RESUMO
Objective: To compare the efficacy of remimazolam and propofol on hemodynamics and quality of early postoperative recovery in elderly patients with frailty undergoing endoscopic retrograde cholangiopancreatography (ERCP). Methods: A total of 108 elderly patients with frailty (aged≥75 years) undergoing elective ERCP in the General Hospital of Northern Theater Command were prospectively enrolled from November 2022 to May 2023. According to the different anesthetic drugs used, the patients were divided into two groups by random number table method: remimazolam group (group R) and propofol group (group P). The group R was given remimazolam 0.15-0.20 mg/kg and alfentanil 5.0 µg/kg for anesthesia onset, and then was pumped remimazolam 0.4-0.8 mg·kg-1·h-1 and alfentanil 0.5 µg·kg-1·min-1 to maintain sedation. The group P was given propofol 1.0-1.5 mg/kg and alfentanil 5.0 µg/kg, and was pumped propofol 2.0-6.0 mg·kg-1·h-1 and alfentanil 0.5 µg·kg-1·min-1. The primary outcome was the incidence of intraoperative hypotension [mean arterial pressure (MAP)<65 mmHg (1 mmHg=0.133 kPa) or MAP>20% decrease from baseline value] and severe hypotension (MAP<55 mmHg) in both groups, and other outcomes included: MAP, heart rate, pulse oxygen saturation (SpO2) and bispectral index (BIS) values of patients at each time of before anesthesia induction (T0), 1 min after anesthesia induction (T1), endoscope through the oropharynx (T2), immediate lithotomy (T3), endoscope withdrawal from the oropharynx (T4), and patients awake (T5); the use of vasoactive drug during operation; the incidence of bradycardia, hypoxemia and injection pain; and the postoperative 15-item Quality of Recovery (QoR-15) score. Results: Group R included 33 males and 20 females, aged (81.5±4.9) years. Group P included 26 males and 29 females, aged (82.3±6.0) years. The incidence of intraoperative hypotension in group R was 24.5% (13/53), which was lower than 43.6% (24/55) in group P (P=0.036), there was no significant difference of the incidence of severe hypotension which was 0 (0/53) and 5.5% (3/55) (P=0.225). Compared with T0, MAP and BIS decreased at T1-T4 (both P<0.05); heart rate and SpO2 decreased at T1-T2 in both groups (both P<0.05). Compared with group P, MAP increased at T1-T4; heart rate, SpO2 and BIS increased in group R (all P<0.05). The use of intraoperative vasoactive drug in group R was (93.9±21.4) µg, lower than (123.3±29.7) µg in group P (P<0.001), and the incidence of bradycardia, hypoxemia and injection pain in group R was 5.7% (3/53), 13.2% (7/53), and 3.8% (2/53), lower than 18.2% (10/55), 30.9% (17/55), and 16.4% (9/55) in group P (all P<0.05). There was no significant difference in the incidence of bucking or involuntary body movement and hiccuping in both groups (both P>0.05). The awakening time in group R was (11.8±3.0) min, longer than (10.3±3.3) min in group P (P=0.016), and the incidence of emergence agitation was 3.8% (2/53), lower than 16.4% (9/55) (P=0.031). There was no significant difference in postanesthesia care unit (PACU) stay duration and the incidence of postoperative nausea and vomiting in both groups (all P>0.05). The postoperative QoR-15 scores at 1 d were (131.9±4.7) and (129.3±5.7) with statistically significant difference (P=0.010), and QoR-15 scores at 3 d were (134.8±3.3) and (133.6±5.0) with no significant difference (P=0.205). Conclusions: Compared with propofol, remimazolam reduces the incidence of intraoperative hypotension, bradycardia, injection pain and the use of intraoperative vasoactive drug on elderly patients with frailty undergoing ERCP. Remimazolam has relatively stable hemodynamics, it prolongs the recovery time but does not significantly affect the quality of early postoperative recovery.
Assuntos
Colangiopancreatografia Retrógrada Endoscópica , Hemodinâmica , Propofol , Humanos , Idoso , Propofol/administração & dosagem , Fragilidade , Período Pós-Operatório , Estudos Prospectivos , Masculino , Período de Recuperação da Anestesia , Feminino , BenzodiazepinasRESUMO
Objective: To explore the effect of high-flow nasal catheter oxygen inhalation in preventing hypoxemia during endoscopic retrograde cholangiopancreatography (ERCP) surgery in elderly patients. Methods: From September 2021 to September 2022, 116 elderly patients (aged ≥ 70 years) who underwent elective ERCP in the Northern Theater General Hospital were prospectively selected, then divided into general nasal catheter oxygen inhalation group [group C, 31 males and 27 females, aged (79.8±6.4) years] and high-flow nasal catheter oxygen inhalation group [group H, 33 males and 25 females, aged (81.4±6.7) years], with 58 patients in each group. All patients were monitored for anesthesia by target-controlled infusion of propofol and remifentanil. The main outcome index was the incidence of intraoperative subclinical hypoxemia (90% ≤ SpO2 < 95%, duration >5 s), hypoxemia (75% < SpO2 < 90%, 5 s < duration ≤ 60 s) and severe hypoxemia (SpO2 < 75% or SpO2 < 90%, duration > 60 s). Secondary observation measures were SpO2 from T0 to T5 (T0, before anesthesia induction; T1, immediately after anesthesia induction; T2, endoscopic introduction; T3, duodenal papula intubation; T4, endoscopic withdrawal; T5, postoperative awakening), the arterial oxygen partial pressure (PaO2), carbon dioxide partial pressure (PaCO2) and pH at T0, 15 min after the induction and T5. Results: The incidence of intraoperative subclinical hypoxemia in group C and group H was 12.0% (7/58) and 3.4% (2/58) respectively, which showed no significant statistical difference (P=0.165) from each other. The incidence of intraoperative hypoxemia in group H was 8.6% (5/58), which was significantly lower than 31.0% (18/58) of group C (P=0.003). Neither group had intraoperative severe hypoxemia. SpO2 of group H were (98.2±0.9)%, (98.2±0.9)%, (97.8±1.7)% and (97.7±1.7)% at T1, T2, T3, T4, which were higher than (96.8±2.1)%, (96.4±3.0)%, (96.1±2.9)% and (96.4±3.4)% in group C (all P<0.05). PaO2 at 15 min after induction in group H was (240.5±46.7) mmHg (1 mmHg=0.133 kPa), which was higher than that of group C (170.6±33.4) mmHg (P<0.001). There was no statistically significant difference in pH and PaCO2 between the two groups of patients at each timepoint. Conclusion: High flow nasal catheter oxygen can effectively reduce the incidence of hypoxemia in ERCP in elderly patients.
Assuntos
Colangiopancreatografia Retrógrada Endoscópica , Oxigênio , Masculino , Idoso , Feminino , Humanos , Colangiopancreatografia Retrógrada Endoscópica/efeitos adversos , Hipóxia/etiologia , Hipóxia/prevenção & controle , Catéteres/efeitos adversos , Anestesia Geral/efeitos adversosRESUMO
OBJECTIVE: The aim of this study was to compare the cytological adequacy rates of different needle passes in ultrasound-guided fine-needle aspiration biopsy of thyroid nodules and, thus, to help establish the criterion for selecting the number of needle passes according to the characteristics of thyroid nodules. PATIENTS AND METHODS: This single-center and randomized prospective study involved 207 consecutive patients with 240 solid or predominantly solid thyroid nodules. These nodules were randomly divided into a 1-pass group, a 2-pass group, and a 3-pass group. Then the nodules were sent for cytopathological diagnosis, and cytological results were classified according to the Bethesda classification. Bethesda I was defined as inadequate, and Bethesda â ¡-â ¥ were defined as adequate. Then the cytological adequacy rates of different groups were compared. RESULTS: In total, 221 nodule specimens were considered as adequate and 19 nodule specimens inadequate. The overall adequacy rate was 92.1%. However, there were no significant differences among the 1, 2, and 3-pass groups in terms of adequacy rates (91.3%, 92.5%, and 92.5%, respectively). CONCLUSIONS: The number of needle passes does not significantly affect the cytological adequacy in ultrasound-guided fine-needle aspiration of solid or predominantly solid thyroid nodules. The cytological adequacy of one-needle pass is comparable to those of two and three-needle passes.
Assuntos
Nódulo da Glândula Tireoide , Humanos , Nódulo da Glândula Tireoide/diagnóstico por imagem , Nódulo da Glândula Tireoide/patologia , Biópsia por Agulha Fina/métodos , Estudos Prospectivos , Ultrassonografia , Ultrassonografia de Intervenção/métodos , Estudos RetrospectivosRESUMO
Objective: To compare the long term and short term outcomes between robotic and open surgery for hilar cholangiocarcinoma radical resection. Methods: This is a single-center and retrospective case-control study. Patients underwent hilar cholangiocarcinoma radical resection between January 2016 and December 2016 at Department of Hepatobiliary Surgery of the General Hospital of the Chinese People's Liberation Army were included. Safety, effectiveness and long-term prognosis of tumors were evaluated. Patients were divided into robotic hilar cholangiocarcinoma radical surgery group (robotic group, n=16) and open hepatic hilar cholangiocarcinoma radical surgery group (open group, n=31) . All cases were confirmed by pathology histological. Age, gender, histology, resection margin status, extent of surgical resection, disease-free survival (DFS) , and overall survival (OS) were retrospectively collected and analyzed.In the follow-up cohort, the primary outcome was patient death and the secondary outcome was tumor recurrence. Continuous variables were expressed as means and medians and were compared using the Student t test if normally distributed or Wilcoxon-Mann-Whitney test for nonparametric variables. Discrete variables were expressed as frequency and percentages and χ(2) or Fisher exact test, when appropriate, were used for comparisons. Kaplan-Meier curves were used to calculate the probability of survival and comparisons were performed using log-rank test. Results: In this study, compared with the open group, the robotic group had a longer operation time ( (338±71) minutes vs. (256±56) minutes, t=4.251, P=0.001) , but the intraoperative blood loss was less (100 ml vs. 200 ml, Z=121.50, P=0.040) , the gastric tube removal time was earlier (3 days vs. 4 days, Z=136.0, P=0.011) , and the postoperative hospital stay was shorter (9 days vs. 12 days, Z=144.50, P=0.040) , and the difference was statistically significant.There was no significant difference in the blood transfusion rate, R0 resection rate, and tumor size between the two groups.The recurrence rates in the robotic group and open surgery were 53.3% and 67.0%, respectively (χ(2)=1.04, P=0.307) .The median survival time of the robotic group and the open group was 22.0 months and 25.0 months. There was no significant difference in the overall survival rate between the two groups (P>0.05) . Conclusion: Compared with laparotomy, robotic HCC radical resection could have an equivalence or non-inferiority approach with acceptable long-term outcome.
Assuntos
Neoplasias dos Ductos Biliares/cirurgia , Tumor de Klatskin/cirurgia , Procedimentos Cirúrgicos Robóticos , Humanos , Laparotomia , Recidiva Local de Neoplasia , Prognóstico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
OBJECTIVE: To observe the effect of the micro ribonucleic acid (miR)-10a on sepsis-induced liver injury in rats through the transforming growth factor-b1 (TGF-b1)/Smad signaling pathway. MATERIALS AND METHODS: The rat model of sepsis was established via cecal ligation and puncture, in which miR-10a was overexpressed and silenced using liposome transfection. The rats were randomly divided into miR-10a mimics group (Mimics group, n=10) and miR-10a inhibitors group (Inhibitors group, n=10), and the sham operation group (Sham group, n=10) was also set up. The transfection efficiency of miR-10a in liver tissues in each group was detected via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), the serum liver function indexes aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were determined. Moreover, the content of the serum reactive oxygen species (ROS), glutathione (GSH), and GSH peroxidase (GSH-Px) was determined using enzyme-linked immunosorbent assay (ELISA). The content of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and myeloperoxidase (MPO) in liver tissues was detected, and the pathological changes in liver tissues were observed through hematoxylin-eosin (HE) staining. Finally, the expression levels of cytochrome P4502E1 (CYP2E1) and TGF-b1/Smad signaling pathway genes and proteins in liver tissues were detected via qRT-PCR and Western blotting. RESULTS: The expression of miR-10a was significantly increased in Mimics group (p<0.05) and extremely low in the Inhibitors group (p<0.05). In Mimics group, the levels of serum AST, ALT, and LDH were significantly increased (p<0.05), the content of ROS, TNF-α, IL-6, and MPO was substantially increased (p<0.05), while that of GSH and GSH-Px notably declined (p<0.05). According to the HE staining results, the liver cells were orderly arranged in the Inhibitors group, and they were disorderly arranged with more inflammatory cells in the Mimics group. The results of the gene and protein assays showed that the expression levels of CYP2E1, TGF-b1, and Smad2 in Mimics group were markedly higher than those in the Sham group (p<0.05), while they displayed the opposite trends in the Inhibitors group (p<0.05). CONCLUSIONS: Silencing miR-10a can inhibit the occurrence of sepsis-induced liver injury in rats by downregulating the TGF-ß1/Smad pathway.
Assuntos
Hepatopatias/metabolismo , MicroRNAs/biossíntese , Sepse/metabolismo , Proteína Smad2/biossíntese , Fator de Crescimento Transformador beta1/biossíntese , Animais , Hepatopatias/etiologia , Hepatopatias/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Ratos , Ratos Sprague-Dawley , Sepse/complicações , Sepse/genética , Transdução de Sinais/fisiologia , Proteína Smad2/genética , Fator de Crescimento Transformador beta1/genéticaRESUMO
Objective: To explore the dynamic features of tumefactive demyelinating lesions (TDLs) in different clinical stages by contrast-enhanced magnetic resonance imaging (MRI). Methods: Thirty-five patients with TDLs proven by pathological studies were prospectively recruited from January 2015 to January 2017.Brain contrast-enhanced MRI of the patients in different clinical stages including acute phase, subacute phase and chronic phase were completed after enrollment.The characteristics of contrast-enhanced MRI in different clinical stages were compared and the evolutional characteristics were summarized. Results: (1) Acute phase (35/35): the patterns of enhancement were patchy (74.3%, n=26), nodule (34.3%, n=12), closed ring (14.3%, n=5) and open ring (11.4%, n=4). (2) Subacute phase (32/35): the patterns of enhancement were open ring (40.6%, n=13), closed ring (31.3%, n=10), patchy (25.0%, n=8) and irregular edge of enhancement (21.9%, n=7). (3) Chronic phase (15/35): the patterns of enhancement were pale patchy (10/15), open ring (5/15) and closed ring (2/15). (4) The proportions of enhancement patterns including patchy, nodule, edge enhancement and pale patchy were significantly different among different clinical stages: â The patchy and nodule were the more common enhancement patterns in acute phase. â¡ The edge enhancement was found mostly in subacute phase. ⢠The pale patchy was found mostly in chronic phase. Conclusions: The manifestation of the lesions on contrast-enhanced MRI may have some characteristics of dynamic evolution according to different clinical stages.The dynamic observation may be helpful for the diagnosis and differential diagnosis of TDLs.
Assuntos
Imageamento por Ressonância Magnética , Encéfalo , Neoplasias Encefálicas , Doenças Desmielinizantes , Humanos , Estudos RetrospectivosAssuntos
Infecções por Bunyaviridae/epidemiologia , Infecções por Bunyaviridae/transmissão , Febre/epidemiologia , Febre/virologia , Phlebovirus , Infecções por Bunyaviridae/diagnóstico , Infecções por Bunyaviridae/virologia , China/epidemiologia , Febre/diagnóstico , Humanos , Índice de Gravidade de DoençaRESUMO
Objective: To explore the clinical characteristics of the neuromyelitis optica spectrum disorders (NMOSD) with the area postrema syndrome as the initial symptom. Methods: A total of 14 cases were enrolled in the study with the diagnose of NMOSD and the area postrema syndrome as the initial symptom. All the clinical data and imaging profiles by the contrasted magnetic resonance imaging (MRI) of the head and spinal cord were collected and analyzed. Results: The median age of onset was (38.1±17.0) years old and the gender ratio of female to male was 10â¶4. The serum aquaporin-4(AQP4)-IgG was positive in 11 subjects and several autoimmune antibodies was positive in 7 subjects. The lesions revealed by MRI of the head mainly located in the area postrema and ependymal periphery which often presented as the linear medullary lesion, while linear lesions over three pieces of vertebra were shown by MRI of the spinal cord which mainly in the grey matter and with a"H" shape around the spinal central canal. Misdiagnose happened in 11 subjects with seven of gastroesophageal reflux disease, two of neurogenic vomiting, one of spinal cord tuberculosis and one of stroke. Conclusions: NMOSD should be considered in patients with unexplained intractable nausea, vomiting and/or hiccups lasted for 48 hours or above, especially in those with positive nervous signs. Contrasted MRI and serum AQP4-IgG need to be performed in the suspected patients. Early detection is crucial for patients with NMOSD.
Assuntos
Aquaporina 4/sangue , Área Postrema/patologia , Autoanticorpos/sangue , Neuromielite Óptica/patologia , Medula Espinal/diagnóstico por imagem , Adulto , Aquaporina 4/metabolismo , Meios de Contraste , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Neuromielite Óptica/sangue , Síndrome , Vômito/etiologia , Adulto JovemRESUMO
Objective: To investigate the clinical features of vestibular migraine with MRI changes. Methods: The clinical data of 150 patients with vestibular migraine completing MRI examination in navy general hospital from August 2008 to August 2016 were retrospectively analyzed.Clinical features of 94 (62.7%) vestibular migraine patients with MRI changes were collected and analyzed according to the medical history, clinical symptoms and signs, as well as the result of auxiliary examinations. Results: The manifestations of MRI in vestibular migraine patients were mainly multiple punctate equal T(1) and long T(2) signals or high signals on FLAIR (fluid attenuated inversion recovery) in subcortical white matter. The average age of the patients at the visit was (52±15) years old, with the male to female ratio of 1â¶3.1. The occurrence of vertigo and migraine were in no particular order, with 54.3% patients presenting migraine several years before vertigo. The duration of vertigo was mostly 24 hours to 72 hours (31.9%). The visual aura (55.3%), photophobia and phonophobia (67.0%) were the most frequently associated symptoms. Conclusions: The vestibular migraine patients with MRI changes have its own specific clinical characteristics and are mainly seen in female with visual aura. The associated symptoms of vestibular migraine appear mostly during the process of vertigo. The pathogenesis of cerebral white matter hyperintensities (WMHs) is not clear.
Assuntos
Imageamento por Ressonância Magnética , Transtornos de Enxaqueca/diagnóstico por imagem , Adulto , Idoso , Epilepsia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vertigem , Substância BrancaRESUMO
In many everyday settings, the relationship between our choices and their potentially rewarding outcomes is probabilistic and dynamic. In addition, the difficulty of the choices can vary widely. Although a large body of theoretical and empirical evidence suggests that dopamine mediates rewarded learning, the influence of dopamine in probabilistic and dynamic rewarded learning remains unclear. We adapted a probabilistic rewarded learning task originally used to study firing rates of dopamine cells in primate substantia nigra pars compacta [Morris G, Nevet A, Arkadir D, Vaadia E, Bergman H (2006) Midbrain dopamine neurons encode decisions for future action. Nat Neurosci 9:1057-1063] for use as a reversal learning task with humans. We sought to investigate how the dopamine depletion in Parkinson's disease (PD) affects probabilistic reward learning and adaptation to a reversal in reward contingencies. Over the course of 256 trials subjects learned to choose the more favorable from among pairs of images with small or large differences in reward probabilities. During a subsequent otherwise identical reversal phase, the reward probability contingencies for the stimuli were reversed. Seventeen PD patients of mild to moderate severity were studied off of their dopaminergic medications and compared to 15 age-matched controls. Compared to controls, PD patients had distinct pre- and post-reversal deficiencies depending upon the difficulty of the choices they had to learn. The patients also exhibited compromised adaptability to the reversal. A computational model of the subjects' trial-by-trial choices demonstrated that the adaptability was sensitive to the gain with which patients weighted pre-reversal feedback. Collectively, the results implicate the nigral dopaminergic system in learning to make choices in environments with probabilistic and dynamic reward contingencies.
Assuntos
Adaptação Psicológica , Deficiências da Aprendizagem/etiologia , Doença de Parkinson/complicações , Aprendizagem por Probabilidade , Reversão de Aprendizagem , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Simulação por Computador , Retroalimentação Psicológica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Psicológicos , Testes Neuropsicológicos , RecompensaRESUMO
Many investigators have reported extensive microglial activation in the mouse substantia nigra and striatum following acute, high-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. Our previous work demonstrated tyrosine hydroxylase (TH)-positive fiber sprouting in the striatum in monkeys that had received a partial dopaminergic lesion using a low-dose, chronic MPTP administration paradigm. To characterize the microglial response, we utilized HLA-DR (LN3) to immunolabel the class II major histocompatibility complex (MHC II). In MPTP-treated monkeys, there was an intense microglial response in the substantia nigra, nigrostriatal tract, and in both segments of the globus pallidus. This response was morphologically heterogeneous, with commingled ramified, activated, and multicellular morphologies throughout the extent of these basal ganglia structures. Surprisingly, there was little evidence of microglial reactivity in the striatum despite evidence of neurodegeneration-by silver labeling and by loss of TH immunolabeling. Moreover, this pattern of microglial reactivity was the same in all animals that had received MPTP and seemed to be independent of the degree of neurotoxin-induced neurodegeneration. Thus, we conclude that microglial reactivity, per se, is not consistently associated with neurodegeneration, but depends on regional differences.
Assuntos
Intoxicação por MPTP/patologia , Microglia/metabolismo , Degeneração Neural/patologia , Animais , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Macaca nemestrina , Masculino , Microglia/efeitos dos fármacos , Degeneração Neural/induzido quimicamente , Doença de Parkinson/patologia , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Dopaminergic lesions result in the acute loss of striatal dopamine content, the loss of tyrosine hydroxylase-immunoreactive fibers, upregulation of preproenkephalin mRNA expression, and compensatory changes in the synthesis and metabolism of dopamine. Despite the severe loss of fine tyrosine hydroxylase-immunoreactive fibers, larger fibers persist. We found that some tyrosine hydroxylase fiber types increase their branching and become thicker after partial lesion. To determine whether the remaining tyrosine hydroxylase fibers were degenerative or part of a compensatory response, we morphologically characterized striatal tyrosine hydroxylase fibers and compared them to silver-stained degenerative structures. Branched and large tyrosine hydroxylase fiber types were nondegenerative. Furthermore, normal preproenkephalin mRNA expression was maintained despite severe overall loss of tyrosine hydroxylase fibers in striatal regions with abundant branching, whereas preproenkephalin mRNA expression increased in severely depleted regions that lacked branched fibers, indicating that branching or sprouting was involved in the compensation for dopamine depletion and the maintenance of normal preproenkephalin expression. In support of compensatory sprouting by tyrosine hydroxylase fibers, mRNA for growth associated protein-43 was upregulated in dopaminergic midbrain cells. We conclude that an important compensatory response to partial dopaminergic depletion is the formation of new branches or sprouting.
Assuntos
Corpo Estriado/fisiologia , Dopamina/metabolismo , Encefalinas/genética , Precursores de Proteínas/genética , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Corpo Estriado/efeitos dos fármacos , Proteína GAP-43/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Macaca nemestrina , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/fisiologia , Fibras Nervosas/ultraestrutura , RNA Mensageiro/genética , Valores de Referência , Transcrição Gênica/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/análiseRESUMO
The site-directed mutated fructosyltransferases (Ftfs) of Streptococcus salivarius ATCC 25975, D312E, D312S, D312N and D312K were all active at 37 degrees C, indicating that Asp-312 present in the 'sucrose box' was not the nucleophilic Asp residue responsible for the formation of a covalent fructosyl-enzyme intermediate required for enzyme activity. Analysis of the kinetic constants of the purified mutated forms of the enzyme showed that Asp-312 was most likely an essential amino acid involved in determining acceptor recognition and/or stabilizing a beta-turn in the protein. In contrast, when the Asp-397 of the Ftf present in the conserved triplet RDP motif of all 60 bacterial and plant family-32 glycosylhydrolases was mutated to a Ser residue, both sucrose hydrolysis and polymerization ceased. Tryptophan emission spectra confirmed that this mutation did not alter protein structure. Comparison of published data from other site-directed mutated enzymes implicated the Asp residue in the RDP motif as the one that may form a transient covalent fructosyl intermediate during the catalysis of sucrose by the Ftf of S. salivarius.
Assuntos
Hexosiltransferases/genética , Mutação , Streptococcus/enzimologia , Streptococcus/genética , Ácido Aspártico/genética , Sequência de Bases , Domínio Catalítico/genética , Primers do DNA/genética , Escherichia coli/genética , Hexosiltransferases/química , Hexosiltransferases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Temperatura , Triptofano/químicaRESUMO
The recombinant fructosyltransferase (Ftf) of Streptococcus salivarius was expressed in Escherichia coli and purified to electrophoretic homogeneity after a combination of adsorption, ion-exchange and gel-filtration chromatography. The N-terminal signal sequence of the Ftf was removed by E. coli at the same site as in its natural host. The purified Ftf exhibited maximum activity at pH 6.0 and 37 degrees C, was activated by Ca2+, but inhibited by the metal ions Cu2+, Zn2+, Hg2+ and Fe3+. The enzyme catalysed the transfer of the fructosyl moiety of sucrose to a number of acceptors, including water, glucose and sucrose via a Ping Pong mechanism involving a fructosyl-enzyme intermediate. While this mechanism of catalysis is utilized by the levansucrases of Bacillus subtilis and Acetobacter diazotrophicus and the values of the kinetic constants for the three enzymes are similar, sucrose was a far more efficient fructosyl-acceptor for the Ftf of S. salivarius than for the two other enzymes.
Assuntos
Hexosiltransferases , Streptococcus/enzimologia , Escherichia coli , Frutanos/metabolismo , Hexosiltransferases/genética , Hexosiltransferases/isolamento & purificação , Hexosiltransferases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
We studied four patients with subacute to chronic varicella zoster virus (VZV) infection of the central nervous system (CNS). VZV infection was verified by detecting antibody to VZV in the cerebrospinal fluid (CSF). VZV caused myelitis in two patients and encephalitis in two patients. In one of the patients with VZV encephalitis, in addition to VZV IgM antibody, VZV DNA was found in the CSF. Among the four patients with VZV infection of the CNS, CSF antibody to VZV was the crucial diagnostic laboratory test which corroborated the clinical features, and indicated that VZV caused neurologic disease. In addition to looking for amplifiable VZV DNA in the CSF of patients with neurologic disease whose clinical and radiologic features suggest VZV infection, we also recommend a search for CSF antibody to VZV, particularly in patients with intervals of weeks to months between zoster and the onset of neurologic disease, or in those patients without rash in whom the tempo of illness is unknown.
Assuntos
Anticorpos Antivirais/líquido cefalorraquidiano , Encefalite Viral/diagnóstico , Herpesvirus Humano 3/imunologia , Mielite/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/líquido cefalorraquidiano , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Adolescente , Adulto , Encefalite Viral/líquido cefalorraquidiano , Encefalite Viral/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mielite/líquido cefalorraquidiano , Mielite/imunologia , Reação em Cadeia da Polimerase , Valor Preditivo dos TestesAssuntos
Fracionamento Celular/métodos , Escherichia coli/química , Proteínas Recombinantes/isolamento & purificação , Técnicas Bacteriológicas , Escherichia coli/genética , Vidro , Hexosiltransferases/genética , Hexosiltransferases/isolamento & purificação , Proteínas Recombinantes/genética , Sonicação , Streptococcus/enzimologia , Streptococcus/genéticaRESUMO
The mammalian striatum is divided into two compartments, the patch (or striosome) and the matrix, which differ on the basis of several cytochemical markers, connection patterns, and time of neurogenesis. In the rat, the patch compartment consists of clusters of neurons isolated by matrix neurons; included in the patch compartment is a rim of neurons subjacent to the corpus callosum and external capsule, called the subcallosal streak. To study the genesis and migration patterns of striatal neurons forming these compartments, we injected pregnant rats with 5-bromo-2'-deoxyuridine (BrdU, which is incorporated into DNA during S-phase mitosis) on embryonic (E) day 14, to label patch neurons, or on E19, to label matrix neurons. Embryos were sacrificed at intervals after injection, for detection of BrdU by immunocytochemistry. Cells labeled at E14 were distributed fairly uniformly in the differentiated portion of the caudate-putamen through E19. However, by the day of birth (P0), E14-labeled cells were clustered into patches and the subcallosal streak. Using double immunocytochemistry for BrdU and for the patch marker substance P, we demonstrated a caudal-rostral gradient in the birth dates of neurons in the patch compartment; E14-labeled cells occupied substance P-labeled patches at the level of the posterior limb of the anterior commissure, but patches further rostral were nearly devoid of E14-labeled cells. The distance between the lateral ventricle and the nearest E14-labeled cells was greater on E19 than on E16 or on P0, suggesting secondary movement of early-born neurons during the process of cluster formation. Neurons labeled at E19 formed the matrix surrounding clusters of unlabeled cells, except in the nucleus accumbens (ventral striatum), where E19-labeled cells formed clusters. The data suggest that the uniformly-distributed population of early-born neurons is disrupted by the invasion of later-born (matrix) neurons, forcing the early-born neurons into clusters which are displaced toward the ventricular surface to form the patch compartment. Early-born neurons adjacent to the external capsule are not displaced, forming the subcallosal streak.
Assuntos
Movimento Celular , Neostriado/embriologia , Animais , Bromodesoxiuridina , Imuno-Histoquímica , Neostriado/química , Neostriado/crescimento & desenvolvimento , Ratos , Substância P/análiseRESUMO
To study the comparative development of the two major neuropeptide genes of the striatum, we used immunocytochemistry to detect immunoreactivity (ir) for substance P and synenkephalin (the N terminus of proenkephalin), and in situ hybridization to detect proenkephalin mRNA. Earliest detection of substance P-ir was in the anlage of the bed nucleus of the stria terminalis (BST, at E15) and in the rostral-lateral caudate-putamen (CPu), at E16. Substance P in the BST was immediately subjacent to the medial ganglionic eminence, while immunoreactivity in the CPu was associated with the lateral ganglionic eminence. Earliest detection of synenkephalin-ir or proenkephalin mRNA was in the caudal-lateral CPu and the adjacent central nucleus of the amygdala (Ce), at E16. Over the next several days, expression of each neuropeptide spread toward the region of first expression of the other neuropeptide. The first overlap of expression of the two neuropeptides was at E18, at the level of the septum. Despite correspondence of substance P-ir and proenkephalin mRNA in patches at P0, very little co-expression of the two neuropeptides was evident in individual neurons. We propose a model in which the CPu develops primarily from the lateral ganglionic eminence, and the extended amygdala develops primarily from the medial ganglionic eminence. Within each structure, two poles of neuropeptide gene expression are established initially: substance P-ir in the rostral CPu and in the rostral-medial pole of the extended amygdala (represented by the BST), and synenkephalin/proenkephalin in the caudal CPu and in the caudal-lateral pole of the extended amygdala (represented by the Ce). A stream of substance P-ir cells connects the two poles of the extended amygdala, in the sublenticular substantia innominata.
Assuntos
Gânglios da Base/embriologia , Substância Inominada/embriologia , Animais , Gânglios da Base/química , Gânglios da Base/citologia , Encefalinas/análise , Imuno-Histoquímica , Precursores de Proteínas/análise , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Substância P/análise , Substância Inominada/química , Substância Inominada/citologiaRESUMO
The earliest detection of the proenkephalin gene was on embryonic day 16 in neuronal cell bodies in the ventrolateral portion of the caudal neostriatum. This expression was identified by both immunocytochemistry for synenkephalin, the nonopioid N-terminus of proenkephalin (1-70), and preproenkephalin in situ hybridization with a complementary DNA probe. Two developmental gradients of preproenkephalin expression and synenkephalin immunoreactivity were observed: (i) a ventrolateral to dorsomedial and caudal to rostral gradient in the rostral caudate-putamen; and (ii) a ventromedial to dorsolateral and rostral to caudal gradient in the caudal caudate-putamen. Ventrolateral to dorsomedial and caudal to rostral developmental gradients of synenkephalin fiber immunoreactivity were also identified in the globus pallidus. Methionine enkephalin immunoreactivity was not consistently detectable until postnatal day 10 and 15 in the rostral and caudal globus pallidus, respectively. A transient patchy distribution of increased preproenkephalin expression from embryonic day 20 through postnatal day 5 occurred. These patches and a subcallosal streak were found to overlap partially with areas of increased tyrosine hydroxylase immunoreactivity by adjacent section analyses. The earliest detection of tyrosine hydroxylase immunoreactivity was found to coincide with that of proenkephalin on embryonic day 16, but in differing regions of the corpus striatum. Tyrosine hydroxylase immunoreactivity in the rostral caudate-putamen preceded, while in the caudal caudate-putamen it followed first expression of the proenkephalin gene. Early proenkephalin expression, by both synenkephalin immunocytochemistry and preproenkephalin in situ hybridization, was also detected in the central nucleus of the amygdala on embryonic day 16 immediately ventral to the area of expression in the caudate-putamen. Preproenkephalin expression in the olfactory tubercle and nucleus accumbens first appeared on embryonic day 20 and expression proceeded in a lateral to dorsomedial gradient continuous with the ventral part of the rostral caudal-putamen. Relatively late detection of methionine enkephalin immunoreactivity in comparison to synenkephalin possibly indicates a developmental delay in the complete enzymatic processing of the proenkephalin precursor. Differing gradients in the ontogeny of preproenkephalin expression in the rostral vs the caudal caudate-putamen suggest possible anatomical and developmental differences of these two regions. Also, transient compartmentalization of preproenkephalin expression and differences in dopaminergic innervation as detected by tyrosine hydroxylase immunoreactivity were further support for the existence of two subsets of proenkephalinergic neurons in the caudate-putamen. Contemporaneous development of preproenkephalin expression and synenkephalin immunoreactivity in the central nucleus of the amygdala with the ventral part of the caudal caudate-putamen also suggested developmental homology.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Envelhecimento/fisiologia , Corpo Estriado/fisiologia , Dopamina/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Encefalina Metionina/análise , Encefalinas/análise , Encefalinas/genética , Precursores de Proteínas/análise , Precursores de Proteínas/genética , Animais , Corpo Estriado/embriologia , Corpo Estriado/crescimento & desenvolvimento , Sondas de DNA , Embrião de Mamíferos , Regulação da Expressão Gênica , Idade Gestacional , Imuno-Histoquímica , Hibridização In Situ , Neurônios/citologia , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/análiseRESUMO
Immunocytochemistry using an antiserum to the C-terminal octapeptide of synenkephalin, proenkephalin(63-70), was performed throughout the rat brain and revealed numerous immunopositive fibers and some cell bodies. The morphology and distribution of synenkephalin immunoreactivity was extremely similar to that of a commercial methionine enkephalin (Met-ENK) antiserum. Colchicine pretreatment allowed the immunostaining of cell bodies not otherwise possible without pretreatment, but did not affect the distribution of immunoreactive fibers. Using 6 microns serial sections, we were able to colocalize synenkephalin and Met-ENK immunoreactivities in gigantocellular neurons of the medullary reticular formation. Preabsorption of the antiserum with [Tyr63]proenkephalin(63-70) octapeptide (YEESHLLA) completely eliminated immunoreactivity in the rat brain, while preabsorption with all other peptides used had no detectable effect. We conclude that our antiserum to synenkephalin is specific for enkephalinergic cell bodies, fibers and terminals. The synenkephalin antiserum used in these studies may have advantages over other antisera utilized for immunocytochemical detection of proenkephalin gene expression.