Root Growth of Arabidopsis thaliana Is Regulated by Ethylene and Abscisic Acid Signaling Interaction in Response to HrpNEa, a Bacterial Protein of Harpin Group

HrpN_Ea is a harpin protein from Erwinia amylovora, a bacterial pathogen that causes fire blight in rosaceous plants. Treating plants with HrpN_Ea stimulates ethylene and abscisic acid (ABA) to induce plant growth and drought tolerance, respectively. Herein, we report that both growth hormones cooperate to mediate the role of HrpN_Ea in promoting root growth of Arabidopsis thaliana seedlings. Root growth is promoted coordinately with elevation in levels of ABA and ethylene subsequent to soaking of germinating seeds of wild-type (WT) Arabidopsis in a solution of HrpN_Ea. However, these responses are arrested by inhibiting WT roots from synthesizing ethylene as well as sensing of ABA and ethylene. The effects of HrpN_Ea on roots are also nullified in ethylene-insensitive etr1-1 and ein5-1 mutants and in the ABA-insensitive mutant abi2-1 of Arabidopsis. These results provide evidence for presence of a relationship between root growth enhancement and signaling by ABA and ethylene in response to HrpN_Ea. Nevertheless, when HrpN_Ea is applied to leaves, ethylene signaling is active in the absence of ABA signaling to promote plant growth. This suggests the presence of a different signaling mechanism in leaves from that in roots. X. Ren and F. Liu contributed equally to this study and are regarded as joint first authors     

Expression,purification and characterization of anti-BAFF antibody secreted from the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

B-Cell activating factor (BAFF) is critical for B cell survival and maturation; excessive expression of it corrupts B-cell tolerance and may lead to autoimmunity. The gene, scFv-Fc, coding for the antibody of BAFF was inserted into the eukaryotic expression vector, pPICZαA, and transformed into Pichia pastoris. A high-level expression strain was obtained using a ‘yeastern blotting’ method. The scFv-Fc antibody was purified and 56 mg was obtained from 1 l of culture supernatant. It retained high binding activity to both soluble BAFF and membrane-bound BAFF.     

A cross-continental test of the Enemy Release Hypothesis: leaf herbivory on Acer platanoides (L.) is three times lower in North America than in its native Europe

Acer platanoides (Norway maple) is a widespread native tree species in Europe. It has been introduced to North America where it has often established dense stands in both secondary woodlands and relatively undisturbed mature woodlands. In Europe A. platanoides is also extending its original range, but generally seems to exist at much lower densities. One explanation for the ‘aggressiveness’ of invasive plants such as A. platanoides is that they have left behind pests and diseases which limit their population densities in their native lands (the enemy release hypothesis or ERH). To assess the ERH for Norway maple, a large network of collaborators assessed leaf herbivory rates in populations throughout Europe and North America. We found significantly lower total leaf herbivory (1.6% ± 0.19, n = 21 vs. 7.4% ± 1.94, n = 34) and lower fungal damage (1.0% ± 0.35, n = 13 vs. 3.7% ± 0.85, n = 34) in North America than in Europe over a 2 year period, which is consistent with the predictions of the Enemy Release Hypothesis. Across years, the average total leaf herbivory was significantly correlated with average annual temperature of the site (P < 0.05), although this was mostly due to sites in Europe (P < 0.001), and not sites in North America (P > 0.05). Furthermore, only populations in Europe showed very high levels of herbivory (e.g., nine sites had total leaf herbivory ranging from 10.0 to 51.2% in at least 1 year) or leaf fungal damage (only one site in North America showed high levels of fungal damage in 1 year), suggesting the possibility of more frequent episodic outbreaks in the native range. Leaf herbivory and fungal damage are only two aspects of consumer pressure and we do not know whether the differences reported here are enough to actually elicit release from top-down population control, but such large scale biogeographic differences in herbivory contribute towards understanding exotic invasions. Jonathan M. Adams and Wei Fang—equally contributed as first authors. A list of the participating members of the Transatlantic Acer platanoides Invasion Network is given in the Appendix 3.     

Use of Galactosyltransferase to Assess the Biological Function of O-linked N-Acetyl- -Glucosamine: A Potential Role for O-GlcNAc during Cell Division

Many cytosolic and nuclear proteins are modified by monomeric O-linked N-acetyl- -glucosamine (O-GlcNAc). The biological functions of this form of glycosylation are unclear but evidence suggests that it heightens regulation of protein function. To assess the biological function of O-GlcNAc addition, we examined the biological effects of galactosyltransferase (GalT) microinjected into the cytoplasm of Xenopus ovarian oocytes. GalT, which catalyzes β1-4-galactose addition to O-GlcNAc, should inhibit deglycosylation and lectin-like interactions requiring unmodified O-GlcNAc residues. Although GalT injection into diplotene-arrested oocytes has no detectable effects on cell viability, it is toxic to oocytes entering meiosis. Cell-cycle-specific toxicity is recapitulated in vitro as GalT inhibits formation of nuclei and microtubule asters from cell-free extracts of ovulated frog eggs. These observations suggest that regulation of O-GlcNAc is important for cell cycle progression and may be important in diseases in which O-GlcNAc metabolism is abnormal. The methods described here outline a viable experimental scheme for ascribing a biological function to this form of glycosylation.     

Synonymous codon usage bias and overexpression of a synthetic gene encoding Interferon α2b in yeast

To achieve higher level expression of Interferon α2b (IFN-α2b) in methylotrophic yeast (Pichia pastoris), a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into P. pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut⁺ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allowed expression of the IFN-α2b transformant up to 810 mg/L as detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/ VSV system, which was 3.3×10⁵ IU/mL. Foundation items: The National ‘973’ Basic Research Program (2002CB111302); The National Natural Science Foundation of China (30370807)     

A new role of Deinococcus radiodurans RecD in antioxidant pathway

In Deinococcus radiodurans, RecBCD holoenzyme is not intact because of the absence of RecB and RecC, but a RecD-like protein does indeed exist. In this work, D. radiodurans recD disruptant was constructed and its possible biological functions were investigated. The results showed that disruption of the recD gene of D. radiodurans resulted in a remarkably increased sensitivity to hydrogen peroxide but had no apparent effect on the resistance to gamma and UV radiation. Furthermore, complementation experiments showed that Escherichia coli RecD, helicase domain or N-terminal domain of D. radiodurans RecD could not individually restore the resistant phenotype to hydrogen peroxide of the recD disruptant, whereas the complete D. radiodurans RecD protein could. Further studies showed that D. radiodurans RecD took part in antioxidant process by stimulating catalase activity and reactive oxygen species scavenging activity in D. radiodurans. These results suggest that D. radiodurans RecD has a new role in the antioxidant pathway.     

Mitochondrial diversity in European and Chinese pigs is consistent with population expansions that occurred prior to domestication

Mitochondrial DNA (mtDNA) diversity in European and Asian pigs was assessed using 1536 samples representing 45 European and 21 Chinese breeds. Diagnostic nucleotide differences in the cytochrome b (Cytb) gene between the European and Asian mtDNA variants were determined by pyrosequencing as a rapid screening method. Subsequently, 637bp of the hypervariable control region was sequenced to further characterize mtDNA diversity. All sequences belonged to the D1 and D2 clusters of pig mtDNA originating from ancestral wild boar populations in Europe and Asia, respectively. The average frequency of Asian mtDNA haplotypes was 29% across European breeds, but varied from 0 to 100% within individual breeds. A neighbour-joining (NJ) tree of control region sequences showed that European and Asian haplotypes form distinct clusters consistent with the independent domestication of pigs in Asia and Europe. The Asian haplotypes found in the European pigs were identical or closely related to those found in domestic pigs from Southeast China. The star-like pattern detected by network analysis for both the European and Asian haplotypes was consistent with a previous demographic expansion. Mismatch analysis supported this notion and suggested that the expansion was initiated before domestication.     

Studies on the interaction mechanism between hexakis(imidazole) manganese(II) terephthalate and DNA and preparation of DNA electrochemical sensor

The interaction between hexakis(imidazole) manganese(II) terephthalate ([Mn(Im)(6)](teph).4H(2)O) and salmon sperm DNA in 0.2M pH 2.30 Britton-Robinson buffer solution was studied by fluorescence spectroscopy and cyclic voltammetry. Increasing fluorescence was observed for [Mn(Im)(6)](2+) with DNA addition, while quenching fluorescence phenomenon appeared for EB-DNA system when [Mn(Im)(6)](2+) was added. There were a couple quasi-reversible redox peaks of [Mn(Im)(6)](2+) from the cyclic voltammogram on the glassy carbon electrode. The peak current of [Mn(Im)(6)](2+) decreased with positive shift of the formal potential in the presence of DNA compared with that in the absence of DNA. All the experimental results indicate that [Mn(Im)(6)](2+) can bind to DNA mainly by intercalative binding mode. The binding ratio of the DNA-[Mn(Im)(6)](2+) association complex is calculated to be 1:1 and the binding constant is 4.44x10(3) M(-1). By using [Mn(Im)(6)](teph).4H(2)O as the electrochemical hybridization indicator, the DNA electrochemical sensor was prepared by covalent interaction and the selectivity of ssDNA modified electrode were described. The results demonstrate the use of electrochemical DNA biosensor in the determination of complementary ssDNA.     

Notch signaling in ocular vasculature development and diseases

Ocular angiogenesis, characterized by the formation of new blood vessels in the avascular area in eyes, is a highly coordinated process involved in retinal vasculature formation and several ocular diseases such as age-related macular degeneration, proliferative diabetic retinopathy and retinopathy of prematurity. This process is orchestrated by complicated cellular interactions and vascular growth factors, during which endothelial cells acquire heterogeneous phenotypes and distinct cellular destinations. To date, while the vascular endothelial growth factor has been identified as the most critical angiogenic agent with a remarkable therapeutic value, the Notch signaling pathway appears to be a similarly important regulator in several angiogenic steps. Recent progress has highlighted the involvement, mechanisms and therapeutic potential of Notch signaling in retinal vasculature development and pathological angiogenesis-related eye disorders, which may cause irreversible blindness.