We have previously shown that a dual system for controlling gene expression that relies both on transcriptional regulation and DNA recombination mediated by the site-directed recombinase, Flp, ...effectively controls the expression of a gene encoding diphtheria toxin (DT-A). In this study, we investigated the use of a chimeric modified enhancer/promoter sequence of the human prostate-specific antigen (PSA) gene to regulate DT-A expression in human prostate cancer cells in culture, in xenografts derived from these cells, and in autochthonous tumors in TRAMP mice. Following adenoviral delivery of DNA encoding PSA promoter-driven Flp recombinase and DT-A, we demonstrate that this transcriptional/DNA recombination control strategy effectively activates DT-A expression in a manner that correlates with the amount of PSA and androgen in cells. Significantly, the size of xenografts was reduced by 50%, and tumor cells in TRAMP mice died following intratumoral injection of DT-A viruses. Direct injection of virally-delivered DT-A into normal mouse prostates resulted in a dramatic reduction in the size of the gland. Our results suggest that the PSA promoter-driven Flp recombinase regulatory system will allow for targeted death of PSA-expressing cells. When combined with newly developed strategies for targeted gene delivery, this approach holds promise as an effective systemically-administered therapy for metastatic prostate cancer.
Many of the genes of Drosophila melanogaster have their transcripts deposited in developing oocytes. These maternally loaded gene products enable an otherwise homo-zygous mutant embryo to survive ...beyond the first stage of development for which the gene product is required. Zygotic mutations that disrupt the Hedgehog signal transduction pathway typically yield a segment polarity 'lawn of denticles' cuticle phenotype. However, an embryo homozygous mutant for a gene can achieve normal embryonic segmentation precluding classification of the gene as a component of the Hh pathway, if wild-type transcripts from the mother are present. This chapter discusses the theory and importance of analyzing germline clone embryos for maternally acting genes involved in Hh signal transduction, and describes in detail the method to generate mutant germline clone embryos.
In today’s dynamic environment, where product demands are highly volatile and unstable, the ability to design and operate manufacturing facilities that are robust with respect to uncertainty and ...variability is becoming increasingly important to the success of any manufacturing firm in order to operate effectively in such an environment. Hence manufacturing facilities must be able to exhibit high levels of robustness and stability in order to deal with changing market demands. In general, Facility Layout Problem (FLP) is concerned with the allocation of the departments or machines in a facility with an objective to minimize the total material handling cost (MHC) of moving the required materials between pairs of departments. Most FLP approaches assume the flow between departments is deterministic, certain and constant over the entire time planning horizon. Changes in product demand and product mix in a dynamic environment invalidate these assumptions. Therefore there is a need for stochastic FLP approaches that aim to assess the impact of uncertainty and accommodate any possible changes in future product demands.This research focuses on stochastic FLP with an objective to present a methodology in the form of a framework that allows the layout designer to incorporate uncertainty in product demands into the design of a facility. In order to accomplish this objective, a measure of impact of this uncertainty is required. Two solution methods for single and multi period stochastic FLPs are presented to quantify the impact of product demand uncertainty to facility layout designs in terms of robustness (MHC) and variability (standard deviation). In the first method, a hybrid (simulation) approach which considers the development of a simulation model and integration of this model with the VIPPLANOPT 2006 algorithm is presented. In the second method, mathematical formulations of analytic robust and stable indices are developed along with the use of VIPPLANOPT for solution procedure. Several case studies are developed along with numerical examples and case studies from the literature are used to demonstrate the proposed methodology and the application of the two methods to address different aspects of stochastic FLP both analytically and via the simulation method. Through experimentation, the proposed framework with solution approaches has proven to be effective in evaluating the robustness and stability of facility layout designs with practical assumptions such as deletion and expansion of departments in a stochastic environment and in applying the analysis results of the analytic and simulation indices to reduce the impact of errors and make better decisions
: We describe fluorescent‐labeled peptide (FLP) studies on living cells. The new technique is nonradioactive and it allows monitoring of the binding and internalization of Vasoactive Intestinal ...Peptide (VIP) in VIP receptor‐expressing cells. The technique is easy to perform and the observed reaction is peptide sequence specific.
The impact of TiN film thickness variations on the effective work function (WF) of poly-Si/TiN/SiO/sub 2/ and poly-Si/TiN/HfSiON interfaces has been investigated. The electrical signatures of these ...gate stacks indicate that the concentration of Hf-Ti and Ti-Si bonds at the (poly-Si/TiN)/HfSiON and (poly-Si/TiN)/SiO/sub 2/ interface plays a significant role on the control of the gate stacks' WF. The density of these interfacial bonds and the related work function changes are correlated to the degree of nucleation of the TiN film on the dielectric.
The catabolite gene activator protein (CAP) and the fumurate nitrate reductase (FNR) are two founder members of the growing CRP-FNR protein superfamily. The consensus FNR binding site ...(TTGAT-N4-ATCAA) closely resembles that of CRP to the extent that both contain a common core motif (NTGAN-N4-NTCAN). The transcription factor FNR plays a role in altering gene expression between aerobic and anaerobic conditions but CRP regulators control the response to glucose starvation. Protein DNA interactions occur between the regulatory protein and DNA at the corresponding promoter using the consensus either CRP or FNR binding sites. Successful transcriptional activation generally requires contact between a DNA-bound activator and RNA polymerase in order to generate on effective complex. CRP promoters are grouped into two classes depending on the location of DNA binding site. Class I promoters contains transcription activator binding sites centered near position -61.5, -71, -82 or -92. Class II promoters the regulator proteins bind to a site centered at or near - 41.5 so three possible contacts, αCTD-AR1, αNTD-AR2 and δ70-AR3, occur between regulator and RNA polymerase. FNR and FLP act as a class II activator.
RNA interference (RNAi) has become an irreplaceable tool for reverse genetics in plants and animals. The universality and specificity of this phenomenon allows silencing of virtually any chosen gene ...to examine its involvement in biological processes. Many strategies exist to reduce the expression of a particular gene using RNAi. Some rely on delivering directly to cells the approximately 21-nucleotide long interfering double-stranded RNA (dsRNA) species that are central mediators of the silencing process. Others rely on the transgenic expression of longer dsRNA molecules, leaving it to the cellular machinery to process these hairpins into short active dsRNA. In this chapter, we describe a transgenic method to deplete a chosen protein from a specific Drosophila tissue following induction of long dsRNA. It was used to uncover the role of lipidic particles in Hedgehog signaling by silencing lipophorin in the fat body (1), and we routinely use it to deplete specific proteins from wing imaginal disc subdomains (2). The method, certainly not restricted to the study of Hedgehog signaling, allows fast and efficient construction of a plasmid incorporating various Drosophila genetic tools to allow heat-shock-induced expression of dsRNA at the desired time and in the desired tissue. For protocols involving injection of in vitro synthesized dsRNA in embryos to study Hedgehog signaling, see for example (3). For genomic screens to identify Hedgehog pathway components in tissue culture cells by transfection of small interfering RNAs, see refs. (4,5).
Fibrillarin is a nucleolar protein known to be involved in the processing of ribosomal RNA precursors. We isolated AtFbr1, a cDNA encoding a homolog of fibrillarin in Arabidopsis. The cDNA is 1.2 kb ...in size and encodes a polypeptide of 310 amino acid residues with a molecular mass of 33 kD. AtFbr1 is expressed at high levels in the flower and root tissue and at a slightly lower level in leaf tissue, whereas it was nearly undetectable in siliques. Expression of AtFbr1 was compared with that of the FLP (fibrillarin-like protein) gene identified by the Arabidopsis genome project. Abscisic acid treatment resulted in the down-regulation of the expression of both AtFbr1 and FLP genes in seedlings, although the degree of suppression was higher for FLP than for AtFbr1. In addition, the expression level of FLP decreased with the age of the seedlings, whereas AtFbr1 did not exhibit any detectable change. The subcellular localization of AtFbrl was studied with an in vivo targeting approach using a fusion protein, and was found to be correctly targeted to the nucleolus in protoplasts when expressed as a green fluorescent fusion protein (GFP). Deletion experiments showed that the N-terminal glycine- and arginine-rich region is necessary and sufficient to target AtFbr1 to the nucleolus.
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