A Src family inhibitor (PP1) potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells

Connexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity. We have recently reported that Cx32 acts as a tumor suppressor gene in renal cancer cells due to the inhibition of Src-dependent signaling. In line with the previous study, here we examined if a Src family inhibitor (PP1) could potentiate tumor-suppressive effect of Cx32 in Caki-2 cell from human renal cell carcinoma. In order to clarify the potentialization of PP1, using Cx32-transfected Caki-2 cells and mock-transfected Caki-2 cells, we estimated difference in cytotoxic effect of PP1 on the two cell clones in vitro as well as in vivo. PP1 showed more cytotoxic effect on Caki-2 cells having Cx32 positive expression than that of Cx32 negative expression at lower doses. This potentialization was also observed in xenograft model of nude mice. The potentialization of the effect mainly depended on the induction of apoptosis but not the control of cell growth. In conjugation with this event, the reduction of anti-apoptotic molecules (Bcl-2 and Bcl-xL) was caused by the combination of Cx32 expression and PP1 treatment in Caki-2 cells. These results suggest that PP1 potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells through the reduction of anti-apoptotic molecules.     

Enhanced conformational diversity search of CDR-H3 in antibodies: role of the first CDR-H3 residue

Through a conformation search by a simulation calculation, the relationships between the amino acid sequences and the conformations of the third complementarity-determining region of the antibody heavy chain (CDR-H3) were investigated to characterize the large conformational varieties of antibodies. Here, we focused on the structural role of the first CDR-H3 residue, and we selected two antibodies, 28B4 and PLG, whose CDR-H3 conformations are significantly different, having Trp and Gly at the first position, respectively. Multicanonical molecular dynamics simulations, with the advantage of enhanced sampling efficiency, were performed for the CDR-H3 fragments of 28B4 and PLG, and a modified CDR-H3 model of 28B4, where the first Trp residue was substituted with Gly. When the first CDR-H3 residue is Trp, almost all of the observed CDR-H3 loops were bent at the first residue. In contrast, when the first residue is Gly, large varieties of loop conformations were observed. The structural role of this Gly residue is discussed from the perspective of the other antibody structures in the database. When the surrounding residues were included in the calculations, CDR-H3 loop structures similar to those in the crystal structures were reproduced as the major conformations for both the 28B4 and PLG antibodies.     

Identification of peptidyl mimics of bioactive gibberellin recognized by an antibody

We screened a phage display peptide library for peptidyl mimotopes of gibberellin against anti-bioactive gibberellin antibody. The peptides obtained were grouped into two homologous sequences and their binding to the antibody was put in competition with free GA(4) but not with GA(4) methylester, suggesting that the peptides behave as mimics of GA(4). As an application, the phage display peptide was shown to work as a tracer for enzyme-linked immunosorbent assay (ELISA) analysis of GA(4).     

Design and synthesis of dysidiolide analogs from vitamin D3: novel class of Cdc25A inhibitors

Potent dysidiolide analogs were synthesized by structural hybridization of dysidiolide and vitamin D(3). These analogs exhibited strong inhibitory activity toward dual-specificity phosphatase Cdc25A (IC(50)=0.44-0.89 microM).     

A specific substrate-inhibitor, a 2'-deoxy-2'-fluorouridine-containing oligoribonucleotide, against human RNase L

We examined the properties of RNA analogues containing 2'-deoxy-2'-alpha-fluorouridine (1) or 2'-O-methyluridine (2) as inhibitors against human RNase L, that cleaves a single-stranded RNA in the presence of 2',5'-linked oligoadenylate (2-5A). The RNA analogue, FF, containing two molecules of 1 in place of uridine efficiently inhibited the RNase L-catalyzed RNA cleavage reaction, whereas the analogue, MM, containing two molecules of 2 was found not to have affinity for the enzyme. The k(cat) value for FF was 1/100 of that for an unmodified RNA, UU, whereas the K(m) value of FF was only twice as great as that of UU. Thus, it was found that the analogue, FF, containing 1 is an efficient inhibitor against human RNase L.     

Concerted changes in the YB2/RYB-a protein and protamine 2 messenger RNA in the mouse testis under heat stress

Translation of a number of mRNAs is under strict regulation via RNA-binding proteins in the spermatogenic cells of testes. A family of Y-box binding proteins represents promising candidates for these presently uncharacterized RNA-binding proteins. The effects of heat stress on the expression of a Y-box binding protein, YB2/RYB-a, and mouse protamine 2 (mP2) were investigated in cultured spermatogenic cells and mouse testes by immunoblot and Northern blot analyses. Localization and alterations in the expression of the YB2/RYB-a protein and the mP2 mRNA in heat-stressed testes were examined by immunohistochemistry and in situ hybridization, respectively. Levels of the YB2/RYB-a protein in spermatogenic cells decreased rapidly as the result of exposure to higher temperature, 37 degrees C or 43 degrees C, compared with the scrotal temperature, 32.5 degrees C, under the culture conditions used. In experimental cryptorchidism, levels of the YB2/RYB-a protein were decreased after Day 10, while the mRNA levels were affected only slightly. The levels of the mP2 mRNA were also decreased and about comparable with those of the YB2/RYB-a protein. Exposure of the lower abdomen to a high temperature, 43 degrees C for 15 min, also damaged the testis and led to a decrease in YB2/RYB-a protein and the mP2 mRNA levels in a coordinated manner. Because YB2/RYB-a is proposed to function as a stabilizer of mP2 mRNA, the perturbation of YB2/RYB-a by heat stress could account for the decline of the mP2 mRNA in elongated spermatids.     

Ceriporic acid C, a hexadecenylitaconate produced by a lignin-degrading fungus, Ceriporiopsis subvermispora

A lignin-degrading basidiomycete, Ceriporiopsis subvermispora produces a series of alkyl- and alkenylitaconates (ceriporic acids). Previously, two alkylitaconic acids with tetradecyl and hexadecyl side chains were isolated and identified as 1-heptadecene-2,3-dicarboxylic acid (ceriporic acid A) and 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B). In the present study, one hexadecenylitaconate (ceriporic acid C) was isolated and its chemical structure was analyzed by glycolation and subsequent (1) trimethylsilation, or (2) acetalation with acetone and acetone-d6. Analyses of the isolated metabolite demonstrated that the hexadecenylitaconic acid was (Z)-1,10-nonadecadiene-2,3-dicarboxylic acid. The structure of the side chain in ceriporic acid C was the same as that of hexadecenylcitraconate, chaetomellic acid B. Thus, it was found that ceriporic acids share close structural similarity with alk(en)yl citraconate derivatives, chaetomellic acids and other lichen lactones, protolichesterinic, lichesterinic, and murolic acids.     

Improvement of shark type I collagen with microbial transglutaminase in urea

In the presence of urea, type I collagen could form a gel with crosslinks with microbial transglutaminase (MTGase). Collagen self-assembly was accelerated with the addition of MTGase. The proportion of reconstructed collagen fibrils was raised with the addition of MTGase. MTGase-treated collagen gel remained gelled at high temperatures at which collagen denatured. By treatment with MTGase, collagen could form the gel under impossible condition to collagen self-assembly, and that denaturation temperature was raised.     

Increased expression of cardiotrophin-1 during ventricular remodeling in hypertensive rats

Cardiotrophin-1 (CT-1) stimulates longitudinal myocardial cell hypertrophy. We examined the expression of CT-1, leukemia inhibitory factor (LIF), and gp130 by competitive RT-PCR and Western blotting in Dahl salt-sensitive (DS) rats with a high-salt diet, which showed a distinct transition from left ventricular hypertrophy (LVH) to congestive heart failure (CHF). The expression levels of CT-1 mRNA and protein were significantly increased at the CHF stage compared with the LVH stage and age-matched Dahl salt-resistant (DR) rats (n = 6 for each group). mRNA expression of LIF was not changed in the left ventricle at any stage by RT-PCR. gp130 mRNA and protein levels of DS rats at 11 and 17 wk were significantly increased compared with age-matched DR rats. The isolated myocyte length of DS rats at 17 wk was the longest among the four groups of rats. The LV end-diastolic dimension (LVDd) of DS rats, determined by echocardiography, was significantly increased at the CHF stage. There was a significant correlation between the CT-1 protein level and LVDd. CT-1 may play a role in ventricular remodeling during transition from LVH to CHF in the rat hypertensive model.