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Trinta a 60% das alergias alimentares em adolescentes e adultos são associadas à alergia ao pólen e estão incluídas na síndrome pólen-frutas (SPF). Esta síndrome é caracterizada por sintomas alérgicos provocados pela ingestão de frutas ou vegetais frescos em pacientes com rinite/rinoconjuntivite alérgica sazonal. Os autores apresentam o caso clínico de um adolescente que após sensibilização primária através de pólens de gramíneas e oliveira manifestou posteriormente, por reatividade cruzada, sintomas de alergia oral com a ingestão de frutas frescas. Após recurso ao método de diagnóstico Immuno-Solid-Phase Allergen Chip (ISAC) verificou-se que as profilinas foram as proteínas responsáveis pela reatividade cruzada.
In adolescents and adults, 30% to 60% of food allergies are associated with pollen allergy and are included in the pollen-food syndrome (PFS). This syndrome is characterized by allergic symptoms elicited by the ingestion of fresh fruits or vegetables in patients with seasonal allergic rhinitis/rhinoconjunctivitis. The authors present the clinical case of an adolescent who, after primary sensitization to grass and olive tree pollens, subsequently manifested by cross-reactivity symptoms of oral allergy with the ingestion of fresh fruit. After diagnostic workup with the Immuno- Solid-phase Allergen Chip (ISAC) assay, profilins were identified as the proteins responsible for the cross-reactivity.
Subject(s)
Humans , Male , Adolescent , Rhinitis, Allergic, Seasonal , Skin TestsABSTRACT
OBJECTIVE@#To investigate the expression of profilin 2 (PFN2) in gastric cancer and assess its potential value as a novel prognostic indicator and a therapeutic target.@*METHODS@#We collected gastric cancer and paired adjacent tissues from 100 patients for immunohistochemical detection of PFN2 expression. According to the expression level of PFN2, the patients were divided into two groups with high (46 cases) and low (48 cases) PNF2 expression in cancer tissues, and also into two groups with high (26 cases) and low (49 cases) PNF2 expression in adjacent tissues. Chi-square test, Spearman correlation and KaplanMeier survival analysis were used to analyze the relationship between PFN2 protein expression level and the patients' clinical parameters. We also tested the effects of PFN2 knockdown and overexpression on the proliferation and migration of MKN-45 cells using Transwell assay and CCK-8 assay.@*RESULTS@#The expression of PFN2 protein was significantly higher in gastric cancer tissues than in adjacent tissues (P < 0.01). PFN2 expression was positively correlated with M-stage of gastric cancer and VEGFR expression in the tumor tissues (P < 0.01). A high expression of PFN2 protein was significantly correlated with a poor prognosis of gastric cancer patients (P < 0.01), and was an independent predictor of the prognosis of gastric cancer. In MKN-45 cells, the cells overexpressing PFN2 showed significantly stronger proliferation and migration abilities than those with PFN2 knockdown (P < 0.001).@*CONCLUSION@#PFN2 protein is highly expressed in gastric cancer tissues to promote the proliferation and migration of the tumor cells. PFN2 may serve as a potential diagnostic marker, a prognostic indicator and a therapeutic target for gastric cancer.
Subject(s)
Humans , Cell Proliferation , Profilins/metabolism , Prognosis , Stomach Neoplasms/pathology , Survival AnalysisABSTRACT
Objective:To explore the effect of miR-3607-3p on the malignant phenotype of cervical cancer cells and related mechanisms.Methods:The OncoLnc bioinformatics website was used to analyze the relationship between the expression of miR-3607-3p and the prognosis of cervical cancer patients. Real time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-3607-3p in human normal cervical epithelial cells (H8) and cervical cancer cell lines (Hela, HCC94, SiHa, C33A). SiHa cells transfected with negative control nucleotide sequence in vitro were defined as negative control (NC) group, and SiHa cells transfected with miR-3607-3p mimicked sequence was defined as miR-3607-3p group. qRT-PCR was used to detect the expression changes of miR-3607-3p in SiHa cells. Cell counting kit-8 (CCK-8) method and scratch test were used to detect the malignant biological behavior changes of SiHa cells. qRT-PCR and Western blot were used to detect the expression changes of profilin 2 (PFN2) gene, and the dual luciferase reporter gene experiment was used to detect the targeting relationship between miR-3607-3p and PFN2. Results:The survival time of patients with high expression of miR-3607-3p was significantly higher than that of patients with low expression of miR-3607-3p ( P<0.01). Compared with H8 cells, the expression of miR-3607-3p was abnormally low in human cervical cancer cell lines ( P<0.05), and the expression of miR-3607-3p was the lowest in the SiHa cell line ( P<0.01). The expression of miR-3607-3p in the NC group and miR-3607-3p group was (1.04±0.31) and (9.28±1.76), respectively. The expression level of miR-3607-3p in SiHa cells transfected with miR-3607-3p mimic sequence was significantly higher than that in NC group, and the proliferation activity and scratch healing rate were significantly lower than that in NC group (all P<0.01). Dual-luciferase reporter gene assay confirmed that miR-3607-3p directly targeted and binded to PFN2 ( P<0.01). qRT-PCR and Western blot results showed that the expression of PFN2 mRNA and protein in miR-3607-3p group was lower than that in NC group; Western blot results showed the expression of proliferating proteins CDK3 and CDK2 in miR-3607-3p group was lower than that in NC group (all P<0.05), and the expression of epithelial phenotype related proteins Claudin-1 and ZO-1 in miR-3607-3p group was higher than that in NC group (all P<0.05). Conclusions:miR-3607-3p is positively correlated with the survival of cervical cancer patients. Up-regulating the expression of miR-3607-3p can inhibit the proliferation and migration of cervical cancer SiHa cells. The mechanism may be related to the targeted inhibition of PFN2.
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BACKGROUND Profilin proteins (PRFs) are small (1215 kD) actin-binding protein, which play a significant role in cytoskeleton dynamics and plant development via regulating actin polymerization. Profilins have been well documented in Arabidopsis, Zea mays L. as well as Phaseolus vulgaris, however no such fully characterization of rice (Oryza sativa L.) profilin gene family has been reported thus far. RESULTS In the present study, a comprehensive genome-wide analysis of rice PRF genes was completed and three members were identified. OsPRF1 and OsPRF2 shared 98.5% similarity (6 nucleotide divergence), but the deduced amino acid sequences of OsPRF1 and OsPRF2 are fully identical. In contrast, the OsPRF3 presents relatively lower similarity with OsPRF1 and OsPRF2. Phylogenetic analysis also support that OsPRF1 has a closer relationship with OsPRF2. Expression pattern analysis revealed the differential expression of OsPRFs in tissues of mature plant, which suggested the potential spatial functional specificity for rice profilin genes. Subcellular localization analysis revealed the OsPRFs were localized in cytoplasm and nucleus and all of them could bind actin monomers. Furthermore, abiotic stresses and hormones treatments assay indicated that the three OsPRF genes could be differentially regulated, suggesting that OsPRF genes might participate in different stress processes in rice. CONCLUSIONS Taken together, our study provides a comprehensive analysis of the OsPRF gene family and will provide a basis for further studies on their roles in rice development and in response to abiotic stresses
Subject(s)
Plant Proteins/genetics , Oryza/genetics , Genome, Plant , Profilins/geneticsABSTRACT
Objective:To obtain recombinant Profilin of silkworm,identify its immunogenicity,predict its B cell epitopes and construct the evolutionary trees. Methods: The nucleotide sequence of Profilin was acquired from NCBI,synthesized it and cloned it into pET-28 vector. Then,the recombinant plasimids were transformed to E. coli BL21. After induced by IPTG,recombinant protein was purified by Affinity chromatography. Furtherly,its allergenicity was identified by Western blot,the potential B cell epitopes was analyzed through DNAStar and build the evolutionary trees by MEGA5. 05. Results: The recombinant protein of Profilin was successfully expressed and purified by affinity chromatography. Besides,the protein contains a high IgE-binding activity with IgE existing in serum of patients allergic to silkworm. Conclusion: The recombinant proflilin has IgE-binding activity, and it is meaningful for fundamental research and specific diagnosis studies of allergic diseases caused by silkworm.
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Toxoplasma gondii is a kind of worldwide obligate intracellular parasitic protozoa,which can live in almost all nucleated cells besides red blood cells and cause zoonosis.Toxoplasma gondii is mainly transmitted by the fecal-oral route;damaged skin,mucous membrane and the placenta are the secondary pathway.After infection,the parasite need to invade host cells and formate parasitophorous vacuole to complete reproductive cycle,while many studies have shown that actin binding protein profilin of Toxoplasma gondii(TgPRF) play important roles in regulating actin polymerization and invading host cells.At the same time,TgPRF can be used as a dominant antigen to induce host immune response,especially innate immunity through Toll like receptor (TLR).In this paper,the structural characteristics of TgPRF and its roles in the invasion of the host and inducing the host immune responses were reviewed,which would provide profound understanding of Toxoplasma gondii pathogenic mechanism and a reference for immune prevention.
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Objective:To discuss the prokaryotic expression system and purification conditions of Toxoplasma gondii profilin-like protein (TgPRF), and to provide basis for the study on anti-tumor immuno-adjuvant. Methods:The coding region of TgPRF gene was amplified with a pair of specific primers which were designed according to the cDNA of tachyzoites of Toxoplasma gondii RH strain.The PCR products were cloned into the pET-28a (+ ) vector after double enzyme digestion. The recombinant pET28a (+ )-TgPRF plasmid was transformed into E.coli DH5αcells.The positive clones were selected by the double restrictive enzyme digestion and sequencing.The correct pET28a (+)-TgPRF plasmid was transformed into E.coli BL21 (DE3)and induced for 4 h by IPTG.The expression of recombinant TgPRF protein was analyzed by SDS-PAGE method;the expression of recombinant protein His-profilin was detected by Western blotting method.Results:The length of product of PCR was 492 bp.The recombinant plasmid pET28a-TgPRF was confirmed by double restriction enzyme digestion and sequencing.The SDS-PAGE results showed that the target protein was expressed in E.coli BL21 (DE3)in bacteria supernatant.The purified TgPRF protein was obtained by Ni-NTA agarose gel column chromatography with the purity>90%.The Western blotting results revealed that the recombinant TgPRF protein could be recognized by Anti-His antibody.Conclusion: The recombinant plasmid pET28a-TgPRF is successfully constructed,and the TgPRF protein is obtained with the soluble prokaryotic expression.
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Objective To observe the splenocytes immune response elicited by different concentrations of recombinant Toxo?plasma gondii profilin(rTgPRF)through the nasal route,and determine the optimal dose. Methods Fifty female BALB/c mice were randomly divided into 5 groups. The immunized groups were intranasally administered with 10,20,30μg or 40μg of rTgPRF that was separately dissolved in 20μl of phosphate?buffered saline(PBS)on days 0,14,and 21 respectively,while the control mice were given PBS solution instead. Two weeks after the last immunization,all mice were killed. Under asceptic conditions,the spleens from the immunized mice were dissected,and then the splenocyte proliferative responses in vitro were tested by CCK?8 kit. The levels of IFN?γ,IL?2,IL?4 and IL?10 of splenocyte culture supernatant were detected by ELISA. Re?sults Compared to the control group,the splenocytes from the 30μg and 40μg groups exhibited a significantly higher prolifer?ative response to rTgPRF(P<0.05),and SI from the 30μg rTgPRF group was higher than that from the 40μg group(P<0.05). The levels of IFN?γin all the immunized groups(P<0.05)and IL?2 in the 20,30μg and 40μg groups were significant?ly stronger than those in the control(P<0.05),and the 30μg group presented the highest concentrations of IFN?γ(P<0.01) and IL?2(P<0.01). There were no statistical differencesa mong the groups in the levels of IL?4 and IL?10. Conclusions The intranasal immunization with rTgPRF can induce the splenocyteproliferation and Th1?type mediated immunity. The best immu?nized dose is confirmed as 30μg.
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Objective:To explore the mechanism of Profilin-1 in regulating eNOS/NO pathway and its role in the development of myocardial hypertrophy.Methods: Spontaneously hypertensive rats (SHR) aged 5 weeks were injected with different adenovirus vectors to induce Profilin-1 expression knockdown (SHR-I) or over express (SHR-H) or to use as control (SHR-C). All these treatment were compared with Wistar-Kyoto rats (SKY) treated with control adenovirus vectors (WKY-C). The same injection was executed at the sixth week during the experiment of 12 weeks. After experiment, the left ventricular weight-to-heart weight ratio (LVW/HW) and left ventricular long axis (LVLA) were measured. Meanwhile, NO contents in blood and myocardium, Profilin-1, eNOS and Caveolin-3 mRNA and protein levels and phosphorylated eNOS (P-eNOS) protein level in myocardium were determined. Results:Compared with WKY-C group, the SHR-C group was statistically higher in LVW/HW (0.79±0.03), LVLA (11.82±0.58 mm) and Profilin-1 mRNA and protein level (P<0.05), but lower in NO content [(18.63±6.23) μmol/L] in blood and [(2.71±0.17) μmol/L] in myocardium), eNOS activity and Caveolin-3 expression (P<0.05). The over expressing Profilin-1 led SHR-H group to a higher value of LVW/HW [(0.93±0.03) mm and LVLA (14.17±0.69) mm] in comparison with SHR-C group (P<0.05), and to a lower value of NO content (in myocardium), eNOS activity and Caveolin-3 expression (P<0.05); however, this phenomenon was reversed by the knockdown Profilin-1 expression (SHR-I group).Conclusions:Profilin-1 expression, being negative in regulating Caveolin-3 expression and eNOS/NO pathway activity, promotes the development of myocardial hypertrophy which can be reversed by Profilin-1 silencing.
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Objective: To explore the mechanism of Profilin-1 in regulating eNOS/NO pathway and its role in the development of myocardial hypertrophy. Methods: Spontaneously hypertensive rats (SHR) aged 5 weeks were injected with different adenovirus vectors to induce Profilin-1 expression knockdown (SHR-I) or over express (SHR-H) or to use as control (SHR-C). All these treatment were compared with Wistar-Kyoto rats (SKY) treated with control adenovirus vectors (WKY-C). The same injection was executed at the sixth week during the experiment of 12 weeks. After experiment, the left ventricular weight-to-heart weight ratio (LVW/HW) and left ventricular long axis (LVLA) were measured. Meanwhile, NO contents in blood and myocardium, Profilin-1, eNOS and Caveolin-3 mRNA and protein levels and phosphorylated eNOS (P-eNOS) protein level in myocardium were determined. Results: Compared with WKY-C group, the SHR-C group was statistically higher in LVW/HW (0.79±0.03), LVLA (11.82±0.58 mm) and Profilin-1 mRNA and protein level (P<0.05), but lower in NO content [(18.63±6.23) μmol/L] in blood and [(2.71±0.17) μmol/L] in myocardium), eNOS activity and Caveolin-3 expression (P<0.05). The over expressing Profilin-1 led SHR-H group to a higher value of LVW/HW [(0.93±0.03) mm and LVLA (14.17±0.69) mm] in comparison with SHR-C group (P<0.05), and to a lower value of NO content (in myocardium), eNOS activity and Caveolin-3 expression (P<0.05); however, this phenomenon was reversed by the knockdown Profilin-1 expression (SHR-I group). Conclusions: Profilin-1 expression, being negative in regulating Caveolin-3 expression and eNOS/NO pathway activity, promotes the development of myocardial hypertrophy which can be reversed by Profilin-1 silencing.
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OBJECTIVE@#To explore the mechanism of Profilin-1 in regulating eNOS/NO pathway and its role in the development of myocardial hypertrophy.@*METHODS@#Spontaneously hypertensive rats (SHR) aged 5 weeks were injected with different adenovirus vectors to induce Profilin-1 expression knockdown (SHR-I) or over express (SHR-H) or to use as control (SHR-C). All these treatment were compared with Wistar-Kyoto rats (SKY) treated with control adenovirus vectors (WKY-C). The same injection was executed at the sixth week during the experiment of 12 weeks. After experiment, the left ventricular weight-to-heart weight ratio (LVW/HW) and left ventricular long axis (LVLA) were measured. Meanwhile, NO contents in blood and myocardium, Profilin-1, eNOS and Caveolin-3 mRNA and protein levels and phosphorylated eNOS (P-eNOS) protein level in myocardium were determined.@*RESULTS@#Compared with WKY-C group, the SHR-C group was statistically higher in LVW/HW (0.79±0.03), LVLA (11.82±0.58 mm) and Profilin-1 mRNA and protein level (P<0.05), but lower in NO content [(18.63±6.23) μmol/L] in blood and [(2.71±0.17) μmol/L] in myocardium), eNOS activity and Caveolin-3 expression (P<0.05). The over expressing Profilin-1 led SHR-H group to a higher value of LVW/HW [(0.93±0.03) mm and LVLA (14.17±0.69) mm] in comparison with SHR-C group (P<0.05), and to a lower value of NO content (in myocardium), eNOS activity and Caveolin-3 expression (P<0.05); however, this phenomenon was reversed by the knockdown Profilin-1 expression (SHR-I group).@*CONCLUSIONS@#Profilin-1 expression, being negative in regulating Caveolin-3 expression and eNOS/NO pathway activity, promotes the development of myocardial hypertrophy which can be reversed by Profilin-1 silencing.
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Objective To observe the change of profilin-1 during the aging of rats' aorta and the anti-aging effect of grape procyanidins (GPC).Methods Young male Wistar rats (9 weeks) and middle rats (12 months) were randomly divided into GPC treatment and control groups respectively.We quantified arterial aging changes through morphological methods.Thoracic aortas were stained with hematoxylin eosin.Serum levels of 3-nitrotyrosine (3-NT),malondialdehyde (MDA),superoxide dismutase (SOD) and nitric oxide (NO) were tested using enzyme-linked immunosorbent assay (ELISA).Western blotting was performed to measure the protein expression of inducible nitric oxide synthase (iNOS) and profilin-1.Results Compared with the young male Wistar rats group,aging change in the aortic morphology of middle rat group were shown by hematoxylin-eosin staining (HE) staining:media thickness (MT) increased [(98.3±0.5)μm vs.(83.1±1.0)μm,P＜0.05],luminal internal diameter (LD) decreased [(15.5 ±0.2) μm vs.(18.2±0.5,P＜0.05)μm,P＜ 0.05],(MT/LD)％ increased [(6.4±0.1) ％ vs.(4.6±0.1)％,P＜0.05],the protein expressions of profilin-1 and iNOS both increased [profilin-1:(1.58 ± 0.09) vs.(1.29 ± 0.04),iNOS:(1.02±0.12) vs.(0.75±0.02),both P＜0.05],levels of NO and SOD in serum decreased [NO:(6.3±0.2)μmol/L vs.(8.4±0.2) μmol/L,SOD:(172.3±1.6) U/ml vs.(189.1±1.5) U/ml,both P＜0.05],the levels of MDA and 3-NT increased[MDA:(11.3±0.3) μmol/L vs.(9.4 ±0.1) μmol/L,3 NT:(40.2±0.3) nmol/L vs.(35.6±0.5) nmol/L,both P＜0.05)].After treatment with GPC,compared with the control group,MT decreased,LD increased and MT/LD (％)decreased in the middle GPC treatment group.The protein expressions of profilin-1 had no changes before and after treatment with GPC both in young and middle groups.After the GPC treatment in middle group,compared with the middle control group,iNOS expression decreased,serum levels of NO and SOD increased,and the levels of MDA and 3-NT decreased significantly (all P ＜0.05).Conclusions Profilin-1 is related with age-related changes in rat aorta.Profilin-1 participates in vascular aging through iNOS induced oxidative stress.GPC may defer vascular aging by inhibiting vascular oxidative stress.
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Many extracellular signaling molecules including hormones, growth factors, neurotransmitters and immunoglobulins elicit intracellular responses by activating phosphatidylinositol-specific phospholipase C (PI-PLC) upon binding to their cell surface receptors. Activated PLC catalyses the hydrolysis of Phosphotidylinositol 4,5- bisphosphate (PIP2) to generate DAG and IP3 , which act as signaling molecules that control various cellular processes. Exploring the mechanism of regulation of PLC activity may lead to understanding various signaling events that regulate cell growth and differentiation. One of the dramatic effects of profilin is inhibition of PIP2 hydrolysis by PLC- γ in eukaryotic cells. In the present study, the effect of profilin on Phosphotidylinositol specific phospholipase C (PIPLC) purified from Bacillus thuringiensis (Bt) was examined. Assay of PI-PLC activity indicated that Bovine profilin activated the hydrolysis of phosphotidylinositol (PI) by BtPI-PLC in a concentration dependent manner under in vitro conditions. A 250 % increase in activity was noted in the presence of profilin but not in presence of phosphoprofilin. In the presence of profilin more proteins are observed in the soluble fraction. In conclusion it can be stated that that profilin activates bacterial PLC activity towards PI hydrolysis.
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Food allergy is an adverse food reaction as a result of immune mechanisms. In a sensitized individual, food allergens activate mast cells and basophils by binding with IgE present on the cell surface, resulting in the release of chemical mediators and various cytokines to cause various clinical symptoms of food allergy. Sensitization to food allergens can occur in the gastrointestinal tract (class 1 food allergy) or as a consequence of cross reactivity to structurally homologous inhalant allergens (class 2 food allergy). The class 1 food allergens are water-soluble glycoproteins with 10-70 kD size that are resistant to heat, acid and enzymes. On the other hand, the class 2 food allergens are highly unstable and degraded by heat or enzymatic digestion. Much progress has been made in identifying and isolating food allergen. Recently cDNAs for many proteins have been isolated and recombinant proteins have been generated. These techniques make it easier to characterize each responsible food allergens. Plant food allergens are classified into families and superfamilies by their structural and functional properties. The most of plant food allergens are the cupin and prolamin superfamilies and the protein families of the plant defense system. The cupin superfamily includes allergenic seed storage proteins of 7s globulin (vicilin) and 11s globulin (legumin). 2s albumin seed storage proteins, the nonspecific lipid transfer proteins, and the cereal alpha-amylase and protease inhibitors belong to the prolamin superfamily. Profilins, heveins, and nonspecific lipid transfer proteins are present in a variety of pollens, nuts, seeds, fruits, and vegetables. These are considered as panallergens, causing a significant degree of IgE-mediated cross-reactivity. Detailed informations about the character of food allergens can be used to develop more sophisticated diagnostic methods and treatment modalities in the near future. Further knowledge of food allergens is also useful to assess the allergenicity of novel protein of genetically mo.
Subject(s)
Humans , Allergens , alpha-Amylases , Basophils , Edible Grain , Classification , Cytokines , Digestion , DNA, Complementary , Food Hypersensitivity , Fruit , Gastrointestinal Tract , Glycoproteins , Hand , Hot Temperature , Immunoglobulin E , Mast Cells , Nuts , Plants , Pollen , Profilins , Protease Inhibitors , Recombinant Proteins , Seed Storage Proteins , VegetablesABSTRACT
Profilin-Ⅰis a small actin monomer-binding protein involved in regulating actin polymerization.It plays an important role in cell proliferation,differention,motility and signals transduction in different cell types including endothelial cells and vascular smooth muscle cells.This article recapitulates the wealth of information on structure,function of profilin-Ⅰand its potential role in the genesis and development of cardiovascular diseases.
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Objective:To clone and express panallergen profilin from the pollen of coco(Cocos nucifera Linnaeus).Methods:RT-PCR and RACE methods were applied to clone the full-length panallergen genes from coco pollen and the sequence was analyzed.The specific primers were designed.The ORF of profilin of coco pollen was amplified with RT-PCR and cloned into the expression vector pET 28a.Expression of the recombinant coco pollen profilin was carried out in E.coli BL21(DE3) and the purification of the recombinant protein was performed via affinity chromatography with Ni2+ coupled to sepharose.IgE reactivity to recombinant coco pollen profilin was investigated by immunoblot.Results:The complete sequence of coco pollen profilin was cloned.The sequence was 608 bp and included an open reading frame(396 bp) coding for 131 amino acids.Sequence analysis showed that the deduced protein was an acidic protein with an estimated molecular mass of 14.19 kD and a pI of 4.61.The GeneBank accession number of the clones was EF173598.After overexpressed in E.coli BL21(DE3),the recombinant protein was purified through affinity chromatography with Ni2+ coupled to sepharose.Immunoassay showed that the recombinant allergen has good IgE binding capacity.Conclusion:The profilin of coco pollen is expressed successfully in BL21(DE3),which will be used as a base for further study on coco pollen related allergy.