Functional characterization and substrate promiscuity of sesquiterpene synthases from Tripterygium wilfordii.

Acyclic terpenes, commonly found in plants, are of high physiological importance and commercial value, and their diversity was controlled by different terpene synthases. During the screen of sesquiterpene synthases from Tripterygium wilfordii, we observed that Ses-TwTPS1-1 and Ses-TwTPS2 promiscuously accepted GPP, FPP, and GGPP to produce corresponding terpene alcohols (linalool/nerolidol/geranyllinalool). The Ses-TwTPS1-2, Ses-TwTPS3, and Ses-TwTPS4 also showed unusual substrate promiscuity by catalyzing GGPP or GPP in addition to FPP as substrate. Furthermore, key residues for the generation of diterpene product, (E, E)-geranyllinalool, were screened depending on mutagenesis studies. The functional analysis of Ses-TwTPS1-1:V199I and Ses-TwTPS1-2:I199V showed that Val in 199 site assisted the produce of diterpene product geranyllinalool by enzyme mutation studies, which indicated that subtle differences away from the active site could alter the product outcome. Moreover, an engineered sesquiterpene high-yielding yeast that produced 162 mg/L nerolidol in shake flask conditions was constructed to quickly identify the function of sesquiterpene synthases in vivo and develop potential applications in microbial fermentation. Our functional characterization of acyclic sesquiterpene synthases will give some insights into the substrate promiscuity of diverse acyclic terpene synthases and provide key residues for expanding the product portfolio.

Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system.

YfiBNR is a recently identified bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiB(L43P) complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiB(L43P) adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiB(L43P) shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiB(L43P)-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.

[Study on synergic and decreasing toxic effects of mineral water and Chinese herbal compound preparation on cisplatin].

OBJECTIVE: The synergic and decreasing toxic effects of mineral water and Chinese herbal compound preparation (MWCHCP) on cisplatin were investigated in sarcoma 180 (S180) mice. METHOD: The S180 mice were treated for 5 days with intraperitoneal injection of cisplatin(7.33 mg x kg(-1)) and oral administration of MWCHCP(1 925, 3 850, 7 700 mg x kg(-1)). Then the mice were killed and the tumor growth inhibition rate, organ index, diarrhea index were determined. Observe pathological sections of stomach to study the protective effect of MWCHCP. Reverse transcription-polymerase chain reaction (RT-PCR) was applied to investigate the tumour necrosis factor-alpha (TNF-alpha) expression level of the intestine. RESULT: Combining with cisplatin and MWCHCP caused a tendency of increasing the tumor growth inhibition rate and significant attenution of cisplatin-induced diarrhea, visceral organ injury, gastric mucosal injury and decreased TNF-alpha mRNA level of intestine. CONCLUSION: The present findings suggest that MWCHCP increases the inhibition rate of tumor growth of cisplatin and has a beneficial influence on gastrointestinal lesion induced by cisplatin.

High-performance liquid chromatographic method for the determination of cyromazine and melamine residues in milk and pork.

A high-performance liquid chromatography method is described in this paper. The method uses NH(2) column and 97% acetonitrile eluate to determine the insecticide cyromazine and metabolite melamine residues in milk and pork. Samples were treated with NaOH and extracted with acetonitrile containing 20% NH(4)OH. Target analytes of samples were cleaned up and concentrated by C(18) column solid-phase extraction. A separation for cyromazine and melamine was achieved, and respective retention times were 8 and 12 min. The calibration curves for cyromazine and melamine were linear in a concentration range of 0.01-1.0 microg/mL, with correlation coefficients of 0.9999 and 0.9997, respectively. The limit of detection of both compounds was 0.2 ng, and the limit of quantitation was 0.02 mg/kg. Recoveries of cyromazine and melamine at fortified levels of 0.02, 0.05, and 0.1 mg/kg ranged from 84.5-90.8%, and 83.6-91.3%, respectively, with coefficient of variation of 3.1-7.8%.

[Sorption characteristics of veterinary antibiotics chlortetracycline on manure].

The sorption characteristics of high-use tetracycline veterinary antibiotics-chlortetracycline (CTC) on pig manure and chicken manure were investigated in laboratory according to the OECD guideline 106. The results showed that CTC was strongly and rapidly adsorbed to the two manures and Freundlich model could describe preferably the CTC nonlinear sorption isotherms. The adsorbed CTC was difficult to be desorbed by rain with desorption H of 1.522 for pig manure and 1.329 for chicken manure, respectively. Only 18.3%-20.4%, 18.7%-19.4% and 55.7%-57.6% adsorbed CTC were desorbed by methanol, 3 mol/L NaCl and 3 mol/L MgCl2. The sorption of CTC on manure were affected by solution pH and ionic strength, the amount of adsorbed CTC on manure decreased with an increase of pH value and ionic strength, and greater sorption effect in the Ca systems than the Na systems was observed. These results suggest that organic matter and cation exchange mainly contribute to the sorption of CTC on manure.

[Subchronic exposure to acrylamide affects reproduction and testis endocrine function of rats].

OBJECTIVE: To explore the effect of subchronic exposure to acrylamide on the reproduction and testis endocrine function of rats. METHODS: Forty healthy adult male SD rats were randomly divided into 4 groups of equal number, exposed to acrylamide at the dose of 0, 4, 10 and 18 mg/(kg x d) respectively for 9 weeks, and then subjected to the determination of the hindlimb landing foot splay, sperm vitality and morphology, the activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) in the testis homogenate, and the levels of testosterone (T) and estradiol (E2) in the serum and testis homogenate. Based on the primary Leydig cell culture models exposed to acrylamide of 0, 0.1, 0.75, 4 and 8 mmol/L, the activity of Leydig cells was measured by the CCK-8 method. RESULTS: Following acrylamide exposure, the hindlimb landing foot splay increased markedly with dose increase (P < 0.01). The rates of sperm vitality were (6.86 +/- 5.46)%, (65.43 +/- 5.16)%, (60.86 +/- 4.26)% and (46.86 +/- 2.73)% in the exposed groups, significantly lower than in the control (P < 0.01); the rates of abnormal sperm were (39.00 +/- 10.95)%, (35.43 +/- 7.54)%, (45.71 +/- 13.28)% and (56.71 +/- 17.01)%, significantly increased in the 10 and 18 mg/(kg x d) groups (P < 0.05); ACP activities were (82.93 +/- 11.05), (73.52 +/- 8.77), (77.67 +/- 3.04) and (68.56 +/- 3.09) U/g prot, showing a decreasing tendency, while ALP activities were (0.96 +/- 0.15), (1.07 +/- 0.22), (1.12 +/- 0.22) and (0.74 +/- 0.10) U/g prot, displaying a tendency of first increasing and then decreasing. Both ACP and ALP activities were inhibited significantly in the 18 mg/(kg x d) group as compared with the control (P < 0.05). A marked reduction was noted in T levels in the serum, (13.44 +/- 4.76), (7.69 +/- 3.84), (5.23 +/- 1.42) and (1.36 +/- 0.86) ng/ml, as well as in the testis homogenate, (4.95 +/- 1.64), (3.01 +/- 0.76), (2.44 +/- 0.91) and (0.85 +/- 0.49) ng/mg prot, (P < 0.01), but no significant changes were observed in 17beta-E2 levels. After 24 hours exposure to acrylamide, the optical densities were 0.82 +/- 0.06, 0.56 +/- 0.07, 0.44 +/- 0.06, 0.26 +/- 0.03 and 0.45 +/- 0.21, showing an evident inhibition of the activity of Leydig cells at the dose of 0.1, 0.75, 4 and 8 mmol/L (P < 0.01). CONCLUSION: Subchronic exposure to acrylamide could affect the normal development of sperm, cause changes of the activity of some enzymes in the testis and significantly influence hindlimb motor coordination. Acrylamide directly damages Leydig cells and affects the endocrine function of the testis.

[Toxicity of acrylamide on male reproduction].

Acrylamide is a common chemical material, extensively used in industry and scientific experiments. Recently, it has been reported that starchy food cooked at high temperature can produce acrylamide. Acrylamide monomer has several toxic effects and the extensive concern for its toxicity has arisen with the finding of acrylamide formation in some processed foods. Researches have shown that acrylamide monomer can cause reproductive toxicity, including toxic effects on male reproductive behavior, male reproductive endocrine function and spermatogenesis. The mechanisms may include the effects of acrylamide on Leydig cells, the formation of motor protein/ chromosomal/DNA alkylation and damage by oxidative stress.