CHO-K1 host cell engineering strategy enabling the establishment of strains producing higher yields of recycling antibodies

As described elsewhere (Biotechnol Bioeng 2010, 2013), our DXB11 (dhfr–) host cell engineering strategy achieved high cell viability for a prolonged period (more than 1 month) and enhanced mAb productivity (\u3e100 pg/cell/day) by nutritional control. Introduction of taurine transporter (TAUT) into DXB11 parent cells increased glutamine uptake and accelerated glutathione metabolism. By forcing the overexpression of TAUT, we were able to control DXB11 host cell functions and thereby increase the monoclonal antibody (mAb) titer up to 8.1 g/L/31 days under conventional 1-L bioreactor fed-batch conditions. Furthermore, the mAb produced by the DXB11/TAUT cells was comparable in quality to the mAb produced by the parent cells. In this study, we used CHO-K1 host cells and a chemically defined medium (CDM) for the development of cell lines producing recycling antibodies (rcAb). CDM adaptation and TAUT overexpression improved CHO-K1 cell performance. Rapid-growth CHO-K1/TAUT cells were developed, and these enabled the establishment of strains that produced higher yields of rcAb than did CHO-K1 parent cell (p \u3c 0.05). Viable cell density of these CHO-K1/TAUT/rcAb strains increased not only under shaker passage culture conditions (p \u3c 0.01) but also under shaker fed-batch culture conditions (p \u3c 0.01). These results suggest that our TAUT overexpression strategy also has a unique potential for the improvement of CHO-K1 host cells as well as DXB11 host cells

Dxb11/taut host cell engineering strategy enabling the establishment of strains producing the highest yield of advanced recycling antibodies

Innovation in monoclonal antibody (mAb) production for clinical use continues to be driven by cell engineering strategies to increase mAb yield and to control the complexity of advanced recycling antibodies (rcAbs). rcAbs offer significant advantages for efficacy by dissociating the antigens in endosomes and recycling free antibodies back to plasma. We were able to reduce the instability in an rcAb-producing CHO cell line whereby mAb titer during the stage of cell line development (CLD) was decreased and improve cell culture optimization. In this study, we used a chemically defined medium (CDM) and CDM-adapted transporter-overexpressing DXB11 host cells for CLD of two different types of rcAb (rcAb-1, rcAb-2). These not only have pH-dependent antigen-binding but also a distinct mechanism of mAb uptake into cells. As described before, taurine transporter (TAUT) overexpression was able to improve DXB11 cell performance. DXB11/TAUT host cells were further developed with CDM, and these enabled the establishment of strains that produced higher yields of rcAb than did DXB11 parent cells. Yields of these DXB11/TAUT/rcAb-1 strains increased up to 7.0 g/L/17 days under 1-L bioreactor fed-batch conditions. In contrast, the mAb yields of DXB11/rcAb-1 were up to 3.5 g/L/17 days. In addition, the mAb properties of DXB11/TAUT/rcAb-1 were comparable to those of DXB11/rcAb-1. These results suggest that our TAUT overexpression strategy has a unique potential for improvement of DXB11 host cells and is useful for the CLD of advanced antibodies with increased complexity. Since our CLD of DXB11/TAUT/rcAb-2 also shows significant promise, we plan to adopt the DXB11/TAUT host cell as our Super CHO cell for future development of advanced antibody drugs

Comparative survey of go/no-go results to identify the inhibitory control ability change of Japanese children

This research, conducted in 1998 and 2008, uses go/no-go data to investigate the fundamentals of cognitive functioning in the inhibitory control ability of Japanese children. 844 subjects from kindergarten to junior high school participated in go/no-go task experiments. Performance of go/no-go tasks, which are frequently used to investigate response inhibition, measures a variety of cognitive components besides response inhibition. With normal brain development, the ability to inhibit responses improves substantially in adolescence. An increase over time in the error rate during the go/no-go tasks of subjects of the same age indicates that these processes are not functioning properly. Comparisons between the 1998 and 2008 data revealed several differences in error rates. In 2008, there were increases in the number of errors in groups from each age range. The comparison also revealed that overall error rates peaked at later ages in the 2008 subjects. Taken together, these results show changing conditions in the inhibitory function of the prefrontal cortex. However, the reason for these changing conditions remains unclear. While a lifestyle questionnaire revealed several differences in factors such as bedtimes and hours spent watching TV, analysis did not reveal a significant correlation.ArticleBioPsychoSocial Medicine.8(1):14(2014)journal articl

Differential Changes of Aorta and Carotid Vasodilation in Type 2 Diabetic GK and OLETF Rats: Paradoxical Roles of Hyperglycemia and Insulin

We investigated large vessel function in lean Goto-Kakizaki diabetic rats (GK) and Otsuka Long-Evans Tokushima Fatty diabetic rats (OLETF) with possible roles of hyperglycemia/hyperosmolarity and insulin. Both young and old GK showed marked hyperglycemia with normal insulin level and well-preserved endothelium-dependent and endothelium-independent vasodilation in aorta and carotid artery. There were significant elevations in endothelial/inducible nitric oxide synthase (eNOS/iNOS) and inducible/constitutive heme oxygenase (HO-1/HO-2) in GK. The endothelium-dependent vasodilation in GK was inhibited partly by NOS blockade and completely by simultaneous blocking of HO and NOS. In contrast, OLETF showed hyperinsulinemia and mild hyperglycemia but significant endothelium dysfunction beginning at early ages with concomitantly reduced eNOS. Insulin injection corrected hyperglycemia in GK but induced endothelium dysfunction and intima hyperplasia. Hyperglycemia/hyperosmolarity in vitro enhanced vessel eNOS/HO. We suggest that hyperinsulinemia plays a role in endothelium dysfunction in obese diabetic OLETF, while hyperglycemia/hyperosmolarity-induced eNOS/HO upregulation participates in the adaptation of endothelium function in lean diabetic GK

Canonical versus non-canonical transsynaptic signaling of neuroligin 3 tunes development of sociality in mice

社会性の発達を調節する新たな機構を発見. 京都大学プレスリリース. 2021-03-26.Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality

Effects of inorganic mercury and methylmercury on osteoclasts and osteoblasts in the scales of the marine teleost as a model system of bone

To evaluate the effects of inorganic mercury (InHg) and methylmercury (MeHg) on bone metabolism in a marine teleost, the activity of tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) as indicators of such activity in osteoclasts and osteoblasts, respectively, were examined in scales of nibbler fish (Girella punctata). We found several lines of scales with nearly the same TRAP and ALP activity levels. Using these scales, we evaluated the influence of InHg and MeHg. TRAP activity in the scales treated with InHg (10-5 and 10-4 M) and MeHg (10-6 to 10-4 M) during 6 hrs of incubation decreased significantly. In contrast, ALP activity decreased after exposure to InHg (10-5 and 10-4 M) and MeHg (10-6 to 10-4 M) for 18 and 36 hrs, although its activity did not change after 6 hrs of incubation. As in enzyme activity 6 hrs after incubation, mRNA expression of TRAP (osteoclastic marker) decreased significantly with InHg and MeHg treatment, while that of collagen (osteoblastic marker) did not change significantly. At 6 hrs after incubation, the mRNA expression of metallothionein, which is a metal-binding protein in osteoblasts, was significantly increased following treatment with InHg or MeHg, suggesting that it may be involved in the protection of osteoblasts against mercury exposure up to 6 hrs after incubation. To our knowledge, this is the first report of the effects of mercury on osteoclasts and osteoblasts using marine teleost scale as a model system of bone. © 2014 Zoological Society of Japan