School formation characteristics and stimuli based modeling of tetra fish.

Self-organizing motion is an important yet inadequately understood phenomena in the field of collective behavior. For birds flocks, insect swarms, and fish schools, group behavior can provide a mechanism for defense against predators, better foraging and mating capabilities and increased hydro/aerodynamic efficiency in long-distance migration events. Although collective motion has received much scientific attention, more work is required to model and understand the mechanisms responsible for school initiation and formation, and information transfer within these groups. Here we investigate schooling of black tetra (Gymnocorymbus ternetzi) fish triggered by startle stimuli in the form of approaching objects. High-speed video and tagging techniques were used to track the school and individual members. We then measured several variables including reaction times, group formation shapes, fish velocity, group density, and leadership within the group. These data reveal three things: (1) information propagates through the group as a wave, indicating that each fish is not reacting individually to the stimulus, (2) the time taken for information to transfer across the group is independent of group density, and (3) information propagates across large groups faster than would be expected if the fish were simply responding to the motion of their nearest neighbor. A model was then built wherein simulated fish have a simple 'stimuli/escape' vector based on a hypothetical field of vision. The model was used to simulate a group of individual fish with initial conditions, size, and stimuli similar to the biological experiments. The model revealed similar behavior to the biological experiments and provide insights into the observed patterns, response times, and wave speeds.

Bacterial-mediated DNA delivery to tumour associated phagocytic cells.

Phagocytic cells including macrophages, dendritic cells and neutrophils are now recognised as playing a negative role in many disease settings including cancer. In particular, macrophages are known to play a pathophysiological role in multiple diseases and present a valid and ubiquitous therapeutic target. The technology to target these phagocytic cells in situ, both selectively and efficiently, is required in order to translate novel therapeutic modalities into clinical reality. We present a novel delivery strategy using non-pathogenic bacteria to effect gene delivery specifically to tumour-associated phagocytic cells. Non-invasive bacteria lack the ability to actively enter host cells, except for phagocytic cells. We exploit this natural property to effect 'passive transfection' of tumour-associated phagocytic cells following direct administration of transgene-loaded bacteria to tumour regions. Using an in vitro-differentiated human monocyte cell line and two in vivo mouse models (an ovarian cancer ascites and a solid colon tumour model) proof of delivery is demonstrated with bacteria carrying reporter constructs. The results confirm that the delivery strategy is specific for phagocytic cells and that the bacterial vector itself recruits more phagocytic cells to the tumour. While proof of delivery to phagocytic cells is demonstrated in vivo for solid and ascites tumour models, this strategy may be applied to other settings, including non-cancer related disease.

Natural killer cells protect mice from DSS-induced colitis by regulating neutrophil function via the NKG2A receptor.

Natural killer (NK) cells are traditionally considered in the context of tumor surveillance and infection defense but their role in chronic inflammatory disorders such as inflammatory bowel disease is less clear. Here, we investigated the role of NK cells in dextran sodium sulfate (DSS)-induced colitis in mice. Depletion of NK cells impairs the survival of mice with colitis and is linked with dramatic increases in colonic damage, leukocyte infiltration, and pro-inflammatory profiles. Mice depleted of NK cells had increased numbers of neutrophils in colons and mesenteric lymph nodes, compared with control mice, in addition to acquiring a hyper-activation status. In vitro and in vivo studies demonstrate that NK cells downregulate pro-inflammatory functions of activated neutrophils, including reactive oxygen species and cytokine production, by direct cell-to-cell contact involving the NK cell-inhibitory receptor NKG2A. Our results indicate an immunoregulatory mechanism of action of NK cells attenuating DSS-induced colitis neutrophil-mediated inflammation and tissue injury via NKG2A-dependent mechanisms.

Use of bioluminescence imaging to track neutrophil migration and its inhibition in experimental colitis.

Inflammatory bowel disease (IBD) is associated with neutrophil infiltration into the mucosa and crypt abscesses. The chemokine interleukin (IL)-8 [murine homologues (KC) and macrophage inflammatory protein (MIP)-2] and its receptor CXCR2 are required for neutrophil recruitment; thus, blocking this engagement is a potential therapeutic strategy. In the present study, we developed a preclinical model of neutrophil migration suitable for investigating the biology of and testing new drugs that target neutrophil trafficking. Peritoneal exudate neutrophils from transgenic ß-actin-luciferase mice were isolated 12h after intraperitoneal injection with thioglycollate, and were assessed phenotypically and functionally. Exudate cells were injected intravenously into recipients with dextran sodium sulphate (DSS)-induced colitis followed by bioluminescence imaging of whole-body and ex vivo organs at 2, 4 and 16-22h post-transfer. Anti-KC antibody or an isotype control were administered at 20 µg/mouse 1h before transfer, followed by whole-body and organ imaging 4h post-transfer. The peritoneal exudate consisted of 80% neutrophils, 39% of which were CXCR2(+) . In vitro migration towards KC was inhibited by anti-KC. Ex vivo bioluminescent imaging showed that neutrophil trafficking into the colon of DSS recipients was inhibited by anti-KC 4h post-cell transfer. In conclusion, this study describes a new approach for investigating neutrophil trafficking that can be used in preclinical studies to evaluate potential inhibitors of neutrophil recruitment.

The endocrine regulation of aging in Caenorhabditis elegans.

In recent years, there has been significant growth in our understanding of the regulation of longevity. The most notable change is the identification and detailed description of a number of molecular pathways modulating the rate of aging. A good portion of this new data has come from studies using the genetic model organism Caenorhabditis elegans. In this review, we provide an overview of physiological systems that are involved in the modulation of aging in C. elegans, then discuss the known endocrine signaling systems that are likely to couple these systems together. Finally, we present a working model describing how aging may be regulated as a coordinated system, communicating through endocrine signals.

A myosin II mutation uncouples ATPase activity from motility and shortens step size.

It is thought that Switch II of myosin, kinesin and G proteins has an important function in relating nucleotide state to protein conformation. Here we examine a myosin mutant containing an S456L substitution in the Switch II region. In this protein, mechanical activity is uncoupled from the chemical energy of ATP hydrolysis so that its gliding velocity on actin filaments is only one-tenth of that of the wild type. The mutant spends longer in the strongly bound state and exhibits a shorter step size, which together account for the reduction in in vitro velocity. This is the first single point mutation in myosin that has been found to affect step size.

Variable surface loops and myosin activity: accessories to a motor.

The catalytic head of myosin is a globular structure that has historically been divided into three segments of 25, 50, and 20 kDa. The solvent-exposed, proteolytically-sensitive surface loops of myosin that join these three segments are highly variable in their sequences. While surface loops have not traditionally been thought to affect enzymatic activities, these loops lie near the ATP and actin-binding sites and have been implicated in the modulation of myosin's kinetic activities. In this work we review the wealth of data regarding the loops that has accumulated over the years and discuss the roles of the loops in contributing to the different activities displayed by different myosin isoforms.

myo-inositol 1,4,6-trisphosphorothioate and myo-inositol 1,3, 6-trisphosphorothioate: partial agonists with very low intrinsic activity at the platelet myo-inositol 1,4,5-trisphosphate receptor.

Racemic mixtures and enantiomerically pure D-isomers of both myo-inositol 1,3,6-trisphosphorothioate [Ins(1,3,6)PS(3)] and myo-inositol 1,4,6-trisphosphorothioate [Ins(1,4,6)PS(3)], prepared by total synthesis, were examined in Ca(2+) flux and binding assays. Both D-Ins(1,3,6)PS(3) and D-Ins(1,4,6)PS(3) were shown to be low intrinsic activity partial agonists at the platelet myo-inositol 1,4, 5-trisphosphate [Ins(1,4,5)P(3)] receptor, releasing less than 20% of the Ins(1,4,5)P(3)-sensitive Ca(2+) store. D-Ins(1,4,6)PS(3) displaced specifically bound [(3)H]Ins(1,4,5)P(3) from rat cerebellar membranes, although displacement was some 34-fold weaker than by D-Ins(1,4,5)P(3). D-Ins(1,4,6)PS(3) displaced [(3)H]Ins(1,4, 5)P(3) from cerebellar membranes with roughly twice the affinity of DL-Ins(1,4,6)PS(3) (IC(50) value = 1.4 +/- 0.35 microM compared with 2.15 +/- 0.13 microM), whereas D-Ins(1,3,6)PS(3) displaced [(3)H]Ins(1,4,5)P(3) with roughly twice the affinity of DL-Ins(1,3, 6)PS(3) (IC(50) value = 17.5 +/- 5.8 microM compared with 34 +/- 10 microM), confirming that the activity of both these phosphorothioates resides in their D-enantiomers. Increasing concentrations of either D-Ins(1,3,6)PS(3) or D-Ins(1,4,6)PS(3) were able to partially antagonize Ca(2+) release induced by submaximal concentrations of Ins(1,4,5)P(3), an inhibition that could be overcome by increasing the concentration of Ins(1,4,5)P(3), suggesting competition for binding at the Ins(1,4,5)P(3)-R. The only low-efficacy partial agonists at the Ins(1,4,5)P(3)-R discovered to date have been phosphorothioates; the novel D-Ins(1,3,6)PS(3) and D-Ins(1,4,6)PS(3) can now be added to this small group of analogs. However, D-Ins(1,4,6)PS(3) has a relatively high affinity for the Ins(1,4,5)P(3)-R but maintains the lowest efficacy of all the partial agonists thus far identified. As such, it may be a useful tool for pharmacological intervention in the polyphosphoinositide pathway and an important lead compound for the development of further Ins(1,4,5)P(3)-R antagonists.

The sequence of the myosin 50-20K loop affects Myosin's affinity for actin throughout the actin-myosin ATPase cycle and its maximum ATPase activity.

We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ADP release [Murphy, C. T., and Spudich, J. A. (1998) Biochemistry 37, 6738-44], while loop 2 affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-9]. In the study described here, we have found that the kcat of the actin-activated ATPase activity is changed by the loop 2 substitutions in a manner that reflects the relative actin-activated ATPase activities of the donor myosins. Additionally, changes in loop 2 affect the affinity of myosin for actin both in the presence and in the absence of nucleotides. Pre-steady-state studies together with the ATPase and affinity data suggest that while loop 2 does not affect interactions between myosin and nucleotide, it plays a role in determining the affinity of myosin for actin in various nucleotide states and in the rate-limiting transition allowing phosphate release.

Expression of inducible nitric oxide synthase activity in human colon epithelial cells: modulation by T lymphocyte derived cytokines.

BACKGROUND: Nitric oxide (NO) synthesis and inducible nitric oxide synthase (iNOS) expression are increased in colonic biopsy specimens from patients with ulcerative colitis, but the cellular source of NO production is not known. AIMS: To examine the distribution of iNOS in human colonic mucosa and to explore the ability of T lymphocyte derived cytokines to regulate iNOS expression and activity in human colonic epithelial cells. METHODS: iNOS expression was examined using immunohistochemistry in colonic biopsy samples from 12 patients with ulcerative colitis and three with infectious colitis and compared with 10 normal controls. In vitro iNOS expression and activity were determined in HT-29 cell cultures; nitrite levels were measured using a fluorescent substrate, iNOS mRNA expression by northern blot analysis, and iNOS protein expression by western blot analysis. RESULTS: No iNOS expression was detected (10 of 10) in non-inflamed mucosa derived from normal controls. In 11 of 12 cases of newly diagnosed ulcerative colitis, iNOS protein was expressed in the epithelial cells, while no other positive cells were found in the lamina propria. Similar iNOS labelling was found in colonic biopsy samples from patients with infectious colitis in the acute phase, but when re-examined in samples from patients in total remission, no iNOS staining was observed. Both interleukin (IL)-13 and IL-4, but not IL-10, are potent inhibitors of iNOS expression and activity induced by an optimal combination of cytokines, namely IL-1 alpha, tumour necrosis factor alpha and interferon gamma. CONCLUSIONS: The data suggest that the epithelium is the major source of iNOS activity in ulcerative colitis and that IL-13 and IL-4 may act as intrinsic regulators of NO generation in intestinal inflammation.

Dictyostelium myosin 25-50K loop substitutions specifically affect ADP release rates.

While most of the sequence of myosin's motor domain is highly conserved among various organisms and tissue types, the junctions between the 25 and 50 kDa domains and the 50 and 20 kDa domains are strikingly divergent. The 50-20K loop is positioned to interact with actin, while the 25-50K loop is situated nearer the ATP binding site [Rayment, I., et al. (1993) Science 261, 50-58]. Chimeric studies of the 50-20K loop [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-569; Rovner, A. S., et al. (1995) J. Biol. Chem. 270 (51), 30260-30263] have shown that this loop affects actin activation of ATPase activity. Given the function of myosin as a molecular motor, it was proposed that the 25-50K loop might specifically alter ADP release [Spudich, J. A. (1994) Nature 374, 515-518]. Here we study the role of this loop by engineering chimeras containing the Dictyostelium myosin heavy chain with loops from two enzymatically diverse myosins, rabbit skeletal and Acanthamoeba. The chimeric myosins complement the myosin null phenotype in vivo, bind nucleotide normally, interact normally with actin, and display wild-type levels of actin-activated ATPase activity. However, the rate of ADP release from the myosins, normally the slowest step involved in motility, was changed in a manner that reflects the activity of the donor myosin. In summary, studies of Dictyostelium myosin heavy chain chimeras have shown that the 50-20K sequence specifically affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994)] while the 25-50K sequence helps determine the rate of ADP release.

Structural analogues of D-myo-inositol-1,4,5-trisphosphate and adenophostin A: recognition by cerebellar and platelet inositol-1,4,5-trisphosphate receptors.

Adenophostins A and B, which are metabolic products of the fungus Penicillium brevicompactum, are potent agonists at the D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptor. In the current study, adenophostin A was approximately 50-fold more potent than Ins(1,4,5)P3 at both releasing Ca2+ from the intracellular stores of permeabilized platelets and displacing [3H]Ins(1,4,5)P3 from its receptor on rat cerebellar membranes. Various analogues bearing structural features found in the adenophostins and/or Ins(1, 4,5)P3 were examined to elucidate the molecular basis for the observed enhanced potency. 2-AMP did not induce Ca2+ release from permeabilized platelets or have any effect on Ins(1,4,5)P3-induced Ca2+ release. Two carbohydrate-based analogues, (2-hydroxyethyl)-alpha-D-glucopyranoside-2',3,4-trisphosphate and alpha,alpha'-trehalose-3,4,3',4'-tetrakisphosphate, could induce release of Ca2+ and displace [3H]Ins(1,4,5)P3 from its binding site on rat cerebellar membranes, although both were less potent than Ins(1,4,5)P3. In common with adenophostin A, release of Ca2+ from the intracellular stores could be inhibited by heparin, and both analogues were metabolically resistant. This study is the first to demonstrate the activity of a synthetic disaccharide at the Ins(1,4, 5)P3 receptor and that the Ins(1,4,5)P3 receptor is capable of accommodating an increased steric bulk. The minimal importance of the 2-hydroxyl group of Ins(1,4,5)P3 (occupied by the pyranoside oxygen in adenophostin) was confirmed by comparing the activity of DL-scyllo-Ins(1,2,4)P3 [which differs from Ins(1,4,5)P3 solely by the orientation of this hydroxyl group] with that of Ins(1,4,5)P3. An analogue of this compound, namely, DL-6-CH2OH-scyllo-Ins(1,2,4)P3, which possesses an equatorial hydroxymethyl group analogous to the 5'-hydroxymethyl group of adenophostin, was found to be equipotent to Ins(1,4,5)P3, demonstrating the tolerance of the Ins(1,4,5)P3 receptor to additional steric bulk at this position.

Enantiomers of myo-inositol-1,3,4-trisphosphate and myo-inositol-1,4,6 -trisphosphate: stereospecific recognition by cerebellar and platelet myo-inositol-1,4,5-trisphosphate receptors.

The naturally occurring tetrakisphosphate myo-inositol-1,3,4, 6-tetrakisphosphate [Ins(1,3,4,6)P4] was able to release Ca2+ from the intracellular stores of permeabilized rabbit platelets but was 40-fold less potent than D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. The Ca2+ releasing activity of Ins(1,3,4,6)P4 was rationalized by envisaging two alternative receptor binding orientations in which the vicinal D-1,6-bisphosphate of Ins(1,3,4,6)P4 mimics the D-4,5-bisphosphate in the Ins(1,4,5)P3 binding conformation. This rationalization predicted that Ins(1,4,5)P3 regioisomers [i.e, D-myo-inositol -1,4,6-trisphosphate [D-Ins(1,4,6)P3] and D-myo-inositol-1,3,6 -trisphosphate [D-Ins(1,3,6)P3]] should also possess Ca(2+)-releasing activity. The unambiguous total synthesis of the enatiomers of Ins(1,4,6)P3 [i.e., D-Ins(1,4,6)P3 and D-Ins(3,4,6)P3] and the enatiomers of Ins(1,3,4)P3 [i.e., D-Ins(1,3,6)P3 and D-Ins(1,3,4)P3] allowed an examination of this prediction. D-Ins(1,4,6)P3 released Ca2+ from the intracellular stores of permeabilized platelets and was only 2-3-fold less potent than Ins(1,4,5)P3. D-Ins(1,3,6)P3 [alternative nomenclature, L-Ins(1,3,4)P3] also released Ca2+ but was 12-fold less potent than Ins(1,4,5)P3. Both D-Ins(1,4,6)P3 and D-Ins(1,3,6)P3 displaced specifically bound [3H]Ins(1,4,5)P3 from the Ins(1,4,5)P3 receptor on rat cerebellar membranes. In contrast, however, D-Ins(3,4,6)P3 [alternative nomenclature, L-Ins(1,4,6)P3] and D-Ins(1,3,4)P3 neither possessed Ca(2+)-releasing activity nor displaced [3H]Ins(1,4,5)P3. The ability of D-Ins(1,3,6)P3 to release Ca2+ in permeabilized platelets is in contrast to its apparent lack of Ca(2+)-mobilizing activity previously reported in rat basophilic leukemic cells. The possibility that this is a reflection of the different Ins(1,4,5)P3 receptor subtypes possessed by these two cell types is discussed.

A role for protein phosphorylation in modulating Ca2+ elevation in rabbit platelets treated with thapsigargin.

The effect of modifying protein kinase and phosphatase activity on Ca2+ influx induced by inhibition of Ca(2+)-ATPase activity has been investigated in rabbit platelets. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) or inhibition of phosphatase type 1/2A (PP1/2A) activity with calyculin A caused a dose-dependent inhibition of cytosolic Ca2+ elevation in thapsigargin (Tg)-treated platelets and decreased Ca2+ influx into platelets at a time when Ca2+ channels had already been opened by pretreatment of cells with Tg. In addition, both activation of PKC and inhibition of PP1/2A activity caused a dose-dependent inhibition of bivalent cation (Mn2+) influx (acting as a surrogate for Ca2+ influx) in Tg-treated platelets. Inhibition of cyclo-oxygenase activity caused a small decrease in [Ca2+]i elevation in Tg-treated platelets, but had no effect on the ability of PMA or calyculin A to inhibit Tg-induced [Ca2+]i elevation Unexpectedly, PMA inhibited Tg-induced [Ca2+]i elevation in the absence of extracellular Ca2+, and in agreement calyculin A decreased [Ca2+]i elevation almost to basal levels. The results from this study were confirmed with another Ca(2+)-ATPase inhibitor, namely 2,5-di(tert-butyl)hydroquinone (tBHQ). These findings therefore suggest that modification of phosphorylation of target protein(s) on serine/threonine amino acid residues plays a role in the regulation of both Ca2+ influx and in the filling state of the intracellular Ca2+ pool in platelets treated with Tg.

The whoosh and trickle of calcium signalling.

The importance of phospholipase C catalysed hydrolysis of phosphatidylinositol-(4,5)bisphosphate (PtdIns(4,5)P2) to inositol-(1,4,5)trisphosphate (Ins(1,4,5)P3) and sn-1,2-diacylglycerol in the signal transduction pathways of eukaryote cells, in response to extracellular stimuli, is now widely recognised. Although nearly 60 naturally occurring inositol phosphates have been identified in mammalian cells, mobilisation of Ca2+ from the intracellular stores has been most commonly attributed to the generation of Ins(1,4,5)P3 [1]. However, there is increasing evidence for the presence of ryanodine receptors (RyR) in non-excitable cells and for cADP-ribose (cADPr) as the signalling molecule responsible for Ca2+ release via the RyR. But what is the purpose for the co-existence of these two intracellular Ca2+ channels in non-excitable cells and why are they so heterogeneous in their distribution? These questions were explored at the recent International Symposium Calcium Signalling in Inflammatory Cells. Depletion of the intracellular Ca2+ pools is followed by entry of Ca2+ into the cell across the plasma membrane, but the mechanism(s) underlying this 'capacitative Ca2+ entry' is not well understood. Many potential signalling pathways which may account for capacitative Ca2+ entry have been proposed although none have been unanimously accepted. New developments in the elucidation of the mechanism responsible for capacitative Ca2+ entry and how Ca2+ entry is regulated, together with progress in the characterisation of plasma membrane Ca2+ entry channels were also discussed at this symposium.

Nicotine increases intracellular calcium in rat hippocampal neurons via voltage-gated calcium channels.

The effect of nicotinic receptor activation on intracellular calcium concentrations ([Ca2+]i) was quantitated in populations of cultured hippocampal neurons loaded with Fura-2. Nicotine (50 microM) and cytisine (50 microM) increased [Ca2+]i by 100%. This response was abolished in the presence of the nicotinic antagonist methyllycaconitine (MLA) whereas KCl-evoked increases in [Ca2+]i were insensitive to MLA. Glial cultures were unaffected by nicotine, although they did respond to glutamate with increased [Ca2+]i. In hippocampal neurons, responses to nicotinic agonists and KCl were dependent on the presence of extracellular Ca2+ and were similarly sensitive (85% inhibition) to CdCl2. These results are consistent with the presence of functional nicotinic receptors on hippocampal neurons. The receptors appear to elevate [Ca2+]i by promoting the influx of extracellular Ca2+ through voltage-gated calcium channels.

Role of type 1 and type 2A phosphatases in signal transduction of platelet-activating-factor-stimulated rabbit platelets.

Calyculin A, the potent inhibitor of type 1 (PP1) and type 2A (PP2A) phosphatases, has been employed in order to investigate the role of endogenously activated PP1/PP2A in the signal-transduction pathway of platelet-activating-factor (PAF)-stimulated platelets. Calyculin A alone caused an increase in protein phosphorylation in unstimulated platelets, with the detection of a number of newly phosphorylated proteins, whereas in PAF-stimulated platelets phosphorylation of the major substrates of protein kinase C and myosin light-chain kinase were no longer transient, but phosphorylation was sustained. PP1/PP2A appear to play a role in Ca2+ homoeostasis, as inhibition of PP1/PP2A caused an inhibition of Ca2+ mobilization and Ca2+ influx through the plasma membrane in PAF-stimulated platelets. The effect of calyculin A on Ca2+ mobilization correlated with the observed inhibition of the production of the signal molecule Ins(1,4,5)P3. The release reaction (which is a Ca(2+)-dependent event) was also inhibited by calyculin A. The results are discussed in relation to the possible role of protein kinase C in mediating the events leading to the effects observed with calyculin A.

Tyrosine-kinase activity in rabbit platelets stimulated with platelet-activating factor. The effect of inhibiting tyrosine kinase with genistein on platelet-signal-molecule elevation and functional responses.

The temporal relationship of tyrosine phosphorylation of proteins in platelet-activating-factor-(PAF)-stimulated rabbit platelets was characterised by Western blotting using a monoclonal anti-phosphotyrosine antibody, demonstrated to be specific for detecting only tyrosine phosphorylated proteins. In addition, the protein tyrosine kinase (PTKase) inhibitor genistein, was used to investigate the role of endogenously activated PTKase(s) in the regulation of receptor-stimulated changes in both signal molecule production and in platelet functional responses. Several tyrosine phosphorylated protein bands (52-62 kDa) were observed in unstimulated platelets, however, within 5 s of PAF stimulation, two further groups of tyrosine phosphorylated protein bands were observed (35-45 kDa and 66-90 kDa) and within 30 s of PAF stimulation a further group was detected (90-150 kDa). Under conditions where intracellular Ca2+ was chelated with acetoxymethyl 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA-AM) and extracellular Ca2+ was chelated with EGTA, the number of tyrosine-phosphorylated bands was greatly reduced. Tyrosine phosphorylation of the proteins induced by PAF stimulation were differentially inhibited by treatment with genistein. Genistein inhibited PAF-induced elevation of the signal molecule inositol 1,4,5-trisphosphate and also inhibited both mobilization of Ca2+ and the influx of Ca2+ through the plasma membrane. These results suggest a role for endogenously activated PTKase(s) in the early stages of signal transduction in PAF-stimulated platelets. Moreover, inhibition of genistein-sensitive PTKase(s) also caused an inhibition of PAF-induced thromboxane B2 generation, dense-granule release and platelet aggregation, indicating a role for PTKase(s) in the regulation of platelet functional responses. Platelets stimulated with alpha-thrombin, ionomycin and 12-O-tetradecanylphorbol 13-acetate gave a similar pattern of phosphorylated proteins to PAF-stimulated platelets, however, whereas genistein inhibited protein phosphorylation, it had no significant effect on functional responses in platelets stimulated with these agents, suggesting that an alternative signalling pathway exists.

Evidence for the presence and location of annexins in human platelets.

In this study the identity of annexins in human platelets has been determined together with their ability to be released by agents which induce platelet degranulation. The presence of proteins cross-reacting to antibodies against annexins I and V was detected in human platelets. However, the study provided evidence that these annexins are not located on the surface of the plasma membrane in a Ca++ dependent manner. Moreover, activation of platelets with several agents which induced platelet degranulation did not cause release of annexins I or V as determined by both immunoblotting and ELISA.