Purpose: To develop and validate an optical imaging nanoprobe for the discrimination of epidermal growth factor (EGF) receptor (EGFR)^overexpressing tumors from surrounding normal tissues that also expresses EGFR. Experimental Design: Near-infrared (NIR) quantum dots (QD) were coupled to EGF using thiol-maleimide conjugation to create EGF-QD nanoprobes. In vitro binding affinity of these nanoprobes and unconjugated QDs was evaluated in a panel of cell lines, with and without anti-EGFR antibody pretreatment. Serial optical imaging of HCT116 xenograft tumors was done after systemic injection of QD and EGF-QD. Results: EGF-QD showed EGFR-specific binding in vitro. In vivo imaging showed three distinct phases, tumor influx (f3 min), clearance (f60 min), and accumulation (1-6 h), of EGF-QD nanoprobes. Both QD and EGF-QD showed comparable nonspecific rapid tumor influx and clearance followed by attainment of an apparent dynamic equilibrium at f60 min. Subsequently (1-6 h), whereas QD concentration gradually decreased in tumors, EGF-QDs progressively accumulated in tumors. On delayed imaging at 24 h, tumor fluorescence decreased to near-baseline levels for both QD and EGF-QD. Ex vivo whole-organ fluorescence, tissue homogenate fluorescence, and confocal microscopic analyses confirmed tumor-specific accumulation of EGF-QD at 4 h. Immunofluorescence images showed diffuse colocalization of EGF-QD fluorescence within EGFR-expressing tumor parenchyma compared with patchy perivascular sequestration of QD. Conclusion: These results represent the first pharmacokinetic characterization of a robust EGFR imaging nanoprobe. The measurable contrast enhancement of tumors 4 h after systemic administration of EGF-QD and its subsequent normalization at 24 h imply that this nanoprobe may permit quantifiable and repetitive imaging of EGFR expression.One of the most promising biological targets for cancer therapy is the epidermal growth factor (EGF) receptor (EGFR), a transmembrane glycoprotein that controls pleiotropic biological phenomena, including proliferation, angiogenesis, tissue invasion, and metastasis (1, 2). Although EGFR is ubiquitously expressed in normal tissues, it is preferentially overexpressed on the surface of many tumors and downstream signaling from this receptor renders them resistant to standard therapies (3,4). Targeted therapies that selectively inhibit this receptor have found widespread clinical applicability (4) but there are few reliable methods to predict response to therapy or gauge treatment response over time (5). Noninvasive imaging techniques that can discriminate between EGFR-overexpressing tumors and surrounding normal tissues that also express EGFR may facilitate repetitive and quantitative imaging of EGFR during a course of treatment.Although several studies have been reported on the imaging of EGFR expression, they predominantly use radiolabeled probes (6 -11). Alternatively, optical imaging using fluorescent techniques (12 -14) offers a convenient means of mapping molecular profiles noninv...

Using fluorescent differential display, we identified, from ‫ف‬ 8000 displayed bands, a DNA fragment showing rapid induction in response to red light irradiation. This EARLY-PHYTOCHROME-RESPONSIVE1 gene ( EPR1 ) encodes a novel nucleuslocalized MYB protein harboring a single MYB domain that is highly similar to the circadian oscillator proteins CCA1 and LHY. EPR1 is regulated by both phytochrome A and phytochrome B, and the red-light induction of EPR1 is not inhibited by cycloheximide, demonstrating that EPR1 represents a primary phytochrome-responsive gene. Our results show that EPR1 overexpression results in enhanced far-red light-induced cotyledon opening and delayed flowering. In wild-type Arabidopsis plants grown in continuous light, the EPR1 transcript exhibits circadian rhythmicity similar to that of CCA1 and LHY . Moreover, EPR1 suppresses its own expression, suggesting that this protein is part of a regulatory feedback loop. Constitutive expression of CCA1 and LHY results in the loss of EPR1 rhythmicity, whereas increased levels of EPR1 have no effect on the central oscillator. We propose that EPR1 is a component of a slave oscillator that contributes to the refinement of output pathways, ultimately mediating the correct oscillatory behavior of target genes.

Phytochrome A (PhyA)-regulated genes in 6-d-old etiolated seedlings of Arabidopsis Landsberg erecta were identified by fluorescent differential display. To screen for PhyA-regulated genes, mRNA fingerprints of the wild type and the phyA-201 mutant were compared from samples prepared 4 h after far-red light irradiation. Approximately 30,000 bands of cDNA were displayed by fluorescent differential display, and 24 differentially expressed bands were observed. Sequence analysis revealed that they represent 20 distinct genes. Among them, 15 genes were confirmed as PhyA regulated by northern-blot (or reverse transcription-polymerase chain reaction) analysis. Thirteen up-regulated genes included 12 known genes that encode nine photosynthetic proteins, two enzymes involved in the biosynthesis of chlorophyll, one DNA damage repair/toleration-related protein, and one unknown gene. Two down-regulated genes were identified as encoding a xyloglucan endotransglycosylase-related protein and a novel member of the ASK protein kinase family. In the phyA-201 mutant and the phyA-201phyB-1 double mutant, expression of all of these genes was photoreversibly up- or down-regulated by type II phytochromes. The results indicate that modes of photoperception differ between PhyA and PhyB, but that both types of phytochromes have overlapping effects on the photoregulation of gene expression.

Nicotine biosynthesis in Nicotiana tabacum is under genetic control by the A and B loci. Plants containing semi-dominant mutations at both the A and B loci (i.e. aabb genotype) have lower nicotine levels, reduced nicotine biosynthetic enzyme activities, and reduced mRNA levels of the corresponding biosynthetic genes. The A and B loci therefore appear to be coordinate regulators of several nicotine biosynthetic genes and define a group of co-regulated genes called the A-B regulon. To investigate the composition of genes in the A-B regulon, a fluorescent differential display (FDD) screen was used to randomly sample the transcriptomes of wild type and mutant aabb roots. This resulted in the isolation of 64 FDD clones, representing 49 unique genes or gene families. Four genes associated with nicotine biosynthesis were identified, whereas most of the other FDD clones were homologous with an assortment of stress response genes. Thirty-three genes or gene families showed reproducible aabb genotype effects, representing seven distinct mRNA expression patterns in response media treatments that increase the mRNA levels of known alkaloid biosynthetic genes. Thus, the A and B loci regulate the mRNA levels of some target genes differently than others. Eleven genes or gene families showed only treatment-specific effects, representing four mRNA accumulation patterns. These results indicate the A-B regulon is complex network of differentially expressed stress response genes, only a small subset of which are involved in nicotine biosynthesis.

The screening for mutants and their subsequent molecular analysis has permitted the identification of a number of genes of Arabidopsis involved in the development and functions of the gynoecium. However, these processes remain far from completely understood. It is clear that in many cases, genetic redundancy and other factors can limit the efficiency of classical mutant screening. We have taken the alternative approach of a reverse genetic analysis of gene function in the Arabidopsis gynoecium. A high-throughput fluorescent differential display screen performed between two Arabidopsis floral homeotic mutants has permitted the identification of a number of genes that are specifically or preferentially expressed in the gynoecium. Here, we present the results of this screen and a detailed characterization of the expression profiles of the genes identified. Our expression analysis makes novel use of several Arabidopsis floral homeotic mutants to provide floral organ-specific gene expression profiles. The results of these studies permit the efficient targeting of effort into a functional analysis of gynoecium-expressed genes.The gynoecium is the fourth and innermost whorl of a typical bisexual flower. It is composed of the female reproductive organs, or carpels, and encloses the ovules, which develop into seeds after fertilization. The gynoecium may be composed of simple, unfused carpels, although in most species it is syncarpic, i.e. composed of several carpels fused together. The gynoecium functions to protect the ovules and to allow the operation of pollen-pistil incompatibility mechanisms. After fertilization, it develops into a fruit that participates in seed dissemination.In Arabidopsis, the gynoecium is a complex syncarpic structure. This first develops as an open-ended tube from a primordial dome in the center of the floral meristem. A vertical septum then forms internally from either side of the gynoecial tube, and the two halves of this septum fuse to divide the structure into two loculi. Placental tissues develop in the zones where the vertical septum and gynoecial wall meet to generate two rows of ovule primordia within each loculus. Each ovule consists of a seven-celled embryo sac of the Polygonum type (Fahn, 1975), together with a small nucellus and two covering integuments. Cell division occurring at the distal end of the gynoecial cylinder forms the style and stigma tissues. The stigma consists of a pappillate epidermal cell layer with a modified external wall and cuticle. This tissue receives and permits the germination of compatible pollen grains. After the penetration of the stigma by pollen tubes, a transmitting tissue in the style and vertical septum functions to guide the pollen tubes toward the ovules where fertilization takes place. After fertilization, the Arabidopsis gynoecium develops into a two-chambered, capsular fruit, termed a silique. This structure opens at maturity to release its seeds by rupture along four zones of dehiscence in the silique wall situated on either side of the vertic...