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.     

Zonation of labeling of lipogenic acetyl-CoA across the liver: implications for studies of lipogenesis by mass isotopomer analysis

Measurement of fractional lipogenesis by condensation polymerization methods assumes constant enrichment of lipogenic acetyl-CoA in all hepatocytes. mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) represent such methods and are based on the combinatorial analyses of mass isotopomer distributions (MIDs) of fatty acids and sterols. We previously showed that the concentration and enrichment of [13C]acetate decrease markedly across the dog liver because of the simultaneous uptake and production of acetate. To test for zonation of the enrichment of lipogenic acetyl-CoA, conscious dogs, prefitted with transhepatic catheters, were infused with glucose and [1,2-13C2]acetate in a branch of the portal vein. Analyses of MIDs of fatty acids and sterols isolated from liver, bile, and plasma very low density lipoprotein by a variant of ISA designed to detect gradients in precursor enrichment revealed marked zonation of enrichment of lipogenic acetyl-CoA. As control experiments where no zonation of acetyl-CoA enrichment would be expected, isolated rat livers were perfused with 10 mm [1,2-13C2]acetate. The ISA analyses of MIDs of fatty acids and sterols from liver and bile still revealed a zonation of acetyl-CoA enrichment. We conclude that zonation of hepatic acetyl-CoA enrichment occurs under a variety of animal models and physiological conditions. Failure to consider gradients of precursor enrichment can lead to underestimations of fractional lipogenesis calculated from the mass isotopomer distributions. The degree of such underestimation was modeled in vitro, and the data are reported in the companion paper (Bederman, I. R., Kasumov, T., Reszko, A. E., David, F., Brunengraber, H., and Kelleher, J. K. (2004) J. Biol. Chem. 279, 43217-43226).     

Molecular interactions that confer latency to transforming growth factor-beta

A major point of regulation of transforming growth factor-beta (TGF-beta) function is through control of activation of the latent TGF-beta complex, which consists of the latency associated peptide (LAP) secreted in non-covalent association with mature TGF-beta. Activation involves proteolysis, dissociation, or altered binding of LAP. However, the mechanism by which LAP confers latency to TGF-beta is poorly understood. Previously, we identified a conserved sequence near the N terminus of LAP as a site of thrombospondin-1 (TSP1) binding to the latent complex. Now we show that expression of the TGF-beta1-latent complex deleted in the TSP1 binding site ((54)LSKL) of LAP (DeltaLSKL LAP) results in secretion of LAP, but not of mature TGF-beta. DeltaLSKL LAP also fails to bind soluble or immobilized TGF-beta1. Consistent with an inability to bind the mature domain, DeltaLSKL LAP is unable to confer latency to TGF-beta, suggesting that the LSKL sequence is important, not only for TSP1 binding and activation, but also for binding to the mature domain. We identified the sequence (94)RKPK in the receptor-binding region of mature TGF-beta1 as the binding site for LAP. Peptides of the RKPK sequence bind LAP and inhibit LAP/TGF-beta association. RKPK peptides also activate latent TGF-beta, presumably by disrupting LAP-mature TGF-beta interactions. These studies provide a molecular basis for both latency and activation by TSP1 through the LSKL sequence of LAP binding to the RKPK sequence of mature TGF-beta.     

Magnetic bead hybridization to detect enterotoxigenic Escherichia coli strains associated with cattle in environmental water sources

A magnetic capture hybridization - polymerase chain reaction (MCH-PCR) method was used to increase the detection sensitivity of the enterotoxin gene LTIIa, used as a biomarker for waste in environmental samples. The samples were collected from cow lagoons of different farms and from environmental waters. Total DNA was extracted from colonies grown on mTEC medium or directly from environmental samples. The cow-specific Escherichia coli LTIIa gene was used as a DNA marker. A LTIIa-specific oligonucleotide probe was designed to capture the LTIIa marker during the MCH, followed by PCR. Varying levels of humic acid were added to the DNA extracts to evaluate the sensitivity and effectiveness of MCH-PCR. The minimal detection limit of MCH-PCR for the LTIIa gene was 2.5 ag/muL DNA. In the presence of humic acid, MCH-PCR was able to increase the detection sensitivity 10 000-fold over that of conventional PCR. The MCH-PCR could also detect one cell with the LTIIa DNA marker in a 1-L seeded environmental water sample. Results in this study indicate that MCH-PCR is more sensitive than nested PCR in testing environmental samples.     

The concentrations of hexadecane and inorganic nutrients modulate the production of extracellular membrane-bound vesicles, soluble protein, and bioemulsifier by Acinetobacter venetianus RAG-1 and Acinetobacter sp. strain HO1-N

In the present study, we addressed the possibility that the production of both bioemulsifiers and membrane-bound vesicles may be a common feature of the growth of Acinetobacter spp. on alkanes, and we determined the extent to which the release of extracellular products by these organisms is regulated by the concentrations of the alkane substrate and inorganic nutrients. To accomplish this objective, we grew Acinetobacter venetianus RAG-1 and Acinetobacter sp. strain HO1-N with different concentrations of nutrients and assayed for extracellular products. The results indicated that the release of vesicles, soluble protein, and bioemulsifier was promoted in various degrees by higher concentrations of hexadecane and inorganic nutrients, while the specific activities of the bioemulsifiers were enhanced with lower nutrient concentrations. Based on our findings, we propose that under conditions of nutrient excess, these strains produce membrane-bound vesicles to function in "luxury uptake" of the alkane substrate for delivery and storage in the form of inclusions. Under the same conditions, soluble bioemulsifier and protein may perform auxiliary roles in cell desorption and (or) alkane uptake. With low concentrations of nutrients, the decreased production of vesicles, protein, and bioemulsifier and the increased activity of the emulsifier may represent a mechanism for reducing biosynthetic demands and conserving cellular material.     

An analysis of replicative senescence in dermal fibroblasts derived from chronic leg wounds predicts that telomerase therapy would fail to reverse their disease-specific cellular and proteolytic phenotype

The accumulation of senescent fibroblasts within tissues has been suggested to play an important role in mediating impaired dermal wound healing, which is a major clinical problem in the aged population. The concept that replicative senescence in wound fibroblasts results in reduced proliferation and the failure of refractory wounds to respond to treatment has therefore been proposed. However, in the chronic wounds of aged patients the precise relationship between the observed alteration in cellular responses with aging and replicative senescence remains to be determined. Using assays to assess cellular proliferation, senescence-associated staining beta-galactosidase, telomere length, and extracellular matrix reorganizational ability, chronic wound fibroblasts demonstrated no evidence of senescence. Furthermore, analysis of in vitro senesced fibroblasts demonstrated cellular responses that were distinct and, in many cases, diametrically opposed from those exhibited by chronic wound fibroblasts. Forced expression of telomerase within senescent fibroblasts reversed the senescent cellular phenotype, inhibiting extracellular matrix reorganizational ability, attachment, and matrix metalloproteinase production and thus produced cells with impaired key wound healing properties. It would appear therefore that the distinct phenotype of chronic wound fibroblasts is not simply due to the aging process, mediated through replicative senescence, but instead reflects disease-specific cellular alterations of the fibroblasts themselves.     

Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.     

A <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Isolate Possessing a Spore-Associated Filament

We report on a novel bacterium, isolated during a screen for environmental isolates of Bacillus thuringiensis, that possesses a novel filamentous structure. Nucleotide sequence from the isolates 16S rRNA gene places the bacterium unambiguously within the Bacillus thuringiensis/Bacillus cereus group. Phase-contrast and electron microscopy indicate the presence of both a parasporal body and a long filament which are retained after sporulation. The filament is shown to consistently arise from the end of the exosporium and next to the parasporal body. Upon spore germination, the parasporal body/filament complex is retained on the cell wall of the resulting bacterium.Received: 4 March 2003 / Accepted: 18 April 2003     

A Bacillus thuringiensis isolate possessing a spore-associated filament

We report on a novel bacterium, isolated during a screen for environmental isolates of Bacillus thuringiensis, that possesses a novel filamentous structure. Nucleotide sequence from the isolate's 16S rRNA gene places the bacterium unambiguously within the Bacillus thuringiensis/Bacillus cereus group. Phase-contrast and electron microscopy indicate the presence of both a parasporal body and a long filament which are retained after sporulation. The filament is shown to consistently arise from the end of the exosporium and next to the parasporal body. Upon spore germination, the parasporal body/filament complex is retained on the cell wall of the resulting bacterium.