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OBJECTIVES: Breast cancer survivors have reported worse health-related quality of life (HRQoL) outcomes on some subscales when compared with members of the general population. However, the increased attention to breast cancer survivorship should have improved the HRQoL of these survivors. Our aim was to examine whether physical and mental component scores (PCS-12 and MCS-12) using the Short Form (SF-12) questionnaire were different for racial/ethnic minorities, specifically for Black and Hispanic women relative to White women. Furthermore, we stratified the data by age group to evaluate these racial/ethnic differences in HRQoL of breast cancer survivors. STUDY DESIGN: Cross-sectional study. METHODS: Pooled cross-sectional analyses using data from the Medical Expenditure Panel Survey between 2008 and 2016 were conducted. Pooled ordinary least squares (OLS) regression was used to examine the racial/ethnic differences in PCS-12 and MCS-12 scores of breast cancer survivors. Furthermore, stratified analyses by age group were conducted to evaluate racial/ethnic differences in HRQoL by the age of breast cancer survivors. RESULTS: After adjusting for confounders, there was no association between race/ethnicity and PCS-12 scores. However, Hispanic breast cancer survivors had statistically significantly lower MCS-12 scores (by 1.9 points [95% confidence interval {CI}: -3.53 to -0.37]) when compared with White breast cancer survivors. For PCS-12, after stratifying by age, the adjusted analyses showed no significant differences in PCS-12 scores when White female breast cancer survivors were compared with the other racial/ethnic categories. On the other hand, Black female survivors aged <50 years had 4.3 points (95% CI: 0.46-8.13) higher MCS-12 scores when compared with their White counterparts, while Hispanic breast cancer survivors aged <50 years had 3.1 points (95% CI: -0.40-6.69) higher MCS-12 scores relative to White women. Furthermore, among female breast cancer survivors aged ≥50 years, Hispanic women had 3.2 points (95% CI: -4.98 to -1.40) lower MCS-12 scores than White women. CONCLUSION: Our study generated findings showing the racial/ethnic differences in HRQoL of breast cancer survivors and presented results stratified by age group. These findings provide the much-needed rationale for targeted and racial/ethnic-specific HRQoL improvement strategies among breast cancer survivors.
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Neoplasias de la Mama , Supervivientes de Cáncer , Estudios Transversales , Femenino , Gastos en Salud , Humanos , Calidad de Vida , Encuestas y CuestionariosRESUMEN
Heart rate (HR) is a valuable and widespread measure for physical training programs, although its description of conditioning is limited to the cardiac response to exercise. More comprehensive measures of exercise adaptation include cardiac output (QÌ), stroke volume (SV) and oxygen uptake (VÌO2), but these physiological parameters can be measured only with cumbersome equipment installed in clinical settings. In this work, we explore the ability of pulse transit time (PTT) to represent a valuable pairing with HR for indirectly estimating QÌ, SV and VÌO2 non-invasively. PTT was measured as the time interval between the peak of the electrocardiographic (ECG) R-wave and the onset of the photoplethysmography (PPG) waveform at the periphery (i.e. fingertip) with a portable sensor. Fifteen healthy young subjects underwent a graded incremental cycling protocol after which HR and PTT were correlated with QÌ, SV and VÌO2 using linear mixed models. The addition of PTT significantly improved the modeling of QÌ, SV and VÌO2 at the individual level ([Formula: see text] for SV, 0.548 for QÌ, and 0.771 for VÌO2) compared to predictive models based solely on HR ([Formula: see text] for SV, 0.503 for QÌ, and 0.745 for VÌO2). While challenges in sensitivity and artifact rejection exist, combining PTT with HR holds potential for development of novel wearable sensors that provide exercise assessment largely superior to HR monitors.
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Ejercicio Físico/fisiología , Oxígeno/metabolismo , Fotopletismografía , Análisis de la Onda del Pulso , Volumen Sistólico , Adulto , Transporte Biológico , Femenino , Voluntarios Sanos , Humanos , MasculinoRESUMEN
Fetal heart rate variability (fHRV) is useful for noninvasive assessment of the status of the autonomic nervous system of the developing fetus. In this pilot study we acquired fetal magnetocardiograms (fMCG) in a magnetically shielded environment. Each recording was of 5-minute duration and was subsequently repeated in a high-frequency noise environment to examine the feasibility of conducting future recordings in clinical environments that lack facilities for magnetic shielding. The fMCG (n=17) were recorded at 9 spatial locations above the pregnant abdomen at 26 to 35 weeks gestational age (GA) by a second-order SQUID gradiometer. The signal-to-noise was adequate for reliable QRS detection even in the noisy environment, especially for GA >/= 30. The total spectral power of the RR-series, as well as band powers at low (0.05 to 0.25 Hz) and high (0.25 to 1.00 Hz) frequencies independently exhibited an increasing trend with GA. There was no evidence of bias in spectral power due to lack of shielding. These results provide experimental evidence supporting further studies in magnetically unshielded environments and may have an important implication for future clinical use of fMCG in the assessment of fHRV.
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The heart rate variability of fetuses under stress from maternal uterine contractions conveys critical information to clinicians and also provides theoretical clues about heart rate regulatory mechanisms. According to the polyvagal theory, the deceleration of fetal heart rate under stress is caused by the withdrawal of vagal tone. Recovery is mediated by its reestablishment. An implication of this mechanism is that the respiratory sinus arrhythmia (RSA) is elevated after the deceleration and subsequent recovery. Findings from our clinical data on fetuses support this theory. The data also indicate that in the instance of a late deceleration the RSA may not be elevated after recovery.
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Fetal magnetocardiography is a useful technique for non-invasive diagnostics of the electrophysiological activity of the fetal heart. The purpose of this study was to acquire fetal magnetocardiograms (FMCG) in a magnetically shielded environment as well as in a high-frequency noise environment, implement a QRS detection algorithm, and evaluate its performance. The FMCG were recorded over a period of 5 minutes at several sensor locations above the pregnant abdomen by second-order SQUID gradiometers. Quantitative analysis of FMCG with normal pregnancies and gestational ages 26-35 weeks showed improved QRS detection performance with signal pre-processing and parameter tuning.
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OBJECTIVE: To assess the relation between usual nutrient intake and subsequently diagnosed age-related nuclear lens opacities. SUBJECTS: Four hundred seventy-eight nondiabetic women aged 53 to 73 years from the Boston, Mass, area without previously diagnosed cataracts sampled from the Nurses' Health Study cohort. METHODS: Usual nutrient intake was calculated as the average intake from 5 food frequency questionnaires that were collected during a 13- to 15-year period before the evaluation of lens opacities. The duration of vitamin supplement use was determined from 7 questionnaires collected during this same period. We defined nuclear opacities as a nuclear opalescence grade of 2.5 or higher using the Lens Opacification Classification System III. RESULTS: The prevalence of nuclear opacification was significantly lower in the highest nutrient intake quintile category relative to the lowest quintile category for vitamin C (P<.001), vitamin E (P =.02), riboflavin (P =.005), folate (P =.009), beta-carotene (P =.04), and lutein/zeaxanthin (P =.03). After adjustment for other nutrients, only vitamin C intake remained significantly associated (P =.003 for trend) with the prevalence of nuclear opacities. The prevalence of nuclear opacities was significantly lower (P<.001) in the highest vitamin C intake quintile category relative to the lowest quintile category (odds ratio, 0.31; 95% confidence interval, 0.16-0.58). There were also statistically significant trends of decreasing prevalence of nuclear opacities with increasing duration of use of vitamin C (P =.004 for trend), vitamin E (P =.03 for trend), and multivitamin (P =.04 for trend) supplements, but only duration of vitamin C supplement use remained significantly associated with nuclear opacities after mutual adjustment for use of vitamin E (P =.05 for trend) or multivitamin (P =.02 for trend) supplements. The prevalence of nuclear opacities was significantly lower (P =.004) for women who used a vitamin C supplement for 10 or more years relative to women who never used vitamin C supplements (odds ratio, 0.36; 95% confidence interval, 0.18-0.72). Plasma measures of vitamins C and E taken at the eye examination were also inversely associated with the prevalence of nuclear opacities. CONCLUSION: These results provide additional evidence that antioxidant nutrients play a role in the prevention of age-related nuclear lens opacities.
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Envejecimiento/efectos de los fármacos , Antioxidantes/administración & dosificación , Ácido Ascórbico/administración & dosificación , Catarata/prevención & control , Encuestas sobre Dietas , Núcleo del Cristalino/efectos de los fármacos , Anciano , Ácido Ascórbico/sangre , Catarata/sangre , Catarata/epidemiología , Estudios de Cohortes , Registros de Dieta , Conducta Alimentaria , Femenino , Ácido Fólico/administración & dosificación , Humanos , Luteína/administración & dosificación , Persona de Mediana Edad , Oportunidad Relativa , Prevalencia , Riboflavina/administración & dosificación , Vitamina E/administración & dosificación , Salud de la Mujer , Xantófilas , Zeaxantinas , beta Caroteno/administración & dosificación , beta Caroteno/análogos & derivadosRESUMEN
Dietary restriction can effectively extend lifespan and retard many age-related debilities. One hypothesis to explain the beneficial effects of dietary restriction is that it prolongs maintenance of cellular homeostasis by limiting endogenous oxidative stress and preserves oxidative defense mechanisms during aging. Ascorbate, a primary antioxidant, may play a major role in preventing oxidative damage. Ascorbate levels were determined in dietary restricted (R) and control (C) Emory mice, a strain which develops age-related cataract due in part to oxidative damage to lens proteins. Mice which consumed a diet restricted by 40% in calories had lower ascorbate concentrations in plasma, liver and kidney. Nevertheless, R animals showed significantly delayed progression of cataract which extended over the entire second half of life. The R diet did not result in different ascorbate levels in this lens. Aging was associated with a decrease in ascorbate in all the examined tissues except lens of both the R and C groups. It is not clear from these data that ascorbate is a prominent factor in the delay of cataract formation or other debilities in R Emory mice. However, it also appears unlikely that lens ascorbate is cataractogenic.
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Ácido Ascórbico/metabolismo , Catarata/prevención & control , Dieta , Envejecimiento/metabolismo , Animales , Catarata/metabolismo , Cristalino/metabolismo , RatonesRESUMEN
The Emory mouse is the best model for age-related cataract. In this work we compare the effects of feeding a control diet (C) with a diet restricted (R) by 40% relative to C animals. In the R animals, median lifespan was extended by 40%. The proportion of R mice with advanced cataract was lower than C mice as early as 5 months of age. The mean grade of cataract was lower in R animals, beginning at 11 months and continuing until the end of the study. Ascorbate levels in R plasma and liver were 41-56% of C animals. There was no difference between diet groups with respect to lens ascorbate. Aging was associated with a decrease in ascorbate in lenses and kidneys in C and R mice. By 22 months, R animals had 48% higher liver glutathione levels than C mice. Liver glutathione levels were maximal at 12 months. Plasma glucose levels were > 27% lower in R animals at 6.5 and 22 months, and there was a 14% increase in glucose levels upon aging for both diet groups. In R mice, glycohemoglobin levels were 51% lower and tail collagen breaktime was decreased by 40%, even in younger animals. Collagen breaktime increased > 360% upon aging for both diet groups. Rates of production of urinary oxo8dG and oxo8G were higher in R animals compared with C animals, and increased upon aging. C animals exhibited more cancer and dermatological lesions, but less tail tip necrosis and inflamed genitals than R mice. These data allow evaluation of several theories of aging.
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Catarata/epidemiología , Ingestión de Energía , Longevidad , Animales , Ácido Ascórbico/metabolismo , Catarata/fisiopatología , Colágeno/metabolismo , ADN/metabolismo , Glucosa/metabolismo , Glutatión/metabolismo , Hemoglobina Glucada/metabolismo , Ratones , Ratones Endogámicos , Oxidación-Reducción , Prevalencia , ARN/metabolismo , Piel/patología , Cola (estructura animal)/metabolismoRESUMEN
A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein.
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Epítopos , Virus de la Hepatitis E/inmunología , Proteínas Virales de Fusión/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cobayas , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fragmentos de Péptidos/inmunología , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genéticaRESUMEN
Mucins present in the tracheobronchial secretions are responsible for the viscoelastic properties of the mucus. Any changes in the mucin structure may alter the physical properties of mucus and hence its function. Previous studies from this laboratory have reported the isolation and characterization of a major mucin component (HTM-1) and a minor, novel mucin component (HTM-2) from the tracheobronchial secretions of cystic fibrosis (CF) individuals. In the present study, the macromolecular properties of the CF mucin components HTM-1 and HTM-2 were further investigated using biophysical methods. Dynamic light scattering studies showed that CF HTM-1 and HTM-2 had a greater extended structure in buffer containing 0.10 and 0.15 M NaCl than that observed in the presence of 0.03 M NaCl. Also, CF HTM-1 had a compact configuration in the presence of 5 and 10 mM Ca2+, while under similar experimental conditions, the structure of CF HTM-2 was unaffected, indicating differences in the macromolecular properties of CF mucin components. Fluorescent probe binding studies revealed that CF HTM-1 had more hydrophobic probe binding domains than those observed for CF HTM-2. In summary, both biochemical and biophysical characterization suggests structural differences between the CF HTM-1 and HTM-2 components.
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Fibrosis Quística/metabolismo , Mucinas/química , Bronquios/metabolismo , Cloruro de Calcio , Colorantes Fluorescentes , Humanos , Luz , Mucinas/aislamiento & purificación , Mucinas/metabolismo , Naftalenosulfonatos , Dispersión de Radiación , Cloruro de Sodio , Tráquea/metabolismoRESUMEN
Five synthetic peptides were prepared based on the nucleotide sequence of open reading frames 2 and 3 encoded in the hepatitis E virus (HEV) genome and were used to develop an enzyme immunoassay (EIA) for the detection of anti-HEV activity in sera. Three different approaches were employed to ascertain the optimal preparation of these peptides as an immunodiagnostic reagent, including (1) a mixture of unconjugated peptides, (2) conjugating individual peptides to bovine serum albumin (BSA) followed by mixing each conjugate at various concentrations, and (3) mixing the peptides before conjugation with BSA to create an artificial antigen complex. The third method was superior in discriminating anti-HEV activity in sera previously tested by Western blot (WB). A frequency distribution of optical density values demonstrated that the peptide-based EIA was able to readily discriminate anti-HEV positive sera from sera devoid of anti-HEV activity. To confirm anti-HEV activity a neutralization test was developed using a mixture of 5 unconjugated peptides. With the exception of sera containing high levels of anti-HEV activity, all sera were neutralized greater than 50%. Strong sera required a higher dilution before a 50% neutralization was achieved. The sensitivity of the WB compared to EIA was 89.5% with and overall concordance of 94.8%. The peptide-EIA was used to determine anti-HEV activity in sera collected from various populations worldwide. In six outbreaks of ET-NANB hepatitis in various geographic regions, anti-HEV activity was demonstrated in 78-100% of cases. The peptide-EIA also detected anti-HEV activity in 14 out of 14 follow-up sera obtained 4-6 months after onset of disease and in 2 of 2 of these patients 5 yr after the acute episode. Anti-HEV activity was found in 8.5% of sera obtain from a healthy population residing in an HEV endemic region and 0.5% in two non-endemic regions (P < 0.001). These data demonstrate that a synthetic peptide-based EIA is sensitive for detecting anti-HEV activity in the sera of patients with acute hepatitis E, convalescents, and among healthy individuals.
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Anticuerpos Antihepatitis/aislamiento & purificación , Virus de la Hepatitis E/inmunología , Hepatitis E/microbiología , Técnicas para Inmunoenzimas , Enfermedad Aguda , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Western Blotting , Niño , Preescolar , Brotes de Enfermedades , Hepatitis E/sangre , Hepatitis E/epidemiología , Hepatitis E/inmunología , Humanos , Lactante , Persona de Mediana Edad , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Sensibilidad y EspecificidadRESUMEN
Restriction of dietary calorie intake is associated with life extension and with the delay of age-related disorders. Preliminary studies demonstrated that by feeding the Emory mouse a diet restricted by 21% in calories cataract and insolubilization of protein could also be delayed. To observe the effects of calorie restriction over prolonged portions of adulthood, Emory mice were fed the control diet (C) or a diet restricted by 40% in calories (R). Feeding the R diet was associated with delayed formation or progress of cataract over virtually the entire second half of life. At 11 months of age, bilateral grade 5 cataracts were present in 17% and 2% of C and R lenses, respectively. At 22 months of age, bilateral grade 5 cataracts were present in 90% and 18% of C and R lenses, respectively. The distribution of alpha-, beta-, and gamma- crystallins in the water-soluble, urea-soluble, and SDS-soluble fractions indicates more similarities than differences between C and R lenses with a specific grade of cataract or of a given age. However, there were significant and abrupt (after grade 4 cataract) losses of particular gamma-crystallins; gamma-crystallins which were not prominent at earlier stages became the major gamma-crystallin moieties. Losses of alpha-crystallins were also noted upon cataract formation or aging in most of the fractions. Aggregates including gamma- and alpha-crystallins also accumulate faster in the C group.
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Catarata/metabolismo , Cristalinas/metabolismo , Dieta , Ingestión de Energía , Cristalino/metabolismo , Envejecimiento/fisiología , Animales , Catarata/etiología , Electroforesis en Gel de Poliacrilamida , Metabolismo Energético , Incidencia , Ratones , Ratones Mutantes , Dodecil Sulfato de Sodio , Solubilidad , Urea , AguaRESUMEN
OBJECTIVES: To determine whether common epitopes are shared by respiratory and reproductive tract mucins and to compare the expression of cross-reactive mucin subtypes in human endocervix and endometrium. DESIGN: An immunohistochemical study of mucin expression using a panel of monoclonal and polyclonal antimucin antibodies and timed endocervical and endometrial biopsies. SETTING: University of Oklahoma Health Sciences Center, a tertiary care referral center. PATIENTS: Twenty-eight women who underwent laparoscopy, laparotomy, or hysterectomy. MAIN OUTCOME MEASURES: The expression of human tracheal mucin subspecies (types I to V) in endocervix (n = 3) and endometria (n = 25). RESULTS: Of the five mucin subspecies, type I mucin was localized to the squamous epithelium of endocervix and both glands and stroma of endometrium. Both tissues failed to react with type II mucin. Type III mucin was localized to differentiated cells of the squamous epithelium of endocervix and the glandular endometrium. Type IV mucin was specific to endometrium and was localized both in endometrial glands and stroma with no reactivity with endocervix. Type V mucin was expressed in both cervical and endometrial stroma and glands. CONCLUSIONS: Human respiratory and reproductive tract mucins share common peptide and carbohydrate epitopes. Human endocervix and endometria express a unique pattern of mucin antigens. Because of their restricted specificity, these monoclonal antibodies could provide new tools to investigate normal and aberrant expression of reproductive tract mucin subtypes in tissues and secretions.
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Cuello del Útero/inmunología , Endometrio/inmunología , Epítopos/análisis , Mucinas/inmunología , Tráquea/inmunología , Adulto , Animales , Anticuerpos Monoclonales , Epitelio/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/inmunologíaRESUMEN
In a population of 188 nondiabetic patients with early cataracts or nuclear brunescence, we assessed the degree to which contrast sensitivity function (CSF) provided more information about a patient's visual disability than high contrast visual acuity measurements. Data collected included LOCS II cataract classification, Bailey-Lovie visual acuity (LogMAR score), Lotmar interferometric visual acuity (LI VA), and distance contrast sensitivity function (CSF) using the Vistech 6500. Generalized least squares regression models in which CS was the dependent variable and either LogMAR score or LI VA was among the independent variables were used to ascertain whether CSF provided additional information about visual disability to that provided by LogMAR score or LI VA. Contrast sensitivity function was decreased only by nuclear opalescence at high frequencies (12 to 18 cpd); for all other cataract types and nuclear color, CSF testing provided no more information about cataract-related visual loss than LI VA or LogMAR score. Measurement of CSF using the Vistech 6500 system in patients with early cataracts provides information on visual dysfunction beyond that provided by LogMAR score or LI VA only in patients with nuclear opalescence, and that may not be clinically significant.
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Catarata/fisiopatología , Sensibilidad de Contraste/fisiología , Agudeza Visual/fisiología , Adulto , Anciano , Catarata/clasificación , Femenino , Humanos , Cristalino/fisiopatología , Masculino , Persona de Mediana EdadRESUMEN
Contrast sensitivity function (CSF) was assessed in a population of diabetics with moderate cataracts to determine if CSF testing provides more information about visual dysfunction than Snellen or Lotmar interferometric visual acuity. With the Lens Opacities Classification Systems Version II (LOCS II) of cataract classification it was possible to grade accurately the type and severity of cataract and nuclear brunescence. The presence of statistically significant relationships between increasing LOCS II classification (worsening cataract) and diminished function, even when the regression model was controlled for Snellen visual acuity, supports the thesis that CSF measurements do provide more information about cataract related visual loss than Snellen acuity alone. Statistically significant (p < or = 0.05) relationships existed between different morphological types of cataract, nuclear colour, and CSF at specific frequencies. The frequencies affected differed with cataract type or nuclear colour, and with distance and near CSF.
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Catarata/fisiopatología , Sensibilidad de Contraste , Diabetes Mellitus/fisiopatología , Adulto , Factores de Edad , Anciano , Percepción de Color , Complicaciones de la Diabetes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores Sexuales , Agudeza VisualRESUMEN
Acetone is one of the most commonly used industrial solvents. Recent literature indicated that in guinea pigs, but not rabbits, acetone is cataractogenic and that elevated acetone exposure is also associated with depressed aqueous ascorbate levels. Other work indicated that aqueous and lens levels of ascorbate are closely linked and that depressed ascorbate status is related to cataract. Taken together, these papers suggested that acetone exposure, depressed ascorbate levels, and cataract are related, possibly causally. While the possibility that acetone is cataractogenic presented a major health concern, it also presented an opportunity to develop a new model of cataract in which hypotheses regarding anticataractogenic effects of ascorbate could be tested. Albino hairless guinea pigs are immunocompetent animals derived from albino Hartley guinea pigs. Animals were fed diets containing low (4.9 mg/day) and high (55 mg/day) levels of ascorbate. This resulted in distinct groups of animals, one with high tissue ascorbate levels and the other with low, but nonscorbutic ascorbate levels. The tissue levels of ascorbate and the relationship between tissue ascorbate levels and dietary intake indicate that with respect to ascorbate uptake, transport, and concentration, these animals are identical to the standard albino Hartley animals. Daily exposure to acetone was extended for 6 months, with a total applied dose of 65 ml. Absorption of the solvent was maximized by the use of hairless animals. Despite exposure of the animals to higher levels of acetone, in no case (n = 20) were cataracts observed over a 2-year period. This is consistent with results using rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)
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Acetona/toxicidad , Ácido Ascórbico/farmacocinética , Catarata/inducido químicamente , Catarata/metabolismo , Administración Tópica , Animales , Disponibilidad Biológica , Dieta , Cobayas , Cristalino/metabolismoRESUMEN
A sensitive, specific procedure was developed for detecting Escherichia coli O157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37 degrees C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli O157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes O157:H7 and O26:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli O157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli O157:H7. Most-probable-number determinations revealed E. coli O157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.
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Infecciones por Escherichia coli/microbiología , Escherichia coli/aislamiento & purificación , Microbiología de Alimentos , Hemorragia Gastrointestinal/microbiología , Animales , Anticuerpos Monoclonales , Bovinos , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Humanos , Carne/microbiología , Leche/microbiologíaRESUMEN
A minor mucin glycoprotein component (HTM-2) was purified from the tracheobronchial secretions of two cystic fibrosis patients using a protocol established in our laboratory. The secretions were solubilized in 0.1 M Tris-HCl buffer (pH 7.5) containing 0.22 M potassium thiocyanate and fractionated on a Bio-Gel A-5m column, followed by digestion with DNAase, rechromatography on the same column and chromatography on hydroxyapatite which resolved the major mucin (HTM-1) from the minor mucin component (HTM-2). The mucin component HTM-2 was further purified using Superose 6 chromatography. SDS-composite gel (2% polyacrylamide + 0.5% agarose) and 6% polyacrylamide gel electrophoresis showed that the purified HTM-2 was totally free of low-molecular-weight contaminants. Equilibrium density sedimentation centrifugation of purified HTM-2 using CsCl gradients also showed the absence of proteoglycans and other low-molecular-weight proteins. Comparison of carbohydrate and amino acid compositions of the two mucin components indicated that HTM-2 was quite different from the major mucin, HTM-1, reported earlier from our laboratory (Biochemistry, 24, 7334, 1985). This suggested that HTM-2 has a different polypeptide core and is perhaps a different gene product. The effects of 6 M guanidine-HCl and different concentrations of NaCl on the molecular size of HTM-2 and its ability to form aggregates was also investigated using the technique of static light scattering. In buffer containing 6 M guanidine-HCl, HTM-2 had a weight-average molecular weight of approximately 4.5 x 10(6). However, in the presence of buffer containing 0.03, 0.10 or 0.15 M NaCl, the molecular weight of HTM-2 was estimated to be approximately 11 x 10(6). These data suggest aggregation of HTM-2 in the presence of a range of NaCl concentrations. In contrast to HTM-1, which is a more anionic glycoprotein, the apparent molecular size of HTM-2 did not decrease at the higher NaCl concentration.
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Bronquios/química , Fibrosis Quística/metabolismo , Mucinas/análisis , Tráquea/química , Aminoácidos/análisis , Humanos , Peso Molecular , Cloruro de Sodio/farmacologíaRESUMEN
A monoclonal antibody (MAb 4E8C12) specific for Escherichia coli O157:H7 and O26:H11 was produced by immunizing BALB/c mice with a rough strain of E. coli O157:H7. The antibody reacted strongly by a direct enzyme-linked immunosorbent assay with each of 36 strains of E. coli O157:H7. No cross-reactivity was observed with strains of Salmonella spp., Yersinia enterocolitica, Shigella dysenteriae, Proteus spp., Escherichia hermanii, Klebsiella pneumoniae, Campylobacter jejuni, Serratia marcescens, Citrobacter spp., Enterobacter cloacae, Hafnia alvei, Aeromonas hydrophila, and all except five strains of E. coli other than serotype O157:H7 (including strains of serotype O157 but not H7). The E. coli strains (all of serotype O26:H11) that reacted with the antibody were enterohemorrhagic E. coli (EHEC) that were isolated from patients with hemolytic uremic syndrome or hemorrhagic colitis and produced verotoxin similar to that of E. coli O157:H7. MAb 4E8C12 belongs to the subclass immunoglobulin G2a and has a kappa light chain. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of E. coli of different serotypes followed by Western immunoblot analysis revealed that MAb 4E8C12 reacted specifically with two proteins of EHEC strains of serotypes O157:H7 and O26:H11 with apparent molecular weights of 5,000 to 6,000. These proteins appeared to be markers specific for EHEC strains of serotypes O157:H7 and O26:H11. This MAb, because of its specificity, may be a useful reagent of an immunoassay for the rapid detection of these types of EHEC isolates in clinical and food specimens.