Target-specific Manner Research Articles

Folate receptor-mediated intracellular delivery of recombinant caspase-3 for inducing apoptosis

Recombinant reversed caspase-3 (rev-caspase-3) is a pro-apoptotic gene capable of intracellular autocatalytic processing, which leads to programmed cell death. Folate receptor-specific intracellular delivery of the rev-caspase-3 gene into KB cells over-expressing folate receptors was explored by employing the folate–poly(ethylene glycol)–polyethylenimine (FOL–PEG–PEI) conjugate as a nonviral polymeric carrier. Using luciferase as a reporter gene, the conditions for formulation of DNA/polymer polyplexes were pre-optimized to attain the highest folate receptor-mediated gene transfection efficiency. FOL–PEG–PEI conjugate complexed with rev-caspase-3 plasmid in an optimized condition gave rise to a great increase in expression and activation of exogenous rev-caspase-3 in KB cells when pretreated with doxorubicin. The synthesized conjugate exhibited higher transfection efficiency than other commercially available transfection agents due to a unique mechanism of folate-receptor mediated endocytic gene transfer. The transfected cells showed a significant extent of apoptosis by rev-caspase-3. This study suggests the potential of using folate-receptor-mediated delivery of rev-caspase-3 gene for inducing tumor cell death in a target-specific manner.

Target-specific gene silencing by siRNA plasmid DNA complexed with folate-modified poly(ethylenimine)

A target-specific delivery system of green fluorescent protein (GFP) small interfering RNA (siRNA) plasmid DNA was developed by using folate-modified cationic polyethylenimine (PEI). A GFP siRNA plasmid vector (pSUPER-siGFP), which inhibits the synthesis of GFP, was constructed and used for suppressing GFP expression in folate receptor over-expressing cells (KB cells) in a target-specific manner. A PEI–poly(ethylene glycol)–folate (PEI–PEG–FOL) conjugate was synthesized as a pSUPER-siGFP plasmid gene carrier. KB cells expressing GFP were treated with various formulations of pSUPER-siGFP/PEI–PEG–FOL complexes to inhibit expression of GFP. The formulated complexes were characterized under various conditions. Their GFP gene inhibition and cellular uptake behaviors were explored by confocal microscopy and flow cytometry analysis. pSUPER-siGFP/PEI–PEG–FOL complexes inhibited GFP expression of KB cells more effectively than pSUPER-siGFP/PEI complexes with no folate moieties and showed far reduced extent of inhibition for folate receptor deficient cells (A549 cells). The results indicated that folate receptor-mediated endocytosis was a major pathway in the process of cellular uptake, suggesting that targeted delivery of siRNA vector could be achieved to a specific cell.

In vivo tumor targeting of ODN-PEG-folic acid/PEI polyelectrolyte complex micelles

A tumor-targeting antisense oligodeoxynucleotide (ODN) delivery system based on polyelectrolyte complex (PEC) micelles is demonstrated. ODN-PEG-folic acid (ODN-PEG-FA) was synthesized using a heterofunctional PEG linker. The PEC micelles for the targeted ODN delivery to tumor cells were produced by ionic interactions between the ODN-PEG-FA and polyethylenimine (PEI). The in vivo targeting properties of the PEC micelles were assessed using a mouse tumor model. The size of ODN-PEG-FA/PEI PEC micelles was 92.3 nm with a relatively narrow distribution. Cellular uptake of the ODN-PEG-FA/PEI PEC micelles by folic acid receptor over-expressing cells (KB) was greatly enhanced compared to that of ODN-PEG/PEI PEC micelles. When the ODN-PEG-FA/PEI PEC micelles were systemically administered to the mice bearing KB cell xenograft tumor, ODN was accumulated to the solid tumor in a target specific manner. This study suggests that the PEC micelles with a receptor-recognizable targeting ligand on the surface have potential for passive and active targeted delivery of ODN drugs to cancer cells.

Recent Advances in Chemical Genomics

Chemical genomics, which utilizes specially designed small chemical compounds early in the discovery phase of new drugs to explore the life science at various levels, can address biological questions that are not amenable to genetic manipulation or functional genomics/proteomics approaches. Following the development of HT phenotypic assays and DNA expression analysis, the integration of cell-based assays with activity / affinity-based approaches allows us to interrogate the cells by analyzing phenotypic alterations, changes of transcript signature or detecting the differences in protein expression levels. Furthermore, activity / affinity-based techniques directly provide a druggable subset of gene products, which interact with small molecules, greatly reducing the complexity of analyzing the proteome. In this paper, we give an account of the recent advances (approaches and strategies) in the field of chemical genomics, and discuss how these approaches enable the investigator to obtain a novel therapeutically relevant target as well as drug candidates acting on them in a target-specific manner. This novel post-genomic discovery strategy, where target identification/ validation is carried out by interactions with small molecules, could significantly reduce the time-scale for early drug discovery, and increase the success rate of finding novel, druggable targets, as well as more specific drug candidates.

Rapid Dopaminergic Signaling by Interneurons That Contain Markers for Catecholamines and GABA in the Feeding Circuitry of Aplysia

Consummatory feeding behaviors in Aplysia californica are controlled by a polymorphic central pattern generator (CPG) circuit. Previous investigations have demonstrated colocalization of markers for GABA and catecholamines within two interneurons, B20 and B65, that participate in configuring the functional output of this CPG. This study examined the contributions of GABA and dopamine (DA) to rapid synaptic signaling from B20 and B65 to follower cells that implement their specification of motor programs. Pharmacological tests did not substantiate the participation of GABA in the mediation of the excitatory postsynaptic potentials (EPSPs) from either B20 or B65. However, GABA and the GABA(B) receptor agonist baclofen were found to modify these signals in a target-specific manner. Several observations indicated that DA acts as the neurotransmitter mediating fast EPSPs from B20 to two radula closer motor neurons B8 and B16. In both motor neurons, application of DA produced depolarizing responses associated with decreased input resistance and increased excitation. B20-evoked EPSPs in both follower cells were occluded by exogenous dopamine and blocked by the DA antagonist sulpiride. While dopamine occlusion and sulpiride block of convergent signaling to B8 from B65 resembled that of B20, both of these actions were less potent on the rapid signaling from B65 to the multifunctional and widely acting interneuron B4/5. These findings indicate that dopamine mediates divergent (B20 to B16 and B8) and convergent (B20 and B65 to B8) rapid EPSPs from two influential CPG interneurons in which it is colocalized with GABA-like immunoreactivity.

Deubiquitinating enzymes: their functions and substrate specificity.

Conjugation of one or more molecules of ubiquitin to target proteins can signify one of several fates, including degradation by the 26S proteasome, or trafficking via the secretory or endocytic pathways. Whereas much attention in recent years has focussed on the mechanisms of forming these different ubiquitin conjugates, far less is known about the removal of ubiquitin, which is performed by deubiquitinating enzymes (DUBs). While it has been appreciated for some 10 years that DUBs constitute large gene families in eukaryotes, and known for much longer that ubiquitination is a reversible process, information on the exact role of DUBs has been slow in coming. This review will attempt to summarise results from the last few years that shows that DUBs are an essential regulatory step of both protein degradation by the proteasome, and of other ubiquitin-dependent processes, by virtue of their ability to regulate protein ubiquitination in a target-specific manner.

The AF-1 Domain of the Orphan Nuclear Receptor NOR-1 Mediates Trans-activation, Coactivator Recruitment, and Activation by the Purine Anti-metabolite 6-Mercaptopurine

NOR-1/NR4A3 is an "orphan member" of the nuclear hormone receptor superfamily. NOR-1 and its close relatives Nurr1 and Nur77 are members of the NR4A subgroup of nuclear receptors. Members of the NR4A subgroup are induced through multiple signal transduction pathways. They have been implicated in cell proliferation, differentiation, T-cell apoptosis, chondrosarcomas, neurological disorders, inflammation, and atherogenesis. However, the mechanism of transcriptional activation, coactivator recruitment, and agonist-mediated activation remain obscure. Hence, we examined the molecular basis of NOR-1-mediated activation. We observed that NOR-1 trans-activates gene expression in a cell- and target-specific manner; moreover, it operates in an activation function (AF)-1-dependent manner. The N-terminal AF-1 domain delimited to between amino acids 1 and 112, preferentially recruits the steroid receptor coactivator (SRC). Furthermore, SRC-2 modulates the activity of the AF-1 domain but not the C-terminal ligand binding domain (LBD). Homology modeling indicated that the NOR-1 LBD was substantially different from that of hRORbeta, a closely related AF-2-dependent receptor. In particular, the hydrophobic cleft characteristic of nuclear receptors was replaced with a very hydrophilic surface with a distinct topology. This observation may account for the inability of this nuclear receptor LBD to efficiently mediate cofactor recruitment and transcriptional activation. In contrast, the N-terminal AF-1 is necessary for cofactor recruitment and can independently conscript coactivators. Finally, we demonstrate that the purine anti-metabolite 6-mercaptopurine, a widely used antineoplastic and anti-inflammatory drug, activates NOR-1 in an AF-1-dependent manner. Additional 6-mercaptopurine analogs all efficiently activated NOR-1, suggesting that the signaling pathways that modulate proliferation via inhibition of de novo purine and/or nucleic acid biosynthesis are involved in the regulation NR4A activity. We hypothesize that the NR4A subgroup mediates the genotoxic stress response and suggest that this subgroup may function as sensors that respond to genotoxicity.

Multiplexed gene expression analysis using the invader RNA assay with MALDI-TOF mass spectrometry detection.

A mass spectrometric approach for measuring gene expression levels has been developed. This technique utilizes a signal amplification system and analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Signal amplification from the targeted RNA employs a recently developed invasive cleavage assay that does not require prior PCR amplification. The assay uses a set of target-specific probes (oligonucleotides), which hybridize to the RNA being measured to create an overlap structure with a single-stranded flap. This flap is enzymatically cleaved and accumulates linearly in a target-specific manner. The products of the reaction, short DNA oligomers, are well suited for quantitative detection by MALDI-TOF mass spectrometry. Multiplexing is achieved by designing the assays so that reaction products for different mRNA targets have discrete masses that can be resolved in a single mass spectrum. Simultaneous analysis of human cytokine in vitro transcripts IL-1beta, TNF-alpha, and IL-6, with GAPDH as a reference standard, was used as a model system to demonstrate this novel method of gene expression analysis.

Antisense inhibition of delta-opioid receptor gene function in vivo by peptide nucleic acids.

Peptide nucleic acids (PNA) are synthetic analogs of DNA that hybridize to complementary oligonucleotide sequences with exceptional affinity and target specificity. The stability of PNA in biological fluids together with the unique hybridization characteristics of these structures suggests that PNA may have considerable potential as antisense agents for experimental use in vivo. To test this hypothesis, we attempted to modulate supraspinal delta-opioid receptor function in rats using PNA sequences designed to be complementary to a region of the rat delta-opioid receptor. Repeated i.c.v. administration of PNA over a period of 5 days significantly inhibited the antinociceptive response and locomotor response to selective delta-opioid receptor agonists. PNA attenuated delta-opioid receptor function in a sequence-specific, target-specific, and reversible manner characteristic of the functional inhibition caused by an antisense mechanism. There were no apparent toxicities arising from the PNA treatment based on the behavior of the animals and inspection of the treated tissues. Saturation binding studies on brain homogenates did not reveal any significant difference in receptor B(max) between treatment groups. However, [(35)S]guanosine-5'-O-(3-thio)triphosphate binding assays demonstrated a significant decrease in agonist efficacy in homogenates prepared from antisense-treated rats. Taken together, these results demonstrate that peptide nucleic acids are effective antisense agents in vivo and suggest that PNA may be a useful alternative to phosphodiester or phosphorothioate oligonucleotides, or variants thereof, for determination of gene function in vivo.

Enhancement by Mg2+ of domain specificity in Ca2+‐dependent interactions of calmodulin with target sequences

Mg2+ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca2+ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca2+-dependent target interactions. The binding of Mg2+ does, however, produce significant spectroscopic changes in residues located in the Ca2+-binding loops, and the Mg-calmodulin complex is significantly different from apo-calmodulin in loop conformation. Direct measurement of Mg2+ binding constants, and the effects of Mg2+ on Ca2+ binding to calmodulin, are consistent with specific binding of Mg2+, in competition with Ca2+. Mg2+ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg2+ has a direct role in conferring stability on both domains of apo-calmodulin. Apo-calmodulin binds typical target sequences from skeletal muscle myosin light chain kinase and neuromodulin with Kd approximately 70-90 nM (at low ionic strength). These affinities are virtually unchanged by 5 mM Mg2+, in marked contrast to the strong enhancement of peptide affinity induced by Ca2+. Under conditions of stimulation and increased [Ca2+], Mg2+ has a role in directing the mode of initial target binding preferentially to the C-domain of calmodulin, due to the opposite relative affinities for binding of Ca2+ and Mg2+ to the two domains. Mg2+ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca2+ threshold for enzyme activation and increases the importance of a partially Ca2+-saturated calmodulin-target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca2+-dependent target interactions.

Antigen-Specific Cytolysis by Neutrophils and NK Cells Expressing Chimeric Immune Receptors Bearing ζ or γ Signaling Domains

AbstractTCR- and IgG-binding Fc receptors (FcγR) mediate a variety of critical biologic activities including cytolysis via the structurally related ζ- and γ-chains. In previous studies, we have described chimeric immune receptors (CIR) in which the ligand-binding domain of a heterologous receptor or Ab is fused directly to the cytoplasmic domain of the TCR ζ-chain. Such ζ-CIRs efficiently trigger cytotoxic function of both T and NK cells in a target-specific manner. In this report, we compared the ability of both ζ- and γ-CIRs to activate the cytolytic function of two distinct classes of FcγR-bearing effectors, NK cells and neutrophils. Mature neutrophils expressing ζ- and γ-CIR were generated in vivo from murine hemopoietic stem cells following transplantation of syngeneic mice with retrovirally transduced bone marrow or in vitro from transduced human CD34+ progenitors following differentiation. Both ζ- and γ-based CIRs were capable of activating target-specific cytolysis by both NK cells and neutrophils, although the ζ-CIR was consistently more efficient. The experimental approach described is a powerful one with which to study the role of nonlymphoid effector cells in the host immune system and permits the rational design of immunotherapeutic strategies that rely on harnessing multiple immune cell functions via CIR-modified hemopoietic stem cells or progenitors.

A target-derived chemoattractant controls the development of the corticopontine projection by a novel mechanism of axon targeting

Abstract Here, we review our studies in rats of target recognition by developing cortical axons focusing on their innervation of the basilar pons, a major hindbrain target. The corticopontine projection develops by a ‘delayed interstitial budding’ of collaterals from layer 5 corticospinal axons, rather than by a direct ingrowth of primary axons or by bifurcation of the growth cone. Branches form de novo from the axon cylinder in the pathway overlying the basilar pons and extend directly into it. Cocultures of cortex and basilar pons in 3-dimensional collagen matrices show that a diffusible chemotropic signal released by the basilar pons directs the growth of collateral branches from layer 5 axons in a target and neuron specific manner. ‘Delayed’ co-cultures suggest that a diffusible, pontine-derived signal also initiates the selective branching of layer 5 axons. In vivo experiments support this chemotropic mechanism. First, corticospinal axons form collateral branches at novel locations directly over ectopic aggregations of basilar pontine neurons induced by X-irradiation; no branches form at positions that would normally overlie the appropriate region of basilar pons which is absent because of the X-irradiation. Thus, the basilar pons, rather than local cues in the axon pathway, appears to control the location of corticospinal axon branching. Second, in a series of experiments in which different subsets of corticospinal axons are prevented from innervating the basilar pons, remaining corticospinal axons extend collaterals in a directed manner to regions of the basilar pons deprived of cortical input, a behavior consistent with a response to a diffusible chemoattractant emanating from these regions. In conclusion, our findings suggest that a diffusible, target-derived chemotropic molecule(s) underlies target recognition in this developing system by initiating the formation and directing the growth of pontine collateral branches of primary layer 5 corticospinal axons.