Search

There are numerous reports of enhanced or emerged visual arts abilities in patients with semantic impairment. These reports led to the theory that a loss of function on the language side of the brain can result in changes of ability to draw and/or to paint. Further, the left posterior middle temporal gyrus (l-pMTG) has been revealed to contribute to the higher control semantic mechanisms with objects recognition and integration of visual information, within a widely distributed network of the left hemisphere. Nevertheless, the theory has not been fully studied in neural bases. The aim of this study is to examine role of the l-pMTG on shape recognition and its reconstruction within drawing behavior, by using a combining method of the repetitive transcranial magnetic stimulation (rTMS) and functional near-infrared spectroscopy (fNIRS). Eighteen healthy participants received a low frequency inhibitory rTMS to their l-pMTG during the drawing task of the Benton Visual Retention Test (BVRT). There was a significant decrease of the mean accuracy of reproductions in the Complex designs of the BVRT, compared to the Simple and Medium designs. The fNIRS data showed strong negative correlations with the results of the BVRT. Though our hypothesis had a contradiction that rTMS would have inhibited the brain activity in the stimulated site, the results suggest that shape recognition and its reconstruction such as the BVRT require neural activations of the l-TL as well as that of the l-pMTG.

In small and large spinal dorsal root ganglion neurons, subtypes of voltage-gated sodium channels, such as NaV1.7, NaV1.8, and NaV1.9 are expressed with characteristically localized and may play different roles in pain transmission and intractable pain development. Selective stimulation of each specific subtype in vivo may elucidate its role of each subtype in pain. So far, this has been difficult with current technology. However, Optogenetics, a recently developed technique, has enabled selective activation or inhibition of specific neural circulation in vivo. Moreover, optogenetics had even been used to selectively excite NaV1.8-expressing dorsal root ganglion neurons to induce nocifensive behavior. In recent years, genetic modification technologies such as CRISPR/Cas9 have advanced, and various knock-in mice can be easily generated using such technology. We aimed to investigate the effects of selective optogenetic activation of NaV1.7-expressing afferents on mouse behavior. We used CRISPR/Cas9-mediated homologous recombination to generate bicistronic NaV1.7–iCre knock-in mice, which express iCre recombinase under the endogenous NaV1.7 gene promoter without disrupting NaV1.7. The Cre-driver mice were crossed with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7iCre/+;Ai32/+, NaV1.7iCre/iCre;Ai32/+, NaV1.7iCre/+;Ai32/Ai32, and NaV1.7iCre/iCre;Ai32/Ai32 mice. Compared with wild–type mice behavior, no differences were observed in the behaviors associated with mechanical and thermal stimuli exhibited by mice of the aforementioned genotypes, indicating that the endogenous NaV1.7 gene was not affected by the targeted insertion of iCre. Blue light irradiation to the hind paw induced paw withdrawal by mice of all genotypes in a light power-dependent manner. The threshold and incidence of paw withdrawal and aversive behavior in a blue-lit room were dependent on ChR2 expression level; the strongest response was observed in NaV1.7iCre/iCre;Ai32/Ai32 mice. Thus, we developed a non-invasive pain model in which peripheral nociceptors were optically activated in free-moving transgenic NaV1.7–ChR2 mice.

NAD(P)H quinone oxidoreductase 1 (NQO1) is a flavoprotein that catalyzes two-electron reduction of quinone to hydroquinone by using nicotinamide adenine dinucleotide (NADPH), and functions as a scavenger for reactive oxygen species (ROS). The function of NQO1 in the immune response is not well known. In the present study, we demonstrated that Nqo1-deficient T cells exhibited reduced induction of T helper 17 cells (Th17) in vitro during Th17(23)- and Th17(β)- skewing conditions. Nqo1-deficient mice showed ameliorated symptoms in a Th17-dependent autoimmune Experimental autoimmune encephalomyelitis (EAE) model. Impaired Th17-differentiation was caused by overproduction of the immunosuppressive cytokine, IL-10. Increased IL-10 production in Nqo1-deficient Th17 cells was associated with elevated intracellular Reactive oxygen species (ROS) levels. Furthermore, overproduction of IL-10 in Th17 (β) cells was responsible for the ROS-dependent increase of c-avian musculoaponeurotic fibrosarcoma (c-maf) expression, despite the lack of dependency of c-maf in Th17(23) cells. Taken together, the results reveal a novel role of NQO1 in promoting Th17 development through the suppression of ROS mediated IL-10 production.

Recent studies have shown that colorectal serrated lesions, which include sessile serrated adenomas (SSAs) and traditional serrated adenomas (TSAs), are precursors of colorectal cancer. However, the molecular mechanisms underlying the carcinogenesis, particularly in TSAs, remain largely uncharacterized. To clarify their molecular and clinicopathological characteristics, we performed mutation and methylation analyses of cancer-associated genes in 78 serrated lesions, including TSAs, SSAs and microvesicular hyperplastic polyps. Target exon sequence analysis was performed with 39 genes, including genes known to be frequently mutated in colorectal cancers and/or serrated lesions. We also used bisulfite pyrosequencing to assess the methylation status of various cancer-associated genes and marker genes of the CpG island methylator phenotype (CIMP). The prevalence of mutations in genes associated with Wnt signaling was significantly higher in TSAs than SSAs (65% vs. 28%, p < 0.01). Among those, RNF43 mutations were observed in 38% of TSAs and 17% of SSAs. In immunohistochemical studies of 39 serrated lesions, the prevalence of abnormal nuclear β-catenin accumulation was significantly higher in TSAs (57%) than SSAs (8%) (P = 0.01). SMOC1 methylation was detected in 54% of TSAs but in no SSAs (p < 0.01). Additionally, SMOC1 methylation was more prevalent among TSAs with KRAS mutation (82%) than with BRAF mutation (38%, p = 0.03). Lesions with CIMP-high or RNF43 mutations were detected only in TSAs with BRAF mutation, suggesting two distinct carcinogenic pathways in TSAs. Mutations in genes associated with Wnt signaling play a greater role in the carcinogenesis of TSAs than SSAs.

Despite recent advances in clinical treatment, pancreatic cancer remains a highly lethal malignancy. In order to improve the survival rate of patients with pancreatic cancer, the development of non-invasive diagnostic methods using effective biomarkers is urgently needed. Here, we developed a highly sensitive method to detect DNA methylation in cell-free (cf)DNA samples based on the enrichment of methyl-CpG binding (MBD) protein coupled with a digital PCR method (MBD-ddPCR). Five DNA methylation markers for the diagnosis of pancreatic cancer were identified through DNA methylation microarray analysis in 37 pancreatic cancers. The sensitivity and specificity of the five markers were validated in another independent cohort of pancreatic cancers (100% and 100%, respectively; n = 46) as well as in The Cancer Genome Atlas data set (96% and 90%, respectively; n = 137). MBD-ddPCR analysis revealed that DNA methylation in at least one of the five markers was detected in 23 (49%) samples of cfDNA from 47 patients with pancreatic cancer. Further, a combination of DNA methylation markers and the KRAS mutation status improved the diagnostic capability of this method (sensitivity and specificity, 68% and 86%, respectively). Genome-wide MBD-sequencing analysis in cancer tissues and corresponding cfDNA revealed that more than 80% of methylated regions were overlapping; DNA methylation profiles of cancerous tissues and cfDNA significantly correlated with each other (R = 0.97). Our data indicate that newly developed MBD-ddPCR is a sensitive method to detect cfDNA methylation and that using five marker genes plus KRAS mutations may be useful for the detection of pancreatic cancers.

Calcium-binding proteins regulate ion metabolism and the necessary signaling pathways for the maturational events of sperm. Our aim is to identify the novel calcium-binding proteins in testis. The gene EFCAB2 (GenBank NM_026626.3, NP_080902.1) was not previously examined, and its properties and exact mechanisms of action are unknown. In this study, we performed phylogenetic and structure prediction analyses of EFCAB2, which displays definitive structural features. Additionally, the distribution, localization, and calcium binding ability of mouse EFCAB2 were investigated. Results revealed extensive conservation of EFCAB2 among different eukaryotic orthologs. The constructed 3D model predicted that mouse EFCAB2 contains seven α-helices and two EF-hand motifs. The first EF-hand motif is located in N-terminal, while the second is located in C-terminal. By aligning the 3D structure of Ca2+-binding loops from EFCAB2 with calmodulin, we predicted six residues that might be involved in Ca2+ binding. The distribution of the Efcab2 mRNA, as determined by northern blotting, was detected only in the testis among mouse tissues. Native and recombinant EFCAB2 protein were detected by western blotting as one band at 20 kDa. In situ hybridization and immunohistochemical analyses showed its localization specifically in spermatogenic cells from primary spermatocytes to elongate spermatids within the seminiferous epithelium, but neither spermatogonia nor somatic cells were expressed. Moreover, EFCAB2 was specifically localized to the principal piece of cauda epididymal sperm flagellum. Furthermore, the analyses of purified recombinant EFCAB2 by Stains-all, ruthenium red staining, and by applying in vitro autoradiography assay showed that the physiological function of this protein is Ca2+ binding. These results suggested that EFCAB2 might be involved in the control of sperm flagellar movement. Altogether, here we describe about EFCAB2 as a novel calcium-binding protein in mouse testis and sperm.

Vascular endothelial growth factor receptor 3 (Vegfr3) has been widely used as a marker for lymphatic and vascular endothelial cells during mouse embryonic development and in adult mouse, making it valuable for studying angiogenesis and lymphangiogenesis under normal and pathological conditions. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a membrane-localized fluorescent reporter protein, Gap43-Venus, under the control of the Vegfr3 regulatory sequence. Vegfr3-Gap43-Venus BAC Tg recapitulated endogenous Vegfr3 expression in vascular and lymphatic endothelial cells during embryonic development and tumor development. Thus, this Tg mouse line contributes a valuable model to study angiogenesis and lymphangiogenesis in physiological and pathological contexts.

The transcription factor MAFB is an important regulator of the development and differentiation of various organs and tissues. Previous studies have shown that MAFB is expressed in embryonic and adult mouse testes and is expected to act as the downstream target of retinoic acid (RA) to initiate spermatogenesis. However, its exact localization and function remain unclear. Here, we localized MAFB expression in embryonic and adult testes and analyzed its gene function using Mafb-deficient mice. We found that MAFB and c-MAF are the only large MAF transcription factors expressed in testes, while MAFA and NRL are not. MAFB was localized in Leydig and Sertoli cells at embryonic day (E) 18.5 but in Leydig cells, Sertoli cells, and pachytene spermatocytes in adults. Mafb-deficient testes at E18.5 showed fully formed seminiferous tubules with no abnormal structure or differences in testicular somatic cell numbers compared with those of control wild-type mice. Additionally, the expression levels of genes related to development and function of testicular cells were unchanged between genotypes. In adults, the expression of MAFB in Sertoli cells was shown to be stage specific and induced by RA. By generating Mafbfl/fl CAG-CreER™ (Mafb-cKO) mice, in which Cre recombinase was activated upon tamoxifen treatment, we found that the neonatal cKO mice died shortly upon Mafb deletion, but adult cKO mice were alive upon deletion. Adult cKO mice were fertile, and spermatogenesis maintenance was normal, as indicated by histological analysis, hormone levels, and germ cell stage-specific markers. Moreover, there were no differences in the proportion of seminiferous stages between cKO mice and controls. However, RNA-Seq analysis of cKO Sertoli cells revealed that the down-regulated genes were related to immune function and phagocytosis activity but not spermatogenesis. In conclusion, we found that MAFB is dispensable for fetal testis morphogenesis and spermatogenesis maintenance in adult mice, despite the significant gene expression in different cell types, but MAFB might be critical for phagocytosis activity of Sertoli cells.

Mouse embryonic stem cells (ESCs) are pluripotent stem cells, which have the ability to differentiate into all three germ layers: mesoderm, endoderm, and ectoderm. Proper levels of phosphorylated extracellular signal-regulated kinase (pERK) are critical for maintaining pluripotency, as elevated pERK evoked by fibroblast growth factor (FGF) receptor activation results in differentiation of ESCs, while, conversely, reduction of pERK by a MEK inhibitor maintains a pluripotent ground state. However, mechanisms underlying proper control of pERK levels in mouse ESCs are not fully understood. Here, we find that Klf5, a Krüppel-like transcription factor family member, is a component of pERK regulation in mouse ESCs. We show that ERK signaling is overactivated in Klf5-KO ESCs and the overactivated ERK in Klf5-KO ESCs is suppressed by the introduction of Klf5, but not Klf2 or Klf4, indicating a unique role for Klf5 in ERK suppression. Moreover, Klf5 regulates Spred1, a negative regulator of the FGF-ERK pathway. Klf5 also facilitates reprogramming of EpiSCs into a naïve state in combination with a glycogen synthase kinase 3 inhibitor and LIF, and in place of a MEK inhibitor. Taken together, these results show for the first time that Klf5 has a unique role suppressing ERK activity in mouse ESCs.

Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) chain. In cartilage, CS plays important roles as the main component of the extracellular matrix (ECM), existing as side chains of the major cartilage proteoglycan, aggrecan. Six glycosyltransferases are known to coordinately synthesize the backbone structure of CS; however, their in vivo synthetic mechanism remains unknown. Previous studies have suggested that two glycosyltransferases, Csgalnact1 (t1) and Csgalnact2 (t2), are critical for initiation of CS synthesis in vitro. Indeed, t1 single knockout mice (t1 KO) exhibit slight dwarfism and a reduction in CS content in cartilage compared with wild-type (WT) mice. To reveal the synergetic roles of t1 and t2 in CS synthesis in vivo, we generated systemic single and double knockout (DKO) mice and cartilage-specific t1 and t2 double knockout (Col2-DKO) mice. DKO mice exhibited postnatal lethality, whereas t2 KO mice showed normal size and skeletal development. Col2-DKO mice survived to adulthood and showed severe dwarfism compared with t1 KO mice. Histological analysis of epiphyseal cartilage from Col2-DKO mice revealed disrupted endochondral ossification, characterized by drastic GAG reduction in the ECM. Moreover, DKO cartilage had reduced chondrocyte proliferation and an increased number of apoptotic chondrocytes compared with WT cartilage. Conversely, primary chondrocyte cultures from Col2-DKO knee cartilage had the same proliferation rate as WT chondrocytes and low GAG expression levels, indicating that the chondrocytes themselves had an intact proliferative ability. Quantitative RT-PCR analysis of E18.5 cartilage showed that the expression levels of Col2a1 and Ptch1 transcripts tended to decrease in DKO compared with those in WT mice. The CS content in DKO cartilage was decreased compared with that in t1 KO cartilage but was not completely absent. These results suggest that aberrant ECM caused by CS reduction disrupted endochondral ossification. Overall, we propose that both t1 and t2 are necessary for CS synthesis and normal chondrocyte differentiation but are not sufficient for all CS synthesis in cartilage.

Spaceflight entails various stressful environmental factors including microgravity. The effects of gravity changes have been studied extensively on skeletal, muscular, cardiovascular, immune and vestibular systems, but those on the nervous system are not well studied. The alteration of gravity in ground-based animal experiments is one of the approaches taken to address this issue. Here we investigated the effects of centrifugation-induced gravity changes on gene expression of brain-derived neurotrophic factor (BDNF) and serotonin receptors (5-HTRs) in the mouse brain. Exposure to 2g hypergravity for 14 days showed differential modulation of gene expression depending on regions of the brain. BDNF expression was decreased in the ventral hippocampus and hypothalamus, whereas increased in the cerebellum. 5-HT1BR expression was decreased in the cerebellum, whereas increased in the ventral hippocampus and caudate putamen. In contrast, hypergravity did not affect gene expression of 5-HT1AR, 5-HT2AR, 5-HT2CR, 5-HT4R and 5-HT7R. In addition to hypergravity, decelerating gravity change from 2g hypergravity to 1g normal gravity affected gene expression of BDNF, 5-HT1AR, 5-HT1BR, and 5-HT2AR in various regions of the brain. We also examined involvement of the vestibular organ in the effects of hypergravity. Surgical lesions of the inner ear's vestibular organ removed the effects induced by hypergravity on gene expression, which suggests that the effects of hypergravity are mediated through the vestibular organ. In summary, we showed that gravity changes induced differential modulation of gene expression of BDNF and 5-HTRs (5-HT1AR, 5-HT1BR and 5-HT2AR) in some brain regions. The modulation of gene expression may constitute molecular bases that underlie behavioral alteration induced by gravity changes.

Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial disease in humans. The role of Th17 immunity in the pathogenesis of intracellular bacteria, such as MAC, is not currently understood. Transcription factor RAR-related orphan receptor gamma t (RORγt) is known as the master regulator for Th17 cell development. Here, we investigated the role of RORγt in host responses against MAC infection. Wild-type (WT) mice and RORγt-overexpressing mice were infected with MAC via intratracheal inoculation. Systemic MAC growth was not different between WT mice and RORγt-overexpressing mice. However, neutrophilic pulmonary inflammation following MAC infection was enhanced in RORγt-overexpressing mice compared with that in WT mice. The cytokine expression shifted toward a Th17 phenotype in the lungs of RORγt-overexpressing mice following MAC infection; the levels of IL-6 and IL-17 were significantly higher in the lung of these mice than in WT mice. In addition to the increase in IL-17 single-positive T cells, T cells producing both IL-17 and interferon-γ were elevated in the lung of RORγt-overexpressing mice following MAC infection. These findings suggest that RORγt overexpression-mediated Th17 bias contributes to local inflammation rather than systemic responses, by regulating neutrophil recruitment into the sites of infection during MAC infection.

Fibroblast growth factor 5 (Fgf5) has been widely used as a marker for the epiblast in the postimplantation embryo and epiblast stem cells (mEpiSCs) in the mouse, making it valuable for study of differentiation of various tissues and epiblast cells in vivo and in vitro. Here, we report for the first time the generation of Fgf5-P2A-Venus BAC transgenic (Tg) mice and show that the BAC Tg can recapitulate endogenous Fgf5 expression in epiblast and visceral endodermal cells of E6.5 and 7.5 embryos. We also show that Fgf5-P2A-Venus BAC Tg mEpiSCs in the undifferentiated state expressed abundant Venus, and upon reprogramming into naïve state, Venus was suppressed. Furthermore, while most Tg mEpiSCs expressed Venus abundantly, surprisingly the Tg mEpiSCs contained a minor subpopulation of Venus-negative cells that were capable of conversion to Venus-positive cells, indicating that even Fgf5 expression shows dynamic heterogeneity in mEpiSCs. Taken together, Fgf5-P2A-Venus BAC Tg mice and mEpiSCs generated in this study will be useful for developmental biology as well as stem cell biology research.

Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1-Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.

The MAF family transcription factors are homologs of v-Maf, the oncogenic component of the avian retrovirus AS42. They are subdivided into 2 groups, small and large MAF proteins, according to their structure, function, and molecular size. MAFK is a member of the small MAF family and acts as a dominant negative form of large MAFs. In previous research we generated transgenic mice that overexpress MAFK in order to suppress the function of large MAF proteins in pancreatic β-cells. These mice developed hyperglycemia in adulthood due to impairment of glucose-stimulated insulin secretion. The aim of the current study is to examine the effects of β-cell-specific Mafk overexpression in endocrine cell development. The developing islets of Mafk-transgenic embryos appeared to be disorganized with an inversion of total numbers of insulin+ and glucagon+ cells due to reduced β-cell proliferation. Gene expression analysis by quantitative RT-PCR revealed decreased levels of β-cell-related genes whose expressions are known to be controlled by large MAF proteins. Additionally, these changes were accompanied with a significant increase in key β-cell transcription factors likely due to compensatory mechanisms that might have been activated in response to the β-cell loss. Finally, microarray comparison of gene expression profiles between wild-type and transgenic pancreata revealed alteration of some uncharacterized genes including Pcbd1, Fam132a, Cryba2, and Npy, which might play important roles during pancreatic endocrine development. Taken together, these results suggest that Mafk overexpression impairs endocrine development through a regulation of numerous β-cell-related genes. The microarray analysis provided a unique data set of differentially expressed genes that might contribute to a better understanding of the molecular basis that governs the development and function of endocrine pancreas.

To elucidate the pure impact of microgravity on small mammals despite uncontrolled factors that exist in the International Space Station, it is necessary to construct a 1 g environment in space. The Japan Aerospace Exploration Agency has developed a novel mouse habitat cage unit that can be installed in the Cell Biology Experiment Facility in the Kibo module of the International Space Station. The Cell Biology Experiment Facility has a short-arm centrifuge to produce artificial 1 g gravity in space for mouse experiments. However, the gravitational gradient formed inside the rearing cage is larger when the radius of gyration is shorter; this may have some impact on mice. Accordingly, biological responses to hypergravity induced by a short-arm centrifuge were examined and compared with those induced by a long-arm centrifuge. Hypergravity induced a significant Fos expression in the central nervous system, a suppression of body mass growth, an acute and transient reduction in food intake, and impaired vestibulomotor coordination. There was no difference in these responses between mice raised in a short-arm centrifuge and those in a long-arm centrifuge. These results demonstrate the feasibility of using a short-arm centrifuge for mouse experiments.

Our recent prospective study found equivalent accuracy of magnifying chromoendoscopy (MC) and endoscopic ultrasonography (EUS) for diagnosing the invasion depth of colorectal cancer (CRC); however, whether these tools show diagnostic differences in categories such as tumor size and morphology remains unclear. Hence, we conducted detailed subset analysis of the prospective data.In this multicenter, prospective, comparative trial, a total of 70 patients with early, flat CRC were enrolled from February 2011 to December 2012, and the results of 66 lesions were finally analyzed. Patients were randomly allocated to primary MC followed by EUS or to primary EUS followed by MC. Diagnoses of invasion depth by each tool were divided into intramucosal to slight submucosal invasion (invasion depth

To investigate the diagnostic performance of diffusion-weighted imaging (DWI) and contrast-enhanced imaging in combination with T2-weighted imaging (T2WI) for magnetic resonance imaging (MRI) evaluation of intrapelvic recurrence of gynecological malignancies.Sixty-two patients with suspected intrapelvic recurrence of gynecological malignancies underwent pelvic MRI including T2WI DWI, and contrast-enhanced imaging. Diagnostic performance for detection of local recurrence, pelvic lymph node and bone metastases, and peritoneal lesions was evaluated by consensus reading of two experienced radiologists using a 5-point scoring system, and compared among T2WI with unenhanced T1-weighted imaging (T1WI) (protocol A), a combination of protocol A and DWI (protocol B), and a combination of protocol B and contrast-enhanced imaging (protocol C). Final diagnoses were obtained by histopathological examinations, radiological imaging and clinical follow-up for at least 6 months. Receiver operating characteristic (ROC) analysis and McNemar test were employed for statistical analysis.Locally recurrent disease, lymph node recurrence, peritoneal dissemination and bone metastases were present in 48.4%, 29.0%, 16.1%, and 6.5% of the patients, respectively. The patient-based sensitivity, specificity, accuracy, and area under the ROC curve (AUC) for detection of intrapelvic recurrence were 55.0, 81.8, 64.5% and 0.753 for protocol A, 80.0, 77.3, 79.0% and 0.838 for protocol B, and 80.0, 90.9, 83.9% and 0.862 for protocol C, respectively. The sensitivity, accuracy, and AUC were significantly better for protocols B and C than for protocol A (p

Gravity change affects many immunological systems. We investigated the effects of hypergravity (2G) on murine thymic cells. Exposure of mice to 2G for three days reduced the frequency of CD4+CD8+ thymocytes (DP) and mature medullary thymic epithelial cells (mTECs), accompanied by an increment of keratin-5 and keratin-8 double-positive (K5+K8+) TECs that reportedly contain TEC progenitors. Whereas the reduction of DP was recovered by a 14-day exposure to 2G, the reduction of mature mTECs and the increment of K5+K8+ TEC persisted. Interestingly, a surgical lesion of the inner ear's vestibular apparatus inhibited these hypergravity effects. Quantitative PCR analysis revealed that the gene expression of Aire and RANK that are critical for mTEC function and development were up-regulated by the 3-day exposure and subsequently down-regulated by the 14-day exposure to 2G. Unexpectedly, this dynamic change in mTEC gene expression was independent of the vestibular apparatus. Overall, data suggest that 2G causes a temporary reduction of DP and a persistent reduction of mature mTECs in a vestibular system-dependent manner, and also dysregulates mTEC gene expression without involving the vestibular system. These data might provide insight on the impact of gravity change on thymic functions during spaceflight and living.

Human natural killer-1 (HNK-1) carbohydrate (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-R) is highly expressed in the brain and required for learning and neural plasticity. We previously demonstrated that expression of the HNK-1 epitope is mostly abolished in knockout mice for GlcAT-P (B3gat1), a major glucuronyltransferase required for HNK-1 biosynthesis, but remained in specific regions such as perineuronal nets (PNNs) in these mutant mice. Considering PNNs are mainly composed of chondroitin sulfate proteoglycans (CSPGs) and regulate neural plasticity, GlcAT-P-independent expression of HNK-1 in PNNs is suggested to play a role in neural plasticity. However, the function, structure, carrier glycoprotein and biosynthetic pathway for GlcAT-P-irrelevant HNK-1 epitope remain unclear. In this study, we identified a unique HNK-1 structure on aggrecan in PNNs. To determine the biosynthetic pathway for the novel HNK-1, we generated knockout mice for GlcAT-S (B3gat2), the other glucuronyltransferase required for HNK-1 biosynthesis. However, GlcAT-P and GlcAT-S double-knockout mice did not exhibit reduced HNK-1 expression compared with single GlcAT-P-knockout mice, indicating an unusual biosynthetic pathway for the HNK-1 epitope in PNNs. Aggrecan was purified from cultured cells in which GlcAT-P and -S are not expressed and we determined the structure of the novel HNK-1 epitope using liquid chromatography/mass spectrometry (LC/MS) as a sulfated linkage region of glycosaminoglycans (GAGs), HSO3-GlcA-Gal-Gal-Xyl-R. Taken together, we propose a hypothetical model where GlcAT-I, the sole glucuronyltransferase required for synthesis of the GAG linkage, is also responsible for biosynthesis of the novel HNK-1 on aggrecan. These results could lead to discovery of new roles of the HNK-1 epitope in neural plasticity.

Recent studies on the large Maf transcription factors have shown that Mafb and Mafa have respective and distinctive roles in β-cell development and maturation. However, whether this difference in roles is due to the timing of the gene expression (roughly, expression of Mafb before birth and of Mafa after birth) or to the specific function of each gene is unclear. Our aim was to examine the functional differences between these genes that are closely related to β cells by using an in vivo model of β-like cell generation. We monitored insulin gene transcription by measuring bioluminescence emitted from the liver of insulin promoter-luciferase transgenic (MIP-Luc-VU) mice. Adenoviral gene transfers of Pdx1/Neurod/Mafa (PDA) and Pdx1/Neurod/Mafb (PDB) combinations generated intense luminescence from the liver that lasted for more than 1 week and peaked at 3 days after transduction. The peak signal intensities of PDA and PDB were comparable. However, PDA but not PDB transfer resulted in significant bioluminescence on day 10, suggesting that Mafa has a more sustainable role in insulin gene activation than does Mafb. Both PDA and PDB transfers ameliorated the glucose levels in a streptozotocin (STZ)-induced diabetic model for up to 21 days and 7 days, respectively. Furthermore, PDA transfer induced several gene expressions necessary for glucose sensing and insulin secretion in the liver on day 9. However, a glucose tolerance test and liver perfusion experiment did not show glucose-stimulated insulin secretion from intrahepatic β-like cells. These results demonstrate that bioluminescence imaging in MIP-Luc-VU mice provides a noninvasive means of detecting β-like cells in the liver. They also show that Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb.

The postnatal proliferation and maturation of insulin-secreting pancreatic β-cells are critical for glucose metabolism and disease development in adults. Elucidation of the molecular mechanisms underlying these events will be beneficial to direct the differentiation of stem cells into functional β-cells. Maturation of β-cells is accompanied by increased expression of MafA, an insulin gene transcription factor. Transcriptome analysis of MafA knockout islets revealed MafA is required for the expression of several molecules critical for β-cell function, including Glut2, ZnT8, Granuphilin, Vdr, Pcsk1 and Urocortin 3, as well as Prolactin receptor (Prlr) and its downstream target Cyclin D2 (Ccnd2). Inhibition of MafA expression in mouse islets or β-cell lines resulted in reduced expression of Prlr and Ccnd2, and MafA transactivated the Prlr promoter. Stimulation of β-cells by prolactin resulted in the phosphorylation and translocation of Stat5B and an increased nuclear pool of Ccnd2 via Prlr and Jak2. Consistent with these results, the loss of MafA resulted in impaired proliferation of β-cells at 4 weeks of age. These results suggest that MafA regulates the postnatal proliferation of β-cells via prolactin signaling.

Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. UTF1 is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the UTF1 gene during mouse development. We found that homozygous UTF1 mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of UTF1 expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of UTF1 expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the UTF1 gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the UTF1 genes.

In diabetes research, bioluminescence imaging (BLI) has been applied in studies of β-cell impairment, development, and islet transplantation. To develop a mouse model that enables noninvasive imaging of β cells, we generated a bacterial artificial chromosome (BAC) transgenic mouse in which a mouse 200-kbp genomic fragment comprising the insulin I gene drives luciferase expression (Ins1-luc BAC transgenic mouse). BLI of mice was performed using the IVIS Spectrum system after intraperitoneal injection of luciferin, and the bioluminescence signal from the pancreatic region analyzed. When compared with MIP-Luc-VU mice [FVB/N-Tg(Ins1-luc)VUPwrs/J] expressing luciferase under the control of the 9.2-kbp mouse insulin I promoter (MIP), the bioluminescence emission from Ins1-luc BAC transgenic mice was enhanced approximately 4-fold. Streptozotocin-treated Ins1-luc BAC transgenic mice developed severe diabetes concomitant with a sharp decline in the BLI signal intensity in the pancreas. Conversely, mice fed a high-fat diet for 8 weeks showed an increase in the signal, reflecting a decrease or increase in the β-cell mass. Although the bioluminescence intensity of the islets correlated well with the number of isolated islets in vitro, the intensity obtained from a living mouse in vivo did not necessarily reflect an absolute quantification of the β-cell mass under pathological conditions. On the other hand, adenovirus-mediated gene transduction of β-cell-related transcription factors in Ins1-luc BAC transgenic mice generated luminescence from the hepatic region for more than 1 week. These results demonstrate that BLI in Ins1-luc BAC transgenic mice provides a noninvasive method of imaging islet β cells and extrapancreatic activity of the insulin gene in the liver under normal and pathological conditions.

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt)) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt) embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt) ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt) ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.