Rad9, Rad17, TopBP1 and Claspin Play Essential Roles in Heat-Induced Activation of ATR Kinase and Heat Tolerance

Hyperthermia is widely used to treat patients with cancer, especially in combination with other treatments such as radiation therapy. Heat treatment per se activates DNA damage responses mediated by the ATR-Chk1 and ATM-Chk2 pathways but it is not fully understood how these DNA damage responses are activated and affect heat tolerance. By performing a genetic analysis of human HeLa cells and chicken B lymphoma DT40 cells, we found that heat-induced Chk1 Ser345 phosphorylation by ATR was largely dependent on Rad9, Rad17, TopBP1 and Claspin. Activation of the ATR-Chk1 pathway by heat, however, was not associated with FancD2 monoubiquitination or RPA32 phosphorylation, which are known as downstream events of ATR kinase activation when replication forks are stalled. Downregulation of ATR, Rad9, Rad17, TopBP1 or Claspin drastically reduced clonogenic cell viability upon hyperthermia, while gene knockout or inhibition of ATM kinase reduced clonogenic viability only modestly. Suppression of the ATR-Chk1 pathway activation enhanced heat-induced phosphorylation of Chk2 Thr68 and simultaneous inhibition of ATR and ATM kinases rendered severe heat cytotoxicity. These data indicate that essential factors for activation of the ATR-Chk1 pathway at stalled replication forks are also required for heat-induced activation of ATR kinase, which predominantly contributes to heat tolerance in a non-overlapping manner with ATM kinase.     

Gene-Gene Combination Effect and Interactions among ABCA1, APOA1, SR-B1, and CETP Polymorphisms for Serum High-Density Lipoprotein-Cholesterol in the Japanese Population

Background/Objective

Gene-gene interactions in the reverse cholesterol transport system for high-density lipoprotein-cholesterol (HDL-C) are poorly understood. The present study observed gene-gene combination effect and interactions between single nucleotide polymorphisms (SNPs) in ABCA1, APOA1, SR-B1, and CETP in serum HDL-C from a cross-sectional study in the Japanese population.

Methods

The study population comprised 1,535 men and 1,515 women aged 35–69 years who were enrolled in the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study. We selected 13 SNPs in the ABCA1, APOA1, CETP, and SR-B1 genes in the reverse cholesterol transport system. The effects of genetic and environmental factors were assessed using general linear and logistic regression models after adjusting for age, sex, and region.

Principal Findings

Alcohol consumption and daily activity were positively associated with HDL-C levels, whereas smoking had a negative relationship. The T allele of CETP, rs3764261, was correlated with higher HDL-C levels and had the highest coefficient (2.93 mg/dL/allele) among the 13 SNPs, which was statistically significant after applying the Bonferroni correction (p<0.001). Gene-gene combination analysis revealed that CETP rs3764261 was associated with high HDL-C levels with any combination of SNPs from ABCA1, APOA1, and SR-B1, although no gene-gene interaction was apparent. An increasing trend for serum HDL-C was also observed with an increasing number of alleles (p<0.001).

Conclusions

The present study identified a multiplier effect from a polymorphism in CETP with ABCA1, APOA1, and SR-B1, as well as a dose-dependence according to the number of alleles present.     

Anti-Influenza Activity of C60 Fullerene Derivatives

The H1N1 influenza A virus, which originated in swine, caused a global pandemic in 2009, and the highly pathogenic H5N1 avian influenza virus has also caused epidemics in Southeast Asia in recent years. Thus, the threat from influenza A remains a serious global health issue, and novel drugs that target these viruses are highly desirable. Influenza A RNA polymerase consists of the PA, PB1, and PB2 subunits, and the N-terminal domain of the PA subunit demonstrates endonuclease activity. Fullerene (C₆₀) is a unique carbon molecule that forms a sphere. To identify potential new anti-influenza compounds, we screened 12 fullerene derivatives using an in vitro PA endonuclease inhibition assay. We identified 8 fullerene derivatives that inhibited the endonuclease activity of the PA N-terminal domain or full-length PA protein in vitro. We also performed in silico docking simulation analysis of the C₆₀ fullerene and PA endonuclease, which suggested that fullerenes can bind to the active pocket of PA endonuclease. In a cell culture system, we found that several fullerene derivatives inhibit influenza A viral infection and the expression of influenza A nucleoprotein and nonstructural protein 1. These results indicate that fullerene derivatives are possible candidates for the development of novel anti-influenza drugs.     

Polysaccharides of Soybean Seeds

Methylation of hot-water-extract fraction of soybean seed polysaccharides by the Haworth’s method and then by the Purdie’s method followed by fractional extraction afforded a fully methylated arabinogalactan which gave on complete hydrolysis 2,3,5-tri-, and 2,3-di-O-methyl l-arabinose and 2,3,6-tri-, and 2,6-di-O-methyl d-galactose in the approximate proportion of 1 : 1 : 3 ~ 4: 1, and on controlled acid methanolysis 2,3,5-tri-, and 2,3-di-O-methyl l-arabinose in about equal proportion. Residual undegraded polysaccharide gave mainly 2,3,6-tri-O-methyl d-galactose on remethylation and hydrolysis.     

Heat-induced Gelation of Myosin from Leg and Breast Muscles of Chicken

Heat-induced gelation of myosin from leg and breast muscles of chicken was studied in 0.6 m KC1. Gel strength of breast myosin was higher than that of leg myosin between pH 5.2 and 6.0. Turbidity of breast myosin increased below pH 6.0 but that of leg myosin did not increase at pH 5.7. Turbidity of leg myosin was higher than that of breast myosin below pH 5.6. Viscosity of breast myosin increased between pH 5.5 and 6.0 as the pH decreases, although that of leg myosin decreased. The breast myosin assembled to form long filaments at pH 5.7, but leg myosin failed to form long filaments. At pH 5.4, breast myosin filaments became longer and leg myosin assembled into filaments though they were shorter than breast myosin filaments. The strength of heat-induced gel formed from the filamentous leg and breast myosins at acidic region was not influenced by F-actin. These results indicate that the strength of heat-induced gel of both myosins is closely related to their morphological properties.     

Studies on Rice Glutelin

The complete amino acid analysis of the whole glutelin preparation from rice endosperm was performed. The recoveries were 101.59% for amino acid residues and 101.68% for nitrogen, and the standard deviations for four determinations on the 22 and 70 hr hydrolyzates were very small. The features of the amino acid composition of the protein were as follows; (1) the high contents of dicarboxylic amino acids, particularly glutamic acid, (2) about 60% of these dicarboxylic amino acids was in the amide form, and (3) the significantly low contents of tryptophan, methionine and half cystine. The amino acid analyses of the two kinds of the subunits of glutelin, the neutral major one and the basic minor one, were also carried out. There were some significant differences between the two subunits, for instance, in the contents of glutamic acid, tryptophan, glycine, half cystine, methionine and lysine. However, the composition of whole glutelin seemed to be settled predominantly by that of the major subunit.     

Isolation and characterization of bacterial strains with the ability to utilize high concentrations of levulinic acid,a platform chemical from inedible biomass

Nineteen levulinic acid (LA)-utilizing bacteria were isolated from environmental samples. Following examination of the use of 80 g/L LA by some isolated strains, Brevibacterium epidermidis LA39–2 consumed 62.6 g/L LA following 8 days incubation. The strain also utilized both 90 and 100 g/L LA, with consumption ratio of 84.3 and 53.3%, respectively, after 10 days incubation.     

Partial Purification and Some Properties of 7-Keto-8-aminopelargonic Acid Synthetase,an Enzyme Involved in Biotin Biosynthesis

7-Keto-8-aminopelargonic acid synthetase (KAPA synthetase) which catalyzes the formation of KAPA from pimelyl CoA and l-alanine, and is involved in biotin biosynthesis, was partially purified from a cell-free extract of Bacillus sphaericus by a procedure involving ammonium sulfate fraction ation, protamine treatment, and DEAE-cellulose column chromatography. The reaction product was bioautographically confirmed to be KAPA. Some properties of the enzyme were also investigated. Among the amino acids, only l-alanine was active as a substrate, condensing with pimelyl CoA, The reaction required pyridoxal phosphate but the other vitamin B₆ compounds were inert. Typical inhibitors of pyridoxal phosphate enzymes showed marked inhibition to the reaction. Various amino acids such as l-cysteine, glycine, d-alanine, l-serine, l-histidine, and d-histidine were also found to be inhibitory.