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.

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.

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.

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.

Direct Comparison of Manganese Detoxification/Efflux Proteins and Molecular Characterization of ZnT10 Protein as a Manganese Transporter.

Manganese homeostasis involves coordinated regulation of specific proteins involved in manganese influx and efflux. However, the proteins that are involved in detoxification/efflux have not been completely resolved nor has the basis by which they select their metal substrate. Here, we compared six proteins, which were reported to be involved in manganese detoxification/efflux, by evaluating their ability to reduce manganese toxicity in chicken DT40 cells, finding that human ZnT10 (hZnT10) was the most significant contributor. A domain swapping and substitution analysis between hZnT10 and the zinc-specific transporter hZnT1 showed that residue Asn(43), which corresponds to the His residue constituting the potential intramembranous zinc coordination site in other ZnT transporters, is necessary to impart hZnT10's unique manganese mobilization activity; residues Cys(52) and Leu(242) in transmembrane domains II and V play a subtler role in controlling the metal specificity of hZnT10. Interestingly, the His → Asn reversion mutant in hZnT1 conferred manganese transport activity and loss of zinc transport activity. These results provide important information about manganese detoxification/efflux mechanisms in vertebrate cells as well as the molecular characterization of hZnT10 as a manganese transporter.

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.

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.

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.

Properties of Zip4 accumulation during zinc deficiency and its usefulness to evaluate zinc status: a study of the effects of zinc deficiency during lactation.

Systemic and cellular zinc homeostasis is elaborately controlled by ZIP and ZnT zinc transporters. Therefore, detailed characterization of their expression properties is of importance. Of these transporter proteins, Zip4 functions as the primarily important transporter to control systemic zinc homeostasis because of its indispensable function of zinc absorption in the small intestine. In this study, we closely investigated Zip4 protein accumulation in the rat small intestine in response to zinc status using an anti-Zip4 monoclonal antibody that we generated and contrasted this with the zinc-responsive activity of the membrane-bound alkaline phosphatase (ALP). We found that Zip4 accumulation is more rapid in response to zinc deficiency than previously thought. Accumulation increased in the jejunum as early as 1 day following a zinc-deficient diet. In the small intestine, Zip4 protein expression was higher in the jejunum than in the duodenum and was accompanied by reduction of ALP activity, suggesting that the jejunum can become zinc deficient more easily. Furthermore, by monitoring Zip4 accumulation levels and ALP activity in the duodenum and jejunum, we reasserted that zinc deficiency during lactation may transiently alter plasma glucose levels in the offspring in a sex-specific manner, without affecting homeostatic control of zinc metabolism. This confirms that zinc nutrition during lactation is extremely important for the health of the offspring. These results reveal that rapid Zip4 accumulation provides a significant conceptual advance in understanding the molecular basis of systemic zinc homeostatic control, and that properties of Zip4 protein accumulation are useful to evaluate zinc status closely.

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.

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.

Soybean extracts increase cell surface ZIP4 abundance and cellular zinc levels: a potential novel strategy to enhance zinc absorption by ZIP4 targeting.

Dietary zinc deficiency puts human health at risk, so we explored strategies for enhancing zinc absorption. In the small intestine, the zinc transporter ZIP4 functions as an essential component of zinc absorption. Overexpression of ZIP4 protein increases zinc uptake and thereby cellular zinc levels, suggesting that food components with the ability to increase ZIP4 could potentially enhance zinc absorption via the intestine. In the present study, we used mouse Hepa cells, which regulate mouse Zip4 (mZip4) in a manner indistinguishable from that in intestinal enterocytes, to screen for suitable food components that can increase the abundance of ZIP4. Using this ZIP4-targeting strategy, two such soybean extracts were identified that were specifically able to decrease mZip4 endocytosis in response to zinc. These soybean extracts also effectively increased the abundance of apically localized mZip4 in transfected polarized Caco2 and Madin-Darby canine kidney cells and, moreover, two apically localized mZip4 acrodermatitis enteropathica mutants. Soybean components were purified from one extract and soyasaponin Bb was identified as an active component that increased both mZip4 protein abundance and zinc levels in Hepa cells. Finally, we confirmed that soyasaponin Bb is capable of enhancing cell surface endogenous human ZIP4 in human cells. Our results suggest that ZIP4 targeting may represent a new strategy to improve zinc absorption in humans.

Validation of Patient Selection for Endovascular Aneurysm Repair or Open Repair of Abdominal Aortic Aneurysm ­ Single-Center Study.

BACKGROUND: To validate the criteria for endovascular aneurysm repair (EVAR) or open repair of abdominal aortic aneurysm (AAA) at Nagoya University Hospital, the results of both treatments were retrospectively compared. METHODS AND RESULTS: Patient selection for EVAR was primarily based on suitable anatomy, minimum age 75 years, and significant comorbidity. From June 2007 to April 2014, 426 patients were treated via EVAR (EVAR group) and 346 patients were treated with open surgery (OS group). The mortality rates of the EVAR and OS groups were not significantly different (0.2% vs. 1.1%; P=0.33). Patient age, operation time, amount of bleeding, and duration of hospital stay were significantly lower in the EVAR group compared with the OS group. The incidence of comorbidity was higher in the EVAR group compared with the OS group. The incidence of early postoperative complications was significantly higher in the OS group, whereas the incidence of late complications for both groups was similar. The cumulative aneurysm-related survival rates were similar (98.9% vs. 98.5%; P=0.767). The cumulative survival rates and reintervention-free rates at 5 years were lower for the EVAR group (76% vs. 89%, P=0.019; 81% vs. 89%, P=0.046). CONCLUSIONS: Patient selection practices and criteria for EVAR and open repair at Nagoya University Hospital are generally acceptable.

Fate of ischemic limbs in patients with Buerger's disease based on our 30-year experience: does smoking have a definitive impact on the late loss of limbs?

PURPOSE: We herein review the long-term results of our series of critical ischemic limbs caused by Buerger's disease. METHODS: A retrospective review of 103 patients with critical limb ischemia who were diagnosed with Buerger's disease by Shionoya's criteria between 1980 and 2010. RESULTS: The age of onset was 38.0 ± 9.7 years (mean ± SD). The median follow-up was 97 months. Thirty-three patients had pain at rest, and 65 patients presented with ischemic ulcers in their toes and/or fingers. Gangrene was present in five patients. Sixteen patients achieved remission with medical therapy and smoking cessation. Sixty-six patients underwent sympathectomy. Bypass to the crural arteries was performed in 23 limbs, with assisted primary patency rates of 67.0 and 45.6 % at five and 10 years. Graft failure caused major amputation in two out of 10 smokers, but no limbs were lost among the 12 ex-smokers. Therapeutic angiogenesis using cell transplantation led to improvement in three ex-smokers; however, amputation was unavoidable in the one smoker who was treated. The limb salvage rate at 20 years was 90 % in ex-smokers and 69 % in smokers, which was not significantly different. CONCLUSIONS: This study demonstrated no significant difference in the limb salvage rate between ex-smokers and smokers. Our results do not support any advantageous effect of smoking cessation on the long-term remission in patients with Buerger's disease.

Regulation of tissue factor pathway inhibitor-2 (TFPI-2) expression by lysine-specific demethylase 1 and 2 (LSD1 and LSD2).

Tissue factor pathway inhibitor-2 (TFPI-2) is a major inhibitor of extracellular matrix degradation. Decreases in TFPI-2 contribute to malignant tumor cell production, and TFPI-2 is a presumed tumor suppressor. TFPI-2 gene transcription is regulated by two epigenetic mechanisms: DNA methylation of the promoter and K4 methylation of histone 3 (H3). Lysine-specific demethylase 1 (LSD1) and LSD2 demethylate H3K4me2/1. LSD1 has been implicated in TFPI-2 regulation through both epigenetic mechanisms, but the involvement of LSD2 remains unknown. We prepared a monoclonal anti-LSD2 antibody that clearly distinguishes LSD2 from LSD1. Knockdown of LSD1 or LSD2 by siRNAs increased TFPI-2 protein and mRNA. Simultaneous knockdown of both LSD1 and LSD2 showed additive effects. Bisulfite sequencing revealed that CpG sites in the TFPI-2 promoter region were unmethylated. These results indicate that LSD2 also contributes to TFPI-2 regulation through histone modification, and that further studies of the involvement of LSD2 in tumor malignancy are warranted.

Possible involvement of Notch signaling in the pathogenesis of Buerger's disease.

PURPOSE: Under pathological conditions, the Notch signal pathway is involved in the inflammatory process in arteriosclerosis, atherosclerosis and angiogenesis under ischemic conditions. The purpose of this study was to observe whether or not Buerger's disease is associated with Notch signal activation. METHODS: All the patients were diagnosed between 1980 and 2009 at Nagoya University Hospital. Twenty-two specimens from 12 patients with Buerger's disease (TAO) and 13 specimens from nine patients with arteriosclerosis obliterans (ASO) were analyzed by immunohistochemistry for Notch1, Jagged-1 (a Notch ligand) and Hes-1 (a Notch 1 target transcription factor). RESULTS: Notch1 and Jagged-1 were highly expressed in the endothelium in the new vasa vasorum and in the smooth muscle cells in the media of specimens from both groups. These Notch-related proteins were also remarkably expressed in inflammatory cells in the intima of specimens from TAO patients. Fewer inflammatory cells expressed Notch-related proteins in atheromatous plaques (Notch1 (%): 8.4 ± 0.76 versus 1.3 ± 0.43, P < 0.001; Jagged-1(%): 9.3 ± 1.1 versus 5.2 ± 1.1, P = 0.03). Indeed, Hes-1, which is a transcription factor downstream of Notch1, was remarkably expressed in the endothelium of new capillary vessels and inflammatory cells in TAO patients. Notch1-positive mononuclear cells were also seen in the thrombus in samples from the TAO group. CONCLUSIONS: Our findings are the first demonstration that Notch signal activation in inflammatory cells may be involved in the pathophysiological mechanism underlying Buerger's disease.

Development of an immuno-affinity column for ochratoxin analysis using an organic solvent-tolerant monoclonal antibody.

Two kinds of monoclonal antibodies (MoAbs), OCA-10A and OCA-1B, were prepared based on their specificity to ochratoxin A (OTA) and ochratoxin B (OTB) and on their tolerance to 40% methanol. In an indirect competitive enzyme-linked immunosorbent assay, the half maximal inhibitory concentration (IC(50)) value of OCA-10A was 27ng/mL for OTA and 17ng/mL for OTB, and that of OCA-1B was 28ng/mL for OTA and 13ng/mL for OTB. Immuno-affinity columns (IACs) using these MoAbs were prepared with agarose gel beads. The IAC with OCA-1B showed a NaCl-dependent binding ability to OTA and OTB, while interestingly, the IAC with OCA-10A bound to them without NaCl. The IAC with OCA-10A showed a high methanol tolerance when compared with existing IACs, as expected from the high methanol tolerance of OCA-10A itself. Such tolerance was maintained for the application of the cocoa extract with 70% methanol and the wheat extract with 60% acetonitrile, while the tolerance was slightly altered by interference from the cocoa extract. Examinations with organic solvents at higher concentrations than the allowable level in existing IACs showed that OTA and OTB spiked with wheat, cocoa and red wine could be purified with high recovery. The newly developed IAC is expected to show sufficient clean-up ability for food analyses.

Establishment of a monitoring system to detect inhibition of mRNA processing.

A number of proteins complete mRNA processing in the nucleus, thus, inhibitor of mRNA processing is worth finding to analyze the mechanism of mRNA maturation in detail. Here, we established a monitoring system for mRNA processing using a test compound, spliceostatin A (SSA), which inhibits mRNA splicing. This system should serve to facilitate the discovery of novel compounds from natural resources that inhibit mRNA processing.