IFI27, a novel epidermal growth factor-stabilized protein, is functionally involved in proliferation and cell cycling of human epidermal keratinocytes.

OBJECTIVES: IFI27 is highly expressed in psoriatic lesions but its function has not been known. The present study aimed to explore its role in proliferation of epidermal keratinocytes. MATERIALS AND METHODS: IFI27 knockdown and over-expression in keratinocytes were used to compare their proliferation, by MTT assay, apoptosis (by annexin V binding) and cell cycle progression by flow cytometry. Formation of cyclin A/CDK1 complex was examined by a co-immunoprecipitaion method. Anti-proliferation effects of IFI27 were also examined in vivo by topical application of IFI27 siRNA on imiquimod-induced psoriatic lesions, in a mouse model. RESULTS: Epidermal growth factor was demonstrated to increase IFI27 expression by prolonging half-life of IFI27 protein. The IFI27 knockdown in keratinocytes reduced the proliferation rate, but had no effect on apoptosis nor on apoptosis-related genes. Interestingly, IFI27 knockdown resulted in S-phase arrest that was found to be associated with increased Tyr15 phosphorylation of CDK1, reduced CDC25B and reduced formation of cyclin A/CDK1 complex. In addition, IFI27 knockdown was also shown to activate p53 by Ser15 phosphorylation and increase p21 expression. Topical application of IFI27 siRNA on imiquimod-induced psoriatic lesion in a mouse model reduced epidermal thickness, formation of rete ridges and PCNA expression. CONCLUSIONS: Our study demonstrates for the first time, that cell function of IFI27 is involved in proliferation of skin keratinocytes both in vitro and in vivo. It suggests that IFI27 might be a suitable target for development of a novel anti-psoriasis therapy.

Trophic size-structure of sailfish Istiophorus platypterus in eastern Taiwan estimated by stable isotope analysis.

To examine trophic dynamics over different size classes, an isotopic study of sailfish Istiophorus platypterus life-history stages was carried out. Samples were collected from eastern Taiwan and the South China Sea during April 2009 and February 2012. A total of 263 samples (111-245 cm, lower jaw fork length, LLJFL ) were examined for changes in trophic structure in relation to LLJFL by using stable isotope analysis of carbon (δ(13) C) and nitrogen (δ(15) N). The δ(15) N values for I. platypterus ranged from 7·51 to 14·19‰ (mean ± s.d. = 12·06 ± 1·16‰) and the δ(13) C values ranged from -22·04 to -15·48‰ (mean ± s.d. = -17·62 ± 1·10‰). The δ(15) N values were positively dependent on LLJFL (r(2) = 0·377), whereas δ(13) C were negatively dependent on LLJFL (r(2) = 0·063). There were significantly different seasonal changes in nitrogen and carbon isotopic concentration, but no significant differences in concentrations between eastern Taiwan and the South China Sea were reported. The trophic level (TL ) of each LLJFL class was correlated, starting from 2·84 TL for size class I (LLJFL < 140 cm) and reaching 5·03 TL for size class VI (LLJFL > 221 cm). The mean ± s.d. TL was 4·43 ± 0·19 for all samples. The results reveal that I. platypterus occupies a wide range of trophic levels and different size classes occupy different trophic positions in the pelagic ecosystem.

Regulation of ovarian cancer progression by microRNA-187 through targeting Disabled homolog-2.

MicroRNAs (miRNAs) play important roles in tumorigenesis by regulating oncogenes and tumor-suppressor genes. In this study, miR-187 and miR-200a were found to be expressed at higher levels in ovarian cancers than in benign tumors. In patients with ovarian cancer, however, higher levels of miR-187 and miR-200a expression were paradoxically associated with better OS and recurrence-free survival. Further, multivariate analysis showed that miR-187 served as an independent prognostic factor for patients with ovarian cancer (n=176). Computational prediction and microarray results indicated that miR-187 directly targeted Disabled homolog-2 (Dab2), and luciferase reporter assays confirmed that the target site of miR-187 was located at the 3'-UTR of the Dab2 gene. Generally considered as a tumor-suppressor gene, Dab2 may actually promote tumor progression in advanced cancers through epithelial-to-mesenchymal transition (EMT). Ectopic expression of miR-187 in cancer cells promoted cell proliferation, but continued overexpression of miR-187 suppressed Dab2 and inhibited migration. Suppression of miR-187 upregulated Dab2, which, by inhibiting E-cadherin levels while stimulating vimentin and phospho-FAK levels, promoted EMT. Reduced ovarian cancer Dab2 histoscores correlated with high miR-187 levels and improved outcomes of patients. Collectively, these results demonstrate distinct dual roles of Dab2 in cell proliferation and tumor progression. In the initial steps of tumorigenesis, upregulated miR-187 suppresses Dab2, promoting cell proliferation. During the later stages, however, continued increased levels of miR-187 inhibits the Dab2-dependent EMT that is associated with tumor invasiveness, which is presumed to be the reason why cancers with high miR-187 levels were associated with better survivals.

Expression of cathepsin S and its inhibitor stefin A in sinonasal inverted papilloma.

BACKGROUND: Dysregulation of cysteinyl cathepsins and their inhibitors, cystatins (stefins), were implied in progression of tumorgenesis; nevertheless, their role in sinonasal inverted papilloma (IP) is still unrecognized. METHODS: The differential expression of cathepsins and stefins in IP and normal tissues were revealed by data of human Affymetrix U133A gene chips, real-time polymerase chain reaction (PCR) and immunohistochemistry. RESULTS: Among the cathepsins and stefins family, expression of cathepsin S and stefin A were most differentially expressed (down- and up-regulated, respectively) in IP tissue as compared with normal tissues. Their expression levels were validated by real-time PCR, which showed the expression level of cathepsin S was significantly down-regulated, whereas the expression of stefin A was significantly up-regulated in IP tissue compared to normal sinus mucosa. Using immunohistochemistry, expression of cathepsin S was observed in stromal and epithelial area macrophages of normal sinus mucosa, but no obvious expression of cathepsin S was found in IP tissue. In contrast, over-expression of stefin A was present in nearly all layers of the proliferative squamous cells of IP, but expression of stefin A was only detected in a scattered area of normal sinus mucosa. CONCLUSION: Down-regulation of cathepsin S and up-regulation of its endogenous inhibitor, stefin A, were found in IP tissues as compared with their expression level in normal sinus mucosa tissues. The biological significance of inverse expression of both stefin A and cathepsin S in sinonasal IP need further investigation in the future.

Acute gastroenteritis caused by multiple enteric pathogens in children.

Of 303 children hospitalized with acute non-bloody, non-mucoid diarrhoea, 69 (22.8%) had polymicrobial infection, including 52 (17.2%) multiple viral infection and 17 (5.6%) viral and bacterial co-infection. Rotavirus had the most important role in both categories; thus the control of rotavirus infection is crucial for maintaining children's health in Taiwan.

The presence of cytokine-suppressive CD4+CD25+ T cells in the peripheral blood and synovial fluid of patients with rheumatoid arthritis.

CD4+CD25+ T cells have been shown to play a regulatory or suppressive role in the immune response and are possibly relevant to the pathogenesis of autoimmune diseases. In the present study, we attempted to investigate the levels of CD4+CD25+ T cells in the peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and the effects of CD4+CD25+ T cells on the in vitro cytokine production by stimulated SF mononuclear cells (SFMC). The results showed that RA patients had similar frequencies of CD4+CD25+ T cells in PB, expressed as a percentages of the lymphocyte population, as did healthy subjects (mean +/- SD: 10.52 +/- 5.87% versus 11.11 +/- 4.58%., respectively). But in contrast to PB, the SF of RA patients contained significantly higher levels of CD4+CD25+ T cells (17.77 +/- 7.92% versus 10.52 +/- 5.87%, respectively. P < 0.001). When cocultured in vitro with SFMC, CD4+CD25+ T cells purified from either PB or SF were found to exert a considerable suppressive effect on the production of cytokines including TNF-alpha, IFN-gamma and interleukin-10 (IL-10). The percentages of inhibition of each cytokines ranged from 41.8 to 98.4% (mean, 80.0%) for TNF-alpha, 42.8 to 98.9% (mean, 83.2%) for IFN-gamma and 59.3 to 96.6% (mean, 80.0%) for IL-10. Because both pro-inflammatory and anti-inflammatory cytokines were suppressed by CD4+CD25+ T cells, whether CD4+CD25+ T cells might play a beneficial role in the suppression of sustained inflammation in rheumatoid synovium remains to be elucidated.

Transcription of Epstein-Barr virus-encoded nuclear antigen 1 promoter Qp is repressed by transforming growth factor-beta via Smad4 binding element in human BL cells.

In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp. Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity. Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta. The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody. The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment. This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis. Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells.

Additive effect of Sp1 and Sp3 in regulation of the ED-L1E promoter of the EBV LMP 1 gene in human epithelial cells.

The ED-L1E promoter of the LMP 1 gene is a GC box-containing promoter. To test if Sp1/Sp3 are important for modulating ED-L1E promoter activity through the GC box, site-specific mutation and deletion constructs carrying a reporter gene were transfected into NPCTW076 and C33A cells. Results showed that deletion or mutation of the GC box abolished ED-L1E activity. Association of Sp1/Sp3 with the GC box was confirmed by electrophoretic mobility shift and supershift assays using Sp1- and Sp3-specific antibodies. Transfection of Sp1- and Sp3-expressing vectors into NPCTW076 and Sp-deficient Drosophila SL2 cells activated ED-L1E in a dose-dependent fashion and showed an additive effect. Data suggest that both factors may function as transcriptional activators and regulate the ED-L1E promoter in human epithelial cells.

Identification of a novel promoter located within the Bam HI Q region of the Epstein-Barr virus genome for the EBNA 1 gene.

EBNA 1 is the only antigen expressed in both Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma cells. Previous studies showed that the mRNA of EBNA 1 in these two tumor tissues was initiated from a promoter located in the Bam HI F fragment (Fp) on the viral genome. Two regulatory elements located in the downstream Bam HI Q region include an EBNA 1 binding site and a positive regulatory region between the Fp and the EBNA1 binding site. This data strongly suggested that a cellular factor(s) may modulate the usage of the Fp. To locate the shortest responsible viral sequence, we constructed a series of luciferase gene and chloramphenicol acetyltransferase (CAT) gene plasmids that contained various portions of the Bam HI F/Q region. Plasmid DNA was then introduced into cells to examine the promoter activity of each construct. By this method, we identified a 186-bp fragment within the Bam HI Q region that possessed the highest activity. This promoter was designated as Qp and found to be orientation-dependent and down-regulated by EBNA 1 in both the type I BL cells and human epithelial cells. Furthermore, RNase protection assay showed that a transcription initiation site was located at nucleotide 62,416 of the EBV genome. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis further confirmed that the transcript was initiated from the Qp, and not the Fp. Therefore, our data suggested that a novel promoter, Qp, located within the Bam HI Q existed for the EBNA 1 expression in the latently infected type 1 BL cells. The biological significance of the selection of the Qp needs further investigation.

Detection of mutations in the p53 gene in human head and neck carcinomas by single strand conformation polymorphism analysis.

Using the polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis, we have examined the highly conserved regions of the p53 gene in 58 biopsy samples of head and neck tumors. Mutations were found in 13/58 (23%) tumor specimens, but not in 6 normal tissues. Ten of 13 mutations were due to single base changes and the remaining 3 were 1- or 8-base deletion mutants. These mutations were clustered in exons 5 and 7 and resulted in amino acid changes. Our results seem to indicate that mutations in the p53 gene contribute to a significant number of cases of the head and neck tumors including 20% of nasopharyngeal carcinoma biopsies. The relationship of Epstein-Barr virus or human papillomavirus and p53 gene mutations in this group of cancers was also analyzed and discussed.

Cloning and characterization of the latent membrane protein (LMP) of a specific Epstein-Barr virus variant derived from the nasopharyngeal carcinoma in the Taiwanese population.

A DNA fragment containing Epstein-Barr virus (EBV) terminal fragment sequence was obtained from a genomic library of nasopharyngeal carcinoma (NPC). One of the clones (clone 1510) contained the gene encoding latent membrane protein (LMP). Sequence analysis revealed that this gene had 95% homology with the LMP sequence of the B95-8 strain. Among the sequence variations, there was a change from G to T at nucleotide position 169,426, resulting in the loss of an XhoI site in exon 1 of the LMP gene. A pair of primers bracketing the XhoI site were designed to synthesize the EBV DNA fragment from nucleotides 169,081-169,577 by using the polymerase chain reaction (PCR) method. The PCR products were then subject to XhoI digestion and to DNA sequencing analysis. This restriction enzyme site polymorphism along with the sequence variations were also observed in 50 biopsy tissues as well as in the throat washings of 6 out of 20 healthy individuals that we examined, indicating that the EBV strain predominantly existing in these biopsy tissues was different from strains of B95-8, Jijoye or nude mouse passaged cells (C15) with an African origin, but closely resembled other nude mouse passaged CAO cells which were originally derived from China. Balb/c 3T3 cells carrying this NPC-LMP gene showed a transformed cell morphology and were tumorigenic in nude mice. The relationship between this unique type of EBV and NPC has yet to be established.