👤 Nayuta Nakazawa

Methylprednisolone (mPSL) pulse therapy is an essential treatment for systemic lupus erythematosus (SLE); however, it carries a risk of osteonecrosis of the femoral head (ONFH). The pathogenesis of ONFH involves neutrophil extracellular trap (NET)-mediated microcirculation disorders. In BALB/c mice with imiquimod (IMQ)-induced lupus, mPSL pulse elevated serum levels of prenylcysteine oxidase 1 (PCYOX1), an enzyme that produces NET inducers hydrogen peroxide and farnesal, resulting in increased NETs in vivo. Although ischemia was observed in the femoral head, IMQ + mPSL-treated BALB/c mice did not develop ONFH. PCYOX1 is abundant in very-low-density lipoproteins. This study aimed to demonstrate that hyperlipidemia exacerbates NET-mediated microcirculation disorders and leads to ONFH development following mPSL pulse in lupus mice. To address this, ApoE mutant hyperlipidemic and BALB/c mice with IMQ-induced lupus received mPSL pulse. NET-forming neutrophils in peripheral blood were detected by flow cytometry. ONFH was assessed microscopically. As a result, IMQ + mPSL-treated ApoE mutant but not BALB/c mice developed ONFH, exhibiting higher levels of PCYOX1 and NET-forming neutrophils in circulation. In addition, NET-forming neutrophils accumulated in the vessels surrounding the femoral head, accompanied by osteocyte necrosis. This study demonstrated that mPSL pulse in lupus mice with hyperlipidemia enhanced PCYOX1 levels and NET formation, resulting in ONFH development, suggesting that hyperlipidemia may be a risk factor for ONFH following mPSL pulse therapy in SLE. Show less

APOE polymorphisms are major genetic risk factors of Alzheimer's disease (AD). Compared with APOE3/E3, the APOE4/E4 genotype is associated with a > 14-fold increased risk. Therefore, we hypothesized that conversion of APOE4 to APOE3 would ameliorate AD-related pathologies. Accordingly, we generated a knock-in mouse model harboring an APOE4-FLEx (Flip-Excision) 4-to-3 construct enabling postnatal Cre-mediated APOE4-to-APOE3 switching. This construct comprised an APOE3 exon inserted in a reverse orientation downstream of the APOE4 exon, flanked by alternating loxP and mutant loxP sites, allowing Cre-mediated FLEx switching from APOE4-to-APOE3. For in vitro validation, HEK293T cells were transfected with APOE4-FLEx 4-to-3 plasmid, followed by AAV8-mediated iCre delivery. For in vivo studies, endogenous Apoe was replaced with the APOE4-FLEx 4-to-3 construct to generate APOE4-FLEx 4-to-3 knock-in mice, which were crossed with tamoxifen-inducible Rosa26-CreERT2 mice to yield Cre: APOE4-FLEx 4-to-3 double-knock-in mice. Tamoxifen was administered to induce APOE switching. Cre expression successfully induced APOE4-to-APOE3 switching in vitro. Tamoxifen administration in Cre: APOE4-FLEx 4-to-3 mice triggered APOE4-to-APOE3 switching in the liver, demonstrating the feasibility of postnatal isoform switching. However, brain APOE protein levels were below the detection limit. Investigation of the underlying cause involving transcript analysis revealed aberrant retention of intron 3 (APOE-I3). This abnormal splicing probably contributed to the decreased expression of fully spliced, translation-competent (mature) APOE mRNA, driving the subsequent protein reduction. Although APOE expression across organs in APOE4-FLEx 4-to-3 mice requires further optimization, our findings demonstrate that Cre-mediated FLEx switching can serve as a potential strategy to induce APOE genotype switching in vivo. Show less

Transcriptional factors, such as Snail, Slug, and Smuc, that cause epithelial-mesenchymal transition are thought to regulate the expression of Ezrin, Radixin, and Moesin (ERM proteins), which serve as anchors for efflux transporters on the plasma membrane surface. Our previous results using lung cancer clinical samples indicated a correlation between Slug and efflux transporter MRP2. In the current study, we aimed to evaluate the relationships between MRP2, ERM proteins, and Slug in lung cancer cells. HCC827 cells were transfected by Mock and Slug plasmid. Both mRNA expression levels and protein expression levels were measured. Then, the activity of MRP2 was evaluated using CDCF and SN-38 (MRP2 substrates). HCC827 cells transfected with the Slug plasmid showed significantly higher mRNA expression levels of MRP2 than the Mock-transfected cells. However, the mRNA expression levels of ERM proteins did not show a significant difference between Slug-transfected cells and Mock-transfected cells. Protein expression of MRP2 was increased in Slug-transfected cells. The uptake of both CDCF and SN-38 was significantly decreased after transfection with Slug. This change was abrogated by treatment with MK571, an MRP2 inhibitor. The viability of Slug-transfected cells, compared to Mock cells, significantly increased after incubation with SN-38. Thus, Slug may increase the mRNA and protein expression of MRP2 without regulation by ERM proteins in HCC827 cells, thereby enhancing MRP2 activity. Inhibition of Slug may reduce the efficacy of multidrug resistance in lung cancer. Show less

Industrial yeasts are generally unable to sporulate but treatment with the immunosuppressive drug rapamycin restores this ability in a sake yeast strain Kyokai no. 7 (K7), Saccharomyces cerevisiae. This finding suggests that TORC1 is active under sporulation conditions. Here, using a reporter gene assay, Northern and Western blots, we tried to gain insight into how TORC1 function under nitrogen starvation conditions in K7 cells. Similarly to a laboratory strain, RPS26A transcription was repressed and Npr1 was dephosphorylated in K7 cells, indicative of the expected loss of TORC1 function under nitrogen starvation. The expression of nitrogen catabolite repression-sensitive genes, however, was not induced, the level of Cln3 remained constant, and autophagy was more slowly induced than in a laboratory strain, all suggestive of active TORC1. We conclude that TORC1 activity is partially reduced under nitrogen starvation conditions in K7 cells. Show less

The hepatocyte paraffin 1 (Hep Par 1) antibody is widely used as a hepatocyte marker, recognizing carbamoyl phosphate synthetase 1 (CPS1), an essential component of the urea cycle. Various missense, nonsense, and frameshift mutations occur in the CPS1 gene. In neonatal patients with homozygous CPS1 deficiency (CPS1D), urea cycle defects with resulting severe hyperammonemia can be fatal, though liver transplantation provides a complete cure for CPS1D. We performed Hep Par 1 immunostaining in the explanted livers of 10 liver transplant patients with CPS1D. Seven were negative for Hep Par 1 in the hepatocytes and the other three showed normal diffuse granular cytoplasmic staining. As expected, all three Hep Par 1-positive patients had at least one missense mutation, and all four patients who had only nonsense or frameshift mutations were Hep Par 1-negative. The other three patients were unexpectedly negative for Hep Par 1, even though each had one missense mutation. These results suggest that CPS1D can be related to the loss of Hep Par 1 reactivity due to the loss of protein production, a one amino acid substitution resulting in an abortive protein product, or both. Hep Par 1 immunohistochemistry can be used as a simple method to confirm CPS1D. Show less

Industrial yeasts, including a sake yeast strain Kyokai no. 7 (K7), are generally unable to sporulate. Previously, we have reported that in K7 (Saccharomyces cerevisiae) cells, deletion of the G1 cyclin gene CLN3, a key activator of the cell cycle, allows the cells to induce IME1 transcription and sporulate under sporulation conditions. Here we show that treatment with the immunosuppressive drug rapamycin also restores sporulation competence in K7 cells. Moreover, sporulation was observed after rapamycin treatment in other industrial yeasts, namely bottom fermenting yeast strains and a wine yeast strain, which are not able to sporulate under normal sporulation conditions. These findings suggest that activation of TORC1 under sporulation conditions leads to sporulation incompetence in these yeasts. Thus, rapamycin treatment will be useful to restore sporulation competence in industrial yeasts. Show less

Industrial yeasts, including a sake yeast Kyokai no. 7 (K7), are generally unable to sporulate. In K7 (Saccharomyces cerevisiae) cells, IME1 transcription was not induced under sporulation conditions, and K7 cells partially restored sporulation ability when transformed with a multicopy plasmid bearing IME1. However, the mechanisms of sporulation incompetence in industrial yeasts are poorly understood. We demonstrated that the deletion of the G1 cyclin CLN3, a key activator of the cell cycle, allows K7 cells to induce IME1 transcription and sporulate under sporulation conditions. In K7 cells, CLN3 mRNA and protein were not down-regulated despite sporulation conditions. Moreover, using a two-hybrid assay, we found that Ime1-Ume6 interaction was promoted in Cln3-deficient K7 cells. Thus, Cln3 is involved in the mechanism underlying sporulation incompetence by inhibiting IME1 transcription and the Ime1-Ume6 interaction. Based on these findings, we hypothesize that the absence of transmission of nutrient starvation signals to CLN3 leads to sporulation incompetence in K7 cells. Show less

CPS1 is a mitochondrial matrix enzyme that catalyzes the first committed step of the urea cycle, the primary system for removing nitrogen produced by protein metabolism using N-acetylglutamate. Patients with CPS1 deficiency have severe hyperammonemia that results in serious neurologic sequelae and sometimes death. LT has been indicated for neonatal-onset CPS1 deficiency. This study retrospectively reviewed five children with a diagnosis of CPS1 deficiency who underwent LDLT from heterozygous donors. Between November 2005 and May 2010, 124 children underwent LDLT with an overall patient and graft survival of 91.0%. Five patients were indicated for LDLT because of CPS1 deficiency. All recipients achieved resolution of their metabolic derangement, without donor complication, with a normal feeding regimen without medication for their original metabolic liver disease. LDLT, even from heterozygous donors, appears to be a feasible option, associated with a better quality of life for treating patients with CPS1 deficiency. Long-term observation may therefore be necessary to collect sufficient data to confirm the efficacy of this treatment modality. Show less

The Wnt pathway is involved in carcinogenesis and three regulatory genes of the Wnt pathway, APC, beta-catenin and Axin are mutated in some primary human cancers. Mutations in these genes can impair the down regulation of beta-catenin, which results in the stabilization of beta-catenin, accumulation of free beta-catenin and subsequent activation of the Wnt pathway. To clarify the genetic alterations of components of the Wnt pathway in oral squamous cell carcinoma (SCC), we examined mutations in the APC, beta-catenin and Axin genes and subcellular localization of beta-catenin. 20 oral SCC tissues and four cell lines derived from oral SCC were used. Mutational analysis was performed by a single-strand conformation polymorphism (SSCP) method and direct sequencing analysis. The samples were also examined by immunohistochemical staining and immunoblot analysis. In 3 of 4 cell lines, mutations were observed in the APC and Axin1 genes without amino acid substitutions. In a clinical sample, a mutation in the Axin1 gene was detected; a T insertion at codon 250 resulted in the formation of a stop codon at codon 259. In addition, cytoplasmic accumulation of beta-catenin was observed in 3 (75%) of 4 cell lines and 18 (90%) of 20 cancer tissue samples. The Axin1 gene may be one of the mutational target in oral SCC. In addition, the cytoplasmic accumulation of beta-catenin is a common characteristic of oral SCC, but is not closely associated with mutational alterations in the APC, beta-catenin and Axin1 genes. Show less

Notch signaling is required for multiple aspects of cardiovascular development, including arterial-venous differentiation, septation and cushion formation. Despite recognition of the importance of the Notch pathway in normal cardiovascular development, the proximate downstream effectors are not yet known. Likely candidate effectors are members of the hairy and enhancer of split related (hesr) family of bHLH transcription factors. However, mutational analysis of individual hesr genes has so far failed to elucidate their role in all Notch-mediated cardiovascular signaling events. An example of this is evident for mutants of gridlock, the zebrafish counterpart of mouse hesr2, which have vascular defects, whereas mouse hesr2 mutants have only cardiac defects. One possible explanation for these differences could be functional redundancy between hesr family members. Here, we report that mice lacking the hesr1 gene are viable and fertile, whereas knockout mouse of both hesr1 and hesr2 is embryonic lethal at 11.5 days postcoitum (dpc) and recapitulates most of the known cardiovascular phenotypes of disrupted Notch pathway mutants including defects in arterial-venous specification, septation and cushion formation. Taken together, our results demonstrate a requirement for hesr1 and hesr2 in mediating Notch signaling in the developing cardiac and vascular systems. Show less

Genes involved in the Notch signaling pathway have been shown to be critical regulators of cardiovascular development. In vitro studies have revealed that the Notch signaling pathway directly regulates transcription of hairy and enhancer of split-related (hesr) genes, encoding basic helix-loop-helix transcription factors. To assess the functional role of hesr genes in cardiovascular development, we generated mice with a targeted disruption of the hesr2 gene and used echocardiography to analyze heart function of the mutant mice. In the early postnatal period, a majority of hesr2 homozygous mice die as a result of congestive heart failure accompanied by pronounced heart enlargement. Transthoracic echocardiography on 5-day-old homozygous mice revealed tricuspid and mitral valve regurgitation and a dilated left ventricular chamber with markedly diminished fractional shortening of the left ventricle. The hemodynamic anomalies were accompanied by morphological changes, such as dysplastic atrioventricular (AV) valves, a perimembranous ventricular septal defect, and a secundum atrial septal defect. AV valve regurgitations attributable to dysplasia of the AV valves were most likely responsible for the heart dysfunction in hesr2 homozygous mice. These observations indicate that the Notch signaling target hesr2 plays an important role in the formation and function of the AV valves. In addition, hesr2 activity may be important for proper development of cardiomyocytes, thereby assuring normal left ventricular contractility. Because of the unique spectrum of cardiac anomalies expressed by hesr2-null mice, they represent a useful model system for elucidating the genetic basis of heart dysfunction. Show less