DUSP3 regulates phosphorylation-mediated degradation of occludin and is required for maintaining epithelial tight junction.

BACKGROUND: Tight junctions (TJ) are multi-protein complexes that hold epithelial cells together and form structural and functional barriers for maintaining proper biological activities. Dual specificity phosphatase 3 (DUSP3), a suppressor of multiple protein tyrosine (Tyr) kinases, is decreased in lung cancer tissues. Here we demonstrated the role of DUSP3 in regulation of epithelial TJ. METHODS: Barrier functions of TJ were examined in wild-type or DUSP3-deficient lung epithelial cells. Animal and clinical data were analyzed for the association between DUSP3 deficiency and lung cancer progression. Proximity ligation assay, immunoblotting, and phosphatase assay were performed to study the effect of DUSP3 on the TJ protein occludin (OCLN). Mutations of Tyr residues on OCLN showed the role of Tyr phosphorylation in regulating OCLN. RESULTS: Compared to those of the DUSP3-expressing cells, we found the expression and distribution of ZO-1, a TJ-anchoring molecule, were abnormal in DUSP3-deficient cells. OCLN had an increased phosphorylation level in DUSP3-deficient cells. We identified that OCLN is a direct substrate of DUSP3. DUSP3 regulated OCLN ubiquitination and degradation through decreasing OCLN tyrosine phosphorylation directly or through suppressing focal adhesion kinase, the OCLN kinase. CONCLUSION: Our study revealed that DUSP3 is an important TJ regulatory protein and its decrease may be involved in progression of epithelial cancers.

EGFR Mutation-Harboring Lung Cancer Cells Produce CLEC11A with Endothelial Trophic and Tumor-Promoting Activities.

The formation of new blood vessels in solid tumors is regulated by various endothelial trophic factors. We identified that CLEC11A, an extracellular C-type lectin, was over-expressed in lung cancer cell lines harboring mutated EGFR. CLEC11A expression was also frequently elevated in lung adenocarcinoma (LAC) tissues with EGFR mutation. CLEC11A-expressing H1299 cells formed larger tumors in nude mice than did the control cells. The CLEC11A-expressing tumors contained more CD31-positive cells, suggesting that they had a higher angiogenic activity. CLEC11A per se did not induce blood vessel formation, but enhanced angiogenesis triggered by VEGF-A or basic FGF in vivo. Additionally, the expression of small hairpin RNA against CLEC11A (shCLEC11A) in HCC827 LAC cells suppressed their tumorigenic ability. Purified CLEC11A exhibited a chemotactic ability, which is dependent on its integrin-binding RGD and LDT motifs, toward endothelial cells. This chemotactic activity was not affected by the presence of a VEGFR inhibitor. Conditioned medium produced by HCC827-shCLEC11A cells had diminished chemotactic ability toward endothelial cells. CLEC11A treatments increased the levels of active integrin ß1 that were not associated with activation of focal adhesion kinases in endothelial cells. Our results indicated that CLEC11A was a factor of angiogenic potential and was involved in lung cancer tumorigenesis.

NEAP/DUSP26 suppresses receptor tyrosine kinases and regulates neuronal development in zebrafish.

Expression of neuroendocrine-associated phosphatase (NEAP, also named as dual specificity phosphatase 26, [DUSP26]) is restricted to neuroendocrine tissues. We found that NEAP, but not its phosphatase-defective mutant, suppressed nerve growth factor (NGF) receptor TrkA and fibroblast growth factor receptor 1 (FGFR1) activation in PC12 cells upon NGF stimulation. Conversely, suppressing NEAP expression by RNA interference enhanced TrkA and FGFR1 phosphorylation. NEAP was capable of de-phosphorylating TrkA and FGFR1 directly in vitro. NEAP-orthologous gene existed in zebrafish. Morpholino (MO) suppression of NEAP in zebrafish resulted in hyper-phosphorylation of TrkA and FGFR1 as well as abnormal body postures and small eyes. Differentiation of retina in zebrafishes with NEAP MO treatment was severely defective, so were cranial motor neurons. Taken together, our data indicated that NEAP/DUSP26 have a critical role in regulating TrkA and FGFR1 signaling as well as proper development of retina and neuronal system in zebrafish.

[UV-Vis spectrum characteristics of phycocyanin in water from Taihu lake].

The present paper analyzed the UV-Vis spectrum characteristics of phycocyanin extracted from 75 water samples around Meiliang Bay of Taihu Lake, China in spring, summer and autumn, 2011, taking standard sample of phycocyanin, Micro-cystic aeruginosa and Anabaena cultured indoor as the reference, and discussed the difference and relation of spectrum among water samples, standard sample and single algae samples. According to the number of absorption peak in the wavelength range from 500 to 700 nm, phycocyanin spectrum of water sampling in Taihu Lake can be divided into three patterns: no peak, single peak and two peaks. In the first pattern, the absorbance changed smoothly and no absorption peak was observed around 620 nm. Depending on the absorption difference in the wavelength range from 300 to 450 nm, this pattern can be divided into type I and type II. Type I only had a absorption peak near 260 nm, with the similar spectrum of chromophoric dissolved organic matter (CDOM) in the wavelength range from 250 to 800 nm. Type II had absorption peak respectively near 260 and 330 nm. In single peak pattern and two peaks pattern, significant absorption peak of phycocyanin appeared around 620 nm. Compared to the other patterns, single peak pattern was more similar to that of standard sample and single algae samples, but different in their maximum absorption peaks position and relative absorption intensity in the wavelength range of 250 approximately 300, 300 approximately 450 and 500 approximately 700 nm, because of different algae species and purity after extraction. In the two peaks pattern, another absorption peak appeared at 670nm, with the absorption shoulder from 350 to 450 nm, and shared the absorption characteristics of phycocyanin and chlorophyll complex protein. The research can provide a basic support for the phycocyanin quantitation and blooms monitoring in Taihu Lake.

New classes of mind bomb-interacting proteins identified from yeast two-hybrid screens.

Notch signaling pathway defines an evolutionarily conserved mechanism in cell-fate determination in a broad spectrum of developmental processes through local cell interactions. mind bomb (mib) encodes an E3 ubiquitin ligase that is involved in Notch activation through Delta ubiquitylation and internalization. To further dissect the function of Mib, two yeast two-hybrid screens for zebrafish Mib/Mib2-binding proteins with different strategies have been performed. 81 putative interesting proteins were discovered and classified into six groups: ubiquitin proteasome pathway, cytoskeleton, trafficking, replication/transcription/translation factors, cell signaling and others. Confirmed by coimmunoprecipitation (Co-IP), Mib interacted with four tested proteins: ubiquitin specific protease 1 (Usp1), ubiquitin specific protease 9 (Usp9), tumor-necrosis-factor-receptor-associated factor (TRAF)-binding domain (Trabid)/zinc finger, RAN-binding domain containing 1 (Zranb1) and hypoxia-inducible factor 1, alpha subunit inhibitor (Hif1an)/factor inhibiting HIF 1 (Fih-1). Usp1, Usp9, Trabid and Fih-1 also bound to zebrafish Mib2, a Mib homolog with similar structural domains and functions. Both Mib and Mib2 can ubiquitylate Trabid and Fih-1, indicating a potential regulating role of Mib and Mib2 on Trabid and Fih-1 and, furthermore, the possible involvement of Notch signaling in hypoxia-regulated differentiation, tumorigenesis and NF-κB pathway. Finally, functions of confirmed Mib/Mib2-interacting proteins are collated, summarized and hypothesized, which depicts a regulating network beyond Notch signaling.

[Baseline correction of spectrum for the inversion of chlorophyll-a concentration in the turbidity water].

Suspended particle material is the main factor affecting remote sensing inversion of chlorophyll-a concentration (Chla) in turbidity water. According to the optical property of suspended material in water, the present paper proposed a linear baseline correction method to weaken the suspended particle contribution in the spectrum above turbidity water surface. The linear baseline was defined as the connecting line of reflectance from 450 to 750 nm, and baseline correction is that spectrum reflectance subtracts the baseline. Analysis result of field data in situ of Meiliangwan, Taihu Lake in April, 2011 and March, 2010 shows that spectrum linear baseline correction can improve the inversion precision of Chl a and produce the better model diagnoses. As the data in March, 2010, RMSE of band ratio model built by original spectrum is 4.11 mg x m(-3), and that built by spectrum baseline correction is 3.58 mg x m(-3). Meanwhile, residual distribution and homoscedasticity in the model built by baseline correction spectrum is improved obviously. The model RMSE of April, 2011 shows the similar result. The authors suggest that using linear baseline correction as the spectrum processing method to improve Chla inversion accuracy in turbidity water without algae bloom.

A novel lignan glycoside with antioxidant activity from Tinospora sagittata var. yunnanensis.

A novel lignan glysocide, namely sagitiside A (1), together with two known ones, (+)-lyoniresinol-2α-O-ß-D-glucopyranoside (2) and (+)-5'-methoxyisolariciresinol 3α-O-ß-D-glucopyranoside (3), was isolated from the 95% ethanol extract of dry roots of Tinospora sagittata var. yunnanensis. The structure of the new compound (1) was determined based on MS, 1D and 2D NMR spectral data. Compounds 1-3 showed antioxidant activity with EC(50) values 55, 75 and 80 µM by 1,1-diphenyl-2-picryhydrazyl (DPPH) radical-scavenging assay.

A novel 18-norclerodane diterpenoid from the roots of Tinospora sagittata var. yunnanensis.

A novel 18-nor-clerodane diterpenoid named sagitone (1) was isolated from the 95% ethanol extract of dry roots of Tinospora sagittata var. yunnanensis together with the five known diterpenoids columbin (2), palmatoside C (3), fibleucin (4), tinophylloloside (5) and epitinophylloloside (6). The structure of the new compound 1 was determined based on MS, IR, 1D and 2D NMR spectral data. The compounds 1-6 did not show significant cytotoxic activity against cancer cell lines K562 and HL-60.

[Studies on the chemical constituents from the roots of Tinospora sagittata var. yunnanensis (II)].

OBJECTIVE: To study the chemical constituents from the roots of Tinospora sagittata var. yunnanensis. METHODS: Various chromatographic techniques were used to isolate and purify the constituents. The structures of these compounds were elucidated on the basis of spectral analysis. RESULTS: Seven compounds were isolated from the plant and their structures were identified as tinophylloloside (1), epitinophylloloside (2), 2-deoxycrustecdysone (3), polypodine B (4) , (+)-5'-methoxyisolariciresinol 3alpha-O-beta-D-glucopyranoside (5), geniposide (6), adenine (7). CONCLUSION: All compounds are isolated from the plant for the first time,and compounds 4, 5, 6 and 7 are isolated firstly from the genus.

Anti-mitochondrial antibodies and primary biliary cirrhosis in TGF-beta receptor II dominant-negative mice.

Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver, characterized by lymphocytic infiltrates in portal tracts, selective destruction of biliary epithelial cells, and anti-mitochondrial Abs (AMAs). The elucidation of early events in the induction of tissue inflammation and autoimmunity in PBC has been hampered by the cryptic onset of the disease, the practical limitations in accessing the target tissue, and the lack of a suitable animal model. We demonstrate in this study that a mouse transgenic for directed expression of a dominant-negative form of TGF-beta receptor type II (dnTGFbetaRII), under the direction of the CD4 promoter, mimics several key phenotypic features of human PBC, including spontaneous production of AMAs directed to the same mitochondrial autoantigens, namely PDC-E2, BCOADC-E2, and OGDC-E2. The murine AMAs also inhibit PDC-E2 activity. Moreover, there is lymphocytic liver infiltration with periportal inflammation analogous to the histological profile in human PBC. Additionally, the serum cytokine profile of affected mice mimics data in human PBC. The concomitant presence of these immunopathological features in the transgenic mice suggests that the TGF-betaRII pathway is implicated in the pathogenesis of PBC. Finally, these data point away from initiation of autoimmunity by mechanisms such as molecular mimicry and more toward activation of an intrinsically self-reactive T cell repertoire in which necessary regulatory T cell influences are lacking.

Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference inhibits proliferation of EBV-positive Burkitt's lymphoma cells.

PURPOSE: Epstein-Barr virus (EBV) is associated with the development of several lymphoid and epithelial malignancies, including Burkitt's lymphoma. The EBV latent protein, EBV Nuclear Antigen 1 (EBNA1), is detectable in almost all types of EBV-associated tumors and is essential for replication and maintenance of the latent episome of EBV. We here examined whether the RNA interference (RNAi) technique could be employed to suppress expression of EBNA1 in EBV-positive Burkitt's lymphoma cells. METHODS: A Raji cell line expressing small hairpin RNAs (shRNAs) against EBNA1 was established and EBNA1 mRNA level was determined by real-time RT-PCR analysis. We investigated the effects of EBNA1 silence on lymphoma cell growth and cell cycle progression. RESULTS: Transfection of an EBNA1 RNAi plasmid resulted in substantial loss of EBNA1 mRNA and significantly inhibited proliferation of Raji cells relative to the control plasmid case. Suppression of EBNA1 was also associated with downregulation of EBV oncogene EBNA2, a decreased PCNA labeling index and increased G0/G1 fraction in cell cycle analysis. CONCLUSIONS: These findings point to potential therapeutic applications for vector-mediated siRNA delivery to control EBV-associated malignant disorders.

Increased levels of chemokine receptor CXCR3 and chemokines IP-10 and MIG in patients with primary biliary cirrhosis and their first degree relatives.

Infiltrating memory T cells play an important role in the destruction of the biliary tract in primary biliary cirrhosis (PBC) and inflammatory chemokines control lymphocyte traffic through their interactions with T cell chemokine receptors. In the present study, we measured plasma levels of chemokines interferon-gamma-inducible protein-10 (IP-10) and monokine induced by gamma interferon (MIG), and also studied the expression of CXCR3 chemokine receptors in 105 subjects, including 53 patients with PBC, 26 first degree relatives and 26 healthy controls. Interestingly, plasma IP-10 and MIG levels in PBC were increased significantly compared to controls and appeared to increase with disease progression. By immunohistochemistry, IP-10 and MIG expressions were evident in the portal areas in PBC. Further, the frequency of CXCR3-expressing cells in peripheral blood was also significantly higher in PBC, and CXCR3-positive cells were also found in the portal areas of diseased livers, primarily on CD4+ cells. Finally, the daughters and sisters of PBC patients also demonstrated increased plasma levels of IP-10 and MIG, but, in contrast, displayed normal frequency of CXCR3+ expressing peripheral blood lymphocytes. Our data imply a role for specific chemokine-chemokine receptor interactions in the pathogenesis of PBC and also highlight the familial risk factor.

Paraquat induces selective dopaminergic nigrostriatal degeneration in aging C57BL/6 mice.

BACKGROUND: Paraquat (PQ; 1, 1'-dimethyl-4, 4'-bipyridinium), a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant MPTP (1-methyl-1, 2, 3, 6-tetrahydropyridine), has been suggested as a potential etiologic factor for the development of Parkinson's disease (PD). Aging is an accepted risk factor for idiopathic Parkinson's disease. The aim of this study was to test the hypothesis that paraquat could induce PD-like nigrostriatal dopaminergic degeneration in aging C57BL/6 mice. METHODS: Senile male C57BL/6 mice were intraperitoneally injected with either saline or PQ at 2-day intervals for a total of 10 doses. Locomotor activity and performance on the pole test were measured 7 days after the last injection and animals were sacrificed one day later. Level of dopamine (DA) and its metabolites levels in the striatum were measured by high-performance liquid chromatography with an electrochemical detector (HPLC-ECD), and numbers of tyrosine hydroxylase (TH) positive neurons were estimated using immunohistochemistry. RESULTS: Locomotor activities were significantly decreased and the behavioral performance on the pole test were significantly impaired in the PQ treated group. Level of DA and its metabolites levels in the striatum were declined by 8 days after the last injection. Immunohistochemical analyses showed that PQ was associated with a reduction in numbers of tyrosine hydroxylase positive neurons. CONCLUSIONS: Long-term repeated exposes to PQ can selectively impair the nigrostriatal dopaminergic system of senile mice, suggesting that PQ could play an important role in the pathogenesis of Parkinson's disease (PD). Our results also validate a novel model of PD induced by exposure to a toxic environmental agent.

Detection of JC virus DNA sequences in colorectal cancers in Japan.

JC virus (JCV), a ubiquitous polyoma virus that commonly infects humans, was first identified as the etiologic agent for the fetal demyelinating disease, progressive multifocal leukoencephalopathy. Recently, a number of reports have documented detection of JCV in samples derived from several types of neural as well as non-neural human tumors. It has been suggested that oncogenicity of JCV depends on a T antigen having a strict structural homology to the T antigen of simian virus 40. To clarify whether JCV might have a potential role with regard to colorectal cancers, we investigated the presence of its genome in a series of cases along with colorectal adenomas and normal colonic mucosa, targeting T antigen, VP and agnoprotein by nested polymerase chain reaction and Southern blotting and T antigen by immunohistochemistry. While VP and agnoprotein were not found in any of the samples examined, T antigen was detected in 6 of 23 colorectal cancers (26.1%) and 1 of 21 adenomas (4.8%), but none of 20 samples of normal colonic mucosa. No clear and diffuse staining with anti-T-antigen antibodies (1:100) could be detected, and there was no correlation with CD20-positive cells, which might have indicated JCV latent infection of B lymphocytes. Presence of T antigen did not influence clinicopathological variables, including survival. In one colonic cancer case positive for T antigen together with lymph node metastasis, DNA extracted from cancer cells in the lymph node revealed no detection of T antigen. Our results are in the intermediate position between the high T antigen rate (81%) in one report and the lack of it (0%) in another focused on colon cancers. It was concluded that T antigen might be integrated in cancer cells in approximately one fourth of Japanese colon cancer cases without clear and diffuse expression of the protein, suggesting a possible role in oncogenesis which might involve a hit-and-run mechanism.

Cytoplasmic expression of c-erbB2 in non-small cell lung cancers.

The aim of this study was to investigate the expression of c-erbB2 in non-small cell lung cancers (NSCLCs) with attention both to membranous and cytoplasmic reaction, and to try to elucidate the meaning of cytoplasmic expression of c-erbB2 in NSCLCs. Immunohistochemical c-erbB2 expression and related clinico-pathological features were examined in 312 surgically resected patient tissues of NSCLCs, including 175 cases of adenocarcinoma and 137 cases of squamous cell carcinoma. Immunostaining of inner- and ecto-domain of c-erbB2, mRNA expression and the quantitation of soluble c-erbB2 in cultured media were performed in five NSCLC cell lines. Cytoplasmic expression of c-erbB2 was observed more frequently than membranous, both in patient tissues and cell lines. Neither membranous nor cytoplasmic expression of c-erbB2 was significantly correlated with short outcome in NSCLCs. Membranous c-erbB2 was expressed by both inner and ecto-domain, while cytoplasmic c-erbB2 was expressed by either or both inner and ecto-domain. c-erbB2 mRNA was produced in most cell lines; however, the soluble form was only detectable in a cell line that only presented a membranous c-erbB2. In conclusion, cytoplasmic c-erbB2 of NSCLCs was not a full-length protein only expressed in cellular membrane, but reflected degenerated c-erbB2 fragments with less functional ability.