Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells

Recombinant immunoglobulin G (IgG) aggregates are formed during their production. However, the process underlying intracellular/extracellular aggregation in cell culture conditions is not well understood, and no effective method exists to assess IgG aggregates. Here, we establish an approach to detect intracellular aggregates using AF.2A1, a small artificial protein that binds to non-native IgG conformers and aggregates. Fluorescent-labeled AF.2A1 is prepared via conjugation and transfected into antibody-producing Chinese hamster ovary (CHO) cells. Micrographic images show intracellular IgG aggregates in CHO cells. The relative amount of intracellular aggregates (versus total intracellular IgG) differed depending on the type of additives used during cell culture. Interestingly, the relative amount of intracellular aggregates moderately correlates with that of in vitro extracellular IgG aggregates, suggesting they are secreted. This method will allow the investigation of antibody aggregation in cells, and may guide the production of therapeutic antibodies with high yield/quality

Effects of Nanosecond Laser Fabrication on Bioactivity of Pure Titanium

AbstractWe are developing surface modification techniques for dental implants with the aim of reducing the time required to realize good adhesion between bone and implant surfaces. A nanosecond Nd:YVO4 laser was used to modify the surfaces of commercially pure titanium (CP Ti) disks and their bioactivities were then evaluated. The surfaces of the CP Ti disks were covered by lines after laser treatment. This treatment created complex microasperities of titania with rutile and anatase crystal structures. This results in the formation of hydroxyapatite on surfaces immersed in 1.5-times concentrated simulated body fluid for 7 days, whereas no hydroxyapatite was observed on conventionally polished surfaces that were immersed under the same conditions. This indicates that laser treatment improves the bioactivity of CP Ti, which is a critical property for osseointegrated implants

System Design of an Autonomous Underwater Robot “DaryaBird”

Various kinds of robots have been developed parallel with the progress of computers and information processing technology, and the operations in the extreme environments, such as disaster areas, space and ocean, are getting one of the practical solutions for those hazardous missions. The underwater robots are one of the extreme environment robots and expected as one of solutions for underwater activities i.e., maintenance of underwater structures, observations, scientific research, where research area is getting wide and deep and also underwater structures are getting large-scale and deep-depth. Their efficiencies have been investigated during recent decades and are proven by ocean experiments. However, the robotic system including the support vessels is still big scale, and not so easy to handle by a few researchers. In this paper, we describe the design of an underwater robot “DaryaBird” developed aiming at handy, small underwater robots which can be operated by a few researchers. In addition, experimental results and mission strategies for AUVC 2010 are reported.AUVSI & ONR\u27s 13th AUVSI 2010 : Association for Unmanned Vehicle Systems International (AUVSI) North America 2010, Aug 24-27, 2010, Denver, CO., US

Effect of Initial Grain Size on Dynamically Recrystallized Grain Size in AZ31 Magnesium Alloy

The effect of initial grain size on dynamically recrystallized grain size (DRX grain size) is examined in AZ31 magnesium alloy with four kinds of initial grain sizes. When the average grain size after the high-temperature compression test is plotted against Zenner-Hollomon parameter (Z-parameter), initial grain size dependence on DRX grain size appears only in the specimen with large initial grain size in the low Z region, but not in the specimen with small grain size or in high Z region. When the grain size measured only in recrystallized region is plotted against Z-parameter, initial grain size dependence on DRX grain size does not appear. From these results, it is concluded that there is no initial grain size dependence on DRX grain size when the DRX proceeds to some degree, because the DRX grain size becomes constant for a given Zvalue. The initial grain size dependence on DRX grain size observed in the low Z region would be related to the rotation of basal plane perpendicular to the compression axis during deformation before the DRX grains have been developed sufficiently

RecR forms a ring-like tetramer that encircles dsDNA by forming a complex with RecF

In the RecFOR pathway, the RecF and RecR proteins form a complex that binds to DNA and exerts multiple functions, including directing the loading of RecA onto single-stranded (ss) DNA regions near double-stranded (ds) DNA–ssDNA junctions and preventing it from forming a filament beyond the ssDNA region. However, neither the structure of the RecFR complex nor its DNA-binding mechanism was previously identified. Here, size-exclusion chromatography and small-angle X-ray scattering data indicate that Thermus thermophilus (tt) RecR binds to ttRecF to form a globular structure consisting of four ttRecR and two ttRecF monomers. In addition, a low resolution model shows a cavity in the central part of the complex, suggesting that ttRecR forms a ring-like tetramer inside the ttRecFR complex. Mutant ttRecR proteins lacking the N- or C-terminal interfaces that are required for tetramer formation are unable to form a complex with ttRecF. Furthermore, a ttRecFR complex containing the DNA-binding deficient ttRecR K23E/R27E double mutant, which contains mutations lying inside the ring, exhibits significantly reduced dsDNA binding. Thus, we propose that the ring-like ttRecR tetramer has a key role in tethering the ttRecFR complex onto dsDNA and that the ring structure may function as a clamp protein

Atomic Bomb Fallout and “Black rain” at Manose District (Hirama-cho) Located Northeast of Nagasaki City

Plutonium atomic bomb (A-bomb) was exploded at 503 meters height above Nagasaki City on August 9, 1945. Many studies on radioactive contamination, radiation dose, effect to people and so on have been conducted until now. Local fallout from the A-bomb mainly spread a lot in the east direction centering on Nishiyama area located about 4 km east of the hypocenter. Like the Hiroshima A-bomb, black rain accompanied by radioactive materials fell shortly after the explosion, but relationship between local fallout fall zone and black rain fall area has not been sufficiently elucidated, which has enhanced the concerns on radiation exposure to the A-bomb survivor. Concerning the black rain due to the Nagasaki A-bomb, it has been said that black rain did not fall across a wide area in a comprehensive way, except for the heavily contaminated Nishiyama area, but fell at various areas with conditions of light rain and/or soaking after the explosion. Among many villages or towns, especially in the Manose District where is a small intermountain village about 7.5 km northeast of the hypocenter, it had always been heard and known that the residents living there encountered black rain and experienced hair loss. In this district, the hair loss of inhabitants occurred actually with high probability, but its reason has not been elucidated well. Therefore, in order to reevaluate the local fallout level in the Manose District by the A-bomb, residual long-lived radionuclides 137Cs and Pu isotopes were measured for soil samples collected at the Manose District including the surrounding areas in July, 2011. As a result, accumulated levels of 137Cs and 239,240Pu in the Manose District were not so high, each being nearly background (BG) level. The dispersion of local fallout Pu from the A-bomb was reconfirmed by 240Pu/239Pu atomic ratios measured in soil samples at the same time, and was found to be primarily limited in the east direction, especially around the Nishiyama area. Those results were consistent with results obtained so far. Further researches are needed for understanding scientifically the cause of the high incidence rate of hair loss in this district.This research was supported by JSPS KAKENHI Grants Numbers 23406002 (April 2011-March 2014) and 26257501 (April 2014-March 2018)

Identification of key yeast species and microbe–microbe interactions impacting larval growth of Drosophila in the wild

自然界で動物の成長を支える共生微生物叢 --中心的な役割を担う共生酵母・細菌の同定--. 京都大学プレスリリース. 2023-12-28.Microbiota consisting of various fungi and bacteria have a significant impact on the physiological functions of the host. However, it is unclear which species are essential to this impact and how they affect the host. This study analyzed and isolated microbes from natural food sources of Drosophila larvae, and investigated their functions. Hanseniaspora uvarum is the predominant yeast responsible for larval growth in the earlier stage of fermentation. As fermentation progresses, Acetobacter orientalis emerges as the key bacterium responsible for larval growth, although yeasts and lactic acid bacteria must coexist along with the bacterium to stabilize this host–bacterial association. By providing nutrients to the larvae in an accessible form, the microbiota contributes to the upregulation of various genes that function in larval cell growth and metabolism. Thus, this study elucidates the key microbial species that support animal growth under microbial transition

The process of displacing the single-stranded DNA-binding protein from single-stranded DNA by RecO and RecR proteins

The regions of single-stranded (ss) DNA that result from DNA damage are immediately coated by the ssDNA-binding protein (SSB). RecF pathway proteins facilitate the displacement of SSB from ssDNA, allowing the RecA protein to form protein filaments on the ssDNA region, which facilitates the process of recombinational DNA repair. In this study, we examined the mechanism of SSB displacement from ssDNA using purified Thermus thermophilus RecF pathway proteins. To date, RecO and RecR are thought to act as the RecOR complex. However, our results indicate that RecO and RecR have distinct functions. We found that RecR binds both RecF and RecO, and that RecO binds RecR, SSB and ssDNA. The electron microscopic studies indicated that SSB is displaced from ssDNA by RecO. In addition, pull-down assays indicated that the displaced SSB still remains indirectly attached to ssDNA through its interaction with RecO in the RecO-ssDNA complex. In the presence of both SSB and RecO, the ssDNA-dependent ATPase activity of RecA was inhibited, but was restored by the addition of RecR. Interestingly, the interaction of RecR with RecO affected the ssDNA-binding properties of RecO. These results suggest a model of SSB displacement from the ssDNA by RecF pathway proteins

Increased oxidative stress precedes the onset of high-fat diet-induced insulin resistance and obesity

金沢大学医薬保健研究域医学系Insulin resistance is a key pathophysiological feature of metabolic syndrome. However, the initial events triggering the development of insulin resistance and its causal relations with dysregulation of glucose and fatty acids metabolism remain unclear. We investigated biological pathways that have the potential to induce insulin resistance in mice fed a high-fat diet (HFD). We demonstrate that the pathways for reactive oxygen species (ROS) production and oxidative stress are coordinately up-regulated in both the liver and adipose tissue of mice fed an HFD before the onset of insulin resistance through discrete mechanism. In the liver, an HFD up-regulated genes involved in sterol regulatory element binding protein 1c-related fatty acid synthesis and peroxisome proliferator-activated receptor α-related fatty acid oxidation. In the adipose tissue, however, the HFD down-regulated genes involved in fatty acid synthesis and up-regulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Furthermore, increased ROS production preceded the elevation of tumor necrosis factor-α and free fatty acids in the plasma and liver. The ROS may be an initial key event triggering HFD-induced insulin resistance. © 2008 Elsevier Inc. All rights reserved