Functional impact of transposable elements using bioinformatic analysis and a comparative genomic approach

A dual coding event, which is the translation of different isoforms from a single gene, is one of the special patterns among the alternative splicing events. This is an important mechanism for the regulation of protein diversity in human and mouse genomes. Although the regulation for dual coding events has been characterized in a few genes, the individual mechanism remains unclear. Numerous studies have described the exonization of transposable elements, which is the splicing mediated insertion of transposable element sequence fragments into mature mRNAs. Therefore, in this study, we investigated the number of transposable element (TE)-derived dual coding genes in human, chimpanzee and mouse genomes. TE fusion exons appeared in the dual coding regions of 309 human genes. Functional protein domain alterations by TE-derived dual coding events were observed in 129 human genes. Comparative TE-derived dual coding events were also analyzed in chimpanzee and mouse orthologs. Seventy chimpanzee orthologs had TE-derived dual coding events, but mouse orthologs did not have any TE-derived dual coding events. Taken together, our analyses listed the number of TE-derived dual coding genes which could be investigated by experimental analysis and suggested that TE-derived dual coding events were major sources for the functional diversity of human genes, but not mouse genes.     

Effect of Ischemic Neuronal Insults on Amyloid Precursor Protein Processing

The nature of the association between ischemic stroke and Alzheimer’s disease (AD) at the cellular and molecular level is still unknown. We evaluated the effect of ischemic neuronal insults on the regulation of amyloid precursor protein (APP) processing. We used an in vitro model of cerebral ischemia (oxygen-glucose deprivation) to evaluate the effect of ischemic neuronal insults on the amyloidogenic and non-amyloidogenic pathways using human neuroblastoma cell line and primary cultured cells of transgenic mice which expressed human APP (Tg2576). Ischemic neuronal insults increased the production of Aβ in Tg2576 primary culture cells compared to controls. A disintegrin and metalloprotease 10 (ADAM 10) was markedly increased in early stage of ischemic insults, which was followed by decreased level of ADAM 10 expression in later stage. The protein and mRNA expression of β-site cleavage enzyme (BACE) and BACE activity was not significantly different between the group of ischemic insults and control. By contrast, the activity of γ-secretase was significantly increased after 4 h of ischemic insults, as compared to controls. The present study showed that the ischemic neuronal insults increased the production of Aβ by influencing APP metabolism, which may link the role of ischemic insults to the pathogenesis of AD.     

Isolation of epithelial stem cells from dermis by a three-dimensional culture system

Skin is a representative self-renewing tissue containing stem cells. Although many attempts have been made to define and isolate skin-derived stem cells, establishment of a simple and reliable isolation procedure remains a goal to be achieved. Here, we report the isolation of cells having stem cell properties from mouse embryonic skin using a simple selection method based on an assumption that stem cells may grow in an anchorage-independent manner. We inoculated single cell suspensions prepared from mouse embryonic dermis into a temperature-sensitive gel and propagated the resulting colonies in a monolayer culture. The cells named dermis-derived epithelial progenitor-1 (DEEP) showed epithelial morphology and grew rapidly to a more than 200 population doubling level over a period of 250 days. When the cells were kept confluent, they spontaneously formed spheroids and continuously grew even in spheroids. Immunostaining revealed that all of the clones were positive for the expression of cytokeratin-8, -18, -19, and E-cadherin and negative for the expression of cytokeratin-1, -5, -6, -14, -20, vimentin, nestin, a ckit. Furthermore, they expressed epithelial stem cell markers such as p63, integrin beta1, and S100A6. On exposure to TGFbeta in culture, some of DEEP-1 cells expressed alpha-smooth muscle actin. When the cells were transplanted into various organs of adult SCID mice, a part of the inoculated cell population acquired neural, hepatic, and renal cell properties. These results indicate that the cells we isolated were of epithelial stem cell origin and that our new approach is useful for isolation of multipotent stem cells from skin tissues.     

Regulation of osteoclast differentiation by the redox-dependent modulation of nuclear import of transcription factors

This study sought to characterize the reduced glutathione (GSH)/oxidized GSSG ratio during osteoclast differentiation and determine whether changes in the intracellular redox status regulate its differentiation through a RANKL-dependent signaling pathway. A progressive decrease of the GSH/GSSG ratio was observed during osteoclast differentiation, and the phenomenon was dependent on a decrease in total glutathione via downregulation of expression of the gamma-glutamylcysteinyl synthetase modifier gene. Glutathione depletion by L-buthionine-(S,R)-sulfoximine (BSO) was found to inhibit osteoclastogenesis by blocking nuclear import of NF-kappaB and AP-1 in RANKL-propagated signaling and bone pit formation by increasing BSO concentrations in mature osteoclasts. Furthermore, intraperitoneal injection of BSO in mice resulted in an increase in bone density and a decrease of the number of osteoclasts in bone. Conversely, glutathione repletion with either N-acetylcysteine or GSH enhanced osteoclastogenesis. These findings indicate that redox status decreases during osteoclast differentiation and that this modification directly regulates RANKL-induced osteoclastogenesis.     

Role of nuclear chromogranin B in inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ mobilization

Recently, secretory granule Ca(2+) storage protein chromogranin B (CGB) was shown to be present in the nucleoplasm proper in a complex structure that consists of the inositol 1,4,5-trisphosphate receptor (IP(3)R)/Ca(2+) channels and the phospholipids. Further, the amounts of IP(3)Rs present in the nucleus of bovine chromaffin cells were shown to be comparable to that of the endoplasmic reticulum. Therefore, we investigated here the potential contribution of nuclear CGB on the IP(3)-dependent Ca(2+) mobilization in the nucleus, using both neuroendocrine PC12 and nonneuroendocrine NIH3T3 cells. Chromogranin A (CGA) expression in the NIH3T3 cells, which do not contain intrinsic chromogranins, increased the IP(3)-induced Ca(2+) releases in the nucleus by 45%, while CGB expression in the same cells increased the IP(3)-induced Ca(2+) releases in the nucleus by 80%. Microinjection of IP(3) into the nucleus of CGB-expressing NIH3T3 cells increased the IP(3)-dependent nuclear Ca(2+) mobilization approximately 3-fold, whereas in CGA-expressing cells it remained the same as that of control cells. In contrast, inhibition of CGA expression in PC12 cells by siRNA treatment decreased the IP(3)-induced Ca(2+) releases in the nucleus by 17%, while inhibition of CGB expression decreased the IP(3)-induced Ca(2+) releases in the nucleus by 55%. Microinjection of IP(3) into the nucleus of siCGB-treated PC12 cells decreased the IP(3)-dependent nuclear Ca(2+) mobilization by approximately 75%, whereas in siCGA-treated cells it remained the same as that of control cells. Given the presence of CGB in the nucleus, these results further highlight the critical contribution of nuclear CGB in the IP(3)-induced Ca(2+) release in the nucleus.     

Length–weight and length–length relationships for 10 species of scorpionfishes (Scorpaenidae) on the south coast of Korea

The relationships are given between total length and wet weight, and between total length and standard length for 10 scorpionfishes from the south coast of Korea.     

Targeted delivery of paclitaxel using folate-conjugated heparin-poly(β-benzyl-l-aspartate) self-assembled nanoparticles

A self-assembled nanoparticulate system composed of a folate-conjugated heparin-poly(β-benzyl-l-aspartate) (HP) amphiphilic copolymer was proposed for targeted delivery of the antineoplastic drug paclitaxel (PTX). PTX was incorporated into three types of heparin-based nanoparticles, including HP, folate-conjugated HP (FHP), and folate-polyethylene glycol (PEG)-conjugated HP (FPHP), using a simple dialysis method. The PTX-loaded nanoparticles were then characterized according to particle size (140-190 nm) and size distribution, drug-loading content and efficiency, and in vitro release behavior. In the cellular uptake study using KB cells positive for the folate-receptor (FR), FHP and FPHP nanoparticles showed a much higher cellular uptake than did unconjugated HP nanoparticles. Specifically, when the PEG spacer was inserted between the folate ligand and heparin backbone, FPHP nanoparticles had a greater cellular uptake than did FHP nanoparticles. The in vitro cytotoxicity of PTX-loaded HP, FHP, and FPHP nanoparticles was studied in KB cells and FR-negative A549 cells. Compared with the cytotoxicity in A549 cells, PTX-loaded FHP and FPHP nanoparticles exhibited more potent cytotoxicity in KB cells than did PTX-loaded HP nanoparticles and free-PTX, suggesting that the presence of folate enhanced intracellular uptake via FR-mediated endocytosis. In addition, FPHP nanoparticles exhibited much greater cytotoxicity in KB cells than did FHP nanoparticles. These results suggest that PTX-loaded folate-conjugated HP nanoparticles are a potentially useful delivery system for cancer cells positive for the folate-receptor.     

Probiotic suppression of the H. pylori-induced responses by conjugated linoleic acids in a gastric epithelial cell line

Conjugated linoleic acids (CLA) produced by Lactobacillus acidophilus was reported to decrease the activation of nuclear factor-kappa B. CLA was suggested as one of the anti-inflammatory molecular mechanisms of probiotics. In the present study, the effects of CLA on H. pylori-induced multiple responses were evaluated. IL-8, TNF-α and iNOS were measured in mRNA and/or protein levels in AGS cells after pretreatment with CLA or CLA-containing conditioned medium (CM) produced by Lactobacillus acidophilus or Lactobacillus plantarum. The increased expressions of IL-8 mRNA/protein and TNF-α mRNA were significantly suppressed by pretreatment with CM or CLA. The levels of IL-8 protein and TNF-α mRNA were suppressed by CM pretreatment better than CLA. The expression of iNOS mRNA was also significantly inhibited by CM pretreatment. These results suggest that the suppression of multiple mediators by CLA-containing CM plays a key role in the anti-inflammatory and anti-carcinogenic effects of probiotics on H. pylori infection.     

A sweetpotato SRD1 promoter confers strong root-, taproot-, and tuber-specific expression in Arabidopsis, carrot, and potato

Harvestable, starch-storing organs of plants, such as fleshy taproots and tubers, are important agronomic products that are also suitable target organs for use in the molecular farming of recombinant proteins due to their strong sink strength. To exploit a promoter directing strong expression restricted to these storage organs, we isolated the promoter region (3.0 kb) of SRD1 from sweetpotato (Ipomoea batatas cv. ‘White Star’) and characterized its activity in transgenic Arabidopsis, carrot, and potato using the β-glucuronidase (GUS) gene (uidA) as a reporter gene. The SRD1 promoter conferred root-specific expression in transgenic Arabidopsis, with SRD1 promoter activity increasing in response to exogenous IAA. A time-course study of the effect of IAA (50 μM) revealed a maximum increase in SRD1 promoter activity at 24 h post-treatment initiation. A serial 5′ deletion analysis of the SRD1 promoter identified regions related to IAA-inducible expression as well as regions containing positive and negative elements, respectively, controlling the expression level. In transgenic carrot, the SRD1 promoter mediated strong taproot-specific expression, as evidenced by GUS staining being strong in almost the entire taproot, including secondary phloem, secondary xylem and vascular cambium. The activity of the SRD1 promoter gradually increased with increasing diameter of the taproot in the transgenic carrot and was 10.71-fold higher than that of the CaMV35S promoter. The SRD1 promoter also directed strong tuber-specific expression in transgenic potato. Taken together, these results demonstrate that the SRD1 promoter directs strong expression restricted to the underground storage organs, such as fleshy taproots and tubers, as well as fibrous root tissues.