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PubMed1993

Agrin and the molecular choreography of synapse formation.

High concentrations of neurotransmitter receptors characterize neuromuscular junctions as well as neuron-neuron synapses in the brain and periphery. Synaptic function is critically dependent upon this marshalling of neurotransmitter receptors to the post-synaptic membrane. This review discusses agrin's role in orchestrating the molecular topography of the post-synaptic apparatus at nerve-muscle synapses and the emerging evidence suggesting a role for agrin in synaptogenesis in the brain.

Agrin↗
PubMed1992

RNA splicing regulates agrin-mediated acetylcholine receptor clustering activity on cultured myotubes.

Agrin is a component of the synaptic basal lamina that induces the clustering of acetylcholine receptors (AChRs) on muscle fibers. A region near the carboxyl terminus of the protein exists in four forms that are generated by alternative RNA splicing. All four alternatively spliced forms of agrin are active in inducing AChR clusters on rat primary and C2-derived muscle fibers. In contrast, only two forms of the protein, each containing an 8 amino acid insert, are capable of inducing clusters on myotubes of S27 cells, a C2 variant that has defective proteoglycans. These two forms are also most active in inducing clusters on chick myotubes. This pattern of differential activity suggests that RNA splicing of agrin transcripts and interactions with proteoglycans or other components of basal lamina have important roles in regulating the localization of neurotransmitter receptors at synaptic sites.

Agrin↗
PubMed2005

Pathophysiological roles of extracellular nucleotides in glial cells: differential expression of purinergic receptors in resting and activated microglia.

Microglial cells are the major cellular elements with immune function inside the CNS and play important roles in orchestrating inflammatory brain response to hypoxia and trauma. Although a complete knowledge of the endogenous factors leading to a prompt activation of microglia is not yet available, activation of P2 purinoreceptors by extracellular ATP has been indicated as a primary factor in microglial response. A still unresolved question, however, is which subtype(s) of P2 receptors mediate(s) the response to ATP. By a combination of RT-PCR, Western blotting, and single-cell calcium imaging, we assessed the presence and the activity of P2 receptor subtypes in the mouse microglial cell line N9. All members of the P2 receptor family, including the recently reported receptor for sugar nucleotides (P2Y(14)), were found to be present in these cells at mRNA and/or protein level. The functionality of the receptors was assessed by analysis of the calcium responses evoked by specific agonists both in N9 cells and in primary microglia from rat brain. Interestingly, a different functional profile of P2 receptors was observed in resting or in LPS-activated N9 cells. Overnight exposure to LPS increased P2Y(6) and P2Y(14), decreased P2X(7), and left unchanged P2Y(1) and P2Y(2,4) receptor activity. The change in the P2 receptor profile in activated cells suggests selective roles for specific P2 receptor subtypes in microglial activation triggered by LPS. We speculate that modulation of microglial cell function via subtype-selective P2 receptor ligands may open up new strategies in the therapeutic management of inflammatory neurological diseases characterized by abnormal microglia response.

Analysis of Variance↗
PubMed2000

Inhibition of cellular action of thrombin by N3-cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist.

A growing body of evidence suggests an important contribution of the cellular actions of thrombin to thrombosis and restenosis following angioplasty. Recently we reported on SCH 79797 (N3-cyclopropyl-7-¿[4-(1-methylethyl)phenyl]methyl¿-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine) and its analogs as new potent, nonpeptide thrombin receptor antagonists. This study further characterizes the biochemical and pharmacological actions of pyrroloquinazoline inhibitors of protease activated receptor-1 (PAR-1) in human platelets and coronary artery smooth muscle cells (hCASMC). SCH 79797 and its N-methyl analog (SCH 203099) inhibited binding of a high-affinity thrombin receptor-activating peptide ([(3)H]haTRAP, Ala-Phe(p-F)-Arg-ChA-HArg-[(3)H]Tyr-NH(2)) to PAR-1 with IC(50) values of 70 and 45 nM, respectively. SCH 79797 inhibited [(3)H]haTRAP binding in a competitive manner. SCH 79797 and SCH 203099 inhibited alpha-thrombin- and haTRAP-induced aggregation of human platelets, but did not inhibit human platelet aggregation induced by the tethered ligand agonist for protease-activated receptor-4 (PAR-4), gamma-thrombin, ADP, or collagen. SCH 203099 inhibited surface expression of P-selectin induced by haTRAP and thrombin, and it did not increase P-selectin expression or prevent thrombin cleavage of the receptor. Thrombin and TFLLRNPNDK-NH(2) (TK), a PAR-1-selective agonist, produced transient increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in hCASMC. This increase in [Ca(2+)](i) was inhibited effectively by SCH 79797. However, the Ca(2+) transients induced by SLIGKV-NH(2,) a PAR-2-selective agonist, were not inhibited by SCH 79797. Thrombin- and TK-stimulated [(3)H]thymidine incorporation also was inhibited completely by SCH 79797. The results of this study demonstrate that SCH 79797 and SCH 203099 are potent, selective antagonists of PAR-1 in human platelets and hCASMC. These data also suggest that the thrombin stimulation of Ca(2+) transients and mitogenesis in hCASMC is mediated primarily through activation of PAR-1.

Apoptosis Regulatory Proteins↗
PubMed2002

Amelioration by beraprost sodium, a prostacyclin analogue, of established renal dysfunction in rat glomerulonephritis model.

Effects of beraprost sodium, a chemically stable prostacyclin analogue, on renal dysfunction in an experimental rat model of glomerulonephritis were investigated. Beraprost sodium (30, 100 and 300 microg/kg) was orally given twice daily from the late stage of nephritis in which renal dysfunction was already developed. Beraprost sodium treatment inhibited the increase in urinary protein, serum creatinine and blood urea nitrogen, and the decrease in creatinine clearance. The elevation of serum creatinine was also inhibited by predonisolone (1 mg/kg). However, captopril (25, 50 and 100 mg/kg) and dipyridamole (20 and 60 mg/kg) failed to inhibit the elevation of serum creatinine. In the beraprost sodium-treated nephritic rats, the increase in mRNA levels for monocyte chemoattractant protein-1 (MCP-1) and collagen in the kidney was inhibited. These results suggest that beraprost sodium ameliorates developed renal dysfunction and is possibly an effective agent for the treatment of human glomerulonephritis.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

Identification and characterization of a novel splice variant of MuSK.

MuSK is a receptor tyrosine kinase that initiates the formation of neuromuscular junctions in response to agrin. Little is known about the ligand-induced activation and kinase-dependent signalling that leads to the clustering of acetylcholine receptors. The ectodomain of these molecule is composed of four Ig-like domains. We describe here the isolation of a novel MuSK splice variant that lacks the third Ig-like domain in its ectodomain. The corresponding RNA is the result of alternative splicing which eliminates two exons. There is 10 times less mRNA for this shorter form than for the long form of MuSK and both forms are regulated coordinately. They decrease strongly after birth and are elevated in denervated muscle. Gene transfer by muscle injection of MuSK DNA into individual muscle fibers demonstrates that kinase-induced acetylcholine receptor clustering caused by overexpression of the two kinases does not depend on the presence of the third Ig-like domain.

Alternative Splicing↗
PubMed2002

Human Ntera-2/D1 neuronal progenitor cells endogenously express a functional P2Y1 receptor.

We report here that human Ntera-2/D1 (NT-2) cells, an undifferentiated committed neuronal progenitor cell line, endogenously express a functional P2Y(1) receptor, while other P2Y subtypes, except perhaps P2Y(4), are not functionally expressed. Quantitative RT-PCR analysis showed that NT-2 cells abundantly express mRNA for P2Y(1) and P2Y(11) receptors, while P2Y(2) and P2Y(4) receptors were detected at considerably lower levels. Western blot analysis also demonstrated expression of P2Y(1) receptors and Galpha(q/11) subunits. Various nucleotides induced intracellular Ca(2+) mobilisation in NT-2 cells in a concentration-dependent manner with a rank order potency of 2-MeSADP > 2-MeSATP > ADP > ATP > UTP > ATPgammaS, a profile resembling that of human P2Y(1) receptors. Furthermore, P2Y(1) receptor-specific (A3P5P) and P2Y-selective (PPADS, suramin) antagonists inhibited adenine nucleotide-induced Ca(2+) responses in a concentration-dependent manner, consistent with expression of a P2Y(1) receptor. Moreover, of seven adenine nucleotides tested, only Bz-ATP and ATPgammaS elicited small increases in cAMP formation suggesting that few, if any, functional P2Y(11) receptors were expressed. P2Y(1) receptor-selective adenine nucleotides, including 2-MeSADP and ADP, also induced concentration-dependent phosphorylation and hence, activation of the extracellular-signal regulated protein kinases (ERK1/2). NT-2 cells, therefore, provide a useful neuronal-like cellular model for studying the precise signalling pathways and physiological responses mediated by a native P2Y(1) receptor.

Adenosine Diphosphate↗
PubMed2000

P2Y receptors contribute to ATP-induced increases in intracellular calcium in differentiated but not undifferentiated PC12 cells.

ATP-induced Ca2+ transients were examined in individual PC12 cells of a well defined clone, before and after treatment with nerve growth factor (NGF) to induce a neurone-like phenotype. Using reverse transcriptase PCR these cells were found to express mRNA for several P2 receptors. In undifferentiated cells the ATP-induced Ca2+ response was entirely dependent on Ca2+ influx, could not be mimicked by UTP, alpha,beta-methylene ATP or dibenzoyl ATP or be blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). ATP had no significant effect on levels of cyclic AMP or inositol 1,4,5-trisphosphate (InsP3). These results suggest that in undifferentiated PC12 cells ATP mainly acts on a P2X receptor, possibly the P2X4 subtype. After treatment with NGF for 7 days the ATP response was increased and partially sensitive to PPADS. A component of the ATP-induced Ca2+ increase was due to mobilisation of intracellular Ca2+ stores and another to capacitative Ca2+ entry. UTP caused an increase in intracellular Ca2+, and InsP3 formation could be stimulated by ATP and UTP. ATP also caused a small increase in cyclic AMP, but this was abolished in the presence of indomethacin. Thus, after NGF treatment ATP acts partially via a P2Y receptor, possibly the P2Y2 subtype.

Adenosine Triphosphate↗
PubMed2002

Mucosal type mast cells express complement receptor type 2 (CD21).

Fragments of complement component C3 generated upon activation of the cascade play an important role in the induction and regulation of immune responses. Receptors interacting with various fragments of this versatile complement protein are expressed on a wide variety of cell types, including lymphocytes, macrophages, dendritic cells, follicular dendritic cells, granulocytes, erythrocytes and consequently, C3-products may influence several biological functions at different sites of the body, where complement activation occurs. Regarding the expression of various C3-receptors on mast cells, mainly rodent serosal type mastocytes have been investigated so far. It has been known for a long time that C3a triggers the release of mediators of immediate type hypersensitivity via binding to serosal-type cells. Complement receptor type 1 (CR1/CD35) and type 2 (CR2/CD21) interacting with the larger activation products, such as C3b and C3d, have so far been shown on serosal type mast cells only. In this study, the expression of CR1/2 on mucosal type mast cells is demonstrated. Using mouse CR1/2 specific single chain antibodies and the natural ligand C3d in cytofluorimetric measurements, we show that the rat mucosal mast cell line RBL-2H3 and mouse bone marrow-derived mast cells (BMMC) express CD21. RT-PCR experiments carried out with mouse CR1 and CR2 specific primers show CD21, but not CD35 specific products in BMMC. It is also demonstrated that, in contrast to serosal type mast cells, mucosal mastocytes do not express CD19. In an attempt to reveal the possible function of CR2 on mucosal type mast cells, the effect of receptor-clustering was tested regarding degranulation, Ca-response and IL-6 production, but no CR2-mediated change was detected in any of these processes.

Animals↗
PubMed1996

The measurement of tissue protein turnover.

Tissue protein turnover can be assessed by a number of semi-, quantitative and qualitative methods. There are a number of static indices of the state of turnover of protein, for example amount of RNA per DNA or protein, the state of aggregation of ribosomes (i.e. the polyribosome index), the abundance of mRNA for particular proteins, and the enzymatic activity of proteins such as proteases, ribonuclease, etc. In addition, the concentration of particular amino acids such as glutamine or non-re-utilizable amino acids, formed post-translationally, such as 3-methylhistidine or hydroxyproline, are able to provide snapshot indices. However, since turnover is a dynamic process it should, ideally, be probed using methods such as the incorporation of tracer amino acids into protein or the dilution of tracer amino acids in the free pool by protein breakdown. The combination of tracer and tissue or limb balance methods is especially powerful since all the dynamic processes can potentially be quantified. The use of stable isotopes to label metabolic tracers has dramatically increased the feasibility of carrying out measurements of protein synthesis and breakdown and there has been a substantial growth in the application of the methods to a wide variety of tissues sampled by biopsy or at operation. Summaries of a number of currently feasible methods are provided, together with commentary on the relative efficacy of the methods and of the instrumental techniques required. There is also a discussion of suitable tracer labels and amino acids, plus a summary of the most reliable current values for protein turnover in a variety of tissues. The review also contains descriptions of potential methods which have not yet been applied in human beings but which are feasible, given the current recent increases in the accuracy and sensitivity of instrumentation for measurement of stable isotope labelling.

Amino Acids↗
PubMed2006

Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains.

During inflammation, leukocytes bind to the adhesion receptors ICAM-1 and VCAM-1 on the endothelial surface before undergoing transendothelial migration, also called diapedesis. ICAM-1 is also involved in transendothelial migration, independently of its role in adhesion, but the molecular basis of this function is poorly understood. Here we demonstrate that, following clustering, apical ICAM-1 translocated to caveolin-rich membrane domains close to the ends of actin stress fibres. In these F-actin-rich areas, ICAM-1 was internalized and transcytosed to the basal plasma membrane through caveolae. Human T-lymphocytes extended pseudopodia into endothelial cells in caveolin- and F-actin-enriched areas, induced local translocation of ICAM-1 and caveolin-1 to the endothelial basal membrane and transmigrated through transcellular passages formed by a ring of F-actin and caveolae. Reduction of caveolin-1 levels using RNA interference (RNAi) specifically decreased lymphocyte transcellular transmigration. We propose that the translocation of ICAM-1 to caveola- and F-actin-rich domains links the sequential steps of lymphocyte adhesion and transendothelial migration and facilitates lymphocyte migration through endothelial cells from capillaries into surrounding tissue.

Actins↗
PubMed2004

Lack of ATP-evoked GABA and glutamate release in the hippocampus of P2X7 receptor-/- mice.

In this study we revealed the participation of P2X(7) receptors in the modulation of electrical stimulation and ATP-evoked GABA and glutamate release from mouse hippocampal slices. Whereas the uptake of radioactivity was not changed, the electrical stimulation-induced release of both [(3)H]glutamate and [(3)H]GABA was decreased in the hippocampus of P2X(7) receptor-deficient mice. ATP (10 mM) elicited [(3)H]glutamate and [(3)H]GABA efflux in wild-type mice, which was inhibited by the non-selective P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium (30 microM) and the P2X(7)-selective antagonist Brilliant Blue G (1 microM). The ATP-evoked release of both GABA and glutamate was virtually absent in the knockout mice. These results indicate that ATP facilitates GABA and glutamate release in the hippocampus by a mechanism involving P2X(7)R.

Adenosine Triphosphate↗
PubMed1993

Peripheral blood lymphocytes express the platelet-type thrombin receptor.

Northern blot analysis of human mononuclear cells indicated that the platelet thrombin receptor may be expressed by lymphocytes. In order to investigate this, we prepared affinity purified rabbit antibodies against the thrombin receptor which bound platelets and blocked thrombin activation. Using flow cytometry on peripheral blood cells, we found that the vast majority of NK cells (CD16/CD56 positive) and a fraction of CD3/CD4 positive T cells expressed the thrombin receptor. B cells, neutrophils and monocytes were negative. These data suggest that potentially thrombin may play a direct role in regulating NK and T cell function.

Antibodies↗
PubMed1985

Cryo-electron microscopy of helical particles TMV and T4 polyheads.

Vitrified Tobacco Mosaic Virus (TMV) and T4 polyhead suspensions have been studied by cryo-electron microscopy. The order of TMV particles is preserved to distances better than 0.3 nm when vitrified. In spite of the high beam sensitivity of unstained biological material, the resolution of images extends to 1.15 nm. Radial density distributions calculated from images of TMV and the helical aggregate of TMV protein (TMVP) show that the RNA is likely visualized in the virus. However, due to the imaging conditions of unstained vitrified specimens, the position of the RNA is erroneous. While TMV and TMVP do not show any departure from cylindrical symmetry, T4 polyheads are sensitive to the thickness of the ice layer in which they are embedded. To avoid flattening, T4 polyheads have to be embedded in an ice layer thicker than their diameter.

Freezing↗
PubMed2006

Postembryonic epigenesis of Vasa-positive germ cells from aggregated hemoblasts in the colonial ascidian, Botryllus primigenus.

We investigated whether Vasa was a germline-specific marker in the colonial ascidian Botryllus primigenus, and whether it was inducible epigenetically in the adult life span. We cloned a Botryllus Vasa homologue (BpVas). The deduced open reading frame encoded 687 amino acid residues. It was expressed specifically by germline cells such as the loose cell mass, oogonia and juvenile oocytes in the ovary, and the primordial testis (compact cell mass), spermatogonia and juvenile spermatocytes in the testis. The loose cell mass, the most primitive germline cells, showed an ultrastructure of undifferentiated cells known as hemoblasts. The hemoblasts did not contain electron-dense materials or a mitochondrial assembly in the cytoplasm. These organelles appeared later in the oogonia and oocytes. When the loose cell mass and developing germ cells were eliminated by extirpating all zooids and buds from the colonies, BpVas transcripts disappeared completely from the vascularized colonies. After 14 days, when the colonies regenerated by vascular budding, BpVas-positive cells reappeared in some cases, and in 30 day colonies, BpVas-positive germ cells were observed in all the regenerated colonies. These results show that in B. primigenus, germ cells are inducible de novo from the Vasa-negative cells even at postembryonic stages.

Amino Acid Sequence↗
PubMed2005

A novel nitric oxide-releasing statin derivative exerts an antiplatelet/antithrombotic activity and inhibits tissue factor expression.

BACKGROUND: NO-releasing statins are new chemical entities, combining HMG-CoA reductase inhibition and slow NO release, that possess stronger anti-inflammatory and antiproliferative activities than the native statins. OBJECTIVE: We evaluated the antithrombotic effects of nitropravastatin (NCX-6550) by assessing its activity on platelet activation and tissue factor (TF) expression by mononuclear cells in vitro and in vivo. METHODS AND RESULTS: In vitro, NCX-6550 inhibited (1) U46619- and collagen-induced platelet aggregation in buffer and plasma; (2) collagen-induced P-selectin expression in whole blood and (3) platelet adhesion to collagen-coated coverslips under high shear stress. These effects were displayed at concentrations of NCX-6550 ranging from 25 to 100 mum, and were totally reverted by the guanylylcyclase inhibitor ODQ (10 microm). Equimolar concentrations of pravastatin had no influence on these parameters of platelet function. LPS- and PMA-induced TF expression by blood mononuclear cells was also inhibited by NCX-6550 (IC50 13 microm), but not by pravastatin, as assessed by functional and immunological assays and by real-time PCR. In a mouse model of platelet pulmonary thromboembolism, induced by the i.v. injection of collagen plus epinephrine, pretreatment with NCX-6550 (24-48 mg kg(-1)) significantly reduced platelet consumption, lung vessel occlusion and mortality. Moreover, nitropravastatin markedly inhibited the generation of procoagulant activity by spleen mononuclear cells and peritoneal macrophages in mice treated with LPS. In these in vivo models too, pravastatin failed to affect platelet activation and monocyte/macrophage procoagulant activity. CONCLUSIONS: Our results show that nitropravastatin exerts strong antithrombotic effects in vitro and in vivo, and may represent an interesting antiatherothrombotic agent for testing in acute coronary syndromes.

Animals↗
PubMed1997

Evidence for a NO synthase in porcine platelets which is stimulated during activation/aggregation.

We tried to characterize the porcine platelet nitric oxide (NO) synthase and its L-arginine (L-arg)/NO metabolism. Using RT-PCR we could show a constitutive endothelial NOS (ecNOS) and an inducible NOS (iNOS) similar mRNA in platelets. The NOS protein could be evidenced by an ecNOS specific antibody which also bound in platelets. This finding could be confirmed by Western blot showing an ecNOS in the membrane but not the cytosolic fraction; iNOS protein could not be detected. Using NADPH-diaphorase staining we could show NO synthase in preactivated platelets but not in resting platelets, indicating that the platelet NOS may be activated during platelet activation/aggregation. Porcine L-arg plasma levels (9.31 x 10(-5) mol/l +/- 10%) could be shown to be in the same range as human plasma levels. Moreover, we could show that the NO precursor L-arg and hydroxy-L-arginine (OHarg) concentration dependently inhibited collagen induced platelet aggregation. Summarizing these results confirm the existence of and further characterize porcine platelet NO synthases.

Animals↗
PubMed1980

Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid.

The two envelope glycoproteins and the viral nucleocapsid of the coronavirus A59 were isolated by solubilization of the viral membrane with Nonidet P-40 at 4 degrees C followed by sucrose density gradient sedimentation. Isolated E2 consisted of rosettes of peplomers, whereas E1, the membrane glycoprotein, was irregular and amorphous. Under certain conditions significant interactions occurred between components of Nonidet P-40-disrupted virions. Incubation of the Nonidet P-40-disrupted virus at 37 degrees C resulted in formation of a complex between one of the viral glycoproteins, E1, and the viral nucleocapsid. This was caused by a temperature-dependent conformational change in E1, resulting in aggregation of E1 and interaction with the viral RNA in the nucleocapsid. E1 also bound rRNA. The E1-nucleocapsid complexes can be distinguished on sucrose and Renografin density gradients from native viral nucleocapsids. The separation of the membrane glycoprotein E1 from the peplomeric glycoprotein E2 permitted preparation of antisera against these isolated proteins. A model is proposed for the arrangement of the three major structural proteins in the coronavirus A59 virion in relation to the viral envelope and RNA.

Capsid↗