Expression analysis of gibberellin-regulated protein in peach by reverse transcription-quantitative PCR.

Gibberellin-regulated protein (GRP) is a fruit severe allergen. The amounts of GRP expression normalized against actin in peach were determined by reverse transcription-quantitative PCR (RT-qPCR). The results were consistent with those determined by enzyme-linked immunosorbent assay (ELISA). The GRP expression was more evident in flesh than peel and increased rapidly in the maturing period. This approach is applicable to estimate the amount of GRP in other plants.

Intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) of the major salivary glands: an assessment of the optimal number and combination of b-values.

Purpose: To examine the optimal number and combination of b-values in intravoxel incoherent motion (IVIM) diffusionweighted imaging (DWI) of the major salivary glands. Material and methods: IVIM-DWI was performed on 10 healthy volunteers using 13 b-values (low b-values: 0-100 s/mm2; high b-values: 200-1000 s/mm2). The IVIM parameters and apparent diffusion coefficient of the bilateral major salivary glands were calculated using 13 b-values and were considered the standard values. We sequentially reduced the number of b-values to 10, 8, 6, and 5. The parameters in each combination were calculated. The standard values were compared with the parameters from each reduced b-value in IVIM-DWI. The Wilcoxon signed-rank test was used to determine whether there were any differences between the parameters in each combination. Bonferroni correction was conducted for multiple comparisons. Results: There were no significant differences between the standard values and parameters from the 2 combinations of 6 b-values. However, significant differences were observed between the standard values and parameters from some combinations of only 2 low or only 2 high b-values. Conclusions: IVIM-DWI of the major salivary glands could be performed using a minimum of 6 b-values. However, they should contain 3 low and 3 high b-values.

Generalized allergic reaction in response to exercise due to strawberry gibberellin-regulated protein: a case report.

BACKGROUND: The Rosaceae family includes fruits, such as peach, apple, Japanese apricot, cherry (Prunoideae subfamily), and strawberry (Rosoideae subfamily). The allergens responsible for Rosaceae fruit allergies have been reported to include Bet v 1 and profilin, which mainly cause oral symptoms, and lipid transfer protein (LTP). Recently, gibberellin-regulated protein (GRP) has been identified as an allergen that induces generalized symptoms in peach-, orange-, and plum-related allergies. Most patients with food allergies induced by GRP show allergic symptoms accompanied by cofactors, such as exercise or drugs. To date, there are very few reports of generalized symptoms induced by strawberry. CASE PRESENTATION: We evaluated the reactivity of strawberry GRP in a 15-year-old boy who was confirmed to have generalized symptoms induced by strawberry with exercise using an oral food challenge test (OFCT). The patient's serum exhibited a strong positive reaction to strawberry GRP but not to peach GRP or peach LTP. The patient's basophils reacted to strawberry and peach GRP but not to peach LTP. CONCLUSIONS: Strawberry GRP may be a causative component for strawberry with exercise-induced generalized symptoms in this patient. This is the first study to investigate the role of GRP in strawberry with cofactor-induced allergic symptoms. Further epidemiological and clinical researches are necessary to improve diagnostic and therapeutic approaches for patients with strawberry allergy.

Determination of Severe Peach Allergens, Gibberellin-Regulated Protein, and Lipid Transfer Protein, Using Monoclonal Antibodies.

In this study, monoclonal antibodies against two major fruit allergens-gibberellin-regulated protein (GRP) and lipid transfer protein (LTP)-were established. Sandwich enzyme-linked immunosorbent assays (ELISAs) for the quantification of peach GRP and LTP were constructed using these antibodies. Both ELISAs reacted with the respective antigens when heated at 100ºC for 20 min, but not when reduced with sodium sulfite, indicating that GRP and LTP are heat-stable, while disulfide bonds play an important role in their native steric structures. GRP and LTP in peaches and peach-containing foods were quantified by these ELISAs. In both cases, there were few differences among peach cultivars normally available on the market; however, concentrations were higher when the peach was ripe. GRP was localized in the pulp of the peach, while LTP was present in the peel. They could be quantified in peach-containing beverages, as well as in dried and canned peaches. GRP in Japanese apricots could also be determined using this ELISA, as its amino acid sequence is the same as that of peach GRP. Then, high concentrations of GRP were detected in umeboshi, a traditional Japanese pickled apricot. Peach leaves were found to have a high LTP content, accordingly, LTP was also observed in lotions containing peach leaf extract. The ability to quantitatively detect GRP and LTP in this study will, therefore, contribute to the improvement of component-resolved diagnoses and quality of life in patients allergic to peaches.

Investigation of the sensitization rate for gibberellin-regulated protein in patients with Japanese cedar pollinosis.

BACKGROUND: Pollen-food allergy syndrome (PFAS) usually manifests as an itching sensation in the mouth and throat immediately after eating fresh fruits and vegetables. However, some patients with PFAS experience systemic symptoms including anaphylaxis. In Europe, cypress gibberellin-regulated protein (GRP) has been noted to cause allergenicity and exhibit cross-reactivity with peach GRP. Japanese cedar (Cryptomeria japonica), classified in the cypress family, is the primary causative substance among all environmental allergens in Japan. However, studies on the prevalence of GRP sensitization in patients with cedar pollinosis are lacking. OBJECTIVE: This study examined the prevalence of GRP sensitization in patients with Japanese cedar pollinosis. METHODS: We enrolled 52 patients who had requested sublingual immunotherapy treatment with mild-to-severe rhinitis during spring, and had a JCP-specific immunoglobulin E (IgE) levels of >0.7 UA/mL. Peach GRP was purified using affinity chromatography with a monoclonal antibody column. Specific IgE levels to peach GRP were measured using an enzyme-linked immunosorbent assay. Samples exhibiting absorbance at 450 nm of over mean plus three standard deviations of the negative control value were defined as positive. Sera from three patients with severe peach allergy were used as positive controls. RESULTS: Eleven sera from 52 patients with JCP-induced allergic rhinitis were positive for peach GRP. CONCLUSION: Twenty percent of patients with cedar pollinosis were sensitized to peach GRP. Well-powered studies are needed to clarify whether these patients are at an increased risk for systemic symptoms or whether they primarily demonstrate only localized symptoms.

Investigation of the sensitization rate for gibberellin-regulated protein in patients with Japanese cedar pollinosis

Background Pollen-food allergy syndrome (PFAS) usually manifests as an itching sensation in the mouth and throat immediately after eating fresh fruits and vegetables. However, some patients with PFAS experience systemic symptoms including anaphylaxis. In Europe, cypress gibberellin-regulated protein (GRP) has been noted to cause allergenicity and exhibit cross-reactivity with peach GRP. Japanese cedar (Cryptomeria japonica), classified in the cypress family, is the primary causative substance among all environmental allergens in Japan. However, studies on the prevalence of GRP sensitization in patients with cedar pollinosis are lacking.Objective This study examined the prevalence of GRP sensitization in patients with Japanese cedar pollinosis.Methods We enrolled 52 patients who had requested sublingual immunotherapy treatment with mild-to-severe rhinitis during spring, and had a JCP-specific immunoglobulin E (IgE) levels of >0.7 UA/mL. Peach GRP was purified using affinity chromatography with a monoclonal antibody column. Specific IgE levels to peach GRP were measured using an enzyme-linked immunosorbent assay. Samples exhibiting absorbance at 450 nm of over mean plus three standard deviations of the negative control value were defined as positive. Sera from three patients with severe peach allergy were used as positive controls.Results Eleven sera from 52 patients with JCP-induced allergic rhinitis were positive for peach GRP.Conclusion Twenty percent of patients with cedar pollinosis were sensitized to peach GRP. Well-powered studies are needed to clarify whether these patients are at an increased risk for systemic symptoms or whether they primarily demonstrate only localized symptoms (AU)

Simultaneous Detection of Six Different Types of Pesticides by an Immunosensor Based on Surface Plasmon Resonance.

Six pesticides, azoxystrobin, boscalid, chlorfenapyr, imazalil, isoxathion, and nitenpyram, were simultaneously detected by using a surface plasmon resonance (SPR) immunosensor. The working ranges were 3.5 - 19 ng/mL for azoxystrobin, 4.5 - 50 ng/mL for boscalid, 2.5 - 25 ng/mL for chlorfenapyr, 5.5 - 50 ng/mL for imazalil, 3.5 - 50 ng/mL for isoxathion, and 8.5 - 110 ng/mL for nitenpyram. They showed adequate recovery results in tomato samples: 104 - 116% for azoxystrobin, 94 - 101% for boscalid, 90 - 112% for chlorfenapyr, 96 - 106% for imazalil, 107 - 119% for isoxathion, and 104 - 109% for nitenpyram. The correlation coefficient with liquid chromatography (HPLC or LC-MS/MS) using vegetable samples also agreed well: 0.91 - 0.99 as R2 without strong bias, except for nitenpyram for which the SPR immunosensor sensitivity was too low. The SPR immunosensor will have high applicability for pesticide residue analyses in vegetable samples.

Development of a direct competitive enzyme-linked immunosorbent assay for determination of the fungicide mepanipyrim and its metabolite.

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for determination of anilinopyrimidine fungicide mepanipyrim in vegetables. Two derivatives of mepanipyrim and mepanipyrim propanol type metabolite which carried carboxy acid were synthesized and conjugated with keyhole limpet hemocyanin. BALB/c mice were immunized to prepare anti-mepanipyrim monoclonal antibodies (MoAbs) by obtained conjugates. The dc-ELISAs based on the prepared MoAbs, MPP107 and MPP204, showed working ranges between 0.12 and 1.8 ng/mL with mepanipyrim for MPP107, 0.12 and 2.4 ng/mL with mepanipyrim for MPP204, and 0.2 ng/mL and 5.7 ng/mL with the mepanipyrim propanol type for MPP204. The dc-ELISAs showed the sufficient sensitivity to determine the mepanipyrim residues for the MRLs of 1-15 mg/kg among the majority of vegetables and fruits in Japan. Recovery and/or correlation results from HPLC suggested that the dc-ELISAs would be applicable to the residue analysis of mepanipyrim and its propanol type in vegetables.

Development of Enzyme-Linked Immunosorbent Assay for Analysis of Total Aflatoxins Based on Monoclonal Antibody Reactive with Aflatoxins B1, B2, G1 and G2.

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for the determination of total amount of aflatoxin B1, B2, G1 and G2 (AFB1, AFB2, AFG1 and AFG2), using a mouse monoclonal antibody that shows similar reactivity to each of these AFs. The working range of the developed dc-ELISA was 50-230 pg/mL for AFB1, 50-270 pg/mL for AFB2, 60-390 pg/mL for AFG1 and 65-700 pg/mL for AFG2. The recovery of AFs from spiked roasted peanuts was 98％. Further, when 4 samples actually contaminated with AFB1, AFB2, AFG1 and AFG2 were examined, the results of dc-ELISA were highly correlated with the values assigned by the Food Analysis Performance Assessment Scheme. The developed dc-ELISA appears to be suitable for the determination of total AFs at concentrations around the maximum permitted level (10 µg/kg for all foods) in Japan.

Evaluation of the roles of the cytosolic N-terminus and His-rich loop of ZNT proteins using ZNT2 and ZNT3 chimeric mutants.

The physiological roles of Zn transporter (ZNT) proteins are being increasingly recognized, and three dimensional structures of ZNT bacterial homologs have facilitated our understanding of their biochemical characteristics at the molecular level. However, the biological role of the unique structural features of vertebrate ZNTs, which are absent in their bacterial homologues, is not completely understood. These ZNT sequences include a cytosolic His-rich loop between transmembrane helices IV and V and the cytosolic N-terminus. This study investigated the contribution of these features to zinc transport by ZNT proteins. The importance of the His residues in the cytosolic His-rich loop was investigated using ZNT2 Ala substitution and deletion mutants. The presence of His residues was not essential for zinc transport, even though they possibly participate in modulation of zinc transport activity. Furthermore, we determined the role of the N-terminus by characterizing ZNT2 and ZNT3 domain-swapped and deletion mutants. Unexpectedly, the N-terminus was also not essential for zinc transport by ZNT2 and the domain-swapped ZNT2 mutant, in which the cytosolic His-rich loop was substituted with that of ZNT3. These results provide molecular insights into understanding the roles of the cytosolic parts of ZNT2, ZNT3, and probably other members of their subgroup.

Development of an Immunosensor Based on Surface Plasmon Resonance for Simultaneous Residue Analysis of Three Pesticides -Boscalid, Clothianidin, and Nitenpyram- in Vegetables.

A simultaneous immunosensor based on surface plasmon resonance (SPR) was developed for determination of 3 pesticides -boscalid, clothianidin and nitenpyram- instead of the direct competitive enzyme-linked immunosorbent assays (dcELISAs) widely used as individual determination methods. Carboxy groups that introduced compounds to their pesticides were designed, and conjugates of them and bovine serum albumin were immobilized onto separate channels of the same sensor chip. When a mixture of 3 monoclonal antibodies reacted to each pesticide, and 3 pesticides were injected into the SPR immunosensor, each channel showed specific reactivity at 15 - 93 ng mL-1 for boscalid, 6.7 - 27 ng mL-1 for clothianidin, and 7.3 - 62 ng mL-1 for nitenpyram. Recovery tests using vegetables spiked with a mixture of 3 pesticides showed good results: 75 - 90%, 88 - 104%, and 72 - 105%, respectively, with a high correlation to results of the dcELISAs. The SPR immunosensor would be useful for the determination of pesticide residues in vegetables.

The impact of inflow treatment for claudicants with both aortoiliac and femoropopliteal occlusive disease.

PURPOSE: This study evaluated the early and long-term results of the sole use of endovascular treatment in the treatment of inflow lesions in claudicants with both aortoiliac and femoropopliteal (FP) lesions. METHODS: A retrospective study that included 100 limbs in 73 patients was performed. The patency rates of aortoiliac artery stents, the continued clinical improvement rates, the risk factors for persistent disabling claudication after inflow revascularization, and the rates of freedom from additional FP procedures were examined. RESULT: After inflow revascularization, almost complete relief from intermittent claudication was seen in 79 % of the limbs, while 21 % of the limbs continued to suffer from disabling claudication. A multivariate analysis showed that a run-off score of ≥7 was an independent predictor for persistent disabling claudication after aortoiliac revascularization [hazard ratio (HR) 5.11, 95 % confidence interval (CI) 1.34-19.45; P = 0.02]. The primary patency rates at 1, 3, 5, and 6 years were 96, 96, 96 and 89 %, respectively. The secondary patency rate at 6 years was 100 %. The continued clinical improvement rates at 1, 3, 5, and 6 years were 81, 78, 78 and 72 %, respectively. The rates of freedom from additional FP procedures at 1, 3, 5, and 6 years were 97, 90, 90, and 90 %, respectively. CONCLUSIONS: Aortoiliac endovascular revascularization is effective treating claudicants with both aortoiliac and femoropopliteal lesions. Furthermore, a run-off score of ≥7 appears to be a potential predictor for persistent disabling claudication.

The isoflavone fraction from soybean presents mRNA maturation inhibition activity.

Recent findings indicate that mRNA splicing inhibitors can be potential anticancer candidates. We have previously established a screening system which monitors mRNA processing in order to identify mRNA processing inhibitors. Among a number of dietary resources, isoflavone fractions showed an inhibitory effect of mRNA processing. These findings demonstrate that a variety of dietary sources have an impact on mRNA biogenesis.

Efficacy of periodically rotated overlapping parallel lines with enhanced reconstruction (PROPELLER) for shoulder magnetic resonance (MR) imaging.

OBJECTIVES: To elucidate the utility of PROPELLER for motion artefact reduction on shoulder MRI and to examine the influence of streak artefacts on diagnosis of clinical images. METHODS: 15 healthy volunteers and 48 patients underwent shoulder MRI with/without PROPELLER (coronal oblique proton density-fast spin echo [PD-FSE], sagittal oblique T2-FSE). In a volunteer study, all sequences were performed in both static and exercise-loaded conditions. Two radiologists graded artefacts and delineation of various anatomical structures in the volunteer study and motion and streak artefacts in the clinical study. Mean scores were compared between sequences with/without PROPELLER. In the clinical study, mean scores of motion artefacts were compared with mean scores of streak artefacts. Wilcoxon signed-rank test was used for all comparisons. RESULTS: In both studies, PROPELLER significantly reduced motion artefacts (P<0.05). In the volunteer study, it significantly improved delineations in sagittal oblique images in the exercise-loaded condition (P<0.05). In the clinical study, streak artefacts appeared dominantly on images with PROPELLER (P<0.05), but influenced diagnosis to a lesser extent than motion artefacts. CONCLUSION: PROPELLER can reduce motion artefacts in shoulder MRI. While it does cause streak artefacts, it affects diagnosis to a lesser extent.

Efficacy of the radial acquisition regime (RADAR) for acquiring head and neck MR images.

OBJECTIVE: To investigate the efficacy of the radial acquisition regime (RADAR) for acquiring head and neck MR images. METHODS: 15 healthy volunteers underwent imaging with 4 sequences [fast spin echo T2 weighted imaging (FSE-T2WI), RADAR T2 weighted imaging (RADAR-T2WI), single-shot echo planar imaging diffusion-weighted imaging (SS-EPI-DWI) and RADAR diffusion-weighted imaging (RADAR-DWI)]. Both standard images and images during periodic mouth motion were acquired. Two radiologists scored the overall image artefacts and detectability of several anatomical structures without knowledge of sequence type. For each sequence, image distortion was quantitatively compared by the anteroposterior to right-left ratio of several anatomical structures. The mean scores of artefacts and distortion of several anatomical structures were compared using the multiple comparison test. The detectabilities were compared using the Wilcoxon signed-rank test. RESULTS: Regardless of mouth motion, RADAR-T2WI was significantly superior to FSE-T2WI in artefacts and oral-area detectability (p < 0.01), and RADAR-DWI was significantly superior to SS-EPI-DWI in terms of artefacts (p < 0.01). In terms of image distortion, RADAR-DWI was significantly superior to SS-EPI-DWI (p < 0.01). CONCLUSION: RADAR-T2WI could replace FSE-T2WI as a conventional T2WI protocol for the head and neck. For the RADAR-DWI sequence, validation studies are needed. Advances in knowledge: RADAR-T2WI was superior to FSE-T2WI with regard to artefacts and detectability, and RADAR-DWI was superior in terms of artefacts compared with SS-EPI-DWI.

Novel mutations in SLC30A2 involved in the pathogenesis of transient neonatal zinc deficiency.

BACKGROUND: Infants are vulnerable to zinc deficiency. Thus, abnormally low breast milk zinc levels cause transient neonatal zinc deficiency (TNZD) in breast-fed infants. TNZD has been considered to be rare because of a paucity of citations in the published literature. However, recent studies of affected mothers identified four missense mutations in the solute carrier family 30 member 2 gene (SLC30A2), which encodes the zinc transporter, ZnT2. METHODS: Genetic analyses of SLC30A2/ZnT2 in three Japanese mothers secreting low-zinc milk (whose infants developed TNZD) were performed. The effects of identified mutations were examined in a cell-based assay. Furthermore, 31 single-nucleotide polymorphisms (SNPs) in SLC30A2/ZnT2 were evaluated for their potential involvement in low-zinc levels in milk. RESULTS: Each mother had a different novel heterozygous mutation in SLC30A2/ZnT2. One mutation reduced splicing efficiency of the SLC30A2/ZnT2 transcript, and all ZnT2 mutants were defective in zinc transport and were unstable in cells. Moreover, four SNPs caused a significant loss of zinc-transport activity, similar to that in disease-causing ZnT2 mutants. CONCLUSION: Our results indicate that many SLC30A2/ZnT2 mutations cause or potentially cause TNZD. Genetic information concerning TNZD pathogenesis is limited, and our results suggest that the TNZD frequency may be higher than previously thought.

Development of a Surface Plasmon Resonance-Based Immunosensor for Detection of 10 Major O-Antigens on Shiga Toxin-Producing Escherichia coli, with a Gel Displacement Technique To Remove Bound Bacteria.

A surface plasmon resonance-based immunosensor (SPR-immunosensor) was developed for the detection of Shiga toxin-producing Escherichia coli (STEC) belonging to the O-antigen groups O26, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The polyclonal antibodies (PoAbs) generated against each of the STEC O-antigen types in rabbits were purified and were immobilized on the sensor chip at 0.5 mg/mL. The limit of detection for STEC O157 by the SPR-immunosensor was found to be 6.3 × 10(4) cells for 75 s. Each of the examined 10 O-antigens on the STECs was detected by the corresponding PoAb with almost no reaction to the other PoAbs. The detected STECs were sufficiently removed from the PoAbs using gelatin or agarose gel without deactivation of the PoAbs, enabling repeatable use of the sensor chip. The developed SPR-immunosensor can be applied for the detection of multiple STEC O-antigens. Furthermore, the new antigen removal technique using the gel displacement approach can be utilized with various immunosensors to improve the detection of pathogens in clinical and public health settings.

Postoperative Outcomes of Hybrid Repair in the Treatment of Aortic Arch Aneurysms.

BACKGROUND: The advent of thoracic endovascular aneurysm repair (TEVAR) has bought about a tremendous revolution in the treatment strategy for aortic arch aneurysms. We reviewed our experience using TEVAR with the hybrid approach in the treatment of aortic arch aneurysms to evaluate its feasibility, safety, and effectiveness. METHODS: Between October 2008 and July 2014, 61 consecutive patients (51 men; mean age 75.8 ± 7.7 years; range, 43-85 years) underwent elective treatment for aortic arch aneurysms with the hybrid approach. The 61 patients were separated into 2 groups. Thirty-five patients underwent total debranching TEVAR for zone 0 (debranching TEVAR group), 26 patients underwent long elephant trunk (ET) followed by secondary retrograde TEVAR (ET group). Preoperative, perioperative, and follow-up data were collected retrospectively in the database. RESULTS: The technical success rate was 100%. The paraplegia rates in total debranching TEVAR and long ET TEVAR and were 2.9% and 3.8%, respectively. The stroke rates in total debranching TEVAR and long ET TEVAR were 11.4% and 7.7%, respectively. The overall 30-day mortality and in-hospital mortality rates for all 61 patients were 0% and 3.4% (n = 2; both were in the total debranching TEVAR group), respectively. There were no perioperative type 1 or 3 endoleaks that required secondary intervention. The mean hospital stay was 15.8 days. The median follow-up was 309 ± 303 days. No aneurysm-related deaths occurred during follow-up. CONCLUSIONS: The hybrid approach can be safely performed with good technical success and good midterm results. In future, new alternative devices for aortic arch pathologies, such as a branched stent graft that eliminates extra-anatomic bypass, should be developed.