A SURVEY FOR 1,3,6,7-TETRAHYDROXY-C-GLYCOSYLXANTHONES EMPHASIZING THE “PRIMITIVE” LEPTOSPORANGIATE FERNS AND THEIR ALLIES

A survey for 1,3,6,7-tetrahydroxy-C-glycosylxanthones of representative species within the primitive vascular plants, emphasizing the leptosporangiate ferns, has indicated a limited distribution of these compounds within three leptosporangiate families: Hymenophyllaceae, Aspleniaceae and Marsileaceae. In the Hymenophyllaceae the distribution of these compounds appears to be a useful criterion for segregating species of Mecodium from other species of Hymenophyllum (sensu lato) and suggests that the tubulate vs. the valvate indusial condition may not be an ideal character for separating all species of Hymenophyllum (s.l.) from those of Trichomanes (s.l.). These compounds appear useful for delimiting several species of Elaphoglossum section Pachyglossa and support a relationship among the Aspleniaceae, Athyriaceae, and Elaphoglossaceae. Their presence in Marsilea also raises questions as to the origin of this group of plants.     

Studies on the characteristics of alginate gels in relation to their use in separation and immobilized applications

Studies on diffusion of NAD and hemoglobin from calcium and barium gels are reported where alginate grade, concentration, and gel dimensions were varied. These show that NAD diffusion characteristics are unaffected by alginate and ion concentrations; however, hemoglobin diffusion is affected by alginate concentration. Both hemoglobin and NAD diffusion patterns were shown to be affected by alginate gel dimensions. Studies are reported that show that polymannuronic alginate gels posses good porosity characteristics while polyguluronic alginates from gels with lower porosity, specifically with respect to high-molecular-weight compounds. These findings are discussed with the view to the use of alginate gels for immobilization, solids separation, and diffusion chromatography techniques.     

Platelet-derived growth factor and transforming growth factor-beta regulate plasminogen activator inhibitor-1 synthesis in vascular smooth muscle cells

Bovine vascular smooth muscle cells (SMC) were examined for production of plasminogen activator inhibitor-1 (PAI-1) which may play a key role in regulating the fibrinolytic system. Growth-arrested SMC released active PAI (101 arbitrary units (AU)/10(6) cells/24 h) and a latent form of PAI (880 AU/10(6) cells/24 h) into the conditioned medium (CM). The levels of PAI were significant since 880 AU of PAI could inhibit approximately 1 microgram of tissue plasminogen activator. The extracellular matrix of SMC also contained PAI activity; however, the level was 17-fold less than that observed in the CM. SMC-PAI was a rapid inhibitor of tissue plasminogen activator (kass greater than 10(7) M-1 S-1) and was identified as a 45-kDa protein immunologically related to endothelial cell PAI-1. PAI-1 comprised 20 and 30%, respectively, of the newly synthesized protein detected in the CM and extracellular matrix of SMC. The SMC growth modulators, platelet-derived growth factor and transforming growth factor-beta, induced PAI-1 activity and protein synthesis by 2- and 3-fold, respectively, in a dose- and time-dependent manner. The increases in PAI-1 activity and protein synthesis were ascribed to elevated levels of PAI-1 mRNA as judged by Northern blot analysis of total RNA prepared from control and platelet-derived growth factor- and transforming growth factor-beta-treated cells. Increases in PAI-1 mRNA levels were evident 1 h after growth factor treatment and were maximal after 4 h. PAI-1 mRNA levels were unaffected by cycloheximide treatment. The results indicate that SMC synthesize and release PAI-1 which could regulate the normal fibrinolytic environment of the arterial wall. During atherosclerosis or after vascular injury increases in platelet-derived or locally produced mitogens may stimulate further PAI-1 synthesis and generate a prothrombotic state.     

The inhibition of tissue type plasminogen activator by plasminogen activator inhibitor-1. The effects of fibrinogen, heparin, vitronectin, and lipoprotein(a).

Plasminogen activator inhibitor-1 (PAI-1) regulates fibrinolysis by inhibiting tissue type plasminogen activator (t-PA). Fibrinogen, heparin, and vitronectin enhance the rate of inhibition of t-PA by PAI-1. Kinetic studies indicate that both fibrinogen and heparin increase the second-order inhibition constant by a maximum of approximately 4-fold, whereas vitronectin increases the rate constant by a maximum of approximately 6-fold. The dissociation constants of fibrinogen, heparin, and vitronectin for the inhibition reaction were 200 nM, 20 nM, and 600 pM, respectively. In addition, PAI-1 inhibition of t-PA may be regulated by the presence of lipoprotein(a) (Lp(a)). Previous studies demonstrated that Lp(a) competes with plasminogen for the active site of fibrinogen- and heparin-bound t-PA. Kinetic studies described here demonstrate that Lp(a) prevents the inhibition of t-PA by PAI-1 in the presence of fibrinogen and heparin, but has no effect on the reaction in the presence of vitronectin or in the absence of either fibrinogen or heparin. The data suggest that fibrinogen and heparin may enhance the rate of inhibition through an interaction with t-PA, and that vitronectin may enhance the inhibition through an interaction with PAI-1. In addition, these experiments indicate that Lp(a) may regulate fibrinolysis by competing with PAI-1 and plasminogen for fibrinogen- and heparin-bound t-PA. These data suggest that PAI-1 inhibition of t-PA in vivo is primarily mediated via interaction with fibrinogen, heparin, vitronectin, and Lp(a), and therefore, the functional levels of PAI-1 activity in the vasculature may be regulated by the presence of these components.     

Plasminogen activator inhibitor-1 binds to fibrin and inhibits tissue-type plasminogen activator-mediated fibrin dissolution.

Plasminogen activator inhibitor-1 (PAI-1) accumulates within thrombi and forming whole blood clots. To explore this phenomenon at the molecular level, PAI-1 binding to fibrin was examined. The experiments were performed by adding 125I-PAI-1, which retains its complete tissue-type plasminogen (t-PA) inhibitory activity, to fibrin matrices formed in 2-cm2 tissue culture wells. Guanidine HCl-activated PAI-1 binding was reversible and was inhibited in the presence of excess, unlabeled PAI-1. Activated 125I-PAI-1 recognized 2 sites on fibrin: a very small number of high affinity sites (Kd less than 1 nM) and principally a large number of low affinity sites with an approximate Kd of 3.8 microM. Latent PAI-1 bound to fibrin at a site indistinguishable from the lower affinity site recognized by activated PAI-1. Fibrin, pretreated with activated PAI-1, was protected from t-PA-mediated plasmin degradation in a PAI-1 dose-responsive manner (IC50 = 12.3 nM). Clot protection correlated with partial occupancy of the low affinity PAI-1 binding site on fibrin and was due to the formation of sodium dodecyl sulfate-stable, PAI-1.t-PA complexes. Latent PAI-1 (27 nM) did not protect the fibrin from dissolution. The localization of PAI-1 to a thrombus by virtue of its fibrin binding potential could result in significant protection of the thrombus from the degradative effects of the fibrinolytic system.     

Factors affecting chloride conductance in apical membrane vesicles from human placenta

Summary Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. ³⁶Cl^– uptake into these vesicles was studied in the presence of an outwardly directed Cl^– gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 m valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl^– were examined in the presence and absence of 5 m valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC₅₀ values of 0.83±0.35 m and 3.43±0.37 m, respectively, in the absence of valinomycin. Although valinomycin reduced ³⁶Cl^– uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC₅₀ of 5.6±2.1 m. Under these conditions, NPA was without effect at concentrations below 100 m. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced ³⁶Cl^– uptake significantly at 100 m concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed.The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl^– uptake with IC₅₀ values of 37.6±14.9 m and 4.59±0.51 m, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl^– gradient and this channel is sensitive to Cl^– channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.This work was supported in part by the Cystic Fibrosis Association of Ireland and Eolas, The Irish Science and Technology Agency. The technical assistance of Mr. Cormac O' Connell in the preparation of the electron micrographs and of Mr. Roddy Monks in the flow cytometric analysis is gratefully acknowledged.     

Comparative biodistribution and antibody-dependent cellular cytotoxicity of native and heavy chain chimeric antibody.

We have recently chimerized the heavy chain of the pan-carcinoma monoclonal antibody (mAb) B72.3. Studies were undertaken to compare the IgG1 chimeric antibody, B72.3-1-3 with native murine B72.3 (nB72.3). Using fluorescence-activated cell sorting analysis, B72.3-1-3 demonstrated specific binding to fresh LS174T tumor cells. Biodistribution of 131I B72.3-1-3 was similar to 131I nB72.3 in nude mice bearing LS174T xenografts. Peak radiolocalization indices were noted on day 6 for B72.3-1-3 and day 8 for nB72.3. Both antibodies were capable of imaging LS174T tumors by radioimmunoscintigraphy. Antibody-dependent cellular cytotoxicity of LS174T by human peripheral blood lymphocytes was tested in 8h 51Cr release assays. With either no antibody or nB72.3, lymphocytes were not capable of killing LS174T cells. However, B72.3-1-3 at a concentration of 5 and 50 micrograms/ml mediated significant lysis of tumor cells by human lymphocytes. These results suggest that chimeric antibodies retain their binding properties to tumor cells and display biodistribution patterns similar to their unmodified counterparts. Such modifications may reduce the deleterious human antimouse antibody response to murine mAbs as well as augment antibody-dependent cellular cytotoxicity of tumor cells by human effectors.