Coagulation of β-conglycinin, glycinin and isoflavones induced by calcium chloride in soymilk.
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ABSTRACT:
SUBMITTER: Hsiao YH
PROVIDER: S-EPMC4542527 | biostudies-other | 2015
REPOSITORIES: biostudies-other
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Project description:The family 16 endo-beta-1,3 glucanase from the extremophilic archaeon Pyrococcus furiosus is a laminarinase, which in 7.9 M GdmCl (guanidinium chloride) maintains a significant amount of tertiary structure without any change of secondary structure. The addition of calcium to the enzyme in 7.9 M GdmCl causes significant changes to the near-UV CD and fluorescence spectra, suggesting a notable increase in the tertiary structure which leads to a state comparable, but not identical, to the native state. The capability to interact with calcium in 7.9 M GdmCl with a consistent recovery of native tertiary structure is a unique property of this extremely stable endo-beta-1,3 glucanase. The effect of calcium on the thermodynamic parameters relative to the GdmCl-induced equilibrium unfolding has been analysed by CD and fluorescence spectroscopy. The interaction of calcium with the native form of the enzyme is studied by Fourier-transform infrared spectroscopy in the absorption region of carboxylate groups and by titration in the presence of a chromophoric chelator. A homology-based model of the enzyme is generated and used to predict the putative binding site(s) for calcium and the structural interactions potentially responsible for the unusual stability of this protein, in comparison with other family 16 glycoside hydrolases.
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Project description:ObjectiveChloride (Cl-) secretion is crucial for intestinal fluid secretion. Therefore, effects of the polyphenol Resveratrol (RSV) on Cl- secretion have been investigated. In a previous study, we observed effects of RSV on forskolin-induced Cl- secretion in the porcine jejunum but not the ileum although RSV itself induced a transepithelial ion current that may represent Cl- secretion in the ileum. The aim of this study was to gain further insights regarding the effects of RSV on characteristics of Cl- secretion in the porcine ileum using the Ussing chamber technique (recording of short circuit currents (Isc) as a measure for epithelial net ion transfer).ResultsRSV increased the Isc in the porcine ileum but not in the porcine jejunum as is already known. This increase was absent in a Cl--free buffer system, indicating that RSV indeed induces Cl- secretion. However, the carbachol-induced Isc was significantly inhibited by RSV indicating an inhibition of Ca2+-induced Cl- secretion. The cellular basis for these contradictory, segment specific results of RSV on Cl- secretion has to be subjected to further studies. The results also underline, that is difficult to generalize effects of RSV between different intestinal locations, organs, cell culture models or species.
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Project description:BackgroundMalignant arrhythmias require drug therapy. However, most of the currently available antiarrhythmic drugs have significant side effects. Ginsenoside Rg2 exhibits excellent cardioprotective effects and appears to be a promising candidate for cardiovascular drug development. So far, the oral toxicity and antiarrhythmic effects of Rg2 have not been evaluated.MethodsAcute oral toxicity of Rg2 was assessed by the Limit Test method in mice. Subchronic oral toxicity was determined by repeated dose 28-day toxicity study in rats. Antiarrhythmic activities of Rg2 were evaluated in calcium chloride-induced arrhythmic rats. Antiarrhythmic mechanism of Rg2 was investigated in arrhythmic rats and H9c2 cardiomyocytes.ResultsThe results of toxicity studies indicated that Rg2 exhibited no single-dose (10 g/kg) acute oral toxicity. And 28-day repeated dose treatment with Rg2 (1.75, 3.5 and 5 g/kg/d) demonstrated minimal, if any, subchronic toxicity. Serum biochemical examination showed that total cholesterol in the high-dose cohort was dramatically decreased, whereas prothrombin time was increased at Day 28, suggesting that Rg2 might regulate lipid metabolism and have a potential anticoagulant effect. Moreover, pretreatment with Rg2 showed antiarrhythmic effects on the rat model of calcium chloride induced arrhythmia, in terms of the reduced duration time, mortality, and incidence of malignant arrhythmias. The antiarrhythmic mechanism of Rg2 might be the inhibition of calcium influx through L-type calcium channels by suppressing the phosphorylation of Ca2+/calmodulin-dependent protein kinase II.ConclusionOur findings support the development of Rg2 as a promising antiarrhythmic drug with fewer side effects for clinical use.
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Project description:BACKGROUND:Local injection of BaCl2 is an established model of acute injury to study the regeneration of skeletal muscle. However, the mechanism by which BaCl2 causes muscle injury is unresolved. Because Ba2+ inhibits K+ channels, we hypothesized that BaCl2 induces myofiber depolarization leading to Ca2+ overload, proteolysis, and membrane disruption. While BaCl2 spares resident satellite cells, its effect on other tissue components integral to contractile function has not been defined. We therefore asked whether motor nerves and microvessels, which control and supply myofibers, are injured by BaCl2 treatment. METHODS:The intact extensor digitorum longus (EDL) muscle was isolated from male mice (aged 3-4 months) and irrigated with physiological salt solution (PSS) at 37 °C. Myofiber membrane potential (Vm) was recorded using sharp microelectrodes while intracellular calcium concentration ([Ca2+]i) was evaluated with Fura 2 dye. Isometric force production of EDL was measured in situ, proteolytic activity was quantified by calpain degradation of αII-spectrin, and membrane disruption was marked by nuclear staining with propidium iodide (PI). To test for effects on motor nerves and microvessels, tibialis anterior or gluteus maximus muscles were injected with 1.2% BaCl2 (50-75 μL) in vivo followed by immunostaining to evaluate the integrity of respective tissue elements post injury. Data were analyzed using Students t test and analysis of variance with P ≤ 0.05 considered statistically significant. RESULTS:Addition of 1.2% BaCl2 to PSS depolarized myofibers from - 79 ± 3 mV to - 17 ± 7 mV with a corresponding rise in [Ca2+]i; isometric force transiently increased from 7.4 ± 0.1 g to 11.1 ± 0.4 g. Following 1 h of BaCl2 exposure, 92 ± 3% of myonuclei stained with PI (vs. 8 ± 3% in controls) with enhanced cleavage of αII-spectrin. Eliminating Ca2+ from PSS prevented the rise in [Ca2+]i and ameliorated myonuclear staining with PI during BaCl2 exposure. Motor axons and capillary networks appeared fragmented within 24 h following injection of 1.2% BaCl2 and morphological integrity deteriorated through 72 h. CONCLUSIONS:BaCl2 injures myofibers through depolarization of the sarcolemma, causing Ca2+ overload with transient contraction, leading to proteolysis and membrane rupture. Motor innervation and capillarity appear disrupted concomitant with myofiber damage, further compromising muscle integrity.
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Project description:Exosomes might have an unimproved potential to serve as effective delivery vehicles. However, when exosomes are developed for therapeutic applications, a method to enhance their delivery is important. This study aimed to evaluate wheather calcium chloride (CaCl2) or other chloride compounds could enhance exosome delivery to various cells without causing toxicity. Exosomes were purified from human serum by using the ExoQuick exosome precipitation kit. Isolated exosomes were mixed with CaCl2 at concentrations ranging from 100 μM to 1 mM, and then washed using Amicon filter for treating the cells. The delivery efficiency of exosomes and the viability of the cells [HEK 293 (human kidney cells) and H9C2 (rat cardiomyocytes)] were evaluated. Cellular uptake of exosomes was observed using a confocal microscope based on PKH26 labeling of exosomes. CaCl2 increased the delivery of exosomes in a dose- and treatment time-dependent manner. In HEK 293 cells, a CaCl2 concentration of 400 μM and exposure time of 12 h increased the delivery of exosomes by >20 times compared with controls. In H9C2 cells, a CaCl2 concentration of 400 μM and exposure time of >24 h increased the delivery of exosomes by >400 times compared with controls. The viability of both cell lines was maintained up to a CaCl2 concentration of 1 mM. However, cobalt chloride, cupric chloride, and magnesium chloride did not change the delivery of exosomes in both cell lines. These results suggest that the use of CaCl2 treatment might be a useful method for enhancing the delivery of exosomes.
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Project description:A urease positive marine actinobacterium Brevibacterium lines was demonstrated to form and dissolve calcite precipitation in conditions with different concentration of Ca2+. Next-generation sequencing (NGS) was used to analyze the transcriptome of B. lines under 0, 50 and 150 mM Ca2+ after 24 h incubation to discover the differentially expressed genes involved. Results provide insight into the molecular response of B. lines stressed with different concentration of Ca2+.
2020-01-16 | GSE93668 | GEO
Project description:Myotonic dystrophy type 1 (DM1) involves misregulated alternative splicing for specific genes. We used exon or nucleotide deletion to mimic altered splicing of genes central to muscle excitation-contraction coupling processes in mice. Mice with forced-skipping of exon 29 in CaV1.1 calcium channel combined with loss of ClC-1 chloride channel function showed a markedly reduced lifespan, whereas other combinations of splicing mimics did not affect survival. The Ca2+/Cl- bi-channelopathy mice exhibited myotonia, weakness, and impairment of mobility and respiration. Chronic administration of the calcium channel blocker verapamil rescued survival and improved force generation, myotonia, and respiratory function. These results suggest that Ca2+/Cl- bi-channelopathy contributes to muscle impairment in DM1 and is potentially mitigated by common clinically available calcium channel blockers.
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Project description:Aims/hypothesis AGEs are considered environmental contributors of type 1 diabetes, but the exact role AGEs play early in pathogenesis of the disease, remains unidentified. We aimed to reduce AGEs with the pharmacotherapy alagebrium chloride in MIN6N8 cells and early in life in NOD mice to determine its impact on beta cell immunogenicity, function, and disease progression. Methods MIN6N8 cells were cultured with AGEs with or without short-term alagebrium therapy to determine the effect on ER stress and beta cell antigen presentation via a reporter assay for protein responses in the unfolded protein response, the enzymatic activity of endoplasmic reticulum aminopeptidase-1 (ERAP1) and the immunopeptidome. To determine the effect of short-term alagebrium therapy on beta cells in vivo, female NOD mice were treated with alagebrium and insulin secretion, insulitis, and immune cell repertoire were studied. Adoptive transfer studies and diabetes progression studies were used to consider the impact of alagebrium on immune cell function and disease outcome. Results In MIN6N8 cells, alagebrium therapy inhibited the induction of endoplasmic reticulum (ER) stress and the activity of ERAP1 by AGE-modified proteins. Alagebrium treated-MIN6N8 cells did not change the MHC Class I presentation of known beta cell antigens but did induce proteomic changes related to ER homeostatic pathways. Prior to overt autoimmune diabetes, female NOD mice treated with alagebrium for 30-40 days had improved insulin secretion, reduced insulitis and amplified proportions of pancreatic CD8+ T cells, mature B cells and F4/80+ macrophages. Splenocytes from alagebrium-treated mice adoptively transferred disease to NODscid recipients and maintained interferon production in vitro. While partial protection of islets by alagebrium was seen following the adoptive transfer of activated NOD G9C8 transgenic TCR CD8+ T cells, alagebrium therapy in NOD mice did not reduce diabetes progression. Conclusions/interpretation Our data suggests that in experimental models of diabetes, short-term alagebrium treatment allows for beta cell improvement, and maintenance of immune cell function, which does not fully mitigate diabetes progression.
2021-06-29 | PXD025998 | Pride
Project description:Autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis. Dysfunction of autophagy is associated with the progression of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Although one of the typical features of brain aging is an accumulation of redox-active metals that eventually lead to neurodegeneration, a plausible link between trace metal-induced neurodegeneration and dysregulated autophagy has not been clearly determined. Here, we used a cupric chloride-induced neurodegeneration model in MN9D dopaminergic neuronal cells along with ultrastructural and biochemical analyses to demonstrate impaired autophagic flux with accompanying lysosomal dysfunction. We found that a surge of cytosolic calcium was involved in cupric chloride-induced dysregulated autophagy. Consequently, buffering of cytosolic calcium by calbindin-D28K overexpression or co-treatment with the calcium chelator BAPTA attenuated the cupric chloride-induced impairment in autophagic flux by ameliorating dysregulation of lysosomal function. Thus, these events allowed the rescue of cells from cupric chloride-induced neuronal death. These phenomena were largely confirmed in cupric chloride-treated primary cultures of cortical neurons. Taken together, these results suggest that abnormal accumulation of trace metal elements and a resultant surge of cytosolic calcium leads to neuronal death by impairing autophagic flux at the lysosomal level.
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Project description:Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca(2+)-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca(2+), Sr(2+), and Ba(2+), and discovered that Mg(2+) competes with Ca(2+) in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore-as revealed by the permeability ratios of these anions-appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1.
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