatment of HEK293 cells expressing the delta-opioid receptor with agonist [d-Pen(2,5)]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr(358) and Ser(363), were phosphorylated, with Ser(363) being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser(363) as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr(358) and Ser(363), with Thr(358) being involved in the receptor internalization.
Assuntos
Receptores Opioides delta/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , D-Penicilina (2,5)-Encefalina/farmacologia , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Receptores Opioides delta/genética , Serina/metabolismo , Relação Estrutura-Atividade , Treonina/metabolismoRESUMO
Similar to other G protein-coupled receptors, rapid phosphorylation of the delta-opioid receptor in the presence of agonist has been reported. Hence, agonist-induced desensitization of the delta-opioid receptor has been suggested to be via the receptor phosphorylation, arrestin-mediated pathway. However, due to the highly efficient coupling between the delta-opioid receptor and the adenylyl cyclase, the direct correlation between the rates of receptor phosphorylation and receptor desensitization as measured by the adenylyl cyclase activity could not be established. In the current studies, using an ecdysone-inducible expression system to control the delta-opioid receptor levels in HEK293 cells, we could demonstrate that the rate of deltorphin II-induced receptor desensitization is dependent on the receptor level. Only at receptor concentrations =90 fmol/mg of protein were rapid desensitizations (t(12) <10 min) observed. Apparently, deltorphin II-induced receptor desensitization involves cellular events in addition to receptor phosphorylation. Mutation of Ser(363) in the carboxyl tail of the delta-opioid receptor to Ala completely abolished the deltorphin II-induced receptor phosphorylation but not the desensitization response. Although the magnitude of desensitization was attenuated, the rate of deltorphin II-induced receptor desensitization remained the same in the S363A mutant as compared with wild type. Also, the S363A mutant could internalize in the presence of deltorphin II. Only when the agonist-induced clathrin-coated pit-mediated receptor internalization was blocked by 0.4 m sucrose that the deltorphin II-induced receptor desensitization was abolished in the S363A mutant. Similarly, 0.4 m sucrose could partially block the agonist-induced rapid desensitization in HEK293 cells expressing the wild type delta-opioid receptor. Taken together, these data supported the hypothesis that rapid desensitization of the delta-opioid receptor involves both the phosphorylation and the internalization of the receptor.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Oligopeptídeos/farmacologia , Receptores Opioides delta/metabolismo , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Substituição de Aminoácidos , Western Blotting , Linhagem Celular , Vesículas Revestidas por Clatrina/efeitos dos fármacos , Vesículas Revestidas por Clatrina/metabolismo , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Humanos , Cinética , Mutagênese Sítio-Dirigida , Oligopeptídeos/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Receptores Opioides delta/agonistas , Receptores Opioides delta/genética , Serina/genética , Serina/metabolismo , Sacarose/farmacologiaRESUMO
Previously, we reported that the time course for the rapid phosphorylation rate of mu-opioid receptor expressed in human embryonic kidney (HEK)293 cells did not correlate with the slow receptor desensitization rate induced by [D-Ala(2),N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO). However, others have suggested that receptor phosphorylation is the trigger for mu-opioid receptor desensitization. In this study, we demonstrated the relatively slow rate of receptor desensitization could be attributed partially to the recycling of internalized receptor as determined by fluorescence-activated cell-sorting analysis. However, the blockade of the endocytic and Golgi transport events in HEK293 cells with monensin and brefeldin A did not increase the initial rate of receptor desensitization. But the desensitization rate was increased by reduction of the mu-opioid receptor level with beta-furnaltrexamine (betaFNA). The reduction of the receptor level with 1 microM betaFNA significantly increased the rate of etorphine-induced receptor desensitization. By blocking the ability of receptor to internalize with 0.4 M sucrose, a significant degree of receptor being rapidly desensitized was observed in HEK293 cells pretreated with betaFNA. Hence, mu-opioid receptor is being resensitized during chronic agonist treatment. The significance of resensitization of the internalized receptor in affecting receptor desensitization was demonstrated further with human neuroblastoma SHSY5Y cells that expressed a low level of mu-opioid receptor. Although DAMGO could not induce a rapid desensitization in these cells, in the presence of monensin and brefeldin A, DAMGO desensitized the mu-opioid receptor's ability to regulate adenylyl cyclase with a t(1/2) = 9.9 +/- 2.1 min and a maximal desensitized level at 70 +/- 4.7%. Furthermore, blockade of receptor internalization with 0.4 M sucrose enhanced the DAMGO-induced receptor desensitization, and the inclusion of monensin prevented the resensitization of the mu-opioid receptor after chronic agonist treatment in SHSY5Y cells. Thus, the ability of the mu-opioid receptor to resensitize and to recycle, and the relative efficiency of the receptor to regulate adenylyl cyclase activity, contributed to the observed slow rate of mu-opioid receptor desensitization in HEK293 cells.
Assuntos
Receptores Opioides mu/metabolismo , Brefeldina A/farmacologia , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Ionóforos/farmacologia , Monensin/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Receptores Opioides mu/agonistas , Transdução de Sinais/efeitos dos fármacosRESUMO
The involvement of a conserved serine (Ser196 at the mu-, Ser177 at the delta-, and Ser187 at the kappa-opioid receptor) in receptor activation is demonstrated by site-directed mutagenesis. It was initially observed during our functional screening of a mu/delta-opioid chimeric receptor, mu delta2, that classical opioid antagonists such as naloxone, naltrexone, naltriben, and H-Tyr-Tic[psi,CH2NH]Phe-Phe-OH (TIPPpsi; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing the chimeric receptor. Antagonists also activated the G protein-coupled inward rectifying potassium channel (GIRK1) in Xenopus oocytes coexpressing the mu delta2 opioid receptor and the GIRK1 channel. By sequence analysis and back mutation, it was determined that the observed antagonist activity was due to the mutation of a conserved serine to leucine in the fourth transmembrane domain (S196L). The importance of this serine was further demonstrated by analogous mutations created in the mu-opioid receptor (MORS196L) and delta-opioid receptor (DORS177L), in which classical opioid antagonists could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing either MORS196L or DORS177L. Again, antagonists could activate the GIRK1 channel coexpressed with either MORS196L or DORS177L in Xenopus oocytes. These data taken together suggest a crucial role for this serine residue in opioid receptor activation.