Unusual (2R,6R)-bicyclo[3.1.1]heptane ring construction in fungal α-trans-bergamotene biosynthesis.

Bergamotenes are bicyclo[3.1.1]heptane sesquiterpenes found abundantly in plants and fungi. Known bergamotene derivatives all possess (2S,6S)-bergamotene backbone. In this study, two (+)-α-trans-bergamotene derivatives (1 and 2) with unusual (2R,6R) configuration were isolated and elucidated from marine fungus Nectria sp. HLS206. The first (+)-α-trans-bergamotene synthase NsBERS was characterized using genome mining and heterologous expression-based strategies. Based on homology search, we characterized another (+)-α-trans-bergamotene synthase LsBERS from Lachnellula suecica and an (+)-α-bisabolol synthase BcBOS from Botrytis cinerea. We proposed that the cyclization mechanism of (+)-α-trans-bergamotene involved endo-anti cyclization of left-handed helix farnesyl pyrophosphate by (6R)-bisabolyl cation, which was supported by molecular docking. The biosynthesis-based volatiles (3-6) produced by heterologous fungal expression systems elicited significant electroantennographic responses of Helicoverpa armigera and Spodoptera frugiperda, respectively, suggesting their potential in biocontrol of these pests. This work enriches diversity of sesquiterpenoids and fungal sesquiterpene synthases, providing insight into the enzymatic mechanism of formation of enantiomeric sesquiterpenes.

Combinatorial mutation on the ß-glycosidase specific to 7-ß-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast.

Taxol is a "blockbuster" antitumor drug produced by Taxus species with extremely low amount, while its analogue 7-ß-xylosyl-10-deacetyltaxol is generally much higher in the plants. Both the fungal enzymes LXYL-P1-1 and LXYL-P1-2 can convert 7-ß-xylosyl-10-deacetyltaxol into 10-deacetyltaxol for Taxol semi-synthesis. Of them, LXYL-P1-2 is twice more active than LXYL-P1-1, but there are only 11 significantly different amino acids in terms of the polarity and acidic-basic properties between them. In this study, single and multiple site-directed mutations at the 11 sites from LXYL-P1-1 to LXYL-P1-2 were performed to define the amino acids with upward bias in activities and to acquire variants with improved catalytic properties. Among all the 17 mutants, E12 (A72T/V91S) was the most active and even displayed 2.8- and 3-fold higher than LXYL-P1-2 on ß-xylosidase and ß-glucosidase activities. The possible mechanism for such improvement was proposed by homology modeling and molecular docking between E12 and 7-ß-xylosyl-10-deacetyltaxol. The recombinant yeast GS115-P1E12-7 was constructed by introducing variant E12, the molecular chaperone gene pdi and the bacterial hemoglobin gene vhb. This engineered yeast rendered 4 times higher biomass enzyme activity than GS115-3.5K-P1-2 that had been used for demo-scale fermentation. Thus, GS115-P1E12-7 becomes a promising candidate to replace GS115-3.5K-P1-2 for industrial purpose.

Chrysoxanthones A⁻C, Three New Xanthone⁻Chromanone Heterdimers from Sponge-Associated Penicillium chrysogenum HLS111 Treated with Histone Deacetylase Inhibitor.

By treating with histone-deacetylase inhibitor valproate sodium, three new heterdimeric tetrahydroxanthone⁻chromanone lactones chrysoxanthones A⁻C (1⁻3), along with 17 known compounds were isolated from a sponge-associated Penicillium chrysogenum HLS111. The planar structures of chrysoxanthones A⁻C were elucidated by means of spectroscopic analyses, including MS, 1D, and 2D NMR. Their absolute configurations were established by electronic circular dichroism (ECD) calculations. Chrysoxanthones A⁻C exhibited moderate antibacterial activities against Bacillus subtilis with minimum inhibitory concentration (MIC) values of 5⁻10 µg/mL.

Aberrant CXCR4 and ß-catenin expression in osteosarcoma correlates with patient survival.

To determine the clinical significance of C-X-C chemokine receptor type 4 (CXCR4) and ß-catenin in osteosarcoma, their protein expression levels were assessed in 96 osteosarcoma and 20 osteochondroma cases using immunohistochemistry. Additionally, CXCR4 and ß-catenin mRNA expression levels were measured in 16 fresh osteosarcoma and 16 adjacent healthy tissue samples using fluorescent reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In the osteosarcoma samples, the positive CXCR4 protein expression rate was significantly higher than the rate in the osteochondroma samples (68.75 vs. 20.00%; P<0.01). Furthermore, ß-catenin protein expression was detected in 61.46% of osteosarcoma cases and 25.00% of osteochondroma cases. Similarly, the RT-qPCR data identified increased CXCR4 and ß-catenin mRNA expression levels in the osteosarcoma compared with adjacent control tissues. It was determined that CXCR4 (P<0.01) and ß-catenin (P<0.05) expression were significantly associated with the clinical Enneking stage, metastasis and survival of osteosarcoma. Furthermore, multivariate analysis identified CXCR4 and ß-catenin protein expression levels, as well as clinical stage and metastasis, as significant risk factors for survival in patients with osteosarcoma (P<0.05). In conclusion, the present study determined that CXCR4 and ß-catenin are abnormally expressed in osteosarcoma tissues, and, therefore, may be important during osteosarcoma progression.

SDF-1/CXCR4 promotes F5M2 osteosarcoma cell migration by activating the Wnt/ß-catenin signaling pathway.

Osteosarcoma (OS), the most common primary malignant bone tumor in children and adolescents, lacks an effective therapy. Stromal cell-derived factor (SDF-1) and its receptor, CXCR4, play multiple roles in migration, proliferation, and survival of different tumor cells. This study aimed to investigate whether the functional SDF-1/CXCR4 signaling mediates chemotaxis in F5M2 OS cells as well as the underlying mechanisms. Immunohistochemistry and immunofluorescence microscopy were used. RNA expression was detected by real-time quantitative polymerase chain reaction, and protein expression was examined by Western blotting. Migration assays were carried out in F5M2 cells. The results showed that the expression of CXCR4 and ß-catenin mRNA and protein was significantly higher in OS tissues compared to the surrounding non-neoplastic tissues. SDF-1 promoted F5M2 cell migration by activating the AKT and Wnt/ß-catenin signaling pathway, which was abrogated by preincubation with AMD3100 and LY294002. In conclusion, SDF-1/CXCR4 axis-promoted F5M2 cell migration was regulated by the Wnt/ß-catenin signaling pathway.

LC-MS/MS determination of pantoprazole in human plasma and its application / 中国药学杂志

OBJECTIVE: To develop an LC-MS/MS method with direct precipitation of sample protein to determine the concentration of pantoprazole in human plasma. METHODS: LC-MS/MS equipment with ESI source and WATERS XTerra® RP18 (4.6 mm × 100 mm, 3.5 μm) column were used in the experiment. The column temperature was set at 35°C, 5 mmol · L-1 ammonium acetate-acetonitrile (40:60, V/V) was used as mobile phase and the flow rate was 1.0 mL · min-1. The injection volume was 2 μL. Omeprazole was taken as the internal standard. Pantoprazole and omeprazole were detected with proton adducts at m/z 384.1 → 200.1 and m/z 346.1 → 198.0 in multiple reaction monitoring (MRM) positive mode, respectively. RESULTS: The calibration curve of pantoprazole was linear in the range of 5-6000 ng · mL-1 (r > 0.9900). The intra-day and inter-day relative standard deviations were both less than 6.5%, and the relative errors were within ±7%. The mean extract recoveries were more than 98%. In the stability studies, pantoprazole was found to be stable in plasma under various storage conditions. CONCLUSION: The method is rapid, simple, accurate, selective, and reproducible for the determination of pantoprazole in human plasma. The method is successfully applied to a pharmacokinetics study of pantoprazole sodium enteric-coated tablets in healthy volunteers.

Rapid determination of eplerenone in human plasma by LC-MS/MS and its application to pharmacokinetic study / 中国药学杂志

OBJECTIVE: To study the pharmacokinetics of single-and multiple-dose of eplerenone tablets in Chinese healthy volunteers. METHODS: Twelve volunteers were administered a single dose of eplerenone at 25, 50, and 100 mg according to 3 × 3 Latin square design. In multiple-dose study, 10 volunteers were administered eplerenone of 50 mg per day for 6 d. Eplerenone concentration in plasma was determined by LC-MS/MS. RESULTS: The main pharmacokinetic parameters of single-dose eplerenone of 25, 50 and 100 mg in 12 volunteers were as follows: ρmax (450.2±146.2), (765.2±258.2), and (1262±428) μg·L-1, tmax (1.71±0.33), (2.06±1.01) and (2.79±1.48) h, t1/2(2.50±0.39), (2.69±0.55) and (2.84±0.53) h, AUC0-24h (2410±778), (4403±1522) and (8202±2398) μg·h·L-1, AUC0-∞ (2429±774), (4426±1523) and (8246±2407) μg·h·L-1. In the multiple-dose study, the main pharmacokinetic parameters after the first dose and those at steady state were as follows: tmax (3.40±1.27) and (2.65±1.00) h, t1/2(3.03±0.66) and (3.22±0.62) h, ρmax (690.7±207.4) and (743.2±192.3), μg·L-1, ρSSmin(12.81±9.64) μg·L-1, ρavss (219.0±59.6) μg·L-1, and AUCSS (5256±1431) μg·h·L-1 CONCLUSION: The pharmacokinetics of eplerenone in human is linear in the range of 25-100 mg. There is no drug accumulation after multiple dosing. Copyright 2012 by the Chinese Pharmaceutical Association.

Role of NMDA receptor-dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries.

Excitotoxic neuronal damage caused by overactivation of N-methyl-D-aspartate glutamate receptors (NMDARs) is thought to be a principal cause of neuronal loss after stroke and brain trauma. Here we report that activation of sterol regulatory element binding protein-1 (SREBP-1) transcription factor in affected neurons is an essential step in NMDAR-mediated excitotoxic neuronal death in both in vitro and in vivo models of stroke. The NMDAR-mediated activation of SREBP-1 is a result of increased insulin-induced gene-1 (Insig-1) degradation, which can be inhibited with an Insig-1-derived interference peptide (Indip) that we have developed. Using a focal ischemia model of stroke, we show that systemic administration of Indip not only prevents SREBP-1 activation but also substantially reduces neuronal damage and improves behavioral outcome. Our study suggests that agents that reduce SREBP-1 activation such as Indip may represent a new class of neuroprotective therapeutics against stroke.

[An improved liquid nitrogen freezing method for establishing canine models of femoral head necrosis].

OBJECTIVE: To establish an canine model of femoral head osteonecrosis using an improved liquid nitrogen freezing method. METHODS: Sixteen adult canines were divided into 4 groups at random and subjected to instant freezing of the unilateral femoral head with liquid nitrogen. The dogs were observed at 2, 4, 8, and 16 weeks after the operation for radiographic, gross and pathological changes of the femoral head. RESULTS: Significant radiographic, gross and pathological changes occurred in the femoral head after the freezing. CONCLUSION: Improved liquid nitrogen freezing of the femoral head provides a simple and convenient method for establishing animal models of femoral head osteonecrosis.

[Expressions of c-Fos and NADPH-d in the related brainstem during vestibular compensation].

OBJECTIVE: To study the mechanism of vestibular compensation and to observe the changes of c-Fos and NADPH-d expressions in the brainstem of the vestibular deafferentation rats in static status or following angular acceleration stimulation. METHODS: Totally 60 SD rats were randomly divided into control group (labyrinthine intact), complete unilateral vestibular deafferentation (UVD) group, simultaneous complete bilateral vestibular deafferentation (BVD) group (n = 20 in each group). Subgroups (n = 10 in each subgroup) were set for static status or following angular acceleration stimulation in each group. Double labeling with histochemistry-immunohistochemistry was performed to observe c-Fos/NADPH-d neurons. RESULTS: No positive c-Fos/NADPH-d expression was observed in the both sides of medial vestibular nucleus (MVN) and prepositus hypoglossi (PrH) of normal rats in static status and BVD rats whether following canal rotation or not. c-Fos/ NADPH-d expression was observed in the ipsilesional MVN and the contralesional PrH of UVD rats. However, c-Fos/NADPH-d were detected in both sides of MVN and PrH in UVD rats and normal rats following angular acceleration stimulation. CONCLUSION: In the ipsilesional MVN and the contralesional PrH, c-Fos plays an important role in vestibular compensation, in which nitric oxide acts as a key neurotransmitter.

[Clinical analysis of normal subjects and dizzy patients with postural sway test].

OBJECTIVE: To study the characteristics of the normal subjects with postural sway test and discuss the value of postural sway test in the diagnosis of dizzy patients. METHODS: Totally 112 normal subjects, 72 patients with peripheral vertigo, and 30 patients with central vertigo were examined using a stabilometer (EAB-100, Anima Co., Japan). Items include patterns, length of locus, envelope area, deflection average center displacement, and romberg rate were recorded and compared. RESULTS: The postural sway was classified into the following five patterns: centripetal, forward and backward, right and left, diffuse, and multicentric. Centripetal pattern was the most prominent figure pattern in the normal subjects, while diffuse pattern was the most prominent figure pattern in the patients with central vertigo. When the normal subjects closed their eyes, age was linearly correlated with the length of locus, envelope area, rectangel area, and unit area path length (P < 0.05). Sex was not correlated to all of these parameters. The length of locus and envelope area in the patient groups were significantly increased when compared with the normal subjects (P < 0.0167). Romberg rate was not significantly different between the patients and the normal subjects. CONCLUSIONS: The pattern of postural sway is useful for the diagnosis of vertigo. Age is an important factor that affects the balance function. Sex seems has no influence. Visual input plays an important role in maintaining postural balance. Stabilometer is a useful tool for evaluating balance function, and postural sway test may be a valuable assistant examination.

[Establishment and characteristics of a human chordoma cell line].

OBJECTIVE: To present an established human chordoma cell line for chordoma research. METHODS: The specimens pathologically identified as chordoma were cultured, using primary tissue culture in vitro. The surviving cells were analyzed by morphology, histochemical stain, cell cycling analysis, karyotype analysis, electron microscopic observation, heterotransplantation and study of invasive capacity in vitro. RESULTS: The newly established cell line CM-319 has been maintained in continual cultures for over 100 generations in two years. Its morphological observation, histochemical staining properties, electron microscopic observation and heterotransplantation showed the common characteristics of chordoma. The doubling time of cells was about 33 hours. Cell cycle analysis showed: G(1) 55.6%, G(2) 21.9% and S 22.5%, G(2)/G(1) = 1.90. Chromosome analysis showed a hypotriploid feature and the success rate of heterotransplantation was 100%. It is capable of invasion in vitro. CONCLUSION: CM-319, as a cell line derived from human chordoma cells, may serve for further studies of chordoma.

[Relationship between expression of BLCAP protein and malignancy of osteosarcoma].

AIM: To study the relationship between expression of bladder cancer-associated protein (BLCAP) and malignancy of osteosarcoma. METHODS: SABC method was applied to study the expression of BLCAP protein in osteosarcoma, according to clinical results, to analyze the prognosis value of BLCAP protein in osteosarcoma. RESULTS: The positive rate of BLCAP protein in primary osteosarcoma was 65.6%,much higher than that in recurrent osteosarcoma, which was 25.0%. CONCLUSION: It may be helpful for evaluating the prognosis of osteosarcoma to study the relationship between expression of BLCAP protein and malignancy of osteosarcoma.