Amplification of the active site of BnLIP3 gene of Brassica napus L. during germination

Lipases are useful enzymes that are responsible for the hydrolysis of triacylglycerides and play an important role in plant growth. In this study, we report a rapid molecular method to amplify a partial sequence of the lipase class 3 family designated BnLIP3 gene of Brassica napus L. in order to follow its expression and analyze its role during seed germination. Therefore, we conceived PCR homologous primers to amplify the active site encoding region of the BnLIP3 family genes. Subsequently, to sequence determination of the 582 bp fragment, we deduced BnLIP3 specific primers for a nested RTPCR application. The deduced 194 amino acid sequence (Genbank 1160264) was found to share 85% of identity with lipase from Arabidopsis thaliana class 3 family. The GxSxG consensus motif near the catalytic triad at the active serine site was also identified. The peptidic sequence showed little homology with mammalian and microbial lipases. RT-PCR analysis indicated that BnLIP3 gene was expressed during B. napus seed germination.Keywords: Brassica napus L., GxSxG lipase, germination, BnLIP3, RT-PCR.African Journal of Biotechnology Vol. 12(25), pp. 3905-391

Cloning, Sequence Analysis and Expression Patterns during Seed Germination of a Rapeseed (Brassica napus L.) G-x-S-x-G-motif Lipase Gene

[EN] Lipases catalyze the hydrolysis of ester bonds in triacylglycerides, generating glycerol and free fatty acids. These enzymes are encoded by extremely complex gene families, and appear to fulfil many different biological functions. Although they are present in all types of organisms, available information on plant lipases is still very limited, as compared to their bacterial and animal counterparts. A full-length clone, BnLIP, encoding a putative lipase, has been isolated by PCR amplification of Brassica napus genomic DNA, with oligonucleotide primers derived from the sequence of an Arabidopsis thaliana homologue. The clone included an open reading frame of 1581 bp encoding a polypeptide of 526 amino acids, with a calculated molecular mass of 59.5 kDa. Analysis of the deduced protein sequence, sequence alignment with homologous proteins from related plant species, and a phylogenetic analysis revealed that the BnLIP protein belongs to the classical GxSxG-motif lipase family. RTPCR assays indicated that the BnLIP gene is expressed specifically, but only transiently, during seed germination: the lipase mRNA was not present at detectable levels in ungerminated seeds, was detected only three days after seed imbibition, but its levels decreased rapidly afterwards. No expression was observed in roots, stems or leaves of adult plants. This expression pattern suggests that BnLIP is one of the lipases involved in the hydrolysis of triacylglycerides stored in rapeseed seeds, ultimately providing nutrients and energy to sustain seedling growth until photosynthesis is activatedGlaied Ghram, I.; Belguith, H.; Ben Mustapha, M.; Himila, I.; Bouhaouala, B.; Vicente, O.; Ben Hamida, J. (2016). Cloning, Sequence Analysis and Expression Patterns during Seed Germination of a Rapeseed (Brassica napus L.) G-x-S-x-G-motif Lipase Gene. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 44(2):437-444. doi:10.15835/nbha4421047243744444

Amplification of the active site of BnLIP3 gene of Brassica napus L. during germination

Lipases are useful enzymes that are responsible for the hydrolysis of triacylglycerides and play an important role in plant growth. In this study, we report a rapid molecular method to amplify a partial sequence of the lipase class 3 family designated BnLIP3 gene of Brassica napus L. in order to follow its expression and analyze its role during seed germination. Therefore, we conceived PCR homologous primers to amplify the active site encoding region of the BnLIP3 family genes. Subsequently, to sequence determination of the 582 bp fragment, we deduced BnLIP3 specific primers for a nested RTPCR application. The deduced 194 amino acid sequence (Genbank 1160264) was found to share 85% of identity with lipase from Arabidopsis thaliana class 3 family. The GxSxG consensus motif near the catalytic triad at the active serine site was also identified. The peptidic sequence showed little homology with mammalian and microbial lipases. RT-PCR analysis indicated that BnLIP3 gene was expressed during B. napus seed germination.Glaied Ghram, I.; Belguith, H.; Messaoudi, A.; Fattouch, S.; Vicente Meana, Ó.; Ben Hamida, J. (2013). Amplification of the active site of BnLIP3 gene of Brassica napus L. during germination. African Journal of Biotechnology. 12(25):3905-3913. doi:10.5897/AJB12.2861S39053913122

The preparation of graft copolymers of cellulose and cellulose derivatives using ATRP under homogeneous reaction conditions

In this comprehensive review, we report on the preparation of graft-copolymers of cellulose and cellulose derivatives using atom transfer radical polymerization (ATRP) under homogeneous conditions. The review is divided into four sections according to the cellulosic material that is graft-copolymerised; (i) cellulose, (ii) ethyl cellulose, (iii) hydroxypropyl cellulose and (iv) other cellulose derivatives. In each section, the grafted synthetic polymers are described as well as the methods used for ATRP macro-initiator formation and graft-copolymerisation. The physical properties of the graft-copolymers including their self-assembly in solution into nanostructures and their stimuli responsive behaviour are described. Potential applications of the self-assembled graft copolymers in areas such as nanocontainers for drug delivery are outline

Indocyanine Green Loaded Biocompatible Nanoparticles: Stabilisation of Indocyanine Green (ICG) Using Biocompatible Silica-Poly (&epsilon-Caprolactone) Grafted Nanocomposites

Indocyanine green (ICG) is a chemically labile compound which needs to be stabilized in aqueous media to be used in biomedical applications. In the present study, poly(ε-caprolactone) (PCL), a semi-crystalline polyester, was used to encapsulate and stabilize ICG in a hydrophobic environment. A hydrophobic and biocompatible nanocomposite was obtained by the process of encapsulating inorganic silica. ICG was embedded in the hydrophobic polymer coating by starting from a well-defined silica (Si) core of either 80 nm or 120 nm diameter, which served as a template for a ‘grafting from’ approach using ε-caprolactone. The obtained nanocomposite Si grafted PCL/ICG was based on silica nanoparticles grafted with PCL, in which ICG was adsorbed. The nanoparticles were characterized by IR spectroscopy, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The change in the surface charge and the colloidal stability of the nanoparticles was followed by zeta potential measurements. This approach of synthesizing nanocomposite-based ICG demonstrates a new route to stabilize ICG. We synthesized biocompatible nanoparticles containing a high ICG concentration and exhibiting excellent stability to aqueous decomposition

Photoreaction of a Hydroxyalkyphenone with the Membrane of Polymersomes: A Versatile Method To Generate Semipermeable Nanoreactors

Block copolymer vesicles can be turned into nanoreactors when a catalyst is encapsulated in these hollow nanostructures. However the membranes of these polymersomes are most often impermeable to small org. mols., while applications as nanoreactor, as artificial organelles, or as drug-delivery devices require an exchange of substances between the outside and the inside of polymersomes. Here, a simple and versatile method is presented to render polymersomes semipermeable. It does not require complex membrane proteins or pose requirements on the chem. nature of the polymers. Vesicles made from three different amphiphilic block copolymers (α,ω-hydroxy-end-capped poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA), α,ω-acrylate-end-capped PMOXA-b-PDMS-b-PMOXA, and poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PB)) were reacted with externally added 2-hydroxy-4'-2-(hydroxyethoxy)-2-methylpropiophenone under UV-irradn. The photoreactive compd. incorporated into the block copolymer membranes independently of their chem. nature or the presence of double bonds. This treatment of polymersomes resulted in substantial increase in permeability for org. compds. while not disturbing the size and the shape of the vesicles. Permeability was assessed by encapsulating horseradish peroxidase into vesicles and measuring the accessibility of substrates to the enzyme. The permeability of photoreacted polymersomes for ABTS, AEC, pyrogallol, and TMB was detd. to be between 1.9 and 38.2 nm s-1. It correlated with the hydrophobicity of the compds. Moreover, fluorescent dyes were released at higher rates from permeabilized polymersomes compared to unmodified ones. The permeabilized nanoreactors retained their ability to protect encapsulated biocatalysts from degrdn. by proteases. [on SciFinder(R)

Photoreaction of a Hydroxyalkyphenone with the Membrane of Polymersomes: A Versatile Method To Generate Semipermeable Nanoreactors

Block copolymer vesicles can be turned into nanoreactors when a catalyst is encapsulated in these hollow nanostructures. However the membranes of these polymersomes are most often impermeable to small organic molecules, while applications as nanoreactor, as artificial organelles, or as drug-delivery devices require an exchange of substances between the outside and the inside of polymersomes. Here, a simple and versatile method is presented to render polymersomes semipermeable. It does not require complex membrane proteins or pose requirements on the chemical nature of the polymers. Vesicles made from three different amphiphilic block copolymers (α,ω-hydroxy-end-capped poly­(2-methyl-2-oxazoline)-<i>block</i>-poly­(dimethylsiloxane)-<i>block</i>-poly­(2-methyl-2-oxazoline) (PMOXA<i>-b-</i>PDMS<i>-b-</i>PMOXA), α,ω-acrylate-end-capped PMOXA<i>-b-</i>PDMS<i>-b-</i>PMOXA, and poly­(ethylene oxide)-<i>block</i>-poly­(butadiene) (PEO-<i>b</i>-PB)) were reacted with externally added 2-hydroxy-4′-2-(hydroxyethoxy)-2-methylpropiophenone under UV-irradiation. The photoreactive compound incorporated into the block copolymer membranes independently of their chemical nature or the presence of double bonds. This treatment of polymersomes resulted in substantial increase in permeability for organic compounds while not disturbing the size and the shape of the vesicles. Permeability was assessed by encapsulating horseradish peroxidase into vesicles and measuring the accessibility of substrates to the enzyme. The permeability of photoreacted polymersomes for ABTS, AEC, pyrogallol, and TMB was determined to be between 1.9 and 38.2 nm s^–1. It correlated with the hydrophobicity of the compounds. Moreover, fluorescent dyes were released at higher rates from permeabilized polymersomes compared to unmodified ones. The permeabilized nanoreactors retained their ability to protect encapsulated biocatalysts from degradation by proteases