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1.
J Am Soc Nephrol ; 30(10): 1910-1924, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31416818

RESUMO

BACKGROUND: Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified role within the target cell for FSGS, the kidney podocyte. METHODS: We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6 mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. RESULTS: Compared with wild-type cells, TRPC6-/- podocytes are less motile and more adhesive, with an altered actin cytoskeleton. We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- and actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, and motility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton, motility, and adhesion-characteristics of TRPC6-/- podocytes. CONCLUSIONS: Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.


Assuntos
Calpaína/fisiologia , Adesão Celular , Movimento Celular , Citoesqueleto/fisiologia , Podócitos/fisiologia , Podócitos/ultraestrutura , Canal de Cátion TRPC6/fisiologia , Animais , Camundongos , Camundongos Knockout
2.
Nature ; 451(7174): 69-72, 2008 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-18172497

RESUMO

Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these channels. Here we show the activation of TRPC5 (canonical TRP 5) homomultimeric and TRPC5-TRPC1 heteromultimeric channels by extracellular reduced thioredoxin, which acts by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis, an inflammatory joint disease that disables millions of people worldwide. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, that endogenous TRPC5-TRPC1 channels of the cells are activated by reduced thioredoxin, and that blockade of the channels enhances secretory activity and prevents the suppression of secretion by thioredoxin. The data indicate the presence of a previously unrecognized ion-channel activation mechanism that couples extracellular thioredoxin to cell function.


Assuntos
Canais de Cátion TRPC/agonistas , Canais de Cátion TRPC/metabolismo , Tiorredoxinas/farmacologia , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Linhagem Celular , Dissulfetos/química , Dissulfetos/metabolismo , Condutividade Elétrica , Humanos , Oxirredução/efeitos dos fármacos , Técnicas de Patch-Clamp , Coelhos , Canais de Cátion TRPC/química , Tiorredoxinas/química
3.
Mol Biol Rep ; 40(1): 7-12, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23114911

RESUMO

The objective of this study was to investigate the single nucleotide polymorphisms (SNPs) within bovine binding lectin-liver (A) gene (MBL1) and to explore its correlation analysis with milk somatic cell score (SCS) which reflects mastitis resistance in cattle. Through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), created restriction site-PCR (CRS-PCR) and DNA sequencing methods, three allelic variant corresponding to the G→A mutation at c.1252G>A in intron1, G→A mutation at c.2534G>A and T→C mutation at c.2569T>C in exon2 of bovine MBL1 gene, could be detected, respectively. The c.2534G>A was a nonsynonymous mutation, resulting in Valine (Val) to Isoleucine (Ile) amino acid replacement (p.Val24Ile). The correlation analysis between the MBL1 SNPs gene and milk SCS were analyzed and a significant correlation with milk SCS was detected in c.2534G>A. The value of milk SCS for individuals with genotype GG was significantly lower than those of genotype GA and AA. Results showed that genotype GG with the lowest milk SCS was favorable for mastitis resistance, whereas genotype AA with the highest milk SCS was easily for mastitis susceptibility. Although more investigations are needed to better clarify the role of these SNPs on mastitis resistance, MBL1 polymorphism appears to be a promising indirect marker to improve dairy mastitis resistance traits in cattle.


Assuntos
Bovinos/genética , Lectina de Ligação a Manose/genética , Leite/citologia , Polimorfismo de Nucleotídeo Único , Alelos , Animais , Feminino , Frequência do Gene , Genótipo , Mastite Bovina/genética , Mastite Bovina/metabolismo , Polimorfismo de Fragmento de Restrição
4.
Mol Biol Rep ; 40(2): 1947-54, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23143182

RESUMO

This study was designed to investigate the candidate single nucleotide polymorphisms (SNPs) in the exon's region of bovine diacylglycerol O-acyltransferase (DGAT1) gene using bioinformatics and experimental methods. A total of 17 SNPs were screened from public data resources and DNA sequencing. Three SNPs (c.572A>G, c.1241C>T and c.1416T>G) of these candidate SNPs were genotyped by created restriction site-polymerase chain reaction (CRS-PCR) methods. The gene-specific SNP markers and their effects on meat and carcass fatness quality traits were evaluated in Chinese commercial cattle. The c.572A>G and c.1416T>G significantly effected on backfat thickness, longissimus muscle area, marbling score, fat color and Warner-Bratzler shear force. No significant association was detected between the c.1241C>T and measured traits. Results from this study suggested that the SNP markers may be effective for the marker-assisted selection of meat and carcass fatness quality traits, and added new evidence that DGAT1 gene is an important candidate gene for the improvement of meat and carcass fatness quality in beef cattle industry.


Assuntos
Tecido Adiposo/anatomia & histologia , Adiposidade/genética , Diacilglicerol O-Aciltransferase/genética , Carne/normas , Músculo Esquelético/anatomia & histologia , Tecido Adiposo/enzimologia , Animais , Dorso/anatomia & histologia , Sequência de Bases , Bovinos/anatomia & histologia , Bovinos/genética , Bovinos/metabolismo , Qualidade dos Alimentos , Frequência do Gene , Estudos de Associação Genética , Músculo Esquelético/enzimologia , Polimorfismo de Nucleotídeo Único
5.
Yi Chuan ; 35(6): 771-7, 2013 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-23774022

RESUMO

As a useful tool for genetic engineering, piggyBac (PB) transposons have been widely used in more than one species of transgenosis or generating mutation studies. At present, the studies about PB transposons in cattle were few. In order to get the PB transposon integration sites and summarize its characteristics in bovine genome, donor plasmid of PB[CMV-EGFP] and helper-dependent plasmid of pcDNA-PBase were constructed and transferred into bovine fibroblasts by Amaxa basic nucleofector kit for primary mammalian fibroblasts. Cell clones stably transfected were obtained after screening by G-418. Genomic DNA of transgenic cells was extracted and the integration sites of PB transposon were detected by genome walking technology. Eight integration sites were obtained in bovine genome, although only 5 sites were mapped on chromosomes 1, 2, 11, and X chromosome. We found that PB transposon was inserted into the "TTAA" location and integrated into the intergenic non-regulatory sites between two genes. Analysis of the composition of the five bases, which was close to the side of the PB integration sites "TTAA", showed that PB 5' tended to be inserted into region rich in GC (62.5%). From the study, we got that transposition occurred in cattle genome by PB transposons and the integration site information acquired from the research will provide theoretical references for bovine study by PB transposon.


Assuntos
Bovinos/genética , Elementos de DNA Transponíveis , Animais , Genoma , Plasmídeos , Transfecção
6.
Biomolecules ; 13(6)2023 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-37371532

RESUMO

An elevated level of circulating homocysteine (Hcy) has been regarded as an independent risk factor for cardiovascular disease; however, the clinical benefit of Hcy lowering-therapy is not satisfying. To explore potential unrevealed mechanisms, we investigated the roles of Ca2+ influx through TRPC channels and regulation by Hcy-copper complexes. Using primary cultured human aortic endothelial cells and HEK-293 T-REx cells with inducible TRPC gene expression, we found that Hcy increased the Ca2+ influx in vascular endothelial cells through the activation of TRPC4 and TRPC5. The activity of TRPC4 and TRPC5 was regulated by extracellular divalent copper (Cu2+) and Hcy. Hcy prevented channel activation by divalent copper, but monovalent copper (Cu+) had no effect on the TRPC channels. The glutamic acids (E542/E543) and the cysteine residue (C554) in the extracellular pore region of the TRPC4 channel mediated the effect of Hcy-copper complexes. The interaction of Hcy-copper significantly regulated endothelial proliferation, migration, and angiogenesis. Our results suggest that Hcy-copper complexes function as a new pair of endogenous regulators for TRPC channel activity. This finding gives a new understanding of the pathogenesis of hyperhomocysteinemia and may explain the unsatisfying clinical outcome of Hcy-lowering therapy and the potential benefit of copper-chelating therapy.


Assuntos
Cobre , Células Endoteliais , Humanos , Cobre/farmacologia , Cobre/metabolismo , Células Endoteliais/metabolismo , Células HEK293 , Proteínas de Transporte , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Cálcio/metabolismo
7.
Biochem Biophys Res Commun ; 424(2): 279-84, 2012 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-22750002

RESUMO

Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cationic channel in the TRP channel family. The channel activity can be regulated by reactive oxygen species (ROS) and cellular acidification, which has been implicated to the pathogenesis of diabetes and some neuronal disorders. However, little is known about the effect of redox-active metal ions, such as copper, on TRPM2 channels. Here we investigated the effect of divalent copper on TRPM2. TRPM2 channel was over-expressed in HEK-293 cells and the whole-cell current was recorded by patch clamp. We found the whole-cell current evoked by intracellular ADP-ribose was potently inhibited by Cu(2+) with a half maximal inhibitory concentration (IC(50)) of 2.59 µM. The inhibitory effect was irreversible. The single channel activity was abolished in the outside-out patches, and intracellular application of Cu(2+) did not prevent the channel activation, suggesting that the action site of Cu(2+) is located in the extracellular domains of the channel. TRPM2 current was also blocked by Hg(2+), Pb(2+), Fe(2+) and Se(2+). We concluded that Cu(2+) is a potent TRPM2 channel blocker. The sensitivity of TRPM2 channel to heavy metal ions could be a new mechanism for the pathogenesis of some metal ion-related diseases.


Assuntos
Cátions Bivalentes/farmacologia , Cobre/farmacologia , Canais de Cátion TRPM/antagonistas & inibidores , Adenosina Difosfato Ribose/metabolismo , Adenosina Difosfato Ribose/farmacologia , Cátions Bivalentes/metabolismo , Cobre/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Ferro/metabolismo , Ferro/farmacologia , Chumbo/metabolismo , Chumbo/farmacologia , Mercúrio/metabolismo , Mercúrio/farmacologia , Técnicas de Patch-Clamp , Selênio/metabolismo , Selênio/farmacologia
8.
Mol Biol Rep ; 39(6): 6625-31, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22327776

RESUMO

Bovine mastitis is a very complex and common disease of dairy cattle and a major source of economic losses to the dairy industry worldwide. In this study, the bovine breast cancer 1, early onset gene (BRCA1) was taken as a candidate gene for mastitis resistance. The main object of this study was to investigate whether the BRCA1 gene was associated with mastitis in cattle. Through DNA sequencing, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Created Restriction Site PCR (CRS-PCR) methods, three SNPs (G22231T, T25025A, and C28300A) were detected and twenty-four combinations of these SNPs were observed. The single SNP and their genetic effects on somatic cell score (SCS) were evaluated and a significant association with SCS was found in C28300A. The mean of genotype EE was significantly lower than those of genotypes EF and FF. The results of combined genotypes analysis of three SNPs showed that BBDDFF genotype with the highest SCS were easily for the mastitis susceptibility, whereas AACCEE genotype with the lowest SCS were favorable for the mastitis resistance. The information provided in the present study will be very useful for improving mastitis resistance in dairy cattle by marker-assisted selection.


Assuntos
Genes BRCA1 , Mastite Bovina/genética , Leite/citologia , Polimorfismo de Nucleotídeo Único , Animais , Bovinos , Contagem de Células , Resistência à Doença/genética , Feminino , Frequência do Gene , Estudos de Associação Genética , Marcadores Genéticos , Predisposição Genética para Doença , Genótipo , Análise de Sequência de DNA
9.
Life Sci ; 306: 120834, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-35902031

RESUMO

Mitochondria play a pivotal role in cellular function, not only acting as the powerhouse of the cell, but also regulating ATP synthesis, reactive oxygen species (ROS) production, intracellular Ca2+ cycling, and apoptosis. During the past decade, extensive progress has been made in the technology to assess mitochondrial functions and accumulating evidences have shown that mitochondrial dysfunction is a key pathophysiological mechanism for many diseases including cardiovascular disorders, such as ischemic heart disease, cardiomyopathy, hypertension, atherosclerosis, and hemorrhagic shock. The advances in methodology have been accelerating our understanding of mitochondrial molecular structure and function, biogenesis and ROS and energy production, which facilitates new drug target identification and therapeutic strategy development for mitochondrial dysfunction-related disorders. This review will focus on the assessment of methodologies currently used for mitochondrial research and discuss their advantages, limitations and the implications of mitochondrial dysfunction in cardiovascular disorders.


Assuntos
Doenças Cardiovasculares , Doenças Mitocondriais , Apoptose , Doenças Cardiovasculares/metabolismo , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo
10.
Commun Biol ; 5(1): 353, 2022 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-35418663

RESUMO

Drung cattle (Bos frontalis) have 58 chromosomes, differing from the Bos taurus 2n = 60 karyotype. To date, its origin and evolution history have not been proven conclusively, and the mechanisms of chromosome fusion and environmental adaptation have not been clearly elucidated. Here, we assembled a high integrity and good contiguity genome of Drung cattle with 13.7-fold contig N50 and 4.1-fold scaffold N50 improvements over the recently published Indian mithun assembly, respectively. Speciation time estimation and phylogenetic analysis showed that Drung cattle diverged from Bos taurus into an independent evolutionary clade. Sequence evidence of centromere regions provides clues to the breakpoints in BTA2 and BTA28 centromere satellites. We furthermore integrated a circulation and contraction-related biological process involving 43 evolutionary genes that participated in pathways associated with the evolution of the cardiovascular system. These findings may have important implications for understanding the molecular mechanisms of chromosome fusion, alpine valleys adaptability and cardiovascular function.


Assuntos
Centrômero , Genoma , Animais , Bovinos , Cariótipo , Cariotipagem , Filogenia
11.
Mol Biol Rep ; 38(8): 5307-12, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21253852

RESUMO

The effect of castration on carcass quality was investigated by ten Chinese Simmental calves. Five calves were castrated randomly at 2 months old and the others were retained as normal intact bulls. All animals were slaughtered at 22 months old. The results showed that bulls carcass had higher weight (P < 0.05), dressing percentages and bigger longissimus muscle areas (P < 0.05) than steers. But steer meat had lower shear force values and was fatter (P < 0.05) than bull. Furthermore, in order to discover genes that were involved in determining steer meat quality, we compared related candidate gene expression in longissimus muscle between steer (tester) and bull (driver) using suppressive subtractive hybridization. Ten genes were identified as preferentially expressed in longissimus muscle of steer. The expression of four selected differentially expressed genes was confirmed by quantitative real-time PCR. Overall, a 1.96, 2.41, 2.89, 2.41-fold increase in expression level was observed in steer compared with bull for actin, gamma 2, smooth muscle, tropomyosin-2, insulin like growth factor 1 and hormone-sensitive lipase, respectively. These results implied that these differentially expressed genes could play an important role in the regulation of steer meat quality.


Assuntos
Castração , Bovinos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Carne/normas , Músculo Esquelético/metabolismo , Animais , DNA Complementar/genética , Masculino , Hibridização de Ácido Nucleico , Característica Quantitativa Herdável
12.
Mol Biol Rep ; 38(7): 4705-8, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21153925

RESUMO

Thyroid hormones play an important role in regulating metabolism and can affect homeostasis of fat deposition. The gene encoding thyroglobulin (TG), producing the precursor for thyroid hormones, has been proposed as a positional and functional candidate gene for a QTL with an effect on fat deposition. In the present study, we identified 6 novel SNPs at the 3' flanking region of the TG gene. The SNP marker association analysis indicated that the T354C, G392A, A430G and T433G SNP markers were significantly associated with marbling score (P < 0.05). Animals with the new homozygote genotype had higher marbling score than those with the other genotypes. Otherwise, the linkage disequilibrium analysis indicated that these four SNPs were completely linked (r (2) = 1). Results from this study suggest that TG gene-specific SNP may be a useful marker for meat quality traits in future marker assisted selection programs in beef cattle.


Assuntos
Bovinos/genética , Estudos de Associação Genética , Carne/normas , Polimorfismo de Nucleotídeo Único/genética , Característica Quantitativa Herdável , Tireoglobulina/genética , Animais , Sequência de Bases , Frequência do Gene/genética , Marcadores Genéticos , Genótipo , Dados de Sequência Molecular , Fenótipo
13.
Waste Manag Res ; 29(12): 1262-70, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21447611

RESUMO

Waste vinegar residue, the by-product of vinegar processing, was used as substrate for phytase production from Aspergillus ficuum NTG-23 in solid-state fermentation to investigate the potential for the efficient re-utilization or recycling of waste vinegar residue. Statistical designs were applied in the processing of phytase production. First, a Plackett-Burman (PB) design was used to evaluate eleven parameters: glucose, starch, wheat bran, (NH(4))(2)SO(4), NH(4)NO(3), tryptone, soybean meal, MgSO(4)·7H(2)O, CaCl(2)·7H(2)O, FeSO(4)·7H(2)O, incubation time. The PB experiments showed that there were three significant factors: glucose, soybean meal and incubation time. The closest values to the optimum point were then derived by steepest ascent path. Finally, a mathematical model was created and validated to explain the behavioural process after these three significant factors were optimized using response surface methodology (RSM). The best phytase activity was attained using the following conditions: glucose (7.2%), soybean meal (5.1%), and incubation time (271 h). The phytase activity was 7.34-fold higher due to optimization by PB design, steepest ascent path design and RSM. The phytase activity was enhanced 0.26-fold in comparison with the results by the second step of steepest ascent path design. The results indicate that with waste vinegar residue as a substrate higher production of phytase from Aspergillus ficuum NTG-23 could be obtained through an optimization process and that this method might be applied to an integrated system for recycling of the waste vinegar residue.


Assuntos
6-Fitase/metabolismo , Ácido Acético/química , Aspergillus/metabolismo , Gerenciamento de Resíduos/métodos , Ácido Acético/metabolismo , Modelos Biológicos , Resíduos
14.
PLoS One ; 16(2): e0247234, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606763

RESUMO

Elevated reactive oxygen species (ROS) in type 2 diabetes cause cellular damage in many organs. Recently, the new class of glucose-lowering agents, SGLT-2 inhibitors, have been shown to reduce the risk of developing diabetic complications; however, the mechanisms of such beneficial effect are largely unknown. Here we aimed to investigate the effects of dapagliflozin on cell proliferation and cell death under oxidative stress conditions and explore its underlying mechanisms. Human proximal tubular cells (HK-2) were used. Cell growth and death were monitored by cell counting, water-soluble tetrazolium-1 (WST-1) and lactate dehydrogenase (LDH) assays, and flow cytometry. The cytosolic and mitochondrial (ROS) production was measured using fluorescent probes (H2DCFDA and MitoSOX) under normal and oxidative stress conditions mimicked by addition of H2O2. Intracellular Ca2+ dynamics was monitored by FlexStation 3 using cell-permeable Ca2+ dye Fura-PE3/AM. Dapagliflozin (0.1-10 µM) had no effect on HK-2 cell proliferation under normal conditions, but an inhibitory effect was seen at an extreme high concentration (100 µM). However, dapagliflozin at 0.1 to 5 µM showed remarkable protective effects against H2O2-induced cell injury via increasing the viable cell number at phase G0/G1. The elevated cytosolic and mitochondrial ROS under oxidative stress was significantly decreased by dapagliflozin. Dapagliflozin increased the basal intracellular [Ca2+]i in proximal tubular cells, but did not affect calcium release from endoplasmic reticulum and store-operated Ca2+ entry. The H2O2-sensitive TRPM2 channel seemed to be involved in the Ca2+ dynamics regulated by dapagliflozin. However, dapagliflozin had no direct effects on ORAI1, ORAI3, TRPC4 and TRPC5 channels. Our results suggest that dapagliflozin shows anti-oxidative properties by reducing cytosolic and mitochondrial ROS production and altering Ca2+ dynamics, and thus exerts its protective effects against cell damage under oxidative stress environment.


Assuntos
Compostos Benzidrílicos/farmacologia , Glucosídeos/farmacologia , Túbulos Renais Proximais/patologia , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citosol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/genética , Fenantridinas/metabolismo , Isoformas de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/metabolismo , Transportador 2 de Glucose-Sódio/genética , Transportador 2 de Glucose-Sódio/metabolismo
15.
Expert Opin Drug Discov ; 16(4): 447-461, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33003971

RESUMO

INTRODUCTION: Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease (ESRD), and 40% of patients with diabetes develop DKD. Although some pathophysiological mechanisms and drug targets of DKD have been described, the effectiveness or clinical usefulness of such treatment has not been well validated. Therefore, searching for new targets and potential therapeutic candidates has become an emerging research area. AREAS COVERED: The pathophysiological mechanisms, new drug targets and potential therapeutic compounds for DKD are addressed in this review. EXPERT OPINION: Although preclinical and clinical evidence has shown some positive results for controlling DKD progression, treatment regimens have not been well developed to reduce the mortality in patients with DKD globally. Therefore, the discovery of new therapeutic targets and effective target-based drugs to achieve better and safe treatment are urgently required. Preclinical screening and clinical trials for such drugs are needed.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Descoberta de Drogas/métodos , Falência Renal Crônica/prevenção & controle , Animais , Nefropatias Diabéticas/complicações , Nefropatias Diabéticas/fisiopatologia , Progressão da Doença , Desenvolvimento de Medicamentos , Humanos , Falência Renal Crônica/etiologia
16.
Biochem Genet ; 48(9-10): 751-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20549332

RESUMO

The objective of this study was to identify genetic polymorphisms of the CACNA2D1 gene and to analyze associations between SNPs and carcass and meat quality traits in cattle. Through PCR-RFLP and DNA sequencing methods, a new allelic variant corresponding to the A --> G mutation (aspartic to glycine amino acid replacement) of the bovine CACNA2D1 gene was detected. Two alleles and three genotypes (AA, AG, and GG) were defined. Genetic character indicated that the A526745G locus showed moderate polymorphism and was in Hardy-Weinberg equilibrium. Gene-specific SNP marker association analysis showed that the A526745G mutant was significantly associated with carcass weight, dressing percentage, meat percentage, and backfat thickness. The results add new evidence that CACNA2D1 is an important candidate gene for the selection of carcass and meat quality traits in the cattle industry.


Assuntos
Canais de Cálcio/genética , Bovinos/anatomia & histologia , Bovinos/genética , Carne/normas , Polimorfismo de Nucleotídeo Único , Animais , Cruzamento , Frequência do Gene , Marcadores Genéticos/genética , Genótipo
17.
Stem Cell Res Ther ; 11(1): 395, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32928296

RESUMO

BACKGROUND: Bone mesenchymal stem cells (MSCs) can promote liver regeneration and inhibit inflammation and hepatic fibrosis. MSCs also can serve as a vehicle for gene therapy. Smad7 is an essential negative regulatory gene in the TGF-ß1/Smad signalling pathway. Activation of TGF-ß1/Smad signalling accelerates liver inflammation and fibrosis; we therefore hypothesized that MSCs overexpressing the Smad7 gene might be a new cell therapy approach for treating liver fibrosis via the inhibition of TGF-ß1/Smad signalling. METHODS: MSCs were isolated from 6-week-old Wistar rats and transduced with the Smad7 gene using a lentivirus vector. Liver cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl4) for 8 weeks. The rats with established liver cirrhosis were treated with Smad7-MSCs by direct injection of cells into the main lobes of the liver. The expression of Smad7, Smad2/3 and fibrosis biomarkers or extracellular matrix proteins and histopathological change were assessed by quantitative PCR, ELISA and Western blotting and staining. RESULTS: The mRNA and protein level of Smad7 in the recipient liver and serum were increased after treating with Smad-MSCs for 7 and 21 days (P < 0.001). The serum levels of collagen I and III and collagenase I and III were significantly (P < 0.001) reduced after the treatment with Smad7-MSCs. The mRNA levels of TGF-ß1, TGFBR1, α-SMA, TIMP-1, laminin and hyaluronic acid were decreased (P < 0.001), while MMP-1 increased (P < 0.001). The liver fibrosis score and liver function were significantly alleviated after the cell therapy. CONCLUSIONS: The findings suggest that the MSC therapy with Smad7-MSCs is effective in the treatment of liver fibrosis in the CCl4-induced liver cirrhosis model. Inhibition of TGF-ß1 signalling pathway by enhancement of Smad-7 expression could be a feasible cell therapy approach to mitigate liver cirrhosis.


Assuntos
Cirrose Hepática Experimental , Células-Tronco Mesenquimais , Animais , Terapia Genética , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/terapia , Cirrose Hepática Experimental/patologia , Células-Tronco Mesenquimais/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteína Smad7/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
18.
Br J Pharmacol ; 176(19): 3845-3856, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31271653

RESUMO

BACKGROUND AND PURPOSE: Mibefradil, a T-type Ca2+ channel blocker, has been investigated for treating solid tumours. However, its underlying mechanisms are still unclear. Here, we have investigated the pharmacological actions of mibefradil on Orai store-operated Ca2+ channels. EXPERIMENTAL APPROACH: Human Orai1-3 cDNAs in tetracycline-regulated pcDNA4/TO vectors were transfected into HEK293 T-REx cells with stromal interaction molecule 1 (STIM1) stable expression. The Orai currents were recorded by whole-cell and excised-membrane patch clamp. Ca2+ influx or release was measured by Fura-PE3/AM. Cell growth and death were monitored by WST-1, LDH assays and flow cytometry. KEY RESULTS: Mibefradil inhibited Orai1, Orai2, and Orai3 currents dose-dependently. The IC50 for Orai1, Orai2, and Orai3 channels was 52.6, 14.1, and 3.8 µM respectively. Outside-out patch demonstrated that perfusion of 10-µM mibefradil to the extracellular surface completely blocked Orai3 currents and single channel activity evoked by 2-APB. Intracellular application of mibefradil did not alter Orai3 channel activity. Mibefradil at higher concentrations (>50 µM) inhibited Ca2+ release but had no effect on cytosolic STIM1 translocation evoked by thapsigargin. Inhibition on Orai channels by mibefradil was structure-related, as other T-type Ca2+ channel blockers with different structures, such as ethosuximide and ML218, had no or minimal effects on Orai channels. Moreover, mibefradil inhibited cell proliferation, induced apoptosis, and arrested cell cycle progression. CONCLUSIONS AND IMPLICATIONS: Mibefradil is a potent cell surface blocker of Orai channels, demonstrating a new pharmacological action of this compound in regulating cell growth and death, which could be relevant to its anti-cancer activity.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Mibefradil/farmacologia , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI2/antagonistas & inibidores , Cálcio/análise , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/química , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Humanos , Mibefradil/química , Proteína ORAI1/metabolismo , Proteína ORAI2/metabolismo , Imagem Óptica
19.
J Histochem Cytochem ; 56(8): 733-43, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18443363

RESUMO

Somatostatin mediates inhibitory functions through five G protein-coupled somatostatin receptors (sst1-5). We used immunohistochemistry, immunofluorescence, and RT-PCR to determine the presence of somatostatin receptors sst1, sst2A, sst2B, sst3, sst4, and sst5 in normal and IgA nephropathy human kidney. All somatostatin receptors were detected in the thin tubules (distal convoluted tubules and loops of Henle) and thick tubules (proximal convoluted tubules) in the tissue sections from nephrectomy and biopsy samples. Immunopositive sst1 and sst4 staining was more condensed in the cytoplasm of tubular epithelial cells. In normal kidney tissue sections, podocytes and mesangial cells in the glomeruli stained for sst1, sst2B, sst4 and sst5, and stained weakly for sst3. In IgA kidney tissue, the expression of somatostatin receptors was significantly increased with particular immmunopositive staining for sst1, sst2B, sst4, and sst5 within glomeruli. In the epithelial cells, the staining for sst2B and sst4 in proximal tubules and sst1, sst2B, and sst5 in distal tubules was increased. The mRNA expression of sst1-5 was also detected by RT-PCR. Somatostatin and all five receptor subtypes were ubiquitously distributed in normal kidney and IgA nephropathy. The increased expression of somatostatin receptors in IgA nephropathy kidney might be the potential pathogenesis of inflammatory renal disease.


Assuntos
Glomerulonefrite por IGA/metabolismo , Rim/metabolismo , Receptores de Somatostatina/biossíntese , Somatostatina/biossíntese , Humanos , Imuno-Histoquímica , RNA Mensageiro/biossíntese , Receptores de Somatostatina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Circ Res ; 98(11): 1381-9, 2006 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-16675717

RESUMO

In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1-phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5-TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation.


Assuntos
Canais de Cálcio/fisiologia , Movimento Celular/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Esfingosina/análogos & derivados , Movimento Celular/fisiologia , Células Cultivadas , Espaço Extracelular/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lisofosfolipídeos/metabolismo , Toxina Pertussis/farmacologia , Receptores de Superfície Celular/metabolismo , Veia Safena/metabolismo , Esfingosina/metabolismo , Esfingosina/farmacologia , Canais de Cátion TRPC/química , Canais de Cátion TRPC/efeitos dos fármacos , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPC/fisiologia
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