Prevention of growth arrest-induced cell death of vascular smooth muscle cells by a product of growth arrest-specific gene, gas6

AbstractWe have purified Gas6 as a growth-potentiating factor for vascular smooth muscle cells (VSMCs) [Nakano, T. et al. (1995) J. Biol. Chem. 270, 5702-57051. However, specific production of Gas6 in growth-arrested cells raises an intriguing question as to the physiological function of Gas6. In this study, we found that serum-starved VSMCs secreted some survival factors and depletion of the factors induced cell death of VSMCs. Finally, we demonstrated that cell death was prevented by the addition of Gas6, suggesting that one of the major biological activity of Gas6 is protection of growth-arrested VSMCs from death

Uropathogenic specific protein gene, highly distributed in extraintestinal uropathogenic Escherichia coli, encodes a new member of H-N-H nuclease superfamily

BACKGROUND: The uropathogenic specific protein (Usp) and three OrfU proteins (OrfU1, OrfU2 and OrfU3) are encoded in the putative small pathogenicity island which is closely associated with Uropathogenic Escherichia coli. Although homology search revealed that Usp and OrfUs have a homology with nuclease-type bacteriocins, which possess H-N-H nuclease motif, and immunity proteins respectively, the molecular activity of these proteins was never investigated. In this study, we try to over-express Usp in E. coli, purify Usp and characterize its molecular activity. METHOD: Recombinant Usp protein was expressed in E. coli BL21(DE3) cells together with 6× Histidine tagged OrfU1 (OrfU1-His) protein, and purified with affinity chromatography using Ni(2+) chelating agarose. The nuclease activity of the purified Usp was examined in vitro by using plasmid DNA as a substrate. The importance of H-N-H motif in nuclease activity of Usp was examined by site-directed mutagenesis study. RESULTS: We revealed that pET expression vector encoding Usp alone could not be maintained in E. coli BL21(DE3), and insertion of the orfUs as well as usp in the constructed plasmid diminished the toxic effect, suggesting that co-expressed OrfUs masked the activity of Usp. To purify Usp protein, we employed the expression vector encoding untagged Usp together with OrfU1-His. A tight complex formation could be observed between Usp and OrfU1-His, which allowed the purification of Usp in a single chromatographic step: binding of Usp/OrfU1-His complex to Ni(2+) chelating agarose followed by elution of Usp from the complex with denaturing reagent. The purified free Usp was found to have the nuclease activity, and the activity was constitutively higher than Usp/OrfU1-His complex. H-N-H motif, which is found in various types of nucleases including a subfamily of nuclease-type bacteriocin, had been identified in the C-terminal region of Usp. Site-directed mutagenesis study showed that the H-N-H motif in Usp is indispensable for its nuclease activity. CONCLUSION: This is the first evidence of the molecular activity of the new member of H-N-H superfamily and lays the foundation for the biological characterization of Usp and its inhibitor protein, OrfUs

Psychosocial twin cohort studies in Japan: the Keio Twin Research Center (KoTReC)

The Keio Twin Research Center (KoTReC) was established in 2009 at Keio University to combine two longitudinal cohort projects — the Keio Twin Study (KTS) for adolescence and adulthood and the Tokyo Twin Cohort Project (ToTCoP) for infancy and childhood. KoTReC also conducted a two-time panel study of self-control and psychopathology in twin adolescence in 2012 and 2013 and three independent anonymous cross-sectional twin surveys (ToTcross) before 2012 — the ToTCross, the Junior and Senior High School Survey and the High School Survey. This article introduces the recent research designs of KoTReC and its publications

Molecular Network Associated with MITF in Skin Melanoma Development and Progression

Various environmental and genetic factors affect the development and progression of skin cancers including melanoma. Melanoma development is initially triggered by environmental factors including ultraviolet (UV) light, and then genetic/epigenetic alterations occur in skin melanocytes. These first triggers alter the conditions of numerous genes and proteins, and they induce and/or reduce gene expression and activate and/or repress protein stability and activity, resulting in melanoma progression. Microphthalmia-associated transcription factor (MITF) is a master regulator gene of melanocyte development and differentiation and is also associated with melanoma development and progression. To find better approaches to molecular-based therapies for patients, understanding MITF function in skin melanoma development and progression is important. Here, we review the molecular networks associated with MITF in skin melanoma development and progression

Genetic screening for malignant hyperthermia and comparison of clinical symptoms in Japan

Malignant hyperthermia (MH) is an anaesthetic complication that causes an abnormal hypermetabolic state. RYR1 encoding ryanodine receptors of the sarcoplasmic reticulum and CACNA1S encoding α subunits of dihydropyridine receptors are known to be associated with MH pathogenicity. We performed genetic screening using next-generation sequencing to evaluate the prevalence of genes associated with MH pathogenicity and clinical symptoms. This was a retrospective cohort study wherein next-generation sequencing data of 77 families diagnosed with MH predisposition by calcium-induced calcium release (CICR) tests from 1995 to 2019 was used to search for RYR1 and CACNA1S variants. Furthermore, the clinical symptoms and predisposition tests in participants with RYR1 and CACNA1S variants were compared. In the 77 families, 44.2%, 7.8%, and 48.1% individuals had RYR1, CACNA1S, and neither RYR1 nor CACNA1S variants, respectively. Clinically significant differences were found in the maximum body temperature, maximum elevated body temperature for 15 min, creatinine kinase level, and CICR rate between the RYR1 and CACNA1S groups. The prevalence of pathogenic CACNA1S variants appears to be prominent in Japan. The severity of clinical symptoms and the CICR rate were greater in individuals with RYR1 variants than in those with CACNA1S variants, likely due to more direct regulation of calcium levels by ryanodine receptors than by dihydropyridine receptors. Genetic analysis of MH in future studies may help identify other genes associated with MH, which will further clarify the relationship between genotypes and MH symptoms and contribute to safer anaesthesia practice.This study was supported by a Grant-in-Aid for Young Scientists (grant number: 17K16733 to Y.N. and 20K17783 to R.K.) from the Japan Society for the Promotion of Science and by the Takeda Science Foundation (H.K.)

Cytotoxic T Lymphocytes Regenerated from iPS Cells Have Therapeutic Efficacy in a Patient-Derived Xenograft Solid Tumor Model

Current adoptive T cell therapies conducted in an autologous setting are costly, time consuming, and depend on the quality of the patient's T cells. To address these issues, we developed a strategy in which cytotoxic T lymphocytes (CTLs) are regenerated from iPSCs that were originally derived from T cells and succeeded in regenerating CTLs specific for the WT1 antigen, which exhibited therapeutic efficacy in a xenograft model of leukemia. In this study, we extended our strategy to solid tumors. The regenerated WT1-specific CTLs had a strong therapeutic effect in orthotopic xenograft model using a renal cell carcinoma (RCC) cell line. To make our method more generally applicable, we developed an allogeneic approach by transducing HLA-haplotype homozygous iPSCs with WT1-specific TCR α/β genes that had been tested clinically. The regenerated CTLs antigen-specifically suppressed tumor growth in a patient-derived xenograft model of RCC, demonstrating the feasibility of our strategy against solid tumors