Effect of Rabbit Anti-Asialo-GM1 (GA1) Polyclonal Antibodies on Neuromuscular Transmission and Acetylcholine-Induced Action Potentials: Neurophysiological and Immunohistochemical Studies

We produced anti-asialo-GM1 (GA1) polyclonal antibodies by sensitizing New Zealand rabbits with GA1 and investigated the epitopes and pathogenic role of anti-GA1 antibodies that appeared in serum. The serum blocked neuromuscular transmission, but not acetylcholine (ACh)-induced potentials, in muscle-spinal cord cocultured cells. The effect was complement independent. The antibodies inhibited voltage-gated Ca2+ channel (VGCC). The epitopes recognized by the antibodies were located in the outer membrane of Schwann cells and motor axons of Wistar rat ventral roots and on motor axons extended from spinal cord to muscle cells in muscle-spinal cocultured cells. The ACh-induced potential was not reduced by the addition of sera, suggesting the blockade is presynaptic. Thus, anti-GA1 antibodies may block neuromuscular transmission by suppressing VGCC on axonal terminals of motor nerves.     

Signal transduction of betacellulin in growth and migration of vascular smooth muscle cells

Epidermal growth factor (EGF) family ligands have been implicated in cardiovascular diseases because of their enhanced expression in vascular lesions and their promoting effects on growth and migration of vascular smooth muscle cells (VSMCs). Betacellulin (BTC), a novel EGF family ligand, has been shown to be expressed in atherosclerotic lesions and to be a potent growth factor of VSMCs. However, the molecular mechanisms downstream of BTC involved in mediating vascular remodeling remain largely unknown. Therefore, the aim of this study was to examine the effects of BTC on signal transduction, growth, and migration in VSMCs. We found that BTC stimulated phosphorylation of EGF receptor (EGFR) at Tyr1068, which was completely blocked by an EGFR kinase inhibitor, AG-1478. BTC also phosphorylated ErbB2 at Tyr877, Tyr1112, and Tyr1248 and induced association of ErbB2 with EGFR, suggesting their heterodimerization in VSMCs. In postreceptor signal transduction, BTC stimulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Akt, and p38 mitogen-activated protein kinase (MAPK). Moreover, BTC stimulated proliferation and migration of VSMCs. ERK and Akt inhibitors suppressed migration markedly and proliferation partially, whereas the p38 inhibitor suppressed migration partially but not proliferation. In addition, we found the presence of endogenous BTC in conditioned medium of VSMCs and an increase of BTC on angiotensin II stimulation. In summary, BTC promotes growth and migration of VSMCs through activation of EGFR, ErbB2, and downstream serine/threonine kinases. Together with the expression and processing of endogenous BTC in VSMCs, our results suggest a critical involvement of BTC in vascular remodeling. epidermal growth factor receptors; ErbB2; migration; signal transduction     

Methylmalonate-semialdehyde dehydrogenase is induced in auxin-stimulated and zinc-stimulated root formation in rice

Proteins induced in rice by auxin and zinc were determined by proteome analysis. Cultured suspension cells of rice were treated with 2,4-dichlorophenoxyacetic acid and ZnSO₄ and then proteins were separated by two-dimensional polyacrylamide gel electrophoresis; seven proteins were found to be induced by auxin and zinc. Of these seven, methylmalonate-semialdehyde dehydrogenase (MMSDH) was elevated by treatment with auxin alone. MMSDH was detected in cultured suspension cells, root and leaf sheath, but not in leaf blades. MMSDH responded to auxin and gibberellin, but did not respond to brassinolide and cytokinin. Furthermore, the amount of MMSDH in slr1, a constitutive gibberellin response mutant, was 2-fold that of wild type. MMSDH mRNA and protein were stimulated in root formation induced by auxin and/or zinc over a 4-week period. These results suggest that MMSDH may be necessary for root formation in rice induced by auxin and/or zinc.Abbreviations BA 6-Benzylaminopurine - BL Brassinolide - CBB Coomassie Brilliant Blue - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA Gibberellic acid - IAA Indole-3-acetic acid - MALDI-TOF MS Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry - MMSDH Methylmalonate-semialdehyde dehydrogenase - 2D-PAGE Two-dimensional polyacrylamide gel electrophoresis - PVDF Polyvinylidene difluoride     

Vasopressin phosphorylates HSP27 in aortic smooth muscle cells

Administration of arginine vasopressin (AVP) time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) at Ser-15 and Ser-85 in smooth muscle of aorta in vivo. The AVP-induced phosphorylation of HSP27 at Ser-15 and Ser-85 was inhibited by a V1a receptor antagonist but not by a V2 receptor antagonist. In cultured aortic smooth muscle A10 cells, AVP markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85. The AVP-induced phosphorylation of HSP27 was attenuated by SB203580 and PD169316, inhibitors of p38 mitogen-activated protein (MAP) kinase, but not by PD98059, a MEK inhibitor. These results strongly suggest that AVP phosphorylates HSP27 via p38 MAP kinase in aortic smooth muscle cells.     

Interaction of the N-terminal domain of Escherichia coli heat-shock protein ClpB and protein aggregates during chaperone activity

The Escherichia coli heat-shock protein ClpB reactivates protein aggregates in cooperation with the DnaK chaperone system. The ClpB N-terminal domain plays an important role in the chaperone activity, but its mechanism remains unknown. In this study, we investigated the effect of the ClpB N-terminal domain on malate dehydrogenase (MDH) refolding. ClpB reduced the yield of MDH refolding by a strong interaction with the intermediate. However, the refolding kinetics was not affected by deletion of the ClpB N-terminal domain (ClpBDeltaN), indicating that MDH refolding was affected by interaction with the N-terminal domain. In addition, the MDH refolding yield increased 50% in the presence of the ClpB N-terminal fragment (ClpBN). Fluorescence polarization analysis showed that this chaperone-like activity is explained best by a weak interaction between ClpBN and the reversible aggregate of MDH. The dissociation constant of ClpBN and the reversible aggregate was estimated as 45 muM from the calculation of the refolding kinetics. Amino acid substitutions at Leu 97 and Leu 110 on the ClpBN surface reduced the chaperone-like activity and the affinity to the substrate. In addition, these residues are involved in stimulation of ATPase activity in ClpB. Thus, Leu 97 and Leu 110 are responsible for the substrate recognition and the regulation of ATP-induced ClpB conformational change.     

Predominant role of basicity of leaving group in alpha-effect for nucleophilic ester cleavage

It has been found that alpha-effects in nucleophilic reactions, unexpectedly large nucleophilicity due to adjacent unpaired electrons, are strongly dependent on the structure of substrate. The nucleophilic cleavages of 4-nitrobenzoate esters and 4-methylbenzoate esters by HOO- have been systematically investigated in detail. When the leaving groups of substrates are sufficiently good (aryl, 2,2,2-trifluoroethyl, and 2,2-dichloroethyl esters), alpha-effect is evident. However, this effect drastically decreases as the leaving group gets poorer, and is only marginal for the cleavages of 2-fluoroethyl and methyl esters. In the nucleophilic cleavages by salicylaldoxime and acetohydroxamic acid, alpha-effect is also notable only for the esters having good leaving groups. These enormous dependences of alpha-effects on the substrate-structure have been interpreted in terms of the difference in the position of transition-state in the reaction coordinate.     

Distribution and phylogenetic analysis of family 19 chitinases in Actinobacteria

In organisms other than higher plants, family 19 chitinase was first discovered in Streptomyces griseus HUT6037, and later, the general occurrence of this enzyme in Streptomyces species was demonstrated. In the present study, the distribution of family 19 chitinases in the class Actinobacteria and the phylogenetic relationship of Actinobacteria family 19 chitinases with family 19 chitinases of other organisms were investigated. Forty-nine strains were chosen to cover almost all the suborders of the class Actinobacteria, and chitinase production was examined. Of the 49 strains, 22 formed cleared zones on agar plates containing colloidal chitin and thus appeared to produce chitinases. These 22 chitinase-positive strains were subjected to Southern hybridization analysis by using a labeled DNA fragment corresponding to the catalytic domain of ChiC, and the presence of genes similar to chiC of S. griseus HUT6037 in at least 13 strains was suggested by the results. PCR amplification and sequencing of the DNA fragments corresponding to the major part of the catalytic domains of the family 19 chitinase genes confirmed the presence of family 19 chitinase genes in these 13 strains. The strains possessing family 19 chitinase genes belong to 6 of the 10 suborders in the order Actinomycetales, which account for the greatest part of the Actinobacteria: Phylogenetic analysis suggested that there is a close evolutionary relationship between family 19 chitinases found in Actinobacteria and plant class IV chitinases. The general occurrence of family 19 chitinase genes in Streptomycineae and the high sequence similarity among the genes found in Actinobacteria suggest that the family 19 chitinase gene was first acquired by an ancestor of the Streptomycineae and spread among the Actinobacteria through horizontal gene transfer.     

Site-directed mutagenesis establishes aspartic acids-227 and -342 as essential for enzyme activity in an isomalto-dextranase from Arthrobacter globiformis

Isomalto-dextranase, from Arthrobacter globiformis T6, is a member of the glycoside hydrolase family 27. However, the alignments of the whole amino acid sequence are distinct from other members of this family. The enzymes cleave the glycosidic bond of the substrate in two different manners: either retaining or inverting the anomeric configuration. We believe that a retaining enzyme is involved in a two-step, double-displacement mechanism utilizing active site carboxylic acids as the nucleophile and general acid/base catalysts in the hydrolytic reaction. The critical amino acid residues at the isomalto-dextranase active site that catalyzes the hydrolysis reaction of dextran have been identified and the roles of nine amino acid residues (D107, D163, D227, D295, D340, D342, D373, D396, and E420) in the isomalto-dextranase from A. globiformis analyzed by site-directed mutagenesis. Of 15 mutant enzymes that were prepared, eight had reduced activities for dextran hydrolysis. Aspartic acids-227 and -342, which are part of the apparent catalytic dyad, were essential for hydrolase activity toward dextran.     

Anti-lipid deposition effect of HMG-CoA reductase inhibitor, pitavastatin, in a rat model of hypertension and hypercholesterolemia

Since the rat is an atherosclerosis-resistant species, the study of atherosclerosis using rats is limited. The present study was undertaken to develop an atherosclerotic model in rats, to investigate the effect of nitric oxide (NO) inactivation and hyperlipidemia, and to evaluate the effect of pitavastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitor, on NO inactivation and on hyperlipidemia-induced changes in the cardiovascular system. Four-month-old male spontaneously hypertensive hyperlipidemic rats (SHHR) and Sprague-Dawley (SD) rats were used to study 1) the effect of the period of treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mg/L) on high fat diet (HFD)-treated SHHR and SD rats, and 2) the effect of pitavastatin (Pit, 0.3 mg/kg/day) on the changes in the aorta of L-NAME- and HFD-treated SHHR and SD rats. L-NAME administration for 1 month then HFD feeding for 2 months markedly increased the deposition of lipids and the thickness of the endothelium in SHHR. Continuous L-NAME treatment with HFD produced severe injury and stripped of endothelium in both strains. The plasma total cholesterol of L-NAME + HFD-treated and L-NAME + HFD + Pit-treated SHHR was significantly higher than that of control SHHR. Lipid deposition, however, was comparatively less in the aorta of L-NAME + HFD + Pit-treated SHHR. The concentration of cholesterol in the aorta of control SHHR was significantly lower than that in the aorta of L-NAME + HFD-treated SHHR, whereas that of L-NAME + HFD + Pit-treated SHHR was the same as that in control SHHR. These data indicated that Pit blocked lipid deposition in the aorta of L-NAME + HFD treated SHHR without changing plasma lipid profiles. In conclusion, NO inactivation and HFD induce lipid deposition in the endothelium, and the HMG-CoA reductase inhibitor blocks the deposition in SHHR.