Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, 29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and 29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.     

Thy-1 regulates fibroblast focal adhesions, cytoskeletal organization and migration through modulation of p190 RhoGAP and Rho GTPase activity

The precise biological role of Thy-1, a glycophosphatidyl-inositol (GPI)-linked cell surface glycoprotein in non-caveolar lipid raft microdomains, remains enigmatic. Evidence suggests that Thy-1 affects intracellular signaling through src-family protein kinases, and modulates adhesive and migratory events, such as thymocyte adhesion and neurite extension. Primary fibroblasts sorted based on presence or absence of cell surface Thy-1 display strikingly distinct morphologies and differ with respect to production of and response to cytokines and growth factors. It is unclear the extent to which Thy-1 mediates these differences. Findings reported here indicate a novel role for Thy-1 in regulating the activity of Rho GTPase, a critical regulator of cellular adhesion and cytoskeletal organization. Endogenous or heterologous Thy-1 expression promotes focal adhesion and stress fiber formation, characteristic of increased Rho GTPase activity, and inhibits migration. Immunoblotting following transfection of RFL6 fibroblasts with Thy-1 demonstrates that Thy-1 expression inhibits src-family protein tyrosine kinase (SFK) activation, resulting in decreased phosphorylation of p190 Rho GTPase-activating protein (GAP). This results in a net increase in active Rho, and increased stress fibers and focal adhesions. We therefore conclude that Thy-1 surface expression regulates fibroblast focal adhesions, cytoskeletal organization and migration by modulating the activity of p190 RhoGAP and Rho GTPase.     

The alpha4 and alpha7 subunits and assembly of the 20S proteasome

The presence of a high-K_m hexokinase activity was tested in both dog and boar spermatozoa. Hexokinase kinetics from dog extracts showed the presence of a specific activity (dog-sperm glucokinase-like protein, DSGLP), in the range of glucose concentrations of 4–10 mM, whereas boar sperm did not show any DSGLP activity. Furthermore, dog-sperm cells, but not those of boar, showed the presence of a protein which specifically reacted against a rat-liver anti-glucokinase antibody. This protein also had a molecular weight equal to that observed in rat-liver extracts, suggesting a close similarity between both the proteins. This glucokinase-like protein was distributed in the peri- and post-acrosomal zones of the head, and the midpiece and principal piece of tail of dog spermatozoa. These results indicate that dog spermatozoa have functional high-K_m hexokinase activity, which could contribute to a very fine regulation of their hexose metabolism. This strict regulation could ultimately be very important in optimizing dog-sperm function along its life-time.     

Light-response quantitative trait loci identified with composite interval and eXtreme array mapping in Arabidopsis thaliana

Genetic analysis of natural variation in ecotypes of Arabidopsis thaliana can facilitate the discovery of new genes or of allelic variants of previously identified genes controlling physiological processes in plants. We mapped quantitative trait loci (QTL) for light response in recombinant inbred lines (RILs) derived from the Columbia and Kashmir accessions via two methods: composite interval mapping and eXtreme array mapping (XAM). After measuring seedling hypocotyl lengths in blue, red, far-red, and white light, and in darkness, eight QTL were identified by composite interval mapping and five localized near photoreceptor loci. Two QTL in blue light were associated with CRY1 and CRY2, two in red light were near PHYB and PHYC, and one in far-red light localized near PHYA. The RED2 and RED5 QTL were verified in segregating lines. XAM was tested for the identification of QTL in red light with pools of RILs selected for extreme phenotypes. Thousands of single feature polymorphisms detected by differential DNA hybridized to high-density oligo-nucleotide arrays were used to estimate allele frequency differences between the pools. The RED2 QTL was identified clearly; differences exceeded a threshold of significance determined by simulations. The sensitivities of XAM to population type and size and genetic models were also determined by simulation analysis.     

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.