RESUMO
Antibody display methods are increasingly being used to produce human monoclonal antibodies for disease therapy. Rapid screening and isolation of specific human antibody genes are valuable for producing human monoclonal antibodies showing high specificity and affinity. In this report, we describe a novel mammalian cell display method in which whole human IgG is displayed on the cell surface of CHO cells. Cells expressing antigen-specific human monoclonal IgGs with high affinity on the cell surface after normal folding and posttranscriptional modification were screened using a cell sorter. The membrane-type IgG-expressing CHO cells were then converted to IgG-secreting cells by transfection with a plasmid coding Cre recombinase. This mammalian cell display method was applied to in vitro affinity maturation of monoclonal C9 IgG specific to the human high-affinity IgE receptor (FcεRIα). The CDR3 of the C9 heavy chain variable region gene was randomly mutated and inserted into pcDNA5FRT/IgG. A C9 IgG (CDRH3r)-expressing CHO cell display library consisting of 1.1×10(6) independent clones was constructed. IgG-displaying cells showing high reactivity to FcεRIα antigen were screened by the cell sorter, resulting in the establishment of a CHO cell line producing with higher reactivity than the parent C9 IgG.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Técnicas de Visualização da Superfície Celular/métodos , Sequência de Aminoácidos , Animais , Células Produtoras de Anticorpos/metabolismo , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Conversão Gênica , Humanos , Imunoglobulina G/metabolismo , Dados de Sequência Molecular , Biblioteca de Peptídeos , Receptores de IgE/química , Receptores de IgE/metabolismo , Recombinação Genética/genética , TransgenesRESUMO
Electrolyzed reduced water (ERW) contains a large amount of molecular hydrogen and a small amount of Pt nanoparticles (Pt NPs). We have found that ERW significantly extended the lifespan of Caenorhabditis elegans in a novel culture medium designated Water Medium. In this study, we found that synthetic Pt NPs at ppb levels significantly extended the nematode lifespan and scavenged reactive oxygen species (ROS) in the nematode induced by paraquat treatment. In contrast, a high concentration of dissolved molecular hydrogen had no significant effect on the lifespan of the nematode. These findings suggest that the Pt NPs in ERW, rather than the molecular hydrogen, extend the longevity of the nematode, at least partly by scavenging ROS.
Assuntos
Caenorhabditis elegans/metabolismo , Meios de Cultura/química , Viabilidade Microbiana , Nanopartículas/química , Platina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Água/metabolismo , Animais , Fenômenos Fisiológicos Bacterianos , Caenorhabditis elegans/efeitos dos fármacos , Eletrólise , Hidrogênio/análise , Nanopartículas/administração & dosagem , Estresse Oxidativo , Platina/administração & dosagem , Platina/química , Água/administração & dosagem , Água/químicaRESUMO
AIM: In pregnant women with epilepsy, it is essential to balance maternal safety and the potential teratogenicity of anticonvulsants. Recently, growing evidence has indicated that valproic acid (VPA) can produce postnatal congenital malformations and impair cognitive function. However, the mechanisms underlying cognitive dysfunction in long-term prognoses remain unclear. METHODS: Pregnant Wistar rats received daily intraperitoneal injections of VPA (200 mg/kg/day) from embryonic day 12.5 until birth. On postnatal day (PD) 149, the rats received an injection of bromodeoxyuridine (BrdU). On PD 150, the rats were subjected to the open field (OF), elevated plus-maze (EPM), and Y-maze tests. After behavioral testing, perfusion fixation was performed and the brain was dissected for immunohistochemistry. RESULTS: A significant marked decrease was seen in the number of BrdU-positive cells in the dentate gyrus of offspring of VPA-treated dams compared to those of control. However, no significant differences in hyperactivity were found based on the results of the OF test among the offspring on PD 150 of 200 VPA-treated dams. In addition, no significant differences were seen in the EPM test. CONCLUSION: The behavioral abnormality observed in young offspring of VPA-treated dams was not significantly different from that of controls in adult offspring on PD 150. However, compared with controls, the number of BrdU-positive cells in VPA-treated rats was halved. The findings suggest that the behavioral abnormality seems to improve as they grow, even if some structural abnormalities may remain in the central nervous system.
Assuntos
Efeitos Tardios da Exposição Pré-Natal , Ácido Valproico , Animais , Feminino , Hipocampo , Humanos , Neurogênese , Gravidez , Prognóstico , Ratos , Ratos Wistar , Ácido Valproico/toxicidadeRESUMO
Electrolyzed reduced water (ERW) has attracted much attention because of its therapeutic effects. In the present study, a new culture medium, which we designated Water medium, was developed to elucidate the effects of ERW on the lifespan of Caenorhabditis elegans. Wild-type C. elegans had a significantly shorter lifespan in Water medium than in conventional S medium. However, worms cultured in ERW-Water medium exhibited a significantly extended lifespan (from 11% to 41%) compared with worms cultured in ultrapure water-Water medium. There was no difference between the lifespans of worms cultured in ERW-S medium and ultrapure water-S medium. Nematodes cultured in ultrapure water-Water medium showed significantly higher levels of reactive oxygen species than those cultured in ultrapure water-S medium. Moreover, ERW-Water medium significantly reduced the ROS accumulation induced in the worms by paraquat, suggesting that ERW-Water medium extends the longevity of nematodes at least partly by scavenging ROS.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , Eletrólise , Longevidade/efeitos dos fármacos , Água/química , Água/farmacologia , Animais , Caenorhabditis elegans/metabolismo , Meios de Cultura/química , Meios de Cultura/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
In pregnant women with epilepsy, it is imperative to balance the safety of the mother and the potential teratogenicity of anticonvulsants, which could cause impairments such as intellectual disability and cleft lip. In this study, we examined behavioral and hippocampal neurogenesis alterations in male offspring of rats exposed to valproic acid (VPA) during pregnancy. Pregnant Wistar rats received daily intraperitoneal injections of VPA (100â¯mg/kg/day or 200â¯mg/kg/day) from embryonic day 12.5 until birth. At postnatal day 29, animals received an injection of bromodeoxyuridine (BrdU). At postnatal day 30, animals underwent the open field (OF), elevated plus-maze, and Y-maze tests. After behavioral testing, animals were decapitated, and their brains were dissected for immunohistochemistry. Of the offspring of the VPA200 mothers, 66.6% showed a malformation. In the OF test, these animals showed locomotor hyperactivity. In the elevated plus-maze, offspring of VPA-treated mothers spent significantly more time in the open arms, irrespective of the treatment dose. The number of BrdU-positive cells in the dentate gyrus of the offspring of VPA-treated mothers increased significantly in a dose-dependent manner compared with the control. A significant positive correlation between spontaneous locomotor activity in the OF and BrdU-positive cell counts was observed across groups. In conclusion, VPA administration during pregnancy results in malformations and attention-deficit/hyperactivity disorder-like behavioral abnormalities in the offspring. An increase in cell proliferation in the hippocampus may underlie the behavioral changes observed. Repeated use of high doses of VPA during pregnancy may increase the risk of neurodevelopmental abnormalities dose dependently and should be carefully considered.
Assuntos
Giro Denteado/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Ácido Valproico/efeitos adversos , Animais , Transtorno do Deficit de Atenção com Hiperatividade/etiologia , Transtorno do Espectro Autista/etiologia , Comportamento Animal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Hipocampo/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Ratos , Ratos Wistar , Ácido Valproico/metabolismo , Ácido Valproico/farmacologiaRESUMO
The blood-brain barrier (BBB) plays important roles in both the physiological and pharmacological state of the brain. Transiently enhancing the permeability of the BBB may allow use of more types of medications for neuropsychiatric diseases. Several studies have demonstrated that seizures cause a transient decrease in BBB integrity. We studied the timing of BBB changes following seizures and the role of astrocytes in this process. Rats received 10 applications of electroconvulsive stimulation (ECS). They were then infused with sodium fluorescein, a fluorescent substance that rarely passes the BBB, via the inferior vena cava. After 120min of circulation, the amount of sodium fluorescein in the brain was measured by two methods in vivo fluorescence imaging (total radiant efficiency) and the brain concentration of sodium fluorescein. To assess any changes to the BBB, we measured S100Β in serum, which is a standard marker of BBB breakdown that is expressed by astrocytes. We also examined ultrastructural changes following ECS. Total radiant efficiency and the brain concentration of sodium fluorescein were significantly increased in treated rats compared to controls when sodium fluorescein was injected immediately after ECS but not when the injection was performed more than 15 min after ECS. Astrocytic endfeet showed swelling around brain capillaries following ECS. In conclusion, ECS transiently enhances the permeability of the BBB, which may be accompanied by changes in astrocytic endfeet.
Assuntos
Astrócitos/fisiologia , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar/fisiologia , Eletrochoque , Animais , Astrócitos/ultraestrutura , Barreira Hematoencefálica/ultraestrutura , Feminino , Fluoresceína/farmacocinética , Corantes Fluorescentes/farmacocinética , Microscopia Eletrônica , Atividade Motora , Imagem Óptica , Distribuição Aleatória , Ratos Wistar , Subunidade beta da Proteína Ligante de Cálcio S100/sangue , Convulsões/metabolismo , Convulsões/patologia , Espectrometria de FluorescênciaRESUMO
Aging-related neurodegenerative disorders are closely associated with mitochondrial dysfunction and oxidative stresses and their incidence tends to increase with aging. Brain is the most vulnerable to reactive species generated by a higher rate of oxygen consumption and glucose utilization compared to other organs. Electrochemically reduced water (ERW) was demonstrated to scavenge reactive oxygen species (ROS) in several cell types. In the present study, the protective effect of ERW against hydrogen peroxide (H2O2) and nitric oxide (NO) was investigated in several rodent neuronal cell lines and primary cells. ERW was found to significantly suppress H2O2 (50-200 µM) induced PC12 and SFME cell deaths. ERW scavenged intracellular ROS and exhibited a protective effect against neuronal network damage caused by 200 µM H2O2 in N1E-115 cells. ERW significantly suppressed NO-induced cytotoxicity in PC12 cells despite the fact that it did not have the ability to scavenge intracellular NO. ERW significantly suppressed both glutamate induced Ca(2+) influx and the resulting cytotoxicity in primary cells. These results collectively demonstrated for the first time that ERW protects several types of neuronal cells by scavenging ROS because of the presence of hydrogen and platinum nanoparticles dissolved in ERW.
Assuntos
Doenças Neurodegenerativas/prevenção & controle , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Purificação da Água/métodos , Água/administração & dosagem , Animais , Linhagem Celular Tumoral , Eletroquímica/métodos , Peróxido de Hidrogênio/antagonistas & inibidores , Camundongos , Neurônios/metabolismo , Óxido Nítrico/antagonistas & inibidores , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Água/químicaRESUMO
Insulin-producing cells express limited activities of anti-oxidative enzymes. Therefore, reactive oxygen species (ROS) produced in these cells play a crucial role in cytotoxic effects. Furthermore, diabetes mellitus (DM) development is closely linked to higher ROS levels in insulin-producing cells. Hita Tenryosui Water(®) (Hita T. W., Hita, Japan) and Nordenau water (Nord. W., Nordenau, Germany), referred to as natural reduced waters (NRWs), scavenge ROS in cultured cells, and therefore, might be a possibility as an alternative to conventional pharmacological agents against DM. Therefore, this study aimed to investigate the role of NRWs in alloxan (ALX)-induced ß-cell apoptosis as well as in ALX-induced diabetic mice. NRWs equally suppressed DNA fragmentation levels. Hita T. W. and Nord. W. ameliorated ALX-induced sub-G(1) phase production from approximately 40% of control levels to 8.5 and 11.8%, respectively. NRWs restored serum insulin levels (p < 0.01) and reduced blood glucose levels (p < 0.01) in ALX-induced mice. Hita T. W. restored tissue superoxide dismutase (SOD) (p < 0.05) activity but not tissue catalase activity. Hita T. W. did not elevate SOD or catalase activity in HIT-T15 cells. Nord. W. restored SOD (p < 0.05) and catalase (p < 0.05) activity in both cultured cells and pancreatic tissue to normal levels. Even though variable efficacies were observed between Hita T. W. and Nord. W., both waters suppressed ALX-induced DM development in CD-1 male mice by administering NRWs for 8 weeks. Our results suggest that Hita T. W. and Nord. W. protect against ALX-induced ß-cell apoptosis, and prevent the development of ALX-induced DM in experimental animals by regulating ALX-derived ROS generation and elevating anti-oxidative enzymes. Therefore, the two NRWs tested here are promising candidates for the prevention of DM development.
RESUMO
It has been demonstrated that hydrogen peroxide (H(2)O(2)) is directly associated with elevated matrix metalloproteinase-2 (MMP-2) expression in several cell lines. Electrochemically reduced water (ERW), produced near the cathode during electrolysis, and scavenges intracellular H(2)O(2) in human fibrosarcoma HT1080 cells. RT-PCR and zymography analyses revealed that when HT1080 cells were treated with ERW, the gene expression of MMP-2 and membrane type 1 MMP and activation of MMP-2 was repressed, resulting in decreased invasion of the cells into matrigel. ERW also inhibited H(2)O(2)-induced MMP-2 upregulation. To investigate signal transduction involved in MMP-2 downregulation, mitogen-activated protein kinase (MAPK)-specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (MAPK/extracellular regulated kinase kinase 1 inhibitor) and c-Jun NH(2)-terminal kinase inhibitor II, were used to block the MAPK signal cascade. MMP-2 gene expression was only inhibited by SB203580 treatment, suggesting a pivotal role of p38 MAPK in regulation of MMP-2 gene expression. Western blot analysis showed that ERW downregulated the phosphorylation of p38 both in H(2)O(2)-treated and untreated HT1080 cells. These results indicate that the inhibitory effect of ERW on tumor invasion is due to, at least in part, its antioxidative effect.
RESUMO
Electrolyzed reduced water, which is capable of scavenging reactive oxygen species, is attracting recent attention because it has shown improved efficacy against several types of diseases including diabetes mellitus. Alloxan produces reactive oxygen species and causes type 1 diabetes mellitus in experimental animals by irreversible oxidative damage to insulin-producing ß-cells. Here, we showed that electrolyzed reduced water prevented alloxan-induced DNA fragmentation and the production of cells in sub-G1 phase in HIT-T15 pancreatic ß-cells. Blood glucose levels in alloxan-induced type 1 diabetes model mice were also significantly suppressed by feeding the mice with electrolyzed reduced water. These results suggest that electrolyzed reduced water can prevent apoptosis of pancreatic ß-cells and the development of symptoms in type 1 diabetes model mice by alleviating the alloxan-derived generation of reactive oxygen species.