RESUMEN
Immune checkpoint blockade has shown significant clinical benefit in multiple cancer indications, but many patients are either refractory or become resistant to the treatment over time. HER2/neu oncogene overexpressed in invasive breast cancer patients associates with more aggressive diseases and poor prognosis. Anti-HER2 mAbs, such as trastuzumab, are currently the standard of care for HER2-overexpressing cancers, but the response rates are below 30% and patients generally suffer relapse within a year. In this study we developed a bispecific antibody (BsAb) simultaneously targeting both PD1 and HER2 in an attempt to combine HER2-targeted therapy with immune checkpoint blockade for treating HER2-positive solid tumors. The BsAb was constructed by fusing scFvs (anti-PD1) with the effector-functional Fc of an IgG (trastuzumab) via a flexible peptide linker. We showed that the BsAb bound to human HER2 and PD1 with high affinities (EC50 values were 0.2 and 0.14 nM, respectively), and exhibited potent antitumor activities in vitro and in vivo. Furthermore, we demonstrated that the BsAb exhibited both HER2 and PD1 blockade activities and was effective in killing HER2-positive tumor cells via antibody-dependent cellular cytotoxicity. In addition, the BsAb could crosslink HER2-positive tumor cells with T cells to form PD1 immunological synapses that directed tumor cell killing without the need of antigen presentation. Thus, the BsAb is a new promising approach for treating late-stage metastatic HER2-positive cancers.
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Anticuerpos Biespecíficos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Neoplasias/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor ErbB-2/antagonistas & inhibidores , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Diallyl disulfide (DADS) is a garlic-derived organosulfur compound. The current study is designed to evaluate the protective effects of DADS against ethanol-induced oxidative stress, and to explore the underlying mechanisms by examining the HO-1/Nrf-2 pathway. METHODS: We investigated whether or not DADS could activate the HO-1 in normal human liver cell LO2, and then evaluated the protective effects of DADS against ethanol-induced damage in LO2 cells and in acute ethanol-intoxicated mice. The biochemical parameters were measured using commercial kits. HO-1 mRNA level was determined by RT-PCR. Histopathology and immunofluorescence assay were performed with routine methods. Protein levels were measured by western blot. RESULTS: DADS significantly increased the mRNA and protein levels of HO-1, stimulated the nuclear translocation of Nrf-2 and increased the phosphorylation of MAPK in LO2 cells. The nuclear translocation of Nrf-2 was abrogated by MAPK inhibitors. DADS significantly suppressed ethanol-induced elevation of lactate dehydrogenase (LDH) and aspartate transaminase (AST) activities, decrease of glutathione (GSH) level, increase of malondialdehyde (MDA) levels, and apoptosis of LO2 cells, which were all blocked by ZnPPIX. In mice, DADS effectively suppressed acute ethanol-induced elevation of aminotransferase activities, and improved liver histopathological changes, which might be associated with HO-1 activation. CONCLUSION: These results demonstrate that DADS could induce the activation of HO-1/Nrf-2 pathway, which may contribute to the protective effects of DADS against ethanol-induced liver injury. GENERAL SIGNIFICANCE: DADS may be beneficial for the prevention and treatment of ALD due to significant activation of HO-1/Nrf-2 pathway.
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Compuestos Alílicos/farmacología , Disulfuros/farmacología , Etanol/toxicidad , Hemo-Oxigenasa 1/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: To study the protective impact of tea polyphenols (TP) on the injury of fibrinolytic functions induced by high-methionine dietary in rats. METHODS: 50 male Wistar rats were divided by stratified based on body weight into 5 groups with 10 in each group: namely control group, model group, low-dose TP group, medium-dose TP group and high-dose TP group. The rats in model group and TP groups were fed with 3% methionine dietary, control group rats with routine diet. In addition, rats in low-dose, medium-dose and high-dose TP groups were treated with TP at 50, 100 and 200 mg/kg dosage respectively by gavages every day, control group and model group rats were given with same amount distilled water. The animals were sacrificed after 8 weeks. The levels of tissue-type plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1) in plasma were determined by ELISA assays, mRNA levels of t-PA and PAI-1 in aortic arch were detected by RT-PCR, t-PA and PAI-1 expression in aortic arch were detected by immunohistochemistry strept-avidin-biotin complex (SABC). RESULTS: After experiment, the t-PA expression of aortic arch in control group, model group, low-dose TP group, medium-dose TP group and high-dose TP group were 133.03 ± 10.14, 95.46 ± 11.08, 111.97 ± 11.91, 130.23 ± 10.80, 139.39 ± 9.41 (F = 14.15, P < 0.01), respectively, and the PAI-1 expression were 90.91 ± 8.67, 166.76 ± 12.18, 139.63 ± 12.71, 134.66 ± 13.19, 109.49 ± 10.82 (F = 31.44, P < 0.01). The t-PA concentration of plasma were (10.69 ± 1.26), (6.13 ± 0.92), (8.56 ± 1.19), (9.69 ± 0.92), (11.97 ± 1.08) ng/ml, respectively (F = 41.98, P < 0.01), and the PAI-1 concentration of plasma were (6.31 ± 0.81), (16.98 ± 1.27), (11.39 ± 0.82), (8.46 ± 0.67), (8.08 ± 0.91) ng/ml, respectively (F = 207.74, P < 0.01). The mRNA levels of t-PA in aortic arch were 1.12 ± 0.02, 0.75 ± 0.14, 1.01 ± 0.09, 0.95 ± 0.08, 1.05 ± 0.13 (F = 5.77, P < 0.05), and the mRNA levels of PAI-1 in aortic arch were 1.25 ± 0.11, 1.74 ± 0.06, 1.23 ± 0.05, 1.09 ± 0.14, 1.23 ± 0.04 (F = 23.56, P < 0.01). CONCLUSION: The results indicate that TP seems to have regulatory function on transcription and protein levels of t-PA and PAI-1, in addition to maintaining the balance between PAI-1 and t-PA and healing the injury of fibrinolytic functions in rats induced by high-methionine dietary.
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Fibrinólisis/efectos de los fármacos , Metionina/efectos adversos , Polifenoles/farmacología , Animales , Dieta , Masculino , Inhibidor 1 de Activador Plasminogénico/sangre , Ratas , Ratas Wistar , Té/química , Activador de Tejido Plasminógeno/sangreRESUMEN
OBJECTIVE: To study the effects of 1-bromopropane (1-BP) on the functions of learning-memory and the central cholinergic system in rats. METHODS: Forty male Wistar rats were randomly divided into four groups: low 1-BP group (200 mg/kg), middle 1-BP group (400 mg/kg), high 1-BP group (800 mg/kg) and control group, and the exposure time was 7 days. The Morris water maze (MWM) test was applied to evaluate the learning-memory function in rats. After the MWM test, the rats were sacrificed, the cerebral cortex and hippocampus were quickly dissected and homogenized in ice bath. The activity of acetylcholine esterase (AChE) and choline acetyltransferase (ChAT) in supernatant of homogenate were detected. RESULTS: The latency and swim path-length of rats in middle and high 1-BP groups prolonged significantly in place navigation test and the efficiency of searching strategy obviously decreased, as compared with control group (P < 0.05 or P < 0.01). In spatial probe test, the number of crossing platform in three 1-BP groups decreased significantly, as compared with control group (P < 0.05 or P < 0.01). The cortical AChE activity of rats in middle and high 1-BP groups was significantly higher than that of control and low 1-BP group (P < 0.05 or P < 0.01). The AChE activity in rat hippocampus of high 1-BP group obviously increased, as compared with control group as compared with control group (P < 0.05). There was no significant difference of cortical ChAT activity between three 1-BP groups and control group (P > 0.05). In the hippocampus, there was no difference of ChAT activity among the groups (P > 0.05). CONCLUSION: 1-BP exposure could significantly influence the learning-memory function in rats due to the increase of AChE activity.
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Acetilcolinesterasa/metabolismo , Corteza Cerebral/efectos de los fármacos , Hipocampo/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Animales , Corteza Cerebral/enzimología , Colina O-Acetiltransferasa/metabolismo , Hipocampo/enzimología , Hidrocarburos Bromados/toxicidad , Masculino , Ratas , Ratas WistarRESUMEN
OBJECTIVE: To investigate the effects of tacrolimus (FK506) on behavioral function and heat-shock proteins (HSP70) expression in nervous tissues of acrylamide (ACR)-induced rats. METHODS: Totally 40 health Wistar rats were randomly divided into control group, model group, low and high doses of FK506 groups. All four groups were treated five times per week for four weeks. Gait score was measured every week. And rats were sacrificed on day 28, the cerebrum, spinal cord and sciatic nerve were dissected, and homogenized in ice bath, then the levels of HSP70 and Bcl-2, Bax were analyzed by western bloting. RESULTS: Compared with the ACR model group, the gait score in low and high doses of FK506 groups decreased by 30.1% and 47.7% respectively in the 4th week. In the cerebrum and sciatic nerve pellet, the level of HSP70 in the FK506 groups increased by 11.6%, 33.3% and 56.3%, 58.5% (P < 0.01), but no significant changes existed in spinal cord. The level of Bcl-2 in the sciatic nerve pellet increased by 39.1% (P < 0.01) but no significant changes existed in the cerebrum and spinal cord from low dose of FK506 group. And the level of Bax in the spinal cord pellet markedly increased by 46.8% but not in cerebrum and sciatic nerve pellet; Whereas in the tissues mentioned above, the levels of Bcl-2 were enhanced remarkably by 16.3%, 14.8% and 56.0% (P < 0.01) in the high dose of FK506 group. And the level of Bax in the cerebrum and spinal cord pellet markedly increased by 16.4% and 40.2% but not in sciatic nerve. The values of Bcl-2/Bax in low and high doses of FK506 groups clearly increased by 15.9%, 33.3%, 36.9% and 30.1%, 49.1%, 60.1% (P < 0.01). CONCLUSION: The administration of FK506 has dramatically neuroprotective effects against the development of ACR neuropathy, which may be related to up-regulating the expression of HSP70 and Bcl-2 with down-regulating the expression of Bax.
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Acrilamida/envenenamiento , Proteínas HSP70 de Choque Térmico/metabolismo , Tejido Nervioso/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tacrolimus/uso terapéutico , Proteína X Asociada a bcl-2/metabolismo , Animales , Masculino , Tejido Nervioso/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Ratas , Ratas WistarRESUMEN
OBJECTIVE: To investigate the effects of garlic oil (GO) on n-hexane metabolized to 2, 5-hexanedione (2, 5-HD) in mice. METHODS: Adult healthy Kunming-mice were treated with n-hexane and GO. The serum was obtained and extracted with ethyl acetate, and the levels of the serum 2, 5-HD were determined by gas chromatography. RESULTS: (1) The concentration of 2, 5-HD in serum increased firstly after a single exposure to n-hexane (4 000 mg/kg). The peak value occurred at 10 hours after n-hexane treatment, but could hardly be detected at 20 h. (2) There was no 2, 5-HD in serum of control mice. The content of 2, 5-HD in serum increased along with the exposure dose of n-hexane. The serum 2, 5-HD contents of the 2000, 4000 and 6000 mg/kg groups mice were 8.04, 16.68 and 22.38 microg/ml at 8 h in pretreated mice, respectively, and showed significant dose-effect relationship. (3) When the different age mice were exposed to the same dose of n-hexane, the contents of 2, 5-HD in serum were significantly different after 8 hours (P<0.05). The serum 2, 5-HD level of the 5 weeks old mice (22.83 microg/ml) was much higher than the 4 (19.59 microg/ml) and 6 (16.42 microg/ml) weeks old mice. (4) When the different gender mice were exposed to the same dose of n-hexane, the concentration of 2, 5-HD in serum of female mice (13.22 microg/ml) was higher than that of the female mice (10.34 microg/ml, P<0.05). (5) GO significantly inhibited the increase of the serum 2, 5-HD levels of both the pretreatment and post-treatment groups treated with 80 mg/kg n-hexane respectively, but the pretreatment with GO exhibited the more suppressive effects than the post-treatment (P>0.05). Compared with the n-hexane group, the concentrations of serum 2, 5-HD in GO-pretreated groups mice decreased by 16.2%, 20.8%, 22.8% (P<0.05) and 32.1% (P<0.01), respectively, and showed significant dose-effect relationship. CONCLUSION: The serum content of 2, 5-HD, the metabolite of n-hexane, is different in different genders and age mice after exposed to the same dose of n-hexane. GO can effectively inhibit the production of n-hexane metabolized to 2, 5-HD in mice serum.
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Compuestos Alílicos/química , Hexanos/farmacocinética , Hexanonas/sangre , Sulfuros/química , Animales , Biotransformación/efectos de los fármacos , Femenino , Masculino , RatonesRESUMEN
The protective effects of diallyl trisulfide (DATS) on acute ethanol-induced liver injury were investigated. Mice were pretreated with DATS (30mg/kgbw) for 7d before being exposed to ethanol (4.8g/kgbw). The biochemical indices (aspartate amino transferase, AST; alanine amino transferase, ALT; triglyceride, TG) were examined to evaluate the protective effects. Mitochondria were isolated for the mitochondrial permeability transition (MPT), membrane potential (DeltaPsi(m)) and adenosine nucleotide pool assay. The lipid peroxidation (malondialdehyde, MDA), non-enzymatic antioxidant (glutathione, GSH) and enzymatic antioxidants (superoxide dismutase, SOD; catalase, CAT; glutathione reductase, GR; glutathione peroxidase, GSH-Px) were measured both in the liver homogenate and isolated mitochondria. Acute ethanol exposure resulted in the significant increase of the ALT, AST and TG levels and hepatic mitochondria dysfunction shown as MPT, and the decreases of DeltaPsi(m), ATP and energy charge (EC). However, DATS pretreatment dramatically attenuated these adverse effects. Beside this, DATS was found to significantly inhibit the increase of the hepatic and mitochondrial MDA levels, which were decreased by 33.3% (P<0.01) and 39.0% (P<0.01), respectively. In addition, DATS pretreatment markedly suppressed the ethanol-induced decrease of the hepatic GSH level and increased the mitochondrial GSH level. Moreover, the activities of the hepatic antioxidant enzymes (SOD, CAT, and GR) and the mitochondrial antioxidant enzymes (SOD, GR, and GSH-Px) were significantly boosted. Thus, we concluded that DATS dramatically attenuated acute ethanol-induced liver injury and mitochondrial dysfunction. The increase of the hepatic and mitochondrial GSH levels and the elevation of the antioxidant enzymes activities should account for the preventive effects.
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Compuestos Alílicos/farmacología , Antioxidantes/farmacología , Depresores del Sistema Nervioso Central/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Etanol/toxicidad , Mitocondrias Hepáticas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Sulfuros/farmacología , Nucleótidos de Adenina/metabolismo , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Glutatión/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/ultraestructura , Permeabilidad/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Triglicéridos/metabolismoRESUMEN
The skin-core evolvement of the carbon fibers was studied as a function of heat-treatment temperature though the analysis of Raman spectroscopy of the carbon fibers surface and core. It was found that the change of the Raman spectra of the carbon fibers core was similar to that on the surface with the increase in heat-treatment temperature. At 1600 degrees C, the Rs and Rc values were almost equal, indicating that the degrees of the graphitization of the carbon fibers surface and core were almost uniform. The Rs and Rc values decreased dramatically with the increase in heat-treatment temperature, and Rs decreased more. At 2800 degrees C, the Rs value came to 0.429, lowered 77.2%, while the Rc value then came to 1.101, lowered 38.7% only. It implied that the graphitization degree of the carbon fibers was enhanced with increasing the heat treatment temperature, and that of carbon fibers surface was enhanced more. The graphite characters of the carbon of the carbon fibers surface were different from that of the carbon fibers core. The former is close to soft carbon, which is easy to graphitize, while the latter is close to hard carbon, which is difficult to graphitize, and it may be resin carbon Skin-core structure gene Rsc (= Rs/Rc) which denoted the skin-core degree of the carbon fibers was first brought forward and adopted. The Rsc value is between 0 and 1. When the Rsc value is equal to 1, the carbon fibers are homogenous. When the Rsc value is close to zero, there are serious skin-core structures in the carbon fibers. The Rsc value reduced linearly with the increase in heat-treatment temperature, indicating that the homogeneous degrees of the carbon fibers decreased and the skin-core degrees of the carbon fibers increased. The crystallite size of the carbon fibers surface and core increased gradually with the increase in heat-treatment temperature, but the surface's increased more quickly, indicating that the carbon of the carbon fibers surface was easier to graphitize than the carbon fibers core. Serious skin-core structure was one of the reasons that caused the reducing of the carbon fibers' tensile strength.
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OBJECTIVE: To investigate the dynamic changes of neurofilament contents in rat's spinal cord induced by 2, 5-hexanedione (2, 5-HD), and explore the molecular mechanism of n-hexane neuropathy. METHODS: Male Wistar rats were administered at a dosage of 400 mg/kg/day 2, 5-HD for 2, 4 and 8 weeks respectively. HD-induced neurological defects were detected and quantified using gait score, and the relative lev-els of NF-H, NF-M, and NF-L in spinal cords of rats were determined by Western Blotting. RESULTS: Exposure to 2, 5-HD produced progressive gait abnormalities, which suggested that the rat model of 2, 5-HD-induced neurotoxicity was established successfully. Western-Blotting results showed that NFs content in spinal cord demonstrated a progressive decline as the intoxication continued. In the supernatant fraction, compared to the controls, NF-H con-tent decreased by 15.7%, 57.0%, and 58.0% respectively after 2, 4, and 8-week treatment with 2, 5-HD (P < 0.01); accordingly, NF-M decreased by 36.0%, 61.3%, and 65.2% respectively (P < 0.01); NF-L decreased by 20.8%, 43.9%, and 44.3% respectively (P < 0.01). In the pellet fraction, the contents of NF-H in groups of 4 and 8 weeks' exposure to HD decreased by 35.6% and 43.2%, respectively (P < 0.01), and those of NF-L decreased by 26.4% and 42.1%, respectively (P < 0.01) when compared to the control. Further-more, NF-M contents in groups of 2, 4 and 8 weeks' exposure decreased by 23.3%, 33.9%, and 63.7% respectively (P < 0.01). The NFs level in spinal cords was highly correlated with gait abnormality of treated rats as the intoxication went on. Multiple correlation coefficients of NF-H, NF-M, and NF-L content with gait score of HD-treated rat were 0.8912, 0.9282 and 0.8981 (P < 0.01) respectively. CONCLUSION: The declines of NFs are high-ly related to neurobehavioral abnormality of 2, 5-HD-treated animals, and involved in the development of n-hexane neuropathy.
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Hexanonas/toxicidad , Proteínas de Neurofilamentos/metabolismo , Médula Espinal/metabolismo , Animales , Modelos Animales de Enfermedad , Marcha/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Médula Espinal/efectos de los fármacosRESUMEN
Protein kinase B (PKB/Akt) plays important roles in the regulation of lipid homeostasis, and impairment of Akt activity has been demonstrated to be involved in the development of non-alcoholic fatty liver disease (NAFLD). Previous studies suggest that cytochrome P4502E1 (CYP2E1) plays causal roles in the pathogenesis of alcoholic fatty liver (AFL). We hypothesized that Akt activity might be impaired due to CYP2E1-induced oxidative stress in chronic ethanol-induced hepatic steatosis. In this study, we found that chronic ethanol-induced hepatic steatosis was accompanied with reduced phosphorylation of Akt at Thr308 in mice liver. Chronic ethanol exposure had no effects on the protein levels of phosphatidylinositol 3 kinase (PI3K) and phosphatase and tensin homologue deleted on chromosome ten (PTEN), and led to a slight decrease of phosphoinositide-dependent protein kinase 1 (PDK-1) protein level. Ethanol exposure resulted in increased levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE)-Akt adducts, which was significantly inhibited by chlormethiazole (CMZ), an efficient CYP2E1 inhibitor. Interestingly, N-acetyl-L-cysteine (NAC) significantly attenuated chronic ethanol-induced hepatic fat accumulation and the decline of Akt phosphorylation at Thr308. In the in vitro studies, Akt phosphorylation was suppressed in CYP2E1-expressing HepG2 (CYP2E1-HepG2) cells compared with the negative control HepG2 (NC-HepG2) cells, and 4-HNE treatment led to significant decrease of Akt phosphorylation at Thr308 in wild type HepG2 cells. Lastly, pharmacological activation of Akt by insulin-like growth factor-1 (IGF-1) significantly alleviated chronic ethanol-induced fatty liver in mice. Collectively, these results indicate that CYP2E1-induced oxidative stress may be responsible for ethanol-induced suppression of Akt phosphorylation and pharmacological modulation of Akt in liver may be an effective strategy for the treatment of ethanol-induced fatty liver.
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Citocromo P-450 CYP2E1/metabolismo , Hígado Graso Alcohólico/metabolismo , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Enfermedad Crónica , Hígado Graso Alcohólico/patología , Células Hep G2 , Humanos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , FosforilaciónRESUMEN
OBJECTIVE: To prepare a neutralizing monoclonal antibody (McAb) against vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Extracellular immunoglobulin (Ig)-like domain III of KDR (KDR III) was expressed in E. coli and purified by affinity chromatograph. Monoclonal antibody against KDR III was prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [(3)H]-TdR incorporation assay were also used to detect the activity of anti-KDR McAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on VEGF-induced mitogenesis of human endothelial cells. RESULTS: McAb Ycom1D3 against KDR III was prepared which bound specifically to both the soluble KDR III and the cell-surface expressed KDR. It effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated activation of KDR expression on human endothelial cells. Furthermore, Ycom1D3 efficiently neutralized VEGF-induced mitogenesis of human umbilical vascular endothelial cells. CONCLUSION: McAb Ycom1D3 against KDR III may suppress the action of VEGF by blocking native vascular endothelial growth factor receptor KDR. It has potential clinical applications in the treatment of cancers and other diseases where pathological angiogenesis is involved.
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Anticuerpos Monoclonales/farmacología , Células Endoteliales/citología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Neovascularización Fisiológica , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: To investigate the dynamic alterations of neurofilament subunits (NF) in sciatic nerve of hens with organophosphorus ester induced the delayed neurotoxicity or neuropathy (OPIDN). METHODS: Hens with OPIDN were produced by giving 30 mg/kg methamidophos subcutaneously to the 10-month-old Roman hens daily for 15 days, and sacrificed after manifesting neurotoxic clinical signs on the 2nd, 10th, and 23rd day respectively. The sciatic nerves were dissected, homogenized and centrifuged. The levels of NF in supernatant and pellet of sciatic nerves were examined by Western blotting respectively at different time from 2 to 23 days. RESULTS: Integrated optional density (IOD) of high molecular weight neurofilament (NF-H) in sciatic nerve pellet of hens on the day 2, 10, 23 after appearance of OPIDN were 145,117 +/- 17,038, 55,917 +/- 17,333 and 45,038 +/- 6,662 respectively. As compared with the control group (78,875 +/- 22,569), the contents of NF-H in pellet were increased by 84% on day 2, and decreased by 29% and 43% on day 10 and 23 respectively. IOD of NF-H in supernatant of sciatic nerves were 4,709 +/- 1,739, 12,337 +/- 3,205 and 16,745 +/- 931, which were reduced significantly as compared with the control (44,083 +/- 6,895) at three different times. There was no significant difference in IOD of middle molecular weight neurofilament (NF-M) between control group (27,925 +/- 2,660) and on day 2 (31,493 +/- 4,625) in pellet. Those were 19,367 +/- 2,746 and 6,612 +/- 1,119 respectively on day 10 and day 23 in pellet of hen's sciatic nerve, which were much less than that in control. Little were detected in supernatant on day 10, and the IOD of NF-M were 3,196 +/- 269 and 5,206 +/- 1,292 on day 2 and day 23 respectively, which were lessened by 81% and 70% as compared with the control (17,243 +/- 3,232). In sciatic nerve pellet of hens, IOD of low molecular weight neurofilament (NF-L) on day 2 was 39,211 +/- 3,800, which was much higher than that in the control (28,749 +/- 9,319). There were no significant differences between IOD on day 10 (27,974 +/- 3,611), day 23 (21,507 +/- 2,286) and the control. There was no detection both on day 2 and 10 in supernatant of sciatic nerve, and IOD of NF-L were 5,962 +/- 1,929 on day 23, which were reduced significantly compared with the control (11,897 +/- 352). CONCLUSION: The alterations of NF in sciatic nerve might contribute to the occurrence and development of OPIDN.
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Proteínas de Neurofilamentos/metabolismo , Compuestos Organotiofosforados/toxicidad , Nervio Ciático/efectos de los fármacos , Animales , Pollos , Femenino , Insecticidas/toxicidad , Nervio Ciático/metabolismo , Nervio Ciático/patología , Pruebas de ToxicidadRESUMEN
OBJECTIVE: To investigate the dynamic changes of alpha-tubulin, beta-tubulin and beta-actin in sciatic nerve of hen with organophosphorus ester-induced delayed neuropathy (OPIDN). METHODS: OPIDN was induced in 10-month-old Roman hens by daily subcutaneous administration of 30 mg/kg methamidophos for 15 days. Hens were sacrificed 2, 10, and 23 days respectively after manifesting neuropathy. The sciatic nerves were dissected, homogenized and used for the determination of the alpha-tubulin, beta-tubulin and beta-actin levels by western blotting. RESULTS: The levels of alpha-tubulin in supernatant of sciatic nerves were decreased by 6%, 15% and 25% respectively on day 2, 10 and 23 respectively, while those in pellet remained almost unchanged. beta-tubulin were decreased by 27%, 6%, 19% in pellet and 1%, 21%, 22% in supernatant of sciatic nerves on 2, 10 and 23 days. Beta-actin level in pellet of sciatic nerve increased by 24%, 48% and 17% on day 2, 10 and 23, and little changes were observed in supernatant. CONCLUSION: Methamidophos may induced changes of alpha-tubulin, beta-tubulin and beta-actin levels in sciatic nerve of hen, which may be one of the mechanism of the contribution to the occurrence and development of OPIDN.
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Insecticidas/toxicidad , Compuestos Organotiofosforados/toxicidad , Nervio Ciático/metabolismo , Actinas/metabolismo , Animales , Pollos , Femenino , Nervio Ciático/efectos de los fármacos , Tubulina (Proteína)/metabolismoRESUMEN
Tri-ortho-cresyl phosphate (TOCP) is an organophosphorus ester. It is capable of producing organophosphorus ester induced delayed neurotoxicity (OPIDN) in human being and sensitive animals, which is characterized by ataxia that progresses to paralysis after 1-3 weeks following exposure to some organophosphorus ester. In present study, 18 adult hens were divided randomly into three groups, i.e. two experimental groups and control group (n = 6 each group). All hens were 10 months old and weighted 1.5-2.0 kg. The hens in two experimental groups were treated with TOCP by gavage at single dosages of 375 and 750 mg/kg respectively. TOCP was dissolved in corn oil and administered at 0.65 ml/kg. Six control hens received an equivalent volume of corn oil by gavage. All hens were sacrificed after 21 days of treatment and the sciatic nerves were dissected, homogenized and used for the determination of cytoskeletal proteins by western blotting. The levels of neurofilament (NF) subunits were decreased both in supernatant and pellet of sciatic nerves, and the most noticeable decrease in levels of NF subunits protein was observed in neurofilament medium (NF-M). Compared to the control hens, neurofilament heavy (NF-H) level decreased by 36 and 38% (P < 0.01) in the pellet and by 27 and 26% (P < 0.05) in the supernatant of sciatic nerves of hens treated with 375 and 750 mg/kg TOCP respectively. The reduction of NF-M were 36 and 68% (P < 0.01) in pellet, 50 and 67% (P < 0.01) in supernatant at 375 and 750 mg/kg dosage respectively. The neurofilament light (NF-L) lessened slightly, but the relative percentage of integrated optical density (IOD) was no significant alteration when compared to the control hens. There were significant decreases in levels of alpha-tubulin, beta-tubulin in pellet and alpha-tubulin, beta-tubulin, beta-actin in supernatant of sciatic nerves in TOCP-treated hens. Thus, the decreases of cytoskeletal proteins suggested the possible involvement of them in delayed neurotoxicity.
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Proteínas del Citoesqueleto/efectos de los fármacos , Plastificantes/toxicidad , Nervio Ciático/efectos de los fármacos , Tritolilfosfatos/toxicidad , Actinas/metabolismo , Administración Oral , Animales , Biomarcadores/análisis , Pollos , Proteínas del Citoesqueleto/metabolismo , Femenino , Proteínas de Neurofilamentos/efectos de los fármacos , Proteínas de Neurofilamentos/metabolismo , Plastificantes/administración & dosificación , Nervio Ciático/metabolismo , Tritolilfosfatos/administración & dosificación , Tubulina (Proteína)/metabolismoRESUMEN
Although the anticancer effects of garlic and its products have been demonstrated by a variety of studies; however, few studies were conducted to investigate the effects of garlic on the adverse effects of chemo/radiotherapy. In order to clarify the above question and make a more comprehensive understanding of the anticancer effects of garlic, tumor xenograft mice model was established by subcutaneous injection of H22 tumor cells, and was used for the investigation of effects of garlic oil (GO) on the chemo/radiotherapy. In the chemotherapy test, tumor-bearing mice were treated with cyclophosphamide (CTX) or CTX plus GO (25 or 50 mg/kg bw) for 14 d, while the mice received a single 5 Gy total body radiation or radiation plus GO (25 or 50 mg/kg bw) in radiotherapy test. The results showed that GO did not increase the tumor inhibitory rate of CTX/radiation, which indicated that GO could not enhance the chemo/radiosensitivity of cancer cells. However, the decrease of the peripheral total white blood cells (WBCs) count induced by CTX/radiation was significantly suppressed by GO cotreatment. Furthermore, GO cotreatment significantly inhibited the decrease of the DNA contents and the micronuclei ratio of the bone marrow. Lastly, the reduction of the endogenous spleen colonies induced by CTX/radiation was significantly suppressed by GO cotreatment. These findings support the idea that GO consumption may benefit for the cancer patients receiving chemotherapy or radiotherapy.
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Compuestos Alílicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Ajo/química , Enfermedades Hematológicas/tratamiento farmacológico , Aceites de Plantas/farmacología , Sulfuros/farmacología , Animales , Antineoplásicos , Línea Celular Tumoral , Ensayo de Unidades Formadoras de Colonias , Ciclofosfamida/efectos adversos , Modelos Animales de Enfermedad , Enfermedades Hematológicas/etiología , Masculino , Ratones , Pruebas de Micronúcleos , Neoplasias/tratamiento farmacológico , Neoplasias/radioterapia , Bazo/citología , Bazo/metabolismoRESUMEN
To investigate the protective effects and the possible mechanisms of garlic oil (GO) against N-nitrosodiethylamine (NDEA)-induced hepatocarcinoma in rats, Wistar rats were gavaged with GO (20 or 40 mg/kg) for 1 week, and then were gavaged with GO and NDEA (10 mg/kg) for the next 20 weeks. The changes of morphology, histology, the biochemical indices of serum, and DNA oxidative damage of liver were examined to assess the protective effects. Lipid peroxidation (LPO), antioxidant defense system, and apoptosis-related proteins were measured to investigate potential mechanisms. At the end of the study (21 weeks), GO administration significantly inhibited the increase of the nodule incidence and average nodule number per nodule-bearing liver induced by NDEA, improved hepatocellular architecture, and dramatically inhibited NDEA-induced elevation of serum biochemical indices (alanine aminotransferase , aspartate aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase) and hepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in a dose-dependent manner. The mechanistic studies demonstrated that GO counteracted NDEA-induced oxidative stress in rats illustrated by the restoration of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST) levels, and the reduction of the malondialdehyde (MDA) levels in liver. Furthermore, the mRNA and protein levels of Bcl-2, Bcl-xl, andß-arrestin-2 were significantly decreased whereas those of Bax and caspase-3 were significantly increased. These data suggest that GO exhibited significant protection against NDEA-induced hepatocarcinogenesis, which might be related with the enhancement of the antioxidant activity and the induction of apoptosis.
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Compuestos Alílicos/uso terapéutico , Anticarcinógenos/uso terapéutico , Antioxidantes/uso terapéutico , Neoplasias Hepáticas Experimentales/prevención & control , Sulfuros/uso terapéutico , 8-Hidroxi-2'-Desoxicoguanosina , Compuestos Alílicos/farmacología , Animales , Anticarcinógenos/farmacología , Antioxidantes/farmacología , Proteínas Reguladoras de la Apoptosis/análisis , Biomarcadores , Peso Corporal/efectos de los fármacos , Aceite de Maíz/farmacología , Daño del ADN/efectos de los fármacos , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análisis , Dietilnitrosamina/toxicidad , Ensayos de Selección de Medicamentos Antitumorales , Perfilación de la Expresión Génica , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Pruebas de Función Hepática , Neoplasias Hepáticas Experimentales/inducido químicamente , Masculino , Tamaño de los Órganos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Oxidorreductasas/análisis , Distribución Aleatoria , Ratas , Ratas Wistar , Sulfuros/farmacologíaRESUMEN
Accumulating evidences support the important roles of sterol regulatory element-binding protein-1 (SREBP-1) activation in ethanol-induced fatty liver, but the underlying mechanisms for its activation are not fully understood. Recent studies have demonstrated that phosphatidylinositol 3 kinase (PI3K)/Akt pathway activation could enhance SREBP-1 activity. The current study was designed to investigate the potential roles of PI3K/Akt pathway in acute ethanol-induced fatty liver in mice. In the first experiment, mice were treated with ethanol (2.5 or 5 g/kg bw) or isocaloric/isovolumetric maltose-dextrin solution, and sacrificed at several time points after ethanol exposure. As expected, ethanol dose-dependently increased the hepatic triglyceride (TG) levels and the protein levels of the mature form of SREBP-1 (n-SREBP-1). The phosphorylation of Akt and glycogen synthase kinase-3ß (GSK-3ß) was significantly increased in mice treated with ethanol (5 g/kg bw), while the protein levels of PI3K-p85 were significantly reduced. To confirm the roles of PI3K/Akt pathway, mice were then pretreated with wortmannin (0.7 or 1.4 mg/kg bw), a specific PI3K/Akt pathway inhibitor, before exposure to ethanol. Interestingly, a dual effect of wortmannin was observed. Low dose of wortmannin significantly reduced the hepatic TG levels, while high dose of wortmannin aggravated ethanol-induced fatty liver. The ratio of LC3II/LC3I of wortmannin (1.4 mg/kg bw) group mice was significantly increased, while the p62 protein level was significantly decreased compared to those of ethanol group, which indicated that wortmannin (1.4 mg/kg bw) might suppress the lipid degradation by autophagy. These results supported the hypothesis that PI3K/Akt activation might be involved in acute ethanol-induced fatty liver, and PI3K/Akt inhibitors might have therapeutic potential for the treatment of ethanol-induced fatty liver.
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Hígado Graso Alcohólico/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Androstadienos/farmacología , Animales , Etanol , Hígado Graso Alcohólico/patología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/metabolismo , WortmaninaRESUMEN
The quaterisation process of 1,2-dibromoethane and pyridine is in situ traced by electronic absorption spectrum. Two absorption peaks, induced by mono- and bis-pyridinium salt of 1,2-dibromoethane, appear at 429 nm and 313 nm, respectively. To explain the phenomena, several kinds of alkyl bromides with special structures were selected and compared by experimental measurement and theoretical calculation. The results indicate that for mono-pyridinium salt of 1,2-dibromoethane, the electron donor property of ortho-bromine group increases the electron cloud density of the carbon atom associated with pyridinium cation, which induces red-shift of absorption wavelength.
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Electrones , Dibromuro de Etileno/química , Compuestos de Piridinio/química , Absorción , Bromuros/química , Modelos Químicos , Reproducibilidad de los Resultados , Análisis Espectral , Electricidad Estática , TermodinámicaRESUMEN
AIM: To construct and express a single chain antibody (scFv) against human vascular endothelial growth factor (VEGF) receptor KDR and characterize its biological activity. METHODS: The restriction enzyme sites were added to the previously cloned V(H) and V(L) genes of mAb Ycom1D3 against KDR by PCR. The anti-KDR scFv gene was constructed by the splicing overlap extensive (SOE) PCR and then inserted into fusion expression vector pAYZH. The recombinant protein was expressed in E.coli 16C9 and purified with His-tag affinity chromatography. The specificity of the purified scFv was examined by ELISA and FACS. RESULTS: DNA sequencing indicated that the cloned scFv gene consisted of 729 bp, encoding 243 amino acids. After induction in low-phosphate medium of AP5, a new protein band with relative molecular mass (M(r)) of 30 000 appeared on gel of SDS-PAGE and on nitrocellulose membrane of Western blot, which was consistent with the theoretically predicted value. Anti-KDR scFv was expressed in the form of inclusion body, which accounted for 20% of total bacterial protein. ELISA and competitive immunofluorescence binding test showed that the anti-KDR scFv had the same binding activity as mAb Ycom1D3 and that it could block VEGF/KDR interaction effectively. CONCLUSION: Recombinant anti-KDR scFv gene has been successfully constructed and expressed in E.coli 16C9, which lays the foundation for its diagnostic and therapeutic application.
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Anticuerpos/genética , Anticuerpos/inmunología , Células Procariotas/inmunología , Células Procariotas/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Animales , Anticuerpos/aislamiento & purificación , Secuencia de Bases , Unión Competitiva , Escherichia coli/genética , Técnica del Anticuerpo Fluorescente , Expresión Génica , Vectores Genéticos/genética , Humanos , Proteínas Inmovilizadas/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Solubilidad , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
RT-PCR was used to clone DNA fragment of the extracellular domain of 4-1BBL from human THP-1 cells (human monocyte), and the expression vector pAYZ4-1BBL was constructed by cloning the extracellular domain of 4-1BBL into the expression vector pAYZ. The extracellular domain of 4-1BBL was expressed in E. coli 16C9 and purified by affinity chromatography. SDS-PAGE and Western blot analysis showed that the relativae molecular weight of soluble 4-1BBL is 22kD which was consistent with the theoretically predicted value. So far as we know, it is the first time that the soluble expression of 4-1BBL in E. coli was achieved 4-1BBL induced a significant release of IL-2 in stimulated Jurkat cells after 48h incubation, especially in the presence of tumor cell. At the same time the apoptosis level of Jurkat cell reduce more than 50%. In conclusion, 4-1BBL may be useful in cancer immunotherapy.