Cultured gill epithelia as models for the freshwater fish gill

We review recent progress in the development of models for the freshwater teleost gill based on reconstructed flat epithelia grown on permeable filter supports in primary culture. Methods are available for single-seeded insert (SSI) preparations consisting of pavement cells (PVCs) only from trout and tilapia, and double-seeded insert (DSI) preparations from trout, containing both PVCs (85%) and mitochondria-rich cells (MRCs, 15%), as in the intact gill. While there are some quantitative differences, both SSI and DSI epithelia manifest electrical and passive permeability characteristics typical of intact gills and representative of very tight epithelia. Both preparations withstand apical freshwater exposure, exhibiting large increases in transepithelial resistance (TER), negative transepithelial potential (TEP), and low rates of ion loss, but there is only a small active apical-to-basolateral "influx" of Cl(-) (and not of Na(+)). Responses to various hormonal treatments are described (thyroid hormone T3, prolactin, and cortisol). Cortisol has the most marked effects, stimulating Na(+),K(+)-ATPase activity and promoting active Na(+) and Cl(-) influxes in DSI preparations, and raising TER and reducing passive ion effluxes in both epithelia via reductions in paracellular permeability. Experiments using DSI epithelia lacking Na(+) uptake demonstrate that both NH(3) and NH(4)(+) diffusion occur, but are not large enough to account for normal rates of branchial ammonia excretion, suggesting that Na(+)-linked carrier-mediated processes are important for ammonia excretion in vivo. Future research goals are suggested.     

PNAS-4 expression and its relationship to p53 in colorectal cancer

PNAS-4 is a novel pro-apoptotic protein activated during the early response to DNA damage; however, the molecular mechanisms and pathways regulating PNAS-4 expression in tumors are not well understood. We hypothesized that PNAS-4 is a p53 down-stream target gene and designed this study. We searched online for putative p53-binding sites in the entire PNAS-4 gene and did not find any corresponding information. In HCT116 colon cancer cells, after being transfected with small interfering RNA to silence p53, the expressions of PNAS-4 and other known p53 target gene (Apaf1, Bax, Fas and Dr5) were determined by real-time PCR. We found that PNAS-4 was up-regulated while Apaf1, Bax, Fas and Dr5 were down-regulated. We then examined the expression of PNAS-4 and p53 mutation in colorectal cancer patients. PNAS-4 expressed both in colorectal cancers and normal tissues, but compared with paired control, PNAS-4 was up-regulated in cancers (P = 0.018). PNAS-4 overexpression ratios were correlated to the p53 mutant status (P = 0.001). The mean PNAS-4 expression levels of p53 mutant homozygote group and heterozygote group were higher than that of p53 wild type group (P = 0.013). The expression ratios of PNAS-4 (every sample in relative to its paired normal mucosa) were different between negative lymph node metastasis (66% up-regulated, 34% down-regulated) and positive metastasis (42% up-regulated, 58% down-regulated). Taken together, these findings suggested that PNAS-4 was not a p53 target, but overexpression of PNAS-4 was correlated to p53 inactivity in colorectal cancer.     

Identification of a movement protein of the tenuivirus rice stripe virus

Rice stripe virus (RSV) is the type member of the genus Tenuivirus. RSV has four single-stranded RNAs and causes severe disease in rice fields in different parts of China. To date, no reports have described how RSV spreads within host plants or the viral and/or host factor(s) required for tenuivirus movement. We investigated functions of six RSV-encoded proteins using trans-complementation experiments and biolistic bombardment. We demonstrate that NSvc4, encoded by RSV RNA4, supports the intercellular trafficking of a movement-deficient Potato virus X in Nicotiana benthamiana leaves. We also determined that upon biolistic bombardment or agroinfiltration, NSvc4:enhanced green fluorescent protein (eGFP) fusion proteins localize predominantly near or within the walls of onion and tobacco epidermal cells. In addition, the NSvc4:eGFP fusion protein can move from initially bombarded cells to neighboring cells in Nicotiana benthamiana leaves. Immunocytochemistry using tissue sections from RSV-infected rice leaves and an RSV NSvc4-specific antibody showed that the NSvc4 protein accumulated in walls of RSV-infected leaf cells. Gel retardation assays revealed that the NSvc4 protein interacts with single-stranded RNA in vitro, a common feature of many reported plant viral movement proteins (MPs). RSV NSvc4 failed to interact with the RSV nucleocapsid protein using yeast two-hybrid assays. Taken together, our data indicate that RSV NSvc4 is likely an MP of the virus. This is the first report describing a tenuivirus MP.     

重庆市爬行动物物种多样性研究及保护

1996～2004年对重庆市35个县(区、市)进行野外调查采集,结合有关资料记载,重庆市共有爬行动物2目12科38属58种,其中龟鳖目4种,有鳞目蜥蜴亚目12种、蛇亚目42种(含重庆市蛇类新记录3属4种)。建议加强对该市爬行动物物种多样性及其生境的保护,合理利用爬行动物资源。     

Lipidomic changes during different growth stages of Nitzschia closterium f. minutissima

Ultra Performance Liquid Chromatography-Electrospray ionization-Quadrupole-Time of Flight Mass Spectrometry (UPLC-ESI-Q-TOF–MS) is a powerful lipidomic tool. In this study, we developed a UPLC/Q-TOF–MS based method to investigate the lipid metabolomic changes in different growth phases of Nitzschia closterium f. minutissima. The data classification and biomarker selection were carried out by using multivariate statistical analysis, including principal components analysis (PCA), projection to latent structures with discriminant analysis (PLS-DA), and orthogonal projection to latent structures with discriminant analysis (OPLS-DA). We discovered that the intercellular lipid metabolites were significantly different among exponential, early stationary and late stationary phases. Thirty-one lipid molecules were selected and identified as putative biomarkers, including free fatty acid, Harderoporphyrin, phosphatidylglycerol, 1,2-diacyglycerl-3-O-4′-(N,N-trimethy)-homoserine, triacylglycerol, cholesterol, sulfoquinovosyldiacylglycerol, lyso-sulfoquinovosyldiacylglycerol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol and lyso-digalactosyldiacylglycerol. These lipids have been shown previously to function in energy storage, membrane stability and photosynthesis efficiency during the growth of diatoms. Further analysis on the putative biomarkers demonstrated that nitrate starvation played critical role in the transition from exponential phase to stationary phase in N. closterium. This study is the first one to explore the lipidomic changes of microalgae in different growth phases, which promotes better understanding of their physiology and ecology.     

Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.