Bidirectional transbilayer lipid movement in human platelets as vizualized by the fluorescent membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene

Transbilayer movement of the fluorescent membrane probe TMA-DPH [1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene] in the plasma membrane of human platelets was investigated by measuring fluorescence intensity and fluorescence decay. Labeling of unstimulated platelets by TMA-DPH results in a rapid increase in fluorescence intensity, leveling off within 1 min. Dilution of platelets into buffer without TMA-DPH leads to an almost complete rapid efflux of TMA-DPH, indicating that TMA-DPH labels only the outer leaflet of the plasma membrane. Transbilayer movement of the fluorescent probe in unstimulated platelets could be observed upon prolonged incubation and occurs with a t1/2 of 60-90 min. Stimulation of platelets with thrombin directly after the initial rapid uptake of TMA-DPH results in a fast increase in membrane-bound TMA-DPH, fully explained by the increase in plasma membrane caused by secretion of intracellular storage organelles. No indications for increased transbilayer movement of the probe were found, since dilution of thrombin-stimulated TMA-DPH-labeled platelets into buffer without TMA-DPH indicated no uptake of TMA-DPH by intracellular membranes. In contrast to thrombin, stimulation of TMA-DPH-labeled platelets with the Ca2(+)-ionophore ionomycin results in a much larger increase in fluorescence intensity. This process is accompanied by labeling of intracellular membranes as indicated by incomplete efflux of TMA-DPH after dilution of the stimulated platelets. Thus, stimulation of platelets by ionomycin gives rise to rapid and massive inward movement of TMA-DPH (t1/2 approximately 10-12 s). Prolonged incubation of platelets in the absence of any stimulus allows labeling of the total lipid pool, including intracellular membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry?

The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A23187, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca2+ dose-response curves of both activities in intact platelets, obtained by stimulation with A23187 in the presence of Ca2+/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for prothrombinase activity in the presence of saturating concentrations of factors Xa, Va, and prothrombin. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their fragments as judged from protein patterns after gel electrophoresis. Platelet stimulation by A23187 resulted in a fast increase in prothrombinase activity, reaching its maximum level after about 20 seconds. Filamin and talin were completely hydrolysed within 15 s, and myosin was partly degraded between 15 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, prothrombinase activity was generated with a sigmoid time course, the steepest increase being observed between 1 and 2 min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and 1.5 min after platelet activation, while degradation of myosin became visible after 2 to 2.5 min. Dibucaine and tetracaine were both found to be potent stimulators of prothrombinase activity, with half-maximal activities obtained at 0.7 and 2.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of prothrombinase activity closely paralleled that of calpain activity over a time course of 1 hour. Ca2+ titration of intact platelets using A23187 and Ca2+/HEDTA buffers, revealed half-maximal response at about 15 microM free Ca2+ for both calpain and prothrombinase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.     

The distribution of molecular classes of phosphatidylglycerol in the membrane of Acholeplasma laidlawii

A double-label technique has been applied to study the distribution of different molecular classes of phosphatidylglycerol in the membrane of Acholeplasma laidlawii. After growth on oleic acid, 16% of the total phosphatidylglycerol contains two oleic acid residues and 84% contains one oleic acid and one saturated fatty acid. The dioleoyl phosphatidylglycerol is present in equal amounts in the outer and the inner layer of the membrane. Phosphatidylglycerol which is associated with membrane proteins consists exclusively of the class containing only one oleic acid.     

Subcellular localization of DNA-binding protein BA by immunofluorescence and immunoelectron microscopy

Nonhistone protein BA has been shown to decrease in amount in the chromatin of growth- stimulated normal rat liver (Yeoman et al. 1975. Cancer Res. 35:1249-1255) and in mitogen-stimulated normal human lymphocytes (Yeoman et al. 1976. Exp. Cell Res. 100:47- 55.). Subsequently, protein BA was purified and was shown to prefer to bind to double- stranded A-T-rich DNAs (Catino et al. 1978. Biochemistry. 17:983-987.). Immunization of rabbits with highly purified protein BA has resulted in the production of a specific antibody. A specific immunoreactivity for chromosomal protein BA has been demonstrated by immunoelectrophoresis and double antibody immunoprecipitation analysis with rabbit anti-BA immunoglobulin and IgG fractions. Light microscope examination of normal rat liver crysections by the indirect immunofluorescence procedure has demonstrated a cytoplasmic as well as a nuclear localization for protein BA with a pronounced perinucleolar fluorescence. Immunoelectron microscopy employing the peroxidase antiperoxidase method of antigen localization has confirmed the immunofluorescence data and has show a heterochromatin localization for protein BA. The relationship of the localization of protein BA to gene control in quiescent cells or to configurations of heterochromatin as well as the marked reduction in the amounts of protein BA which occur in stimulated growth states remains to be defined.     

Nuclear and cytoplasmic maturation of sow oocytes are not synchronized by specific meiotic inhibition with roscovitine during in vitro maturation

The effect of roscovitine exposure prior to IVM was studied on cumulus expansion, on changes of cumulus-oocyte contacts and on nuclear and cytoplasmic maturation of sow oocytes. It was hypothesized that delayed nuclear maturation and prolonged contact with cumulus cells allows prolonged cytoplasmic differentiation and therefore improves oocyte developmental potential. Cumulus-oocyte complexes (COCs) were exposed for 22 h or 44 h to 0, 25 or 50 microM of roscovitine and subsequently cultured for 22, 29 or 44 h without roscovitine. COCs were examined for cumulus expansion and oocytes for nuclear status and dynamics of transzonal microfilaments. Oocyte developmental potential was assessed by blastocyst formation after IVF. Fifty muM of roscovitine inhibited cumulus expansion for the first 22 h of culture, and maintained oocytes in meiotic arrest for 44 h. Roscovitine treatment during 22 h prior to culture for 44 h without roscovitine did not increase embryo development, but oocytes cultured for 66 h without roscovitine had reduced blastocyst formation. Oocytes cultured for 29 h after roscovitine exposure showed reduced blastocyst rates compared with their counterparts cultured for 44 h. Roscovitine treatment during 44 h prior to culture for 22 h or 44 h without roscovitine reduced embryo development. Transzonal microfilaments were reduced after culture with roscovitine, and disappeared during culture without roscovitine. It is concluded that prolonged contact with cumulus cells does not improve oocyte developmental potential. Furthermore, it is suggested that nuclear and cytoplasmic maturation in vitro cannot be seen as two independent processes.