[Establishment of real time PCR for detecting plasma cell free DNA of rats and its significance].

OBJECTIVE: Overtraining is a serious problem in sports, assessed by comprehensive multi-index evaluation, but so far there is still no sensitive, specific monitoring indicator or simple evaluation method to evaluate it. This research established a method for detecting plasma cell free DNA (cfDNA) of rats by real time PCR and discuss edits significance: a new molecular marker of overtraining? METHODS: Twelve male SD rats were randomly divided into control group and overtraining group. The overtraining group rats were undertaken overtraining on a motor-driven treadmill for 5 weeks, while the control group rats kept quiescent. All the rats were drawn blood at pre-and after-5 weeks to detect plasma levels of cfDNA, testosterone (T) and corticosterone (Cort) as well as peroxidation/antioxidation parameters (T-AOC, MDA, SOD, GSH-Px) and creatin kinase (CK). RESULTS: (1) Plasma cfDNA of rat was detected specifically by our real time PCR. (2) Compared with control group rats, the plasma cfDNA of overtraining rats increased obviously (about 5.43 fold). (3) Plasma cfDNA was related to plasma T, Cort, T/C ratio and MDA (correlation coefficent were -0.729, 0.854, -0.655 and 0.720, respectively) rather than plasma T-AOC, GSH-Px, SOD and CK. CONCLUSION: (1) A real time PCR method was established successfully to determine plasma cfDNA of rat. (2) A remarkable raises of plasma levels of cfDNA were found in overtraining rats which were associated with T, Cort and T/C, suggested that plasma cfDNA might be a new molecular marker of overtraining. (3) The increase of plasma cfDNA of overtraining rat might correlate with enhanced oxidative stress induced by overtraining instead of muscle damage.

[Cloning and analysis of six full-length cDNA similar to sheep KAP6-1 from cashmere goat].

Cashmere is the fiber of kings, produced from the cashmere goat (Capra hircus). Cashmere fabric has little squama. Due to its lightness cashmere feels smooth and silky. An intriguing feature of cashmere structure is multiplicity of the hair keratin proteins with distinctive amino acid compositions. To study the role and regulation of one of these keratins, we used the SMART ( switching mechanism at 5' end of RNA transcript) technology to construct a cDNA library of skin tissue from an Inner Mongolia male cashmere goat. A total of 636 cDNA sequences were obtained by randomly sequenced from 5' of cDNA library. Sequence comparison with the GenBank database confirmed that there are 41 sequences showed high nucleotide sequence identity in the coding region with sheep keratin associated protein 6-1 (KAP6-1). They represent six different cDNAs (The accession numbers in GenBenk are AY310749, AY310750, AY310751, AY310752, AY310753 and AY310754, respectively). They are full-length cDNAs according to the known KAP6-1 genomic gene sequences of sheep. From the nucleotide sequences the open reading frames were identified, that encoded six basic proteins of 82, 84, 71, 71, 83, 83 amino acids, respectively, with a combined glycine and tyrosine content of about 60%. Compared analysis showed that the KAPs from goats shared more than 55.4% identities in nucleotide sequences and more than 79.8% identities in amino acid sequences with each other, and shared highest identities (81.9% approximately 98.8%) with KAP6-1 from sheep. The KAP6s from different animal species shared more than 50% identities in amino acid sequences with each other.