Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins--towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation.

BACKGROUND: Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins. RESULTS: The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin ß-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit:H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform. CONCLUSION: Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.

Suppression of lipopolysaccharide-induced inflammatory responses in RAW 264.7 murine macrophages by aqueous extract of Clinopodium vulgare L. (Lamiaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: The wild basil Clinopodium vulgare L. is commonly used in Bulgarian folk medicine for treatment of irritated skin, mastitis- and prostatitis-related swelling, as well as for some disorders accompanied with significant degree of inflammation (e.g. gastric ulcers, diabetes, and cancer). AIM OF STUDY: To determine the effect of aqueous extract of Clinopodium vulgare L. on LPS-induced inflammatory responses of murine RAW 264.7 macrophages. MATERIALS AND METHODS: Cell cytotoxicity was evaluated by MTT assay. Protein expression levels were monitored by Western blot analysis. Production of NO and PGE(2) was measured by the Griess colorimetric method and enzyme immunoassay, respectively. Activation of MMP-9 was visualized by gelatin zymography. Cytokine levels were determined by BioPlex assay. Intracellular ROS and free radical scavenging potential were measured by DCFH-DA and DPPH method, respectively. Xanthine oxidase activity was evaluated spectrophotometrically. RESULTS: The extract suppresses NF-kappaB activation by preventing I kappa-B phosphorylation and inhibits the phosphorylation of p38 and SAPK/JNK MAPKs. It down-regulates iNOS expression which manifests as a drastic decrease of NO production, inhibits MMP-9 activation, but does not affect COX-2 protein levels and reduces only slightly the released PGE(2). Secretion of IL-1 beta and Il-10 is greatly reduced, whereas suppression of TNF-alpha and GM-CSF production is less dramatic. The extract has strong free radical scavenging properties and exerts inhibitory effect on xanthine oxidase activity, which lowers the levels of intracellular ROS. CONCLUSION: The study provides evidence for the anti-inflammatory potential of Clinopodium vulgare L. aqueous extract.

A novel cyclization reaction between 2,3-bis(trimethylsilyl)buta-1,3-diene and acyl chlorides with straightforward formation of polysubstituted furans.

A novel cyclization process of 2,3-bis(trimethylsilyl)buta-1,3-diene with various acyl chlorides in the presence of aluminium trichloride affords 2,5-disubstituted or 2,3,5-trisubstituted furans in short reaction time; a subsequent acylation process of the furan ring occurs if the reaction time is prolonged.

Fetuin-A gene expression, synthesis and release in primary human hepatocytes cultured in a galactosylated membrane bioreactor.

This paper reports on human hepatocytes cultured in a galactosylated membrane bioreactor in order to explore the modulation of the effects of a pro-inflammatory cytokine, Interleukin-6 (IL-6) on the liver cells at molecular level. In particular the role of IL-6 on gene expression and production of a glycoprotein, fetuin-A produced by hepatocytes, was investigated by culturing hepatocytes in the membrane bioreactor, both in the absence and presence of IL-6 (300 pg/ml). IL-6 modulated the fetuin-A gene expression, synthesis and release by primary human hepatocytes cultured in the bioreactor. A 75% IL-6-induced reduction of fetuin-A concentration in the medium was associated with a 60% increase of C-reactive protein in the same samples. Real-time-PCR demonstrated an 8-fold IL-6-induced reduction of fetuin-A gene expression. These results demonstrate that the hepatocyte galactosylated membrane bioreactor is a valuable tool to study IL-6 effects and gave evidence, for the first time, that IL-6 down-regulates the gene expression and synthesis of fetuin-A by primary human hepatocytes. The human hepatocyte bioreactor behaves like the in vivo liver, reproducing the same hepatic acute-phase response that occurs during the inflammatory process.

Novel membranes and surface modification able to activate specific cellular responses.

The design of new polymeric biomaterials together with new strategies to modify membrane surface are crucial to optimise cell-biomaterial interactions in vivo and in vitro biohybrid systems. In this study we report on the novel semipermeable membranes synthesised from a polymeric blend of modified polyetheretherketone and polyurethane able to support the long-term maintenance and differentiation of human liver cells and on the surface modification of polyethersulfone membranes by plasma polymerisation of acrylic acid monomers and by immobilization of arginine-glycine-aspartic acid (RGD) peptide through a hydrophilic "spacer arm" molecule. The performance of the modified and unmodified membranes was tested by evaluation of the liver function expression of primary human hepatocytes in terms of albumin production, protein secretion and drug biotransformation.

Galactose derivative immobilized glow discharge processed polyethersulfone membranes maintain the liver cell metabolic activity.

New strategies aimed to surface modification of polymeric membranes are crucial to optimise cell-biomaterial interactions in vivo and in vitro biohybrid systems. In this paper, we investigated the surface modification of Polyethersulfone (PES) membranes by plasma polymerisation of acrylic acid monomers (PES-pdAA) and by immobilization of galactonic acid through a hydrophilic "spacer arm" molecule (PES-pdAA-SA-GAL). The modification steps were characterised by high resolution X-ray photoelectron spectroscopy. The performance of modified and unmodified membranes was evaluated by assessing the expression of liver specific biotransformation functions of pig and human hepatocytes. Human liver cells cultured on PES-pdAA-SA-GAL membranes displayed an enhanced albumin production, urea synthesis and protein secretion for 24 days of culture. The immobilisation of galactose derivative units on the membrane allowed specific interactions with hepatocytes biomimicking the cellular microenvironment and produced an improvement of the long-term maintenance and differentiation of human hepatocytes.

Bioprospecting in plants for engineered proteins.

For more than two decades, bioengineered plants have produced protein therapeutics for human and animal use. Almost all proteins produced by other existing systems, including antibodies, vaccines and plasma proteins, have now been manufactured in plants. Considering the limitations of microbial and mammalian reactor-based protein-production technologies and the impending bottleneck in manufacturing capacity, plants are now emerging as an attractive alternative system with which to supply the growing need for protein-based therapeutics. However, full realization of the promise of plant-derived engineered proteins requires that we confront the dual challenges of bioequivalence and product consistency, challenges that are largely related to post-translational protein modifications (PTMs) that are crucial to the structure and function of most eukaryotic proteins. Among the protein PTMs, the foremost challenge for bioactivity and acceptance by the pharmaceutical and biotechnology industries and regulatory agencies is glycosylation. Advances made in recent years that 'humanize' plant glycosylation pathways combined with the discovery of terminal sialic acids (SAs) in plants now make feasible the bioengineering in plants of glycoproteins that have mammalian-like glycosylation.

Biotransformation and liver-specific functions of human hepatocytes in culture on RGD-immobilized plasma-processed membranes.

In this paper we report on the metabolic response of human hepatocytes grown on polyethersulfone membranes surface modified with a plasma-deposited acrylic acid coating and RGD peptide covalently immobilized through a "spacer arm" molecule. The modified surfaces were characterized by means of X-ray photoelectron spectroscopy and water contact angle measurements. The performance of modified and unmodified membranes was evaluated by assessing the expression of liver specific and biotransformation functions of human hepatocytes. Diclofenac, a non-steroidal anti-inflammatory drug, was used to investigate the biotransformation functions. Surface-modified membranes elicit specific cellular responses and induce hepatocytes to enhance the synthesis rate of albumin and urea, particularly in the presence of diclofenac. Also the biotransformation functions were expressed at high levels.

Mexican-American parental involvement with a Texas elementary school.

A sample of 403 Mexican-American parents of elementary school age children were surveyed regarding their involvement in school. Helping children with school work, attending parent-teacher conferences, and fundraising were identified by the parents as activities in which they most frequently participated.