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Longipetalol A (1) is an unprecedented highly modified triterpenoid with a unique 1,2-seco-3-(2-oxo-phenylethyl)-17α-13,30-cyclodammarane skeleton, featuring an acetal-lactone fragment. It was isolated from Dichapetalum longipetalum along with two additional derivatives, namely, longipetalols B (2) and C (3). Their structures were elucidated using spectroscopic analyses combined with single-crystal X-ray diffraction. Compounds 1, 2, and 3 exhibited inhibitory effects on nitric oxide production in lipopolysaccharide-induced RAW264.7 macrophages.

Background: To examine the expression of cysteine-rich 61 (Cyr61/CCN1) protein in laryngeal squamous- cell carcinoma (LSCC) tissues, and its relationship with the tumor epithelial-mesenchymal transition (EMT), invasion, metastasis, and prognosis. Materials and Methods: Immunohistochemistry was used to detect the expressions of Cyr61, Vimentin (Vim), and E-cadherin (E-cad) in 88 cases of LSCC tissues and 30 cases of tumor-adjacent normal tissues. Vim and E-cad were used as mesenchymal and epithelial markers, respectively, to determine the relationship between Cyr61 expression and the EMT of LSCC cells. In addition, clinical and histopathological data were combined to analyze the relationship between the positive-expression rates of Cyr61, Vim and E-cad and LSCC invasion, metastasis and prognosis. Results: In LSCC tissues, Vim expression rate was significantly higher than that of the tumor-adjacent tissues, whereas E-cad expression rate was significantly lower than that of the tumor-adjacent tissues. The Vim expression rate was significantly higher in stages T3 and T4 than in stages T1 and T2 LSCC tissues, whereas E-cad expression rate was significantly lower in stages T3 and T4 than in stages T1 and T2 LSCC tissues. Compared to the group without lymph node metastasis, the Vim expression rate was significantly higher and the E-cad expression rate was significantly lower in the group with lymph node metastasis. The expression rate of Cyr61 was significantly higher in LSCC tissues than in the tumor-adjacent normal tissues. In addition, the Cyr61 expression rate was higher in stages T3 and T4 than in stages T1 and T2 LSCC, and higher in the group with lymph node metastasis than in the group without lymph node metastasis. The Vim expression rate was significantly higher in the Cyr61 positive group than in the Cyr61 negative group, whereas the E-cad expression rate was significantly higher in the Cyr61 negative group than in the Cyr61 positive group. Survival analysis indicated that survival rates of Cyr61 positive, Vim positive and E-cad negative groups were significantly lower than that of Cyr61 negative, Vim negative and E-cad positive groups, respectively. Conclusions: Cyr61 expression is closely associated with LSCC invasion and lymph node metastasis. Overexpression of Cyr61 may induce EMT and therefore leads to LSCC invasion and metastasis and poor prognosis. Cyr61 may become a new maker for clinical prediction of LSCC invasion and metastasis and a new target for LSCC treatment.

Three new polyunsaturated lipids, (6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraen-3-one (1), (6Z,9Z,12Z,15Z)-1-bromooctadeca-6,9,12,15-tetraen-3-one (2), and (Z)-ethyl docos-5-enoate (3), together with two known polyunsaturated lipids, 4(Z),7(Z),10(Z)-tridecatrienoic acid (4) and (6Z,9Z,12Z,15Z)-octadeca-1,6,9,12,15-pentaen-3-one (5), were isolated from the marine sponge Haliclona sp., which was collected from Guangxi, using HSCCC and HPLC methods. Chemical structures of the five compounds were elucidated by spectroscopic techniques.

Two new compounds, rabdosia acids A (1) and B (2), were isolated from the petroleum ether extract of Rabdosia lophanthoides. The structures of these compounds were elucidated on the basis of spectroscopic methods (IR, HR-ESI-MS, 1D NMR, and 2D NMR).

A chemical investigation of marine sponge Jaspis stellifera, collected from the South China Sea, led to the isolation of four new isomalabaricane-type triterpenoids, jaspiferins C-F (1-4). The structures of those compounds were elucidated by extensive spectroscopic methods. Jaspiferin C (1), which has the six-membered carbon ring at the side chain, was discovered for the first time from the isomalabaricane-type triterpenoids. The hypothesis of a biogenetic pathway to generate jaspiferin C (1) was depicted.

A phytochemical investigation on gorgonian Muricella sp. from East China Sea resulted in the isolation of eight eunicellin diterpenoids including two new ones, muricellins A-B (1, 2). Chemical structures of these compounds were elucidated by spectroscopic techniques (1D and 2D NMR and MS) and by comparison with data reported in the literature. Anti-rheumatoid arthritis activities of 1, 3, 4, and 6 have been evaluated.

In this and subsequent articles, we intend to explore Rosa's conjecture that every tree is graceful [1]. We define the concept of joint sum of graceful trees and study its operational properties. We shall prove the gracefulness of a certain family of trees.

This article is a continuation of the article “The Joint Sum of Graceful Trees.” In this and subsequent articles, we intend to explore Rosa's conjecture that every tree is graceful [1]. Using the concept of radical product of graceful trees, we prove the gracefulness of a certain family of trees. Some of the results we obtain here are more general than those of Ma [2] and Zeng [3].

The objective of the study was to explore the inhibitive role of cyclin D1 gene silence in laryngeal squamous cell carcinoma by lentivirus-mediated RNA interference.Cd1-RNAi-Lentivirus and the control lentivirus (GFP-Lentivirus) were transfected into Hep-2 cells. Reverse transcriptase polymerase chain reaction was performed to explore the cyclin D1 expression level in Cd1-RNAi-Lentivirus-transfected Hep 2 cells. The apoptosis and viability of Cd1-RNAi-Lentivirus-treated Hep-2 cells were measured with flow cytometry and methyl thiazolyl tetrazoliym assay. In an animal experiment, 10 mice bearing Hep-2 cell tumor were intratumorally injected with Cd1-RNAi-Lentivirus; and the other 10 mice were injected with GFP-Lentivirus. Terminal deoxytransferase-mediated dUTP nick end labeling stains and transmission electron microscope were used to observe the apoptosis in the xenografts.Cyclin D1 was knocked down after Cd1-RNAi-Lentivirus was transfected into Hep-2 cells. The proliferative ability of Hep-2 cells was significantly inhibited by Cd1-RNAi-Lentivirus, and a significant apoptosis of Hep-2 cells was also observed after Cd1-RNAi-Lentivirus transfection. The average weight and volume of tumors in the Cd1-RNAi-Lentivirus-treated group were significantly lower than those in the control group (P.01). The significant apoptosis was detected with terminal deoxytransferase-mediated dUTP nick end labeling stain and transmission electron microscope.The present findings suggest that cyclin D1 gene silence by lentivirus-mediated RNA interference can inhibit growth and promote apoptosis of laryngeal squamous cell carcinoma.

Objective: To identify the regulatory mechanisms of Toll- like receptor (TLR)-associated genes in chronic rhinosinusitis (CRS) with nasal polyps (NP) using gene microarray analyses. Methods: We pooled: (1) NP biopsy specimens from 10 nonatopic CRS patients and (2) healthy mucosal tissue from 10 additional nonatopic healthy patients (controls). These pooled samples were evaluated by gene microarrays that included 125 genes for TLRs and associated signaling elements. To validate gene product expressions, 20 NP and 15 normal nasal turbinate tissues were evaluated for TLR-9 expression by immunohistochemical staining and Western blots using samples from gland cells, epithelial cells, and mononuclear cells cytologically identified by HE staining. Results: In pooled NP samples compared to pooled controls, 4 genes were upregulated (≧2-fold higher expression) and 19 were downregulated (≤0.5-fold lower expression). TLR-9 was an upregulated gene in NP tissue. Compared to control tissue, there were significantly higher percentages of TLR-9 positively stained NP gland cells, epithelial cells, and mononuclear cells (p < 0.001). On Western blots, while both normal and NP tissues expressed TLR-9 protein, the expression was significantly more pronounced for NP tissue (p < 0.001). Conclusions: Inflammation associated with CRS may be due to dysregulated innate immune elements, particularly TLR-9 and its associated signal transduction elements, which may impact upon prolonged activation of adaptive immune responses in the sinonasal mucosa.

To investigate the expression of integrin alpha-v subunit in laryngeal and hypopharyngeal squamous cell carcinomas (LHSCC) and to correlate the expression ratio with clinic and pathologic features of LHSCC, meanwhile, to investigate the relationship between the expression of integrin alpha-v subunit and tumor angiogenesis.A tissue microarray of LHSCC was designed and made. Using this microarray, the expression of integrin alpha-v subunit in LHSCC was studied by immunohistochemistry, and the expression disparity in different clinic and pathologic staging of LHSCC was analyzed. The immunohistochemical staining of CD105 was done in the same microarray, the intratumor microvessel density (IMVD) was calculated by CD105 staining. The relationship between integrin alpha-v subunit expression and the IMVD was analyzed.In primary cancer tissue, the expression ratio of integrin alpha-v subunit was 68.0% (51/75), significantly higher than normal tissue beside cancer (10.3%, 3/29, chi2 = 28.68, P0.001); the expression ratio of integrin alpha-v subunit in lymph node metastatic carcinoma was 100.00% (20/20), significantly higher than normal tissue (chi2 = 38.77, P0.001) and primary cancer tissue (chi2 = 12.69, P0.05); in group with lymph node metastasis, the expression ratio of integrin alpha-v subunit was significantly higher than group without lymph node metastasis (chi2 = 10.87, P0.001); the IMVD of the group with integrin alpha-v subunit positive expression was significantly higher than the group with integrin alpha-v subunit negative expression (P0.001).There was significant relationship between integrin alpha-v subunit expression and lymphatic metastasis of LHSCC. Overexpression of the integrin alpha-v subunit may have contributed to the tumor angiogenesis and lead to lymphatic metastasis. Integrin alpha-v subunit may become a novel lymphatic metastasis marker of LHSCC.

The objective of this study was to investigate the role of alpha v-integrin subunit (ITGAV, CD51) in invasion and metastasis of the laryngeal and hypopharyngeal squamous cell carcinomas and to evaluate whether an antisense oligonucleotide sequence (ASONs) targeting ITGAV gene can result in proliferative inhibition and induce to apoptosis of laryngeal carcinoma cell lines (Hep-2). Firstly, a tissue microarray contained 75 primary carcinomas, 29 non-cancerous normal tissues and 20 metastatic lymph nodes was constructed and used to detect the expression of ITGAV by immunohistochemistry. The changes of ITGAV expression from each group were assessed and correlated to the clinical parameters of the patients. Secondly, the ASONs targeting ITGAV gene was transfected into Hep-2 cells in vitro. The proliferative ability of the cells after transfection was measured by MTT assay and the apoptosis was detected using flow cytometry. Results showed that the expression of ITGAV was significantly correlated with differentiation and lymph node metastasis of these cancers. In vitro test showed that the proliferative ability of Hep-2 cells was significantly inhibited by ASONs in a way of concentration- and time-depending mode, and a significant apoptosis of Hep-2 cells was also observed after ASONs transfection. In conclusion, the expression of ITGAV was significantly correlated with differentiation and metastasis of the laryngeal and hypopharyngeal carcinomas; down-regulation of ITGAV gene could inhibit proliferation of Hep-2 cells and induce to its apoptosis. These results suggest that ITGAV gene may become a promising prognostic marker and new treatment target for these cancers.

Objective: The purpose of this study was to explore the inhibiting role of adenoviral-mediated (Ad)-tissue factor pathway inhibitor (TFPI)-2 gene in the growth of laryngeal squamous cell carcinoma (LSCC). Study Design: The nude mice bearing LSCC were prepared by intracutaneous injection of Hep-2 cell. Gene therapy was performed by injecting adenoviruses carrying TFPI-2 gene around tumors in the animal model. Methods: Eighteen nude mice bearing Hep-2 cell tumor were randomly separated into the treated group and control group. The former were injected with recombinant adenovirus Ad-TFPI-2 around the tumor, and the later were injected with equivalent Ad-LacZ. After treatment, differences of tumor weight, volume, and ultrastructure of tumor cells between these two groups were observed by measuring and using transmission electron microscope. Apoptosis in Hep-2 xenotransplants was detected using the terminal deoxy-transferase-mediated dUTP nick end labeling. In addition, the expressions of TFPI-2 protein and proliferating cell nuclear antigen (PCNA) in tumor tissues were detected using Western blot and immunohistochemistry, respectively. Results: The average weight and volume of tumor in the treated group were significantly lower than that in the control group (P < .01). The PCNA index was obviously lower in the tumors treated group than that in the control group (P < .01). In addition, cell apoptosis was observed in xenotransplants of the treated group but not in the control group. Conclusion: Peritumor injection of Ad-TFPI-2 can inhibit growth of LSCC in nude mice model, and TFPI-2 might be a desirable gene for gene therapy in LSCC.

To investigate the inhibitory effects of adenovirus transduced TFPI-2 gene on the growth of laryngeal squamous carcinoma.Recombinant adenoviruses carrying human TFPI-2 gene were amplified and identified. The nude mouse model of laryngeal squamous carcinoma was established by intracutaneous injection of Hep-2 cells. Mice in the treated group were injected with recombinant adenoviruses with Ad-TFPI-2 (adenoviruses-TFPI-2) in peritumor tissue while mice in control group were injected with equivalent null plasmids. After treatment, the tumor weight and volume of tumor in each mouse were measured respectively. The morphological changes of tumor cells were observed using transmission electron microscope and proliferating cell nuclear antigen (PCNA) expression was examined using immunohistochemistry.The Ad-TFPI-2 virus titer was 2.8 x 10(12) PFU/ml after amplification. The average tumor weight and volume in Ad-TFPI-2 treated group were (1.20 +/- 0.34) cm3 and the volume (1.52 +/- 0.39) g, which were significantly lower than the tumor weight (2. 08 +/- 0.52) cm3 and (2.67 +/- 0.47) g in the control group (P0. 01). Apoptosis was observed in the tumors of Ad-TFPI-2 treated group. The PCNA index in Ad-TFPI-2 group was (54.9% +/- 12.4%), which was obviously lower than that (75.8% +/- 11.2%)in control group (P0.01).Peritumor injection of Ad-TFPI-2 can inhibit the growth of laryngeal squamous carcinoma in nude mouse model.

To evaluate the inhibitory effect of endostatin on tumor growth of human laryngeal squamous carcinoma in nude mice and to explore the possible mechanism of the inhibition and the possible way of biological therapy.Nude mice model bearing laryngocarcinoma was established by using human laryngeal squamous carcinoma cell line ( Hep-II). The animals were given endostatin (20 mg x kg(-1) x d(-1)) or PBS, for 21 consecutive days. The volumes of the subcutaneous tumor were observed. The microstructure in which the general 2-step immuohistochemical examination was adopted and ultra-microstructural changes of carcinoma after administration of endostatin were observed under light and electron microscopes for pathology examination.The differences were statistically significant for the net mice weight, tumor weight, and tumor volume and weight/net mice weight between the treatment group and the control group. The restrained percentage of tumor was 45.9%. The necrosis and apoptosis of the tumor cell and the angiogenesis reduction were found under light and electron microscope in the treatment group. The expression of MVD, PCNA and VEGF of the treatment group is lower than that of the control group, and T test showed that P0.01, P0.05, P0.05 respectively, the differences were statistically significant.These studies showed that endostatin could significantly restrain the development of laryngocarcinoma. The mechanism may be due to the effect of antiangiogesis.

Matrilysin (matrix metalloproteinases-7, MMP-7) might play an important role in invasion and metastasis of tumor and is thought to be related to prognosis of many kinds of carcinomas. This study was designed to investigate the significance of MMP-7 expression in the invasion and metastasis of laryngeal cancer and the relationship of MMP-7 expression with the prognosis of laryngeal cancer.The expression of MMP-7 in 70 samples with paraffin-embedded laryngeal cancer and corresponding para-tumor normal mucosas were using immunohistochemical technique. The mRNA expression of MMP-7 were examined by reverse transcription-polymerase chain reaction (RT-PCR) in 24 frozen specimens of laryngeal cancer and corresponding para-tumor normal mucosas.(1) The immunohistochemical staining of MMP-7 was observed in 77.1% (54/70) of cancer tissues, which was significantly higher than that (5.7%, 4/70) of para-tumor normal mucosas (P0.01). The mRNA expression of MMP-7 was detected in 75.0% (18/24) of cancer tissues, which was also significantly higher than that (4.2%, 1/24) of para-tumor normal mucosas (P0.01). (2) The expression of MMP-7 in protein level was hot only higher in T3-T4 group than that in T1-T2 group (P0.01) but also higher in nodal metastases group than that in group without nodal metastases (P0.01). (3) The mRNA expression of MMP-7 was significantly higher in T3-T4 group than that in T1-T2 group (2.908 +/- 0.891 vs.1.662 +/- 0.508, P0.01) and was significantly higher in nodal metastases group than that in group without nodal metastases (2.958 +/- 0.827 vs. 1.887 +/- 0.769,P0.01). (4) Kaplan-Meier survival curve showed that the prognosis of group with MMP-7 protein high expression was poorer than that in group of MMP-7 protein weak or negative expression (log-rank=4.7559, 8.9513; P=0.0292, 0.0028). No significant difference was found between group with MMP-7 protein weak expression and group with that negative expression (log-rank=0.0314, P= 0.8593).MMP-7 possesses close relationship with the invasion, metastasis, and prognosis of laryngeal cancer, and it may be served as a marker in estimating the invasive and metastatic potency and prognosis of laryngeal cancer.

To study the laryngeal function reconstruction with sternohyvoid muscle after partial laryngectomy to treat T2 phase of glottis cancer.66 patients diagnosed to be at T2 phase of glottis cancer were treated by partial laryngectomy from 1992 to 1998. At the same time, they were rebuilt vocal cords with sternohyvoid muscle.All patients were decannulated from 2 to 3 weeks. They renewed own respiration, swallow and voice function. Overall three-year survival rate was 97.0% and five-year survival rate was 93.8%.New operation is superior than traditional one in improving survival rate and life quality.