A quantitative analysis of microalgal lipids for optimization of biodiesel and omega‐3 production

The lipid characteristics of microalgae are known to differ between species and change with growth conditions. This work provides a methodology for lipid characterization that enables selection of the optimal strain, cultivation conditions, and processing pathway for commercial biodiesel production from microalgae. Two different microalgal species, Nannochloropsis sp. and Chlorella sp., were cultivated under both nitrogen replete and nitrogen depleted conditions. Lipids were extracted and fractionated into three major classes and quantified gravimetrically. The fatty acid profile of each fraction was analyzed using GC–MS. The resulting quantitative lipid data for each of the cultures is discussed in the context of biodiesel and omega‐3 production. This approach illustrates how the growth conditions greatly affect the distribution of fatty acid present in the major lipid classes and therefore the suitability of the lipid extracts for biodiesel and other secondary products. Biotechnol. Bioeng. 2013; 110: 2096–2104. © 2013 Wiley Periodicals, Inc.     

O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues

O-mannosylation is a vital protein modification. In humans, defective O-mannosylation of α-dystroglycan results in severe congenital muscular dystrophies. However, other proteins bearing this modification in vivo are still largely unknown. Here, we describe a highly reliable method combining glycosidase treatment with LC–MS analyses to identify mammalian O-mannosylated proteins from tissue sources. Our workflow identified T-cadherin (H-cadherin, CDH13) as a novel O-mannosylated protein. In contrast to known O-mannosylated proteins, single mannose residues (Man-α-Ser/Thr) are attached to this cell adhesion molecule. Conserved O-glycosylation sites in T-, E- and N-cadherins from different species, point to a general role of O-mannosyl glycans for cadherin function.     

Stable deuterium internal standard for the isotope-dilution LC–MS/MS analysis of elastin degradation

Chemical synthesis of the deuterium isotope desmosine-d₄ has been achieved. This isotopic compound possesses all four deuterium atoms at the alkanyl carbons of the alkyl amino acid substitution in the desmosine molecule and is stable toward acid hydrolysis; this is required in the measurement of two crosslinking molecules, desmosine and isodesmosine, as biomarkers of elastic tissue degradation. The degradation of elastin occurs in several widely prevalent diseases. The synthesized desmosine-d₄ is used as the internal standard to develop an accurate and sensitive isotope-dilution liquid chromatography–tandem mass spectrometry analysis, which can serve as a generalized method for an accurate analysis of desmosine and isodesmosine as biomarkers in many types of biological tissues involving elastin degradation.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

Glyphosate effects on the gene expression of the apical bud in soybean (Glycine max)

Glyphosate is a broad spectrum, non-selective herbicide which has been widely used for weed control. Much work has focused on elucidating the high accumulation of glyphosate in shoot apical bud (shoot apex). However, to date little is known about the molecular mechanisms of the sensitivity of shoot apical bud to glyphosate. Global gene expression profiling of the soybean apical bud response to glyphosate treatment was performed in this study. The results revealed that the glyphosate inhibited tryptophan biosynthesis of the shikimic acid pathway in the soybean apical bud, which was the target site of glyphosate. Glyphosate inhibited the expression of most of the target herbicide site genes. The promoter sequence analysis of key target genes revealed that light responsive elements were important regulators in glyphosate induction. These results will facilitate further studies of cloning genes and molecular mechanisms of glyphosate on soybean shoot apical bud.     

Rhamnogalacturonan II structure shows variation in the side chains monosaccharide composition and methylation status within and across different plant species

A paradigm regarding rhamnogalacturonans II (RGII) is their strictly conserved structure within a given plant. We developed and employed a fast structural characterization method based on chromatography and mass spectrometry, allowing analysis of RGII side chains from microgram amounts of cell wall. We found that RGII structures are much more diverse than so far described. In chain A of wild‐type plants, up to 45% of the l –fucose is substituted by l –galactose, a state that is seemingly uncorrelated with RGII dimerization capacity. This led us to completely reinvestigate RGII structures of the Arabidopsis thaliana fucose‐deficient mutant mur1, which provided insights into RGII chain A biosynthesis, and suggested that chain A truncation, rather than l –fucose to l –galactose substitution, is responsible for the mur1 dwarf phenotype. Mass spectrometry data for chain A coupled with NMR analysis revealed a high degree of methyl esterification of its glucuronic acid, providing a plausible explanation for the puzzling RGII antibody recognition. The β–galacturonic acid of chain A exhibits up to two methyl etherifications in an organ‐specific manner. Combined with variation in the length of side chain B, this gives rise to a family of RGII structures instead of the unique structure described up to now. These findings pave the way for studies on the physiological roles of modulation of RGII composition.     

Long-lasting inhibition of EGFR autophosphorylation in A549 tumor cells by intracellular accumulation of non-covalent inhibitors

In the present study, a small set of reversible or irreversible 4-anilinoquinazoline EGFR inhibitors was tested in A549 cells at early (1 h) and late (8 h) time points after inhibitor removal from culture medium. A combination of assays was employed to explain the observed long-lasting inhibition of EGFR autophosphorylation. We found that EGFR inhibition at 8 h can be due, besides to the covalent interaction of the inhibitor with Cys797, as for PD168393 (2) and its prodrug 4, to the intracellular accumulation of non-covalent inhibitors by means of an active cell uptake, as for 5 and 6. Compounds 5–6 showed similar potency and duration of inhibition of EGFR autophosphorylation as the covalent inhibitor 2, while being devoid of reactive groups forming covalent bonds with protein thiols.     

Biosynthesis of linoleic acid in Tyrophagus mites (Acarina: Acaridae)

We report here that Tyrophagus similis and Tyrophagus putrescentiae (Astigmata: Acaridae) have the ability to biosynthesize linoleic acid [(9Z, 12Z)-9, 12-octadecadienoic acid] via a Δ12-desaturation step, although animals in general and vertebrates in particular appear to lack this ability. When the mites were fed on dried yeast enriched with d₃₁-hexadecanoic acid (16:0), d₂₇-octadecadienoic acid (18:2), produced from d₃₁-hexadecanoic acid through elongation and desaturation reactions, was identified as a major fatty acid component of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in the mites. The double bond position of d₂₇-octadecadienoic acid (18:2) of PCs and PEs was determined to be 9 and 12, respectively by dimethyldisulfide (DMDS) derivatization. Furthermore, the GC/MS retention time of methyl 9, 12-octadecadienoate obtained from mite extracts agreed well with those of authentic linoleic acid methyl ester. It is still unclear whether the mites themselves or symbiotic microorganisms are responsible for inserting a double bond into the Δ12 position of octadecanoic acid. However, we present here the unique metabolism of fatty acids in the mites.     

Midgut aminopeptidase N isoforms from Ostrinia nubilalis: Activity characterization and differential binding to Cry1Ab and Cry1Fa proteins from Bacillus thuringiensis

Aminopeptidase N (APN) isoforms from Lepidoptera are known for their involvement in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis. These enzymes belong to a protein family with at least eight different members that are expressed simultaneously in the midgut of lepidopteran larvae. Here, we focus on the characterization of the APNs from Ostrinia nubilalis (OnAPNs) to identify potential Cry receptors. We expressed OnAPNs in insect cells using a baculovirus system and analyzed their enzymatic activity by probing substrate specificity and inhibitor susceptibility. The interaction with Cry1Ab and Cry1Fa proteins (both found in transgenic insect-resistant maize) was evaluated by ligand blot assays and immunocytochemistry. Ligand blots of brush border membrane proteins showed that both Cry proteins bound mainly to a 150 kDa-band, in which OnAPNs were greatly represented. Binding analysis of Cry proteins to the cell-expressed OnAPNs showed that OnAPN1 interacted with both Cry1Ab and Cry1Fa, whereas OnAPN3a and OnAPN8 only bound to Cry1Fa. Two isoforms, OnAPN2 and OnAPN3b, did not interact with any of these two proteins. This work provides the first evidence of a differential role of OnAPN isoforms in the mode of action of Cry proteins in O. nubilalis.