Kiyoshi Ikeda

Prostaglandins are powerful inducers of NGF and BDNF production in mouse astrocyte cultures

We found that prostaglandin (PG) D2 and PGE2, which are major PGs in the brain of mammals, powerfully induced the secretion of nerve growth factor (NGF) from cultured mouse astrocytes; PGE2 or PGD2 induced an approximately 12‐ or 19‐fold increase in NGF secretion after a 24‐h incubation, respectively. Moreover, it was found that the sequential metabolites of PGD2, PGJ2, Δ12‐PGJ2, and 15‐deoxy‐Δ12,14‐PGJ2, induced the NGF secretion to the culture medium strikingly (60–98‐fold of the control after a 24‐h incubation). NGF secretion induced by the J2 series of PGs was accompanied by the increased expression of NGF mRNA. These PGs also stimulated the secretion/synthesis of brain‐derived neurotrophic factor (BDNF). Our findings suggest that PGs play a neuroprotective role by inducing NGF and BDNF production in the central nervous system.

Up-regulation of the expression of leucine-rich α2-glycoprotein in hepatocytes by the mediators of acute-phase response

n Abstractn n Leucine-rich α2-glycoprotein (LRG) is a plasma protein in which leucine-rich repeats (LRRs) were first discovered. Although the physiological function of LRG is not known, increases in the serum level of LRG have been reported in various diseases. In this study, we found that LRG was induced by recombinant human IL-6 in human hepatoma HepG2 cells. The induction of LRG by IL-6 was up-regulated synergistically with either IL-1β or TNFα in a pattern similar to those for type 1 acute-phase proteins. We also found that lipopolysaccharide (LPS) administered intraperitoneally to mice enhanced dose-dependently the expression of LRG mRNA in the liver as well as those for mouse major acute-phase proteins. These results strongly suggest that LRG was a secretory type 1 acute-phase protein whose expression was up-regulated by the mediator of acute-phase response.n n

The inhibitory mechanism of bovine pancreatic phospholipase A2 by aldehyde terpenoids

Abstract We established the stereoselective synthesis of (E)-3-methoxycarbonyl-2,4,6-trienal compound A and discovered that the compound A showed more powerful inhibitory activity toward phospholipase A 2 (PLA 2 ) from bovine pancreas than manoalide which is a typical PLA 2 inhibitor. As the inhibitory mechanism of PLA 2 by A , the irreversible formation of dihydropyridine derivative resulting from the reaction of A with lysine residues in PLA 2 was proposed based on the model reactions. Furthermore, A was found to selectively modify Lys56 which is included in the interfacial recognition site of this enzyme by the MALDI-TOF-MS peptide mapping analyses.

A Novel Phospholipase A2 Inhibitor with Leucine-rich Repeats from the Blood Plasma of Agkistrodon blomhoffii siniticus SEQUENCE HOMOLOGIES WITH HUMAN LEUCINE-RICH α2-GLYCOPROTEIN

The phospholipase A2(PLA2) inhibitor PLIβ, purified from the blood plasma of Chinese mamushi snake (Agkistrodon blomhoffii siniticus), is a 160-kDa trimer with three 50-kDa subunits; and it inhibits specifically the enzymatic activity of the basic PLA2 from its own venom (Ohkura, N., Okuhara, H., Inoue, S., Ikeda, K., and Hayashi, K. (1997) Biochem. J. 325, 527–531). In the present study, the 50-kDa subunit was found to be glycosylated withN-linked carbohydrate, and enzymatic deglycosylation decreased the molecular mass of the 50-kDa subunit to 39-kDa. One 160-kDa trimer of PLIβ was found to form a stable complex with three basic PLA2 molecules, indicating that one basic PLA2 molecule would bind stoichiometrically to one subunit of PLIβ. A cDNA encoding PLIβ was isolated from a Chinese mamushi liver cDNA library by use of a probe prepared by a polymerase chain reaction on the basis of the partially determined amino acid sequence of the subunit. The cDNA contained an open reading frame encoding a 23-residue signal sequence followed by a 308-residue protein, which contained the sequences of all the peptides derived by lysyl endopeptidase digestion of the subunit. The molecular mass of the mature protein was calculated to be 34,594 Da, and the deduced amino acid sequence contained four potentialN-glycosylation sites. The sequence of PLIβ showed no significant homology with that of the known PLA2inhibitors. But, interestingly, it exhibited 33% identity with that of human leucine-rich α2-glycoprotein, a serum protein of unknown function. The most striking feature of the sequence is that it contained nine leucine-rich repeats (LRRs), each of 24 amino acid residues and thus encompassing over two-thirds of the molecule. LRRs in PLIβ might be responsible for the specific binding to basic PLA2, since LRRs are considered as the motifs involved in protein-protein interactions.

Synthesis of a new phospholipase A2inhibitor of an aldehyde terpenoid and its possible inhibitory mechanism

Abstract 3-(E)-Methoxycarbonyl-2,4,6-trienal compound A was stereoselectively synthesized and found to show strong inhibitory activity toward phospholipase A 2 (PLA 2 ) from bovine pancreas. As the inhibitory mechanism of PLA 2 by A , the irreversible formation of dihydropyridine resulting from the reaction of A with lysine residues in PLA 2 is proposed based on the model reactions.

Specificity of two types of phospholipase A2 inhibitors from the plasma of venomous snakes

Specificity of two different types of phospholipase A2 (PLA2) inhibitory proteins from the blood plasma of venomous snakes was investigated. Two Crotalidae inhibitors, having a carbohydrate recognition domain (CRD) in their sequences, inhibited specifically the group‐II acidic PLA2s of their own snake venom. On the other hand, Elapidae inhibitor, having two tandem patterns of cysteine residues found in proteins of the Ly‐6 superfamily, inhibited not only the group‐I PLA2 from its own snake venom but also the group‐I, ‐II, and ‐III PLA2s from other snake venom. Amino acid sequences of PLA2s that were specifically inhibited by the inhibitors were compared with those of the other PLA2s. A unique aromatic patch structure appeared on the group‐II acidic PLA2s was suggested to be involved in the binding to the Crotalidae inhibitors; and residues located in or close to the Ca2+ binding loop of PLA2, in the binding to the Elapidae inhibitor.

Production of NGF, BDNF and GDNF in mouse astrocyte cultures is strongly enhanced by a cerebral vasodilator, ifenprodil

Ifenprodil, a non-competitive NMDA-receptor antagonist, has been shown to exhibit marked cytoprotective activities in animal models for focal ischemia and Parkinsons disease. To test the hypothesis that the cytoprotective effect is due to the release of neurotrophic factors (NTFs), we examined the effects of ifenprodil on the NTF contents in mouse astrocyte cultures. The results revealed that ifenprodil strongly enhanced the production of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) in these cultures. The ifenprodil-induced NGF secretion was found to be partially mediated by the activation of protein kinase C (PKC) and p42/p44 mitogen-activated protein (MAP) kinase cascade pathways. These findings suggest that the cytoprotective effects of ifenprodil are probably attributed to enhanced secretion of these NTFs from astrocytes.

Identification of β-type phospholipase A2 inhibitor in a nonvenomous snake, Elaphe quadrivirgata

Abstract A novel serum protein inhibiting specifically the enzymatic activity of the basic phospholipase A 2 (PLA 2 ) from the venom of the Chinese mamushi snake ( Agkistrodon blomhoffii siniticus ) was purified from a nonvenomous Colubridae snake, Elaphe quadrivirgata . The purified inhibitor was a 150-kDa glycoprotein having a trimeric structure, composed of two homologous 50-kDa subunits. Their amino acid sequences, containing leucine-rich repeats, were typical of the β-type PLA 2 inhibitor (PLIβ), previously identified from the serum of A. blomhoffii siniticus . The inhibitor inhibited exclusively group II basic PLA 2 s and did not inhibit other kinds of PLA 2 s. This is the first paper reporting the existence of PLIβ in a nonvenomous snake. The existence of PLIβ in the nonvenomous snake reflects that PLIβs are widely distributed over the snake species and participate commonly in regulating the physiological activities of the unidentified target PLA 2 s.

Structure of acidic phospholipase A2 for the venom of Agkistrodon halys blomhoffii at 2.8Åresolution

The crystal structure of acidic phospholipase A2 from the venom of Agkistrodon halys blomhoffii has been determined by molecular replacement methods based on the known structure of Crotalus atrox PLA2, a same group II enzyme. The overall structures, except the calcium-binding regions, are very similar to each other. A calcium ion is pentagonally ligated to two carboxylate oxygen atoms of Asp-49 and each carbonyl oxygen atoms of Tyr-28, Gly-30 and Ala-31. A reason why the former enzyme functions as monomeric form, while the latter one does as dimer, could be presumed by the structural comparison of these calcium-binding regions. Although Gly-32 is usually participated as a ligand in the coordination with calcium ion in group I PLA2, it is characteristically replaced to Ala-31 in the present structure, and thus the coordination geometry of calcium ion is rather different from the usually observed one.

Production of nerve growth factor enhanced in cultured mouse astrocytes by glycerophospholipids, sphingolipids, and their related compounds

The NGF secretion from cultured mouse astrocytes was enhanced by sublethal concentrations of phosphatidic acid (PA), ceramide, or sphingosine (Sph), and concentration dependently by lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC), or sphingosine-1-phosphate (S1P), but was unaffected by any concentrations of phosphatidylcholine (PC), phosphatidylethanolamine (PE) or sphingomyelin (SM). The enhancement of NGF synthesis by Sph was completely inhibited by the addition of ceramide synthase inhibitor, fumonisin B1. LPA and S1P showed similar hyperbolic curves with maximum NGF secretion at concentrations of more than 50xa0μM, but they showed no proliferative effect on quiescent astrocytes. The mechanisms underlying the stimulation of NGF synthesis by 50xa0μM LPA and 50xa0μM S1P were further investigated by using various inhibitors. One of the protein kinase C (PKC) inhibitors, Gö6976, suppressed the LPA- and S1P-stimulated NGF synthesis by 70 and 80%, respectively. LPA and S1P were found to activate common multiple signaling pathways for NGF production, involving the activation of the protein kinase C (PKC), mitogen-activated protein (MAP) kinase, and phosphatidylinositol 3-kinase (PI-3K) pathways.