Phloroglucinol Derivatives from the Fruits of Eucalyptus globulus and Their Cytotoxic Activities.

A new phloroglucinol derivative, named eucalyptin B (1), along with five related known compounds (2 - 6), was isolated from the fruits of Eucalyptus globulus. Their structures were elucidated by means of 1D- and 2D-NMR spectroscopy, with the absolute configuration of 1 determined by electronic circular dichroism (ECD) calculations. All isolated compounds (1 - 6) were evaluated for their cytotoxic activities against lung (A549), breast (4T1), and skin (B16F10) cancer cell lines. On the basis of cell viability assay, the cytotoxic activity of eucalyptin B (1) was further confirmed by apoptosis assay. Additionally, after treatment with eucalyptin B (1), the apoptosis factor proteins (Bcl2 and Bax) and caspase-3 levels in A549 cells were also determined by Western-blot analysis. By cytotoxic assay, eucalyptin B (1) exhibited potent cytotoxicity against A549 cells with an IC50 value of 1.51 µm and induced concentration dependent apoptosis of up to 49%. Additionally, eucalyptin B (1) inhibited 5-fold and increased 10-folds in the level of Bcl2 and Bax, respectively. Furthermore, the 11-fold increase in the level of caspase-3 confirmed eucalyptin B (1) activated caspase dependent apoptosis pathway. In conclusion, the isolated compound eucalyptin B (1) has promising cytotoxic activity in tumor cells, especially in A549.

Differential Immune Responses and Protective Effects in Avirulent Mycobacterial Strains Vaccinated BALB/c Mice.

Screening live mycobacterial vaccine candidates is the important strategy to develop new vaccines against adult tuberculosis (TB). In this study, the immunogenicity and protective efficacy of several avirulent mycobacterial strains including Mycobacterium smegmatis, M. vaccae, M. terrae, M. phlei, M. trivial, and M. tuberculosis H37Ra were compared with M. bovis BCG in BALB/c mice. Our results demonstrated that differential immune responses were induced in different mycobacterial species vaccinated mice. As BCG-vaccinated mice did, M. terrae immunization resulted in Th1-type responses in the lung, as well as splenocytes secreting IFN-γ against a highly conserved mycobacterial antigen Ag85A. M. smegmatis also induced the same splenocytes secreting IFN-γ as BCG and M. terrae did. In addition, M. terrae and M. smegmatis-immunized mice predominantly increased expression of IL-10 and TGF-ß in the lung. Most importantly, mice vaccinated with H37Ra and M. vaccae could provide the same protection in the lung against virulent M. tuberculosis challenge as BCG. The result may have important implications in developing adult TB vaccine.

EB1 promotes Aurora-B kinase activity through blocking its inactivation by protein phosphatase 2A.

EB1 (end-binding protein 1) is a key player in the regulation of microtubule dynamics. In concert with its binding partners, adenomatous polyposis coli and p150(glued), EB1 plays a crucial role in a variety of microtubule-based cellular processes. In this study we have identified in a yeast two-hybrid screen the mitotic kinase and chromosome passenger protein Aurora-B as a binding partner of EB1. GST pull-down and immunoprecipitation experiments reveal a specific interaction between Aurora-B and EB1 both in cells and in vitro. Immunofluorescence microscopy shows that these two proteins colocalize on the central spindle in anaphase and in the midbody during cytokinesis. Kinase assays using both immunoprecipitated and purified Aurora-B demonstrate that EB1 is not a substrate of Aurora-B. Rather, EB1 positively regulates Aurora-B kinase activity. EB1 overexpression remarkably enhances Aurora-B activity and knockdown of its expression impairs Aurora-B activity. Our data further show that EB1 is able to protect Aurora-B from dephosphorylation/inactivation by protein phosphatase 2A (PP2A) by blocking PP2A binding to Aurora-B. These findings establish Aurora-B as an EB1-interacting protein and suggest that EB1 stimulates Aurora-B activity through antagonizing its dephosphorylation/inactivation by PP2A.

[The influence of benign prostatic hyperplasia drugs on incidence and pathology grading of prostate cancer].

OBJECTIVE: To analyze the influence of benign prostatic hyperplasia (BPH) drugs on incidence and pathology grading of prostate cancer in China. METHODS: Retrospectively investigated the history of drug treatment in 1029 cases of BPH in patients from February 1998 to December 2004. According to the history of drug use, the patients were divided into 4 groups: finasteride group, alpha-receptor inhibitor group, finasteride and alpha-receptor inhibitor combination group and control group (untreated group). We gathered pathology sections of patients in all groups, and gave Gleason Score to each. The difference of incidence and pathology grading of prostate cancer were analyzed by Stata 7.0. RESULTS: The incidence of prostate cancer in the population of our study was 13.5%; The incidence in finasteride group, alpha-receptor inhibitor group, combination group and control group was 9.8%, 16.0%, 10.3% and 18.6%, respectively. There was significant difference between the two groups with the use of finasteride and the two groups without it (P < 0.05). In our study, the ratio of middle or high level pathology grading (Gleason ≥ 7) in prostate cancer patients was 58.3%, the ratio of middle or high level pathology grading prostate cancer patients in the four groups was 71.4%, 59.6%, 67.7% and 40.0%, respectively. In the comparison of composition ratio of middle or high level prostate cancer, there was significant difference between the two groups with the use of finasteride and the two groups without it (P < 0.05). CONCLUSIONS: Finasteride can lower the risk of prostate cancer, but increase the pathology grade of the prostate cancer which has occurred in the same time. The alpha-receptor inhibitor does not have the same effect.

Mefloquine Inhibits Esophageal Squamous Cell Carcinoma Tumor Growth by Inducing Mitochondrial Autophagy.

Esophageal squamous cell carcinoma (ESCC) has a worldwide impact on human health, due to its high incidence and mortality. Therefore, identifying compounds to increase patients' survival rate is urgently needed. Mefloquine (MQ) is an FDA-approved anti-malarial drug, which has been reported to inhibit cellular proliferation in several cancers. However, the anti-tumor activities of the drug have not yet been completely defined. In this study, mass spectrometry was employed to profile proteome changes in ESCC cells after MQ treatment. Sub-cellular localization and gene ontology term enrichment analysis suggested that MQ treatment mainly affect mitochondria. The KEGG pathway enrichment map of down-regulated pathways and Venn diagram indicated that all of the top five down regulated signaling pathways contain four key mitochondrial proteins (succinate dehydrogenase complex subunit C (SDHC), succinate dehydrogenase complex subunit D, mitochondrially encoded cytochrome c oxidase III and NADH: ubiquinone oxidoreductase subunit V3). Meanwhile, mitochondrial autophagy was observed in MQ-treated KYSE150 cells. More importantly, patient-derived xenograft mouse models of ESCC with SDHC high expression were more sensitive to MQ treatment than low SDHC-expressing xenografts. Taken together, mefloquine inhibits ESCC tumor growth by inducing mitochondrial autophagy and SDHC plays a vital role in MQ-induced anti-tumor effect on ESCC.

Memory performance of hypercholesterolemic mice in response to treatment with soy isoflavones.

The aim of this study is to investigate the memory performance of hypercholesterolemic mice in response to soy isoflavones (SI) treatment and the mechanism involved. In this study, 64 mice were randomly divided into four groups: control, high lipid diet without SI, high lipid diet with a low SI level (50 mg/kg bw) and high lipid diet with a high SI level (100 mg/kg bw). The experimental period was 30 days. The results indicated that the mice given the different treatments showed the different percentages of good, medium and poor memory performance. chi(2) analysis revealed significant difference in memory performance (P<0.05) between the high lipid diet without SI group and the high lipid diet with a low SI level group or high lipid diet with a high SI level group. Moreover, SI treatment resulted in a decrease in blood cholesterol (TC) level (high lipid diet without SI group versus high lipid diet with a low SI level group or high lipid diet with a high SI level group, P<0.05) and triglyceride (TG) level (high lipid diet without SI group versus high lipid diet with a low SI level group or high lipid diet with a high SI level group, P<0.05). In addition, SI treatment resulted in a significant decrease in acetylcholinesterase (AChE) activity and significant increases in glutamic acid and aspartic acid contents in the frontal cerebral cortex and hippocampus. The results suggest that SI improve the memory performance of hypercholesterolemic mice, and the mechanism underlying the improvement might closely correlate with its roles in decreasing high blood lipid levels and modulating the metabolism of neurotransmitters such as acetylcholine and amino acids in brain areas of hypercholesterolemic mice.

[Study on macrophages infected with Mycobacterium tuberculosis H37Ra in vitro].

AIM: To study the production of nitric oxide and secretion of cytokines after infection of macrophages with Mycobacterium tuberculosis H37Ra. METHODS: 24 hours after infection of RAW264.7 cells with Mycobacterium tuberculosis H37Ra, the production of NO and H(2);O(2); as well as the secretion levels of IL-12 and TNF-α in the supernatants of culture were determined by Griess method, chemical method and ELISA assay respectively. The expression of IL-12 and TNF-α mRNA in macrophages was detected by reverse transcription polymerase chain reaction (RT- PCR). RESULTS: Macrophages infected with Mycobacterium tuberculosis H37Ra produced effectively more NO, H(2);O(2);, and enhanced the release of IL-12, TNF-α and the expression of IL-12, TNF-α mRNA (P<0.05). CONCLUSION: Mycobacterium tuberculosis H37Ra can induce the production of more nitric oxide and cytokines which play important roles in the host immune response.

Small-molecule inhibition of Aurora kinases triggers spindle checkpoint-independent apoptosis in cancer cells.

Aurora kinases are key regulators of mitotic progression and have also been implicated in tumorigenesis. Small molecules that inhibit Aurora kinases have shown impressive anticancer activity in preclinical studies and are currently under clinical evaluation. In this study, our data show that suppression of Aurora activity with a specific inhibitor prevents the proliferation of breast cancer cells. Molecular modeling studies indicate that the Aurora inhibitor suppresses Aurora activity by competitive displacement of ATP. Mechanistically, the Aurora inhibitor causes the accumulation of multinucleated cells, leading to profound apoptosis in the absence of caspase-3 activity. Further studies show that the sensitivity of cancer cells to the Aurora inhibitor is independent of the spindle checkpoint. In addition, the Aurora inhibitor acts synergistically with the vinca alkaloids but not with the taxanes in inhibiting cell proliferation and inducing apoptosis. These results suggest that Aurora inhibitors might be effective in spindle checkpoint-defective cancer cells and a combination of Aurora inhibitors with the vinca alkaloids is a promising approach for cancer chemotherapy.

[Study on the cytotoxicity of spleen lymphocytes and immune mechanisms in mice immunized by Mycobacterium tuberculosis H37Ra].

AIM: To explore the specific cytotoxicity of spleen lymphocytes and the immune mechanisms in mice immunized by Mycobacterium tuberculosis(MTB) H37Ra. METHODS: At the various interval (30 days, 60 days) after the mice were immunized by MTB H37Ra, BCG and PBS, the spleen lymphocytes of the immunized mice were used as the effector cells while the Sp2/0 cells expressing the secreted protein Ag85B were used as the target cells, and the cytotoxicity of spleen lymphocytes in the immunized mice was measured by MTT assay. Spleen lymphocytes were collected at 60 days after immunization and stimulated with PPD, and the expression of perforin, granzyme B on mRNA level was detected by RT-PCR. RESULTS: The cytotoxicity of spleen lymphocytes in the group immunized by MTB H37Ra was significantly higher than that of PBS control group (P<0.05), and slightly higher than that of BCG group. The mRNA expression of perforin, granzyme B in H37Ra group and BCG group was significantly higher than that in PBS control group (P<0.05); the expression of perforin mRNA in H37Ra group was significantly higher than that in BCG group (P<0.05), but there was no obvious difference with regard to the expression of granzyme B mRNA between H37Ra group and BCG group. CONCLUSION: The cytotoxicity of spleen lymphocytes is enhanced after mice were immunized by MTB H37Ra, which may be related to the expression of perforin and granzyme B.

[Separation of proteins by gradient pressurized capillary electrochromatography].

The separation of four proteins including RNase A, cytochrome C, lysozyme and myoglobin was investigated by reversed-phase gradient pressurized capillary electrochromatography (p-CEC) with 1.5 microm non-porous silica C18 stationary phase. This mode was compared with micro-high performance liquid chromatography (mu-HPLC) and the effects of applied voltage, stationary phase and concentration of ion-pairing agent (trifluoroacetic acid, TFA) on the gradient p-CEC were also studied. This separation was performed rapidly on a new CEC instrument Trisep 2010 GV. The results showed that the retention mechanism of proteins in p-CEC mode is based on both chromatographic partitioning and electrophoretic migration. The results also demonstrated that p-CEC may have great potential for fast and efficient separation of proteins.

Separation of ephedrine stereoisomers by molecularly imprinted polymers--influence of synthetic conditions and mobile phase compositions on the chromatographic performance.

Separation of ephedrine stereoisomers by molecularly imprinted polymers was performed to study the factors that affect the selectivity and column efficiency. The polymer synthesized with pentaerythritol triacrylate as the cross-linker and chloroform as the porogen was found to have the best overall separation performance. Investigation of the recognition mechanism by NMR and chromatographic analysis revealed that the major binding forces between the polymer stationary phase and ephedrine are the ionic and hydrogen bonding interactions. Studies of the influence of mobile phase compositions on the HPLC analysis have shown that a methanol-aqueous buffer was the suitable mobile phase for the separation in which pH, ionic strength and water content can be adjusted to optimize the chromatographic analysis.

[Advances in the chiral resolution of organophosphorus compounds by liquid chromatography].

The developments in the chiral resolution of organophosphorus compounds by liquid chromatography in recent years are reviewed. Two different approaches of separation: indirect resolution (chiral derivatization reagent method) and direct resolution (chiral mobile phase additive and chiral stationary phase), focusing on several chiral stationary phases--Pirkle type, cyclodextrin, cellulose, ligand exchange and other chiral stationary phases are presented. The possible mechanism of chiral recognition is discussed. Eighty-five references are involved.