Dendroecological and genetic insights for future management of an old-planted forest of the endangered Mediterranean fir Abies pinsapo

Pinsapo fir (Abies pinsapo Boiss.) is an endangered Mediterranean conifer that has raised strong conservation interest as a paradigmatic example of species characterized by small and fragmented populations. We studied an old reforestation stand composed of A. pinsapo, Pinus nigra and Pinus sylvestris established in the 1910s in central-eastern Spain (about 500 km north of the species native distribution range), with the aim of evaluating the stand’s suitability as an ex situ conservation unit for the fir. To this end, we investigated the long-term performance of the stand and assessed genetic diversity of A. pinsapo. Tree-ring width (TRW) and carbon isotope discrimination (Δ¹³C) were used to characterise growth dynamics and intrinsic water-use efficiency (WUE_i), respectively. Furthermore, 42 pinsapo firs were genotyped at five microsatellite loci to compare their genetic variation with published data on natural populations. A. pinsapo showed ca. two-fold higher radial growth than pines in the last 80 years; however, a growth decrease was observed for all species from the 1990s onwards. Indexed TRW was positively associated with Δ¹³C at the species level, denoting inter-annual growth dependence on water availability. Overall, Δ¹³C was higher for A. pinsapo compared to pines, indicating lower WUE_i, but Δ¹³C significantly decreased over the last 50 years for all species, likely as the result of tighter stomatal regulation of water loss, resulting in WUE_i increases of about 25 %. Recently, however, A. pinsapo showed reduced WUE_i increase in concord with growth slowdown, suggesting a threshold response for stomatal regulation. Although genetic diversity of A. pinsapo was about half of natural populations, the old-planted stand could be important for the conservation of this endemic species considering its good long-term growth and physiology. The latest decrease in performance of A. pinsapo, however, asks for urgent management measures aimed at reducing the competition for water and promoting growth and natural regeneration. This study illustrates the potential of combining tree-ring-based long-term physiological information with genetic data to ascertain the prospects of artificial stands for conservation purposes.     

Initiation and characterization of a diploid cell line from larval tissues ofAedes dorsalis (Meigen)

Summary Mosquito cell cultures were initiated from the minced tissues of newly hatchedAedes dorsalis (Meigen) larvae. Continuous cell division occurred only after an adaptive period of approximately 6 months. Optimal growth of the cells required a relatively low pH of 6.5. Karyological studies showed that the cells have remained diploid (2n=6) for 60 serial passages and that the cultures are free of contaminating cells. The cultures also were shown to be free of bacteria (includingMycoplasma), fungi and virions. Subpopulations (strains) of the original parental cultures have been selected and characterized on the basis of morphology, karyology, growth rate and monolayer formation. These studies were supported in part by funds from the Office of Naval Research, by Research Grant AI03028 from the National Institute of Allergy and Infectious Diseases, and by General Research Support Grant I-SO1-FR-05441 from the National Institutes of Health, U.S. Department of Health, Education and Welfare.     

ATP enhances cyclic AMP accumulation by intact P388 mouse lymphoma cells

One of the characteristics of malignant cells is a poor response to hormones and a low level of cyclic AMP. Whilst this is true of intact P388 mouse lymphoma cells, high levels of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity can be measured in particulate preparations of these cells. When ATP is added to the incubation medium of intact lymphoma cells, the cyclic AMP level is enhanced. This effect of ATP is not mediated by adenosine, nor is it enhanced by NaF. The ATP content of the lymphoma cells is much lower than that of CH23 Chinese hamster fibroblast and PCM3 hybrid cells, whose cyclic AMP levels are not affected by the presence of ATP. This suggests that adenylate cyclase, in the lymphoma cells, is bathed in a pool which is deficient in substrate. The substrate concentration of this pool is thought to be elevated by addition of ATP to the incubation medium with ATP, itself, crossing the plasma membrane.     

Isolation and characterization of Chinese hamster ribosomal DNA clones

We have examined the ribosomal RNA (rRNA) genes of the Chinese hamster ovary (CHO) cell line. A partial EcoRI library of genomic CHO DNA was prepared using lambda Charon-4A. We isolated two recombinants containing the region transcribed as 45S pre-rRNA and 13 kb of external spacer flanking 5' and 3' to the transcribed region. These sequences show restriction site homology with the vast majority of the genomic sequences complementary to rRNA. In addition to this form of rDNA, Southern blot analysis of EcoRI-cut CHO genomic DNA reveals numerous minor fragments ranging from 2 to 19 kb which are complementary to 18S rRNA. We isolated one clone which contains the 18S rRNA gene and sequences 5' which appear to contain length heterogeneity within the non-transcribed spacer region. We have nine additional cloned EcoRI fragments in which the homology with 18S rRNA is limited to a 0.9-kb EcoRI-HindIII fragment. This EcoRI-HindIII fragment is present in each of the cloned EcoRI fragments, and is flanked on both sides by apparently nonribosomal sequences which bear little restriction site homology with each other or the major cloned rDNA repeat.     

Cytotoxic sesquiterpenoids from Dysoxylum parasiticum (Osbeck) Kosterm. stem bark

Two undescribed sesquiterpenoid derivatives, 10β,11-dihydroxy-1β-hydroperoxide-4αH,5αH,7βH-guaiane (1) and 10β-hydroxy-4α,4β-dimethyl-5αH,7αH-eudesm-3-one (2) along with three known of guaiane, aromadendrane, and cadinane sesquiterpenoids (3-5) were isolated from the stem bark of Dysoxylum parasiticum (Osbeck) Kosterm. The chemical structure of the compounds were determined by detailed analysis of spectroscopic data, including MS, IR, 1D, and 2D NMR as well as through comparison with data of known analogues. The isolated compounds were also tested for cytotoxic properties against MCF-7 breast cancer line. Compound 4, was found to exhibit the strongest cytotoxic activity with an IC₅₀ value of 12.17 μM, while the other compounds showed moderate or no activity.     

油菜双单倍体诱导育种技术研究进展

油菜是食用油、优质饲料蛋白的重要来源,杂种优势利用是油菜培育优势性状最重要的手段,且提高亲本的选育效率对优质品种的培育具有积极的推动作用。现有油菜育种技术存在效率低、周期长、盲目性大、应用范围有限等诸多问题,不适于油菜产业快速发展的需求。双单倍体诱导育种技术是近年来新兴的一种快速选育油菜新品种的技术方法。该技术以操作简便、应用范围广、效率高等优势被广泛应用于油菜新品种的选育过程中。从油菜双单倍体诱导技术创新研究的发现、作用表现、诱导机制、作用价值等方面系统地综述了油菜双单倍体诱导技术的研究进展,展望了油菜双单倍体诱导技术的应用前景,以期为未来油菜双单倍体诱导技术以及其他作物诱导系的研究和利用提供参考。     

在食管癌细胞增殖过程中蛋白酶激活受体-2(PAR-2)对细胞周期蛋白D1(CyclinD1)的调控机制*

目的:探讨在食管癌细胞增殖过程中蛋白酶激活受体-2(PAR-2)影响细胞周期蛋白D1(CyclinD1)的机制。方法:方法:使用食管癌EC109细胞株,实验分为:空白对照组、PAR-2激动组(加入激动剂SLIGKV)、PAR-2反激动组(加入反激动剂VKGILS)、PAR-2 shRNA组(PAR-2shRNA成功转染)和MAPK抑制组(加入阻滞剂PD98059);取对数生长期的细胞,以6×10⁴cells/ml的密度接种于培养瓶中,置于孵育箱中培养24 h后使用PBS清洗3次,更换为无血清的1640培养基培养24 h,之后各实验组按实验设计分别加入所需试剂,每组设置3个复孔,于孵育箱中继续培养24 h,采用RT-PCR法检测PAR-2、ERK1、CyclinD1的mRNA的表达水平;采用Western blot法检测PAR-2、ERK1、p-ERK1、CyclinD1的蛋白表达水平。结果:与空白对照组相比,PAR-2激动组PAR-2mRNA、ERK1 mRNA、和CyclinD1 mRN表达明显升高(P＜0.05),PAR-2、p-ERK1和CyclinD1蛋白表达升高(P＜0.05);PAR-2 shRNA组PAR-2mRNA、ERK1 mRNA和CyclinD1 mRNA表达降低(P＜0.05),PAR-2、p-ERK1和CyclinD1蛋白表达降低(P＜0.05);MAPK抑制组ERK1 mRNA和CyclinD1 mRNA表达明显降低(P＜0.05),p-ERK1和CyclinD1表达降低(P＜0.05)。结论: PAR-2可通过MAPK通路调节CyclinD1的表达从而促进食管癌细胞EC109的增殖。     

Dual inhibitors of RAF-MEK-ERK and PI3K-PDK1-AKT pathways: Design,synthesis and preliminary anticancer activity studies of 3-substituted-5-(phenylamino) indolone derivatives

The dysfunction and mutual compensatory activation of RAF-MEK-ERK and PI3K-PDK1-AKT pathways have been demonstrated as the hallmarks in several primary and recurrent cancers. The strategy of concurrent blocking of these two pathways shows clinical merits on effective cancer therapy, such as combinatory treatments and dual-pathway inhibitors. Herein, we report a novel prototype of dual-pathway inhibitors by means of merging the core structural scaffolds of a MEK1 inhibitor and a PDK1 inhibitor. A library of 43 compounds that categorized into three series (Series I–III) was synthesized and tested for antitumor activity in lung cancer cells. The results from structure-activity relationship (SAR) analysis showed the following order of antitumor activity that 3-hydroxy-5-(phenylamino) indolone (Series III)?>?3-alkenyl-5-(phenylamino) indolone (Series I)?>?3-alkyl-5-(phenylamino) indolone (Series II). A lead compound 9za in Series III showed most potent antitumor activity with IC₅₀ value of 1.8?±?0.8?µM in A549 cells. Moreover, antitumor mechanism study demonstrated that 9za exerted significant apoptotic effect, and cellular signal pathway analysis revealed the potent blockage of phosphorylation levels of ERK and AKT in RAF-MEK-ERK and PI3K-PDK1-AKT pathways, respectively. The results reported here provide robust experimental basis for the discovery and optimization of dual pathway agents for anti-lung cancer therapy.