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1.
Int J Mol Sci ; 20(24)2019 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-31847351

RESUMO

Pleurotus ostreatus is a widely cultivated edible fungus around the world. At present, studies on the developmental process of the fruiting body are limited. In our study, we compared the differentially expressed proteins (DEPs) in the stipe and cap of the fruiting body by high-throughput proteomics. GO and pathway analysis revealed the great differences in the metabolic levels, including sucrose and starch metabolism, and sphingolipid signaling and metabolism, and the differences of 16 important DEPs were validated further by qPCR analysis in expression level. In order to control the cap and stipe development, several chemical inducers were applied to the primordium of the fruiting body according to the pathway enrichment results. We found that CaCl2 can affect the primordium differentiation through inhibiting the stipe development. EGTA (ethyleneglycol bis (ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid) treatment confirmed the inhibitory role of Ca2+ in the stipe development. Our study not only shows great metabolic differences during the cap and stipe development but also reveals the underlying mechanism directing the primordium differentiation in the early development of the fruiting body for the first time. Most importantly, we provide a reliable application strategy for the cultivation and improvement of the Pleurotus ostreatus, which can be an example and reference for a more edible fungus.


Assuntos
Cálcio/metabolismo , Carpóforos/metabolismo , Pleurotus/crescimento & desenvolvimento , Pleurotus/metabolismo , Cloreto de Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Ácido Egtázico/farmacologia , Carpóforos/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/fisiologia , Pleurotus/efeitos dos fármacos , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Esfingolipídeos/metabolismo
2.
Vet Res ; 50(1): 110, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856906

RESUMO

Intestinal epithelium functions as a barrier to protect multicellular organisms from the outside world. It consists of epithelial cells closely connected by intercellular junctions, selective gates which control paracellular diffusion of solutes, ions and macromolecules across the epithelium and keep out pathogens. Rotavirus is one of the major enteric viruses causing severe diarrhea in humans and animals. It specifically infects the enterocytes on villi of small intestines. The polarity of rotavirus replication in their target enterocytes and the role of intestinal epithelial integrity were examined in the present study. Treatment with EGTA, a drug that chelates calcium and disrupts the intercellular junctions, (i) significantly enhanced the infection of rotavirus in primary enterocytes, (ii) increased the binding of rotavirus to enterocytes, but (iii) considerably blocked internalization of rotavirus. After internalization, rotavirus was resistant to EGTA treatment. To investigate the polarity of rotavirus infection, the primary enterocytes were cultured in a transwell system and infected with rotavirus at either the apical or the basolateral surface. Rotavirus preferentially infected enterocytes at the basolateral surface. Restriction of infection through apical inoculation was overcome by EGTA treatment. Overall, our findings demonstrate that integrity of the intestinal epithelium is crucial in the host's innate defense against rotavirus infection. In addition, the intercellular receptor is located basolaterally and disruption of intercellular junctions facilitates the binding of rotavirus to their receptor at the basolateral surface.


Assuntos
Enterócitos/virologia , Células Epiteliais/virologia , Mucosa Intestinal/citologia , Rotavirus/classificação , Rotavirus/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura/veterinária , Ácido Egtázico/farmacologia , Enterócitos/efeitos dos fármacos , Miofibroblastos/fisiologia , Suínos , Internalização do Vírus , Replicação Viral
3.
Integr Biol (Camb) ; 11(8): 342-352, 2019 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-31724713

RESUMO

Throughout the body, epithelial tissues contain curved features (e.g. cysts, ducts and crypts) that influence cell behaviors. These structures have varied curvature, with flat structures having zero curvature and structures such as crypts having large curvature. In the ovary, cortical inclusion cysts (CICs) of varying curvatures are found, and fallopian tube epithelial (FTE) cells have been found trapped within these cysts. FTE are the precursor for ovarian cancer, and the CIC niche has been proposed to play a role in ovarian cancer progression. We hypothesized that variations in ovarian CIC curvature that occur during cyst resolution impact the ability of trapped FTE cells to invade into the surrounding stroma. Using a lumen model in collagen gels, we determined that increased curvature resulted in more invasions of mouse FTE cells. To isolate curvature as a system parameter, we developed a novel technique to pattern concave curvatures into collagen gels. When FTE cells were seeded to confluency on curved substrates, increases in curvature increased the number of invading FTE cells and the invasion distance. FTE invasion into collagen substrates with higher curvature depended on matrix metalloproteinases (MMPs), but expression of collagen I degrading Mmps was not different on curved and flat regions. A finite-element model predicted that contractility and cell-cell connections were essential for increased invasion on substrates with higher curvature, while cell-substrate interactions had minimal effect. Experiments supported these predictions, with invasion decreased by blebbistatin, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or N-cadherin-blocking antibody, but with no effect from a focal adhesion kinase inhibitor. Finally, experimental evidence supports that cell invasion on curved substrates occurs in two phases-a cell-cell-dependent initiation phase where individual cells break away from the monolayer and an MMP-dependent phase as cells migrate further into the collagen matrix.


Assuntos
Células Epiteliais/citologia , Tubas Uterinas/patologia , Cistos Ovarianos/patologia , Ovário/patologia , Animais , Caderinas/metabolismo , Adesão Celular , Comunicação Celular , Colágeno/metabolismo , Progressão da Doença , Ácido Egtázico/farmacologia , Tubas Uterinas/metabolismo , Feminino , Análise de Elementos Finitos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Microfluídica , Microscopia Confocal , Neoplasias Ovarianas/patologia , Fenótipo
4.
ACS Appl Mater Interfaces ; 11(43): 39574-39585, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31589019

RESUMO

In the past few decades, intracellular calcium overload has been shown to induce cell death through multiple signaling pathways. In this study, we used BAPTA-AM, a well-known membrane-permeable Ca2+ chelator, to prevent cell injury by allaying the intracellular calcium overload. We explored the clinical potentials of BAPTA-AM-loaded liposome (BAL) in the treatment of the acute liver failure (ALF) mouse model, which is characterized by severe hepatic necrosis and apoptosis. We discovered that BAL can significantly inhibit D-GalN-induced LO2 cell damage as it increased cell viability by 60% and downregulated the LPS-stimulated inflammatory response in RAW 264.7 macrophages by reversing the morphological change and modulating TNF-α and NF-κB expressions. Through systemic administration, BAL can rapidly accumulate in damaged liver tissue and exhibit excellent treatment effects on the D-GalN/LPS-induced ALF mouse model, including elevation of the survival rate (from 10 to 80%), recovery of normal liver indexes and liver health indicators, improvement of liver blood microcirculation (increased the blood flow volume by 80% and flow rate by 60%), and blood coagulation. The underlying hepatoprotective effect of BAL is presumably based on the antinecrosis and antiapoptosis abilities attributed to its inhibition on oxidative stress, restriction on TNF-α receptor, and mitochondria-mediated apoptotic pathway by effectively clearing the overloaded intercellular calcium. BAL holds great potential as a new therapeutic strategy for ALF treatment, and its prominent cell rescue ability provides ample opportunities for the treatment of many other diseases that are characterized by rapid and massive cell damage.


Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Ácido Egtázico/análogos & derivados , Falência Hepática Aguda , Animais , Ácido Egtázico/química , Ácido Egtázico/farmacologia , Lipopolissacarídeos/toxicidade , Lipossomos , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/tratamento farmacológico , Falência Hepática Aguda/metabolismo , Falência Hepática Aguda/patologia , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , NF-kappa B/metabolismo , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismo
5.
EMBO Rep ; 20(12): e47755, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31663248

RESUMO

The spatial and temporal dynamics of cell contractility plays a key role in tissue morphogenesis, wound healing, and cancer invasion. Here, we report a simple optochemical method to induce cell contractions in vivo during Drosophila morphogenesis at single-cell resolution. We employed the photolabile Ca2+ chelator o-nitrophenyl EGTA to induce bursts of intracellular free Ca2+ by laser photolysis in the epithelial tissue. Ca2+ bursts appear within seconds and are restricted to individual target cells. Cell contraction reliably followed within a minute, causing an approximately 50% drop in the cross-sectional area. Increased Ca2+ levels are reversible, and the target cells further participated in tissue morphogenesis. Depending on Rho kinase (ROCK) activity but not RhoGEF2, cell contractions are paralleled with non-muscle myosin II accumulation in the apico-medial cortex, indicating that Ca2+ bursts trigger non-muscle myosin II activation. Our approach can be, in principle, adapted to many experimental systems and species, as no specific genetic elements are required.


Assuntos
Drosophila melanogaster/citologia , Células Epiteliais/fisiologia , Animais , Animais Geneticamente Modificados , Fenômenos Biomecânicos , Quelantes de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Forma Celular/efeitos dos fármacos , Forma Celular/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Miosina Tipo II/fisiologia , Processos Fotoquímicos , Análise de Célula Única , Análise Espaço-Temporal
6.
Toxicon ; 167: 123-133, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31181295

RESUMO

Bacillus thuringiensis crystal (Cry) proteins, used for decades as insecticidal toxins, are well known to be toxic to certain insects, but not to mammals. A novel group of Cry toxins called parasporins possess a strong cytocidal activity against some human cancer cells. Cry41Aa, or parasporin3, closely resembles commercially used insecticidal toxins and yet is toxic to the human hepatic cancer cell line HepG2, disrupting membranes of susceptible cells, similar to its insecticidal counterparts. In this study, we explore the protective effect that the common divalent metal chelator EGTA exerts on Cry41Aa's activity on HepG2 cells. Our results indicate that rather than interfering with a signalling pathway as a result of chelating cations in the medium, the chelator prevented the toxin's interaction with the membrane, and thus the subsequent steps of membrane damage and p38 phosphorylation, by removing cations bound to plasma membrane components. BAPTA and DTPA also inhibited Cry41Aa toxicity but at higher concentrations. We also show for the first time that Cry41Aa induces pore formation in planar lipid bilayers. This activity is not altered by EGTA, consistent with a biological context of chelation. Salt supplementation assays identified Ca2+, Mn2+ and Zn2+ as being able to reinstate Cry41Aa activity. Our data suggest the existence of one or more metal cation-dependent receptors in the Cry41Aa mechanism of action.


Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Membrana Celular/efeitos dos fármacos , Quelantes/farmacologia , Ácido Egtázico/farmacologia , Bicamadas Lipídicas/química , Substâncias Protetoras/farmacologia , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Membrana Celular/química , Células Hep G2 , Humanos , Íons , Modelos Moleculares , Técnicas de Patch-Clamp
7.
J Biol Chem ; 294(30): 11445-11457, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31171717

RESUMO

Most members of the family of proteins containing a transmembrane BAX inhibitor motif (TMBIM) have anti-apoptotic activity, but their in vivo functions and intracellular mechanisms remain obscure. Here, we report that zebrafish Tmbim3a/Grinaa functions in the prevention of cold-induced endoplasmic reticulum (ER) stress and apoptosis. Using a gene-trapping approach, we obtained a mutant zebrafish line in which the expression of the tmbim3a/grinaa gene is disrupted by a Tol2 transposon insertion. Homozygous tmbim3a/grinaa mutant larvae exhibited time-dependently increased mortality and apoptosis under cold exposure (at 16 °C). Mechanistically, using immunofluorescence, fluorescence-based assessments of intracellular/mitochondrial Ca2+ levels, mitochondrial membrane potential measurements, and Ca2+-ATPase assays, we found that cold exposure suppresses sarcoplasmic/ER Ca2+-ATPase (SERCA) activity and induces the unfolded protein response (UPR) and ER stress. We also found that the cold-induced ER stress is increased in homozygous tmbim3a/grinaa mutant embryos. The cold-stress hypersensitivity of the tmbim3a/grinaa mutants was tightly associated with disrupted intracellular Ca2+ homeostasis, followed by mitochondrial Ca2+ overload and cytochrome c release, leading to the activation of caspase 9- and caspase-3-mediated intrinsic apoptotic pathways. Treatment of zebrafish larvae with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester (BAPTA-AM) or with 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of the calcium-releasing protein inositol 1,4,5-trisphosphate receptor (IP3R), alleviated cold-induced cell death. Together, these findings unveil a key role of Tmbim3a/Grinaa in relieving cold-induced ER stress and in protecting cells against caspase 9- and caspase 3-mediated apoptosis during zebrafish development.


Assuntos
Apoptose , Temperatura Baixa , Estresse do Retículo Endoplasmático , Proteínas de Membrana/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Apoptose/efeitos dos fármacos , Compostos de Boro/farmacologia , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Proteínas de Membrana/genética , Mutação , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Frações Subcelulares/metabolismo , Resposta a Proteínas não Dobradas , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética
8.
Cell Physiol Biochem ; 52(6): 1339-1360, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050282

RESUMO

BACKGROUND/AIMS: Melanocortin receptors (MCRs) belong to a hormonal signalling pathway with multiple homeostatic and protective actions. Microvascular and umbilical vein endothelial cells (ECs) express components of the melanocortin system, including the type 1 receptor (MC1R), playing a role in modulating inflammation and vascular tone. Since ECs exhibit a remarkable heterogeneity, we investigated whether human artery ECs express any functional MCR and whether its activation affects cell migration. METHODS: We used reverse transcription real-time PCR to examine the expression of melanocortin system components in primary human artery ECs. We assessed MC1R protein expression and activity by western blot, immunohistochemistry, cAMP production, and intracellular Ca²âº mobilization assays. We performed gap closure and scratch tests to examine cell migration after stimulation with alpha-melanocyte-stimulating hormone (α-MSH), the receptor highest-affinity natural ligand. We assessed differential time-dependent transcriptional changes in migrating cells by microarray analysis. RESULTS: We showed that human aortic ECs (HAoECs) express a functionally active MC1R. Unlike microvascular ECs, arterial cells did not express the α-MSH precursor proopiomelanocortin, nor produced the hormone. MC1R engagement with a single pulse of α-MSH accelerated HAoEC migration both in the directional migration assay and in the scratch wound healing test. This was associated with an enhancement in Ca²âº signalling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in HAoECs undergoing directional migration allowed identifying dynamic co-regulation of genes involved in extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing - which have all well-established influences on EC motility -, without affecting the balance between pro- and anticoagulant genes. CONCLUSION: Our work broadens the knowledge on peripherally expressed MC1R. These results indicate that the receptor is constitutively expressed by arterial ECs and provide evidence of a novel homeostatic function for MC1R, whose activation may participate in preventing/healing endothelial dysfunction or denudation in macrovascular arteries.


Assuntos
Receptor Tipo 1 de Melanocortina/metabolismo , Aorta/citologia , Sinalização do Cálcio/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Antígeno Ki-67/metabolismo , Oligopeptídeos/farmacologia , Receptor Tipo 1 de Melanocortina/genética , alfa-MSH/farmacologia
9.
Mol Carcinog ; 58(8): 1349-1361, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31045290

RESUMO

Prostate cancer (PCa) patients' mortality is mainly attributed to complications caused by metastasis of the tumor cells to organs critical for survival, such as bone. We hypothesized that PCa cell-bone interactions would promote paracrine signaling. A panel of PCa cell lines were cocultured with hydroxyapatite ([HA]; inorganic component of bone) of different densities. Conditioned media (CM) was collected and analyzed for calcium levels and effect on paracrine signaling, cell migration, and viability in vitro and in vivo. Our results showed that calcium levels were elevated in CM from cancer cell-bone cocultures, compared to media or cancer cells alone, and this could be antagonized by ethylene glycol-bis(2-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), a calcium chelator, or knockdown of Snail protein. We also observed increased signal transducer and activator of transcription 3 (STAT3) phosphorylation and paracrine cell proliferation and migration in LNCaP cells incubated with CM from various cell lines; this phosphorylation and cell migration could be antagonized by Snail knockdown or various inhibitors including EGTA, STAT3 inhibitor (WP1066) or cathepsin L inhibitor (Z-FY-CHO). In vivo, higher HA bone density increased tumorigenicity and migration of tumor cells to HA implant. Our study shows that cancer-bone microenvironment interactions lead to calcium-STAT3 signaling, which may present an area for therapeutic targeting of metastatic PCa.


Assuntos
Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Neoplasias da Próstata/patologia , Fator de Transcrição STAT3/metabolismo , Microambiente Tumoral/fisiologia , Animais , Osso e Ossos/patologia , Cálcio/metabolismo , Catepsina L/antagonistas & inibidores , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Durapatita/farmacologia , Ácido Egtázico/farmacologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Piridinas , Interferência de RNA , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais , Fatores de Transcrição da Família Snail/genética , Tirfostinas
10.
Dis Model Mech ; 12(5)2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31043432

RESUMO

DYRK1A is a major causative gene in Down syndrome (DS). Reduced incidence of solid tumors such as neuroblastoma in DS patients and increased vascular anomalies in DS fetuses suggest a potential role of DYRK1A in angiogenic processes, but in vivo evidence is still scarce. Here, we used zebrafish dyrk1aa mutant embryos to understand DYRK1A function in cerebral vasculature formation. Zebrafish dyrk1aa mutants exhibited cerebral hemorrhage and defects in angiogenesis of central arteries in the developing hindbrain. Such phenotypes were rescued by wild-type dyrk1aa mRNA, but not by a kinase-dead form, indicating the importance of DYRK1A kinase activity. Chemical screening using a bioactive small molecule library identified a calcium chelator, EGTA, as one of the hits that most robustly rescued the hemorrhage. Vascular defects of mutants were also rescued by independent modulation of calcium signaling by FK506. Furthermore, the transcriptomic analyses supported the alterations of calcium signaling networks in dyrk1aa mutants. Together, our results suggest that DYRK1A plays an essential role in angiogenesis and in maintenance of the developing cerebral vasculature via regulation of calcium signaling, which may have therapeutic potential for DYRK1A-related vascular diseases.


Assuntos
Vasos Sanguíneos/patologia , Sinalização do Cálcio , Técnicas de Inativação de Genes , Proteínas Quinases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Vasos Sanguíneos/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Encéfalo/patologia , Encéfalo/ultraestrutura , Hemorragia Cerebral/patologia , Ácido Egtázico/farmacologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Desenvolvimento Embrionário/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Harmina/farmacologia , Espaço Intracelular/metabolismo , Mutação/genética , Fenótipo , Transcriptoma/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/antagonistas & inibidores
11.
BMC Genomics ; 20(1): 47, 2019 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651090

RESUMO

BACKGROUND: Bloodstream malaria parasites require Ca++ for their development, but the sites and mechanisms of Ca++ utilization are not well understood. We hypothesized that there may be differences in Ca++ uptake or utilization by genetically distinct lines of P. falciparum. These differences, if identified, may provide insights into molecular mechanisms. RESULTS: Dose response studies with the Ca++ chelator EGTA (ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid) revealed stable differences in Ca++ requirement for six geographically divergent parasite lines used in previous genetic crosses, with the largest difference seen between the parents of the HB3 x Dd2 cross. Genetic mapping of Ca++ requirement yielded complex inheritance in 34 progeny clones with a single significant locus on chromosome 7 and possible contributions from other loci. Although encoded by a gene in the significant locus and a proposed Ca++ target, PfCRT (P. falciparum chloroquine resistance transporter), the primary determinant of clinical resistance to the antimalarial drug chloroquine, does not appear to contribute to this quantitative trait. Stage-specific application of extracellular EGTA also excluded determinants associated with merozoite egress and erythrocyte reinvasion. CONCLUSIONS: We have identified differences in Ca++ utilization amongst P. falciparum lines. These differences are under genetic regulation, segregating as a complex trait in genetic cross progeny. Ca++ uptake and utilization throughout the bloodstream asexual cycle of malaria parasites represents an unexplored target for therapeutic intervention.


Assuntos
Cálcio/metabolismo , Loci Gênicos , Malária Falciparum/parasitologia , Parasitos/genética , Plasmodium falciparum/genética , Animais , Cruzamentos Genéticos , Ácido Egtázico/farmacologia , Feminino , Estudos de Associação Genética , Haplótipos/genética , Padrões de Herança/genética , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Merozoítos/efeitos dos fármacos , Merozoítos/metabolismo , Parasitos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/metabolismo
12.
Glia ; 67(5): 915-934, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30632636

RESUMO

Optogenetics has been widely expanded to enhance or suppress neuronal activity and it has been recently applied to glial cells. Here, we have used a new approach based on selective expression of melanopsin, a G-protein-coupled photopigment, in astrocytes to trigger Ca2+ signaling. Using the genetically encoded Ca2+ indicator GCaMP6f and two-photon imaging, we show that melanopsin is both competent to stimulate robust IP3-dependent Ca2+ signals in astrocyte fine processes, and to evoke an ATP/Adenosine-dependent transient boost of hippocampal excitatory synaptic transmission. Additionally, under low-frequency light stimulation conditions, melanopsin-transfected astrocytes can trigger long-term synaptic changes. In vivo, melanopsin-astrocyte activation enhances episodic-like memory, suggesting melanopsin as an optical tool that could recapitulate the wide range of regulatory actions of astrocytes on neuronal networks in behaving animals. These results describe a novel approach using melanopsin as a precise trigger for astrocytes that mimics their endogenous G-protein signaling pathways, and present melanopsin as a valuable optical tool for neuron-glia studies.


Assuntos
Astrócitos/metabolismo , Rede Nervosa/metabolismo , Neurônios/metabolismo , Optogenética/métodos , Opsinas de Bastonetes/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Compostos Azo/farmacologia , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/citologia , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Pirimidinas/farmacologia , Opsinas de Bastonetes/genética , Potenciais Sinápticos/fisiologia , Triazóis/farmacologia , Xantenos/farmacologia
13.
Cryobiology ; 87: 105-109, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30682339

RESUMO

Present study aimed to determine to what extent freeze-dried spermatozoa were able to withstand high-temperature conditions: transient increase in storage temperature and long-term exposure to room temperature. Mouse spermatozoa were freeze-dried in EGTA/Tris-HCl buffered solution alkalinized using KOH (K-ETBS, pH 7.7), and then stored for up to 7 months at 4 °C or 25 °C. After 2 months' storage, some of the 4°C-stored spermatozoa were exposed to 40 °C for 1 week or 1 month, then again stored at 4 °C for the remaining storage period. Following storage, rehydrated spermatozoa were injected into mouse oocytes. The resulting zygotes were assessed for chromosome damage, in vitro development up to the blastocyst stage, and post-implantation development to normal fetuses on day 18 of gestation. In storage at 4 °C, one-week exposure to 40 °C had no adverse effect on the chromosome integrity and developmental competence compared to non-exposure to 40 °C (continuous storage at 4 °C). In contrast, one-month exposure to 40 °C caused an increasing level of chromosome damage (36%, P < 0.05) and reduced frequencies of blastocysts (54%, P < 0.05) and normal fetuses (36%, P < 0.05) compared to the frequencies obtained by continuous storage at 4 °C (15%, 82% and 52%, respectively). Storage at 25 °C resulted in accumulation of chromosome damage (27%, P < 0.05), leading to decreased blastocyst formation (63%, P < 0.05). But, the frequency of normal fetus (44%) was not significantly different from that obtained by continuous storage at 4 °C. Consequently, mouse spermatozoa freeze-dried in K-ETBS withstood temporary exposure to 40 °C for 1 week. Chromosome damage accumulated in spermatozoa during storage at 25 °C.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Liofilização/métodos , Injeções de Esperma Intracitoplásmicas/métodos , Espermatozoides/transplante , Animais , Blastocisto/citologia , Cromossomos/fisiologia , Ácido Egtázico/farmacologia , Desenvolvimento Embrionário/fisiologia , Feminino , Feto , Temperatura Alta , Hidróxidos/farmacologia , Estudos Longitudinais , Masculino , Camundongos , Oócitos/crescimento & desenvolvimento , Compostos de Potássio/farmacologia
14.
Innate Immun ; 25(1): 3-12, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30426809

RESUMO

Pseudomonas aeruginosa-derived pigment pyocyanin (PCN) has been proved to induce cell apoptosis mediated by the generation of reactive oxygen species (ROS), which has been studied mainly in epithelial cells and neutrophils. However, we previously found that the PCN-producing strain PA14 induces cell apoptosis in human NK cell line NK92 more effectively than in PCN-deficient strain PA14-phZ1/2 via a yet undetermined mechanism. In the current study, we found that PCN-induced NK92 cell apoptosis occurs through mitochondrial damage despite inhibiting intracellular ROS generation. Intracellular Ca2+ ([Ca2+]i) and Bcl-2 family proteins act as important "priming signals" for apoptosis. PCN treatment increased [Ca2+]i in NK92 cells more than twofold after 2 h stimulation, whereas the Ca2+-chelating agent ethylene glycol tetra-acetic acid (EGTA) inhibited apoptosis. PCN triggered the activation of Bim, Bid, Bik, Bak, and phospho-Bad in NK92 cells in a concentration-dependent manner, but these pro-apoptotic Bcl-2 family proteins were not inhibited by EGTA. In this study, we describe the function of PCN in NK92 cells and identify mitochondrial damage as the mechanism underlying the apoptosis. [Ca2+]i and pro-apoptotic Bcl-2 family proteins are novel targets for PCN-induced apoptosis. Clarification of the cytotoxic diversity of PCN provides a new therapeutic target for defense from P. aeruginosa-induced immune cell damage.


Assuntos
Células Matadoras Naturais/fisiologia , Mitocôndrias/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/fisiologia , Piocianina/metabolismo , Apoptose , Cálcio/metabolismo , Linhagem Celular , Ácido Egtázico/farmacologia , Humanos , Espaço Intracelular , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
Cell Rep ; 25(12): 3451-3464.e3, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30566869

RESUMO

A Ca2+ current transient block (ICaTB) by protons occurs at some ribbon-type synapses after exocytosis, but this has not been observed at mammalian hair cells. Here we show that a robust ICaTB occurs at post-hearing mouse and gerbil inner hair cell (IHC) synapses, but not in immature IHC synapses, which contain non-compact active zones, where Ca2+ channels are loosely coupled to the release sites. Unlike ICaTB at other ribbon synapses, ICaTB in mammalian IHCs displays a surprising multi-peak structure that mirrors the EPSCs seen in paired recordings. Desynchronizing vesicular release with intracellular BAPTA or by deleting otoferlin, the Ca2+ sensor for exocytosis, greatly reduces ICaTB, whereas enhancing release synchronization by raising Ca2+ influx or temperature increases ICaTB. This suggests that ICaTB is produced by fast multivesicular proton-release events. We propose that ICaTB may function as a submillisecond feedback mechanism contributing to the auditory nerve's fast spike adaptation during sound stimulation.


Assuntos
Canais de Cálcio/metabolismo , Células Ciliadas Auditivas/metabolismo , Mamíferos/metabolismo , Prótons , Vesículas Sinápticas/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Nervo Coclear/efeitos dos fármacos , Nervo Coclear/fisiologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Exocitose/efeitos dos fármacos , Gerbillinae , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Nifedipino/farmacologia , Rana catesbeiana , Temperatura
16.
Blood Adv ; 2(21): 2848-2861, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30381401

RESUMO

Dyslipidemia is a risk factor for clinically significant thrombotic events. In this condition, scavenger receptor CD36 potentiates platelet reactivity through recognition of circulating oxidized lipids. CD36 promotes thrombosis by activating redox-sensitive signaling molecules, such as the MAPK extracellular signal-regulated kinase 5 (ERK5). However, the events downstream of platelet ERK5 are not clear. In this study, we report that oxidized low-density lipoprotein (oxLDL) promotes exposure of procoagulant phosphatidylserine (PSer) on platelet surfaces. Studies using pharmacologic inhibitors indicate that oxLDL-CD36 interaction-induced PSer exposure requires apoptotic caspases in addition to the downstream CD36-signaling molecules Src kinases, hydrogen peroxide, and ERK5. Caspases promote PSer exposure and, subsequently, recruitment of the prothrombinase complex, resulting in the generation of fibrin from the activation of thrombin. Caspase activity was observed when platelets were stimulated with oxLDL. This was prevented by inhibiting CD36 and ERK5. Furthermore, oxLDL potentiates convulxin/glycoprotein VI-mediated fibrin formation by platelets, which was prevented when CD36, ERK5, and caspases were inhibited. Using 2 in vivo arterial thrombosis models in apoE-null hyperlipidemic mice demonstrated enhanced arterial fibrin accumulation upon vessel injury. Importantly, absence of ERK5 in platelets or mice lacking CD36 displayed decreased fibrin accumulation in high-fat diet-fed conditions comparable to that seen in chow diet-fed animals. These findings suggest that platelet signaling through CD36 and ERK5 induces a procoagulant phenotype in the hyperlipidemic environment by enhancing caspase-mediated PSer exposure.


Assuntos
Plaquetas/metabolismo , Antígenos CD36/metabolismo , Caspases/metabolismo , Fibrina/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Fosfatidilserinas/metabolismo , Animais , Plaquetas/citologia , Antígenos CD36/antagonistas & inibidores , Venenos de Crotalídeos/farmacologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Humanos , Hiperlipidemias/complicações , Hiperlipidemias/patologia , Lectinas Tipo C , Lipoproteínas LDL/farmacologia , Camundongos , Camundongos Knockout , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Ativação Plaquetária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Trombose/etiologia , Trombose/patologia , Quinases da Família src/metabolismo
17.
Neurotoxicology ; 69: 97-107, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30292652

RESUMO

Exposure to insecticides has been found to have deleterious effects on human health. Lambda-cyhalothrin (LCT), a mixture of isomers of cyhalothrin, is a pyrethroid insecticide routinely used in pest control programs. LCT was reported to cause neurotoxic effects in various models. However, the mechanism of underlying effect of LCT on cytotoxicity in normal human brain cells is still elusive. This study examined whether LCT affected Ca2+ homeostasis and Ca2+-related physiology in Gibco® Human Astrocytes (GHA cells), and explored whether BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid), a selective Ca2+ chelator, has protective effects on LCT-treated GHA cells. The data show that LCT (10-15 µM) concentration-dependently induced cytotoxicity in both GHA cells and DI TNC1 normal rat astrocytes but only induced intracellular Ca2+ concentration ([Ca2+]i) rises in GHA cells. In terms of Ca2+ signaling in GHA cells, LCT-induced [Ca2+]i rises were reduced by removing extracellular Ca2+ and were inhibited by store-operated Ca2+ channel modulators (2-APB, econazole or SKF96365). In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished LCT-induced [Ca2+]i rises. Conversely, incubation with LCT abolished thapsigargin-induced [Ca2+]i rises. Regarding cytotoxicity, LCT evoked apoptosis by regulating apoptotic protein expressions (Bax, BCl-2, cleaved caspase-9/-3). This apoptotic response was significantly inhibited by prechelating cytosolic Ca2+ with BAPTA-AM. Together, in GHA cells, LCT induced [Ca2+]i rises by inducing Ca2+ entry via store-operated Ca2+ channels and Ca2+ release from the endoplasmic reticulum. Moreover, BAPTA-AM has a protective effect on inhibiting LCT-activated mitochondrial apoptotic pathway. This study provided new insights into the molecular protective mechanism of LCT-induced cytotoxicity in normal human astrocytes.


Assuntos
Astrócitos/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Inseticidas/toxicidade , Mitocôndrias/efeitos dos fármacos , Nitrilos/toxicidade , Piretrinas/toxicidade , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Astrócitos/metabolismo , Cálcio/metabolismo , Quelantes de Cálcio/farmacologia , Sinalização do Cálcio/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Ácido Egtázico/farmacologia , Humanos , Mitocôndrias/metabolismo , Ratos
18.
J Pharmacol Sci ; 138(3): 167-175, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30322800

RESUMO

Na+, K+-ATPase is a highly expressed membrane protein. Dysfunction of Na+, K+-ATPase has been implicated in the pathophysiology of several neurodegenerative and psychiatric disorders, however, the underlying mechanism of neuronal cell death resulting from Na+, K+-ATPase dysfunction is poorly understood. Here, we investigated the mechanism of neurotoxicity due to Na+, K+-ATPase inhibition using rat organotypic hippocampal slice cultures. Treatment with ouabain, a Na+, K+-ATPase inhibitor, increased the ratio of propidium iodide-positive cells among NeuN-positive cells in the hippocampal CA1 region, which was prevented by MK-801 and d-AP5, specific blockers of the N-methyl-d-aspartate (NMDA) receptor. EGTA, a Ca2+-chelating agent, also protected neurons from ouabain-induced injury. We observed that astrocytes expressed the glutamate aspartate transporter (GLAST), and ouabain changed the immunoreactive area of GFAP-positive astrocytes as well as GLAST. We also observed that ouabain increased the number of Iba1-positive microglial cells in a time-dependent manner. Furthermore, lithium carbonate, a mood-stabilizing drug, protected hippocampal neurons and reduced disturbances of astrocytes and microglia after ouabain treatment. Notably, lithium carbonate improved ouabain-induced decreases in GLAST intensity in astrocytes. These results suggest that glial cell abnormalities resulting in excessive extracellular concentrations of glutamate contribute to neurotoxicity due to Na+, K+-ATPase dysfunction in the hippocampal CA1 region.


Assuntos
Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/patologia , Morte Celular/efeitos dos fármacos , Transportador 1 de Aminoácido Excitatório/metabolismo , Microglia/efeitos dos fármacos , Microglia/patologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Astrócitos/metabolismo , Contagem de Células , Células Cultivadas , Maleato de Dizocilpina/farmacologia , Ácido Egtázico/farmacologia , Técnicas In Vitro , Carbonato de Lítio/farmacologia , Ouabaína/antagonistas & inibidores , Ouabaína/farmacologia , Ratos , Valina/análogos & derivados , Valina/farmacologia
19.
Cryobiology ; 84: 82-90, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30244698

RESUMO

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca2+), mPB1- (modified PBS without Ca2+), mPB1+/EGTA (mPB1+ containing EGTA), mPB1-/EGTA (mPB1- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Ácido Egtázico/farmacologia , Vitrificação , Animais , Quelantes de Cálcio/farmacologia , Feminino , Fertilização In Vitro , Técnicas de Maturação in Vitro de Oócitos , Oócitos/efeitos dos fármacos , Ovinos , Carneiro Doméstico
20.
Biochimie ; 153: 203-209, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30244813

RESUMO

Although 24(S)-hydroxycholesterol (24S-OHC) plays an important role to maintain homeostasis of cholesterol in the brain, it induces neuronal cell death at high concentrations. 24S-OHC-induced cell death was suppressed by γ-tocopherol (γ-Toc) but not by γ-tocotrienol (γ-Toc3) in a similar way to α-tocopherol (α-Toc) and α-tocotrienol (α-Toc3) in human neuroblastoma SH-SY5Y cells. Both γ-Toc and γ-Toc3 significantly inhibited cumene hydroperoxide-induced cell death, as previously shown in the case of α-Toc and α-Toc3. Lipid droplet-like structure formation induced by 24S-OHC was suppressed by neither γ-Toc nor γ-Toc3. The phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) was induced by 24S-OHC, which was suppressed by CaMKII phosphorylation-site inhibitor mM3 but not by calmodulin-binding-site inhibitor KN62. A calcium chelator, BAPTA-AM, inhibited calcium ionophore A23187-induced CaMKII phosphorylation but not 24S-OHC-induced CaMKII phosphorylation. Receptor-interacting protein kinase 1 (RIPK1) phosphorylation induced by 24S-OHC was not inhibited by either mM3 or KN62, suggesting that CaMKII activation does not affect RIPK1 phosphorylation. Knockdown of RIPK1 using siRNA induced not only inhibition of CaMKII phosphorylation but also reduction of total CaMKII protein levels, suggesting that RIPK1 may regulate CaMKII signalling. 24S-OHC-induced RIPK1 phosphorylation was inhibited by neither α-Toc nor α-Toc3. In contrast, CaMKII phosphorylation induced by 24S-OHC was significantly suppressed by α-Toc but not by α-Toc3. These results suggest that CaMKII activation is involved in the mechanism of 24S-OHC-induced cell death and that Toc inhibits the cell death via inhibition of CaMKII activation through a RIPK1 phosphorylation-independent pathway.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Morte Celular/efeitos dos fármacos , Hidroxicolesteróis/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Tocoferóis/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Linhagem Celular Tumoral , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Ativação Enzimática , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/fisiologia
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