Quantification of intervertebral efforts during walking: comparison between a healthy and a scoliotic subject.

The accurate quantification of internal efforts in the human body is still a challenge in biomechanics. The aim of this study is to quantify the intervertebral efforts along the spine during walking, in order to compare the dynamical behaviours between a healthy and a scoliotic subject. Practically, one healthy subject, one scoliotic patient before an instrumentation surgery (Cobb 41 degrees ) and after this instrumentation (Cobb 7.5 degrees ) walked on a treadmill at 4 km/h. The acquisition system included optokinetic sensors, recording the 3D-joint coordinates, a treadmill equipped with strain gauges, measuring the external forces independently applied to both feet, and bi-planar radiographs, enabling the 3D reconstruction of the spine from C7 to L5, using a free form interpolation technique. The intervertebral efforts were computed using an inverse dynamical model of the human body in 3D. As results, significant differences of the spine kinematics were recorded which lead to different internal effort behaviour in magnitude, shift, coordination and pattern when normalized to the subject mass. Particularly, the normalized antero-posterior intervertebral torques are less uniform for the scoliotic patient (from min -2.5 to max 1.9 Nm/kg) than the healthy subject (from -1.5 to 1.5 Nm/kg). This disequilibrium in the left-right balance of the scoliotic patient is a bit rectified after surgery (from -1.3 to 1.1 Nm/kg).

Cutting edge: the nucleotide receptor P2X7 contains multiple protein- and lipid-interaction motifs including a potential binding site for bacterial lipopolysaccharide.

The nucleotide receptor P2X7 has been shown to modulate LPS-induced macrophage production of numerous inflammatory mediators. Although the C-terminal portion of P2X7 is thought to be essential for multiple receptor functions, little is known regarding the structural motifs that lie within this region. We show here that the P2X7 C-terminal domain contains several apparent protein-protein and protein-lipid interaction motifs with potential importance to macrophage signaling and LPS action. Surprisingly, P2X7 also contains a conserved LPS-binding domain. In this report, we demonstrate that peptides derived from this P2X7 sequence bind LPS in vitro. Moreover, these peptides neutralize the ability of LPS to activate the extracellular signal-regulated kinases (ERK1, ERK2) and to promote the degradation of the inhibitor of kappaB-alpha isoform (IkappaB-alpha) in RAW 264.7 macrophages. Collectively, these data suggest that the C-terminal domain of P2X7 may directly coordinate several signal transduction events related to macrophage function and LPS action.

Purinergic receptor modulation of lipopolysaccharide signaling and inducible nitric-oxide synthase expression in RAW 264.7 macrophages.

Previous studies have suggested that the P2Z/P2X7 purinergic receptor can participate in nucleotide-induced modulation of lipopolysaccharide (LPS) stimulated inflammatory mediator production. To test this hypothesis, we evaluated whether antagonism of the P2Z/P2X7 receptor can influence LPS signaling and expression of the inducible form of nitric-oxide synthase (iNOS) in RAW 264.7 macrophages. In the present study, we demonstrate that pretreatment of RAW 264.7 macrophages with a P2Z/P2X7 receptor antagonist, periodate oxidized adenosine 5'-triphosphate (o-ATP), substantially inhibits LPS-stimulated NO production and iNOS expression without altering cell viability. This effect on LPS-induced iNOS expression is mimicked by a pyridoxal-phosphate-based antagonist (pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid) of the P2Z/P2X7 purinergic receptor, indicating that these results are not unique to o-ATP. Additionally, o-ATP prevents cell death induced by P2Z/P2X7 receptor agonists. To ascertain how P2Z/P2X7 receptor antagonists influence LPS signaling, we evaluated the capacity of o-ATP to regulate LPS-mediated activation of the transcription factor, nuclear factor-kappaB, and the mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2. These experiments reveal that pretreatment of RAW 264.7 cells with o-ATP attenuates the LPS stimulation of a nuclear factor-kappaB-like binding activity. Moreover, the activation of ERK1 and ERK2 by LPS, but not by the phorbol ester, phorbol 12-myristate 13-acetate, is also blocked in RAW 264.7 cells by o-ATP pretreatment. In summary, these data suggest that the P2Z/P2X7 receptor modulates LPS-induced macrophage activation as assessed by iNOS expression and NO production. This report implicates the P2Z/P2X7 receptor in the control of protein kinase cascades and transcriptional processes, and these observations are likely to be important for the development of selective purinergic receptor antagonists for the treatment of septic shock.

Regulation of inducible nitric oxide synthase expression by macrophage purinoreceptors and calcium.

Macrophage activation is central to the progression of multiple diseases via the release of inflammatory mediators such as cytokines and nitric oxide. Despite the recognized overlap in the regulatory mechanisms involved in mediator production, little formation exists regarding receptor-initiated signaling pathways that coordinately control multiple end points, such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide production. In this study, the expression of inducible nitric oxide synthase (iNOS) in macrophages is shown to be regulated by calcium and by a purinoreceptor signaling system. The P2Y purinoreceptor partial agonist, 2-methylthio-ATP (2-MeS-ATP), inhibits the expression of iNOS induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) in primary macrophages. Additionally, 2-MeS-ATP attenuates the expression of iNOS in macrophages isolated from CD-1 mice challenged with LPS, and it inhibits LPS-induced TNF-alpha and interleukin-1 alpha (IL-1 alpha) release, thereby preventing endotoxic death. Thus, purinoreceptors and calcium are likely to be critical for macrophage activation and the production of inflammatory mediators stimulated by LPS.

A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins.

In interphase cells, alpha-casein kinase I (alpha-CKI) is found associated with cytosolic vesicular structures, the centrosome, and within the nucleus. To identify the specific vesicular structures with which alpha-CKI is associated, established cell lines and primary rat neurons were immunofluorescently labeled with an antibody raised to alpha-CKI. In nonneuronal cells, alpha-CKI colocalizes with vesicular structures which align with microtubules and are partially coincident with both Golgi and endoplasmic reticulum markers. In neurons, alpha-CKI colocalizes with synaptic vesicle markers. When synaptic vesicles were purified from rat brain, they were highly enriched in a CKI, based on activity and immunoreactivity. The synaptic vesicle-associated CKI is an extrinsic kinase and was eluted from synaptic vesicles and purified. This purified CKI has properties most similar to alpha-CKI. When the activities of casein kinase I or II were specifically inhibited on isolated synaptic vesicles, CKI was shown to phosphorylate a specific subset of vesicle proteins, one of which was identified as the synaptic vesicle-specific protein SV2. As with alpha-CKI, the synaptic vesicle CKI is inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). However, synthesis of PIP2 was detected only in plasma membrane-containing fractions. Therefore, PIP2 may spatially regulate CKI. Since PIP2 synthesis is required for secretion, this inhibition of CKI may be important for the regulation of secretion.

ATP-dependent inositide phosphorylation required for Ca(2+)-activated secretion.

Regulated fusion of secretory granules with the plasma membrane in secretory cells requires ATP, Ca2+ and cytosolic as well as membrane proteins. ATP-dependent steps in Ca(2+)-activated secretion from PC12 cells require three cytosolic PEP proteins (priming in exocytosis proteins, PEP1-3), the identity of which will provide insights into the required ATP-using reactions. PEP3 was recently identified as phosphatidylinositol transfer protein (PtdInsTP), and here we report that PEP1 consists of the type I phosphatidylinositol-4-phosphate 5-kinase (PtdInsP5K). The roles of PEP3/PtdInsTP and PEP1/PtdInsP5K in sequential phosphoinositide recruitment and phosphorylation explains their synergistic activity in ATP-dependent priming. Moreover, inhibition of Ca(2+)-activated secretion by PtdIns(4,5)P2-specific antibodies and phospholipase C implies that 5-phosphorylated inositides play a novel, necessary role in the regulated secretory pathway. The results indicate that lipid kinase-mediated phosphorylation is an important basis for ATP use in the exocytotic pathway.

Type I phosphatidylinositol 4-phosphate 5-kinase isoforms are specifically stimulated by phosphatidic acid.

A phosphatidylinositol 4-phosphate (PIP) kinase was isolated and purified to near homogeneity from bovine erythrocyte membranes. The PIP kinase was extracted from bovine erythrocyte membranes with a high salt wash, followed by phosphocellulose and phenyl-Sepharose chromatography. The predominant protein after phenyl-Sepharose purification had a molecular size of 68 kDa. Renaturation of PIP kinase activity after SDS-PAGE showed that a 68-kDa protein was able to phosphorylate PIP. An antibody developed against the 68-kDa protein Western blots the 68-kDa protein and is able to immunoprecipitate the 68-kDa protein and PIP kinase activity from membrane extracts. Based on functional studies, the 68-kDa protein is indistinguishable from the type I PIP kinase previously characterized from human erythrocyte membranes (Bazenet, C. E., Ruano, A.R., Brockman, J.L., and Anderson, R.A. (1990) J. Biol. Chem. 265, 18012-18022). These studies also show that the type I PIP kinases, but not the type II PIP kinase, are stimulated by phosphatidic acid, suggesting alternative roles for these enzymes. Two immunoreactive isoforms of the type I PIP kinase, of 68 and 90 kDa, were identified in rat brain and partially purified. Both of these isoforms are also stimulated by phosphatidic acid.