New Thymol Derivatives and Cytotoxic Constituents from the Root of Eupatorium cannabinum ssp. asiaticum

Two new thymol (=5‐methyl‐2‐(1‐methylethyl)phenol) derivatives, 8,10‐didehydro‐9‐(3‐methylbutanoyl)thymol 3‐O‐tiglate ( 1 ) and 9‐O‐angeloyl‐8‐methoxythymol 3‐O‐isobutyrate ( 2 ), were isolated from the root of Eupatorium cannabinum ssp. asiaticum, together with six known compounds, 3 – 8 . The structures of 1 and 2 were determined through extensive 1D/2D‐NMR and MS analyses. Among the isolates, 9‐acetoxy‐8,10‐epoxythymol 3‐O‐tiglate ( 3 ) was the most cytotoxic, with IC₅₀ values of 0.02±0.01, 1.02±0.07, and 1.36±0.12 μg/ml, respectively, against DLD‐1, CCRF‐CEM, and HL‐60 cell lines. In addition, 10‐acetoxy‐9‐O‐angeloyl‐8‐hydroxythymol ( 4 ) and eupatobenzofuran ( 6 ) exhibited cytotoxicities, with IC₅₀ values of 1.14±0.16 and 2.63±0.22, and 7.63±0.94 and 2.31±0.14 μg/ml, respectively, against DLD‐1 and CCRF‐CEM cell lines.     

Serum Iron Concentration,but Not Hemoglobin,Correlates with TIMI Risk Score and 6-Month Left Ventricular Performance after Primary Angioplasty for Acute Myocardial Infarction

Objective

Anemia is associated with high mortality and poor prognosis after acute coronary syndrome (ACS). Increased red cell distribution width (RDW) is a strong independent predictor for adverse outcomes in ACS. The common underlying mechanism for anemia and increased RDW value is iron deficiency. It is not clear whether serum iron deficiency without anemia affects left ventricular (LV) performance after primary angioplasty for acute myocardial infarction (AMI). We investigated the prognostic value of serum iron concentration on LV ejection fraction (EF) at 6 months and its relationship to thrombolysis in myocardial infarction (TIMI) risk score in post MI patients.

Methods

We recruited 55 patients who were scheduled to undergo primary coronary balloon angioplasty after AMI and 54 age- and sex-matched volunteers. Serum iron concentration and interleukin-6 levels were measured before primary angioplasty. LVEF was measured by echocardiography at baseline and after 6 months. TIMI risk score was calculated for risk stratification.

Results

Serum iron concentration was significantly lower in those in whom LVEF had not improved ≥10% from baseline (52.7±24.1 versus 80.8±50.8 µg/dl, P = 0.016) regardless of hemoglobin level, and was significantly lower in the AMI group than in the control group (62.5±37.7 versus 103.0±38.1 µg/dl, P<0.001). Trend analysis revealed that serum iron concentration decreased as TIMI risk score increased (P = 0.002). In addition, lower serum iron concentrations were associated with higher levels of inflammatory markers. Multiple linear regression showed that baseline serum iron concentration can predict LV systolic function 6 months after primary angioplasty for AMI even after adjusting for traditional prognostic factors.

Conclusion

Hypoferremia is not only a marker of inflammation but also a potential prognostic factor for LV systolic function after revascularization therapy for AMI, and may be a novel biomarker for therapeutic intervention.     

Inactivation of Cdc13p triggers MEC1-dependent apoptotic signals in yeast

Inactivation of the budding yeast telomere binding protein Cdc13 results in abnormal telomeres (exposed long G-strands) and activation of the DNA damage checkpoint. In the current study, we show that inactivation of Cdc13p induces apoptotic signals in yeast, as evidenced by caspase activation, increased reactive oxygen species production, and flipping of phosphatidylserine in the cytoplasmic membrane. These apoptotic signals were suppressed in a mitochondrial (rho(o)) mutant. Moreover, mitochondrial proteins (e.g. MTCO3) were identified as multicopy suppressors of cdc13-1, suggesting the involvement of mitochondrial functions in telomere-initiated apoptotic signaling. These telomere-initiated apoptotic signals were also shown to depend on MEC1, but not TEL1, and were antagonized by MRE11. Our results are consistent with a model in which single-stranded G-tails in the cdc13-1 mutant trigger MEC1-dependent apoptotic signaling in yeast.     

Cloning of Amb a I (antigen E), the major allergen family of short ragweed pollen.

To determine the structure of Amb a I (previously called antigen E), the major allergen from short ragweed, cDNA from pollen was cloned into lambda gt11 and lambda gt10. One of the three distinct clones isolated from the lambda gt11 library by screening with anti-denatured Amb a I antibodies was used to screen both libraries for other Amb a I sequences. Multiple clones were isolated and sequenced and proved to be highly homologous but nonidentical. The clones could be divided into three groups based on sequence similarity, and in accordance with the International Union of Immunological Societies-approved nomenclature (Marsh, D. G., Goodfriend, L., King, T. P., Lowenstein, H., and Platts-Mills, T. A. E. (1986) Bull. WHO 64, 767-770) they have been designated Amb a I.1, Amb a I.2, and Amb a I.3. Clones within a group have greater than 99% identity, and similarity among groups is 85-90% at the nucleotide level. The amino acid sequence of four peptides (isolated from antigen E obtained from the Research Resources Branch of the National Institutes of Health) containing 132 amino acids was identical to one of the clones (Amb a I.1). The presence of multiple naturally occurring isoelectric forms of Amb a I was demonstrated by two-dimensional gel electrophoresis and Western blotting. Southern blot analysis demonstrates the presence of multiple Amb a I-related sequences in the ragweed genome. Amb a I is therefore not a single molecule but rather a family of closely related proteins.     

Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2

Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).     

The Preliminary Studies on Culture of Unfertilized Ovary of Rice in Vitro

The present paper reports the results of the culture of unfertilized ovaries of rice in vitro. The inducting medium was N6 supplemented with 2 mg/L 2,4-D, 500 mg/L casien hydrolysate and sucrose was 4%. The differentiated medium was N6 supplemented with 2 mg/L Kinetin, 500 mg/L casein hydrolysate and the concentration of the sucrose was 3%. The 4 cultivars and 2 crossed combinations were used as the experimental materials. The experiments were shown the differentiation of the callus occurred amony various cultivars. The induced frequency in the crossed combinations was higher than that in the cultivars. Now 12 green plants and 3 albino plantlets have been obtained. The chromasomes of 11 green plantlets have been examined. Among them, 6 plantlets were haploid (n =12 ) and 5 plantlets were diploid. The embryoids were located in the micropylar end. Some of them possessed the suspensor, similar as zygote embryos. The callus was found from different origin. One of them was originated from haploid tissue derived from the nuclear in the embryo sac. Another was originated from the diploid tissue in the integument or ovary wall. The origin of the callus from the unfertilized ovary was discussed.     

Cell cycle G2/M arrest and activation of cyclin-dependent kinases associated with low-dose paclitaxel-induced sub-G1 apoptosis

Paclitaxel is a potential anti-cancer agent for several malignancies including ovary, breast, and head and neck cancers. This study investigated the kinetics of paclitaxel-induced cell cycle perturbation in two human nasopharyngeal carcinoma (NPC) cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with higher concentrations (0.1 or 1 μM) of paclitaxel showed obvious G2/M arrest and then converted to a cell population with reduced DNA content, which was detected as a sub-G2 peak in the flow cytometric histographs. If a low concentration (5 nM) of paclitaxel was used instead, transient G2/M arrest was observed in NPC cells, which subsequently converted to a sub-G1 form during the treatment period. Internucleosomal fragmentation and chromatin condensation were detectable in these sub-G1 and sub-G2 cells, suggesting that persistent or transient G2/M arrest is a prerequisite step for apoptosis elicited by varying doses of paclitaxel. The levels of cyclins A, B1, D1, E, CDK 1 (CDC 2), CDK 2 and proliferating cell nuclear antigen (PCNA) were unchanged in NPC cells following treatment with any concentration of paclitaxel; however, apoptosis-related cyclin B1-associated CDC 2 kinase was highly activated by paclitaxel even at concentrations as low as 5 nM, which is consistent with the finding that low-dose paclitaxel is also able to induce apoptosis in NPC cells. Activation of cyclin B1-associated CDC 2 kinase seems to be an important G2/M event required for paclitaxel-induced apoptosis, and this activation of cyclin B1/CDC 2 kinase could be attributed to the increased activity of CDK 7 kinase. This revised version was published online in November 2006 with corrections to the Cover Date.     

MicroRNA-33a functions as a bone metastasis suppressor in lung cancer by targeting parathyroid hormone related protein

Background

Bone is a common site of metastasis for lung cancer, and is associated with significant morbidity and a dismal prognosis. MicroRNAs (miRNAs) are increasingly implicated in regulating the progression of malignancies.

Methods

The efficacy of miR-33a or anti-miR-33a plasmid was assessed by Real-time PCR. Luciferase assays were using One-Glo Luciferase Assay System. Measurement of secreted factors was determined by ELISA kit.

Results

We have found that miR-33a, which is downregulated in lung cancer cells, directly targets PTHrP (parathyroid hormone-related protein), a potent stimulator of osteoclastic bone resorption, leading to decreased osteolytic bone metastasis. We also found that miR-33a levels are inversely correlated with PTHrP expression between human normal bronchial cell line and lung cancer cell lines. The reintroduction of miR-33a reduces the stimulatory effect of A549 on the production of osteoclastogenesis activator RANKL (receptor activator of nuclear factor kappa-B ligand) and M-CSF (macrophage colony-stimulating factor) on osteoblasts, while the expression of PTHrP is decreased in A549 cells. miR-33a overexpression also reduces the inhibitory activity of A549 on the production of OPG (osteoprotegerin), an osteoclastogenesis inhibitor. In addition, miR-33a-mediated PTHrP downregulation results in decreased IL-8 secretion in A549, which contributes to decreased lung cancer-mediated osteoclast differentiation and bone resorption.

Conclusions

These findings have led us to conclude that miR-33a may be a potent tumor suppressor, which inhibits direct and indirect osteoclastogenesis through repression of PTHrP.

General significance

miR-33a may even predict a poor prognosis for lung cancer patients.