Serine phosphorylation-dependent coregulation of topoisomerase I by the p14ARF tumor suppressor

p14ARF (ARF) and topoisomerase I play central roles in cancer and have recently been shown to interact. The interaction activates topoisomerase I, an important target for camptothecin-like chemotherapeutic drugs, but the regulation of the interaction is poorly understood. We have used the H358 and H23 lung cancer cell lines and purified recombinant human topoisomerase I to demonstrate that the ARF/topoisomerase I interaction is regulated by topoisomerase I serine phosphorylation, a modification that regulates topoisomerase I activity. Both cell lines express wild-type ARF and topoisomerase I proteins at equivalent levels, but H23 topoisomerase I, unlike that of H358 cells, is largely devoid of serine phosphorylation, has low activity, and complexes poorly with ARF. The ability of H23 topoisomerase I to complex with ARF can be restored by treatment with the serine kinase, casein kinase II. Consistent with these observations, we show that the response of H23 cells to camptothecin treatment is unaffected by changes in intracellular levels of ARF. However, in H358 and PC-3 cells, which express a serine phosphorylated topoisomerase I that complexes with ARF, ectopic overexpression of ARF causes sensitization to camptothecin, and siRNA-mediated down-regulation of endogenous ARF causes desensitization to camptothecin. These biological responses correlate with increased and decreased levels, respectively, of ARF/topoisomerase I complex and DNA-bound topoisomerase I. Thus, ARF is a serine phosphorylation-dependent coregulator of topoisomerase I in vivo, and it regulates cellular sensitivity to camptothecin by interacting with topoisomerase I. Certain cancer associated defects affecting ARF/topoisomerase I complex formation could contribute to cellular resistance to camptothecin.     

Activation of MAPK by potassium bisperoxo(1,10-phenanthroline)oxovanadate (V).

Potassium bisperoxo(1,10-phenantroline)oxovanadate (V) [bpV(phen)] is a potent protein tyrocine phosphatase inhibitor which mediates a variety of biological effects. The aim of these studies was to examine the role(s) of mitogen activated protein kinase (MAPK) pathways in PC12 cell proliferation and toxicity by bpV(phen). BpV(phen) exerts a bimodal effect in PC12 cells: proliferation at low and cell death at higher micromolar concentrations. Activation of MAPK by bpV(phen) depends on time and concentration. The phosphorylation pattern of extracellular regulated kinases (ERK 1/2), c-jun N-terminal activated kinases (JNK) and p38 in PC12 cells is strikingly different. Activation of JNK is sustained in PC12 cells. In contrast, ERK 1/2 activation is transient and treatment with PD98059 indicates that ERK activation by bpV(phen) is partly independent from the ras-MEK pathway. Stability studies of bpV(phen) in DMEM and PBS showed linear relationship with T1/2 about 6 h and 10 days in DMEM and PBS, respectively. Comparison between the time courses of MAPK activation and kinetics of bpV(phen) decomposition as assessed by 51V-NMR analysis show that the initial and maximal phosphorylation signals are produced in the presence of the complex bpV(phen) and not caused by the decomposition products of bpV(phen).     

Studies on the Biomass of Robinia pseudoaeacia Plantation

The average height of 31-aged Robinia pseudoacacia plantation in west mountain of Beijing is 7 in and the mean breastheight diameter of tree is 8.3 cm. The number of tree per hectare appears 1750. The canopy coverage of plantation shows 0.5. Using tile diameter square at tree breast height times tree height (D2H), as an independing variable, to estimate the dry weight of various parts of trees, between them the significient correlations occur. According to the modle of Wstem=58.88(D2H)0.88 (WBranch)=e7.77+0.001(D2H) (Wleaf)=e5.71+0.0008(D2H) (Wroot)＝e7.67+0.0009(D2H) 28.45 t/ha of the stem biomass, 11.6 t/ha of the branch biomass. 0.97 t/ha of the leaf biomass, 7.6 t/ha of the root biomass are estimated in arbor layer. The aboveground and belowground biomass of shrub and herb layers axe 13.71 t/ha and 10.72 t/ha respectively in September, the biomass of shrub and herb layer is 24.43 t/ha. 73.05 t/ha of the total biomass in Robinia pseudoacacia plantation is obtained.     

迁人江淮稻区褐稻虱生物型跟踪监测及分析

该文对近年来褐稻虱迁入虫源的性质进行了探讨,结果表明迁入的虫源在鉴别品种Mudgo上的致害力,生存率、取食量和原始的种群生物型I没有明显的差别;两者在酯酶同工酶谱带数目和RF值也没有差异。迁入的虫源在田间抗性品种上明显表现虫量少。因此,迁入江淮稻区虫源仍是种群生物型I。人工在Mudgo上诱导产生的生物型1再回到感性品种上连续饲养8代,其对Mudgo的致害性降为1.O级,表明了褐稻虱生物型的不稳定性。     

The complete structure of the chloroplast 70S ribosome in complex with translation factor pY

Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo‐EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid‐specific ribosomal proteins. These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid‐specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light‐ and temperature‐dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A‐site and P‐site, where it protects the decoding centre and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non‐rotated state, in which the intersubunit bridges to the large subunit are stabilized.     

Conformation-activity relationship on novel 4-pyridylmethylthio derivatives with antiangiogenic activity

We found 4-pyridylmethylthio derivative 1 to be very effective in using antiangiogenesis activity to prevent proliferation of HUVECs (Human Umbilical Vein Endothelial Cells), which was induced by vascular endothelial growth factor (VEGF). Compound 1 was equally effective in inhibiting VEGF receptor2 tyrosine kinase (KDR, IC₅₀ = 26 nM). We deduced that the inhibition was the result of binding the catalytic domain of VEGF receptor2 tyrosine kinase in a similar fashion to both phthalazine derivative PTK787 2 and anthranylamide derivative AAL993 3. In this report, we will describe the conformational analyses, from ab initio MO calculation and X-ray crystallographic analyses, of compound 1 and the analogs, which include non-active 9, all in comparison with 2 and 3. The conformation–activity relationships suggest that a nonbonded intramolecular interaction between the sulfur and the carbonyl oxygen of 1 was very important in inhibiting KDR.     

Synthesis and biological evaluation of N-mercaptoacylproline and N-mercaptoacylthiazolidine-4-carboxylic acid derivatives as leukotriene A4 hydrolase inhibitors

We studied the synthetic modification of structurally similar N-mercaptoacyl-L-proline and (4R)-N-mercaptoacylthiazolidine-4-carboxylic acid to obtain potent leukotriene A(4) (LTA(4)) hydrolase inhibitors. An N-mercaptoacyl group, (2S)-3-mercapto-2-methylpropionyl group, was effective for both scaffolds. Additional introduction of a large substituent such as 4-isopropylbenzylthio (3f), 4-tert-butylbenzylthio (3l) or 4-cyclohexylbenzylthio group (3m) with (S)-configuration at the C(4) position of proline yielded much more potent LTA(4) hydrolase inhibitors (IC(50); 52, 31, and 34 nM, respectively) than captopril (IC(50); 630,000 nM).     

Microbial communities in oligotrophic Lake Toya,Japan

 The vertical distributions of planktonic bacteria, chroococcoid cyanobacteria, and heterotrophic nanoflagellates (HNF) were examined in Lake Toya, an oligotrophic lake located in Hokkaido, the northern island of Japan, at monthly intervals from May 1993 to May 1994. The abundances of bacteria, cyanobacteria, and HNF during the study period ranged from 10⁴ to 10⁵,10² to 10⁴, 10 to 10² cells ml⁻¹, respectively. The range of bacterial abundances was among the lowest previously reported from other oligotrophic lakes. The vertical distributions of both bacteria and cyanobacteria were influenced by thermal stratification: they were homogeneous throughout the water column during the circulation period (January to April) and heterogeneous during the period of stratification (June to November). During the period of stratification, the cyanobacterial abundance decreased toward the surface in the euphotic zone while the frequency of diving cells (FDC) increased, suggesting that grazing pressure was high near the surface. This hypothesis was supported by the relatively high abundance of HNF at the surface and the negative correlation between HNF and cyanobacterial abundances in the euphotic zone (r = −0.503, n = 33, P < 0.05). On the other hand, multiple regression analysis revealed that 52% of the variation in bacterial abundance in the stratified period can be explained by chlorophyll a concentration, water temperature, and HNF abundance (df = 3, 45; F = 16.2; P < 0.01), suggesting that both substrate limitation and grazing loss by HNF were important factors controlling bacterial abundance in the lake. Received: June 21, 2002 / Accepted: October 16, 2002 Present address: 5-2-2-18-805 Kikusui-motomachi, Shiroishi, Sapporo 003-0825, Japan Present address: School of Environmental Science, University of Shiga Prefecture, 2500 Hassaka-cho, Hikone 522-8533, Japan Tel. +81-749-28-8307; Fax +81-749-28-8463 e-mail: ban@ses.usp.ac.jp Acknowledgments We thank Dr. H. Ueda for encouraging this study and Mr. Haruna and the members of the Plankton Laboratory, Hokkaido University, for their help in sampling. We also thank two anonymous reviewers for their critical comments. Correspondence to:S. Ban