Ramón B. Rodríguez

The use of recombinant gilthead sea bream (Sparus aurata) growth hormone for radioiodination and standard preparation in radioimmunoassay.

A gilthead sea bream growth hormone (sbGH) obtained by cloning and expression of sbGH cDNA was used to develop a sensitive and specific radioimmunoassay (RIA). Iodination of recombinant sbGH (rsbGH) was performed by the classical Chloramine-T method. Specific antiserum, raised in rabbits, was added in a final dilution of 1/36,000. The minimum detectable dose was 30 pg, and the midrange of the assay (ED50) was 275 pg. Intra- and inter-assay coefficients of variation (CV) were 3.3 and 5.8% at ED50 levels. Human GH (hGH), ovine GH (oGH), carp gonadotropin (cGtH), chinook salmon gonadotropin (sGtH), ovine prolactin (oPRL) and recombinant tilapia prolactin (rtiPRL) did not show cross-reactivity. Serial dilutions of chinook salmon GH (sGH) and recombinant rainbow trout GH (rtGH) showed a low but significant cross-reactivity. A good parallelism between rsbGH standard and serial dilutions of native sbGH, plasma and pituitary extracts was observed. In addition, when plasma and pituitary samples were analyzed for GH quantification, non-significant differences were observed within this and previous RIA for native sbGH. Therefore, it appears conclusive that our rsbGH can be used successfully as a standard and radioiodinated hormone in GH assays for gilthead sea bream, which is extensively cultured in the Mediterranean area.

Podocalyxin enhances the adherence of cells to platelets

Abstract.Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Δ) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin αVβ3/αVβ5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid.

A novel homozygous splice junction mutation in GPIIb associated with alternative splicing, nonsense-mediated decay of GPIIb-mRNA, and type II Glanzmann's thrombasthenia

Summary.  This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmanns thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (≤ 10% of control) platelet content of GPIIb–IIIa, confirming it was indeed a type II GT. The molecular genetic analysis of the proband revealed the presence of a homozygous G188A transition in GPIIb. This mutation alters the consensus sequence of the splice donor site of intron 1 changing arginine 63 for lysine (R63K). No other mutation than [G188A]GPIIb was found in the proband and her parents after complete analysis of GPIIb and GPIIIa coding sequences, and the promoter, 3′‐UTR, and intronic flanking regions of GPIIb. The GT phenotype of the proband is the result of a limited availability of GPIIb‐mRNA. The etiopathogenic role of the [G188A]GPIIb mutation is supported by the following observations: (i) both parents, who are heterozygous for the [G188A]GPIIb mutation, show a marked decrease in the platelet content of GPIIb‐mRNA; (ii) exontrap analysis demonstrated that the G188A mutation leads to a marked reduction in the steady‐state level of GPIIb‐mRNA. The reduced availability of platelet GPIIb‐mRNA associated with the G188A mutation seems to be caused by either inefficient RNA splicing or a preferred utilization of alternative intronic donor sites that generate an in‐frame STOP codon with the result of activation of nonsense‐mediated mRNA decay, or both.

Autoradiographic localization of growth hormone binding sites in Sparus aurata tissues using a recombinant gilthead seabream growth hormone

Abstract Growth hormone (GH) binding sites in tissues of gilthead seabream, Sparus aurata , were localized by using an in vitro autoradiographic analysis. Cryomicrotome sections of liver, spleen, kidney, heart and skeletal muscle were mounted on gelatin-coated slides and incubated with a radioiodinated recombinant gilthead seabream GH (0.54 ng/100 μl buffer) for 24 h at 4 °C. Slides were coated with autoradiographic emulsion and exposed for 1–9 weeks. The highest density of GH binding sites was found in liver. This specific binding was evident on hepatocytes. Blood cells in hepatic vascular system also showed GH binding while intrahepatic exocrine pancreas did not. In spleen, a high density of GH binding sites was found surrounding melanomacrophage centers. Splenic ellipsoid cells also showed a strong GH binding while splenic pulp exhibited a weak GH binding. Kidney GH binding sites were present in renal tubules and renal haemopoietic tissue. Cardiac muscle exhibited an intense GH binding. However, GH binding sites were not detected in body skeletal muscle. This study confirms and extends, by using autoradiographic techniques, previous results obtained by radioreceptor assays.