Molecular characterization of the myosatin gene and the effect of fasting on its expression in Chinese perch (Siniperca chuatsi).

Myostatin (MSTN) is an important member of the transforming growth factor-ß (TGF-ß) superfamily and is a muscle growth inhibitor. In the present study, we cloned the Chinese perch MSTN cDNA sequence and analyzed its expression patterns under various conditions. The MSTN full cDNA sequence was 3347 bp long, including an open-reading frame of 1131 bp, which encoded 376 amino acids. Sequence analysis demonstrated that the MSTN shared a highly conserved signal peptide, a TGF-ß functional peptide, a hydrolytic site (RARR), and nine conservative cysteine residues with other members of the TGF-ß superfamily. Sequence alignment and phylogenetic tree analyses indicated that the MSTN had a close relationship with teleostean fish, but they are far separated from mammals. Real-time polymerase chain reaction analysis revealed that the MSTN was strongly expressed in the skeletal muscle and heart tissues. Temporal expression analysis demonstrated that the MSTN gene was expressed in very low levels, from 20 to 90 dph (post-hatching development), and was at its highest level at 150 dph (P < 0.05). The fasting-re-feeding experiment showed that the expression of the MSTN gene was initially decreased in response to a single meal, after seven days of fasting, and subsequently increased significantly, and finally decreased back to its original level. Together, our results provided valuable knowledge regarding the regulation of MSTN gene expression in Chinese perch.

Rapid Muscle Relaxation in Siniperca chuatsi is Coordinated by Parvalbumin (PVALB) and MiR-181a.

Parvalbumins (PVALBs) are particularly abundant in the fast-contracting muscles and correlate positively with muscle relaxation speed in amphibians and fishes. MiRNAs play important roles in diverse biologic processes via binding to the 3' untranslated region (3'UTR) of the target mRNAs. In the study, four PVALB isoforms, named as PVALB1, 2, 3, and 4, were identified in the mandarin fish (Siniperca chuatsi) fast muscle and PVALB4 exhibited the highest expression level among them. By bioinformatics analysis, a putative miR-181a binding site in PVALB4 was detected and the direct interaction between miR-181a and PVALB4 was confirmed with the luciferase reporter assay. Further, when miR-181a was inhibited, it substantially increased PVALB4 mRNA expression level and the muscle relaxation rate in vivo. Taken together, the obtained results suggest that miRNA-181a/PVALB4 is an evolutionarily conserved miRNAtarget pair and their interaction is correlated with muscle relaxation rate in the mandarin fish. Therefore, the study revealed a novel molecular mechanism in the regulation of skeletal muscle relaxation in fish.

Signal transduction pathways in Synechocystis sp. PCC 6803 and biotechnological implications under abiotic stress.

Cyanobacteria have developed various response mechanisms in long evolution to sense and adapt to external or internal changes under abiotic stresses. The signal transduction system of a model cyanobacterium Synechocystis sp. PCC 6803 includes mainly two-component signal transduction systems of eukaryotic-type serine/threonine kinases (STKs), on which most have been investigated at present. These two-component systems play a major role in regulating cell activities in cyanobacteria. More and more co-regulation and crosstalk regulations among signal transduction systems had been discovered due to increasing experimental data, and they are of great importance in corresponding to abiotic stresses. However, mechanisms of their functions remain unknown. Nevertheless, the two signal transduction systems function as an integral network for adaption in different abiotic stresses. This review summarizes available knowledge on the signal transduction network in Synechocystis sp. PCC 6803 and biotechnological implications under various stresses, with focuses on the co-regulation and crosstalk regulations among various stress-responding signal transduction systems.

miR-143: a novel regulator of MyoD expression in fast and slow muscles of Siniperca chuatsi.

The fast and slow skeletal muscle fibers in fish are separated to a much greater degree than in mammals. MyoD is required for the maintenance of normal fiber type balance in muscles. So far, the upstream regulatory factors of MyoD in terms of controlling muscle phenotype are poorly understood. In the present study, we used Siniperca chuatsi as a model system and demonstrated that miR-143 expression was negatively correlated with MyoD expression in the fast and slow muscles of S. chuatsi. The luciferase reporter assay further verified the direct interaction between the miR-143 and MyoD. The miR-143 suppression also led to the significant increase in MyoD and fast myosin heavy chain gene expression in vivo. Taken together, our studies indicated that miRNA participates in controlling the performance of different muscle fiber types in vertebrates.

Systematic identification and differential expression profiling of MicroRNAs from white and red muscles of siniperca chuatsi.

MicroRNAs (miRNAs) participate in the regulation of myogenesis and muscle physiological function. Most skeletal muscles in vertebrates contain a mixture of fibertypes. So far, the regulatory mechanism of the miRNA in terms of controlling muscle phenotype is poorly understood. In the present study, we use Siniperca chuatsi as a model system and demonstrate that miRNAs are involved in regulating the physiological processes and metabolism of different muscle fibers in vertebrates. The miRNA transcriptomes of the white muscle, red muscle, and five other tissues from Siniperca chuatsi were profiled using Solexa deep sequencing. We characterized 186 conserved miRNAs and 3 novel miRNAs from the two small RNA libraries of white and red muscles. Among the 155 miRNAs overlapped between the two libraries, we identified 60 significantly expressed miRNAs between the two types of muscle fibers. Using integrative miRNA target-prediction and network-analysis approaches, an interaction network of differentially expressed and muscle-related miRNAs and their putative targets were constructed. Sch-miR-181a-5p that could act to control the performance of the different muscle fiber types by targeting the myostatin gene was identified.

Characterization and ontogenetic expression analysis of the myosin light chains from the fast white muscle of mandarin fish Siniperca chuatsi.

Three full-length complementary DNA (cDNA) clones were isolated encoding the skeletal myosin light chain 1 (MLC1; 1237 bp), myosin light chain 2 (MLC2; 1206 bp) and myosin light chain 3 (MLC3; 1079 bp) from the fast white muscle cDNA library of mandarin fish Siniperca chuatsi. The sequence analysis indicated that MLC1 and MLC3 were not produced from differentially spliced messenger RNAs (mRNA) as reported in birds and rodents but were encoded by different genes. The MLC2 encodes 170 amino acids, which include four EF-hand (helix-loop-helix) structures. The primary structures of the Ca(2+)-binding domain were well conserved among the MLC2s of seven other fish species. The ontogenetic expression analysis by real-time PCR showed that the three light-chain mRNAs were first detected in the gastrula stage, and their expression increased from the tail bud stage to the larval stage. All three MLC mRNAs showed longitudinal expression variation in the fast white muscle of S. chuatsi, especially MLC1 which was highly expressed at the posterior area. Taken together, the study provides a better understanding about the MLC gene structure and their expression pattern in muscle development of S. chuatsi.

Distribution and expression of recombinant plasmid encoding chicken interleukin-2.

A plasmid DNA that encodes chicken interleukin-2 (pCI-ChIL-2-EGFP) was investigated for its distribution and expression after intramuscular (i.m.) injection in chickens. After the i.m. injection, serum distribution was detectable from 2 h post inoculation (p.i.), peaked at 8 h p.i., and disappeared at 7 days p.i. The plasmid DNA was also observed in several organs including heart, liver, lung, spleen, bursa and inoculated muscle at different time points, but at 19 days p.i. the plasmid DNA was not found in any organ except inoculated muscle. Fluorescence of enhanced green fluorescent protein (EGFP) was found in cytoplasm and nucleus of cultured Vero cells, chicken embryo fibroblasts and peripheral blood lymphocytes, which were transfected in vitro with the plasmid DNA or in vivo with Lipofectamine. The expression profile of the fusion gene (ChIL-2-EGFP) in vivo was measured by RT-PCR, ELISA and fluorescence microscopy. The EGFP expression was detected from 8 h p.i. to 14 days p.i. and peaked at 5 days p.i., when the number of EGFP-expression myocytes was about 5% in the injected site. These results demonstrate that intramuscular administration of plasmid DNA leads to widespread distribution and long-term expression in vivo.

Renal excretion of oxalate in patients with chronic renal failure or nephrolithiasis.

This study was carried out in order to investigate renal oxalate excretion in a group of normal subjects (n = 40), a group of patients with uremia (n = 52) and a group with nephrolithiasis (n = 34). We found that the mean concentrations of oxalate in the 24-hour urine specimens of both patient groups were below the normal range. Although the renal creatinine clearance (CCR) was significantly decreased in some stone patients (n = 14), decreased renal oxalate clearance was noted only in those patients with severe renal failure. Thus, plasma oxalate was found to be elevated only in patients with chronic renal failure (mean +/- SD, 49.7 +/- 12.4 mumol/l), while the normal value was 17.0 +/- 6.7 mumol/l. The mean tubular excretion fraction of oxalate was also found to increase markedly in uremia with a mean of 26.3 +/- 17.3% (in normal subjects, 11.7 +/- 7.5%), but their mean daily urinary excretion of oxalate decreased to 63.2 mumol/day (mean value of 232.6 mumol/day in normal subjects). A positive correlation was observed between oxalate and creatinine, and between oxalate and calcium excretion, which was not found in normal subjects or patients with kidney stones. In nephrolithiasic patients, the daily excretion of oxalate, calcium and phosphate had no discernible increment and the mean excretory ratio of oxalate, calcium or phosphate to creatinine was all within normal limits. But when the CCR of stone patients was below 80 ml/min, their daily excretion of oxalate and calcium decreased significantly (p less than 0.01) and the excretory ratio of phosphate to creatinine markedly increased.(ABSTRACT TRUNCATED AT 250 WORDS)