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BACKGROUND: We evaluate the affect on the hematocrit (Hct) drop and the amount of transfused red blood cells (RBCs) during cardiopulmonary bypass (CPB) in adult cardiac surgery patients due to minimizing the CPB circuit by using integrated components. METHODS: Two hundred and seventy-two patients were included in this retrospective audit. Patients were assigned to three cohorts: the first cohort consisted of patients operated on with a CPB circuit volume of 1630 ml in 2008; the second cohort of such patients in 2010, with 1380 ml; and the third cohort of such patients in 2011, with 1250 ml. RESULTS: There were no significant differences with respect to patient demographics. The priming volume was consecutively significantly reduced; (1635 ± 84 ml, 1384 ± 72 ml and 1256 ± 130 ml, p<0.0001). A trend of decreased amount of RBCs during CPB was visible (cohort 1630: 98 ± 195 ml, cohort 1380: 35 ± 151 ml and cohort 1250: 48 ± 113 ml, p=0.02). Also, the amount of RBCs during the total CPB procedure shows a decreased trend (cohort 1630: 122 ± 230 ml, cohort 1380: 52 ± 180 ml and cohort 1250: 71 ± 156 ml, p=0.04). Blood loss during CPB was significantly lower in cohorts 1380 and 1250 (1630: 922 ± 378 ml, 1380: 706 ± 347 ml and 1250: 708 ± 418 ml, p<0.0001). The volume of diuresis was significantly larger in cohort 1630 (1630: 1166 ± 800 ml, 1380: 477 ± 530 ml and 1250: 523 ± 504 ml, p<0.0001). The Hct drop at the start and end of CPB was significantly reduced comparing cohort 1630 with cohort 1250 (1630: 32 ± 7%, 1380: 30 ± 7% and 1250: 28 ± 10%, p=0.002) at the start of CPB and (1630: 31 ± 7%, 1380: 29 ± 7% and 1250: 28 ± 8%, p=0.016) at the end of CPB. The Hct values at the start and end of CPB were significantly different between the cohorts (1680: 0.23 ± 0.03 L/L - 0.22 ± 0.02 L/L, 1380: 0.25 ± 0.03 L/L - 0.25 ± 0.03 L/L and 1250: 0.25 ± 0.03 L/L- 0.25 ± 0.03 L/L, p= 0.001 and 0.0001). CONCLUSIONS: Minimizing our CPB circuit by using integrated components has affected the drop of Hct and the amount of transfused RBCs.
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Puente Cardiopulmonar , Transfusión de Eritrocitos , Auditoría Médica , Adulto , Anciano , Femenino , Hematócrito , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
The prognosis of adult patients with ALL remains unsatisfactory. AlloSCT is associated with a beneficial GVL response mediated by donor T cells. However, GVHD results in substantial mortality and long-term morbidity. T-cell depletion (TCD) of the graft reduces the severity of GVHD, but is associated with an increased relapse rate after alloSCT. Therefore, early sequential donor lymphocyte infusion (DLI) is likely to be necessary for a successful GVL reaction. Twenty-five adult ALL patients (10 Ph(+)ALL) were eligible for early DLI after initial disease control with myeloablative TCD-alloSCT in first CR (CR1), if active GVHD was absent at 3-6 months after alloSCT. Patients with a sibling donor or an unrelated donor were scheduled for 3.0 × 10(6) CD3(+) cells/kg or 1.5 × 10(6) CD3(+) cells/kg, respectively, at 6 months after alloSCT. Three patients died before evaluation (one early relapse). Five patients had active GVHD. Fourteen of the remaining seventeen patients received DLI (median time-to-DLI: 185 days). Overall, only 17% required long-term systemic immunosuppression for GVHD. With a median follow-up after TCD-alloSCT of 50 months, 2-year survival probability was 68% (95% confidence interval (CI) 49-87%). In conclusion, myeloablative TCD-alloSCT with early sequential DLI is an efficient and safe post-remission treatment for adult ALL patients in CR1.
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Trasplante de Células Madre Hematopoyéticas/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfocitos T/metabolismo , Acondicionamiento Pretrasplante/métodos , Adulto , Femenino , Humanos , Depleción Linfocítica , Transfusión de Linfocitos/métodos , Masculino , Persona de Mediana Edad , Pronóstico , Donantes de Tejidos , Trasplante Homólogo , Adulto JovenRESUMEN
The development of the lactating mammary gland is a complex multifactorial process occurring in mammals during pregnancy. We show here that this process requires NHERF1/EBP50 (Na/H exchanger regulatory factor 1/ERM-binding phosphoprotein 50) expression and that successful lactation depends on NHERF1 allele copy number, with rates of 50 and 20% in NHERF1(+/-) and (-/-) mice, respectively. The prolactin receptor (PRLR)-STAT5 signaling provides the central axis triggering the differentiation of secretory mammary alveolar cells. In successfully lactating glands, NHERF1 is massively upregulated and forms complexes with PRLR, but also with ß-catenin, E-cadherin and ezrin at the alveolar basal membrane, establishing basal polarity. In NHERF1-deficient glands, the basal polarity is disrupted, the PRLR levels and basal membrane localization are abolished, and the downstream STAT5 activation collapses with consequent reduction of milk protein synthesis. NHERF1/EBP50, a protein deregulated in breast cancer, thus emerges as an important physiological mediator of milk secretion, by engagement of PRLR in multimeric complexes at the alveolar basal membrane with subsequent network activation leading to cell differentiation.
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Fosfoproteínas/metabolismo , Receptores de Prolactina/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Alelos , Animales , Cadherinas/metabolismo , Polaridad Celular , Proteínas del Citoesqueleto/metabolismo , Femenino , Genotipo , Lactancia , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Embarazo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/genética , Regulación hacia Arriba , beta Catenina/metabolismoRESUMEN
Mdm2 inhibits the function of the p53 tumor suppressor. Mdm2 is overexpressed in many tumors with wild-type p53 suggesting an alternate mechanism of loss of p53 activity in tumors. An Mdm2-binding protein (MTBP) was identified using a yeast two-hybrid screen. In tissue culture, MTBP inhibits Mdm2 self-ubiquitination, leading to stabilization of Mdm2 and increased degradation of p53. To address the role of MTBP in the regulation of the p53 pathway in vivo, we deleted the Mtbp gene in mice. Homozygous disruption of Mtbp resulted in early embryonic lethality, which was not rescued by loss of p53. Mtbp+/- mice were not tumor prone. When mice were sensitized for tumor development by p53 heterozygosity, we found that the Mtbp+/-p53+/- mice developed significantly more metastatic tumors (18.2%) as compared to p53+/- mice (2.6%). Results of in vitro migration and invasion assays support the in vivo findings. Downmodulation of Mtbp in osteosarcoma cells derived from p53+/- mice resulted in increased invasiveness, and overexpression of Mtbp in Mtbp+/-p53+/- osteosarcoma cells inhibited invasiveness. These results suggest that MTBP is a metastasis suppressor. These results advance our understanding of the cellular roles of MTBP and raise the possibility that MTBP is a novel therapeutic target for metastasis.
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Neoplasias Óseas/patología , Proteínas Portadoras/fisiología , Neoplasias Hepáticas/secundario , Osteosarcoma/secundario , Animales , Southern Blotting , Neoplasias Óseas/metabolismo , Movimiento Celular , Femenino , Silenciador del Gen/fisiología , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Osteosarcoma/metabolismo , Fenotipo , Embarazo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Mdm2, an E3 ubiquitin ligase, negatively regulates the tumour suppressor p53. Loss of Mdm2 in mice results in p53-dependent apoptosis and embryonic lethality. This phenotype was rescued by the p53(515C) allele, which encodes an apoptosis-deficient p53R172P protein. However, these mice died within 2 weeks of birth, due to a severe impairment of progenitor cell expansion during postnatal haematopoiesis and cerebellar development, leading to p53-dependent cell cycle arrest. Loss of Mdm2 led to phosphorylation of the p53R172P protein, p53R172P stability and activation of the cell cycle inhibitor p21 in proliferating cells, but not in differentiated cells, in multiple tissue compartments. Proliferating cells of epithelial origin were not affected. The haematopoietic and neural defects were alleviated in mice lacking Mdm2 and containing one p53(515C) and one p53-null allele, but spermatogenesis was arrested. These findings establish a crucial role for the p53-Mdm2 network in regulating proliferation and progenitor expansion in many cell lineages and have important implications for the use of drugs that aim to disrupt the p53-Mdm2 interaction.
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Proteínas Proto-Oncogénicas c-mdm2/fisiología , Células Madre/citología , Proteína p53 Supresora de Tumor/fisiología , Alelos , Animales , Diferenciación Celular/fisiología , Proliferación Celular , Cerebelo/crecimiento & desarrollo , Cerebelo/patología , Crecimiento/fisiología , Hematopoyesis/fisiología , Masculino , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-mdm2/deficiencia , Transducción de Señal/fisiología , Espermatogénesis/fisiología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
We tested a phenyl mannitol broth containing ceftizoxime and aztreonam (PHMB(+)) for detection of methicillin-resistant Staphylococcus aureus (MRSA) with reference MRSA strains and, subsequently, with clinical samples (n = 1,098). All reference MRSA strains induced color change in PHMB(+) after 24 to 72 h of incubation. In a clinical setting, 40 MRSA strains were detected with PHMB(+), compared with only 23 detected with a routine method. Thus, this selective broth significantly (P < 0.001) improved the rate of MRSA detection.
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Manitol/metabolismo , Resistencia a la Meticilina , Fenolsulfonftaleína/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Aztreonam/farmacología , Técnicas Bacteriológicas , Ceftizoxima/farmacología , Cefalosporinas/farmacología , Medios de Cultivo/química , Humanos , Monobactamas/farmacología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/clasificación , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
The high frequency of single nucleotide polymorphisms (SNPs) in the human genome makes them ideal genetic markers for mapping, diagnosing disease-related alleles, and identifying SNPs that contribute to drug response differences between individuals. Here we report a novel assay utilizing a single nucleotide primer extension (SNuPE) and electrospray ionization mass spectrometry (ESI-MS) detection for the analysis of SNPs. In contrast to most SNuPE genotyping technologies that detect the extended primer product, the novel Survivor assay detects the unreacted dideoxynucleotides (ddNTPs) remaining or surviving in solution following a SNuPE. This assay involves a simple analysis of the same four ddNTP analytes, regardless of the SNP being investigated, and either single or double-stranded DNA can be used to genotype a SNP, without any labeling requirements of the ddNTPs or oligonucleotide primers. We have tested and blindly validated the Survivor assay by genotyping the C/T SNP at -857 of the human TNFalpha promoter gene. The results obtained are in agreement with the control sequencing data. The results demonstrate that the homogeneous Survivor assay with ESI-MS detection offers advantages in simplicity, accuracy, specificity, and sensitivity. Additional advantages of the method include enhanced hybridization efficiencies in this solution-phase assay and the elimination of immobilized primers for the isolation of single-stranded DNA. With a one-well reaction and an automation platform being developed, the Survivor assay provides a powerful new tool for large-scale SNP analysis and screening.
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Oligodesoxirribonucleótidos/genética , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Factor de Necrosis Tumoral alfa/genética , Secuencia de Bases , ADN Polimerasa Dirigida por ADN/metabolismo , Genotipo , Humanos , Datos de Secuencia MolecularRESUMEN
A novel approach to parallel liquid chromatography/ tandem mass spectrometry (LC/MS/MS) analyses for pharmacokinetic assays and for similar quantitative applications is presented. Modest modifications render a conventional LC/MS system capable of analyzing samples in parallel. These modifications involve the simple incorporation of three valves and four LC columns into a conventional system composed of one binary LC pumping system, one autosampler, and one mass spectrometer. An increase in sample throughput is achieved by staggering injections onto the four columns, allowing the mass spectrometer to continuously analyze the chromatographic window of interest Using this approach, the optimized run time is slightly greater than the sum of the widths of the desired peaks. This parallel chromatography unit can operate under both gradient and isocratic LC conditions. To demonstrate the utility of the system, atorvastatin, five of its metabolites, and their deuterated internal standards (IS) were analyzed using gradient elution chromatography conditions. The results from a prestudy assay evaluation (PSAE) tray of standards and quality control (QC) samples from extracted spiked human plasma are presented. The relative standard deviation and the accuracy of the QC samples did not exceed 8.1% and 9.6%, respectively, which is well within the acceptance criteria of the pharmaceutical industry. For this particular analysis, the parallel chromatography system decreased the overall run time from 4.5 to 1.65 min and, therefore, increased the overall throughput by a factor of 2.7 in comparison to a conventional LC/MS/MS analytical method.
RESUMEN
Chloro-S-triazine herbicides [cyanazine (CZ), atrazine (AZ), simazine (SZ)] increase mammary tumors in Crl:CD BR rats but not in F-344 rats or in mice. A nongenotoxic mechanism was investigated since the chloro-S-triazines are negative in short-term tests for genotoxicity. An in vivo battery was used to assess the chloro-S-triazines for estrogenic activity or for their ability to increase prolactin (PRL) levels, both of which play important roles in enhancing mammary gland tumorigenesis in rodents. Ovariectomized (OVX) female rats were treated with AZ, CZ, SZ, or three CZ metabolites for 4 days via intraperitoneal injection. The pattern of responses between the chloro-S-triazines and four controls (estradiol, estriol, haloperidol, reserpine) was compared. For the 6 end-points examined, the responses from rats treated with AZ, CZ, SZ, and the metabolites of CZ most closely matched the responses from the reserpine-treated rats (a PRL rather than estrogenic mechanism). In addition, AZ, CZ, and SZ were tested in several other in vitro models (estrogen/biogenic amine receptor competition assays and a yeast-expressed human estrogen receptor transcription assay) as well as an in vivo 24 h time-course experiment to characterize the CZ-induced increases in PRL levels. AZ, CZ, and SZ are not estrogen receptor (ER) activating compounds based on yeast transactivation and receptor competition data. CZ and AZ demonstrated marginal competition (at mM levels) to the D and alpha2 adrenergic receptors. Ligands to the D2 receptor, but not the alpha2 adrenergic receptor, are known to induce mammary tumors. CZ was also found to produce elevated PRL levels in a time-course similar to that seen with reserpine and haloperidol. Overall, the pattern of responses obtained with the chloro-S-triazines most closely matched the responses observed for reserpine. Taken together, these data suggest chloro-S-triazine-induced mammary tumors in rats are mediated through a PRL mechanism, which is thought to be of low relevance to humans.
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Atrazina/toxicidad , Herbicidas/toxicidad , Neoplasias Mamarias Experimentales/inducido químicamente , Prolactina/sangre , Simazina/toxicidad , Triazinas/toxicidad , Inhibidores de Captación Adrenérgica/farmacología , Animales , Atrazina/metabolismo , Bioensayo , Peso Corporal/efectos de los fármacos , Antagonistas de Dopamina/farmacología , Estradiol/farmacología , Estriol/farmacología , Femenino , Genes Reporteros , Haloperidol/farmacología , Herbicidas/metabolismo , Humanos , Masculino , Ovariectomía , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Reserpina/farmacología , Simazina/metabolismo , Activación Transcripcional , Triazinas/metabolismo , Útero/efectos de los fármacos , Levaduras/genética , Levaduras/metabolismoRESUMEN
Cyanazine is a member of the chloro-s-triazine class of herbicides. Other triazine herbicides have been shown to induce mammary-gland tumors in rats, although the response is unique to the Sprague-Dawley strain. Cyanazine is nongenotoxic. The present study was conducted to evaluate the chronic toxicity and oncogenic potential of cyanazine. Groups of 62 male and female rats were fed diets containing cyanazine at concentrations of 1, 5, 25, or 50 ppm for up to 2 yr. Mean body weight and body weight gain of male and female rats of the 25- and 50-ppm groups were significantly reduced over the course of the study. Food consumption and food efficiency were also reduced in these groups. Survival was not adversely affected in the treatment groups compared to controls. A significant increase in the incidence of masses of the inguinal region was noted among female rats of the 50-ppm group. These masses were correlated with a significant increase in the incidence of female rats with mammary-gland adenocarcinomas and carcinosarcomas. The incidence of rats with malignant mammary-gland tumors was elevated in the 5-, 25-, and 50-ppm groups, although the incidence within the 5-ppm group was within historical controls. There were no other toxicologically significant observations with respect to ophthalmological, clinical laboratory, or pathological evaluations. Under the conditions of this study, the no-observed-adverse-effect level was 5 ppm. Research into the mechanism of action suggests these mammary tumors are mediated through a prolactin mechanism that is thought to be of low relevance to humans.
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Herbicidas/toxicidad , Neoplasias Mamarias Animales/inducido químicamente , Triazinas/toxicidad , Adenocarcinoma/inducido químicamente , Alimentación Animal/análisis , Animales , Bioensayo , Peso Corporal/efectos de los fármacos , Carcinosarcoma/inducido químicamente , Ingestión de Alimentos/efectos de los fármacos , Femenino , Contaminación de Alimentos , Herbicidas/administración & dosificación , Herbicidas/sangre , Herbicidas/orina , Masculino , Ratas , Ratas Sprague-Dawley , Triazinas/administración & dosificación , Triazinas/sangre , Triazinas/orinaRESUMEN
Recently it has been shown that acetonitrile chemical ionization tandem mass spectrometry (CI-MS/MS) is a rapid, on-line means to determine double bond position in fatty acid methyl esters (FAME). The mechanism of this gas phase condensation reaction has been studied. Evidence of the (1-methyleneimino)-1-ethenylium ion (m/z 54), formed upon the reaction of acetonitrile with itself, adding across the double bond in a [2 + 2] cycloaddition reaction is observed. When this nascent complex undergoes collision-induced dissociation, two diagnostic ions emerge. One of these ions results from loss of the hydrocarbon end of the FAME, whereas the other ion results from loss of the methyl ester end, and when considered together, the diagnostic ions localize the positions of the double bonds in the FAME. Several labeling and MS/MS/MS experiments on the two diagnostic ions were performed to determine a plausible fragmentation mechanism of the stable (1-methyleneimino)-1-ethenylium-FAME complex. The first generation product ions, or diagnostic ions, appear to be formed though a charge-driven mechanism, whereas the second generation product ions are formed via charge-remote fragmentations. Plausible mechanisms for the formation and subsequent dissociation of the diagnostic ions are presented for the monounsaturated, diunsaturated, and polyunsaturated (3 or more double bonds) FAME.
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Acetonitrilos/química , Ácidos Grasos Insaturados/química , Cromatografía de Gases y Espectrometría de Masas , Metilación , PolienosRESUMEN
The slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecA PCR ("gold standard") for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screen test detected the penicillin-binding protein 2a (PBP2a) antigen in 87 of 90 genetically diverse methicillin-resistant S. aureus (MRSA) stock culture strains, leading to a sensitivity of 97%. The three discrepant MRSA strains displayed positive results only after induction of the mecA gene by exposure to methicillin. Both mecA PCR and MRSA-Screen displayed negative results among the methicillin-susceptible S. aureus strains (n = 106), as well as for Micrococcus spp. (n = 10), members of the family Enterobacteriaceae (n = 10), Streptococcus pneumoniae (n = 10), and Enterococcus spp. (n = 10) (specificity = 100%). Producing the same PBP2a antigen, all 10 methicillin-resistant Staphylococcus epidermidis strains score positived in both the latex test and the mecA PCR. Consequently, the MRSA-Screen test should be applied only after identification of the MRSA strain to the species level to rule out coagulase-negative staphylococci. In conclusion, due to excellent specificity and sensitivity the MRSA-Screen latex test has the potential to be successfully used for routine applications in the microbiology laboratory.
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Resistencia a la Meticilina , Staphylococcus aureus/efectos de los fármacos , Pruebas de Fijación de Látex , Sensibilidad y Especificidad , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
We report structure determination of an octaene fatty acid, 4,7,10, 13,16,19,22,25-octacosaoctaenoic acid (28:8n-3). The molecular weight and double bond locations were determined using acetonitrile chemical ionization mass spectrometry (MS) and MS/MS and were confirmed by MS of hydrogenated and deuterogenated 28:8 and by argentation thin-layer chromatography. 28:8n-3 was 1.2 +/- 0.1%, in oil derived from the heterotrophic dinoflagellate Crypthecodinium cohnii and a commercial polyunsaturated fatty acid concentrate derived from fish oils (0.16 +/- 0.01%), both components of human dietary supplements. It was not found in whole bovine retina, cultured Y79 human retinoblastoma cells, or neonate baboon cerebral cortex. The long chain polyunsaturates present in the C. cohnii oil suggest a possible route for 28:8n-3 biosynthesis similar to that for biosynthesis of 22:6n-3.
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Dinoflagelados/química , Ácidos Grasos Omega-3/química , Aceites/química , Animales , Bovinos , Corteza Cerebral/química , Aceites de Pescado/química , Cromatografía de Gases y Espectrometría de Masas , Humanos , Biología Marina , Modelos Químicos , Oxidación-Reducción , Retina/químicaRESUMEN
A rapid method is presented for determining the location of double bonds in polyunsaturated fatty acid methyl esters (FAME) using an ion-trap mass spectrometer. The mass spectrum of the chemical ionization reagent acetonitrile in an ion trap includes a m/z 54 ion, identified previously as 1-methyleneimino-1-ethenylium ion. We show that it reacts with double bonds of polyunsaturated FAME to yield a series of covalent product ions all appearing at (M + 54)+. Collisional dissociation of these ions yields diagnostic fragments, permitting unambiguous localization of double bonds. For methylene-interrupted and conjugated FAME, one of these fragments results from loss of the hydrocarbon end of the chain, while the other involves loss of the methyl ester. Major diagnostic-fragment ions for monoene and diene FAME occur as a result of cleavage adjacent to either allylic sites or double bonds in the original analyte and appear at one mass unit above the mass expected for homolytic cleavage. Fragmentation of polyene FAME yields major diagnostic ions resulting from cleavage between double bonds that appear one mass unit lower. The method is shown to produce highly characteristic spectra for FAME with 1 to 6 double bonds. Identification of double-bond position in highly unsaturated fatty acids is demonstrated in a mixture of unknown polyunsaturated FAME from an extract of cultured Y79 human retinoblastoma cells.
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Ácidos Grasos Insaturados/química , Acetonitrilos , Ésteres/química , Humanos , Espectrometría de Masas , Peso Molecular , Células Tumorales CultivadasRESUMEN
Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated. This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required. A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B. gladioli (n = 14), Ralstonia pickettii (n = 6), B. multivorans (n = 2), Stenotrophomonas maltophilia (n = 3), and Pseudomonas aeruginosa (n = 11), were collected from environmental, clinical, and reference sources. In addition, 27 clinical isolates putatively identified as Burkholderia spp. were recovered from the sputum of Dutch CF patients. All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism [RFLP] analysis). The best system for the biochemical identification of B. cepacia appeared to be the API 20NE test. None of the biochemical assays successfully grouped the B. gladioli strains. The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different Burkholderia spp. With this method, B. gladioli was also reliably classified in a separate group. For the laboratory diagnosis of B. cepacia, we recommend parallel cultures on blood agar medium and selective agar plates. Further identification of colonies with a Burkholderia phenotype should be performed with the API 20NE test. For final confirmation of species identities, PCR amplification of the small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended.
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Infecciones por Burkholderia/diagnóstico , Burkholderia/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Burkholderia/clasificación , Burkholderia/genética , Burkholderia cepacia/clasificación , Burkholderia cepacia/aislamiento & purificación , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , ADN Ribosómico/genética , Geografía , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/aislamiento & purificación , ARN Ribosómico 16S/genética , Mapeo Restrictivo , Alineación de Secuencia , Microbiología del SueloRESUMEN
A rapid, high-selectivity method with subfemtomole sensitivity is reported for quantification of alpha-tocopherol in plasma-based gas chromatography/tandem mass spectrometry (GC/MS/MS) using a tabletop quadrupole ion trap mass spectrometer. Sample workup is rapid, consisting of protein precipitation followed by liquid/liquid extraction and O-trimethylsilyl derivatization of alpha-tocopherol (alpha-T-TMS) and an internal standard, 2,2,5,7,8-pentamethyl-6-chromanol (PC-TMS). Rudimentary chromatography was carried out using an 8-m DB-5 capillary column resulting in an analyte retention time of 7.2 min. No interferences from the plasma matrix were observed. The assay has a detection limit of 178 amol (89.6 fg) and a lower limit of quantification of 700 amol (350 fg) of derivatized alpha-tocopherol in diluted plasma; < 30 pL of plasma is estimated to yield sufficient alpha-tocopherol for quantitative analysis at typical concentrations found in humans. A calibration curve constructed from National Institute of Standards and Technology serum standards was linear in the working range of 1.9-1073 ng/mL (0.95-0.54 ng). Within- and between-day precision averaged 5.8% and did not exceed 11.3% for three concentrations of quality control (QC) solutions. The overall accuracy for the QC samples was within 7.2%. Storage studies showed that, alpha-T-TMS and PC-TMS are stable under conditions that might be encountered during analyses. In a test study, plasma kinetic curves for alpha-tocopherol-d6 and alpha-tocopherol-d3 were obtained for a catheterized pregnant ewe and her fetus who were simultaneously given a bolus injection of alpha-tocopherol-d6, to the ewe and alpha-tocopherol-d3 to the fetus. These data show that a tabletop GC ion trap can determine alpha-T-TMS and its isotopomers quantitatively at high selectivity in a complex matrix.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Vitamina E/sangre , Animales , Cromanos/sangre , Deuterio , Femenino , Embarazo , Estándares de Referencia , Reproducibilidad de los Resultados , Ovinos , Compuestos de Trimetilsililo , Vitamina E/normasRESUMEN
Over the past several years, there has been increasing concern that chemicals and pesticides found in the environment may mimic endogenous estrogens, potentially producing adverse effects in wildlife and human populations. Because estrogenicity is one of the primary concerns, a 90-day/one-generation reproduction study with 17 beta-estradiol was designed to set dose levels for future multigenerational reproduction and combined chronic toxicity/oncogenicity studies. The purpose of these studies is to evaluate the significance of a range of responses as well as to provide benchmark data for a risk assessment for chemicals with estrogen-like activities. This 90-day/one-generation reproduction study was conducted in male and female Crl:CD BR rats using dietary concentrations of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol. Endpoints were chosen in order to evaluate both subchronic and reproductive toxicity. In addition, several mechanistic/biochemical endpoints were evaluated for their usefulness in follow-up studies. In the P1 generation, dietary administration of 2.5, 10, and 50 ppm 17 beta-estradiol produced dose-dependent decreases in body weight, body weight gain, food consumption, and food efficiency. At 10 and 50 ppm 17 beta-estradiol, minimal to mild nonregenerative anemia, lymphopenia, decreased serum cholesterol (50 ppm only), and altered splenic lymphocyte subtypes were also observed in the P1 generation. Additionally, at these concentrations, there were changes in the weights of several organs. Evidence of ovarian malfunction, characterized by reduced numbers of corpora lutea and large antral follicles, was observed at 2.5 ppm 17 beta-estradiol and above. Other pathologic changes in males and females fed 10 and 50 ppm 17 beta-estradiol included centrilobular hepatocellular hypertrophy; diffuse hyperplasia of the pituitary gland; feminization of the male mammary glands; mammary gland hyperplasia in females; increased number of cystic follicles in the ovary; hypertrophy of the endometrium and endometrial glands in the uterus; degeneration of seminiferous epithelium; and atrophy of the testes and the accessory sex glands. In the reproduction portion of this study, rats fed 10 or 50 ppm 17 beta-estradiol did not produce litters. While there was no evidence that the 50 ppm treated rats mated, 33.3% of the rats fed 10 ppm mated but did not produce litters. No effects on mating and fertility indices were observed in rats fed 0.05 and 2.5 ppm 17 beta-estradiol. Pup weights at birth were statistically decreased relative to control in the groups fed 0.05 and 2.5 ppm 17 beta-estradiol. Weights of the rats in the 0.05 ppm group recovered by postnatal day 4 and remained similar to control throughout the remainder of the study. The mean gestation length of the 0.05 ppm group was slightly, albeit not statistically significantly, shorter (0.5 days) than that of the control group, which may have contributed to the decrease in birth weight of the 0.05 ppm group. In contrast, the weights of the F1 generation rats fed 2.5 ppm 17 beta-estradiol remained decreased relative to the control group throughout the study. Parental administration of 17 beta-estradiol did not alter anogenital distance in male or female pups. The onset of sexual maturation, as measured by day of preputial separation in males and day of vaginal opening in females, was delayed in male rats fed 2.5 ppm (by 8.2 days) and was hastened in female rats fed 0.05 and 2.5 ppm (by 1.6 and 8.8 days, respectively). The age at vaginal opening ranged from 26 to 37, 26 to 35, and 21 to 25 days for rats fed 0, 0.05, and 2.5 ppm 17 beta-estradiol, respectively. Hence, the range of age at vaginal opening was similar between the control and 0.05 ppm group. The organ weight and pathologic alterations observed in the adult F1 generation rats were similar to those observed in the P1 generation rats. (ABSTRACT TRUNCATED)
Asunto(s)
Estradiol/toxicidad , Feto/efectos de los fármacos , Reproducción/efectos de los fármacos , Animales , Femenino , Masculino , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/patología , Tamaño de los Órganos/efectos de los fármacos , Ovario/efectos de los fármacos , Ovario/patología , Embarazo , Ratas , Testículo/efectos de los fármacos , Testículo/patología , Vagina/efectos de los fármacos , Vagina/patologíaRESUMEN
An inexpensive modification to a gas chromatography injector liner is reported that facilitates continuous admission of analyte into a gas chromatograph/mass spectrometer (GC/MS) for methods development. The MS methods development liner can be made by making simple modifications to commercially available liners and fits into standard injectors in place of the normal liners without any need to break vacuum in the MS. The injector temperature and gas flow rates are adjusted to provide appropriate analyte levels in the MS, which can be admitted under conditions identical with those of real analyses, including co-admission of column bleed. The device is particularly useful for development of tandem MS methods in GC/MS/MS instruments, which are configured with the GC as the sole sample inlet.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/instrumentación , Vitamina E/análisis , Análisis de Inyección de Flujo , Cromatografía de Gases y Espectrometría de Masas/métodos , Programas Informáticos , Temperatura , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/análisisRESUMEN
In the process of evaluating a proprietary compound for weak estrogenic activity, two different types of dosing regimens were used, repeated daily dosing (three times/d) and continuous-release pellets. In studies using the proprietary compound, rats that were dosed via intraperitoneal injection showed no estrogenic responses, while those receiving the test compound via continuous-release pellets displayed several estrogenic responses. Because of the conflicting results, the control pellets were evaluated for estrogenic activity in the same battery of tests using the same number of pellets. In studies using only the control pellets, several estrogenic responses were observed including increased uterine weight, uterine stromal cell proliferation, estrous conversion, uterine progesterone receptor content, and decreased uterine estrogen receptor content. Animals receiving no pellet implant showed no estrogenic responses. In addition, a methylene chloride/DMSO extract of the control pellets promoted expression of a reporter gene controlled by the estrogen receptor and demonstrated competition with 17 beta-estradiol for binding to the human estrogen receptor. It is concluded that component(s) of the control pellets possess weak estrogenic activity.