Spatial and temporal cellular responses to single-strand breaks in human cells

DNA single-strand breaks (SSB) are one of the most frequent DNA lesions produced by reactive oxygen species and during DNA metabolism, but the analysis of cellular responses to SSB remains difficult due to the lack of an experimental method to produce SSB alone in cells. By using human cells expressing a foreign UV damage endonuclease (UVDE) and irradiating the cells with UV through tiny pores in membrane filters, we created SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions. Cellular responses to the SSB were characterized by using antibodies and fluorescence microscopy. Upon UV irradiation, poly(ADP-ribose) synthesis occurred immediately in the irradiated area. Simultaneously, but dependent on poly(ADP-ribosyl)ation, XRCC1 was translocated from throughout the nucleus, including nucleoli, to the SSB. The BRCT1 domain of XRCC1 protein was indispensable for its poly(ADP-ribose)-dependent recruitment to the SSB. Proliferating cell nuclear antigen and the p150 subunit of chromatin assembly factor 1 also accumulated at the SSB in a detergent-resistant form, which was significantly reduced by inhibition of poly(ADP-ribose) synthesis. Our results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB

The effect of leg hyperthermia using far infrared rays in bedridden subjects with type 2 diabetes mellitus

We examined the effect of leg hyperthermia on oxidative stress in bedridden subjects with type 2 diabetes mellitus using 15-min sessions of far infrared rays over a two-week period. Four subjects (male 1, female 3) incapacitated by a stroke were recruited for this study. All patients were admitted to Takahashi Central Hospital and ate the same hospital meals. Fasting plasma glucose, HbA1c, tumor necrosis factor (TNF)alpha, free fatty acid, leptin, adiponectin and plasma 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha) levels as a marker of oxidative stress were measured on admission, just before and 2 weeks after local heating of the leg. Results showed that plasma total 8-epi-PGF2alpha levels were decreased significantly while TNFalpha levels were increased significantly. On the other hand, glucose, HbA1c, free fatty acid, leptin and adiponectin levels were not changed during the study period. These results suggest that repeated leg hyperthermia may protect against oxidative stress.</p

Inhibitory Effect of 1α-Hydroxyvitamin D3 on N-nitrosobis (2-oxopropyl)Amine-induced Cholangiocarcinogenesis in Syrian Hamsters

Sixty-three male 5-week-old Syrian hamsters received the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) s.c. in 5 weekly injections (the first, 70mg/kg body, and the remaining, 20mg/kg each). The hamsters that received BOP were given intragastric administration of 0.2ml of medium chain triglyceride (MCT) with or without 0.04μg of 1α-hydroxyvitamin D3 [1α(OH)D3] through a feeding tube for 12 weeks. Thus, 3 groups were assigned:Group 1;BOP alone (n＝20), Group 2;BOP＋MCT (n＝18) and Group 3;BOP＋1α(OH)D3 (n＝25). The mean body weight of Group 3 was lower than those of Groups 1 and 2 at the end of the experiment (p＜0.001,Tukey-Kramer HSD test). At the end of week 12, all surviving hamsters were put to sleep. The incidences of liver tumors were 80%, 72% and 32% in Groups 1, 2 and 3, respectively. The incidence of tumors in Group 3 was significantly lower than in Group 1 and Group 2 (p＜0.05, χ2-test). All tumors were cholangiocarcinoma. These results indicated that BOP-induced cholangiocarcinogenesis was suppressed by the supplemental administration of 1α(OH)D3

Liver-specific γ-glutamyl carboxylase-deficient mice display bleeding diathesis and short life span

Liver-Specific γ-Glutamyl Carboxylase-Deficient Mice Display Bleeding Diathesis and Short Life Span. Azuma K, Tsukui T, Ikeda K, Shiba S, Nakagawa K, et al. PLOS ONE. 2014. 9(2) doi:10.1371/journal.pone.008864

Glycolysis Inhibition Inactivates ABC Transporters to Restore Drug Sensitivity in Malignant Cells

Cancer cells eventually acquire drug resistance largely via the aberrant expression of ATP-binding cassette (ABC) transporters, ATP-dependent efflux pumps. Because cancer cells produce ATP mostly through glycolysis, in the present study we explored the effects of inhibiting glycolysis on the ABC transporter function and drug sensitivity of malignant cells. Inhibition of glycolysis by 3-bromopyruvate (3BrPA) suppressed ATP production in malignant cells, and restored the retention of daunorubicin or mitoxantrone in ABC transporter-expressing, RPMI8226 (ABCG2), KG-1 (ABCB1) and HepG2 cells (ABCB1 and ABCG2). Interestingly, although side population (SP) cells isolated from RPMI8226 cells exhibited higher levels of glycolysis with an increased expression of genes involved in the glycolytic pathway, 3BrPA abolished Hoechst 33342 exclusion in SP cells. 3BrPA also disrupted clonogenic capacity in malignant cell lines including RPMI8226, KG-1, and HepG2. Furthermore, 3BrPA restored cytotoxic effects of daunorubicin and doxorubicin on KG-1 and RPMI8226 cells, and markedly suppressed subcutaneous tumor growth in combination with doxorubicin in RPMI8226-implanted mice. These results collectively suggest that the inhibition of glycolysis is able to overcome drug resistance in ABC transporter-expressing malignant cells through the inactivation of ABC transporters and impairment of SP cells with enhanced glycolysis as well as clonogenic cells

Structure of Musashi1 in a complex with target RNA: the role of aromatic stacking interactions

Mammalian Musashi1 (Msi1) is an RNA-binding protein that regulates the translation of target mRNAs, and participates in the maintenance of cell ‘stemness’ and tumorigenesis. Msi1 reportedly binds to the 3′-untranslated region of mRNA of Numb, which encodes Notch inhibitor, and impedes initiation of its translation by competing with eIF4G for PABP binding, resulting in triggering of Notch signaling. Here, the mechanism by which Msi1 recognizes the target RNA sequence using its Ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2 has been revealed on identification of the minimal binding RNA for each RBD and determination of the three-dimensional structure of the RBD1:RNA complex. Unique interactions were found for the recognition of the target sequence by Msi1 RBD1: adenine is sandwiched by two phenylalanines and guanine is stacked on the tryptophan in the loop between β1 and α1. The minimal recognition sequences that we have defined for Msi1 RBD1 and RBD2 have actually been found in many Msi1 target mRNAs reported to date. The present study provides molecular clues for understanding the biology involving Musashi family proteins

Isolation and function of mouse tissue resident vascular precursors marked by myelin protein zero

Newly identified tissue-resident vascular precursor cells are recruited into growing vessels and contribute to vasculogenesis in adult mice