RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells

Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major pathways that account for survival after post-replicational DNA damage. TLS functions by filling gaps on a daughter strand that remain after DNA replication caused by damage on the mother strand, while HR can repair gaps and breaks using the intact sister chromatid as a template. The RAD18 gene, which is conserved from lower eukaryotes to vertebrates, is essential for TLS in Saccharomyces cerevisiae. To investigate the role of RAD18, we disrupted RAD18 by gene targeting in the chicken B-lymphocyte line DT40. RAD18(-/-) cells are sensitive to various DNA-damaging agents including ultraviolet light and the cross-linking agent cisplatin, consistent with its role in TLS. Interestingly, elevated sister chromatid exchange, which reflects HR- mediated post-replicational repair, was observed in RAD18(-/-) cells during the cell cycle. Strikingly, double mutants of RAD18 and RAD54, a gene involved in HR, are synthetic lethal, although the single mutant in either gene can proliferate with nearly normal kinetics. These data suggest that RAD18 plays an essential role in maintaining chromosomal DNA in cooperation with the RAD54-dependent DNA repair pathway.     

Purification,crystallization and preliminary X-ray studies of human augmenter of liver regeneration

Human augmenter of liver regeneration has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C222, with unit-cell parameters a=51.7 A, b=78.8 A, c=63.7 A. Diffraction data were collected to 2.80 A with a completeness of 99.9% (99.9% for the last shell), a R(sym) value of 0.092(0.236) and an I/sigma(I) value of 6.2(2.7).     

Raman microspectroscopic study of biomolecular structure inside living adhesive cells

Cells adhesion is very important for many physiological processes. Using advanced Raman microspectroscopic technique, we selected T Leukemia cells (Jurkat) as the materials and obtained simultaneously conformation information of various biomolecules inside the whole living cells. By comparing the Raman microspectroscopic spectra of single and adhesive cancer cells, we found for the first time that when cells adhered, the conformation of the biomolecules (DNA, protein, carbohydrates and lipids) inside the cells had different changes: (i) the backbone of double-stranded DNA maintained orderly B-form or modified B-form conformation, whereas the groups of its deoxyribose and bases were modified; (ii) the conformational changes of the main chain and the side chain in the protein were obviously variant. The lines intensity belonging to a-helix and p-sheet decreased, while that of p-turn increased. Tyrosine and tryptophane residues of the protein changed from "buried state" to "exposed state"; the lines intensi     

Estimation of amino acid pairs sensitive to variants in human phenylalanine hydroxylase protein by means of a random approach

In this data-based theoretical analysis, we use a random approach to estimate amino acid pairs in human phenylalanine 4-hydroxylase (PAH) protein in order to determine which amino acid pairs are more sensitive to 187 variants in human PAH protein. The rationale of this study is based on our hypothesis and previous findings that the harmful variants are more likely to occur at randomly unpredictable amino acid pairs rather than at randomly predictable pairs. This is reasonable to argue as randomly predictable amino acid pairs are less likely to be deliberately evolved, whereas randomly unpredictable amino acid pairs are probably deliberately evolved in connection with protein function. 94.12% of 187 variants occurred at randomly unpredictable amino acid pairs, which accounted for 71.84% of 451 amino acid pairs in human PAH protein. The chance of a variant occurring is five times higher in randomly unpredictable amino acid pairs than in predictable pairs. Thus, randomly unpredictable amino acid pairs are more sensitive to variance in human PAH protein. The results also suggest that the human PAH protein has a natural tendency towards variants.     

Transforming growth factor-beta increases Escherichia coli K1 adherence,invasion, and transcytosis in human brain microvascular endothelial cells

Escherichia coli K1 traversal of the human brain microvascular endothelial cells (HBMEC) that constitute the blood-brain barrier (BBB) is a complex process involving E. coli adherence to and invasion of HBMEC. In this study, we demonstrated that human transforming growth factor-beta-1 (TGF-beta1) increases E. coli K1 adherence, invasion, and transcytosis in HBMEC. In addition, TGF-beta1 increases RhoA activation and enhances actin condensation in HBMEC. We have previously shown that E. coli K1 invasion of HBMEC requires phosphatidylinositol-3 kinase (PI3K) and RhoA activation. TGF-beta1 increases E. coli K1 invasion in PI3K dominant-negative HBMEC, but not in RhoA dominant-negative HBMEC, indicating that TGF-beta1-mediated increase in E. coli K1 invasion is RhoA-dependent, but not PI3K-dependent. Our findings suggest that TGF-beta1 treatment of HBMEC increases E. coli K1 adherence, invasion, and transcytosis, which are probably dependent on RhoA.     

Prediction of amino acid pairs sensitive to mutations in the spike protein from SARS related coronavirus

In this study, we analyzed the amino acid pairs affected by mutations in two spike proteins from human coronavirus strains 229E and OC43 by means of random analysis in order to gain some insight into the possible mutations in the spike protein from SARS-CoV. The results demonstrate that the randomly unpredictable amino acid pairs are more sensitive to the mutations. The larger is the difference between actual and predicted frequencies, the higher is the chance of mutation occurring. The effect induced by mutations is to reduce the difference between actual and predicted frequencies. The amino acid pairs whose actual frequencies are larger than their predicted frequencies are more likely to be targeted by mutations, whereas the amino acid pairs whose actual frequencies are smaller than their predicted frequencies are more likely to be formed after mutations. These findings are identical to our several recent studies, i.e. the mutations represent a process of degeneration inducing human diseases.     

Purification, crystallization and preliminary X-ray studies of a p-nitrophenylphosphatase from Bacillus stearothermophilus

Thermostable p-nitrophenylphosphatase from Bacillus stearothermophilus has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C(2), with unit-cell parameters a = 67.17 A, b = 57.84 A, c = 62.49 A and alpha = 90.0 degrees, beta = 95.4 degrees, gamma = 90.0 degrees. Diffraction data were collected to 1.40 A resolution with a completeness of 94.7% (96.6% for the last shell), an R(fac) value of 0.074 (0.341) and an I/sigma (I) value of 30.1 (2.67).