Maternal Separation Enhances Conditioned Fear and Decreases the mRNA Levels of the Neurotensin Receptor 1 Gene with Hypermethylation of This Gene in the Rat Amygdala

Stress during postnatal development is associated with an increased risk for depression, anxiety disorders, and substance abuse later in life, almost as if mental illness is able to be programed by early life stressors. Recent studies suggest that such “programmed” effects can be caused by epigenetic regulation. With respect to conditioned fear, previous studies have indicated that early life stress influences its development in adulthood, whereas no potential role of epigenetic regulation has been reported. Neurotensin (NTS) is an endogenous neuropeptide that has receptors densely located in the amygdala and hippocampus. Recently, NTS systems have constituted an emerging target for the treatment of anxiety. The aim of the present work is to clarify whether the NTS system is involved in the disturbance of conditioned fear in rats stressed by maternal separation (MS). The results showed that MS enhanced freezing behaviors in fear-conditioned stress and reduced the gene expression of NTS receptor (NTSR) 1 but not of NTS or NTSR2 in the amygdalas of adult rats. The microinjection of a NTSR1 antagonist into the amygdala increased the percentage of freezing in conditioned fear, whereas the microinjection of NTSR1 agonist decreased freezing. These results suggest that NTSR1 in the amygdala may play a role in the effects of MS on conditioned fear stress in adult rats. Moreover, MS increased DNA methylation in the promoter region of NTSR1 in the amygdala. Taken together, MS may leave epigenetic marks in the NTSR1 gene in the amygdala, which may enhance conditioned fear in adulthood. The MS-induced alternations of DNA methylation in the promoter region of NTSR1 in the amygdala may be associated with vulnerability to the development of anxiety disorders and depression in adulthood.     

Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly

CENP-A chromatin forms the foundation for kinetochore assembly. Replication-independent incorporation of CENP-A at centromeres depends on its chaperone HJURP^Scm3, and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURP^Scm3 to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1^KNL2, which is critical for the recruitment of Mis18 and HJURP^Scm3. We identify two novel fission yeast Mis18-interacting proteins (Eic1 and Eic2), components of the Mis18 complex. Eic1 is essential to maintain Cnp1^CENP-A at centromeres and is crucial for kinetochore integrity; Eic2 is dispensable. Eic1 also associates with Fta7^CENP-Q/Okp1, Cnl2^Nkp2 and Mal2^CENP-O/Mcm21, components of the constitutive CCAN/Mis6/Ctf19 complex. No Mis18BP1^KNL2 orthologue has been identified in fission yeast, consequently it remains unknown how the key Cnp1^CENP-A loading factor Mis18 is recruited. Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1^KNL2, thus representing the functional counterpart of Mis18BP1^KNL2 in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3^HJURP Cnp1^CENP-A loading factors. The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.     

Isolation and validation of eight microsatellite loci in a damselfly Ischnura senegalensis (Odonata: Coenagrionidae) by pyrosequencing a bead-enriched library

Eight microsatellite markers for the population genetics and evolutionary ecology of Ischnura senegalensis (Rambur 1842), which shows body color polymorphism in females, were developed using a streptavidin-bead enrichment library and pyrosequence by a next generation sequencer. The number of alleles per locus and effective number of alleles ranged from 3 to 11 and from 1.24 to 5.51, respectively. Observed and expected heterozygosities were 0.18–0.75 and 0.19–0.77, respectively. No linkage disequilibrium between loci was detected. One locus, IsenAC75, deviated significantly from the Hardy–Weinberg equilibrium, and the locus and additional two loci, IsenAC40 and IsenAC8, were suspected for the presence of null alleles. Altogether, these eight microsatellite loci are considered to be useful for population genetic analyses because of the high polymorphic status and independency.     

Confuser® MM can be used as an attractant to enable monitoring of the peach leafminer moth,Lyonetia clerkella (Lepidoptera: Lyonetiidae), in peach orchards treated with a mating disrupter

Sex-pheromone traps baited with a rubber-septum containing 14-methyl-1-octadecene are used to monitor the emergence of adult peach leafminer moths, Lyonetia clerkella (L.) (Lepidoptera: Lyonetiidae), in peach orchards. This compound is also used as a mating disrupter; large amounts of synthetic 14-methyl-1-octadecene can successfully interfere with the mate-finding behavior of the male moths. In peach orchards treated with mating disrupters, however, conventional rubber-septum lures are ineffective, because mating disrupters interfere with male orientation. In this work we demonstrated that a commercially-available mating disrupter, Confuser^® MM, can be used as an attractant in orchards treated with mating disrupters. In an indoor flight tunnel experiment, 33–42 % of males pre-exposed to a mating disrupter responded to one to ten Confuser^® MM dispensers whereas only 8 % responded to a conventional lure made from a rubber-septum impregnated with a relatively small amount of the sex pheromone. The number of male moths captured by traps baited with five dispensers of Confuser^® MM in orchards treated with mating disrupters was positively correlated with the number of males trapped by use of a conventional rubber-septum lure in untreated orchards. This suggests that Confuser^® MM can be used as an attractant for monitoring emergence time and relative abundance in an orchard being treated with a mating disrupter.     

3-(2-Aminocarbonylphenyl)propanoic acid analogs as potent and selective EP3 receptor antagonists. Part 3: Synthesis,metabolic stability,and biological evaluation of optically active analogs

A series of 3-(2-aminocarbonylphenyl)propanoic acid analogs possessing the (1R)-1-(3,5-dimethylphenyl)-3-methylbutylamine moiety on the carboxyamide side chain were synthesized and evaluated for their binding affinity for the EP1-4 receptors and their antagonist activity for the EP3 receptor. Rational drug design based on the structure of the metabolites in human liver microsomes led us to the discovery of another series of analogs. Several compounds were further evaluated for their in vivo efficacy in rats after oral administration and also for their pharmacokinetic profiles including in vitro stability in the liver microsomes.     

Selective detection of 2-nitrobenzenesulfenyl-labeled peptides by matrix-assisted laser desorption/ionization-time of flight mass spectrometry using a novel matrix

The 2-nitrobenzenesulfenyl (NBS) method, which is useful for quantitative proteome analysis, is based on stable isotope labeling of tryptophan residues with NBS chloride ((12)C(6)-NBSCl or (13)C(6)-NBSCl). We found that 3-hydroxy-4-nitrobenzoic acid (3H4NBA) is a more suitable matrix than 2,5-dihydroxybenzoic acid (DHB) for detecting NBS-labeled peptides by MALDI-quadrupole IT (QIT)-TOF MS . Furthermore, NBS-labeled peptides were selectively ionized and detected in a mixture of NBS-labeled and unlabeled peptides. Labeled paired peaks were easily detected without enrichment, nonpaired labeled peaks were clearly distinguished from unlabeled contaminating peptides, and nitrotyrosine-containing peptides were also selectively detected on the 3H4NBA matrix, while by-product-peaks arising from nitrobenzene moieties were suppressed. The use of 3H4NBA as a comatrix with CHCA improved the sensitivity of detection while substantially retaining the selectivity of 3H4NBA. The 3H4NBA matrix offers great advantages in terms of simplicity, sensitivity, and usability when used for the NBS method and for MALDI-TOF MS analysis applied to compounds having a nitrobenzene ring.     

T cell deficiency leads to liver carcinogenesis in azoxymethane-treated rats

There is an increasing amount of evidence suggesting that T cell deficiency contributes to tumor development. However, it is unclear whether T cell deficiency leads to liver and colon carcinogenesis. The aim of this study was to investigate the role of T cells on liver and colon carcinogenesis. Athymic F344/N Jcl-rnu/- (nu/nu) rats and euthymic F344/N Jcl-rnu/+(nu/+) rats were administered the carcinogen azoxymethane (AOM) at a dose of 15 mg/kg body wt once a week for 2 weeks. At 48 weeks after the second carcinogen treatment, the rats were sacrificed, and livers and colons were examined. Apoptosis and cell proliferation were evaluated by DNA fragmentation and proliferating cell nuclear antigen assays, respectively. Wild-type p53 and members of the Jun and Fos oncogene families were detected by Western blotting. AOM treatment induced 100% liver tumor and 63.6% colon tumor incidence in T cell-deficient nu/nu rats, compared with 0% and 38.5% incidence in nu/+ rats. T cell deficiency promoted the inhibitory action of AOM on apoptosis in both liver and colon at 48 weeks. In contrast, T cell deficiency increased cell proliferation after AOM treatment in both tissues. Wild-type p53 was reduced in both tissues of T cell-deficient rats. AOM treatment induced c-Jun and c-Fos expressions in the liver but increased only Fos B in the colon, whereas T cell deficiency enhanced c-Jun overexpression in the liver. These results suggest that T cell deficiency leads to liver carcinogenesis partly by a reduction in wild-type p53 and increasing c-Jun expression in AOM-treated rats.     

Expression of low density lipoprotein receptor-related protein 4 (Lrp4) gene in the mouse germ cells

The low density lipoprotein receptor-related protein 4 gene (Lrp4) was identified by subtractive screening of cDNAs of the migratory primordial germ cells (PGCs) of E8.5-9.5 embryo and E3.5 blastocysts. Lrp4 is expressed in PGCs in the hindgut and the dorsal mesentery of E9.5 embryos, and in germ cells in the genital ridges of male and female E10.5-13.5 embryos. Lrp4 is also expressed in spermatogonia of the neonatal and adult testes and in the immature oocytes and follicular cells of the adult ovary. The absence of Lrp4 expression in the blastocyst, embryonic stem cells and embryonic germ cells suggests the Lrp4 is a molecular marker that distinguishes the germ cells from embryo-derived pluripotent stem cells.     

Rapid and efficient MALDI-TOF MS peak detection of 2-nitrobenzenesulfenyl-labeled peptides using the combination of HPLC and an automatic spotting apparatus.

In this paper, we report MALDI-TOF ms analysis of 2-nitrobenzenesulfenyl (NBS) labeled peptides with the powerful aid of an LC-automatic spotting system. using this approach we analyzed mammalian sera (rat and mouse) as biological samples to demonstrate performance. The labeling was carried out using a binary set of 2-nitrobenzenesulfenyl chloride (heavy and light), which modified tryptophan residues in sample proteins. Approximately 1600 doublet peaks were detected in the mass spectrum, some of which had more than threefold differences in their intensities. systematic separation/spotting followed by mass analysis of the NBS-labeled peptides derived from biological samples is described for the first time. This method has proved to be an effective application of NBS-labeled peptides and can be a powerful technique for quantitative analysis of proteins expressed in biological systems.