Podocalyxin enhances the adherence of cells to platelets.

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-Delta) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin alphaVbeta3/alphaVbeta5, 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.

Production and characterization of murine monoclonal antibodies against human podocalyxin.

Podocalyxin (podxl) is a protein with a peptide bone of approximately 55.5 kDa that undergoes a post-translational glycosylation, yielding a final molecular mass from approximately 145 to approximately 200 kDa. This protein is normally found covering the vascular side of the epithelial glomerular cells, the podocytes, and its presence is essential to maintain a normal renal function. It has also been reported in other cells and tissues although its function has not been yet clarified. The carboxy-terminal intracellular domain of podxl is nearly 100% identical in most species; however, the ectodomain shows considerable variations although the cysteine residues are conserved. Detection of this protein is elusive, most likely due to differences in post-translational modifications. We aimed at producing murine monoclonal antibodies against human podxl. Immunization with Chinese hamster ovarian -hpodxl-green fluorescence protein live cells yielded five different monoclonal antibodies that were characterized by enzyme-linked immunosorbent assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis/western blot, flow cytometry, immunohistochemistry, and immunoprecipitation. The different behavior of these antibodies suggests that some of them may react against epitopes masked by different glycosylated protein moieties.

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

This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (

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.

Cloning, expression, and characterization of a recombinant gilthead seabream growth hormone.

cDNA clones coding for the gilthead seabream (Sparus aurata) growth hormone (sbGH) were isolated from a pituitary expression library using a flounder cDNA probe. The nucleotide sequence of a GH cDNA clone containing an insert of 896 nucleotides was determined. The cDNA encoded a polypeptide of 204 amino acids including a signal peptide of 17 amino acids and contained a 5' and a 3' untranslated region of 48 and 233 nucleotides, respectively. The mRNA determined by Northern blot was approximately 1 kb. Amino acid sequence homologies of 97.1% with red seabream GH, 88.9% with the tuna GH, and 67% with the coho salmon GH was found. Transient expression of a sbGH cDNA was done in HeLa cells by induction with a vaccinia virus system, and the expressed GH was detected by immunofluorescence and immunoprecipitation with a specific antibody to the native sbGH. The sbGH cDNA was expressed in Escherichia coli by using the pGEX-3X and the pET-3a expression systems. The recombinant sbGH expressed in the pET-3a system was similar, if not identical, to the native hormone when analyzed by homologous radioimmunoassay and receptor binding assay.

Cloning of the sole (Solea senegalensis) growth hormone-encoding cDNA.

We report here the complete nucleotide (nt) sequence of a cDNA clone encoding Solea senegalensis growth hormone (sGH) isolated from an expression library prepared from sole pituitary gland poly(A)+RNA. The library was screened using a flounder GH cDNA. The cDNA sequence containing an insert of 769 nt was found to encode a polypeptide of 203 amino acids (aa), including a signal peptide of 17 aa. The 5'- and 3'-untranslated regions of the message are 17 and 119-nt long, respectively. Northern blot hybridization detected a 0.9-kb RNA species. The sGH cDNA sequence shows homologies of 80.9, 76.9, 73.8 and 64.2% with the GH of tuna, gilthead seabream, flounder and rainbow trout.

Protein G-horseradish peroxidase based method for light-microscope immunocytochemistry. Application to the pituitary gland of the killifish, Fundulus heteroclitus.

Horseradish peroxidase-protein G conjugate was used to localize anti-human luteinizing hormone and anti-human chorionic gonadotropin primary antibodies bound to gonadotropins in paraffin and Historesin embedded sections of Fundulus heteroclitus pituitaries. The sensitivity and specificity of this method and those obtained after the avidin-biotin-peroxidase complex, the streptavidin-biotin-peroxidase complex and the peroxidase-antiperoxidase procedures were compared. The protein G-horseradish peroxidase method gave a clear immunostaining of gonadotrophic cells with virtually no background. Detection efficiency was reduced in comparison with the other techniques. As for the embedding medium, hydrophilic resin Historesin considerably enhanced the structural detail and the resolution of the immunostaining at light-microscope level.

[Acute renal failure caused by ceporin, kanamycin and gentamicin]. / Ostraia pochechnaia nedostatochnost', vyzvannaia tseporinom, kanamitsinom i gentamitsinom.

The use of aminoglycosides and cephalosporins is fairly often complicated by acute renal failure (ARF), particularly so if overdoses are used and baseline renal function is impaired. The course of ARF and outcome of treatment have been analyzed in 51 patients. ARF was caused by a nephrotoxic effect of aminoglycosides, cephalosporins or a combination thereof (ARF, type A) in 30 (58.8%) patients, and a combination with other factors (hypotension, arterial hypertension, sepsis) in 21 (41.2%) patients (ARF, type B). Nephrotoxic effect was more commonly produced by a ceporin-gentamicin combination (in 34 (66%) of 51 cases). Nineteen (55.8%) of the 34 patients died, in spite of extracorporeal detoxication treatment (peritoneal dialysis, hemodialysis), which way be attributed to a severe original condition (mostly, due to severe sepsis, original functional renal insufficiency, etc.) rather than the nephrotoxic effect of antibiotics. Hyperazotemia without marked oliguria is a specific feature of ARF, induced by nephrotoxic action of antibiotics. Preventive principles are proposed.