A universal DNA-based protein detection system.

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

Disruption of mouse cytochrome p450 4f14 (Cyp4f14 gene) causes severe perturbations in vitamin E metabolism.

Vitamin E is a family of naturally occurring and structurally related lipophilic antioxidants, one of which, α-tocopherol (α-TOH), selectively accumulates in vertebrate tissues. The ω-hydroxylase cytochrome P450-4F2 (CYP4F2) is the only human enzyme shown to metabolize vitamin E. Using cDNA cloning, cell culture expression, and activity assays, we identified Cyp4f14 as a functional murine ortholog of CYP4F2. We then investigated the effect of Cyp4f14 deletion on vitamin E metabolism and status in vivo. Cyp4f14-null mice exhibited substrate-specific reductions in liver microsomal vitamin E-ω-hydroxylase activity ranging from 93% (γ-TOH) to 48% (γ-tocotrienol). In vivo data obtained from metabolic cage studies showed whole-body reductions in metabolism of γ-TOH of 90% and of 68% for δ- and α-TOH. This metabolic deficit in Cyp4f14(-/-) mice was partially offset by increased fecal excretion of nonmetabolized tocopherols and of novel ω-1- and ω-2-hydroxytocopherols. 12'-OH-γ-TOH represented 41% of whole-body production of γ-TOH metabolites in Cyp4f14(-/-) mice fed a soybean oil diet. Despite these counterbalancing mechanisms, Cyp4f14-null mice fed this diet for 6 weeks hyper-accumulated γ-TOH (2-fold increase over wild-type littermates) in all tissues and appeared normal. We conclude that CYP4F14 is the major but not the only vitamin E-ω-hydroxylase in mice. Its disruption significantly impairs whole-body vitamin E metabolism and alters the widely conserved phenotype of preferential tissue deposition of α-TOH. This model animal and its derivatives will be valuable in determining the biological actions of specific tocopherols and tocotrienols in vivo.

Induced over-expression of the transcription factor OsDREB2A improves drought tolerance in rice.

The DREB and CBF transcription factors play a critical role in plant development and abiotic stress responses and, therefore, represent attractive targets for a molecular plant breeding approach. In this study, the rice OsDREB2A gene was isolated and expressed under the control of a stress-inducible promoter (4ABRC) to improve the abiotic stress tolerance of japonica rice variety TNG67. T2 and T3 transgenic lines over-expressing OsDREB2A were found to have improved survival rates under severe drought and salt stress conditions relative to non-transgenic rice plants or rice plants transformed with the empty vector control. OsDREB2A expression was found to be markedly induced by drought and ABA treatment. The results indicate that the induced over-expression of OsDREB2A driven by the 4ABRC promoter in engineered rice plants may protect cells during stress.

Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model.

To investigate the possibility of using commensal bacteria as signal mediators for inhibiting the disease cholera, we stably transformed Escherichia coli Nissle 1917 (Nissle) to express the autoinducer molecule cholera autoinducer 1 (CAI-1) (shown previously to prevent virulence when present with another signaling molecule, autoinducer 2, at high concentrations) and determined the effect on Vibrio cholerae virulence gene expression and colonization in an infant mouse model. We found that pretreatment of mice for 8 h with Nissle engineered to express CAI-1 (Nissle-cqsA) greatly increased the mice's survival (92%) from ingestion of V. cholerae. Pretreatment with Nissle-cqsA for only 4 h increased survival by 77%, whereas ingesting Nissle-cqsA at the same time as V. cholerae increased survival rates by 27%. Immunostaining revealed an 80% reduction in cholera toxin binding to the intestines of mice pretreated for 8 h with Nissle-cqsA. Further, the numbers of V. cholerae in treated mouse intestines was reduced by 69% after 40 h. This finding points to an easily administered and inexpensive approach where commensal bacteria are engineered to communicate with invasive species and potentially prevent human disease.

Secretion of insulinotropic proteins by commensal bacteria: rewiring the gut to treat diabetes.

Here, we show that commensal bacteria can stimulate intestinal epithelial cells to secrete insulin in response to glucose. Commensal strains were engineered to secrete the insulinotropic proteins GLP-1 and PDX-1. Epithelia stimulated by engineered strains and glucose secreted up to 1 ng ml(-1) of insulin with no significant background secretion.

Fluorescently labeled liposomes for monitoring cholera toxin binding to epithelial cells.

Vibrio cholerae, the causative agent for cholera, expresses a toxin required for virulence consisting of two subunits: the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB is frequently used as an indicator of the presence of pathogenic V. cholerae and binds to the G(M1) ganglioside on the surface of epithelial cells. To study V. cholerae virulence (CTB expression) in the presence of human epithelia, we devised an inexpensive, simple, and rapid method for quantifying CTB bound on epithelial surfaces in microtiter plates. G(M1) ganglioside was incorporated into the lipid bilayer of liposomes both encapsulating the fluorescent dye sulforhodamine B (SRB) and with SRB tagged to lipids in the bilayer (BEGs). In addition, G(M1)-embedded liposomes encapsulating SRB only (EGs) and with SRB in their bilayers only (BGs) were synthesized. The three types of liposomes were compared with respect to their efficacy for both visualizing and quantifying CTB attached to the surface of Caco-2 cells. The BEGs were the most effective overall, providing both visualization under a fluorescence microscope and quantification after lysis in a microtiter plate reader. A limit of detection corresponding to 0.2 8microg/ml applied CTB was attained for the on-cell assay using the microtiter plate reader approach, whereas as low as 2 microg/ml applied CTB could be observed under the fluorescence microscope.

Interrupting Vibrio cholerae infection of human epithelial cells with engineered commensal bacterial signaling.

Vibrio cholerae El Tor serotypes are largely responsible for outbreaks of cholera in the developing world. The infection cycle for some strains of V. cholerae is coordinated, at least in part, through quorum sensing. That is, the expression of virulence genes depends on the concentration of V. cholerae autoinducers cholera autoinducer 1 (CAI-1) and autoinducer 2 (AI-2). High concentrations of CAI-1 and AI-2 have been shown previously to inhibit virulence gene expression. We have demonstrated here that a commensal bacterium, E. coli Nissle 1917 (Nissle), can be engineered to express CAI-1 (Nissle expresses AI-2 natively) and effectively interrupt V. cholerae virulence. We engineered Nissle to express CAI-1 under control of the lac promoter, and demonstrated inhibition of V. cholerae expression of cholera toxin (CT, as indicated by presence of the CT subunit B (CTB)) and of the toxin co-regulated pilus (TCP, as indicated by the relative transcript of TCP subunit A (TCPA)) in both monocultures of V. cholerae and co-cultures with epithelial cells, Nissle, and V. cholerae. In the model system of Caco-2 epithelia incubated with V. cholerae, we demonstrated that co-cultures with Nissle expressing CAI-1 activity reduced CTB binding to Caco-2 cells by 63% over co-cultures with wild-type Nissle. Further, cultures with Nissle expressing CAI-1 had significantly lower TCPA transcription than controls with wild-type Nissle. These results represent a significant step towards a prophylactic method for combating enteric disease through engineered quorum signaling within a commensal bacterial strain.

An efficient field screening procedure for identifying transposants for constructing an Ac/Ds-based insertional-mutant library of rice.

An efficient system was developed, and several variables tested, for generating a large-scale insertional-mutagenesis population of rice. The most important feature in this improved Ac/Ds tagging system is that one can conveniently carry out large-scale screening in the field and select transposants at the seedling stage. Rice was transformed with a plasmid that includes a Basta-resistance gene (bar). After the Ds element is excised during transposition, bar becomes adjacent to the ubiquitin promoter, and the rice plant becomes resistant to the herbicide Basta. In principle, one can plant up to one million plants in the field and select those plants that survive after spraying with Basta. To test the utility of this system, 4 Ds starter lines were crossed with 14 different Ac plants, and many transposants were successfully identified after planting 134,285 F2 plants in the field. Over 2,800 of these transposants were randomly chosen for PCR analysis, and the results fully confirmed the reliability of the field screening procedure.

An efficient method for producing an indexed, insertional-mutant library in rice.

Generation of an indexed, saturated, insertional-mutant library is an aid to understanding the functions of genes in an organism. However, 10 years of work by many investigators have not yet yielded such a library in rice. The major reason is that determining the chromosomal locations of a very large number of random insertion mutants by flanking sequence analysis is highly labor intensive, and therefore, libraries that do exist have not been indexed. We report here an efficient procedure to construct an indexed, region-specific, insertional-mutant library of rice. The procedure makes use of efficient long-PCR-based high-throughput indexing, coupled with a random but anchored population of Ds transposants. Long-PCR indexing allows rapid and simultaneous determination of the chromosomal locations of a large number of mutants that surround a particular anchor line, thus converting a random library into an indexed one. Such a library can be used directly, without the need to screen a large random library for a desired mutant plant.

Regulation of proto-oncogenic dbl by chaperone-controlled, ubiquitin-mediated degradation.

The dbl proto-oncogene product is a prototype of a growing family of guanine nucleotide exchange factors (GEFs) that stimulate the activation of small GTP-binding proteins from the Rho family. Mutations that result in the loss of proto-Dbl's amino terminus produce a variant with constitutive GEF activity and high oncogenic potential. Here, we show that proto-Dbl is a short-lived protein that is kept at low levels in cells by efficient ubiquitination and degradation. The cellular fate of proto-Dbl is regulated by interactions with the chaperones Hsc70 and Hsp90 and the protein-ubiquitin ligase CHIP, and these interactions are mediated by the spectrin domain of proto-Dbl. We show that CHIP is the E3 ligase responsible for ubiquitination and proteasomal degradation of proto-Dbl, while Hsp90 functions to stabilize the protein. Onco-Dbl, lacking the spectrin homology domain, cannot bind these regulators and therefore accumulates in cells at high levels, leading to persistent stimulation of its downstream signaling pathways.

Regulation of the Dbl proto-oncogene by heat shock cognate protein 70 (Hsc70).

The dbl oncogene product is the defining member of a family of onco-proteins known as Dbl guanine nucleotide exchange factors (GEFs) that facilitate the activation of the small GTP-binding proteins Cdc42, Rac, and Rho. Oncogenic activation of proto-Dbl occurs through loss of the amino-terminal 497 residues, rendering the protein constitutively active. Because both onco- and proto-Dbl contain the structural elements required for GEF activity (i.e. the Dbl homology (DH) and pleckstrin homology (PH) domains), it is thought that the amino terminus of proto-Dbl somehow inhibits the biochemical activity of the protein. To better understand the molecular basis of this regulation, we set forth to identify cellular proteins that preferentially bind the proto-oncogenic form of Dbl. We identified the molecular chaperone heat shock cognate protein (Hsc70) as a binding partner that preferentially interacts with the proto-oncogenic form of Dbl. Dbl is complexed with Hsc70 in transfected cells, as well as in native mouse brain extracts. The interaction between Hsc70 and proto-Dbl is mediated by at least two regions in Dbl, the aminoterminal spectrin homology domain (residues 224-417) and the pleckstrin homology domain (residues 711-808). Overexpression of a dominant negative Hsc70 mutant leads to activation of proto-Dbl GEF activity, indicating that the chaperone negatively regulates proto-Dbl function in vivo. We propose that Hsc70 attenuates Dbl activity by maintaining an inactive conformation in which the amino terminus is "folded over" the catalytic DH-PH domain.