Stable integration and expression of wasabi defensin gene in “Egusi” melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot

Production of “Egusi” melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer “Egusi” resistant to these diseases, cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mgl^−l kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and ‘Ejagham’, respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype ‘Ejagham’ were transgenic. A DNA fragment corresponding to the wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1–5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.     

Distribution of rhodanese in different parts of the urogenital systems of sheep at pre- and post-natal stages

The enzyme rhodanese (thiosulfate:cyanide sulfurtransferase) is a ubiquitous enzyme present in all living organisms, from bacteria to humans and plays a central role in cyanide detoxification. The purpose of this investigation is to determine and compare rhodanese activity in different parts of urogenital systems of male and female sheep fetuses at 2.5, 3, 3.5, 4, 4.5, and 5 months of age. The highest activity of rhodanese in male fetus was in kidney cortex, followed by medulla of the kidney. No significant difference was observed in other organs. In female fetus, the highest activity was in kidney cortex followed by oviduct and medulla of kidney. The enzyme activity of tissues increased with age. There was no significant difference (P > 0.05) between male and female fetuses in levels of rhodanese activity of different tissues except in urinary bladder at 2.5 and 3 months and in urethra at 4.5 months of age. The results of this study might indicate the involvement of rhodanese in cyanide detoxification in tissues which are more exposed to cyanide. On the other hand, rhodanese might perform other functions which are specific in these tissues.     

Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression

N‐Glycans of human proteins possess both α2,6‐ and α2,3‐linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3‐linkage due to the absence of α2,6‐sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)‐producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC‐Sambucus nigra (SNA) lectin that preferentially binds α2,6‐linked SA. The presence of α2,6‐linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2‐fold compared to the control. For host cell engineering, the CHOZN^® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single‐cell clones were derived from the enriched population and selected based on FITC‐SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6‐linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6‐linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human‐like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio‐better” protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:334–346, 2015     

A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum

An efficient and reliable transformation system for a very important medicinal plant Papaver bracteatum was developed through optimization of several factors that affect the rate of effective A. rhizogenes-mediated transformation and growth rate of hairy root. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, and three explants types, hypocotyls, leaves and excised shoots were examined. The highest frequency of transformation was achieved using LBA9402 strain in the excised shoots. Several inoculation and co-cultivation media and different concentration of arginine were evaluated using LBA9402 strain and the excised shoots as explant. Interestingly, a drastic increase in the frequency of transformation (47.3 %) was observed when Murashige and Skoog medium containing 1 mM arginine and lacking NH₄NO₃ KH₂PO₄, KNO₃ and CaCl₂ was used. The effect of sucrose concentration and the ratio of NH₄ ⁺: NO₃ ^? on hairy root biomass was examined. Maximum biomass was obtained in 30 g/l sucrose and 20:10 mM ratio of NH₄ ⁺ to NO₃ ^? on MS medium. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR) and Southern hybridization.     

A critical view on social performance assessment at company level: social life cycle analysis of an algae case

The International Journal of Life Cycle Assessment - Social indicators are not easy to be quantitatively analyzed, although at the local scale, the social impacts might be relevant and important....     

Formation of ethyl β-xylopyranoside during simultaneous saccharification and co-fermentation of paper sludge

An unexpected product was detected during simultaneous saccharification and co-fermentation (SSCF) of paper sludge using added commercial cellulase (Spezyme CP) by Saccharomyces cerevisiae RWB222, S. cerevisiae D5A, and Zymomonas mobilis 8b. Based on glycosyl composition analysis, linkage analysis and NMR analysis, the compound was identified as ethyl β-xylopyranoside (EXP). The carbon mass balance analysis showed up to 25% of xylan originally present in paper sludge was converted to EXP. EXP formation was found in simultaneous saccharification of beech wood xylan as well, and later proved to be produced by the Trichoderma reesei derived cellulase and hemicellulase mixture (Spezyme CP) during the course of xylan hydrolysis in the presence of ethanol, and its production increased with an increased concentration of ethanol, xylan, and T. reesei enzyme. Similar condensation reactions were also observed with other alcohols. These alcoholysis reactions were found to be reversible. Thermoanaerobacterium saccharolyticum was found to be able to degrade EXP.     

Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.     

Influence of thidiazuron on callus induction and crocin production in corm and style explants of <Emphasis Type="Italic">Crocus sativus</Emphasis> L.

Crocus sativus L., mostly famous as saffron, has gained more attention due to its crocin (crocetin ester) pigment responsible for its extensive pharmaceutical properties. In this study, we established two different callus cultures from corm and style explants of saffron to find out the best explant as a suitable source for crocin production. Comparative analyses of total phenolic, flavonoid, carotenoid and anthocyanin contents were also performed in the two callus cultures. For callus induction, different combinations of MS medium with name thidiazuron (TDZ), benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination were tested. Of the used media, all the combinations containing TDZ and NAA gave 100% callus induction. HPLC-DAD and HPLC–ESI-MS analysis were used for identification of crocin esters in established callus cultures. The highest amount of 0.35 mg g^?1 DW crocin was detected in style originated calli grown on the medium containing 3 mg L^?1 NAA?+?1 mg L^?1 TDZ while the corm calli showed the most abundant total carotenoid (0.73 mg g^?1 DW), phenolic (15.04 mg gallic acid equivalent g^?1 DW) and flavonoid (0.76 mg rutin equivalent g^?1 DW) contents. In general, style-derived calli showed longer time survival with a fine texture and good quality compared to corm-derived calli.