RESUMEN
Transplant tourism is routinely denounced by influential voices such as the World Health Organization, the Declaration of Istanbul and the Madrid Resolution as an unethical solution to worldwide organ shortages. Instead, it is suggested that national deceased donor schemes and multinational organ-sharing programs are the only acceptable avenues for addressing the organ shortage crisis. The present demand for self-sufficiency in organ supply responds to risks such as poor clinical outcomes, and exploitation of the poor through the various commercial practices of transplant tourism. However, opponents of transplant tourism say little about what governments should do to ensure that their citizens have real and comprehensive access to all forms of transplantation. To address this complex question, we describe a current practice of international transplant medicine in Singapore. It addresses salient concerns with transplant tourism and supports the principle of national self-sufficiency in organ supply, even as its health care system thrives and expands comprehensive transplant services to its citizens by catering to international patients. We offer a critical appraisal of the Singaporean system, and some suggestions to minimize the risk of abuse by international patients or operatives of illegal organ markets.
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Turismo Médico , Trasplante , Humanos , InternacionalidadRESUMEN
This study aimed to characterize antimicrobial resistance and virulence genes in multi-drug resistant enterotoxigenic Escherichia coli (ETEC) isolates (n=117) collected from porcine post-weaning diarrhoea cases in Australia (1999-2005). Isolates were serotyped, antibiogram-phenotyped for 12 antimicrobial agents and genotyped by PCR for 30 plasmid-mediated antimicrobial resistance genes (ARGs), 22 intestinal and 38 extraintestinal E. coli virulence genes (VGs). Nine serogroups were identified, the most prevalent being O149 (46.2%), O141 (11.2%) and Ont (31.6%). None of the isolates showed resistance to ceftiofur or enrofloxacin and 9.4% were resistant to florfenicol. No corresponding extended-spectrum/AmpC ß-lactamase, fluoroquinolone or floR ARGs were detected. An antimicrobial resistance index (ARI) was calculated from the combined data with a weighting for each antimicrobial agent dependent upon its significance to human health. Serogroup O141 isolates had a significantly higher ARI due to an elevated prevalence of aminoglycoside ARGs and possession of more virulence genes (VGs), including ExPEC or EHEC adhesins (bmaE, sfa/focDE, fimH, ihA) in toxin-producing strains that lacked the normally associated F4 and F18 fimbriae. Few associations between ARGs and VGs were apparent, apart from tetC, sfa/focDE and ompT which, for a sub-set of O141 isolates, suggest possible plasmid acquisition from ExPEC. The multi-drug resistant ETEC ARG/VG profiles indicate a high probability of considerable strain and plasmid diversity, reflecting various selection pressures at the individual farm level rather than emergence and lateral spread of MDR resistant/virulent clones.
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Diarrea/veterinaria , Escherichia coli Enterotoxigénica/aislamiento & purificación , Infecciones por Escherichia coli/veterinaria , Enfermedades de los Porcinos/microbiología , Animales , Australia/epidemiología , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , Diarrea/epidemiología , Diarrea/microbiología , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli Enterotoxigénica/efectos de los fármacos , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/patogenicidad , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Pruebas de Sensibilidad Microbiana/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Análisis de Secuencia de ADN , Porcinos , Enfermedades de los Porcinos/epidemiología , Virulencia/genéticaRESUMEN
OBJECTIVE: To describe how various antimicrobials are used in commercial pig herds in Australia and for what disease conditions. PROCEDURE: Managers of large pig herds (> 200 sows) across Australia and their veterinarians participated in an internet-based survey in 2006. Questions were asked about herd management, the occurrence of bacterial diseases and the type and frequency of antimicrobial use. An antimicrobial usage index for each herd was derived as a summary of the risk of selection for antimicrobial resistance. Relationships between responses were explored with univariate and multivariate analysis. RESULTS: Responses were received for 197 herds estimated to represent at least 51% of all large pig herds in Australia. Most piggeries relied on drugs of low importance in human medicine (e.g. tetracyclines, penicillins and sulfonamides). For the two drugs of high importance in human medicine that can be legally prescribed to pigs in Australia, ceftiofur use was reported in 25% of herds and virginiamycin in none. Infections attributed to Lawsonia, Mycoplasma and Escherichia coli motivated the most use of antimicrobials. No useful association was found between management factors and the antimicrobial use index. CONCLUSION: Most antimicrobial use in the Australian pig industry is based on drugs of low importance to public health. Enhanced control of E. coli infections without reliance on antimicrobials would further reduce the risk of selecting for antimicrobial resistance relevant to public health. The amount of variation in the usage index between herds suggests that antimicrobial use should be constantly reviewed on a herd by herd basis.
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Antiinfecciosos/uso terapéutico , Infecciones Bacterianas/veterinaria , Enfermedades de los Porcinos/tratamiento farmacológico , Enfermedades de los Porcinos/microbiología , Crianza de Animales Domésticos/métodos , Animales , Australia , Infecciones Bacterianas/tratamiento farmacológico , Utilización de Medicamentos , Encuestas Epidemiológicas , Internet , Encuestas y Cuestionarios , PorcinosRESUMEN
OBJECTIVE: To investigate the prevalence of Parkinson disease (PD) in Singapore and compare the rates between Singaporean Chinese, Malays, and Indians. METHODS: A three-phase community-based survey among a disproportionate random sample of 15,000 individuals (9,000 Chinese, 3,000 Malays, 3,000 Indians) aged 50 years and above who live in central Singapore was conducted. In phase 1, trained interviewers conducted a door-to-door survey using a validated 10-question questionnaire. In phase 2, medical specialists examined participants who screened positive to any of the questions. Participants suspected to have PD had their diagnosis confirmed in phase 3 by a movement disorders specialist. RESULTS: The participation rate was 67% among 22,279 eligible individuals. Forty-six participants with PD were identified of which 16 were newly diagnosed cases. The prevalence rate of PD for those aged 50 and above in Singapore was 0.30% (95% CI: 0.22 to 0.41), age-adjusted to US 1970 census. The prevalence rates increased significantly with age. The age-adjusted prevalence rates among Chinese (0.33%, 95% CI: 0.22 to 0.48), Malays (0.29%, 95% CI: 0.13 to 0.67), and Indians (0.28%, 95% CI: 0.12 to 0.67) were the same (p = 1.0). CONCLUSIONS: The prevalence of PD in Singapore was comparable to that of Western countries. Race-specific rates were also similar to previously reported rates and similar among the three races. Environmental factors may be more important than racially determined genetic factors in the development of PD.
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Etnicidad/genética , Enfermedad de Parkinson/etnología , Anciano , Anciano de 80 o más Años , China/etnología , Femenino , Encuestas Epidemiológicas , Humanos , India/etnología , Malasia/etnología , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/epidemiología , Prevalencia , Proyectos de Investigación , Muestreo , Singapur/epidemiología , Encuestas y CuestionariosRESUMEN
INTRODUCTION: This study sets out to determine the usefulness of a questionnaire to screen for hearing impairment, assess the psychosocial impact of hearing handicap and survey older persons' attitudes towards hearing aid usage. METHODS: Subjects were recruited from a Geriatric Medicine unit over a six-month period. A questionnaire was administered, followed by an otoscopic examination and audiometric testing. RESULTS: Sixty-three patients were included in the study. Fifty-two (83%) patients had hearing impairment, of which 34 were moderately severe and 18 were mild. Of the six questions used in hearing screening, the question on self-perception was the most specific (91%). Administering the remaining five questions on activities of daily living improved the questionnaire's sensitivity from 58% to 73%, although the specificity was reduced from 91% to 64%. Of the 30 patients with self-perceived and audiometrically-confirmed hearing impairment, about 40% reported negative psychosocial impact as a result of the handicap. 66.7% were not keen to consider using hearing aid, even if recommended. The willingness to use hearing aids was correlated to patients' functional status (p=0.002) but not to the severity of hearing impairment (p=0.157). CONCLUSION: Self-perception of hearing problems in the elderly is a strong indicator of hearing impairment. Introducing additional culturally-relevant questions based on activities of daily living improves the detection rate of hearing impairment. Although hearing loss impacts negatively on psychosocial well-being, most elderly subjects are unwilling to consider the use of hearing aids. There is a need to educate the elderly on the importance of intervention in order to reduce their handicap and improve their quality of life.
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Trastornos de la Audición/diagnóstico , Tamizaje Masivo/métodos , Encuestas y Cuestionarios , Anciano , Anciano de 80 o más Años , Actitud Frente a la Salud , Estudios de Cohortes , Femenino , Audífonos/psicología , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , SingapurRESUMEN
Like most cutting edge medical technology, human stem cell research raises a number of difficult and important ethical issues and concerns, requiring potential benefits to be balanced against the need to protect the rights and welfare of citizens. Much of the debate involves research using embryonic stem (ES) cells, which in turns revolves around the moral status of the human embryo, and the level of respect and protection that should be accorded. This is an especially sensitive issue in pluralistic societies where different, if not conflicting, cultural and religious perspectives exist. Another contentious issue as far as the derivation of ES cells is concerned is the intent involved in producing the embryos, specifically whether it is ethically permissible to allowing embryos to be made solely for the purpose of research. These and several other relevant ethical issues will be discussed, including a comparison of guidelines and positions adopted in different countries.
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Investigaciones con Embriones/ética , Células Madre , HumanosRESUMEN
Information Technology (IT) has transformed the ways modern healthcare systems acquire, store, access and communicate medical information. These developments offer significant benefits to patients and healthcare providers, but they give rise to ethical and legal challenges in the protection of patient privacy and confidentiality. The traditional and humanistic concept of doctor-patient relationship is also under threat as IT is used to bypass the need for personal consultations. One effective approach to continue the use of IT in medicine while minimising its potential hazards is through legal reforms and setting public standards for accessibility and expression of patient autonomy. Ultimately, the role and limitations of IT as a tool to pursue the goals of medicine has to be carefully deliberated, clearly defined and judiciously delineated to ensure its effectiveness and safety.
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Confidencialidad/ética , Atención a la Salud/ética , Ética Médica , Internet/ética , HumanosRESUMEN
One of the consequences of the rapidly increasing numbers of dementia patients in Singapore will be the need for all clinicians (including non-psychiatrists) to be familiar with the fundamentals of how decision-making capacity should be assessed. The clinical settings when the need for such evaluations arise, often involve cognitively or emotionally impaired patients who are required to make treatment, placement, financial or testamentary decisions. The clinician must first diagnose the patient's psychopathology and then go onto testing the functional abilities involved in decision-making. These comprise (1) making and expressing a choice, (2) understanding the relevant information, (3) appreciating the relevance of the information to oneself and (4) reasoning with the given information. The eventual judgement of the patient's decision-making capacity involves the weighing of impairment noted in any of the four decisional abilities against the potential adverse consequences of abiding by the patient's decision. The ethical impulse underlying this manner of judgement balances the respecting of patient's autonomy with protecting the patient from harm. Given the relative complexity of the assessment process, there is merit in developing a semi-structured approach to the evaluation of patients' decision-making capabilities; such an approach can guide a wider group of clinicians and psychologists through the essential steps of the process and thus enable the assessment to be more thorough, as well as fairer, to the patient.
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Toma de Decisiones , Demencia/psicología , Competencia Mental , Demencia/diagnóstico , Humanos , Encuestas y CuestionariosRESUMEN
Singapore's greying population has led to an alarming increase in the prevalence of dementia, and inevitably to a rising number of dementia patients who lack competence and relatives. Although legislative provisions exist in the current Mental Disorders and Treatment Act (MDTA) for the appointment of committees of person and estate for incompetent patients, healthcare professionals continue to face challenging obstacles in their attempts to search for appropriate surrogate decision-makers for such patients. We illustrate, with 2 cases from our clinical experience, the ethical, legal and administrative tangles faced in our search for appropriate surrogate decision-makers. Specific problems, such as administrative ambiguities in applying the MDTA and the impracticalities of a rigorous judicial process in appointing surrogates, are discussed. We propose possible solutions how these obstacles can be effectively tackled, in particular adopting a radical shift from the present judiciary model to a clinical-based model of appointing surrogates for healthcare decisions. This clinical model is based on best interests considerations and emphasies peer review and a multidisciplinary consultative approach in determining the clinical merits of proposed treatment and its impact on quality of life. The ultimate aim of such a change is to facilitate timely medical care while ensuring ethical practices that respect the patient's dignity.
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Ética Clínica , Apoderado , Anciano , Familia , Femenino , Humanos , Consentimiento Informado/ética , Consentimiento Informado/legislación & jurisprudencia , Legislación Médica , Masculino , Grupo de Atención al Paciente , Apoderado/legislación & jurisprudencia , SingapurRESUMEN
For centuries, physicians have been allowed to interfere and overrule patient's preferences with the aim of securing patient benefit or preventing harm. With the radical rise in emphasis on individual control and freedom, medical paternalism no longer receives unquestioned acceptance by society as the dominant mode for decision-making in health care. But neither is a decision-making approach based on absolute patient autonomy a satisfactory one. A more ethical and effective approach is to enhance a patient's autonomy by advocating a medical beneficence that incorporates patients' values and perspectives. This can be achieved through a model for shared decision making, acknowledging that though the final choices reside ultimately in patients, only through physician beneficence can the patient be empowered to make meaningful decisions that serve them best. For such a model to function effectively, the restoration of trust in doctor-patient relationship and the adoption of patient-centred communication are both crucial.
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Paternalismo , Relaciones Médico-Paciente , Autonomía Profesional , Toma de Decisiones , HumanosRESUMEN
INTRODUCTION: Delayed hospital discharge of elderly patients after an acute illness poses significant problems, including bed-blockade, iatrogenesis, and increase in morbidity, dependency and social isolation. The aim of this study was to determine the factors associated with delayed discharge from an acute geriatric unit. MATERIALS AND METHODS: This was a prospective cohort study involving 172 consecutive non-institutionalised patients, 65 years and older, admitted to an acute geriatric unit. The medical, functional, psychological and social variables of each subject were documented and any decline in functional status recorded. Based on the discharge status, patients were dichotomised into the "delayed" and "non-delayed" groups. Initial bivariate analyses of the variables were performed to select those associated with the "delayed discharge" group at the P < 0.05 levels. These variables were in turn entered into a multivariate logistic regression model to identify factors significantly associated with delayed discharge. RESULTS: Forty-eight patients (27.9%) had their discharge from the acute geriatric care unit delayed despite clinically fit to be so. Variables significantly associated with delayed discharge in the multivariate model were: 1) decline in basic activities of daily living (ADL) status [P < 0.001; adjusted odds ratio (AOR), 3.63; 95% confidence interval (CI), 1.33 to 9.86] and 2) need for skilled nursing [P = 0.012; AOR, 4.57; 95% CI, 1.88 to 11.14]. CONCLUSIONS: The study demonstrates the central importance of effectively identifying elderly patients who are at risk of delayed discharge after acute hospitalisation through a multidisciplinary focus on illness-induced functional decline. It is likely therefore that proactive approaches to rehabilitation and caregiver training, and adequate community-based services, may facilitate early and effective return of these patients to their homes, thereby reducing inappropriate use of acute beds.
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Anciano/fisiología , Enfermería Geriátrica/estadística & datos numéricos , Unidades Hospitalarias/estadística & datos numéricos , Alta del Paciente , Actividades Cotidianas , Anciano/psicología , Anciano de 80 o más Años , Ocupación de Camas , Femenino , Enfermería Geriátrica/normas , Humanos , Tiempo de Internación , Masculino , Oportunidad Relativa , Alta del Paciente/normas , Alta del Paciente/estadística & datos numéricos , Estudios Prospectivos , SingapurRESUMEN
The goals of medicine as a profession dedicated to healing and caring of the sick in a dignified manner depend very much on a stable and trusting doctor-patient relationship. In the last decade, rapid changes in the healthcare delivery system and socio-political climate have resulted in considerable strain on this relationship. What is needed is a reiteration of the fiduciary obligation of the physician and the central role of the patient in the relationship. Concrete steps and changes at the institutional and individual levels need to be taken to preserve the element of trust in the patient-physician covenant. Only then can the medical profession retain the moral dimension of its role in society.
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Atención Dirigida al Paciente/tendencias , Relaciones Médico-Paciente , Ética Clínica , Salud Holística , HumanosRESUMEN
There is a growing concern that, like in many developed countries, medical practice in Singapore is fast losing its role and status as a profession. The commodification and industrialisation of health care, and weakening of its ethical foundations are among the main forces threatening to deprofessionalise the practice of medicine. To overcome these challenges, an honest and introspective review of the goals of medicine and an affirmation of the ethical values of medicine are needed in order to reinstate the unique role of medicine in our society. Important steps to take include adopting a patient-centred philosophy and practice culture, promoting and emphasising ethical awareness and sensitivity among physicians, and active participation in constructive dialogues to negotiate the social contract of the profession. A more permanent impact may be achieved through cultivation of medical virtues in physicians, and the integration of core elements of medical professionalism into the ethical systems and mission statement of today's health care organisations.
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Ética Médica , Humanos , Relaciones Interprofesionales , Atención Dirigida al Paciente/normas , Relaciones Médico-Paciente , Ética Basada en PrincipiosRESUMEN
A prospective study was carried out to determine the prevalence of swallowing impairment and feeding dependency in an acute geriatric medicine unit in Singapore and the co-morbidity associated with it. A total of 211 patients were assessed over a 3-month period. Only 7.1% of patients gave a history of swallowing impairment prior to hospitalisation. We found that 29.4% of patients on admission and 28.2% on discharge had swallowing impairment, with a mortality of 8.1%. The prevalence of feeding dependency, as defined by the need for feeding assistance or tube feeding, was 26.5% before admission and 27.8% on discharge. The mode of feeding between the time of admission and discharge was changed in 14.9% of patients in response to the evolving medical condition. Swallowing impairment was significantly associated with the presence of dehydration (RR = 2.82, CI = 1.74-4.57), chest infection on admission (RR = 2.85, CI = 1.85-4.41), development of nosocomial chest infection (RR = 6.75, CI = 2.60-17.5), discharge to institutional care (RR = 2.8, CI = 1.51-3.47) and increased mortality (RR = 3.77, CI = 1.45-9.70). We concluded that swallowing impairment and feeding dependency are common in the elderly admitted to an acute geriatric unit. As elderly patients seldom inform clinicians of any underlying swallowing impairment and in view of the increased morbidity and mortality associated with this disability, it is important to screen for swallowing impairment. The high prevalence of feeding dependency adds to the burden of care in the ill elderly.
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Trastornos de Deglución/epidemiología , Hospitalización , Apoyo Nutricional/estadística & datos numéricos , Anciano , Anciano de 80 o más Años , Costo de Enfermedad , Trastornos de Deglución/diagnóstico , Conducta Alimentaria , Femenino , Humanos , Masculino , Prevalencia , Estudios Prospectivos , Factores de Riesgo , Singapur/epidemiologíaRESUMEN
This article familiarizes the reader with AIDS among Asians and Pacific Islanders (A&PIs) in New York City. The first section describes the epidemiology of AIDS among A&PIs in the United States and in New York City. In the United States 4,131 A&PIs have been diagnosed with AIDS. The age at diagnosis in the United States has been relatively stable over the past decade, although the proportion of diagnoses represented by older A&PIs (aged 40 to 49) has been increasing in New York City. The proportion of heterosexual diagnoses have been relatively stable over the past 6 years, whereas other racial groups have shown increases in the proportion of heterosexual transmissions. Data on defining opportunistic infections suggest that a different clinical pattern exists among A&PIs when compared with other racial groups diagnosed in New York City and the United States whereby A&PIs exhibit the highest proportion of Pneumocystis carinii pneumonia of all groups. The second section provides a discussion of some of the issues facing HIV positive A&PIs and their families in the process of accessing services at community-based HIV service organizations in New York City. One broad concept emerged from the client focus group that served to organize the analysis: family--a compelling case for HIV service organizations to initiate, expand, and maintain family services, however family is defined. The location of A&PI AIDS service organizations and its implications for recentering families in the caregiving process is discussed.
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Síndrome de Inmunodeficiencia Adquirida/etnología , Asiático , Necesidades y Demandas de Servicios de Salud , Infecciones Oportunistas Relacionadas con el SIDA/epidemiología , Infecciones Oportunistas Relacionadas con el SIDA/etnología , Síndrome de Inmunodeficiencia Adquirida/transmisión , Adolescente , Adulto , Anciano , Asia/etnología , Recolección de Datos , Familia , Femenino , Seropositividad para VIH/psicología , Humanos , Masculino , Persona de Mediana Edad , Ciudad de Nueva York/epidemiología , Islas del Pacífico/etnología , Apoyo Social , Estados Unidos/epidemiologíaRESUMEN
We have made antigen-specific cytotoxic reagents by conjugating the chimeric antibody BR96 (chiBR96) to Pseudomonas exotoxin A (PE), as either native PE or a truncated form (LysPE40) devoid of the cell-recognition region (domain I). PE kills cells by ADP-ribosylation of elongation factor 2, thereby inhibiting protein synthesis. Chimeric BR96 immunotoxins were constructed by chemical conjugation of the toxin to Fab', F(ab')2, and intact IgG and purified by anion-exchange and gel-filtration chromatography. Chimeric BR96 [IgG and F(ab')2] immunotoxins were cytotoxic against tumor cell lines displaying the BR96 antigen, with EC50 values ranging from 0.1 to 110 pM. Immunotoxins constructed with chiBR96 Fab' were 50-100-fold less cytotoxic. Competition analysis showed that the immunotoxins were specifically active through their BR96 antigen-binding ability. The binding of chiBR96-PE and chiBR96-LysPE40 to antigen was equivalent to that of BR96 itself and these immunotoxins were found to internalize very rapidly, displaying 90% of their cytotoxicity within 1 h. Binding assays determined that chiBR96 F(ab')2-LysPE40 bound as well as chiBR96-LysPE40; however, chiBR96 Fab'-LysPE40 bound 20-fold less efficiently. The chiBR96 Fab'-LysPE40 internalized similarly to the F(ab')2 or the IgG immunotoxins. Therefore, the chiBR96 Fab'-LysPE40 immunotoxin is less cytotoxic toward target cells because of reduced antigen binding. This is may be due to the monovalent nature of chiBR96 Fab'-LysPE40. This study shows that the monoclonal antibody chiBR96-Pseudomonas exotoxin A immunotoxins can be effective at inhibiting protein synthesis in target cells.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/química , Exotoxinas/toxicidad , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/química , Inmunotoxinas/toxicidad , Pseudomonas , Animales , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Unión Competitiva/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Exotoxinas/química , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/inmunología , Inmunotoxinas/química , Inmunotoxinas/inmunología , Indicadores y Reactivos , Ratones , Proteínas de Neoplasias/biosíntesis , PlásmidosRESUMEN
The effects of pH on the intrinsic fluorescence of purified human erythrocyte glucose transporter (HEGT) were studied to deduce the structure and the ligand-induced dynamics of this protein. D-Glucose increases tryptophan fluorescence of HEGT at a 320-nm peak with a concomitant reduction in a 350-nm peak, suggesting that glucose shifts a tryptophan residue from a polar to a nonpolar environment. Cytochalasin B or forskolin, on the other hand, only produces a reduction at the 350-nm peak. The pH titration of the intrinsic fluorescence of HEGT revealed that at least two tryptophan residues are quenched, one with a pKa of 5.5, the other with a pKa of 8.2, indicating involvement of histidine and cysteine protonation, respectively. D-Glucose abolishes both of these quenchings. Cytochalasin B or forskolin, on the other hand, abolishes the histidine quenching but not the cysteine quenching and induces a new pH quenching with a pKa of about 4, implicating involvement of a carboxyl group. These results, together with the known primary structure and the transmembrane disposition of this protein, predict the dynamic interactions between Trp388 and His337, Trp412 and Cys347, and Trp412 and Glu380, depending on liganded state of HEGT, and suggest the importance of the transmembrane helices 9, 10, and 11 in transport function.
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Eritrocitos/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Secuencia de Aminoácidos , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Conformación Proteica , Espectrometría de Fluorescencia , Triptófano/metabolismoRESUMEN
The secondary structural compositions of the human erythrocyte glucose transporter in proteoliposome vesicles were assessed on the basis of circular dichroism (CD) spectra measured in the absence and in the presence of D-glucose or an inhibitor, cytochalasin B. We designed and used a scattered-light-collecting device, which corrects CD spectra for optical artifacts originating from light scattering. Relative contents of eight types of secondary structure were estimated by using basis spectra generated by the eigenvector method based on CD spectra of 15 proteins of known structure. Results indicate that the glucose transporter is composed of approximately 82% alpha-helices, 10% beta-turns, and 8% other random structure, with no beta-strands. In the presence of an excess of D-glucose, the alpha-helical content is reduced by more than 10% and there is a significant increase in the random structure content. Cytochalasin B does not appear to affect the secondary structural composition of the transporter to any significant degree.
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Membrana Eritrocítica/metabolismo , Proteínas de Transporte de Monosacáridos/sangre , Proteolípidos , Dicroismo Circular , Citocalasina B/farmacología , Glucosa/farmacología , Humanos , Liposomas , Conformación Proteica , Espectrofotometría Ultravioleta/instrumentación , Espectrofotometría Ultravioleta/métodosRESUMEN
Hydrogen exchange kinetic behavior of human erythrocyte glucose transporter protein in vesicles was studied in the absence and in the presence of D-glucose or a well known inhibitor, cytochalasin B. This is to detect a proposed channel of water penetrating into the protein through which the sugar molecule passes and to monitor any conformational changes induced by the substrate or inhibitor. Analyses of the kinetic data revealed several classes of hydrogens which exchange with readily distinguishable rates. Of 660 hydrogens detected per transporter, approximately 30% exchanged with rates generally characterized as those of free amide hydrogens indicating they are interfaced to solvent water. Since the transporter is known to be embedded deep in the hydrophobic area of the membrane with minimum exposure to the outside of the membrane lipid bilayer, a significant portion of these free amide hydrogens must be at the purported channel rather than outside of the membrane. D-Glucose and cytochalasin B affected the exchange kinetics of these presumably channel-associated free amide hydrogens rather differently. D-Glucose reduced the apparent rate constants, but not the total number. Cytochalasin B on the other hand reduced the total number to one-half without significantly changing the apparent rate constants. The remaining 70% of the labeled hydrogens exchanged with much slower rates which vary 10-10,000-fold, indicating that they are internally structured peptide amide and side chain hydrogens. Both D-glucose and cytochalasin B further reduced the rates of these hydrogens, indicating a global stabilization of the protein structure.
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Citocalasina B/farmacología , Membrana Eritrocítica/análisis , Hidrógeno/sangre , Proteínas de Transporte de Monosacáridos/sangre , Deuterio , Glucosa/farmacología , Humanos , Cinética , Conformación Proteica , Espectrofotometría InfrarrojaRESUMEN
Chemical and functional purity of the human erythrocyte glucose transporter preparation obtained by DEAE column chromatography after octyl glucoside solubilization was assessed. The cytochalasin B binding capacity of the preparation indicates that the preparation is 60-85% functional glucose transporter. Gel filtration chromatography on TSK 250 column separates this preparation into at least three major peptide fractions, namely, P0, P1 and P2, with apparent Mr of approx. 80 000, 43 000 and 17 000, respectively. When the preparation is photolabelled with [3H]cytochalasin B prior to the separation only P0 and P1 are labelled. Exposure of the preparation to octyl glucoside or to ultraviolet light irradiation results in an increase in P0 in a time-dependent manner with a concomitant and proportional reduction in P1, without affecting P2 appreciably. For individual preparations, relative abundance of P0 and P1 vary widely in a reciprocal fashion, while that of P2 is practically fixed at approx. 10% of the total protein. The specific activity of cytochalasin B binding of each preparation correlates linearly with the relative abundance of P1 of the preparation, which gives a calculated specific binding activity of 22 nmol/mg protein for this fraction. These results indicate that P1 and P0 are native and denatured transporter, respectively, while P2 is contaminating protein impurities. These results demonstrate that the glucose transporter preparation contains approx. 10% of nontransporter protein impurities, with a varying amount (up to 30%) of denatured transporter, and that the transporter free of the chemical impurities and the denatured transporter can be obtained by a gel filtration chromatography of this preparation.