During the development of any PEGylated protein or peptide, toxicology in relevant species will be conducted prior to human exposure. Normally, comprehensive metabolism data accompany the toxicity ...studies for a small molecule. We have examined whether such studies would be relevant in the safety assessment of PEGylated material. Literature data indicate that the polyethylene glycol (PEG) associated with a biological molecule should provide no extra concern because the exposure-toxicity relationship of PEG in animals and humans has been thoroughly investigated and metabolism/excretion of PEG is well understood. Based on the comparisons of PEG exposure from PEGylated biological products and the exposure of PEG associated with toxicity in humans, the therapeutic index is large (approximately 600-fold or greater). Therefore, assuming that toxicological evaluation of a biological molecule of interest is complete and satisfactory therapeutic windows are achieved, the data contained in this review indicate that the PEG associated with a protein or other biological molecule does not represent an additional unquantified risk to humans.
Main conclusion
Heteromannans are the predominant hemicelluloses in the gametophytic stem of the moss
Hypnodendron menziesii
and occur in the walls of all cell types
Little is known about the ...cell-wall polysaccharides of mosses. Monosaccharide analysis of cell walls isolated from the stem of the umbrella moss
Hypnodendron menziesii
was consistent with heteromannans, probably galactoglucomannans, being the predominant hemicellulosic polysaccharides in the walls. Immunofluorescence and immunogold microscopy with the monoclonal antibody LM21, specific for heteromannans, showed that these polysaccharides were present in the walls of all stem cell types. These cell types, except the hydroids, have secondary walls. Experiments in which sections were pre-treated with 0.1 M sodium carbonate and with the enzyme pectate lyase indicated that the heteromannans have
O
-acetyl groups that limit LM21 binding and the cell walls contain pectic homogalacturonan that masks detection of heteromannans using LM21. Therefore, to fully detect heteromannans in the cell walls, it was essential to use these pre-treatments to remove the
O
-acetyl groups from the heteromannans and pectic homogalacturonan from the cell walls. Fluorescence microscopy experiments with a second monoclonal antibody, LM22, also specific for heteromannans, showed similar results, but the binding was considerably weaker than with LM21, possibly as a result of subtle structural differences in the epitopes of the two antibodies. Although heteromannans occur abundantly in the cell walls of many species in basal lineages of tracheophytes, prior to the present study, research on the distribution of these polysaccharides in the walls of different cell types in mosses was confined to the model species
Physcomitrium patens
.
Purpose
To determine the chemical composition of fruit (apple and orange) and vegetable (carrot) pomaces in order to assess their potential use in value-added products.
Methods
A range of ...bromatological and mass spectrometry-based metabolomic analyses were employed to chemically characterise apple, orange, and carrot pomaces.
Results
Dietary fibre was the most abundant component of the substrates, comprising most by cellulose and pectin. Another major component of the pomaces were soluble sugars. Nevertheless, all pomaces showed low levels of lipids (below 5%) with most of the fatty acids being unsaturated. Besides, each pomace was found to have a more diverse metabolomic composition than earlier described, and the variation between batches was not significantly different for most parameters tested.
Conclusion
This study shows that these food-grade pomaces have good potential for bioconversion into high-value-added products via microbial fermentation, and not solely for the extraction of a small number of chemical compounds. In addition, untargeted metabolomic tools showed great potential in providing a more comprehensive analysis of waste biomass composition, as well as potentially being useful in food and feed safety assessment.
Graphical Abstract
Eliminating the core fucose from the N-glycans of the Fc antibody segment by pathway engineering or enzymatic methods has been shown to enhance the potency of therapeutic antibodies, especially in ...the context of antibody-dependent cytotoxicity (ADCC). However, there is a significant challenge due to the limited defucosylation efficiency of commercially available α-l-fucosidases. In this study, we report a unique α-l-fucosidase (PnfucA) from the bacterium Prevotella nigrescens that has a low sequence identity compared with all other known α-l-fucosidases and is highly reactive toward a core disaccharide substrate with fucose α(1,3)-, α (1,4)-and α(1,6)-linked to GlcNAc, and is less reactive toward the Fuc-α(1,2)-Gal on the terminal trisaccharide of the oligosaccharide Globo H (Bb3). The kinetic properties of the enzyme, such as its Km and kcat, were determined and the optimized expression of PnfucA gave a yield exceeding 30 mg/L. The recombinant enzyme retained its full activity even after being incubated for 6 h at 37 °C. Moreover, it retained 92 and 87% of its activity after freezing and freeze-drying treatments, respectively, for over 28 days. In a representative glycoengineering of adalimumab (Humira), PnfucA showed remarkable hydrolytic efficiency in cleaving the α(1,6)-linked core fucose from FucGlcNAc on the antibody with a quantitative yield. This enabled the seamless incorporation of biantennary sialylglycans by Endo-S2 D184 M in a one-pot fashion to yield adalimumab in a homogeneous afucosylated glycoform with an improved binding affinity toward Fcγ receptor IIIa.Eliminating the core fucose from the N-glycans of the Fc antibody segment by pathway engineering or enzymatic methods has been shown to enhance the potency of therapeutic antibodies, especially in the context of antibody-dependent cytotoxicity (ADCC). However, there is a significant challenge due to the limited defucosylation efficiency of commercially available α-l-fucosidases. In this study, we report a unique α-l-fucosidase (PnfucA) from the bacterium Prevotella nigrescens that has a low sequence identity compared with all other known α-l-fucosidases and is highly reactive toward a core disaccharide substrate with fucose α(1,3)-, α (1,4)-and α(1,6)-linked to GlcNAc, and is less reactive toward the Fuc-α(1,2)-Gal on the terminal trisaccharide of the oligosaccharide Globo H (Bb3). The kinetic properties of the enzyme, such as its Km and kcat, were determined and the optimized expression of PnfucA gave a yield exceeding 30 mg/L. The recombinant enzyme retained its full activity even after being incubated for 6 h at 37 °C. Moreover, it retained 92 and 87% of its activity after freezing and freeze-drying treatments, respectively, for over 28 days. In a representative glycoengineering of adalimumab (Humira), PnfucA showed remarkable hydrolytic efficiency in cleaving the α(1,6)-linked core fucose from FucGlcNAc on the antibody with a quantitative yield. This enabled the seamless incorporation of biantennary sialylglycans by Endo-S2 D184 M in a one-pot fashion to yield adalimumab in a homogeneous afucosylated glycoform with an improved binding affinity toward Fcγ receptor IIIa.
Barley (1,3;1,4)-β-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-β-d-glucanase, enabling the hydrolysis of (1,3;1,4)-β-d-glucans in the cell walls of leaves and ...germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-β-d-glucan endohydrolase (1,3;1,4)-β-d-glucanase isoenzyme EII (HvEII) and (1,3)-β-d-glucan endohydrolase (1,3)-β-d-glucanase isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-β-d-glucanase and one variant that hydrolyzed both (1,3)-β-d-glucans and (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-β-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-β-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-β-d-glucans or (1,3)-β-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-β-d-glucans and (1,3)-β-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.
A series of 3-acylhydrazono-4-hydroxycoumarins were synthesized via condensation of 3-acetyl-4-hydroxycoumarin with appropriate hydrazides. The structures of the newly-synthesized compounds were ...characterized by spectral and elememental analysis or HRMS measurements. Their antioxidant properties were evaluated by using scavenging effects on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical as well as inhibition of lipid peroxidation. Moreover, their ability to inhibit in vitro soybean lipoxygenase has been investigated. They were found to be capable of rapid inactivation of alkylperoxy radicals.
The industrial production of palm oil concurrently generates a substantial amount of empty fruit bunch (EFB) fibers that could be used as a feedstock in a lignocellulose-based biorefinery. Lignin ...byproducts generated by this process may offer opportunities for the isolation of value-added products, such as p-hydroxybenzoate (pBz), to help offset operating costs. Analysis of the EFB lignin by nuclear magnetic resonance (NMR) spectroscopy clearly revealed the presence of bound acetate and pBz, with saponification revealing that 1.1 wt% of the EFB was pBz; with a lignin content of 22.7 %, 4.8 % of the lignin is pBz that can be obtained as a pure component for use as a chemical feedstock. Analysis of EFB lignin by NMR and derivatization followed by reductive cleavage (DFRC) showed that pBz selectively acylates the γ-hydroxyl group of S units. This selectivity suggests that pBz, analogously with acetate in kenaf, p-coumarate in grasses, and ferulate in a transgenic poplar augmented with a feruloyl-CoA monolignol transferase (FMT), is incorporated into the growing lignin chain via its γ-p-hydroxybenzoylated monolignol conjugate. Involvement of such conjugates in palm lignification is proven by the observation of novel p-hydroxybenzoylated non-resinol β–β-coupled units in the lignins. Together, the data implicate the existence of p-hydroxybenzoyl-CoA:monolignol transferases that are involved in lignification in the various willows (Salix spp.), poplars and aspen (Populus spp., family Salicaceae), and palms (family Arecaceae) that have p-hydroxybenzoylated lignins. Even without enhancing the levels by breeding or genetic engineering, current palm oil EFB ‘wastes’ should be able to generate a sizeable stream of p-hydroxybenzoic acid that offers opportunities for the development of value-added products derived from the oil palm industry.
Inhibition of the vascular endothelial growth factor (VEGF) signaling pathway has emerged as one of the most promising new approaches for cancer therapy. We describe herein the key steps starting ...from an initial screening hit leading to the discovery of pazopanib, N 4-(2,3-dimethyl-2H-indazol-6-yl)-N 4-methyl-N 2-(4-methyl-3-sulfonamidophenyl)-2,4-pyrimidinediamine, a potent pan-VEGF receptor (VEGFR) inhibitor under clinical development for renal-cell cancer and other solid tumors.
Collenchyma cells have thickened primary cell walls and provide mechanical support during plant growth. During their development, these cells elongate and their walls thicken considerably. We used ...microscopy and synchrotron small-angle X-ray scattering to study changes in the orientations of cellulose microfibrils that occur during development in the walls of collenchyma cells present in peripheral strands in celery (Apium graveolens) petioles. Transmission electron microscopy showed that the walls consisted of many lamellae (polylamellate), with lamellae containing longitudinally oriented cellulose microfibrils alternating with microfibrils oriented at higher angles. The lamellae containing longitudinally oriented microfibrils predominated at later stages of development. Nevertheless, transmission electron microscopy of specially stained, oblique sections provided evidence that the cellulose microfibrils were ordered throughout development as crossed-polylamellate structures. These results are consistent with our synchrotron small-angle X-ray scattering results that showed the cellulose microfibrils become oriented increasingly longitudinally during development. Some passive reorientation of cellulose microfibrils may occur during development, but extensive reorientation throughout the wall would destroy ordered structures. Atomic force microscopy and field emission scanning electron microscopy were used to determine the orientations of newly deposited cellulose microfibrils. These were found to vary widely among different cells, which could be consistent with the formation of crossed-polylamellate structures. These newly deposited cellulose microfibrils are deposited in a layer of pectic polysaccharides that lies immediately outside the plasma membrane. Overall, our results show that during development of collenchyma walls, the cellulose microfibrils become increasingly longitudinal in orientation, yet organized, crossed-polylamellate structures are maintained.
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