Search

The epigenetic regulatory system significant influences the fate determination of cells during developmental processes. Prdm12 is a transcriptional regulator that modulates gene expression epigenetically. The Prdm12 gene has been shown to be expressed in neural tissues, specifically during development, but its detailed function is not fully understood. This study investigated the function of the Prdm12 gene in P19 mouse embryonic tumor cells as a model for neural differentiation. A decrease in the expression of neuron-specific genes and the alterations of dendrites and axons morphology was confirmed in Prdm12-knockout P19 cells. In addition, almost no astrocytes were generated in Prdm12-knockout P19 cells. Comprehensive gene expression analysis revealed that there was a reduction in the expression of the inhibitory neuron-specific genes Gad1/2 and Glyt2, but not the excitatory neuron-specific gene VGLUT2, in Prdm12-knockout P19 cells. Furthermore, the expression of inhibitory neuron-related factors, Ptf1a, Dbx1, and Gsx1/2, decreased in Prdm12-knockout P19 cells. Gene expression analysis also revealed that the Ptf1a, Hic1, and Foxa1 genes were candidate targets of Prdm12 during neurogenesis. These results suggest that Prdm12 regulates the differentiation of inhibitory neurons and astrocytes by controlling the expression of these genes during the neural differentiation of P19 cells.

Although antibodies have attracted attention as next-generation biopharmaceuticals, the costs of purifying the products and of arranging the environment for cell cultivation are high. Therefore, there is a need to increase antibody efficacy and improve product quality as much as possible. Since antibodies are glycoproteins, their glycan structures have been found to affect the function of antibodies. Especially, afucosylation of the N-linked glycan in the Fc region is known to significantly increase antibody-dependent cellular cytotoxicity. In this study, we established a double-mutant ΔGMDΔGFT in which GDP-mannose 4,6-dehydratase and GDP-fucose transporter were knocked out in Chinese hamster ovary cells, a platform for biopharmaceutical protein production. By adapting ΔGMDΔGFT cells to serum-free medium and constructing suspension-cultured cells, we established host CHO cells with no detected fucosylated glycans and succeeded in production of afucosylated antibodies. We also demonstrated that, in culture in the presence of serum, fucosylation occurs due to contamination from serum components. Furthermore, we found that afucosylation of glycans does not affect cell growth after adaptation to serum-free medium as compared to wild-type CHO cells growth and does not significantly affect the expression levels of other endogenous fucose metabolism-related enzyme genes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00501-3.

Dog is the first animal that was established as a close partner of human beings. Based on the vast genetic diversity and breeding, dogs exhibit unique genetic evolution and diversity from Chihuahua to St. Bernard. The safety tests of the pharmacological products also included domestic dogs as the test subjects. Although the safety confirmation test of chemicals for human use is important, the welfare of experimental animals requires special consideration. In this study, we cultured domestic dog-derived primary fibroblasts isolated from their muscle tissues. Furthermore, we successfully immortalized them through lentivirus-mediated gene transfer of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT). We further demonstrated that the established immortalized domestic dog-derived fibroblasts retained the characteristics of the original parental cells. These cells might act a suitable in vivo model system to replace the implication of animals for evaluating the potential toxicity of pharmacological chemicals. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00504-0.

Perilla frutescens (L.) Britton var. frutescens (egoma in Japan) is a traditional oilseed that has several varieties with different photoperiod responses. Although egoma pomace, industrial waste produced during oil extraction, is a rich source of macro- and micro-nutrients such as protein, fiber, minerals, and polyphenols, it has not yet been used for purposes other than livestock feeding. To find out a better use of perilla pomace and its function, we selected four varieties of egoma originating from different regions with different photoperiod responses: two varieties were from Japan, which are broadly cultivated for oilseed and are highly sensitive to light and temperature. The other two varieties from Nepal, which are tolerant to low light and low temperature. Rosmarinic acid-3-O-glucoside, rosmarinic acid, and apigenin-7-O-glucoside were detected as the main polyphenolic constituents in every variety, while apigenin and luteolin were present only in perilla pomace from Japan. In IgE-sensitized RBL-2H3 cells, polyphenols derived from two varieties of Japan suppressed degranulation of mast cells, but those derived from the two varieties of Nepal did not, indicating that apigenin and luteolin may be in part responsible for the anti-allergic response. In addition, it was found that proteins involved in the degranulation signaling pathway, such as PLCγ2, Syk, and Akt, were less phosphorylated in cells treated with the egoma pomace extracts of Japanese origin. Taken together, pomace from egoma varieties derived from different regions may differently modulate allergic response in part due to the difference in polyphenol composition and may be applied to develop nutraceuticals and functional foods fortified with anti-allergic properties.

We previously reported the successful establishment of multiple immortalized cell lines that preserved the original nature of the primary cells via co-expression of R24C mutant cyclin-dependent kinase 4 (CDK4(R24C)), Cyclin D1, and telomerase reverse transcriptase (TERT). However, as these genes are kind of oncogenes, tools to control their expression levels are favorable. In this study, we describe a new polycistronic lentiviral vector expressing proliferation factors, CDK4(R24C) and Cyclin D1 along with enhanced green fluorescence protein (EGFP) under the control of doxycycline (Dox)-dependent transactivator (rtTA) and tetracycline response element (TRE). By introducing the Dox-inducible lentiviral vector into human airway epithelial cells, we established a novel human airway epithelial cell line harboring polycistronic Dox-inducible CDK4(R24C) and Cyclin D1, referred to as Tet-on K4D cells. We showed that the cell growth of Tet-on K4D cells could be controlled by Dox. Furthermore, expression of K4D genes and rtTA gene can be independently monitored by fluorescent imaging. Cultured airway epithelial cells are useful as a tool for studying the pathogenesis of lung disorders. Altogether, our established human airway epithelial cells could be used for a variety of studies such as lung pathology and biology underlying the differentiation process to form the complex pseudostratified multicellular layers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00477-0.

With the aim of regulating clock gene expression to control cell activities in cell processing engineering, the effect of the combination of residual glucose concentration and subsequent increment by temporal glucose feeding on the oscillation of the expression of clock gene Per2 was investigated employing rat Mesenchymal stem cell (MSC)-like cells having Per2 promoter gene with a destabilized luciferase gene (Per2-dLuc). Two experiments with several initial glucose concentrations and different times of cultures (2 and 5 days) before temporal glucose feeding (0.9 g/L) were employed to realize various concentrations of residual glucose in the medium before the feeding. In these experiments, the lower residual glucose concentrations (0.002–0.02 g/L) before temporal glucose feeding tended to induce the larger amplitude of oscillation of Per2 expression than the higher ones (0.55–0.74 g/L). When the residual glucose concentration before glucose feeding was low (0.014–0.038 g/L), the higher temporal glucose concentration (0.23–0.9 g/L) feeding tended to induce the larger amplitude of oscillation of Per2 expression than the lower ones (0.012–0.023 g/L). Taken together, we found that the amplitude of oscillation of the expression of clock gene Per2 could be controlled by the combination of residual glucose concentration and glucose concentration of subsequent temporal feeding.

We found that strawberry (Fragaria x ananassa) extract has an IgE production suppressive activity and its oral administration improved skin manifestation in atopic dermatitis model mice. In present study, we identified an active substance using the IgE-producing human myeloma cell line U266. Gel filtration experiment indicated that the IgE suppressor was more than 6 kDa in molecular size. In addition, its pectinase treatment inhibited the activity, suggesting that the active substance in strawberry extract is pectin. Among solutions of water-(WP), hexametaphosphate-(HXP), acid-(HP) and alkali soluble pectin (OHP) extracted from strawberry, only OHP suppressed IgE production, and their suppressive activity was cancelled by pectinase treatment. In addition, OHP extracted from apple also inhibited IgE production. Furthermore, OHP also suppressed IgE production and did not affect IgG and IgM production in human peripheral blood mononuclear cells in an in vitro immunization condition. From these results, we concluded that OHP was an IgE suppressor in strawberry extract.

To obtain a large number of human mesenchymal stem cell (hMSCs) for allograft, nonwoven fabrics (NWF) were used as a cell culture scaffold. NWF are three-dimensional fiber aggregates formed by heat bonding and have a high surface area for cell adhesion and elongation. Inoculation hMSC was done to a center of NWF disc (diameter, 15.1 mm; depth, 0.1 mm). A cell suspension inoculum had a volume of 10 µL, which was close to the void volume of the disc, and resulted in a high initial (24 h) cell adhesion efficiency. Use of green fluorescent protein expressing rat MSCs and fluorescence microscopy revealed that adding an additional 10 μL of medium at 0–2 h after the cell inoculation made the initial horizontal distribution of cells in the NWF disc more uniform. Addition of 10 μL of the medium after 1 and 2 h of hMSC inoculation (0.15 × 10(3) cells/cm(2) NWF-fiber) markedly increased the final cell density (21 days) from 2.48 to 7.45 × 10(3) cells/cm(2) NWF-fiber and fold increase in cell density by 16–48-fold. In conclusion, the addition of an additional medium after inoculation made the initial cells distribution in NWF more uniform, which might result in higher final cell density.

Lysozyme is an anti-bacterial protein that is widely distributed in nature. Our previous studies revealed that lysozyme shows anti-inflammatory effect on hyperinflammatory macrophages in vitro. The effect of lysozyme on lipopolysaccharide-induced inflammation model mice was examined in this study. Oral administration of lysozyme at 2250 mg/kg body weight/day (high-dose group) significantly suppressed interleukin (IL)-6 and tumor necrosis factor-α levels in the serum. IL-6 level in the spleen was significantly suppressed by lysozyme at 450 mg/kg body weight/day (middle-dose group) and high-dose group due to the suppression of gene expression level. The gene expression levels of IL-1β and IL-12 were also decreased by lysozyme in the high-dose group. In addition, lysozyme significantly suppressed IL-6 level in the liver in the high-dose group. Our findings suggest that lysozyme mitigates inflammatory condition in vivo by suppressing inflammatory cytokine levels in serum and organs from LPS-induced inflammation model mice.