RESUMO
OBJECTIVE: The objective of this work was to gather an international consensus group to propose a global definition and diagnostic approach of laryngopharyngeal reflux (LPR) to guide primary care and specialist physicians in the management of LPR. METHODS: Forty-eight international experts (otolaryngologists, gastroenterologists, surgeons, and physiologists) were included in a modified Delphi process to revise 48 statements about definition, clinical presentation, and diagnostic approaches to LPR. Three voting rounds determined a consensus statement to be acceptable when 80% of experts agreed with a rating of at least 8/10. Votes were anonymous and the analyses of voting rounds were performed by an independent statistician. RESULTS: After the third round, 79.2% of statements (N = 38/48) were approved. LPR was defined as a disease of the upper aerodigestive tract resulting from the direct and/or indirect effects of gastroduodenal content reflux, inducing morphological and/or neurological changes in the upper aerodigestive tract. LPR is associated with recognized non-specific laryngeal and extra-laryngeal symptoms and signs that can be evaluated with validated patient-reported outcome questionnaires and clinical instruments. The hypopharyngeal-esophageal multichannel intraluminal impedance-pH testing can suggest the diagnosis of LPR when there is >1 acid, weakly acid or nonacid hypopharyngeal reflux event in 24 h. CONCLUSION: A global consensus definition for LPR is presented to improve detection and diagnosis of the disease for otolaryngologists, pulmonologists, gastroenterologists, surgeons, and primary care practitioners. The approved statements are offered to improve collaborative research by adopting common and validated diagnostic approaches to LPR. LEVEL OF EVIDENCE: 5 Laryngoscope, 134:1614-1624, 2024.
Assuntos
Refluxo Laringofaríngeo , Laringe , Humanos , Refluxo Laringofaríngeo/diagnóstico , Otorrinolaringologistas , Impedância Elétrica , Inquéritos e Questionários , Monitoramento do pH EsofágicoRESUMO
OBJECTIVE: This state-of-the-art article reviews the epidemiology, diagnosis, and management of vocal fold leukoplakia, with focus on recent advances. It focuses on the clinical challenges that otolaryngologists face balancing both oncological efficacy and functional outcomes in leukoplakia and presents the current philosophies and techniques to consider when managing such patients. DATA SOURCES: PubMed/MEDLINE. REVIEW METHODS: We conducted a detailed review of publications related to vocal cord and laryngeal leukoplakia, dysplasia, hyperkeratosis, leukoplakia endoscopy, and leukoplakia management focusing specifically on oncologic outcomes, voice preservation, current and emerging diagnosis, and management techniques. CONCLUSIONS: There has been a paradigm shift away from performing "vocal cord stripping" procedures that can cause irreversible hoarseness toward voice preservation surgery while achieving comparable oncologic control. Surgical technical and instrumental developments have been designed to maximally treat superficial disease while preserving underling vibratory mucosa. Recent improvements in histopathological grading systems and advances in biomarker classification may allow for improved oncologic risk stratification. Furthermore, improvements in endoscopic imaging capabilities and contact endoscopy are currently being studied for their potential diagnostic significance. IMPLICATIONS FOR PRACTICE: To optimally manage vocal fold leukoplakia, the otolaryngologist should become familiar with the oncologic implications of the disease and the importance of obtaining pathologic diagnosis to rule out malignancy. In addition, the surgeon should maintain surgical techniques and knowledge of available instruments and lasers that can assist in surgical management while prioritizing the preservation of vibratory tissue and voice quality. Finally, the surgeon and the patient should understand the clinical importance of routine endoscopic surveillance.
Assuntos
Doenças da Laringe , Leucoplasia , Prega Vocal , Humanos , Doenças da Laringe/diagnóstico , Doenças da Laringe/epidemiologia , Doenças da Laringe/terapia , Leucoplasia/diagnóstico , Leucoplasia/epidemiologia , Leucoplasia/terapiaRESUMO
Vocal fold immobility can be either unilateral or bilateral and partial or complete. The aim of this chapter is to discuss the management of unilateral paresis using medialization thyroplasty with or without arytenoid adduction as a means of treating neurogenic causes as opposed to mechanical fixation. Medialization thyroplasty is an open surgical procedure that is performed under local or general anesthesia. Essentially, it aims to close the glottic gap, approximating both vocal folds together and thereby allowing for restoration of the efficiency of the larynx. The glottic gap results from atrophy of the affected vocal fold and in so doing results in glottic insufficiency which causes voice breathiness, strain, fatigue, aspiration, and swallowing difficulties that make up the bulk of symptoms associated with this condition. Unlike injection laryngoplasty, medialization thyroplasty does not increase the "bulk" of the atrophic vocal fold but merely brings the fold closer to its unaffected partner. Besides the obvious lateralization, there is occasionally a third dimensional component to the affected fold. The slipping and prolapse forward of the arytenoid cartilage due to atrophy of the muscles supporting it and the natural declination of the facet joint it rests on cause a vertical drop of the level of the affected vocal fold that may not be remedied with the medialization procedure, hence requiring arytenoid adduction. Although attempts to medialize the vocal fold have been described in the past with limited access, the basic premise of creating a window in the thyroid cartilage remains central. The differences between materials used, their respective strengths and weaknesses, the pitfalls and pearls in achieving a good closure and improvement in voice, swallow, and safety of the airway are all discussed accordingly.
Assuntos
Cartilagem Aritenoide/cirurgia , Laringoplastia , Paralisia das Pregas Vocais/cirurgia , Prega Vocal/inervação , Prega Vocal/fisiopatologia , Humanos , Seleção de Pacientes , Cartilagem Tireóidea/cirurgia , Paralisia das Pregas Vocais/etiologia , Paralisia das Pregas Vocais/patologia , Prega Vocal/patologiaRESUMO
Neurolaryngology as a subspecialty of laryngology has developed considerably in the last four decades with more laryngologists, neurologists, speech and swallow therapists, and neurophysiologists taking interest in the field. The North American and Japanese laryngology societies have increasingly focused on conditions which are mainly concerned with aberrations of the nervous system affecting the larynx directly or indirectly. In the last few years, societies in Europe and the Asia-Pacific have also recognized the need to collaborate both within their organizations and with other societies globally. Cross-border pollination of ideas has increasingly become easier and with the aid of technology - almost seamless with real-time capacity to share operating experience, lectures, and panel discussions. The future advances in neurolaryngology will require incremental improvements in processes of diagnostics, objectivization (where possible) of pathology, standardization of treatments with comparison of results using accepted patient-based tests, investigations and imaging where possible. Ultimately, from the contributions in the previous chapters, it is fairly obvious that many conditions are still poorly understood and therefore management becomes more symptom based rather than dealing with the root cause of the problem. An understanding of the physiology of vocalization, swallow, and breathing beyond a rudimentary acceptance of many towards the vagus nerve and other neural factors may help understand what has otherwise been a rather simplistic approach to one of the most complex parts of the human body, essential to life and equally important - the quality of life. In this chapter, we aim to look at where advances in neurolaryngology may and perhaps will take place. We will look at the potential of better imaging modalities, neurophysiological testing and physiology of the brain. Tests and treatments currently in use may require some refinements or be possibly abandoned and replaced with more effective ones that can demonstrate a difference in the management of various patient groups. The future is hard to predict, and the rate of advancement equally so, but given the rate at which information technology, artificial intelligence, and basic science research are progressing, neurolaryngology may indeed have its welcome boost in the not too distant future.
Assuntos
Neurologia/tendências , Otolaringologia/tendências , Humanos , Neuroimagem/tendências , Monitorização Neurofisiológica/tendênciasRESUMO
PURPOSE: This purpose of this case series is to present the first four cases utilizing micro-phonosurgical instrumentation designed specifically for use with a semi-flexible 'robotic' system-the Medrobotics Flex system and to evaluate the accessibility and feasibility of this platform in the context of transoral robotic surgery (TORS) for laryngeal surgery. METHODS: Four patients (3 female, 1 male; age range 49-79 years) were operated by the senior author at CHL-a tertiary hospital centre between 2016 and 2017. The 'robot' was deployed in all cases to assess its accessibility and ability to perform surgery in the larynx. RESULTS: All four patients were successfully treated using the system along with newly developed instrumentation specifically focused on phonosurgery. CONCLUSION: This series has demonstrated accessibility and ability for laryngeal surgery using a novel semi-rigid operator-controlled 'robotic' system. We encountered no device failures and were able to perform all the selected cases uneventfully.
Assuntos
Doenças da Laringe/cirurgia , Laringe/cirurgia , Procedimentos Cirúrgicos Robóticos/instrumentação , Prega Vocal/cirurgia , Idoso , Estudos de Viabilidade , Feminino , Humanos , Doenças da Laringe/patologia , Laringe/patologia , Masculino , Pessoa de Meia-Idade , Procedimentos Cirúrgicos Robóticos/métodos , Prega Vocal/patologiaRESUMO
The objective of the study was to determine the etiology and subsequent management of patients with unilateral vocal fold immobility (UVFI) and compare our results with other such studies. This was a retrospective case series of all patients that were treated for UVFI at one single tertiary referral centre between 2010 and 2014. The medical records of 161 patients over a 5-year period diagnosed with UVFI were analyzed. We looked at the patient demographics, side of immobility, etiology, management and voice assessment. A total of 21 patients were excluded due to varying reasons including second presentation and incomplete data. Our results demonstrated 37.1% of cases to be due to non-thyroid surgery (mainly vascular or anterior cervical spine surgery) compared to thyroid or parathyroid (18.6%). Carotid endarterectomy was the commonest cause followed by cervical spine discectomy or fusion. Other iatrogenic causes included thoracic surgery either involving the lung or not. Our results are very much in keeping with those seen by our colleagues in North America. A better appreciation of the causes of UVFI especially in cases not performed by otolaryngologists and head and neck surgeons should be highlighted and the necessary steps should be taken to prevent this iatrogenic complication.
Assuntos
Endarterectomia das Carótidas/efeitos adversos , Procedimentos Ortopédicos/efeitos adversos , Complicações Pós-Operatórias , Procedimentos Cirúrgicos Torácicos/efeitos adversos , Paralisia das Pregas Vocais , Bélgica , Endarterectomia das Carótidas/métodos , Feminino , Humanos , Doença Iatrogênica/prevenção & controle , Masculino , Pessoa de Meia-Idade , Pescoço/cirurgia , Procedimentos Ortopédicos/métodos , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/fisiopatologia , Complicações Pós-Operatórias/terapia , Estudos Retrospectivos , Testes de Articulação da Fala/métodos , Doenças da Coluna Vertebral/cirurgia , Procedimentos Cirúrgicos Torácicos/métodos , Paralisia das Pregas Vocais/diagnóstico , Paralisia das Pregas Vocais/etiologia , Paralisia das Pregas Vocais/fisiopatologia , Paralisia das Pregas Vocais/terapia , Prega Vocal/fisiopatologia , Qualidade da VozRESUMO
UNLABELLED: Type I medialization thyroplasty (MT) was introduced by Isshiki more than 40 years ago. It is one of the most widely used surgical options to correct glottic insufficiency. Intraoperatively, the surgeon relies solely on perceptual subjective measures to help to achieve an optimal glottic closure by bringing the affected vocal fold closer to the midline in order to close the glottic gap. One of the challenges of MT is the persistence of symptoms due to incorrect choice of implant size. As of now, no standard objective measure is being used to determine the optimal implant size needed to achieve the glottic closure required. Peak direct subglottic pressure (PDSGP) is one of the aerodynamic objective measurements of vocal efficiency that significantly increases in cases of glottic insufficiency. It is easily measured during MT by inserting a catheter through the cricothyroid membrane. A prospective study was carried out on patients undergoing MT using the Montgomery Implant(®). Choice of implant size was carried out based on the standard perceptual subjective assessment by the operating surgeon and was based on degree of glottic closure and voice quality. PDSGP was recorded for each implant size and then we tested the agreement between the chosen implant size and the lowest PDSGP. The agreement between the implant size of choice and the lowest PDSGP recorded was 62.5 % [CI 44-79 %]. PDSGP was easy to measure and resulted in no complications. PDSGP is a useful tool that could assist in the choice of the correct implant size needed during MT. LEVEL OF EVIDENCE: 4.
Assuntos
Glote/cirurgia , Laringoplastia/métodos , Paralisia das Pregas Vocais/cirurgia , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Próteses e Implantes , Procedimentos de Cirurgia Plástica , Paralisia das Pregas Vocais/etiologia , Prega Vocal/cirurgia , Qualidade da Voz , Adulto JovemRESUMO
Voice rehabilitation after transoral laser microsurgery to the larynx is challenging. We wait at least 6 months before surgical intervention. Only a few patients after total or extended cordectomy requested voice restoration. Subjective perception of voice using the Voice Handicap Index in medialization thyroplasty was significantly better. Medialization thyroplasty with elevation of the fibrous tissue from the inner surface of the thyroid cartilage is critical in achieving success. We preferred the Montgomery Thyroplasty Implant System. Transoral larynx anterior commissure stent placement after laser-assisted sectioning of anterior synechiae with application of mitomycin C is an effective procedure for anterior synechiae.
Assuntos
Carcinoma de Células Escamosas/cirurgia , Glote/cirurgia , Neoplasias Laríngeas/cirurgia , Distúrbios da Voz/reabilitação , Humanos , Laringectomia/métodos , Laringoplastia/métodos , Terapia a Laser/métodos , Microcirurgia/métodos , Fonoterapia , Qualidade da VozRESUMO
We reviewed the results and side-effect profile of the Dysport preparation of botulinum toxin A (BTA) in the management of the adductor spasmodic dysphonia. We performed 272 injection episodes in 68 patients, 42 (62%) female, 26 (38%) male. A total of 116 of these injections were unilateral, and 156 were bilateral; 94% of the injections were considered to have been successful with a voice score of 2 or higher. The mean duration of effect (injection intervals) was 128.8 days in the unilateral cohort and 118.7 days in the bilateral (P > 0.05). We injected a relatively lower dose of BTA for unilateral injection episodes in our institution compared to those reported by others to produce comparable results and side-effect profiles.
Assuntos
Toxinas Botulínicas Tipo A/administração & dosagem , Toxinas Botulínicas Tipo A/uso terapêutico , Disfonia/tratamento farmacológico , Fármacos Neuromusculares/administração & dosagem , Fármacos Neuromusculares/uso terapêutico , Toxinas Botulínicas Tipo A/efeitos adversos , Estudos de Coortes , Feminino , Lateralidade Funcional , Humanos , Injeções/métodos , Masculino , Pessoa de Meia-Idade , Fármacos Neuromusculares/efeitos adversos , Estudos Retrospectivos , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do Tratamento , Voz/efeitos dos fármacosRESUMO
AIMS: To compare voice quality as defined by formant analysis using a sustained vowel in patients who have undergone a partial glossectomy with a group of normal subjects. METHODS & PROCEDURES: The design consisted of a single centre, cross-sectional cohort study. The setting was an Adult Tertiary Referral Unit. A total of 26 patients (19 males) who underwent partial glossectomy and 31 normal volunteers (18 males) participated in the study. Group comparisons using the first three formant frequencies (F1, F2 and F3) using linear predictive coding (Laryngograph Ltd, London, UK) were performed. The existence of any significant difference of F1, F2 and F3 between the two groups using the sustained vowel /i/ and the effects of other factors, namely age, first presentation versus recurrence, site (oral cavity, oropharynx), subsite (anterior two-thirds of the tongue, tongue base), stage, radiation, complication, and neck dissection, were analysed. OUTCOMES & RESULTS: Formant frequencies F1, F2 and F3 were normally distributed. F1 and F2 were significantly different in normal males versus females. F1, F2 and F3 were not different statistically between male and female glossectomees. Comparison of only women showed significant differences between normal subjects and patients in F2 and F3, but none in F1. This was the opposite in men where F1 was significantly different. Age, tumour presentation, site, subsite, radiation and neck dissection showed no significant difference. Postoperative complications significantly affected the F1 formant frequency. CONCLUSIONS: The study found that the formant values in patients following a partial glossectomy were altered significantly as compared with the normal control subjects. Only gender and complications and not the age, site, subsite, radiation and neck dissection were seen to influence the formant scores.
Assuntos
Carcinoma de Células Escamosas/cirurgia , Glossectomia , Neoplasias Bucais/cirurgia , Neoplasias Orofaríngeas/cirurgia , Qualidade da Voz , Adulto , Idoso , Carcinoma de Células Escamosas/fisiopatologia , Estudos Transversais , Feminino , Glossectomia/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/fisiopatologia , Neoplasias Orofaríngeas/fisiopatologia , Período Pós-Operatório , Fatores Sexuais , Acústica da FalaRESUMO
The objective of this study was to assess the difference in voice quality as defined by acoustical analysis using sustained vowel in laryngectomized patients in comparison with normal volunteers. This was designed as a retrospective single center cohort study. An adult tertiary referral unit formed the setting of this study. Fifty patients (40 males) who underwent total laryngectomy and 31 normal volunteers (18 male) participated. Group comparisons with the first three formant frequencies (F1, F2, and F3) using linear predictive coding (LPC) (Laryngograph Ltd, London, UK) was performed. The existence of any significant difference of F1, F2, and F3 between the two groups using the sustained vowel /i/ and the effects of other factors namely, tumor stage (T), chemoradiotherapy, pharyngectomy, cricothyroid myotomy, closure of pharyngoesophageal segment, and postoperative complication were analyzed. Formant frequencies F1, F2, and F3 were significantly different in male laryngectomees compared to controls: F1 (P<0.001, Mann-Whitney U test), F2 (P<0.001, Student's t test), and F3 (P=0.008, Student's t test). There was no significant difference between females in both groups for all three formant frequencies. Chemoradiotherapy and postoperative complications (pharyngocutaneous fistula) caused a significantly lower formant F1 in men, but showed little effect in F2 and F3. Laryngectomized males produced significantly higher formant frequencies, F1, F2, and F3, compared to normal volunteers, and this is consistent with literature. Chemoradiotherapy and postoperative complications significantly influenced the formant scores in the laryngectomee population. This study shows that robust and reliable data could be obtained using electroglottography and LPC in normal volunteers and laryngectomees using a sustained vowel.
Assuntos
Neoplasias Laríngeas/patologia , Neoplasias Laríngeas/cirurgia , Laringectomia , Fonética , Distúrbios da Voz/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Estudos Retrospectivos , Espectrografia do SomRESUMO
The 38-kDa lipoprotein is one of the most potent cell surface immunogens of Mycobacterium tuberculosis in antibody-and T cell-mediated reactions. Using a pure recombinant form of the protein, we have recently shown that it binds phosphate much like that of the phosphate-binding protein (M(r) = 34.4 kDa) that is localized in the periplasm of Escherichia coli and is involved as an initial receptor for active transport of phosphate. The purified 38-kDa protein has been crystallized in 2 forms that are suitable for high-resolution structural analyses. One form belongs to the monoclinic space group P2(1) with unit cell dimensions of a = 67.42 A, b = 113.38 A, c = 42.68 A, and beta = 108.53 degrees. The other is of orthorhombic space group P2(1)2(1)2 with a = 125.46 A, b = 72.27 A, and c = 73.43 A. Both crystal forms diffract to about 2 A resolution on a fine focus rotating anode.
Assuntos
Antígenos de Bactérias/química , Epitopos Imunodominantes/imunologia , Lipoproteínas/química , Mycobacterium tuberculosis/imunologia , Antígenos de Bactérias/imunologia , Cristalização , Cristalografia por Raios X , Epitopos Imunodominantes/química , Lipoproteínas/imunologia , Mycobacterium tuberculosis/químicaRESUMO
The D-glucose/D-galactose-binding protein (M(r) = 33,000) found in the periplasm of bacterial cells serves as the primary high-affinity receptor of active transport for and chemotaxis toward both sugar epimers. This protein from Escherichia coli binds D-glucose with a Kd of 2 x 10(-7) M, which is about 2 times tighter than D-galactose. The 2.0-A resolution crystal structure of the binding protein complexed with D-galactose has been refined to a crystallographic R-factor of 0.167. This structure, combined with that previously refined for the complex with D-glucose [Vyas, N.K., Vyas., M. N., & Quiocho, F. A. (1988) Science 242, 1290-1295], provides understanding, in atomic detail, of recognition of sugar epimers and anomers. In the two complex structures, the sugar ring is positioned identically in the binding site, and each hydroxyl group common to both is involved in very similar cooperative hydrogen-bonding interactions with protein residues and ordered water molecules. Only the beta-anomer of both monosaccharides is bound, with Asp154 OD1 primarily responsible for accepting a hydrogen bond from the anomeric hydroxyl. Recognition of both sugar epimers is accomplished principally by hydrogen bonding of Asp14 OD1 with the equatorial OH4 of D-glucose and OD2 with the axial OH4 of D-galactose. These results are reconciled with equilibrium and fast kinetics data, which indicate binding of both anomers of the two sugars, and further compared with sugar recognition by other periplasmic sugar-binding proteins with specificities for arabinose/galactose/fucose, maltooligosaccharides, and ribose.
Assuntos
Galactose/química , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Bactérias/química , Cristalografia , Escherichia coli , Galactose/metabolismo , Glucose/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/metabolismo , EstereoisomerismoRESUMO
The Fab of a monoclonal anti-carbohydrate antibody, SYA/J6 (IgG3, kappa, murine), raised against the O-polysaccharide antigen of the cell surface lipopolysaccharide of variant Y Shigella flexneri, a Gram negative bacterium, has been crystallized in the unliganded form and in complex with tri- and pentasaccharide antigens. The three crystal forms belong to the tetragonal space group P4(3)2(1)2, or P4(1)2(1)2, with very similar unit cell dimensions and an asymmetric unit that contains one molecule of about 50,000 Daltons, and a fourth crystal form belongs to monoclinic space group P2(1) that contains four molecules of Fab in an asymmetric unit. Whereas diffractions of these crystals on an area detector-rotating anode system extend to only about 3.5 A resolution, those measured using an imaging plate and synchrotron radiation at the Photon Factory facility extend to 2.5 A.
Assuntos
Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Shigella flexneri/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Sequência de Carboidratos , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Lipopolissacarídeos/isolamento & purificação , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/imunologia , Ligação Proteica , Difração de Raios XRESUMO
The primary sequence of the receptor for L-arabinose or Ara-binding protein (ABP) composed of 306 residues is very different from the D-glucose/D-galactose-binding protein (GGBP) which consists of 309 residues. Nevertheless, superimpositioning of the well-refined high resolution structures of ABP in complex with D-galactose and the GGBP in complex with D-glucose shows very similar structures; 220 of the residues (or about 70%) have a root mean square deviation of 2.0 A. From the superpositioning, nine pairs of continuous segments (consisting of 8-51 residues), mainly alpha-helices and beta-strands that form the core of the two lobes of the bilobate proteins were found to exhibit strong sequence homology. The equivalenced structures and aligned sequences show that many of the polar, as well as aromatic residues, in the sugar-binding sites located in the cleft between the two lobes are highly conserved. Surprisingly, however, the exact mode of binding of the D-galactose in ABP is totally different from that of the D-glucose in GGBP. Using the structurally aligned sequences of the ABP and GGBP as a template, we have matched the sequence of the ribose-binding protein (RBP) which consists of 271 residues with the ABP/GGBP pair. Although the nine aligned segments of all three proteins show little sequence identity, they have significant homology. Four additional segments of RBP were matched only with GGBP, leading to the alignment of about 90% of the RBP sequence with the GGBP sequence. Many of the conserved residues in the binding sites of ABP and GGBP matched with similar residues in RBP. Additional observations indicate that the GGBP/RBP pair is more closely related than the ABP/RBP or ABP/GGBP pair. All three binding proteins, which may have diverged from a common ancestor, serve as primary receptors for bacterial high affinity active transport systems. Moreover, GGBP and RBP, but not ABP, also act as receptors for chemotaxis. An exposed site located in one domain, which includes Gly74, for interacting with the trg transmembrane signal transducer that is involved in triggering chemotaxis has been located in the structure of GGBP (Vyas, N.K., Vyas, M.N., and Quiocho, F.A. (1988) Science 242, 1290-1295). Whereas the site is absent in the structure of ABP, it is strongly predicted to be present in RBP which shares the same trg transducer with GGBP. The knowledge-based alignment of RBP further revealed two possible additional peripheral chemotactic sites that show high structural and sequence similarity between GGBP and RBP only. At least one of these sites, together with the one proven to exist in the other domain, could be used by the signal transducer with which both binding proteins interact in a way which the substrate-loaded "closed cleft" structure could be discriminated from the unliganded "open cleft" form by the transducer.
Assuntos
Proteínas de Transporte/genética , Proteínas de Escherichia coli , Proteínas Periplásmicas de Ligação , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Arabinose/metabolismo , Proteínas de Transporte/metabolismo , Galactose/metabolismo , Glucose/metabolismo , Dados de Sequência Molecular , Conformação Proteica , Receptores de Superfície Celular/metabolismo , Ribose/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
Single crystals of the Fab fragment of a murine monoclonal antibody BAT123 (IgG1, kappa) raised against a dominant neutralizing determinant of gp120 of HIV that are suitable for X-ray structural analysis have been obtained. The thick prismatic plate crystals belong to space group P2(1)2(1)2 with unit cell dimensions of a = 177.42 A, b = 37.36 A and c = 73.30 A.
Assuntos
Anticorpos Anti-HIV , Proteína gp120 do Envelope de HIV/imunologia , Animais , Anticorpos Monoclonais , Epitopos/imunologia , Fragmentos Fab das Imunoglobulinas , Camundongos , Camundongos Endogâmicos BALB C , Difração de Raios XRESUMO
We have determined the relative affinities in solution for various metals which bind to the lone calcium-binding site of the D-galactose-binding protein which resembles the EF-hand loop. In order of affinity the metals are: Ca2+ approximately Tb3+ approximately Pb2+ greater than Cd2+ greater than Sr2+ greater than Mg2+ much greater than Ba2+. The binding affinity for calcium (Kd = 2 microM) and the slow off-rate determined for terbium (1 x 10(-3) s-1) and that the metal-binding site is unperturbed by sugar binding argue for a structural role. Furthermore, we have crystallographically refined the structure of the binding protein with the calcium substituted by cadmium, compared it with the calcium-bound structure, and found them to be identical. The results of these structural and solution studies support the hypothesis that for a given metal-binding loop, cation hydration energy, size, and charge are major factors contributing to binding affinity.
Assuntos
Proteínas de Ligação ao Cálcio , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Monossacarídeos , Proteínas Periplásmicas de Ligação , Sítios de Ligação , Proteínas de Transporte/isolamento & purificação , Cátions Bivalentes , Escherichia coli/metabolismo , Galactose/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Térbio/metabolismoRESUMO
D-galactose-binding (or chemoreceptor) protein of Escherichia coli serves as an initial component for both chemotaxis towards galactose and glucose and high-affinity active transport of the two sugars. Well-refined x-ray structures of the liganded forms of the wild-type and a mutant protein isolated from a strain defective in chemotaxis but fully competent in transport have provided a molecular view of the sugar-binding site and of a site for interacting with the Trg transmembrane signal transducer. The geometry of the sugar-binding site, located in the cleft between the two lobes of the bilobate protein, is novel in that it is designed for tight binding and sequestering of either the alpha or beta anomer of the D-stereoisomer of the 4-epimers galactose and glucose. Binding specificity and affinity are conferred primarily by polar planar side-chain residues that form intricate networks of cooperative and bidentate hydrogen bonds with the sugar substrates, and secondarily by aromatic residues that sandwich the pyranose ring. Each of the pairs of anomeric hydroxyls and epimeric hydroxyls is recognized by a distinct Asp residue. The site for interaction with the transducer is about 18 A from the sugar-binding site. Mutation of Gly74 to Asp at this site, concomitant with considerable changes in the local ordered water structures, contributes to the lack of productive interaction with the transmembrane signal transducer.