RESUMO
Toll-like receptor (TLR) agonists or pro-inflammatory cytokines converge to activate the nuclear factor κB (NF-κB) signaling pathway, which provokes inflammatory responses. In the present study, we identified amiodarone hydrochloride as a selective inhibitor of the TLR3-mediated NF-κB signaling pathway by screening the RIKEN NPDepo Chemical Library. In human umbilical vein endothelial cells (HUVEC), amiodarone selectively inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by polyinosinic-polycytidylic acid (Poly(I:C)), but not tumor necrosis factor-α, interleukin-1α, or lipopolysaccharide. In response to a Poly(I:C) stimulation, amiodarone at 20 µM reduced the up-regulation of mRNA expression encoding ICAM-1, vascular cell adhesion molecule-1, and E-selectin. The nuclear translocation of the NF-κB subunit RelA was inhibited by amiodarone at 15-20 µM in Poly(I:C)-stimulated HUVEC. Amiodarone diminished the fluorescent dots of LysoTracker® Red DND-99 scattered over the cytoplasm of HUVEC. Therefore, the present study revealed that amiodarone selectively inhibited the TLR3-mediated NF-κB signaling pathway by blocking the acidification of intracellular organelles.
Assuntos
Amiodarona , NF-kappa B , Humanos , NF-kappa B/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Receptor 3 Toll-Like/metabolismo , Células Endoteliais/metabolismo , Amiodarona/farmacologia , Amiodarona/metabolismo , Células Cultivadas , Transdução de Sinais , Molécula 1 de Adesão de Célula Vascular/metabolismo , Organelas/metabolismo , Concentração de Íons de HidrogênioRESUMO
Energy reserves, primarily stored in the insect's fat body, are essential for physiological processes such as reproduction and cocoon formation. However, whether these processes are mutually constraining is unknown. Here, we showed that cocoon-free silkworms accumulate amino acid constituents of silk proteins in the hemolymph and maintain lipid and sugar reserves in the pupal fat body by repressing the expression of sericin and fibroin genes in the middle and posterior silk glands, respectively, via butterfly pierisin-1A catalytic domain expression. This, in turn, upregulates insulin/insulin-like signaling and target of rapamycin (IIS/TOR) signaling, which enhances vitellogenesis and accelerates ovarian development, thus contributing to increased fecundity. The impacts of semi-starvation on fecundity and egg hatchability were also less pronounced in cocoon-free silkworms compared with wildtype silkworms. These data uncover the resource allocation trade-off between cocoon formation and fecundity and demonstrate that nutritional signaling plays a role in regulating silkworm reproduction.
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INTRODUCTION: This multicenter, randomized, comparative, and investigator-masked crossover clinical trial sought to compare the efficacy and tolerability of fixed combinations of 0.1% brimonidine/0.5% timolol (BTFC) versus 1% dorzolamide/0.5% timolol (DTFC) as adjunctive therapies to prostaglandin analogues. METHODS: A total of 110 patients with open-angle glaucoma or ocular hypertension previously treated with prostaglandin analogue monotherapy were randomized to receive either BTFC or DTFC as adjunctive therapy for 8 weeks. These patients were then crossed over to the alternative treatment arm for another 8 weeks. The reduction in intraocular pressure (IOP) (primary outcome), occurrence of adverse events, ocular discomfort after instillation, and patient preference (secondary outcomes) were recorded through patient interviews. RESULTS: BTFC instillation for 8 weeks reduced IOP by 3.55 mmHg, demonstrating non-inferiority to DTFC instillation (3.60 mmHg; P < 0.0001, mixed-effects model). Although adverse events were rare with both combinations, patients reported greater discomfort with DTFC than with BTFC (P < 0.0001). More patients preferred BTFC (P < 0.0001) over DTFC, as BTFC caused minimal or no eye irritation. CONCLUSION: As BTFC offered better tolerability than DTFC with comparable reduction in IOP, we recommend it as an alternative for patients who experience ocular discomfort with DTFC-prostaglandin analogue combination therapy. TRIAL REGISTRATION NUMBER: jRCTs051190125.
Patients with glaucoma who require further reduction in intraocular pressure while undergoing monotherapy with prostaglandin analogue ophthalmic solution have been prescribed two enhanced treatment options: 0.1% brimonidine/0.5% timolol fixed combination ophthalmic solution (BTFC) and 1% dorzolamide/0.5% timolol fixed combination ophthalmic solution (DTFC). The Aibeta Crossover Study Group in Japan compared the efficacy and tolerability of fixed combinations of BTFC versus DTFC when an additional fixed combination ophthalmic solution was prescribed in patients with open-angle glaucoma or ocular hypertension who had been treated with prostaglandin analogue monotherapy. We recruited 110 patients previously treated with prostaglandin analogue monotherapy at 20 clinical centers in Japan, then randomly assigned them to two alternative treatment groups: the BTFC to DTFC group or the DTFC to BTFC group, as an adjunctive therapy to prostaglandin analogues for total of 16 weeks. We compared the reduction in intraocular pressure, occurrence of side effects, eye discomfort after instillation, and patient preference between BTFC versus DTFC instillations. The intraocular pressure reduction of BTFC instillation was comparable to that of DTFC instillation, showing non-inferiority to DTFC (3.55 mmHg vs. 3.60 mmHg; P < 0.0001, mixed-effects model). Both eye drops caused few side effects; however, patients felt greater eye discomfort with DTFC than with BTFC (P < 0.0001). Because of less eye irritation, more patients preferred BTFC (P < 0.0001) over DTFC. We can recommend using BTFC for patients who feel eye discomfort with DTFCprostaglandin analogue combination therapy.
Assuntos
Glaucoma de Ângulo Aberto , Timolol , Humanos , Timolol/efeitos adversos , Glaucoma de Ângulo Aberto/tratamento farmacológico , Estudos Cross-Over , Anti-Hipertensivos/efeitos adversos , Soluções Oftálmicas/uso terapêutico , Tartarato de Brimonidina/uso terapêutico , Pressão Intraocular , Prostaglandinas Sintéticas/uso terapêutico , Combinação de MedicamentosRESUMO
Growth factors, including fibroblast growth factor-7 (FGF-7), are a group of proteins that stimulate various cellular processes and are often used with carriers to prevent the rapid loss of their activities. Sericin with great biocompatibility has been investigated as a proteinaceous carrier to enhance the stability of incorporated proteins. The difficulties in obtaining intact sericin from silkworm cocoons and the handling of growth factors with poor stability necessitate an efficient technique to incorporate the protein into a sericin-based biomaterial. Here, we report the generation of a transgenic silkworm line simultaneously expressing and incorporating FGF-7 into cocoon shells containing almost exclusively sericin. Growth-factor-functionalized sericin cocoon shells requiring simple lyophilization and pulverization processes were successfully used to induce the proliferation and migration of keratinocytes. Moreover, FGF-7 incorporated into sericin-cocoon powder exhibited remarkable stability, with more than 70% of bioactivity being retained after being stored as a suspension at 25 °C for 3 months. Transgenic sericin-cocoon powder was used to continuously supply biologically active FGF-7 to generate a three-dimensionally cultured keratinocyte model in vitro. The outcomes of this study propound a feasible approach to producing cytokine-functionalized sericin materials that are ready to use for cell cultivation.
Assuntos
Bombyx , Sericinas , Animais , Animais Geneticamente Modificados , Materiais Biocompatíveis/farmacologia , Bioengenharia , Bombyx/genética , Bombyx/metabolismo , Queratinócitos/metabolismo , Pós , Sericinas/metabolismo , Sericinas/farmacologiaRESUMO
Silk fibroin exhibits high biocompatibility and biodegradability, making it a versatile biomaterial for medical applications. However, contaminated silkworm-derived substances in remnant sericin from the filature and degumming process can result in undesired immune reactions and silk allergy, limiting the widespread use of fibroin. Here, we established transgenic silkworms with modified middle silk glands, in which sericin expression was repressed by the ectopic expression of cabbage butterfly-derived cytotoxin pierisin-1A, to produce cocoons composed solely of fibroin. Intact, nondegraded fibroin can be prepared from the transgenic cocoons without the need for sericin removal by the filature and degumming steps that cause fibroin degradation. A wide-angle X-ray diffraction analysis revealed low crystallinity in the transgenic cocoons. However, nondegraded fibroin obtained from transgenic cocoons enabled the formation of fibroin sponges with varying densities by using 1-5% (v/v) alcohol. The effective chondrogenic differentiation of ATDC5 cells was induced following their cultivation on substrates coated with intact fibroin. Our results showed that intact, allergen-free fibroin can be obtained from transgenic cocoons without the need for sericin removal, providing a method to produce fibroin-based materials with high biocompatibility for biomedical uses.
Assuntos
Bombyx , Fibroínas , Sericinas , Animais , Animais Geneticamente Modificados , Materiais Biocompatíveis/química , Bombyx/metabolismo , Fibroínas/química , Sericinas/química , Seda/químicaRESUMO
The silk glands of silkworms produce large quantities of fibroin, which is a protein that can be physically processed and used as a biodegradable carrier for cell growth factors in tissue engineering applications. Meanwhile, protein microcrystals known as polyhedra, which are derived from cypovirus 1, have been used as a vehicle to protect and release encapsulated cell growth factors. We report the generation of transgenic silkworms that express recombinant fibroblast growth factor-7 (FGF-7) fused with the polyhedron-encapsulating signal in polyhedra produced in the middle (MSG) and posterior (PSG) silk glands. Immunofluorescence showed that polyhedra from silk glands are associated with FGF-7. The MSG and PSG from transgenic silkworms were processed into fine powdery materials, from which FGF-7 activity was released to stimulate the proliferation of human keratinocyte epidermal cells. Powders from PSGs exhibited higher FGF-7 activity than those from MSGs. Moreover, PSG powder showed a gradual release of FGF-7 activity over a long period and induced keratinocyte proliferation and differentiation in 3D culture to promote the formation of stratified epidermis expressing positive differentiation marker proteins. Our results indicate that powdery materials incorporating the FGF-7-polyhedra microcrystals from silk glands are valuable for developing cell/tissue engineering applications in vivo and in vitro.
RESUMO
Cypovirus is an insect virus that is encapsulated in stable cubic protein crystals composed of polyhedrin protein produced in virus-infected cells. Molecular technology developed over the last decade is now able to immobilise proteins of interest on polyhedrin crystals. Modified polyhedrin crystals can be used in cell cultures for implantation in animals and vaccines, among other applications. However, this technique does not work for some proteins. Here, we developed and tested an alternative approach for immobilising foreign proteins in polyhedrin crystals using a linker method; diverse proteins, such as fluorescent proteins, enzymes, antibodies, and streptavidin were successfully contained. The immobilised antibodies retained their binding activity on filter paper, implying their potential for new immunochromatography applications. Moreover, this immobilisation method allows enzymes to be collected from one reaction reagent and transferred to another reagent. These results demonstrate the potential of this immobilisation method and the likelihood of expanding the applications of polyhedrin crystals using this approach.
Assuntos
Proteínas Imobilizadas/química , Proteínas de Matriz de Corpos de Inclusão/química , Animais , Engenharia de Proteínas/métodos , Reoviridae/química , Proteínas Virais/química , Proteínas Estruturais Virais/químicaRESUMO
In some adult holometabolous insects, specific epithelial tissues form imaginal discs, small sac-like clusters of cells that form in the larval body. During metamorphosis, in response to hormones, the discs undergo dramatic changes including cell proliferation and differentiation. In Bombyx mori insects, the cuticular protein (CP) genes, which are expressed at prepupal stage in the wing discs, are divided into six groups according to their developmental expression profile and responsiveness to steroid hormones. In this article, we discuss the expression of CP genes in the wing disc, and examine the molecular mechanisms by which metamorphosis and cell transformations are regulated by hormones in insects.
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Bombyx/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Metamorfose Biológica/genética , Asas de Animais/metabolismo , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Ecdisona/metabolismo , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Pupa/genética , Pupa/crescimento & desenvolvimento , Asas de Animais/crescimento & desenvolvimentoRESUMO
The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.
Assuntos
Citocinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Fator de Crescimento Neural/farmacologia , Neurônios/citologia , Proteínas de Matriz de Corpos de Inclusão/genética , Reoviridae/fisiologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Preparações de Ação Retardada , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 8 da Matriz/metabolismo , Nanopartículas , Fator de Crescimento Neural/química , Fator de Crescimento Neural/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas de Matriz de Corpos de Inclusão/metabolismo , Células PC12 , Tamanho da Partícula , Ratos , Reoviridae/genética , Reoviridae/metabolismo , Transdução de SinaisRESUMO
Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.
Assuntos
ADP Ribose Transferases , Animais Geneticamente Modificados , Bombyx , Citotoxinas , Glândulas Exócrinas/metabolismo , Hidrogéis/farmacologia , Proteínas de Insetos , Células-Tronco Embrionárias Murinas/metabolismo , Sericinas , ADP Ribose Transferases/biossíntese , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bombyx/genética , Bombyx/metabolismo , Citocinas/biossíntese , Citotoxinas/biossíntese , Citotoxinas/genética , Citotoxinas/farmacologia , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Proteínas de Insetos/farmacologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Sericinas/biossíntese , Sericinas/genética , Sericinas/farmacologiaRESUMO
Juvenile hormone (JH) postpones metamorphosis of insect larvae until they have attained an appropriate stage and size. Then, during the final larval instar, a drop in JH secretion permits a metamorphic molt that transforms larvae to adults either directly (hemimetaboly) or via a pupal stage (holometaboly). In both scenarios, JH precludes metamorphosis by activating the Kr-h1 gene through a JH receptor, Methoprene-tolerant (Met). Removal of Met, Kr-h1, or JH itself triggers deleterious precocious metamorphosis. Although JH is thought to maintain the juvenile status throughout larval life, various methods of depleting JH failed to induce metamorphosis in early-instar larvae. To determine when does JH signaling become important for the prevention of precocious metamorphosis, we chose the hemimetabolous bug, Pyrrhocoris apterus, and the holometabolous silkworm, Bombyx mori. Both species undergo a fixed number of five larval instars. Pyrrhocoris larvae subjected to RNAi-mediated knockdown of Met or Kr-h1 underwent precocious adult development when treated during the fourth (penultimate) instar, but younger larvae proved increasingly resistant to loss of either gene. The earliest instar developing minor signs of precocious metamorphosis was the third. Therefore, the JH-response genes may not be required to maintain the larval program during the first two larval instars. Next, we examined Bombyx mod mutants that cannot synthesize authentic, epoxidized forms of JH. Although mod larvae expressed Kr-h1 mRNA at severely reduced levels since hatching, they only entered metamorphosis by pupating after four, rarely three instars. Based on findings in Pyrrhocoris and Bombyx, we propose that insect postembryonic development is initially independent of JH. Only later, when larvae gain competence to enter metamorphosis, JH signaling becomes necessary to prevent precocious metamorphosis and to optimize growth.
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Bombyx/crescimento & desenvolvimento , Heterópteros/crescimento & desenvolvimento , Hormônios Juvenis/metabolismo , Metamorfose Biológica/fisiologia , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Primers do DNA/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Larva/fisiologia , Metoprene , Interferência de RNA , Especificidade da EspécieRESUMO
We investigated the effects of nanosized TiO2 particles on the death of mouse leukemia L1210 cells. TiO2 particles suppressed proliferation and induced cell death, as measured by lactate dehydrogenase (LDH) release into the culture medium. Chromatin condensation, which is typical of the initiation of cell death, was observed in approximately 14% cells cultured with titanium dioxide (TiO2) particles for 12 h. Furthermore, giant DNA fragments of approximately 2 Mbp and high-molecular-weight DNA fragments between 100 kbp and 1 Mbp were observed in cells cultured for 18 h with TiO2 particles. These giant and high-molecular-weight DNA fragments were further degraded into smaller DNA fragments, appearing as DNA ladders. Corresponding to the generation of DNA fragments, caspase-3 activity increased in cells treated with TiO2 particles. TiO2 particle-induced LDH release was not inhibited by cytochalasin D, an inhibitor of endocytosis. These results suggest that nanosized TiO2 particles can induce apoptosis associated with DNA fragmentation and caspase-3 activation and that TiO2 particle-induced apoptosis is not caused by endocytosis but is associated with contact of the particles with the cell surface.
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Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Leucemia L1210/patologia , Nanopartículas Metálicas/toxicidade , Animais , Materiais Biocompatíveis/toxicidade , Células Cultivadas , Citocalasina D/farmacologia , Ativação Enzimática , L-Lactato Desidrogenase/metabolismo , Leucemia L1210/tratamento farmacológico , Leucemia L1210/metabolismo , Camundongos , Titânio/toxicidadeRESUMO
Juvenile hormone (JH), a sesquiterpenoid produced by the insect corpus allatum gland (CA), prevents metamorphosis in larvae and stimulates vitellogenesis in adult females. Whether the same JH signaling pathway regulates both processes is presently unknown. Here, we employ the robust JH response during reproduction and development of the linden bug, Pyrrhocoris apterus, to compare the function of key JH-signaling genes encoding the JH receptor, Methoprene-tolerant (Met), its binding partner Taiman (Tai), and a JH-inducible protein, Krüppel-homolog 1 (Kr-h1). RNA interference (RNAi) with Met or Tai, but not Kr-h1, blocked ovarian development and suppressed vitellogenin gene expression in the fat body of females raised under reproduction-inducing conditions. Loss of Met and Tai matched the effects of CA ablation or the natural absence of JH during reproductive diapause. Stimulation of vitellogenesis by treatment of diapausing females with a JH mimic methoprene also required both Met and Tai in the fat body, whereas Kr-h1 RNAi had no effect. Therefore, the Met-Tai complex likely functions as a JH receptor during vitellogenesis. In contrast to Met and Kr-h1 that are both required for JH to prevent precocious metamorphosis in P. apterus larvae, removal of Tai disrupted larval ecdysis without causing premature adult development. Our results show that while Met operates during metamorphosis in larvae and reproduction in adult females, its partner Tai is only required for the latter. The diverse functions of JH thus likely rely on a common receptor whose actions are modulated by distinct components.
Assuntos
Heterópteros/fisiologia , Hormônios Juvenis/metabolismo , Animais , Diapausa de Inseto , Heterópteros/crescimento & desenvolvimento , Heterópteros/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Larva/fisiologia , Muda , Reprodução , Comportamento Sexual Animal , Transdução de SinaisRESUMO
Juvenile hormone (JH) is a sesquiterpenoid of vital importance for insect development, yet the molecular basis of JH signaling remains obscure, mainly because a bona fide JH receptor has not been identified. Mounting evidence points to the basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) domain protein Methoprene-tolerant (Met) as the best JH receptor candidate. However, details of how Met transduces the hormonal signal are missing. Here, we demonstrate that Met specifically binds JH III and its biologically active mimics, methoprene and pyriproxyfen, through its C-terminal PAS domain. Substitution of individual amino acids, predicted to form a ligand-binding pocket, with residues possessing bulkier side chains reduces JH III binding likely because of steric hindrance. Although a mutation that abolishes JH III binding does not affect a Met-Met complex that forms in the absence of methoprene, it prevents both the ligand-dependent dissociation of the Met-Met dimer and the ligand-dependent interaction of Met with its partner bHLH-PAS protein Taiman. These results show that Met can sense the JH signal through direct, specific binding, thus establishing a unique class of intracellular hormone receptors.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Modelos Moleculares , Sesquiterpenos/metabolismo , Transdução de Sinais/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Dimerização , Proteínas de Drosophila/química , Imunoprecipitação , Ligantes , Metoprene/metabolismo , Dados de Sequência Molecular , Mutação/genética , Piridinas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Fatores de Transcrição/metabolismoRESUMO
Recently, our group discovered an alternative titanium dioxide (TiO(2)) activation method that uses ultrasound irradiation (US/TiO(2)) instead of ultraviolet irradiation. The pre-S1/S2 protein from hepatitis B virus, which recognizes liver cells, was immobilized to the surface of TiO(2) nanoparticles using an amino-coupling method. The ability of the protein-modified TiO(2) nanoparticles to recognize liver cells was confirmed by surface plasmon resonance analysis and immuno-staining analyses. After uptake of TiO(2) nanoparticles by HepG2 cancer cells, the cells were injured using this US/TiO(2) method; significant cell injury was observed at an ultrasound irradiation intensity of 0.4 W/cm(2). Together with these results, this strategy could be applied to new cell injuring systems that use ultrasound irradiation in place of photodynamic therapy in the near future.
Assuntos
Nanopartículas , Proteínas/química , Titânio/isolamento & purificação , Linhagem Celular Tumoral , Humanos , Titânio/química , UltrassomRESUMO
During pupal metamorphosis, the anterior silk gland (ASG) of the silkworm, Bombyx mori, undergoes programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). Annexin IX (ANX IX) has been identified as a 20E-inducible gene in dying ASGs, and we show here that its expression is down-regulated in tissues destined to die but not in tissues that survive pupal metamorphosis. ANX IX expression was high in the ASGs during the feeding period, when the ecdysteroid titer was low, and decreased in response to the rising ecdysteroid titer that triggered pupal metamorphosis. Before gut purge, in vitro exposure of the ASGs to 20E levels corresponding to the ecdysteroid concentration present at the time of gut purge caused a decrease in ANX IX messenger RNA levels. Expression profiles of EcR and USP, and the 20E concentration-responses of these genes, indicate the importance of the relative abundance of EcR-A and EcR-B1 isoforms in ANX IX regulation. These results suggest an involvement of ANX IX in the determination of PCD timing by delaying or suppressing the response to the increase in hemolymph ecdysteroid concentration during the prepupal period.
Assuntos
Anexinas/fisiologia , Apoptose/fisiologia , Bombyx/fisiologia , Animais , Anexinas/biossíntese , Primers do DNA/química , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila , Ecdisterona/fisiologia , Reação em Cadeia da Polimerase/métodos , Fatores de Tempo , Fatores de Transcrição/fisiologiaRESUMO
The prothoracic glands of early last (fifth) instar larvae of the silkworm are inactive with regard to ecdysteroidogenesis and unresponsive to prothoracicotropic hormone (PTTH) [J. Insect Physiol. 31 (1985) 455]. In an attempt to elucidate the hormonal mechanisms that cause the inactivity, we compared the effects of PTTH, dibutyryl cyclic AMP (dbcAMP), a cAMP phosphodiesterase inhibitor (IBMX), juvenile hormone analogue (JHA) and 20-hydroxyecdysone (20E) on secretory activity of the third, fourth and fifth instar glands. Among the factors examined, feedback inhibition by 20E was indicated to be the most likely factor. Inhibition was moderate in the third and early fourth instars while 20E strongly inhibited the glands of middle fourth instar larvae. The inhibitory effect of 20E was reduced by removal of the brain and corpora allata. Once the glands were suppressed by 20E to the degree of exhibiting neither secretory activity nor responsiveness to PTTH, dbcAMP or IBMX did not elicit ecdysone secretion at all. Thus the feedback inhibition may shut down ecdysteroidogenesis although it is obscure whether it affects the intracellular transductory cascade from the PTTH receptor through cAMP. Taken together, this evidence suggests that inactivity of the gland in the early fifth instar is brought about by feedback inhibition of the glands by 20E occurring in the late fourth instar, and that this inactivity is maintained by the juvenile hormone found in the early fifth instar.