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1.
Vet Microbiol ; 266: 109333, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35033844

RESUMO

Porcine deltacoronavirus (PDCoV) is an enteropathogen found in many pig producing countries. It can cause acute diarrhea, vomiting, dehydration, and death in newborn piglets, seriously affecting the development of pig breeding industries. To date, our knowledge of the pathogenesis of PDCoV and its interactions with host cell factors remains incomplete. Using Co-IP coupled with LC/MS-MS, we identified 67 proteins that potentially interact with PDCoV in LLC-PK1 cells; five of the identified proteins were chosen for further evaluation (IMMT, STAT1, XPO5, PIK3AP1, and TMPRSS11E). Five LLC-PK1 cell lines, each with one of the genes of interest knocked down, were constructed using CRISPR/cas9. In these knockdown cells lines, only STAT1KD resulted in a significantly greater virus yield. Knockdown of the remaining four genes resulted, to varying degrees, in a lower virus yield that wild-type LLC-PK1 cells. The absence of STAT1 did not significantly affect the attachment of PDCoV to cells, but did result in increased viral internalization. Additionally, PDCoV infection stimulated expression of interferon stimulated genes (ISGs) downstream of STAT1 (IFIT1, IFIT2, RADS2, ISG15, MX1, and OAS1) while knockdown of STAT1 resulted in a greater than 80 % decrease in the expression of all six ISGs. Our findings show that STAT1 interacts with PDCoV, and plays a negative regulatory role in PDCoV infection.


Assuntos
Infecções por Coronavirus , Doenças dos Suínos , Animais , Infecções por Coronavirus/veterinária , Interferons , Células LLC-PK1 , Suínos , Internalização do Vírus
2.
Toxins (Basel) ; 13(11)2021 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-34822571

RESUMO

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.


Assuntos
Túbulos Renais Proximais/fisiopatologia , Micotoxinas/efeitos adversos , Ocratoxinas/efeitos adversos , Estresse Oxidativo , Animais , Linhagem Celular , Humanos , Túbulos Renais Proximais/metabolismo , Células LLC-PK1 , Sus scrofa , Suínos
3.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638861

RESUMO

Among organic-inorganic hybrid molecules consisting of organic structure(s) and metal(s), only few studies are available on the cytotoxicity of nucleophilic molecules. In the present study, we investigated the cytotoxicity of a nucleophilic organotellurium compound, diphenyl ditelluride (DPDTe), using a cell culture system. DPDTe exhibited strong cytotoxicity against vascular endothelial cells and fibroblasts along with high intracellular accumulation but showed no cytotoxicity and had less accumulation in vascular smooth muscle cells and renal epithelial cells. The cytotoxicity of DPDTe decreased when intramolecular tellurium atoms were replaced with selenium or sulfur atoms. Electronic state analysis revealed that the electron density between tellurium atoms in DPDTe was much lower than those between selenium atoms of diphenyl diselenide and sulfur atoms of diphenyl disulfide. Moreover, diphenyl telluride did not accumulate and exhibit cytotoxicity. The cytotoxicity of DPDTe was also affected by substitution. p-Dimethoxy-DPDTe showed higher cytotoxicity, but p-dichloro-DPDTe and p-methyl-DPDTe showed lower cytotoxicity than that of DPDTe. The subcellular distribution of the compounds revealed that the compounds with stronger cytotoxicity showed higher accumulation rates in the mitochondria. Our findings suggest that the electronic state of tellurium atoms in DPDTe play an important role in accumulation and distribution of DPDTe in cultured cells. The present study supports the hypothesis that nucleophilic organometallic compounds, as well as electrophilic organometallic compounds, exhibit cytotoxicity by particular mechanisms.


Assuntos
Derivados de Benzeno/farmacologia , Células Endoteliais/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Compostos Organosselênicos/farmacologia , Telúrio/farmacologia , Animais , Derivados de Benzeno/química , Derivados de Benzeno/metabolismo , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Células LLC-PK1 , Modelos Químicos , Estrutura Molecular , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Compostos Organosselênicos/química , Compostos Organosselênicos/metabolismo , Suínos , Telúrio/química
4.
Peptides ; 146: 170646, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34500007

RESUMO

Megalin-mediated albumin endocytosis plays a critical role in albumin reabsorption in proximal tubule (PT) epithelial cells (PTECs). Some studies have pointed out the modulatory effect of bradykinin (BK) on urinary protein excretion, but its role in PT protein endocytosis has not yet been determined. Here, we studied the possible correlation between BK and albumin endocytosis in PT. Using LLC-PK1 cells, a model of PTECs, we showed that BK specifically inhibited megalin-mediated albumin endocytosis. This inhibitory effect of BK was mediated by B2 receptor (B2R) because it was abolished by HOE140, an antagonist of B2R, but it was not affected by Lys-des-Arg9-BK, an antagonist of B1. BK induced the stall of megalin in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to a decrease in surface megalin expression. In addition, we showed that BK, through B2R, activated calphostin C-sensitive protein kinase C, which mediated its effect on the surface megalin expression and albumin endocytosis. These results reveal an important modulatory mechanism of PT albumin endocytosis by BK, which opens new possibilities to understanding the effect of BK on urinary albumin excretion.


Assuntos
Albuminas/metabolismo , Bradicinina/farmacologia , Endocitose/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Túbulos Renais Proximais/metabolismo , Células LLC-PK1 , Proteína Quinase C/metabolismo , Receptor B2 da Bradicinina/metabolismo , Suínos
5.
Cells ; 10(8)2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34440628

RESUMO

Tubulin post-translational modifications regulate microtubule properties and functions. Mitotic spindle microtubules are highly modified. While tubulin detyrosination promotes proper mitotic progression by recruiting specific microtubule-associated proteins motors, tubulin acetylation that occurs on specific microtubule subsets during mitosis is less well understood. Here, we show that siRNA-mediated depletion of the tubulin acetyltransferase ATAT1 in epithelial cells leads to a prolonged prometaphase arrest and the formation of monopolar spindles. This results from collapse of bipolar spindles, as previously described in cells deficient for the mitotic kinase PLK1. ATAT1-depleted mitotic cells have defective recruitment of PLK1 to centrosomes, defects in centrosome maturation and thus microtubule nucleation, as well as labile microtubule-kinetochore attachments. Spindle bipolarity could be restored, in the absence of ATAT1, by stabilizing microtubule plus-ends or by increasing PLK1 activity at centrosomes, demonstrating that the phenotype is not just a consequence of lack of K-fiber stability. We propose that microtubule acetylation of K-fibers is required for a recently evidenced cross talk between centrosomes and kinetochores.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/enzimologia , Células Epiteliais/enzimologia , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/enzimologia , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Células LLC-PK1 , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/genética , Mitose , Transdução de Sinais , Fuso Acromático/genética , Suínos
6.
Bioorg Med Chem Lett ; 48: 128256, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34256117

RESUMO

Acute kidney injury (AKI) is a common clinical problem that is associated with high mortality due to multiple complex mechanisms. Cisplatin is the most important and highly effective chemotherapeutic agent used for the treatment of various solid tumors; however, it is associated with dose-dependent adverse effects, particularly in the kidney where it can cause severe nephrotoxicity. The pathophysiological basis of cisplatin-induced nephrotoxicity has been investigated over the last few decades, and the key pathological occurrences in cisplatin nephrotoxicity include renal tubular cell injury and death. Necrostatin-1 (Nec-1) has been confirmed to act as a specific and potent small-molecule inhibitor of necroptosis. However, the effects of three structurally distinct necrostatins on cisplatin-induced nephrotoxicity remain ambiguous. The aim of this study was to determine if three types of necrostatins (Nec-1, Nec-3-A, and/or Nec-3-B) can exert protective effects in regard to the AKI induced by cisplatin. Our results indicated that necrostatins can prevent cisplatin induced nephrotoxicity via modulating apoptotic pathways through the suppression of cleaved caspase-3 and also by influencing the function of mitogen-activated protein kinase pathway members, including extracellular signal-regulated kinases, c-Jun N-terminal kinases, and p38, in the renal tubular epithelial cell line LLC-PK1. These findings suggest that necrostatins exert beneficial anti-apoptotic effects in the context of AKI induced by cisplatin.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Imidazóis/farmacologia , Indóis/farmacologia , Inflamação/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antineoplásicos/química , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Imidazóis/química , Indóis/química , Túbulos Renais/efeitos dos fármacos , Células LLC-PK1 , Estrutura Molecular , Necroptose/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Suínos
7.
Biochem Biophys Res Commun ; 565: 50-56, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34090210

RESUMO

Fluorescence polarization microscopy (FPM) can visualize the dipole orientation of fluorescent molecules and has been used for analyzing architectural dynamics of biomolecules including cytoskeletal proteins. To monitor the orientation of target molecules by FPM, target molecules need to be labeled with fluorophores in a sterically constrained manner, so that the fluorophores do not freely rotate. Recently, a versatile probe for such labeling using fluorescent proteins, POLArIS (Probe for Orientation and Localization Assessment, recognizing specific Intracellular Structures of interest), was reported. POLArIS is a fusion protein consisting of a non-immunoglobulin-based recombinant binder Affimer and a green fluorescent protein (GFP), where the Affimer and GFP are rigidly connected to each other. POLArIS probe for molecules of interest can be developed through phage display screening of Affimer. This screening is followed by the rigid connection of fluorescent proteins to the selected Affimers. The Affimer-based POLArIS, however, cannot be used with animal immune libraries for selecting specific binder clones. In addition, multi-color FPM by POLArIS was not available due to the lack of color variations of POLArIS. In this study, we have developed new versions of POLArIS with nanobodies, which are compatible with animal immune libraries, and expanded color variations of POLArIS with cyan/green/yellow/red fluorescent proteins, enabling multi-color orientation imaging for multiple targets. Using nanobody-based POLArIS orientation probes, we performed two-color FPM of F-actin and vimentin in living cells. Furthermore, we made nanobody-based POLArIS probes that have different dipole orientations for adjusting the orientation of fluorescence polarization with respect to the target molecules. These nanobody-based POLArIS with options of colors and dipole orientations will enhance the performance of this probe for broader applications of fluorescence polarization imaging in living cells, tissues, and whole organisms.


Assuntos
Cor , Corantes Fluorescentes/química , Imagem Óptica , Animais , Corantes Fluorescentes/síntese química , Humanos , Células LLC-PK1 , Suínos , Células Tumorais Cultivadas
8.
Am J Physiol Renal Physiol ; 321(2): F179-F194, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34180716

RESUMO

The trafficking of proteins such as aquaporin-2 (AQP2) in the exocytotic pathway requires an active actin cytoskeleton network, but the mechanism is incompletely understood. Here, we show that the actin-related protein (Arp)2/3 complex, a key factor in actin filament branching and polymerization, is involved in the shuttling of AQP2 between the trans-Golgi network (TGN) and the plasma membrane. Arp2/3 inhibition (using CK-666) or siRNA knockdown blocks vasopressin-induced AQP2 membrane accumulation and induces the formation of distinct AQP2 perinuclear patches positive for markers of TGN-derived clathrin-coated vesicles. After a 20°C cold block, AQP2 formed perinuclear patches due to continuous endocytosis coupled with inhibition of exit from TGN-associated vesicles. Upon rewarming, AQP2 normally leaves the TGN and redistributes into the cytoplasm, entering the exocytotic pathway. Inhibition of Arp2/3 blocked this process and trapped AQP2 in clathrin-positive vesicles. Taken together, these results suggest that Arp2/3 is essential for AQP2 trafficking, specifically for its delivery into the post-TGN exocytotic pathway to the plasma membrane.NEW & NOTEWORTHY Aquaporin-2 (AQP2) undergoes constitutive recycling between the cytoplasm and plasma membrane, with an intricate balance between endocytosis and exocytosis. By inhibiting the actin-related protein (Arp)2/3 complex, we prevented AQP2 from entering the exocytotic pathway at the post-trans-Golgi network level and blocked AQP2 membrane accumulation. Arp2/3 inhibition, therefore, enables us to separate and target the exocytotic process, while not affecting endocytosis, thus allowing us to envisage strategies to modulate AQP2 trafficking and treat water balance disorders.


Assuntos
Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Aquaporina 2/metabolismo , Exocitose/fisiologia , Rim/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Membrana Celular/metabolismo , Endocitose/fisiologia , Células LLC-PK1 , Fosforilação , Transporte Proteico/fisiologia , Ratos , Suínos
9.
Vet Res ; 52(1): 86, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127062

RESUMO

Porcine deltacoronavirus (PDCoV) is a newly discovered swine enteropathogenic coronavirus with worldwide distribution. However, efficient strategies to prevent or treat the infection remain elusive. Our in vitro study revealed that ergosterol peroxide (EP) from the mushroom Cryptoporus volvatus has efficient anti-PDCoV properties. The aim of this study is to evaluate the potential of EP as a treatment for PDCoV in vivo and elucidate the possible mechanisms. Seven-day-old piglets were infected with PDCoV by oral administration in the presence or absence of EP. Piglets infected with PDCoV were most affected, whereas administration of EP reduced diarrhea incidence, alleviated intestinal lesion, and decreased viral load in feces and tissues. EP reduced PDCoV-induced apoptosis and enhanced tight junction protein expressions in the small intestine, maintaining the integrity of the intestinal barrier. EP showed immunomodulatory effect by suppressing PDCoV-induced pro-inflammatory cytokines and the activation of IκBα and NF-κB p65, and upregulating IFN-I expression. Knockdown of p38 inhibited PDCoV replication and alleviated PDCoV-induced apoptosis, implying that EP inhibited PDCoV replication and alleviated PDCoV-induced apoptosis via p38/MAPK signaling pathway. Collectively, ergosterol peroxide can protect piglets from PDCoV, revealing the potential of EP for development as a promising strategy for treating and controlling the infection of PDCoV.


Assuntos
Apoptose/efeitos dos fármacos , Infecções por Coronavirus/veterinária , Deltacoronavirus , Ergosterol/análogos & derivados , Doenças dos Suínos/virologia , Junções Íntimas/efeitos dos fármacos , Animais , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Deltacoronavirus/efeitos dos fármacos , Ergosterol/farmacologia , Ergosterol/uso terapêutico , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/virologia , Células LLC-PK1 , Masculino , Suínos , Doenças dos Suínos/tratamento farmacológico
10.
Vet Microbiol ; 257: 109097, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33933854

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that causes gastroenteritis in pigs and no vaccines or antiviral drugs are available. Bile acids are active factors in intestines and influence the replication of enteric viruses. Currently, the role of bile acids on PDCoV replication is unknown. In this study, we tested the effects of different types of bile acids on the replication of PDCoV in cell culture. We found that physiological concentrations of bile acids chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) had antiviral activity against PDCoV in porcine kidney cell line (LLC-PK1) and porcine small intestinal epithelial cell line (IPEC-J2). In IPEC-J2 cells, CDCA and LCA inhibited PDCoV replication at post-entry stages by inducing the production of interferon (IFN)-λ3 and IFN-stimulated gene 15 (ISG15) via G protein-coupled receptor (GPCR). In summary, bile acids CDCA and LCA restricted PDCoV infection and LCA functioned through a GPCR-IFN-λ3-ISG15 signaling axis in IPEC-J2 cells. Our results may open new avenues for the development of antiviral drugs to treat PDCoV infection in pigs.


Assuntos
Ácidos e Sais Biliares/farmacologia , Ácido Quenodesoxicólico/farmacologia , Deltacoronavirus/fisiologia , Ácido Litocólico/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Ácidos e Sais Biliares/química , Deltacoronavirus/efeitos dos fármacos , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno , Interferons/imunologia , Células LLC-PK1 , Suínos , Doenças dos Suínos/virologia
11.
Bioorg Med Chem Lett ; 41: 128012, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33838305

RESUMO

Tacrolimus (FK506), a calcineurin inhibitor, is an effective immunosuppressive agent mainly used to lower the risk of organ rejection after allogeneic organ transplant. However, FK506-associated adverse effects, such as nephrotoxicity, may limit its therapeutic use. In this study, we confirmed that epigallocatechin-3-gallate (EGCG), sanguiin H-6, and gallic acid increased cell survival following FK506-induced cytotoxicity in renal epithelial LLC-PK1. Among these compounds, gallic acid exerted the strongest protective effect, further confirmed in the FK506-induced nephrotoxicity rat model. Additionally, we identified supporting evidence for the nephroprotective function of gallic acid using molecular docking and bioavailability investigations.


Assuntos
Ácido Gálico/farmacologia , Rim/efeitos dos fármacos , Células LLC-PK1/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Syzygium/química , Tacrolimo/antagonistas & inibidores , Animais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ácido Gálico/química , Masculino , Estrutura Molecular , Substâncias Protetoras/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Suínos , Tacrolimo/farmacologia
12.
Vet Microbiol ; 257: 109068, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33894664

RESUMO

Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus (CoV) that continues to spread globally, placing strain on economic and public health. Currently, the pathogenic mechanism of PDCoV remains largely unclear, and effective strategies to prevent or treat PDCoV infection are still limited. In this study, the interaction between autophagy and PDCoV replication in LLC-PK1 cells was investigated. We demonstrated that PDCoV infection induced a complete autophagy process. Pharmacologically induced autophagy with rapamycin increased the expression of PDCoV N, while pharmacologically inhibited autophagy with wortmannin decreased the expression of PDCoV N, suggesting that PDCoV-induced autophagy facilitates virus replication. Further experiments showed that PDCoV infection activated p38 signaling pathway to trigger autophagy. Besides, ergosterol peroxide (EP) alleviated PDCoV-induced activation of p38 to suppress autophagy, thus exerting its antiviral effects. Finally, we employed a piglet model of PDCoV infection to demonstrate that EP prevented PDCoV infection by suppressing PDCoV-induced autophagy via p38 signaling pathway in vivo. Collectively, these findings accelerate the understanding of the pathogenesis of PDCoV infection and provide new insights for the development of EP as an effective therapeutic strategy for PDCoV.


Assuntos
Antivirais/farmacologia , Autofagia , Infecções por Coronavirus/veterinária , Deltacoronavirus/efeitos dos fármacos , Ergosterol/análogos & derivados , Sistema de Sinalização das MAP Quinases , Replicação Viral/efeitos dos fármacos , Animais , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Deltacoronavirus/fisiologia , Ergosterol/farmacologia , Células LLC-PK1 , Suínos , Doenças dos Suínos/virologia
13.
Biol Pharm Bull ; 44(4): 501-506, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33790101

RESUMO

Multidrug and toxic compound extrusion (MATE) transporters are primarily expressed in the kidneys and liver, where they contribute to the excretion of organic cations. Our previous study suggested that pig MATE2 (class III) participates in testosterone secretion from Leydig cells. In humans, it is unclear which MATE class is involved in testosterone transport. In this study, we aimed to clarify whether human MATE1 (hMATE1) or human MATE2K (hMATE2K) mediates testosterone transport. To confirm that testosterone inhibits transporter-mediated tetraethylammonium (TEA) uptake, a cis-inhibition assay was performed using cells that stably expressed hMATE1 or hMATE2K. Docking simulations were performed to characterize differences in the binding of hMATE1 and hMATE2K to testosterone. Transport experiments in LLC-PK1 cells that stably expressed hMATE1 were used to test whether hMATE1 mediates testosterone transport. We detected differences between the amino acid sequences of the substrate-binding sites of hMATE1 and hMATE2K that could potentially be involved in testosterone binding. Testosterone and estradiol inhibited TEA uptake mediated by hMATE1 but not that mediated by hMATE2K. Transport experiments in LLC-PK1 cells indicated that testosterone might be transported via hMATE1. This study suggested that hMATE1, but not hMATE2K, is involved in human testosterone transport.


Assuntos
Proteínas de Transporte de Cátions Orgânicos/metabolismo , Testosterona/farmacologia , Animais , Cimetidina/farmacologia , Estradiol/farmacologia , Células HEK293 , Humanos , Células LLC-PK1 , Modelos Moleculares , Proteínas de Transporte de Cátions Orgânicos/química , Suínos , Tetraetilamônio/metabolismo
14.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669601

RESUMO

Lead (Pb) is one of the major heavy metals that are toxic to vertebrates and usually considered as environmental pollutants. ABCC4/MRP4 is an organic anion transporter that mediates cellular efflux of a wide range of exogenous and endogenous compounds such as cyclic nucleotides and anti-cancer drugs; however, it remains unclear whether ABCC4 and its orthologs function in the detoxification and excretion of toxic lead. In this study, we found that the transcriptional and translational expression of zebrafish abcc4 was significantly induced under lead exposure in developing zebrafish embryos and adult tissues. Overexpression of zebrafish Abcc4 markedly decreased the cytotoxicity and accumulation of lead in pig renal proximal tubule cell line (LLC-PK1 cells). To further understand the functions of zebrafish Abcc4 in lead detoxification, the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system was used to create an abcc4-/- mutant zebrafish line. In comparison with the wild-type (WT) zebrafish, the abcc4-/- mutants showed a higher death rate and lead accumulation upon exposure to lead. Furthermore, a stable abcc4-transgenic zebrafish line was successfully generated, which exerted stronger ability to detoxify and excrete lead than WT zebrafish. These findings indicate that zebrafish Abcc4 plays a crucial role in lead detoxification and cellular efflux and could be used as a potential biomarker to monitor lead contamination in a water environment.


Assuntos
Técnicas de Inativação de Genes , Chumbo/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Transporte Biológico , Regulação da Expressão Gênica no Desenvolvimento , Inativação Metabólica , Células LLC-PK1 , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação/genética , Suínos , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética
15.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33674463

RESUMO

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.


Assuntos
Citoesqueleto de Actina/metabolismo , Polarização de Fluorescência/métodos , Corantes Fluorescentes/metabolismo , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Imagem Molecular/métodos , Estrelas-do-Mar/embriologia , Animais , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Células LLC-PK1 , Suínos
16.
Am J Physiol Renal Physiol ; 320(5): F799-F813, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33749324

RESUMO

Glutamate N-methyl-d-aspartate receptor (NMDAR) hyperfunction is known to contribute to acute renal failure due to ischemia-reperfusion and endotoxemia. d-Serine is a coagonist for NMDAR activation, but whether NMDARs play a role in d-serine-mediated nephrotoxicity remains unclear. Here, we demonstrate that NMDAR blockade ameliorated d-serine-induced renal injury. In NMDAR-expressing LLC-PK1 cells, which were used as a proximal tubule model, d-serine but not l-serine induced cytotoxicity in a dose-dependent manner, which was abrogated by the selective NMDAR blockers MK-801 and AP-5. Time-dependent oxidative stress, evidenced by gradually increased superoxide and H2O2 production, was associated with d-serine-mediated cytotoxicity; these reactive oxygen species could be alleviated not only after NMDAR inhibition but also by NADPH oxidase (NOX) inhibition. Activation of protein kinase C (PKC)-δ and PKC-ζ is a downstream signal for NMDAR-mediated NOX activation because PKC inhibition diminishes the NOX activity that is induced by d-serine. Renal injury was further confirmed in male Wistar rats that intraperitoneally received d-serine but not l-serine. Peak changes in glucosuria, proteinuria, and urinary excretion of lactate dehydrogenase and malondialdehyde were found after 24 h of treatment. Persistent tubular damage was observed after 7 days of treatment. Cotreatment with the NMDAR blocker MK-801 for 24 h abolished d-serine-induced functional insufficiency and tubular damage. MK-801 attenuated renal superoxide formation by lowering NOX activity and protein upregulation of NOX4 but not NOX2. These results reveal that NMDAR hyperfunction underlies d-serine-induced renal injury via the effects of NOX4 on triggering oxidative stress.NEW & NOTEWORTHY Ionotropic N-methyl-d-aspartate receptors (NMDARs) are not only present in the nervous system but also expressed in the kidney. Overstimulation of renal NMDARs leads to oxidative stress via the signal pathway of calcium/protein kinase C/NADPH oxidase in d-serine-mediated tubular cell damage. Intervention of NMDAR blockade may prevent acute renal injury caused by d-serine.


Assuntos
Túbulos Renais Proximais/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Insuficiência Renal/metabolismo , Serina , Animais , Cálcio/metabolismo , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Células LLC-PK1 , Masculino , NADPH Oxidase 4/metabolismo , Estresse Oxidativo , Proteína Quinase C/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Insuficiência Renal/induzido quimicamente , Insuficiência Renal/patologia , Insuficiência Renal/prevenção & controle , Transdução de Sinais , Suínos
17.
Int Immunopharmacol ; 93: 107317, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33493866

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus (CoV) that poses economic and public health burdens. Currently, there are no effective antiviral agents against PDCoV. Cryptoporus volvatus often serves as an antimicrobial agent in Traditional Chinese Medicines. This study aimed to evaluate the antiviral activities of ergosterol peroxide (EP) from C. volvatus against PDCoV infection. The inhibitory activity of EP against PDCoV was assessed by using virus titration and performing Quantitative Reverse transcription PCR (RT-qPCR), Western blotting and immunofluorescence assays in LLC-PK1 cells. The mechanism of EP against PDCoV was analyzed by flow cytometry, RT-qPCR and Western blotting. We found that EP treatment inhibited PDCoV infection in LLC-PK1 cells in a dose-dependent manner. Subsequently, we demonstrated that EP blocked virus attachment and entry using RT-qPCR. Time-of-addition assays indicated that EP mainly exerted its inhibitory effect at the early and middle stages in the PDCoV replication cycle. EP also inactivated PDCoV infectivity directly as well as suppressed PDCoV-induced apoptosis. Furthermore, EP treatment decreased the phosphorylation of IκBα and p38 MAPK induced by PDCoV infection as well as the mRNA levels of cytokines (IL-1ß, IL-6, IL-12, TNF-α, IFN-α, IFN-ß, Mx1 and PKR). These results imply that EP can inhibit PDCoV infection and regulate host immune responses by downregulating the activation of the NF-κB and p38/MAPK signaling pathways in vitro. EP can be used as a potential candidate for the development of a new anti-PDCoV therapy.


Assuntos
Antivirais/farmacologia , Deltacoronavirus/efeitos dos fármacos , Deltacoronavirus/imunologia , Ergosterol/análogos & derivados , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Ergosterol/química , Ergosterol/farmacologia , Proteínas I-kappa B/metabolismo , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Células LLC-PK1 , Polyporaceae , Suínos , Doenças dos Suínos , Fator de Transcrição RelA/metabolismo , Vírion/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
18.
Mol Pharmacol ; 99(3): 217-225, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33495275

RESUMO

Recent studies have revealed that Na/K-ATPase (NKA) can transmit signals through ion-pumping-independent activation of pathways relayed by distinct intracellular protein/lipid kinases, and endocytosis challenges the traditional definition that cardiotonic steroids (CTS) are NKA inhibitors. Although additional effects of CTS have long been suspected, revealing its agonist impact through the NKA receptor could be a novel mechanism in understanding the basic biology of NKA. In this study, we tested whether different structural CTS could trigger different sets of NKA/effector interactions, resulting in biased signaling responses without compromising ion-pumping capacity. Using purified NKA, we found that ouabain, digitoxigenin, and somalin cause comparable levels of NKA inhibition. However, although endogenous ouabain stimulates both protein kinases and NKA endocytosis, digitoxigenin and somalin bias to protein kinases and endocytosis, respectively, in LLC-PK1 cells. The positive inotropic effects of CTS are traditionally regarded as NKA inhibitors. However, CTS-induced signaling occurs at concentrations at least one order of magnitude lower than that of inotropy, which eliminates their well known toxic actions on the heart. The current study adds a novel mechanism that CTS could exert its biased signaling properties through the NKA signal transducer. SIGNIFICANCE STATEMENT: Although it is now well accepted that NKA has an ion-pumping-independent signaling function, it is still debated whether direct and conformation-dependent NKA/effector interaction is a key to this function. Therefore, this investigation is significant in advancing our understanding of the basic biology of NKA-mediated signal transduction and gaining molecular insight into the structural elements that are important for cardiotonic steroid's biased action.


Assuntos
Glicosídeos Cardíacos/farmacologia , Digitoxigenina/farmacologia , Glicosídeos/farmacologia , Ouabaína/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células LLC-PK1 , ATPase Trocadora de Sódio-Potássio/metabolismo , Suínos
19.
Am J Physiol Cell Physiol ; 320(4): C495-C508, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33439776

RESUMO

Claudins are essential for tight junction formation and paracellular transport, and they affect key cellular events including proliferation and migration. The properties of tight junctions are dynamically modulated by a variety of inputs. We previously showed that the inflammatory cytokine tumor necrosis factor-α (TNFα), a major pathogenic factor in kidney disease, alters epithelial permeability by affecting the expression of claudin-1, -2, and -4 in kidney tubular cells. Here, we explored the effect of TNFα on claudin-3 (Cldn-3), a ubiquitous barrier-forming protein. We found that TNFα elevated Cldn-3 protein expression in tubular epithelial cells (LLC-PK1 and IMCD3) as early as 3 h post treatment. Bafilomycin A and bortezomib, inhibitors of lysosomal and proteasomes, respectively, reduced Cldn-3 degradation. However, TNFα caused a strong upregulation of Cldn-3 in the presence of bafilomycin, suggesting an effect independent from lysosomes. Blocking protein synthesis using cycloheximide prevented Cldn-3 upregulation by TNFα, verifying the contribution of de novo Cldn-3 synthesis. Indeed, TNFα elevated Cldn-3 mRNA levels at early time points. Using pharmacological inhibitors and siRNA-mediated silencing, we determined that the effect of TNFα on Cldn-3 was mediated by extracellular signal regulated kinase (ERK)-dependent activation of NF-κB and PKA-induced activation of CREB1. These two pathways were turned on by TNFα in parallel and both were required for the upregulation of Cldn-3. Because Cldn-3 was suggested to modulate cell migration and epithelial-mesenchymal transition (EMT), and TNFα was shown to affect these processes, Cldn-3 upregulation may modulate regeneration of the tubules following injury.


Assuntos
Claudina-3/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células CACO-2 , Movimento Celular/efeitos dos fármacos , Claudina-3/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Células LLC-PK1 , Masculino , Camundongos , Transdução de Sinais , Suínos , Regulação para Cima
20.
Toxins (Basel) ; 13(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33466223

RESUMO

Massive, Africanized honeybee attacks have increased in Brazil over the years. Humans and animals present local and systemic effects after envenomation, and there is no specific treatment for this potentially lethal event. This study evaluated the ability of a new Apilic antivenom, which is composed of F(ab')2 fraction of specific immunoglobulins in heterologous and hyperimmune equine serum, to neutralize A. mellifera venom and melittin, in vitro and in vivo, in mice. Animal experiments were performed in according with local ethics committee license (UFRJ protocol no. DFBCICB072-04/16). Venom dose-dependent lethality was diminished with 0.25-0.5 µL of intravenous Apilic antivenom/µg honeybee venom. In vivo injection of 0.1-1 µg/g bee venom induced myotoxicity, hemoconcentration, paw edema, and increase of vascular permeability which were antagonized by Apilic antivenom. Cytotoxicity, assessed in renal LLC-PK1 cells and challenged with 10 µg/mL honeybee venom or melittin, was neutralized by preincubation with Apilic antivenom, as well the hemolytic activity. Apilic antivenom inhibited phospholipase and hyaluronidase enzymatic activities. In flow cytometry experiments, Apilic antivenom neutralized reduction of cell viability due to necrosis by honeybee venom or melittin. These results showed that this antivenom is effective inhibitor of honeybee venom actions. Thus, this next generation of Apilic antivenom emerges as a new promising immunobiological product for the treatment of massive, Africanized honeybee attacks.


Assuntos
Antivenenos/uso terapêutico , Venenos de Abelha/antagonistas & inibidores , Mordeduras e Picadas/tratamento farmacológico , Meliteno/antagonistas & inibidores , Animais , Anticorpos/sangue , Abelhas , Brasil , Linhagem Celular , Sobrevivência Celular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Hemólise/efeitos dos fármacos , Cavalos , Hialuronoglucosaminidase/antagonistas & inibidores , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Injeções Intradérmicas , Células LLC-PK1 , Dose Letal Mediana , Masculino , Camundongos , Modelos Animais , Testes de Neutralização , Fosfolipases/antagonistas & inibidores , Suínos
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