Synthesis and structure-activity relationship of dehydroxymethylepoxyquinomicin analogues as inhibitors of NF-kappaB functions

We previously found dehydroxymethylepoxyquinomicin (DHMEQ) inhibited NF-kappaB activation and showed anti-inflammatory activity in vivo. Here we designed and synthesized analogues of DHMEQ and tested their biological activity as NF-kappaB inhibitors in human T cell leukemia Jurkat cells. The hydroxyl group at the 2-position of the benzamide moiety was found to be essential for the inhibitory activity. But etherification of this group did not diminish the activity completely. Thus, for further mechanistic studies the hydroxyl group at the 2-position may be useful for extension with a linker and biotin moiety.     

Distribution of the surface antigen HAM-4 and cytoskeleton during reformation of bile-canalicular structures in rat primary cultured hepatocytes.

The distribution of rat bile-canalicular surface antigen (HAM-4 antigen) and cytoskeletal elements (microtubules, actin filaments, and cytokeratin filaments) was examined during the reformation of bile-canalicular structures (BC-structures) in primary cultures of dissociated hepatocytes obtained following collagenase perfusion. HAM-4 antigen, which initially dispersed after cell dissociation, became focused into regions of cell-to-cell contact even before formation of BC-structures. Typical bile-canalicular microvilli also appeared in these regions before the intercellular spaces were completely closed. Finally, after in vitro reformation of BC, HAM-4 antigen was localized specifically at the BC-surface. The process of BC-reformation and the intracellular organization of actin and cytokeratin filaments were not significantly affected by microtubule inhibitors (nocodazole, colcemid, and colchicine). However, the localization of HAM-4 antigen molecules at the surface of BC was disrupted by these inhibitors, suggesting that the distribution of HAM-4 antigen, which represents a marker for the reconstruction of surface polarity, is dependent on microtubule function.     

Human placental ferritin receptor

Brush-border membranes from human placenta were prepared and their purity was clarified by biochemical and morphological methods. Ferritin binding to these prepared membranes was examined using horse spleen 125I-apoferritin, and was found to be completed within 10 min at 37 degrees C and pH 7.5. The amount of ferritin bound to the membranes was found to be proportional to the amount of membrane added and saturable for a given amount of the membrane in the presence of excess ligand. The membranes exhibited specific ferritin binding with a Ka of 2.3 X 10(7) M-1 at pH 7.5. A competitive binding assay indicated that horse spleen 125I-apoferritin binding was inhibited by a 10-fold molar excess of horse spleen ferric ferritin and a 500-fold molar excess of human transferrin. These results suggest that human placental brush-border membranes have specific receptors for horse spleen apoferritin molecules.     

Embryonic expression of beta-actin-lacZ hybrid gene injected into the fertilized ovum of the domestic fowl.

An experiment was carried out to investigate the expression of cloned DNA injected into the germinal disc of the chick fertilized ovum. The beta-actin-lacZ hybrid gene, MiwZ, was injected, in the closed circular form, into the cytoplasm of the germinal disc at the single-cell stage. The embryos were cultured in vitro, then in recipient eggshells up to day 4 of incubation. The survival rate of the embryos at day 4 was 42% (55/130), and the rate of embryos expressing MiwZ was 64% (35/55). Twenty-two embryos expressed the MiwZ in both embryonic and extraembryonic tissues, while the remainder expressed the MiwZ in only extraembryonic tissues. Mosaic expression was observed in most of the embryos expressing MiwZ in embryonic tissues. Expression throughout all tissues of the embryo including blood cells occurred in one case. In this case, the injected DNA was assumed to have integrated at an earlier stage. The results indicate that it is now possible to investigate the promoter activities of introduced exogenous genes as well as the effect of introduced genes on embryogenesis in early chick embryos. This technique may also facilitate the production of transgenic chicks.     

Promotion of collagen production by human fibroblasts with gastric cancer cells in vitro

To elucidate the histogenesis of gastric scirrhous cancer, the promotion of collagen production by normal human skin fibroblasts (HSF-1) with human gastric cancer cells (KATO-III, MKN-45 and MKN-28) was investigated by direct coculture and parabiotic culture. Argyrophilic collagenous fibers were demonstrated among fibroblasts on both direct cocultures and parabiotic cultures of the fibroblasts with gastric cancer cells. Microscopic examination showed that these fibers appeared earlier and were more abundant and thicker in direct cocultures and parabiotic cultures than in single cultures of fibroblasts. Gastric cancer cells in single or parabiotic culture did not form argyrophilic fibers. For quantitative proof of the promotion of collagen production by fibroblasts with gastric cancer cells, hydroxyproline produced by fibroblasts was measured. Much higher fibroblast hydroxyproline values were obtained in parabiotic cultures with gastric cancer cell lines than in single cultures of HSF-1. Moreover, the rate of collagen synthesis by HSF-1 was much higher than that of any gastric cancer cell line tested. These results demonstrate that gastric cancer cells enhance collagen production by fibroblasts in vitro. This finding suggests that they may produce a factor promoting fibroblast collagen synthesis and that this may contribute to the formation of stromal collagen in human gastric scirrhous cancer.     

Genetic linkage analyses of Romano-Ward syndrome (RWS) in 13 Japanese families

Romano-Ward syndrome (RWS) is an autosomal dominant disorder characterized by prolongation of the electrocardiographic QT interval, with clinical manifestations that include recurrent syncope and sudden death from ventricular arrhythmias. Presymptomatic diagnosis is difficult because of the variability in these signs among carriers, but it is important for clinical management to prevent sudden cardiac death. To find an LQT (long QT) locus in Japanese patients and to identify DNA markers useful for presymptomatic diagnosis, linkage analyses were undertaken in 13 Japanese families with RWS patients by means of two DNA markers located on 11p15.5. One of these marker loci, HRAS, was previously reported to be tightly linked to the LQT locus in another ethnic group. Our analyses of homogeneity suggest evidence for genetic heterogeneity of RWS within the Japanese population.