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1.
Clin Otolaryngol ; 43(2): 591-597, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29119679

RESUMO

OBJECTIVES: The surveillance of noise-induced hearing loss (NIHL) according to the Health and Safety Executive (HSE) differs from the medico-legal criteria used to assess NIHL. Our study compares the two systems and proposes a novel method of simplifying the medico-legal criteria and applying it to ascertain noise-induced hearing loss. DESIGN: The anonymised audiograms of a group of 87 industrial workers from a single site were analysed with both methods. RESULTS: The comparison showed approximately one-third of the workers assessed in this study had their noise-induced hearing loss underestimated by the HSE criteria. The majority of these individuals were over 40 years of age. CONCLUSIONS: The HSE criteria for noise-induced hearing loss need review and re-alignment with the medico-legal criteria to address the discrepancy between the two systems.


Assuntos
Perda Auditiva Provocada por Ruído/diagnóstico , Doenças Profissionais/diagnóstico , Adulto , Fatores Etários , Idoso , Audiometria , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ruído Ocupacional , Exposição Ocupacional , Reino Unido , Adulto Jovem
2.
J Laryngol Otol ; 129(12): 1174-81, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26549131

RESUMO

OBJECTIVE: To examine whether distortion product otoacoustic emissions can serve as a replacement for pure tone audiometry in longitudinal screening for occupational noise exposure related auditory deficit. METHODS: A retrospective review was conducted of pure tone audiometry and distortion product otoacoustic emission data obtained sequentially during mandatory screening of brickyard workers (n = 16). Individual pure tone audiometry thresholds were compared with distortion product otoacoustic emission amplitudes, and a correlation of these measurements was conducted. RESULTS: Pure tone audiometry threshold elevation was identified in 13 out of 16 workers. When distortion product otoacoustic emission amplitudes were compared with pure tone audiometry thresholds at matched frequencies, no evidence of a robust relationship was apparent. Seven out of 16 workers had substantial distortion product otoacoustic emissions with elevated pure tone audiometry thresholds. CONCLUSION: No clinically relevant predictive relationship between distortion product otoacoustic emission amplitude and pure tone audiometry threshold was apparent. These results do not support the replacement of pure tone audiometry with distortion product otoacoustic emissions in screening. Distortion product otoacoustic emissions at frequencies associated with elevated pure tone audiometry thresholds are evidence of intact outer hair cell function, suggesting that sites distinct from these contribute to auditory deficit following ototrauma.


Assuntos
Audiometria de Tons Puros/métodos , Perda Auditiva Provocada por Ruído/diagnóstico , Exposição Ocupacional/efeitos adversos , Saúde Ocupacional , Emissões Otoacústicas Espontâneas/fisiologia , Adulto , Idoso , Limiar Auditivo/fisiologia , Bases de Dados Factuais , Feminino , Perda Auditiva Provocada por Ruído/epidemiologia , Humanos , Incidência , Masculino , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Estudos Retrospectivos , Medição de Risco , Índice de Gravidade de Doença , Reino Unido , Adulto Jovem
3.
Acta Otolaryngol ; 127(2): 138-42, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17364344

RESUMO

CONCLUSION: The presence of matrix metalloproteinase (MMP)-8 and MMP-13 was found to be significantly higher in cholesteatoma compared with post-auricular skin. The results show that the control group used has implications for further studies. OBJECTIVES: To compare the presence of MMP-8 and MMP-13 in cholesteatoma, deep meatal and post-auricular skin. Our null hypothesis was that there was no difference in expressions of MMP-8 and MMP-13 in the three groups. MATERIALS AND METHODS: The study was carried out in a secondary care specialist centre and used prospective retrieval of specimens for immunohistological localization of MMP-8 and MMP-13. Eleven patients undergoing cholesteatoma surgery were recruited for the study. Eleven cholesteatoma specimens, 10 deep meatal skin specimens and 10 post-auricular skin specimens were analysed. Specimens were analysed by immunohistochemistry using monoclonal antibodies to MMP-8 and MMP-13. Two observers scored the slides independently in a blind fashion. RESULTS: The presence of MMP-8 and MMP-13 was found to be significantly higher in cholesteatoma compared to post-auricular skin (p=0.02, p=0.03, respectively). There were no significant differences in expression of MMP-8 and MMP-13 between cholesteatoma and deep meatal skin (p=0.08, p=0.09, respectively). There were no significant differences in the control groups.


Assuntos
Colesteatoma da Orelha Média/enzimologia , Orelha Externa/enzimologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 8 da Matriz/metabolismo , Pele/enzimologia , Humanos , Imuno-Histoquímica , Estudos Prospectivos
5.
Arch Dis Child Fetal Neonatal Ed ; 91(5): F365-6, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16923934

RESUMO

Sleeping posture has been implicated in the pathophysiology of sudden infant death syndrome. The effect of supine and lateral sleeping positions on nasal patency was investigated using acoustic rhinometry in 11 healthy newborns. The implications of the findings in sudden infant death syndrome are discussed.


Assuntos
Resistência das Vias Respiratórias/fisiologia , Recém-Nascido/fisiologia , Cavidade Nasal/fisiologia , Postura/fisiologia , Sono/fisiologia , Feminino , Humanos , Cuidado do Lactente/métodos , Masculino , Rinometria Acústica/métodos , Decúbito Dorsal/fisiologia
6.
Clin Otolaryngol ; 30(2): 143-8, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15839866

RESUMO

OBJECTIVES: The aim of this study was to compare postoperative symptoms following coblation tonsillectomy with those experienced following a traditional cold dissection. DESIGN: A prospective randomized controlled trial. SETTING: Secondary otorhinolaryngology care. PARTICIPANTS: Ninety-two adult patients with recurrent tonsillitis meriting tonsillectomy were recruited and randomly allocated into either coblation or cold dissection tonsillectomy groups. MAIN OUTCOME MEASURES: Primary outcomes were post-operative pain, otalgia, swallowing and analgesia use at 6-8 hours, 1, 3, 7 and 14 days post-operative. Secondary outcomes were post-operative day returned to eating and returned to normal activities/work. RESULTS: No significant differences between the two groups (P >or= 0.1) were found in any of the above primary outcomes, apart from swallowing at 6-8 hrs post-operatively where the cold dissection group had less pain. This group also returned earlier to normal eating (P = 0.03). The power of the study was sufficient to show a difference in the visual analogue scores of 2 between groups. CONCLUSIONS: The use of coblation to perform tonsillectomy does not confer any symptomatic benefits to the patient over conventional cold dissection tonsillectomy.


Assuntos
Ablação por Cateter/instrumentação , Complicações Pós-Operatórias/epidemiologia , Tonsilectomia/instrumentação , Adolescente , Adulto , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Índice de Gravidade de Doença , Inquéritos e Questionários , Fatores de Tempo , Tonsilite/cirurgia
7.
Clin Otolaryngol ; 30(6): 517-20, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16402976

RESUMO

OBJECTIVE: To analyse the influence of long-term smoking on the hearing threshold of individuals subjected to occupational noise exposure. DESIGN: Prospective observational cohort study. SETTING: Occupational health examination of noise-exposed employees in the brick manufacturing industry. PARTICIPANTS: A study group of long-term smokers (n = 30) and a control group of non-smokers (n = 58) were identified from a population of 227 male noise-exposed employees. Individuals of both groups were employed for 10 years or more at a single brick manufacturing plant. Data on noise exposure, smoking habits, medical and otological history were collected and standard pure tone audiometry was obtained. Exclusion criteria included asymmetrical or conductive hearing loss, uncontrolled systemic illnesses, history of head injury, chronic middle ear pathology or major ear operations. MAIN OUTCOME MEASURE: Pure tone audiometry. RESULTS: Both groups had similar mean age and total duration of occupational noise exposure. The median age-corrected hearing thresholds at 3 and 4 kHz in the smokers group were significantly higher (approximately 7dB) than those in the non-smokers group. No statistical difference in the hearing thresholds between both groups was found in any other tested frequency (0.5, 1, 2, 6 and 8 kHz). CONCLUSION: Long-term smokers with occupational noise exposure may, on the basis of this limited study, have a higher risk of developing permanent hearing loss at 3 and 4 kHz when compared with non-smokers with a similar occupational history.


Assuntos
Perda Auditiva Provocada por Ruído/fisiopatologia , Fumar/efeitos adversos , Audiometria de Tons Puros , Limiar Auditivo/fisiologia , Cóclea/fisiopatologia , Estudos de Coortes , Materiais de Construção , Indústrias Extrativas e de Processamento , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Ruído Ocupacional , Exposição Ocupacional , Estudos Prospectivos , Fatores de Tempo
8.
J Laryngol Otol ; 118(10): 796-8, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15550187

RESUMO

OBJECTIVES: Pre-admission clinics are traditionally run jointly by nurses and doctors. Within an adult ENT pre-admission clinic, we wished to assess what doctors added to nurses' pre-clerking, to determine whether doctors were actually needed in the clinic. METHODS: Prospective study, looking at how often doctors, seeing patients after ward-based nurses, changed or added to clerking or tests as organized by nurses. RESULTS: Out of 184 patients, doctors changed or added to nurses' clerking or planned investigations in 47 patients (26 per cent), making 64 different changes. The commonest reasons for changes were ordering blood tests (22 changes), chest X-rays (eight), cancelling due to hypertension (seven), altering drug history (five) and requesting electrocardiograms (five changes). CONCLUSION: Most changes made by doctors could be eliminated by designing a pre-admission clinic protocol that could easily be used by nurses. We recommend that all ENT departments consider implementing nurse-led pre-admission clinics.


Assuntos
Corpo Clínico Hospitalar , Otorrinolaringopatias/diagnóstico , Admissão do Paciente , Adulto , Testes Diagnósticos de Rotina/métodos , Humanos , Profissionais de Enfermagem , Recursos Humanos de Enfermagem Hospitalar , Cuidados Pré-Operatórios/métodos , Estudos Prospectivos
9.
J Laryngol Otol ; 118(12): 919-26, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15667676

RESUMO

Retropharyngeal abscess (RPA) is an uncommon condition with the potential for significant morbidity and mortality if not detected early. The authors present a case report of a 19-month-old child who presented with the common clinical features of a retropharyngeal abscess and in whom the diagnosis was not established by examination and ultrasonography. This led to a delay in appropriate management until a computed tomography (CT) scan was performed under general anaesthesia. The scan demonstrated the diagnosis and surgical drainage was performed under the same anaesthetic. The child subsequently made a complete recovery. The investigation and treatment of RPAs is a matter of some debate and the authors review the recent literature to determine the best management strategy.


Assuntos
Abscesso Retrofaríngeo/diagnóstico , Drenagem , Humanos , Lactente , Masculino , Abscesso Retrofaríngeo/microbiologia , Abscesso Retrofaríngeo/terapia , Tomografia Computadorizada por Raios X
10.
Clin Otolaryngol Allied Sci ; 27(6): 505-8, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12472520

RESUMO

Keratinocytes in middle ear cholesteatoma have hyperproliferative properties. There is controversy regarding the role of p53 and its effect on cellular proliferation in cholesteatoma. This study was instituted to examine this. Cholesteatoma and deep meatal skin control specimens were analysed for MIB-1 (n = 7, controls = 7), a marker of cellular proliferation, and p53 (n = 17, controls = 17) expression by immunocytochemistry. Expression of p53 was minimal or absent in both cholesteatoma and controls (P = 0.2). MIB-1 expression was higher, but not significantly so, in cholesteatoma than in controls (P = 0.09). Our study has shown no significant p53 expression in cholesteatoma epithelium. This suggests that there is no dysfunction in the p53-mediated cell cycle control mechanisms in cholesteatoma.


Assuntos
Colesteatoma da Orelha Média/genética , Colesteatoma da Orelha Média/patologia , Queratinócitos/patologia , Proteína Supressora de Tumor p53/análise , Divisão Celular , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise
11.
Otol Neurotol ; 22(5): 579-81, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11568661

RESUMO

HYPOTHESIS: Matrix metalloproteinase 1 (MMP-1) is overexpressed in cholesteatoma. BACKGROUND: Cholesteatoma destroys bone, whereas deep meatal skin does not. MMP-1 is a type I collagenase that may be responsible for this destruction. This prospective study was designed to identify overexpression of MMP-1 by cholesteatoma in comparison with deep meatal skin. METHODS: Ten cholesteatoma specimens and nine deep meatal skin specimens were removed during otologic surgery and then fixed in formalin and embedded in paraffin. Immunocytochemistry studies were performed using a monoclonal antibody to MMP-1. A pathologist assessed the slides in a blinded fashion. Expression of MMP-1 protein in epidermis and in stroma was scored from 0 to 10. Five further cholesteatoma specimens and three deep meatal skin specimens underwent reverse transcriptase polymerase chain reactions to assess messenger ribonucleic acid production. Paired and unpaired Student's t tests were used to assess the difference in expression levels. RESULTS: Cholesteatoma stroma expressed significantly more MMP-1 protein than did deep meatal skin stroma (p = 0.04). MMP-1 was localized to stromal fibroblasts. There was no difference in the epidermal expression levels of the two tissue types (p = 0.42). The reverse transcriptase polymerase chain reaction showed expression at the messenger ribonucleic acid size of MMP-1 (262 base pair) in all cholesteatoma specimens examined. One deep meatal skin specimen showed a weak signal; no signal was seen in the other specimens. CONCLUSIONS: MMP-1 is overexpressed by the stromal fibroblasts present in cholesteatoma as compared with deep meatal skin. It is possible that these cells rather than the keratinocytes are responsible for bone destruction in this disease.


Assuntos
Colesteatoma da Orelha Média/enzimologia , Ossículos da Orelha/enzimologia , Metaloendopeptidases/metabolismo , Técnicas de Cultura de Células , DNA Complementar/metabolismo , Fibroblastos/enzimologia , Humanos , Imuno-Histoquímica , Queratinócitos/enzimologia , Estudos Prospectivos , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/enzimologia , Pele/patologia
12.
Clin Otolaryngol Allied Sci ; 26(3): 227-30, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11437847

RESUMO

Radiotherapy of T1aN0M0 glottic carcinoma results in a local control rate of 80-94%. This homogenous group, which is the earliest recognisable invasive malignancy in the head and neck region, provides a 'unique model' for studying possible biological markers of radiosensitivity. p53 and MIB-1 were investigated as possible markers of radiosensitivity in such a group. In all, 107 patients with T1aN0M0 glottic squamous cell carcinoma treated with radiotherapy were identified. Cases not responsive to radiotherapy were compared with matched radiosensitive controls by immunohistochemistry using monoclonal primary antibodies to MIB-1 (n = 18; controls = 10) and p53 (n = 6; controls = 11). No significant difference in p53 expression was noted between the two groups (P = 0.73). A greater MIB-1 expression was found in the radiosensitive group but only a trend towards significance was observed (P = 0.06). MIB-1 is a potential marker of radiosensitivity. A larger multicentre study is required for a more definitive answer.


Assuntos
Carcinoma de Células Escamosas/radioterapia , Neoplasias Laríngeas/radioterapia , Proteínas Nucleares/análise , Tolerância a Radiação/genética , Proteína Supressora de Tumor p53/análise , Anticorpos Monoclonais , Antígenos Nucleares , Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Genes p53/genética , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/imunologia , Mutação , Prognóstico , Tolerância a Radiação/imunologia , Falha de Tratamento
13.
Indian J Ophthalmol ; 49(3): 173-6, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15887725

RESUMO

PURPOSE: To evaluate the efficacy of topical (1%) and systemic itraconazole against common fungi such as Aspergillus and other filamentous fungi that cause mycotic corneal ulcer. METHODS: A prospective randomised, controlled study was done in 54 clinically suspected cases of fungal keratitis of which 44 were culture proven. Half the cases (n=27) with superficial involvement were treated with only topical itraconazole (1%) and the other half were treated with both topical and systemic itraconazole. RESULTS: Aspergillus, Penicillium and Fusarium were the most common fungi isolated. The ulcer resolved in 42 eyes (77%) and 12 eyes (23%) did not respond well to treatment. Four of 12 non-responding eyes were caused by Fusarium species. CONCLUSION: Itraconazole, given either topically or systemically, is effective in treating mycotic corneal ulcers.


Assuntos
Antifúngicos/uso terapêutico , Úlcera da Córnea/tratamento farmacológico , Infecções Oculares Fúngicas/tratamento farmacológico , Itraconazol/uso terapêutico , Micoses/tratamento farmacológico , Administração Oral , Administração Tópica , Adulto , Antifúngicos/administração & dosagem , Aspergillus/isolamento & purificação , Úlcera da Córnea/microbiologia , Estudos Cross-Over , Infecções Oculares Fúngicas/microbiologia , Feminino , Fusarium/isolamento & purificação , Humanos , Itraconazol/administração & dosagem , Masculino , Pessoa de Meia-Idade , Micoses/microbiologia , Penicillium/isolamento & purificação , Estudos Prospectivos , Resultado do Tratamento
14.
Indian J Otolaryngol Head Neck Surg ; 53(2): 100-4, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23119767

RESUMO

Rhinosporidiosis is one of the common tropical diseases in ENT practice. In the present study, it constitutes about 1% hospital admission for E: surgery per annum. Nasal and extranasal involvement particularly occular involvement was critically examined in this study. Complete excision of rhinosporidial mass under endoscopie guidance for nasal rhinosporidiosis is found to give superior result in term of recurrence.

15.
J Laryngol Otol ; 114(5): 336-8, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10912260

RESUMO

Following informed parental consent 93 children underwent bilateral grommet insertion. Tympanometry was performed pre-operatively, and immediately prior to myringotomy. A standardized anaesthetic was used. At myringotomy the presence or absence of fluid was recorded, as well as the time since induction of the general anaesthetic. A pre-operative type B tympanogram predicted a middle-ear effusion at myringotomy in 92 per cent of patients. A pre-operative type C2 tympanogram predicted a middle-ear effusion at myringotomy in 39 per cent of patients. Sixty tympanograms (30 per cent) changed following a general anaesthetic. Fourteen type B tympanograms changed to type A and eight of these had effusions. The duration of the general anaesthetic did not influence the probability of a middle-ear effusion being present at myringotomy. A pre-operative type B tympanogram is a good predictor of middle-ear fluid. The duration of the general anaesthetic is not significant in predicting the presence of a middle-ear effusion.


Assuntos
Testes de Impedância Acústica/normas , Anestésicos Gerais/efeitos adversos , Óxido Nitroso/efeitos adversos , Otite Média com Derrame/diagnóstico , Anestesia Geral/efeitos adversos , Anestésicos Gerais/administração & dosagem , Pré-Escolar , Feminino , Humanos , Masculino , Óxido Nitroso/administração & dosagem , Estudos Prospectivos
16.
Am J Otol ; 21(4): 482-4, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10912691

RESUMO

OBJECTIVE: To determine the effect of topical adrenaline application after myringotomy and before tympanostomy tube placement on the development of myringosclerosis. STUDY DESIGN: A prospective, randomized, double-blind study, with each patient acting as his or her own control. Ethical approval and full parental consent were obtained. SETTING: Department of Otorhinolaryngology-Head and Neck Surgery in a university teaching hospital. PATIENTS: Fifty children satisfying inclusion and exclusion criteria for first-time tympanostomy tube insertion. THERAPEUTIC INTERVENTION: Myringotomy followed by adrenaline application to incision before tympanostomy tube insertion. Control contralateral ear received saline application after myringotomy. Follow-up examination was done 14 to 21 days after surgery and again after 1 year by a single blinded surgeon. MAIN OUTCOME MEASURE: Comparison of myringosclerosis between adrenaline-treated ears and matched control ears. RESULTS: No difference was found in early morbidity between the two groups of ears. Myringosclerosis after 1 year was not found to have been significantly affected by adrenaline application (p = 0.2) CONCLUSION: The use of adrenaline on the myringotomy site before tympanostomy tube placement was not found to influence early postoperative morbidity or the later development of myringosclerosis.


Assuntos
Epinefrina/administração & dosagem , Ventilação da Orelha Média/efeitos adversos , Otosclerose/etiologia , Otosclerose/prevenção & controle , Vasoconstritores/administração & dosagem , Administração Tópica , Criança , Método Duplo-Cego , Humanos , Incidência , Morbidade , Otosclerose/diagnóstico , Otosclerose/epidemiologia , Estudos Prospectivos
17.
Clin Otolaryngol Allied Sci ; 23(4): 345-7, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9762497

RESUMO

A qualitative and quantitative study of the presence of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9) in cholesteatoma was performed. Ten cholesteatoma and four deep meatal skin specimens were analysed for gelatinase activity at molecular weights corresponding to MMP-2 and MMP-9 using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) Zymography. Gelatinase activity at 72 kDa and 92 kDa was investigated. Western blotting was employed using primary monoclonal antibodies to provide a qualitative assessment of MMP-2 and MMP-9. Non-parametric data analysis using the Mann-Whitney U test did not show a significant difference in expression of MMP-2 (P = 0.51) or MMP-9 (P = 0.14) between the two tissue types. Western blotting showed the presence of both MMP-2 and MMP-9 in the majority of specimens, both cholesteatoma and deep meatal skin.


Assuntos
Colesteatoma/metabolismo , Colagenases/metabolismo , Gelatinases/metabolismo , Metaloendopeptidases/metabolismo , Pele/metabolismo , Western Blotting , Meato Acústico Externo , Eletroforese em Gel de Poliacrilamida , Humanos , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz
18.
Clin Otolaryngol Allied Sci ; 23(6): 515-9, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9884804

RESUMO

Nine cholesteatomas, seven middle ear granulations and five deep meatal skin specimens were analysed for gelatinase activity at molecular weights corresponding to those of matrix metalloproteinase-1 (MMP-1) using SDS PAGE zymography. Gelatinase activity at 41-43 kDa and 45-47 kDa was investigated. Western blotting was employed using a primary monoclonal antibody to MMP-1 to provide a qualitative assessment of MMP-1. Western blotting was also used with a monoclonal antibody to MMP-3 to discover if MMP-3 gelatinase activity occurring around the molecular weight of MMP-1 may have contributed to the results. A significantly higher expression of activity was recorded in cholesteatoma and middle ear granulations at 45-47 kDa in comparison with deep meatal skin. Western blotting indices were to be present in all of the cholesteatoma specimens tested. Only one of the specimens (cholesteatoma) tested showed any MMP-3 presence.


Assuntos
Colesteatoma da Orelha Média/enzimologia , Colagenases/análise , Orelha Média/química , Tecido de Granulação/química , Pele/química , Western Blotting/métodos , Orelha Média/enzimologia , Eletroforese em Gel de Poliacrilamida/métodos , Tecido de Granulação/enzimologia , Humanos , Metaloproteinase 1 da Matriz , Metaloproteinase 3 da Matriz/análise , Peso Molecular , Pele/enzimologia
19.
J Biol Chem ; 272(21): 13629-39, 1997 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-9153212

RESUMO

To investigate the relationship between RNA folding and ribozyme catalysis, we have carried out a detailed kinetic analysis of four structural derivatives of the hairpin ribozyme. Optimal and suboptimal (wild-type) substrate sequences were studied in conjunction with stabilization of helix 4, which supports formation of the catalytic core. Pre-steady-state and steady-state kinetic studies strongly support a model in which each of the ribozyme variants partitions between two major conformations leading to active and inactive ribozyme. substrate complexes. Reaction rates for cleavage, ligation, and substrate binding to both ribozyme conformations were determined. Ligation rates (3 min-1) were typically 15-fold greater than cleavage rates (0.2 min-1), demonstrating that the hairpin ribozyme is an efficient RNA ligase. On the other hand, substrate binding is very rapid (kon = 4 x 10(8) M-1 min-1), and the ribozyme. substrate complex is very stable (KD < 25 pM; koff < 0.01 min-1). Stabilization of helix 4 increases the proportion of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates. These effects can be explained by stabilization of the catalytic core of the ribozyme. Rigorous consideration of conformational isomers and their intrinsic kinetic properties was necessary for development of a kinetic scheme for the ribozyme-catalyzed reaction.


Assuntos
Conformação de Ácido Nucleico , RNA Catalítico/química , Sequência de Bases , Catálise , DNA/química , DNA/metabolismo , Desenho de Fármacos , Cinética , Modelos Químicos , Dados de Sequência Molecular , RNA/química , RNA/metabolismo , RNA Catalítico/metabolismo , Moldes Genéticos
20.
RNA ; 3(1): 1-16, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8990394

RESUMO

RNA transcripts corresponding to the 250-nt 3' untranslated region of the R2 non-LTR retrotransposable element are recognized by the R2 reverse transcriptase and are sufficient to serve as templates in the target DNA-primed reverse transcription (TPRT) reaction. The R2 protein encoded by the Bombyx mori R2 can recognize this region from both the B. mori and Drosophila melanogaster R2 elements even though these regions show little nucleotide sequence identity. A model for the RNA secondary structure of the 3' untranslated region of the D. melanogaster R2 retrotransposon was developed by sequence comparison of 10 species aided by free energy minimization. Chemical modification experiments are consistent with this prediction. A secondary structure model for the 3' untranslated region of R2 RNA from the R2 element from B. mori was obtained by a combination of chemical modification data and free energy minimization. These two secondary structure models, found independently, share several common sites. This study shows the utility of combining free energy minimization, sequence comparison, and chemical modification to model an RNA secondary structure.


Assuntos
Conformação de Ácido Nucleico , DNA Polimerase Dirigida por RNA/metabolismo , RNA/química , Retroelementos/genética , Algoritmos , Animais , Sequência de Bases , Bombyx/genética , Simulação por Computador , Drosophila/classificação , Drosophila/genética , Metilação , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Ligação Proteica , RNA/efeitos dos fármacos , RNA/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Especificidade por Substrato , Temperatura , Moldes Genéticos , Termodinâmica
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