Reversal of anticancer drug resistance by COTC based on intracellular glutathione and glyoxalase I

Suppression of resistance to anticancer drugs by COTC of glyoxalase I (GloI) inhibitor targeting intracellular glutathione (GSH) and GloI was studied. Depletion of the cellular GSH content and inhibition of GloI by COTC increased chemotherapy-mediated apoptosis in apoptosis-resistant pancreatic adenocarcinoma AsPC-1 cells.     

Overexpression of human myotonic dystrophy protein kinase in Schizosaccharomyces pombe induces an abnormal polarized and swollen cell morphology

We expressed human myotonic dystrophy protein kinase (DMPK) in the fission yeast Schizosaccharomyces pombe, in which the overexpression of human DMPK affects cell growth and cell shape. The human DMPK protein has a leucine-rich domain at the N-terminus, a serine/threonine kinase domain in the middle, and a hydrophobic region at the C-terminus. C-Terminus-deleted DMPK produced a middle-swollen phenotype (lemon-like shape), indicating an abnormality in cell division. On the other hand, when both the kinase domain and C-terminus were present, the expression of DMPK resulted in polarized cell growth and multinucleated/branched cells. The lemon-like phenotype seen with the C-terminus-deleted DMPK disappeared when the ATP binding site of DMPK was disrupted by replacing the lysine at amino acid 100 with arginine (K100R mutant). However, polarized and/or multinucleated cells lacking the DMPK N-terminus were not rescued by the K100R mutation. Therefore, we conclude that the N-terminus of DMPK plays an important role in DMPK kinase activity, and that the C-terminus of DMPK determines the intracellular localization of the protein.     

Intracellular localization of homopolymeric amino acid-containing proteins expressed in mammalian cells

Many human proteins have homopolymeric amino acid (HPAA) tracts, which are involved in protein-protein interactions and also have intrinsic polymerization properties. Polyglutamine or polyalanine expansions cause several neurodegenerative diseases. To examine the properties of HPAAs, we expressed 20 kinds of 30-residue HPAA fused to the C terminus of yellow fluorescent protein in mammalian cells. Specific localization was observed depending on the HPAA. Polyarginine and polylysine aggregated in the nucleus. Polyalanine, polyhistidine, polyisoleucine, polyleucine, polymethionine, polyphenylalanine, polythreonine, polytryptophan, and polyvaline localized in the cytoplasm, and some of these HPAAs formed aggregate(s). Hydrophobic HPAAs such as polyisoleucine, polyleucine, polyphenylalanine, and polyvaline were found as one major aggregate or cumulus in the perinuclear region. Western blot analysis indicated that hydrophobic HPAA tracts appear to oligomerize and form high molecular weight complexes. These results indicate that hydrophobicity itself may trigger the oligomerization and aggregation of proteins when overexpressed in cells. Our experiments provide novel insights into the nature of the HPAAs that are often seen in human and other organisms.     

Role of a highly conserved YPITP motif in 2-oxoacid:ferredoxin oxidoreductase: heterologous expression of the gene from Sulfolobus sp.strain 7, and characterization of the recombinant and variant enzymes.

2-Oxoacid:ferredoxin oxidoreductase from Sulfolobus sp. strain 7, an aerobic and thermoacidophilic crenoarchaeon, catalyses the coenzyme A-dependent oxidative decarboxylation of pyruvate and 2-oxoglutarate, a cognate Zn-7Fe-ferredoxin serving as an electron acceptor. It comprises two subunits, a (632 amino acids) and b (305 amino acids). To further elucidate its structure and function, we constructed a gene expression system. The wild-type recombinant enzyme was indistinguishable from the natural one in every criterion investigated. A series of variants was constructed to elucidate the role of the YPITP-motif (residues 253-257) in subunit a, which is conserved universally in the 2-oxoacid:ferredoxin oxidoreductase (OFOR) family. Single amino-acid replacements at Y253 and P257 by other amino acids caused a drastic loss of enzyme activity. T256, the hydroxyl group of which has been proposed to be essential for binding of the 2-oxo group of the substrate in the Desulfovibrio africanus enzyme, was unexpectedly replaceable with Ala, the kcat and Km for 2-oxoglutarate being approximately 33% and approximately 51%, respectively, as compared with that of the wild-type enzyme. Replacement at other positions resulted in a significant decrease in the kcat of the reaction while the Km for 2-oxoacid was only slightly affected. Thus, the YPITP-motif is essential for the turnover of the reaction rather than the affinity toward 2-oxoacid.     

Duration of neutralizing antibody titer after Japanese encephalitis vaccination

In paired serum samples collected from 17 children, we measured neutralizing antibody (NTAb) titers after the second series of routine Japanese encephalitis (JE) vaccination in Japan to estimate the duration of NTAb titer when children did not receive the third series of routine vaccination by applying a random coefficient model. We also measured NTAb titers in adult serum samples to confirm the duration of NTAb titer estimated in the analysis of pediatric serum samples. In the absence of the third series of routine vaccination, 18% (3/17), 47% (8/17), 82% (14/17) and 100% (17/17) of children were estimated to become NTAb negative at 5, 10, 15, and 20 years after the second series of routine vaccination, respectively. Of 38 adults, 39.5% (15/38) became NTAb negative; the percentage was somewhat lower than that of antibody-negative children. The results suggested that JE vaccination schedule should be reevaluated in the future.     

Effect of N-arachidonoyl-l-serine on human cerebromicrovascular endothelium

N-arachidonoyl-l-serine (ARA-S) is an endogenous lipid, chemically related to the endocannabinoid, N-arachidonoyl ethanolamine (i.e., anandamide) and with similar physiologic and pathophysiologic functions. Reports indicate that ARA-S possesses vasoactive and neuroprotective properties resembling those of cannabinoids. However, in contrast to cannabinoids, ARA-S binds weakly to its known classical receptors, CB1 and CB2, and is therefore considered to be a ‘cannabinoid-like’ substance. The originally described ARA-S induced-endothelial-dependent vasorelaxation was not abrogated by CB1, CB2 receptor antagonists or TRPV1 competitive inhibitor. The present report demonstrates that ARA-S enhances the fluorescence staining of both cannabinoid receptors (CB1 and CB2) in human brain endothelial cells (HBEC). This reaction is specific since it was reduced by respective selective receptor antagonist (SR141716A and SR141728A). ARA-S alone or in the presence of ET-1 was shown to alter the cytoskeleton (actin). Both ARA-S stimulated phosphorylation of various kinases (MAPK, Akt, JNK and c-JUN) and alteration of cytoskeleton are mediated via CB1, CB2 and TRPV1 receptors. The findings also showed the involvement of Rho/Rock and PI3/Akt/NO pathways in the ARA-S-induced phosphorylation of kinases and actin reorganization in HBEC. All of the above mentioned ARA-S-induced effects were reduced by the treatment with LY294002 (inhibitor of PI3/Akt kinase), except MAPK kinase. In addition, MAPK, JNK, c-JUN phosphorylation were inhibited by H1152 (inhibitor of Rho/ROCK kinase), except Akt kinase. Furthermore, PI3/Akt pathway was inhibited by pretreatment with l-NAME (inhibitor of NOS). The findings suggest that ARA-S is a modulator of Rho kinase and may play a critical role in the regulation of its activity and subsequent effects on the cytoskeleton and its role in supporting essential cell functions like vasodilation, proliferation and movement.     

Proteomic characterization of inter-alpha inhibitor proteins from human plasma

Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma. Recent studies have implicated a role for IaIp in sepsis, and have demonstrated their potential as biomarkers in sepsis and cancer. For characterization of isolated IaI proteins and contaminating proteins during the last steps of the purification process, SELDI-TOF MS and HPLC-ESI-MS/MS were used. After separation by SDS-PAGE or 2-DE, polypeptide bands of 80, 125 and 250 kDa were excised from gels and digested by trypsin. The tryptic peptides were analyzed by both MS methods. The main contamination during the purification process, a band of 80 kDa, contains mainly IaIp heavy chain (HC) H3. HC H1 and H2 were also found in this band. In addition, some vitamin K-dependent clotting factors and inhibitors and other plasma proteins were identified. The 125-kDa band, representing the pre-alpha inhibitor, was found to contain both bikunin and HC H3. The presence of other HC H1, H2 and the recently described HC H4 was also detected by SELDI-TOF MS. The presence of HC H1, H2, and H3 in the 125-kDa band was confirmed by ESI-MS/MS, but not the presence of the H4. Three polypeptides, H1 and H2 together with bikunin, were identified in the 250-kDa band, representing the ITI, by both MS techniques. Once again, the presence of H4 was detected in this band only by SELDI-TOF MS, but the number of corresponding peptides was still not sufficient for final identification of this polypeptide. The importance of the application of proteomic methods for the proper evaluation of therapeutic drugs based on human plasma is discussed.     

Preparation of conophylline affinity nano-beads and identification of a target protein

Conophylline, a vinca alkaloid extracted from the tropical plant Ervatamia microphylla, has been shown to induce the differentiation of insulin-producing β-cells in cultured cells and in animals. However, its mechanism of action and the molecular target have remained unclear. Therefore, we prepared a fishing probe with conophylline to identify the target protein by using latex nano-beads, which are newly innovated tools for affinity-purification. With these conophylline-linked nano-beads, we found that conophylline directly interacted with ARL6IP. ARL6IP may thus be involved in the mechanism of cellular differentiation of β-cells, and this probe should be useful to find other target proteins.