RESUMO
BACKGROUND: This prospective study was designed to examine the associations of demographic, clinical, psychological and neuroendocrine factors with acute and chronic post-operative pain following partial mastectomy. METHODS: Sixty-four female patients scheduled for partial mastectomy were enrolled. Pre-operative anxiety/depression was assessed, using the Hospital Anxiety and Depression Scale (HADS). Pre-operative 24-h urinary cortisol levels were measured 2 days before surgery. Post-operative pain was examined using a visual analog scale (VAS) for acute pain on 0-2 post-operative day (POD), and a short-form McGill Pain Questionnaire for chronic pain at 6 months after surgery. In the last 29 subjects, post-operative 24-h urinary cortisol levels were also measured on 0 POD and were subjected to correlation analysis. RESULTS: Multivariate logistic regression analysis revealed that lower pre-operative cortisol secretion and greater pre-operative anxiety were significantly associated with an increased risk of moderate to severe acute post-operative pain [Odds Ratio (95% Confidence Interval); 0.96 (0.92-0.98), and 1.24 (1.04-1.54)], and that patients with greater pre-operative anxiety and moderate to severe acute pain were more likely to develop chronic post-operative pain [OR (95% CI); 1.63 (1.23-2.40), and 5.07 (1.30-24.6)]. Correlational analysis demonstrated that the post-operative cortisol level was inversely correlated with pre-operative anxiety and the intensity of acute post-operative pain (r = -0.40, p < 0.05, and r = -0.50, p < 0.01), but not with the intensity of chronic pain. CONCLUSIONS: This study confirms that pre-operative anxiety is associated with both acute and chronic post-operative pain after partial mastectomy. It also suggests that lower perioperative cortisol secretion might be associated with greater acute post-operative pain. SIGNIFICANCE: Although the associations between psychological stress/stress hormone levels and chronic post-operative pain remain to be determined, pre-operative psychological stress and perioperative cortisol levels are correlated with acute post-operative pain.
Assuntos
Dor Aguda/fisiopatologia , Transtornos de Ansiedade/complicações , Ansiedade/psicologia , Dor Crônica/complicações , Depressão/psicologia , Dor Pós-Operatória/etiologia , Humanos , Mastectomia , Medição da Dor , Estudos Prospectivos , Estresse PsicológicoRESUMO
BACKGROUND: In this randomized controlled trial, we examined whether intra- and postoperative infusion of low-dose ketamine decreased postoperative morphine requirement and morphine-related adverse effects as nausea and vomiting after scoliosis surgery. METHODS: After IRB approval and informed consent, 36 patients, aged 10-19 years, undergoing posterior correction surgery for adolescent idiopathic scoliosis, were randomly allocated into two groups: intra- and postoperative ketamine infusion at a rate of 2 µg/kg/min until 48 h after surgery (ketamine group, n = 17) or infusion of an equal volume of saline (placebo group, n = 19). All patients were administered total intravenous anesthesia with propofol and remifentanil during surgery and intravenous morphine using a patient-controlled analgesia device after surgery. The primary outcome was cumulative morphine consumption in the initial 48 h after surgery. Pain scores (Numerical Rating Scale, NRS, 0-10), sedation scales, incidence of postoperative nausea and vomiting (PONV), and antiemetic consumption were recorded by nurses blinded to the study protocol for 48 h after surgery. RESULTS: Patient characteristics did not differ between the two groups. Cumulative morphine consumption for 48 h after surgery was significantly lower in the ketamine group compared to the placebo group (0.89 ± 0.08 mg/kg vs. 1.16 ± 0.07 mg/kg, 95% confidence interval for difference between the means, 0.03-0.48 mg/kg, P = 0.019). NRS pain, sedation scales, and incidence of PONV did not differ between the two groups. Antiemetic consumption was significantly smaller in ketamine group. CONCLUSIONS: Intra- and postoperative infusion of low-dose ketamine reduced cumulative morphine consumption and antiemetic requirement for 48 h after surgery.
Assuntos
Ketamina/administração & dosagem , Escoliose/cirurgia , Adolescente , Criança , Feminino , Humanos , Masculino , Morfina/administração & dosagem , Dor Pós-Operatória/prevenção & controle , Náusea e Vômito Pós-Operatórios/prevenção & controle , Adulto JovemRESUMO
Doxapram is the only dedicated respiratory stimulant used to aid recovery of breathing after major surgery. Doxapram acts on peripheral chemoreceptors and although the central action of doxapram has been suggested, its detailed neuronal mechanism is unknown. We assessed doxapram-induced changes in spontaneous cervical nerve (C4) inspiratory activity and the firing of action potentials in pre-inspiratory and inspiratory neurones in the medulla. Experiments were performed in neonatal rat brainstem-spinal cord preparations, which can produce respiratory rhythm for several hours under in vitro conditions. Doxapram application (for 15 min) increased the frequency and amplitude of C4 activity dose-dependently. Doxapram induced changes in the electrophysiological properties of pre-inspiratory and inspiratory neurones. Our results suggest that respiratory activity enhancement was likely to be induced via effects on the potassium channels of pre-inspiratory and inspiratory neurones and indicate the central actions of doxapram.
Assuntos
Tronco Encefálico/efeitos dos fármacos , Doxapram/farmacologia , Neurônios/efeitos dos fármacos , Mecânica Respiratória/efeitos dos fármacos , Medicamentos para o Sistema Respiratório/farmacologia , Medula Espinal/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Bulbo/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
In order to elucidate the mechanisms of post-exercise acute renal failure, one of the complications of hereditary renal hypouricemia, we have targeted the mouse Slc22a12 gene by the exchange of exons 1-4 with pMC1neo-polyA. The knockout mice revealed no gross anomalies. The concentration ratio of urinary urate/creatinine of the knockout mice was significantly higher than that of wildtype mice, indicating an attenuated renal reabsorption of urate. The plasma levels of urate were around 11 muM and were similar among the genotypes. Although the fractional excretion of urate of knockout mice was tend to higher than that of wildtype mice, the urate reabsorption ability remained in the kidney of knockout mice, indicating a urate reabsorptive transporter other than Urat1.
Assuntos
Camundongos Knockout , Transportadores de Ânions Orgânicos/genética , Alantoína/urina , Animais , Northern Blotting , Western Blotting , Cromatografia Líquida de Alta Pressão , Creatinina/urina , Camundongos , Transportadores de Ânions Orgânicos/metabolismo , Ácido Úrico/sangue , Ácido Úrico/metabolismo , Ácido Úrico/urinaRESUMO
Alternative splicing of the 12-base exon 2 of the adenosine monophosphate deaminase (AMPD) gene is subject to regulation by both cis- and trans-regulatory signals. The extent of exon 2 inclusion is stage- and cell type-specific and is subject to the physiological state of the cell. In adult skeletal muscle, a cell type that regulates the activity of this allosteric enzyme at several levels, the exon 2-plus form of AMPD, predominates. We have performed a systematic analysis of the cis-acting regulatory sequences that reside in the intron immediately downstream of this mini-exon. A complex element comprising sequences that enhance exon 2 inclusion and sequences that counteract this effect resides in the middle of this intron. We demonstrate that the enhancing component is bipartite, with more than a kilobase of sequence separating the two functional sites. The presence of even minimal levels the mini-exon in the fully processed AMPD mRNA requires both of these sites, neither of which appears in any other published splicing enhancer. An RNA binding activity derived from a muscle cell line requires both of the enhancing sites. Mutations in either of the sites that eliminate exon 2 inclusion abrogate this binding activity.
Assuntos
AMP Desaminase/genética , Processamento Alternativo , Elementos Facilitadores Genéticos , Éxons/genética , Íntrons , Células 3T3 , Animais , Sequência de Bases , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Músculos/citologia , Músculos/metabolismo , Mutagênese Sítio-Dirigida , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: A noninvasive bronchoscopic microsampling (BMS) probe was developed to sample biochemical constituents of the epithelial lining fluid in small airways. DESIGN: Observational, controlled study. SETTING: Intensive care unit of academic medical center. PATIENTS AND PROCEDURE: BMS was applied in a control group of seven patients who had hemoptysis or small solitary peripheral nodules but no hypoxemia or other signs of acute inflammation and in four patients with acute respiratory distress syndrome (ARDS), to test whether BMS can ascertain the presence of acute pulmonary inflammation without complications. MEASUREMENTS AND RESULTS: Complications, including a significant decrease in arterial oxygen saturation, were observed neither during nor after BMS. In the ARDS group, albumin, lactate dehydrogenase, interleukin-6, basic fibroblast growth factor, and neutrophil elastase concentrations in epithelial lining fluid were significantly higher (p <.0001, p =.012, p <.0001, p <.0001, and p <.0001, respectively) than in the control group. Serial BMS was safely performed in one patient with ARDS, allowing us to observe a correlation between changes in the concentration of inflammation-related biochemical markers and clinical course of the disease. CONCLUSIONS: These results suggest that BMS is safe and useful to monitor pulmonary biochemical events in ARDS.
Assuntos
Líquido da Lavagem Broncoalveolar/química , Broncoscopia/métodos , Síndrome do Desconforto Respiratório/fisiopatologia , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We examined the alterations of peripheral vascular responses following ischemic insult during perioperative period of esophagectomy. Increase of palm blood flow after vascular occlusion, i.e., reactive hyperemia (RH), measured by near-infrared spectroscopy (NIRS) was employed to assess forearm vascular responses. The measurements of RH were performed in esophagectomized patients (n = 12) before induction of anesthesia and postoperatively until the next day of extubation in comparison with normal volunteers (n = 11). After esophagectomy, the RH, which was comparable with those in volunteers, was depressed by 50% on 1 POD, and did not recover until the third POD. In particular, patients receiving laparoscopy-assisted surgery showed less decrease of RH than those receiving the standard open laparotomy. These results suggest that vascular responses to increase blood flow against ischemic insult is depressed following esophagectomy.
Assuntos
Neoplasias Esofágicas/fisiopatologia , Esofagectomia , Adulto , Idoso , Velocidade do Fluxo Sanguíneo , Neoplasias Esofágicas/cirurgia , Feminino , Antebraço/irrigação sanguínea , Humanos , Laparoscopia , Laparotomia , Masculino , Pessoa de Meia-Idade , Monitorização Fisiológica , Consumo de Oxigênio , Período Pós-Operatório , Espectroscopia de Luz Próxima ao Infravermelho , Resistência VascularRESUMO
A 46-year-old woman with exertional myalgia developed slowly progressive weakness in her lower extremities. She had slight muscle weakness in her facial and upper extremities, and severe muscle weakness and atrophy in lower extremities more marked in the proximal portions. Serum creatine kinase was slightly elevated. After ischemic forearm exercise test, blood ammonia had no elevation although lactate level increased normally. The computed tomography revealed that a characteristic distribution of skeletal muscle involvement with proximal and flexor muscles more severely affected than distal and extensor in the lower extremities. In addition, the left sternocleidomastoid muscle showed marked atrophy with an asymptomatic weakness of over 20 years duration suggesting abnormal development. Needle EMG examination showed a large number of easily recruited, short-duration, low-amplitude motor unit potentials in all extremities. Muscle biopsy showed absence of adenosine monophosphate deaminase activity with normal cytochrome c oxidase and phosphorylase activity. With the muscle enzyme activity assay, adenosine monophosphate deaminase activity was found to be lower than 0.2% of the controls. The DNA analysis revealed that she was compound heterozygote involving two missense mutations (R388W and R425H) in exon 9 and exon 10 of AMPD1 gene. This is the first report of primary myoadenylate deaminase deficiency with progressive weakness and atrophy caused by novel compound heterozygous mutations of AMPD1 gene, and suggests that adenosine monophosphate deaminase is closely related not only to energy metabolism but also to the development of skeletal muscle.
Assuntos
AMP Desaminase/genética , Atrofia Muscular/genética , Mutação de Sentido Incorreto , AMP Desaminase/deficiência , Alelos , Substituição de Aminoácidos , Biópsia , Progressão da Doença , Feminino , Heterozigoto , Humanos , Japão , Pessoa de Meia-Idade , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Atrofia Muscular/enzimologia , Atrofia Muscular/patologia , Paresia/genética , Paresia/patologiaRESUMO
Divalent mercury (Hg(2+)) blocked human skeletal Na(+) channels (hSkM1) in a stable dose-dependent manner (K(d) = 0.96 microM) in the absence of reducing agent. Dithiothreitol (DTT) significantly prevented Hg(2+) block of hSkM1, and Hg(2+) block was also readily reversed by DTT. Both thimerosal and 2,2'-dithiodipyridine had little effect on hSkM1; however, pretreatment with thimerosal attenuated Hg(2+) block of hSkM1. Y401C+E758C rat skeletal muscle Na(+) channels (mu1) that form a disulfide bond spontaneously between two cysteines at the 401 and 758 positions showed a significantly lower sensitivity to Hg(2+) (K(d) = 18 microM). However, Y401C+E758C mu1 after reduction with DTT had a significantly higher sensitivity to Hg(2+) (K(d) = 0.36 microM) than wild-type hSkM1. Mutants C753Amu1 (K(d) = 8.47 microM) or C1521A mu1 (K(d) = 8.63 microM) exhibited significantly lower sensitivity to Hg(2+) than did wild-type hSkM1, suggesting that these two conserved cysteinyl residues of the P-loop region may play an important role in the Hg(2+) block of the hSkM1 isoform. The heart Na(+) channel (hH1) was significantly more sensitive to low-dose Hg(2+) (K(d) = 0.43 microM) than was hSkM1. The C373Y hH1 mutant exhibited higher resistance (K(d) = 1.12 microM) to Hg(2+) than did wild-type hH1. In summary, Hg(2+) probably inhibits the muscle Na(+) channels at more than one cysteinyl residue in the Na(+) channel P-loop region. Hg(2+) exhibits a lower K(d) value (<1. 23 microM) for inhibition by forming a sulfur-Hg-sulfur bridge, as compared to reaction at a single cysteinyl residue with a higher K(d) value (>8.47 microM) by forming sulfur-Hg(+) covalently. The heart Na(+) channel isoform with more than two cysteinyl residues in the P-loop region exhibits an extremely high sensitivity (K(d) < 0. 43 microM) to Hg(+), accounting for heart-specific high sensitivity to the divalent mercury.
Assuntos
Cisteína , Cloreto de Mercúrio/farmacologia , Canais de Sódio/química , Canais de Sódio/fisiologia , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacologia , Substituição de Aminoácidos , Animais , Dissulfetos/farmacologia , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Músculo Esquelético , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Canais de Sódio/genética , Timerosal/farmacologia , TransfecçãoRESUMO
STUDY OBJECTIVE: To examine the effects of additional droperidol or butorphanol to epidural fentanyl infusion on postsurgical analgesia. DESIGN: Prospective, randomized, single blinded clinical study. SETTING: University-affiliated medical center. PATIENTS: 60 ASA physical status I and II patients undergoing anorectal surgery by one surgeon. INTERVENTIONS: Patients were randomly allocated to the following groups according to the medication that was continuously administered into the epidural space: 1) Group C (n = 20) received 0.4 mg fentanyl in 40 ml of 0.125% bupivacaine; 2) Group D (n = 20) received 2.5 mg droperidol and 0.4 mg fentanyl in 40 ml of 0.125% bupivacaine; and 3) Group B (n = 20) received 2 mg butorphanol and 0.4 mg fentanyl in 40 ml of 0.125% bupivacaine over 24 hours postoperatively. MEASUREMENTS AND MAIN RESULT: Postsurgical analgesia and the incidence of fentanyl-related complications, such as nausea/vomiting, somnolence, pruritus, and respiratory depression, were assessed for 24 hours postoperatively. At 16 and 24 hours after surgery, 75% of patients in Group D reported no pain versus 35% in Group C (p < 0.05). In addition, the overall visual analogue scale examined at 24 hours was significantly lower in Group D than that in Group C (22 +/- 17 mm vs. 44 +/- 22 mm, respectively; p < 0.05). Simultaneously, the incidence of postoperative nausea/vomiting was lower in Group D compared with Group C (20% vs. 60%; p < 0.05). On the other hand, butorphanol did not modify the analgesic effects or complications of epidural fentanyl infusion. CONCLUSION: In this study population, additional droperidol, not butorphanol, improved postsurgical analgesia accompanied by less incidence of nausea/vomiting during epidural fentanyl administration.
Assuntos
Adjuvantes Anestésicos/uso terapêutico , Canal Anal/cirurgia , Analgesia Epidural , Analgésicos Opioides/uso terapêutico , Butorfanol/uso terapêutico , Droperidol/uso terapêutico , Fentanila/uso terapêutico , Dor Pós-Operatória/tratamento farmacológico , Reto/cirurgia , Adjuvantes Anestésicos/efeitos adversos , Analgésicos Opioides/efeitos adversos , Análise de Variância , Anestésicos Locais/efeitos adversos , Anestésicos Locais/uso terapêutico , Bupivacaína/efeitos adversos , Bupivacaína/uso terapêutico , Butorfanol/efeitos adversos , Droperidol/efeitos adversos , Feminino , Fentanila/efeitos adversos , Seguimentos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Medição da Dor , Náusea e Vômito Pós-Operatórios/induzido quimicamente , Estudos Prospectivos , Prurido/induzido quimicamente , Respiração/efeitos dos fármacos , Método Simples-Cego , Fases do Sono/efeitos dos fármacosRESUMO
Numerous changes in gene expression are known to occur during replicative senescence, including changes in genes involved in the cell cycle control. In the present study, we have found a severe impairment in the activation of Cdk2 and Cdk4 in response to mitogens in senescent human fibroblasts and determined the molecular basis for this. Although Cdk4 protein was constitutively expressed in senescent cells at the same level as in early-passage young cells, it was found to be complexed with a distinct set of Cdk inhibitors. Cdk4 derived from early passage quiescent cells was effectively activated by incubation with cyclin D1 and Cdk-activating kinase (CAK) in vitro, whereas Cdk4 from senescent cells was not. Cdk2 protein was dramatically decreased in senescent cells and complexed primarily with cyclin D1 and p21. This cyclin D1-bound Cdk2 was not activated by CAK either in vivo or in vitro, implicating cyclin D1 as an inhibitor of Cdk2 activation. Thus, one of the underlying molecular events involved in replicative senescence is the impaired activation of Cdk4 and Cdk2 due to increased binding of p16 to Cdk4 and increased association of Cdk2 with cyclin D1 and p21.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Senescência Celular/fisiologia , Ciclina D1/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Supressoras de Tumor , Proteínas Sanguíneas/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Ciclina D1/farmacologia , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , DNA/biossíntese , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/farmacologia , Fase de Repouso do Ciclo Celular/fisiologia , Pele/citologia , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
In response to DNA damage, mammalian cells adopt checkpoint regulation, by phosphorylation and stabilization of p53, to delay cell cycle progression. However, most cancer cells that lack functional p53 retain an unknown checkpoint mechanism(s) by which cells are arrested at the G(2)/M phase. Here we demonstrate that a human homolog of Cds1/Rad53 kinase (hCds1) is rapidly phosphorylated and activated in response to DNA damage not only in normal cells but in cancer cells lacking functional p53. A survey of various cancer cell lines revealed that the expression level of hCds1 mRNA is inversely related to the presence of functional p53. In addition, transfection of normal human fibroblasts with SV40 T antigen or human papilloma viruses E6 or E7 causes a marked induction of hCds1 mRNA, and the introduction of functional p53 into SV40 T antigen- and E6-, but not E7-, transfected cells decreases the hCds1 level, suggesting that p53 negatively regulates the expression of hCds1. In cells without functional ataxia telangiectasia mutated (ATM) protein, phosphorylation and activation of hCds1 were observed in response to DNA damage induced by UV but not by ionizing irradiation. These results suggest that hCds1 is activated through an ATM-dependent as well as -independent pathway and that it may complement the function of p53 in DNA damage checkpoints in mammalian cells.
Assuntos
Ciclo Celular , Dano ao DNA , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Compartimento Celular , Proteínas de Ciclo Celular , Quinase do Ponto de Checagem 2 , Mapeamento Cromossômico , Cromossomos Humanos Par 22 , Clonagem Molecular , Proteínas de Ligação a DNA , Ativação Enzimática , Humanos , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de TumorRESUMO
BACKGROUND: While permissive hypercapnia is commonly practised in critical care, it remains unclear if the comparable manoeuvres are clinically acceptable during anaesthesia. This retrospective study aimed at describing the anaesthetic implications of hypercapnia associated with deliberate hypoventilation during thoracic surgery in patients with severe emphysema. METHODS: Thirteen patients with emphysema who required thoracic surgery under similar anaesthesia were reviewed: 3 patients were managed to maintain normocapnia (normocapnia group) whereas 10 patients developed hypercapnia (PaCO2 >70 mmHg) as a result of restricting peak airway pressures (hypercapnia group). RESULTS: In the normocapnia group (PaCO2: 45+/-1 mmHg, mean+/-SD), no event which required therapeutic intervention during the surgery was seen, whereas 2 of 3 patients showed postoperative air leakage which persisted over 5 days. In the hypercapnia group, the maximum PaCO2 during anaesthesia ranged between 70 mmHg and 135 mmHg (98-21 mmHg). During anaesthesia, all 10 patients required inotropic support to prevent hypotension, 4 patients required tracheal gas insufflation of oxygen to the operated lung to avoid hypoxaemia and 3 patients required lidocaine to treat ventricular arrhythmia. However, the trachea was extubated in the operation theatre in 9 of 10 patients and no organ dysfunction was observed postoperatively. Four patients showed postoperative air leak on the first postoperative day, one of which persisted over 5 days. CONCLUSION: Although there are some limitations, this preliminary study indicates that hypercapnia around 100 mmHg during anaesthesia for thoracic surgery may not be associated with serious consequences.
Assuntos
Anestesia Geral , Dióxido de Carbono/sangue , Hipercapnia/sangue , Pneumonectomia , Idoso , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Cardiotônicos/uso terapêutico , Cuidados Críticos , Feminino , Seguimentos , Humanos , Hipotensão/prevenção & controle , Hipoventilação/sangue , Complicações Intraoperatórias/prevenção & controle , Intubação Intratraqueal , Lidocaína/uso terapêutico , Masculino , Pessoa de Meia-Idade , Salas Cirúrgicas , Oxigenoterapia , Pneumonectomia/efeitos adversos , Pneumotórax/etiologia , Enfisema Pulmonar/cirurgia , Estudos RetrospectivosRESUMO
Checkpoint genes cause cell cycle arrest when DNA is damaged or DNA replication is blocked. Although a human homolog of Chk1 (hChk1) has recently been reported to be involved in the DNA damage checkpoint through phosphorylation of Cdc25A, B, and C, it is not known at which phase(s) of the cell cycle hChk1 functions and how hChk1 causes cell cycle arrest in response to DNA damage. In the present study, we demonstrate that in normal human fibroblasts (MJ90), hChk1 is expressed specifically at the S to M phase of the cell cycle at both the RNA and protein levels and that it is localized to the nucleus at this time. hChk1 activity, as determined by phosphorylation of Cdc25C, is readily detected at the S to M phase of the cell cycle, and DNA damage induced by UV or ionizing radiation does not enhance the expression of hChk1 or its activity. Furthermore, hChk1 exists in an active form at the S to M phase in fibroblasts derived from patients with ataxia telangiectasia (AT) which lack the functional AT mutated (ATM) gene product, suggesting that hChk1 expression is independent of functional ATM. Taken together with the findings that phosphorylation of Cdc25C on serine 216 is increased at the S to M phase, it is suggested that at this particular phase of the cell cycle, even in the absence of DNA damage, hChk1 phosphorylates Cdc25C on serine 216, which is considered to be a prerequisite for the G2/M checkpoint. Thus, hChk1 may play an important role in keeping Cdc25C prepared for responding to DNA damage by phosphorylating its serine residue at 216 during the S to M phase.
Assuntos
Ciclo Celular , Proteínas Quinases/biossíntese , Proteínas Serina-Treonina Quinases , Proteínas/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/enzimologia , Quinase 1 do Ponto de Checagem , DNA/efeitos da radiação , Dano ao DNA , Proteínas de Ligação a DNA , Indução Enzimática , Fibroblastos/enzimologia , Células HeLa/enzimologia , Humanos , Metáfase , Fosforilação , Isoformas de Proteínas/metabolismo , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Fase S , Proteínas Supressoras de Tumor , Raios Ultravioleta , Raios X , ras-GRF1RESUMO
p57KIP2 is a potent tight-binding inhibitor of several G1 cyclin/cyclin-dependent kinase (Cdk) complexes, and is a negative regulator of cell proliferation. The gene encoding p57KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome (BWS). Previously we demonstrated that p57KIP2 is imprinted and only the maternal allele is expressed in both mice and humans. We also showed mutations found in p57KIP2 in patients with BWS that were transmitted from the patients' carrier mothers, indicating that the expressed maternal allele was mutant and that the repressed paternal allele was normal. In the study reported here, we performed functional analysis of the two mutated p57KIP2 genes. We showed that the nonsense mutation found in the Cdk inhibitory domain in a BWS patient rendered the protein inactive with consequent complete loss of its role as a cell cycle inhibitor and of its nuclear localization. We also showed that the mutation in the QT domain, although completely retaining its cell cycle regulatory activity, lacked nuclear localization and was thus prevented from performing its role as an active cell cycle inhibitor. Consequently, no active p57KIP2 would have existed, which might have caused the disorders in BWS patients.
Assuntos
Síndrome de Beckwith-Wiedemann/genética , Quinases relacionadas a CDC2 e CDC28 , Proteínas Nucleares/genética , Animais , Células COS , Núcleo Celular/enzimologia , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p57 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Escherichia coli/genética , Imunofluorescência , Regulação Bacteriana da Expressão Gênica , Humanos , Mutação , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Plasmídeos/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Although the cyclopentenone prostaglandin A1 (PGA1) is known to arrest the cell cycle at the G1 phase in vitro and to suppress tumor growth in vivo, its relatively weak activity limits its usefulness in cancer chemotherapy. In an attempt to develop antitumor drugs of greater potency and conspicuous biological specificity, we synthesized novel analogs based on the structure of PGA1. Of the newly synthesized analogs, 15-epi-delta7-PGA1 methyl ester (NAG-0092), 12-iso-delta7-PGA1 methyl ester (NAG-0093), and ent-delta7-PGA1 methyl ester (NAG-0022) possess a cross-conjugated dienone structure around the five-member ring with unnatural configurations at C(12) and/or C(15) and were found to be far more potent than native PGA1 in inhibiting cell growth and causing G1 arrest in A172 human glioma cells. These three analogs induced the expression of p21 at both RNA and protein levels in a time- and dose-dependent fashion. Kinase assays with A172 cells treated with these analogs revealed that both cyclin A- and E-dependent kinase activities were markedly reduced, although cyclin D1-dependent kinase activity was unaffected. Immunoprecipitation-Western blot analysis showed that the decrease in cyclin A-dependent kinase activity was due to an increased association of p21 with cyclin A-cyclin-dependent kinase 2 complexes, whereas the decrease in cyclin E-dependent activity was due to a combined mechanism involving reduction in cyclin E protein itself and increased association of p21. Thus, these newly synthesized PGA1 analogs may prove to be powerful tools in cancer chemotherapy as well as in investigations of the structural basis of the antiproliferative activity of A series prostaglandins.
Assuntos
Antineoplásicos/farmacologia , Ciclina E/metabolismo , Glioma/metabolismo , Prostaglandinas A/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Antineoplásicos/química , Divisão Celular/efeitos dos fármacos , Ciclina A/metabolismo , Ciclina D1/metabolismo , Ciclina E/antagonistas & inibidores , Fase G1 , Humanos , Prostaglandinas A/química , Proteínas Quinases/metabolismo , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
We have examined if sevoflurane anaesthesia per se modified the number of circulating leucocytes in humans. Fifty-nine patients undergoing elective surgery were anaesthetized with sevoflurane in oxygen. The inhaled concentration was increased gradually to 5% and maintained for 20 min. Arterial blood samples were obtained before induction of anaesthesia and at 20 min. While the total number of leucocytes remained constant, circulating neutrophils decreased (mean 3370 (SD 1030) mm-3 to 3170 (940) mm-3; P < 0.01) and lymphocytes increased (1870 (520) mm-3 to 2040 (580) mm-3; P < 0.01). We conclude that high concentrations of sevoflurane modified the distribution of leucocytes in anaesthetized patients.
Assuntos
Anestésicos Inalatórios/farmacologia , Contagem de Leucócitos/efeitos dos fármacos , Éteres Metílicos/farmacologia , Adulto , Humanos , Período Intraoperatório , Linfócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , SevofluranoRESUMO
Although crystal structural analysis of cyclin A/cyclin-dependent kinase 2 (Cdk2)/p27 (Russo, A. A., Jeffrey, P. D., Pattern, A. K., Massague, J., and Pavletich, N. P. (1996) Nature 382, 325-331) has suggested that the 310 helix region in Cdk inhibitors of the Cip/Kip family may be involved in the inhibition of cyclin/Cdk activities, there is no biochemical evidence supporting this hypothesis. In the present study, we demonstrated that cyclin and Cdk binding domains of p57 were necessary but were not sufficient in themselves for the inhibition of cyclin A/Cdk2 and cyclin E/Cdk2, and that the 3(10) helix region of this protein is indispensable for the inhibition of these complexes. In contrast, the 3(10) helix regions of p21 and p27 were not required, and cyclin- and Cdk-binding domains alone were sufficient for the inhibition of all cyclin/Cdk complexes examined. Site-directed mutagenesis identified phenylalanine 79 and tyrosine 80 within the 3(10) helix region of p57 as crucial residues for kinase inhibition, supporting the structural evidence that the 3(10) helix binds deep inside the catalytic cleft of Cdk2, mimicking ATP. Mutations within the 3(10) helix region of the p57 molecule completely abolished the ability to arrest the cell cycle at G1 in vivo. These results indicate that this region is specifically utilized by p57 in selectively inhibiting cyclin A or E/Cdk2+ activities. Thus the 3(10) helix motif may confer a specific regulatory mechanism by which p57 differentially regulates Cdk2 and Cdk4 activities.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Inibidores do Crescimento/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae , Sítios de Ligação , Cristalografia por Raios X , Ciclina A/antagonistas & inibidores , Ciclina E/antagonistas & inibidores , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p57 , Inibidores Enzimáticos/química , Escherichia coli , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Conformação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Spodoptera , Relação Estrutura-AtividadeRESUMO
UNLABELLED: Recent reports have discussed the potential risk of transient radicular irritation (TRI) after spinal anesthesia with lidocaine. Because we have not encountered such neurologic sequelae with the high incidence reported, we prospectively examined the incidence of TRI after spinal anesthesia with lidocaine. One thousand forty-five adult patients (aged 47 +/- 15 yr) receiving spinal anesthesia with 3% hyperbaric lidocaine (1.0-1.5 mL) for anorectal surgery were consecutively studied. After the induction of spinal anesthesia, all patients were placed in the prone position for surgery. Patients were evaluated for neurologic symptoms in the buttocks, thighs, or lower extremities using a checklist to standardize data collection. Although there was no complaint of neurologic symptoms on Postoperative Day (POD) 1, four patients (0.4%) reported aching, hypesthesia, numbness, or dull pain of both lower extremities and buttocks by the morning of POD 3. In three patients, the symptoms resolved without any treatment by POD 5, whereas in one patient, numbness of the lower extremities lasted until POD 7. We conclude that a combination of lidocaine with surgical position or leg manipulation during surgery might be a major contributing factor in the development of transient neurologic syndrome. IMPLICATIONS: Recent studies have reported the potential risk of transient neurologic syndrome after lidocaine spinal anesthesia. The current study reports a low incidence of such sequelae in 1045 patients undergoing anorectal surgery during the prone position. Other factors, such as surgical position, may be important in the development of this syndrome.