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.

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.

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.

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.

Comparison of the role of protein kinase C in platelet functional responses induced by three different mechanisms, PAF, ionomycin and arachidonic acid.

The role of protein kinase C (PKC) in modulating platelet activation has been examined in platelets pre-incubated with either the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the non-specific protein kinase inhibitor, staurosporine. In order to determine where in the signal transduction pathway PKC is exerting its effect platelets were activated either with a receptor-operated stimulus platelet activating factor (PAF) or by direct elevation of [Ca2+]i (ionomycin) or with arachidonic acid which is converted into thromboxane B2 (TxB2). In PAF-stimulated platelets activation of PKC inhibited both [Ca2+]i elevation and TxB2 generation but had no effect on 5-hydroxytryptamine (5-HT) release whilst staurosporine increased the duration of [Ca2+]i elevation and potentiated TxB2 generation but inhibited 5-HT release. In ionomycin-stimulated platelets modulation of PKC had no effect on [Ca2+]i elevation but in contrast to PAF-stimulated platelets PKC activation caused potentiation of TxB2 generation and 5-HT release whilst inhibition of PKC caused inhibition of TxB2 generation and 5-HT release. Modulation of PKC did not affect arachidonic acid-induced TxB2 generation. These findings suggest that in receptor activated platelets endogenously activated PKC is exerting a negative feedback role, however, when [Ca2+]i elevation is not modified by PKC activation or inhibition (such as in ionomycin stimulated platelets) the relationship between the state of PKC activation and subsequent platelet functional responses corresponds more closely. The findings from this study suggest a different relationship between PKC and TxB2 generation than between PKC and dense granule release in PAF-stimulated platelets.

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.

Direct measurement of octanol-water partition coefficients by high-pressure liquid chromatography.

A technique is presented for the direct measurement of octanol-water partition coefficients by HPLC. The method involves running solutes in octanol-saturated water as the mobile phase against water-saturated octanol entrained on an inert support. Log P correlates linearly with log tc for a number of standards. The measurable range in log P (so far) is -0.3 to +3.7. A critical review of chromatographic methods in Hansch analysis is given.

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.

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.

Selective inhibition of protein kinase C. Effect on platelet-activating-factor-induced platelet functional responses.

The role of protein kinase C (PKC) in platelet-activating-factor (PAF)-induced platelet activation was examined by using two selective inhibitors of PKC, namely Ro 31-7549/001 and Ro 31-8220/002. Both inhibitors dose-dependently inhibited PAF-induced phosphorylation of the major 40-47 kDa protein substrate of PKC, with 50% inhibition at 4.5 microM-Ro 31-7549/001 and 0.7 microM-Ro 31-8220/002. Inhibition of PKC had no effect on maximal elevation of intracellular Ca2+ [Ca2+]i produced by either a high or a low dose of PAF, but significantly increased the duration of the Ca2+ signal and the thromboxane B2 (TxB2) generation in high-dose PAF-stimulated platelets. The inhibitors also abrogated the effect of the PKC activator phorbol 12-myristate 13-acetate on PAF-induced [Ca2+]i elevation. Sub-maximal PAF-induced dense-granule release and platelet aggregation were dose-dependently inhibited by Ro 31-7549/001 and Ro 31-8220/002. The findings suggest that endogenously activated PKC holds a bifurcating role in PAF-activated platelets, negatively affecting duration of both [Ca2+]i and TxB2 generation, and positively influencing dense-granule release and aggregation.

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.

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.

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.

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.

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.

The relationship between cytosolic Ca2+, sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate elevation in platelet-activating-factor-stimulated rabbit platelets. Influence of protein kinase C on production of signal molecules.

The temporal and dose-response relationships of platelet-activating-factor (PAF)-induced changes in the concentrations of cytosolic Ca2+ ([Ca2+]i), Ins(1,4,5)P3 and 1,2-diacylglycerol (DAG) were examined. In addition, phosphorylation of protein kinase C (PKC) substrate (40-47 kDa protein) was determined. In high-dose PAF-activated platelets, all three signal molecules increased rapidly and transiently, with the peak Ins(1,4,5)P3 concentration preceding maximal elevation of [Ca2+]i by 5 s. In low-dose PAF-activated platelets there were large increases in [Ca2+]i and dense-granule release, without any increase in Ins(1,4,5)P3 and DAG or 40-47 kDa protein phosphorylation. Staurosporine, a non-specific PKC inhibitor, produced enhanced elevations in the concentrations of Ins(1,4,5)P3, DAG and thromboxane B2, and the duration of the Ca2+ signal in platelets stimulated with a high dose, but not a low dose, of PAF. These results suggest there are both phospholipase C-dependent and -independent changes in Ca2+ homoeostasis. Endogenously activated PKC regulates the formation of signal molecules.

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.