Cdh1 Regulates Cell Cycle through Modulating the Claspin/Chk1 and the Rb/E2F1 Pathways

APC/Cdh1 is a major cell cycle regulator and its function has been implicated in DNA damage repair; however, its exact role remains unclear. Using affinity purification coupled with mass spectrometry, we identified Claspin as a novel Cdh1-interacting protein and further demonstrated that Claspin is a novel Cdh1 ubiquitin substrate. As a result, inactivation of Cdh1 leads to activation of the Claspin/Chk1 pathway. Previously, we demonstrated that Rb interacts with Cdh1 to influence its ability to degrade Skp2. Here, we report that Cdh1 reciprocally regulates the Rb pathway through competing with E2F1 to bind the hypophosphorylated form of Rb. Although inactivation of Cdh1 in HeLa cells, with defective p53/Rb pathways, led to premature S phase entry, acute depletion of Cdh1 in primary human fibroblasts resulted in premature senescence. Acute loss of many other major tumor suppressors, including PTEN and VHL, also induces premature senescence in a p53- or Rb-dependent manner. Similarly, we showed that inactivation of the p53/Rb pathways by overexpression of SV40 LT-antigen partially reversed Cdh1 depletion–induced growth arrest. Therefore, loss of Cdh1 is only beneficial to cells with abnormal p53 and Rb pathways, which helps explain why Cdh1 loss is not frequently found in many tumors.     

Effect of monoclonal antibody on expression of lipid metabolism related genes in porcine adipocytes

In order to study the mechanism of monoclonal antibody (McAb) against a porcine 40-kDa adipocyte-specific plasma membrane protein in reducing fat deposition, porcine primary adipocytes were treated with the McAb during the process of adipocyte differentiation; its effect on expression of lipid metabolism related genes was investigated. Adipocytes were treated with 1-methyl-3-isobutylmethylxanthine (IDX) plus 10 μg/mL of the McAb or without McAb. The mRNA levels of adipocyte differentiation related genes (PPARγ and C/EBPα), lipid metabolism related genes (FAS, HSL, CPT-1B, DGAT and A-FABP) and adiponectin gene (AdipoQ) were determined using real-time quantitative PCR. The results showed that the differentiated adipocyte number and triglyceride (TG) content in adipocytes treated with the McAb were lower than that in cells without McAb during the whole process of adipocyte differentiation. The McAb significantly reduced mRNA expression of PPARγ, C/EBPα, FAS, DGAT, A-FABP and adiponectin genes, but increased mRNA expression of HSL and CPT-1B genes during the medium and latter stage of adipocyte differentiation. This suggested that the McAb decreased triglycerol accumulation in adipocyte by both inhibiting adipocyte differentiation and regulating lipid metabolism, especially at the medium and latter stage of porcine adipocyte differentiation.     

The Structural Adaptation of Aerial Parts of Invasive <Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> to Water Regime

Alternanthera philoxeroides has successfully invaded diverse habitats with considerably various water availability, threatening biological diversity in many parts of the world. Because its genetic variation is very low, phenotypic plasticity is believed to be the primary strategy for adapting to the diverse habitats. In the present paper, we investigated the plastic changes of anatomical traits of the aerial parts of A. philoxeroides from flooding to wet then to drought habitat; the results are as follows: A. philoxeroides could change anatomical structures sensitively to adapt to water regime. As a whole, effects of water regime on structures in stem were greater than those in leaf. Except for principal vein diameter and stoma density on leaf surfaces, all other structural traits were significantly affected by water regime. Among which, cuticular wax layer, collenchyma cell wall, phloem fiber cell wall, and hair density on both leaf surfaces thickened significantly with decrease of water availability, whereas, pith cavity and vessel lumen in stem lessened significantly; wet habitat is vital for the spread of A. philoxeroides from flooding to drought habitat and vice versa, because in this habitat, it had the greatest structural variations; when switching from flooding to wet then to drought habitat, the variations of cuticular wax layer, collenchyma cell wall, phloem fiber cell wall, pith cavity area ratio, diameter of vessel lumen, and hair density on both leaf surfaces, played the most important role. These responsive variables contribute most to the adaptation of A. philoxeroides to diverse habitats with considerably various water availability.     

Reduction in SBPase Activity by Antisense RNA in Transgenic Rice Plants: Effect on Photosynthesis,Growth, and Biomass Allocation at Different Nitrogen Levels

Rice cultivar zhonghua11 (Oryza sativa L. ssp. japonica) plants with decreased sedoheptulose-1, 7-bisphosphatase (SBPase) were obtained by transformation with the rice SBPase antisense gene under the control of the maize ubiquitin promoter. The transgenic and wild-type plants were grown at different nitrogen levels (0.1, 1, or 10 mM NH₄NO₃). Growth rates of the seedlings were measured by the changes in dry weight, and the photosynthetic carbon reduction activities and the potential efficiency of photosystem II were measured by CO₂ assimilation and F _v/F _m, respectively. At low N, there are strong effects on growth and photosynthesis when SBPase was reduced by genetic manipulation. Decreased SBPase activity led to a decrease in the amount of starch accumulated in the leaves at all N levels and the decrease was much more prominent in low N than that in high N, but the starch allocation between shoot and root was unaltered. The analysis of chlorophyll fluorescence and SBPase activity indicated that the decrease of growth and photosynthesis at different N levels were not related to the function of PSII but to the activity of SBPase. Western blot analysis showed the content of SBPase in thylakoid membranes was much more than in the stroma fractions in transgenic plants at low N. Results suggested that low N in addition to a 34% decrease in SBPase activity is sufficient to diminish photosynthesis and limit biomass production. Decreased SBPase activity may reduce the N use efficiency of photosynthesis and growth and alter biomass allocation.     

Palmitoylation modification of Gαo depresses its susceptibility to GAP-43 activation

Interaction between GAP-43 (growth associated protein-43) and Gα_o (alpha subunit of Go protein) influences the signal transduction pathways leading to differentiation of neural cells. GAP-43 is known to increase guanine nucleotide exchange by Gα_o, which is a major component of neuronal growth cone membranes. However, it is not clear whether GAP-43 stimulation is related to the Gα_o palmitoylation or the conversion of Gα_o from oligmers to monomers, which was shown to be a necessary regulatory factor in GDP/GTP exchange of Gα_o. Here we expressed and purified GAP-43, GST-GAP-43 and Gα_o proteins, detected their stimulatory effect on [³⁵S]-GTPγS binding of Gα_o. It was found that the EC₅₀ of both GAP-43 and GST-GAP-43 activation were tenfold lower in case of depalmitoylated Gα_o than palmitoylated Gα_o. Non-denaturing gel electrophoresis and p-PDM cross-linking analysis revealed that addition of GST-GAP-43 induced disassociation of depalmitoylated Gα_o from oligomers to monomers, but did not influence the oligomeric state of palmitoylated Gα_o, which suggests that palmitoylation is a key regulatory factor in GAP-43 stimulation on Gα_o. These results indicated the interaction of GAP-43 and Gα_o could accelerate conversion of depalmitoylated Gα_o but not palmitoylated Gα_o from oligomers to monomers, so as to increase the GTPγS binding activity of Gα_o. Results here provide new evidence about how signaling protein palmitoylation is involved in the G-protein-coupled signal transduction cascade, and give a useful clue on the participation of GAP-43 in G-protein cycle by its preferential activation of depalmitoylated Gα_o.     

Hitchhiking of Cu/Zn Superoxide Dismutase to Peroxisomes – Evidence for a Natural Piggyback Import Mechanism in Mammals

Most newly synthesized peroxisomal proteins are imported in a receptor-mediated fashion, depending on the interaction of a peroxisomal targeting signal (PTS) with its cognate targeting receptor Pex5 or Pex7 located in the cytoplasm. Apart from this classic mechanism, heterologous protein complexes that have been proposed more than a decade ago are also to be imported into peroxisomes. However, it remains still unclear if this so-called piggyback import is of physiological relevance in mammals. Here, we show that Cu/Zn superoxide dismutase 1 (SOD1), an enzyme without an endogenous PTS, is targeted to peroxisomes using its physiological interaction partner 'copper chaperone of SOD1' (CCS) as a shuttle. Both proteins have been identified as peroxisomal constituents by 2D-liquid chromatography mass spectrometry of isolated rat liver peroxisomes. Yet, while a major fraction of CCS was imported into peroxisomes in a PTS1-dependent fashion in CHO cells, overexpressed SOD1 remained in the cytoplasm. However, increasing the concentrations of both CCS and SOD1 led to an enrichment of SOD1 in peroxisomes. In contrast, CCS-mediated SOD1 import into peroxisomes was abolished by deletion of the SOD domain of CCS, which is required for heterodimer formation. SOD1/CCS co-import is the first demonstration of a physiologically relevant piggyback import into mammalian peroxisomes.     

Non-Additive Effects of Water and Nitrogen Addition on Ecosystem Carbon Exchange in a Temperate Steppe

Changes in precipitation and nitrogen (N) deposition can influence ecosystem carbon (C) cycling and budget in terrestrial biomes, with consequent feedbacks to climate change. However, little is known about the main and interactive effects of water and N additions on net ecosystem C exchange (NEE). In a temperate steppe of northern China, a field-manipulated experiment was conducted to evaluate the responses of NEE and its components to improve N and water availability from 2005 to 2008. The results showed that both water and N additions stimulated gross ecosystem productivity (GEP), ecosystem respiration (ER), and NEE. Water addition increased GEP by 17%, ER by 24%, and NEE by 11% during the experimental period, whereas N addition increased GEP by 17%, ER by 16%, and NEE by 19%. The main effects of both water and N additions changed with time, with the strongest water stimulation in the dry year and a diminishing N stimulation over time. When water and N were added in combination, there were non-additive effects of water and N on ecosystem C fluxes, which could be explained by the changes in species composition and the shifts of limiting resources from belowground (water or N) to aboveground (light). The positive water and N additions effects indicate that increasing precipitation and N deposition in the future will favor C sequestration in the temperate steppe. The non-additive effects of water and N on ecosystem C fluxes suggest that multifactor experiments are better able to capture complex interactive processes, thus improving model simulations and projections.     

HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS¹⁶²) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS¹⁶² as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS¹⁶² is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS¹⁶² is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS¹⁶² is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.