Intracellular Ca Levels Research Articles

Inhibition of forward and reverse transport of Ca2+ via Na+/Ca2+ exchangers (NCX) prevents sperm capacitation

BackgroundWhile calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na+/Ca2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were evaluated.ResultsPartial and complete blockage of Ca2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation.ConclusionsInhibition of outflux and influx of Ca2+ triggered similar effects, thus indicating that both forward and reverse Ca2+ transport through NCX exchangers are essential for sperm capacitation.

Regulatory effects of Trichinella spiralis serpin-type serine protease inhibitor on endoplasmic reticulum stress and oxidative stress in host intestinal epithelial cells

Endoplasmic reticulum stress (ERS) and oxidative stress (OS) are adaptive responses of the body to stressor stimulation. Although it has been verified that Trichinella spiralis (T. spiralis) can induce ERS and OS in the host, their association is still unclear. Therefore, this study explored whether T. spiralis-secreted serpin-type serine protease inhibitor (TsAdSPI) is involved in regulating the relationship between ERS and OS in the host intestine. In this study, mice jejunum and porcine small intestinal epithelial cells (IECs) were detected using qPCR, western blotting, immunohistochemistry (IHC), immunofluorescence (IF), and detection kits. The results showed that ERS- and OS-related indexes changed significantly after TsAdSPI stimulation, and Bip was located in IECs, indicating that TsAdSPI could induce ERS and OS in IECs. After the use of an ERS inhibitor, OS-related indexes were inhibited, suggesting that TsAdSPI-induced OS depends on ERS. When the three ERS signalling pathways, ATF6, IRE1, and PERK, were sequentially suppressed, OS was only regulated by the PERK pathway, and the PERK-eif2α-CHOP-ERO1α axis played a key role. Similarly, the expression of ERS-related indexes and the level of intracellular Ca2+ were inhibited after adding the OS inhibitor, and the expression of ERS-related indexes decreased significantly after inhibiting calcium transfer. This finding indicated that TsAdSPI-induced OS could affect ERS by promoting Ca2+ efflux from the endoplasmic reticulum. The detection of the ERS and OS sequences revealed that OS occurred before ERS. Finally, changes in apoptosis-related indexes were detected, and the results indicated that TsAdSPI-induced ERS and OS could regulate IEC apoptosis. In conclusion, TsAdSPI induced OS after entering IECs, OS promoted ERS by enhancing Ca2+ efflux, and ERS subsequently strengthened OS by activating the PERK-eif2α-CHOP-ERO1α axis. ERS and OS induced by TsAdSPI synergistically promoted IEC apoptosis. This study provides a foundation for exploring the invasion mechanism of T. spiralis and the pathogenesis of host intestinal dysfunction after invasion.

Elaidic acid leads to mitochondrial dysfunction via mitochondria-associated membranes triggers disruption of mitochondrial calcium fluxes

Elaidic acid (EA) stimulation can lead to endoplasmic reticulum stress (ERS), accompanied by a large release of Ca2+, and ultimately the activation of NLRP3 inflammasome in Kupffer cells (KCs). Mitochondrial instability or dysfunction may be the key stimulating factors to activate NLRP3 inflammasome, and sustained Ca2+ transfer can result in mitochondrial dysfunction. We focused on KCs to explore the damage to mitochondria by EA. After EA stimulation, cells produced an oxidative stress (OS) response with a significant increase in ROS release. Immunoprecipitation experiments and the addition of inhibitors revealed that the increase in the level of intracellular Ca2+ led to Ca2+ accumulation in the mitochondrial matrix via mitochondria-associated membranes (MAMs). This was accompanied by a significant release of mROS, loss of MMP and ATP, and a significant increase in mitochondrial permeability transition pore opening, ultimately leading to mitochondrial instability. These findings confirmed the mechanism that EA induced mitochondrial Ca2+ imbalance in KCs via MAM, ultimately leading to mitochondrial dysfunction. Meanwhile, EA induced OS and the decrease of MMP and ATP in rat liver, and significant lesions were found in liver mitochondria. Swelling of the inner mitochondrial cristae and mitochondrial vacuolization occurred, with a marked increase in lipid droplets.

CaMKIIα signaling is required for the neuroprotective effects of Dl‐3‐n‐butylphthalide in Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) is the most common form of neurodegenerative disease and most anti‐AD drugs have failed in clinical trials, hence it is urgent to find effective potential drugs against AD. DL‐3‐n‐Butylphthalide (NBP) is a compound extracted from celery seed and is a multiple‐target drug, and we have demonstrated NBP was effective for improving cognitive and global functioning in patients with subcortical vascular cognitive impairment without dementia and exhibited good safety. Even though there are several studies that have demonstrated the neuroprotective effects of NBP on cognitive impairment, and the mechanisms of NBP remains relatively unexplored.MethodCell viabilities was observed by MTT. Apoptosis and the level of intracellular Ca2+ were examined by flow cytometry. The activation of CaMKIIα signaling was detected by western blotting and immunofluorescence.ResultIn this study, we found that NBP could alleviated the increase of intracellular Ca2+ and reverse down‐regulation of Ca2+/calmodulin‐dependent protein kinase alpha (CaMKIIα) signaling, and rescue neuronal apoptosis in SH‐SY5Y cells treated by Aβ oligomers. Further, we found that these neuroprotection of NBP on neuronal damage and CaMKIIα signaling were abolished when CaMKIIα expression was knock‐downed or its activity was inhibited.ConclusionThus, our findings suggested that CaMKIIα signaling was required for the neuroprotective effects of NBP in AD, and provide an improved basis for elucidation of the mechanism and treatment of NBP in AD.

Neuroglobin protects against cerebral ischemia/reperfusion injury in rats by suppressing mitochondrial dysfunction and endoplasmic reticulum stress-mediated neuronal apoptosis through synaptotagmin-1.

Cerebral ischemia/reperfusion (I/R) injury remains a grievous health threat, and herein effective therapy is urgently needed. This study explored the protection of neuroglobin (Ngb) in rats with cerebral I/R injury. The focal cerebral I/R rat models were established by middle cerebral artery occlusion (MCAO) and neuronal injury models were established by oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. The brain injury of rats was assessed. Levels of Ngb, Bcl-2, Bax, endoplasmic reticulum stress (ERS)-related markers, and Syt1 were measured by immunofluorescence staining and Western blotting. The cytotoxicity in neurons was assessed by lactate dehydrogenase (LDH) release assay. Levels of intracellular Ca2+ and mitochondrial function-related indicators were determined. The binding between Ngb and Syt1 was detected by co-immunoprecipitation. Ngb was upregulated in cerebral I/R rats and its overexpression alleviated brain injury. In OGD/R-induced neurons, Ngb overexpression decreased LDH level and neuronal apoptosis, decreased Ca2+ content, and mitigated mitochondrial dysfunction and ERS-related apoptosis. However, Ngb silencing imposed the opposite effects. Importantly, Ngb could bind to Syt1. Syt1 knockdown partially counteracted the alleviation of Ngb on OGD/R-induced injury in neurons and cerebral I/R injury in rats. Briefly, Ngb extenuated cerebral I/R injury by repressing mitochondrial dysfunction and endoplasmic reticulum stress-mediated neuronal apoptosis through Syt1.

The NMDA receptor regulates integrin activation, ATP release and arterial thrombosis through store-operated Ca2+ entry in platelets.

Platelet activation and thrombus formation is crucial for hemostasis, but also trigger arterial thrombosis. Calcium mobilization plays an important role in platelet activation, because many cellular processes depend on the level of intracellular Ca2+ ([Ca2+](i)), such as integrin activation, degranulation, cytoskeletal reorganization. Different modulators of Ca2+ signaling have been implied, such as STIM1, Orai1, CyPA, SGK1, etc. Also, the N-methyl-D-aspartate receptor (NMDAR) was identified to contribute to Ca2+ signaling in platelets. However, the role of the NMDAR in thrombus formation is not well defined. In vitro and in vivo analysis of platelet-specific NMDAR knock-out mice. In this study, we analyzed Grin1fl/fl-Pf4-Cre+ mice with a platelet-specific knock-out of the essential GluN1 subunit of the NMDAR. We found reduced store-operated Ca2+ entry (SOCE), but unaltered store release in GluN1-deficient platelets. Defective SOCE resulted in reduced Src and PKC substrate phosphorylation following stimulation of glycoprotein (GP)VI or the thrombin receptor PAR4 followed by decreased integrin activation but unaltered degranulation. Consequently, thrombus formation on collagen under flow conditions was reduced ex vivo, and Grin1fl/fl-Pf4-Cre+ mice were protected against arterial thrombosis. Results from human platelets treated with the NMDAR antagonist MK-801 revealed a crucial role of the NMDAR in integrin activation and Ca2+ homeostasis in human platelets as well. NMDAR signaling is important for SOCE in platelets and contributes to platelet activation and arterial thrombosis. Thus, the NMDAR represents a novel target for anti-platelet therapy in cardiovascular disease (CVD).

Mechanism of Molecular Activity of Yolkin—a Polypeptide Complex Derived from Hen Egg Yolk—in PC12 Cells and Immortalized Hippocampal Precursor Cells H19-7

Food-derived bioactive peptides able to regulate neuronal function have been intensively searched and studied for their potential therapeutic application. Our previous study showed that a polypeptide complex yolkin, isolated from hen egg yolk as a fraction accompanying immunoglobulin Y (IgY), improved memory and cognitive functions in rats. However, the mechanism activated by the yolkin is not explained. The goal of the present study was to examine what molecular mechanism regulating brain-derived neurotrophic factor (BDNF) expression is activated by the yolkin complex, using in vitro models of PC12 cell line and fetal rat hippocampal cell line H19-7. It was shown that yolkin increased the proliferative activity of rat hippocampal precursor cells H19-7 cells and upregulated the expression/production of BDNF in a cyclic adenosine monophosphate (cAMP)-response element-binding protein (CREB)-dependent manner. Additionally the upregulation of carboxypeptidase E/neurotrophic factor–α1 (CPE/(NF-α1) expression was shown. It was also determined that upregulation of CREB phosphorylation by yolkin is dependent on cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) signaling pathway activation. Moreover, the impact of yolkin on the level of intracellular Ca2+, nitric oxide, and activation of extracellular signal-regulated kinases 1/2 (ERK 1/2 kinase) was excluded. These results emphasize that yolkin can act comprehensively and in many directions and may participate in the regulation of neurons’ survival and activity. Therefore, it seems that the yolkin specimen can be used in the future as a safe, bioavailable, natural nutraceutical helping to improve the cognition of older people.

Post-translational modification-dependent regulation of CaMKII-delta conformation in live cardiac myocytes.

Calcium/calmodulin dependent protein kinase II (CaMKII) is a key regulator of numerous functions in cardiac myocytes including Ca regulation, contraction, and transcriptional control. Overactivation of CaMKII due to post-translational modifications like autophosphorylation at Thr287 can autonomously activate and prolong CaMKII activation, even after Ca/CaM dissociates, and thereby promote cardiac pathologies such as heart failure and arrhythmias. The specific molecular mechanisms by which CaMKII PTMs autonomously activate and prolong kinase activity are not fully understood. We found using a novel intermolecular FRET-based approach in live myocytes in a recent study that PTM-dependent alteration in CaMKII's affinity for CaM is one molecular basis by which CaMKII becomes autonomously activated. Here, we investigate how the conformational state of CaMKII in the autonomously activated state may be affected by PTMs using our FRET-based CaMKIIδ conformational state reporter, Camui (RFP-CaMKIIδ-GFP), and its phosphomimetic (T287D) and phosphoresistant (T287A) mutant variants, expressed via adenoviral infection in intact cardiac myocytes. We also conducted parallel in vitro biochemical experiments using HEK cell lysates. We found that the phosphomimetic T287D Camui exhibited prolonged durations in the active open conformational state following pacing-induced activation relative to WT or phosphoresistant T287A Camui. Moreover, the prolonged autonomous conformational state persisted even after intracellular Ca levels returned to diastolic levels at which CaM dissociates. These data suggest that PTM-dependent regulation of the conformational state of CaMKII may serve as an additional molecular mechanism for autonomous CaMKII activation that may function in tandem with PTM-dependent alterations in CaMKII's affinity for CaM.

Excessive neutrophil extracellular trap formation induced by Porphyromonas gingivalis lipopolysaccharide exacerbates inflammatory responses in high glucose microenvironment.

Neutrophil extracellular trap (NET) is a novel defense strategy of neutrophils and found to be induced by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) or high glucose. The aim of this study was to investigate the roles and mechanisms of NET formation in high glucose inflammatory microenvironment. NETs induced by 1 μg/ml P. gingivalis LPS and/or 25 mM glucose were visualized using a fluorescence microscopy and the levels of extracellular DNA were determined by a microplate reader. The bactericidal efficiency of NETs was assessed by quantifying the survival P. gingivalis in neutrophils. The levels of NLRP3 and IL-1β in THP-1 derived-macrophages, and the expressions of p-PKC βII, p-MEK1/2, p-ERK1/2, ORAI1 and ORAI2 in neutrophils were detected by Western blot. Moreover, levels of intracellular Ca2+ and reactive oxygen species (ROS) in neutrophils were explored by flow cytometry. P. gingivalis LPS enhanced the formation of NETs and increased the levels of extracellular DNA in high glucose microenvironment (p < 0.05). Compared with normal glucose inflammatory microenvironment, quantities of extra- and intracellular viable P. gingivalis in neutrophils exposed to NETs induced in high glucose inflammatory one were increased (p < 0.05) and the expressions of NLRP3 and IL-1β were dramatically increased in macrophages co-cultured with NETs from high glucose inflammatory microenvironment (p < 0.05). In addition, levels of ROS, intracellular Ca2+, p-PKC βII, p-MEK1/2, p-ERK1/2, ORAI1 and ORAI2 were increased in neutrophils stimulated with both high glucose and P. gingivalis LPS compared with the single stimulus groups (p < 0.05). In high glucose inflammatory microenvironment, formation of NETs was enhanced via oxidative stress, which failed to reverse the decreased bactericidal capacity in high glucose microenvironment, and instead aggravated the subsequent inflammatory responses.

Ca2+-regulated enzymes calpain and calcineurin in neurodegenerative processes and prospects for neuroprotective pharmacotherapy

Calcium (Ca2+) and Ca2+-regulated enzymes calpain and calcineurin are the key molecules of signaling mechanisms in neurons and ensure the normal course of intracellular neurochemical and neurophysiological processes. The imbalance and increase in the intracellular level of Ca2+ correlates with the activation of calpain and calcineurin. Inactivation of endogenous inhibitors and/or absence of exogenous pharmacological inhibitors of these enzymes may induce a cascade of intracellular mechanisms that are detrimental to the structural integrity and functional activity of neurons. The interrelated processes of Ca2+ imbalance, dysregulation of calpain and calcineurin are directly related to the development of intracellular pathophysiological reactions leading to the degeneration and death of selective neuronal populations in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The review briefly presents the characteristics of calpain and calcineurin, their interrelated role in the neurodegeneration processes. Data on the efficiency of the exogenous inhibitors (in vivo, in vitro) point out the potential role of pharmacological regulation of calpain and calcineurin for neuroprotection.

Enhancement of Fungal Enzyme Production by Radio-Frequency Electromagnetic Fields.

Enzyme production by microorganisms on an industrial scale has demonstrated technical bottlenecks, such as low efficiency in enzyme expression and extracellular secretion. In this study, as a potential tool for overcoming these technical limits, radio-frequency electromagnetic field (RF-EMF) exposure was examined for its possibility to enhance production of an enzyme, α-amylase, in a filamentous fungus, Aspergillus oryzae. The RF-EMF perfectly resonated at 2 GHz with directivity radiation pattern and peak gain of 0.5 dB (0.01 Watt). Total protein concentration and activity of α-amylase measured in media were about 1.5-3-fold higher in the RF-EMF exposed (10 min) sample than control (no RF-EMF) during incubation (the highest increase after 16 h). The level of α-amylase mRNA in cells was approximately 2-8-fold increased 16 and 24 h after RF-EMF exposure for 10 min. An increase in vesicle accumulation within fungal hyphae and the transcription of some genes involved in protein cellular trafficking was observed in RF-EMF-exposed samples. Membrane potential was not changed, but the intracellular Ca2+ level was elevated after RF-EMF exposure. Our results suggest that RF-EMF can increase the extracellular level of fungal total proteins and α-amylase activity and the intracellular level of Ca2+.

Cannabidiol prevents methamphetamine-induced neurotoxicity by modulating dopamine receptor D1-mediated calcium-dependent phosphorylation of methyl-CpG-binding protein 2.

In the past decade, methamphetamine (METH) abuse has sharply increased in the United States, East Asia, and Southeast Asia. METH abuse not only leads to serious drug dependence, but also produces irreversible neurotoxicity. Currently, there are no approved pharmacotherapies for the treatment of METH use disorders. Cannabidiol (CBD), a major non-psychoactive (and non-addictive) cannabinoid from the cannabis plant, shows neuroprotective, antioxidative, and anti-inflammatory properties under METH exposure. At present, however, the mechanisms underlying these properties remain unclear, which continues to hinder research on its therapeutic potential. In the current study, computational simulations showed that CBD and METH may directly bind to the dopamine receptor D1 (DRD1) via two overlapping binding sites. Moreover, CBD may compete with METH for the PHE-313 binding site. We also found that METH robustly induced apoptosis with activation of the caspase-8/caspase-3 cascade in-vitro and in-vivo, while CBD pretreatment prevented these changes. Furthermore, METH increased the expression of DRD1, phosphorylation of Methyl-CpG-binding protein 2 (MeCP2) at serine 421 (Ser421), and level of intracellular Ca2+ in-vitro and in-vivo, but these effects were blocked by CBD pretreatment. The DRD1 antagonist SCH23390 significantly prevented METH-induced apoptosis, MeCP2 phosphorylation, and Ca2+ overload in-vitro. In contrast, the DRD1 agonist SKF81297 markedly increased apoptosis, MeCP2 phosphorylation, and Ca2+ overload, which were blocked by CBD pretreatment in-vitro. These results indicate that CBD prevents METH-induced neurotoxicity by modulating DRD1-mediated phosphorylation of MeCP2 and Ca2+ signaling. This study suggests that CBD pretreatment may resist the effects of METH on DRD1 by competitive binding.

Neutrophil extracellular traps induced by Porphyromonas gingivalis lipopolysaccharide modulate inflammatory responses via a Ca2+-dependent pathway

ObjectiveThe aim of this study was to explore the effects and underlying mechanisms of Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) on the formation of neutrophil extracellular traps (NETs). DesignNETs induced by 1 μg/ml P. gingivalis LPS were observed by a fluorescence microscope and quantified by a microplate reader. Quantities of extra- and intracellular P. gingivalis in neutrophils were determined to assay the bactericidal efficiency of NETs. Intracellular Ca2+ levels in neutrophils were explored by flow cytometry. Expressions of phospho-tumor progression locus 2 (p-TPL2), phospho-mitogen-activated protein kinase kinase1/2 (p-MEK1/2), phospho-extracellular signal-regulated kinase1/2 (p-ERK1/2), ORAI1, ORAI2 and peptidylarginine deiminase 4 (PAD4) were detected by Western blot. In addition, neutrophil elastase activities in NETs incubated with macrophages were assayed to evaluate their clearance. ResultsP. gingivalis LPS contributed to the formation of NETs and the increased levels of extracellular DNA (p < 0.05), which enhanced bactericidal activity of neutrophils (p < 0.05). Levels of intracellular Ca2+, p-TPL2, p-MEK1/2, p-ERK1/2, ORAI1, ORAI2 and PAD4 were increased in P. gingivalis LPS-treated neutrophils compared with control group (p < 0.05). In addition, inhibition of intracellular Ca2+ by two Ca2+ chelators, and PAD4 knockdown resulted in decreased levels of extracellular DNA (p < 0.05). After co-culture of NETs with macrophages, neutrophil elastase activities were decreased (p < 0.05). ConclusionP. gingivalis LPS induced the formation of NETs via a Ca2+-TPL2-MEK-ERK-PAD4 signaling pathway, which contribute to the elimination of P. gingivalis.

Interaction between nanometer calcium oxalate and renal epithelial cells repaired with carboxymethylated polysaccharides.

Injury of renal tubular epithelial cells (HK-2) is an important cause of kidney stone formation. In this article, the repairing effect of polysaccharide (PCP0) extracted from the traditional Chinese medicine Poria cocos and its carboxymethylated derivatives on damaged HK-2 cells was studied, and the differences in adhesion and endocytosis of the cells to nanometer calcium oxalate monohydrate (COM) before and after repair were explored. Sodium oxalate (2.8mmol/L) was used to damage HK-2 cells to establish a damage model, and then Poria cocos polysaccharides (PCPs) with different carboxyl (COOH) contents were used to repair the damaged cells. The changes in the biochemical indicators of the cells before and after the repair and the changes in the ability to adhere to and internalize nano-COM were detected. The natural PCPs (PCP0, COOH content=2.56%) were carboxymethylated, and three carboxylated modified Poria cocos with 7.48% (PCP1), 12.07% (PCP2), and 17.18% (PCP3) COOH contents were obtained. PCPs could repair the damaged HK-2 cells, and the cell viability was enhanced after repair. The cell morphology was gradually repaired, the proliferation and healing rate were increased. The ROS production was reduced, and the polarity of the mitochondrial membrane potential was restored. The level of intracellular Ca2+ ions decreased, and the autophagy response was weakened. The cells repaired by PCPs inhibited the adhesion to nano-COM and simultaneously promoted the endocytosis of nano-COM. The endocytic crystals mainly accumulated in the lysosome. Inhibiting adhesion and increasing endocytosis could reduce the nucleation, growth, and aggregation of cell surface crystals, thereby inhibiting the formation of kidney stones. With the increase of COOH content in PCPs, its ability to repair damaged cells, inhibit crystal adhesion, and promote crystal endocytosis all increased, that is, PCP3 with the highest COOH content showed the best ability to inhibit stone formation.

Intracellular Ca2+ Signaling and Calcium Release-Activated Calcium Modulator ORAI1 Are Associated With CD4+ T Lymphocytes in Dairy Cows.

The nutritional status of dairy cows and the metabolism of specific nutrients are critical regulators of immune cell function. Around the time of parturition, mobilization of body lipid and muscle helps compensate for the decrease in nutrient intake and the increased requirements of the mammary gland for lactation. An end-result of these processes is the marked increase in circulating concentrations of fatty acids (FA), which are a major risk factor for immune dysfunction. In food animal species such as dairy cows, any disturbance in nutritional or immunological homeostasis leads to deleterious feedback loops that can further risk health, efficiency of nutrient use, and compromise availability of safe and nutritious dairy foods for humans. Despite substantial progress with respect to regulation of innate immunity, such knowledge for adaptive immunity is scarce. To help bridge this gap in knowledge, we sought to study the role of calcium release-activated calcium modulator ORAI1 activation in T cells systemic immune function in vivo. CD4+ T cells were isolated from peripheral blood of dairy cows diagnosed as healthy or with ketosis, a common metabolic disorder of FA metabolism. Results revealed that levels of intracellular Ca2+ and reactive oxygen species (ROS) along with the abundance of store-operated Ca2+ entry (SOCE) moiety increased during ketosis. Further, plasma concentrations of inflammatory cytokines were elevated, the balance of Th17/Treg cells was disrupted, mitochondrial function impaired, and the abundance of mitophagy-related proteins in CD4+ T cells altered during ketosis. Molecular characterization of the direct effects of FA was evaluated in CD4+ T cells isolated from the spleen of 1-day-old calves. Enhanced supply of FA increased intracellular Ca2+ and ROS concentrations, upregulated the abundance of proteins associated with mitochondrial dynamics and ORAI1. Intermediates of mitophagy accumulated and the balance of Th17/Treg cells also was affected by the supply of FA. These negative effects were attenuated by silencing or inhibition of ORAI1 in CD4+ T cells. Together, data indicated that physiological states that lead to increases in systemic concentrations of FA could impact adaptive immunity negatively through ORAI1 regulated intracellular Ca2+, ROS balance, and increased effector functions of Th17 cells.

GM2/GM3 controls the organizational status of CD82/Met microdomains: further studies in GM2/GM3 complexation

At cell surface gangliosides might associate with signal transducers proteins, grown factor receptors, integrins, small G-proteins and tetraspanins establishing microdomains, which play important role in cell adhesion, cell activation, motility, and growth. Previously, we reported that GM2 and GM3 form a heterodimer that interacts with the tetraspanin CD82, controlling epithelial cell mobility by inhibiting integrin-hepatocyte growth factor-induced cMet tyrosine kinase signaling. By using molecular dynamics simulations to study the molecular basis of GM2/GM3 interaction we demonstrate, here, that intracellular levels of Ca2+ mediate GM2/GM3 complexation via electrostatic interaction with their carboxyl groups, while hydrogen bonds between the ceramide groups likely aid stabilizing the complex. The presence of GM2/GM3 complex alters localization of CD82 on cell surface and therefore downstream signalization. These data contribute for the knowledge of how glycosylation may control signal transduction and phenotypic changes.

МЕХАНИЗМЫ МИТОХОНДРИАЛЬНОЙ ДИСФУНКЦИИ НЕЙРОНОВ ПРИ ВОЗДЕЙСТВИИ МЫШЬЯКА И АЛЮМИНИЯ (ОБЗОР)

Exposure to neurotropic chemicals (aluminum, arsenic, etc.) as a result of pollution of environmental objects can cause disruption of the bioenergetics of nerve cells. Objectives. To analyze and summarize the literature data on the mechanisms of the effects of arsenic and aluminum on the structure and functions of neuronal mitochondria. Sources of data: literature sources reflecting the mechanisms of the influence of these neurotoxicants on neuronal mitochondria. Methods. The basis of this study was the review of literature on this topic. Results. The influence of arsenic compounds on nerve cells causes mitochondrial dysfunction due to the activation of oxidative stress, an increase in the intracellular level of Ca&lt;sup&gt;2+&lt;/sup&gt;, a decrease in the mitochondrial membrane potential and the level of calpain 1, but aluminum compounds increase the formation of reactive oxygen species (ROS) and disrupt the activity of cytochrome c-oxidase and the energy-producing function of mitochondria in various types of neurons. Mitochondrial dysfunction, caused when exposed to these metals is accompanied by the decrease in ROS resynthesis and the activation of oxidative stress, which in its turn decreases energy generation in mitochondria still to a greater extent according to the mechanism of a vicious circle («CIRCULUS VITIOSUS»). Conclusions. The presented information deepens our knowledge about the mechanisms of neuronal bioenergetics disorders under the influence of arsenic and aluminum compounds, which is the basis for further research in order to develop effective methods of prevention, detoxification and antioxidant therapy for acute and chronic arsenic and aluminum poisoning and to implement the results obtained in practical healthcare.

Enhanced Responsive Formation of Extracellular Traps in Macrophages Previously Exposed to Porphyromonas gingivalis.

Tolerance is defined to be a hyporesponsive state following repeated stimulations with bacteria or their virulence factors and has potential impacts on the development of periodontitis. Recently, macrophages have been reported to release chromatin and antimicrobial peptides to form extracellular traps upon bacterial or chemical stimulations. Thus, we explored the roles and mechanisms of tolerance induced by Porphyromonas gingivalis (P. gingivalis) in macrophage extracellular traps (METs). Tolerance in peritoneal macrophages from mice was triggered by repeated P. gingivalis stimulation. METs were observed using fluorescence microscopy, and the levels of extracellular DNA were determined by microplate reader assays. The expression of p-RAF, p-MEK, and p-ERK was examined by Western blot, and reactive oxygen species (ROS) production was explored using flow cytometry. Moreover, the levels of intracellular Ca2+ were also determined by confocal microscopy to identify the possible mechanisms related to the changes in METs in P. gingivalis-pretreated macrophages. Repeated P. gingivalis stimulation contributed to the formation of METs and increased levels of extracellular DNA (p < 0.05). ROS generation and RAF/MEK/ERK phosphorylation were decreased in P. gingivalis-pretreated macrophages compared with non-pretreated cells (p < 0.05), which was inconsistent with the changes in METs. However, in P. gingivalis-pretreated macrophages, the levels of intracellular Ca2+ were significantly increased compared with the single stimulation group. Additionally, inhibition of intracellular Ca2+ resulted in a decrease in the levels of extracellular DNA in P. gingivalis-pretreated cells (p < 0.05). Taken together, P. gingivalis-pretreated macrophages released more METs, possibly related to the increased levels of intracellular Ca2+.

Clinical effect, changes in the lipid structure of the erythrocyte membrane and microcirculation in patients with arterial hypertension in combination with coronary heart disease under the influence of laser therapy

To evaluate the clinical effect of LT in patients with hypertension in combination with coronary artery disease and trace its relationship with the dynamics of the structure of the lipid bilayer of the erythrocyte membrane and changes in the microvascular bed. We examined 65 male patients (mean age 50.9±6.3 years) with II-III degree AH in combination with coronary artery disease with angina pectoris. Among them, 40 patients received a 10-day course of LT, and 25 patients underwent simulated laser irradiation. At the initial stage and after 1 month, all patients underwent a bicycle exercise test, a study of the lipid composition of the erythrocyte membrane, including the main fractions of phospholipids and free cholesterol, as well as the level of intracellular Ca2+ and lipid peroxidation products - malondialdehyde and diene conjugates. Microcirculation was assessed using the method of conjunctival biomicroscopy. One month after the course of LT, patients showed a significant increase in exercise tolerance by 37.8%, a decrease in systolic blood pressure with a standard load by 9.9%. The improvement of the clinical picture occurred against the background of a decrease in the activity of lipid peroxidation and structural changes in the cell membrane: an increase in polyunsaturated fractions of phospholipids and a decrease in the cholesterol content, as well as a decrease in the Ca2+ level in the cell from 0.23 [0.19; 0.32] to 0.20 [0.16; 0.26] mmol/l. The results of the analysis of conjunctival biomicroscopy demonstrated a statistically significant decrease in the ratio of arteriolovenular calibers, a limitation of the severity of sludge syndrome by 59%, and an almost twofold (from 3.9±0.52 to 7.2±1.23 cap/mm2) increase in capillary density. The data obtained showed that in patients with hypertension in combination with coronary artery disease, LT causes positive changes in the lipid structure of the cell membrane and microcirculation parameters, which is accompanied by a hypotensive effect and an improvement in the clinical and functional state of patients.

Molecular mechanisms underlying the calcium-mediated uptake of hematite nanoparticles by the ciliate Tetrahymena thermophila

In aquatic ecosystems, the calcium (Ca) concentration varies greatly. It is well known that Ca affects the aggregation of nanoparticles (NPs) and thus their bioaccumulation. Nevertheless, Ca also plays critical roles in various biological processes, whose effects on NP accumulation in aquatic organisms remain unclear. In this study, the effects of Ca on the uptake of polyacrylate-coated hematite NPs (HemNPs) by the aquatic ciliate Tetrahymena thermophila were investigated. At all of the tested Ca concentrations, HemNPs were well dispersed in the experimental medium, excluding the possibility of Ca to influence HemNP bioaccumulation by aggregating the NPs. Instead, Ca was shown to induce the clathrin-mediated endocytosis and phagocytosis of HemNPs. Manipulation of the Ca speciation in the experimental medium as well as the influx and intracellular availability of Ca in T. thermophila indicated that HemNP uptake was regulated by the intracellular Ca level. The results of the proteomics analyses further showed that the binding of intracellular Ca to calmodulin altered the activity of proteins involved in clathrin-mediated endocytosis (calcineurin and dynamin) and phagocytosis (actin). Overall, the biologically inductive effects of Ca on NP accumulation in aquatic organisms should be considered when evaluating the environmental risks of NPs.