Conditional transformation of mouse pancreatic epithelial cells: an in vitro model for analysis of genetic events in pancreatocarcinogenesis

Pancreatic ductal adenocarcinomas arise through the accumulation of certain genetic alterations including ras, p16, p53, and DPC4. We found that activation of ras and inactivation of p53 could cooperatively induce in vitro tumorigenicity in conditionally immortalized pancreatic epithelial (IMPE) cells. IMPE cells were established from transgenic mice bearing a temperature-sensitive mutant SV40 Large T (LT) antigen. IMPE cells grew continuously under permissive conditions (33 degrees C with interferon-gamma), but rapidly suffered growth arrest under non-permissive conditions (39 degrees C without interferon-gamma). The cells showed strong expression of E-cadherin and beta-catenin as epithelial markers, and cytokeratin 19, a specific ductal cell marker. Cell proliferation under permissive conditions was associated with down-regulation of p21 expression through inactivation of p53 after overexpression of LT antigen. Intriguingly, the shift from the permissive to non-permissive culture conditions caused G2/M arrest of IMPE cells. Although the cells did not form colonies when cultured in soft agar without activation of ras, cells with ras activation via an adenovirus vector formed colonies under permissive conditions. These findings suggest that activation of ras and inactivation of p53 can cooperatively induce anchorage-independent growth of IMPE cells. This cell line might be useful for studying the processes involved in pancreatocarcinogenesis.     

Enhancement of the inhibitory activity of oatp antisense oligonucleotides by incorporation of 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) without a loss of subtype selectivity

Antisense oligonucleotides (AONs) that specifically target the genes of rat organic anion transporting polypeptide (oatp) subtypes were selected by using antisense in vitro selection (AIVS) and a conventional gene alignment program (GAP). When we incorporated several of our original 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) residues into AONs, which were designed as gapmers containing a series of 2'-deoxynucleotides in the center, at both the 3' and 5' ends, the inhibitory activity of these oatp AONs was enhanced and their inhibition was mediated by RNase H cleavage. Moreover, these ENA AONs did not lose their oatp selectivity. These strategies of using AIVS and GAP to select AONs followed by incorporation of ENA residues were effective for synthesizing oatp subtype-specific AONs.     

Effects of acetic acid treatment on plant chromosome structures analyzed by atomic force microscopy

Acetic acid treatment has been frequently used to remove cellular contaminants from plant chromosome samples for structural analyses by scanning electron microscopy and atomic force microscopy (AFM). We evaluated the effects of various concentrations of acetic acid treatments on barley chromosome structures by using AFM. The long-term 45% acetic acid treatment significantly damaged the chromosome structures, although the treatment effectively removed the cellular contaminants. On the other hand, the treatment with 15% acetic acid could not obtain sufficiently clean chromosome samples and the chromosome surface structures could not be observed. In contrast, we obtained clean chromosome preparation without severe damage by using an intermediate concentration (30%) of acetic acid treatment. In the centromeric region, we could observe fiber structures with a width of 100 nm, which were composed of ca. 50-nm granules and aligned to the axes of chromosomes. Thus, AFM analysis of chromosomes appropriately treated with acetic acid will provide important insights into the organization of higher-order structures of plant chromosomes.     

T-0901317, a synthetic liver X receptor ligand,inhibits development of atherosclerosis in LDL receptor-deficient mice

Liver X receptors (LXR alpha and LXR beta) are nuclear receptors, which are important regulators of cholesterol and lipid metabolism. LXRs control genes involved in cholesterol efflux in macrophages, bile acid synthesis in liver and intestinal cholesterol absorption. LXRs also regulate genes participating in lipogenesis. To determine whether the activation of LXR promotes or inhibits development of atherosclerosis, T-0901317, a synthetic LXR ligand, was administered to low density lipoprotein receptor (LDLR)(-/-) mice. T-0901317 significantly reduced the atherosclerotic lesions in LDLR(-/-) mice without affecting plasma total cholesterol levels. This anti-atherogenic effect correlated with the plasma concentration of T-0901317, but not with high density lipoprotein cholesterol, which was increased by T-0901317. In addition, we observed that T-0901317 increased expression of ATP binding cassette A1 in the lesions in LDLR(-/-) mice as well as in mouse peritoneal macrophages. T-0901317 also significantly induced cholesterol efflux activity in peritoneal macrophages. These results suggest that LXR ligands may be useful therapeutic agents for the treatment of atherosclerosis.     

Collagen XVIII,a basement membrane heparan sulfate proteoglycan,interacts with L-selectin and monocyte chemoattractant protein-1

Leukocyte infiltration during inflammation is mediated by the sequential actions of adhesion molecules and chemokines. By using a rat ureteral obstruction model, we showed previously that L-selectin plays an important role in leukocyte infiltration into the kidney. Here we report the purification, identification, and characterization of an L-selectin-binding heparan sulfate proteoglycan (HSPG) expressed in the rat kidney. Partial amino acid sequencing and Western blotting analyses showed that the L-selectin-binding HSPG is collagen XVIII, a basement membrane HSPG. The binding of L-selectin to isolated collagen XVIII was specifically inhibited by an anti-L-selectin monoclonal antibody, EDTA, treatment of the collagen XVIII with heparitinase or heparin but not by chemically desulfated heparin. A cell binding assay showed that the L-selectin-collagen XVIII interaction mediates cell adhesion. Interestingly, collagen XVIII also interacted with a chemokine, monocyte chemoattractant protein-1, and presented it to a monocytic cell line, THP-1, which enhanced the alpha(4)beta(1) integrin-mediated binding of the THP-1 cells to vascular cell adhesion molecule-1. Thus, collagen XVIII may provide a link between selectin-mediated cell adhesion and chemokine-induced cellular activation and accelerate the progression of leukocyte infiltration in renal inflammation.     

Cloning of the genes for the pituitary glycoprotein hormone alpha and follicle-stimulating hormone beta subunits in the Japanese crested ibis,Nipponia nippon

We have isolated a part of the gene for the pituitary glycoprotein hormone common alpha subunit (PGHalpha) and the whole gene for the follicle-stimulating hormone beta subunit (FSHbeta) in the Japanese crested ibis (Nipponia nippon), a critically endangered bird species in East Asia. The nucleotide sequence of a part of the PGHalpha gene (5026 bp) contained three exons holding the whole coding and 3' untranslated regions, but lacked a 5' untranslated region. Its exon-intron structure was similar to that in mammals, but different from that in teleosts in the location of the second intron. For the FSHbeta gene, the nucleotide sequence of 7633 bp was assembled from two phage clones. The exon-intron structure of three exons and two introns was similar to that observed in mammals and teleosts. In the putative promoter region of the ibis FSHbeta gene, a progesterone responsive element (PRE)-like sequence and two AP-1 responsive element-like sequences reported in the ovine FSHbeta gene were not conserved in complete form. The increased number of ATTTA motifs in the putative 3' untranslated region in comparison with those in Japanese quail and chicken FSHbeta cDNA suggested that more rapid degradation of FSHbeta mRNA occurs in this species. Deduced amino acid sequences of the ibis PGHalpha and FSHbeta showed high similarities with those of the corresponding subunits of other avian species. This is the first report on the genomic sequences of the PGHalpha and FSHbeta in an avian species.     

Susceptibility to kainate-induced seizures under dietary zinc deficiency

Zinc homeostasis in the brain is altered by dietary zinc deficiency, and its alteration may be associated with the etiology and manifestation of epileptic seizures. In the present study, susceptibility to kainate-induced seizures was enhanced in mice fed a zinc-deficient diet for 4 weeks. When Timm's stain was performed to estimate zinc concentrations in synaptic vesicles, Timm's stain in the brain was attenuated in the zinc-deficient mice. In rats fed the zinc-deficient diet for 4 weeks, susceptibility to kainate-induced seizures was also enhanced. When the release of zinc and neurotransmitters in the hippocampal extracellular fluid of the zinc-deficient rats was studied using in vivo microdialysis, the zinc concentration in the perfusate was less than 50% of that of the control rats and the increased levels of zinc by treatment with kainate were lower than the basal level in control rats, suggesting that vesicular zinc is responsive to dietary zinc deficiency. The levels of glutamate in the perfusate of the zinc-deficient rats were more increased than in the control rats, whereas the levels of GABA in the perfusate were not at all increased in the zinc-deficient rats, unlike in the control rats. The present results demonstrate an enhanced release of glutamate associated with a decrease in GABA concentrations as a possible mechanism for the increased seizure susceptibility under zinc deficiency.     

A C35 carotenoid biosynthetic pathway

Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C(30) carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C(35) backbone. The products of condensation of farnesyldiphosphate and GDP, C(35) structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C(40) or C(30) pathways accepted and converted the C(35) substrate, thus creating a C(35) carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures.