Chemical modification of triplex-forming oligonucleotide to promote pyrimidine motif triplex formation at physiological pH

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in wide variety of potential applications, such as artificial regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis in vivo. Stabilization of pyrimidine motif triplex at physiological pH is, therefore, crucial for improving its potential in various triplex-formation-based strategies in vivo. To this end, we investigated the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid modification of triplex-forming oligonucleotide (TFO), in which 2'-O and 4'-C of the sugar moiety were bridged with the methylene chain and 3'-O was replaced by 3'-NH, on pyrimidine motif triplex formation at physiological pH. The modification not only significantly increased the thermal stability of the triplex but also increased the binding constant of triplex formation about 15-fold. The increased magnitude of the binding constant was not significantly changed when the number and position of the modification in TFO changed. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the modified TFO in the free state resulting from the bridging of different positions of the sugar moiety with an alkyl chain and the increased hydration of the modified TFO in the free state caused by the introduction of polar nitrogen atoms may significantly increase the binding constant at physiological pH. The study on the TFO viability in human serum showed that the modification significantly increased the resistance of TFO against nuclease degradation. This study presents an effective approach for designing novel chemically modified TFOs with higher binding affinity of triplex formation at physiological pH and higher nuclease resistance under physiological condition, which may eventually lead to progress in various triplex-formation-based strategies in vivo.     

Analysis of peptide uptake and location of root hair‐promoting peptide accumulation in plant roots

Peptide uptake by plant roots from degraded soybean‐meal products was analyzed in Brassica rapa and Solanum lycopersicum. B. rapa absorbed about 40% of the initial water volume, whereas peptide concentration was decreased by 75% after 24 h. Analysis by reversed‐phase HPLC showed that number of peptides was absorbed by the roots during soaking in degraded soybean‐meal products for 24 h. Carboxyfluorescein‐labeled root hair‐promoting peptide was synthesized, and its localization, movement, and accumulation in roots were investigated. The peptide appeared to be absorbed by root hairs and then moved to trichoblasts. Furthermore, the peptide was moved from trichoblasts to atrichoblasts after 24 h. The peptide was accumulated in epidermal cells, suggesting that the peptide may have a function in both trichoblasts and atrichoblasts. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Restoration of Dioxin-Induced Damage to Fetal Steroidogenesis and Gonadotropin Formation by Maternal Co-Treatment with α-Lipoic Acid

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an endocrine disruptor, causes reproductive and developmental toxic effects in pups following maternal exposure in a number of animal models. Our previous studies have demonstrated that TCDD imprints sexual immaturity by suppressing the expression of fetal pituitary gonadotropins, the regulators of gonadal steroidogenesis. In the present study, we discovered that all TCDD-produced damage to fetal production of pituitary gonadotropins as well as testicular steroidogenesis can be repaired by co-treating pregnant rats with α-lipoic acid (LA), an obligate co-factor for intermediary metabolism including energy production. While LA also acts as an anti-oxidant, other anti-oxidants; i.e., ascorbic acid, butylated hydroxyanisole and edaravone, failed to exhibit any beneficial effects. Neither wasting syndrome nor CYP1A1 induction in the fetal brain caused through the activation of aryl hydrocarbon receptor (AhR) could be attenuated by LA. These lines of evidence suggest that oxidative stress makes only a minor contribution to the TCDD-induced disorder of fetal steroidogenesis, and LA has a restorative effect by targeting on mechanism(s) other than AhR activation. Following a metabolomic analysis, it was found that TCDD caused a more marked change in the hypothalamus, a pituitary regulator, than in the pituitary itself. Although the components of the tricarboxylic acid cycle and the ATP content of the fetal hypothalamus were significantly changed by TCDD, all these changes were again rectified by exogenous LA. We also provided evidence that the fetal hypothalamic content of endogenous LA is significantly reduced following maternal exposure to TCDD. Thus, the data obtained strongly suggest that TCDD reduces the expression of fetal pituitary gonadotropins to imprint sexual immaturity or disturb development by suppressing the level of LA, one of the key players serving energy production.     

IRAK-4-dependent degradation of IRAK-1 is a negative feedback signal for TLR-mediated NF-kappaB activation

The activation of interleukin 1 receptor-associated kinase (IRAK)-1 is a key event in the transmission of signals from Toll-like receptors (TLRs). The catalytic activity of the protein kinase is not essential for its ability to activate nuclear factor (NF) kappaB, because transfection of a kinase-dead mutant of IRAK-1 (IRAK-1KD) is able to activate NF-kappaB in HEK293T cells. In the present study, we observed that the effect of IRAK-1KD was impaired by simultaneous expression of IRAK-4. The effect of IRAK-4 was accompanied by the phosphorylation and degradation of IRAK-1KD. Expression of IRAK-4KD instead of IRAK-4 did not cause these events. In IRAK-4-deficient Raw264.7 macrophages that were prepared by introducing short-hairpin RNA probes, the basal level of IRAK-1 was increased markedly. Stimulation of these cells with TLR ligands did not cause the degradation of IRAK-1, which was clearly observed in the parent cells. These results suggested that the expression of IRAK-4 alone is sufficient to cause the degradation of IRAK-1; the autophosphorylation of IRAK-1 is not necessary to terminate the TLR-induced activation of NF-kappaB. IRAK-4 has an ability to induce the degradation of IRAK-1 in addition to its role as an activator of IRAK-1.     

Phylogenetic analysis of long-chain hydrocarbon-degrading bacteria and evaluation of their hydrocarbon-degradation by the 2,6-DCPIP assay

Thirty-six bacteria that degraded long-chain hydrocarbons were isolated from natural environments using long-chain hydrocarbons (waste car engine oil, base oil or the c-alkane fraction of base oil) as the sole carbon and energy source. A phylogenetic tree of the isolates constructed using their 16S rDNA sequences revealed that the isolates were divided into six genera plus one family (Acinetobacter, Rhodococcus, Gordonia, Pseudomonas, Ralstonia, Bacillus and Alcaligenaceae, respectively). Furthermore, most of the isolates (27 of 36) were classified into the genera Acinetobacter, Rhodococcus or Gordonia. The hydrocarbon-degradation similarity in each strain was confirmed by the 2,6-dichlorophenol indophenol (2,6-DCPIP) assay. Isolates belonging to the genus Acinetobacter degraded long-chain normal alkanes (n-alkanes) but did not degrade short-chain n-alkanes or cyclic alkanes (c-alkanes), while isolates belonging to the genera Rhodococcus and Gordonia degraded both long-chain n-alkanes and c-alkanes.     

Restoration of spermatogenesis and fertility in azoospermic mutant mice by suppression and reelevation of testosterone followed by intracytoplasmic sperm injection.

Advances in assisted reproduction techniques such as in vitro fertilization and intracytoplasmic sperm injection have made paternity possible for many patients with male infertility. However, at least some sperm or spermatids are required for these techniques to be successful, and patients incapable of producing spermatids cannot be helped. Male mice homozygous for the mutant juvenile spermatogonial depletion (jsd) gene show spermatogonial arrest and an elevated intratesticular testosterone level like many other experimental infertility models such as those with iradiation- or chemotherapy-induced testicular damage. In this category of infertile males, suppression of the testosterone level induces spermatogonial differentiation to the stage of spermatocytes but no further. In the present study with jsd mutant mice, we induced spermatogenesis first to spermatocytes and then to elongated spermatids by suppression of testosterone levels with a GnRH antagonist, Nal-Glu, at a dose of 2500 microg kg(-1) day(-1) for 4 wk and then withdrawal of Nal-Glu. Spermatids were seen in the cross-sections of seminiferous tubules in all mice treated by administration and subsequent withdrawal of Nal-Glu. Four weeks after withdrawal of Nal-Glu, some of the germ cells differentiated into elongated spermatids. Supplementation with testosterone and Nal-Glu after 4 wk of treatment with Nal-Glu alone also induced spermatogenesis similar to the induction by withdrawal of Nal-Glu. Thus, we ascribe the restoration of the differentiation of spermatocytes to spermatids to reelevation of the testosterone level. Furthermore, we successfully rescued male sterility in jsd mice by subsequent intracytoplasmic sperm injection using the elongated spermatids induced by the programmed hormone therapy.     

A long Y chromosome in man

Summary An unusually long Y chromosome was described in the phenotypically normal father and paternal grandfather of a girl with Down's syndrome, and likewise in a male infant with multiple malformations and his father, normal in phenotype. Measurements revealed that the long Y chromosome corresponded in length to autosomes of group 16–18.Information was obtained to show that the increased length of the Y chromosome was an inheritable character, and that a long Y chromosome was not always associated with an abnormal phenotype (or phenotypes).Contribution No. 585 from the Zoological Institute, Hokkaido University.     

Antiobesity and emetic effects of a short-length peptide YY analog and its PEGylated and alkylated derivatives

Neuropeptide Y2 receptor (Y2R) agonism is an important anorectic signal and a target of antiobesity drug discovery. Recently, we synthesized a short-length Y2R agonist, PYY-1119 (4-imidazolecarbonyl-[d-Hyp²⁴,Iva²⁵,Pya(4)²⁶,Cha^27,36,γMeLeu²⁸,Lys³⁰,Aib³¹]PYY(23–36), 1) as an antiobesity drug candidate. Compound 1 induced marked body weight loss in diet-induced obese (DIO) mice; however, 1 also induced severe vomiting in dogs at a lower dose than the minimum effective dose administered to DIO mice. The rapid absorption of 1 after subcutaneous administration caused the severe vomiting. Polyethylene glycol (PEG)- and alkyl-modified derivatives of 1 were synthesized to develop Y2R agonists with improved pharmacokinetic profiles, i.e., lower maximum plasma concentration (C_max) and longer time at maximum concentration (T_max). Compounds 5 and 10, modified with 20?kDa PEG at the N-terminus and eicosanedioic acid at the Lys³⁰ side chain of 1, respectively, showed high Y2R binding affinity and induced significant body weight reduction upon once-daily administration to DIO mice. Compounds 5 and 10, with their relatively low C_max and long T_max, partially attenuated emesis in dogs compared with 1. These results indicate that optimization of pharmacokinetic properties of Y2R agonists is an effective strategy to alleviate emesis induced by Y2R agonism.     

Densification of cyanobacteria from a lake leading to production of β‐cyclocitral and related volatile organic compounds and species change

The purpose of the present study was to demonstrate that the lysis with the blue color formation was caused by densification of the cyanobacteria, and related events of the species change in the cyanobacteria were induced by the resulting volatile organic compounds (VOCs), particularly β‐cyclocitral. In order to obtain a high cell density of cyanobacteria in the laboratory, a concentration technique (graduated cylinder method) using the buoyancy of the gas vesicles was successfully used. The collected scum contained mainly Dolichospermum spp. and Microcystis, and the dispersed cyanobacteria were concentrated in the surface layer after several hours and the concentration ratio became approximately 10. The concentrated cyanobacteria were gradually lysed, while some of the cyanobacteria sank to the bottom, which finally died and disappeared. This method has the additional advantage that it is possible to visualize the entire lysis process. During the concentration process, β‐cyclocitral and its oxidation products together with β‐ionone were significantly detected. Because β‐cyclocitral was easily oxidized to the corresponding carboxylic acid, the pH of the water in the graduated cylinder decreased to approximately 6. Under favorable conditions, lysis with the blue color from phycocyanin could be observed due to the acid stress. Overall, the results of the present study were consistent with the hypothesis that VOCs were produced when the cyanobacteria are highly dense, and that the lysis with the blue color formation occurs due to the higher density.