Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter

Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis might be reducing liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4⁺ T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.     

Spatiotemporal Expression of tmie in the Inner Ear of Rats during Postnatal Development

The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6. Transmembrane inner ear (tmie) is the causative gene and its mutation through deletion of a 40-kilobase genomic region including tmie leads to deafness. The function of Tmie is unknown. To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie in the rat cochlea by using a Tmie-specific antibody. Results showed that tmie expression was prominent in early postnatal rat cochleas in the stereocilia bundles of hair cells. The Tmie signal spread from the stereocilia to the hair cell body region and on to organ of Corti cells. No Tmie signal was observed in cell nuclei; Tmie was localized to the cytoplasm. Because Tmie is predicted to have 1 or 2 transmembrane domains, we postulate that it is localized to membrane-based organelles or the plasma membrane. Our results imply that Tmie exists in the cytoplasm and may have a key role in the maturation and structure of stereocilia bundles in developing hair cells. After hair cell maturation, Tmie is thought to be involved in the maintenance of organ of Corti cells.Circling is often observed in mouse and rat deafness mutants and is commonly suggested to be a consequence of inner ear defects that impair vestibular systems.^3,12,14 The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6; these mice have abnormal circling behavior, suggesting a balance disorder, and profound deafness.^6,7 The most notable pathologic phenotypes of circling mice are the almost completely degenerated cochlea and remarkably reduced cellularity in spiral ganglion neurons. The causative gene for circling is transmembrane inner ear (tmie), with a 40-kilobase genomic deletion including tmie.¹ tmie is also the causative gene of the spinner (sr/sr) mouse, which has phenotypes similar to circling mice, although the mutation patterns are different.⁸ Spinner mice also show circling behavior, hearing loss, imbalance, and swimming inability. In addition, spinner mice have 2 mutations in the tmie gene: the 40-kb genomic deletion including tmie and a point mutation that leads to a truncated protein.⁸In humans, 7 different homozygous recessive mutations in TMIE currently are known to exist in affected members of consanguineous families segregating severe-to-profound prelingual deafness, consistent with linkage to DFNB6.^9,10 Although the functions of murine Tmie and human TMIE are unknown, this protein appears to be important for normal hearing and vestibular function.In a previous study, we produced transgenic mice overexpressing tmie that resulted in phenotypic rescue of circling.¹¹ Normal expression of transgenic tmie induced phenotypic rescue in circling homozygous mutants, although some mice did not show amelioration of abnormal behavior, hearing ability, or tissue morphology in the inner ear. Therefore the Tmie protein is required for normal inner ear function in mouse.¹¹To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie. Knowing when, where, and to what extent this protein is produced in the developing inner ear will provide important clues to protein function. In adult mouse and rat, tmie is expressed in various tissues.^2,13 Whether Tmie plays an important role in those tissues is uncertain, because circling mice that lack the entire tmie gene have no noteworthy problems in any tissues except those of the inner ear systems.⁶In this study, we were interested in the postnatal stages before and after the onset of hearing (around postnatal day [P] 12) in rats; therefore, the postnatal period P0 to19 was studied. Although all the cells that form the mature cochlea are present at birth, important conformational changes occur during this period, including the formation of the tunnel of Corti and the establishment or retraction of neuronal connections. The expression pattern of tmie in the developing inner ear during early postnatal development has not been investigated previously. Here we document our use of a Tmie-specific antibody to elucidate the spatial and temporal expression of tmie in the rat inner ear during postnatal development.     

2.45 GHz radiofrequency fields alter gene expression in cultured human cells

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.     

Proteome analysis of developing mice diastema region

Different from humans, who have a continuous dentition of teeth, mice have only three molars and one incisor separated by a toothless region called the diastema in the hemi mandibular arch. Although tooth buds form in the embryonic diastema, they regress and do not develop into teeth. In this study, we evaluated the proteins that modulate the diastema formation through comparative analysis with molar-forming tissue by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteome analysis. From the comparative and semi-quantitative proteome analysis, we identified 147 up- and 173 down-regulated proteins in the diastema compared to the molar forming proteins. Based on this proteome analysis, we selected and evaluated two candidate proteins, EMERIN and RAB7A, as diastema tissue specific markers. This study provides the first list of proteins that were detected in the mouse embryonic diastema region, which will be useful to understand the mechanisms of tooth development.     

Developmental regulations of Perp in mice molar morphogenesis

Angiogenesis, a complex biologic process, is regulated by a large number of angiogenic factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). Whether Bone morphogenetic proteins-2 (BMP-2), the osteoinductive factor, could significantly reinforce the effect of VEGF and FGF-2 on angiogenesis has not been studied in detail. To study the positive effects of multiple growth factors on angiogenesis, HUVECs were treated with BMP-2, VEGF, or FGF-2 singly and in binary and ternary combinations. This study further investigates the optimal timing of the ternary combination of BMP-2, VEGF and FGF-2 for angiogenesis in the chorioallantoic membrane (FGF-2 CAM). Results of single applications of BMP-2, VEGF, or FGF-2 suggested that HUVECs angiogenesis could be promoted in a dose-dependent manner and that the optimal concentration of BMP, VEGF and FGF-2 was 10, 50 and 1 ng/mL, respectively. These results indicated that the angiogenic activity of VEGF and FGF-2 was amplified by combining with BMP-2. The ternary combination of BMP-2, VEGF and FGF-2 exhibited a positive and synergistic effect on HUVECs angiogenesis, with the lower concentrations of each factor (1 ng/mL of BMP-2, 25 ng/mL of VEGF and 0.1 ng/mL of FGF-2) being sufficient to show synergistic promotion. When VEGF and FGF-2 were added in the initial activation stage and BMP-2 was added in the maturation stage, both HUVECs angiogenesis in vitro and CAM angiogenesis in vivo could be enhanced more effectively. These results could provide a basis for the controlled release systems capable of delivering multiple factors sequentially to promote angiogenesis in tissue engineering.     

Serum amyloid A1 is involved in amyloid plaque aggregation and memory decline in amyloid beta abundant condition

Transgenic Research - Alzheimer's disease (AD) is a neurodegenerative disorder, characterized by cognitive impairment, progressive neurodegeneration, and amyloid-β (Aβ) lesion. In the...     

Stage-dependent gene expression profiles during natural killer cell development

Natural killer (NK) cells develop from hematopoietic stem cells (HSCs) in the bone marrow. To understand the molecular regulation of NK cell development, serial analysis of gene expression (SAGE) was applied to HSCs, NK precursor (pNK) cells, and mature NK cells (mNK) cultured without or with OP9 stromal cells. From 170,464 total individual tags from four SAGE libraries, 35,385 unique genes were identified. A set of genes was expressed in a stage-specific manner: 15 genes in HSCs, 30 genes in pNK cells, and 27 genes in mNK cells. Among them, lipoprotein lipase induced NK cell maturation and cytotoxic activity. Identification of genome-wide profiles of gene expression in different stages of NK cell development affords us a fundamental basis for defining the molecular network during NK cell development.     

Therapeutic Non-Toxic Doses of TNF Induce Significant Regression in TNFR2-p75 Knockdown Lewis Lung Carcinoma Tumor Implants

Tumor necrosis factor-alpha (TNF) binds to two receptors: TNFR1/p55-cytotoxic and TNFR2/p75-pro-survival. We have shown that tumor growth in p75 knockout (KO) mice was decreased more than 2-fold in Lewis lung carcinoma (LLCs). We hypothesized that selective blocking of TNFR2/p75 LLCs may sensitize them to TNF-induced apoptosis and affect the tumor growth. We implanted intact and p75 knockdown (KD)-LLCs (>90%, using shRNA) into wild type (WT) mice flanks. On day 8 post-inoculation, recombinant murine (rm) TNF-α (12.5 ng/gr of body weight) or saline was injected twice daily for 6 days. Tumor volumes (tV) were measured daily and tumor weights (tW) on day 15, when study was terminated due to large tumors in LLC+TNF group. Tubular bones, spleens and peripheral blood (PB) were examined to determine possible TNF toxicity. There was no significant difference in tV or tW between LLC minus (-) TNF and p75KD/LLC-TNF tumors. Compared to 3 control groups, p75KD/LLC+TNF showed >2-5-fold decreases in tV (p<0.001) and tW (p<0.0001). There was no difference in tV or tW end of study vs. before injections in p75KD/LLC+TNF group. In 3 other groups tV and tW were increased 2.7-4.5-fold (p<0.01, p<0.0002 and p<0.0001). Pathological examination revealed that 1/3 of p75KD/LLC+rmTNF tumors were 100% necrotic, the remaining revealed 40-60% necrosis. No toxicity was detected in bone marrow, spleen and peripheral blood. We concluded that blocking TNFR2/p75 in LLCs combined with intra-tumoral rmTNF injections inhibit LLC tumor growth. This could represent a novel and effective therapy against lung neoplasms and a new paradigm in cancer therapeutics.     

Activation of ERK accelerates repair of renal tubular epithelial cells,whereas it inhibits progression of fibrosis following ischemia/reperfusion injury

Extracellular signal-regulated kinase (ERK) signals play important roles in cell death and survival. However, the role of ERK in the repair process after injury remains to be defined in the kidney. Here, we investigated the role of ERK in proliferation and differentiation of tubular epithelial cells, and proliferation of interstitial cells following ischemia/reperfusion (I/R) injury in the mouse kidney. Mice were subjected to 30 min of renal ischemia. Some mice were administered with U0126, a specific upstream inhibitor of ERK, daily during the recovery phase, beginning at 1 day after ischemia until sacrifice. I/R caused severe tubular cell damage and functional loss in the kidney. Nine days after ischemia, the kidney was restored functionally with a partial restoration of damaged tubules and expansion of fibrotic lesions. ERK was activated by I/R and the activated ERK was sustained for 9 days. U0126 inhibited the proliferation, basolateral relocalization of Na,K-ATPase and lengthening of primary cilia in tubular epithelial cells, whereas it enhanced the proliferation of interstitial cells and accumulation of extracellular matrix. Furthermore, U0126 elevated the expression of cell cycle arrest-related proteins, p21 and phospholylated-chk2 in the post-ischemic kidney. U0126 mitigated the post-I/R increase of Sec10 which is a crucial component of exocyst complex and an important factor in ciliogenesis and tubulogenesis. U0126 also enhanced the expression of fibrosis-related proteins, TGF-β1 and phosphorylated NF-κB after ischemia. Our findings demonstrate that activation of ERK is required for both the restoration of damaged tubular epithelial cells and the inhibition of fibrosis progression following injury.