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1.
Molecules ; 24(22)2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31744088

RESUMO

The modulators of farnesoid X receptor (FXR), a bile acid receptor, regulate various biological processes including bile acid metabolism, and are associated with the control of fatty liver and osteoporosis. Thus, the control of FXR activity and development of FXR modulators are critical not only for research, but also for clinical application. In this study, we synthesized novel FXR agonists 1-4 possessing isoxazole and N-substituted benzimidazole moieties, and compared their effects on osteoblast differentiation with the known FXR agonists, chenodeoxycholic acid and a synthetic compound, GW4064. Two (3 and 4) of the four novel FXR agonists 1-4 showed high specificities for FXR. Computer-assisted modeling suggested that the binding of the FXR agonist 3 with ligand binding domain of FXR was similar to GW4064. FXR was expressed in mouse bone marrow-derived mesenchymal stem cell (MSC)-like ST2 cells (ST-2 MSCs). The FXR agonists activated the BMP-2-induced differentiation of ST-2 MSCs into osteoblasts and enhanced the expression of RUNX2. Moreover, the potency of the FXR agonist 3 was comparable to GW4064 in promoting osteoblast differentiation of ST-2 MSCs. These results indicate that FXR activation enhanced the BMP-2-induced differentiation of MSCs into osteoblasts through activating RUNX2 expression. FXR could be a potential therapeutic target for the treatment of bone diseases such as osteoporosis.

2.
J Agric Food Chem ; 67(36): 10107-10115, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31434473

RESUMO

We examined the antiobesity effect of a limonoid 7-deacetoxy-7-oxogedunin, named CG-1, purified from the seeds of Carapa guianensis, Meliaceae, known as andiroba in high-fat-diet (HFD)-fed mice. C57BL/6 mice were fed a low-fat diet or an HFD and orally administered CG-1 (20 mg/kg) for 7 weeks. CG-1 lowered the body weight gain and improved the serum triglyceride level and insulin sensitivity in HFD-fed mice. The expression level of the adipogenesis-related genes was lowered by CG-1 in the visceral white adipose tissue (vWAT). The mRNA expression level of the macrophage-related genes decreased in vWAT following the administration of CG-1 to HFD-fed mice. It is noteworthy that CG-1 activated the brown adipose tissue (BAT) with enhanced expression of uncoupling protein 1 and increased the rectal temperature in HFD-fed mice. These results indicate that the limonoid CG-1 decreased body weight gain and ameliorated hypertriglyceridemia and insulin resistance with the activation of BAT in HFD-fed mice.


Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Fármacos Antiobesidade/administração & dosagem , Resistência à Insulina , Limoninas/administração & dosagem , Meliaceae/química , Obesidade/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Tecido Adiposo Marrom/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Obesidade/metabolismo , Obesidade/fisiopatologia , Sementes/química , Triglicerídeos/sangue , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Ganho de Peso/efeitos dos fármacos
3.
Molecules ; 24(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035366

RESUMO

Limonoids are phytochemicals with a variety of biological properties. In the present study, we elucidated the molecular mechanism of suppression of adipogenesis in adipocytes by a limonoid, 7-deacetoxy-7-oxogedunin (CG-1) from Carapa guianensis (Meliaceae), known as andiroba. CG-1 reduced the accumulation of intracellular triglycerides in a concentration-dependent manner. The expression levels of the adipogenic, lipogenic, and lipolytic genes were decreased by CG-1 treatment, whereas the glycerol release level was not affected. When CG-1 was added into the medium during days 0-2 of 6-days-adipogenesis, the accumulation of intracellular lipids and the mRNA levels of the adipogenesis-related genes were decreased. In addition, the phosphorylation level of insulin receptor substrate-1 (IRS-1) and Akt in the early phase of adipocyte differentiation (within 1 day after initiating adipocyte differentiation) was reduced by CG-1. Furthermore, insulin-activated translocation of glucose transporter 4 to the plasma membranes in adipocytes was suppressed by CG-1, followed by decreased glucose uptake into the cells. These results indicate that an andiroba limonoid CG-1 suppressed the accumulation of intracellular lipids in the early phase of adipocyte differentiation through repression of IRS-1/Akt-mediated glucose uptake in adipocytes.


Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Transportador de Glucose Tipo 4/genética , Glucose/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Limoninas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células 3T3-L1 , Adipogenia/efeitos dos fármacos , Animais , Transportador de Glucose Tipo 4/metabolismo , Limoninas/química , Meliaceae/química , Camundongos , Estrutura Molecular
4.
Exp Cell Res ; 380(2): 171-179, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31039349

RESUMO

Classically activated macrophages (CAMs) play a crucial protective role in the host by killing the invading pathogens. However, excessive activation of CAMs causes chronic inflammation leading to host tissue damage. Thus, control of macrophage activity is necessary to prevent chronic inflammation. To date, regulation of CAMs in the development of chronic inflammatory diseases has not been elucidated. In this study, we investigated the effect of a pan-caspase inhibitor, zVAD-fmk, in cell death in lipopolysaccharide (LPS)-activated macrophages, CAMs. Necrostatin-1, an inhibitor of necroptosis, inhibited zVAD-fmk-induced cell death in CAMs. The expression of mixed lineage kinase domain-like protein (MLKL) involved in the necroptosis pathway was up-regulated by LPS in CAMs. zVAD-fmk enhanced the phosphorylation of MLKL in CAMs. Moreover, inhibition of activation of mitogen activated protein kinase p38 and generation of reactive oxygen species (ROS) reduced zVAD-fmk-induced cell death in CAMs. Inhibition of ROS generation decreased the activation of MLKL and p38 in zVAD-fmk-treated CAMs. These results, taken together, indicate that zVAD-fmk-induced cell death occurred by necroptosis through ROS-mediated activation of MLKL and p38 in CAMs. Elucidation of the molecular mechanism underlying zVAD-fmk-induced necroptosis in CAMs might help in better understanding its significance in chronic inflammatory diseases.

5.
Bioorg Med Chem ; 27(11): 2220-2227, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31029550

RESUMO

Antagonizing transcriptional activity of farnesoid X receptor (FXR) in the intestine has been reported as an effective means for the treatment of nonalcoholic fatty liver disease, type 2 diabetes and obesity. We describe herein that the building blocks necessary to maintain the antagonism of our chemotype were investigated in order to modulate in vivo pharmacokinetic behavior and the tissue distribution without blunting the activity against FXR. A comprehensive understanding of the structure-activity relationship led to analog 30, which is superior to 12 in terms of its pharmacokinetic profiles by oral administration and its tissue distribution toward target tissues (liver and ileum) in rats while preserving the in vitro activity of 12 against FXR. Thus, 30 should be a candidate compound to investigate the effects of inhibiting FXR activity while simultaneously improving the outcome of nonalcoholic fatty liver disease, type 2 diabetes and obesity.

6.
Sci Rep ; 9(1): 1931, 2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760783

RESUMO

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the production of PGD2 in adipocytes and is selectively induced by a high-fat diet (HFD) in adipose tissue. In this study, we investigated the effects of HFD on obesity and insulin resistance in two distinct types of adipose-specific L-PGDS gene knockout (KO) mice: fatty acid binding protein 4 (fabp4, aP2)-Cre/L-PGDS flox/flox and adiponectin (AdipoQ)-Cre/L-PGDS flox/flox mice. The L-PGDS gene was deleted in adipocytes in the premature stage of the former strain and after maturation of the latter strain. The L-PGDS expression and PGD2 production levels decreased in white adipose tissue (WAT) under HFD conditions only in the aP2-Cre/L-PGDS flox/flox mice, but were unchanged in the AdipoQ-Cre/L-PGDS flox/flox mice. When fed an HFD, aP2-Cre/L-PGDS flox/flox mice significantly reduced body weight gain, adipocyte size, and serum cholesterol and triglyceride levels. In WAT of the HFD-fed aP2-Cre/L-PGDS flox/flox mice, the expression levels of the adipogenic, lipogenic, and M1 macrophage marker genes were decreased, whereas those of the lipolytic and M2 macrophage marker genes were enhanced or unchanged. Insulin sensitivity was improved in the HFD-fed aP2-Cre/L-PGDS flox/flox mice. These results indicate that PGD2 produced by L-PGDS in premature adipocytes is involved in the regulation of body weight gain and insulin resistance under nutrient-dense conditions.

7.
ACS Med Chem Lett ; 9(2): 78-83, 2018 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-29456791

RESUMO

Farnesoid X receptor (FXR) plays a major role in the control of cholesterol metabolism. Antagonizing transcriptional activity of FXR is an effective means to treat the relevant metabolic syndrome. Some of antagonists so far have the charged functions; however, they may negatively affect the pharmacokinetics. We describe herein a structure-activity relationship (SAR) exploration of nonacidic FXR antagonist 6 focusing on two regions in the structure and biological evaluation of nonacidic 10 with the characteristic N-acylated piperidine group obtained from SAR studies. As the robust affinity to FXR is feasible with our nonacidic analogue, 10 is among the most promising candidates for in vivo testing.

8.
Nutrients ; 10(2)2018 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-29373533

RESUMO

Plant flavonoids have a variety of biological properties. In a previous study, we found that the tea of the Asian dayflower, Commelina communis L., decreased the body weight gain in high-fat diet-fed mice. In this study, we studied the anti-adipogenic ability of a flavonoid orientin that is found in abundance in C. communis. Orientin repressed the accumulation of intracellular triglyceride (TG) in mouse adipocyte 3T3-L1 cells. The treatment with orientin also decreased the mRNA levels of the genes involved in adipogenesis, lipogenesis, lipolysis, and TG synthesis, and reduced the release of glycerol. Orientin lowered the expression of CCAAT/enhancer binding protein (C/EBP) δ in the early stage of adipogenesis, leading to a decrease in the expression of the adipogenic master transcription factors such as peroxisome proliferator-activated receptor (PPAR) γ and C/EBPα. Moreover, the anti-adipogenic effect of orientin repressed the phosphorylation of Akt and subsequent phosphorylation of forkhead box protein O1 (FOXO1), which inhibits the transcription of the Ppar gene. These results indicate that a plant flavonoid orientin suppressed the expression of the Pparγ gene through repression of C/ebpδ expression and inhibition of the phosphoinositide 3-kinase /Akt-FOXO1 signaling in adipocytes.


Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Proteína delta de Ligação ao Facilitador CCAAT/antagonistas & inibidores , Flavonoides/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glucosídeos/farmacologia , PPAR gama/antagonistas & inibidores , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/antagonistas & inibidores , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Células 3T3-L1 , Adipócitos/enzimologia , Adipócitos/metabolismo , Animais , Proteína delta de Ligação ao Facilitador CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteína Forkhead Box O1/antagonistas & inibidores , Proteína Forkhead Box O1/metabolismo , Glicerol/metabolismo , Lipólise/efeitos dos fármacos , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triglicerídeos/metabolismo
9.
Neurochem Res ; 43(2): 488-499, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29204750

RESUMO

6-Hydroxydopamine (6-OHDA) induces the production of reactive oxygen species (ROS) that are associated with various neurodegenerative diseases such as Parkinson's disease. 3,3',4',7-Tetrahydroxyflavone (fisetin), a plant flavonoid has a variety of physiological effects such as antioxidant activity. In this study, we investigated the molecular mechanism of the neuroprotective effects of fisetin against 6-OHDA-induced cell death in human neuroblastoma SH-SY5Y cells. 6-OHDA-mediated cell toxicity was reduced in a fisetin concentration-dependent manner. 6-OHDA-mediated elevation of the expression of the oxidative stress-related genes such as hemeoxygenase-1, NAD(P)H dehydrogenase quinone 1, NF-E2-related factor 2, and γ-glutamate-cysteine ligase modifier was suppressed by fisetin. Fisetin also lowered the ratio of the proapoptotic Bax protein and the antiapoptotic Bcl-2 protein in SH-SY5Y cells. Moreover, fisetin effectively suppressed 6-OHDA-mediated activation of caspase-3 and caspase-9, which leads to the cell death, while, 6-OHDA-induced caspase-3/7 activity was lowered. Furthermore, fisetin activated the PI3K-Akt signaling, which inhibits the caspase cascade, and fisetin-mediated inhibition of 6-OHDA-induced cell death was negated by the co-treatment with an Akt inhibitor. These results indicate that fisetin protects 6-OHDA-induced cell death by activating PI3K-Akt signaling in human neuronal SH-SY5Y cells. This is the first report that the PI3K-Akt signaling is involved in the fisetin-protected ROS-mediated neuronal cell death.


Assuntos
Apoptose/efeitos dos fármacos , Flavonoides/farmacologia , Neuroblastoma/tratamento farmacológico , Oxidopamina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Flavonoides/química , Humanos , Neuroblastoma/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
Biochem Biophys Res Commun ; 490(2): 393-399, 2017 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-28623133

RESUMO

Prostaglandin (PG) D2 enhanced lipid accumulation in adipocytes. However, its molecular mechanism remains unclear. In this study, we investigated the regulatory mechanisms of PGD2-elevated lipid accumulation in mouse adipocytic 3T3-L1 cells. The Gi-coupled DP2 (CRTH2) receptors (DP2R), one of the two-types of PGD2 receptors were dominantly expressed in adipocytes. A DP2R antagonist, CAY10595, but not DP1 receptor antagonist, BWA868C cleared the PGD2-elevated intracellular triglyceride level. While, a DP2R agonist, 15R-15-methyl PGD2 (15R) increased the mRNA levels of the adipogenic and lipogenic genes, and decreased the glycerol release level. In addition, the forskolin-mediated increase of cAMP-dependent protein kinase A (PKA) activity and phosphorylation of hormone-sensitive lipase (HSL) was repressed by the co-treatment with 15R. Moreover, the lipolysis was enhanced in the adipocyte-differentiated DP2R gene-knockout mouse embryonic fibroblasts. These results indicate that PGD2 suppressed the lipolysis by repression of the cAMP-PKA-HSL axis through DP2R in adipocytes.


Assuntos
Adipócitos/metabolismo , Lipólise , Prostaglandina D2/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Células 3T3-L1 , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Receptores Imunológicos/genética , Receptores de Prostaglandina/genética , Transdução de Sinais
11.
J Agric Food Chem ; 64(51): 9607-9615, 2016 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-27977180

RESUMO

Citrus limonoids are secondary metabolites and exhibit a variety of biological activities. In this study, we elucidated the suppression of adipogenesis by a Citrus limonoid kihadanin B and determined its molecular mechanism in mouse 3T3-L1 adipocytes. Kihadanin B was purified from the peels of immature Citrus unshiu by HPLC, and its chemical structure was determined by NMR and mass spectrometry. Kihadanin B reduced the lipid accumulation with the reduction of the expression levels of the adipogenic and lipogenic genes, but did not affect lipolysis in adipocytes. Phosphorylation levels of Akt and a forkhead transcriptional factor, FOXO1, a repressor of PPARγ, were lowered by kihadanin B. Furthermore, kihadanin B increased the binding level of FOXO1 to the PPARγ gene promoter in adipocytes. These results indicate that a Citrus limonoid kihadanin B repressed the adipogenesis by decreasing lipid accumulation through the suppression of the Akt-FOXO1-PPARγ axis in 3T3-L1 adipocytes.


Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Citrus/química , Proteína Forkhead Box O1/metabolismo , Limoninas/farmacologia , PPAR gama/metabolismo , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteína Forkhead Box O1/genética , Frutas/química , Limoninas/química , Camundongos , PPAR gama/genética , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-akt/genética
12.
PLoS One ; 11(9): e0163640, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27669565

RESUMO

Baicalein has been used as a Chinese medicine, and is an abundant plant flavonoid present in fruits and vegetables. Here, we examined the effects of baicalein in adipogenesis and investigated its molecular mechanism in adipocytes. Baicalein lowered the intracellular lipid accumulation and decreased the transcription levels of the adipocyte-specific genes in mouse 3T3-L1 adipocytes. Glucose uptake mediated by glucose transporter 4 (GLUT4) was reduced, causing down-regulation of the intracellular lipid accumulation. These reductions were also observed even when baicalein was added in only early stage of adipogenesis (0-2 days) of 6-day-adipogenesis. Chromatin immunoprecipitation assay showed that baicalein decreased the binding level of C/EBPα protein to the promoter region of the GLUT4 gene. Phosphorylation of Akt at 1 h after the initiation of adipogenesis was inhibited by the treatment with baicalein. Inhibition during only the first 1.5 h after the initiation of adipogenesis by baicalein or an Akt inhibitor was enough to decrease the lipid contents in the cells undergoing adipocyte differentiation for 6 days. These results indicate that baicalein decreased the intracellular lipid accumulation by down-regulation of glucose uptake via repression of Akt-C/EBPα-GLUT4 signaling in the very early stage of adipogenesis of 3T3-L1 adipocytes.

13.
Biol Pharm Bull ; 39(6): 969-76, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27251498

RESUMO

Macrophages play pivotal roles in inflammatory responses. Previous studies showed that various natural products exert antiinflammatory effects by regulating macrophage activation. Recent studies have shown that shikonin (SHK) and its derivatives (ß-hydroxyisovalerylshikonin, acetylshikonin, and isobutylshikonin), which are 1,4-naphthoquinone pigments extracted from the roots of Lithospermum erythrorhizon, have various pharmacological, including antiinflammatory and antitumor, effects. Even though there have been many studies on the antiinflammatory activities of SHK derivatives, only a few have described their direct effects on macrophages. We investigated the effects of SHK derivatives on lipopolysaccharide (LPS)-treated macrophages. Low doses of SHK derivatives induced significant macrophage cytotoxicity (mouse macrophage-like J774.1/JA-4 cells and mouse peritoneal macrophages) in the presence of LPS. SHK activated caspases-3 and -7, which led to DNA fragmentation, but this cytotoxicity was prevented through a pan-caspase inhibitor in LPS-treated JA-4 cells. Maximal cytotoxic effects were achieved when SHK was added immediately before LPS addition. These results indicate that SHK derivatives induce caspase-dependent apoptotic cell death of LPS-treated macrophages and suggest that SHK acts during an early stage of LPS signaling.


Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Naftoquinonas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Células Cultivadas , Fragmentação do DNA , Feminino , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C
14.
Mol Cell Endocrinol ; 431: 1-11, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27132806

RESUMO

Triiodothyronine (T3) enhanced the expression of adipogenic and lipogenic genes with elevation of the intracellular lipids through thyroid hormone receptor (TR) α in mouse 3T3-L1 cells. However, the transcription of the SREBP-1c and HSL genes was decreased by T3. Such T3-mediated alterations were negated by TRα siRNA. Chromatin immunoprecipitation assay showed that the binding of TRα to the TR-responsive element (TRE) of the FAS promoter was elevated by T3. In contrast, the ability of TRα to bind to the TRE of the SREBP-1c promoter was decreased by T3. In addition, the binding of SREBP-1c to the SRE of the HSL promoter was lowered by T3. These results indicate that T3 increased the accumulation of intracellular lipids by enhancing the expression of the FAS gene through direct binding of TRα to the FAS promoter and simultaneously lowered the amount of lipolysis via reduced binding of T3-decreased SREBP-1c to the HSL promoter.


Assuntos
Adipócitos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Receptores alfa dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/metabolismo , Células 3T3-L1 , Animais , Linhagem Celular , Lipídeos/genética , Camundongos , Regiões Promotoras Genéticas/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Transcrição Genética/genética , Tri-Iodotironina/genética
15.
PLoS Negl Trop Dis ; 10(1): e0004339, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26731263

RESUMO

The metabolic pathway of purine nucleotides in parasitic protozoa is a potent drug target for treatment of parasitemia. Guanosine 5'-monophosphate reductase (GMPR), which catalyzes the deamination of guanosine 5'-monophosphate (GMP) to inosine 5'-monophosphate (IMP), plays an important role in the interconversion of purine nucleotides to maintain the intracellular balance of their concentration. However, only a few studies on protozoan GMPR have been reported at present. Herein, we identified the GMPR in Trypanosoma brucei, a causative protozoan parasite of African trypanosomiasis, and found that the GMPR proteins were consistently localized to glycosomes in T. brucei bloodstream forms. We characterized its recombinant protein to investigate the enzymatic differences between GMPRs of T. brucei and its host animals. T. brucei GMPR was distinct in having an insertion of a tandem repeat of the cystathionine ß-synthase (CBS) domain, which was absent in mammalian and bacterial GMPRs. The recombinant protein of T. brucei GMPR catalyzed the conversion of GMP to IMP in the presence of NADPH, and showed apparent affinities for both GMP and NADPH different from those of its mammalian counterparts. Interestingly, the addition of monovalent cations such as K+ and NH4+ to the enzymatic reaction increased the GMPR activity of T. brucei, whereas none of the mammalian GMPR's was affected by these cations. The monophosphate form of the purine nucleoside analog ribavirin inhibited T. brucei GMPR activity, though mammalian GMPRs showed no or only a little inhibition by it. These results suggest that the mechanism of the GMPR reaction in T. brucei is distinct from that in the host organisms. Finally, we demonstrated the inhibitory effect of ribavirin on the proliferation of trypanosomes in a dose-dependent manner, suggesting the availability of ribavirin to develop a new therapeutic agent against African trypanosomiasis.


Assuntos
GMP Redutase/metabolismo , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Antimetabólitos/farmacologia , GMP Redutase/genética , Regulação Enzimológica da Expressão Gênica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Proteínas Recombinantes , Ribavirina/farmacologia , Especificidade da Espécie , Temperatura Ambiente , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo
16.
Biochem Biophys Rep ; 5: 328-334, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28955839

RESUMO

Macrophages play an important role in immune and inflammatory responses, and have been extensively studied in vitro using culture media such as RPMI1640 medium, Dulbecco's modified Eagle medium (DMEM), and Ham's F-12 medium (F-12). We found that the activation phenotypes of a murine macrophage-like cell line, J774.1/JA-4, were obviously different in two distinct culture media (F-12 and DMEM), both of which were supplemented with 10% of the same fetal bovine serum (FBS). Among these phenotypes, nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression, induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), were remarkably different. iNOS expression was higher in the macrophages cultured in DMEM than in F-12 for 20 h, while no significant differences were shown in NO production between in F-12 and DMEM. It might be the reason why DMEM have reduced NO production by the induced iNOS. Besides, [Formula: see text]-generating activity, and production of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in the activated macrophages were also different between the cultures in F-12 and DMEM. These results suggest that F-12 and DMEM contain certain components responsible for modification of macrophage activation processes and/or macrophage functions. Our present results provide evidence that the choice of culture medium is important in the study and analysis of macrophage activation.

17.
Biol Pharm Bull ; 38(9): 1255-64, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26119425

RESUMO

A simple and rapid assay method for analysis of the metabolic activity of viable but non-culturable (VBNC) Salmonella was established. An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost its culturability during incubation with 1-10 mM H2O2 in Luria-Bertani (LB) medium. To assess the viability of the bacteria, we measured 3 different metabolic activities: Respiratory activity by 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) reduction, glucose uptake assessed with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and DNA synthesis activity evaluated by 5-ethynyl-2'-deoxyuridine (EdU) incorporation. These activities were analyzed by both confocal laser-scanning microscopy and flow cytometry, together with colony-formation assays on LB-agar plates. The results showed that some of the H2O2-treated SE cells were in the VBNC state and that the extent of H2O2-induced decrease in each metabolic activity varied according to the activity. That is, glucose-uptake activity was not markedly changed, being kept at the highest level; whereas the respiratory activity was less than that of the glucose-uptake, and DNA synthesis activity was the lowest among them. These results suggest that the VBNC state might be characterized by different metabolic activities that vary and correspond to the kind and strength of the stress, threatening bacterial survival in an adverse environment.


Assuntos
Salmonella enteritidis/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , DNA Bacteriano/metabolismo , Desoxiglucose/análogos & derivados , Desoxiglucose/farmacologia , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Citometria de Fluxo , Glucose/metabolismo , Peróxido de Hidrogênio/farmacologia , Microscopia Confocal , Salmonella enteritidis/efeitos dos fármacos
18.
J Agric Food Chem ; 63(20): 4979-87, 2015 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-25945786

RESUMO

3,7,3',4'-Tetrahydroxyflavone (fisetin) is a flavonoid found in vegetables and fruits having broad biological activities. Here the effects of fisetin on adipogenesis and its regulatory mechanism in mouse adipocytic 3T3-L1 cells are studied. Fisetin inhibited the accumulation of intracellular lipids and lowered the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein (C/EBP) α and fatty acid-binding protein 4 (aP2) during adipogenesis. Moreover, the mRNA levels of genes such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase involved in the fatty acid biosynthesis (lipogenesis) were reduced by the treatment with fisetin. The expression level of the glucose transporter 4 (GLUT4) gene was also decreased by fisetin, resulting in down-regulation of glucose uptake. Furthermore, fisetin inhibited the phosphorylation of the mammalian target of rapamycin (mTOR) and that of p70 ribosomal S6 kinase, a target of the mTOR complex, the inhibition of which was followed by a decreased mRNA level of the C/EBPα gene. The results obtained from a chromatin immunoprecipitation assay demonstrated that the ability of C/EBPα to bind to the GLUT4 gene promoter was reduced by the treatment with fisetin, which agreed well with those obtained when 3T3-L1 cells were allowed to differentiate into adipocytes in medium in the presence of rapamycin, an inhibitor for mTOR. These results indicate that fisetin suppressed the accumulation of intracellular lipids by inhibiting GLUT4-mediated glucose uptake through inhibition of the mTOR-C/EBPα signaling in 3T3-L1 cells.


Assuntos
Adipócitos/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Flavonoides/farmacologia , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 4/genética , Camundongos , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética
19.
Biol Pharm Bull ; 38(1): 7-16, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25744452

RESUMO

Heme oxygenase-1 (HO-1) catabolizes the degradation of heme into bilirubin, carbon monoxide, and iron ions. The HO-1 products provide antioxidant cytoprotection in addition to having potent antiinflammatory and immunomodulatory functions. HO-1 is induced by its substrate heme and environmental factors including oxidative and heat stresses. Although previous studies reported that lipopolysaccharide (LPS) induced the expression of both the HO-1 gene and its protein in macrophages, the major regulators of HO-1 expression remain unknown. To identify these regulators, we used two types of cell, the murine macrophage-like cell line J774.1/JA-4 and its LPS-resistant mutant, LPS1916. Based on a comparison of the results obtained with these cells, we found that nitric oxide (NO) was closely linked to the induction of HO-1. Real-time polymerase chain reaction (PCR) showed that the time course for inducible HO-1 mRNA by LPS or LPS+interferon (IFN)-γ was similar to that for inducible NO synthase (iNOS) mRNA. Furthermore, the expression of iNOS mRNA and protein increased earlier than that of HO-1 mRNA and protein. N-Nitro-L-arginine methyl ester, an NO synthase inhibitor, reduced both HO-1 expression and NO production in LPS+IFN-γ-treated JA-4 cells. Furthermore, NOC-12, an NO donor, significantly induced HO-1 expression not only in JA-4 but also in LPS1916 cells. Reactive oxygen species (ROS) scavengers, such as superoxide dismutase and catalase, did not affect HO-1 protein expression in LPS+IFN-γ-treated JA-4 cells. These results suggest that, among ROS, NO plays an important role in HO-1 induction in activated macrophages treated with LPS+IFN-γ.


Assuntos
Heme Oxigenase-1/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Óxido Nítrico/metabolismo , Animais , Linhagem Celular , Heme Oxigenase-1/genética , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/genética , Camundongos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Superóxidos/metabolismo
20.
Biol Pharm Bull ; 37(10): 1617-25, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25109306

RESUMO

An environmental isolate of Salmonella enterica serovar Enteritidis (SE) clone, SE Cl#15-1, loses its culturability during 72-h culture in M9 minimal medium containing 0.8% glucose, a concentration twice higher than that in normal M9 medium, whereas the bacterium retains its culturability in normal M9 medium. Live/dead analysis using the 5-cyano-2,3-di(p-tolyl) tetrazolium chloride (CTC)-reduction assay revealed that SE cells cultured in M9 medium containing 0.8% glucose died with time when in the "viable but non-culturable" (VBNC) state. Assay of the culturability of SE cells in the used supernatant (0.4 spent M9 or 0.8 spent M9) also indicated that 0.8 spent M9 soon showed a lethal effect on intact SE cells. These results suggest that large amounts of glucose metabolites might have been responsible for the toxicity. Analysis of the 0.8 spent M9 revealed that formate rapidly accumulated in the medium. The pH of the medium rapidly dropped to 4.7, leading to conversion of formate to formic acid, which might have damaged the bacterial cell membrane. These results suggest that the excessive amount of glucose in the M9 medium might have injured SE cells in the VBNC state by being metabolized to formic acid and other acidic compounds.


Assuntos
Meios de Cultura/farmacologia , Glucose/administração & dosagem , Viabilidade Microbiana/efeitos dos fármacos , Salmonella/efeitos dos fármacos , Salmonella/crescimento & desenvolvimento , Contagem de Colônia Microbiana/métodos
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