A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarcinoma breast cancer cells

In vitro assays involving primary cells are used routinely to evaluate organ-specific toxic effects, for instance, the use of primary hepatocytes to evaluate hepatotoxicity. A major drawback of an in vitro system is the lack of multiple organ interactions as observed in a whole organism. A novel cell culture system, the integrated discrete multiorgan cell culture system (IdMOC), is described here. The IdMOC is based on the "wells within a well" concept, consisting of a cell culture plate with larger, containing wells, within each of which are multiple smaller wells. Cells from multiple organs can be cultured initially in the small wells (one organ per well, each in its specialized medium). On the day of toxicity testing, a volume of drug-containing medium is added to the containing well to flood all inner wells, thereby interconnecting all the small wells. After testing, the overlying medium is removed and each cell type is evaluated for toxicity using appropriate endpoints. We report here the application of IdMOC in the evaluation of the cytotoxicity of tamoxifen, an anticancer agent with known human toxicity, on primary cells from multiple human organs: liver (hepatocytes), kidney (kidney cortical cells), lung (small airway epithelial cells), central nervous system (astrocytes), blood vessels (aortic endothelial cells) as well as the MCF-7 human breast adenocarcinoma cells. IdMOC produced results that can be used for the quantitative evaluation of its anticancer effects (i.e., cytotoxicity towards MCF-7 cells) versus its toxicity toward normal organs (i.e., liver, kidney, lung, CNS, blood vessels).     

Detection of von Willebrand factor-cleaving protease (ADAMTS-13) in human platelets

The hemostatic activity of von Willebrand factor (vWF) is strongly dependent on its multimeric structure, with the highest activity in 'unusually large' multimers secreted from endothelial cells. The multimeric structure is regulated by a plasma protease, vWF-cleaving protease (vWF-CP, or ADAMTS-13). ADAMTS-13 mRNA is variably expressed in liver and other tissues. Because 15-25% of circulating vWF is stored in platelets, the presence and function of ADAMTS-13 in platelets are important issues. Here we report ADAMTS-13 expression in human platelets. Western blot analysis and flow cytometric analysis on permeabilized platelets revealed the presence of ADAMTS-13 protein. Real-time PCR demonstrated that ADAMTS-13 mRNA is present in platelets of six healthy volunteers, with little quantitative difference. The presence of ADAMTS-13 in platelets may imply the functional role of this enzyme in the local regulation of platelet function at the site of vascular injury or thrombus formation, and provide a useful tool for the analysis of structure and function of this enzyme.     

Induction of cytokines by toxins that have an identical RNA N-glycosidase activity: Shiga toxin, ricin, and modeccin

Shiga toxin (Stx) has an A1-B5 subunit structure, and the A subunit is an RNA N-glycosidase that inhibits cellular protein synthesis. We previously reported that in Caco-2 cells Stx induced cytokines and that the RNA N-glycosidase activity was essential for the cytokine induction. It is known that the binding of the Stx-B subunit to its receptor glycolipid, Gb3, mediates an A subunit-independent signal in some types of cells, but the involvement of this signal in the cytokine induction is unclear. In this study, we investigated whether RNA N-glycosidase itself induces cytokines. IL-8 production was enhanced by Stx, ricin, and modeccin, three toxins that inhibit protein synthesis through an identical RNA N-glycosidase activity, but not by two other types of protein synthesis inhibitors, diphtheria toxin and cycloheximide. The RNA N-glycosidase-type toxins showed a similar induction pattern of cytokine mRNAs. Brefeldin A, a Golgi apparatus inhibitor, completely suppressed the cytokine induction by the toxins. Analysis by using inhibitors of toxin binding and also Stx-B subunit showed that the cytokine-inducing activity was independent of Gb3-mediated signaling. These results indicate that RNA N-glycosidase itself induces the cytokine production and that intracellular transport of toxins through the Golgi apparatus is essential for the activity.     

Optimum amyloid fibril formation of a peptide fragment suggests the amyloidogenic preference of beta2-microglobulin under physiological conditions

Beta(2)-microglobulin (beta(2)m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Although full-length beta(2)m readily forms amyloid fibrils in vitro by seed-dependent extension with a maximum at pH 2.5, fibril formation under physiological conditions as detected in patients has been difficult to reproduce. A 22-residue K3 peptide of beta(2)m, Ser(20)-Lys(41), obtained by digestion with Acromobacter protease I, forms amyloid fibrils without seeding. To obtain further insight into the mechanism of fibril formation, we studied the pH dependence of fibril formation of the K3 peptide and its morphology using a ThT fluorescence assay and electron microscopy, respectively. K3 peptide formed amyloid fibrils over a wide range of pH values with an optimum around pH 7 and contrasted with the pH profile of the seed-dependent extension reaction of full-length beta(2)m. This suggests that once the rigid native-fold of beta(2)m is unfolded and additional factors triggering the nucleation process are provided, full-length beta(2)m discloses an intrinsic potential to form amyloid fibrils at neutral pH. The fibril formation was strongly promoted by dimerization of K3 through Cys(25). The morphology of the fibrils varied depending on the fibril formation conditions and the presence or absence of a disulfide bond. Various fibrils had the potential to seed fibril formation of full-length beta(2)m accompanied with a characteristic lag phase, suggesting that the internal structures are similar.     

Orally active factor Xa inhibitor: synthesis and biological activity of masked amidines as prodrugs of novel 1,4-diazepane derivatives

Factor Xa (fXa) is a serine protease, which plays a pivotal role in the coagulation cascade. To improve the oral anticoagulant activity of fXa inhibitors containing a 1,4-diazepane moiety as the P4 part, a prodrug strategy was examined. Among the compounds evaluated in this study, amidoxime prodrugs bearing an ester moiety, such as compounds 21 and 30, showed effective oral anticoagulant activity in mice.     

Specific gene silencing in the pre-implantation stage mouse embryo by an siRNA expression vector system

Recently, small interfering RNAs (siRNAs) have become a powerful and widely used tool for the analysis of gene function in mammalian cells. Here we report that the microinjection of an siRNA expression vector into the nucleus is an efficient and powerful method of specific gene silencing in pre-implantation mouse embryos. We used this method to examine the expression of two genes EGFP and Oct4. Vectors encoding siRNAs targeted against EGFP or Oct4 were injected into the pronucleus or nucleus of zygotes, which were then cultured until the blastocyst stage. When the effects of RNAi were examined in blastocyst stage eggs, there was robust inhibition of the gene product in a concentration-dependent manner at both the mRNA and the protein level. The expression of other endogenous genes was not affected, showing the specificity of the vector-mediated RNAi. In addition, this method was effective for inhibiting maternally expressed mRNA. To demonstrate that RNAi of Oct4 induced a similar phenotype to that of Oct4-null embryos, the blastocysts were further cultured in ES medium. After the fourth day of culture, the embryos either had outgrown only a layer of trophoblast cells or showed developmental arrest at the blastocyst stage (>90%). Moreover, concomitant with Oct4 suppression at the blastocyst stage, we observed inhibition of Fgf4, a gene that is known to be induced downstream of Oct4 expression. Taken together, these results demonstrate that the use of siRNA expression vector is a powerful way to achieve gene silencing in the pre-implantation stage embryo.     

Polyhistidine tract expansions in HOXA1 result in intranuclear aggregation and increased cell death

HOXA1 gene is part of a cluster of homeotic selector genes that regulates the anteroposterior patterning of mammals during embryonic development. HOXA1 encodes two alternatively spliced mRNAs with two isoforms, A and B, the former contains the homeodomain and expressed in early embryonic development. HOXA1 contains a string of 10 histidine repeats. However, individuals heterozygous for 7, 9, 11, and 12 histidine repeat variants were present among the Japanese population, notably in some autism cases. To determine the biological implications of the different polyhistidine repeat lengths, we expressed these variants in COS-7 and a human neuroblastoma cell line (SK-N-SH). Expression of expanded variants of HOXA1 isoform A, containing 11 and 12 polyhistidine, resulted in early and great degree of protein aggregation in the nucleus. This aggregation resulted in accelerated cell death in cells expressing 11 and 12 expanded variants compared to those transfected with 7 and 10 polyhistidine variants. Furthermore, we showed that these aggregates were ubiquitinated and were inhibited by a histidine-modifying compound, DEPC. These data suggest that HOXA1 protein with polyhistidine tract expansions misfold, aggregate, and have a toxic effect on cell.     

Association of soluble epoxide hydrolase gene polymorphism with insulin resistance in type 2 diabetic patients

The insulin resistance found in diabetes is influenced by vascular tone and local blood flow. Endothelial-derived hyperpolarizing factor (EDHF) functions as a potent vasodilator to regulate vascular tone, and its production is regulated by soluble epoxide hydrolase (sEH). In this study, we examined the genotype distribution and allele frequency of sEH gene G860A (Arg287Gln) polymorphism in Japanese subjects (n=499) (non-diabetic subjects, n=205; type 2 diabetic patients, n=294). Also, to accurately evaluate insulin resistance, we performed the euglycemic hyperinsulinemic clamp test for each type 2 diabetic patient (n=86) from whom agreement was obtained, and then examined a possible association of sEH gene G860A polymorphism with insulin resistance status. There was no significant difference in genotype distribution and allele frequency between non-diabetic subjects and type 2 diabetic patients. Interestingly, however, there was close association of sEH gene G860A (Arg287Gln) polymorphism with insulin resistance in type 2 diabetic patients, which was not observed in non-diabetic subjects. These results suggest that sEH and EDHF play some important role in the pathogenesis of insulin resistance found in type 2 diabetes.