RESUMO
Objective: To evaluate the clinical outcomes of computer aided design and computer aided manufacturing (CAD/CAM) ceramic endocrowns in endodontically treated posterior teeth after five years by a retrospective study. Methods: Patients who received CAD/CAM ceramic endocrowns after endodontically treatment in Department of Endodontics, School of Stomatology, The Fourth Military Medical University between January 2016 and June 2017 were invited for this clinical study. Clinical performance was evaluated in the aspect of color match, anatomic form,marginal adaptation, restoration integrity and secondary caries. Survival rate of the restorations was calculated by the use of Kaplan-Meier method. Log-rank test was applied as well for the sake of analyzing the effect of tooth position, sex and materials to the survival rate of the restorations. Results: Seventy-four patients, 25 men and 49 women with age of (38.8±10.2) years, participated in this study for a total of 101 CAD/CAM ceramic endocrowns after observation period of (62.8±12.0) months. There were 8 failed cases among 101 restorations, 5 were loss of retention, 2 were ceramic fracture and 1 was secondary caries respectively. In particular, 93% (89/96) restorations got score A on anatomic form and 95% (91/96) restorations got score A on marginal adaptation, while 38% (36/96) restorations showed the good color match compared with the abutment teeth. The estimated cumulative survival rate of CAD/CAM ceramic endocrowns in endodontically treated posterior teeth after 5 years was 93.0% (95%CI: 87.9%-98.1%). The single-factor Log-rank analysis demonstrated that there was no statistically significant difference in the survival rate of CAD/CAM ceramic endocrowns among men and women, premolars and molars, position in the dental arch, or different materials (χ²<0.01, P=0.957; χ²=0.64, P=0.422; χ²=0.69, P=0.407; χ²=0.88, P=0.349). Conclusions: Based on this clinical study, the clinical performance of CAD/CAM ceramic endocrowns in endodontically treated posterior teeth after five years is reliable, which could be a general option to restore nonvital teeth.
Assuntos
Coroas , Porcelana Dentária , Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Porcelana Dentária/uso terapêutico , Estudos Retrospectivos , Planejamento de Prótese Dentária , Análise do Estresse Dentário , Teste de Materiais , Desenho Assistido por Computador , CerâmicaRESUMO
BACKGROUND: Nomograms are rarely employed to estimate the survival of patients with advanced and metastatic pancreatic cancer (PC). Herein, we developed a comprehensive approach to using a nomogram to predict survival probability in patients with advanced and metastatic PC. METHODS: A total of 323 patients with advanced and metastatic PC were identified from the Chinese People's Liberation Army (PLA) General Hospital. A baseline nomogram was constructed using baseline variables of 323 patients. Additionally, 233 patients, whose tumors showed initial responses to first-line chemotherapy, were enrolled in the chemotherapy response-based model. 128 patients and 108 patients with advanced and metastatic PC from January 2019 to April 2021 were selected for external validating baseline model and chemotherapy response-based model. The 1-year and 2-year survival probability was evaluated using multivariate COX regression models. The discrimination and calibration capacity of the nomograms were assessed using C-statistic and calibration plots. The predictive accuracy and net benefit of the nomograms were evaluated using ROC curve and DCA, respectively. RESULTS: In the baseline model, six variables (gender, KPS, baseline TB, baseline N, baseline WBC and baseline CA19-9) were used in the final model. In the chemotherapy response-based model, nine variables (KPS, gender, ascites, baseline N, baseline CA 19-9, baseline CEA, change in CA 19-9 level at week, change in CEA level at week and initial response to chemotherapy) were included in the final model. The C-statistics of the baseline nomogram and the chemotherapy response-based nomogram were 0.67 (95% CI, 0.62-0.71) and 0.74 (95% CI, 0.69-0.77), respectively. CONCLUSION: These nomograms were constructed to predict the survival probability of patients of advanced and metastatic PC. The baseline model and chemotherapy response-based model performed well in survival prediction.
Assuntos
Nomogramas , Neoplasias Pancreáticas/mortalidade , Albuminas/uso terapêutico , Antineoplásicos/uso terapêutico , Antígeno CA-19-9/sangue , Antígeno Carcinoembrionário/sangue , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Combinação de Medicamentos , Feminino , Humanos , Avaliação de Estado de Karnofsky , Masculino , Pessoa de Meia-Idade , Ácido Oxônico/uso terapêutico , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Probabilidade , Modelos de Riscos Proporcionais , Curva ROC , Fatores Sexuais , Taxa de Sobrevida , Tegafur/uso terapêutico , GencitabinaRESUMO
OBJECTIVE: To understand the cognition and behavior of drug safety in Beijing middle school students and provide advice for relevant education. METHODS: A cross-sectional survey using paper questionnaires was carried out on the student body of nine Beijing middle schools. Multi-stage proportionate stratified cluster sampling was adopted to enroll participants. In addition to demographic questions, the questionnaire included 17 questions assessing the cognition and behavior of safe drug use, prioritizing questions that aligned with the health education guideline for primary and secondary school students from Chinese Ministry of Education. Descriptive statistical methods were applied using the SAS 9.2 software. RESULTS: Of the 4 220 students investigated, 2 097(49.7%) were males and 2 123(50.3%) were females. The average age was (14.3±1.7) years. 2 030(48.1%) students were from downtown areas, 1 511(35.8%) were from urban-rural linking areas and 679(16.1%) were from rural areas. Half (51.5%) of the respondents were junior high school students, and the others were from senior high schools (34.2%) and vocational high schools (14.3%). Most of the students (89.6%) lived off campus. The awareness rate of drug safety knowledge was 74.4%, the median score of drug safety behavior was 4 points (full score was 5 points) and there was a statistically positive correlation between the two (Spearman's correlation coefficient was 0.156, P<0.001). Both the awareness rates and the drug safety behavior scores were statistically different among the students in different regions, different school types and different residence types (P<0.001). Multiple factors analysis demonstrated the correlation between the cognition degrees of both drug safety knowledge, behavior and the above factors. Of all the students, 80.4% agreed that any drug could have adverse drug reactions; 40.5% were aware that antibiotics couldn't kill viruses; as many as 49.6% mistook aspirin as antibiotic; 97.4% would read drug instructions before taking them; Only 42.4% put expired drugs into special recycling bins; 49.8% would deviate from the suggested dosage and frequency of their medication when they were sick with common diseases. CONCLUSION: Overall, the cognition of drug safety in Beijing middle school students is good, but problems still exist in medication adherence, the management of expired drugs and the antibiotics cognition, which need to be fixed through specific, pointed way of education. And more efforts should be made to improve the cognition in rural regions, vocational high schools and on campus students.
Assuntos
Cognição , Instituições Acadêmicas , Estudantes , Adolescente , Pequim , Criança , Estudos Transversais , Feminino , Educação em Saúde , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Masculino , População Rural , Inquéritos e QuestionáriosRESUMO
OBJECTIVE: To study the association between statins use and liver-injury through prescription sequence symmetry analysis(PSSA)and evaluate the feasibility of the method to be used in Chinese Medical Insurance Database. METHODS: The data of the patients who prescribed both statins and liver-proactive drugs in Chinese Basic Medical Insurance Database in 2013 were selected as study subjects to calculate the adjusted sequence ratio(ASR)with signal detection methods to determine the study parameters and investigate the potential association between statins use and liver-injury. RESULTS: In 5 649 individuals which met the inclusion criteria, the washout period was set as one month and interval period was set as 60 days. The overall ASR of statins was 1.471(95%CI: 1.395-1.550), the ASR of atorvastatin was 1.419(95% CI: 1.335-1.508), the ASR of simvastatin was 1.307(95%CI: 1.164-1.467). The positive signal was strong in 30 days interval period. CONCLUSIONS: PSSA indicated that there might be potential association between statins use and liver-injury, especially the uses of atorvastatin and simvastatin. This signal detection method may be a fast and effective method in drug safety evaluation and can be used in Chinese Medical Insurance Database.
Assuntos
Atorvastatina/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Sinvastatina/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/epidemiologia , Bases de Dados Factuais , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Fígado/efeitos dos fármacos , Medicamentos sob PrescriçãoRESUMO
The aim of this study was to explore the therapeutic effect of Pleurotus eryngii cellulose on experimental fatty liver in rats. Rats were fed high-fat fodder to establish a rat fatty liver model, and were then fed different concentrations of Pleurotus eryngii cellulose for six weeks. Lipitor was used as a positive control. Measured levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), and total triglyceride (TG); the activity of malondialdehyde (MDA), superoxide dismutase (SOD), hepatic lipase (HL), and lipoprotein lipase; and liver histopathological changes. Successfully established rat fatty liver model after feeding high-fat fodder for one week. A diet of P. eryngii cellulose for six weeks significantly reduced ALT, AST, TC, and TG levels in rat serum (P < 0.01); TC and AST levels in P. eryngii cellulose high-dose group and Lipitor group were not significantly different from those of the control (P > 0.05). SOD activity increased significantly, while MDA and HL activity decreased (P < 0.05); fatty degeneration and fat accumulation both decreased in hepatic tissue. Hepatic protection of P. eryngii cellulose showed dose-related effect. P. eryngii cellulose can affect lipid metabolism, having therapeutic effects on fatty liver in rats.
Assuntos
Celulose/farmacologia , Fígado Gorduroso/tratamento farmacológico , Metabolismo dos Lipídeos/efeitos dos fármacos , Pleurotus , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Celulose/uso terapêutico , Colesterol/sangue , Modelos Animais de Doenças , Fígado Gorduroso/sangue , Fígado/efeitos dos fármacos , Masculino , Ratos , Triglicerídeos/sangueRESUMO
The purpose of this study was to identify critical genes associated with septic multiple trauma by comparing peripheral whole blood samples from multiple trauma patients with and without sepsis. A microarray data set was downloaded from the Gene Expression Omnibus (GEO) database. This data set included 70 samples, 36 from multiple trauma patients with sepsis and 34 from multiple trauma patients without sepsis (as a control set). The data were preprocessed, and differentially expressed genes (DEGs) were then screened for using packages of the R language. Functional analysis of DEGs was performed with DAVID. Interaction networks were then established for the most up- and down-regulated genes using HitPredict. Pathway-enrichment analysis was conducted for genes in the networks using WebGestalt. Fifty-eight DEGs were identified. The expression levels of PLAU (down-regulated) and MMP8 (up-regulated) presented the largest fold-changes, and interaction networks were established for these genes. Further analysis revealed that PLAT (plasminogen activator, tissue) and SERPINF2 (serpin peptidase inhibitor, clade F, member 2), which interact with PLAU, play important roles in the pathway of the component and coagulation cascade. We hypothesize that PLAU is a major regulator of the component and coagulation cascade, and down-regulation of PLAU results in dysfunction of the pathway, causing sepsis.
Assuntos
Regulação da Expressão Gênica , Traumatismo Múltiplo/genética , Mapas de Interação de Proteínas/genética , Sepse/genética , Humanos , Metaloproteinase 8 da Matriz/biossíntese , Traumatismo Múltiplo/complicações , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sepse/complicações , Ativador de Plasminogênio Tipo Uroquinase/biossínteseRESUMO
We summarize our recent progress in the development of the optical coherence tomography (OCT) systems suitable for clinical diagnosis and the preliminary results for in vivo diagnosis of epithelial cancers (e.g., bladder cancers). The endoscopic spectral-domain OCT system allows simultaneous, real-time, cross-sectional OCT images of tissue structure and functions (i.e., local Doppler blood flow) of biological tissue for enhanced diagnosis. A new approach to use spectral demodulation of elastic scattering is discussed for potential cancer grading. The transverse and axial resolutions of the OCT scopes are 12 microm and 10 microm, respectively. Results of the preliminary clinical studies show that unlike animal carcinogenesis models, bladder cancers in humans are more complicated in terms of epithelial backscattering changes: some lesions exhibit enhanced backscattering; some show reduced scattering owing to complex surface condition changes such as asperities or invaginations induced by tumorigenesis (e.g., papillary transitional cell cancers). Nevertheless, promising results can be provided by incorporating other diagnostic parameters such as changes in local vasculature and urothelial heterogeneity.
Assuntos
Endoscópios , Aumento da Imagem/instrumentação , Neoplasias Epiteliais e Glandulares/diagnóstico , Tomografia de Coerência Óptica/instrumentação , Neoplasias da Bexiga Urinária/diagnóstico , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Aumento da Imagem/métodos , Ratos , Tomografia de Coerência Óptica/métodosRESUMO
PURPOSE: We describe the technique of fluorescence image guided optical coherence tomography (FG-OCT). We examined its ability to enhance specificity and sensitivity for the noninvasive diagnosis of early bladder cancer. MATERIALS AND METHODS: Transitional cell carcinoma was developed in 54 Fisher 344 female rats by intravesical methyl-nitroso-urea instillations. Two or three rats were diagnosed sequentially by 5-ALA (5-aminolevulinic acid hydrochloride) induced fluorescence imaging, cross-sectional OCT and histological microscopy weekly during weeks 11 to 33 following initial methyl-nitroso-urea instillation to track the course of carcinogenesis. RESULTS: The specificity of fluorescence detection was significantly enhanced by FG-OCT (53% and 93%, respectively, p <0.0001). The sensitivity of fluorescence detection and FG-OCT was 79% and 100%, respectively. CONCLUSIONS: FG-OCT cystoscopy has the potential to diagnose early bladder cancer with high sensitivity and specificity with drastically decreased imaging time compared to that of white light guided OCT cystoscopy.
Assuntos
Carcinoma de Células de Transição/diagnóstico , Fluorescência , Tomografia de Coerência Óptica , Neoplasias da Bexiga Urinária/diagnóstico , Tecido Adiposo/patologia , Animais , Carcinoma Papilar/diagnóstico , Carcinoma de Células de Transição/patologia , Modelos Animais de Doenças , Reações Falso-Positivas , Feminino , Hiperplasia/diagnóstico , Mucosa/patologia , Ratos , Ratos Endogâmicos F344 , Sensibilidade e Especificidade , Fatores de Tempo , Tomografia de Coerência Óptica/métodos , Neoplasias da Bexiga Urinária/patologia , Urotélio/patologiaRESUMO
We report an experimental study of the possibility of high-speed optical coherence tomography (OCT) for high-resolution imaging characterization of detrusor dynamic morphophysiology and analysis of the mechanisms that lead to geriatric incontinence (GI). The spontaneous contractility of intact fresh rabbit bladders was imaged with two-dimensional (2D) OCT ex vivo at up to 8 frames/s. The time-lapse 2D OCT images were postprocessed by image segmentation and fast-Fourier-transform analysis to characterize the dynamic morphological changes of the bladder contractility. In addition, we studied young and aging rat bladders to analyze the differences in dynamics. Preliminary results of our ex vivo study reveal that time-lapse OCT can track the contractile waves of bladders at high spatial resolution and characterize their dynamic morphophysiology in terms of amplitude, phase, and frequency. The results suggest that time-lapse OCT has the potential to act as a detrusor optical biopsy to enhance the diagnosis of detrusor dysfunction and thus of the mechanisms that lead to GI.
Assuntos
Algoritmos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Vídeo/métodos , Tomografia de Coerência Óptica/métodos , Bexiga Urinária/citologia , Bexiga Urinária/fisiologia , Animais , Inteligência Artificial , Estudos de Viabilidade , Aumento da Imagem/instrumentação , Microscopia de Vídeo/instrumentação , Contração Muscular/fisiologia , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tomografia de Coerência Óptica/instrumentaçãoRESUMO
We report an experimental study of the possibility of enhancing early bladder cancer diagnosis with fluorescence-image-guided endoscopic optical coherence tomography (OCT). After the intravesical instillation of a 10% solution of 5-aminolevulinic acid, simultaneous fluorescence imaging (excitation of 380-420 nm, emission of 620-700 nm) and OCT are performed on rat bladders to identify the photochemical and morphological changes associated with uroepithelial tumorigenesis. The preliminary results of our ex vivo study reveal that both fluorescence and OCT can identify early uroepithelial cancers, and OCT can detect precancerous lesions (e.g., hyperplasia) that fluorescence may miss. This suggests that a cystoscope combining 5-aminolevulinic acid fluorescence and OCT imaging has the potential to enhance the efficiency and sensitivity of early bladder cancer diagnosis.
Assuntos
Cistoscopia , Fluorescência , Tomografia de Coerência Óptica , Neoplasias da Bexiga Urinária/diagnóstico , Animais , RatosRESUMO
Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an alpha,alpha-1,1-glycosidic linkage. This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates, and lower and higher plants, where it may serve as a source of energy and carbon. In yeast and plants, it may also serve as a signaling molecule to direct or control certain metabolic pathways or even to affect growth. In addition, it has been shown that trehalose can protect proteins and cellular membranes from inactivation or denaturation caused by a variety of stress conditions, including desiccation, dehydration, heat, cold, and oxidation. Finally, in mycobacteria and corynebacteria, trehalose is an integral component of various glycolipids that are important cell wall structures. There are now at least three different pathways described for the biosynthesis of trehalose. The best known and most widely distributed pathway involves the transfer of glucose from UDP-glucose (or GDP-glucose in some cases) to glucose 6-phosphate to form trehalose-6-phosphate and UDP. This reaction is catalyzed by the trehalose-P synthase (TPS here, or OtsA in Escherichia coli ). Organisms that use this pathway usually also have a trehalose-P phosphatase (TPP here, or OtsB in E. coli) that converts the trehalose-P to free trehalose. A second pathway that has been reported in a few unusual bacteria involves the intramolecular rearrangement of maltose (glucosyl-alpha1,4-glucopyranoside) to convert the 1,4-linkage to the 1,1-bond of trehalose. This reaction is catalyzed by the enzyme called trehalose synthase and gives rise to free trehalose as the initial product. A third pathway involves several different enzymes, the first of which rearranges the glucose at the reducing end of a glycogen chain to convert the alpha1,4-linkage to an alpha,alpha1,1-bond. A second enzyme then releases the trehalose disaccharide from the reducing end of the glycogen molecule. Finally, in mushrooms there is a trehalose phosphorylase that catalyzes the phosphorolysis of trehalose to produce glucose-1-phosphate and glucose. This reaction is reversible in vitro and could theoretically give rise to trehalose from glucose-1-P and glucose. Another important enzyme in trehalose metabolism is trehalase (T), which may be involved in energy metabolism and also have a regulatory role in controlling the levels of trehalose in cells. This enzyme may be important in lowering trehalose concentrations once the stress is alleviated. Recent studies in yeast indicate that the enzymes involved in trehalose synthesis (TPS, TPP) exist together in a complex that is highly regulated at the activity level as well as at the genetic level.
Assuntos
Trealose/biossíntese , Parede Celular/metabolismo , Glucosiltransferases/metabolismo , Hidrólise , Cinética , Estrutura Molecular , Trealose/análogos & derivados , Trealose/química , Trealose/metabolismoRESUMO
The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli ( approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and UDP-glucose, with ADP-glucose, GDP-glucose and UDP-glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP-glucose as the glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.
Assuntos
Glucosiltransferases/química , Glucosiltransferases/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/química , Sequência de Aminoácidos , Western Blotting , Cromatografia , Clonagem Molecular , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Glucose-6-Fosfato/metabolismo , Heparina/metabolismo , Cinética , Modelos Químicos , Dados de Sequência Molecular , Fases de Leitura Aberta , Peptídeos/química , Ligação Proteica , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Fatores de TempoRESUMO
A series of trehalose-based oligosaccharides were isolated from the cytoplasmic fraction of Mycobacterium smegmatis and purified by gel-filtration and paper chromatography and TLC. Their structures were determined by HPLC and GLC to determine sugar composition and ratios, MALDI-TOF MS to measure molecular mass, methylation analysis to determine linkages, (1)H-NMR to obtain anomeric configurations of glycosidic linkages, and exoglycosidase digestions followed by TLC to determine sequences and anomeric configurations of the monosaccharides. Six different oligosaccharides were identified all with trehalose as the basic structure and additional glucose or galactose residues attached in various linkages. One of these oligosaccharides is the disaccharide trehalose (Glcalpha1-1alphaGlc), which is present in substantial amounts in these cells and also in other mycobacteria. Two other oligosaccharides, the tetrasaccharides Glcalpha1-4Glcalpha1-1alphaGlc6-1alphaGal and Galalpha1-6Galalpha1-6Glcalpha1-1alphaGlc, have not previously been isolated from natural sources or synthesized chemically. The fourth oligosaccharide, Glcbeta1-6Glcbeta1-6Glcalpha1-1alphaGlc, has been isolated from corynebacteria, but not reported in other organisms. Two other oligosaccharides, Glcalpha1-4Glcalpha1-1alphaGlc, which has been synthesized chemically and isolated from insects but not previously reported in mycobacteria, and Glcbeta1-6Glcalpha1-1alphaGlc, which was previously isolated from Mycobacterium fortuitum and yeast, were also characterized. Another trisaccharide found in the cytosol has been partially characterized as arabinosyl-1-4trehalose, but neither the anomeric configuration nor the D or L configuration of the arabinose is known. In analogy with sucrose and its higher homologs, raffinose and stachyose, which may act as protective agents during maturation drying in plants, these trehalose homologs may also have a protective role in mycobacteria, perhaps during latency.
Assuntos
Mycobacterium smegmatis/química , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Trealose/química , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Citoplasma/química , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
d-Arabinose is a major sugar in the cell wall polysaccharides of Mycobacterium tuberculosis and other mycobacterial species. The reactions involved in the biosynthesis and activation of d-arabinose represent excellent potential sites for drug intervention since d-arabinose is not found in mammalian cells, and the cell wall arabinomannan and/or arabinogalactan appear to be essential for cell survival. Since the pathway involved in conversion of d-glucose to d-arabinose is unknown, we incubated cells of Mycobacterium smegmatis individually with [1-(14)C]glucose, [3,4-(14)C]glucose, and [6-(14)C]glucose and compared the specific activities of the cell wall-bound arabinose. Although the specific activity of the arabinose was about 25% lower with [6-(14)C]glucose than with other labels, there did not appear to be selective loss of either carbon 1 or carbon 6, suggesting that arabinose was not formed by loss of carbon 1 of glucose via the oxidative step of the pentose phosphate pathway, or by loss of carbon 6 in the uronic acid pathway. Similar labeling patterns were observed with ribose isolated from the nucleic acid fraction. Since these results suggested an unusual pathway of pentose formation, labeling studies were also done with [1-(13)C]glucose, [2-(13)C]glucose, and [6-(13)C]glucose and the cell wall arabinose was examined by NMR analysis. This method allows one to determine the relative (13)C content in each carbon of the arabinose. The labeling patterns suggested that the most likely pathway was condensation of carbons 1 and 2 of fructose 6-phosphate produced by the transaldolase reaction with carbons 4, 5, and 6 (i.e., glyceraldehyde 3-phosphate) formed by fructose-1,6 bisphosphate aldolase. Cell-free enzyme extracts of M. smegmatis were incubated with ribose 5-phosphate, xylulose 5-phosphate, and d-arabinose 5-phosphate under a variety of experimental conditions. Although the ribose 5-phosphate and xylulose 5-phosphate were converted to other pentoses and hexoses, no arabinose 5-phosphate (or free arabinose) was detected in any of these reactions. In addition, these enzyme extracts did not convert arabinose 5-phosphate to any other pentose or hexose. In addition, incubation of [(14)C]glucose 6-phosphate and various nucleoside triphosphates (ATP, CTP, GTP, TTP, and UTP) with cytosolic or membrane fractions from the mycobacterial cells did not result in formation of a nucleotide form of arabinose, although other radioactive sugars including rhamnose and galactose were found in the nucleotide fraction. Furthermore, no radioactive arabinose was found in the nucleotide fraction isolated from M. smegmatis cells grown in [(3)H]glucose, nor was arabinose detected in a large-scale extraction of the sugar nucleotide fraction from 300 g of cells. The logical conclusion from these studies is that d-arabinose is probably produced from d-ribose by epimerization of carbon 2 of the ribose moiety of polyprenylphosphate-ribose to form polyprenylphosphate-arabinose, which is then used as the precursor for formation of arabinosyl polymers.
Assuntos
Arabinose/biossíntese , Glucose/metabolismo , Mycobacterium smegmatis/enzimologia , Radioisótopos de Carbono , Sistema Livre de Células , Ativação EnzimáticaRESUMO
Enterobacter cloacae has been implicated as one of the causative agents in neonatal infection and causes a septicemia thought to be initiated via the gastrointestinal tract. The adhesion of radiolabeled E. cloacae to HT-29 cells was concentration and temperature dependent and was effectively blocked by unlabeled bacteria or by millimolar concentrations of alpha-mannosides and micromolar concentrations of high-mannose oligosaccharides. A variety of well-characterized mannose oligosaccharides were tested as inhibitors of adhesion. The best inhibitor was the Man9(GlcNAc)2-tyrosinamide, which was considerably better than other tyrosinamide-linked oligosaccharides such as Man7(GlcNAc)2, Man6(GlcNAc)2 or Man5(GlcNAc)2. Further evidence that the bacteria preferred Man9(GlcNAc)2 structures was obtained by growing HT-29 cells in the presence of glycoprotein processing inhibitors that block mannosidase I and increase the amount of protein-bound Man9(GlcNAc)2 at the cell surface. Such cells bound 1.5- to 2-fold more bacteria than did control cells. The adhesin involved in binding to high-mannose structures was purified from isolated pili. On sodium dodecyl sulfate-gels, a 35-kDa protein was identified by its specific binding to a mannose-containing biotinylated albumin. The amino acid sequences of several peptides from the 35-kDa subunit showed over 85% identity to FimH, the mannose-specific adhesin of Salmonella typhimurium. Pili were labeled with 125I and examined for the ability to bind to HT-29 cells. Binding showed saturation kinetics and was inhibited by the addition of Man9(GlcNAc)2-tyrosinamide but not by oligosaccharides with fewer mannose residues. Polyclonal antibody against this 35-kDa protein also effectively blocked adhesion of pili or E. cloacae, but no effect was observed with nonspecific antibody. These studies demonstrate that the 35-kDa pilus subunit is a lectin whose specificity is directed toward Man, (GlcNAc)2 oligosaccharides.
Assuntos
Adesinas Bacterianas/metabolismo , Adesinas de Escherichia coli , Aderência Bacteriana/fisiologia , Enterobacter cloacae/patogenicidade , Proteínas de Fímbrias , Fímbrias Bacterianas/metabolismo , Lectinas Tipo C , Lectinas de Ligação a Manose , Receptores de Superfície Celular/metabolismo , 1-Desoxinojirimicina/farmacologia , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Alcaloides/farmacologia , Sequência de Aminoácidos , Sequência de Carboidratos , Membrana Celular/metabolismo , Células Cultivadas , Fímbrias Bacterianas/química , Indolizinas/farmacologia , Lectinas/metabolismo , Receptor de Manose , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Fragmentos de Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/antagonistas & inibidores , Análise de Sequência , Homologia de Sequência de Aminoácidos , Swainsonina/farmacologiaRESUMO
Homonojirimycin (HNJ) and N-methylhomonojirimycin (MHNJ) were tested as inhibitors of the purified glycoprotein processing enzymes, glucosidase I and glucosidase II. MHNJ was a reasonably good inhibitor of glucosidase I (Ki = 1 x 10(-6) M) and was about three times as effective on this enzyme as was HNJ. On the other hand, HNJ inhibited glucosidase II with a Ki of about 1 x 10(-6) M, whereas MHNJ was three times less effective (Ki = 3 x 10(-5) M). However, the butyl derivative of HNJ had very low activity toward these two processing glucosidases. HNJ and its methyl derivative were also tested in vivo using influenza virus-infected MDCK cells, and measuring the inhibition of N-linked oligosaccharide processing of the viral envelope glycoproteins. With 100 micrograms/ml of MHNJ in the medium, essentially all of the N-linked oligosaccharide chains of the virus were of the "high-mannose" type with the major structure being characterized as Glc3Man9(GlcNAc)2. Similar results were obtained with HNJ although this compound was less effective in vivo as well as in vitro. These results are in keeping with these inhibitors being effective at the glucosidase I step. Both inhibitors were also tested in MDCK cell cultures to determine whether they affected the in vivo synthesis of proteins, or of lipid-linked saccharides. In contrast to deoxynojirimycin, which has been reported to inhibit the formation of lipid-linked saccharides, no effects were seen on either the incorporation of mannose into lipid-linked saccharides or the incorporation of leucine into protein.
Assuntos
Glicoproteínas/metabolismo , Inibidores de Glicosídeo Hidrolases , Oligossacarídeos/metabolismo , Piperidinas/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , 1-Desoxinojirimicina/análogos & derivados , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Glucosamina/análogos & derivados , Imino Piranoses , Orthomyxoviridae/metabolismo , Proteínas do Envelope Viral/metabolismo , alfa-GlucosidasesRESUMO
A number of unusual and rare carbohydrates were tested as potential inhibitors of various glycosidases, as well as inhibitors of N-linked oligosaccharide processing. The best inhibitors of several arylglycosidases and of glucosidase I were L-xylulose and L-fructose. Both of these sugars showed some inhibitory activity towards yeast alpha-glucosidase but were inactive against beta-glucosidase and other arylglycosidases. The inhibition of yeast alpha-glucosidase by L-xylulose was of a competitive nature and required a concentration of 1 x 10(-5) M for 50% inhibition. Both L-xylulose and L-fructose also inhibited the purified soybean glucosidase I, with 50% inhibition occurring at about 1 x 10(-4) M, but showed no inhibitory activity against soybean glucosidase II. When influenza virus-infected MDCK cells were raised in the presence of L-xylulose, there was a dose-dependent inhibition in the formation of complex types of oligosaccharides on the viral glycoproteins consistent with the inhibition of the processing glucosidase I. This inhibition resulted in the occurrence of oligosaccharides on the viral glycoproteins that were characterized as Glc3Man9(GlcNAc)2 structures. L-Fructose also inhibited glycoprotein processing in cell culture, and the inhibition resulted in the formation of similar oligosaccharides to those seen with L-xylulose. However, L-fructose was a poorer inhibitor than L-xylulose and required much higher concentrations for the same degree of inhibition. Neither of these compounds inhibited protein synthesis or the formation of lipid-linked saccharides in culture MDCK cells, even when tested at concentrations of 5 mg/ml (about 30 mM) of culture media.
Assuntos
Frutose/farmacologia , Glicoproteínas/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Xilulose/farmacologia , Animais , Bovinos , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Glicoproteínas/metabolismo , Glicosídeo Hidrolases/antagonistas & inibidores , Orthomyxoviridae/metabolismo , Proteínas Virais/antagonistas & inibidoresRESUMO
Calystegines, polyhydroxy nortropane alkaloids, are a recently discovered group of plant secondary metabolites believed to influence rhizosphere ecology as nutritional sources for soil microorganisms and as glycosidase inhibitors. Evidence is presented that calystegines mediate nutritional relationships under natural conditions and that their biological activities are closely correlated with their chemical structures and stereochemistry. Assays using synthetic (+)- and (-)-enantiomers of calystegine B2 established that catabolism by Rhizobium meliloti, glycosidase inhibition, and allelopathic activities were uniquely associated with the natural, (+)-enantiomer. Furthermore, the N-methyl derivative of calystegine B2 was not catabolized by R. meliloti, and it inhibited alpha-galactosidase, but not beta-glucosidase, whereas the parent alkaloid inhibits both enzymes. This N-methyl analog therefore could serve to construct a cellular or animal model for Fabry's disease, which is caused by a lack of alpha-galactosidase activity.
Assuntos
Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Nortropanos/isolamento & purificação , Nortropanos/farmacologia , Plantas Medicinais/química , alfa-Galactosidase/antagonistas & inibidores , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , DNA Bacteriano/biossíntese , Glicosídeo Hidrolases/antagonistas & inibidores , Raízes de Plantas/química , Pseudomonas/efeitos dos fármacos , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/metabolismo , Sementes/química , Sinorhizobium meliloti/efeitos dos fármacos , Sinorhizobium meliloti/crescimento & desenvolvimento , Sinorhizobium meliloti/metabolismo , Alcaloides de Solanáceas , Relação Estrutura-AtividadeRESUMO
A number of antibiotics were tested as potential inhibitors of the purified trehalose-P synthase of Mycobacterium smegmatis. Of about 30 compounds tested, 4 (cathomycin, circulin, diumycin, and moenomycin) were active against this enzyme. Thus each of these compounds inhibited the formation of trehalose-P by the purified trehalose-P synthase when either UDP-glucose or GDP-glucose was used as the glucosyl donor. However, preincubation of the synthase with heparin, a polyanion activator of the enzyme when UDP-glucose is used as the substrate, prevented the inhibition by these various antibiotics. Fifty percent inhibition by diumycin and moenomycin occurred at a concentration of about 50 microg/ml (Ki of about 1 x 10(-5) M), but 50% inhibition by cathomycin and circulin required substantially higher concentrations (about 50 to 200 microg/ml). The inhibition by cathomycin, diumycin, and moenomycin was of the competitive type, whereas that by circulin was noncompetitive in nature. However, the inhibition was of a complex nature and the data suggest two different binding sites for these inhibitors. Photoaffinity labeling of the synthase with an azido-UDP-[32P]glucose probe was effectively blocked by diumycin, moenomycin, or cathomycin indicating that these inhibitors do interact at the substrate binding site. These antibiotics also inhibited the growth of M. smegmatis when added to cells innoculated into trypticase soy broth. The inhibition of growth was concentration-dependent and directly proportional to the size of the bacterial innoculum. These antibiotics, however, did not inhibit protein synthesis nor did they inhibit the incorporation of mannose into lipid-linked saccharides.
Assuntos
Antibacterianos/farmacologia , Ciclotídeos , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Mycobacterium/enzimologia , Proteínas de Bactérias/biossíntese , Bambermicinas/farmacologia , Heparina/farmacologia , Cinética , Mycobacterium/efeitos dos fármacos , Mycobacterium/crescimento & desenvolvimento , Novobiocina/farmacologia , Fosfolipídeos/farmacologiaRESUMO
The trehalose-P synthase was purified to near homogeneity from the cytoplasmic fraction of Mycobacterium smegmatis. At the final stage of purification, the enzyme preparation showed one major band of 59 kDa on SDS gels. The 59 kDa band became labeled with N3-UDP[32P]-glucose, and this labeling was inhibited in a concentration-dependent manner by either unlabeled UDP-glucose or GDP-glucose. The native enzyme also had a molecular weight of about 60 kDa by gel filtration, indicating that the active enzyme is a monomer. The 59 kDa protein was subjected to endoproteinase Lys-C digestion, and three peptides isolated by HPLC were sequenced. The sequences of 56 amino acids in these three peptides showed 60% identity to the trehalose-P synthases of Saccharomyces cerevesiae and Schizosaccharomyces pombe. The purified mycobacterial enzyme catalyzed the synthesis of trehalose-P from glucose-6-P and a variety of nucleoside diphosphate glucose derivatives, depending on whether a polyanion was absent or present. Thus, UDP-glucose and GDP-glucose were the best glucosyl donors, but maximum activity with UDP-glucose required the presence of a polyanion such as heparin, whereas activity with GDP-glucose was relatively independent of polyanion. The presence of heparin in the incubation mixture increased the affinity of the enzyme for UDP-glucose by a factor of 100, or more. However, the affinity for GDP-glucose was only twofold better in the presence of heparin. The purified synthase also utilized ADP-glucose and CDP-glucose, but the K(m) for these glucosyl donors was quite high even in the presence of polyanion. The effect of heparin on UDP-glucose activity was dose-dependent and maximum at about 1-2 micrograms of heparin/incubation. However, the size of the heparin molecule (i.e., the number of monosaccharide residues) was critical for activation, and only those heparins with 18 or more monosaccharide units were effective in stimulating activity.