RESUMO
IL-17-producing T-helper 17 (Th17) cells are involved in the pathogenesis of autoimmune disorders such as rheumatoid arthritis (RA). Here, we show that a methoxyflavanone from the Asian medicinal herb Perilla frutescens (termed Perilla-derived methoxyflavanone, PDMF) suppresses Th17 response and collagen-induced arthritis (CIA), an animal model of RA. We found that co-stimulation with PDMF suppressed Th17 cell differentiation and inhibited IL-17A secretion by differentiated Th17 cells. In vivo administration of PDMF to a CIA mouse model significantly ameliorated the development of RA-like joint symptoms, accompanied by decreased IL-17A production. Mechanistically, PDMF neither suppresses Th17-inducing IL-6 signaling nor reciprocally expands regulatory T (Treg) cells, but rather negatively regulates T-cell receptor (TCR) signaling-driven activation of Akt, which is another positive regulator of Th17 cell differentiation. These results suggest that PDMF is useful in preventing RA and the pro-inflammatory Th17 response.
Assuntos
Artrite Experimental , Artrite Reumatoide , Perilla frutescens , Plantas Medicinais , Animais , Camundongos , Artrite Experimental/induzido quimicamente , Artrite Experimental/tratamento farmacológico , Interleucina-17 , Modelos Animais de DoençasRESUMO
Cellular senescence is an inherent tumor suppressive process, and cancer-targeted senescence induction represents an attractive anti-tumor strategy. Here, we show that a methoxyflavanone derivative (Perilla-derived methoxyflavanone, PDMF) from the Asian medicinal herb, Perilla frutescens, induces cellular senescence in A549 human adenocarcinoma cells but not in normal human bronchial epithelial (NHBE) cells. We also provide evidence that PDMF preferentially activates the p53-p21 pathway in A549 cells, and that p53 is essential for its pro-senescent activity.
Assuntos
Adenocarcinoma de Pulmão , Perilla frutescens , Células A549 , Senescência Celular , Células Epiteliais/metabolismo , Humanos , Perilla frutescens/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Sarcopenia is an age-related skeletal muscle atrophy. Exercise is effective in improving sarcopenia via two mechanisms: activation of skeletal muscle satellite cells (SCs) and stimulation of muscle protein synthesis. In contrast, most nutritional approaches for improving sarcopenia focus mainly on muscle protein synthesis, and little is known about SC activation. Here, we investigated the effect of lemon myrtle extract (LM) on SC activation both in vitro and in vivo. Primary SCs or myoblast cell lines were treated with LM or its derived compounds, and incorporation of 5-bromo-2'-deoxyuridine, an indicator of cell cycle progression, was detected by immunocytochemistry. We found that LM significantly activated SCs (p < 0.05), but not myoblasts. We also identified casuarinin, an ellagitannin, as the active compound in LM involved in SC activation. The structure−activity relationship analysis showed that rather than the structure of each functional group of casuarinin, its overall structure is crucial for SC activation. Furthermore, SC activation by LM and casuarinin was associated with upregulation of interleukin-6 mRNA expression, which is essential for SC activation and proliferation. Finally, oral administration of LM or casuarinin to rats showed significant activation of SCs in skeletal muscle (p < 0.05), suggesting that LM and casuarinin may serve as novel nutritional interventions for improving sarcopenia through activating SCs.
Assuntos
Taninos Hidrolisáveis , Myrtaceae/química , Extratos Vegetais , Células Satélites de Músculo Esquelético , Animais , Células Cultivadas , Taninos Hidrolisáveis/farmacologia , Extratos Vegetais/farmacologia , Ratos , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismoRESUMO
Melilotus indicus, is a traditional medicine used as analgesic and emollient. Although Melilotus indicus extract (MIE) has recently been shown to suppress growth of several tumor cell lines, information regarding its antitumor mechanism is completely unknown. Here, we report the mechanism underlying the effects of MIE on human hepatocellular carcinoma cells, specifically HepG2, and SNU-182 cells. Methanolic MIE impaired the proliferation, and induced cell death in both HepG2 and SNU-182 cells but not in normal hepatic L-02 cells. Mechanistically, flow cytometric analysis revealed that MIE induces apoptosis in HepG2, and SNU-182 cells. However, MIE-induced apoptosis were not affected by a pan caspase inhibitor z-VAD-fmk as well as MIE did not stimulate caspase activation. Furthermore we found that MIE-induced apoptosis could be attributed to a mechanism involving mitochondria-mediated pathways evidenced by decrease in the mitochondrial membrane potential (ΔΨm), increase in the Bax/Bcl-2 ratio, and translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. Suppression in AIF expression by siRNA reduced MIE-induced apoptosis which suggested the dependency of MIE on AIF to induce apoptosis in hepatocellular carcinoma cells. To the best of our knowledge this is the first report elucidating the anticancer mechanism of MIE. Our findings suggested that MIE might be a good extract for developing anticancer drugs for human hepatocellular carcinoma treatment.
RESUMO
Anti-cancer tyrosine kinase inhibitors (TKIs) are effective in many types of cancers including non-small cell lung cancer, while appearance of TKI-resistant tumors suggests a need for the development of their potentiation strategies. We have previously shown that a methoxyflavanone derivative from the Asian medicinal herb Perilla frutescens (Perilla-derived methoxyflavanone; PDMF) shows a prominent anti-tumor activity against A549 human lung adenocarcinoma. Here we show that PDMF and anti-cancer TKIs (nilotinib, bosutinib, dasatinib, and ponatinib) synergistically suppress proliferation of A549 cells. Flow cytometric analysis indicated that co-stimulation with nilotinib (4 µM) and PDMF induced G2/M cell cycle arrest in low PDMF doses (10-50 µM), whereas this combination triggered de novo G1 arrest in higher PDMF dosages (50-125 µM). We also found that co-administration with nilotinib and PDMF significantly suppressed in vivo tumorigenicity of A549 cells in athymic nude mice.
RESUMO
Perilla frutescens is an Asian dietary herb consumed as an essential seasoning in Japanese cuisine as well as used for a Chinese medicine. Here, we report that a newly found methoxyflavanone derivative from P. frutescens (Perilla-derived methoxyflavanone, PDMF; 8-hydroxy-5,7-dimethoxyflavanone) shows carcinostatic activity on human lung adenocarcinoma, A549. We found that treatment with PDMF significantly inhibited cell proliferation and decreased viability through induction of G2/M cell cycle arrest and apoptosis. The PDMF stimulation induces phosphorylation of tumor suppressor p53 on Ser15, and increases its protein amount in conjunction with up-regulation of downstream cyclin-dependent kinase inhibitor p21Cip1/Waf1 and proapoptotic caspases, caspase-9 and caspase-3. We also found that small interfering RNA knockdown of p53 completely abolished the PDMF-induced G2/M cell cycle arrest, and substantially abrogated its proapoptotic potency. These results suggest that PDMF represents a useful tumor-preventive phytochemical that triggers p53-driven G2/M cell cycle arrest and apoptosis.
RESUMO
Perilla frutescens is a dietary leafy herb consumed as a traditional Japanese condiment as well as used for Chinese medicine with anti-inflammatory activity. Here we report a hitherto-unrecognized P. frutescens phytochemical that potently suppresses IgE-mediated type I hypersensitivity reactions. Structural analysis reveals that the purified anti-allergic compound (Perilla-derived methoxyflavanone, PDMF) is identified as 8-hydroxy-5,7-dimethoxyflavanone. PDMF significantly inhibits IgE-mediated histamine release from RBL-2H3 rat basophilic leukemia cells as compared with those seen in known P. frutescens-derived anti-inflammatory polyphenols. We also show that oral administration of PDMF not only suppresses passive cutaneous anaphylaxis, but also prevents allergic rhinitis-like nasal symptoms in a murine model of Japanese cedar pollinosis. Mechanistically, PDMF negatively regulates Akt phosphorylation and intracellular Ca2+ influx, both of which are essential for mast cell secretory granule translocation and its exocytosis upon high-affinity IgE receptor (FcεRI) cross-linking. These results represent PDMF as a new potent anti-allergic phytochemical useful for prevention of IgE-driven hypersensitivity reactions.
Assuntos
Antialérgicos/administração & dosagem , Medicamentos de Ervas Chinesas/administração & dosagem , Flavanonas/administração & dosagem , Hipersensibilidade Imediata/prevenção & controle , Imunoglobulina E/imunologia , Perilla frutescens/química , Plantas Medicinais/química , Rinite Alérgica Sazonal/tratamento farmacológico , Animais , Antialérgicos/química , Antialérgicos/isolamento & purificação , Antialérgicos/farmacologia , Linhagem Celular Tumoral , Cryptomeria/imunologia , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Feminino , Flavanonas/química , Flavanonas/isolamento & purificação , Flavanonas/farmacologia , Liberação de Histamina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Ratos , Receptores de IgE/imunologia , Rinite Alérgica Sazonal/prevenção & controleRESUMO
The high prevalence of Japanese cedar pollinosis in Japan is associated with a negative impact on the quality of life of patients, as well as significant loss of productivity among the workforce in early spring, thus representing a serious social problem. Furthermore, the prevalence is increasing, and has risen by more than 10% in this decade. Cry j 1 and Cry j 2 were identified as the major allergens in Japanese cedar pollen (JCP), and in 2004, the existence of other major and minor allergens were revealed by a combination of two-dimensional electrophoresis and immunoblotting analysis. Allergenome analysis identified a chitinase, a lipid transfer protein, a serine protease, and an aspartic protease as novel IgE-reactive allergens in patients with JCP allergy. Thaumatin-like protein (Cry j 3) was shown to be homologous to Jun a 3, a major allergen from mountain cedar pollen. Isoflavone reductase-like protein was also characterized in a study of a JCP cDNA library. The characterization of component allergens is required to clarify the sensitizer or cross-reactive elicitor allergens for component-resolved diagnosis (CRD). Increasing evidence from numerous clinical trials indicates that CRD can be used to design effective allergen-specific immunotherapy. In this review, we summarize the eight characterized JCP allergens and discuss the impact of CRD and characterization of novel allergens on allergen-specific immunotherapy.
Assuntos
Alérgenos/imunologia , Cryptomeria/efeitos adversos , Pólen/imunologia , Rinite Alérgica Sazonal/diagnóstico , Rinite Alérgica Sazonal/imunologia , Antígenos de Plantas/imunologia , Reações Cruzadas/imunologia , Dessensibilização Imunológica , Humanos , Imunização , Imunoglobulina E/imunologia , Japão , Medicina de Precisão , Prevalência , Rinite Alérgica Sazonal/epidemiologia , Rinite Alérgica Sazonal/terapiaRESUMO
We applied Chrysanthemum flower oil (CFO) to a hyperuricemia model by feeding rats a hyperuricemia-inducing diet (HID) and investigated its effect on serum uric acid (SUA) levels and its mode of action. CFO is the oily fraction that contains polyphenols derived from chrysanthemum flowers. Oral administration of CFO to HID-fed rats significantly decreased their SUA levels. It also inhibited xanthine oxidase activities in the liver and increased urine uric acid levels. The effects of CFO on the renal gene expressions that accompanied the induction of hyperuricemia were comprehensively confirmed by DNA microarray analysis. The analysis showed up-regulation of those genes for uric acid excretion by CFO administration. These results suggest that CFO suppresses the increase in SUA levels via two mechanisms: suppression of uric acid production by inhibition of xanthine oxidase in the liver and acceleration of its excretion by up-regulation of uric acid transporter genes in the kidney.
Assuntos
Chrysanthemum/química , Flores/química , Hiperuricemia/tratamento farmacológico , Hiperuricemia/genética , Análise de Sequência com Séries de Oligonucleotídeos , Óleos de Plantas/farmacologia , Animais , Bovinos , Hiperuricemia/sangue , Hiperuricemia/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Óleos de Plantas/administração & dosagem , Óleos de Plantas/uso terapêutico , Ratos , Ácido Úrico/sangue , Xantina Oxidase/metabolismoRESUMO
Lactic acid bacteria (LAB) represent an attractive delivery vehicle for oral allergy vaccine because of their safety as a food microorganism as well as their potent adjuvant activity triggering anti-allergic immune response. Here, we report the generation of recombinant LAB expressing a major Japanese cedar pollen allergen Cry j 1 (Cry j 1-LAB), and their prophylactic effect in vivo. To facilitate heterologous expression, the codon usage in the Cry j 1 gene was optimized for the host LAB strain Lactobacillus plantarum by the recursive PCR-based exhaustive site-directed mutagenesis. Use of the codon-optimized Cry j 1 cDNA and a lactate dehydrogenase gene fusion system led to a successful production of recombinant Cry j 1 in L. plantarum NCL21. We also found that oral vaccination with the Cry j 1-LAB suppressed allergen-specific IgE response and nasal symptoms in a murine model of cedar pollinosis.
Assuntos
Alérgenos , Cryptomeria/genética , Cryptomeria/imunologia , Lactobacillus/genética , Pólen , Rinite Alérgica Sazonal/prevenção & controle , Administração Oral , Alérgenos/genética , Alérgenos/imunologia , Animais , Antialérgicos/imunologia , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Imunoglobulina E/sangue , Camundongos , Modelos Animais , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Pólen/genética , Pólen/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologiaRESUMO
We tested the effect of oral administration of fermented sake lees with lactic acid bacteria (FESLAB) on a murine model of allergic rhinitis upon immunization and nasal sensitization with ovalbumin (OVA). We used Lactobacillus paracasei NPSRIk-4 (isolated from sake lees), and L. brevis NPSRIv-8 (from fermented milk) as starter strains to produce the FESLAB. Oral FESLAB administration resulted in the development of significantly fewer sneezing symptoms than those seen in sham control animals given sterile water. We also found that FESLAB suppressed the allergen-induced degranulation of RBL2H3 rat basophilic leukemia cells.
Assuntos
Basófilos/citologia , Degranulação Celular , Fermentação , Imunoglobulina E/imunologia , Lactobacillus/metabolismo , Rinite/prevenção & controle , Vinho , Animais , Basófilos/imunologia , Linhagem Celular Tumoral , Suplementos Nutricionais , Feminino , Hipersensibilidade/complicações , Camundongos , Ratos , Rinite/complicações , Rinite/imunologiaRESUMO
The fungal strain Mortierella alliacea YN-15 is a promising industrial producer of polyunsaturated fatty acids (PUFAs), in particular arachidonic acid. In order to more efficiently produce PUFAs, the metabolism of an externally supplied plant oil, α-linolenic acid (ALA)-rich linseed triacylglycerol (TAG), was examined, and time-dependent changes in the composition of its lipid and fatty acid metabolites were traced. Addition of linseed TAG to growing cultures resulted in a transient increase in extracellular 1,2-diacylglycerol (DAG), and even more so of 1,3-DAG, in the mycelia. This was followed by a decrease in both DAGs and an increase in TAG. Eicosapentaenoic acid (EPA), a desaturated and elongated product of ALA, accumulated to a greater extent in cellular phospholipids than in neutral lipids. Moreover, the addition of ALA in free fatty acid form to the culture led to the generation of EPA. However, EPA production was not observed upon addition of ALA-rich 1,2- or 1,3-DAG, indicating that fatty acids released from exogenous lipids were used for resynthesis of mycelial TAG. These results suggested that TAG might be hydrolyzed by extracellular lipases, whereas its synthesis might be catalyzed by intracellular enzymes. Appropriate regulation of such enzymes might be an effective strategy to enhance PUFA production under plant oil supplementation.
Assuntos
Ácidos Graxos Insaturados/biossíntese , Ácidos Graxos Insaturados/metabolismo , Metabolismo dos Lipídeos/fisiologia , Mortierella/metabolismo , Calibragem , Técnicas de Cultura de Células/normas , Células Cultivadas , Meios de Cultura/farmacologia , Diglicerídeos/análise , Diglicerídeos/metabolismo , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Fungos/metabolismo , Óleo de Semente do Linho/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Mortierella/efeitos dos fármacos , Mortierella/crescimento & desenvolvimento , Óleos de Plantas/química , Óleos de Plantas/isolamento & purificação , Óleos de Plantas/metabolismo , Fatores de TempoRESUMO
Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal rhinitis and conjunctivitis in Japan, and an understanding of its full allergen repertoire is prerequisite for the development of future molecular diagnostics and immunotherapeutic strategies. Here we report the identification of a new C. japonica pollen IgE-binding antigen (CJP-8) homologous to lipid transfer proteins (LTPs), a class of plant cross-reactive allergens found in foods, latex, and pollen grains. The cjp-8 cDNA encodes a 165-amino acid polypeptide possessing the conserved eight cysteines characteristic of plant LTP family members. Escherichia coli-expressed recombinant CJP-8 (r-CJP-8) reacted with IgE antibody from Japanese cedar pollinosis patients at a 37.5% frequency (6/16).
Assuntos
Antígenos de Plantas/imunologia , Proteínas de Transporte/imunologia , Cryptomeria/imunologia , Imunoglobulina E/imunologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Sequência de Aminoácidos , Antígenos de Plantas/genética , Clonagem Molecular , Cryptomeria/genética , Cisteína/imunologia , Humanos , Dados de Sequência Molecular , Proteínas de Plantas/genética , Rinite Alérgica Sazonal/imunologia , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens. OBJECTIVES: We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family. METHODS: We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis. RESULTS: cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63. CONCLUSIONS: We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis.
Assuntos
Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/imunologia , Cryptomeria/imunologia , Pólen/imunologia , Sequência de Aminoácidos , Anticorpos/imunologia , Antígenos de Plantas/biossíntese , Antígenos de Plantas/metabolismo , Ácido Aspártico Proteases/antagonistas & inibidores , Ácido Aspártico Proteases/metabolismo , Biocatálise/efeitos dos fármacos , Western Blotting , Domínio Catalítico/genética , Clonagem Molecular , Cryptomeria/genética , Precursores Enzimáticos/metabolismo , Hemoglobinas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Dados de Sequência Molecular , Filogenia , Pólen/química , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rinite Alérgica Sazonal/imunologia , Homologia de Sequência de AminoácidosRESUMO
In marine aquaculture, rotifers and Artemia nauplii employed as larval fish feed are often nutritionally enriched with forage such as yeast and algal cells supplemented with polyunsaturated fatty acids and xanthophylls, which are required for normal growth and a high survival ratio of fish larvae. To reduce the enrichment steps, we propose here the use of a marine thraustochytrid strain, Schizochytrium sp. KH105, producing docosahexaenoic acid, docosapentaenoic acid, canthaxanthin, and astaxanthin. The KH105 cells prepared by cultivation under optimized conditions were successfully incorporated by rotifers and Artemia nauplii. The contents of docosahexaenoic acid surpassed the levels required in feed for fish larvae, and the enriched Artemia showed an increased body length. The results demonstrate that we have developed an improved method of increasing the dietary value of larval fish feed.
Assuntos
Ração Animal , Suplementos Nutricionais , Células Eucarióticas/metabolismo , Ácidos Graxos Insaturados/administração & dosagem , Peixes/crescimento & desenvolvimento , Peixes/microbiologia , Xantofilas/administração & dosagem , Animais , Ácidos Graxos Insaturados/metabolismo , Larva/crescimento & desenvolvimento , Xantofilas/metabolismoRESUMO
BACKGROUND: Japanese cedar pollinosis is a severe allergic disease in Japan. The most effective means of decreasing allergic inflammation reactions is still avoidance of the aeroallergen. Recently, a novel air purification system using positively and negatively charged cluster ions was developed to create comfortable living environments. We aimed to assess the ability of existing technology to lower allergenicity of Japanese cedar pollen. METHODS: A Japanese cedar pollen extract was nebulized from the top of a cylindrical container with 2 or 4 ion-generating devices. The extract in a mist was passed through the space filled with or without plasma cluster ions for 90 s, and the ion-treated or nontreated extract was then collected in a Petri dish at the bottom of the container. RESULTS: The ion-exposed extract was significantly diminished in its reactivities to anti-Cry j 1 or anti-Cry j 2 antiserum and to human allergic sera IgE on ELISA. SDS-PAGE analysis revealed that ion exposure induced protein degradation in the pollen extract. Similarly, the ion treatment impaired about 80% of the binding to pooled sera IgE from patients allergic to Japanese cedar pollen on ELISA inhibition. Furthermore, intracutaneous and conjunctival reaction tests showed a remarkable diminution in the allergenicity of the ion-irradiated extract. CONCLUSION: Ion irradiation resulted in a remarkable decrease in in vitro and in vivo allergenicities of atomized Japanese cedar pollen extracts.
Assuntos
Ionização do Ar , Alérgenos/imunologia , Cedrus/imunologia , Pólen/imunologia , Poluição do Ar em Ambientes Fechados/prevenção & controle , Humanos , Extratos Vegetais/imunologiaRESUMO
BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is one of the most prevalent sources of the allergens that elicit rhinitis and conjunctivitis. Only Cry j 1 and Cry j 2 have been well characterized as the major allergens of this pollen. OBJECTIVES: The aims of this study were to complete the repertoire of C. japonica pollen allergens, to investigate their variability with respect to IgE-reactive patterns and to identify the isoforms of Cry j 1 and Cry j 2 by proteome analysis. METHODS: Proteins in C. japonica pollen separated on two-dimensional (2-D) polyacrylamide gel electrophoresis were immunodetected with IgE in sera of 40 subjects allergic to C. japonica pollen. Mass fingerprinting was used to elucidate the diversity of the major allergens. RESULTS: 2-D immunolabeling with individual patients' sera showed the distinguishable IgE-binding patterns inlaid with 4-87 spots from a total of 131 IgE-binding protein spots. At least 12 Cry j 1 (27.5-75% of IgE-binding frequency) and 3 Cry j 2 (32.5-40%) isoforms were localized. In total, 31 spots were found to be more reactive than the highest IgE-reactive isoform of Cry j 2. CONCLUSIONS: The proteomics approaches showed great interindividual variation of IgE-binding patterns to C. japonica proteins and contributed to the repertoire of numerous C. japonica allergens other than Cry j 1 and Cry j 2. Protein microsequencing demonstrated more complicated multiplicity in Cry j 1 than previously known and new isoforms in Cry j 2.
Assuntos
Alérgenos/imunologia , Alérgenos/isolamento & purificação , Cryptomeria/imunologia , Imunoglobulina E/imunologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Alérgenos/química , Sequência de Aminoácidos , Antígenos de Plantas , Cryptomeria/química , Eletroforese em Gel Bidimensional , Feminino , Humanos , Immunoblotting , Imunoglobulina E/química , Masculino , Dados de Sequência Molecular , Proteínas de Plantas/química , Pólen/química , Isoformas de Proteínas , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A major part of the palmitic acid (C16:0) generated by fatty acid synthase is converted into stearic acid (C18:0) via carbon chain elongation. Here, we describe the cloning and expression of a rat hepatic enzyme, rELO2, responsible for the elongation of C16:0, presumably at the condensing reaction. Heterologous expression experiments in a yeast, Saccharomyces cerevisiae, demonstrated the elongation activity of rELO2 on C16:0 and to a lesser extent, C18:0 and fatty acids with low desaturation degree. This was distinct from that rELO1, a rat homolog of HELO1, which preferably catalyzed the elongation of mono- and polyunsaturated fatty acids of C16-C20. The Northern analysis showed that the expression of rELO2, but not rELO1, in hepatocytes was activated by the cycles of fasting and refeeding rats on a fat-free diet. Under these conditions, the rELO1 was expressed constitutively in various tissues but the rELO2 transcripts were detected predominantly in liver.