Selection of housekeeping genes for normalization by real-time RT-PCR: analysis of Or-MYB1 gene expression in Orobanche ramosa development.

Real-time PCR has become the method of choice for accurate and in-depth expression studies of candidate genes. To avoid bias, real-time PCR is referred to one or several internal control genes that should not fluctuate among treatments. A need for reference genes in the parasitic plant Orobanche ramosa has emerged, and the studies in this area have not yet been evaluated. In this study, the genes 18S rRNA, Or-act1, Or-tub1, and Or-ubq1 were compared in terms of expression stability using the BestKeeper software program. Among the four common endogenous control genes, Or-act1 and Or-ubq1 were the most stable in O. ramosa samples. In parallel, a study was carried out studying the expression of the transcription factor Or-MYB1 that seemed to be implicated during preinfection stages. The normalization strategy presented here is a prerequisite to accurate real-time PCR expression profiling that, among other things, opens up the possibility of studying messenger RNA levels of low-copy-number-like transcription factors.

Analysis of swine beta1 integrin (CD29) epitopes through monoclonal antibodies developed using two immunization strategies.

This report describes the production and characterization of monoclonal antibodies (MAbs) to swine beta1 integrin subunit (CD29) using two different immunization strategies. MAb GP4B4 was developed from a mouse immunized with porcine peripheral blood mononuclear cells (PBMC), while MAbs GP1A5, GP1A6, and GP4A1 were produced by immunizing mice with a porcine CD29 recombinant protein (rpCD29) developed in our laboratory, which includes the ligand binding site. GP4B4 and UCP1D2 (specific to porcine CD29) immunoprecipitated two bands of approximately 115 and 150 kDa under reducing conditions. The molecule recognized by these two antibodies was studied using flow cytometry and was found in practically all cells studied, displaying a similar reaction pattern. Western blot assays performed with rpCD29 indicated that MAbs GP1A5, GP1A6, and GP4A1 recognized the 30-kDa band for this recombinant protein, confirming their specificity for the beta1, integrin subunit. Immunohistochemical analyses of these MAbs disclosed a morphological pattern associated with smooth muscle, epithelium, and myeloid cells, as expected in MAbs recognizing CD29. This MAb panel could be useful as a general anti-CD29 reagent and would allow further research into this important integrin in swine.

Molecular cloning, expression pattern and chromosomal mapping of pig CD9 antigen.

CD9 is a member of the transmembrane-4 superfamily of surface molecules that seems to have a relevant role in cell migration and adhesion, as well as malignant progression. This work describes the isolation of the cDNA coding for the porcine CD9 molecule. Pig CD9 cDNA was isolated from a smooth muscle cDNA library and contains a 678-bp open reading frame with its predicted polypeptide sequence of 226 amino acids. The deduced amino acid sequence conserves the main characteristics of TM4 proteins, including the presence of four transmembrane domains. Like their homologous molecules from other species, pig CD9 has two extracellular regions of a different size with the minor loop bearing two possible glycosylation sites. The pig CD9 gene was localized to chromosome 5q25 by using a somatic cell hybrid panel. Analysis of CD9 expression in different porcine cells and tissues demonstrated that CD9 mRNA is ubiquitously expressed.

Swine platelet antigens: section report.

A total of 14 antibodies were found to label resting and/or activated swine platelets. Six recognized CD previously characterized for swine (CD29, CD41/61 and CD46). One had been characterized for human cells (CD47). Two antibodies with CD14 and SLA class I specificity suggested by the donor as well as five blind antibodies were also positive on platelets. One antibody appeared to recognize the swine homologue to human CD47, and four remained unclustered.

[Evaluation of medical reports received from the second level of care]. / Valoración de los informes médicos recibidos del segundo nivel.

OBJECTIVES: To quantify the monthly inter-clinic rate (MIR), the medical reports received, their quality and the period of delay until the first second-level consultation. DESIGN: Cross-sectional, descriptive study. SETTING: Rural health centre. SAMPLE: 498 first consultations requested of the second level by three family medicine lists between June and November 1999. 132 were excluded for presenting a criterion of exclusion (consultations within the second level, check-ups, appointments missed by the patient and consultations that could not be recorded), which left a sample size of 366. MEASUREMENTS AND RESULTS: Mean MIR was 34 . Ophthalmology (21.9%), gynaecology (15.3%) and traumatology (13.9%) were the most commonly requested services. 69 reports (18.8%) were received, leaving 297 (81.2%) not received. Pneumology (100%) and internal medicine (81.8%) services sent through most reports, whereas haematology and rehabilitation (0%) sent through least. The mean score on the reports was 8 +/- 2 out of a maximum of 10. Mean delay was 73 +/- 46 days. CONCLUSION: The monthly inter-clinic rate found was within the figures cited in the literature. The percentage of reports received was way below what was found in the literature, but their quality was good.

Valoración de los informes médicos recibidos del segundo nivel / Evaluation of the medical reports received from the second care level

Objetivos. Cuantificar la tasa de interconsulta mensual (TIM), porcentaje de informes médicos recibidos, calidad de éstos y tiempo de demora de la primera consulta al segundo nivel. Diseño. Estudio descriptivo, transversal. Emplazamiento. Centro de salud rural. Muestra. Las 498 primeras consultas solicitadas al segundo nivel de 3 cupos de médicos de familia desde junio a noviembre de 1999. Se excluyeron 132 por presentar algún criterio de exclusión (consultas entre el segundo nivel, revisiones, consultas a las que el paciente no acudió y aquellas que no se pudieron registrar), quedando un tamaño muestral de 366 (n).Mediciones y resultados. La TIM media fue del 34 . Los servicios de oftalmología (21,9 por ciento), ginecología (15,3 por ciento) y traumatología (13,9 por ciento) fueron los más solicitados. Se recibieron 69 informes (18,8 por ciento), quedando 297 (81,2 por ciento) sin recibir. Los servicios de neumología (100 por ciento) y medicina interna (81,8 por ciento) fueron los que más informes remitieron, mientras que hematología y rehabilitación (0 por ciento) fueron los que menos. La puntuación media de los informes fue de 8 ñ 2 sobre un máximo de 10. La demora media fue de 73 ñ 46 días. Conclusión. La TIM se encuentra entre los valores descritos en la bibliografía. El porcentaje de informes recibidos es muy inferior a lo hallado en la bibliografía, pero su calidad es buena (AU)

Molecular cloning and characterization of the pig homologue to human CD29, the integrin beta1 subunit.

BACKGROUND: CD29 is the beta1 subunit, a member of the integrin gene superfamily that function as receptor for cell adhesion molecules of the extracellular matrix. Porcine integrin beta1 subunit is involved in rejection of pig-to-human tissue xenografts as target of the natural antibodies present in the human serum. Moreover since CD29, as part of the beta1 integrins very late antigen 4 (VLA-4) and VLA-6, is involved in homing and differentiation of haematopoietic progenitor cells, its characterization in pig is critical to study the interaction of porcine adhesion molecules with human ligands in the induction of donor-specific tolerance toward porcine antigens, a process extremely desirable to prevent rejection of xenogeneic organs. METHODS: The porcine CD29 cDNA has been isolated from a cDNA library and its structure determined. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the expression of CD29 in different tissues. RESULTS: The nucleotide sequence of the porcine cDNA includes an open reading frame encoding a polypeptide of 798 amino acids. Expression analysis showed that porcine CD29 is expressed in all lymphoid tissues tested and, in lower amounts, in nonlymphoid tissues. Pig CD29 deduced amino acid sequence displays extensive conservation compared with CD29 sequences from other species and a common structural feature with all the other CD29 molecules analyzed in mammals, including the 12 potential N-glycosilation sites. Punctual changes between human and swine CD29 molecule into the ligand binding domain, and/or into the regulatory domain, suggest potential differences between human and porcine CD29 relative to the human CD29 ligands. CONCLUSIONS: Cloning of the swine CD29 gene offers a new tool for an alternative protocol of removing xenoreactive antibodies in the recipient. In addition, the determination of the differences between human and swine CD29 will help to understand the adhesion molecule-ligand interactions and their function across the swine-human barrier in xenotransplantation.