The molecular mechanism of embryonic stem cell pluripotency maintenance

In vitro cultured embryonic stem （ES） cells are derived from the inner cell mass （ICM） of pre-implantation embryos, and are capable of giving rise to all cell and tissue types of the three germ layers upon being injected back into blastocysts. These ceils are therefore said to possess pluripotency that can be maintained infinitely in culture under optimal conditions. Such pluripotency maintenance is believed to be due to the symmetrical cleavage of the cells in an undifferentiated state. The pluripotency of ES cells is the basis for their various practical and potential applications. ES cells can be used as donor cells to generate knockout or transgenic animals, as in vitro models of mammalian development, and as cell resources for cell therapy in regenerative medicine. The further success in these applications, particularly in the last two, is dependent on the establishment of a culture system with components in the medium clearly defined and the subsequent procedures for controlled differentiation of the cells into specific lineages. In turn, elucidating the molecular mechanism for pluripotency maintenance of ES cells is the prerequisite. This paper summarizes the recent progresses in this area, focusing mainly on the LIF/STAT3, BMPs/Smads, canonical Wnt, TGFβ/activin/nodal, PI3K and FGF signaling pathways and the genes such as oct4, nanog that are crucial in ES cell pluripotency maintenance. The regulatory systems of pluripotency maintenance in both mouse and human ES cells are also discussed. We believe that the cross-talkings between these signaling pathways, as well as the regulatory system underlying pluripotency maintenance will be the main focus in the area of ES cell researches in the future.     

Phosphodiesterase 4 and compartmentalization of cyclic AMP signaling

Cyclic AMP (cAMP), as a second messenger, plays a critical role in cellular signaling transduction. However, it is not clear how this apparently identical cAMP signal induces divergent physiological re- sponses. The potential explanation that cAMP signaling is compartmentalized was proposed by Buxton and Brunton twenty years ago. Compartmentalization of cAMP signaling allows spatially distinct pools of protein kinase A (PKA) to be differently activated. Research on cAMP signaling has regained impetus in many fields of life sciences due to the progress in understanding cAMP signaling complexity and functional diversity. The cAMP/PKA signaling compartments are maintained by A-kinase anchoring proteins (AKAPs) which bind PKA and other signaling proteins, and by PDEs which hydrolyse cAMP and thus terminate PKA activity. PDE4 enzymes belong to PDE superfamily and stand at a crossroad that allows them to integrate various signaling pathways with that of cAMP in spatially distinct com- partments. In the current review, the nomenclature, taxonomy and gene expression of PDE4, and the system and region of its effect are described. In addition, the idiographic molecules, mechanisms, and regulation models of PDE4 are summarized. Furthermore, the important roles PDE4 plays in the matu- ration of rat granulosa cells and cAMP signaling compartmentalization are discussed.     

An energy conduction model for cell image segmentation

Cell image segmentation is an essential step in cytopathological analysis.Although their execution speed is fast,the results of cell image segmentation by conventional pixel-based,edge-based and continuity-based methods are often coarse.Fine structures in a cell image can be obtained with a method that quickly adjusts the threshold levels.However,the processing time of such a method is usually long and the final results may be sensitive to intensity differences and other factors.In this article,a new energy model is proposed that synthesizes a differential equation from the conventional and level set methods,and utilizes the nonuniformity property of cell images (e.g.cytoplasms are more uneven than the background).The feasibility and robustness of the proposed model was demonstrated by processing relatively complicated background images of both simulated and real cell images.     

Systemic defense signaling in tomato

The wound-inducible expression of proteinase inhibitors （PIs） genes in tomato provides a powerful model system to elucidate the signal transduction pathway of systemic defense response. An increasing body of evidence indicates that systemin and jasmonic acid （JA） work in the same signaling pathway to activate the expression of Pls and other defense-related genes. However, little is known about how systemin and JA interact to regulate cell to cell communication over long distances. Genetic analysis of the systemin/JA signaling pathway in tomato plants provides a unique opportunity to dissect the mechanism by which peptide and oxylipin signals interact to coordinate systemic expression of defense-related genes. Previously, it has been proposed that systemin is the long-distance mobile signal for systemic expression of defense related genes. However, recent genetic approach provided new evidence that jasmonic acid, rather than systemin, functions as the systemic wound signal, and that the peptide systemin works to regulate the biosynthesis of JA.     

A CHASE domain containing protein kinase OsCRL4, represents a new AtCRE1-like gene family in rice

AtCRE1 is known to be a cytokinin receptor inArabidopsis. The AtCRE1 protein contains CHASE domain at the N-terminal part, followed by a transmitter (histidine kinase) domain and two receiver domains. The N-terminal CHASE domain of AtCRE1 contains putative recognition sites for cytokinin. Five CHASE domains containing proteins were found in rice, OsCRLla, OsCRLlb, OsCRL2, OsCRL3, and OsCRL4. OsCRL1a, OsCRL1b, OsCRL2 and OsCRL3 contain the four domains existing in CRE1, whereas OsCRL4 only contains the CHASE domain and a putative Ser/Thr protein kinase domain The authors cloned the encoding gene OsCRL4 and found that it represents a new member of the cytokinin receptor protein in rice.     

Protein tyrosine phosphatase is possibly involved in cellular signal transduction and the regulation of ABA accumulation in response to water deficit in Maize L.coleoptile

Water deficit-induced ABA accumulation is an ideal model or “stimulus-response”system to investigate cellular stress signaling in plant cels,using such a model the cellular stress signaling triggered by water deficit was investigated in Maize L.coleoptile.Water deficit-induced ABA accumulation was sensitively blocked by NaVO3,a potent inhibitor both to plasma membrane H^ -ATPase(PM-H^ -ATPase)and protein tyrosine phosphatase(PTPase).However,while PM-H^ -ATPase activity was unaffected under water deficit and PM-H^ -ATPase activator did not induce an ABA accumulation instead of water deficit,water deficit induced an increase in the protein phosphatase activity,and furthermore,ABA accumulation was inhibited by PAO,a specific inhibitor of PTPase.These results indicate that protein phosphtases may be involved in the cellular signaling in response to water deficit.Further studies identifiled at least four species of protein phosphtase as assayed by using pNPP as substrate,among which one component was especially sensitive to NaVO3.The NaVO3-sensitive enzyme was purified and finally showed a protein band about 66kD on SDS/PAGE.The purified enzyme showed a great activity to some specific PTPase substrates at pH 6.0.In addition to NaVO3,the enzyme was also sensitive to some other PTPase inhibitors such as Zn^2 and MO3^3 ,but not to Ca^2 and Mg^2 ,indicating that it might be a protein tyrosine phosphatase.Interestingly,the purified enzyme could be deactivated by some reducing agent DTT.which was previously proved to be an inhibitor of water deficit-induced ABA accumulation.This result further proved that PTPase might be involved in the cellular signaling of ABA accumulation in response to water deficit.     

Promoter of soybean early nodulin gene<Emphasis Type="Italic">enod2B</Emphasis> is induced by rhizobial Nod factors in transgenic rice

Nod factors, which are signaling molecules produced by Rhizobia, are the principal determinants of host specificity in Rhizobium-legume symbiosis. Nod factors can elicit a number of characteristic developmental responses in the roots of legumes, such as depolarization of the membrane potential in epidermal cells, specific expression of early nodulin genes and changes in the flux of calcium in root hairs, deformation of root hairs, cell division in the root cortex and formation of the nodule primordinm. Whether the rice plant can respond to signaling molecules (i.e. Nod factors) is an important question, as it could establish the potential for symbiotic nitrogen fixation in rice. The promoter of the soybean (Glycine max) early nodulin gene Gmenod2B fused to the β-glucuronidase (GUS) reporter gene was used as a molecular marker to explore whether Nod factors can be recognized by rice cells as signaling molecules. Transgenic rice plants harboring the chimeric gene Gmenod2BP-GUS were obtained via an Agrobacterium tumefaciens-mediated system. NodNGR factors produced by a broad-host-range Rhizobium strain NGR234(pA28) were used as probes to investigate the activity of the Gmenod2B promoter in rice. Our results showed that the early nodulin gene Gmenod2B promoter was induced by NodNGR factors in transgenic rice, and that it was specifically expressed in rice plant roots. Moreover, GUS gene expression driven by the Gmenod2B promoter in transgenic rice was regulated by nitrogen status. These findings indicated that rice possessed the ability to respond to Nod factor signals, and that this signal transduction system resulted in activation of the Gmenod2B promoter. Thus, we predict that the Nod-factor inducible nodulin expression system, which is similar to Rhizobium-legume symbiosis, may also exist in rice.     

C-di-GMP signaling and implications for pathogenesis of Mycobacterium tuberculosis

C-di-GMP is a ubiquitous bacterial second messenger that regulates a wide range of bacterial physiological processes including biofilm formation, virulence, motility and cell differentiation. Here, we have summarized our current knowledge on the upstream signaling factors and downstream effectors of c-di-GMP in addition to the interaction between c-di-GMP and eukaryotic organisms. New discoveries in these areas have enriched our understanding of the diversity of c-di-GMP signaling pathways and provide important clues for us to explore the roles of c-di-GMP signaling in human pathogens such as Mycobacterium tuberculosis.     

Protein phosphorylation is involved in the water stress-induced ABA accumulation in the roots ofMalus hupehensis Rehd

Water stress-induced ABA accumulation plays a key role in the root to shoot communication and/or the cell to cell signaling under the soil stresses. The signaling of the water stress itself that leads to the accumulation, however, is less known. In this study, we subjected the roots of Malus hupehensis seedlings to water stress treatment and investigated the ABA accumulation in relation to protein phosphorylation. Our results showed that ABA accumulation could be substantially triggered in 40 min and reached 4 folds in 100 min after treatment with 30% PEG 6000 (polyethylene glycol). The water stress treatment also led to a substantial enhancement of total kinase activity, assessed with histone-Ⅲ as substrate, in 15 min and a maximum enhancement in 30 min before it declined to initial level. The Ca²⁺-dependent kinase activity showed a similar, if not more sensitive, trend. When the roots were fed with labeled ³²P- ATP, water stress enhanced the labeling of proteins, which showed a maximum labeling at 40 min. Two inhibitors of protein kinases, Quercetin and H7, effectively diminished or completely blocked the ABA accumulation under the stress treatment. It is therefore suggest that protein phosphorylation is involved in the signaling of the water stress-induced ABA accumulation.     

Identification of CRE1 as a cytokinin receptor from Arabidopsis

Cytokinins are a class of plant hormones that are central to the regulation of cell division and differentiation in plants. It has been proposed that they are detected by a two-component system, because overexpression of the histidine kinase gene CKI1 induces typical cytokinin responses and genes for a set of response regulators of two-component systems can be induced by cytokinins. Two-component systems use a histidine kinase as an environmental sensor and rely on a phosphorelay for signal transduction. They are common in microorganisms, and are also emerging as important signal detection routes in plants. Here we report the identification of a cytokinin receptor. We identified Arabidopsis cre1 (cytokinin response 1) mutants, which exhibited reduced responses to cytokinins. The mutated gene CRE1 encodes a histidine kinase. CRE1 expression conferred a cytokinin-dependent growth phenotype on a yeast mutant that lacked the endogenous histidine kinase SLN1 (ref. 10), providing direct evidence that CRE1 is a cytokinin receptor. We also provide evidence that cytokinins can activate CRE1 to initiate phosphorelay signalling.     

Direct control of shoot meristem activity by a cytokinin-activating enzyme

The growth of plants depends on continuous function of the meristems. Shoot meristems are responsible for all the post-embryonic aerial organs, such as leaves, stems and flowers. It has been assumed that the phytohormone cytokinin has a positive role in shoot meristem function. A severe reduction in the size of meristems in a mutant that is defective in all of its cytokinin receptors has provided compelling evidence that cytokinin is required for meristem activity. Here, we report a novel regulation of meristem activity, which is executed by the meristem-specific activation of cytokinins. The LONELY GUY (LOG) gene of rice is required to maintain meristem activity and its loss of function causes premature termination of the shoot meristem. LOG encodes a novel cytokinin-activating enzyme that works in the final step of bioactive cytokinin synthesis. Revising the long-held idea of multistep reactions, LOG directly converts inactive cytokinin nucleotides to the free-base forms, which are biologically active, by its cytokinin-specific phosphoribohydrolase activity. LOG messenger RNA is specifically localized in shoot meristem tips, indicating the activation of cytokinins in a specific developmental domain. We propose the fine-tuning of concentrations and the spatial distribution of bioactive cytokinins by a cytokinin-activating enzyme as a mechanism that regulates meristem activity.     

关于用尾穗苋黄化苗子叶苋红合成法进行细胞分裂素活力测定的问题

尾穗苋(Amaranthus Candatus L.)种子在发芽75小时以后,其黄化苗子叶对光照失去敏感性。无论给予连续或间断光照,均未引起苋红的合成。但激动素却仍能使黄化苗子叶正常合成苋红。且合成苋红的光密度值与激动素浓度的关系曲线,在实验室自然光照条件下与在暗室中的规律基本相同。用已知浓度的激动素测定其活力对光下制作的标准曲线进行检测后表明,光下所制作的标准曲线能用于细胞分裂素的活力测定。因此,可初步认为,能够在正常光照条件下进行细胞分裂素的活力测定。     

半夏组织培养中不同植物生长调节物质对形态发生的效应

半夏叶片、叶柄离体培养，探讨不同植物生长调节物质对其形态发生的效应。结果表明：ＮＡＡ、２，４－Ｄ有利于愈伤组织形成，且附加２，４－Ｄ形成愈伤组织最快；诱导率最高；ＮＡＡ和低浓度２，４－Ｄ有利于根分化，附加ＮＡＡ根生长最快，高浓度２，４＋６－ＢＡ或ＫＴ抑制根的形成，６－ＢＡ或ＫＴ有利于芽的分化，６－ＢＡ促进芽的分化效果显著。     

匙唇兰次生培养研究

研究了匙唇兰的类原生质体次生培养.建立了其原球茎次生培养的可行性方法,选择营养液中细胞分裂素与生长素的种类与含量.匙唇兰幼苗的侧芽与顶芽在改良MS(1949)培养基中离体培养6到10周即可形成类原生质体.检测不同的细胞分裂素与生长素配比对匙唇兰原球茎次生培养的效果.用Minitab14.12统计分析软件做方差分析.其结果表明,细胞分裂素6-BA与生长素NAA双因素的效应以及交互效应都显著.细胞分裂素6-BA有利于原球茎的次生扩繁与增殖,生长素NAA刺激生根,有利于幼苗的形成.新鲜椰乳对原球茎的增殖以及幼苗生根都有特殊的良好效果,其对照试验结果明显优于其它没有添加新鲜椰乳的配方.最佳增殖配方是细胞分裂素6-BA 2.0 mg.L-1和生长素NAA0.5 mg.L-1.诱导幼苗形成的最佳配方是细胞分裂素6-BA 2.0 mg.L-1和NAA1.0 mg.L-1.     

A novel tobacco gene coding for a product similar to bacterial two-component regulators

The two-component signaling system has been studied in bacteria. It takes part in signal transduction of adaptive behavior. Recent studies have shown that a similar two-component system is also present in eukaryotes. Examples of this areETRl andCKLl genes which may involve the signal transduction of plant hormone ethylene and cytokinin respectively. The cloning and characterization of a novel gene (NTHKl) fragment from tobacco are presented. Its partial sequence codes for a product which shows similarity to many two-component signaling proteins. Southern blot analysis indicated that there are 2 to 3 copies ofNTHKl gene in tobacco genome (allotetraploid). Homologous genes may also exist in other plants such as Arabidopsis, soybean and spinach. The expression ofNTHKl gene has also been analyzed in tobacco. Further studies on the isolation of full-length cDNA ofNTHKl gene will elucidate more clearly its function in signal perception and transduction.