Calmodulin Levels Research Articles

PRP4 Promotes Skin Cancer by Inhibiting Production of Melanin, Blocking Influx of Extracellular Calcium, and Remodeling Cell Actin Cytoskeleton.

Pre-mRNA processing factor 4B (PRP4) has previously been shown to induce epithelial-mesenchymal transition (EMT) and drug resistance in cancer cell lines. As melanin plays an important photoprotective role in the risk of sun-induced skin cancers, we have investigated whether PRP4 can induce drug resistance and regulate melanin biosynthesis in a murine melanoma (B16F10) cell line. Cells were incubated with a crucial melanogenesis stimulator, alpha-melanocyte-stimulating hormone, followed by transfection with PRP4. This resulted in the inhibition of the production of melanin via the downregulation of adenylyl cyclase-cyclic adenosine 3′,5′-monophosphate (AC)–(cAMP)–tyrosinase synthesis signaling pathway. Inhibition of melanin production by PRP4 leads to the promotion of carcinogenesis and induced drug resistance in B16F10 cells. Additionally, PRP4 overexpression upregulated the expression of β-arrestin 1 and desensitized the extracellular calcium-sensing receptor (CaSR), which in turn, inhibited the influx of extracellular Ca2+ ions. The decreased influx of Ca2+ was confirmed by a decreased expression level of calmodulin. We have demonstrated that transient receptor potential cation channel subfamily C member 1 was involved in the influx of CaSR-induced Ca2+ via a decreasing level of its expression. Furthermore, PRP4 overexpression downregulated the expression of AC, decreased the synthesis of cAMP, and modulated the actin cytoskeleton by inhibiting the expression of Ras homolog family member A (RhoA). Our investigation suggests that PRP4 inhibits the production of melanin in B16F10 cells, blocks the influx of Ca2+ through desensitization of CaSR, and modulates the actin cytoskeleton through downregulating the AC–cAMP pathway; taken together, these observations collectively lead to the promotion of skin carcinogenesis.

Study on a Plasmonic Tilted Fiber Grating-Based Biosensor for Calmodulin Detection.

Tilted fiber Bragg grating, which has the advantages of both fiber Bragg grating and long-period fiber grating, has been widely studied for sensing in many fields, especially in the field of biochemistry. Calmodulin, which has a wide distribution in eukaryotes, can regulate several enzymes such as adenylate cyclase and guanylate cyclase and mediates several cellular processes such as cell proliferation and cyclic nucleotide metabolism. The abnormal levels of calmodulin in the body will result in serious effects from metabolism to nerve growth and memory. Therefore, it is important to measure the calmodulin concentration in the body. In this work, we propose and experimentally demonstrate a plasmonic tilted fiber Bragg grating-based biosensor for calmodulin detection. The biosensor was made using an 18° tilted fiber Bragg grating with a 50 nm-thick gold nanofilm coating the surface of the fiber, and transient receptor potential channels were bonded onto the surface of the gold nanofilm to serve as bio-detectors for calmodulin detection. Experimental results showed that the limit of detection using our biosensor was 0.44 nM. Furthermore, we also demonstrated that the interaction between calmodulin and transient receptor potential channels was quite weak without calcium in the solution, which agrees with the biology. Our proposed biosensor has a simple structure, is easy to manufacture, and is of small size, making it a good choice for real-time, label-free, and microliter-volume biomolecule detection.

Involvement of mitochondrial fission in renal tubular pyroptosis in mice exposed to high and environmental levels of glyphosate combined with hard water

Chronic interstitial nephritis in agricultural communities (CINAC) has reached epidemic proportions. The combination of glyphosate and hard water has been postulated to play a potent aetiological role in CINAC. Therefore, dynamin-related protein 1 (Drp1)-mediated aberrant mitochondrial fission and subsequent activation of the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (Nlrp3)/caspase1 pathway may be involved in the pathogenesis of nephropathy. In the present study, mice were sub-chronically exposed to high doses and environmental levels of glyphosate (100 mg/kg body weight (mg/kg·bw) glyphosate in Roundup and 0.7 mg/L pure glyphosate, respectively) and hard water (2500 mg/L CaCO3 and 250 mg/L Ca2+, respectively) in drinking water. Moreover, Mdivi-1 (Md-1, 10 mg/kg·bw) was intraperitoneally injected to inhibit Drp1 on the basis of the high-dose experiment. Histopathological examination, biochemical analysis, ELISA, western blotting and fluorescent staining were used to analyse renal structure, renal tubular pyroptosis and mitochondrial fission/fusion alterations. The results showed dramatic proximal tubular injury, particularly in the combined groups. Moreover, significant increases in the protein expression levels of calmodulin (CaM), calmodulin-dependent protein kinase II (CaMKII), Drp1/p-Drp1-Ser616 and the Txnip/Nlrp3/caspase1 signalling pathway, and alterations in oxidative stress were observed in the combined groups, and these effects were attenuated by the Drp1 inhibitor Md-1. Intriguingly, there may be a synergistic effect of glyphosate and hard water on renal injury. Taken together, these results suggest that the combination of glyphosate and hard water, even at environmental exposure levels, enhances pyroptosis and ongoing tubulointerstitial inflammation through excessive Drp1-mediated mitochondrial fission.

Mycorrhiza-induced plant defence responses in trifoliate orange infected by Phytophthora parasitica

Arbuscular mycorrhizal fungi (AMF) reduce disease incidence of host plants through the competition of carbon sources and direct inhibition of pathogens, as well as through induction of biochemical and molecular responses. However, it is not known whether AMF enhance the resistance to Phytophthora parasitica-induced root rot in citrus and what the underlying mechanisms are. This study was carried out to analyze roles of Funneliformis mosseae (a mycorrhizal fungus) in plant defence responses of Poncirus trifoliata infected by P. parasitica. A week after the pathogen infection, mycorrhizal seedlings possessed higher expression of root mitogen-activated protein kinase 3 (PtMAPK3) regardless of P. parasitica infection. F. mosseae induced higher root salicylic acid (SA) concentrations, accompanied with up-regulation of SA synthesis genes (PtPAL1 and PtEPS1), regardless of being infected with P. parasitica or not. Jasmonic acid (JA) synthesis genes were down-regulated by mycorrhization in the absence of P. parasitica and up-regulated (except for PtAOC) by mycorrhization under P. parasitica infection. Moreover, F. mosseae stimulated higher expression of pathogenesis-related protein gene 1 (PtPR1), PtPR4, and PtPR5, especially under P. parasitica infection. F. mosseae inoculation increased levels of root lignin, calmodulin, and total soluble phenol and activities of root chitinase, phenylalanine ammonialyase, and β-1,3-glucanase, and decreased concentrations of root nitric oxide with or without P. parasitica infection. These results implied that F. mosseae elicited MAPKs cascades as well as SA- and calmodulin-mediated signal pathways to activate disease-defence genes, proteins, and compounds to early-warn P. parasitica infection for enhancing tolerance of root rot in trifoliate orange.

Calmodulin/CaMKII-γ mediates prosurvival capability in apicidin-persistent hepatocellular carcinoma cells via ERK1/2/CREB/c-fos signaling pathway.

Calmodulin (CaM), a Ca2+ binding protein, plays a critical role in cancer initiation and progression through binding and activating numerous target proteins, including Ca2+ /calmodulin-dependent protein kinase (CaMK) family proteins. However, the mechanisms underlying the effects of CaM/CaMKs on the survival capability of liver cancer cells is unclear, and this study investigates this mechanism in apicidin-persistent HA22T cells. CaM level was upregulated, especially in the cytosol, in apicidin-persistent HA22T cells than in parental HA22T cells and was positively associated with cell proliferation and migration capacity of apicidin-persistent HA22T cells. Further, the expression of CaM-activated CaMKs-dependent signaling cascades, includingCaMKK2, CaMKIV, CaMKII-γ, and p-CaMKIIwas observed in apicidin-persistent HA22T cells, which were transiently activated by mitogen-activated protein kinase oncogenic signaling, such as CREB, ERK1/2, and c-fos. Furthermore, a specific CaM inhibitor trifluoperazine reduced the levels of p-CREB, p-ERK1/2, and c-fos in apicidin-persistent HA22T cells than in parental HA22T cells. Additionally, inhibition of CaM also suppressed CaM-induced Bcl-XL (an antiapoptotic protein) expression in apicidin-persistent HA22T cells. Our finding emphasizes an essential role of CaM/CaMKs in augmentation of the survival capability of apicidin-persistent liver cancer cells and suggests that CaM inhibition significantly attenuates CaM-induced tumor growth and abrogates antiapoptotic function and also offers a promising therapeutic target for cancer treatment.

Calmodulin level is significantly increased in the cerebrospinal fluid of patients with sporadic Creutzfeldt-Jakob disease.

Human prion diseases (PrDs) are a group of fatal and transmissible neurodegenerative disorders that are diagnosed definitively in post mortem brains. Calmodulin (CaM) is a ubiquitous calcium-binding protein. Increased brain CaM level has been reported in prion-infected rodent models and some scrapie-infected cells. However, the putative alteration of CaM in cerebrospinal fluid (CSF) of human PrDs is uncertain. Here, we try to figure out the profiles of CSF CaM in sporadic Creutzfeldt-Jacob disease. Cerebrospinal fluid samples of 40 Chinese patients with probable sporadic Creutzfeldt-Jacob disease (sCJD) and 40 cases without sCJD (non-PrDs) were recruited in this study. The presence of CaM in the CSF was assessed by Western blot, while total tau levels were measured using an enzyme-linked immunosorbent assay kit. In addition, the presence of CaM in another CSF panel consisting of 30 definite sCJD cases and 30 non-PrD cases was evaluated using CaM-specific Western blot analysis. Cerebrospinal fluid CaM positivity was observed in 28/40 cases of probable sCJD and in 9/40 non-PrD cases. The CSF tau levels in the probable sCJD cases were markedly higher than those in the non-PrD cases. Logistic regression established a significant correlation between CSF CaM signal and total CSF tau level. Similar results were observed in the panel of cases with definite sCJD: the rates of CSF CaM positivity in the definite sCJD cases and the non-PrD cases were 22/30 and 6/30, respectively. Although CSF CaM positivity might not be a sCJD-specific phenomenon, a significantly high rate of CaM-positive CSF in sCJD cases, especially in those with high CSF tau levels, rendered it a valuable diagnostic biomarker for sCJD.

ITRAQ-based comparative proteomic analysis in different developmental stages of Echinococcus granulosus.

Cystic echinococcosis, caused by infection with the larval stage of the cestode Echinococcus granulosus, is a chronic zoonosis. The lifecycle of the E. granulosus parasite includes three consecutive stages that require specific gene regulation or protein expression to survive environmental shifts between definitive hosts and intermediate hosts. The aim of the present study is to screen and analyze the stage differential antigens to be considered for vaccine development against E. granulosus. By using the iTRAQ (isobaric tags for relative and absolute quantification) method, the differentially expressed proteins were selected from the three consecutive developmental stages of E. granulosus: oncosphere, adult tapeworms, and protoscolex. Through a bioinformatics analysis including Clusters of Orthologous Groups (COG), Gene Ontology (GO), and pathway metabolic annotation, we identified some proteins of interest from each stage. The results showed that a large number of differentially expressed proteins (375: oncosphere vs. adult, 346: oncosphere vs. protoscolex, and 391: adult vs. protoscolex) were identified from the three main lifecycle stages. Analysis of the differential protein pathways showed that these differential proteins are mainly enriched in metabolic pathways, Huntington’s diseases, Alzheimer’s diseases, and ribosome metabolic pathways. Interestingly, among these differential proteins, expression levels of paramyosin, HSP60, HSP70, HSP90, cathepsin L1, cathepsin D, casein kinase, and calmodulin were significantly higher in the oncosphere than in the adult or protoscolex (p < 0.05). We hope our findings will help to identify potential targets for diagnosis or for therapeutic and prophylactic intervention.

Modulation of calcium-binding proteins expression and cisplatin chemosensitivity by calcium chelation in human breast cancer MCF-7 cells

Cisplatin (CDDP) is currently one of the most effective FDA-approved treatments for breast cancer. Previous studies have shown that CDDP-induced cell death in human breast cancer (MCF-7) cells is associated with disruption of calcium homeostasis. However, whether the sensitivity of breast cancer cells to cisplatin is associated with dysregulation of the expression of calcium-binding proteins (CaBPs) remains unknown. In this study, we evaluated the effect of the intracellular calcium chelator (BAPTA-AM) on viability of MCF-7 cells in the presence of toxic and sub-toxic doses of cisplatin. Furthermore, this study assessed the expression of CaBPs, calmodulin, S100A8, and S100A14 in MCF-7 cells treated with cisplatin. Cell viability was determined using MTT-based in vitro toxicity assay. Intracellular calcium imaging was done using Fluo-4 AM, a cell-permeant fluorescent calcium indicator. Expression of CaBPs was tested using real-time quantitative PCR. Exposure of cells to increasing amounts of CDDP correlated with increasing fluorescence of the intracellular calcium indicator, Fluo-4 AM. Conversely, treating cells with cisplatin significantly decreased mRNA levels of calmodulin, S100A8, and S100A14. Treatment of the cells with calcium chelator, BAPTA-AM, significantly enhanced the cytotoxic effects of sub-toxic dose of cisplatin. Our results indicated a statistically significant negative correlation between calmodulin, S100A8, and S100A14 expression and sensitivity of breast cancer cells to a sub-toxic dose of cisplatin. We propose that modulating the activity of calcium-binding proteins, calmodulin, S100A8, and S100A14, could be used to increase cisplatin efficacy, lowering its treatment dosage while maintaining its chemotherapeutic value.

Lactobacillus plantarum TWK10-fermented soymilk improves cognitive function in type 2 diabetic rats.

The brain is especially sensitive to diabetes-induced damage. Chronic hyperglycemia can potentially lead to brain dysfunctions, affecting spatial learning and memory. The type 2 diabetes (T2D) rats were administered TWK10-fermented soy milk water extract (WE) and ethanol extract (EE) for 6 weeks. WE and EE treatment attenuated T2D-induced alteration in cognitive function assessed using the Morris water maze. Moreover, administration of WE and EE significantly elevated superoxide dismutase activity (166.96% and 181.21%, P < 0.05, respectively) and reduced malondialdehyde concentration (35.03% and 43.97%, P < 0.05, respectively) in the hippocampus of the rats. Additionally, the calmodulin level and nitric oxide concentration were regulated by WE and EE. This study provides scientific evidence that WE and EE enhance anti-oxidative enzyme activity, which subsequently regulates factors associated with cognitive function in T2D rats. © 2020 Society of Chemical Industry.

The Regulating Mechanism of Chrysophanol on Protein Level of CaM-CaMKIV to Protect PC12 Cells Against Aβ25-35-Induced Damage.

ObjectiveTo investigate the neuroprotective effect of chrysophanol (CHR) on PC12 treated with Aβ25-35, and the involved mechanism.MethodsAfter the establishment of an AD cell model induced by Aβ25-35, the cell survival rate was detected by MTT, cell apoptosis was assayed by Hoechst 33342 staining, mRNA expressions of calmodulin (CaM), calcium/calmodulin-dependent protein kinase kinase (CaMKK), calcium/calmodulin-dependent protein kinase IV (CaMKIV) and tau (MAPT; commonly known as tau) were determined by qRT-PCR, and protein levels of CaM, CaMKK, CaMKIV, phospho-CaMKIV (p-CaMKIV), tau and phospho-tau (p-tau) were detected by Western blot analysis.ResultsWhen pretreated with CHR before exposure to Aβ25-35, PC12 cells showed that increased cell viability and reduced apoptosis. The qRT-PCR results indicated that the deposition of Aβ25-35 triggers a decrease in levels of CaM, CaMKK, CaMKIV, and tau in PC12 cells. In addition, Western blot results also suggested that Aβ25-35 decreases the protein expression of CaM, CaMKK, CaMKIV, p-CaMKIV, and the ratio of p-tau to tau in PC12 cells. However, the above effects were significantly alleviated after the treatment of CHR.ConclusionCHR plays a neuroprotective role in AD though decreasing the protein level of CaM-CaMKK-CaMKIV and the expression of p-tau downstream.

Calmodulin inhibition as a mode of action of antifungal imidazole pharmaceuticals in non-target organisms

To improve assessment of risks associated with pharmaceutical contamination of the environment, it is crucial to understand effects and mode of action of drugs in non-target species. The evidence is accumulating that species with well-conserved drug targets are prone to be at risk when exposed to pharmaceuticals. An interesting group of pharmaceuticals released into the environment is imidazoles, antifungal agents with inhibition of ergosterol synthesis as a primary mode of action in fungi. However, imidazoles have also been identified as competitive antagonists of calmodulin (CaM), a calcium-binding protein with phylogenetically conserved structure and function. Therefore, imidazoles would act as CaM inhibitors in various organisms, including those with limited capacity to synthesize sterols, such as arthropods. We hypothesized that effects observed in crustaceans exposed to imidazoles are related to the CaM inhibition and CaM-dependent nitric oxide (NO) synthesis. To test this hypothesis, we measured (i) CaM levels and its gene expression, (ii) NO accumulation and (iii) gene expression of NO synthase (NOS1 and NOS2), in the cladoceran Daphnia magna exposed to miconazole, a model imidazole drug. Whereas significantly increased CaM gene expression and its cellular allocation were observed, supporting the hypothesized mode of action, no changes occurred in either NO synthase expression or NO levels in the exposed animals. These findings suggest that CaM inhibition by miconazole leads to protein overexpression that compensates for the loss in the protein activity, with no measurable downstream effects on NO pathways. The inhibition of CaM in D. magna may have implications for effect assessment of exposure to mixtures of imidazoles in aquatic non-target species.

Allopregnanolone Modulates GABAAR-Dependent CaMKIIδ3 and BDNF to Protect SH-SY5Y Cells Against 6-OHDA-Induced Damage.

Allopregnanolone (APα), as a functional neurosteroid, exhibits the neuroprotective effect on neurodegenerative diseases such as Parkinson’s disease (PD) through γ-aminobutyric acid A receptor (GABAAR), but it has not been completely understood about its molecular mechanisms. In order to investigate the neuroprotective effect of APα, as well as to clarify its possible molecular mechanisms, SH-SY5Y neuronal cell lines were incubated with 6-hydroxydopamine (6-OHDA), which has been widely used as an in vitro model for PD, along with APα alone or in combination with GABAAR antagonist (bicuculline, Bic), intracellular Ca2+ chelator (EGTA) and voltage-gated L-type Ca2+ channel blocker (Nifedipine). The viability, proliferation, and differentiation of SH-SY5Y cells, the expression levels of calmodulin (CaM), Ca2+/calmodulin-dependent protein kinase II δ3 (CaMKIIδ3), cyclin-dependent kinase-1 (CDK1) and brain-derived neurotrophic factor (BDNF), as well as the interaction between CaMKIIδ3 and CDK1 or BDNF, were detected by morphological and molecular biological methodology. Our results found that the cell viability and the number of tyrosine hydroxylase (TH), bromodeoxyuridine (BrdU) and TH/BrdU-positive cells in 6-OHDA-treated SH-SY5Y cells were significantly decreased with the concomitant reduction in the expression levels of aforementioned proteins, which were ameliorated following APα administration. In addition, Bic could further increase the number of TH or BrdU-positive cells as well as the expression levels of aforementioned proteins except for TH/BrdU-double positive cells, while EGTA and Nifedipine could attenuate the expression levels of CaM, CaMKIIδ3 and BDNF. Moreover, there existed a direct interaction between CaMKIIδ3 and CDK1 or BDNF. As a result, APα-induced an increase in the number of TH-positive SH-SY5Y cells might be mediated through GABAAR via Ca2+/CaM/CaMKIIδ3/BDNF (CDK1) signaling pathway, which would ultimately facilitate to elucidate PD pathogenesis and hold a promise as an alternative therapeutic target for PD.

Mycorrhiza Regulates Signal Substance Levels and Pathogen Defense Gene Expression to Resist Citrus Canker

Citrus canker is a quarantined disease, severely harming citrus plants. Soil beneficial arbuscular mycorrhizal fungi (AMF) can provide a biological control pathway to resist pathogens. This work was to test changes of signal substances including hydrogen peroxide (H2O2), nitric oxide (NO), calmodulin (CaM), salicylic acid (SA) and jasmonic acid (JA) and the pathogen defense gene expression in roots of AMF (Paraglomus occultum) and non-AMF trifoliate orange (Poncirus trifoliata) seedlings after infected by a expressions citrus canker pathogen (Xanthomonas axonopodis pv. Citri, Xac). AMF inoculation significantly improved plant height, stem diameter and leaf number. Xac infection dramatically decreased root H2O2, NO, and SA levels, but increased root CaM and JA concentrations in non-AMF seedlings. There were higher H2O2 and CaM levels and lower JA levels in Xac-infected seedlings than in non-Xac-infected seedlings under mycorrhization. Under non-Xac infection, mycorrhizal treatment reduced root H2O2, NO, and SA but increased CaM and JA levels. However, under Xac infection, mycorrhizal inoculation distinctly accelerated root H2O2, NO, CaM, and SA accumulation, accompanied with up-regulated expression levels of root PtEPS1 (enhance pseudomonas susceptibility 1) and PtPR4 (pathogenesis related gene 4), indicating that Xac stimulated mycorrhizal roles in enhancing resistance of citrus canker. Such results imply that citrus plants with pre-inoculated AMF had stronger resistance to Xac infection through increasing signal substrate accumulation and pathogen defense gene expressions.&#x0D; &#x0D; *********&#x0D; In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue.&#x0D; *********

Effect of hydrogen sulfide on the intimal hyperplasia of vein grafts in rabbits

Objective To observe the effect of exogenous hydrogen sulfide (H2S) on the intimal hyperplasia of vein grafts in rabbits and and its possible molecular mechanism. Methods Forty New Zealand rabbits were randomly divided into 5 group, including blank control group (Ctr group); two weeks of simple transplantation; four weeks of simple transplantation; transplantation + NaHS 2 weeks group (NaHS: 30 μmol/kg. 3 days, 2 weeks of continuous administration), transplantation + NaHS 4 weeks group (NaHS: 30 μmol/kg. 3 days, 4 weeks of continuous administration). Rabbit jugular vein transplantation animal model was established. Administration by intraperitoneal injection once 3 days. Serum H2S level was detected by enzyme linked immunosorbent assay (ELISA). The structure and hyperplasia of vascular tissue were observed by hematoxylin-eosin staining (HE) staining. real-time quantitative polymerase chain reaction (Real-time PCR) was used to determine the expression levels of cystathionine-gamma-lyase (CSE) mRNA. CSE, Calmodulin (p-CaM/ CaM)and Cyclin D1 protein levels were measured by Western blotting. Results After NaHS treatment, the intimal hyperplasia of vein grafts was relieved. Compared with the corresponding group, the serum H2S level increased in the transplantation + NaHS 2 weeks group and transplantation + NaHS 4weeks group (33.65±4.10 vs. 23.56±4.82, P<0.05; 36.50±4.62 vs. 23.72±3.95, P<0.05). And the expression of CSE mRNA and protein increased significantly (mRNA: 0.54±0.04 vs. 0.15±0.05, P<0.05; 1.08±0.13 vs. 0.11±0.02, P<0.05; Protein: 0.56±0.06 vs. 0.47±0.03, P<0.05; 1.14±0.07 vs. 0.38±0.04, P<0.05). The expression of p-CaM/CaM and Cyclin D1 protein decreased significantly (p-CaM: 0.25±0.05 vs. 0.52±0.06, P<0.05; 1.21±0.09 vs. 2.39±0.15, P<0.05; CaM: 0.42±0.04 vs. 0.73±0.07, P<0.05, 0.47±0.05 vs. 1.17±0.06 P<0.05; Cyclin D1: 0.28±0.06 vs. 0.95±0.11, P<0.05; 0.29±0.04 vs. 1.02±0.10, P<0.05). Conclusion Exogenous hydrogen sulfide could inhibit the intima proliferation of vein grafts which related to the reduction of the expression of p-CaM/CaM and Cyclin D1. Key words: Hydrogen sulfide; Vein grafts; Transplantation; Intima hyperplasia

Anthocyanins Extracted from Aronia melanocarpa Protect SH-SY5Y Cells against Amyloid-beta (1-42)-Induced Apoptosis by Regulating Ca2+ Homeostasis and Inhibiting Mitochondrial Dysfunction.

We investigated the cytoprotective effects of anthocyanins in Aronia melanocarpa against apoptosis induced by Aβ1-42, a key mediator of AD pathophysiology. We measured intracellular calcium with a colorimetric kit, cellular apoptosis with DAPI, intracellular ROS with the fluorescent marker 2,3-dimethoxy-1,4-naphthoquinone, mitochondrial membrane potential with JC-1, and ATP with a colorimetric kit. Gene transcription and protein expression levels of calmodulin, cytochrome c, caspase-9, cleaved caspase-3, Bcl-2, and Bax were analyzed by RT-PCR and Western blotting. The results showed that pretreatment with anthocyanins significantly inhibited Aβ1-42-induced apoptosis, decreased intracellular calcium and ROS, and increased ATP and mitochondrial membrane potential. RT-PCR and Western blotting revealed that anthocyanins upregulated the gene transcription and protein expression of calmodulin and Bcl-2 and downregulated those of cytochrome c, caspase-9, cleaved caspase-3, and Bax. A. melanocarpa anthocyanins protected SH-SY5Y cells against Aβ1-42-induced apoptosis by regulating Ca2+ homeostasis and apoptosis-related genes and inhibiting mitochondrial dysfunction.

336 - Oxidation of Methionine 77 in calmodulin alters mouse growth and behavior

Calmodulin is a ubiquitous signaling protein that upon binding calcium interacts with its intracellular targets, of which over 350 have been described. Methionine 77 in calmodulin can be stereospecifically oxidized to methionine sulfoxide by mammalian methionine sulfoxide reductase A. Whether this has in vivo significance is unknown, we therefore created a mutant mouse in which wild type calmodulin-1 was replaced by a calmodulin containing a mimic of methionine sulfoxide at residue 77. Total calmodulin levels were unchanged in the homozygous M77Q mutant, which is viable and fertile. No differences were observed on learning tests, including the Morris water maze and associative learning. Cardiac stress test results were also the same for mutant and wild type mice. However, young male and female mice were 20% smaller than wild type mice, although food intake was normal for their weight. Young M77Q mice were notably more active and exploratory than wild type mice. This behavior difference was objectively documented on the treadmill and open field tests. The mutant mice ran 20% longer on the treadmill than controls, and in the open field test, the mutant mice explored more than controls and exhibited reduced anxiety. These phenotypic differences bore a similarity to those observed in mice lacking calcium/calmodulin kinase Iiα (CaMKIIα). We then showed that M77Q calmodulin was less effective in activating CaMKIIα than wild type calmodulin. Thus, characterization of the phenotype of a mouse expressing a constitutively active mimic of calmodulin led to the identification of the first calmodulin target that can be differentially regulated by the oxidation state of Met77. We conclude that reversible oxidation of methionine 77 in calmodulin by MSRA can regulate cellular function.

Oxidation of Methionine 77 in Calmodulin Alters Mouse Growth and Behavior.

Methionine 77 in calmodulin can be stereospecifically oxidized to methionine sulfoxide by mammalian methionine sulfoxide reductase A. Whether this has in vivo significance is unknown. We therefore created a mutant mouse in which wild type calmodulin-1 was replaced by a calmodulin containing a mimic of methionine sulfoxide at residue 77. Total calmodulin levels were unchanged in the homozygous M77Q mutant, which is viable and fertile. No differences were observed on learning tests, including the Morris water maze and associative learning. Cardiac stress test results were also the same for mutant and wild type mice. However, young male and female mice were 20% smaller than wild type mice, although food intake was normal for their weight. Young M77Q mice were notably more active and exploratory than wild type mice. This behavior difference was objectively documented on the treadmill and open field tests. The mutant mice ran 20% longer on the treadmill than controls and in the open field test, the mutant mice explored more than controls and exhibited reduced anxiety. These phenotypic differences bore a similarity to those observed in mice lacking calcium/calmodulin kinase IIα (CaMKIIα). We then showed that MetO77 calmodulin was less effective in activating CaMKIIα than wild type calmodulin. Thus, characterization of the phenotype of a mouse expressing a constitutively active mimic of calmodulin led to the identification of the first calmodulin target that can be differentially regulated by the oxidation state of Met77. We conclude that reversible oxidation of methionine 77 in calmodulin by MSRA has the potential to regulate cellular function.

Mechanism of nitric oxide and acid-sensing ion channel 1a modulation of panic-like behaviour in the dorsal periaqueductal grey of the mouse

Predators induce defensive responses and fear behaviours in prey. The rat exposure test (RET) is frequently used as an animal model of panic. Nitric oxide (NO) which has been reported to be activated by the NMDA receptor, in turn mediates calcium/calmodulin-dependent protein kinase II (CaMKII) signalling pathways in defensive responses. ACCN2, the orthologous human gene of acid-sensing ion channel 1a (ASIC1a), is also associated with panic disorder; however, few studies have focused on the role of ASIC1a in the modulation of panic and calcium/CaMKII signalling by NO. In the present study, NG-nitro-L-arginine-methyl-ester (L-NAME; non-selective NOS inhibitor), S-nitroso-N-acetyl-D,L-penicillamine (SNAP; NO donor), and psalmotoxin (PcTx-1; selective ASIC1a blocker) were administered to the dorsal periaqueductal grey (dPAG) before the predator stimulus, and the roles of NO in the expression of ASIC1a, phosphorylation of CaMKIIα (p-CaMKIIα) and expression of calmodulin (CaM) were investigated. The effects of ASIC1a, p-CaMKIIα and CaM regulation were also examined. Our results showed that intra-dPAG infusion of L-NAME weakened panic-like behaviour and decreased ASIC1a, p-CaMKIIα and CaM expression levels, whereas intra-dPAG infusion of SNAP enhanced panic-like behaviour and increased ASIC1a, p-CaMKIIα and CaM levels. Intra-dPAG infusion of PcTx-1 also weakened panic-like behaviour and decreased p-CaMKIIα expression level. Taken together, these results indicate that NO and ASIC1a are involved in the modulation of RET-induced panic-like behaviour in the dPAG. NO regulates the calcium/CaMKII signalling pathways, and ASIC1a participates in this regulation.

Failure to upregulate calmodulin underlies the suppressed KCa3.1 function and enhanced sensitivity to adenosine in CD8+ T cells of head and neck cancer patients

Abstract The limited ability of the cytotoxic CD8+ T cells to infiltrate solid tumors presents a major roadblock to developing effective immunotherapy. Adenosine (Ado) accumulates in high concentrations in solid tumors where it contributes to the suppression of T cell function. We have previously shown that Ado reduces the chemotactic ability of peripheral blood CD8+ T cells (PBTs) from head and neck squamous cell carcinoma (HNSCC) patients by suppressing KCa3.1 channel function. Herein, we conducted experiments to elucidate the mechanism of KCa3.1 dysregulation in HNSCC PBTs. KCa3.1 channels are calcium-activated and require binding of calmodulin (CAM). PBTs were isolated from HNSCC patients and healthy donors (HD), and activated with CD3/CD28 antibodies. CAM levels decreased post-activation in HNSCC PBTs (by ~24%, n=7) while they increased in HD PBTs (by ~37%, n=6, p=0.001). To study whether CAM downregulation contributes to KCa3.1 dysfunction, we transfected HD PBTs with siRNA against CAM (siCam). siCam transfection decreased CAM expression and KCa3.1 currents, but not KCa3.1 expression. We then studied whether CAM downregulation affects the T cell chemotactic response to Ado in HD PBTs. Control (scrRNA-transfected) HD PBTs migrated towards CXCL12 in the presence of Ado, but downregulation of CAM abrogated their chemotactic ability in the presence of Ado (~71% inhibition, n=8, p&amp;lt;0.012). Activation of KCa3.1 channels with 1-EBIO restored the ability of siCam-transfected PBTs to migrate towards CXCL12 in the presence of Ado. Our data suggest that downregulation of CAM decreases KCa3.1 activity and suppresses chemotaxis in HNSCC PBTs which may contribute to their limited ability to effectively penetrate Ado-rich tumors.

Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Increases Small-Conductance Ca2+-Activated K+ Current in Patients with Chronic Atrial Fibrillation.

BackgroundIncreased small-conductance Ca2+-activated K+ current (SK), abnormal intracellular Ca2+ handling, and enhanced expression and activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been found in clinical and/or experimental models of atrial fibrillation (AF), but the cumulative effect of these phenomena and their mechanisms in AF are still unclear. This study aimed to test the hypothesis that CaMKII increases SK current in human chronic AF.Material/MethodsRight atrial appendage tissues from patients with either sinus rhythm (SR) or AF and neonatal rat atrial myocytes were used. Patch clamp, qRT-PCR, and Western blotting techniques were used to perform the study.ResultsCompared to SR, the apamin-sensitive SK current (IKAS) was significantly increased, but the mRNA and protein levels of SK1, SK2, and SK3 were significantly decreased. In AF, the steady-state Ca2+ response curve of IKAS was shifted leftward and the [Ca2+]i level was significantly increased. CaMKII inhibitors (KN-93 or autocamtide-2-related inhibitory peptide (AIP)) reduced the IKAS in both AF and SR. The inhibitory effect of KN-93 or AIP on IKAS was greater in AF than in SR. The expression levels of calmodulin, CaMKII, and autophosphorylated CaMKII at Thr287 (but not at Thr286) were significantly increased in AF. Furthermore, KN-93 inhibited the expression of (Thr287)p-CaMKII and SK2 in neonatal rat atrial myocytes.ConclusionsSK current is increased via the enhanced activation of CaMKII in patients with AF. This finding may explain the difference between SK current and channels expression in AF, and thus may provide a therapeutic target for AF.