Myogenetic oligodeoxynucleotide (myoDN) recovers the differentiation of skeletal muscle myoblasts deteriorated by diabetes mellitus.

Skeletal muscle wasting in patients with diabetes mellitus (DM) is a complication of decreased muscle mass and strength, and is a serious risk factor that may result in mortality. Deteriorated differentiation of muscle precursor cells, called myoblasts, in DM patients is considered to be one of the causes of muscle wasting.We recently developed myogenetic oligodeoxynucleotides (myoDNs), which are 18-base single-strand DNAs that promote myoblast differentiation by targeting nucleolin. Herein, we report the applicability of a myoDN, iSN04, to myoblasts isolated from patients with type 1 and type 2 DM. Myogenesis of DM myoblasts was exacerbated concordantly with a delayed shift of myogenic transcription and induction of interleukins. Analogous phenotypes were reproduced in healthy myoblasts cultured with excessive glucose or palmitic acid, mimicking hyperglycemia or hyperlipidemia. iSN04 treatment recovered the deteriorated differentiation of plural DM myoblasts by downregulating myostatin and interleukin-8 (IL-8). iSN04 also ameliorated the impaired myogenic differentiation induced by glucose or palmitic acid. These results demonstrate that myoDNs can directly facilitate myoblast differentiation in DM patients, making them novel candidates for nucleic acid drugs to treat muscle wasting in patients with DM.ArticleFrontiers in Physiology. 12 : 679152 (2021)journal articl

Berberine and palmatine inhibit the growth of human rhabdomyosarcoma cells

Published online: 29 Aug 2019A natural isoquinoline alkaloid, berberine, has been known to exhibit anti-tumor activity in various cancer cells via inducing cell cycle arrest. However, it has not been investigated whether berberine and its analogs inhibit the growth of rhabdomyosarcoma (RMS), which is the most frequent soft tissue tumor in children. The present study examined the anti-tumor effects of berberine and palmatine on expansions of three human embryonal RMS cell lines; ERMS1, KYM1, and RD. Intracellular incorporation of berberine was relatively higher than that of palmatine in every RMS cell line. Berberine significantly inhibited the cell cycle of all RMS cells at G(1) phase. On the other hand, palmatine only suppressed the growth of RD cells. Both of berberine and palmatine strongly inhibited the growth of tumorsphere of RD cells in three-dimensional culture. These results indicate that berberine derivatives have the potential of anti-tumor drugs for RMS therapy.ArticleBIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY. 84(1):63-75 (2020)journal articl

Identification of the Myogenetic Oligodeoxynucleotides (myoDNs) That Promote Differentiation of Skeletal Muscle Myoblasts by Targeting Nucleolin

Herein we report that the 18-base telomeric oligodeoxynucleotides (ODNs) designed from the Lactobacillus rhamnosus GG genome promote differentiation of skeletal muscle myoblasts which are myogenic precursor cells. We termed these myogenetic ODNs (myoDNs). The activity of one of the myoDNs, iSN04, was independent of Toll-like receptors, but dependent on its conformational state. Molecular simulation and iSN04 mutants revealed stacking of the 13–15th guanines as a core structure for iSN04. The alkaloid berberine bound to the guanine stack and enhanced iSN04 activity, probably by stabilizing and optimizing iSN04 conformation. We further identified nucleolin as an iSN04-binding protein. Results showed that iSN04 antagonizes nucleolin, increases the levels of p53 protein translationally suppressed by nucleolin, and eventually induces myotube formation by modulating the expression of genes involved in myogenic differentiation and cell cycle arrest. This study shows that bacterial-derived myoDNs serve as aptamers and are potential nucleic acid drugs directly targeting myoblasts.ArticleFRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY. 8:616706 (2021)journal articl

Expression of Intercellular Adhesion Molecule-1 in the Livers of Rats Treated with Diethylnitrosamine

It has been reported that levels of soluble intercellular adhesion molecule-1 (ICAM-1) in the blood are elevated in hepatocellular carcinoma patients. In the present study, serial observations of the localization of ICAM-1 in the liver were made by light and electron microscopy in rats with carcinogen-induced cancer. Male Fisher rats were given diethylnitrosamine (DEN) orally in their drinking water. Rats were sacrificed at 6, 8, 12, or 14 weeks after the start of DEN administration and the liver tissue was collected. ICAM-1 expression in liver was assessed using indirect immunoperoxidase staining with anti-rat ICAM-1 antibody. Although ICAM-1 expression by endothelial cells in livers of DEN-treated rats was lower than in the control group at 8 weeks, it was higher in the membrane and cytoplasm of hepatocytes. The expression of ICAM-1 in mesenchymal cells was decreased, paralleling development of cellular atypia, whereas in hepatocyte membranes and cytoplasm it was increased in these atypia. ICAM-1 was localized to the cytoplasm of cancer cells, but to the membrane of hepatocytes in the treated livers at 14 weeks. Furthermore, the levels of ICAM-1 in mesenchymal cells tended to be lower in the cancerous area than in the atypical hyperplastic nodule, and were reduced as the density of cell atypia increased, in comparison to cells in areas without cancerous nodules. We concluded that ICAM-1 may be influenced the development of cancer induced in the rat liver by a chemical carcinogen

Repeated Pancreatectomy for Recurrent Pancreatic Carcinoma after Pylorus-Preserving Pancreatoduodenectomy: Report of Two Patients

Repeated pancreatectomy for pancreatic carcinoma is extremely rare. We report two such patients who underwent pancreatectomy for carcinoma developing in the pancreatic remnant after pylorus-preserving pancreatoduodenectomy (PpPD) for invasive pancreatic ductal carcinoma. One patient underwent PpPD for invasive pancreatic ductal carcinoma and received adjuvant chemotherapy. Follow-up computed tomography (CT) demonstrated a low-density mass in the remnant pancreas, which was diagnosed as a carcinoma by endoscopic ultrasound-guided fine-needle aspiration cytology 5 years 10 months after PpPD. She underwent curative resection of the remnant pancreas and is alive and well 13 months after the second operation. The other patient underwent PpPD for invasive pancreatic ductal carcinoma. Follow-up CT showed a low-density mass in the remnant pancreas after 2 years 11 months. He received systemic chemotherapy with S-1 for 3 months. The tumor shrank, and the patient underwent curative resection of the remnant pancreas 3 years 1 month after the initial operation. Repeated pancreatectomy may provide a chance of long survival for patients with carcinoma developing in the remnant pancreas after pancreatectomy if the recurrence occurring at long term is limited to the remnant pancreas

Incorporation of metabolic activation potentiates cyclophosphamide-induced DNA damage response in isogenic DT40 mutant cells

Elucidating the DNA repair pathways that are activated in the presence of genotoxic agents is critical to understand their modes of action. Although the DT40 cell-based DNA damage response (DDR) assay provides rapid and sensitive results, the assay cannot be used on genotoxic compounds that require metabolic activation to be reactive. Here, we applied the metabolic activation system to a DDR and micronucleus (MN) assays in DT40 cells. Cyclophosphamide (CP), a well-known cross-linking agent requiring metabolic activation, was preincubated with liver S9 fractions. When DT40 cells and mutant cells were exposed to the preactivated CP, CP caused increased cytotoxicity in FANC-, RAD9-, REV3- and RAD18-mutant cells compared to isogenic wild-type cells. We then performed a MN assay on DT40 cells treated with preactivated CP. An increase in the MN was observed in REV3- and FANC-mutant cells at lower concentrations of activated CP than in the parental DT40 cells. These results demonstrated that the incorporation of metabolic preactivation system using S9 fractions significantly potentiates DDR caused by CP in DT40 cells and their mutants. In addition, our data suggest that the metabolic preactivation system for DDR and MN assays has a potential to increase the relevance of this assay to screening various compounds for potential genotoxicity

Convenient, multi-well plate-based DNA damage response analysis using DT40 mutants is applicable to a high-throughput genotoxicity assay with characterization of modes of action

Chemists continually synthesize myriad new chemicals (~2000/yr), some of which make their way into the environment or otherwise pose possible threats to humans who potentially become exposed to the compounds. Regulators must determine whether these, along with the glut (~80,000) of existing, chemicals are toxic and at what exposure levels. An important component of this determination is to ascertain the mode of action (MOA) of each compound as it relates to the pathway the compound uses to induce genotoxicity. Several assays have traditionally been used to reveal these effects to the genome: the Ames test, tests with yeast and mammalian cell lines, and animal studies. Previously, we described a new multi-well plate-based method which makes use of the DT40 isogenic cell line and its dozens of available mutants knocked out in DNA repair and cell cycle pathways and we now provide a detailed protocol of the further improvement of the assay. Although the DT40 line has existed for some time and has been used in numerous studies of DNA repair pathways, little use has been made of this valuable resource for toxicological investigations. Our method introduces the XTT dye scheme determination of cell survival in a manner that greatly increases throughput and reduces cost while maintaining reasonable sensitivity. Although this new genotoxicity assay requires validation with many more mutagens before becoming an established, regulatory decision-making analysis tool, we believe that this method will be very advantageous if eventually added to the repertoire of those investigating MOAs of potentially genotoxic substances

A case of isolated hypocalciuric hypercalcemia and type 2 diabetes mellitus followed by Grave\u27s disease

A 73-year-old woman was found to have diabetes mellitus along with elevated levels of calcium and alkaline phosphatase. She had also sometimes exhibited slight elevation of parathyroid hormone (97 pg/ml) while receiving treatment for hypertension and heart failure with atrial fibrillation, which had been detected by the Department of Cardiology at 71 years of age. She was admitted with elevated blood glucose (422 mg/dl) and hemoglobin A1c (12%) levels, as well as for further assessment of hyperparathyroidism. Her diabetes was treated with intensive insulin therapy (insulin lispro at 24 U per day). Assessment of hypercalcemia revealed that urinary calcium excretion was extremely low (0 mg/day) and the fractional excretion of Ca (FECa) was 0%. Familial hypocalciuric hypercalcemia was suspected. No abnormalities of the parathyroid gland were found by ultrasonography and 99mTc+Tl scintigraphy. The patient and her family members did not have any mutations in the extracellular domain or transmembrane domain of the calcium-sensing receptor, which has been reported to play an important role in Ca binding and intracellular signaling. However, the patient did have a missense mutation of codon 990 (R990G) in the intracellular domain, which has been reported to be a site of polymorphism, as well as a mutation of intron 6 (16 base pairs downstream from the exon-intron boundary: IVS6+16). One year later, she developed Grave\u27s disease that presented with fatigue and weight loss

〈Originals〉Relationship between clinical manifestations and CMR findings in cardiac sarcoidosis

[Abstract] Background : Different cardiac manifestations/events have been reported in patients with sarcoidosis ; however, factors related to the onsetof manifestations in cardiac sarcoidosis (CS) remain unclear. Late gadolinium-enhancement cardiac magnetic resonance (LGE-CMR) has been used to detect myocardial damage/fibrosis. Therefore, we examined the characteristics of clinical manifestations in CS patients using LGE-CMR. Methods : Thirty-three consecutive CS patients underwent CMR. According to the mural location of LGE ; mid-wall, epicardial, or transmural, the LGE pattern was classified into three groups : A : LGE existing at one location only, B: LGE at any two locations, C : LGE at all locations. CMR findings were also analyzed in relation to each clinical manifestation. Results : Myocardial damage was detected by LGE imaging in 91% of the patients and was most frequently observed in the basal septal myocardium. %LGE area was negatively correlated with LV ejection fraction (EF) (P <0.01). Significantly decreased LV EF and increased %LGE area were observed in Group B and C. In addition, all patients in group C exhibited a clinical manifestation. The patients with high-grade atrioventricular block showed the increased %LGE area (P < 0.01). Although occurrence of ventricular tachycardia was not associated with any changes in LV EF, mass, and %LGE area, hospitalization for heart failure was associated with reduced LV EF, and increased mass and %LGE area (P<0.05 for all).Conclusions : Different clinical manifestations in CS have been associated with the development of myocardial fibrosis/damage and resultant myocardial dysfunction shown by CMR. CMR may be useful for characterizing the pathophysiology of respective clinical manifestations/events