Recognition of forked and single-stranded DNA structures by human RAD18 complexed with RAD6B protein triggers its recruitment to stalled replication forks

Post-replication DNA repair facilitates the resumption of DNA synthesis upon replication fork stalling at DNA damage sites. Despite the importance of RAD18 and polymerase η (Polη) for post-replication repair (PRR), the molecular mechanisms by which these factors are recruited to stalled replication forks are not well understood. We present evidence that human RAD18 complexed with RAD6B protein preferentially binds to forked and single-stranded DNA (ssDNA) structures, which are known to be localized at stalled replication forks. The SAP domain of RAD18 (residues 248–282) is crucial for binding of RAD18 complexed with RAD6B to DNA substrates. RAD18 mutated in the SAP domain fails to accumulate at DNA damage sites in vivo and does not guide DNA Polη to stalled replication forks. The SAP domain is also required for the efficient mono-ubiquitination of PCNA. The SAP domain mutant fails to suppress the ultraviolet (UV)-sensitivity of Rad18 -knockout cells. These results suggest that RAD18 complexed with RAD6B is recruited to stalled replication forks via interactions with forked DNA or long ssDNA structures, a process that is required for initiating PRR.     

The role of ferritin in the intracellular distribution of gallium 67

The binding of gallium 67 or iron 59 to ferritin in vitro was investigated using equilibrium dialysis. Gallium 67 did not bind to apo-ferritin until the protein was transformed into ferritin in the presence of iron citrate. Apotransferrin inhibited the binding of ⁶⁷Ga to ferritin, especially in the presence of sodium bicarbonate and citrate, thus indicating that ⁶⁷Ga has not gained access to ferritin from its complex with transferrin. Similar inhibition was observed for ferritin-⁵⁹Fe. The release of ⁵⁹Fe from its transferrin complex was enhanced by ATP, citrate, or ascorbic acid, while these reagents did not stimulate the dissociation of ⁶⁷Ga from its transferrin complex. On the other hand, ⁶⁷Ga injected intravenously in vivo was not found in the ferritin fractions of rat liver, kidney, and tumor. The difference between experimental results in vivo and in vitro supports the hypothesis that ⁶⁷Ga in the cytoplasm is not labile enough to be bound to ferritin. We have indicated a significant role of ferritin in distinguishing between ⁶⁷Ga and ⁵⁹Fe in the cell, and provided some clues to interpret the chemical forms of ⁶⁷Ga in the cytoplasm.     

Defective development of the gall bladder and cystic duct in Lgr4‐ hypomorphic mice

Leucine‐rich repeat (LRR) ‐containing G protein coupled receptor (LGR) family members are characterized by the presence of a seven‐transmembrane domain and LRR motifs. We describe a new function for Lgr4 in the development of the gall bladder and cystic duct and in the epithelium–mesenchyme interaction. Lgr4 expression was observed in the gall bladder epithelium when the gall bladder primordium elongated ventrally. Although Lgr4 hypomorphic mutant (Lgr4^Gt/Gt) embryos developed a normal gall bladder bud at embryonic day (E) 10.25, no further elongation was observed at later stages. At E12.5, the mesenchyme surrounding the gall bladder had completely disappeared in Lgr4^Gt/Gt embryos, while the gall bladder remained unelongated. Neighboring tissues such as liver and pancreas were unaffected, as revealed by expression of marker genes. This is the first report of a mutant mouse that lacks a gall bladder and cystic duct without affecting the other tissues that derive from the same hepatic diverticulum. Developmental Dynamics 238:993–1000, 2009. © 2009 Wiley‐Liss, Inc.     

Production of antitumor substances from shochu distillation remnants

This study aimed to develop a new processing method for the effective use of rice shochu distillation remnants. We examined the inhibitory effects on the growth of human lung carcinoma cells in the medium of rice shochu distillation remnants with various fungi. Interestingly, high inhibitory effects on the growth of human lung carcinoma cells in the medium of rice shochu distillation remnants with Aspergillus oryzae were obtained, although no inhibitory effect was observed in the case of synthetic medium. We therefore fractionated the medium of rice shochu distillation remnants with A. oryzae using anion-exchange and reverse-phase chromatography. Furthermore, we attempted to determine the chemical structure of compounds that showed high inhibitory effects on the growth of tumor cells. The chemical structure of 1-hydroxy-6-(1-methylpropyl)-3-(2-methylproryl)-2(1H)-pyrazinone was revealed on the basis of liquid and gas mass spectroscopies. This compound should be completely safe based on toxic test results using model mice.     

Inconsistency between hepatic expression and serum concentration of transthyretin in mice humanized at the transthyretin locus

Human transthyretin (TTR) has about 110 variants, more than 90 of which are associated with human amyloidosis. Several groups have generated transgenic mice that carry various mutant TTR genes. However, formation of mouse/human TTR heterotetramers has been shown to be inhibitory to dissociation and subsequent amyloid formation. To avoid the effect of mouse Ttr and produce humanized mice carrying different TTR variants at high efficiency, we first produced a null allele in the mouse transthyretin locus using targeting vector that contained a neomycin resistance gene flanked by lox71 and loxP. Then, through Cre‐mediated recombination, we created a replacement allele that carried either a human normal (Val30) or mutant (Met30) TTR cDNA. This replacement resulted in a humanized TTR mouse with similar tissue‐specific profile of human TTR as that of the endogenous mouse Ttr gene. The expression levels of human TTR mRNA and protein in the liver of homozygous human TTR (Val30/Val30) mice were about twice those of heterozygous mouse/human TTR (+/Val30) mice. However, the serum human TTR levels in the Val30/Val30 mice were much less than those in the +/Val30 mice. This contradictory expression was due to unstable Val30 tetramers caused by low binding affinity to mouse retinol binding protein.     

Naso-maxillary deformity due to frontonasal expression of human transthyretin gene in transgenic mice

BACKGROUND: Retinoic acid, a metabolic product of retinol, is essential for craniofacial morphogenesis. Transthyretin (TTR) is a plasma protein delivering retinol to tissues. We produced several transgenic mouse lines using the human mutant TTR (hTTRMet30) gene to establish a mouse model of familial amyloidotic polyneuropathy. One of the lines showed an autosomal dominant inheritance of naso-maxillary deformity termed Nax. RESULTS: The Nax malformation was characterized by a hypoplastic developmental defect of the frontonasal region. Homozygous mice with higher transgene expressions showed more severe phenotypes, but a subline, in which the copy number and expression of the transgene was reduced, showed a normal phenotype, indicating that the hTTRMet30 expression caused the malformation. Nax mice began to express the hTTRMet30 gene in the nasal placode from embryonic day 10.5 (E10.5), which was 2 days earlier than in the other transgenic lines with a normal phenotype. Excessive cell death was observed in the nasal placode of the E10.5 Nax embryos. In addition, the forced expression of hTTRMet30 in the nasal placode of transgenic mice resulted in similar phenotypes. CONCLUSION: The expression of the hTTRMet30 gene in the nasal placode at E10.5 induced apoptotic cell death, leading to hypoplastic deformity in the frontonasal region.