The Intron 4 Polymorphism in the Calcium-Sensing Receptor Gene in Diabetes Mellitus and its Chronic Complications, Diabetic Nephropathy and Non-Diabetic Renal Disease

Background/Aims: Calcium-Sensing Receptor (CaSR) significantly affects calcium-phosphate metabolism in kidneys, and it is implicated in the pathogenesis of diabetes mellitus (DM) due to its expression in pancreatic F-cells. The role of CaSR as one of the players in pathogenesis of chronic kidney disease (CKD) has been speculated. Methods: 158 Type 2 diabetic patients divided into three groups according to occurrence and type of kidney complications, 66 nondiabetic patients CKD, and 93 healthy subjects were enrolled into the study to analyze the role of two CaSR polymorphisms (in the codon 990 and in the intron 4) in ethiopathogenesis of DM and CKD. The Type 2 diabetic groups consisted of 48 patients without any kidney abnormalities, 58 patients with diabetic nephropathy (DN), and 52 patients with nondiabetic renal disease (NDRD). The distribution of genotype and allele frequencies was studied using PCR with the TaqMan Discrimination Assay or followed by the Restriction Fragment Length Polymorphism method, respectively. Results: We have found that the intron 4 polymorphism is a risk factor for the development of DM and CKD, except DN, while the codon 990 does not show any disease association. Conclusion: We conclude that CaSR is a general factor in pancreas and kidney pathologies. i 2014 S. Karger AG, Base

Comprehensive Mass Spectrometric Analysis of Snake Fruit: Salak (Salacca zalacca)

Snake fruit (Salacca zalacca (Gaert.) Voss) is a fruit species traditionally cultivated in Indonesia and other Southeast Asian countries. The edible parts of the fruits contain a certain amount of total phenolic, flavonoid, and monoterpenoid compounds, proving them to be their perfect sources. The main goal of this work was to detect, quantify, and identify various phenolic compounds present in snake fruit pulp. Ultrahigh performance liquid chromatography coupled to a Q-Orbitrap tandem mass spectrometer was able to detect 19 phenolic compounds in the salak pulp, including 5 flavanols, 6 phenolic acids, 2 flavonols, 1 flavone, and also 5 presumably new phenolic compounds. Among the detected compounds, 11 were reported and quantified for the first time in salak pulp. Chlorogenic acid was by far the most predominant phenolic compound. The next relatively abundant compounds in snake fruit were epicatechin, isoquercetin, neochlorogenic acid, ferulic acid, gallic acid and procyanidine B2 (levels at ca 5–10 μg/g in MeOH extract), syringic acid, and caffeic acid (levels at ca 1 μg/g in H2O extract). A significant total phenolic content (257.17 μL/mL) and antioxidant activities (10.56 μM TE/g of fruit pulp) were determined. In conclusion, S. zalacca fruit has potential to serve as a natural source of phenolic compounds with antioxidative activities which may be associated with their health benefits

The Intron 4 Polymorphism in the Calcium-Sensing Receptor Gene in Diabetes Mellitus and its Chronic Complications, Diabetic Nephropathy and Non-Diabetic Renal Disease

Background/Aims: Calcium-Sensing Receptor (CaSR) significantly affects calcium-phosphate metabolism in kidneys, and it is implicated in the pathogenesis of diabetes mellitus (DM) due to its expression in pancreatic F-cells. The role of CaSR as one of the players in pathogenesis of chronic kidney disease (CKD) has been speculated. Methods: 158 Type 2 diabetic patients divided into three groups according to occurrence and type of kidney complications, 66 nondiabetic patients CKD, and 93 healthy subjects were enrolled into the study to analyze the role of two CaSR polymorphisms (in the codon 990 and in the intron 4) in ethiopathogenesis of DM and CKD. The Type 2 diabetic groups consisted of 48 patients without any kidney abnormalities, 58 patients with diabetic nephropathy (DN), and 52 patients with nondiabetic renal disease (NDRD). The distribution of genotype and allele frequencies was studied using PCR with the TaqMan Discrimination Assay or followed by the Restriction Fragment Length Polymorphism method, respectively. Results: We have found that the intron 4 polymorphism is a risk factor for the development of DM and CKD, except DN, while the codon 990 does not show any disease association. Conclusion: We conclude that CaSR is a general factor in pancreas and kidney pathologies. i 2014 S. Karger AG, Base

Avenanthramides: Unique Bioactive Substances of Oat Grain in the Context of Cultivar, Cropping System, Weather Conditions and Other Grain Parameters

Our study was focused on the evaluation of the content of a wider spectrum of eight avenanthramides (AVNs) as unique components of oat grain under the effects of four selected factors (cultivar, locality, cropping system, and year). The weather effects on changes in the AVN content and their relationship to other important parameters of oat grain were further evaluated in more detail. A sensitive UHPLC system coupled with a QExactive Orbitrap mass spectrometer was used for AVN quantification. AVNs confirmed a high variability (RDS = 72.7–113.5%), which was dominantly influenced by the locality and year factors. While most AVN types confirmed mutually high correlations (r = 0.7–0.9), their correlations with the other 10 grain parameters were lower (r ≤ 0.48). Their significant correlations (0.27–0.46) with β-D-glucan could be used in perspective in breeding programs for the synergetic increase of both parameters. PCA analysis and Spearman correlations based on individual cultivars confirmed a significant effect of June and July precipitation on the increase of Σ AVNs. However, the results also indicated that higher precipitation can generate favorable conditions for related factors, such as preharvest sprouting evoking a direct increase of AVNs synthesis in oat grain

Specific Avenin Cross-Reactivity with G12 Antibody in a Wide Range of Current Oat Cultivars

Current clinical studies confirm that the consumption of oats for people suffering from celiac disease is safe. Some studies have confirmed different levels of immunoreactive gluten epitopes of oats in different cultivars, while others explain these differences due to contamination with gluten-rich species or as random cross-reactivity ELISA of homologous oat epitopes with anti-wheat gliadin antibodies. The aim of our two-year study was therefore to map cross-reactive oat epitopes in a set of 132 oat cultivars using a G12-based ELISA kit. The results were focused on the varietal and annual level of cross-reactivity (interference) of avenin epitopes with the G12 antibody on the identification of potential cultivars with significantly different interferences and assessing the degree of risk of possible false-contamination with external gluten. Although repeated evaluations confirmed high year-to-year variability (RSD ≥ 30%) in approximately 2/3 of the cultivars, the content of interfering avenin epitopes with G12 did not exceed the considered safe limit (20 mg·kg−1) for celiacs. At the same time, not only annual but, above all, significant cultivar dependences in the interference of avenins to the G12 antibody were demonstrated. Genetic dependence was further confirmed in connection with the proven avenin polymorphism as well as immunoblotting with the identification of interfering peptides with the G12 antibody in the 25 and 30 kDa regions. It was the occurrence of two bands around 30 kDa that predominantly occurred in oat cultivars with a relatively higher content of cross-reactive avenins (12–16 mg·kg−1). Due to the fact that the contents of interfering avenins ranged in several cultivars even over 16 mg·kg−1, the choice of a suitable oat cultivar may be crucial for gluten-free food producers, as it reduces the risk of a possible false-response of the commercial ELISA kits when checking the real-gluten contamination

Avenanthramides: Unique Bioactive Substances of Oat Grain in the Context of Cultivar, Cropping System, Weather Conditions and Other Grain Parameters

Our study was focused on the evaluation of the content of a wider spectrum of eight avenanthramides (AVNs) as unique components of oat grain under the effects of four selected factors (cultivar, locality, cropping system, and year). The weather effects on changes in the AVN content and their relationship to other important parameters of oat grain were further evaluated in more detail. A sensitive UHPLC system coupled with a QExactive Orbitrap mass spectrometer was used for AVN quantification. AVNs confirmed a high variability (RDS = 72.7–113.5%), which was dominantly influenced by the locality and year factors. While most AVN types confirmed mutually high correlations (r = 0.7–0.9), their correlations with the other 10 grain parameters were lower (r ≤ 0.48). Their significant correlations (0.27–0.46) with β-D-glucan could be used in perspective in breeding programs for the synergetic increase of both parameters. PCA analysis and Spearman correlations based on individual cultivars confirmed a significant effect of June and July precipitation on the increase of Σ AVNs. However, the results also indicated that higher precipitation can generate favorable conditions for related factors, such as preharvest sprouting evoking a direct increase of AVNs synthesis in oat grain

Pepsin digest of wheat gliadin fraction increases production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB signaling pathway and an NLRP3 inflammasome activation.

Celiac disease (CD) is a gluten-responsive, chronic inflammatory enteropathy. IL-1 cytokine family members IL-1β and IL-18 have been associated with the inflammatory conditions in CD patients. However, the mechanisms of IL-1 molecule activation in CD have not yet been elucidated. We show in this study that peripheral blood mononuclear cells (PBMC) and monocytes from celiac patients responded to pepsin digest of wheat gliadin fraction (PDWGF) by a robust secretion of IL-1β and IL-1α and a slightly elevated production of IL-18. The analysis of the upstream mechanisms underlying PDWGF-induced IL-1β production in celiac PBMC show that PDWGF-induced de novo pro-IL-1β synthesis, followed by a caspase-1 dependent processing and the secretion of mature IL-1β. This was promoted by K+ efflux and oxidative stress, and was independent of P2X7 receptor signaling. The PDWGF-induced IL-1β release was dependent on Nod-like receptor family containing pyrin domain 3 (NLRP3) and apoptosis-associated speck like protein (ASC) as shown by stimulation of bone marrow derived dendritic cells (BMDC) from NLRP3(-/-) and ASC(-/-) knockout mice. Moreover, treatment of human PBMC as well as MyD88(-/-) and Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF)(-/-) BMDC illustrated that prior to the activation of caspase-1, the PDWGF-triggered signal constitutes the activation of the MyD88/TRIF/MAPK/NF-κB pathway. Moreover, our results indicate that the combined action of TLR2 and TLR4 may be required for optimal induction of IL-1β in response to PDWGF. Thus, innate immune pathways, such as TLR2/4/MyD88/TRIF/MAPK/NF-κB and an NLRP3 inflammasome activation are involved in wheat proteins signaling and may play an important role in the pathogenesis of CD

PDWGF stimulates BMDC to IL-1β production through NLRP3 and ASC.

<p>(<b>A</b>) BMDC from WT, NLRP3−/− and ASC−/− mice were exposed to PDWGF (100 µg/ml) alone for 24 h; or first PDWGF was added for 21.5 h, the subsequently ATP (2 mM) was added for additional 2.5 h. IL-1β was measured in culture supernatants. (<b>B</b>) Flow-cytometric evaluation of PDWGF-induced maturation assessed by CD40, CD80, and CD86 expression on BMDC from WT and NLRP3−/− mice. WT and NLRP3−/− BMDC were cultured with 100 µg/ml of PDWGF (green), as well as 0.1 µg/ml of LPS (red) or 100 µg/ml of OVA (grey-filled) as positive and negative controls, respectively. Isotype controls are represented in black overlays. (<b>C</b>) Cells were preincubated with caspase-1 inhibitor Z-YVAD-fmk for 30 min, and then exposed to PDWGF in combination with ATP. Production of IL-1β was measured in culture supernatants. Results are expressed as mean ± SD from 4 independent experiments. The levels of significance for KO BMDC vs. WT BMDC are indicated as follows: *P<0.05, **P<0.01, and ***P<0.001.</p

TLR signaling is required for pro-IL-1β synthesis in response to PDWGF.

<p>WT BMDC and MyD88−/−, TRIF−/−, and IL-1R−/− KO BMDC were treated with PDWGF alone or in combination with ATP and (<b>A</b>) IL-1β production was evaluated after 24 h. (<b>B</b>) Cell lysates were evaluated for <i>de novo</i> synthesis of pro-IL-1β (<b>C</b>) WT BMDC and TLR2−/−, TLR4−/−, and TLR2/4−/− KO BMDC were treated with PDWGF alone or in combination with ATP, and IL-1β production was evaluated after 24 h. (<b>D</b>) Cell lysates were evaluated for <i>de novo</i> synthesis of pro-IL-1β. Data in (A) and (C) are expressed as mean ± SD from 5 independent experiments. *P<0.05, **P<0.01 vs. WT BMDC. Blots in (B) and (D) are representative from 3 independent experiments. β-actin was used as a loading control. (<b>E</b>) Celiac PBMC were treated with PDWGF alone or in combination with anti-TLR4 or anti-TLR2 Ab. IL-1β secretion was evaluated after 24 h. LPS was used as a positive control. Mean ± SD, 8 independent experiments. **P<0.01, ***P<0.001 vs. cells without anti-TLR Ab.</p

PDWGF-induced IL-1β production from celiac patient PBMC is modulated by K+ efflux, but is independent of the P2X7 receptor; as shown by (A) ELISA, mean ± SD, n = 10, ***P<0.001 vs. PDWGF-treated cells; and by (B) Western blot.

<p>Representative blots from 5 independent experiments are shown. (<b>C</b>) Inhibition of ROS modulate PDWGF-induced IL-1β secretion, mean ± SD, n = 10; as well as (D) pro-IL-1β production from PBMC of CD patients. Representative blots from 3 independent experiments are shown. β-actin was used as a loading control. ***P<0.001 vs. PDWGF-treated cells.</p