Insights into the susceptibility of raspberries to Drosophila suzukii oviposition

The spotted wing drosophila (SWD) causes massive losses in red raspberry (Rubus idaeus L.) cultivation by direct oviposition leading to damages of the soft skin fruits. Knowledge of the fly's host preference could help farmers in managing the pest. We used a laboratory-based oviposition assay for screening the germplasm of Rubus to ascertain whether the spotted wing drosophila prefers certain cultivars to others for oviposition and if preference is based on citric acid and Brix content or firmness. Correlation analyses of evaluated characters with no-choice tests results in 3 years obtained no correlation between citric acid, Brix content and oviposition. Primocane raspberries were lower affected by SWD than floricane raspberries. The Rubus hybrid cultivar “Dorman Red” and the primocane cultivar “Pokusa” showed the lowest oviposition rate compared to the other 58 evaluated genotypes. We found that oviposition correlates to firmness of the investigated cultivars, which strongly indicates that host preference is partly connected to that character in raspberries. The results are discussed regarding the use of Rubus genetic resources in breeding and integrated pest management to control spotted wing drosophila in the field.     

Isolation and Characterization of Mustard (Sinapis alba L.) Seed Storage Proteins

A total storage protein fraction was prepared from mustard (Sinapis alba L.) seeds via isolated protein bodies and characterized by sedimentation, immunological, and electrophoretic techniques. Mustard seed storage protein consists of three fractions (1) a “legumin-like” 13-S complex composed of two pairs of disulfide-linked polypeptides (16.5 + 28.5 kDa and 19.5 + 34 kDa, respectively) and two single polypeptides (18 kDa and 26 kDa), (2) a “vicilin-like” 9-S complex composed of two glycoproteins (64 kDa and 77 kDa), and (3) two small polypeptides (10 kDa and 11 kDa) which probably represent the 1.7-S complex found in other Cruciferae. In contrast to related species, no glycosylated polypeptide was found in the 13-S complex. Immunological relationships were found between the paired polypeptides of the 13-S complex but not between polypeptides of the 13-S complex and polypeptides of the 9-S complex. Pulse-chase labeling and in vitro translation of polysomal RNA from young embryos demonstrated that the polypeptides of the 13-S complex originate from high molecular mass precursors, except for the 18 kDa polypeptide which appears to be synthesized in its final size. The amino-acid composition of the major polypeptides of the mustard storage protein is given.     

Production of HIV-1 p24 protein in transgenic tobacco plants

The production of antigens for vaccines in plants has the potential as a safe and cost-effective alternative to traditional production systems. Toward the development of a plant-based expression system for the production of human immunodeficiency virus type I (HIV-1) p24 capsid protein, the p24 gene was introduced into the genome of tobacco plants using Agrobacterium tumefaciens-mediated gene transfer. Southern blot analyses confirmed the presence of the p24 coding sequence within the genome of transgenic lines. Western blot analysis of protein extracts from transgenic plants identified plant-expressed p24 protein that cross-reacted with a p24-specific monoclonal antibody, thus confirming the maintenance of antigenicity. Quantification of the p24 protein using enzyme-linked immunosorbent assay (ELISA) estimated yields of approx 3.5 mg per g of soluble leaf protein. Similar accumulation levels of p24 were also detected in T1 plants, confirming that the p24 gene is transmitted stably. Our results indicate that plant-based transgenic expression represents a viable means of producing p24 for the development of HIV vaccine and for use in HIV diagnostic procedures.     

Inhibition of P38 MAPK Reduces Tumor Conditioned Medium-Induced Angiogenesis in Co-Cultured Human Umbilical Vein Endothelial Cells and Fibroblasts

Tumor conditioned medium (CM) has been widely used to stimulate endothelial cells to form capillary-like structures in in vitro angiogenesis models. We report herein the effect of HT1080 and A549 CM after they were mixed with microvascular endothelial cells medium-2 (EGM-2) on angiogenesis in human umbilical vein endothelial cells (HUVECs). Both HT1080 and A549 CM decreased HUVEC proliferation, to different extents. While A549 CM significantly increased capillary-like structure formation in a co-culture system, no effect of HT1080 was apparent. Inhibition of p38 mitogen-activated protein kinase (MAPK) blocked both basal and A549 CM induced capillary-like structure formation, but inhibition of extracellular signal-regulated kinases (ERK) and that of c-Jun N-terminal protein kinases (JNK) MAPK had no such effect. Activation of ERK MAPK was inhibited by both CMs, whereas p38 MAPK was inactivated by HT1080 and activated by A549 CM and a control. Neither CM had an effect on JNK MAPK. The results suggest that p38 MAPK played a critical role in capillary-like structure formation in the co-culture, partly via promotion of apoptosis in HUVECs.     

Molecular phylogeny and systematics of drosophila retrotransposons and retroviruses

Full classification of Drosophila melanogaster retrotransposons with long terminal repeats (LTR-retrotransposons) has been recomposed, and their evolutional analysis in sequenced genomes of different species of drosophila and other arthropods has been carried out. D. melanogaster LTR-retrotransposons are divided into three groups: gypsy (one, two, or three open reading frames (ORFs)), copia (one ORF), and BEL (one ORF). The gypsy group is divided into three subgroups. Subgroup I is underrepresented by retrotransposons-retroviruses with three ORFs and their derivatives, which have lost the env gene (ORF3). Subgroup II is underrepresented by retrotransposons with two ORFs, and subgroup III is underrepresented by retrotransposons with one ORF. A comparative analysis of homologs of gypsy group LTR-retrotransposons evidences that subgroups I and II are represented only in the genomes of Lepidoptera and Diptera. The gypsy group of LTR-retrotransposons with one and two ORFs is found in almost all genomes of arthropods. Most of the families of D. melanogaster gypsy group LTR-retrotransposons have close homologs in the genomes of other species of drosophila. A degree of identity of retrotransposons sequences is correlated with a degree of relation between species of drosophila, indicating vertical transmission of retrotransposons. Obvious cases of horizontal transfer of some mobile elements have been detected including retrotransposons without the env gene. Homologs of distinct ORFs of retrotransposons—genes gag and env—have been found. Gene-homolog of the gag gene—Grp (CG5680)—is under purifying selection, so it has an important function in drosophila genome.     

A functional variant in ST2 gene is associated with risk of hypertension via interfering MiR‐202‐3p

Recent studies have suggested that interleukin 1 receptor‐like 1 (ST2) plays a critical role in pathogenesis of several cardiovascular disease conditions. In this study, we examined association of 13 single nucleotide polymorphisms (SNPs) of ST2 gene with essential hypertension (EH) risk in 1151 patients with EH and 1135 controls. Our study showed that variants rs11685424, rs12999364 and rs3821204 are highly associated with an increase in risk of EH, while rs6543116 is associated with a decrease risk of EH. Notably, in silico analyses suggested the G>C change of rs3821204, which located within the 3′UTR of soluble ST2 mRNA, disrupted a putative binding site for miR202‐3p. Functional analyses suggested that miR‐202‐3p significantly decreased soluble ST2‐G mRNA stability and inhibited its endogenous expression. Furthermore, we found increased plasma‐soluble ST2 (sST2) level was highly associated with CC genotype of rs3821204 in vivo. Taken together, our findings provide the first evidence that genetic variants in ST2 gene are associated with EH risk and variant rs3821204 may influence the development of EH by controlling sST2 expression.     

A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study

Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in drosophila.Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent.To obtain information about the IDR regions, we studied full-length SuFu’s structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.     

Long-term engraftment of p18INK4C-deficient hematopoietic stem cells is enhanced in the sublethally-irradiated recipients

Non-myeloablative regimens for host conditioning have been widely used in clinical hematopoietic stem cell transplantation due to their reduced toxicity on the recipients. But a milder conditioning regimen may require a higher engrafting ability of donor stem cells in competing with endogenous stem cells. Thus, new strategies for enhancing the competitiveness of donor stem cells in non-myeloablative recipients would have important implications for current clinical stem cell transplantation. It is known that the absence of p18 ^INK4C (p18) gene can enhance the self-renewal potential of hematopoietic stem cells (HSCs). We applied the approach of competitive bone marrow transplantation to evaluate the impact of p18 gene deletion on long-term engraftment of HSCs in sublethally irradiated hosts. We found that p18 ^−/− HSCs had a significant advantage over wild-type HSCs during long-term engraftment in the mouse recipients that received a sub-lethal irradiation (5-Gy). The engraftment efficiency of p18 ^−/− HSCs in the sub-lethally irradiated recipients was similar to that in the lethally irradiated (10-Gy) recipients. Our current study demonstrates that enhanced engraftment of donor HSCs in the absence of p18 does not strictly depend on the dose of irradiation used for host conditioning. Therefore, p18 might serve as a potential drug target for increasing the efficacy of stem cell transplant in the patients that are preconditioned with either a myeloablative or non-myeloablative regimen.     

Human ribosomal protein S13 promotes gastric cancer growth through down‐regulating p27Kip1

Our previous works revealed that human ribosomal protein S13 (RPS13) was up‐regulated in multidrug‐resistant gastric cancer cells and overexpression of RPS13 could protect gastric cancer cells from drug‐induced apoptosis. The present study was designed to explore the role of RPS13 in tumorigenesis and development of gastric cancer. The expression of RPS13 in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemical staining and Western blot analysis. It was found RPS13 was expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa. RPS13 was then genetically overexpressed in gastric cancer cells or knocked down by RNA interference. It was demonstrated that up‐regulation of RPS13 accelerated the growth, enhanced in vitro colony forming and soft agar cologenic ability and promoted in vivo tumour formation potential of gastric cancer cells. Meanwhile, down‐regulation of RPS13 in gastric cancer cells resulted in complete opposite effects. Moreover, overexpression of RPS13 could promote G1 to S phase transition whereas knocking down of RPS13 led to G1 arrest of gastric cancer cells. It was further demonstrated that RPS13 down‐regulated p27^kip1 expression and CDK2 kinase activity but did not change the expression of cyclin D, cyclin E, CDK2, CDK4 and p16^INK4A. Taken together, these data indicate that RPS13 could promote the growth and cell cycle progression of gastric cancer cells at least through inhibiting p27^kip1 expression.     

Analysis of Npl4 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Npl4 in ERAD

Npl4 is a 67 kDa protein forming a stable heterodimer with Ufd1, which in turn binds the ubiquitous p97/VCP ATPase. According to a widely accepted model, VCP^Ufd1–Npl4 promotes the retrotranslocation of emerging ER proteins, their ubiquitination by associated ligases, and handling to the 26S proteasome for degradation in a process known as ERAD (ER-associated degradation). Using a series of Npl4 deletion mutants we have revealed that the binding of Ufd1 to Npl4 is mediated by two regions: a conserved stretch of amino acids from 113 to 255 within the zf-Npl4 domain and by the Npl4 homology domain between amino acids 263 and 344. Within the first region, we have identified two discrete subdomains: one involved in Ufd1 binding and one regulating VCP binding. Expression of any one of the mutants failed to induce any changes in the morphology of the ER or Golgi compartments. Moreover, we have observed that overexpression of all the analyzed mutants induced mild ER stress, as evidenced by increased Grp74/BiP expression without associated XBP1 splicing or induction of apoptosis. Surprisingly, we have not observed any accumulation of the typical ERAD substrate αTCR. This favors the model where the Ufd1–Npl4 dimer forms a regulatory gate at the exit from the retrotranslocone, rather than actively promoting retrotranslocation like the p97VCP ATPase.     

Hairless regulates p53 target genes to exert tumor suppressive functions in glioblastoma

Glioblastoma (GBM) is the most common malignant brain tumor and is associated with a poor prognosis, with most patients living less than a year after diagnosis. Given that GBM nearly always recurs after conventional treatments, there is an urgent need to identify novel molecular targets. Hairless (HR) is a nuclear factor enriched in the skin and has been previously implicated in hair cycling. HR is also highly expressed in the brain, but its significance is unknown. We found that human hairless gene (HR) expression is significantly decreased in all GBM subtypes compared with normal brain tissue and is predictive of prognosis, which suggests that loss of HR expression can contribute to GBM pathogenesis. HR was recently discovered to bind to and regulate p53 responsive elements, and thus we hypothesized that HR may have a tumor suppressive function in GBM by modulating p53 target gene expression. We found that HR indeed regulates p53 target genes, including those implicated in cell cycle progression and apoptosis in the GBM-derived U87 cell line, and restoring HR expression triggered G2/M arrest and apoptosis. An analysis of sequenced genomes from patients with GBM revealed 10 HR somatic mutations in patients with glioma, two of which are located in the histone demethylase domain of HR. These two mutations, P996S and K1004N, were reconstructed and found to have impaired p53 transactivating properties. Collectively, the results of our study suggest that HR has tumor suppressive functions in GBM, which may be clinically relevant and a potential avenue for therapeutic intervention.     

Syntaxin 17 cycles between the ER and ERGIC and is required to maintain the architecture of ERGIC and Golgi

Background information. Syntaxin 17 is a SNARE (soluble N‐ethylmaleimide‐sensitive‐factor‐attachment protein receptor) protein that predominantly localizes to the ER (endoplasmic reticulum) and to some extent in the ERGIC (ER—Golgi intermediate compartment). Syntaxin 17 has been suggested to function as a receptor at the ER membrane that mediates trafficking between the ER and post‐ER compartments. It has a unique 33 amino acid luminal tail whose function is not known. Here we have investigated the structural requirements for localization of syntaxin 17 to the ERGIC and its role in trafficking. Results. Deletion analysis showed that syntaxin 17 required its cytoplasmic domain to exit the ER and localize to the ERGIC. Mutation of a conserved tyrosine residue in the cytoplasmic domain resulted in reduced localization of syntaxin 17 in the ERGIC and ER‐exit sites, suggesting the presence of a tyrosine‐based ER export motif. Syntaxin 17 also required its C‐terminal tail to localize to the ERES (ER exit sites) and ERGIC. Knockdown of syntaxin 17 destabilized the ERGIC organization and also caused fragmentation of the Golgi complex. Syntaxin 17 showed direct interaction with transmembrane proteins p23 and p25 (cargo receptors that cycle between the ER and Golgi) with the help of its C‐terminal tail. Overexpression of syntaxin 17 redistributed β‐COP (β‐coatomer protein) which required its C‐terminal tail. Overexpression of syntaxin 17 also blocked the anterograde transport of VSVG (vesicular stomatitis virus G‐protein) in the ERGIC. Conclusions. We show that syntaxin 17 has a tyrosine‐based motif which is required for its incorporation into COPII (coatomer protein II) vesicles, exit from the ER and localization to the ERGIC. Our results suggest that syntaxin 17 cycles between the ER and ERGIC through classical trafficking pathways involving COPII and COPI (coatomer protein I) vesicles, which requires its unique C‐terminal tail. We also show that syntaxin 17 is essential for maintaining the architecture of ERGIC and Golgi.     

Immunological characterization of chlorophyll a/b-binding proteins of barley thylakoids

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCI) using a photosystem (PS) I preparation (PSI-200) wild-type from barley (Hordeum vulgare L. cv. Svaløf's Bonus) as the antigen. These antibodies cross-reacted with a minor light-harvesting chlorophyll a/b-protein of PSII (Chla/b-P1=CP29), but not with the major one, LHCII (=Chla/b-P2^**). Similarly, a monoclonal antibody to Chla/b-P1, elicited by a PSII preparation as the antigen, cross-reacted with LHCI, but not LHCII. This explains why an antigen consisting of LHCII, free of LHCI, but contaminated with Chla/b-P1, can elicit antibodies which cross-react with LHCI. Immunoblot assays showed that LHCI and Chla/b-P1 have at least two epitopes in common. Immunogold labelling of thin-sectioned wild-type thylakoids confirmed a preferential localisation of Chla/b-P1 in grana partition membranes and LHCI in stroma lamellae. The presence of LHCI was demonstrated in barley mutants lacking the PSI reaction centre (viridis-zb ⁶³) and chlorophyll b (chlorina-f2), and was correlated with the presence of long-wavelength (730 nm) fluorescence emission at 77 K. The mutant viridis-k ²³, which has a 77 K long-wavelength fluorescence peak at 720 nm, was shown by immune-blot assay to lack LHCI, although Chla/b-P1 was present.Abbreviations Chl-P chlorophyll-protein - CM Carlsberg Monoclonal - Da dalton - LHC light-harvesting complex - PAGE polyacrylamide gel electrophoresis - PSI, II photosystem I, II - PSI-200 PSI containing LHCI polypeptides - SDS sodium dodecyl sulphate     

Subcellular localization and mechanisms of nucleocytoplasmic distribution of p202, an interferon-inducible candidate for lupus susceptibility

Increased expression of p202 (52 kDa), an interferon (IFN)-inducible murine protein, in splenic cells (B- and T-cells) derived from female mice of the lupus-prone strains is correlated with increased susceptibility to develop systemic lupus erythematosus. However, the molecular mechanisms remain unclear. Our previous studies have indicated that, in IFN-treated fibroblasts, p202 is detected both in the cytoplasm and in the nucleus. Moreover, in the cytoplasm, a fraction of p202 associates with a membranous organelle. Here we report that, in the cytoplasm, a fraction of p202 associated with mitochondria. Additionally, we found that the constitutive p202 is primarily detected in the cytoplasm. Remarkably, the IFN treatment of cells potentiated nuclear accumulation of p202. Our observations are consistent with the possibility that IFN signaling regulates p202 levels as well as its nucleocytoplasmic distribution. These observations will serve as a basis to elucidate the molecular mechanisms by which p202 contributes to lupus susceptibility.     

果蝇心脏发育标记基因Kruppel的多克隆抗体制备

Kruppel在果蝇发育过程中起着重要的调控作用。为了进一步研究Kruppel的功能,需要制备Kruppel蛋白及其抗体.对已有的Kruppel序列进行分析,选取适当区域进行引物设计,从果蝇心脏cDNA文库中PCR扩增得到Kruppel部分编码区序列,并其连接到pET-28a载体上。将重组质粒（pET-28a-Kruppel）转化rosetta受体菌,通过IPTG（Isopropylβ-D-thiogalactoside）诱导表达融合蛋白,用镍柱进行亲和纯化。将纯化得到的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western blot检测抗体的效价。     

Strong Heterogeneity in Nucleotidic Composition and Codon Bias in the Pea Aphid (<Emphasis Type="Italic">Acyrthosiphon pisum)</Emphasis> Shown by EST-Based Coding Genome Reconstruction

The aim of this study was to analyze patterns of nucleotidic composition and codon usage in the pea aphid genome (Acyrthosiphon pisum). A collection of 60,000 expressed sequence tags (ESTs) in the pea aphid has been used to automatically reconstruct 5809 coding sequences (CDSs), based on similarity with known proteins and on coding style recognition. Reconstructions were manually checked for ribosomal proteins, leading to tentatively reconstruct the nea-complete set of this category. Pea aphid coding sequences showed a shift toward AT (especially at the third codon position) compared to drosophila homologues. Genes with a putative high level of expression (ribosomal and other genes with high EST support) remained more GC3-rich and had a distinct codon usage from bulk sequences: they exhibited a preference for C-ending codons and CGT (for arginine), which thus appeared optimal for translation. However, the discrimination was not as strong as in drosophila, suggesting a reduced degree of translational selection. The space of variation in codon usage for A. pisum appeared to be larger than in drosophila, with a substantial fraction of genes that remained GC3-rich. Some of those (in particular some structural proteins) also showed high levels of codon bias and a very strong preference for C-ending codons, which could be explained either by strong translational selection or by other mechanisms. Finally, genomic traces were analyzed to build 206 fragments containing a full CDS, which allowed studying the correlations between GC contents of coding and those of noncoding (flanking and introns) sequences.     

Polyethism within hunters of the ponerine ant,Ectatomma ruidum Roger (Formicidae,Ponerinae)

Summary During laboratory experiments, two categories of prey-foraging workers were found inEctatomma ruidum: stingers and transporters. When numerous live drosophila were offered to the ants, one group of hunters specialized in killing the prey and another in transporting simultaneously the dead drosophila to the nest. Sometimes, there was a transfer of prey by a stinger towards a transporter, after an active soliciting of the transporter by antennation or by using the forelegs. We found high positive correlations between the colony size and the number of ants in each subcaste. A negative correlation existed between the colony size and the proportion of hunters. However, the proportion of workers in the two behavioral subcastes of hunters was stable in spite of differences in colony size. The phylogenetic interest of this type of cooperative predation is discussed.