A growth QTL (<Emphasis Type="Italic">Pbwg1</Emphasis>) region of mouse chromosome 2 contains closely linked loci affecting growth and body composition

Previous QTL studies have identified 24 QTLs for body weight and growth from 3 to 10 weeks after birth in an intersubspecific backcross mouse population between C57BL/6J and wild Mus musculus castaneus that has 60% of the body size of C57BL/6J. The castaneus allele at the most potent QTL (Pbwg1) on proximal chromosome 2 retards growth. In this study we have developed a congenic strain with a 44.1-Mb interval containing the castaneus allele at Pbwg1 by recurrent backcrossing to C57BL/6J. The congenic mouse developed was characterized by significantly higher body weight gain between 1 and 3 weeks of age and lower weight of white fat pads at 10 weeks of age than C57BL/6J. However, no clear difference in body weight at 1–10 weeks of age was observed between congenic and C57BL/6J strains. QTL analysis with 269 F₂ mice between the two strains did not identify any QTLs for body weight at 1, 3, 6, and 10 weeks of age, but it discovered eight closely linked QTLs affecting body weight gain from 1 to 3 weeks of age, lean body weight, weight of white fat pads, and body length within the Pbwg1 region. The castaneus alleles at all fat pad QTLs reduced the phenotypes, whereas at the remaining growth and body composition QTLs, they increased the trait values. These results illustrate that Pbwg1, which initially appeared to be a single locus, was resolved into several loci with opposite effects on the composition traits of overall body weight. This gives a reason for the loss of the Pbwg1 effect found in the original backcross population. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acetylation of Histone H2AX at Lys 5 by the TIP60 Histone Acetyltransferase Complex Is Essential for the Dynamic Binding of NBS1 to Damaged Chromatin

The association and dissociation of DNA damage response (DDR) factors with damaged chromatin occurs dynamically, which is crucial for the activation of DDR signaling in a spatiotemporal manner. We previously showed that the TIP60 histone acetyltransferase complex acetylates histone H2AX, to facilitate H2AX exchange at sites of DNA damage. However, it remained unclear how the acetylation of histone H2AX by TIP60 is related to the DDR signaling. We found that the acetylation but not the phosphorylation of H2AX is essential for the turnover of NBS1 on damaged chromatin. The loss of H2AX acetylation at Lys 5 by TIP60 in cells disturbed the accumulation of NBS1 at sites of DNA damage. Although the phosphorylation of H2AX is also reportedly required for the retention of NBS1 at damage sites, our data indicated that the acetylation-dependent NBS1 turnover by TIP60 on damaged chromatin restricts the dispersal of NBS1 foci from the sites of DNA damage. These findings indicate the importance of the acetylation-dependent dynamic binding of NBS1 to damaged chromatin, created by histone H2AX exchange, for the proper accumulation of NBS1 at DNA damage sites.     

Synthesis, pharmacophore modeling and in vitro activity of 10,11-dihydrodibenzo[b,f]oxepine-4-carboxamide derivatives as novel and potent antagonists of the prostaglandin EP4 receptor

The construction of a EP(4) antagonists pharmacophore model and the discovery of a highly potent oxepinic series of EP(4) antagonists is discussed. Compound 1a exhibits an excellent selectivity profile toward EP(2) receptor subtype and low cytochrome P450 inhibition potential.     

Stable genetic transformation of Larix gmelinii L. by particle bombardment of zygotic embryos

We report a new protocol for the stable transformation of Larix gmelinii. Thirty mature zygotic embryos precultured for 3 days on solid medium supplemented with benzyladenine were bombarded with plasmids pUC-GHG (GUS, HPT, and GFP genes) or pBI221-HPT (HPT and GUS genes). After a 2-month culture on selection medium, hygromycin-resistant calli appeared on the surfaces of the necrotic embryos. The frequencies of embryos with resistant calli were 18.4% and 17.4% in the transformations with pUC-GHG and pBI221-HPT DNA, respectively. More than 20 adventitious shoots formed from each of the transgenic calli. Of 17 elongated shoots selected for culturing on a rooting medium, five shoots rooted after 2 months. Expression of the GFP and GUS genes was detected in the resistant tissues by microscopic observations and by a histological GUS activity assay, respectively. PCR and Southern analysis confirmed the stable insertion of the introduced DNA into the genome.     

Cloning and characterization of a cDNA encoding beta-amyrin synthase from petroleum plant Euphorbia tirucalli L

Euphorbia tirucalli L., known as the petroleum plant, produces a large amount of triterpenes, such as beta-amyrin. Degenerate RT-PCR based on the sequences conserved among known beta-amyrin synthases led to cloning of a putative triterpene synthase cDNA, EtAS, from leaves of E. tirucalli. The deduced amino acid sequence of the EtAS cDNA showed the highest identity of 82% to the Panax ginseng beta-amyrin synthase. Heterologous expression of the EtAS ORF in the methylotrophic yeast, Pichia pastoris, resulted in production of beta-amyrin, revealing that the EtAS cDNA codes for a beta-amyrin synthase. This is the first report of a gene involved in the triterpene synthetic pathway from Euphorbiaceae plants.     

Novel matrix metalloproteinase inhibitors: generation of lead compounds by the in silico fragment-based approach

Generation of structurally new matrix metalloproteinase inhibitors was successfully carried out using an in silico technique. In order to identify the small fragment interacting with residues in the S1' pocket of MMP-1 through hydrogen bonds, we performed in silico screening using the LUDI program. As a result, acetyl-L-alanyl-(N-methyl)amide (Ac-L-Ala-NHMe) was selected to link with another fragment, hydroxamic acid that interacted with catalytic zinc. By this approach, the L-glutamic acid derivative 2b was discovered to be a new type of matrix metalloproteinase inhibitor. Further transformation to reduce its peptidic nature and improve activity yielded nonpeptidic lead compounds as inhibitors of MMP-1, -2, -3, and -9.     

Expression of Reg/PAP family members during motor nerve regeneration in rat

In this study, we examined the expression of mRNAs for Regenerating gene (Reg)/pancreatitis-associated protein (PAP) family members following hypoglossal nerve injury in rats. In addition to four rat family members (RegI, Reg-2/PAP I, PAP II, and PAP III) that had been identified, we newly cloned and sequenced a type-IV Reg gene in rats. Among these five family members, the expression of Reg-2/PAP I mRNA was predominantly enhanced in injured motor neurons after axotomy. Furthermore, a marked induction of PAP III mRNA was observed in the distal part of the injured nerve. A polyclonal antibody was raised against PAP III, and a Western blotting analysis using this antibody confirmed an increased level of PAP III protein in the injured nerve. These results suggest that Reg family members would be new mediators among injured neurons and glial cells, and may play pivotal roles during nerve regeneration.     

Further mapping of quantitative trait loci for postnatal growth in an inter-sub-specific backcross of wild Mus musculus castaneus and C57BL/6J mice

We performed a quantitative trait locus (QTL) analysis of eight body weights recorded weekly from 3 weeks to 10 weeks after birth and two weight gains recorded between 3 weeks and 6 weeks, and between 6 weeks and 10 weeks in an inter-sub-specific backcross population of wild Mus musculus castaneus mice captured in the Philippines and the common inbred strain C57BL/6J ( M. musculus domesticus ), to elucidate the complex genetic architecture of body weight and growth. Interval mapping identified 17 significant QTLs with main effects on 11 chromosomes. In particular, the main effect of the most potent QTL on proximal chromosome 2 increased linearly with age, whereas other QTLs exerted effects on either the early or late growth period. Surprisingly, although wild mice displayed 60% of the body size of their C57BL/6J counterparts, the wild-derived allele enhanced growth at two QTLs. Interestingly, five of the 17 main-effect QTLs identified had significant epistatic interaction effects. Five new epistatic QTLs with no main effects were identified on different chromosomes or regions. For one pair of epistatic QTLs, mice that were heterozygous for the wild-derived allele at one QTL and homozygous for that allele at another QTL exhibited the most rapid growth in all four possible genotypic combinations. Out of the identified QTLs, several showed significant sex-specific effects.     

Induced synchrony in Cryptococcus neoformans after release from G2-arrest

Cryptococcus neoformans was grown first to OD 4 under moderate aeration, then diluted 2.5 times with fresh medium, and grown under limited aeration for 5 h. Oxygen concentration decreased from 5-6 mg l(-1) to 1.5 mg l(-1) 1 h after the shift to limited aeration, and remained at a similar level thereafter. In all the eleven strains examined the shift caused unbudded G(2)-arrest in more than half of the cells. In three strains more than 80% of the cells were arrested in unbudded G(2), and, therefore they were selected for synchrony experiments. After being shifted to extensive aeration again, the cells resumed growth by synchronous budding, followed by synchronous nuclear division. This method has turned out to be a good tool to prepare synchronized culture in C. neoformans, especially when a large amount of synchronized cells is needed. This is worthy of attention, since synchronous cultures after release from G(2)-arrest have not been reported yet in any yeast species.