IRAG2 Interacts with IP3-Receptor Types 1, 2, and 3 and Regulates Intracellular Ca2+ in Murine Pancreatic Acinar Cells

The inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is also known as Jaw1 or lymphoid-restricted membrane protein (LRMP) and shares homology with the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1). IRAG1 interacts with inositol trisphosphate receptors (IP3 receptors /IP3R) via its coiled-coil domain and modulates Ca2+ release from intracellular stores. Due to the homology of IRAG1 and IRAG2, especially in its coiled-coil domain, it is possible that IRAG2 has similar interaction partners like IRAG1 and that IRAG2 also modulates intracellular Ca2+ signaling. In our study, we localized IRAG2 in pancreatic acinar cells of the exocrine pancreas, and we investigated the interaction of IRAG2 with IP3 receptors and its impact on intracellular Ca2+ signaling and exocrine pancreatic function, like amylase secretion. We detected the interaction of IRAG2 with different subtypes of IP3R and altered Ca2+ release in pancreatic acinar cells from mice lacking IRAG2. IRAG2 deficiency decreased basal levels of intracellular Ca2+, suggesting that IRAG2 leads to activation of IP3R under unstimulated basal conditions. Moreover, we observed that loss of IRAG2 impacts the secretion of amylase. Our data, therefore, suggest that IRAG2 modulates intracellular Ca2+ signaling, which regulates exocrine pancreatic function.

Dynamics of diurnal cortisol and alpha-amylase secretion and their associations with PTSD onset in recent interpersonal trauma survivors

Background Dysfunction in major stress response systems during the acute aftermath of trauma may contribute to risk for developing posttraumatic stress disorder (PTSD). The current study investigated how PTSD diagnosis and symptom severity, depressive symptoms, and childhood trauma uniquely relate to diurnal neuroendocrine secretion (cortisol and alpha-amylase rhythms) in women who recently experienced interpersonal trauma compared to non-traumatized controls (NTCs). Method Using a longitudinal design, we examined diurnal cortisol and alpha-amylase rhythms in 98 young women (n = 57 exposed to recent interpersonal trauma, n = 41 NTCs). Participants provided saliva samples and completed symptom measures at baseline and 1-, 3-, and 6-month follow-up. Results Multilevel models (MLMs) revealed lower waking cortisol predicted the development of PTSD in trauma survivors and distinguished at-risk women from NTCs. Women with greater childhood trauma exposure exhibited flatter diurnal cortisol slopes. Among trauma-exposed individuals, lower waking cortisol levels were associated with higher concurrent PTSD symptom severity. Regarding alpha-amylase, MLMs revealed women with greater childhood trauma exposure exhibited higher waking alpha-amylase and slower diurnal alpha-amylase increase. Conclusions Results suggest lower waking cortisol in the acute aftermath of trauma may be implicated in PTSD onset and maintenance. Findings also suggest childhood trauma may predict a different pattern of dysfunction in stress response systems following subsequent trauma exposure than the stress system dynamics associated with PTSD risk; childhood trauma appears to be associated with flattened diurnal cortisol and alpha-amylase slopes, as well as higher waking alpha-amylase.

Glyoxalase-I Is Upregulated in Acute Cerulein-Induced Pancreatitis: A New Mechanism in Pancreatic Inflammation?

Inflammation caused by oxidative stress (ROS) demonstrates an essential mechanism in the pathogenesis of acute pancreatitis (AP). Important sources for ROS comprise the reactive compound methylglyoxal (MGO) itself and the MGO-derived formation of advanced glycation end-products (AGEs). AGEs bind to the transmembrane receptor RAGE and activate NF-κB, and lead to the production of pro-inflammatory cytokines. MGO is detoxified by glyoxalase-I (Glo-I). The importance of Glo-I was shown in different models of inflammation and carcinogenesis. Nevertheless, the role of Glo-I and MGO in AP has not been evaluated so far. This study analyzed Glo-I in cerulein-(CN)-induced AP and determined the effects of Glo-I knockdown, overexpression and pharmacological modulation. Methods: AP was induced in C57BL6/J mice by i.p. injection of CN. Glo-I was analyzed in explanted pancreata by Western Blot, qRT-PCR and immunohistochemistry. AR42J cells were differentiated by dexamethasone and stimulated with 100 nM of CN. Cells were simultaneously treated with ethyl pyruvate (EP) or S-p-bromobenzylglutathione-cyclopentyl-diester (BrBz), two Glo-I modulators. Knockdown and overexpression of Glo-I was achieved by transient transfection with Glo-I siRNA and pEGFP-N1-Glo-I-Vector. Amylase secretion, TNF-α production (ELISA) and expression of Glo-I, RAGE and NF-κB were measured. Results: Glo-I was significantly upregulated on protein and mRNA levels in CN-treated mice and AR42J cells. Dexamethasone-induced differentiation of AR42J cells increased the expression of Glo-I and RAGE. Treatment of AR42J cells with CN and EP or BrBz resulted in a significant reduction of CN-induced amylase secretion, NF-κB, RAGE and TNF-α. Overexpression of Glo-I led to a significant reduction of CN-induced amylase levels, NF-κB expression and TNF-α, whereas Glo-I knockdown revealed only slight alterations. Measurements of specific Glo-I activity and MGO levels indicated a complex regulation in the model of CN-induced AP. Conclusion: Glo-I is overexpressed in a model of CN-induced AP. Pharmacological modulation and overexpression of Glo-I reduced amylase secretion and the release of pro-inflammatory cytokines in AP in vitro. Targeting Glo-I in AP seems to be an interesting approach for future in vivo studies of AP.

Expression of antibody fragments in Saccharomyces cerevisiae strains evolved for enhanced protein secretion

Monoclonal antibodies, antibody fragments and fusion proteins derived thereof have revolutionized the practice of medicine. Major challenges faced by the biopharmaceutical industry are however high production costs, long processing times and low productivities associated with their production in mammalian cell lines. The yeast Saccharomyces cerevisiae, a well-characterized eukaryotic cell factory possessing the capacity of post­translational modifications, has been industrially exploited as a secretion host for production of a range of products, including pharmaceuticals. However, due to the incompatible surface glycosylation, few antibody molecules have been functionally expressed in S. cerevisiae. Here, three non-glycosylated antibody fragments from human and the Camelidae family were chosen for expression in a S. cerevisiae strain (HA) previously evolved for high α-amylase secretion. These included the Fab fragment Ranibizumab (Ran), the scFv peptide Pexelizumab (Pex), and a nanobody consisting of a single V-type domain (Nan). Both secretion and biological activities of the antibody fragments were confirmed. In addition, the secretion level of each protein was compared in the wild type (LA) and two evolved strains (HA and MA) with different secretory capacities. We found that the secretion of Ran and Nan was positively correlated with the strains’ secretory capacity, while Pex was most efficiently secreted in the parental strain. To investigate the mechanisms for different secretion abilities in these selected yeast strains for the different antibody fragments, RNA-seq analysis was performed. The results showed that several bioprocesses were significantly enriched for differentially expressed genes when comparing the enriched terms between HA.Nan vs. LA.Nan and HA.Pex vs. LA.Pex, including amino acid metabolism, protein synthesis, cell cycle and others, which indicates that there are unique physiological needs for each antibody fragment secretion.

Evaluation of Enogen® Feed Corn on growth performance and carcass characteristics of finishing pigs

Enogen® Feed Corn (EFC; Syngenta Seeds, LLC, Downers Grove, IL) hybrids contain a trait for expression of heat-stable alpha amylase in the grain. Alpha amylase is an enzyme responsible for breakdown of starch in the small intestine; supplementation of exogenous alpha amylase to pigs may result in greater starch digestibility and thus improved gain efficiency. A total of 288 pigs (Line 600 × 241, DNA, Columbus, NE; initially 41.6 kg) were utilized in an 82-d trial to determine if replacing conventional yellow dent corn (CONV) with EFC in diets with or without distillers dried grains with solubles (DDGS) influences growth performance and carcass characteristics. Pens of pigs were randomly assigned to 1 of 4 dietary treatments balancing for initial BW. There were 9 pens per treatment with 8 pigs per pen (an equal number of barrows and gilts per pen). Treatments were arranged in a 2 × 2 factorial with main effects of corn source (CONV or EFC) and DDGS (0 or 25%). Experimental diets were fed in meal form in 3 phases: d 0 to 29, 29 to 47, and 47 to 82. Pigs were weighed approximately every 2 wk and at the beginning of each phase. On d 82, pigs were transported to a commercial abattoir for processing and carcass data collection. Data were analyzed using PROC GLIMMIX procedure of SAS with pen as the experimental unit. There were no corn source by DDGS interactions (P > 0.05) observed for overall performance or carcass characteristics. Overall, average daily gain (ADG) was marginally greater (P < 0.089) for pigs fed EFC than CONV with no evidence (P > 0.196) for difference in average daily feed intake (ADFI), gain efficiency (G:F), hot carcass weight (HCW), or other carcass traits. Addition of DDGS decreased (P < 0.047) overall ADG and G:F. Pigs fed DDGS had marginally lower (P < 0.071) HCW, less (P < 0.050) backfat depth, greater (P < 0.026) loin depth, and greater (P < 0.020) percentage lean and carcass fat iodine value (IV). In summary, addition of 25% DDGS to the diet decreased ADG and increased carcass fat IV. Pigs fed EFC tended to have improved overall ADG; however, G:F and carcass characteristics were not different between corn sources. These results suggest that EFC, although not beneficial, may be used as a substitute for CONV without any deleterious effects on growth performance. Further research should be conducted to understand if addition of EFC to swine diets could be beneficial in younger pigs exhibiting decreased pancreatic α-amylase secretion following weaning, or whether heat treatment of diets, such as pelleting, may influence the response to EFC.

Pitfalls in AR42J-model of cerulein-induced acute pancreatitis

Background AR42J are immortalized pancreatic adenocarcinoma cells that share similarities with pancreatic acinar cells. AR42J are often used as a cell-culture model of cerulein (CN)-induced acute pancreatitis (AP). Nevertheless, it is controversial how to treat AR42J for reliable induction of AP-like processes. Gene knockout and/or overexpression often remain challenging, as well. In this study, we demonstrate conditions for a reliable induction of proinflammatory markers upon CN treatment in AR42J and high transfection efficacy using Glyoxalase-I (Glo-I) as a target of interest. Methods Effects of dexamethasone (dexa) and CN on cell morphology and amylase secretion were analyzed via ELISA of supernatant. IL-6, TNF-α and NF-κB-p65 were measured via qRT-PCR, ELISA and Western Blot (WB). Transfection efficacy was determined by WB, qRT-PCR and immune fluorescence of pEGFP-N1-Glo-I-Vector and Glo-I-siRNA. Results Treatment of AR42J with 100 nm dexa is mandatory for differentiation to an acinar-cell-like phenotype and amylase production. CN resulted in secretion of amylase but did not influence amylase production. High levels of CN-induced amylase secretion were detected between 3 and 24 hours of incubation. Treatment with LPS alone or in combination with CN did not influence amylase release compared to control or CN. CN treatment resulted in increased TNF-α production but not secretion and did not influence IL-6 mRNA. CN-induced stimulation of NF-κB was found to be highest on protein levels after 6h of incubation. Transient transfection was able to induce overexpression on protein and mRNA levels, with highest effect after 12 to 24 hours. Gene-knockdown was achieved by using 30 pmol of siRNA leading to effective reduction of protein levels after 72 hours. CN did not induce amylase secretion in AR42J cell passages beyond 35. Conclusion AR42J cells demonstrate a reliable in-vitro model of CN-induced AP but specific conditions are mandatory to obtain reproducible data.

Influence of Non-Thermal Atmospheric Pressure Plasma Jet on Extracellular Activity of α-Amylase in Aspergillus oryzae

In a previous study, we found that plasma can enhance spore germination and α-amylase secretion in A. oryzae, a beneficial fungus used in fermentation. To confirm this, in the current study, we investigated the effects of plasma on development and α-amylase secretion using an enlarged sample size and a different plasma source: a plasma jet. There was a ~10% (p < 0.01) increase in spore germination upon non-thermal atmospheric pressure plasma jet (NTAPPJ) treatment for 5 min and 10 min, as compared with the control (no plasma treatment). The activity of α-amylase detected in potato dextrose broth (PDB) media during incubation was significantly elevated in plasma-treated samples, with a more obvious increase upon 10 min and 15 min treatments and 24–96 h incubation periods. The levels of the oxidation reduction potential (ORP) and NOX (nitrogen oxide species) were higher in the plasma-treated samples than in the control samples, suggesting that these two variables could serve as standard indicators for enhancing α-amylase activity after plasma treatment. Genome sequencing analysis showed approximately 0.0016–0.0017% variations (changes in 596–655 base pairs out of a total of 37,912,014 base pairs) in the genomic DNA sequence of A. oryzae after plasma treatment. Our results suggest that NATPPJ can enhance the spore germination and extracellular activity of α-amylase, probably by increasing the levels of ORP and NOX to an optimum level.

Isolation, Screening and Time Course Study of Amylase-producing Fungi from Garri Processing Environment

Amylases (E.C.3.2.1.1) are enzymes which catalyze the breakdown of -1,4 glycosidic linkages of starch to simple sugars and different monomeric products. Microbial amylases are essential enzymes compared with animals and plants amylases in industrial applications. Thus, in southern Nigeria, garri processing waste-water from mills poses a serious environmental challenge and this situation could be exploited by utilizing microorganisms colonizing the area to manufacture microbial products. This present study was aimed at evaluating the potential of fungi isolated from garri processing environment for amylase secretion and conduct a time course study of the enzyme production. Fungi were isolated from the soil and waste water from garri processing sites using Standard Microbiological Procedures. The five fungi isolated from garri processing environmental samples were screened using Lugols iodine. Three fungal isolates were then selected on the hydrolysis of starch in qualitative zonation agar plates but one isolate named B among them was better in starch hydrolysis related to highest clear zone plates. After qualitative screening, the three hyper production amylase extracted fungi were identified according to the morphological characteristics. The hyper producing amylase isolate B was then identified as Aspergillus flavus (RCBBR_AEAFUN2) and recorded as a novel strain in southern Nigeria according to molecular characteristics, which was selected for further studies. The current results of time course study showed that the maximum growth (2.453 g) occurred after 24 h of incubation at pH 4.5 while maximum enzyme production (2.3 U/ml/min) was obtained after 96 h of incubation at pH 3.5. Therefore, the hyper producing amylase from garri processing sites, Aspergillus flavus (RCBBR_AEAFUN2) novel strain has great prospects for future biotechnological and industrial applications and help in ameliorating the environmental pollution posed by the waste-water.

Leucine improves α-amylase secretion through the general secretory signaling pathway in pancreatic acinar cells of dairy calves

The present study aimed to elucidate the mechanisms by which leucine impacts the secretion of pancreatic enzymes, especially amylase, by studying the proteomics profiles of pancreatic acinar (PA) cells from dairy cows. PA cells, the experimental model, were treated with four concentrations of leucine (0, 0.23, 0.45, and 0.90 mM). The abundance of different proteins in the four leucine treatment groups was detected. Label-free proteomic analysis enabled the identification of 1,906 proteins in all four treatment groups, and 1,350 of these proteins showed common expression across the groups. The primary effects of leucine supplementation were increased ( P < 0.05) citrate synthase and ATPase activity, which enlarged the cytosolic ATP pool, and the upregulation of secretory protein 61 (Sec61) expression, which promoted protein secretion. In summary, these results suggest that leucine increases citrate synthase in the TCA cycle and ATPase activity and promotes the Sec signaling pathway to increase the exocrine function of PA cells.