Platelet plasminogen activator inhibitor: purification and characterization of interaction with plasminogen activators and activated protein C

Plasminogen activator inhibitor (PAI) was purified in active form from porcine platelets under nondenaturing conditions. The purified inhibitor (Mr 47,000) reacts with tissue-type plasminogen activator (t-PA), urokinase (UK), and activated protein C (APC) to yield both SDS-stable complexes and a modified PAI of slightly reduced molecular weight. The second-order rate constants for the inhibition of t-PA and UK by PAI are 3.5 X 10(7) and 3.4 X 10(7) M-1 s-1, respectively. Activated protein C reacts with PAI with a second-order rate constant of 1.1 X 10(4) M-1 s-1. This rate is not accelerated by protein S, phospholipid, and calcium, or heparin. It is concluded that (1) PAI can function as both inhibitor and substrate of its target proteases, (2) if APC promotes fibrinolysis via inactivation of PAI, then APC must be present in concentrations several orders of magnitude greater than t-PA, or the interaction of APC and PAI must be accelerated by presently unknown mechanisms, and (3) in the absence of heparin, platelet PAI is the most rapid inhibitor of APC yet described.     

Effect of micelle diameter on tryptophan dynamics in an amphipathic helical peptide in phosphatidylcholine

The effect of dimyristoylphosphatidylcholine (DMPC) on the conformation and environment of the single tryptophan residue of a model amphipathic helical polypeptide has been investigated by fluorescence quenching with a water-soluble, neutral quencher (acrylamide) and multiple-frequency phase fluorometry. The peptide H-Ser-Ser-Ala-Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Ly s-Glu- Ala-Phe-Ser-Ser-Ser-OH [18As; Kanellis, P., Romans, A.Y., Johnson, B.J., Kercret, H., Chiovetti, R., Jr., Allen, T.M., & Segrest, S.P. (1980) J. Biol. Chem. 255, 11464] was synthesized by solid-phase techniques. Peptide was incubated at 26 degrees C with DMPC at various peptide:lipid weight ratios. The diameter of the resulting disk-shaped micelles increases with increasing lipid concentration from 12.0 +/- 0.4 nm at a 1:1 weight ratio of peptide to lipid to a maximum of 48.7 +/- 1.0 nm at a 1:13 ratio. At a weight ratio of 1:5, the average diameter is 22.7 +/- 0.6 nm. Decreasing the peptide:lipid ratio of the micelle resulted in a blue-shift in the fluorescence emission maximum (from 337 nm at 1:1 to 334 nm at 1:5), an increase in the fluorescence lifetime of the tryptophan measured by the phase shift method at 18 MHz (from 3.12 ns at 1:1 to 3.61 ns at 1:5), a decrease in the rate of fluorescence quenching by acrylamide (from 0.87 x 10(9) M-1 s-1 at 1:1 to 0.42 x 10(9) M-1 s-1 at 1:5), and an increase in the activation energy for quenching (from 6.7 kcal/mol at 1:1 to 12.7 kcal/mol at 1:5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies to confirm the source of 11ß-hydroxyandrostenedione

In a longitudinal study of 82 children we found a gradual rise in median plasma concentrations of 11ß-hydroxyandrostenedione (11ß-OH-A4) from 2.5 to 6.4 nmol/1 during childhood which was similar in both sexes. This could reflect changes in adrenal function during the adrenarche and sexual maturation. Plasma concentrations of 11ß-OH-A4 in adults follow the patterns of cortisol secretion. In patients with diseases of the adrenal cortex, the plasma concentrations of 11ß-OH-A4 were consistent with the pathology of each condition. In women with polycystic ovaries (PCO) undergoing gonadotrophic stimulation for in vitro fertilization and embryo transfer, 11ß-OH-A4 (median = 3.8 nmol/l), testosterone and androstenedione, were raised when compared to women with normal ovaries (11ß-OH-A4 median = 2.6 nmol/l). Follicular fluid has concentrations of 11ß-OH-A4 six to twelve times greater than plasma levels and in women with PCO, 11ß-OH-A4 concentrations were lower than in women with normal ovaries, which is consistent with an inhibition of ovarian 11ß-hydroxylase. Granulosa cells in vitro demonstrated the production of 11ß-OH-A4 by side chain cleavage of cortisol. These data support an adrenal source for 11ß-OH-A4 but the raised plasma concentrations in women with polycystic ovary syndrome (PCOS) may reflect the excess androgen output from the ovary. 11ß-OH-A4 may therefore be an additional marker for ovarian dysfunction.     

The kinetics of generation of influenza-specific cytotoxic T-lymphocyte precursor cells

A dramatic increase in the number of restimulatable (memory) cytolytic T-cell precursors (CTLps) has been demonstrated to occur 2 to 4 days postimmunization. By 4 or at the latest 6 days following in vivo priming, the frequency of restimulatable influenza-specific CTLps has reached that characteristic of a memory response. This indicates that the proliferative events which give rise to memory CTLps occur earlier than might have been expected and in fact are completed prior to the cessation of othe primary CTL response. Such information may have important significance to future immunomodulatory efforts aimed at perturbing the establishment of T-cell memory in vivo.     

Response of Peyer's patch lymphocyte subsets to Giardia muris infection in BALB/c mice. II. B-cell subsets: enteric antigen exposure is associated with immunoglobulin isotype switching by Peyer's patch B cells

The aim of this study was to quantify the response of Peyer's patch B cells, surface IgA-bearing (sIgA) B cells, and surface IgM-bearing (sIgM) B cells to Giardia muris infection. Following infection of a cohort of immunocompetent BALB/c mice with G. muris cysts, Peyer's patch cell suspensions were prepared at serial time points during the infection, incubated with fluorescein-conjugated monoclonal antibodies directed against murine leukocytes, B cells, sIgA B cells, sIgM B cells, or T cells, and analyzed by flow cytometry. Of total Peyer's patch leukocytes, the percentages of B cells, sIgA B cells, and sIgM B cells in uninfected BALB/c mice were 64.7 +/- 2.0% (mean +/- SEM), 30.3 +/- 1.5%, and 52.5 +/- 2.4%, respectively. The total number of Peyer's patch leukocytes increased significantly (1.8 X) during G. muris infection, and returned to control levels as the infection was cleared. The percentages of Peyer's patch T and total B cells did not change significantly during Giardia infection. However, sequential changes were observed in the percentages and numbers of sIgM and sIgA B cells during the infection. Peyer's patch sIgM B cells rapidly increased in percentage and number, reaching maximum levels 1 week after cyst inoculation. After remaining constant the first week, the number of Peyer's patch sIgA B cells increased during the second week of G. muris infection, reaching a maximum level 11-14 days after cyst inoculation. The data support the hypothesis that immunoglobulin isotype switching in Peyer's patches is induced by antigen exposure.     

Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.     

Leg chaetotaxy and the classification of the Uropodina (Acari: Mesostigmata)

Details of the segmental chaetotaxy of the legs of 47 species of Uropodina are given. On the basis of the ontogenetic development of the chaetotaxy, the Uropodina may be divided into two groups which coincide with the concepts of the Lower (Polyaspidoidea) and Higher (Uropodoidea) Uropodina of certain authors. Chaetotactic criteria do not support the classification of the Polyaspidini and Trachyuropodini sensu Hirschmann and Z-Nicol within the Oplitinae Hirschmann & Z-Nicol, the Diarthrophallini within the Uroactiniinae Hirschmann & Z-Nicol or the genus Trachytes within the Uropodini.
A critical appraisal is given of the classification of the Uropodidae (based on "Gangmerk-male") by Hirschmann and Z-Nicol.     

The identification of a distinct export step following the biosynthesis of leukotriene C4 by human eosinophils

We have examined the requirements for the export of leukotriene C4 (LTC4) from cultured human eosinophils. To define saturability and kinetics of LTC4 export, eosinophils were interacted with leukotriene A4 (LTA4) at 37 degrees C, and the methanolic extracts of the cell-associated and extracellular compartments were then analyzed for LTC4 content by reverse phase high performance liquid chromatography with on-line monitoring of absorbance at 280 nm. When LTA4 was added at concentrations from 0 to 100 microM for 10 min at 37 degrees C, the amount of LTC4 released extracellularly became constant at an LTA4 concentration of 7.5 microM or greater even though the amount of intracellular LTC4 continued to increase. When eosinophils were incubated with 50 microM LTA4 for 0-60 min at 37 degrees C and then held at 0 degrees C for the remainder of the 60-min interval, 54.2 and 77.3% (n = 3), respectively, of the total LTC4 was released extracellularly after 15 and 30 min of incubation at 37 degrees C. Eosinophils incubated with 50 microM LTA4 at 0 degrees C for 1 h synthesized 290 pmol of LTC4 (n = 3) which was approximately half-maximal, all of which was retained intracellularly. We utilized the time and temperature dependence of LTC4 export to preload eosinophils with both LTC4 and leukotriene C5 (LTC5) by sequentially supplying them with specific substrates. With increasing concentrations of intracellular LTC5, there was dose-dependent inhibition of the subsequent release of LTC4 at 37 degrees C, with the sum of the released glutathionyl leukotrienes remaining constant. In addition, only minimal competition for LTC4 release occurred when cells were preloaded with both LTC4 and the conjugate of 1-chloro-2,4-dinitrobenzene and reduced glutathione, S-(dinitrophenyl)glutathione. The criteria of saturability, time dependence of LTC4 release at 37 degrees C, competition of LTC4 with LTC5 for release, and the inhibition of LTC4 release at 0 degrees C establish the export of LTC4 from cells as a novel and specific biochemical step distinct from both LTA4 uptake and the conjugation of LTA4 with reduced glutathione by LTC4 synthase to form LTC4.