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1.
Pestic Biochem Physiol ; 159: 1-8, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400771

RESUMO

We examined the molecular regulation of porphyrin biosynthesis and protective responses in transgenic rice (Oryza sativa) expressing Bradyrhizobium japonicum Fe-chelatase (BjFeCh) after treatment with acifluorfen (AF). During the photodynamic stress imposed by AF, transcript levels of BjFeCh in transgenic plants increased greatly; moreover, transcript levels of OsFeCh2 remained almost constant, whereas in wild type (WT) plants they were considerably down-regulated. In the heme branch, transgenic plants exhibited greater levels of OsFC and HO transcripts than WT plants in the untreated stems as well as in the AF-treated leaves and stems. Both WT and transgenic plants treated with AF substantially decreased transcript levels for all the genes in the chlorophyll branch, with less decline in transgenic plants. After AF treatment, ascorbate (Asc) content and the redox Asc state greatly decreased in leaves of WT plants; however, in transgenic plants both parameters remained constant in leaves and the Asc redox state increased by 20% in stems. In response to AF, the leaves of WT plants greatly up-regulated CatA, CatB, and GST compared to those of transgenic plants, whereas, in the stems, transgenic plants showed higher levels of CatA, CatC, APXb, BCH, and VDE. Photochemical quenching, qP, was considerably dropped by 31% and 18% in WT and transgenic plants, respectively in response to AF, whereas non-radiative energy dissipation through non-photochemical quenching increased by 77% and 38% in WT and transgenic plants, respectively. Transgenic plants treated with AF exhibited higher transcript levels of nucleus-encoded photosynthetic genes, Lhcb1 and Lhcb6, as well as levels of Lhcb6 protein compared to those of WT plants. Our study demonstrates that expression of BjFeCh in transgenic plants influences not only the regulation of porphyrin biosynthesis through maintaining higher levels of gene expression in the heme branch, but also the Asc redox function during photodynamic stress caused by AF.


Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/enzimologia , Ferroquelatase/metabolismo , Nitrobenzoatos/farmacologia , Oryza/metabolismo , Porfirinas/biossíntese , Proteínas de Bactérias/genética , Ferroquelatase/genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Plantas Geneticamente Modificadas
2.
J Photochem Photobiol B ; 196: 111497, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31154276

RESUMO

Systematic identification and quantification of active radical sites in a small molecule, pyrazolium 3,5-dinitrobenzoate:3,5-dinitrobenzoic acid as well as in the stable free radical (DPPH•) were carried out by Fukui functions calculation using DFT functional with B3LYP/6-311++G(d,p) level of basis set. Bioactive Lewis acid-base compound, pyrazolium 3,5-dinitrobenzoate:3,5-dinitrobenzoic acid (PDNB:DNBA) has been synthesized and crystallized by slow evaporation - solution method at 30 °C. Various functional groups and the structural arrangements were ascertained from spectral and XRD analyses, respectively. UV-vis spectral analysis was used to find out the stability of the anticipated drug for about 60 min using methanol as a solvent. Stabilization of the compound was linked to the presence of enormous N-H…O, O-H…O and C-H…O hydrogen bonding interactions identified through Hirshfeld surface analysis. Chemical stability and reactivity of the drug were validated from theoretical optimization and HOMO-LUMO analysis. Active nucleophilic, electrophilic and radical sites of PDNB:DNBA were also identified from molecular electrostatic potential analysis. Inhibition of growth of pathogens in screening experiments by the proposed drug attests its suitability in biological applications. Antioxidant activity of the compound, PDNB:DNBA, endorses its aptness for scavenging reactive radicals. Fluorimetry experiments confirm hyperchromism in DNA binding analysis proving groove mode of binding. Molecular docking explored the various modes of intermolecular interactions of the drug with microbes as well as DNA.


Assuntos
DNA/química , Nitrobenzoatos/química , Pirazóis/química , Animais , Antioxidantes/química , Aspergillus niger/efeitos dos fármacos , Sítios de Ligação , Bovinos , DNA/metabolismo , Conformação Molecular , Simulação de Acoplamento Molecular , Nitrobenzoatos/metabolismo , Nitrobenzoatos/farmacologia , Conformação de Ácido Nucleico , Teoria Quântica , Eletricidade Estática
3.
Pain ; 160(5): 1166-1174, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30913166

RESUMO

Recent studies have made significant progress in identifying distinct populations of peripheral neurons involved in itch transmission, whereas the cellular identity of spinal interneurons that contribute to itch processing is still a debate. Combining genetic and pharmacological ablation of spinal excitatory neuronal subtypes and behavioral assays, we demonstrate that spinal somatostatin-positive (SOM) excitatory interneurons transmit pruritic sensation. We found that the ablation of spinal SOM/Lbx1 (SOM) neurons caused significant attenuation of scratching responses evoked by various chemical pruritogens (chemical itch). In an attempt to identify substrates of spinal itch neural circuit, we observed that spinal SOM neurons partially overlapped with neurons expressing natriuretic peptide receptor A (Npra), the receptor of peripheral itch transmitter B-type natriuretic peptide. Spinal SOM neurons, however, did not show any overlap with itch transmission neurons expressing gastrin-releasing peptide receptor in the dorsal spinal cord, and the gastrin-releasing peptide-triggered scratching responses were intact after ablating spinal SOM neurons. Dual ablation of SOM and Npra neurons in the spinal cord reduced chemical itch responses to a greater extent than ablation of SOM or Npra neurons alone, suggesting the existence of parallel spinal pathways transmitting chemical itch. Furthermore, we showed that SOM peptide modulated itch processing through disinhibition of somatostatin receptor 2A-positive inhibitory interneuron. Together, our findings reveal a novel spinal mechanism for sensory encoding of itch perception.


Assuntos
Interneurônios/metabolismo , Prurido/induzido quimicamente , Prurido/patologia , Somatostatina/metabolismo , Medula Espinal/patologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Inibidores da Angiogênese/farmacologia , Animais , Cloroquina/toxicidade , Modelos Animais de Doenças , Técnicas In Vitro , Interneurônios/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Nitrobenzoatos/farmacologia , Técnicas de Patch-Clamp , Proteínas Proto-Oncogênicas c-fos/metabolismo , Somatostatina/genética , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , p-Metoxi-N-metilfenetilamina/toxicidade , Proteínas tau/genética , Proteínas tau/metabolismo
4.
Eur J Med Chem ; 166: 22-31, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30684868

RESUMO

Involved in the tyrosine degradation pathway, 4-hydroxyphenylpyruvate dioxygenase (HPPD) is an important target for treating type I tyrosinemia. To discover novel HPPD inhibitors, we proposed a hydrophobicity-oriented drug design (HODD) strategy based on the interactions between HPPD and the commercial drug NTBC. Most of the new compounds showed improved activity, compound d23 being the most active candidate (IC50 = 0.047 µM) with about 2-fold more potent than NTBC (IC50 = 0.085 µM). Therefore, compound d23 is a potential drug candidate to treat type I tyrosinemia.


Assuntos
4-Hidroxifenilpiruvato Dioxigenase/antagonistas & inibidores , Cicloexanonas/farmacologia , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Nitrobenzoatos/farmacologia , 4-Hidroxifenilpiruvato Dioxigenase/química , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Domínio Catalítico , Cicloexanonas/química , Cicloexanonas/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Nitrobenzoatos/química , Nitrobenzoatos/metabolismo , Relação Estrutura-Atividade
6.
Cell Physiol Biochem ; 50(4): 1460-1473, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30359963

RESUMO

BACKGROUND/AIMS: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. METHODS: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (Vmem) and swelling-dependent chloride (Cl-) currents (IClswell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. RESULTS: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl- current under isotonic conditions resembling features of the swelling-activated Cl- current (IClswell). The cell membrane potential (Vmem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na+-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. CONCLUSION: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.


Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Tamanho Celular/efeitos dos fármacos , Glicina/farmacologia , Sistema A de Transporte de Aminoácidos/antagonistas & inibidores , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Cloretos/metabolismo , Ciclopentanos/farmacologia , Soluções Hipotônicas/farmacologia , Indanos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Microglia/citologia , Microglia/metabolismo , Nitrobenzoatos/farmacologia , Técnicas de Patch-Clamp
7.
Cell Physiol Biochem ; 45(3): 867-882, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29421809

RESUMO

BACKGROUND/AIMS: In the human genome, more than 400 genes encode ion channels, which are ubiquitously expressed and often coexist and participate in almost all physiological processes. Therefore, ion channel blockers represent fundamental tools in discriminating the contribution of individual channel types to a physiological phenomenon. However, unspecific effects of these compounds may represent a confounding factor. Three commonly used chloride channel inhibitors, i.e. 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS), 5-nitro-2-[(3-phenylpropyl) amino]benzoic acid (NPPB) and the anti-inflammatory drug niflumic acid were tested to identify the lowest concentration effective on Cl- channels and ineffective on K+ channels. METHODS: The activity of the above mentioned compounds was tested by whole cell patch-clamp on the swelling-activated Cl- current ICl,swell and on the endogenous voltage-dependent, outwardly rectifying K+ selective current in human kidney cell lines (HEK 293/HEK 293 Phoenix). RESULTS: Micromolar (1-10 µM) concentrations of DIDS and NPPB could not discriminate between the Cl- and K+ selective currents. Specifically, 1 µM DIDS only affected the K+ current and 10 µM NPPB equally affected the Cl- and K+ currents. Only relatively high (0.1-1 mM) concentrations of DIDS and prolonged (5 minutes) exposure to 0.1-1 mM NPPB preferentially suppressed the Cl- current. Niflumic acid preferentially inhibited the Cl- current, but also significantly affected the K+ current. The endogenous voltage-dependent, outwardly rectifying K+ selective current in HEK 293/HEK 293 Phoenix cells was shown to arise from the Kv 3.1 channel, which is extensively expressed in brain and is involved in neurological diseases. CONCLUSION: The results of the present study underscore that sensitivity of a given physiological phenomenon to the Cl- channel inhibitors NPPB, DIDS and niflumic acid may actually arise from an inhibition of Cl- channels but can also result from an inhibition of voltage-dependent K+ channels, including the Kv 3.1 channel. The use of niflumic acid as anti-inflammatory drug in patients with concomitant Kv 3.1 dysfunction may result contraindicated.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Canal de Potássio Kv1.3/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Potássio/metabolismo , Animais , Cloretos/metabolismo , Células Epiteliais/citologia , Células HEK293 , Humanos , Túbulos Renais Proximais/citologia , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Camundongos , Células NIH 3T3 , Ácido Niflúmico/química , Ácido Niflúmico/farmacologia , Nitrobenzoatos/química , Nitrobenzoatos/farmacologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo
8.
J Med Microbiol ; 67(1): 129-138, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29214974

RESUMO

PURPOSE: Ctn[15-34], a carboxyl-terminal fragment of crotalicidin (a cathelicidin from the venom gland of a South American rattlesnake), has shown antifungal activity against clinical and standard strains of Candida species. The aim of the present work was to investigate the underlying mechanisms of the candidicidal activity of Ctn[15-34]. METHODOLOGY: The time-kill profile and drug synergism were evaluated by means of a microdilution assay and multi-parametric flow cytometry. The presumptive interaction of Ctn[15-34] with lipid membranes was estimated in vitro with a lipid-mimic compound, the chromogenic substance 4-nitro-3-(octanoyloxy)benzoic acid (4N3OBA).Results/Key findings. The absorbance increment (at 425 nm) indicated a concentration- and time-dependent in-solution association between Ctn[15-34] and 4N3OBA. The interaction of Ctn[15-34] with Candida cells was confirmed by flow cytometric measurements with the 5(6)-carboxyfluorescein-labelled peptide (CF-Ctn[15-34]). Analysis of the killing time of Candida exposed to Ctn[15-34] and amphotericin B (AMB) showed that both the peptide and polyene drug reduce the number of c.f.u. but in mechanistically different ways. The Ctn[15-34] peptide alone caused yeast cell membrane disruption, which was confirmed by lactate dehydrogenase leakage and biomarkers of cell death mediated by necrosis. CONCLUSION: Overall, Ctn[15-34] displays a synergistic antifungal activity with AMB, an effect that can be further developed into a multi-target therapeutic option with other antimycotics currently in use.


Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Catelicidinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Anfotericina B/farmacologia , Candidíase/tratamento farmacológico , Sinergismo Farmacológico , Fluoresceínas/farmacologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Nitrobenzoatos/farmacologia
9.
Int J Mol Med ; 41(3): 1331-1338, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29286070

RESUMO

When treating glaucoma, excessive scar tissue reactions reduce the postoperative survival rate of the filtering bleb. Accumulating evidence has demonstrated that the proliferation, migration and extracellular matrix (ECM) synthesis of fibroblasts are important molecular mechanisms underlying scar formation. Recent evidence has demonstrated that chloride channels play an important role in controlling cell proliferation, apoptosis, migration and the cell cycle process in several cell types, but the effects of chloride channels on conjunctival fibroblasts have not be studied. The aim of the present study was to investigate the effects of the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) on cell proliferation, apoptosis, migration, cell cycle progression and ECM synthesis in human conjunctival fibroblasts (HConFs), and to further investigate the mechanism of resistance to scar formation following glaucoma filtration surgery. HConFs were exposed to NPPB or lubiprostone. Cell proliferation and viability was evaluated using the Cell Counting Kit-8. Cell migration was measured using Transwell migration and scratch­wound assays. Flow cytometry was used to study apoptosis and cell cycle progression. Quantitative polymerase chain reaction and western blot analyses were performed to determine mRNA and protein expression levels, respectively. Following NPPB treatment, HConFs exhibited reduced proliferation and migration, along with increased apoptosis. NPPB also inhibited cell cycle progression by arresting cells in the G0̸G1 phase and reducing collagen I and fibronectin expression, as well as the phosphorylation of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT). However, lubiprostone treatment exerted the opposite effects on HConFs. Therefore, NPPB treatment inhibited proliferation, migration, cell cycle progression and synthesis of the ECM, while promoting apoptosis in HConFs, by inhibiting the PI3K̸AKT signaling pathway.


Assuntos
Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Túnica Conjuntiva/citologia , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Nitrobenzoatos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibronectinas/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos
10.
J Neurosci Res ; 96(1): 117-127, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28699288

RESUMO

The mechanism of brain edema is complex and still remains unclear. Our aim was to investigate the regional differences of cell volume and intracellular Ca2+ concentration ([Ca2+ ]i ) dynamics during hypotonic stress in male mouse hemi-brain slices. Brain slices were loaded with the fluorescence Ca2+ indicator fura-2, and cell volume and [Ca2+ ]i in the lateral cerebral cortex (LCC) and hippocampal CA1 (CA1) region were measured simultaneously during exposure to hypotonic stress using Ca2+ insensitive (F360) and Ca2+ sensitive fluorescence (F380), respectively. Brain cell swelling induced by hypotonic stress was followed by a regulatory volume change that coincided with an increase in [Ca2+ ]i . The degrees of change in cell volume and [Ca2+ ]i were significantly different between the LCC and CA1. The increase in cell volume and [Ca2+ ]i in the LCC, but not in the CA1, was decreased by the transient receptor potential channel blockers LaCl3 and GdCl3 . The increase in [Ca2+ ]i in both the LCC and CA1, was significantly decreased by the intracellular Ca2+ modulators thapsigargin and xestospongin C. The K+ channel activator isoflurane and Cl- channel blocker NPPB significantly decreased [Ca2+ ]i in the LCC. This study demonstrated that, between cells located in the LCC and in the CA1, the characteristics of brain edema induced by hypotonic stress are different. This can be ascribed to the different contribution of volume sensitive G-protein coupled receptor and stretch sensitive Ca2+ channels.


Assuntos
Região CA1 Hipocampal/metabolismo , Cálcio/metabolismo , Tamanho Celular , Córtex Cerebral/metabolismo , Espaço Intracelular/metabolismo , Pressão Osmótica/fisiologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Isoflurano/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nitrobenzoatos/farmacologia , Técnicas de Cultura de Órgãos , Pressão Osmótica/efeitos dos fármacos
11.
Adv Exp Med Biol ; 959: 49-64, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28755183

RESUMO

Untreated HT1 rapidly degenerates into very severe liver complications often resulting in liver cancer. The molecular basis of the pathogenic process in HT1 is still unclear. The murine model of FAH-deficiency is a suitable animal model, which represents all phenotypic and biochemical manifestations of the human disease on an accelerated time scale. After removal of the drug 2-(2-N-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), numerous signaling pathways involved in cell proliferation, differentiation and cancer are rapidly deregulated in FAH deficient mice. Among these, the Endoplasmic reticulum (ER) pathway, the heat stress response (HSR), the Nrf2, MEK and ERK pathways, are highly represented. The p21 and mTOR pathways critical regulators of proliferation and tumorigenesis have also been found to be dysregulated. The changes in these pathways are described and related to the development of liver cancer.


Assuntos
Hidrolases/metabolismo , Hepatopatias/metabolismo , Fígado/metabolismo , Tirosinemias/metabolismo , Animais , Cicloexanonas/farmacologia , Humanos , Hepatopatias/etiologia , Nitrobenzoatos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tirosinemias/complicações , Tirosinemias/tratamento farmacológico
12.
Adv Exp Med Biol ; 959: 93-100, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28755187

RESUMO

Hereditary tyrosinemia type 1 (HT1) is characterized by severe progressive liver disease and renal tubular dysfunction. Kidney involvement is characterized by hypophosphatemic rickets and Fanconi syndrome. Different animal models were useful to investigate the pathophysiology of the disease and the effects of NTBC therapy on liver and kidney function. NTBC has revolutionized the prognosis of HT1 and its acute and chronic effects on renal tubular function have been proved, with normalization of tubular function within a few weeks, particularly hypophosphatemia and proteinuria. NTBC therapy is highly effective in improving renal function both at short and long-term. However, its efficacy critically depends on the age at start of treatment with normal outcome in patients diagnosed at birth by newborn screening.


Assuntos
Cicloexanonas/farmacologia , Cicloexanonas/uso terapêutico , Nefropatias/tratamento farmacológico , Nefropatias/etiologia , Rim/efeitos dos fármacos , Nitrobenzoatos/farmacologia , Nitrobenzoatos/uso terapêutico , Tirosinemias/complicações , Tirosinemias/tratamento farmacológico , Animais , Humanos , Rim/patologia , Nefropatias/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Hepatopatias/tratamento farmacológico , Hepatopatias/etiologia , Hepatopatias/patologia
13.
Adv Exp Med Biol ; 959: 175-185, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28755195

RESUMO

The discovery that a natural product leptospermone had herbicidal activity formed the starting point for chemical synthesis to find more activity and selectivity. A series of molecules called triketones were found to possess good activity and 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC) was selected for toxicology testing. NTBC fed at low doses to rats and dogs caused keratopathy, which on cessation of the diet recovered. Mice, rabbits and monkeys fed NTBC did not show this response. Research discovered that NTBC caused tyrosinaemia which was due to inhibition of the enzyme 4-hydroxyphenylpyruvate dioxygenase in both mammals and plants thereby finding a novel target for killing plants. NTBC was also used sucessfully as a drug to treat a rare inborn error of metabolism, tyrosinaemia type I, in collaboration with Professor's Sven Lindstedt and Elisabeth Holme. Understanding the mechanism of toxicity of NTBC led to novel herbicide discovery and saved the lives of children with acute tyrosinaemia type I.


Assuntos
Cicloexanonas/farmacologia , Cicloexanonas/uso terapêutico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/fisiologia , Nitrobenzoatos/farmacologia , Nitrobenzoatos/uso terapêutico , Tirosinemias/tratamento farmacológico , Animais , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Humanos , Tirosina/metabolismo , Tirosinemias/metabolismo
14.
Adv Exp Med Biol ; 959: 205-213, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28755198

RESUMO

This chapter provides a clinical perspective on the challenges that stand between current clinical practice and a cure for hepatorenal tyrosinemia (HT1). HT1 has been transformed in the last 50 years from an aggressive often undiagnosed childhood disease causing liver failure or liver cancer, with infant death in most patients, to a condition that is detectable at birth, and for which treatment with nitisinone (NTBC) and diet can prevent detectable liver or kidney abnormalities. What challenges remain? The properties of the affected metabolic pathway and the broad spectrum of severity seen in untreated patients are incompletely understood but potentially important for patients. Available treatments have potential complications, including liver transplantation (risks of surgery and of immunosuppression to prevent rejection), nitisinone and diet therapy (hypertyrosinemia, corneal opacities, nutritional imbalances and possibly developmental delay). The detection of liver cancer is imperfect and laborious. The effects of tyrosinemia during pregnancy are little-known. Although animal models of HT1 are becoming standard research tools in cell replacement and gene modification therapy, these techniques are not currently applicable to HT1 itself. Treatment adherence is variable, causing concern about long term outcome for some patients. Around the world, there are great disparities in the diagnosis and treatment of HT1. Most affected individuals are born in places where newborn screening for HT1 is not performed and where appropriate treatment is not available. We hope that this list will help to focus on some of these remaining obstacles to a cure for HT1.


Assuntos
Tirosinemias/dietoterapia , Tirosinemias/tratamento farmacológico , Animais , Cicloexanonas/farmacologia , Cicloexanonas/uso terapêutico , Dieta/métodos , Dietoterapia/métodos , Humanos , Recém-Nascido , Nefropatias/dietoterapia , Nefropatias/tratamento farmacológico , Nefropatias/etiologia , Falência Hepática/dietoterapia , Falência Hepática/tratamento farmacológico , Falência Hepática/etiologia , Neoplasias Hepáticas/dietoterapia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Transplante de Fígado/métodos , Triagem Neonatal/métodos , Nitrobenzoatos/farmacologia , Nitrobenzoatos/uso terapêutico , Tirosinemias/complicações
15.
Neurochem Res ; 42(9): 2551-2559, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28401401

RESUMO

Anion channels and connexin hemichannels are permeable to amino acid neurotransmitters. It is hypothesized that these conductive pathways release GABA, thereby influencing ambient GABA levels and tonic GABAergic inhibition. To investigate this, we measured the effects of anion channel/hemichannel antagonists on tonic GABA currents of rat hippocampal neurons. In contrast to predictions, blockade of anion channels and hemichannels with NPPB potentiated tonic GABA currents of neurons in culture and acute hippocampal slices. In contrast, the anion channel/hemichannel antagonist carbenoxolone (CBX) inhibited tonic currents. These findings could result from alterations of ambient GABA concentration or direct effects on GABAA receptors. To test for effects on GABAA receptors, we measured currents evoked by exogenous GABA. Coapplication of NPPB with GABA potentiated GABA-evoked currents. CBX dose-dependently inhibited GABA-evoked currents. These results are consistent with direct effects of NPPB and CBX on GABAA receptors. GABA release from hippocampal cell cultures was directly measured using HPLC. Inhibition of anion channels with NPPB or CBX did not affect GABA release from cultured hippocampal neurons. NPPB reduced GABA release from pure astrocytic cultures by 21%, but the total GABA release from astrocytes was small compared to that of mixed cultures. These data indicate that drugs commonly used to antagonize anion channels and connexin hemichannels may affect tonic currents via direct effects on GABAA receptors and have negligible effects on ambient GABA concentrations. Interpretation of experiments using NPPB or CBX should include consideration of their effects on tonic GABA currents.


Assuntos
Conexinas/antagonistas & inibidores , Conexinas/fisiologia , Antagonistas de Receptores de GABA-A/farmacologia , Receptores de GABA-A/fisiologia , Canais de Ânion Dependentes de Voltagem/antagonistas & inibidores , Canais de Ânion Dependentes de Voltagem/fisiologia , Aminobenzoatos/farmacologia , Animais , Animais Recém-Nascidos , Carbenoxolona/farmacologia , Células Cultivadas , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Masculino , Nitrobenzoatos/farmacologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/farmacologia
16.
Bioorg Chem ; 70: 27-33, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27863748

RESUMO

A series of novel cholinesterase inhibitors containing nitrobenzoate core structure were synthesized by a facile and efficient method. The structure of the novel compounds were fully characterized and confirmed by analytical as well as spectroscopic methods. Compound indicated as 2f was found to possess the best cholinesterase inhibitory activities of all the evaluated compounds. Results suggest that 2f is a selective acetylcholinesterase inhibitor, although it also inhibits butyrylcholinesterase at higher concentration. Kinetics inhibition result suggest that 2f is a mixed-mode inhibitor of acetylcholinesterase. In addition, it was found to have low cytotoxicity. Molecular docking on compound 2f was carried out to rationalize the observed in vitro enzymatic assay results. Most importantly, the potential of nitrobenzoate derivatives as cholinesterase inhibitor was shown through this study. In summary, we discovered nitrobenzoates as a new scaffold that may eventually yield useful compounds in treatment of Alzheimer's disease.


Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Nitrobenzoatos/química , Nitrobenzoatos/farmacologia , Acetilcolinesterase/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Animais , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Linhagem Celular , Electrophorus , Humanos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade
17.
Cancer Lett ; 386: 179-188, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27894959

RESUMO

PURPOSE: Cholangiocarcinoma (CCA) is a malignancy of the biliary epithelium that is associated with low five-year survival. The apelin receptor (APLNR), which is activated by the apelin peptide, has not been studied in CCA. The purpose of this study is to determine if inhibition of the apelin/APLNR axis can inhibit CCA growth. METHODS: Immunohistochemistry, rtPCR, immunofluorescence, flow cytometry, and ELISA was used to measure APLNR expression in human CCA cells and tissues. Mz-ChA-1 cells were treated with increasing concentrations of apelin and ML221, an APLNR antagonist. Expression of proliferative and angiogenic genes were measured via rtPCR. In vivo, Mz-ChA-1 cells were injected into the flanks of nu/nu mice, which were treated with ML221 (150 µg/kg) via tail vein injection. RESULTS: Expression of the apelin/APLNR axis was increased in CCA. In vitro, CCA proliferation and angiogenesis was inhibited by ML221 treatment. ML221 treatment significantly decreased tumor growth in nu/nu mice. CONCLUSION: The apelin/APLNR axis regulates CCA proliferation and angiogenesis. Inhibition of the apelin/APLNR axis decreases tumor growth in our xenograft model. Targeting APLNR signaling has the potential to serve as a novel, tumor directed therapy for CCA.


Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neovascularização Patológica , Nitrobenzoatos/farmacologia , Piranos/farmacologia , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Adulto , Idoso de 80 Anos ou mais , Animais , Apelina , Receptores de Apelina , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Asian J Androl ; 19(4): 418-424, 2017 Jul-Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27270342

RESUMO

Human spermatozoa encounter an osmotic decrease from 330 to 290 mOsm l-1 when passing through the female reproductive tract. We aimed to evaluate the role of chloride channels in volume regulation and sperm motility from patients with asthenozoospermia. Spermatozoa were purified using Percoll density gradients. Sperm volume was measured as the forward scatter signal using flow cytometry. Sperm motility was analyzed using computer-aided sperm analysis (CASA). When transferred from an isotonic solution (330 mOsm l-1 ) to a hypotonic solution (290 mOsm l-1 ), cell volume was not changed in spermatozoa from normozoospermic men; but increased in those from asthenozoospermic samples. The addition of the chloride channel blockers, 4,4'-diisothiocyanatostilbene-2,2'- isulfonic acid (DIDS) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) to the hypotonic solution caused the normal spermatozoa to swell but did not increase the volume of those from the asthenozoospermic semen. DIDS and NPPB decreased sperm motility in both sets of semen samples. The inhibitory effect of NPPB on normal sperm motility was much stronger than on spermatozoa from the asthenozoospermic samples. Both sperm types expressed ClC-3 chloride channels, but the expression levels in the asthenozoospermic samples were much lower, especially in the neck and mid-piece areas. Spermatozoa from men with asthenozoospermia demonstrated lower volume regulating capacity, mobility, and ClC-3 expression levels (especially in the neck) than did normal spermatozoa. Thus, chloride channels play important roles in the regulation of sperm volume and motility and are downregulated in cases of asthenozoospermia.


Assuntos
Astenozoospermia/metabolismo , Canais de Cloreto , Motilidade Espermática , Espermatozoides/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Adulto , Envelhecimento , Tamanho Celular , Canais de Cloreto/antagonistas & inibidores , Regulação para Baixo , Humanos , Soluções Hipotônicas , Masculino , Nitrobenzoatos/farmacologia , Concentração Osmolar , Análise do Sêmen , Espermatozoides/ultraestrutura
19.
Sci Rep ; 6: 31472, 2016 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-27510727

RESUMO

Multidrug resistance is a major limitation for microtubule-binding agents in cancer treatment. Here we report a novel microtubule inhibitor (2-morpholin-4-yl-5-nitro-benzoic acid 4-methylsulfanyl-benzyl ester, IMB5046), its cytotoxicity against multidrug-resistant cell lines and its antitumor efficacy in animal models. IMB5046 disrupted microtubule structures in cells and inhibited purified tubulin polymerization in vitro. It bound to the colchicine pocket of tubulin. IMB5046 displayed potent cytotoxicity against multiple tumor cell lines with an IC50 range of 0.037-0.426 µM. Notably, several multidrug-resistant cell lines which were resistant to colchicine, vincristine and paclitaxel remained sensitive to IMB5046. IMB5046 was not a P-glycoprotein substrate. IMB5046 blocked cell cycle at G2/M phase and induced cell apoptosis. Microarray assay indicated that the differentially expressed genes after IMB5046 treatment were highly related to immune system, cell death and cancer. In a mouse xenograft model IMB5046 inhibited the growth of human lung tumor xenograft by 83% at a well-tolerated dose. It is concluded that IMB5046 is a tubulin polymerization inhibitor with novel chemical structure and can overcome multidrug resistance. It is a promising lead compound for cancer chemotherapy, especially for treatment of multidrug-resistant tumors.


Assuntos
Benzoatos/administração & dosagem , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Morfolinas/administração & dosagem , Neoplasias/tratamento farmacológico , Nitrobenzoatos/administração & dosagem , Moduladores de Tubulina/administração & dosagem , Células A549 , Animais , Benzoatos/química , Benzoatos/farmacologia , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Células HT29 , Humanos , Camundongos , Morfolinas/química , Morfolinas/farmacologia , Células NIH 3T3 , Neoplasias/genética , Nitrobenzoatos/química , Nitrobenzoatos/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Mol Pharmacol ; 90(3): 275-85, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27413118

RESUMO

Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated but ATP-gated chloride channel. Previous studies suggested that VX-770 [ivacaftor, N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide], a CFTR potentiator now used in clinics, increases the open probability of CFTR by shifting the gating conformational changes to favor the open channel configuration. Recently the chloride channel blocker and CFTR potentiator 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) has been reported to enhance CFTR activity by a mechanism that exploits the ATP hydrolysis-driven, nonequilibrium gating mechanism unique to CFTR. Surprisingly however, NPPB increased the activity of nonhydrolytic G551D-CFTR, the third most common disease-associated mutation. Here, we further investigated the mechanism of NPPB's effects on CFTR gating by assessing its interaction with well-studied VX-770. Interestingly, once G551D-CFTR was maximally potentiated by VX-770, NPPB further increased its activity. However, quantitative analysis of this drug-drug interaction suggests that this pharmacologic synergism is not due to independent actions of NPPB and VX-770 on CFTR gating; instead, our data support a dependent mechanism involving two distinct binding sites. This latter idea is further supported by the observation that the locked-open time of a hydrolysis-deficient mutant K1250A was shortened by NPPB but prolonged by VX-770. In addition, the effectiveness of NPPB, but not of VX-770, was greatly diminished in a mutant whose second nucleotide-binding domain was completely removed. Interpreting these results under the framework of current understanding of CFTR gating not only reveals insights into the mechanism of action for different CFTR potentiators but also brings us one step forward to a more complete schematic for CFTR gating.


Assuntos
Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Nitrobenzoatos/farmacologia , Quinolonas/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Sinergismo Farmacológico , Modelos Biológicos , Mutação
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