Dynamic redistribution of glycoprotein Ib/IX on surface-activated platelets. A second look

The present study has re-evaluated the mobility of glycoprotein Ib/IX (GPIb/IX), the von Willebrand factor receptor, on surface-activated platelets. A previous report employing immunogold cytochemistry with monoclonal and polyclonal antibodies specific for GPIb/IX concluded that the receptor remained stabilized in plasma membranes and did not move during platelet attachment and spreading on formvar grids, despite the observation that immunogold particles marking GPIb/IX were missing from peripheral margins and pseudopods of the surface-activated platelets. Addition of thrombin to surface-activated, spread platelets freed GPIb/IX from its anchor to the membrane and stimulated movement of receptor-ligand complexes into caps over centers of spread platelets. In our investigation, surface-activated platelets, stimulated or not by thrombin, were fixed in a higher concentration of glutaraldehyde than used by the earlier workers before exposure to monoclonal or polyclonal antibody to GPIb/IX, after incubation with the antibody, but before treatment with the immunogold marker, protein A gold (PAG), or after both antibody and PAG. When fixed before exposure to antibody and PAG, GPIb/IX receptors were dispersed evenly over dendritic and spread platelets from edge to edge, including peripheral margins and pseudopods. Thrombin had no influence on distribution of the receptors. Exposure to antiglycocalicin antibody before fixation caused movement of GPIb/IX receptors from peripheral margins of spread cells and pseudopods of dendritic forms. Thrombin treatment did not enhance the movement. Fixation after exposure of surface-activated platelets, treated or not with thrombin, to antibody and PAG caused movement of GPIb/IX receptors into caps over cell centers. Results indicate that central movement of GPIb/IX receptors is unrelated to surface activation, spreading, or thrombin stimulation. Rather, the translocation is caused by the antiglycocalicin antibody and accentuated by PAG.     

Identification, characterization, and In situ detection of a fruit-body-specific hydrophobin of Pleurotus ostreatus

Hydrophobins are small (length, about 100 +/- 25 amino acids), cysteine-rich, hydrophobic proteins that are present in large amounts in fungal cell walls, where they form part of the outermost layer (rodlet layer); sometimes, they can also be secreted into the medium. Different hydrophobins are associated with different developmental stages of a fungus, and their biological functions include protection of the hyphae against desiccation and attack by either bacterial or fungal parasites, hyphal adherence, and the lowering of surface tension of the culture medium to permit aerial growth of the hyphae. We identified and isolated a hydrophobin (fruit body hydrophobin 1 [Fbh1]) present in fruit bodies but absent in both monokaryotic and dikaryotic mycelia of the edible mushroom Pleurotus ostreatus. In order to study the temporal and spatial expression of the fbh1 gene, we determined the N-terminal amino acid sequence of Fbh1. We also synthesized and cloned the double-stranded cDNA corresponding to the full-length mRNA of Fbh1 to use it as a probe in both Northern blot and in situ hybridization experiments. Fbh1 mRNA is detectable in specific parts of the fruit body, and it is absent in other developmental stages.     

Effects of exogenous recombinant ovine interferon tau on circulating concentrations of progesterone, cortisol, luteinizing hormone, and antiviral activity; interestrous interval; rectal temperature; and uterine response to oxytocin in cyclic ewes

Interferon tau (IFN tau) is the conceptus-produced antiluteolytic signal in ruminants. Three experiments examined the effects of s.c. administration of recombinant ovine (ro)IFN tau on interestrous interval (IEI), oxytocin (OT)-induced uterine prostaglandin F2alpha metabolite (PGFM) production, rectal temperature (RT), respiration rate (RR), and plasma concentrations of progesterone, cortisol, LH, and antiviral activity (AVA) in plasma and uterine flushings. In experiment I, 20 ewes were treated s.c. with either 0, 1, 2, or 4 mg/day roIFN tau (0.7 x 10(8) U/mg; 5 ewes/dosage) from Days 11 to 15 of the estrous cycle (estrus = Day 0) and were challenged with OT (30 IU) on Day 15. Jugular blood samples were collected at -10, 0, 10, 20, 30, 40, 50, and 60 min relative to the OT challenge and assayed for PGFM. Recombinant oIFN tau increased IEI (16.7, 18.7, and 22.6 +/- 0.6 days for 0, 2, and 4 mg roIFN tau, respectively, p < 0.01). Recombinant oIFN tau did not affect peak PGFM response to OT (2309 +/- 172 pg/ml; p > 0.1). However, the 4 mg/day dosage delayed the time to peak PGFM (32.4 vs. 47.5 +/- 3.4 min; p < 0.01, 0 vs. 4 mg) and resulted in approximately 200% higher concentrations of PGFM at 60 min post-OT (0 vs. 4 mg/day, p < 0.07). Experiment II was similar to experiment I, except that only the 0- and 4-mg/day dosages of roIFN tau were administered. Ewes were hysterectomized on Day 16, and assay of uterine flushes detected no AVA from ewes treated with either 0 or 4 mg/day roIFN tau. In experiment III, 20 ewes were treated s.c. with either 0, 2, 4, or 6 mg roIFN tau on Day 12. Blood samples, RT, and RR were obtained at frequent intervals for 24 h, and plasma was assayed for progesterone, cortisol, LH, and AVA. Plasma AVA, which increased in a dose-dependent manner, was detectable within 60 min and remained elevated at 24 h compared to control values. RT (elevated 0.5-1.0 degrees C), RR, and cortisol increased in response to all dosages of roIFN tau, with peak values occurring 150-180 min postinjection. For all dosages of roIFN tau, plasma progesterone declined from 120 to 360 min posttreatment and then returned to pretreatment values by 24 h (p < 0.01) as compared to controls. Overall, exogenous roIFN tau altered uterine PGFM response to OT from a pulse to a gradual and sustained elevation and extended IEI with only a transient decline in progesterone and mild hyperthermia, effects that are not expected to compromise pregnancy.