ABSTRACT
Viperin is an interferon (IFN)-inducible multifunctional protein. Recent evidence from high-throughput analyses indicates that most IFN-inducible proteins, including viperin, are intrinsically expressed in specific tissues; however, the respective intrinsic functions are unknown. Here we show that the intrinsic expression of viperin regulates adipose tissue thermogenesis, which is known to counter metabolic disease and contribute to the febrile response to pathogen invasion. Viperin knockout mice exhibit increased heat production, resulting in a reduction of fat mass, improvement of high-fat diet (HFD)-induced glucose tolerance, and enhancement of cold tolerance. These thermogenic phenotypes are attributed to an adipocyte-autonomous mechanism that regulates fatty acid ß-oxidation. Under an HFD, viperin expression is increased, and its function is enhanced. Our findings reveal the intrinsic function of viperin as a novel mechanism regulating thermogenesis in adipose tissues, suggesting that viperin represents a molecular target for thermoregulation in clinical contexts.
Subject(s)
Adipose Tissue/metabolism , Gene Expression Regulation , Proteins/genetics , Thermogenesis/genetics , Adipocytes/metabolism , Animals , Energy Metabolism/genetics , Male , Mice , Mice, KnockoutABSTRACT
LPS induces inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, and causes an inflammatory response. The development of small molecules that have suppressive effect on those inflammatory cytokines is a desirable strategy for the treatment of inflammatory diseases. We synthesized 12 novel compounds with 4-amino-N-(4-(benzo[d]oxazol-2-ylamino)phenyl)butanamide moiety and evaluated their biological activities. Among them, 4 compounds (compound 5d, 5c, 5f, 5m and synthetic intermediate 4d) showed potent inhibition activities on IL-1ß and IL-6 mRNA expression in vitro. Further, in vivo activity was evaluated with two compounds (5f and 4d) and mRNA levels of IL-1ß, IL-6, and TNF-α were significantly decreased without hepatotoxicity. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective for suppression of representative inflammatory cytokines.
Subject(s)
Benzoxazoles , Inflammation , Interleukin-6 , RNA, Messenger , Tumor Necrosis Factor-alpha , Benzoxazoles/pharmacology , Cytokines/genetics , Cytokines/metabolism , Humans , Inflammation/drug therapy , Inflammation/genetics , Inflammation/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Lipopolysaccharides/administration & dosage , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Development of small molecules that inhibit inflammatory cytokines is a desirable strategy for the treatment of inflammatory diseases such as rheumatoid arthritis (RA). Following up a previous study, we synthesized 10 novel compounds with a 2,5-diaminobenzoxazole moiety and evaluated their biological activities. Among them, compound 3e showed potent inhibitory activity on Interleukin 6 (IL-6)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling inhibition (71.5%), and 3a showed excellent inhibitory activity on Interleukin 1 (IL-1ß) (92.1%). To test in vivo anti-inflammatory activity, compounds 3a and 3e were administered by intraperitoneal (IP) injection after subcutaneous (SC) injection of zymosan A into the right footpad of mice. Inflammation on the footpad was reduced after administration of compounds 3a and 3e. Especially, compound 3a showed a significant ameliorative effect on zymosan-induced inflammation. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective on the RA animal model through inhibition of the IL-6/STAT3 signaling pathway. Since drugs developed with small molecule inhibitors have several advantages over biological drugs, further study on these compounds is needed for the development of potent SMI drugs on RA.
Subject(s)
Arthritis, Rheumatoid , STAT3 Transcription Factor , Animals , Arthritis, Rheumatoid/chemically induced , Arthritis, Rheumatoid/drug therapy , Cytokines/metabolism , Inflammation/drug therapy , Interleukin-6/metabolism , Mice , STAT3 Transcription Factor/metabolismABSTRACT
We used antioxidant-containing nanoparticles (NPs) to treat acute hearing loss. Alpha-lipoic acid (ALA) served as the antioxidant; we employed Pluronic F127 to fabricate NPs. In vitro, ALA-NPs protected cells of the organ of Corti in HEI-OC1 mice, triggering nuclear translocation of NRF2 and increases in the levels of antioxidant proteins, including Nrf2, HO-1, SOD-1, and SOD-2. In vivo, the hearing of mice that received ALA-NP injections into the middle ear cavity was better preserved after induction of ototoxicity than in control animals. The cochlear Nrf2 level increased in test mice, indicating that the ALA-NPs protected hearing via the antioxidant mechanism observed in vitro. ALA-NPs effectively protected against acute hearing loss by activating the Nrf2/HO-1 pathway.
Subject(s)
Hearing Loss/drug therapy , Nanoparticles/chemistry , Poloxamer/chemistry , Thioctic Acid/administration & dosage , Thioctic Acid/therapeutic use , Tympanic Membrane/pathology , Animals , Antioxidants/pharmacology , Cell Death/drug effects , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Disease Models, Animal , Hearing Loss/pathology , Male , Mice, Inbred C57BL , NF-E2-Related Factor 2/metabolism , Nanoparticles/ultrastructure , Thioctic Acid/pharmacologyABSTRACT
To evaluate the effect of drug hydrophobicity on nanoparticle delivery in vivo, we conducted a comparative study using different photosensitizer-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Chlorin e6 (Ce6) and pheophorbide a (Pba) with similar structure but different hydrophobicity were loaded into PLGA-NPs separately. We observed release profiles and photodynamic effects in vitro from the resulting Ce6- and Pba-PLGA-NPs. After intravenous injection into SCC7 tumor-bearing mice, biodistribution and accumulation of two drugs in tumor tissue were observed by real-time fluorescence imaging. Finally, in vivo photodynamic therapy with Ce6- and Pba-PLGA-NPs provided different therapeutic results according to imaging data. The results demonstrated that drug hydrophobicity is an important factor in nanoparticle drug delivery and should be considered for efficient drug delivery in vivo.
Subject(s)
Nanoparticles/chemistry , Photosensitizing Agents/chemistry , Animals , Cell Line, Tumor , Cell Survival/physiology , Chlorophyll/analogs & derivatives , Chlorophyll/chemistry , Chlorophyllides , Drug Delivery Systems/methods , Flow Cytometry , Hydrophobic and Hydrophilic Interactions , Mice , Photochemotherapy , Polylactic Acid-Polyglycolic Acid Copolymer/chemistry , Porphyrins/chemistryABSTRACT
For efficient drug delivery, we introduce a click-chemistry-mediated two-step tumor-targeting strategy for nanoparticles (NPs). We modified HER2-binding trastuzumab with trans-cyclooctene (TCO-Trb), and fabricated tetrazine-modified NPs containing the anticancer drug, SN38 (SN38-Tz-NPs). To target tumor cells with the Tz-NPs, the tumor cells are first treated with TCO-Trb. The TCO-Trb binds HER2s and presents multiple TCO groups on the cell surface. Subsequently, the cells are treated with SN38-Tz-NPs that can bind the cell surface via click chemistry between Tz and TCO. This click chemistry-mediated binding resulted in enhanced tumor-targeting of Tz-NPs to the target tumor cells. In our study, this strategy was performed and analyzed in vitro and in vivo, and the results show that this is a promising strategy for tumor-targeted drug delivery by NPs.
Subject(s)
Cyclooctanes/chemistry , Drug Delivery Systems/methods , Irinotecan/chemistry , Nanoparticles/chemistry , Neoplasms, Experimental/drug therapy , Trastuzumab/chemistry , Animals , Cell Line, Tumor , Click Chemistry/methods , Irinotecan/administration & dosage , Irinotecan/pharmacokinetics , Mice, Inbred BALB C , Mice, Nude , Nanoparticles/administration & dosage , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Protein Binding , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Tissue Distribution , Topoisomerase I Inhibitors/administration & dosage , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/pharmacokinetics , Trastuzumab/administration & dosage , Trastuzumab/metabolism , Trastuzumab/pharmacokineticsABSTRACT
Nanoparticles have been widely used as drug carriers, and finding new materials for them is important for efficient drug delivery. Herein, we developed a new nanoparticle using emulsan and flax seed oil. Emulsan is one of the representative biosurfactants obtained from Acinetobacter calcoaceticus RAG-1. The resulting nanoparticles have an emulsan shell and a hydrophobic oil core, into which pheophorbide a (Pba) was loaded as a model drug. The nanoparticles were about 165.7â¯nm and were stably dispersed in an aqueous condition for more than one week. They demonstrated fast uptake in SCC7 mouse squamous cell carcinoma cells and killed the tumor cells after laser irradiation due to the photodynamic effect of Pba. After injection into SCC7 tumor-bearing mice via the tail vein, the particles showed longer blood circulation and 3.04-fold higher tumor accumulation in tissue than free Pba. These results demonstrate that emulsan-based nanoparticles have promising potential in drug delivery.
Subject(s)
Carcinoma, Squamous Cell/diagnostic imaging , Drug Delivery Systems , Nanoparticles/chemistry , Polysaccharides, Bacterial/chemistry , Skin Neoplasms/diagnostic imaging , Animals , Cell Line, Tumor , Drug Carriers/chemistry , Mice , Mice, Inbred C3H , Optical ImagingABSTRACT
Herein, we report the development of self-assembled nanoparticles using rhamnolipid, a biosurfactant. Rhamnolipid is produced by Pseudomonas aeruginosa, and has an amphiphilic structure that is suitable for the formation of a nanoparticle shell. These rhamnolipid nanoparticles were loaded with pheophorbide a (Pba), a hydrophobic photosensitizer. The resulting nanoparticles had about 136.1-nm-diameter spherical shapes and had excellent water solubility without aggregation for one month. These nanoparticles showed fast uptake into SCC7 tumor cells and induced photodynamic damage upon laser irradiation. After intravenous injection to SCC7 tumor-bearing mice, their long blood circulation time and high accumulation in tumor tissue were observed in real-time fluorescence imaging. Upon laser irradiation, these rhamnolipid nanoparticles showed complete tumor suppression by photodynamic therapy in vivo. These promising results demonstrate the potential of rhamnolipid nanoparticles for drug delivery, and suggest that further attention to rhamnolipid research would be fruitful.
Subject(s)
Drug Delivery Systems , Glycolipids/chemistry , Nanoparticles/chemistry , Photochemotherapy , Animals , Cell Death/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Chlorophyll/analogs & derivatives , Chlorophyll/chemistry , Chlorophyll/pharmacology , Mice , Nanoparticles/ultrastructure , Singlet Oxygen/chemistry , Tissue Distribution/drug effectsABSTRACT
We fabricated poly (ethylene glycol)-block-polycaprolactone (PEG-b-PCL) nanoemulsion for drug delivery and photodynamic therapy. PEG-b-PCL effectively stabilized the interface between water and soybean oil, and the resulting nanoemulsion was about 220.3 nm in diameter with spherical shape. For photodynamic therapy (PDT), chlorin e6 (Ce6) was loaded into the nanoemulsion as a photosensitizer (PS). These chlorin e6-loaded PEG-PCL nanoemulsions (Ce6-PCL-NEs) showed efficient cellular uptake and, upon laser irradiation, generated singlet oxygen to kill tumor cells. Particularly, Ce6-PCL-NEs showed prolonged blood circulation and about 60% increased tumor accumulation compared to free Ce6 after intravenous injection to 4T1 tumor-bearing mice. These results demonstrate the promising potential of Ce6-PCL-NEs for efficient PDT and in vivo drug delivery to tumor tissue.
Subject(s)
Drug Carriers/chemistry , Emulsions , Lactones/chemistry , Nanoparticles , Polyethylene Glycols/chemistry , Porphyrins/administration & dosage , Radiation-Sensitizing Agents/administration & dosage , Animals , Cell Survival/drug effects , Cells, Cultured , Chlorophyllides , Dose-Response Relationship, Drug , Drug Delivery Systems , Emulsions/chemistry , Mice , Models, Animal , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Photochemotherapy , Porphyrins/chemistry , Porphyrins/pharmacokinetics , Radiation-Sensitizing Agents/chemistry , Tissue DistributionABSTRACT
Flower-like gold nanoparticles, so called gold nanoflowers (AuNFs), were synthesized through the reduction of HAuC4 with ascorbic acid in the presence of chitosan polymers. Chitosan-mediated AuNFs exhibited the distinct SERS signals of 2-chlorothiophenol (CTP) due to the presence of many interstitial gaps (so called hot spots) on the surface. For the facile silica coating, the AuNFs were conjugated with terminal carboxylate groups of (3-glycidyloxypropyl)trimethoxysilane (GPTMS), consequently forming alkoxy-terminated AuNFs which could facilely participate in the sol-gel reaction for silica coating. The resulting core-shell particles, i.e., CTP-adsorbed AuNFs with silica coating, exhibited the distinct SERS signals of CTP embedded within silica layer, warranting the effectiveness of this chemical strategy for spectroscopic labeling of Raman probes.
Subject(s)
Gold/chemistry , Metal Nanoparticles/chemistry , Nanotechnology , Silanes/chemistry , Silicon Dioxide/chemistry , Spectrum Analysis, Raman/methods , Capsules , Chlorides/chemistry , Gels , Gold Compounds/chemistry , Models, Molecular , Molecular Conformation , Oxidation-ReductionABSTRACT
In a recent study, we isolated the diketopiperazine disulfide glionitrin A from the co-culture broth of a mine drainage-derived fungus (Aspergillus fumigatus KMC901) and bacterium (Sphingomonas KMK001). Here, we investigated the antitumor activity of glionitrin A and its underlying molecular mechanisms in human prostate cancer DU145 cells. Glionitrin A showed significant cytotoxicity, promoting cell cycle arrest and apoptosis. Glionitrin A-treated cells exhibited elevated levels of phospho-histone 2AX (Ser139), a marker of DNA damage, and accumulated in both S phase and G2/M phase due to the activation of checkpoints associated with the ataxia-telangiectasia-mutated and ataxia-telangiectasia-mutated-Rad3-related Chk1/2 pathway downstream of p53-binding protein 1 phosphorylation at Ser1778. In addition, glionitrin A induced apoptosis through both caspase-dependent and -independent pathways. Glionitrin A activated caspase-8, -9 and -3 and also released endonuclease G from the mitochondria to the nucleus in a dose-dependent manner. Our in vivo study performed in nude mice bearing xenografts of DU145 cells showed that glionitrin A dramatically reduced the tumor volume by an average of 38.2% (5 mg/kg, per os (p.o.)) and 71.3% (10 mg/kg, p.o.) at 27 d after the beginning of treatment. Taken together, these findings provide a detailed description of the mechanism underlying the biological activity of the new natural product glionitrin A, which has the potential to be developed as an anti-prostate cancer agent.
Subject(s)
Antineoplastic Agents/therapeutic use , Ataxia Telangiectasia Mutated Proteins/metabolism , Biological Products/therapeutic use , Checkpoint Kinase 2/metabolism , Diketopiperazines/therapeutic use , Prostatic Neoplasms/drug therapy , Protein Kinases/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Aspergillus fumigatus , Biological Products/pharmacology , Caspase 1/metabolism , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Checkpoint Kinase 1 , DNA Damage , Diketopiperazines/pharmacology , Endodeoxyribonucleases/metabolism , Heterografts , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Phosphorylation , Prostatic Neoplasms/metabolism , Sphingomonas , Tumor Suppressor p53-Binding Protein 1ABSTRACT
In this work, dual-functional composite particles possessing fluorescence and temperature-sensitive functionalities were developed in the form of QD-bound hydrogels for biomedical applications. First, the surface of silica nanoparticles (SNPs) was functionalized with olefin silanes, followed by hydrogel encapsulation through a radical polymerization. The encapsulated hydrogels were poly(N- isopropylacrylamide-co-acrylic acid) P(NIPAM-co-AAc) copolymer, showing the sensitive volume changes corresponding to the alternating temperature changes between 25 degrees C and 45 degrees C. At an optimal pH5, the hydrogel-encapsulated SNPs (SiO2@hydrogel) were effectively anchored by amino quantum dots (amino-QDs) through electrostatic (attractive) interactions between carboxylate groups of hydrogels and amine groups of QDs. QD-bound hydrogels with co-monomer ratio of [NIPAM:AAc = 83:17 wt%] exhibited the higher PL intensity than other samples with [NIPAM:AAc = 96:4 wt% or 91:9 wt%], indicating that higher fraction of carboxylate groups by AAc induced the effective bounding with QDs possessing positively charged amine groups.
Subject(s)
Hydrogels/chemistry , Nanotechnology , Quantum Dots/chemistry , Temperature , Acrylamides/chemistry , Alkenes/chemistry , Methacrylates/chemistry , Organosilicon Compounds/chemistry , Polymers/chemistry , Silicon Dioxide/chemistry , Spectrometry, FluorescenceABSTRACT
Silica nanoparticles (SNPs) were functionalized by aminosilanes of different chain lengths, such as (3-aminopropyl) trimethoxysilane (APTMS), [3-(2-aminoethylamino)propyl] trimethoxysilane (AEAPTMS), and N-(3-trimethoxysilylpropyl)diethylenetriamine (TMSP), followed by the electrostatic conjugation with carboxyl quantum dots (QDs). The resulting QD-anchored SNPs (Q-SNPs) were treated with a silicate solution to afford thin silica-coated fluorescent nanoparticles. The Q-SNPs prepared using AEAPTMS exhibited the highest photoluminescence (PL) intensity compared to those prepared using APTMS and TMSP. Moreover, the conjugation of amine-terminated SNPs with carboxyl QDs was found to be very strong under acidic pH conditions. The silicate-coated Q-SNPs exhibited a long-lasting PL intensity compared to the pristine Q-SNPs because the silica coating prevented the oxidative degradation and/or detachment of the anchored QDs from the SNPs. Such a protective coating strategy would be a useful guideline to prepare stable nanostructured materials for prolonged applications. The composite particles were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), and photoluminescence (PL) spectroscopy.
Subject(s)
Fluorescent Dyes/chemistry , Nanoparticles/chemistry , Quantum Dots/chemistry , Silanes/chemistry , Silicates/chemistry , Nanotechnology , Organosilicon Compounds/chemistry , Polyamines/chemistryABSTRACT
ABSTRACT: 18 F-FP-CIT PET/CT is a useful diagnostic tool for differentiating between idiopathic Parkinson disease and atypical Parkinson syndrome by visualizing the striatum, where the nerve endings of nigrostriatal dopaminergic neurons are located. We present an unusual accumulation of 18 F-FP-CIT in the infarct and peri-infarct brain area of an 83-year-old man who was referred for the management of suspected cerebral infarction due to sudden dysarthria and delirium.
Subject(s)
Parkinson Disease , Male , Humans , Aged, 80 and over , Parkinson Disease/complications , Parkinson Disease/diagnostic imaging , Positron Emission Tomography Computed Tomography , Tropanes , Cerebral Infarction/complications , Cerebral Infarction/diagnostic imaging , Tomography, Emission-Computed, Single-Photon , Dopamine Plasma Membrane Transport ProteinsABSTRACT
Here, we report that gomisin J and gomisin N, dibenzocyclooctadiene type lignans isolated from Schisandra chinensis, inhibit Wnt/ß-catenin signaling in HCT116 cells. Gomisins J and N appear to inhibit Wnt/ß-catenin signaling by disrupting the interaction between ß-catenin and its specific target DNA sequences (TCF binding elements, TBE) rather than by altering the expression of the ß-catenin protein. Gomisins J and N inhibit HCT116 cell proliferation by arresting the cell cycle at the G0/G1 phase. The G0/G1 phase arrest induced by gomisins J and N appears to be caused by a decrease in the expression of Cyclin D1, a representative target gene of the Wnt/ß-catenin signaling pathway, as well as Cdk2, Cdk4, and E2F-1. Therefore, gomisins J and N, the novel Wnt/ß-catenin inhibitors discovered in this study, may serve as potential agents for the prevention and treatment of human colorectal cancers.
Subject(s)
Cyclooctanes/pharmacology , Lignans/pharmacology , Polycyclic Compounds/pharmacology , Wnt Proteins/antagonists & inhibitors , beta Catenin/antagonists & inhibitors , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Cyclin D1/biosynthesis , Cyclin D1/genetics , G1 Phase/drug effects , HCT116 Cells , Humans , Regulatory Sequences, Nucleic Acid , Resting Phase, Cell Cycle/drug effects , TCF Transcription Factors/metabolism , Transcription, Genetic/drug effects , Wnt Proteins/metabolism , beta Catenin/metabolismABSTRACT
Metabolic reprogramming is an important cancer hallmark. However, the mechanisms driving metabolic phenotypes of cancer cells are unclear. Here, we show that the interferon-inducible (IFN-inducible) protein viperin drove metabolic alteration in cancer cells. Viperin expression was observed in various types of cancer and was inversely correlated with the survival rates of patients with gastric, lung, breast, renal, pancreatic, or brain cancer. By generating viperin knockdown or stably expressing cancer cells, we showed that viperin, but not a mutant lacking its iron-sulfur cluster-binding motif, increased lipogenesis and glycolysis via inhibition of fatty acid ß-oxidation in cancer cells. In the tumor microenvironment, deficiency of fatty acids and oxygen as well as production of IFNs upregulated viperin expression via the PI3K/AKT/mTOR/HIF-1α and JAK/STAT pathways. Moreover, viperin was primarily expressed in cancer stem-like cells (CSCs) and functioned to promote metabolic reprogramming and enhance CSC properties, thereby facilitating tumor growth in xenograft mouse models. Collectively, our data indicate that viperin-mediated metabolic alteration drives the metabolic phenotype and progression of cancer.
Subject(s)
Interferons , Neoplasms , Humans , Mice , Animals , Interferons/genetics , Interferons/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Neoplasms/pathology , Glycolysis , Neoplastic Stem Cells/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Tumor MicroenvironmentABSTRACT
Phytochromes enable plants to sense light information and regulate developmental responses. Phytochromes interact with partner proteins to transmit light signals to downstream components for plant development. PIRF1 (phytochrome-interacting ROP guanine-nucleotide exchange factor (RopGEF 1)) functions as a light-signaling switch regulating root development through the activation of ROPs (Rho-like GTPase of plant) in the cytoplasm. In vitro pulldown and yeast two-hybrid assays confirmed the interaction between PIRF1 and phytochromes. PIRF1 interacted with the N-terminal domain of phytochromes through its conserved PRONE (plant-specific ROP nucleotide exchanger) region. PIRF1 also interacted with ROPs and activated them in a phytochrome-dependent manner. The Pr form of phytochrome A enhanced the RopGEF activity of PIRF1, whereas the Pfr form inhibited it. A bimolecular fluorescence complementation analysis demonstrated that PIRF1 was localized in the cytoplasm and bound to the phytochromes in darkness but not in light. PIRF1 loss of function mutants (pirf1) of Arabidopsis thaliana showed a longer root phenotype in the dark. In addition, both PIRF1 overexpression mutants (PIRF1-OX) and phytochrome-null mutants (phyA-211 and phyB-9) showed retarded root elongation and irregular root hair formation, suggesting that PIRF1 is a negative regulator of phytochrome-mediated primary root development. We propose that phytochrome and ROP signaling are interconnected through PIRF1 in regulating the root growth and development in Arabidopsis.
Subject(s)
Arabidopsis Proteins/metabolism , GTP Phosphohydrolase Activators/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Phytochrome/metabolism , Plant Roots/growth & development , Arabidopsis/anatomy & histology , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Flowers/metabolism , Guanine Nucleotide Exchange Factors/genetics , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Light , Phytochrome/genetics , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/physiology , Two-Hybrid System TechniquesABSTRACT
The ankyrin domain is one of the most common protein motifs in eukaryotic proteins. Repeated ankyrin domains are ubiquitous and their mediation of protein-protein interactions is involved in a number of physiological and developmental responses such as the cell cycle, signal transduction and cell differentiation. A novel putative phytochrome-interacting ankyrin repeat protein 2 (PIA2) containing three repeated ankyrin domains was identified in Arabidopsis. An in vitro pull-down and phosphorylation assay revealed that PIA2 is phosphorylated and interacts directly with oat phytochrome A. The N-terminal domain of PIA2 was specifically phosphorylated, whereas interactions between the domains of PIA2 and phytochrome A had no Pr/Pfr preference. PIA2 was ubiquitously expressed in most tissues and was localized in both the nucleus and the cytoplasm independent of treatment with light of specific wavelengths. Anthocyanin accumulation in seedlings grown under far-red light, a typical phenotype of wild-type plants, was reduced in a loss-of-function mutant of PIA2 (pia2), whereas anthocyanin accumulation was increased in an overexpressing plant (PIA2-OX). The gene expression of UDP-flavonoid-3'-glucosyl-transferase (UF3GT), a major enzyme in the anthocyanin biosynthesis processes, was decreased in pia2 knockout plants suggesting that decreased anthocyanin was because of the decreased expression of UF3GT. Our results suggest that PIA2 plays a role in the anthocyanin biosynthesis during seedling development as a novel phytochrome-interacting protein.
Subject(s)
Ankyrin Repeat , Anthocyanins/biosynthesis , Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Phytochrome A/metabolism , Amino Acid Sequence , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant , Gene Knockout Techniques , Molecular Sequence Data , Mutagenesis, Insertional , Phosphorylation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Seedlings/genetics , Seedlings/metabolismABSTRACT
Fluorescence pulse width can provide size information on the fluorescence-emitting particle, such as the nuclei of propidium iodide-stained cells. To analyze nuclear size in the present study, rather than perform the simple doublet discrimination approach usually employed in flow cytometric DNA content analyses, we assessed the pulse width of the propidium iodide fluorescence signal. The anti-cancer drug etoposide is reportedly cytostatic, can induce a strong G2/M arrest, and results in nuclear enlargement. Based on these characteristics, we used etoposide-treated HCT116 cells as our experimental model system. The fluorescence pulse widths (FL2-W) of etoposide-treated (10 microM, 48 h) cells were distributed at higher positions than those of vehicle control, so the peak FL2-W value of etoposide-treated cells appeared at 400 while those of vehicle control cells appeared at 200 and 270. These results were consistent with our microscopic observations. This etoposide-induced increase in FL2-W was more apparent in G2/M phase than other cell cycle phases, suggesting that etoposide-induced nuclear enlargement preferentially occurred in G2/M phase cells rather than in G0/G1 or S phase cells.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cell Nucleus/drug effects , Etoposide/pharmacology , Flow Cytometry/methods , Blotting, Western , Cell Cycle/drug effects , Cell Survival/drug effects , Cells, Cultured , Fluorescence , HCT116 Cells , Humans , Organ Size/drug effectsABSTRACT
The global burden of bone-related diseases is increasing in the aging society; thus, improved bone targeted imaging for their early identification and treatment are needed. In this study, we screened novel peptide ligands for hydroxyapatite, a major inorganic component of teeth and bones, and identified a peptide enabling in vivo bone targeting and real-time fluorescence bone detection. To isolate peptides highly specific for hydroxyapatite, we used negative and positive selection from a randomized 8-mer peptide phage library and identified hydroxyapatite-specific peptides (HA-pep2, HA-pep3, and HA-pep7). Among these three peptides, HA-pep3 showed the highest binding capacity and superior dissociation constant towards hydroxyapatite surfaces over time (~ 88.3% retained on hydroxyapatite after two weeks). Furthermore, HA-pep3 was highly specific for hydroxyapatite compared to other calcium salt-based materials. Using this superior specificity, HA-pep3 showed higher accumulation in skull, spine, and joints in comparison with scrambled control peptide during real-time whole-body imaging. Ex vivo analysis of the major organs and bone from mice demonstrated that the fluorescence intensity in bone was about 3.32 folds higher in the case of HA-pep3 than the one exhibited by the scrambled control peptide. Our study identified a novel approach for targeting ligands for bone specific imaging and can be useful for drug delivery applications.