RESUMO
The cJun N-terminal kinase 1 (JNK1) is implicated in diet-induced obesity. Indeed, germline ablation of the murine Jnk1 gene prevents diet-induced obesity. Here we demonstrate that selective deficiency of JNK1 in the murine nervous system is sufficient to suppress diet-induced obesity. The failure to increase body mass is mediated, in part, by increased energy expenditure that is associated with activation of the hypothalamic-pituitary-thyroid axis. Disruption of thyroid hormone function prevents the effects of nervous system JNK1 deficiency on body mass. These data demonstrate that the hypothalamic-pituitary-thyroid axis represents an important target of metabolic signaling by JNK1.
Assuntos
Sistema Hipotálamo-Hipofisário/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Hipófise/metabolismo , Glândula Tireoide/metabolismo , Animais , Ingestão de Alimentos , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Quinase 8 Ativada por Mitógeno/genética , Obesidade/metabolismo , Transdução de SinaisRESUMO
Obesity is characterized by a dysregulated immune system, which may causally associate with insulin resistance and type 2 diabetes. Despite widespread use of nonobese diabetic (NOD) mice, NOD with severe combined immunodeficiency (scid) mutation (SCID) mice, and SCID bearing a null mutation in the IL-2 common γ chain receptor (NSG) mice as animal models of human diseases including type 1 diabetes, the underlying metabolic effects of a genetically altered immune system are poorly understood. For this, we performed a comprehensive metabolic characterization of these mice fed chow or after 6 wk of a high-fat diet. We found that NOD mice had â¼50% less fat mass and were 2-fold more insulin sensitive, as measured by hyperinsulinemic-euglycemic clamp, than C57BL/6 wild-type mice. SCID mice were also more insulin sensitive with increased muscle glucose metabolism and resistant to diet-induced obesity due to increased energy expenditure (â¼10%) and physical activity (â¼40%) as measured by metabolic cages. NSG mice were completely protected from diet-induced obesity and insulin resistance with significant increases in glucose metabolism in peripheral organs. Our findings demonstrate an important role of genetic background, lymphocytes, and cytokine signaling in diet-induced obesity and insulin resistance.
Assuntos
Resistência à Insulina/fisiologia , Interleucina-2/metabolismo , Linfócitos/metabolismo , Camundongos Endogâmicos NOD/metabolismo , Obesidade/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/efeitos adversos , Metabolismo Energético/fisiologia , Glucose/metabolismo , Técnica Clamp de Glucose/métodos , Insulina/metabolismo , Linfócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Obesidade/fisiopatologia , Transdução de Sinais/fisiologiaRESUMO
Salmonella infection is the one of the major causes of food borne illnesses including fever, abdominal pain, diarrhea, and nausea. Thus, early detection of Salmonella contamination is important for our healthy life. Conventional detection methods for the food contamination have limitations in sensitivity and rapidity; thus, the early detection has been difficult. Herein, we developed a bioelectronic nose using a carbon nanotube (CNT) field-effect transistor (FET) functionalized with Drosophila odorant binding protein (OBP)-derived peptide for easy and rapid detection of Salmonella contamination in ham. 3-Methyl-1-butanol is known as a specific volatile organic compound, generated from the ham contaminated with Salmonella. We designed and synthesized the peptide based on the sequence of the Drosophila OBP, LUSH, which specifically binds to alcohols. The C-terminus of the synthetic peptide was modified with three phenylalanine residues and directly immobilized onto CNT channels using the π-π interaction. The p-type properties of FET were clearly maintained after the functionalization using the peptide. The biosensor detected 1 fM of 3-methyl-1-butanol with high selectivity and successfully assessed Salmonella contamination in ham. These results indicate that the bioelectronic nose can be used for the rapid detection of Salmonella contamination in food.
Assuntos
Nariz Eletrônico , Contaminação de Alimentos/análise , Nanotubos de Carbono/química , Peptídeos/química , Receptores Odorantes/química , Salmonella/isolamento & purificação , Transistores EletrônicosRESUMO
The cytoplasmic polyadenylation element binding protein CPEB1 (CPEB) regulates germ cell development, synaptic plasticity, and cellular senescence. A microarray analysis of mRNAs regulated by CPEB unexpectedly showed that several encoded proteins are involved in insulin signaling. An investigation of Cpeb1 knockout mice revealed that the expression of two particular negative regulators of insulin action, PTEN and Stat3, were aberrantly increased. Insulin signaling to Akt was attenuated in livers of CPEB-deficient mice, suggesting that they might be defective in regulating glucose homeostasis. Indeed, when the Cpeb1 knockout mice were fed a high-fat diet, their livers became insulin-resistant. Analysis of HepG2 cells, a human liver cell line, depleted of CPEB demonstrated that this protein directly regulates the translation of PTEN and Stat3 mRNAs. Our results show that CPEB regulated translation is a key process involved in insulin signaling.
Assuntos
Insulina/metabolismo , Fígado/metabolismo , PTEN Fosfo-Hidrolase/genética , Biossíntese de Proteínas/genética , Fator de Transcrição STAT3/genética , Fatores de Transcrição/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Animais , Linhagem Celular , Dieta Hiperlipídica , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Insulina/genética , Resistência à Insulina/genética , Redes e Vias Metabólicas , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/metabolismo , Polirribossomos/genética , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/deficiência , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Obesity is a major cause of insulin resistance, and weight loss is shown to improve glucose homeostasis. But the underlying mechanism and the role of inflammation remain unclear. Male C57BL/6 mice were fed a high-fat diet (HFD) for 12 wk. After HFD, weight loss was induced by changing to a low-fat diet (LFD) or exercise with continuous HFD. The weight loss effects on energy balance and insulin sensitivity were determined using metabolic cages and hyperinsulinemic euglycemic clamps in awake mice. Diet and exercise intervention for 3 wk caused a modest weight loss and improved glucose homeostasis. Weight loss dramatically reduced local inflammation in skeletal muscle, liver, and heart but not in adipose tissue. Exercise-mediated weight loss increased muscle glucose metabolism without affecting Akt phosphorylation or lipid levels. LFD-mediated weight loss reduced lipid levels and improved insulin sensitivity selectively in liver. Both weight loss interventions improved cardiac glucose metabolism. These results demonstrate that a short-term weight loss with exercise or diet intervention attenuates obesity-induced local inflammation and selectively improves insulin sensitivity in skeletal muscle and liver. Our findings suggest that local factors, not adipose tissue inflammation, are involved in the beneficial effects of weight loss on glucose homeostasis.
Assuntos
Tecido Adiposo/patologia , Inflamação/patologia , Resistência à Insulina/fisiologia , Obesidade/patologia , Redução de Peso/fisiologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Western Blotting , Composição Corporal/fisiologia , Dieta , Metabolismo Energético/fisiologia , Interleucinas/sangue , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Músculo Esquelético/patologia , Miocárdio/patologia , Condicionamento Físico Animal/fisiologia , Transdução de Sinais/fisiologiaRESUMO
AIM: To test the transactivation domain-mediated control of glucose homeostasis by the tumor suppressor p53. BACKGROUND: The tumor suppressor p53 has a critical role in maintenance of glucose homeostasis. Phosphorylation of Ser18 in the transaction domain of p53 controls the expression of Zpf385a, a zinc finger protein that regulates adipogenesis and adipose function. This results suggest that the transactivation domain of p53 is essential to the control of glucose homeostasis. MATERIALS AND METHODS: Mice with mutations in the p53 transactivation domain were examined for glucose homeostasis as well as various metabolic parameters. Glucose tolerance and insulin tolerance tests were performed on age matched wild type and mutant animals. In addition, mice expressing increased dosage of p53 were also examined. RESULTS: Mice with a mutation in p53Ser18 exhibit reduced Zpf385a expression in adipose tissue, adipose tissue-specific insulin resistance, and glucose intolerance. Mice with relative deficits in the transactivation domain of p53 exhibit similar defects in glucose homeostasis, while "Super p53" mice with an increased dosage of p53 exhibit improved glucose tolerance. CONCLUSION: These data support the role of an ATM-p53 cellular stress axis that helps combat glucose intolerance and insulin resistance and regulates glucose homeostasis.
RESUMO
TRPM2 Ca(2+)-permeable cation channel is widely expressed and activated by markers of cellular stress. Since inflammation and stress play a major role in insulin resistance, we examined the role of TRPM2 Ca(2+) channel in glucose metabolism. A 2-h hyperinsulinemic euglycemic clamp was performed in TRPM2-deficient (KO) and wild-type mice to assess insulin sensitivity. To examine the effects of diet-induced obesity, mice were fed a high-fat diet for 4-10 mo, and metabolic cage and clamp studies were conducted in conscious mice. TRPM2-KO mice were more insulin sensitive partly because of increased glucose metabolism in peripheral organs. After 4 mo of high-fat feeding, TRPM2-KO mice were resistant to diet-induced obesity, and this was associated with increased energy expenditure and elevated expressions of PGC-1α, PGC-1ß, PPARα, ERRα, TFAM, and MCAD in white adipose tissue. Hyperinsulinemic euglycemic clamps showed that TRPM2-KO mice were more insulin sensitive, with increased Akt and GSK-3ß phosphorylation in heart. Obesity-mediated inflammation in adipose tissue and liver was attenuated in TRPM2-KO mice. Overall, TRPM2 deletion protected mice from developing diet-induced obesity and insulin resistance. Our findings identify a novel role of TRPM2 Ca(2+) channel in the regulation of energy expenditure, inflammation, and insulin resistance.
Assuntos
Metabolismo Energético/fisiologia , Glucose/metabolismo , Canais de Cátion TRPM/fisiologia , Animais , Western Blotting , Composição Corporal/fisiologia , Peso Corporal/fisiologia , Calmodulina/metabolismo , Calorimetria Indireta , Gorduras na Dieta/farmacologia , Técnica Clamp de Glucose , Teste de Tolerância a Glucose , Imunoprecipitação , Inflamação/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina/fisiologia , Camundongos , Camundongos Knockout , Miocárdio/enzimologia , Miocárdio/metabolismo , Consumo de Oxigênio/fisiologia , Fosforilação , RNA/biossíntese , RNA/genética , Superóxido Dismutase/metabolismoRESUMO
To study the metabolic activity of NF-kappaB, we investigated phenotypes of two different mouse models with elevated NF-kappaB activities. The transcriptional activity of NF-kappaB is enhanced either by overexpression of NF-kappaB p65 (RelA) in aP2-p65 mice or inactivation of NF-kappaB p50 (NF-kappaB1) through gene knock-out. In these models, energy expenditure was elevated in day and night time without a change in locomotion. The mice were resistant to adulthood obesity and diet-induced obesity without reduction in food intake. The adipose tissue growth and adipogenesis were inhibited by the elevated NF-kappaB activity. Peroxisome proliferator-activator receptor gamma expression was reduced by NF-kappaB at the transcriptional level. The two models exhibited elevated inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) in adipose tissue and serum. However, insulin sensitivity was not reduced by the inflammation in the mice on a chow diet. On a high fat diet, the mice were protected from insulin resistance. The glucose infusion rate was increased more than 30% in the hyperinsulinemic-euglycemic clamp test. Our data suggest that the transcription factor NF-kappaB promotes energy expenditure and inhibits adipose tissue growth. The two effects lead to prevention of adulthood obesity and dietary obesity. The energy expenditure may lead to disassociation of inflammation with insulin resistance. The study indicates that inflammation may prevent insulin resistance by eliminating lipid accumulation.
Assuntos
Metabolismo Energético/fisiologia , Resistência à Insulina/fisiologia , NF-kappa B/fisiologia , Adipogenia/genética , Adipogenia/fisiologia , Tecido Adiposo Branco/metabolismo , Animais , Western Blotting , Composição Corporal/genética , Composição Corporal/fisiologia , Temperatura Corporal , Peso Corporal/genética , Peso Corporal/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Ingestão de Alimentos/genética , Ingestão de Alimentos/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Metabolismo Energético/genética , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Teste de Tolerância a Glucose , Inflamação/genética , Inflamação/metabolismo , Resistência à Insulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/genética , Obesidade/induzido quimicamente , Obesidade/genética , PPAR gama/genética , PPAR gama/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Obesity and type 2 diabetes are characterized by insulin resistance. Mice lacking the protein-tyrosine phosphatase PTP1B in all tissues are hypersensitive to insulin but also have diminished fat stores. Because adiposity affects insulin sensitivity, the extent to which PTP1B directly regulates glucose homeostasis has been unclear. We report that mice lacking PTP1B only in muscle have body weight and adiposity comparable to those of controls on either chow or a high-fat diet (HFD). Muscle triglycerides and serum adipokines are also affected similarly by HFD in both groups. Nevertheless, muscle-specific PTP1B(-/-) mice exhibit increased muscle glucose uptake, improved systemic insulin sensitivity, and enhanced glucose tolerance. These findings correlate with and are most likely caused by increased phosphorylation of the insulin receptor and its downstream signaling components. Thus, muscle PTP1B plays a major role in regulating insulin action and glucose homeostasis, independent of adiposity. In addition, rosiglitazone treatment of HFD-fed control and muscle-specific PTP1B(-/-) mice revealed that rosiglitazone acts additively with PTP1B deletion. Therefore, combining PTP1B inhibition with thiazolidinediones should be more effective than either alone for treating insulin-resistant states.
Assuntos
Deleção de Genes , Glucose/metabolismo , Homeostase , Músculo Esquelético/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Adiposidade/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Creatina Quinase/metabolismo , Homeostase/efeitos dos fármacos , Insulina/farmacologia , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Rosiglitazona , Transdução de Sinais/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Triglicerídeos/metabolismoRESUMO
Odorant-binding proteins (OBPs) primarily function in the transport of hydrophobic odorants. In this study, OBPs originating from rat and pig were cloned into a mammalian expression vector, pcDNA3, and expressed in HEK-293 cells, and their specificity for odorants and olfactory receptors was examined. Results suggest that OBPs have a high affinity for the olfactory receptors when both the OBP and receptor originate from the same species. The rat OBPs were bound not only to the rat olfactory receptor I7 but also to the odorant specific to I7. The solubility of the odorant was increased by both OBP2 and OBP3, which originate from rat, but with different efficiencies. These results demonstrate that OBPs specifically interact with odorants as well as olfactory receptors, and these interactions can influence the sensitivity of olfactory receptor-based biosensors.
Assuntos
Técnicas Biossensoriais/métodos , Odorantes/análise , Receptores Odorantes/química , Animais , Linhagem Celular , Humanos , Ratos , Receptores Odorantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato , SuínosRESUMO
Various nanobiosensors composed of biomaterials and nanomaterials have been developed, due to their demonstrated advantage of showing high performance. Among various biomaterials for biological recognition elements of the nanobiosensor, sensory receptors, such as olfactory and taste receptors, are promising biomaterials for developing nanobiosensors, because of their high selectivity to target molecules. Field-effect transistors (FET) with nanomaterials such as carbon nanotube (CNT), graphene, and conducting polymer nanotube (CPNT), can be combined with the biomaterials to enhance the sensitivity of nanobiosensors. Recently, many efforts have been made to develop nanobiosensors using biomaterials, such as olfactory receptors and taste receptors for detecting various smells and tastes. This review focuses on the biomaterials and nanomaterials used in nanobiosensor systems and studies of various types of nanobiosensor platforms that utilize olfactory receptors and taste receptors which could be applied to a wide range of industrial fields, including the food and beverage industry, environmental monitoring, the biomedical field, and anti-terrorism.
Assuntos
Materiais Biocompatíveis/química , Técnicas Biossensoriais/instrumentação , Nanoestruturas/química , Nanotecnologia/instrumentação , Transistores Eletrônicos , Condutividade Elétrica , Desenho de Equipamento , Humanos , Proteínas Imobilizadas/metabolismo , Polímeros/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores Odorantes/metabolismo , Olfato , Propriedades de Superfície , PaladarRESUMO
As a posterior ocular disease, wet age-related macular degeneration (WAMD) has been known to be related to vision loss, accompanying ocular complications. The intravitreous injection of VEGF antibodies has been reported to be an effective treatment to relieve symptoms of WAMD. However, the limitations of this treatment are high costs and invasiveness. For this reason, oral delivery route can be considered as a cost-effective way and the safest method to deliver drug molecules to the eyes. Accordingly, ursodeoxycholic acid (UDCA) was included in the oral formulation as the potential substance for the cure of WAMD in the animal model. Various pharmacological activities, such as antioxidant or anti-inflammatory effects, have been reported for UDCA and recent reports support the effects of UDCA in ocular treatment. However, due to poor water solubility and low pKa (around 5.0), it has been challenging to formulate aqueous solution of UDCA in the neutral pH range. In the present study, we confirmed the aqueous solubility of the oral UDCA formulation and performed a preclinical study, including pharmacokinetic profiling and WAMD model efficacy study in mice after oral administration of the drug solution. The results demonstrated that the formulation improved bioavailability of UDCA and efficiently delivered UDCA to the eye tissues after oral absorption. UDCA formulation was found to have inhibitory effects of choroidal neovascularization with a functional recovery in mice retinas. Taken together, our results suggest that the oral UDCA formulation could be used as a potent supplement for the cure of WAMD and related retinal diseases.
RESUMO
Odorant-binding proteins are low molecular weight, soluble proteins that are secreted by glands of the nasal cavity. Their function is known to be the transport of hydrophobic odorants. This feature is important to artificial olfactory biosensors, which operate in the aqueous phase. In this study, one of rat odorant-binding proteins, OBP3, was inserted into a mammalian expression vector pcDNA3, expressed, and secreted from human embryonic kidney-293 (HEK-293) cells. The his(6) tag and signal peptide of the prelysozyme (plys) were fused with OBP3 for the detection and secretion of the proteins, respectively. The secretion level of OBP3 was maximal at 3h of incubation time. The secreted OBP3 increased the solubility of a hydrophobic odorant, octanal, which is the specific odorant of rat olfactory receptor I7. The secreted OBP3 also bound to olfactory receptor I7. These interactions consequently increased the cellular signal intensity stimulated by the odorant in the cells expressing olfactory receptor I7. Our findings indicate that odorant-binding protein can be effectively used to increase the sensitivity of olfactory receptor-based biosensors.
Assuntos
Bioensaio/métodos , Rim/metabolismo , Odorantes/análise , Receptores Odorantes/metabolismo , Bioensaio/instrumentação , Linhagem Celular , Humanos , Proteínas Recombinantes de Fusão/análise , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Cameleon is a genetically engineered Ca2+ sensing molecule consisting of two variants of the green fluorescent protein (GFP), calmodulin and calmodulin-binding protein, M13. HEK-293 cells stably expressing three types of cameleons, yellow cameleon-2, cameleon-3er, and cameleon-2nu, were constructed, and the expression and localization of these cameleons were confirmed by fluorescent imaging. Among the cameleons, the yellow cameleon-2 was selected for analyzing the change in Ca2+ induced by the olfactory receptor-mediated signal transduction, because it is localized in the cytosol and binds to cytosolic Ca2+ ions. Cells stably expressing yellow cameleon-2 were transfected with each of the test olfactory receptor genes, odr-l0 and 17, and the expression of the olfactory receptor genes were examined using immunocytochemical methods and RT-PCR. Stimulating each olfactory receptor with its specific odorant caused an increase in the intracellular Ca2+ level, which was measured using yellow cameleon-2. These results demonstrate that yellow cameleon-2 can be conveniently used to examine the function of the olfactory receptors expressed in heterologous cells.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Receptores Odorantes/fisiologia , Cálcio/metabolismo , Linhagem Celular , Humanos , Imuno-Histoquímica , Receptores Odorantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
A multiplexed bioelectronic sensor was developed for the purpose of rapid, on-site, and simultaneous detection of various target molecules. Olfactory and taste receptors were produced in Escherichia coli, and the reconstituted receptors were immobilized onto a multi-channel type carbon nanotube field-effect transistor. This device mimicked the human olfactory/taste system and simultaneously measured the conductance changes with high sensitivity and selectivity following treatment with various odor and taste molecules commonly known to be indicators of food contamination. Various pattern recognition of odorants and tastants was available with a customized platform for the simultaneous measurement of electrical signals. The simple portable bioelectronic device was suitable for efficient monitoring of food freshness and is expected to be used as a rapid on-site sensing platform with various applications.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas Imobilizadas/metabolismo , Receptores Odorantes/metabolismo , Olfato , Paladar , Transistores Eletrônicos , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Aditivos Alimentares/análise , Contaminação de Alimentos/análise , Humanos , Proteínas Imobilizadas/química , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestrutura , Odorantes/análise , Receptores Odorantes/químicaRESUMO
Odors are perceived differently as a function of individual human experience, and communicating about odors between individuals is therefore very difficult. There is a need to classify and standardize odors, but appropriate tools have not yet been developed. A bioelectronic nose mimics human olfaction and detects target molecules with high sensitivity and selectivity. This new tool has great potential in many applications and is expected to accelerate odor classification and standardization. In particular, a multiplexed bioelectronic nose can provide complex odor information using pattern recognition techniques, and could even reproduce odors via an integrated olfactory display system. We expect that a bioelectronic nose will be a useful tool for odor standardization by providing codes for odors that enable us to communicate odor information.
Assuntos
Técnicas Biossensoriais , Nariz Eletrônico , Modelos Neurológicos , Odorantes/análise , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/normas , Humanos , Nanotecnologia , Padrões de ReferênciaRESUMO
An olfactory receptor protein of C. elegans, ODR-10, was expressed in Escherichia coli as a fusion protein, with GST and 6x His-tag. The expression of the target protein was analyzed by SDS-PAGE and Western blot, and was confirmed to be expressed at the membrane fraction of the host E. coli. The surface of a quartz crystal microbalance (QCM) was coated with crude membrane extracts, containing the expressed receptor protein, and the interaction between the olfactory receptor and various odorant molecules examined. Compared with other odorants, diacetyl (2,3-butanedione), known as a natural ligand for the ODR-10 receptor, interacted most strongly with the expressed protein. Various concentrations of diacetyl were applied to the expressed ODR-10 receptor, and the response of the QCM showed a linear relationship to the logarithmic value of the odorant concentration. This piezoelectric biosensor system, using olfactory receptor proteins expressed in E. coli, can be used in diagnostics, toxic chemical detection and the quality control of food.
Assuntos
Técnicas Biossensoriais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Receptores Odorantes/química , Receptores Odorantes/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Escherichia coli/genética , Odorantes/análise , Receptores Odorantes/genéticaRESUMO
There have been many trials to visualize smell using various techniques in order to objectively express the smell because information obtained from the sense of smell in human is very subjective. So far, well-trained experts such as a perfumer, complex and large-scale equipment such as GC-MS, and an electronic nose have played major roles in objectively detecting and recognizing odors. Recently, an optoelectronic nose was developed to achieve this purpose, but some limitations regarding the sensitivity and the number of smells that can be visualized still persist. Since the elucidation of the olfactory mechanism, numerous researches have been accomplished for the development of a sensing device by mimicking human olfactory system. Engineered olfactory cells were constructed to mimic the human olfactory system, and the use of engineered olfactory cells for smell visualization has been attempted with the use of various methods such as calcium imaging, CRE reporter assay, BRET, and membrane potential assay; however, it is not easy to consistently control the condition of cells and it is impossible to detect low odorant concentration. Recently, the bioelectronic nose was developed, and much improved along with the improvement of nano-biotechnology. The bioelectronic nose consists of the following two parts: primary transducer and secondary transducer. Biological materials as a primary transducer improved the selectivity of the sensor, and nanomaterials as a secondary transducer increased the sensitivity. Especially, the bioelectronic noses using various nanomaterials combined with human olfactory receptors or nanovesicles derived from engineered olfactory cells have a potential which can detect almost all of the smells recognized by human because an engineered olfactory cell might be able to express any human olfactory receptor as well as can mimic human olfactory system. Therefore, bioelectronic nose will be a potent tool for smell visualization, but only if two technologies are completed. First, a multi-channel array-sensing system has to be applied for the integration of all of the olfactory receptors into a single chip for mimicking the performance of human nose. Second, the processing technique of the multi-channel system signals should be simultaneously established with the conversion of the signals to visual images. With the use of this latest sensing technology, the realization of a proper smell-visualization technology is expected in the near future.
RESUMO
An olfactory receptor protein of rats, I7, was expressed on the surface of human embryonic kidney (HEK)-293 cells. For targeting and detecting the protein, rho-tag import sequence was fused with the I7 protein. The olfactory receptor was expressed on the plasma membrane of HEK-293 cells, and stable cell lines regulated by an inducer were obtained. The expression on the cell surface was confirmed by immunocytochemical and Western blotting methods, and the binding of specific odorant molecules to the olfactory receptor was measured using quartz crystal microbalance (QCM). The results for QCM coated with cells containing the olfactory receptor showed that the expressed protein I7 strongly interacted with octyl aldehyde (octanal), which is an odorant specific to the I7 protein. Several other odorants were tested, and the results showed that I7 interacted differently with them. The QCM response to the serial concentrations of octyl aldehyde showed that the response is dose dependent. All these results indicate that the I7 receptor protein expressed on the surface of the heterologous cell system is sensitive to the specific odorant and can be used for the quantitative measurement of the odorant.
Assuntos
Técnicas Biossensoriais/instrumentação , Receptores Odorantes/genética , Animais , Clonagem Molecular , Eletrodos , Técnicas de Transferência de Genes , Humanos , Ligantes , Ratos , Receptores Odorantes/metabolismoRESUMO
In the human smell sensing system, there are about 390 kinds of olfactory receptors (ORs) which bind to various odorants with different affinities and specificities. Characterization and odorant binding pattern analysis of the ORs are essential for understanding of human olfaction and to mimic the olfactory system in various applications. Although various cell-based odorant screening systems have been developed for this purpose, many human ORs (hORs) still remain orphan because of the time-consuming and labor-intensive experimental procedures of the available screening methods. In this study, we constructed an ion channel-coupled hOR for simple odorant detection by rapidly visualizing the odorant response to overcome the limitations of conventional screening systems. The hORs were coupled to the Kir6.2 potassium channel and the fusion proteins were expressed in HEK293 cells. In this system, when an odorant binds to the hORs coupled to the ion channel, a conformational change in the OR occurs, which consequently opens the ion channel to result in ion influx into the cell. This ion influx was then visualized using a membrane potential dye. Cells expressing ion channel-coupled hORs showed high sensitivity and selectivity to their specific odorants, and the odorant-hOR binding pattern was visualized to identify the response of individual hORs to various odorants, as well as the response of various hORs to various odorants. These results indicate that the ion channel-coupled hOR system can be effectively used not only for simple and fast high-throughput odorant screening, but also to visualize the odorant-hOR response pattern.