Hepatic cysteine sulphinic acid decarboxylase depletion and defective taurine metabolism in a rat partial nephrectomy model of chronic kidney disease.

BACKGROUND: Taurine depletion occurs in patients with end-stage chronic kidney disease (CKD). In contrast, in the absence of CKD, plasma taurine is reported to increase following dietary L-glutamine supplementation. This study tested the hypothesis that taurine biosynthesis decreases in a rat CKD model, but is rectified by L-glutamine supplementation. METHODS: CKD was induced by partial nephrectomy in male Sprague-Dawley rats, followed 2 weeks later by 2 weeks of 12% w/w L-glutamine supplemented diet (designated NxT) or control diet (NxC). Sham-operated control rats (S) received control diet. RESULTS: Taurine concentration in plasma, liver and skeletal muscle was not depleted, but steady-state urinary taurine excretion (a measure of whole-body taurine biosynthesis) was strongly suppressed (28.3 ± 8.7 in NxC rats versus 78.5 ± 7.6 µmol/24 h in S, P < 0.05), accompanied by reduced taurine clearance (NxC 0.14 ± 0.05 versus 0.70 ± 0.11 ml/min/Kg body weight in S, P < 0.05). Hepatic expression of mRNAs encoding key enzymes of taurine biosynthesis (cysteine sulphinic acid decarboxylase (CSAD) and cysteine dioxygenase (CDO)) showed no statistically significant response to CKD (mean relative expression of CSAD and CDO in NxC versus S was 0.91 ± 0.18 and 0.87 ± 0.14 respectively). Expression of CDO protein was also unaffected. However, CSAD protein decreased strongly in NxC livers (45.0 ± 16.8% of that in S livers, P < 0.005). L-glutamine supplementation failed to rectify taurine biosynthesis or CSAD protein expression, but worsened CKD (proteinuria in NxT 12.5 ± 1.2 versus 6.7 ± 1.5 mg/24 h in NxC, P < 0.05). CONCLUSION: In CKD, hepatic CSAD is depleted and taurine biosynthesis impaired. This is important in view of taurine's reported protective effect against cardio-vascular disease - the leading cause of death in human CKD.

Cloning a peanut resveratrol synthase gene and its expression in purple sweet potato.

A resveratrol synthase gene was cloned from the peanut plant (Arachis hypogaea) by RT-PCR and was transformed into purple sweet potato (Ipomoea batatas) by Agrobacterium-mediated transformation. Stem sections were infected with bacterial solution of OD(600) = 0.4 for 20 min and then cocultured for 2 days. Infected explants were cultured on MS media containing 50 mg/l kanamycin, 0.02 mg/l NAA and 1 mg/l 6-BA for bud induction or containing 75 mg/l kanamycin, 1.0 mg/l NAA and 0.1 mg/l 6-BA for root formation. The bud and root induction rates were 37.5 and 25.0%, respectively. 105 regenerated plants were obtained, with 11 positive plants by PCR and Southern blotting analyses. A high level of resveratrol glucoside (340 µg/g dry weight), but no resveratrol, was detected in the transformed plants by HPLC. This study also provides a stable genetic transformation and plant regeneration method for metabolic modification of purple sweet potato.

Cloning, microbial expression and structure-activity relationship of polyphenol oxidases from Camellia sinensis.

Polyphenol oxidase (PPO) can be used for organic synthesis and degradation of wastes and dyes in industries. Lack of enzyme sources is a major barrier for its application. A PPO gene, with a full length of 1.8kb without introns, was cloned by PCR from genomic DNA of five common cultivars of Camellia sinensis. They had a 98.2-99.9% degree of identity in nucleotides and 94.7-96.1% in amino acids and encoded a polypeptide of 599 amino acids with a signal peptide targeting the chloroplast and three Cu-binding domains. The mature PPO showed high expression and enzyme activity after refolding the inclusion bodies in Escherichia coli BL21 (DE3) using pET30c expression vector, but low expression in Pichia pastoris GS115 using both the secretory and non-secretory vectors pPICZalphaA and pPICZA. The expression of PPO mutants demonstrated that the signal sequences prevented recombinant gene expression in E. coli. PPO activity was not affected by the C-terminus and was slightly inhibited by the CuC domain. Other domains were important for its activity. A 3.1-fold increase in PPO activity over non-recombinant controls was obtained by expressing the PPO fragment without signal sequences and the CuC domain in E. coli BL21 (DE3) using the pET30c vector.