Identification and characterization of new long conserved noncoding sequences in vertebrates

Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENU-induced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results suggest that mutations occur with equal frequency in LCNS but are eliminated by natural selection during the course of evolution.     

Isolation and characterization of androgen-dependent non-histone chromosomal protein from dorsolateral prostate of rats

Rat prostate contains a unique androgen-dependent non-histone protein (Matuo et al. (1)). The non-histone protein was isolated in homogeneous form by extraction of nuclei from the dorsolateral prostate with 0.35 M NaCl in the presence of 1 mM PMSF and chromatography on a CM-Sepharose column. The final fraction was greater than 98% pure as judged by electrophoreses in SDS- and acid/urea-gels. The purified protein had a molecular weight of approximately 20,000, and an isoelectric point of approximately 11.5. Its absorption peak was at 276 nm and A(1%, 276nm)=9.3. The protein is characterized by the absence of cysteine, histidine and tryptophan, and by the high content of methionine, tyrosine and phenylalanine.     

TAK-599, a novel N-phosphono type prodrug of anti-MRSA cephalosporin T-91825: synthesis,physicochemical and pharmacological properties

Crystalline 1 (TAK-599) is a novel N-phosphono prodrug of anti-methicillin-resistant Staphylococcus aureus (MRSA) cephalosporin 2a (T-91825) that has high affinity for penicillin-binding protein (PBP) 2' (IC(50); 0.90 microg/mL) and shows potent in vitro anti-MRSA activity (MIC against MRSA N133; 1.56 microg/mL), comparable to that of vancomycin (1.56 microg/mL). Although 2a had insufficient water solubility (2.3 mg/mL) for parenteral administration, 1 showed excellent water solubility (>100 mg/mL, pH 7) as well as good chemical stability in the solid state and solution. In pharmacokinetic studies, when 1 was administered intravenously to rats and monkeys, it was rapidly converted into 2a in the blood. These results show that 1 (TAK-599) is a highly promising parenteral cephalosporin targeted for MRSA infection.     

Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells

Sperm cryopreservation provides an economical means of storing genetically engineered mouse strains in resource facilities. In general, relatively high fertilization rates are obtained for frozen/thawed sperm of the CBA/JN, DBA/2N, and C3H inbred strains and some F1 hybrid strains. However, the fertilization rate for frozen/thawed sperm of C57BL/6, which is the main strain of genetically engineered mice, remains very low. Therefore, it is necessary to establish an in vitro fertilization (IVF) method for cryopreserved C57BL/6 sperm that can obtain a high rate of fertilization after thawing. In the present study, we focused on the effects of methyl-beta-cyclodextrin (MBCD) on the fertilizing ability of frozen/thawed C57BL/6 sperm. Our results have shown that the highest fertilization rate for frozen/thawed sperm was obtained with MBCD at 1.0 mM for 30 min (63.7% +/- 11.0%), but the effects were attenuated by long-term incubation for 120 min at 1.0 or 2.0 mM. The embryos with frozen/thawed sperm showed good developmental potential, and the offspring had normal fertility. The efflux of cholesterol from frozen/thawed sperm was increased by MBCD in a dose-dependent manner and occurred much earlier and to a greater extent than bovine serum albumin. The localization of cholesterol labeled by filipin in the sperm plasma membrane was drastically decreased by MBCD. In summary, we suggest that MBCD is useful for developing an IVF method for frozen/thawed C57BL/6 mouse sperm achieving a high fertilization rate, being involved in the capacity to sequester cholesterol from sperm membrane.     

Co-precipitation molecules hemopexin and transferrin may be key molecules for fibrillogenesis in TTR V30M amyloidogenesis

The disease model of familial amyloidotic polyneuropathy—7.2-hMet30 mice—manifests amyloid deposition that consists of a human amyloidogenic mutant transthyretin (TTR) (TTR V30M). Our previous study found amyloid deposits in 14 of 27 7.2-hMet30 mice at 21–24 months of age. In addition, non-fibrillar TTR deposits were found in amyloid-negative 7.2hMet30 mice. These results suggested that TTR amyloidogenesis required not only mutant TTR but also an additional factor (or factors) as an etiologic molecule. To determine the differences in serum proteome in amyloid-positive and amyloid-negative mice in the 7.2-hMet30 model, we used proteomic analyses and studied serum samples obtained from these mice. Hemopexin (HPX) and transferrin (Tf) were detected in the serum samples from amyloid-positive mice and were also found in amyloid deposits via immunohistochemistry, but serum samples from amyloid-negative mice did not contain HPX and Tf. These two proteins were also not detected in non-fibrillar TTR deposits. In addition, in silico analyses suggested that HPX and Tf facilitate destabilization of TTR secondary structures and misfolding of TTR. These results suggest that HPX and Tf may be associated with TTR amyloidogenesis after fibrillogenesis in vivo.     

Correction to: Food conditions,competitive regime,and female social relationships in Japanese macaques: within-population variation on Yakushima

Primates - In our published paper (Hanya et al. 2008), we found an error in a figure described in the     

Octanoate inhibits very low-density lipoprotein secretion in primary cultures of chicken hepatocytes

The effects of octanoate, a medium-chain fatty acid, on very low-density lipoprotein (VLDL) secretion in primary cultures of chicken hepatocytes were compared with those of palmitate. Palmitate added to the incubation media at concentrations up to 0.36 mM increased intracellular triacylglycerol (TG) accumulation and VLDL-TG secretion in a concentration-dependent manner, whereas the addition of octanoate alone (0.21-0.6 mM) did not change these parameters. VLDL-TG secretion from hepatocytes cultured in media to which 0.6 or 1.0 mM octanoate had been added in the presence of 0.21 mM palmitate was significantly lower than that obtained under control incubation conditions (0.21 mM palmitate only). The addition of 1.0 mM octanoate to the incubation media with or without 0.21 mM palmitate decreased VLDL apolipoprotein B (apoB) secretion. These results demonstrate that the addition of octanoate to primary cultures of chicken hepatocytes reduces VLDL secretion in respect of both TG and apoB secretion. It is suggested that medium-chain fatty acids are a factor modulating VLDL secretion, which plays a key role in fat deposition in chickens.