cAMP inhibition of Akt is mediated by activated and phosphorylated Rap1b

Rap1b has been implicated in the transduction of the cAMP mitogenic signal. Rap1b is phosphorylated and activated by cAMP, and its expression in cells where cAMP is mitogenic leads to an increase in G(1)/S phase entry and tumor formation. The PCCL3 thyroid follicular cells represent a differentiated and physiologically relevant system that requires thyrotropin (TSH), acting via cAMP, for a full mitogenic response. In this model system, cAMP stimulation of DNA synthesis requires activation and phosphorylation of Rap1b by the cAMP-dependent protein kinase A (PKA). This scenario presents the challenge of identifying biochemical processes involved in the phosphorylation-dependent Rap1b mitogenic action. In thyroid cells, Akt has been implicated in the stimulation of cell proliferation by TSH and cAMP. However, the mechanism(s) by which cAMP regulates Akt activity remains unclear. In this study we show that in PCCL3 cells 1) TSH inhibits Akt activity via cAMP and PKA; 2) Rap1b is required for cAMP inhibition of Akt; and 3) transduction of the cAMP signal into Akt requires activation as well as phosphorylation of Rap1b by PKA.     

Biochemical, Conformational, and Immunogenic Analysis of Soluble Trimeric Forms of Henipavirus Fusion Glycoproteins

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are paramyxoviruses discovered in the mid- to late 1990s that possess a broad host tropism and are known to cause severe and often fatal disease in both humans and animals. HeV and NiV infect cells by a pH-independent membrane fusion mechanism facilitated by their attachment (G) and fusion (F) glycoproteins. Here, several soluble forms of henipavirus F (sF) were engineered and characterized. Recombinant sF was produced by deleting the transmembrane (TM) and cytoplasmic tail (CT) domains and appending a glycosylphosphatidylinositol (GPI) anchor signal sequence followed by GPI-phospholipase D digestion, appending a trimeric coiled-coil (GCNt) domain (sF_GCNt), or deleting the TM, CT, and fusion peptide domain. These sF glycoproteins were produced as F₀ precursors, and all were apparent stable trimers recognized by NiV-specific antisera. Surprisingly, however, only the GCNt-appended constructs (sF_GCNt) could elicit cross-reactive henipavirus-neutralizing antibody in mice. In addition, sF_GCNt constructs could be triggered in vitro by protease cleavage and heat to transition from an apparent prefusion to postfusion conformation, transitioning through an intermediate that could be captured by a peptide corresponding to the C-terminal heptad repeat domain of F. The pre- and postfusion structures of sF_GCNt and non-GCNt-appended sF could be revealed by electron microscopy and were distinguishable by F-specific monoclonal antibodies. These data suggest that only certain sF constructs could serve as potential subunit vaccine immunogens against henipaviruses and also establish important tools for further structural, functional, and diagnostic studies on these important emerging viruses.     

Lipopolysaccharide-induced cytokine production in peripheral blood mononuclear cells: intracellular localization of tumor necrosis factor α and interleukin 1β detected with a three-color immunofluorescence technique

 Lipopolysaccharide (LPS) can induce monocytes to produce various cytokines such as tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β). In the present study, the kinetics of both intracellular and extracellular accumulation of TNFα and IL-1β in LPS stimulated mononuclear cell (MNC) cultures has been determined. A three-color-immunofluorescence technique was used to detect intracellular accumulation of cytokines. Intracellular accumulation of TNFα in monocytes starts shortly after initiation of the culture; i.e., TNFα is detectable after 1 h, reaching a peak level after 3–4 hours with 50–65% of monocytes staining positive. In parallel with its increased intracellular presence, TNFα was also found in the culture supernatant. The intracellular accumulation of IL-1β in monocytes became detectable after 2 h of culture in the presence of LPS. After 4 h, a plateau was reached, with 90% of the monocytes being positive. In parallel, but with a little delay, IL-1β could be detected in the culture supernatant. TNFα and IL-1β can be produced simultaneously in the same monocytes as was shown by a three-color-immunofluorescence technique. It is concluded that TNFα and IL-1β are good parameters for the early measurement of monocyte activation and that both the intracellular accumulation in monocytes and the amount of secreted cytokines can be used for such a purpose. The intracellular accumulation in monocytes can be measured by the three-color-immunofluorescence technique described. Accepted: 27 August 1996     

昆虫的化学感觉机理

昆虫是通过化学感觉器与其周围环境中的大量化学信息发生联系的。通过特定的化学感觉机制 ,昆虫可感知来自种内和种间 ,以及无机环境中的各种化学信息 ,并由此而作出相应的行为反应 ,从而为其自身寻找适宜的食物、配偶以及生存与繁殖场所 (如躲避天敌、避免或减少竞争等等 ) ,达到最大的繁殖成功。阐明昆虫的化学感觉机理 ,不仅可在理论上进一步加深对昆虫与植物、昆虫与昆虫相互关系的了解 ,而且可在实践上为开发害虫治理的新途径提供理论指导。本文将根据目前的最新研究成果 ,主要就昆虫的化学感觉机理 ,包括嗅觉和味觉机理作一综述 ,以期…     

Cellular Networks in Epidermal Peels of Onion

Cellular networks ill epidermal peels of onion bulb can be distinguished by first removal of superficially attached cytoplasmic constituents with Triton phos- phate buffer and then by staining with Coomassie blue R 250(Fig. 4). Two distinct kinds of networks can be further recognized by treatment with colchicine and cytochalasins: one thinner network underneath the periphery of plasmalemma can be abolished by colchicine (Fig. 7, 8); and the other thicker one which associates tangentially with the nucleus was more distinctive after cytochalasin B treatment (Fig. 5, 6). Discussion is made regarding these two networks in wall-enclosed plant cells as revealed by the present technic.     

Study of Diurnal Changes in Photosynthetic Rate and Quantum Efficiency of Grapevine Leaves and Their Utilization in Canopy Management

Photosynthetic rate and quatum efficiency of grapevine (Vitis vinifera L. cv. Sauvignon blanc) leaves were measured under the field with ample soil water supply, and in phytotron with ample supply of water and mineral nutrients, constant air humidity and CO2 concentration, and optimum air temperature, respectively. Under field conditions CO2 assimilation quantum efficiency of leaves reached its maximum in the morning, which was followed by continuous decrease and midday depression. The leaves intercepting more light energy in the morning showed a higher quantum efficiency. Those leaves subjected continuously to strong irradiance exhibited a more obvious and longer midday depression. Reduction of leaf light interception around midday could reduce midday depression. Shaded leaves had a higher quantum efficiency than leaves under direct sunlight. The diurnal changes in photosynthetic rate and quantum efficiency of leaves were shown to be closely related to the variations in mesophyll resistance to CO2. In phytotron experiments the photosynthetic quantum efficiency of leaves was reduced after a certain period of illumination not only at 1200 μmol · m-2 · s-1 PFD, higher than the saturating light of vine leaves (≈1000 μmol · m-2 · s-1), which was caused by "photoinhibition”, but also at 800 and 200μmol · m-2 · s-1, which was similar to "photoinhibition”. But photosynthetic quantum efficiency of leaves exposed continuously to a very weak PFD (100 μmol · m -2 · s-1) remained contant. The diurnal changes in mesophyll resistance to CO2 of vine leaves could be partly related to photoinhibition. It is considered that, under field conditions without soil water limitation, midday depression of vine leaf photosynthesis could be a result of an increase of the mesophyll resistance induced by multiple effects of strong light, high temperature and low humidity. A higher light interception by canopy plane in the morning may be advantageous to exploit higher photosynthetic potentiality of leaves, but a lower light interception in the middle of day may reduce midday depression. The north-south orientation plane can provide optimum light regime and improve photosynthetic environment in vineyards.     

Put the metal to the petal: metal uptake and transport throughout plants

Compared to other organisms, plants have expanded families of transporters that are involved in the uptake and efflux of metals. Fortunately, in many cases, the examination of double mutants has been sufficient to overcome the challenge of studying functionally redundant gene families. Plants that lack two heavy-metal-transporting P-type ATPase family members (HMA2 and HMA4) reveal a function for these transporters in Zn translocation from roots to shoots. Likewise, the phenotype of plants that lack two natural resistance associated macrophage protein (NRAMP) homologs (NRAMP3 and NRAMP4) implicate these metal uptake proteins in the mobilization of vacuolar Fe stores during seed germination. Most families of metal transporters are ubiquitous but the Yellow Stripe1-Like (YSL) family is plant specific and YSL family members have been implicated in the transport of metals that are complexed with a plant specific chelator called nicotianamine (NA).     

Development of 25 near-isogenic lines (NILs) with ten BPH resistance genes in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.): production,resistance spectrum,and molecular analysis

Key message

A first set of 25 NILs carrying ten BPH resistance genes and their pyramids was developed in the background of indica variety IR24 for insect resistance breeding in rice.

Abstract

Brown planthopper (Nilaparvata lugens Stal.) is one of the most destructive insect pests in rice. Development of near-isogenic lines (NILs) is an important strategy for genetic analysis of brown planthopper (BPH) resistance (R) genes and their deployment against diverse BPH populations. A set of 25 NILs with 9 single R genes and 16 multiple R gene combinations consisting of 11 two-gene pyramids and 5 three-gene pyramids in the genetic background of the susceptible indica rice cultivar IR24 was developed through marker-assisted selection. The linked DNA markers for each of the R genes were used for foreground selection and confirming the introgressed regions of the BPH R genes. Modified seed box screening and feeding rate of BPH were used to evaluate the spectrum of resistance. BPH reaction of each of the NILs carrying different single genes was variable at the antibiosis level with the four BPH populations of the Philippines. The NILs with two- to three-pyramided genes showed a stronger level of antibiosis (49.3–99.0%) against BPH populations compared with NILs with a single R gene NILs (42.0–83.5%) and IR24 (10.0%). Background genotyping by high-density SNPs markers revealed that most of the chromosome regions of the NILs (BC₃F₅) had IR24 genome recovery of 82.0–94.2%. Six major agronomic data of the NILs showed a phenotypically comparable agronomic performance with IR24. These newly developed NILs will be useful as new genetic resources for BPH resistance breeding and are valuable sources of genes in monitoring against the emerging BPH biotypes in different rice-growing countries.

     

The reduction of Raf-1 protein by phosphorothioate ODNs and siRNAs targeted to the same two mRNA sequences

Two sets of 20-mer phosphorothioate-modified oligodeoxynucleotide DNAs (sODN) and 21-mer or 22-mer small interfering RNAs (siRNAs), targeted to the same coding sites in raf-1 mRNA, were compared for their abilities to reduce the amount of endogenously expressed Raf-1 protein in T24 cells. The amount of Raf-1 protein was monitored by careful quantitation of Western blots. We found that the siRNAs were somewhat less effective than the S-ODNs in reducing the Raf-1 protein level 20 hours after a 4-hour transfection. The siRNA duplexes were characterized by circular dichroism (CD) spectra, and melting temperatures (Tm) were obtained for the siRNA duplexes and DNA x RNA hybrids formed by the S-ODNs. The S-ODNs differed in their effectiveness, the S-ODN that formed the more stable hybrid being the more effective in reducing the Raf-1 protein level, but the two siRNAs were equally effective despite a difference in Tm of about 20 degrees C. Finally, the siRNAs and S-ODNs had a comparable nonspecific effect on a nontargeted (Bcl-2) protein. Our data add to others in the literature that show it can be difficult to select siRNAs that are more effective than antisense ODNs in downregulating endogenously expressed proteins.