[Utilization of used fungus-growing materials of Gastrodia elata].

This study aims to explore the resource utilization of used fungus-growing materials produced in the cultivation of Gastrodia elata. To be specific, based on the production practice, this study investigated the recycling mechanism of used fungus-growing materials of G. elata by Phallus inpudicus. To screen edible fungi with wide adaptability, this study examined the allelopathic effects of Armillaria mellea secretions on P. impudicus and 6 kinds of large edible fungi and the activities of enzymes related to degradation of the used fungus-growing materials of G. elata. The results showed that P. impudicus can effectively degrade cellulose, hemicellulose, and lignin in used fungus-growing materials of G. elata. The cellulase activity of A. mellea was significantly higher than that of P. impudicus, and the activities of lignin peroxidase, polyphenol oxidase, and xylanase of P. impudicus were significantly higher than those of A. mellea, which was the important reason why A. mellea and P. impudicus used different parts and components of the used fungus-growing materials to absorb carbon sources and develop ecological niche differences. The growth of P. impudicus was significantly inhibited on the used fungus-growing materials of G. elata. The secretions of A. mellea had allelopathic effects on P. impudicus and other edible fungi, and the allelopathic effects were related to the concentration of allelopathy substances. The screening result showed that the growth and development of L. edodes and A. auricular were not significantly affected by 30% of A. mellea liquid, indicating that they had high resistance to the allelopathy of A. mellea. The results showed that the activities of extracellular lignin peroxidase, polyphenol oxidase, and xylanase of the two edible fungi were similar to those of P. impudicus, and the cellulase activity was higher than that of P. impudicus. This experiment can be further verified by small-scale production tests.

[Methods for synchronous detection of 14 mycotoxins in Pseudostellariae Radix and investigation on its contamination].

This study aimed to establish a method for synchronous detection of 14 mycotoxins in Pseudostellariae Radix and investigate its contamination with mycotoxins, so as to provide technical guidance for monitoring the quality of Chinese medicinal materials and medication safety. The sample was extracted with 80% acetonitrile in an oscillator for 1 h, purified using the modified QuEChERS purifying agent(0.1 g PSA + 0.3 g C_(18) + 0.3 g MgSO_4), and separated on a Waters HSS T3 chromatographic column(2.1 mm×100 mm, 1.8 µm). The gradient elution was carried out with 0.1% formic acid in water and acetonitrile, followed by the scanning in the multi-reaction monitoring(MRM) mode and the analysis of mycotoxin contamination in 26 Pseudostellariae Radix samples. The recovery rates of the established method were within the range of 82.17%-113.6%, with the RSD values less than 7% and the limits of quantification(LOQ) being 0.019-0.976 µg·kg~(-1). The detection rate of 14 mycotoxins in 26 batches of medicinal materials was 53.85%. The detection rate of sterigmatocystin(ST) was the highest, followed by those of zearalenone(ZEN), aflatoxin G_2(AFG_2), fumonisin B_1(FB_1), HT-2 toxin, and nivalenol(NIV). Their respective detection rates were 38.46%, 26.92%, 23.08%, 11.54%, 11.54%, and 7.69%, with the pollution ranges being 1.48-69.65, 0.11-31.05, 0.11-0.66, 0.28-0.83, 20.86-42.56, and 0.46-1.84 µg·kg~(-1), respectively. The established method for the detection of 14 mycotoxins is accurate, fast and reliable. The research results have very important practical significance for guiding the monitoring and prevention and control of exogenous fungal contamination of Chinese medicinal materials.

Clinical significance of serum expression of GROß in esophageal squamous cell carcinoma.

AIM: To determine the association between serum levels of growth-related gene product ß (GROß) and clinical parameters in esophageal squamous cell carcinoma (ESCC). METHODS: Using enzyme-linked immunosorbent assay, serum GROß levels were measured in ESCC patients (n = 72) and healthy volunteers (n = 83). The association between serum levels of GROß and clinical parameters of ESCC was analyzed statistically. RESULTS: The serum GROß levels were much higher in ESCC patients than in healthy controls (median: 645 ng/L vs 269 ng/L, P < 0.05). Serum GROß levels were correlated positively with tumor size, lymph node metastasis, and tumor-node-metastasis (TNM) staging, but not with gender or the histological grade of tumors in ESCC patients. The sensitivity and specificity of the assay for serum GROß were 73.61% and 56.63%, respectively. CONCLUSION: GROß may function as an oncogene product and contribute to tumorigenesis and metastasis of ESCC.

[PAR-1 regulation of intracellular Ca²(+) mobilization in pulmonary giant cell carcinoma cell line PLA801D/PLA801C].

OBJECTIVES: To investigate molecular mechanisms of PAR-1 regulation on intracellular Ca²(+) mobilization in lung giant cell carcinoma cells in vitro and its involvement in tumor metastasis. METHODS: Free intracellular Ca²(+) ([Ca²(+)]i) was measured in lung giant cell carcinoma PLA801C and PLA801D cells by confocal microscopy. Sense and anti-sense PAR-1 expression vectors were transfected into PLA801C (C+)and PLA801D(D-) cells, respectively. The effects of PAR-1 expression were investigated by thrombin and TRAP-induced mobilization of [Ca²(+)]i in the C+ and D-cells. RESULTS: There were significant differences of the mean values of [Ca²(+)]i between PLA801D (59.55) and PLA801C cells (35.46, P < 0.01). The mean [Ca²(+)]i of C+ cells (45.77) was significantly higher than that of its control CV cells (35.46, P < 0.05), and the mean [Ca²(+)]i of D-cells (48.42) was significantly lower than that of its control DV cells (59.55, P < 0.05). The peaks of [Ca²(+)]i of C+ and CV cells were 48.19 ± 9.84 and 45.64 ± 9.87 (P < 0.05) respectively at 80 s and 100 s after thrombin treatment, but were 111.31 ± 25.00 and 52.93 ± 11.21 (P < 0.05) respectively at 60 s after TRAP treatment. The peaks of [Ca²(+)]i of D- and DV cells were 40.71 ± 5.89 and 61.07 ± 21.36 (P < 0.05) respectively at 60 s after thrombin treatment, but were 84.98 ± 11.23 and 102.58 ± 21.48 (P < 0.05) respectively at 40 s after TRAP treatment. CONCLUSIONS: The high metastatic potential of PLA801D and PLA801C may be related to [Ca²(+)]i of the tumor cells. PAR-1 may play an important role in the metastasis of lung giant cell carcinoma cells by up-regulating the intracellular Ca²(+).

Cytotoxic diterpenoids from Euphorbia helioscopia.

Four new jatrophane-type diterpenoids (1-4), together with 16 known related compounds, were isolated from the Chinese medicinal plant Euphorbia helioscopia. The structures of 1-4 were determined on the basis of spectroscopic and chemical methods. Cytotoxicity of the isolated compounds against HeLa and MDA-MB-231 cells was evaluated, with only helioscopinolide A (5) and euphornin (3a) being active.

Ingenane diterpenoids from Euphorbia esula.

An extensive study of metabolites present in Euphorbia esula led to isolation of 16 ingenane diterpenoids 1-16 together with the known ingenane derivative 17 and four known cycloartane triterpenoids. Their structures were elucidated on the basis of spectroscopic studies and comparison with known related compounds. All the compounds were assayed for their inhibitory activity against human HeLa cervical cancer cell line.

[Functional aspects of protease-activated receptor 1 in promoting metastasis of lung cancer].

OBJECTIVE: To study the functional aspects of protease-activated receptor 1 (PAR-1) gene involved in tumor metastasis. METHODS: Two human lung giant cell carcinoma cell lines PLA801C (low metastasis potential) and PLA801D (high metastasis potential) were chosen as in-vitro human cancer model systems. Sense and anti-sense expression constructs of PAR-1 gene (pC/PAR1s and pC/PAR1as) were transfected into PLA-801C and PLA-801D cells by lipofection. PAR-1 expression was determined by RT-PCR and western blot analysis. MTT growth, flow cytometry analysis, fibronectin adhesion, and matrigel invasion assays were used to study the effect of PAR-1 expression on the proliferation, adhesion, and invasion of the transfected cells. RESULTS: Appropriate up-regulation or down-regulation of protein expression of PAR-1 was observed in both transfected cell lines (PLA801C and PLA801D) to express PAR-1s or PAR-1as, respectively. Expression of the sense PAR-1 markedly increased cellular proliferation, adhesion and invasion of PLA-801C cells. In contrast, anti-sense PAR-1 significantly inhibited cell growth, adhesion and invasion capabilities, along with cell arrest at G0/G1 phase of the PLA-801D cells. CONCLUSIONS: Successful up- and down- regulation of expression of PAR-1 can be achieved by in-vitro transfection of sense and antisense PAR-1 constructs. PAR-1 may enhance metastasis of lung cancer through its regulation of cellular proliferation, adhesion and invasion. Down-regulation of expression of PAR-1 may provide a new therapeutic strategy against lung carcinoma.

[Expression of PAR-1 in human lung carcinoma and its relationship with tumor metastatic potential].

OBJECTIVE: To explore the correlation between expression of PAR-1 and metastasis of human lung carcinoma. METHODS: Expression levels of PAR-1 were examined in surgically resected lung carcinoma specimens and corresponding lymph nodes by RT-PCR and immunohistochemistry, combined with morphometric methodology and clinicopathologic profiles. RESULTS: Strong PAR-1 staining was detected in the periphery of carcinoma nests, adenocarcinomatous emboli, foci of atypical adenomatous hyperplasia adjacent to the adenocarcinoma and atypical proliferation of duct epithelium of bronchial mucous glands. The expression rates of PAR-1 were 73.8% (59/80) and 63.9% (23/36) by immunohistochemistry and RT-PCR respectively. The percentage of PAR-1 protein expression cells was significantly higher in tumors with metastasis (85.7%, 48/56) than those without (45.8%, 11/24). Morphometric study demonstrated that there were significant differences of PAR-1 protein expression levels between tumors with metastatic and those without, primary and metastatic carcinomas, primary carcinomas and benign lung tissues adjacent to the carcinoma. No significant correlation was found between PAR-1 expression level and tumor size, histological types and tumor grades. The positive rate of PAR-1 mRNA expression in the metastatic group was significantly higher than that of the non-metastatic group (78.3%, 18/23 v.s. 38.5%, 5/13). CONCLUSION: PAR-1 expression may play an important role in determining the malignant phenotypes of lung cancers and significantly contribute to their initiation, progression and metastasis.

[Differentially expressed genes in human giant-cell lung cancer lines with different metastatic potentials].

OBJECTIVE: To screen genes differentially expressed in two human giant-cell lung cancer lines of same origin but with different metastasis potentials. METHODS: Suppression subtractive hybridization (SSH) was done twice on two giant-cell lung cancer lines, PLA-801C and PLA-801D (hereafter abbreviated as C and D), of same origin but with low (C) and high (D) metastatic potentials. In the first round, SSH C was used as tester and D as driver, while in the second round, the tester and driver were interchanged. The sequences acquired from both rounds of SSH were spotted on glass slides respectively and screened by hybridizing with two-color fluorescence probes. Clones that had different expression levels on chips were also confirmed by RNA dot blot or Northern blot. RESULTS: There were 16 sequences with high expression in C as compared to those in D, and 79 sequences with high expression in D compared to those in C. After sequencing, most of them were found to be highly homologous to those encoding the following proteins: (1) cytokines and their receptors; (2) kinases and related proteins; (3) other proteins including enzymes, heat shock proteins, receptors, proteins of cell skeleton and mitochondria, products of oncogenes, etc; (4) some proteins deduced from gene sequences with yet unknown functions. CONCLUSION: The alterations in expression of some known genes, including HSP70, AXL receptor tyrosine kinase and 14-3-3zeta, might have impact on metastasis of giant-cell lung cancer. Whether some differentially expressed genes newly revealed are metastasis-related needs further study.

[The specific killing of human melanoma cells by replication selective adenovirus].

OBJECTIVE: To construct replication selective adenovirus AdhepE1 targeting human melanoma and observe its specific killing of human melanoma cells in vitro. METHODS: Adenovirus E1 region, the murine tyrosinase promoter and enhancer DNA sequences were acquired respectively by PCR cloning. The shuttle plasmid of replication-selective adenovirus targeting human melanoma was constructed by DNA recombination. Replication-selective adenovirus AdhepE1 was generated by homologous recombination. The human melanoma cell line SK-Mel-1 and hepatocellular carcinoma cell line HepG2 were attacked separately by lower dose of AdhepE1. Change of cell morphology was observed and the surviving cells were calculated. The expression of E1A was assayed by RT-PCR to verify the specific-replication of AdhepE1. RESULTS: Replication selective adenovirus AdhepE1 targeting human melanoma was acquired by PCR. Human melanoma cell line SK-Mel-1 was sensitive to oncolytic killing of AdhepE1 whereas HepG2 was little responsive. The results of RT-PCR suggested that AdhepE1 replicated specifically in human melanoma cells. CONCLUSION: AdhepE1 can selectively kill human melanoma cells.