ABSTRACT
Tryptophan hydroxylase I (TPH1) catalases the 5-hydroxylation reaction of L-Trp, which is the rate-limiting step in the synthesis of serotonin. Serotonin, a major component of Bufonis venenum, is involved in numerous physiological functions as an important neurotransmitter. In this study, BbgTPH1 cDNA was cloned from the parotid gland of Bufo bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pMAL-BbgTPH1, and the induced conditions to express the recombinant BbgTPH1 in E. coli TB1 cells were optimized. The full length of BbgTPH1 is 1984 bp (GenBank accession No. JQ768313) with a 1443 bp open reading frame (ORF) encoding a 480 amino acid residues. The deduced protein molecular weight is 55.2 kDa and its theoretical isoelectric point is 5.58. The sequence includes conserved domain and special signal sequence of the aromatic amino acid hydroxylase (AAAH) superfamily. Homologous alignment showed that BbgTPH1 shared a high homology with other species. Phylogenetic tree showed the closest relative to BbgTPH1 was Xenopus tropical-TPH1. The best induction conditions of recombinant BbgTPH1 were 0.5 mmol·L-1 IPTG at 20℃ for 8 h. The function of BbgTPH1 was identified by in vitro enzymatic reaction and the recombinant BbgTPH1 was able to produce 5-hydroxytryptophan by catalyzing tryptophan. This study represents the first time of cloning and identification of the function of TPH1 in Bufo genus. The results of this study will be an important foundation for future studies of biosynthesis of bufotenines in the parotid gland of B. bufo gargarizans.
ABSTRACT
Objective: To investigate the influence of unilateral internal carotid artery (ICA) stenosis (70-99%) or occlusion evaluated by color Doppler ultrasound (CDU) and transcranial Doppler (TCD) on ICA hemodynamics of contralateral carotid artery (50-69% stenosis) in patients with bilateral infernal carotid artery stenosis. Methods: A total of 102 consecutive patients with bilateral ICA stenosis (unilateral stenosis 50-69%, contralateral stenosis 70-99% or occlusion) were enrolled from June 2005 to June 2011 and they were used as study group (group A). The patients were screened by color Doppler flow imaging (CDFI) and TCD, and they were confirmed as bilateral ICA stenosis by DSA. At the same time, 89 patients with unilateral ICA stenosis 50-69% and normal contralateral ICA were used as control group (group B). The differences of ICA stenosis 50-69% in the two groups and the peak systolic velocity (PSV), end-diastolic flow velocity (EDV), resistance index (RI) and pulsatility index (PI) of anterior cerebral artery (ACA) in both groups were compared. Group A was redivided into an anterior communicating antery patent group (A1 group, n = 54) and an anterior communicating artery occluded group (A2 group, n = 48) according to the results of TCD and DSA examinations. The differences of the hemodynamic parameters of the 50-69% stenotic ICA and ACA stenosis, and the correlations of the hemodynamic changes in both groups were further compared. Results: Circled digit oneThe PSV and EDV of unilateral stenotic ICA stenosis and ACA with 50-69% stenosis in group A were significantly higher than those in group B (all P < 0.05), RI and PI were significantly lower (P = 0.001, P = 0.000). Circled digit twoPSV and EDV of unilateral ICA and ACA with 50-69% stenosis in group A1 were higher than those in group A2 (all P < 0.01), but RI of ICA and PI of ACA in group A1 were lower than those in group A2 (P = 0.000, P = 0.000), the differences were statistically significant. Circled digit threeThe velocity of ICA was positively correlated with that of ACA (r = 0.327, 0.422, P = 0.007, P = 0.000). Conclusion: When one side of internal carotid artery is severely stenotic, the measured value of velocity of the other side of ICA (50-69% stenosis) is significantly higher than the diagnostic criteria of the actual lesion. The anterior communicating artery patency is the basis of hemodynamic changes of the bilateral stenotic ICA. The identification of the degree of bilateral ICA stenosis should be evaluated comprehensively in combination with the extracranial and intracranial hemodynamic changes.
ABSTRACT
<p><b>AIM</b>To study the mechanism of dracorhodin perchlorate-induced Hela cell apoptosis.</p><p><b>METHODS</b>Cell viability was measured by MTT method. Morphological changes were observed by phase contrast microscopy and Hoechst 33258 staining. DNA fragmentation was assayed by agarose gel electrophoresis. Protein expression was detected by Western blot analysis.</p><p><b>RESULTS</b>Dracorhodin perchlorate induced Hela cell apoptosis. The apoptosis was partially reversed by caspase-1, -3, -8, -9 and caspase family inhibitors. Treatment of Hela cells with dracorhodin perchlorate for 12 h increased the protein expression ratio of Bax/Bcl-XL; procaspase-3, -8, ICAD and PARP were cleaved to smaller molecules.</p><p><b>CONCLUSION</b>Dracorhodin perchlorate induced Hela cell death via alteration of Bax/Bcl-XL ratio and activation of caspases.</p>