Metabolic engineering of Arabidopsis for butanetriol production using bacterial genes

Includes bibliographical references (pages 119-120).1,2,4-butanetriol (butanetriol) is a useful precursor for the synthesis of the energetic material butanetriol trinitrate and several pharmaceutical compounds. Bacterial synthesis of butanetriol from xylose or arabinose takes place in a pathway that requires four enzymes. To produce butanetriol in plants by expressing bacterial enzymes, we cloned native bacterial or codon optimized synthetic genes under different promoters into a binary vector and stably transformed Arabidopsis plants. Transgenic lines expressing introduced genes were analyzed for the production of butanetriol using gas chromatography coupled to mass spectrometry (GC-MS). Soil-grown transgenic plants expressing these genes produced up to 20 µg/g of butanetriol. To test if an exogenous supply of pentose sugar precursors would enhance the butanetriol level, transgenic plants were grown in a medium supplemented with either xylose or arabinose and the amount of butanetriol was quantified. Plants expressing synthetic genes in the arabinose pathway showed up to a forty-fold increase in butanetriol levels after arabinose was added to the medium. Transgenic plants expressing either bacterial or synthetic xylose pathways, or the arabinose pathway showed toxicity symptoms when xylose or arabinose was added to the medium, suggesting that a by-product in the pathway or butanetriol affected plant growth. Furthermore, the metabolite profile of plants expressing arabinose and xylose pathways was altered. Our results demonstrate that bacterial pathways that produce butanetriol can be engineered into plants to produce this chemical. This proof-of-concept study for phytoproduction of butanetriol paves the way to further manipulate metabolic pathways in plants to enhance the level of butanetriol production.Published with support from the Colorado State University Libraries Open Access Research and Scholarship Fund

Therapeutic effect of antimyeloma antibodies conjugated with gold nanoparticles on the growth of myeloma cell line

Nanobiotechnology is a field of biomedical application of nanosize system which is a rapidly developing area within nanotechnology among these nonmaterial, gold nanoparticles (AuNPs) which receive a significant attention due to their unique physical, chemical and biological properties. AuNPs and bio-conjugated AuNPs have been widely used in cancer treatment besides other application on cancer detection and diagnosis. In this study the potential therapeutic effects of polyclonal Antimyeloma antibody (AbMM) conjugated to AuNPs in comparison with naked (AbMM) or AuNPs alone in modulation of proliferation capacity in vitro and different stages of MM cell cycle have been evaluated besides evaluation of their effects on tumor growth delay. Effect of AuNPs , (AbMM) and (Nanogold -Antimyeloma Antibodies conjugate) (gold-AbMM) on growth of myeloma cells showed decreasing in multiple myeloma SP2OR (MM) viability with increasing dose of these treatments compared to that of control also a significant enhancement in the apoptosis after conjugation of Nanogold to the Antimyeloma was observed. The induction of apoptosis with gold-AbMM was significantly higher than the MM cells exposed to only AbMM or AuNPs. The study concluded that the efficacy of (gold-AbMM) on induced myeloma growth inhibition is better than that of individual AuNPs and AbMM

N-(4,6-Dimethyl­pyrimidin-2-yl)-1,3-benzothia­zol-2-amine

In the title compound, C13H12N4S, an amino N atom is connected to a 1,3-benzothia­zole fused-ring system and a dimethyl-substituted pyrimidine ring, these components being aligned [inter­planar dihedral angle = 1.9 (1)°]. The secondary amino N atom forms an inter­molecular N—H⋯N hydrogen bond to an N atom of the fused ring of an adjacent mol­ecule, generating a centrosymmetric cyclic hydrogen-bonded dimer [graph set R 2 2(8)]

Arabidopsis thaliana PGR7 Encodes a Conserved Chloroplast Protein That Is Necessary for Efficient Photosynthetic Electron Transport

A significant fraction of a plant's nuclear genome encodes chloroplast-targeted proteins, many of which are devoted to the assembly and function of the photosynthetic apparatus. Using digital video imaging of chlorophyll fluorescence, we isolated proton gradient regulation 7 (pgr7) as an Arabidopsis thaliana mutant with low nonphotochemical quenching of chlorophyll fluorescence (NPQ). In pgr7, the xanthophyll cycle and the PSBS gene product, previously identified NPQ factors, were still functional, but the efficiency of photosynthetic electron transport was lower than in the wild type. The pgr7 mutant was also smaller in size and had lower chlorophyll content than the wild type in optimal growth conditions. Positional cloning located the pgr7 mutation in the At3g21200 (PGR7) gene, which was predicted to encode a chloroplast protein of unknown function. Chloroplast targeting of PGR7 was confirmed by transient expression of a GFP fusion protein and by stable expression and subcellular localization of an epitope-tagged version of PGR7. Bioinformatic analyses revealed that the PGR7 protein has two domains that are conserved in plants, algae, and bacteria, and the N-terminal domain is predicted to bind a cofactor such as FMN. Thus, we identified PGR7 as a novel, conserved nuclear gene that is necessary for efficient photosynthetic electron transport in chloroplasts of Arabidopsis

AGO1 and AGO2 Act Redundantly in miR408-Mediated Plantacyanin Regulation

Background: In Arabidopsis, AGO1 and AGO2 associate with small RNAs that exhibit a Uridine and an Adenosine at their 59 end, respectively. Because most plant miRNAs have a 59U, AGO1 plays many essential roles in miRNA-mediated regulation of development and stress responses. In contrast, AGO2 has only been implicated in antibacterial defense in association with miR393*, which has a 59A. AGO2 also participates in antiviral defense in association with viral siRNAs. Principal Findings: This study reveals that miR408, which has a 59A, regulates its target Plantacyanin through either AGO1 or AGO2. Indeed, neither ago1 nor ago2 single mutations abolish miR408-mediated regulation of Plantacyanin. Only an ago1 ago2 double mutant appears compromised in miR408-mediated regulation of Plantacyanin, suggesting that AGO1 and AGO2 have redundant roles in this regulation. Moreover, the nature of the 59 nucleotide of miR408 does not appear essential for its regulatory role because both a wildtype 59A-MIR408 and a mutant 59U-MIR408 gene complement a mir408 mutant. Conclusions/Significance: These results suggest that miR408 associates with both AGO1 and AGO2 based on criteria that differ from the 59 end rule, reminiscent of miR390-AGO7 and miR165/166-AGO10 associations, which are not based on the nature of the 59 nucleotide

Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer

Background Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer. Methodology In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens. Results Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002). Conclusion Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer

Bacteria-responsive microRNAs regulate plant innate immunity by modulating plant hormone networks

MicroRNAs (miRNAs) are key regulators of gene expression in development and stress responses in most eukaryotes. We globally profiled plant miRNAs in response to infection of bacterial pathogen Pseudomonas syringae pv. tomato (Pst). We sequenced 13 small-RNA libraries constructed from Arabidopsis at 6 and 14 h post infection of non-pathogenic, virulent and avirulent strains of Pst. We identified 15, 27 and 20 miRNA families being differentially expressed upon Pst DC3000 hrcC, Pst DC3000 EV and Pst DC3000 avrRpt2 infections, respectively. In particular, a group of bacteria-regulated miRNAs targets protein-coding genes that are involved in plant hormone biosynthesis and signaling pathways, including those in auxin, abscisic acid, and jasmonic acid pathways. Our results suggest important roles of miRNAs in plant defense signaling by regulating and fine-tuning multiple plant hormone pathways. In addition, we compared the results from sequencing-based profiling of a small set of miRNAs with the results from small RNA Northern blot and that from miRNA quantitative RT-PCR. Our results showed that although the deep-sequencing profiling results are highly reproducible across technical and biological replicates, the results from deep sequencing may not always be consistent with the results from Northern blot or miRNA quantitative RT-PCR. We discussed the procedural differences between these techniques that may cause the inconsistency

Effects of elevated seawater pCO2 on gene expression patterns in the gills of the green crab, Carcinus maenas

Background: The green crab Carcinus maenas is known for its high acclimation potential to varying environmental abiotic conditions. A high ability for ion and acid-base regulation is mainly based on an efficient regulation apparatus located in gill epithelia. However, at present it is neither known which ion transport proteins play a key role in the acid-base compensation response nor how gill epithelia respond to elevated seawater pCO2 as predicted for the future. In order to promote our understanding of the responses of green crab acid-base regulatory epithelia to high pCO2, Baltic Sea green crabs were exposed to a pCO2 of 400 Pa. Gills were screened for differentially expressed gene transcripts using a 4,462-feature microarray and quantitative real-time PCR. Results: Crabs responded mainly through fine scale adjustment of gene expression to elevated pCO2. However, 2% of all investigated transcripts were significantly regulated 1.3 to 2.2-fold upon one-week exposure to CO2 stress. Most of the genes known to code for proteins involved in osmo- and acid-base regulation, as well as cellular stress response, were were not impacted by elevated pCO2. However, after one week of exposure, significant changes were detected in a calcium-activated chloride channel, a hyperpolarization activated nucleotide-gated potassium channel, a tetraspanin, and an integrin. Furthermore, a putative syntaxin-binding protein, a protein of the transmembrane 9 superfamily, and a Cl-/HCO3 - exchanger of the SLC 4 family were differentially regulated. These genes were also affected in a previously published hypoosmotic acclimation response study. Conclusions: The moderate, but specific response of C. maenas gill gene expression indicates that (1) seawater acidification does not act as a strong stressor on the cellular level in gill epithelia; (2) the response to hypercapnia is to some degree comparable to a hypoosmotic acclimation response; (3) the specialization of each of the posterior gill arches might go beyond what has been demonstrated up to date; and (4) a re-configuration of gill epithelia might occur in response to hypercapnia