Enhancing the organic solvent resistance of ω-amine transaminase for enantioselective synthesis of (R)-(+)-1(1-naphthyl)-ethylamine.

BACKGROUND: Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. An R-selective ω-amine transaminase from Aspergillus terreus (AtATA) exhibited activity toward 1-acetylnaphthalene. However, AtATA displayed unsatisfactory organic solvent resistance, which is required to enhance the solubility of the hydrophobic substrate 1-acetylnaphthalene. So, improving the tolerance of enzymes in organic solvents is essential. MAIN METHODS AND RESULTS: The method of regional random mutation combined with combinatorial mutation was used to improve the resistance of AtATA in organic solvents. Enzyme surface areas are structural elements that undergo reversible conformational transitions, thus affecting the stability of the enzyme in organic solvents. Herein, three surface areas containing three loops were selected as potential mutation regions. And the "best" mutant T23I/T200K/P260S (M3) was acquired. In different concentrations of dimethyl sulfoxide (DMSO), the catalytic efficiency (kcat /Km ) toward 1-acetylnaphthalene and the stability (half-life t1/2 ) were higher than the wild-type (WT) of AtATA. The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics (MD) simulations under 15%, 25%, 35%, and 45% DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine ((R)-NEA), which was an intermediate of Cinacalcet Hydrochloride for the treatment of secondary hyperthyroidism and hypercalcemia. Moreover, in a 20-mL scale-up experiment, 10 mM 1-acetylnaphthalene can be converted to (R)-NEA with 85.2% yield and a strict R-stereoselectivity (enantiomeric excess (e.e.) value >99.5%) within 10 h under 25% DMSO. CONCLUSION: The beneficial mutation sites were identified to tailor AtATA's organic solvents stability via regional random mutation. The "best" mutant T23I/T200K/P260S (M3) holds great potential application for the synthesis of (R)-NEA.

[Regulatory Effect of Chinese Drugs for Stasis Removing and Collaterals Dredging on the Expres- sions of podocin and CD2AP in Podocyte Slit Diaphragm of Diabetic Nephropathy Rats].

OBJECTIVE: To observe the regulatory effect of Chinese drugs for stasis removing and collaterals dredging (CDSRCD) on the expressions of podocin and CD2AP in podocyte slit diaphragm (SD) of diabetic nephropathy (DN) rats. METHODS: DN rat model was duplicated in 40 male Sprague- Dawley rats by feeding high fat high glucose diet combined with intraperitoneally injecting 1 % streptozoto- cin (STZ, 35 mg/kg). Totally 36 successfully modeled rats were divided into the model group, the CD- SRCD group,- and the irbesartan group according to random digit table, 12 in each group. Besides, anoth- er 10 normal rats were recruited as a normal group. Rats in the CDSRCD group and the irbesartan group were intragastrically fed with CDSRCD and irbesartan respectively. Rats in the normal group and the mod- el group were fed with equal volume of distilled water at the same time. 24 h urine protein quantitation was detected using ELISA at various time points. Body weight (BW) , kidney weight ( KW), kidney index (KI) , fasting blood glucose (FBG) , serum creatinine (SCr), blood urea nitrogen (BUN), and uric acid (UA) in each group were detected after 16 weeks of intervention. The pathomorphological changes of re- nal tissue were observed under light microscope and electron microscope respectively. The protein and mRNA expressions of podocin and CD2AP were detected by Western blot and Real-time PCR respectively. RESULTS: (1) Compared with the normal group, 24 h urine protein quantitation significantly increased at week 4, 8, 12, and 16, respectively (P <0. 01). BW was decreased; KI and levels of FBG, SCr, BUN, and UA all increased after modeling (P <0. 01). Compared with the model group, 24 h urine protein quan- titation significantly decreased in the CDSRCD group and the irbesartan group at week 4, 8, 12, and 16, respectively (P <0. 01). Besides, it was more obviously reduced in the CDSRCD group than in the irbe- sartan group (P <0. 05, P <0.01). BUN level obviously decreased both in the CDSRCD group and the irbesartan group after modeling (P <0. 05, P <0. 01). (2) Results of renal pathology showed that disar- ranged renal structure, obviously thickened basement membrane, severely proliferated mesenteria, widely fused foot processes in the model group. All these pathological changes were attenuated in the CD- SRCD group and the irbesartan group to some degree. (3) Results of Western blot and Real-time PCR showed, compared with the normal group, protein and mRNA expressions of podocin and CD2AP decreased in the model group (P <0. 01). Compared with the model group, protein and mRNA expressions of podocin and CD2AP increased in the CDSRCD group and the irbesartan group (P <0. 01). Protein and mRNA expressions of podocin and CD2AP increased more in the CDSRCD group than in the irbesartan group (P <0. 05). CONCLUSIONS: CDSRCD could protect renal function by lowering urinary protein in DN rats, improve renal pathological changes. Its mechanism might be related to up-regulating mRNA and protein expressions of podocin and CD2AP.

Pharmacokinetics of rapamycin-eluting stents in miniswine coronary model.

BACKGROUND: The results of clinical trials of rapamycin-eluting stents reduce restenosis have been quite promising. The main purpose of this study was to characterize the in vivo pharmacokinetics of high dose rapamycin (Rapa)-eluting stents in a miniswine coronary model. METHODS: Ten miniswines underwent placement of 18 high dose Rapa-eluting stents in the left anterior descending and right coronary arteries. At the planned times of the 1.5th, 12th, 24th hour, 3th, 7th and 28th day, the animals (n = 1, 1, 2, 2, 2, and 2, respectively) were euthanized after completion of coronary angiography. Blood samples were obtained at 0, 10, 20, 30 minutes; 1, 2, 6, 24 hours; and 3, 7, 28 days to determine systemic Rapa levels. Rapa levels in whole blood, arterial wall, heart, renal and liver tissues were determined by high-performance liquid chromatography/mass spectroscopy. RESULTS: Peak whole blood concentration (Cmax), time to peak concentration (tmax), elimination half-life (t1/2beta), area under the curve (AUC), and apparent systemic clearance (Cl/F) were (10.91 +/- 1.28) ng/ml, (2.0 +/- 0.2) hours, (7.25 +/- 0.63) hours, (1.15 +/- 0.11) ng x h x ml(-1), and (180 +/- 12) ml x h(-1) x kg(-1), respectively. More than 95% Rapa detected is localized in the coronary artery surrounding the stent and heart. CONCLUSION: Stent-based delivery of Rapa via a copolymer stent is feasible and safe. This strategy holds promise for the prevention of stent restenosis.