Biology subjects

Kubota, T.

Publications and source records attributed to Kubota, T..

4 recordsLinked to original sources

bioRxiv2022

Metabolic differences and differentially expressed genes between C57BL/6J and C57BL/6N mice substrains

C57BL/6J (B6J) and C57BL/6N (B6N) mice are the most frequently used substrains in C57BL/6 (B6) inbred mice, serving as physiological models for in vivo studies and as background strains to build transgenic mice. However, little attention has been paid to the phenotypic differences between B6J and B6N mice, and they have often been attributed to mutations in the nicotinamide nucleotide transhydrogenase (Nnt) gene, which was found only in B6J. Nevertheless, phenotypic differences between the two that cannot be explained by Nnt mutations alone, especially in metabolic traits, indicate the presence of genetic variants associated with metabolism other than Nnt. We aimed to identify these genetic differences between B6J and B6N mice. Our results provide insights into differentially expressed genes (DEGs) in adipose tissues, skeletal muscle, liver, hypothalamus, and hippocampus, and phenotypic differences in metabolic traits between B6J and B6N. B6J mice had significantly lower body weight than B6N mice, regardless of a normal or high-fat diet. Blood insulin levels in B6J mice were significantly lower than those in B6N mice and glucose levels during dietary obesity were higher in B6J mice. Metabolic assessments revealed greater physical activity, less food intake, and higher energy expenditure in B6J mice than in B6N mice. Among the DEGs that were highly expressed in B6J mice compared to B6N mice, three genes--insulin degrading enzyme, adenylosuccinate synthase 2, and ectonucleotide triphosphate diphosphohydrolase 4--and the DEGs that had lower expression in B6J mice compared to B6N mice--Nnt, WD repeat and FYVE domain containing 1, and dynein light chain Tctex-type 1--were overlapped in all seven tissues. Our study provides insights into DEGs between B6J and B6N, which will be useful for substrain selection for mouse experiments, avoiding erroneous experimental results, and reviewing the results of studies that have used B6J, B6N, or mixed substrains.

genetics↗

bioRxiv2021

Structural analysis and construction of thermostable antifungal chitinase

Chitin is a biopolymer of N-acetyl-D-glucosamine with {beta}-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the {beta}-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzymes thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual applicable conditions. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. Based on the crystal structure and sequence alignment among other chitinases, we rationally introduced proline residues, a disulfide bond, and salt bridge in the chitinase using protein engineering methods. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. IMPORTANCEWe solved the crystal structure of the chitinase from Gazyumaru (Ficus microcarpa) latex exhibiting antifungal activity. Furthermore, we demonstrated that the thermostable mutant enzyme with a melting temperature (Tm) 6.9 {degrees}C higher than wild type (WT) and a half-life at 60{degrees}C that is 15 times longer than WT was constructed through 10 amino acid substitutions, including five proline residues substitutions, making disulfide bonding, and building a salt bridge network in the enzyme. These mutations do not affect its high antifungal activity and chitinase activity, and the principle for the construction of the thermostable chitinase was well explained by its crystal structure. Our results provide a useful strategy to enhance the thermostability of this enzyme family and to use the thermostable mutant as a seed for antifungal agents for practical use.

biochemistry↗

bioRxiv2021

Spontaneous modulations of high frequency cortical activity

ObjectiveWe clarified the clinical and mechanistic significance of physiological modulations of high-frequency broadband cortical activity associated with spontaneous saccadic eye movements during a resting state. MethodsWe studied 30 patients who underwent epilepsy surgery following extraoperative electrocorticography and electrooculography recordings. We determined whether high-gamma activity at 70-110 Hz preceding saccade onset would predict upcoming ocular behaviors. We assessed how accurately the model incorporating saccade-related high-gamma modulations would localize the primary visual cortex defined by electrical stimulation. ResultsThe whole-brain level dynamic atlas demonstrated transient high-gamma suppression in the striatal region before saccade onset and high-gamma augmentation subsequently involving the widespread posterior brain regions. More intense striatal high-gamma suppression predicted the upcoming saccade directed to the ipsilateral side and lasting longer in duration. The bagged-tree-ensemble model demonstrated that intense saccade-related high-gamma modulations localized the visual cortex with an accuracy of 95%. ConclusionsWe successfully animated the neural dynamics supporting saccadic suppression, a principal mechanism minimizing the perception of blurred vision during rapid eye movements. The primary visual cortex per se may prepare actively in advance for massive image motion expected during upcoming prolonged saccades. SignificanceMeasuring saccade-related electrocorticographic signals may help localize the visual cortex and avoid misperceiving physiological high-frequency activity as epileptogenic. Highlights- The whole-brain level dynamic atlas animated spontaneous high gamma modulations associated with saccadic eye movements. - Preceding high gamma activity in the striatal cortex predicted the direction and duration of the upcoming saccades. - Saccade-related high-gamma modulations localized the stimulation-defined visual cortex with an accuracy of 95%.

neuroscience↗

bioRxiv2018

A transgenic mutant mouse line accompanied by the complete deletion of interleukin-33 showed insulin and leptin resistances

Interleukin (IL) -33 has been identified as a member of the IL-1 family. Members of the IL-1 family have been reported to be involved in the regulation of energy homeostasis and glucose metabolism. Homozygous transgenic mutant mice of FLP14 line, that we previously generated, unexpectedly developed mature-onset obesity and diabetes. Through genetic investigations, we found that insertion of the transgenes had resulted in complete deletion of the Il33 gene. These obese male homozygous mutant mice exhibited hyperphagia with hyperleptinemia and insulin resistance caused by increased hepatic gluconeogenesis and decreased glucose uptake in skeletal muscle. As a result of examining preobese male homozygous mutant mice to investigate with the exclusion of the effect of obesity, hyperphagia with hyperleptinemina and insulin resistance caused by decreased glucose uptake in skeletal muscle were already observed, but the increased hepatic glucose production was not. To investigate whether the insulin resistance was caused by deletion of the Il33 gene, we treated these preobese homozygous mutant mice with recombinant IL-33 protein and noted a significant improvement in insulin resistance. Thus, insulin resistance in these homozygous mutant mice was caused, at least in part, by IL-33 deficiency, suggesting a favorable role of IL-33 for glucose metabolism in the skeletal muscle.

molecular biology↗