Mucin genes in horse airways: MUC5AC, but not MUC2, may play a role in recurrent airway obstruction.

REASONS FOR PERFORMING STUDY: Increased mucin gene expression may be an important cause of mucus accumulation observed in recurrent airway obstruction (RAO)-affected horses. To date, however, no mucin gene sequences are available for the horse. OBJECTIVES: To identify equine homologues of gel-forming mucins and investigate their expression at different airway generations of healthy and RAO-affected horses. METHODS: Two equine homologues were identified by cloning and sequencing fragments of equine (eq)MUC5AC and eqMUC2. RESULTS: Semiquantitative RT-PCR on RNA from airways (generations 1, 5, 10, 15; small airways and parenchyma), stomach (glandular), and colon revealed that eqMUC5AC is expressed in equine stomach and in all of the airway samples. In contrast, eqMUC2 steady-state mRNA levels were detected in colon and very faintly in stomach, but not in airway tissue. EqMUC5AC expression was also compared to that of ZO-1, a tight junction protein, and eqMUC5AC/ZO-1 ratios were higher in RAO-affected compared to control horses at all airway generations. CONCLUSIONS: That eqMUC5AC is expressed in horse airways, but any expression of MUC2 is undetectable and unlikely to be of physiological consequence. POTENTIAL RELEVANCE: EqMUC5AC up-regulation may be a primary mechanism responsible for mucus hypersecretion and accumulation in RAO.

Reducción de la prevalencia de los defectos de cierre del tubo neural mediante un programa regional de cribado con alfafetoproteína en suero materno / Reduction in the prevalence of neural tube closure defects through a regional screening programe with alpha-fetoprotein in maternal serum

Objetivo: Optimación de un programa regional de cribado con alfafetoproteína sérica materna con objeto de que la prevalencia de los defectos de cierre del tubo neural en el Principado de Asturias sea próxima a cero.Sujetos y métodos: Las participantes han sido gestantes pertenecientes a las diferentes áreas de salud del Principado de Asturias independientemente de su procedencia, sanidad pública o privada. Se estudiaron a 63.163 embarazadas en 11 años. Todas las técnicas bioquímicas se realizaron en el Laboratorio de Bioquímica del Hospital San Agustín.Resultados: Se ha conseguido optimizar el programa de cribado con alfafetoproteína en suero materno con porcentajes muy bajos de falsos positivos y amniocentesis realizadas, el 1,9 y el 0,5 por ciento, respectivamente. Se ha observado que las anencefalias presentan valores de alfafetoproteína en suero materno significativamente más elevados que las espinas bífidas abiertas, y se ha conseguido disminuir la prevalencia de este tipo de malformaciones en el Principado de Asturias de una media de 1,3 a prácticamente cero.Conclusiones: La puesta en funcionamiento de un programa de estas características requiere un equipo multidisciplinario para que los resultados obtenidos sean satisfactorios. Después de varios años de desarrollo los resultados obtenidos han convencido a los obstetras y médicos generalistas de la utilidad de incorporar dicho programa al protocolo del embarazo. Este programa ha servido, además, para obtener un conocimiento exhaustivo de este tipo de malformaciones en el Principado de Asturias. (AU)

Utilidad de la alfafetoproteína sérica materna como parámetro de riesgo del embarazo / Utility of maternal serum alpha-fetoprotein as a risk marker of the pregnancy

Objetivos: Demostrar que las embarazadas con valores elevados, sin justificación aparente, de alfafetoproteína sérica tienen un riesgo mayor de resultados perinatales adversos.Sujetos y métodos: Se estudiaron 43.424 gestantes desde el segundo trimestre del embarazo hasta el parto. Se calculó el riesgo relativo entre los valores de alfafetoproteína en suero materno (AFPSM) y los resultados perinatales siguientes: partos pretérminos, muertes fetales anterior y posterior a la semana 28 de gestación, y recién nacidos con bajo peso.Resultados: Se estudió la influencia de las concentraciones de AFPSM sobre 4 resultados perinatales adversos, observándose en todos ellos una diferencia significativa entre el grupo de gestantes considerado control (AFPSM 2,5 MDM). Se observó un mayor riesgo relativo en las gestantes con muerte fetal anterior a la semana 28 de gestación.Conclusiones: Se ha comprobado que existe una relación entre los valores elevados de AFPSM y el riesgo de un resultado perinatal adverso. Sin embargo, la AFPSM no se puede considerar un marcador de cribado adecuado, por su baja sensibilidad, para seleccionar gestantes con un riesgo elevado de un resultado adverso. (AU)

Optimization of nucleated red blood cell (NRBC) recovery from maternal blood collected using both layers of a double density gradient.

The isolation of fetal nucleated red blood cells (NRBC) from maternal blood represents a promising approach to non-invasive prenatal diagnosis. However, the number of fetal NRBC in maternal circulation is quite low and therefore difficult to isolate. An enrichment procedure in which both layers from a double density 1.077/1.107 g/ml gradient are collected was optimized, followed by MACS selection using non-commercial monoclonal antibodies. The influence of the delay in processing maternal blood on the NRBC distribution in both interfaces of the gradient was also studied in cord blood and peripheral maternal blood samples. A significant increase in the number of NRBC isolated from maternal blood was achieved by collecting both layers of the double density gradient compared with the previous protocol in which only the lower layer was recovered. Cord blood samples showed significant differences in the number of NRBC recovered when processed at 24 instead of within 3 h. This effect was also observed in the number of NRBC collected only from the upper layer of peripheral maternal blood samples. Therefore, in order to minimize the target cell losses, it is advisable to process the maternal blood samples as soon as possible.

Year-long validation study and reference values for urinary amino acids using a reversed-phase HPLC method.

BACKGROUND: Reversed-phase HPLC (RP-HPLC) has become an alternative to ion-exchange chromatography for amino acid analysis in biological fluids. However, validation studies for its urine application are limited, and the corresponding reference values have not been reported extensively. We studied the long-term performance of a commercial HPLC method for urine amino acid analysis and established specific age-related reference values for urine amino acid excretion. METHODS: Method performance was continuously assessed by recovery and precision studies with urine samples and controls, respectively. Healthy individuals were prospectively analyzed throughout a 5-year period. Excretion of individual amino acids, expressed as mmol/mol of creatinine, was included in six age-related groups for random urine samples (0-1 month, 1-12 months, 1-3 years, 3-8 years, 8-16 years, and >16 years) and in two groups for 24-h urine collections (8-16 years and >16 years). RESULTS: Over a 1-year period, CVs for retention times were <0.5% and 3.3% for within- and between-run imprecision, respectively. For amino acid concentrations, within-run CVs were 2.9-17% and between-run CVs were 7.1-46% for the same period. Amino acid recoveries were 78-122%. Reference intervals for 35 amino acids were calculated and compared with the concentrations observed in patients diagnosed with specific pathologies. A few statistically significant differences were found between the reference intervals derived using random and 24-h urine collections. CONCLUSIONS: Long-term reliability of the RP-HPLC method for urine amino acid analysis has been demonstrated. Representative age-related reference intervals for the RP-HPLC method in both random urine and 24-h urine collections have been established, and their feasibility for diagnosis of aminoaciduria has been shown. These intervals could serve as a guide for laboratories changing to HPLC methods.

GCDFP-70 protein in cyst fluid identified as albumin and used to classify cysts in women with breast gross cystic disease.

We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to study cyst fluids from women with breast gross cystic disease. The subjects could be classified into two categories according to the concentrations of protein GCDFP-70 in the cyst fluid: those with Type I cysts had a very low content of this protein; those with Type II cysts had very high concentrations. Analysis of the amino acid sequence of GCDFP-70 from both cyst fluid types confirmed that this protein is human plasma albumin. The average concentration of albumin found in Type I cyst fluids was 0.32 g/L and that corresponding to Type II was 10.16 g/L. Thus, albumin quantification from cyst fluids or analysis by either polyacrylamide or agarose gel electrophoresis provides a simple procedure for classifying these fluids, yielding results that correlate well with previous classifications based on other measurements such as sodium, potassium, and chloride concentrations. This albumin-based quantification method may improve the classification of breast cysts and might be useful in further studies on functional changes in the cysts and their relationship to breast cancer.

IgA-CK-BB complex with CK-MB electrophoretic mobility can lead to erroneous diagnosis of acute myocardial infarction.

This patient, on admission, presented with a tentative diagnosis of myocardial infarction: the electrocardiogram showed a nonspecific ST-segment and T-wave abnormalities, and total creatine kinase (CK; EC 2.7.3.2) activity was slightly increased (238 U/L). However, a high electrophoretic value for CK-MB (50% of total CK activity) and the electrophoretic pattern of lactate dehydrogenase (EC 1.1.1.27) isoenzymes ruled out myocardial infarction. The isoenzyme migrating as CK-MB was found later to contain no immunologically normal CK-M subunits, and it was bound to IgA. A mixture of the patient's serum and a human serum control containing all CK isoenzymes showed altered electrophoretic mobility only for CK-BB, indicating that the patient's serum contained antibodies to the B unit of CK. Elution from a Sephadex G-200 column showed that the peak at which most of the anodic CK was eluted corresponded to a molecular mass of approximately 200 kDa. Evidently this atypical isoenzyme was an IgA-CK-BB complex. Because this macro CK type 1 can mimic CK-MB, it may therefore be a source of confusion.

[Changes in the prolactin levels after administration of ethanol in diestrus 2]. / Alteración de los niveles de prolactina tras la administración de etanol en diestro 2.

The effect of ethanol administered at 18.00 h. of diestrus 2 on prolactin secretion has been studied in rats. Serum levels and pituitary content of prolactin were measured at 30, 60, 90 and 120 min. after administration of different doses of ethanol (0.5, 2 and 4 g/kg). Moreover, the variation of the prolactin release was determined at different hours of the rat estrous cycle, after administration of a single dose of ethanol (2 g/kg) at 18.00 h of diestrus. 2. Serum prolactin levels were significantly elevated after the preovulatory administration of ethanol with all the doses tested. The hyperprolactinemia appeared 60 min after ethanol treatment and the high prolactin levels were maintained during all the estrous cycles, especially in the proestrus day. The normal levels were re-established the 5th day after treatment. The ethanol produced a byphasic effect on pituitary prolactin content. During the first post-treatment hours, the pituitary prolactin concentration decreased with respect to the control group, but 24 hours after treatment, these values were increased and the normal concentration was restored 36 hours after treatment.

Rapid turbidimetric determination of serum C3c and C4 by end point centrifugal analysis.

An end point turbidimetric method for the determination of C3c and C4 in serum using a centrifugal analyzer (Cobas Bio) is described. Several analytical factors were evaluated -pH, temperature, PEG and antibody concentration. Wide variations of temperature and pH did not significantly affect the turbidimetric reaction. A 20 g/L PEG concentration and 25-fold antiserum dilution were found satisfactory for the analysis. Precision of the assay was good and comparison with a RID method yielded an r value of 0.97. The procedure is simple and reliable.