Potent inhibitors of protein kinase C have been isolated from sheep brain by DEAE-cellulose, phenyl-Sepharose CL-4B and Mono Q anion-exchange chromatography. Analysis by one-and two-dimensional SDS/polyacrylamide gel electrophoresis showed the purified preparation to contain three bands ranging over 29 -33 kDa in molecular mass, each consisting of several charge isomers with similar pl values (5.4-5.7). Peptide mapping, amino acid analysis and sequencing suggested that the proteins are related, with the possibility that some species are distinct gene products. The concentration of inhibitor proteins required for half-maximal inhibition of protein kinase C activity is 1.7 pM. Inhibitory activity could not be affected by increasing the substrate, cofactor or ATP concentration in the standard protein kinase C assay, but was abolished by heat treatment. The inhibitor preparation did not affect the binding of phorbol dibutyrate to protein kinase C and could inhibit phosphorylation over a wide range of calcium concentrations. Inhibitory activity could be removed by immunoprecipitation of the purified inhibitor proteins with polyclonal antibodies raised against synthetic peptides, the sequences corresponding to those of peptide fragments obtained from protein digests. Amino acid sequence analysis of the inhibitors confirms they are novel proteins although similarities exist with a neuronal specific protein termed 14-3-3 and the carboxy terminus of the calcium-lipid binding series (endonexin/cdlpactin/lipocortin).A common mechanism for transducing signals from hormones, neurotransmitters, antigens and growth factors etc. into cells is by phospholipase hydrolysis of inositol phospholipids [l]. One product of this receptor-mediated hydrolysis, inositol 1,4,5-trisphosphate, releases calcium from intracellular stores while the second product 1,2-diacylglycerol, remains in membranes and initiates the activation of protein kinase C. Signal transduction through phosphorylation by this kinase is separate from and often synergistic to the calcium signalling pathway for the control of various cellular functions.Protein kinase C is known to be ubiquitous in tissues and mediates a range of physiological processes such as secretion from endocrine and exocrine tissues, transmitter release in central and peripheral nervous systems, smooth muscle contraction, lymphocyte mitogenesis as well as modulating membrane ion conductance [2]. Protein kinase C consists of a family of highly similar but distinct proteins [3,4]; differences in substrate and cofactor specificities [5, 61 and tissue expression [7] are known to occur for the different isoforms. This may in part account for the wide range of physiological activities attributed to protein kinase C.Activation of protein kinase C in vivo and its dependence on cofactors is now well documented [8]. It can also be activated directly by phorbol esters and is now accepted to be The amino acid sequence of the 17-kDa kinase C inhibitor protein, isolated by McDonald and Walsh, has been shown to ...

Background-Ginseng is a commonly used nutraceutical. Intriguingly, existing literature reports both wound-healing and antitumor effects of ginseng extract through opposing activities on the vascular system. To elucidate this perplexity, we merged a chemical fingerprinting approach with a deconstructional study of the effects of pure molecules from ginseng extract on angiogenesis. Methods and Results-A mass spectrometric compositional analysis of American, Chinese and Korean, and Sanqi ginseng revealed distinct "sterol ginsenoside" fingerprints, especially in the ratio between a triol, Rg1, and a diol, Rb1, the 2 most prevalent constituents. Using a Matrigel implant model and reconstituting the extracts using distinct ratios of the 2 ginsenosides, we demonstrate that the dominance of Rg1 leads to angiogenesis, whereas Rb1 exerts an opposing effect. Rg1 also promoted functional neovascularization into a polymer scaffold in vivo and the proliferation of, chemoinvasion of, and tubulogenesis by endothelial cells in vitro, an effect mediated through the expression of nitric oxide synthase and the phosphatidylinositol-3 kinase3 Akt pathway. In contrast, Rb1 inhibited the earliest step in angiogenesis, the chemoinvasion of endothelial cells. Conclusions-The present study explains, for the first time, the ambiguity about the effects of ginseng in vascular pathophysiology based on the existence of opposing active principles in the extract. We also unraveled a speciogeographic variation impinging on the compositional fingerprint that may modulate the final phenotype. This emphasizes the need for regulations standardizing herbal therapy, currently under the Dietary Supplement and Health Education Act. Furthermore, we propose that Rg1 could be a prototype for a novel group of nonpeptide molecules that can induce therapeutic angiogenesis, such as in wound healing. Key Words: ginsenosides Ⅲ angiogenesis Ⅲ nitric oxide T he use of botanicals as alternative medicine has seen a resurgence of use in the United States and Europe. In 2001, $17.8 billion was spent on dietary supplements, $4.2 billion of it for herbal remedies. 1 It is estimated that Ͼ12.1% of adults in the United States used herbal medicines in 1997. 2 Interestingly, in the United States, herbal medicines are regulated as dietary supplements and fall under the purview of the 1994 Dietary Supplement and Health Education Act (DSHEA) and thus can be marketed without prior approval by the Food and Drug Administration (FDA). 3 Ginseng is a key component in traditional Chinese medicine and is also one of the most extensively used botanical products in the West, which is evident from annual sales of US $0.225 to $0.3 billion. 4 Named by the botanist Carl Meyer, the genus Panax derives its name from the Greek pan (all) and akos (healing). 4 The activity of ginseng extract has been studied extensively. Ambiguously, ginseng extract has been reported to promote wound healing 5 and inhibit tumor growth 6,7 through opposing effects on angiogenesis. To elucidate this perplexity in...

For the widely distributed P2Y receptors for nucleotides, the transductional and functional responses downstream of their coupling to G proteins are poorly characterized. Here we describe apoptotic induction and the associated differential stimulation of mitogenactivated protein (MAP) kinase family members by the human P2Y 1 receptor. The potent P2Y 1 receptor agonist, 2-methylthio-ADP (2-MeSADP), stimulated the extracellular-signal regulated kinases (ERK1/2) (EC 50 ϳ5 nM) as well as several, but not all isoforms detected, of the stress-activated protein kinase (SAPK) family. Phosphoisoforms of p38 were unaffected. The induced kinase activity was blocked by the P2Y 1 receptor-selective antagonist, adenosine-2-phosphate-5-phosphate, but unaffected by pertussis toxin. In addition, the endogenous ligand ADP, and significantly also 2-MeSATP, induced concentration-dependent phosphorylation changes in the same MAP kinase family members. The sustained activation of ERK1/2 was associated with Elk-1 phosphorylation that was abolished by the MEK1 inhibitor, PD 98059. However, the concomitant transient activation of the SAPKs was not sufficient to induce c-Jun or ATF-2 phosphorylation. The transient phase of the ERK activity was partially inhibited either by the phosphatidylinositol 3-kinase inhibitor, LY 294002, or the PKC inhibitor, Gö 6976. In addition, the Src inhibitor, PP1, or expression of dominant negative Ras also attenuated the transient phase of ERK phosphorylation. In contrast, inhibition of Ras or Src had no effect on the sustained ERK activity, which was critically dependent on phosphatidylinositol 3-kinase. The transient SAPK activity was suppressed by expression of a dominant negative form of MKK4. Furthermore, this kinase-deficient mutant inhibited 2-MeSADP-induced caspase-3 stimulation and the associated decrease in cell number. In conclusion, adenosine di-and triphosphate stimulation of the human P2Y 1 receptor can transiently activate the Ras-ERK cascade via the cooperative effects of phosphatidylinositol 3-kinase, Src and PKC. The sustained ERK stimulation, via a Ras-insensitive pathway, culminates in Elk-1 activation without inducing a proliferation effect. The transient SAPK activity did not evoke transcription factor phosphorylation but was required for the P2Y 1 receptor-mediated apoptotic function.Extracellular nucleotides can interact with cell surface P2 receptors both in the central nervous system and in peripheral tissues to produce a broad range of physiological effects. The P2 family is divided into two main types as follows: the P2X receptors are ligand-gated ion channels, and the P2Y receptors are G protein-coupled (1, 2). Part of the present study describes the signaling pathways of the P2Y 1 receptor, the first member of the P2Y family to be identified (3). The P2Y 1 receptor is widely distributed and has been described in mammalian heart, vascular, liver, kidney, prostate, gastrointestinal, pulmonary, connective, and immune tissues (4, 5). It has also been identified in skeletal muscle ...

A potent inhibitor of protein kinase C (PKC), inhibitor protein-1 (KCIP-l), isolated from sheep brain has been shown to consist of eight isoforms by reverse-phase HPLC. Direct protein sequence analysis has revealed these to be the same as those of 14-3-3 protein, described as an activator of tyrosine and tryptophan hydroxylases involved in neurotransmitter biosynthesis. The N-termini of KCIP-1 isoforms were shown to be acetylated, and secondary structure predictions revealed a high degree of a-helix with an amphipathic nature. KCIP-1 showed no inhibitory activity towards protein kinase M (the catalytic fragment of PKC) and had no effect on the activities of three other protein kinases, CAMP-dependent protein kinase, Ca2 c calmoddin-dependent protein kinase I1 and casein kinase 2. Four forms of KCIP-1 were shown to be substrates for PKC in vitro, but none were phosphorylated by the other protein kinases mentioned above.Protein kinase C (PKC), a calcium and phospholipid-dependent enzyme, is activated by diacylglycerol, hydrolyzed from inositol phospholipids by phospholipase C in response to a variety of extracellular signals [l]. This results in the phosphorylation of a wide range of proteins leading to the regulation of many physiological processes. A large family of PKC isoforms with multiple subspecies have now been identified which show subtle individual characteristics and specificity for substrates [2] which may suggest different roles for some of the isoforms.The role of PKC in regulating cellular function has been studied using specific activators (phorbol esters) which substitute for the physiological second messenger diacylglycerol [3], as well as naturally occurring and synthetic inhibitors. H-7 and K-252 inhibit the enzyme by competing with ATP [4,5] but the use of these compounds is limited in studies of regulatory mechanisms by their lack of specificity towards PKC. One of the most potent PKC inhibitors recently described is the microbial alkaloid staurosporine, with a Ki of 2.7 nM [6]. Endogenous sphingosine and lysosphingolipids may play a role in cellular regulation and have been proposed to act as negative effectors of PKC [7].There have been few reports of mammalian proteins that have been shown to have potent inhibitory activity towards PKC. We recently described the isolation and characterization of a PKC inhibitor protein from sheep brain named protein Correspondence to A. Aitken, Laboratory of Protein Structure,