Characterization of a novel chromodomain-containing gene from the silkworm, Bombyx mori

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein across different eukaryotic species, and is crucial in the establishment and maintenance of heterochromatin. HP1 proteins have two distinct functional domains, an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD), which are required for the selective binding of HP1 proteins to modified histones. During our screen for HP1-like proteins in the Bombyx mori genome, we found a novel silkworm gene, Bombyx mori chromodomain protein 1 (BmCdp1), encoding a putative chromobox protein with only two CDs. The BmCdp1 family proteins are closely related to the HP1 proteins, and most of them belong to insect lineages. qRT-PCR analysis indicated that BmCdp1 mRNA was most abundantly expressed in early embryos, and relatively higher expression was observed in larval testes, hemocytes, and pupal ovaries. Western blot and immunostaining experiments showed that BmCdp1 was localized mainly in the nucleus of BmN4 cells. We searched BmCdp1-bound loci in the Bombyx genome by ChIP-seq analysis using Flag-tagged BmCdp1-expressing BmN4 cells. Combined with ChIP-qPCR experiments, we identified two reliable BmCdp1-bound loci in the genome. siRNA-mediated knockdown of BmCdp1 in BmN4 cells and early embryos did not affect the expression of the gene located close to the BmCdp1-bound locus.     

Organization and expression of the chicken N-myc gene.

We cloned the chicken N-myc gene and analyzed its structure and expression. We found that it consisted of three exons with coding regions in exons 2 and 3. Comparison to mammalian N-myc genomic sequence indicated that nucleotide sequences of the 5'-flanking region, noncoding exon 1, and introns were not conserved, but coding and 3' noncoding sequences showed significant homology to mammalian N-myc. Alignment of deduced amino acid sequences of chicken and mammalian N-myc proteins revealed nine conserved domains interrupted by different lengths of nonhomologous sequences. Two of the domains were specific to N-myc proteins, and the other seven were common to c-myc proteins. Northern blot (immunoblot) and in situ hybridization analyses of 3.5-day-old chicken embryos revealed that high-level expression of the N-myc gene was confirmed to certain tissues, e.g., the central nervous system, neural crest derivatives, and mesenchyme of limb buds. In the beak and limb primordia, N-myc expression in the mesenchyme was higher toward the distal end, suggesting possible involvement in positional assignment of the tissue within the rudimentary structures.     

Preservation of microplate-attached human hepatoma cells and their use in cytotoxicity tests

We investigated the feasibility of hypothermic- orcryogenically-preserved human hepatoma Hep G2 cell preculturedin 96-well plates in cytotoxicity testings. First, we observedthat microplates precoated with both collagen (CN) and pronectin (PN) showed significantly improved living cell adhesion (71.0 ± 5.5%) after 48 hr of cryopreservation with 10%-DMSO containing culture medium, whereas non-coated surfaces gave very low living cell adhesion (33.5 ± 2.1%). Hypothermic preservation was most suitable for short-term storage, and cryogenic preservation at –20 °C allowed cells to be used within a week of the storage period. Only cryopreservation in a deep freezer (–85 °C) gave satisfactory results in much longer period of storage. Second, we evaluated the cytotoxicity of ten chemicals during 48 hr of exposure using hypothermically – (4 °C for 2 days) or cryogenically – (–85 °C for 7 days) preserved cells cultured inCN/PN-precoated microplates in comparison with results fromfreshly inoculated cells. Although almost the same LD₅₀values were obtained, LD₁₀ values of relatively hydrophilic chemicals obtained with cryopreserved cell were significantly lowered. These results shown that CN/PN-precoating is effective in keeping cells attached even in recultivation of preserved cells and that the toxicities of relatively hydrophilic chemicals tend to be overestimated when we use preserved cells in that manner.     

Triazoyl–phenyl linker system enhancing the aqueous solubility of a molecular probe and its efficiency in affinity labeling of a target protein for jasmonate glucoside

In methods employing molecular probes to explore the targets of bioactive small molecules, long or rigid linker moieties are thought to be critical factors for efficient tagging of target protein. We previously reported the synthesis of a jasmonate glucoside probe with a highly rigid linker consisting of a triazoyl–phenyl (TAzP) moiety, and this probe demonstrated effective target tagging. Here we compare the TAzP probe with other rigid or flexible probes with respect to target tagging efficiency, hydrophobic parameters, aqueous solubility, and dihedral angles around the biaryl linkage by a combination of empirical and calculation methods. The rigid biaryl linkage of the TAzP probe has a skewed conformation that influences its aqueous solubility. Such features that include rigidness and good aqueous solubility resulted in highly efficient target tagging. These findings provide a promising guideline toward designing of better linkers for improving molecular probe performance.     

Great apes’ understanding of biomechanics: eye-tracking experiments using three-dimensional computer-generated animations

Primates - Visual processing of the body movements of other animals is important for adaptive animal behaviors. It is widely known that animals can distinguish articulated animal movements even...     

Novel proteins interacting with peroxisomal protein receptor PEX7 in Arabidopsis thaliana

Peroxisomal matrix protein transport relies on 2 cytosolic receptors, PEX5 and PEX7, which import peroxisomal targeting signal type 1 (PTS1) and PTS2-containing proteins, respectively. To better understand the transport mechanism of PEX7, we isolated PEX7 complexes using proteomics. We identified PEX5 as well as PTS1- and PTS2-containing proteins within the complex, thereby confirming the interaction between PEX5 and PEX7 during cargo transport that had been previously characterized by biochemical approaches. In addition, a chaperone T-complex and 2 small Rab GTPases were identified. We recently reported that the RabE1c is involved in the degradation of the PEX7 when abnormal PEX7 is accumulated on the peroxisomal membrane. This study expands our knowledge on the transport machinery via PEX7 by identifying both known and novel PEX7-interacting proteins and thus is helpful for further investigation of the regulation of the peroxisomal protein receptor during its translocation.     

Molecular genetic system for regenerative studies using newts

Urodele newts have the remarkable capability of organ regeneration, and have been used as a unique experimental model for more than a century. However, the mechanisms underlying regulation of the regeneration are not well understood, and gene functions in particular remain largely unknown. To elucidate gene function in regeneration, molecular genetic analyses are very powerful. In particular, it is important to establish transgenic or knockout (mutant) lines, and systematically cross these lines to study the functions of the genes. In fact, such systems have been developed for other vertebrate models. However, there is currently no experimental model system using molecular genetics for newt regenerative research due to difficulties with respect to breeding newts in the laboratory. Here, we show that the Iberian ribbed newt (Pleurodeles waltl) has outstanding properties as a laboratory newt. We developed conditions under which we can obtain a sufficient number and quality of eggs throughout the year, and shortened the period required for sexual maturation from 18 months to 6 months. In addition, P. waltl newts are known for their ability, like other newts, to regenerate various tissues. We revealed that their ability to regenerate various organs is equivalent to that of Japanese common newts. We also developed a method for efficient transgenesis. These studies demonstrate that P. waltl newts are a suitable model animal for analysis of regeneration using molecular genetics. Establishment of this experimental model will enable us to perform comparable studies using these newts and other vertebrate models.     

Fecal metabolite of a gnotobiotic mouse transplanted with gut microbiota from a patient with Alzheimer’s disease

ABSTRACT

Studies of Alzheimer’s disease are based on model mice that have been altered by transgenesis and other techniques to elicit pathogenesis. However, changes in the gut microbiota were recently suggested to diminish cognitive function in patients, as well as in model mice. Accordingly, we have created model mice of the human gut microbiota by transplanting germ-free C57BL/6N mice with fecal samples from a healthy volunteer and from an affected patient. These humanized mice were stably colonized and reproduced the bacterial diversity in donors. Remarkably, performance on Object Location Test and Object Recognition Test was significantly reduced in the latter than in the former at 55 weeks of age, suggesting that gut microbiota transplanted from an affected patient affects mouse behavior. In addition, metabolites related to the nervous system, including γ-aminobutyrate, taurine, and valine, were significantly less abundant in the feces of mice transplanted with microbiota from the affected patient.