观赏植物花期调控途径及其分子机制

开花期控制对观赏植物的生产和应用具有重要意义。目前关于高等植物成花机理的研究已经取得了突破性进展,为观赏植物花期调控开辟了新途径。该文总结了观赏植物花期调控的途径和方法,并对改良观赏植物花期的技术思路做了初步分析。通过与高等植物成花机制研究的对比分析发现,观赏植物开花机理的研究已有了长足发展,一些观赏植物的转基因研究也取得了丰硕成果。利用分子设计育种途径改良观赏植物的开花期,突破了传统方法的局限性,其研究和应用前景非常广阔。     

Ala99ser mutation in RIα Regulatory Subunit of protein kinase A causes reduced kinase activation by cAMP and arrest of hormone-dependent breast cancer cell growth

Expression of the RI regulatory subunit of protein kinase A type I is increased in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. Ala99 (the pseudophosphorylation site) of human RI was replaced with Ser (RI-p) for the structure-function analysis of RI. MCF-7 hormone- dependent breast cancer cells were transfected with an expression vector for the wild-type RI or mutant RI-p. Overexpression of RI-P resulted in suppression of protein kinase A type II, the isozyme of type I kinase, production of kinase exhibiting reduced cAMP activation, and inhibition of cell growth showing an increase in G0/G1 phase of the cell cycle and apoptosis. The wild-type RI overexpression had no effect on protein kinase A isozyme distribution or cell growth. Overexpression of protein kinase A type II regulatory subunit, RII, suppressed RI and protein kinase A type I and inhibited cell growth. These results show that the growth of hormone-dependent breast cancer cells is dependent on the functional protein kinase A type I.     

Sclerite calcification and reef-building in the fleshy octocoral genus Sinularia (Octocorallia: Alcyonacea)

Alcyonacean octocorals in tropical reefs are usually not considered as reef builders. Some Sinularia species, however, are capable of consolidating sclerites at the colony base to form spiculite. Nanwan Bay, southern Taiwan, features both fossilized and recently formed boulders composed of spiculite, thus demonstrating the role of Sinularia in contributing to the reef structure. Section radiography of an 18.5 kg spiculite boulder demonstrated a regular density banding of 3–6-mm intervals. Core survey indicated spiculite coverage of 25–30% on the live reef and of 30–40% on the uplifted boulders. Cores taken from living Sinularia revealed a distinct transition from discrete sclerites to compact spiculite and amorphous calcium carbonate cementing the sclerites. In the widespread S. gibberosa, sclerite formation appeared to start intracellularly, followed by a prolonged extracellular calcification process. At the calcification site, multiple sclerocytes formed expanded pseudopod-like membranes that interconnected, forming multicellular vesicles (MCVs) around the sclerites. The MCVs and the pseudopods disappeared at sclerite maturation, followed by degradation of the sclerocytes around the mature sclerites. At the colony base, granular vesicles were distributed among the sclerites, indicating a cementing process in progress. These findings suggest that colonies of Sinularia are able to cement sclerites and consolidate them at their base into spiculite, thus making them reef builders.     

Long term stabilization of expanding aortic aneurysms by a short course of cyclosporine A through transforming growth factor-beta induction

Abdominal aortic aneurysms (AAAs) expand as a consequence of extracellular matrix destruction, and vascular smooth muscle cell (VSMC) depletion. Transforming growth factor (TGF)-beta 1 overexpression stabilizes expanding AAAs in rat. Cyclosporine A (CsA) promotes tissue accumulation and induces TGF -beta1 and, could thereby exert beneficial effects on AAA remodelling and expansion. In this study, we assessed whether a short administration of CsA could durably stabilize AAAs through TGF-beta induction. We showed that CsA induced TGF-beta1 and decreased MMP-9 expression dose-dependently in fragments of human AAAs in vitro, and in animal models of AAA in vivo. CsA prevented AAA formation at 14 days in the rat elastase (diameter increase: CsA: 131.9±44.2%; vehicle: 225.9±57.0%, P = 0.003) and calcium chloride mouse models (diameters: CsA: 0.72±0.14 mm; vehicle: 1.10±0.11 mm, P = .008), preserved elastic fiber network and VSMC content, and decreased inflammation. A seven day administration of CsA stabilized formed AAAs in rats seven weeks after drug withdrawal (diameter increase: CsA: 14.2±15.1%; vehicle: 45.2±13.7%, P = .017), down-regulated wall inflammation, and increased αSMA-positive cell content. Co-administration of a blocking anti-TGF-beta antibody abrogated CsA impact on inflammation, αSMA-positive cell accumulation and diameter control in expanding AAAs. Our study demonstrates that pharmacological induction of TGF-beta1 by a short course of CsA administration represents a new approach to induce aneurysm stabilization by shifting the degradation/repair balance towards healing.     

Comprehensive proteomic mass spectrometric characterization of human cannabinoid CB2 receptor

The CB1 and CB2 cannabinoid receptors belong to the GPCR superfamily and are associated with a variety of physiological and pathophysiological processes. Both receptors, with several lead compounds at different phases of development, are potentially useful targets for drug discovery. For this reason, fully elucidating the structural features of these membrane-associated proteins would be extremely valuable in designing more selective, novel therapeutic drug molecules. As a first step toward obtaining information on the structural features of the drug-receptor complex, we describe the full mass spectrometric (MS) analysis of the recombinant human cannabinoid CB2 receptor. This first complete proteomic characterization of a GPCR protein beyond rhodopsin was accomplished by a combination of several LC/MS approaches involving nanocapillary liquid chromatography, coupled with either a quadrupole-linear ion trap or linear ion trap-FTICR mass spectrometer. The CB2 receptor, with incorporated N-terminal FLAG and C-terminal HIS6 epitope tags, was functionally expressed in baculovirus cells and purified using a single step of anti-FLAG M2 affinity chromatography. To overcome the difficulties involved with in-gel digestion, due to the highly hydrophobic nature of this membrane-associated protein, we conducted in-solution trypsin and chymotrypsin digestions of purified and desalted samples in the presence of a low concentration of CYMAL5. This was followed by nanoLC peptide separation and analysis using a nanospray ESI source operated in the positive mode. The results can be reported confidently, based on the overlapping sequence data obtained using the highly mass accurate LTQ-FT and the 4000 Q-Trap mass spectrometers. Both instruments gave very similar patterns of identified peptides, with full coverage of all transmembrane helices, resulting in the complete characterization of the cannabinoid CB2 receptor. Mass spectrometric identification of all amino acid residues in the cannabinoid CB2 receptor is a key step toward the "Ligand Based Structural Biology" approach developed in our laboratory for characterizing ligand binding sites in GPCRs using a variety of covalent cannabinergic ligands.     

Cloning and expression profiling of small RNAs expressed in the mouse ovary

Small noncoding RNAs have been suggested to play important roles in the regulation of gene expression across all species from plants to humans. To identify small RNAs expressed by the ovary, we generated mouse ovarian small RNA complementary DNA (srcDNA) libraries and sequenced 800 srcDNA clones. We identified 236 small RNAs including 122 microRNAs (miRNAs), 79 piwi-interacting RNAs (piRNAs), and 35 small nucleolar RNAs (snoRNAs). Among these small RNAs, 15 miRNAs, 74 piRNAs, and 21 snoRNAs are novel. Approximately 70% of the ovarian piRNAs are encoded by multicopy genes located within the repetitive regions, resembling previously identified repeat-associated small interference RNAs (rasiRNAs), whereas the remaining approximately 30% of piRNA genes are located in nonrepetitive regions of the genome with characteristics similar to the majority of piRNAs originally cloned from the testis. Since these two types of piRNAs display different structural features, we categorized them into two classes: repeat-associated piRNAs (rapiRNAs, equivalent of the rasiRNAs) and non-repeat-associated piRNAs (napiRNAs). Expression profiling analyses revealed that ovarian miRNAs were either ubiquitously expressed in multiple tissues or preferentially expressed in a few tissues including the ovary. Ovaries appear to express more rapiRNAs than napiRNAs, and sequence analyses support that both may be generated through the "ping-pong" mechanism. Unique expression and structural features of these ovarian small noncoding RNAs suggest that they may play important roles in the control of folliculogenesis and female fertility.     

Effects of different irrigation modes on winter wheat grain yield and water- and nitrogen use efficiency

Taking the widely planted winter wheat cultivar Tainong 18 as test material, a field experiment was conducted to study the effects of different irrigation modes on the winter wheat grain yield and water- and nitrogen use efficiency in drier year (2009-2010) in Tai' an City of Shandong Province, China. Five treatments were installed, i. e., irrigation before sowing (CK), irrigation before sowing and at jointing stage (W1), irrigation before sowing and at jointing stages and at over-wintering stage with alternative irrigation at milking stage (W2), irrigation before sowing and at jointing and flowering stages (optimized traditional irrigation mode, W3), and irrigation before sowing and at over-wintering, jointing, and milking stages (traditional irrigation mode, W4). The irrigation amount was 600 m3 hm(-2) one time. Under the condition of 119.7 mm precipitation in the winter wheat growth season, no significant difference was observed in the grain yield between treatments W2 and W4, but the water use efficiency was significantly higher in W2 than in W4. Comparing with treatment W3, treatments W2 and W4 had obviously higher grain yield, but the water use efficiency had no significant difference. The partial factor productivity from N fertilization was the highest in W2 and W4, and the NO3(-)-N accumulation amount in 0-100 cm soil layer at harvest was significantly higher in W2 than in W3 and W4, suggesting that W2 could reduce NO3(-)-N leaching loss. Under the conditions of our experiment, irrigation before sowing and jointing stages and at over-wintering stage with alternative irrigation at milking stage was the optimal irrigation mode in considering both the grain yield and the water- and nitrogen use efficiency.     

Functional Dissection and Hierarchy of Tubulin-folding Cofactor Homologues in Fission Yeast

We describe the isolation of fission yeast homologues of tubulin-folding cofactors B (Alp11) and E (Alp21), which are essential for cell viability and the maintenance of microtubules. Alp11^B contains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21^E does not. Both mammalian and yeast cofactor E, however, do contain leucine-rich repeats. Immunoprecipitation analysis shows that Alp11^B interacts with both α-tubulin and Alp21^E, but not with the cofactor D homologue Alp1, whereas Alp21^E also interacts with Alp1^D. The cellular amount of α-tubulin is decreased in both alp1 and alp11 mutants. Overproduction of Alp11^B results in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of α-tubulin. Both full-length Alp11^B and the C-terminal third containing the CLIP-170 domain localize in the cytoplasm, and this domain is required for efficient binding to α-tubulin. Deletion of alp11 is suppressed by multicopy plasmids containing either alp21⁺ or alp1⁺, whereas alp21 deletion is rescued by overexpression of alp1⁺ but not alp11⁺. Finally, the alp1 mutant is not complemented by either alp11⁺ or alp21⁺. The results suggest that cofactors operate in a linear pathway (Alp11^B-Alp21^E-Alp1^D), each with distinct roles.     

Determinants of HMGB proteins required to promote RAG1/2-recombination signal sequence complex assembly and catalysis during V(D)J recombination

Efficient assembly of RAG1/2-recombination signal sequence (RSS) DNA complexes that are competent for V(D)J cleavage requires the presence of the nonspecific DNA binding and bending protein HMGB1 or HMGB2. We find that either of the two minimal DNA binding domains of HMGB1 is effective in assembling RAG1/2-RSS complexes on naked DNA and stimulating V(D)J cleavage but that both domains are required for efficient activity when the RSS is incorporated into a nucleosome. The single-domain HMGB protein from Saccharomyces cerevisiae, Nhp6A, efficiently assembles RAG1/2 complexes on naked DNA; however, these complexes are minimally competent for V(D)J cleavage. Nhp6A forms much more stable DNA complexes than HMGB1, and a variety of mutations that destabilize Nhp6A binding to bent microcircular DNA promote increased V(D)J cleavage. One of the two DNA bending wedges on Nhp6A and the analogous phenylalanine wedge at the DNA exit site of HMGB1 domain A were found to be essential for promoting RAG1/2-RSS complex formation. Because the phenylalanine wedge is required for specific recognition of DNA kinks, we propose that HMGB proteins facilitate RAG1/2-RSS interactions by recognizing a distorted DNA structure induced by RAG1/2 binding. The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA.