Dynamics of strand passage catalyzed by topoisomerase II

DNA topoisomerase II is a homodimeric molecular machine that uses ATP hydrolysis to untangle DNA by passing one double-stranded DNA duplex (T-segment) through another double-stranded duplex (G-segment). However, despite extensive studies, the dynamics of ATP-dependent T-transport is still not very clear. Here, based on the proposal that transport of the T-segment through the transiently cleaved G-segment and the opened C-gate of the enzyme is via a free diffusion mechanism, the dynamics of T-transport are studied theoretically. Our results show that, to complete passage of the strand with nearly 100% efficiency, the C-gate is required to open by a width that is only slightly larger than the width of DNA duplex and for a time shorter than 100 μs in the presence of several k _B T binding affinities of the T-segment for the B′ domains. The results are implied by our understanding of the opening and closing dynamics of the C-gate. Moreover, the dependence of chemomechanical coupling efficiency on degrees of DNA supercoiling by gyrases can also be explained by using our results. On the basis of these theoretical results and previous experimental data, a modified two-gate model for chemomechanical coupling of the topoisomerase II enzyme is proposed.     

Contrasting leaf characteristics of trees and lianas in secondary and mature forests in southwestern China

We compared variation in sun-canopy leaf anatomy, morphology and photosynthetic rates of coexisting woody species (trees and lianas) in an 8-year-old secondary forest (SF) and mature forest (MF) in the wet season in Xishuangbanna, SW China. Variability of leaf traits of 66 species within growth-form groups in each forest was quantified using coefficients of variation (CV). For the mean values, the woody species in the SF had significantly higher leaf thickness and stomatal density, but lower nonmesophyll/mesophyll ratios than those in the MF. The average leaf area and leaf mass area (LMA) in the studied woody species did not change greatly during the successional process, but differed significantly between the growth forms, with trees having higher values than lianas. The light-saturated photosynthetic rate per unit leaf area (A _a) of the woody species in the SF ranged from 11.2 to 34.5 μmol m⁻² s⁻¹, similarly to pioneer tree species from literature data in southeast Asia. The A _a and photosynthetic nitrogen-use efficiency (PNUE) were significantly higher than those in the MF; whereas A _a in the MF ranged between 9 to 21 μmol m⁻² s⁻¹, with similar values between lianas and trees. For all woody species in both SF and MF, there were no significant differences in the average values of the CV of all measured variables for both lianas and trees. However, considerable variation in leaf anatomy, morphology, and photosynthetic rates within both growth forms and forests existed, as well as substantial variation in leaf size and stomatal density. We concluded that the tropical woody species formed a heterogeneous functional group in terms of leaf morphology and physiology in both secondary and mature forests.     

3-Hydroxyflavone inhibits endogenous Aurora B and induces growth inhibition of cancer cell line

The Aurora kinases play a critical role in mitosis and have been suggested as promising targets for cancer therapy due to their frequent overexpression in a variety of tumors. Compared with established inhibitors of cell division such as the anti-tubulins, novel agents target mitotic enzymes and show similar efficacy but with fewer side effects. Several small-molecule inhibitors of Aurora kinases have been developed as anticancer agents, some of which have progressed to early clinical evaluation. Here we identified 3-hydroxyflavone as a novel Aurora B inhibitor through high throughput screening. 3-Hydroxyflavone showed potent inhibition to Aurora B with the IC₅₀ on a nanomolar basis in the enzyme-based kinase activity assay. In the cell-based western blotting analysis, 3-hydroxyflavone dramatically decreased the phosphorylation level of Histone H3 on the site of serine 10, demonstrating the potent endogenous Aurora B activity inhibition in cell level. The followed cell image analysis provided the consist result. To make it clear whether 3-hydroxyflavone inhibited Aurora B by direct binding or not, SPR analysis was carried out to measure the affinity of interaction between Aurora B protein and 3-hydroxyflavone and the result proved the binding with high affinity. Usually Aurora activity suppression induced cancer cell proliferation inhibition. Colony formation and cell viability with/without treatment of 3-hydroxyflavone were measured using CCK-8. The growth suppression under 3-hydroxyflavone present and the growth recovery after being released gave strong evidence that presence of 3-hydroxyflavone efficiently inhibited the fast growth of cancer cells.     

Identification and characterization of adipose triglyceride lipase (ATGL) gene in birds

Adipose triglyceride lipase (ATGL) is a triglyceride hydrolysis lipase and is generally related to lipid metabolism in animals. The ATGL gene was well studied in mammals, however very less was known in birds that differed significantly with mammals for lipid metabolism. In this study, cloning, mRNA real time and association analysis was performed to characterize the ATGL gene in birds. Results showed that the obtained ATGL gene cDNA of parrot, quail, duck were 1,651 bp (NCBI accession number: GQ221784), 1,557 bp (NCBI accession number: GQ221783) and 1,440 bp each, encoded 481-, 482- and 279-amino acid (AA) peptide, respectively. The parrot ATGL (pATGL) gene was found to predominantly express in breast muscle and leg muscle, and very higher ATGL mRNA level was also found in heart, abdominal fat and subcutaneous fat. The quail ATGL (qATGL) gene was also predominantly expressed in breast muscle and leg muscle, and then to a much lesser degree in heart. The duck ATGL (dATGL) gene was found to predominantly express in subcutaneous fat and abdominal fat, quite higher ATGL mRNA was also found in heart, spleen, breast muscle and leg muscle. Blast analyses indicated the high homology of ATGL and its patatin region, and moreover, and the active serine hydrolase motif (“GASAG” for “GXSXG”) and the glycine rich motif (“GCGFLG” for “GXGXXG”) were completely conservative among 14 species. Association analyses showed that c.950+24C>A, c.950+45C>G, c.950+73G>A, c.950+83C>T and c.950+128delA of chicken ATGL gene (cATGL) were all significantly or highly significantly with cingulated fat width (CFW) (P < 0.05 or P < 0.01), and c.777−26C>A, c.950+45C>G, c.950+73G>A and c.950+118C>T were all significantly or highly significantly with pH value of breast muscle (BMPH) (P < 0.05).     

Generation and analysis of ESTs from the grass carp, Ctenopharyngodon idellus

Grass carp, Ctenopharyngodon idellus (Valenciennes, 1844), is an economically important species widely cultured in the world, but its genome research resources are largely lacking. The objectives of this study were to construct normalized cDNA libraries for efficient EST analysis, to generate ESTs from these libraries, and to identify EST-related molecular markers such as microsatellites and single nucleotide polymorphisms (SNPs) for genetic analysis of this species. A total of 6,269 ESTs were generated representing 4,815 unique sequences, from which 105 putative microsatellites and 5,228 SNPs were identified. These genome resources provide the material basis for future genetic and functional analyses in this species.     

Matched pairs of human prostate stromal cells display differential tropic effects on LNCaP prostate cancer cells

Prostate stromal cells may play binary roles in the process of prostate cancer development. As the first to be encountered by infiltrating prostate cancer cells, prostate stromal cells form the first defense line against prostate cancer progression and metastasis. However, interaction between prostate cancer and stromal cells may facilitate the formation of a tumor microenvironment favoring cancer cell growth and survival. To establish an experimental system for studying the interaction between cancer and stromal cells, we isolated three matched pairs of normal and cancer-associated human prostate stromal clones. In this report, we describe the morphologic and behavioral characteristics of these cells and their effect on LNCaP prostate cancer cells in co-culture. Unlike LNCaP prostate cancer cells, the isolated prostate stromal clones are large fibroblast-like cells with a slow proliferation rate. Growth and survival of these clones are not affected by androgens. The stromal cells display high resistance to serum starvation, while cancer-associated stromal clones have differentiated survival ability. In co-culture experiments, the stromal cells protected some LNCaP prostate cancer cells from death by serum starvation, and cancer-associated stromal clones showed more protection. This work thus established a panel of valuable human prostate stromal cell lines, which could be used in co-culture to study the interaction between prostate cancer and prostate stromal cells.     

Improvement of Cephalosporin C Production by Recombinant DNA Integration in <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>

Cephalosporins are widely used as anti-infectious β-lactam antibiotics in clinic. For the purpose of increasing the yield of cephalosporin C (CPC) fermentation, especially in an industrial strain, A. chrysogenum genes cefEF and cefG, which encode the ultimate and penultimate steps in CPC biosynthesis, cefT, which encodes a CPC efflux pump, and vgb, which encodes a bacterial hemoglobin gene were transformed in various combinations into an industrial strain of A. chrysogenum. Both PCR and Southern blotting indicated that the introduced genes were integrated into the chromosome of A. chrysogenum. Carbon monoxide difference spectrum absorbance assay was performed and the result showed that Vitreoscilla hemoglobin was successfully expressed in A. chrysogenum and had biological activity. HPLC analysis of fermentation broth of recombinant A. chrysogenum showed that most transformants had a higher CPC production level than the parental strain. Multiple transformants containing an additional copy of cefG showed a significant increase in CPC production. However, cefT showed little effect on CPC production in this high producer. The highest improvement of CPC titer was observed in the mutant with an extra copy of cefG + cefEF + vgb whose CPC production was increased by 116.3%. This was the first report that three or more genes were introduced simultaneously into A. chrysogenum. Our results also demonstrated that the combination of these genes had a synergy effect in a CPC high producer.