Transcriptional regulatory region and DNA methylation analysis of TNNI1 gene promoters in Gaoyou duck skeletal muscle (Anas platyrhynchos domestica).

1. The slow skeletal muscle troponin I (TNNI1) gene has been found to be specifically expressed in slow muscle fibres and plays an important role in muscle development. The aim of this study was to determine the active control area of duck TNNI1 and identify the potential cis-regulatory elements in the promoter. 2. In this study, the TNNI1 promoter was first cloned by genome walking and the sequences were analysed using bioinformatics software. Firefly luciferase reporter gene vectors, driven by a series of constructs with progressive deletions, were used to identify the core transcriptional regulatory region of the duck TNNI1 gene. The methylation status of the CpG island in the TNNI1 promoter was detected in skeletal muscle on embryonic days 21 and 27, by bisulphite sequencing PCR (BSP). 3. The results showed two CpG islands presented in the promoter region, with one of the CpG islands located in the core transcriptional regulatory region (-2078/-885 bp). The total methylation levels of the 14 CpG sites were not altered between breast and leg muscles on embryonic days 21 and 27. However, four CpG sites (loci of positions 4, 11, 13, and 14) showed dramatically different methylation levels between breast and leg muscles at embryonic days 21 and 27. Analysis showed that multiple CpG sites had a significant correlation between the methylation levels of the CpG sites and mRNA expressions in skeletal muscle. Multiple transcription factor binding sites including Sp1, c-Myc, Oct-1 and NF-kB motifs were identified and might be responsible for transcriptional regulation of the TNNI1 gene. 4. These findings contribute to further understanding of the fundamental mechanism for transcriptional regulation of the TNNI1 gene in ducks.

[Determination of sialic acids in serum of lung cancer with ultrafiltration-capillary electrophoresis].

A method for the determination of sialic acids in serum with ultrafiltration-capillary electrophoresis is described and the operation was optimized. Sialic acids were directly separated and analyzed with UV detection at 195 nm and without pre-or post-column derivatization. The recovery of N-acetylneuraminic acid (NANA) was 92.6%, the concentration and mass detection limit of NANA were 9.6 mumol/L and 39 fmol respectively. This method was used for the determination of NANA level in the serum of 11 lung cancer patients and 30 normal adults. The results showed that the average concentration of NANA in the serum of patients was much higher than that of normal adults with P < 0.001. The results were also compared with those obtained by the traditional colorimetric method, with good linear relationship of r = 0.983 at n = 10. It is concluded that the method described in this paper is simple and sensitive, and is suitable for basic research and clinical applications to malignant tumors.