Simultaneous determination of ascorbic acid and dehydroascorbic acid in fish tissues by high-performance liquid chromatography

An high-performance liquid chromatographic method with post-column derivatization has been developed for the simultaneous determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fish tissues. Extracted AA and DHAA were separated by a Shim-pack SCR-101H column within 20 min, reacted with sodium hydroxide containing sodium borohydride and monitored at 300 nm. The detection limits for both AA and DHAA were 0.1 μg/ml.     

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation. This receptor-induced inhibition of proliferation was blocked by a kinase-deficient MKK4 and by the p38 MAPK inhibitor SB203580. Additionally, transfection of constitutively activated Galphaq into cells also led to inhibition of proliferation in a JNK- and p38 MAPK-dependent manner. These results demonstrate that the alpha1B-adrenergic receptor/Galphaq signaling inhibits cell proliferation through pathways involving JNK and p38 MAPK.     

Effects of recombinant human macrophage colony-stimulating factor on atherosclerotic lesions established in the aorta of high cholesterol-fed rabbits

Anti-atherosclerotic effects of human macrophage colony-stimulating factor were investigated using rabbits fed a high cholesterol diet. Rabbits fed a diet containing 2% cholesterol for 59 days developed hyperlipidemia and atheromatous aortic plaques. They were then administered 80 microg/kg/day of either macrophage colony-stimulating factor or human serum albumin, as a control, for the next 12 weeks. Compared with the control group, rabbits treated with macrophage colony-stimulating factor had significantly fewer plaques on the inner surface of the thoracic and abdominal aortae, and half the sectional area of thickened intima in the aortic arch, as well as in the thoracic and abdominal aortae. Macrophage colony-stimulating factor also decreased the cholesterol content of the atherosclerotic lesions. Serobiochemical analyses revealed that macrophage colony-stimulating factor increased the levels of high density lipoprotein-cholesterol significantly, without influencing other lipid parameters such as the level of low density lipoproteins. The effects of macrophage colony-stimulating factor were evident until the fourth week of drug injection, at which time anti-human macrophage colony-stimulating factor antibodies were clearly induced in the serum. These results indicate that exogenously administered macrophage colony-stimulating factor suppresses atherosclerotic lesions induced by a high cholesterol diet by activating lipid metabolism in vivo.     

A high molecular weight glutamyl endopeptidase and its endogenous inhibitors from cucumber leaves

We purified a glutamyl endopeptidase that is a major foliar endopeptidase in cucumber. The endopeptidase had a molecular mass of 400 kDa, consisted of four subunits of 97 kDa, and was inactivated by SH-modifying reagents. Its optimum pH and optimum temperature were 8.0 and 30-37 degrees C, respectively. An internal amino acid sequence of the endopeptidase was highly homologous to a partial sequence of unidentified proteins deduced from genetic information for Arabidopsis thaliana, soybean and rice, but not to the sequences of bacterial glutamyl endopeptidases or animal proteases. Therefore, the unidentified proteins might be glutamyl endopeptidases and be widely distributed only among plant species. The activity of the cucumber glutamyl endopeptidase was inhibited by at least three inhibitors existing in cucumber leaves. One of the inhibitors was a competitive inhibitor of 25 kDa, which did not significantly inhibit commercial endopeptidases derived from animals and microorganisms. This suggests that the cucumber glutamyl endopeptidase might be controlled by endogenous inhibitors in vivo.     

Regulation of Rac and Cdc42 pathways by G(i) during lysophosphatidic acid-induced cell spreading

The pertussis toxin-sensitive G protein, G(i), has been implicated in lysophosphatidic acid-induced cell mitogenesis and migration, but the mechanisms remain to be detailed. In the present study, we found that pertussis toxin blocks lysophosphatidic acid-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of constitutively active mutants of Rho family small GTP-binding proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that G(i)-induced cell spreading is mediated through the Rac and Cdc42 pathway. Transfection of constitutively active mutants of G alpha(i) and G alpha(11) and G beta gamma subunits enhanced spreading of pertussis toxin-treated cells. G beta(1) with G gamma(12), a major G gamma form in fibroblasts, was more effective for increasing cell spreading than G beta(1)gamma(2) or G beta(1) plus G gamma(12)S2A, a mutant in which Ser-2, a phosphorylation site for protein kinase C, is replaced with alanine. In addition, a protein kinase C inhibitor diminished G beta(1)gamma(12)-induced cell spreading, suggesting a role for phosphorylation of the protein. These findings indicate that both G alpha(i) and G beta gamma stimulate Rac and Cdc42 pathways with lysophosphatidic acid-induced cell spreading on fibronectin.     

Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade

Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Galpha(q), but not by Gbetagamma, were dependent on phospholipase C and c-Src. Galpha(q) stimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gbetagamma activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gbetagamma-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Galpha(q) and Gbetagamma stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.     

A TGACGT motif in the 5'-upstream region of alpha-amylase gene from Vigna mungo is a cis-element for expression in cotyledons of germinated seeds

Alpha-amylase is expressed at high levels in cotyledons of germinated seeds of Vigna mungo. The mRNA for alpha-amylase appeared in cotyledons of the seeds at 1 d after imbibition started (DAI). Two TGACGT motifs at -445 and at -125 in the promoter region of the gene interacted with nuclear proteins from cotyledons of dry seeds and the activities were detected until 3 DAI. A transient assay with particle bombardment showed that the downstream region from -135 in the promoter was required for high level expression in the cotyledons and the activity was reduced by mutation of the TGACGT motif at -125. The activities to bind the TGACGT motifs were detected in the axes of the seeds at 1 DAI but disappeared at 4 DAI, although the mRNA for alpha-amylase in the axes appeared at 4 DAI and increased in level by 6 DAI. A transient assay experiment showed that a positive regulatory element for the expression in the axes was located in the region from -630 to -453. These results indicated that the TGACGT motif at -125 was required for high level expression of the gene in the cotyledons of the germinated seeds.     

Involvement of histone phosphorylation in thymocyte apoptosis by protein phosphatase inhibitors

Incubation of rat thymocytes with the inhibitors of protein phosphatase such as calyculin A and okadaic acid resulted in an increase in DNA fragmentation. These effects were dependent on the concentration of the inhibitors and the incubation time. Analyses of the fragmented DNA revealed the production of approximately 50 kbp of DNA and a 180 bp DNA ladder. In addition, a laser scanning-microscopic analysis showed that these compounds caused nuclear condensation. Thus, these results demonstrated that protein phosphatase inhibitors induced thymocyte apoptosis. The inhibitors of protein phosphatase increased the phosphorylation of proteins of approximately 15 kDa. The phosphorylation of proteins preceded the DNA fragmentation induced by these inhibitors. Judging from acetic acid-urea-Triton X-100 gel electrophoresis, the phosphorylated proteins were histone H1 and H2A/H3. Therefore, these results suggest that phosphorylation of histones triggers the DNA fragmentation of thymocytes undergoing apoptosis.     

Identification of the calmodulin-binding domain of neuron-specific protein kinase C substrate protein CAP-22/NAP-22. Direct involvement of protein myristoylation in calmodulin-target protein interaction

Various proteins in the signal transduction pathways as well as those of viral origin have been shown to be myristoylated. Although the modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is involved in the synaptogenesis and neuronal plasticity, binds calmodulin, but the protein lacks any canonical calmodulin-binding domain. In the present report, we show that CAP-23/NAP-22 isolated from rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin. Myristoylated and non-myristoylated recombinant proteins were produced in Escherichia coli, and their calmodulin-binding properties were examined. Only the former bound to calmodulin. Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated. The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain. Phosphorylation of a single serine residue in the N-terminal domain (Ser5) by protein kinase C abolished the binding. Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was also found myristoylation-dependent, suggesting the importance of myristoylation in protein-protein interactions.