N-Terminal Acetylation of Two Major Latex Proteins from Arabidopsis thaliana Using Electrospray Ionization Tandem Mass Spectrometry

The primary structure of two proteins named major latex protein in Arabidopsis thaliana were characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometer and Nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) after two-dimensional gel electrophoresis separation. We revealed that the two proteins with the same N termini and the N-terminal alanine were acetylated after methionine cleavage by fragmentation of three doubly charged peptides using a quadrupole-time of flight 2 tandem mass spectrometer. It was worth noting that one peptide with sodium addition and acetylation was sequenced. It is usually difficult to analyze the peptide sequence of sodium adduct due to the 22-Da increment. The two proteins are highly homologous, and both their N-terminal and C-terminal peptides were sequenced. Of the two proteins, gi|15236568 (spot A) appears only in the seeding stage and flower organ, but gi|15236566 (spot B) appears throughout the whole life of A. thaliana. The biological mechanism of the two proteins and the function of N-terminal acetylation remain to be elucidated. This study showed that ESI-MS/MS was a powerful tool for the characterization of N-terminal acetylation of proteins.     

Molecular characterization of heat shock protein 70 genes in the liver of three warm freshwater fishes with differential tolerance to microcystin-LR

Heat shock protein 70 (HSP70) protect cell from oxidative stress by preventing the irreversible loss of vital proteins and facilitating their subsequent regeneration. Silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idellus), and Nile tilapia (Oreochromis nilotica) are three warm freshwater fishes with differential tolerance to microcystin-LR (MC-LR). Full-length cDNAs encoding the HSP70 were cloned from the livers of the three fishes. The HSP70 cDNAs of silver carp, grass carp, and Nile tilapia were 2356, 2348, and 2242 bp in length and contained an open-reading frame of 1950 bp (encoding a polypeptide of 649 amino acids), 1950 bp (649 amino acids), and 1917 bp (638 amino acids), respectively. Like mammalian HSP70, the HSP70 of the three fish was also composed of an ATPase domain from residues 1 to 383 (44 kDa), substrate peptide binding domain from residues 384 to 544 (18 kDa), and a C-terminus domain from residues 545 to 649 (10 kDa). The relatively high conservation of HSP70 sequences among different vertebrates is consistent with their important role in fundamental cellular processes. Using beta-actin as an external control, RT-PCR within the exponential phase was conducted to determine the constitutive and inducible expression level of HSP70 gene among the three fishes (6-12 g) intraperitoneally injected with MC-LR (50 μg kg(-1) body weight). Both constitutive and inducible liver mRNA levels of the fish HSP70 genes showed positive relationships with their tolerance to MC-LR: highest in Nile tilapia, followed by silver carp, and lowest in grass carp. The differential expression pattern of liver HSP70 genes in the three fish indicated a potential role of HSP70 in the detoxification process of MC-LR.     

The effect of electrical field strength on activation and development of cloned caprine embryos

The activation procedure used in nuclear transfer (NT) is one of the critical factors affecting the efficiency of animal cloning. The purpose of this study was to compare the effect of two electrical field strengths (EFS) for activation on the developmental competence of caprine NT embryos reconstructed from ear skin fibroblasts of adult Alpine does. The NT embryos were obtained by transfer of the quiescent fibroblasts at the fourth passage into the enucleated metaphase II (M II) oocytes. Four to five hours after electrical fusion, the NT-embryos were activated by EFS either at 1.67 or at 2.33 kV/cm and immediately incubated in 6-DMAP (2 mM) for 4 h. The cleavage rate of the NT-embryos activated with 2.33 kV/cm was greater than that activated with 1.67 kV/cm after in vitro culture for 18 h (65.6% versus 19.6%, p < 0.001). No pregnancy was found in 14 recipient does after transferring 51 NT embryos at 1-2 cell stages activated with 1.67 kV/cm. In contrast, two of the seven recipients were pregnant and gave birth to three kids after transferring 61 NT embryos at 1-2 cell stages activated by 2.33 kV/cm. The birth weights of three cloned kids were within the normal range of Alpine goats. However, one kid died 1h after birth while the remaining two are still healthy. DNA analysis by polymerase chain reaction (single-strand conformation polymorphism, SSCP) confirmed that the three kids were genetically identical to the nuclear donor.     

Allelopathic potential of sweet potato (Ipomoea batatas) germplasm resources of Yunnan Province in southwest China

A laboratory bioassay was conducted to determine the allelopathic potentials of aqueous extracts from either roots or leaves of seventeen sweet potato [Ipomoea batatas L. (Lam)] cultivars (SP0, SP1, SP2, SP3, SP4, SP5, SP6, SP7, SP9, SP10, SP11, SP13, SP14, SP15, SP16, SP18, and SP19). Most inhibitory rates on Lactuca sativa calculated for leaf or root extracts from the seventeen sweet potato cultivars exhibited positive values and significantly increased with increasing concentration. Germination was totally inhibited at a concentration of 0.05 g·mL^?1 for leaf water extracts of SP13, SP15, SP18 and at a concentration of 0.05 g·mL^?1 for both leaf and root water extracts of SP19. Inhibition of root length was clearly greater than inhibition of shoot length for both leaf and root water extracts. Biomass inhibition increased with increasing concentration, but some cultivars showed stimulatory effects at low concentrations, and inhibition was generally more pronounced for root water extracts than for leaf water extracts. Moreover, most synthetical inhibitory rates for both leaf and root water extracts from the seventeen cultivars exhibited positive values and significantly increased with increasing concentration. Comparing the synthetical inhibitory rates for both leaf and root water extracts among the seventeen cultivars, SP19, SP6, SP11, and SP7 had the highest allelopathic inhibition. The inhibitory activity on germination index was the greatest, followed by germination rate, root length, biomass, and shoot length in all bioassays. Inhibition by leaf water extracts was generally greater than inhibition by root water extracts, except in the case of shoot length or biomass. Overall, we conclude that all seventeen sweet potato cultivars have strong inhibitory effects on L. sativa, but that these effects vary with cultivar and plant part, with SP19, SP6, SP11, and SP7 exhibiting the highest rates of allelopathic inhibition.     

SNP mapping of QTL affecting growth and fatness on chicken GGA1

An F2 chicken population was established from a crossbreeding between a Xinghua line and a White Recessive Rock line. A total of 502 F2 chickens in 17 full-sib families from six hatches was obtained, and phenotypic data of 488 individuals were available for analysis. A total of 46 SNP on GGA1 was initially selected based on the average physical distance using the dbSNP database of NCBI. After the polymorphism levels in all F0 individuals (26 individuals) and part of the F1 individuals (22 individuals) were verified, 30 informative SNP were potentially available to genotype all F2 individuals. The linkage map was constructed using Cri-Map. Interval mapping QTL analyses were carried out. QTL for body weight (BW) of 35 d and 42 d, 49 d and 70 d were identified on GGA1 at 351–353 cM and 360 cM, respectively. QTL for abdominal fat weight was on GGA1 at 205 cM, and for abdominal fat rate at 221 cM. Two novel QTL for fat thickness under skin and fat width were detected at 265 cM and 72 cM, respectively.     

Incidence and Risk of Treatment-Related Mortality with mTOR Inhibitors Everolimus and Temsirolimus in Cancer Patients: A Meta-Analysis

Background

Two novel mammalian targets of rapamycin (mTOR) inhibitors everolimus and temsirolimus are now approved by regulatory agencies and have been widely investigated among various types of solid tumors, but the risk of fatal adverse events (FAEs) with these drugs is not well defined.

Methods

We searched PubMed, EMBASE, and Cochrane library databases for relevant trials. Eligible studies included prospective phase II and III trials evaluating everolimus and temsirolimus in patients with all malignancies and data on FAEs were available. Statistical analyses were conducted to calculate the summary incidence, RRs and 95% confidence intervals (CIs) by using either random effects or fixed effect models according to the heterogeneity of the included studies.

Results

A total of 3322 patients with various advanced solid tumors from 12 trials were included. The overall incidence of mTOR inhibitors associated FAEs was 1.8% (95%CI: 1.3–2.5%), and the incidences of everolimus related FAEs were comparable to that of temsirolimus (1.7% versus 1.8%). Compared with the controls, the use of mTOR inhibitors was associated with an increased risk of FAEs, with a RR of 3.24 (95%CI: 1.21–8.67, p = 0.019). On subgroup analysis, a non-statistically significant increase in the risk of FAEs was found according to different mTOR inhibitors, tumor types or controlled therapy. No evidence of publication bias was observed.

Conclusion

With the present evidence, the use of mTOR inhibitors seems to increase the risk of FAEs in patients with advanced solid tumors. More high quality trials are still needed to investigate this association.     

The LRRK2-related Roco kinase Roco2 is regulated by Rab1A and controls the actin cytoskeleton

We identify a new pathway that is required for proper pseudopod formation. We show that Roco2, a leucine-rich repeat kinase 2 (LRRK2)-related Roco kinase, is activated in response to chemoattractant stimulation and helps mediate cell polarization and chemotaxis by regulating cortical F-actin polymerization and pseudopod extension in a pathway that requires Rab1A. We found that Roco2 binds the small GTPase Rab1A as well as the F-actin cross-linking protein filamin (actin-binding protein 120, abp120) in vivo. We show that active Rab1A (Rab1A-GTP) is required for and regulates Roco2 kinase activity in vivo and that filamin lies downstream from Roco2 and controls pseudopod extension during chemotaxis and random cell motility. Therefore our study uncovered a new signaling pathway that involves Rab1A and controls the actin cytoskeleton and pseudopod extension, and thereby, cell polarity and motility. These findings also may have implications in the regulation of other Roco kinases, including possibly LRRK2, in metazoans.     

Centrosomal protein FOR20 is essential for S-phase progression by recruiting Plk1 to centrosomes

Centrosomes are required for efficient cell cycle progression mainly by orchestrating microtubule dynamics and facilitating G1/S and G2/M transitions. However, the role of centrosomes in S-phase progression is largely unknown. Here, we report that depletion of FOR20 (FOP-related protein of 20 kDa), a conserved centrosomal protein, inhibits S-phase progression and prevents targeting of Plk1 (polo-like kinase 1) to centrosomes, where FOR20 interacts with Plk1. Ablation of Plk1 also significantly induces S-phase defects, which are reversed by ectopic expression of Plk1, even a kinase-dead mutant, but not a mutant that fails to localize to centrosomes. Exogenous expression of centrosome-tethered Plk1, but not wild-type Plk1, overrides FOR20 depletion-induced S-phase defects independently of its kinase activity. Thus, these data indicate that recruitment of Plk1 to centrosomes by FOR20 may act as a signal to license efficient progression of S-phase. This represents a hitherto uncharacterized role of centrosomes in cell cycle regulation.     

Detecting Protein Candidate Fragments Using a Structural Alphabet Profile Comparison Approach

Predicting accurate fragments from sequence has recently become a critical step for protein structure modeling, as protein fragment assembly techniques are presently among the most efficient approaches for de novo prediction. A key step in these approaches is, given the sequence of a protein to model, the identification of relevant fragments - candidate fragments - from a collection of the available 3D structures. These fragments can then be assembled to produce a model of the complete structure of the protein of interest. The search for candidate fragments is classically achieved by considering local sequence similarity using profile comparison, or threading approaches. In the present study, we introduce a new profile comparison approach that, instead of using amino acid profiles, is based on the use of predicted structural alphabet profiles, where structural alphabet profiles contain information related to the 3D local shapes associated with the sequences. We show that structural alphabet profile-profile comparison can be used efficiently to retrieve accurate structural fragments, and we introduce a fully new protocol for the detection of candidate fragments. It identifies fragments specific of each position of the sequence and of size varying between 6 and 27 amino-acids. We find it outperforms present state of the art approaches in terms (i) of the accuracy of the fragments identified, (ii) the rate of true positives identified, while having a high coverage score. We illustrate the relevance of the approach on complete target sets of the two previous Critical Assessment of Techniques for Protein Structure Prediction (CASP) rounds 9 and 10. A web server for the approach is freely available at http://bioserv.rpbs.univ-paris-diderot.fr/SAFrag.     

Low concentrations of NaHSO3 increase photosynthesis, biomass, and attenuate photoinhibition in Satsuma mandarin (Citrus unshiu Marc.) plants

Spraying low concentrated (0.5–5.0 mM) solutions of NaHSO₃ on Satsuma mandarin (Citrus unshiu Marc.) leaves resulted in enhancement (maximal about 15 % at 1 mM NaHSO₃) of net photosynthetic rate (P _N) for 6 d. The potential photochemical efficiency of photosystem 2 (PS2, F_v/F_m) and the quantum yield of PS2 electron transport (Φ_PS2) were increased under strong photon flux density (PFD). The slow phase of millisecond delayed light emission (ms-DLE) was increased, showing that the transmembrane proton motive force related to photophosphorylation was enhanced. We also observed that low concentrations of NaHSO₃ promoted the production of ATP in irradiated leaves. We suggest that the increase in P _N in Satsuma mandarin leaves caused by low concentrations of NaHSO₃ solution may have been due to the stimulation of photophosphorylation and, hence, the increase in photochemical efficiency through speeding-up of PS2 electron transport. Photoinhibition of photosynthesis in leaves was modified by NaHSO₃ treatment under high PFD. Hence the increase in leaf dry mass seems to be associated with the mitigation of photoinhibition caused by strong PFD.