RESUMEN
We developed a highly sensitive assay for detecting protein-protein interaction using chimeric receptors comprising two molecules of interest in the extracellular domain and interferon alpha and beta receptor subunit 1 or 2 (IFNAR1/2) in the intracellular domain. This intracellular IFNAR1/2 reconstitution system (IFNARRS) proved markedly more sensitive than the NanoBiT system, currently considered one of the best detection systems for protein interaction. Employing chimeric receptors with extracellular domains from the IFNγ or IL-2 receptor and the intracellular domains of IFNAR1/2, the IFNARRS system effectively identifies low IFNγ or IL-2 levels. Cells stably expressing these chimeric receptors responded to IFNγ secreted by activated T cells following various stimuli, including a specific peptide-antigen. The activation signals were further enhanced by the expression of relevant genes, such as costimulators, via IFN-stimulated response elements in the promoters. Besides IFNγ or IL-2, the IFNARRS system demonstrated the capability to detect other cytokines by using the corresponding extracellular domains from these target cytokine receptors.
Asunto(s)
Interferón gamma , Interleucina-2 , Receptor de Interferón alfa y beta , Linfocitos T , Humanos , Receptor de Interferón alfa y beta/metabolismo , Receptor de Interferón alfa y beta/genética , Linfocitos T/metabolismo , Linfocitos T/inmunología , Interleucina-2/metabolismo , Interferón gamma/metabolismo , Receptores de Interleucina-2/metabolismo , Receptores de Interleucina-2/genética , Unión Proteica , Activación de Linfocitos , Células HEK293RESUMEN
Coronavirus disease 2019 (COVID-19) has become a serious public health problem worldwide. In general, healthcare workers are considered to be at higher risk of COVID-19 infection. However, the prevalence of COVID-19 among healthcare workers in Japan is not well characterized. In this study, we aimed to examine the seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies among 2160 healthcare workers in hospitals and clinics that are not designated to treat COVID-19 patients in Japan. The prevalence of SARS-CoV-2 immunoglobulin G was 1.2% in August and October 2020 (during and after the second wave of the pandemic in Japan), which is relatively higher than that in the general population in Japan (0.03-0.91%). Because of the higher risk of COVID-19 infection, healthcare workers should be the top priority for further social support and vaccination against SARS-CoV-2.
Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos Antivirales , Personal de Salud , Hospitales Generales , Humanos , Japón/epidemiología , Estudios SeroepidemiológicosRESUMEN
The extrusion of protons is considered a very general parameter of the activation of many kinds of membrane or intracellular molecules, such as receptors, ion channels, and enzymes. We found that pepstatin A caused a reproducible, concentration-related increase in the extracellular acidification rate in two microglial cell lines, Ra2 and 6-3. Washing abolished pepstatin A-induced acidification immediately. However, pepstatin A did not cause the extracellular acidification in other cell types, such as CHO, C6 glioma, and NIH3T3 cells. These observations strongly suggest that pepstatin A interacts with certain membrane proteins specific to both Ra2 and 6-3 cells from outside. N-methylmaleimide and N,N'-dicyclohexylcarbodiimide, inhibitors of H(+)-ATPase, were found to reduce pepstatin A-induced response strongly, while bafilomycin A1, a vacuolar H(+)-ATPase inhibitor, vanadate, a P-type H(+)-ATPase inhibitor, and NaN3, an F1 ATPase inhibitor, virtually did not. 5-(N-ethyl-N-isopropyl) amiloride, an inhibitor of Na(+)/H(+) exchanger isoform 1, greatly enhanced pepstatin-induced response, while amiloride did not. Zn(2+), a voltage-dependent proton channel blocker, did not affect pepstatin-induced response neither. Staurosporine, a nonspecific inhibitor of protein kinase C, inhibited pepstatin A-induced response, while chelerythrine, more selective inhibitor of protein kinase C, greatly enhanced it. H-7 and H-8 did not affected the response. These findings suggest that pepstatin A induces extracellular acidification in microglia cell lines, Ra2 and 6-3, through an N-methylmaleimide- and N,N'-dicyclohexylcarbodiimide-sensitive, but bafilomycin A1-insensitive, ATPase, which seems to be distinct from protein kinase C-dependent process.
Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Espacio Extracelular/efectos de los fármacos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Microglía/efectos de los fármacos , Pepstatinas/farmacología , Inhibidores de Proteasas/farmacología , Animales , Células Cultivadas , Cricetinae , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Espacio Extracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Microglía/enzimología , RatasRESUMEN
Prolonged dietary restriction has been reported to suppress age-induced phenomena. In order to investigate how prolonged caloric restriction reduces age-related deterioration of hippocampal synaptic transmission, we compared the levels of major hippocampal polyunsaturated fatty acids, arachidonic acid and docosahexaenoic acid between 4- and 26-month-old rats. The Ca(2+) responses upon perfusion of NMDA or 30 mM K(+) between 4- and 26-month-old rats with prolonged dietary restriction were also compared using the fluorescent probe Fura-2. A decrease in membrane arachidonic acid is thought to be a major causal factor in the age-related impairment of long-term potentiation. Long-term caloric restriction seems to increase arachidonic acid levels regardless of age. However, there is no significant difference of hippocampal arachidonic acid levels between in freely feeding 4- and 26-month-old rats. Similar results were obtained from the measurement of hippocampal docosahexaenoic acid levels. Under caloric restriction, the 500 microM N-methyl-D-aspartate-induced Ca(2+) response was greatly reduced by aging, while the 30 mM K(+)-induced Ca(2+) response was not affected. In our preliminary data, the amplitude of the population spike after tetanic stimulation did not differ between 4- and 26-month-old rats under caloric restriction, while 50 microM of 2-amino-5-phosphonovaleric acid, a N-methyl-D-aspartate antagonist, markedly inhibited a potentiation of the population spike in 4-month-old rats, but with negligible inhibition in 26-month-old rats. From these results, an age-related impairment of hippocampal excitatory synaptic transmission may not be solely due to the reduction of membrane arachidonic acid. Caloric restriction might prevent age-related reduction in hippocampal synaptic transmission by enhancing non-N-methyl-D-aspartate mechanisms.