Preliminary studies of the effects of an induced phosphate deficiency on carbohydrate-nitrogen ratios inAlternanthera philoxeroides [Centrospermae: Amaranthacea] and on the feeding ofAgasicles hygrophila [Col.: Chrysomelidae]

In no-choice tests but not in choice tests, alligatorweed flea beetles,Agasicles hygrophila Selman & Vogt (Col.: Chrysomelidae) exposed to alligatorweed plants grown with two levels of mineral nutrition fed significantly more on those grown in full mineral nutrient than those grown with deficient phosphate. Chemical analysis showed that the full nutrient plants had more ethanol-soluble nitrogen compounds but less total carbohydrate than the phosphate-deficient plants. The response of beetles to phosphate-deficient alligatorweed may thus result from a change in the carbohydrate-nitrogen composition of host plants, though further investigation is needed for confirmation. Flea beetle response was identical to terminal and mature leaf tissue of full nutrient plants.     

Cytophysiological characteristics of Arabidopsis thaliana cultivated cells with disable perception of ethylene signal by the ETR1 receptor

Contradictory data about ethylene influence on cell growth and division prompted us to investigate cytophysiological characteristics of suspension cultures of Arabidopsis thaliana of wild type Col-0 and ert1-1 mutant carrying a point mutation in the site of ethylene binding by the ETR1 receptor. Some cytophysiological characteristics of the etr1-1 cultivated cells differed from those of Col-0: the growth rate of mutant cells was less and cell sizes were smaller, the culture was committed to the formation of tracheary elements (TE), had a pronounced modal class of nuclei (54%) with the amount of DNA 8C and a tendency to expand the ploidy toward 32C. Despite the absence of ethylene perception by the ETR1 receptor, the cell culture of mutant responded to treatment with ethylene by growth acceleration, an increase in cell viability and in the number of cells in the S-phase of the cell cycle. The inhibitor of ethylene binding to receptors, 1-methylcyclopropene, suppressed growth and viability of the cells of both genotypes. In the etr1-1 cell culture, the inhibitor reduced the number of S-phase nuclei and activated TE formation. All data obtained indicate that ethylene perception and transduction of ethylene signal are required for the maintenance of cell viability and active in vitro growth. It is supposed that the functional activity of the ETR1 receptor is necessary for optimal cell expansion, whereas other receptors are responsible for cell proliferation.     

Multi-lineage differentiation of human mesenchymal stromal cells on the biophysical microenvironment of cell-derived matrix

We obtained fibroblast- (FDM) and preosteoblast- (PDM) derived matrices in vitro from their respective cells. Our hypothesis was that these naturally occurring cell-derived matrices (CDMs) would provide a better microenvironment for the multi-lineage differentiation of human mesenchymal stromal cells (hMSCs) than those based on traditional single-protein-based platforms. Cells cultured for 5–6 days were decellularized with detergents and enzymes. The resulting matrices showed a fibrillar surface texture. Under osteogenic conditions, human bone-marrow-derived stromal cells (HS-5) exhibited higher amounts of both mineralized nodule formation and alkaline phosphatase (ALP) expression than those cultured on plastic or gelatin. Osteogenic markers (Col I, osteopontin, and cbfa1) and ALP activity from cells cultured on PDM were notably upregulated at 4 weeks. The use of FDM significantly improved the cellular expression of chondrogenic markers (Sox 9 and Col II), while downregulating that of Col I at 4 weeks. Both CDMs were more effective in inducing cellular synthesis of glycosaminoglycan content than control substrates. We also investigated the effect of matrix surface texture on hMSC (PT-2501) differentiation; soluble matrix (S-matrix)-coated substrates exhibited a localized fibronectin (FN) alignment, whereas natural matrix (N-matrix)-coated substrates preserved the naturally formed FN fibrillar alignment. hMSCs cultured for 4 weeks on N-matrices under osteogenic or chondrogenic conditions deposited a greater amount of calcium and proteoglycan than those cultured on S-matrices as assessed by von Kossa and Safranin O staining. In contrast to the expression levels of lineage-specific markers for cells cultured on gelatin, FN, or S-matrices, those cultured on N-matrices yielded highly upregulated levels. This study demonstrates not only the capacity of CDM for being an effective inductive template for the multi-lineage differentiation of hMSCs, but also the critical biophysical role that the matrix fibrillar texture itself plays on the induction of stem cell differentiation.     

Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation

Key message

Human glucocerebrosidase with vacuolar anchoring domains was targeted to protein storage vacuoles (PSVs) of Arabidopsis seeds, but unexpectedly via the Golgi complex. PSV-targeting to effectively avoid problematic N-glycans is protein dependent.

Abstract

Plant-specific N-glycosylation patterns elaborated within the Golgi complex are a major limitation of using plants to produce biopharmaceuticals as the presence of β1,2 xylose and/or α1,3 fucose residues on the recombinant glycoprotein can render the product immunogenic if administrated parenterally. A reporter protein fused to a vacuolar membrane targeting motif comprised of the BP-80 transmembrane domain (TMD), and the cytoplasmic tail (CT) of α-tonoplast intrinsic protein (α-TIP) is delivered to protein storage vacuoles (PSVs) of tobacco seeds by ER-derived transport vesicles that bypass the Golgi complex. This prompted us to investigate whether a pharmaceutical glycoprotein is targeted to PSVs using the same targeting sequences, thus avoiding the unwanted plant-Golgi-specific complex N-glycan modifications. The human lysosomal acid β-glucosidase (glucocerebrosidase; GCase) (EC 3.2.1.45) fused to the BP-80 TMD and α-TIP CT was produced in Arabidopsis thaliana wild-type (Col-0) seeds. The chimeric GCase became localized in PSVs but transited through the Golgi complex, as indicated by biochemical analyses of the recombinant protein’s N-glycans. Our findings suggest that use of this PSV-targeting strategy to avoid problematic N-glycan maturation on recombinant therapeutic proteins is not consistently effective, as it is likely protein- and/or species-specific.     

Expression pattern analysis of Will Die Slowly genes in Arabidopsis

Leaf senescence is a developmental programmed cell death (PCD) that occurs as a response to external and internal signals. Several factors, such as the environment, plant hormones, and senescence-associated genes, regulate leaf senescence. In Drosophila melanogaster, Will Die Slowly (WDS) is a WD-repeat protein, which is closely related to PCD. Phylogenetic analysis indicated that eight genes are highly homologous to D. melanogaster WDS (DmWDS) in Columbia ecotype (Col) of Arabidopsis thaliana. In this study, the expression patterns of three close homologues of DmWDS named WDS1, WDS2, and WDS3 were investigated. Real-time PCR revealed the spatio-temporal expression levels of these three genes. No tissue-specific expression of the three AtWDS genes was observed. These genes were expressed at every growth stage; the variation in their expression was similar: the expression of the three AtWDS reached the peak in leaves at the 37 days after sowing, at the time when the first pod initially appeared on the plant and the leaf 7 show 25 to 50% yellow; and the expression of the three AtWDS reached the peak in flowers at the 43.5 days after sowing, at the time when 50% of the flowers bloomed. In addition, the expression level of the three AtWDS peaked at the 48 h after the plants were treated with 0.5 mM salicylic acid (SA). WDS3 also exhibited a high expression at 24, 48, and 72 h. Taken together; these results suggest that AtWDS genes may be involved in plant PCD     

Pyramiding Bt genes for increasing resistance of cotton to two major lepidopteran pests: Spodoptera litura and Heliothis armigera

To more effectively control two major cotton insects (cotton bollworm and Spodoptera litura) and improve the efficacy of the pest resistance management, novel transgenic plants expressing Bacillus thuringiensis Cry9C gene were generated, and gene stacking strategy was incorporated. Initially, a binary plasmid vector harboring Cry9C gene was introduced into an elite cotton cultivar Simian-3 by Agrobacterium-mediated transformation. Integration and expression of the Cry9C genes in three transgenic lines were confirmed by PCR and RT-PCR. Among these transgenic lines, T0 generation of line 16 (L-16) with normal phenotypes were selected by ELISA assays for its highest expression level of Cry9C. In T1 population of L-16, the expression level of Cry9C ranged from 29 to 45 μg/g fresh leaf. The following insect bioassays demonstrated that transgenic S3-35S::Cry9C cotton plants exhibited moderate toxicity to Heliothis armigera but strong toxicity to S. litura compared with the transgenic plants expressing Cry 1Ac gene. For incorporation of gene staking strategy, Cry9C gene and Cry 2A or Cry 1Ac were pyramided, respectively by sexual crossing. The expression of Cry9C protein in all F1 progenies had a similar level as the parent plants indicating the high heritability of Bt genes in transgenic progenies. Progenies from both Cry9C × Cry 2A and Cry9C × Cry 1Ac exhibited higher resistance to S. litura compared with their parents. Together our data demonstrated that our newly generated transgenic plants represent a reservoir of novel insect-resistant materials in cotton breeding, and the successful incorporation of gene pyramiding technology can provide a new solution of developing multiple resistance management strategies.     

Magainin 2 Induces Bacterial Cell Death Showing Apoptotic Properties

Magainin 2 is pore-forming antimicrobial peptide on lipid matrix of bacterial membrane, secreted from the skin of the African clawed frog Xenopus laevis. The aim of this study was to investigate a new concept for antibacterial mechanisms called bacterial apoptosis-like cell death. We examined the morphological changes induced by magainin 2 in Escherichia coli, regarding apoptosis. Specifically, phosphatidylserine externalization from the inner to outer membrane surface was detected by Annexin V staining, and DNA fragmentation and chromatin condensation was detected by TUNEL and DAPI assay. We also found much mechanistic evidence to support the hypothesis that magainin 2 induces bacterial apoptosis-like death—including disturbance of membrane detected by DiBAC₄(3), caspase activation observed by FITC-VAD-FMK staining, and analyzing the role of RecA in bacterial apoptosis-like death through the RecA expression assay by Western blot—in E. Coli when treated with magainin 2. On the basis of these results, magainin 2 exerts antibacterial activity with a new mechanism which is bacterial apoptosis-like death. Searching antimicrobial agents with novel mechanisms of action can be an effective strategy to coping with the emergence of new resistance mechanisms. Magainin 2 deserves further research as a potential antimicrobial therapeutic agent.     

Biological control of puncturevine,Tribulus terrestris in california after twenty years of activity of introduced weevils

Fifteen years of field tests were conducted in California on 1,200 plots infested with the annual puncturevine (Tribulus terrestris L.) in order to examine the effect of 2 introduced weevils,Microlarinus lareynii (Jacquelin du Val) andM. lypriformis Wollaston (Col.: Curculionidae), on puncturevine. Since 5 of the 6 series of plots marked a substantial reduction in viable seed production and puncturevine coverage, it is suggested that the 2 introduced weevils curtailed the pucturevine population's capacity to compensate in the natural situation and significantly contributed to the decline in puncturevine density.     

Abnormalities of blood selenium and glutathione peroxidase activity in patients with acquired immunodeficiency syndrome and aids-related complex

Severe protein-calorie malnutrition is common in patients with AIDS and could contribute to the progressive deterioration characteristic of that disease. Selenium deficiency could also have a negative impact on immune function and other organ functions vital for recovery from infectious diseases. Therefore, to assess any role for selenium in AIDS, we determined plasma and erythrocyte selenium levels and glutathione peroxidase activity in 13 patients with AIDS compared to 8 patients with AIDS-related complex (ARC) and 14 healthy controls. Plasma selenium levels were significantly reduced in AIDS patients compared to controls (p<.0001) and to ARC (p<.02). Erythrocyte selenium levels in both AIDS and ARC were also reduced compared to controls (p<.02), but not to each other. Glutathione peroxidase activity in AIDS was 28.9±1.4 U/g Hb vs 38.4±6.9 in ARC (p=NS) and 52.3±1.7 in controls (p<.0001 vs AIDS;p<.02 vs ARC). When all groups were combined, there were significant correlations between total lymphocyte count and both plasma selenium (r=.53;p<.002) and erythrocyte glutathione peroxidase activity (r=.65;p<.0001). In addition, strong correlations were noted between plasma selenium and serum albumin (r=.68;p<.0001), plasma selenium and glutathione peroxidase (r=.77;p<.0001), and glutathione peroxidase and hematocrit (r=.66;p<.0001). In AIDS or ARC, no correlations between selenium with disease duration or weight loss were present. We conclude that, in comparison to normals, patients manifesting infection with human immunodeficiency virus have evidence of selenium deficiency as determined by diminished plasma and erythrocyte levels and glutathione peroxidase activity. These abnormalities are most marked in patients with AIDS, but are also present in patients with AIDS-related complex. Selenium deficiency has important implications for the progression and pathogenesis of clinical disease in AIDS.     

Streptosporangium subfuscum sp. nov., isolated from the rhizosphere of marigold (Tagetes erecta L.)

A Gram-stain positive, filamentous bacterial strain, designated strain NEAU-TWSJ13^T, was isolated from the rhizosphere of a marigold (Tagetes erecta L.) plant collected in Heilongjiang Province, northeast China, and characterized using a polyphasic approach. The strain was observed to form abundant aerial hyphae differentiated into spherical sporangia. 16S rRNA gene sequence similarity studies showed that strain NEAU-TWSJ13^T belongs to the genus Streptosporangium, being most closely related to Streptosporangium fragile DSM 43847^T (98.6 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that it formed a phyletic line with S. fragile DSM 43847^T, Streptosporangium jomthongense NBRC 110047^T (98.4 % 16S rRNA gene similarity) and Streptosporangium violaceochromogenes DSM 43849^T (97.6 % 16S rRNA gene similarity). A combination of DNA–DNA hybridization results and some phenotypic characteristics indicated that strain NEAU-TWSJ13^T can be distinguished from S. fragile DSM 43847^T and S. jomthongense NBRC 110047^T. Moreover, strain NEAU-TWSJ13^T can also be differentiated from S. violaceochromogenes DSM 43849^T and other Streptosporangium species showing high 16S rRNA gene sequence similarity (>98.0 %) by morphological and physiological characteristics. Therefore, it is proposed that strain NEAU-TWSJ13^T represents a novel species of the genus Streptosporangium, for which the name Streptosporangium subfuscum sp. nov. is proposed. The type strain is NEAU-TWSJ13^T ( = CGMCC 4.7146^T = DSM = 46724^T).     

Distribution of antigenic determinants recognized by three monoclonal antibodies (Q2/70, Q5/6 and Q5/13) on human Ia-like alloantigens and on their subunits

The distribution of antigenic determinants recognized by the anti-Ia-like antigen monoclonal antibodies (MoAb) Q2/70, Q5/6 and Q5/13 on molecules coded for by theDR locus and by non-DR loci was investigated using a binding assay with¹²⁵I-labeled Ia-like antigens isolated from four B lymphoid cell lines. The determinants reacting with the MoAb Q2/70 and Q5/13 are expressed on all DR alloantigens tested and on BR4X7 specificities, while those reacting with the MoAb Q5/6 are not detectable on DRw7 and BR4X7 molecules. None of the monoclonal antibodies reacted with DC1 molecules. The MoAb Q5/6 and Q5/13 reacted with the isolatedβ subunit of the Ia-like antigenic complex, while the MoAb Q2/70 did not react with the isolated chains.     

Mechanism of bFGF-binding Peptide Reversing Adriamycin Resistance in Human Gastric Cancer Cells

Development of drug resistance is a challenging problem in cancer chemotherapy. It has been shown that basic fibroblast growth factor (bFGF) plays an important role in an epigenetic mechanism of drug resistance. We have isolated a bFGF binding peptide P7 with inhibitory activity against bFGF-induced proliferation of human gastric cancer cells by screening a phage display library. In this study, we found that P7 peptide also has efficacy of reversing bFGF-induced resistance to Adriamycin (ADM) in human gastric cancer cells. Further investigations with SGC-7901 cells revealed that inhibition of Akt activation triggered by bFGF, and reversal of bFGF-induced up-regulation of Bcl-2 and XIAP and down-regulation of Bax, contribute to P7 peptide counteracting the anti-apoptotic effect of bFGF, and further reversing bFGF-induced resistance to ADM. The results suggested that the bFGF-binding peptide may have therapeutic potential of drug resistance in gastric cancer.     

Knockdown of LYRM1 Rescues Insulin Resistance and Mitochondrial Dysfunction Induced by FCCP in 3T3-L1 Adipocytes

LYR motif-containing 1 (LYRM1) was recently discovered to be involved in adipose tissue homeostasis and obesity-associated insulin resistance. We previously demonstrated that LYRM1 overexpression might contribute to insulin resistance and mitochondrial dysfunction. Additionally, knockdown of LYRM1 enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We investigated whether knockdown of LYRM1 in 3T3-L1 adipocytes could rescue insulin resistance and mitochondrial dysfunction induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to further ascertain the mechanism by which LYRM1 is involved in obesity-associated insulin resistance. Incubation of 3T3-L1 adipocytes with 1 µM FCCP for 12 h decreased insulin-stimulated glucose uptake, reduced intracellular ATP synthesis, increased intracellular reactive oxygen species (ROS) production, impaired insulin-stimulated Glucose transporter type 4 (GLUT4) translocation, and diminished insulin-stimulated tyrosine phosphorylation of Insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Protein Kinase B (Akt). Knockdown of LYRM1 restored insulin-stimulated glucose uptake, rescued intracellular ATP synthesis, reduced intracellular ROS production, restored insulin-stimulated GLUT4 translocation, and rescued insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt in FCCP-treated 3T3-L1 adipocytes. This study indicates that FCCP-induced mitochondrial dysfunction and insulin resistance are ameliorated by knockdown of LYRM1.