Calcineurin B-like interacting protein kinase OsCIPK23 functions in pollination and drought stress responses in rice （Oryza sativa L.）

Droughtis very harmful to grain yield due to its adverse effect on reproduction,especially on pollination proeess in rice.However,the molecular basis of such an effect still remains largely unknown.Here,wereport the role of amember of CBL(Calcineurin B-Like)Interacting Protein Kinase(CIPK)family,OsCIPK23,in pollination and stress responses in dee.Molecular analyses revealed that it is mainly expressed in pistil and anther but up-regulated by pollination,as well as by treatments of various abiotic stresses and phytohormones.RNA interference-mediated suppression of OsCIPK23 expression significantly reduced seed set and conferred a hypersensitive response to drought stress,indicating its possible roles in pollination and drought stress.In consistent,overexpression of OsCIPK23 induced the expression of seVeral drought tolerance related genes.Taken together,these results indicate that OsCIPK23 is a multistress induced gene and likely mediatesa signaling pathway commonly shared by both pollination and drought stress responses in rice.     

Identification of quantitative trait loci for four morphologic traits under water stress in rice （Oryza sativa L.）

Late season drought coinciding with the rice booting to heading stage affects the development of plant height,panicle exsertion,and flag leaf size,and causes significant yield loss.In this study,a recombinant inbred line population derived from a cross between paddy and upland cultivars was used for data collection of the morphologic traits under well water and drought stress conditions.bought stress was applied at the stage of panicle initiation in the field in 2002 and at the booting stage in PVC pipes in 2003.The data from stress con ditions and their ratios(tait measured under stress condition/trait measured under well water condition)or differences(trait measured under stress condition minus trait measured under well water condition)were used for OTL analysis.Totally,17 and 36 QTLs for these traits were identified in 2002 and 2003,respectively,which explained a range of 2.58%-29.82%Of the phenotypic variation.Among them,six QTLs were commonly identified in the two years,suggesting that the drought stress in the two years was different.The genetic basis of these traits will provide useful information for improving rice late season drought resistance,and their application as indirect indices in rice late season drought resistance screening was also discussed.     

Leaf surface factors of transgenic Bt cotton associated with the feeding behaviors of cotton aphids: A case study on non-target effects

The present paper reports case study results of the risk assessment of transgenic Bt cotton on a non-target pest, cotton aphid, Aphis gossypii. Several types of techniques, i.e., electrical penetration graph (EPG), light and electron microscopy, bioassays and chemical analysis, were applied to investigate physical and chemical leaf factors of 2 transgenic Bt cotton lines (GK12 and GK19) and their pa-rental non-Bt cotton line (Simian3) associated with searching and feeding behaviors of cotton aphids on leaves or leaf extracts of cotton plants. EPG results showed that there were some differences among behaviors of cotton aphids on 2 Bt cotton and 1 non-Bt cotton lines. Cotton aphids performed similarly to leaf surface extracts from 3 cotton lines; and leaf surface chemicals, mainly volatiles and waxes, were almost identical in the components and concentrations among the cotton lines. However, three cotton lines were quite different from each other in the densities of certain kinds of covering trichomes. Therefore, the relationships between the physical characteristics and the searching behaviors of cotton aphids on the three cotton lines were constructed as the regression equations. Glandular trichomes and covering trichomes with 5 branches influenced the cotton aphids' searching behaviors effectively; and other trichomes with other branches affected aphids in varying ways. These results demonstrated that leaf surface physical factors of transgenic Bt cotton lines different from their parental non-Bt line could affect the penetration behaviors of non-target cotton aphids. Cotton aphids penetrate and feed more easily on two Bt cotton lines than on the non-Bt cotton line.     

Differences of ecological functions inside and outside the wetland nature reserves in Sanjiang Plain, China

Conservation effectiveness of wetland nature reserves is determined by both the management intensity inside the reserves and the hydrological status outside the reserves. Therefore, differences of ecological functions inside and outside the reserves are an integrated indictor for assessing conservation effectiveness. Based on the land use map created from Landsat-TM satellite image and 1:50000 Digital Elevation Model (DEM) data in the study area, the catchments that belong to the wetland reserves were determined as their hydrological sensitive zones by SWAT (Soil and Water Assessment Tool) hydrological model. The ecological function indices of wetland reserves and their corresponding hydrological sensitive zones were calculated through expert consultation and value assessment on wetland ecosystem service functions. The results showed that the ecological functions of national wetland reserves were better than those of local reserves in general. However, the wetland ecological functions of hydrological sensitive zones of the former were not always better than those of the latter. Meanwhile, clustering analysis showed that the wetland ecological functions of several adjacent reserves in Nongjiang-Bielahonghe watershed were similar. But correlation analysis found that there existed a remarkable positive correlation between the wetland ecological function indices of local reserves and their hydrological sensitive zones.     

Identification and Characterization of Novel MicroRNAs from Schistosoma japonicum

Background

Schistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs.

Methodology and Results

We have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18–26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host.

Conclusions

Authentic miRNAs were identified for the first time in S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development.     

Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice

Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27(-/-) mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27(-/-) mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27(-/-) mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27(-/-)and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27(-/-) mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1alpha were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27(-/-) mice. Remarkably, Fsp27(-/-) MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity.     

Activation-dependent hindrance of photoreceptor G protein diffusion by lipid microdomains

The dynamics of G protein-mediated signal transduction depend on the two-dimensional diffusion of membrane-bound G proteins and receptors, which has been suggested to be rate-limiting for vertebrate phototransduction, a highly amplified G protein-coupled signaling pathway. Using fluorescence recovery after photobleaching (FRAP), we measured the diffusion of the G protein transducin alpha-subunit (Galpha(t)) and the G protein-coupled receptor rhodopsin on disk membranes of living rod photoreceptors from transgenic Xenopus laevis. Treatment with either methyl-beta-cyclodextrin or filipin III to disrupt cholesterol-containing lipid microdomains dramatically accelerated diffusion of Galpha(t) in its GTP-bound state and of the rhodopsin-Galphabetagamma(t) complex but not of rhodopsin or inactive GDP-bound Galphabetagamma. These results imply an activity-dependent sequestration of G proteins into cholesterol-dependent lipid microdomains, which limits diffusion and exclude the majority of free rhodopsin and the free G protein heterotrimer. Our data offer a novel demonstration of lipid microdomains in the internal membranes of living sensory neurons.     

Biochemical and structural characterization of the intracellular mannanase AaManA of Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase family belonging to clan GH-A

An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-beta-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9 angstroms resolution showed that AaManA adopted a (beta/alpha)8-barrel fold. Two catalytic residues were identified: Glu151 at the C terminus of beta-stand beta4 and Glu231 at the C terminus of beta7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr95 and Cys150. We propose a model of substrate binding in AaManA in which Glu282 interacts with the axial OH-C(2) in-2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.     

Rationally designed anti-HIV peptides containing multifunctional domains as molecule probes for studying the mechanisms of action of the first and second generation HIV fusion inhibitors

We have previously shown that the first generation human immunodeficiency virus (HIV) fusion inhibitor T20 (Fuzeon) contains a critical lipid-binding domain (LBD), whereas C34, another anti-HIV peptide derived from the gp41 C-terminal heptad repeat, consists of an important pocket-binding domain (PBD), and both share a common 4-3 heptad repeat (HR) sequence (Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612-9620). T1249, the second generation HIV fusion inhibitor, has both LBD and PBD but a different HR sequence, suggesting that these three anti-HIV peptides may have distinct mechanisms of action. Here we rationally designed a set of peptides that contain multiple copies of a predicted HR sequence (5HR) or the HR sequence plus either LBD (4HR-LBD) or PBD (PBD-4HR) or both (PBD-3HR-LBD), and we compared their anti-HIV-1 activity and biophysical properties. We found that the peptide 5HR exhibited low-to-moderate inhibitory activity on HIV-1-mediated cell-cell fusion, whereas addition of LBD and/or PBD to the HR sequence resulted in a significant increase of the anti-HIV-1 activity. The peptides containing PBD, including PBD-4HR and PBD-3HR-LBD, could form a stable six-helix bundle with the N-peptide N46 and effectively blocked the gp41 core formation, whereas the peptides containing LBD, e.g. 4HR-LBD and PBD-3HR-LBD, could interact with the lipid vehicles. These results suggest that the HR sequence in these anti-HIV peptides acts as a structure domain and is responsible for its interaction with the HR sequence in N-terminal heptad repeat, whereas PBD and LBD are critical for interactions with their corresponding targets. T20, C34, and T1249 may function like 4HR-LBD, PBD-4HR, and PBD-3HR-LBD, respectively, to interact with different target sites for inhibiting HIV fusion and entry. Therefore, this study provides important information for understanding the mechanisms of action of the peptic HIV-1 fusion inhibitors and for rational design of novel antiviral peptides against HIV and other viruses with class I fusion proteins.