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HER1-and HER2-targeted drugs are effective in cancer therapy, especially against lung, breast and colon malignancies; however, resistance of cancer cells to HER1-and HER2-targeted therapies is becoming a serious problem. The avidity/affinity constant (K A ) and growth inhibitory effect of anti-HER3 rat monoclonal antibodies (mAb, Ab1∼Ab6) in the presence of therapeutic mAb or low-molecular-weight inhibitors against HER family proteins were analyzed by flow cytometry-based Scatchard plots (Splot) and cell proliferation assay. The K A of Ab3 and Ab6, but not Ab1 or Ab4, split into dual (high and low) modes of K A , and Ab6 exhibited greater anti-proliferative effects against LS-174T colon cancer cells in the presence of Pertuzumab (anti-HER2 mAb). A high K A by Ab6 and Ab6-mediated increased growth inhibition were observed against NCI-H1838 lung or BT474 breast cancer cells, respectively, in the presence of Panitumumab (anti-HER1 mAb) or Perutuzumab. A high K A by Ab6 and Ab6-mediated increased anti-proliferative effects against NCI-H1838 or BT474 were also respectively observed in the presence of Erlotinib (HER1 inhibitor) or Lapatinib (HER1/HER2 inhibitor). In HER1-knockout (KO) NCI-H1838, the reactivity and K A of Ab4 increased compared with in parent NCI-H1838. In HER1-KO or HER3-KO SW1116 colon cancer cells, dual modes of K A with Pertuzumab were noted, and the combination Ab6 and Pertuzumab promoted growth inhibition of HER1-KO, but not of parent SW1116., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Takashi Masuko reports financial support was provided by Healios KK., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disorder which manifests progressive renal cyst formation and leads to end-stage kidney disease. Around 85% of cases are caused by PKD1 heterozygous mutations, exhibiting relatively poorer renal outcomes than those with mutations in other causative gene PKD2. Although many disease models have been proposed for ADPKD, the pre-symptomatic pathology of the human disease remains unknown. To unveil the mechanisms of early cytogenesis, robust and genetically relevant human models are needed. Here, we report a novel ADPKD model using kidney organoids derived from disease-specific human induced pluripotent stem cells (hiPSCs). Importantly, we found that kidney organoids differentiated from gene-edited heterozygous PKD1-mutant as well as ADPKD patient-derived hiPSCs can reproduce renal cysts. Further, we demonstrated the possibility of ADPKD kidney organoids serving as drug screening platforms. This newly developed model will contribute to identifying novel therapeutic targets, extending the field of ADPKD research., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2020 Elsevier Inc. All rights reserved.)

AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Citrullinemia type 1 (CTLN1) is a urea cycle disorder (UCD) caused by mutations of the ASS1 gene, which is responsible for production of the enzyme argininosuccinate synthetase (ASS), and classically presented as life-threatening hyperammonemia in newborns. Therapeutic options are limited, and neurological sequelae may persist. To understand the pathophysiology and find novel treatments, induced pluripotent stem cells (iPSCs) were generated from a CTLN1 patient and differentiated into hepatocyte-like cells (HLCs). CTLN1-HLCs have lower ureagenesis, recapitulating part of the patient's phenotype. l-arginine, an amino acid clinically used for UCD treatment, improved this phenotype in vitro. Metabolome analysis revealed an increase in tricarboxylic acid (TCA) cycle metabolites in CTLN1, suggesting a connection between CTLN1 and the TCA cycle. This CTLN1-iPSC model improves the understanding of CTLN1 pathophysiology and can be used to pursue new therapeutic approaches., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Colorectal carcinoma (CRC) is one of the most frequently diagnosed cancers and the leading cause of cancer-related death for both men and women. Recent studies have revealed that a small sub-population of cancer cells, termed cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), are endowed with tumor-initiating ability, self-renewal ability and differentiation ability. CSCs/CICs are resistant to current therapies including chemotherapy and radiotherapy. Thus, CSCs/CICs are responsible for recurrence and metastasis, and eradication of CSCs/CICs is essential to cure cancer. In this study, we isolated CR-CSCs/CICs as sphere-cultured cells and found that a product derived from LY6/PLAUR domain containing 3 (LYPD3) is preferentially expressed in CSCs/CICs. Gene overexpression and gene knockdown experiments revealed that LYPD3 has a role in the maintenance of CR-CSCs/CICs. The findings provide a novel molecular insight into CR-CSCs/CICs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) is one of the most common malignancy, and the prognosis is not still satisfactory due to treatment resistance, recurrence and distant metastasis. Cancer stem cells (CSCs)/cancer-initiating cells (CICs) is endowed with higher tumor-initiating ability, self-renewal ability and differentiation ability, and CSCs/CICs are resistant to treatments. Thus, CSCs/CICs are thought to be responsible for recurrence and distant metastasis, and eradication of CSCs/CICs is essential to cure CRCs. However, the molecular mechanisms of CSCs/CICs are remain unknown, and we aimed to elucidate molecular aspects of CR-CSCs/CICs in this study. We screened the transcriptome data of primary human CR-CSCs/CICs that we previously established, and found that LEM domain containing 1 (LEMD1) is preferentially expressed in CR-CSCs/CICs. LEMD1 belongs to cancer-testis (CT) antigen, and has five transcript variants (variant 1 [V1] - variant 5 [V5]). We found that LEMD1 V1, V2 and V3 is expressed in testis and CR-CSCs/CICs, whereas LEMD1 V4 and V5 is ubiquitously expressed. LEMD1 gene knockdown experiments using siRNAs and gene overexpression experiments revealed that LEMD1 has a role in the maintenance of CR-CSCs/CICs. These observations indicate that CR-CSC/CIC-specific LEMD1 variants are reasonable target of CR-CSC/CIC-targeted therapy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Incretin secretion and effect on insulin secretion are not fully understood in patients with type 2 diabetes. We investigated incretin and insulin secretion after meal intake in obese and non-obese Japanese patients with type 2 diabetes compared to non-diabetic subjects. Nine patients with type 2 diabetes and 5 non-diabetic subjects were recruited for this study. Five diabetic patients were obese (BMI > or = 25) and 4 patients were non-obese (BMI < 25). In response to a mixed meal test, the levels of immunoreactive insulin during 15-90 min and C-peptide during 0-180 min in non-obese patients were significantly lower than those in obese patients. Total GLP-1 and active GIP levels showed no significant difference between obese and non-obese patients throughout the meal tolerance test. In addition, there were no significant differences between diabetic patients and non-diabetic subjects. In conclusion, incretin secretion does not differ between Japanese obese and non-obese patients with type 2 diabetes and non-diabetic subjects., (2010 Elsevier Inc. All rights reserved.)

Hippocampal cholinergic neurostimulating peptide (HCNP), which enhances acetylcholine synthesis and induces cholinergic phenotype development of the septohippocampal system, is derived from HCNP precursor protein (HCNPpp), also known as phosphatidylethanolamine binding protein (PEBP) and Raf kinase inhibitor protein (RKIP). Our previous study demonstrated that expression of HCNPpp mRNA was decreased in the hippocampi of autopsied brains of Alzheimer's disease (AD) patients, indicating the association of HCNP with the pathogenesis of AD. To clarify the involvement of gene variations in the promoter region of the gene encoding HCNPpp in this mRNA reduction, we analyzed DNA polymorphisms or mutations within this gene promoter region in AD patients by direct sequencing. The promoter was found to contain a CpG island without a TATA box, an element of housekeeping gene promoters. Moreover, no disease-specific polymorphisms or mutations were identified, suggesting that the decrease of mRNA can be ascribed to transcriptional or posttranscriptional changes in activity.

Extracellular signal-regulated kinase (ERK) is a member of the MAP kinase family, and can regulate several cellular responses. The isoforms ERK1 and ERK2 have markedly similar amino acid sequences, but exhibit distinctive physiological functions. As well as ERK2, ERK1 was auto- and mono-phosphorylated at Tyr204 in the activation loop during Escherichia coli production, resulting in basal level activity, approximately 500-fold less compared with fully-active ERK1 dual-phosphorylated at Thr202 and Tyr204. Crystal structure demonstrated that the mono-phosphorylated ERK1 kinase possessed a novel conformation distinguishable from the un-phosphorylated (inactive) and the dual-phosphorylated (full-active) forms. The characteristic structural features in both the C-helix and the activation loop likely contribute to the basal activity of the mono-phosphorylated ERK1. The structural dissection of ERK1 compared to ERK2 suggests that the structural differences in the D-motif binding site and in the backside binding site are putative targets for development of selective ERK1/ERK2 inhibitors.

Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of beta-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a beta-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of beta-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of beta-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted beta-cell differentiation (mainly from duct cells) and proliferation of pre-existing beta-cells, resulting in an increase of the beta-cell mass that improved glucose tolerance in diabetic mice.

The tyrosine kinase Fyn is a member of the Src kinase family. Besides the role of Fyn in T cell signal transduction in concert with Lck, its excess activity in the brain is involved with conditions such as Alzheimer's and Parkinson's diseases. Therefore, inhibition of Fyn kinase may help counteract these nervous system disorders. Here, we solved the crystal structure of the human Fyn kinase domain complexed with staurosporine, a potent kinase inhibitor, at 2.8 A resolution. Staurosporine binds to the ATP-binding site of Fyn in a similar manner as in the Lck- and Csk-complexes. The small structural differences in the staurosporine-binding and/or -unbinding region among the three kinase domains may help obtaining the selective inhibitors against the respective kinases.

Non-alcoholic steatohepatitis (NASH) may progress to liver cirrhosis, and NASH patients with liver cirrhosis have a risk of development of hepatocellular carcinoma. Peroxisome proliferator-activated receptor (PPAR) gamma ligand has recently been reported to have improved the condition of patients with NASH. The aim of this study was to investigate whether pioglitazone, a PPARgamma ligand, has any influence on the animal model of NASH as well as isolated hepatic stellate cells. In vivo, the effects of pioglitazone were examined using the choline-deficient L-amino acid-defined (CDAA)-diet liver fibrosis model. After two weeks, pioglitazone improved hepatic steatosis, prevented liver fibrosis, and reduced preneoplastic lesions in the liver after 10 weeks. Pioglitazone reduced the expression of TIMP-1 and TIMP-2 mRNA without changing MMP-13 mRNA expression compared to the liver fed a CDAA diet alone. In vitro, pioglitazone prevented the activation of hepatic stellate cells resulting in reducing the expression of type I procollagen, MMP-2, TIMP-1, and TIMP-2 mRNA with increased MMP-13 mRNA expression. These results indicate that pioglitazone may be one of the candidates for the benefit drugs for the liver disease of patients with NASH.

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.

The t(8;13) translocation, found in a rare and aggressive type of stem cell myeloproliferative disorder, leads to the generation of a fusion protein between the N-terminal gene product of fused in myeloproliferative disorders (FIM)/ZNF198 and the fibroblast growth factor receptor 1 (FGFR1) kinase domain. The chimeric protein was reported to have constitutively activated tyrosine kinase activity. However, little is known about a role of FIM in hematopoietic cell regulation. Here we show that FIM protein is ubiquitously expressed in mouse embryonic tissues but much less in hematopoietic cells. We also show that forced expression of FIM inhibits the emergence of hematopoietic cells in the cultured mouse aorta-gonad-mesonephros (AGM) region on embryonic day (E) 11.5, where definitive hematopoiesis is first found during embryogenesis. These results suggest that the expression level of FIM determines the development of hematopoiesis during mouse ontogeny.

Differentiation of hematopoietic stem cells into blood cells is controlled by several transcription factors. Recently, we identified a putative transcription factor, ELYS (for embryonic large molecule derived from yolk sac), using a subtraction strategy. During mouse embryogenesis, ELYS transcripts were predominantly expressed in hematopoietic tissues, such as the yolk sac, aorta-gonad-mesonephros (AGM), and liver. Here, we report the cloning and characterization of the mouse ELYS gene. The ELYS gene spanned approximately 60kb encoding 36 exons, and was assigned between D1Mit315 and D1Mit458 markers in chromosome 1. The transcription initiation site was identified as the G residue located 670bp upstream of the translation start codon. A region downstream of the transcriptional start site contributed to high promoter activity. This region contained potential DNA elements for transcription factors such as GATA-1, -2, -3, heat shock factor (HSF) 2, and NF-kappaB, which are known to play important roles in hematopoietic events.

Recent studies showed that the function of some amino acids is not only nutritional but also pharmacological. However, the effects of amino acids on liver fibrosis and hepatic stellate cell (HSC) remain unclear. In this research, as a result of screening of amino acids using liver fibrosis induced by DMN administration, L-cysteine was selected as a suppressor of liver fibrosis. Furthermore, the number of activated HSCs, which increased in the fibrotic liver after DMN administration, was decreased in L-cysteine-fed rats. Treatment of freshly isolated HSCs with L-cysteine resulted in inhibition of the increase in smooth muscle alpha-actin (alphaSMA) expression by HSCs and BrdU incorporation into the activated HSCs. These findings suggest that L-cysteine is effective against liver fibrosis. The mechanism of inhibition of fibrosis in the liver is surmized to be direct inhibition of activated HSC proliferation and HSC transformation by L-cysteine.

Recent studies have suggested the existence of progenitors common to hematopoietic and endothelial cells, called hemangioblasts, in, for instance, embryonic dorsal aorta. To identify a membrane-bound or secretory molecule regulating early hematopoiesis, we screened a cDNA library from dorsal aortas of embryonic day (E) 10.5 mice by a signal sequence trap method and obtained a clone encoding a sialoprotein, endomucin-1. Immunohistochemistry revealed that the endomucin-1 transcript was specifically expressed in the endothelial cells of dorsal aorta of E10.5 mouse embryo. Overexpression of endomucin-1 strongly inhibited adhesion and aggregation of cells, including cultured endothelial cells from E10.5 dorsal aorta. These data suggest that endomucin-1 may play a role in detachment of hematopoietic cells from endothelium during early hematopoiesis., (Copyright 2001 Academic Press.)

Hepatic fibrosis results from an imbalance between fibrogenesis and fibrolysis in the liver. It remains uninvestigated whether Kupffer cells produce matrix metalloproteinase-13 (MMP-13), which mainly hydrolyzes extracellular matrix (ECM). We sought to determine the role of Kupffer cells in fibrogenesis/fibrolysis. In vivo, we used the rat model of pig serum-induced liver fibrosis. A subset was treated with gadolinium chloride (GdCl(3)), which specifically acts on Kupffer cells. Administration of GdCl(3) remarkably decreased the hydroxyproline content of the liver and increased the expression of MMP-13 mRNA in the liver without a difference in procollagen type I and tissue inhibitors of metalloproteinase-1 (TIMP-1) mRNA expression on Northern blot analysis with the elimination of ED2-positive cells. In vitro, addition of GdCl(3) to isolated Kupffer cells showed increased type I collagen-degrading activity in a dose-dependent manner as well as MMP-13 mRNA expression on Northern blot analysis. It is concluded that Kupffer cells are a major source of MMP-13 and modulation of Kupffer cells by GdCl(3) prevents liver fibrosis with increased expression of MMP-13 mRNA and protein, whereas procollagen type I and TIMP-1 mRNA, which encode two major effectors of fibrogenesis, were unchanged. This is the first report showing that Kupffer cells produce interstitial collagenase (MMP-13) resulting in the reduction of ECM. This discovery may provide new insights into therapy for hepatic fibrosis., (Copyright 2000 Academic Press.)

Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes characterized by autosomal dominant inheritance, early-onset, and impaired insulin secretion. The type 3 and type 5 forms of MODY result from mutations in the genes encoding the transcription factor, hepatocyte nuclear factor (HNF)-1alpha and HNF-1beta, respectively. The mechanism by which mutations in one allele of the HNF-1 gene impair pancreatic beta cell function is unclear. We studied the effects of wild-type and four mutant (L12H, R263C, P379fsdelCT, and L584S585fsinsTC) HNF-1alpha, which were identified in Japanese subjects with MODY3 on human insulin gene transcription. Both wild-type (WT) HNF-1alpha and HNF-1beta bound to the oligonucleotide containing the A3 element sequence in the human insulin promoter and transactivated the insulin-luciferase reporter gene by 30- and 31-fold, respectively. In contrast, binding of L12H, R263C and L584S585fsinsTC-HNF-1alpha to the probe was impaired. Transactivation activity by the four mutant HNF-1alpha was reduced (4.3 to 43.3% of WT). These data suggest that the insulin gene is a candidate target gene of HNF-1alpha/HNF-1beta and the impairment of insulin gene transcription by mutations in the HNF-1 gene might be involved in the pathogenesis of MODY., (Copyright 1999 Academic Press.)

Antibody to the hypervariable region 1 of hepatitis C virus (HCV) is thought to have neutralizing activity. The complexity of hypervariable region 1 quasispecies was compared between immune complexed and non-immune complexed HCV particles. Immune complexes and non-immune complexes, including intact HCV virions, were separated by differential flotation centrifugation and immunoprecipitation, and immune complexes were observed in 9 of 11 patients with chronic hepatitis C. Considerable differences in both hypervariable region 1 quasispecies and predominant clones were demonstrated between immune complexes and non-immune complexes in 4 patients and not in 5 patients. These results suggest that the specificity of antibody to hypervariable region 1 is related to the formation of immune complexes and that escape mutants with highly different quasispecies are resistant to neutralization by antibody to hypervariable region 1.

We studied the effects of a tumor necrosis factor-alpha (TNF-alpha) production inhibitor E3330, [(2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquinolyl)]-2-nonyl-2-+ ++propenoic acid] on liver regeneration in rats subjected to 70% partial hepatectomy. The BrdU labeling index was 53.5 +/- 3.8% 24 h after partial hepatectomy alone and was significantly inhibited by E3330 (25 and 50 mg/ kg) in a dose-dependent manner (43.3 +/- 2.1% and 19.0 +/- 10.3%, respectively; p < 0.05, p < 0.01). In these 3 groups, TNF-alpha protein levels in liver tissue were 187 +/- 18, 123 +/- 47, and 89 +/- 33 pg/mg total protein, respectively, with a significant decrease by E3330 administration (p < 0.01). TNF-alpha mRNA expression in liver tissue was also reduced by E3330. These results suggest that TNF-alpha promotes liver regeneration after partial hepatectomy.

The complexity of hypervariable region 1 (HVR1) quasispecies of hepatitis C virus (HCV) in serum and peripheral blood mononuclear cells (PBMCs) was assessed at several time points in 11 patients with chronic hepatitis C. Polymerase chain reaction and single-strand conformation polymorphism analysis revealed that four patients (36%) showed serial changes in the complexity of HVR1 quasispecies, which were identical in both serum and PBMCs; two patients (18%) showed both serial changes in quasispecies and differences in quasispecies between serum and PBMCs; and five patients (45%) showed neither serial changes in nor quasispecies differences between serum and PBMCs. These results demonstrate both the existence of different quasispecies between serum and PBMCs and the infection of PBMCs by HCV.