RESUMO
Bromosulphophthalein and N-ethylmaleimide, inhibitors of glutathione S-transferase (EC 2.5.1.18 RX: glutathione R. transferase), have been used to identify variant forms of the erythrocyte enzyme. One 'atypical' sample was detected and was shown to have appreciably different kinetic and stability properties. These inhibitors may be useful in surveys of variation in this group of enzymes.
Assuntos
Eritrócitos/enzimologia , Glutationa Transferase/metabolismo , Adulto , Etilmaleimida/farmacologia , Feminino , Glutationa Transferase/antagonistas & inibidores , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Cinética , Masculino , Sulfobromoftaleína/farmacologiaRESUMO
The developmental expression of Cu,Zn superoxide dismutase in human lung and erythrocytes has been studied using activity measurements, immunoblotting and immunohistochemistry. Enzyme activity in erythrocytes increased significantly during gestation but no developmental trend was seen in lung. Immunoblotting identified a single enzyme form that was present in a variety of tissues and immunohistochemistry showed the enzyme to have widespread distribution in lung tissue. These data indicate that Cu,Zn superoxide dismutase is consistently expressed during human development and that, unlike in other species, no late-fetal surge in expression occurs.
Assuntos
Eritrócitos/enzimologia , Pulmão/embriologia , Superóxido Dismutase/metabolismo , Citosol/enzimologia , Feto , Idade Gestacional , Histocitoquímica , Humanos , Soros Imunes , Técnicas Imunoenzimáticas , Pulmão/citologia , Pulmão/enzimologia , Superóxido Dismutase/sangueRESUMO
The developmental expression of the basic, near-neutral and acidic isoenzymes of glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) has been studied in heart and diaphragm. Neither these enzymes nor the putative muscle-specific GST4 isoenzyme demonstrated any developmental trends in expression. In vitro hybridisation and SDS-discontinuous polyacrylamide gel electrophoresis were used to show that the GST4 isoenzyme is a homodimer composed of monomers that have a slightly larger molecular weight than the near-neutral isoenzyme. The sensitivity of GST4 to inhibitors also appeared similar to that of the GST1 2 isoenzyme. Immunodiffusion and immunoblotting techniques were used to show that the acidic enzyme in muscle is immunologically identical to that in other tissues.
Assuntos
Diafragma/crescimento & desenvolvimento , Feto/enzimologia , Glutationa Transferase/metabolismo , Coração/crescimento & desenvolvimento , Isoenzimas/metabolismo , Desenvolvimento Muscular , Miocárdio/enzimologia , Cromatografia , Citosol/enzimologia , Diafragma/embriologia , Diafragma/enzimologia , Eletroforese em Gel de Poliacrilamida , Idade Gestacional , Glutationa Transferase/antagonistas & inibidores , Coração/embriologia , Humanos , Concentração de Íons de Hidrogênio , Imunoensaio , Imunodifusão , Lactente , Recém-Nascido , Isoenzimas/antagonistas & inibidoresRESUMO
Adsorption of 125I-labelled rabbit IgG to plastics (cellulose nitrate, polyallomer, polystyrene, polyvinyl) was primarily dependent on initial antibody concentration and, to a lesser extent, on time allowed for adsorption. The highest concentration tested (100 mug/ml) gave the highest quantities adsorbed after 18 h at room temperature. This concentration, however, gave the lowest percent adsorption (6.5 to 12.0%) of the initial amount. IgG concentration of 10 mug/ml resulted in 25.0 to 65.1% adsorption over the same time period; at 1 mug/ml, 47.0 to 96.6% of the initial amount was adsorbed. All of the plastics tested adsorbed IgG to approximately the same degree, with the exception of cellulose nitrate. This plastic adsorbed 32 to 49% less than the others, under maximal adsorption conditions.
Assuntos
Imunoglobulinas , Plásticos , Adsorção , Animais , Celulose , Técnicas Imunológicas , Coelhos , RadioimunoensaioRESUMO
Certain serum samples produce high background in Western and direct immunoblot assays that detect human serum IgG against specific antigens. We determined that this was due to a reaction between endogenous IgG and the membrane blocking agent (we refer to this as blocking-specific background). Using milk as blocking agent, we screened 107 sera by Western immunoblot or checkerboard immunoblot assays, and found that 6.5% of sera had background intensities sufficient to interfere with the interpretation of final results. Blocking-specific background was also observed using bovine serum albumin and other animal protein-based blocking agents. As the primary antibody in these immunoblot assays was human IgG, we investigated human serum albumin as a blocking agent; this approach eliminated the problem of blocking-specific background.
Assuntos
Immunoblotting/métodos , Imunoglobulina G/análise , Coloração e Rotulagem , Reações Cruzadas , Proteínas do Leite , Albumina Sérica/química , Soroalbumina Bovina/químicaRESUMO
Human Factor VIII associated von Willebrand factor (VIII:vWF) binds to human platelets in vitro only in the presence of a mediator such as ristocetin, thrombin or ADP. Studies reported here were designed to determine if human platelets will adhere to solid-phase VIII:vWF. Human VIII:vWF was purified from a phosphate precipitate of A1(OH)3 absorbed plasma using 4% agarose and DEAE cellulose. Purified VIII:vWF (90 units of VIII:vWF activity/mg) was coated on dialysis membranes using ultrafiltration (final concentration of 0.4 units/cm2). Membranes (0.5 cm2) were held stationary in human citrated PRP suspension or washed platelet suspensions and stirred continuously for 5 minutes at 37 degrees C. The membranes were then rinsed in phosphate buffered saline, fixed, stained, and examined by light and scanning electron microscopy. Abundant normal platelets adhered to VIII:vWF-coated membranes, while minimal adhesion was seen on uncoated membranes and membranes coated with albumin. Adhesion occurred without ristocetin, thrombin, ADP or other agonist and in the presence of Ca+2/Mg+2 ions. Preincubation of the VIII:vWF coated membranes with monospecific rabbit anti-VIII:vWF inhibited the adhesion reaction. However, preincubation of VIII:vWF coated membranes with naturally occurring human anti-FVIIIc antibodies failed to interfere with platelet adhesion. Platelets from a patient with Bernard-Soulier Syndrome (BSS) which did not bind human VIII:vWF in the presence of ristocetin or aggregate with bovine cryoprecipitate also did not adhere to VIII:vWF-coated membranes.
Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Fator VIII/metabolismo , Adesividade Plaquetária , Trombocitemia Essencial/sangue , Fator de von Willebrand/metabolismo , Humanos , SíndromeRESUMO
In this paper, we develop a method to predict the energy deposition in tumours undergoing ultrasound-hyperthermia treatments. Energy depositions are calculated using an exact, analytic solution to the problem of ultrasound scattering from a spherical tumour. The biological tissues are modelled as solid, lossy elastic media so that (i) transverse-wave modes, in addition to longitudinal-wave modes, are considered, and (ii) mode coupling is fully accounted for during the scattering. The model tumour is of arbitrary size and no restrictions are placed on its material parameters relative to the surrounding tissue. Simpler analytic results are given for tumours that differ in densities and rigidities only slightly from the surrounding tissue. We briefly discuss how the above analysis could be extended to more complex systems, i.e., irregularly shaped tumours.
Assuntos
Hipertermia Induzida , Modelos Teóricos , Neoplasias/terapia , Terapia por Ultrassom , Humanos , Cinética , Matemática , Neoplasias/fisiopatologia , Espalhamento de RadiaçãoRESUMO
Against a background of growing interest in more sensitive assays for quantifying various acute phase proteins, we evaluated the performance of recently developed tests for C-reactive protein (CRP), serum amyloid A (SAA) and mannose-binding protein (MBP) on the Behring nephelometer II (BNII). Sample results outside the calibration ranges of 3.5 to 220 mg/L for CRP, 3.3 to 215 mg/L for SAA and 0.09 to 5.6 mg/L for MBP were automatically re-measured at another dilution. The lower limits of detection were 0.01, 0.7 and 0.01 mg/L for CRP, SAA and MBP, respectively. The coefficients of variation (CV) for intra- (n > or = 20) and inter- (n > or = 15) assay precision were < 5.2% and < 8.5%, respectively, for the three proteins at concentrations representing low, normal and high. Linearity for each method was within 5% of the expected values throughout the calibration range. We observed no significant interference from bilirubin (up to 300 mg/L) or haemoglobin (up to 10 g/ L) for the three tests. Method comparison studies performed for CRP and SAA yielded the following results: y (CRP on BNII) = 0.75x (ELISA, Hemagen) -0.25 mg/L (r = 0.981, Sy/x = 2.1 mg/L; y (SAA on BNII) = 1.44x (ELISA, Hemagen) -9.9 mg/L (r = 0.972, Sy/x = 6.9 mg/L), where ELISA is enzyme-linked immunosorbent assay. Reference intervals established in 261 adult blood donors (aged 36.2 +/- 9.0 years) were found to be log-normal with 2.5th, 50th, and 97.5th centiles of < 0.17, 1.00 and 10.1 mg/L for CRP, < 0.84, 2.10 and 9.70 mg/L for SAA; and 0.30, 1.28 and 4.10 mg/L for MBP. We observed no relationship with CRP concentration and age; however, SAA levels increased with age while MBP levels decreased. The BNII provides a simple, rapid and sensitive system for measuring CRP, SAA and MBP in human serum.
Assuntos
Apolipoproteínas/análise , Proteína C-Reativa/análise , Proteínas de Transporte/sangue , Nefelometria e Turbidimetria/métodos , Proteína Amiloide A Sérica/análise , Adulto , Ensaio de Imunoadsorção Enzimática , Estudos de Avaliação como Assunto , Humanos , Soros Imunes , Lectinas de Ligação a Manose , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Young physicians represent a sizeable constituency of the medical professions, but they are facing increased socioeconomic pressures. The large number of women physicians continues to increase, thus changing the demographics of the young physician population. Rising economic pressures have become harsh realities for all of them. Increasing medical school debts, the cost of establishing and building a practice, and heightening competition resulting from an increased supply of physicians are all factors that influence their practice decisions. It has become impossible for young physicians to claim, "All I want to do is practice medicine."
Assuntos
Médicos/estatística & dados numéricos , Administração da Prática Médica , Adulto , Escolha da Profissão , Coleta de Dados , Emprego/estatística & dados numéricos , Previsões , Humanos , Renda , Fatores Socioeconômicos , Estados UnidosAssuntos
Assistência Integral à Saúde , Continuidade da Assistência ao Paciente , Dietética , Idoso , Serviços de Saúde Comunitária , Diabetes Mellitus/dietoterapia , Feminino , Insuficiência Cardíaca/dietoterapia , Humanos , Falência Renal Crônica/dietoterapia , Massachusetts , Casas de Saúde , Fenômenos Fisiológicos da Nutrição , Equipe de Assistência ao Paciente , Instituições de Cuidados Especializados de EnfermagemAssuntos
Biologia Celular , Crescimento , Animais , Diferenciação Celular , Membrana Celular/análise , Movimento Celular , MorfogêneseAssuntos
Catolicismo , Hospitais Religiosos/normas , Assistência Centrada no Paciente , Altruísmo , Capitação , Ética Institucional , Humanos , Cobertura do Seguro , Liderança , Programas de Assistência Gerenciada/economia , Programas de Assistência Gerenciada/organização & administração , Programas de Assistência Gerenciada/tendências , Política Organizacional , Estados UnidosRESUMO
Bacterial endotoxins (lipopolysaccharides) were found to suppress X-ray-induced malignant transformation of C3H/10T1/2 cells. Endotoxins were effective if present either throughout the 6-week transformation assay period, or for the final 4-week phase, but not when present only for the initial 2-week phase. Neither growth nor survival of C3H/10T1/2 cells, or a radiation-transformed cell line derived from them, were affected by endotoxins. Also, the endotoxins did not affect the formation of foci by the radiation transformed cells when these cells were co-cultured with untransformed cells. These results suggest that endotoxins exert their effect directly upon the transformation process itself, perhaps at a 'late' step in the conversion of an untransformed to a transformed cell.
Assuntos
Transformação Celular Neoplásica/efeitos da radiação , Endotoxinas/farmacologia , Animais , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Relação Dose-Resposta a Droga , CamundongosRESUMO
Peroxidase-conjugated antibodies were found to be as sensitive as those conjugated to fluorescein-isothiocyanate (FITC) for identification of selected enterovirus types by the indirect technique. Peroxidase conjugates, however, were found to give fewer nonspecific reactions. The reasons for the higher specificity of the immunoperoxidase technique appear to be related to the relative size, charge, and uniformity of the preparations. Monomers of peroxidase-conjugated globulin are larger than those of FITC conjugates, but the latter readily form aggregates. This was shown by chromatography on Sepharose 6B columns: various FITC-conjugated globulins eluted before those conjugated to peroxidase and before (125)I-labeled immunoglobulin G. The net charge of the conjugates was determined by adsorption to ion-exchange columns. FITC-labeled globulins had a negative net charge, eluting at pH 5.1 from diethylamino-ethyl-Sephadex A-50 columns. Peroxidase conjugates were not retained by either cationic or anionic exchangers at pH values ranging from 4.0 to 10.5. Further, the fluorescein/protein ratios of FITC conjugates from different commercial sources and of those prepared in the laboratory were found to be variable; higher fluorescein/protein ratios (>2:1) give a higher degree of nonspecific reactions, whereas the peroxidase/protein ratio does not appear to affect specificity. These characteristics of peroxidase conjugates make the immunoperoxidase technique easier to standardize and more reliable for enterovirus identification than the immunofluorescence technique.
Assuntos
Enterovirus/isolamento & purificação , Animais , Anticorpos Anti-Idiotípicos , Anticorpos Antivirais , Linhagem Celular , Enterovirus Humano B/isolamento & purificação , Imunofluorescência , Glutaral , Cabras/imunologia , Haplorrinos , Soros Imunes , Indicadores e Reagentes , Radioisótopos do Iodo , Rim , Peroxidases , Poliovirus/isolamento & purificação , Coelhos/imunologia , Sódio/metabolismo , Tensoativos , Cultura de VírusRESUMO
Rapidly rising health care costs, a growing surplus of physicians, and recent federal legislation have created a more competitive health care market and have led to rapid growth in the number of health maintenance organizations, or HMOs. HMOs may place psychiatrists and their patients at a disadvantage because they generally restrict coverage of mental illness, use mental health practitioners other than psychiatrists, and lack provision for treating chronic and indigent mentally ill patients. In this review article, the authors describe the characteristics of HMOs, review the historical, legislative, and economic forces that have contributed and will contribute to their explosive growth, and outline measures that psychiatrists must take to ensure that mental health care is not compromised in HMOs and other price-competitive health care delivery systems.
Assuntos
Sistemas Pré-Pagos de Saúde/tendências , Serviços de Saúde Mental/organização & administração , Controle de Custos/tendências , Atenção à Saúde/tendências , Previsões , Psiquiatria , Qualidade da Assistência à Saúde/tendências , Estados UnidosRESUMO
Recent guidelines established by the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and the U.S. Centers for Disease Control and Prevention (CDC) recommend the use of a two-test protocol for the serologic diagnosis of Lyme disease (LD). The two-test protocol relies on a sensitive screening test, which is followed by specific immunoglobulin M (IgM) and/or IgG immunoblotting (IB), depending on the date of disease onset, of all samples with equivocal and positive screening test results. We evaluated a commercially available IgM-IgG enzyme-linked immunosorbent assay (ELISA) and separate IB tests for IgM and IgG antibodies to Borrelia burgdorferi as candidate assays for the two-test protocol. Serum samples obtained from healthy controls (n = 29), from patients with diagnoses or laboratory findings associated with serologic cross-reactivity to LD (n = 24), and from patients with well-documented early- and late-stage LD provided by the CDC and the College of American Pathologists (n = 53) were examined to determine each assay's individual sensitivity and specificity. No false-positive results were detected among the healthy controls by either ELISA or IB, whereas four false-positive ELISA results were recorded within the cross-reactive group. None of these sera, however, were positive for either IgM or IgG reactivity according to IB band criteria. With regard to the patients with LD, we determined the sensitivity and specificity of the ELISA to be 96 and 100%, respectively, compared with the reference data provided for these specimens. When we compared our IB results with data from CDC, the assay sensitivity and specificity were 80 and 96.2%, respectively, for IgM and 81.8 and 95.8%, respectively, for IgG. Pursuant to this evaluation we assessed the suitability of the two-test protocol by performing a retrospective analysis using clinical history to define samples as positive or negative for LD. We determined clinical sensitivity and specificity for all study subjects (n = 112) to be 50 and 100%, respectively. A reduction in the clinical sensitivity of the two-test protocol was associated with a lack of antibody response or seroconversion in LD patients treated with antibiotics. We conclude that the CDC-ASTPHLD guidelines provide useful criteria for test performance and interpretation aimed at standardizing the serologic diagnosis of LD.
Assuntos
Doença de Lyme/diagnóstico , Guias de Prática Clínica como Assunto , Testes Sorológicos/normas , Grupo Borrelia Burgdorferi/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Doença de Lyme/sangueRESUMO
A quantitative enzyme-linked immunosorbent assay was used for identification of selected enteroviruses: poliovirus type 1, echovirus type 6, coxsackievirus A type 9, and coxsackievirus B types 1 through 6. Partially purified viral antigens or virus-specific antibodies were adsorbed to polystyrene spectrophotometer cuvettes, which permitted the assays to be reported and compared in terms of enzyme units specifically reacting. Both the adsorbed antigen and the adsorbed antibody methods were approximately equal in terms of sensitivity and specificity of reaction. By use [14C]leucine-labeled enteroviruses, the amount of virus that bound to the plastics used was shown to be dependent on the purity of the virus preparation used, but it was higher than the amount that was bound by plastics coated with viral antibody. Diluents which contained 0.15% (vol/vol) Tween 20 and 2.0% (wt/vol) bovine serum albumin in phosphate-buffered saline, pH 7.2, were found to be the most effective in inhibiting nonspecific adsorption of immunoreagents. However, the presence of these inhibitors in phosphate-buffered saline solutions also caused desorption of virus or viral antibody during immunoassays; the amount of virus desorption varied with the type of preparation used, and antibody desorption was dependent on the concentration of antibody initially used for adsorption. For specific identification of a given enterovirus type by the enzyme-linked immunosorbent assay method used, approximately 10(5) plaque-forming units of virus per assay tube were required.
Assuntos
Enterovirus/classificação , Ensaio de Imunoadsorção Enzimática/métodos , Técnicas Imunoenzimáticas/métodos , Echovirus 6 Humano/classificação , Enterovirus Humano B/classificação , Poliovirus/classificaçãoRESUMO
OBJECTIVE: To measure detection of clinical benign prostatic hyperplasia (BPH) in a general medicine practice. DESIGN: Self-administered questionnaire and retrospective ambulatory medical record review. SETTING: Hospital-based general medicine practice. PATIENTS: Two hundred and four men aged 50 years and older. MEASUREMENTS AND MAIN RESULTS: Clinical information was obtained from a self-administered questionnaire containing the American Urological Association symptom index and the BPH Impact Index bother scale, and from retrospective review of ambulatory medical records for the previous 24 months. Thirty percent of patients had moderate to severe urinary tract symptoms, and 67% of these individuals were bothered by the symptoms. Only 52% with moderate to severe symptoms recalled any discussion with their primary care physician about their symptoms. There was medical record documentation of a review of urinary tract symptoms in only 18% and a prostate examination in only 64%. Patients with more symptoms and bother tended to recall a discussion of urinary tract symptoms with their physician. However, moderate to severe symptoms and bother were not associated with increased documentation of a history of urinary tract symptoms or prostate examination. CONCLUSIONS: Clinical BPH was underdetected in a general medicine practice. Because many men do not complain to their physicians about urinary tract symptoms and reduced quality of life, perhaps primary care physicians should pay more attention to recognizing this common condition of older men.
Assuntos
Hiperplasia Prostática/diagnóstico , Medicina de Família e Comunidade , Humanos , Masculino , Auditoria Médica , Pessoa de Meia-Idade , Relações Médico-Paciente , Hiperplasia Prostática/complicações , Hiperplasia Prostática/epidemiologia , Estudos Retrospectivos , Inquéritos e Questionários , Transtornos Urinários/etiologiaRESUMO
The circadian spontaneous locomotor activity of rats exposed to 0.1 mg lead/kg, po from 3 days until 4 and 6 weeks of age was similar to that of controls. However, hyperactivity during initial hours of recording was observed in rats that were treated with lead (Pb) until 8 weeks of age. When treatment was discontinued for 2 weeks, previously Pb-exposed rats had a tendency to be hypoactive. The elevated locomotor activity in 8-week-old lead-treated rats was not accompanied by any significant changes of noradrenaline levels in the cerebral cortex or hippocampus. Alterations in noradrenaline content of the hippocampus were, however, observed in rats that had been treated with Pb for 4 and 6 weeks. The turnover rate of noradrenaline in the hippocampus was also found to be significantly reduced following treatment for 6 weeks. Regional distribution of Pb in the brains of lead-exposed rats showed a large accumulation of the metal in the hippocampus. The alterations of the noradrenergic function in the hippocampus may be associated with the preferential storage of lead in this region.