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BACKGROUND AND AIM: Phytoestrogens are traditionally used for cardiovascular risks but direct effects on the ischemic heart remain unclear. Plants with phytoestrogens are used for reducing menopausic symptoms and they could also be cardioprotectives. Here we investigated whether maca (Lepidium meyenii) contains isoflavones and prevents cardiac stunning, in comparison to soy isoflavones. EXPERIMENTAL PROCEDURE: Both products were orally and daily administered to rats during 1 week before exposing isolated hearts to ischemia/reperfusion (I/R). Young male (YM), female (YF) and aged female (AgF) rats treated with maca (MACA, 1 g/kg/day) or soy isoflavones (ISOF, 100 mg/kg/day) were compared to acute daidzein (DAZ, 5 mg/kg i.p.) and non-treated rat groups. Isolated ventricles were perfused inside a calorimeter to simultaneously measure contractile and calorimetrical signals before and during I/R. RESULTS AND CONCLUSIONS: Maca has genistein and daidzein. MACA and ISOF improved the post-ischemic contractile recovery (PICR) and muscle economy (P/Ht) in YM and YF hearts, but not in AgF hearts. DAZ improved PICR and P/Ht more in YM than in YF. The mKATP channels blockade reduced both PICR and P/Ht in DAZ-treated YM hearts, without affecting them in ISOF or MACA-treated YM hearts. In MACA treated YF hearts, the simultaneous blockade of NOS and mKATP channels, or the mNCX blockade reduced cardioprotection. Results show that subacute oral treatment with maca or with soy isoflavones was strongly preventive of cardiac ischemic dysfunction, more than the acute administration of a pure isoflavone (daidzein, genistein). Maca induced synergistic and complex mechanisms which prevented mitochondrial calcium overload.
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This paper presents a new assay to determine the activity of the lysosomal enzyme α-N-acetylgalactosaminidase (Naga, EC 3.2.1.49) in human serum. It is based on the use of a new chromogenic substrate, DNP-α-GalNAc (2,4-dinitrophenyl-N-acetyl-α-D-galactosaminide) and is performed at pH 4.3 and 37 °C. This allows continuous monitoring of the absorbance of the released DNP. The assay can be performed with a standard spectrophotometer. Compared to established methods using an endpoint assay with MU-α-GalNAc (4-methylumbelliferyl-GalNAc), the present method gives a ca. 3-fold higher specific activity, while only one tenth of the serum concentration in the assay is required. Hence, the assay is at least 30-fold more sensitive than that with MU-α-GalNAc. The pH dependence of the reaction with DNP-α-GalNAc in the pH 3.5 to 6.5 region, while using 4% serum in the assay, shows only one peak around pH 4. This pH optimum is similar to that reported with MU-α-GalNAc. In the accompanying paper (Albracht and Van Pelt (2017) Multiple exo-glycosidases in human serum as detected with the substrate DNP-α-GalNAc. II. Three α-N-acetylgalactosaminidase-like activities in the pH 5 to 8 region. Biochim. Biophys. Acta 159 (2017) Part I and II), the method is used to show that, under special assay conditions, three more Naga-like activities can be uncovered in human serum.
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The aim of this study was to evaluate the effects of differential concentration of dimethylsulphoxide (DMSO) on the morphology, cell viability, mRNA, and protein expression of stem cells obtained from the intraoral area. Stem cells derived obtained from gingiva were cultured in a growth medium in the presence of DMSO at concentrations ranging from 0.01 to 10%. The morphology and cellular viability were evaluated on days 1, 3, 5, 7 and 10. Quantitative polymerase chain reaction was used to evaluate the mRNA levels of collagen I and Runt-related transcription factor 2 (Runx2). Immunofluorescent assays were performed for Runx2 and collagen I, and protein expressions were measured, including those of Runx2 and collagen I using western blot analysis. Cells in the control group showed normal fibroblast morphology in the growth media. Cells from the higher DMSO concentration were significantly different compared to the control. The decrease in cell viability was noted in the higher concentration. A notable change in collagen I expression was noted at the higher concentrations of DMSO groups. Based on these findings, it was concluded that DMSO may have detrimental effects on the cell morphology and viability of mesenchymal stem cells. The results also suggest that DMSO has toxic effects via reduced collagen I expression.
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BACKGROUND: Proliferation of hepatic stellate cells (HSCs) play pivotal role in the progression of hepatic fibrosis consequent to chronic liver injury. Silibinin (SBN), a flavonoid compound, has shown to possess cell cycle arresting potential against many actively proliferating cancers cell lines. The objective of this study was to evaluate the anti-proliferative and cell cycle arresting properties of SBN in rapidly proliferating human hepatic stellate LX-2 cell line. METHODS: LX-2 cells were fed with culture medium supplemented with different concentrations of SBN (10, 50 and 100 µM). After 24 and 96 h of treatment, total cell number was determined by counting. Cytotoxicity was evaluated by trypan blue dye exclusion test. The expression profile of cMyc and peroxisome proliferator-activated receptor-γ (PPAR-γ) protein expressions was evaluated by Western blotting. Oxidative stress marker genes profile was quantified using qPCR. The migratory response of HSCs was observed by scrape wound healing assay. RESULTS: SBN treatments significantly inhibit the LX-2 cell proliferation (without affecting its viability) in dose dependent manner. This treatment also retards the migration of LX-2 cells toward injured area. In Western blotting studies SBN treatment up regulated the protein expressions of PPAR-γ and inhibited cMyc. CONCLUSION: The present study shows that SBN retards the proliferation, activation and migration of LX-2 cells without inducing cytotoxicity and oxidative stress. The profound effects could be due to cell cycle arresting potential of SBN.
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OBJECTIVES: Amyloid-beta (Aß) peptide deposition into insoluble plaques is a pathological hallmark of Alzheimer's disease (AD), but soluble oligomeric Aß is considered to be more potent and has been hypothesized to directly impair learning and memory. Also, evidences from some clinical studies indicated that Aß oligomer formation is the major cause for early AD onset. However, the biochemical mechanism involved in the oligomer-induced toxicity is not very well addressed. So, thise present study was undertaken to study the effects of single intracerebroventricular (icv) injection of protofibrillar Aß 1-42 on the behavioral and biochemical profile in rats. METHODS: Rats were divided into two groups (n = 8 per group): (1) sham control group and (2) Aß 1-42 injected group. A single dose of protofibrillar Aß 1-42 (5 ul) through icv injection was bilaterally administered into the dorsal hippocampus, while sham control animals were administered with 5 µl of vehicle. RESULTS: The results demonstrated that the protofibrillar Aß significantly inhibited long-term memory retention and increased anxiety levels as shown by the behavioral studies. The amyloid deposits were present inside the brain even six weeks after injection as confirmed by thioflavin-T staining and the neurodegeneration induced by these deposits was confirmed by Nissl's staining in hippocampal and cortical regions. The amyloid aggregates induced reactive oxygen species (ROS) production, acetylcholinesterase activity, nitrite levels, lipid peroxidation, and inhibited antioxidant enzyme activity in hippocampus, cortex, and striatum regions of rat brain after six weeks. DISCUSSION: The present study indicated that protofibrillar Aß 1-42 injection altered long term memory, induced anxiety-like behavior and also developed Alzheimer's disease like pathology in rats.
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Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/complicaciones , Péptidos beta-Amiloides/toxicidad , Ansiedad/etiología , Trastornos de la Memoria/etiología , Estrés Oxidativo/fisiología , Fragmentos de Péptidos/toxicidad , Acetilcolinesterasa/metabolismo , Enfermedad de Alzheimer/mortalidad , Animales , Antioxidantes/metabolismo , Peso Corporal/efectos de los fármacos , Modelos Animales de Enfermedad , Inyecciones Intraventriculares , Peroxidación de Lípido/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Nitritos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Agregado de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Estadísticas no Paramétricas , Factores de TiempoRESUMEN
The aim of the present work was to investigate the potential of the new and innovative artificial barrier, Permeapad™, when exposed to surfactants and co-solvents, often employed for poorly water soluble compounds. The barrier was in addition also exposed to fasted and fed state simulated intestinal fluids versions 1 and 2 (FaSSIF and FeSSIF), all of which the Permeapad™ barrier was compatible with based upon relative comparison of the permeability of the hydrophilic marker calcein in phosphate buffer. The new barrier therefore holds a huge potential due to its functional stability and robustness. It can be used as a standard tool to investigate permeability of drugs in the presence of different surfactants and co-solvents, from DMSO stock solutions at even high concentrations and for the evaluation of permeability in the presence of biomimetic media (BMM).
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Biomimética , Secreciones Intestinales/metabolismo , Fluoresceínas/metabolismo , Absorción Intestinal , Permeabilidad , Solventes/metabolismo , Tensoactivos/metabolismoRESUMEN
Using an in vitro random screening of small-molecule compounds, we discovered cis-diamminedichloroplatinum(II) (cisplatin), an anticancer agent, as a potential inhibitor of collagen fibril-formation. The inhibitory effect was found only when cisplatin was dissolved in dimethylsulphoxide (DMSO), indicating that the active species were cisplatin derivatives formed in the DMSO solution. The cisplatin derivatives inhibited the formation of collagen fibrils in vitro without affecting the triple-helical conformation of the collagen molecules. Incubation with the cisplatin solution in DMSO also inhibited in situ deposition of collagen fibrils in a human umbilical vein endothelial cell (HUVEC) culture. In addition, the derivatization of cisplatin in DMSO abolished the cytotoxicity of the original compound. The platinum complex was further revealed to interact with specific sites on the collagen triple helix, and the binding sites were suggested to contain His and/or Met residues. Mass spectrometry analysis of the cisplatin solution in DMSO and a structure-activity relationship study strongly suggested that the active compound is [Pt(NH3 )2 (Cl)(DMSO)](+) . This platinum complex will be useful for investigating molecular mechanisms of collagen self-assembly and for drug development for the treatment of fibrotic diseases.
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Antineoplásicos/química , Cisplatino/química , Cisplatino/farmacología , Colágeno/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Cisplatino/metabolismo , Colágeno/química , Complejos de Coordinación/química , Complejos de Coordinación/metabolismo , Complejos de Coordinación/farmacología , Dimetilsulfóxido/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Microscopía Confocal , Modelos Moleculares , Nefelometría y Turbidimetría , Unión Proteica , Relación Estructura-ActividadRESUMEN
Mechanistic target of rapamycin (mTOR) is a master regulator of cell growth through its ability to stimulate ribosome biogenesis and mRNA translation. In contrast, the p53 tumor suppressor negatively controls cell growth and is activated by a wide range of insults to the cell. The mTOR and p53 signaling pathways are connected by a number of different mechanisms. Chemotherapeutics that inhibit ribosome biogenesis often induce nucleolar stress and activation of p53. Here we have investigated how the p53 response to nucleolar stress is affected by simultaneous mTOR inhibition in osteosarcoma and glioma cell lines. We found that inhibitors of the mTOR pathway including rapamycin, wortmannin, and caffeine blunted the p53 response to nucleolar stress induced by actinomycin D. Synthetic inhibitors of mTOR (temsirolimus, LY294.002 and PP242) also impaired actinomycin D triggered p53 stabilization and induction of p21. Ribosomal protein (RPL11) is known to be required for p53 protein stabilization following nucleolar stress. Treatment of cells with mTOR inhibitors may lead to reduced synthesis of RPL11 and thereby destabilize p53. We found that rapamycin mimicked the effect of RPL11 depletion in terms of blunting the p53 response to nucleolar stress. However, the extent to which the levels of p53 and RPL11 were reduced by rapamycin varied between cell lines. Additional mechanisms whereby rapamycin blunts the p53 response to nucleolar stress are likely to be involved. Indeed, rapamycin increased the levels of endogenous MDM2 despite inhibition of its phosphorylation at Ser-166. Our findings may have implications for the design of combinatorial cancer treatments with mTOR pathway inhibitors.
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Nucléolo Celular/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Ribosómicas/metabolismo , Estrés Fisiológico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dactinomicina/farmacología , Humanos , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Estrés Fisiológico/efectos de los fármacos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The effectiveness of current treatment for age related macular degeneration (AMD) by targeting one molecule is limited due to its multifactorial nature and heterogeneous pathologies. Treatment strategy to target multiple signaling pathways or pathological components in AMD pathogenesis is under investigation for better clinical outcome. Inhibition of the redox function of apurinic endonuclease 1/redox factor-1 (APE1) was found to suppress endothelial angiogenesis and promote neuronal cell recovery, thereby may serve as a potential treatment for AMD. In the current study, we for the first time have found that a specific inhibitor of APE1 redox function by a small molecule compound E3330 regulates retinal pigment epithelium (RPEs) cell response to oxidative stress. E3330 significantly blocked sub-lethal doses of oxidized low density lipoprotein (oxLDL) induced proliferation decline and senescence advancement of RPEs. At the same time, E3330 remarkably decreased the accumulation of intracellular reactive oxygen species (ROS) and down-regulated the productions of monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF), as well as attenuated the level of nuclear factor-κB (NF-κB) p65 in RPEs. A panel of stress and toxicity responsive transcription factors that were significantly upregulated by oxLDL was restored by E3330, including Nrf2/Nrf1, p53, NF-κB, HIF1, CBF/NF-Y/YY1, and MTF-1. Further, a single intravitreal injection of E3330 effectively reduced the progression of laser-induced choroidal neovascularization (CNV) in mouse eyes. These data revealed that E3330 effectively rescued RPEs from oxidative stress induced senescence and dysfunctions in multiple aspects in vitro, and attenuated laser-induced damages to RPE-Bruch׳s membrane complex in vivo. Together with its previously established anti-angiogenic and neuroprotection benefits, E3330 is implicated for potential use for AMD treatment.
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Benzoquinonas/administración & dosificación , Neovascularización Coroidal/tratamiento farmacológico , ADN-(Sitio Apurínico o Apirimidínico) Liasa/antagonistas & inhibidores , Fármacos Neuroprotectores/administración & dosificación , Estrés Oxidativo/efectos de los fármacos , Propionatos/administración & dosificación , Epitelio Pigmentado de la Retina/metabolismo , Animales , Senescencia Celular/efectos de los fármacos , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inyecciones Intravítreas , Ratones , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/patologíaRESUMEN
Receptor-ligand binding is an essential interaction for biological function. Oxidative stress can modify receptors and/or membrane lipid dynamics, thus altering cell physiological functions. The aim of this study is to analyze how oxidative stress may alter receptor-ligand binding and lipid domain distribution in the case of progesterone-induced blocking factor/progesterone-induced blocking factor-receptor. For membrane fluidity regionalization analysis of MEC-1 lymphocytes, two-photon microscopy was used in individual living cells. Lymphocytes were also double stained with AlexaFluor647/progesterone-induced blocking factor and Laurdan to evaluate -induced blocking factor/progesterone-induced blocking factor-receptor distribution in the different membrane domains, under oxidative stress. A new procedure has been developed which quantitatively analyzes the regionalization of a membrane receptor among the lipid domains of different fluidity in the plasma membrane. We have been able to establish a new tool which detects and evaluates lipid raft clustering from two-photon microscopy images of individual living cells. We show that binding of progesterone-induced blocking factor to progesterone-induced blocking factor-receptor causes a rigidification of plasma membrane which is related to an increase of lipid raft clustering. However, this clustering is inhibited under oxidative stress conditions. In conclusion, oxidative stress decreases membrane fluidity, impairs receptor-ligand binding and reduces lipid raft clustering.
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Linfocitos B/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Microdominios de Membrana/efectos de los fármacos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Proteínas Gestacionales/química , Receptores de Citocinas/química , Análisis de la Célula Individual/métodos , Factores Supresores Inmunológicos/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Linfocitos B/citología , Linfocitos B/metabolismo , Carbocianinas/química , Línea Celular Tumoral , Colorantes Fluorescentes/química , Humanos , Lauratos/química , Fluidez de la Membrana/efectos de los fármacos , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Estrés Oxidativo , Proteínas Gestacionales/metabolismo , Unión Proteica , Receptores de Citocinas/metabolismo , Coloración y Etiquetado/métodos , Factores Supresores Inmunológicos/metabolismoRESUMEN
Phosphodiesterase type 4 inhibitors (PDE4-Is) have received increasing attention as cognition-enhancers and putative treatment strategies for Alzheimer's disease (AD). By preventing cAMP breakdown, PDE4-Is can enhance intracellular signal transduction and increase the phosphorylation of cAMP response element-binding protein (CREB) and transcription of proteins related to synaptic plasticity and associated memory formation. Unfortunately, clinical development of PDE4-Is has been seriously hampered by emetic side effects. The new isoform-specific PDE4D-I, GEBR-7b, has shown to have beneficial effects on memory at non-emetic doses. The aim of the current study was to investigate chronic cognition-enhancing effects of GEBR-7b in a mouse model of AD. To this extent, 5-month-old (5M) APPswe/PS1dE9 mice received daily subcutaneous injections with GEBR-7b (0.001 mg/kg) or vehicle for a period of 3 weeks, and were tested on affective and cognitive behavior at 7M. We demonstrated a cognition-enhancing potential in APPswe/PS1dE9 mice as their spatial memory function at 7M in the object location test was improved by prior GEBR-7b treatment. APPswe/PS1dE9 mice displayed lower levels of CREB phosphorylation, which remained unaltered after chronic GEBR-7b treatment, and higher levels of tau in the hippocampus. Hippocampal brain-derived neurotrophic factor levels and synaptic densities were not different between experimental groups and no effects were observed on hippocampal GSK3ß and tau phosphorylation or Aß levels. In conclusion, GEBR-7b can enhance spatial memory function in the APPswe/PS1dE9 mouse model of AD. Although the underlying mechanisms of its cognition-enhancing potential remain to be elucidated, PDE4D inhibition appears an interesting novel therapeutic option for cognitive deficits in AD.
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Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Hipocampo/efectos de los fármacos , Iminas/farmacología , Aprendizaje por Laberinto/efectos de los fármacos , Memoria/efectos de los fármacos , Morfolinas/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large , Guanilato-Quinasas/metabolismo , Hipocampo/metabolismo , Iminas/uso terapéutico , Proteínas de la Membrana/metabolismo , Ratones , Morfolinas/uso terapéutico , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Fosforilación/efectos de los fármacosRESUMEN
OBJECTIVE(S): The examination of the possibility of applying lower CPA- concentrations and obtaining the similar results to those using higher concentrations; as it is shown, the toxicity of the CPAs used in vitrification approach will diminish. MATERIALS AND METHODS: Following vitrification/warming, oocytes were subjected to PZD/ICSI. SRs, FRs, and DRs were recorded. SRs and DRs of the embryos were monitored after vitrification/warming. IHC studies were done. Data were analyzed in comparison to the data of Exp. (experimental groups) applying 1.5 M CPA- concentrations (largely-used concentration). RESULTS: The data of oocytes exposed to 1.25 M concentrated CPAs were in consistency with those exposed to 1.5 M and fresh oocytes in terms of SRs, FRs and DRs. Normal spindle and chromatin configuration is in consistence between the two experimental groups, but lower in comparison with control group. The lower the concentrations were, the less SRs, FRs, DRs were. Also, spindle organizations were more normal in comparison with the experimental groups as the concentrations decreased. The results of DRs for embryos which were exposed to 1.25 and 1.0 M concentrated CPAs were close to those vitrified with 1.5 M and fresh embryos but IHC observations in the three Exp. were significantly lower than those of fresh embryos. The results of 7.5 M concentrated CPAs solutions were significantly lower than those of the control group 1.5, 1.25 and 1.0 M treated. CONCLUSIONS: Vitrification by cryotop technology using minimal volume approach increases both cooling and warming rates, therefore, the CPAs limited reduction to 1.25 and 1.0 M instead of using 1.5 M for oocytes and embryos cryotop-vitrification procedure, may be a slight adjustment.
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OBJECTIVE(S): The examination of the possibility of applying lower CPA- concentrations and obtaining the similar results to those using higher concentrations; as it is shown, the toxicity of the CPAs used in vitrification approach will diminish. MATERIALS AND METHODS: Following vitrification/warming, oocytes were subjected to PZD/ICSI. SRs, FRs, and DRs were recorded. SRs and DRs of the embryos were monitored after vitrification/warming. IHC studies were done. Data were analyzed in comparison to the data of Exp. (experimental groups) applying 1.5 M CPA- concentrations (largely-used concentration). RESULTS: The data of oocytes exposed to 1.25 M concentrated CPAs were in consistency with those exposed to 1.5 M and fresh oocytes in terms of SRs, FRs and DRs. Normal spindle and chromatin configuration is in consistence between the two experimental groups, but lower in comparison with control group. The lower the concentrations were, the less SRs, FRs, DRs were. Also, spindle organizations were more normal in comparison with the experimental groups as the concentrations decreased. The results of DRs for embryos which were exposed to 1.25 and 1.0 M concentrated CPAs were close to those vitrified with 1.5 M and fresh embryos but IHC observations in the three Exp. were significantly lower than those of fresh embryos. The results of 7.5 M concentrated CPAs solutions were significantly lower than those of the control group 1.5, 1.25 and 1.0 M treated. CONCLUSIONS: Vitrification by cryotop technology using minimal volume approach increases both cooling and warming rates, therefore, the CPAs limited reduction to 1.25 and 1.0 M instead of using 1.5 M for oocytes and embryos cryotop-vitrification procedure, may be a slight adjustment.
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Selective cyclooxygenase 2 (COX2) inhibitors (COXIBs) are effective anti-inflammatory and analgesic drugs with improved gastrointestinal (GI) safety compared to nonselective nonsteroidal anti-inflammatory drugs known as traditional (tNSAIDs). However, their use is associated with a cardiovascular (CV) hazard (i.e. increased incidence of thrombotic events and hypertension) due to the inhibition of COX2-dependent vascular prostacyclin. Aiming to design COX2-selective inhibitors with improved CV safety, new NO-releasing COXIBs (NO-COXIBs) have been developed. In these hybrid drugs, the NO-mediated CV effects are expected to compensate for the COXIB-mediated inhibition of prostacyclin. This study evaluates the potential CV beneficial effects of VA694, a promising NO-COXIB, the anti-inflammatory effects of which have been previously characterized in several in vitro and in vivo experimental models. When incubated in hepatic homogenate, VA694 acted as a slow NO-donor. Moreover, it caused NO-mediated relaxant effects in the vascular smooth muscle. The chronic oral administration of VA694 to young spontaneously hypertensive rats (SHRs) significantly slowed down the age-related development of hypertension and was associated with increased plasma levels of nitrates, stable end-metabolites of NO. Furthermore, a significant improvement of coronary flow and a significant reduction of endothelial dysfunction were observed in SHRs submitted to chronic administration of VA694. In conclusion, VA694 is a promising COX2-inhibiting hybrid drug, showing NO releasing properties which may mitigate the CV deleterious effects associated with the COX2-inhibition.
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Antiinflamatorios no Esteroideos/farmacología , Inhibidores de la Ciclooxigenasa 2/farmacología , Factores Relajantes Endotelio-Dependientes/administración & dosificación , Endotelio/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Nitratos/farmacología , Óxido Nítrico/administración & dosificación , Pirroles/farmacología , Animales , Antiinflamatorios no Esteroideos/química , Presión Sanguínea/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Inhibidores de la Ciclooxigenasa 2/química , Endotelio/patología , Factores Relajantes Endotelio-Dependientes/farmacología , Hipertensión/sangre , Masculino , Nitratos/sangre , Nitratos/química , Óxido Nítrico/farmacología , Nitritos/sangre , Pirroles/química , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Flujo Sanguíneo Regional/efectos de los fármacosRESUMEN
The native HIV-1 envelope spike exists as trimers on the virion surface. Therefore antibodies elicited following immunisation with trimeric envelope vaccines may be directed against epitopes found on functional envelope spikes. Novel monoclonal antibodies were produced by priming mice with plasmid DNA expressing either HIV-1 CN54 or ZM96 gp140 sequences, and boosting with CN54 trimeric rgp140. Spleen cells were fused with NS-0 cells and hybridomas were screened for production of antibodies which bound trimeric rgp140. 18 monoclonal antibodies (MAbs) were isolated from 16 colonies, which were characterised by binding in ELISA and Western blotting, neutralisation of pseudotyped viruses and isotype. Specificity for the third variable (V3) region was detected in 5 of these antibodies, suggesting that the trimeric protein did not alter the main focus of the response compared with monomeric protein. 3 MAbs were identified as neutralising in a pseudotype assay, all of which were mapped to the V3 region. Although cross clade binding of antibodies was detected the neutralisation was C clade specific.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/aislamiento & purificación , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Western Blotting , Ensayo de Inmunoadsorción Enzimática , VIH-1/genética , Ratones , Pruebas de Neutralización , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for induction with rifampicin were created. Williams' medium E showed the best results on morphology and viability of the hepatocytes and was therefore used for the following induction experiments. Cells cultured in Dulbecco's Modified Eagle Medium were not inducible. Incubation with rifampicin increased the CYP activity in two different hepatocyte preparations in a dose dependent manner (EC50=1.20 µM and 6.06 µM; Emax=4.1- and 3.4-fold induction). No increase in CYP activity was detected after incubation with dexamethasone or phenobarbital. The hepatocyte culture conditions established in this study proved to be valuable for investigation of the induction of equine CYPs. In further studies, other equine drugs can be evaluated for CYP induction with this in vitro system.
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Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Evaluación Preclínica de Medicamentos/veterinaria , Hepatocitos/efectos de los fármacos , Drogas Veterinarias/farmacología , Animales , Antiinflamatorios/efectos adversos , Antiinflamatorios/farmacología , Antibióticos Antituberculosos/efectos adversos , Antibióticos Antituberculosos/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cumarinas/metabolismo , Medio de Cultivo Libre de Suero/metabolismo , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , Dexametasona/efectos adversos , Dexametasona/farmacología , Inducción Enzimática/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/metabolismo , Caballos , Hipnóticos y Sedantes/efectos adversos , Hipnóticos y Sedantes/farmacología , Inmunohistoquímica/veterinaria , Indicadores y Reactivos/metabolismo , Cinética , Fenobarbital/efectos adversos , Fenobarbital/farmacología , Rifampin/efectos adversos , Rifampin/farmacología , Drogas Veterinarias/efectos adversosRESUMEN
The ability of symmetrically substituted long chain polymethylene tetramines, methoctramine (1) and its analogs 2-4 to kill cancer cells was studied. We found that an elevated cytotoxicity was correlated with a 12 methylene chain length separating the inner amine functions (6-12-6 carbon backbone), together with the introduction of diphenylethyl moieties on the terminal nitrogen atoms (compound 4) of a tetramine backbone. Compound 4 triggered dissipation of mitochondrial transmembrane potential and increased intracellular peroxide levels, leading to a caspase-independent HeLa cell death associated with a rapid activation of autophagy. The antioxidant N-acetylcysteine inhibited cell death and activation of autophagy, indicating a link between oxidative stress and autophagy. Autophagy was rapidly triggered even by tetramines 2 and 3, indicating that is related to their polyamine structure. Autophagy did not protect HeLa cells against cytotoxicity elicited by compound 4. The present study shows that, by modifications of the methoctramine structure, it is possible to design polyamine derivatives highly cytotoxic against tumor cells and that the appropriate design of molecules bearing polyamine-like structures leads to powerful inducers of autophagy.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Neoplasias/patología , Poliaminas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HT29 , Células HeLa , Humanos , Estructura Molecular , Poliaminas/síntesis química , Poliaminas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Risk assessment of soils is usually based on chemical measurements and assuming accidental soil ingestion and evaluating induced toxic and carcinogenic effects. Recently biological tools have been coupled to chemical-based risk assessment since they integrate the biological effects of all xenobiotics in soils. We employed integrated monitoring of soils based on chemical analyses, risk assessment and in vitro models in the highly urbanized semirural area of the Olona Valley in northern Italy. Chemical characterization of the soils indicated low levels of toxic and carcinogenic pollutants such as PAHs, PCDD/Fs, PCBs and HCB and human risk assessment did not give any significant alerts. HepG2 and BALB/c 3T3 cells were used as a model for the human liver and as a tool for the evaluation of carcinogenic potential. Cells were treated with soil extractable organic matters (EOMs) and the MTS assay, LDH release and morphological transformation were selected as endpoints for toxicity and carcinogenicity. Soil EOMs induced dose-dependent inhibition of cell growth at low doses and cytotoxicity after exposure to higher doses. This might be the result of block of cell cycle progression to repair DNA damage caused by oxidative stress; if this DNA damage cannot be repaired, cells die. No significant inductions of foci were recorded after exposure to EOMs. These results indicate that, although the extracts contain compounds with proven carcinogenic potential, the levels of these pollutants in the analyzed soils were too low to induce carcinogenesis in our experimental conditions. In this proposed case study, HepG2 cells were found an appropriate tool to assess the potential harm caused by the ingestion of contaminated soil as they were able to detect differences in the toxicity of soil EOMs. Moreover, the cell transformation assay strengthened the combined approach giving useful information on carcinogenic potential of mixtures.
Asunto(s)
Células Hep G2/efectos de los fármacos , Contaminantes del Suelo/toxicidad , Animales , Células 3T3 BALB/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Daño del ADN/efectos de los fármacos , Humanos , Técnicas In Vitro , Italia , Ratones , Medición de Riesgo , Suelo/químicaRESUMEN
The environmental dioxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is classified as a Group 1 human carcinogen and teratogenic agent. We hypothesize that TCDD-induced oxidative stress may also interfere with mitochondrial ATP-sensitive potassium channels (mitoKATP), which are known to regulate and to be regulated by mitochondrial redox state. We investigated the effects of an acute treatment of male Wistar rats with TCDD (50 µg/kg i.p.) and measured the regulation of cardiac mitoKATP. While the function of cardiac mitochondria was slightly depressed, mitoKATP activity was 52% higher in animals treated with TCDD. The same effects were not observed in liver mitochondria isolated from the same animals. Our data also shows that regulation of mitochondrial ROS production by mitoKATP activity is different in both groups. To our knowledge, this is the first report to show that TCDD increases mitoKATP activity in the heart, which may counteract the increased oxidative stress caused by the dioxin during acute exposure.
Asunto(s)
Carcinógenos/toxicidad , Dioxinas/toxicidad , Canales KATP/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Canales de Potasio/metabolismo , Animales , Masculino , Mitocondrias Hepáticas/metabolismo , Estrés Oxidativo , Ratas , Ratas WistarRESUMEN
We evaluated whether the nanoformulation of curcumin could be more effective than free curcumin against arsenic-induced immune dysfunction in rats. Curcumin was encapsulated in polylactic-co-glycolic acid (PLGA). Nanocurcumin (CUR-NP) exhibited a spherical shape with the mean particle size of 130.8 nm. Rats were randomly divided into five groups of six each. Group I was kept as the control. In Group II, rats were exposed to sodium arsenite (25 ppm) daily through drinking water for 42 days. Groups III, IV and V were treated with arsenic as in Group II, however, they were administered with nanoparticle, curcumin (100 mg/kg bw) and CUR-NP (100 mg/kg bw), respectively, by oral gavage during the last 14 days of arsenic exposure. At term, serum and spleen were collected. Immune dysfunction was evaluated by assessing cellular and humoral immunities. Arsenic significantly decreased the splenic lymphocyte proliferation in response to the antigen -- Keyhole Limpet Hemocyanin (KLH) and mitogen -- concanavalin-A. Arsenic reduced both the delayed type hypersensitivity response and secondary antibody (IgG) response to KLH. It also reduced the lipopolysaccharide-stimulated nitric oxide production in splenic lymphocytes. Free curcumin and CUR-NP treatment significantly attenuated these arsenic-mediated effects. However, the magnitude of the effects indicates that CUR-NP has better ameliorative potential than free curcumin at the equivalent dose level.