A simple, dual direct expression plasmid system in prokaryotic and mammalian cells.

We introduce a simple, dual direct cloning plasmid system (pgMAX-II) for gene expression analysis in both prokaryotic () and mammalian cells. This system, which uses a prokaryotic expression unit adapted from the pgMAX system and a mammalian promoter, is effective for subcloning using the DNA topoisomerase II toxin CcdB. Given that molecular biological cloning systems broadly rely on for rapid growth, the proposed concept may have wide applicability beyond mammalian cells.

Enhanced β-adrenergic response in mice with dominant-negative expression of the PKD2L1 channel.

Polycystic kidney disease (PKD) is the most common genetic cause of kidney failure in humans. Among the various PKD-related molecules, PKD2L1 forms cation channels, but its physiological importance is obscure. In the present study, we established a transgenic mouse line by overexpressing the dominant-negative form of the mouse PKD2L1 gene (i.

TP53/p53-FBXO22-TFEB controls basal autophagy to govern hormesis.

Preconditioning with a mild stressor such as fasting is a promising way to reduce severe side effects from subsequent chemo- or radiotherapy. However, the underlying mechanisms have been largely unexplored. Here, we demonstrate that the TP53/p53-FBXO22-TFEB (transcription factor EB) axis plays an essential role in this process through upregulating basal macroautophagy/autophagy.

Requirement of the Ca channel β subunit for sympathetic PKA phosphorylation.

Facilitation of cardiac function in response to signals from the sympathetic nervous system is initiated by the phosphorylation of L-type voltage-dependent Ca channels (VDCCs) by protein kinase A (PKA), which in turn is activated by β-adrenoceptors. Among the five subunits (α, β, α/δ, and γ) of VDCCs, the α subunit and the family of β subunits are substrates for PKA-catalyzed phosphorylation; however, the subunit responsible for β-adrenergic augmentation of Ca channel function has yet to be specifically identified. Here we show that the VDCC β subunit is required for PKA phosphorylation upon sympathetic acceleration.

Two mouse models carrying truncating mutations in Magel2 show distinct phenotypes.

Schaaf-Yang syndrome (SYS) is a neurodevelopmental disorder caused by truncating variants in the paternal allele of MAGEL2, located in the Prader-Willi critical region, 15q11-q13. Although the phenotypes of SYS overlap those of Prader-Willi syndrome (PWS), including neonatal hypotonia, feeding problems, and developmental delay/intellectual disability, SYS patients show autism spectrum disorder and joint contractures, which are atypical phenotypes for PWS. Therefore, we hypothesized that the truncated Magel2 protein could potentially produce gain-of-function toxic effects.

Ganglioside Synthase Knockout Reduces Prion Disease Incubation Time in Mouse Models.

Localization of the abnormal and normal isoforms of prion proteins to detergent-resistant membrane microdomains, lipid rafts, is important for the conformational conversion. Lipid rafts are enriched in sialic acid-containing glycosphingolipids (namely, gangliosides). Alteration in the ganglioside composition of lipid rafts can affect the localization of lipid raft-associated proteins.

Contribution of rat embryonic stem cells to xenogeneic chimeras in blastocyst or 8-cell embryo injection and aggregation.

The ultimate goal of regenerative medicine is the transplantation of a target organ generated by the patient's own cells. Recently, a method of organ generation using pluripotent stem cells (PSCs) and blastocyst complementation was reported. This approach is based on chimeric animal generation using an early embryo and PSCs, and the contribution of PSCs to the target organ is key to the method's success.

A domain responsible for spontaneous conversion of bank vole prion protein.

Bank vole is a small rodent that shows high susceptibility to infection with diverse prion strains. To determine whether the increased susceptibility of bank voles to prion diseases can be attributed to the intrinsic nature of bank vole prion protein (PrP) or to host factors other than PrP, we produced transgenic mice overexpressing bank vole PrP. These transgenic mice spontaneously developed neurological illness with spongiform changes and the accumulation of abnormal PrP in the brain.

Ubiquitin-Specific Protease 2 Modulates the Lipopolysaccharide-Elicited Expression of Proinflammatory Cytokines in Macrophage-like HL-60 Cells.

We investigated the regulatory roles of USP2 in mRNA accumulation of proinflammatory cytokines in macrophage-like cells after stimulation with a toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Human macrophage-like HL-60 cells, mouse macrophage-like J774.1 cells, and mouse peritoneal macrophages demonstrated negative feedback to USP2 mRNA levels after LPS stimulation, suggesting that USP2 plays a significant role in LPS-stimulated macrophages.

Macrophage ubiquitin-specific protease 2 modifies insulin sensitivity in obese mice.

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing , a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes.

Animal ethics and welfare education in wet-lab training can foster residents' ethical values toward life.

Live animals are used in surgical skills training in wet lab, which has undeniable effectiveness for the development of future surgeons. However, where such training is provided, animal welfare is a major consideration. Increasingly, institutions that offer wet-lab training are incorporating animal ethics and welfare-related content into their training courses, but the effectiveness of such animal ethics education has yet to be evaluated quantitatively.

SCF(Fbxo22)-KDM4A targets methylated p53 for degradation and regulates senescence.

Recent evidence has revealed that senescence induction requires fine-tuned activation of p53, however, mechanisms underlying the regulation of p53 activity during senescence have not as yet been clearly established. We demonstrate here that SCF(Fbxo22)-KDM4A is a senescence-associated E3 ligase targeting methylated p53 for degradation. We find that Fbxo22 is highly expressed in senescent cells in a p53-dependent manner, and that SCF(Fbxo22) ubiquitylated p53 and formed a complex with a lysine demethylase, KDM4A.

Immunosuppressive and Prometastatic Functions of Myeloid-Derived Suppressive Cells Rely upon Education from Tumor-Associated B Cells.

Myeloid-derived suppressive cells (MDSC) have been reported to promote metastasis, but the loss of cancer-induced B cells/B regulatory cells (tBreg) can block metastasis despite MDSC expansion in cancer. Here, using multiple murine tumor models and human MDSC, we show that MDSC populations that expand in cancer have only partially primed regulatory function and limited prometastatic activity unless they are fully educated by tBregs. Cancer-induced tBregs directly activate the regulatory function of both the monocyte and granulocyte subpopulations of MDSC, relying, in part, on TgfβR1/TgfβR2 signaling.

Modified autonomic regulation in mice mutated in the β4 subunit of the lh/lh calcium channel.

Genetic analyses have revealed an important association between P/Q-type calcium channel activities and hereditary neurological disorders. The P/Q-type channels are composed principally of heterologous multimeric subunits including CaV2.1 and CaVβ4.

A novel transgenic mouse model carrying human Tribbles related protein 3 (TRB3) gene and its site specific phenotype.

Tribbles related protein 3 (TRB3) pseudokinase plays a crucial role in cell proliferation, migration and morphogenesis during development. In our recent study, an introduction of human TRB3 gene into mouse mammary tumor cells caused an increase of proliferation of tumor cells and their nuclear size. In the current study, to examine whether this gene causes de novo morphological changes in a specific organ site we have developed a novel variation of the transgenic mouse model that conditionally expresses human TRB3 (hTRB3) gene using Cre-recombinase (Cre)/loxP recombination system.

A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high-grade serous carcinoma development.

Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter.

Colony-stimulating factor-1 signaling suppresses renal crystal formation.

We recently reported evidence suggesting that migrating macrophages (Mϕs) eliminate renal crystals in hyperoxaluric mice. Mϕs can be inflammatory (M1) or anti-inflammatory (M2), and colony-stimulating factor-1 (CSF-1) mediates polarization to the M2Mϕ phenotype. M2Mϕs promote renal tissue repair and regeneration, but it is not clear whether these cells are involved in suppressing renal crystal formation.

Beneficial effects of Brazilian propolis on type 2 diabetes in ob/ob mice: Possible involvement of immune cells in mesenteric adipose tissue.

The anti-diabetic effects of Brazilian propolis were examined using ob/ob mice. Although repeated injection of an ethanol extract of Brazilian propolis (100 mg/kg, ip, twice a week for 12 weeks) did not affect body weight gain and food intake of ob/ob mice, blood glucose and plasma cholesterol levels were significantly attenuated. Moreover, the propolis extract partially restored glucose tolerance and insulin resistance, indicating anti-diabetic properties of the extract.

Ubiquitin-specific protease 2-69 in macrophages potentially modulates metainflammation.

Macrophages play a critical role in chronic inflammation and metabolic diseases. We identified a longer splice variant of ubiquitin specific protease (USP) 2-69 as a novel molecule that modulates pathways implicated in metabolic disorders. Expression levels of aP2/FABP4 and PAI-1/SERPINE1 genes were increased by 4- and 1.

Improvement of spontaneous alternation behavior deficit by activation of α4β2 nicotinic acetylcholine receptor signaling in the ganglioside GM3-deficient mice.

We have reported that in ganglioside GM3-deficient (GM3(-/-)) mice, spontaneous alternation behavior assessed by a Y-maze task was significantly lower, and total arm entries were significantly higher than in wild-type mice. The objective of the present study was to examine the role of nicotinic acetylcholine receptor (nAChR) signaling in impairment of spontaneous alternation behavior of GM3(-/-) mice. Nicotine treatment (0.

Targeted disruption of fad24, a regulator of adipogenesis, causes pre-implantation embryonic lethality due to the growth defect at the blastocyst stage.

In previous studies, we identified a novel gene, factor for adipocyte differentiation 24 (fad24), which plays an important role during the early stages of adipogenesis in mouse 3T3-L1 cells. Moreover, overexpression of fad24 increased the number of smaller adipocytes in white adipose tissue and improved glucose metabolic activity in mice, thus indicating that fad24 functions as a regulator of adipogenesis in vivo. However, the physiological roles of fad24 in vivo are largely unknown.

Phenotypic Characterization of LEA Rat: A New Rat Model of Nonobese Type 2 Diabetes.

Animal models have provided important information for the genetics and pathophysiology of diabetes. Here we have established a novel, nonobese rat strain with spontaneous diabetes, Long-Evans Agouti (LEA) rat derived from Long-Evans (LE) strain. The incidence of diabetes in the males was 10% at 6 months of age and 86% at 14 months, while none of the females developed diabetes.

Exclusive expression of VMAT2 in noradrenergic neurons increases viability of homozygous VMAT2 knockout mice.

The vesicular monoamine transporter 2 (VMAT2) translocates monoamine neurotransmitters from the neuronal cytoplasm into synaptic vesicles. Since VMAT2-/- mice die within a few days of birth, it is difficult to analyze the detailed VMAT2 functions using these mice. In this study, we generated human VMAT2 transgenic mice that expressed VMAT2 in noradrenergic neurons with the aim to rescue the lethality of VMAT2 deletion.

Impairment of neuropsychological behaviors in ganglioside GM3-knockout mice.

The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. Although its expression is tightly controlled during early embryo development and postnatal development and maturation in the brain, the physiological role of ganglioside GM3 in the regulation of neuronal functions has not been elucidated. In the present study, we examined motor activity, cognitive and emotional behaviors, and drug administration in juvenile GM3-knockout (GM3-KO) mice.