Plant Glycosides Research Articles

Discovery of α-l-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42

Enzymes of the glycoside hydrolase family 42 (GH42) are widespread in bacteria of the human gut microbiome and play fundamental roles in the decomposition of both milk and plant oligosaccharides. All GH42 enzymes characterized so far have β-galactosidase activity. Here, we report the existence of a GH42 subfamily that is exclusively specific for α-l-arabinopyranoside and describe the first representative of this subfamily. We found that this enzyme (BlArap42B) from a probiotic Bifidobacterium species cannot hydrolyze β-galactosides. However, BlArap42B effectively hydrolyzed paeonolide and ginsenoside Rb2, plant glycosides containing an aromatic aglycone conjugated to α-l-arabinopyranosyl-(1,6)-β-d-glucopyranoside. Paeonolide, a natural glycoside from the roots of the plant genus Paeonia, is not hydrolyzed by classical GH42 β-galactosidases. X-ray crystallography revealed a unique Trp345-X12-Trp358 sequence motif at the BlArap42B active site, as compared with a Phe-X12-His motif in classical GH42 β-galactosidases. This analysis also indicated that the C6 position of galactose is blocked by the aromatic side chains, hence allowing accommodation only of Arap lacking this carbon. Automated docking of paeonolide revealed that it can fit into the BlArap42B active site. The Glcp moiety of paeonolide stacks onto the aromatic ring of the Trp252 at subsite +1 and C4-OH is hydrogen bonded with Asp249 Moreover, the aglycone stacks against Phe421 from the neighboring monomer in the BlArap42B trimer, forming a proposed subsite +2. These results further support the notion that evolution of metabolic specialization can be tracked at the structural level in key enzymes facilitating degradation of specific glycans in an ecological niche.

Lactobacillus acidophilus Metabolizes Dietary Plant Glucosides and Externalizes Their Bioactive Phytochemicals.

ABSTRACTTherapeutically active glycosylated phytochemicals are ubiquitous in the human diet. The human gut microbiota (HGM) modulates the bioactivities of these compounds, which consequently affect host physiology and microbiota composition. Despite a significant impact on human health, the key players and the underpinning mechanisms of this interplay remain uncharacterized. Here, we demonstrate the growth of Lactobacillus acidophilus on mono- and diglucosyl dietary plant glycosides (PGs) possessing small aromatic aglycones. Transcriptional analysis revealed the upregulation of host interaction genes and identified two loci that encode phosphotransferase system (PTS) transporters and phospho-β-glucosidases, which mediate the uptake and deglucosylation of these compounds, respectively. Inactivating these transport and hydrolysis genes abolished or severely reduced growth on PG, establishing the specificity of the loci to distinct groups of PGs. Following intracellular deglucosylation, the aglycones of PGs are externalized, rendering them available for absorption by the host or for further modification by other microbiota taxa. The PG utilization loci are conserved in L. acidophilus and closely related lactobacilli, in correlation with versatile growth on these compounds. Growth on the tested PG appeared more common among human gut lactobacilli than among counterparts from other ecologic niches. The PGs that supported the growth of L. acidophilus were utilized poorly or not at all by other common HGM strains, underscoring the metabolic specialization of L. acidophilus. These findings highlight the role of human gut L. acidophilus and select lactobacilli in the bioconversion of glycoconjugated phytochemicals, which is likely to have an important impact on the HGM and human host.

Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.)

Abstract β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p -nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a V max of 131.6 μmol min −1 mg −1 protein, K m of 0.158 mM, K cat of 144.8 s −1 , K cat / K m of 917.4 mM −1 s −1 , and K m / V max of 0.0012 mM min mg μmole −1 , using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10 −5 s −1 , and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material.

A Novel Fingerprinting Method for Glycosides in Nakshatra Plants by HPTLC

Nakshatra plants are medicinally important and used from olden days onwards to treat various diseases. In the present study, a HPTLC method was developed for separation of glycosides in plants. The powdered drug of Nakshatra plant was extracted with ethanol and identification was done chemically by Molisch's, Fehling's, Bial's test and separation of glycosides was performed densitometrically at λ=254-700 nm using as a mobile phase, ethyl acetate: methanol: water (20:2.5:2.5). The results of qualitative analysis revealed the presence of carbohydrates, reducing sugars and pentose. HPTLC method achieved a good separation of glycosides at 254 nm, 304 nm, 354 nm, 404 nm with recorded peak areas and Rf values. The comparative common compound of sample glycosides was also done.

Overview of Important Medicinal Plants at Fars Province, Iran

Brachychiton rupestris and Brachychiton luridum are deciduous trees belonging to family Malvaceae. Several members of the genus Brachychiton and its related genus, Sterculia, are used in folk medicine for the treatment of infections. Herein, we investigated the antimicrobial activity of the total extracts, n-hexane, dichloromethane as well as the ethyl acetate fractions of the leaves of both species using agar well diffusion method in an effort to better consolidate the scientific basis for their traditional uses. The study was carried out against two Gram-positive and two Gram-negative bacteria together with two fungi. The extracts and fractions showed mean inhibition zones diameter in the range of 10.97-14.98 mm with minimum inhibitory concentration values ranging between 39.06 and 625 μg/mL against the susceptible bacteria and fungi. The ethyl acetate fraction of B. luridum exhibited notable activity against Staphylococcus aureus while the total extract showed moderate activity against Salmonella typhi. Regarding B. rupestris, the total extract, n-hexane and ethyl acetate fractions showed moderate activity against Escherichia coli, Candida albicans, and Salmonella typhi, respectively. Other extracts and fractions showed weak or no activity against the tested microorganisms. Qualitative phytochemical screening of both plants was performed to preliminary unveil the classes of secondary metabolites present in the leaves. The results indicated the existence of flavonoids, tannins, sterols, triterpenes, carbohydrates and/or glycosides in both plants.

The Effects of Gender and Multiple Oral Dosing on the Pharmacokinetics and Bioavailability of Morroniside in Beagle Dogs: A Pilot Study

Morroniside is a well-known iridoid plant glycoside. Attempts were made to study the effects of gender and multiple oral dosing on the pharmacokinetics and bioavailability of morroniside in beagle dogs. The concentration of morroniside in plasma was determined using a LC-MS/MS method. Main pharmacokinetic parameters were estimated by DAS 2.0 pharmacokinetic software. Statistical analysis was performed using student t-test with P-values less than 0.05 as the level of significance. No significant gender difference was observed in the pharmacokinetic behaviour of morroniside and no differences were observed in the pharmacokinetic parameters following single and multiple administrations of morroniside. The absolute bioavailability after the oral administration of morroniside in beagle dogs was found to be 7.47±0.65%.

In vitro effects of 1α,25(OH)2D3-glycosides from Solbone A (Solanum glaucophyllum leaves extract; Herbonis AG) compared to synthetic 1α,25(OH)2D3 on myogenesis

The presence of glycoside derivatives of 1α,25(OH)2D3 endows plants to gradual release of the free bioactive form of 1α,25(OH)2D3 from its glycoconjugates by endogenous animal tissue glycosidases. This results in increased half-life of the hormone in blood when purified plant fractions are administered for therapeutic purposes. In this work, we evaluated the role 1α,25(OH)2D3-glycosides enriched natural product (Solbone A) from Solanum glaucophyllum leaf extract compared with synthetic 1α,25(OH)2D3 on myogenic differentiation in C2C12 myoblasts. For these, differentiation markers and myogenic parameters were studied in C2C12 myoblasts. Results showed that Solbone A, likewise the synthetic hormone, increased creatine kinase activity at day 2 after differentiation induction (60%, p<0.05). Solbone A and synthetic 1α,25(OH)2D3 increased vitamin D3 receptor protein expression at 10nM (50% and 30%, respectively) and the transcription factor myogenin (80%, p<0.05). However, tropomyosin expression was not affected by both compounds. In addition, myosin heavy chain (MHC) protein expression was increased 30% at day 2 of differentiation. Solbone A or synthetic 1α,25(OH)2D3 had no effects on myogenin nor MHC cell localization. Cellular mass increased with myogenesis progression, being Solbone A more effective than synthetic 1α,25(OH)2D3. Finally, Solbone A, as well as synthetic 1α,25(OH)2D3, augmented the index fusion of cultured muscle fibers. In conclusion, these results demonstrated that Solbone A exhibit at least equal or greater effects on early myoblast differentiation as synthetic hormone, suggesting that plant glycosides could be an effective, accessible and cheaper substitute for synthetic 1α,25(OH)2D3 to promote muscle growth.

МАСС-СПЕКТРОМЕТРИЯ СУПРАМОЛЕКУЛЯРНЫХ КОМПЛЕКСОВ ГЛИЦИРРИЗИНОВОЙ КИСЛОТЫ И СТРЕПТОМИЦИНА

Глицирризиновая кислота (ГК), выделенная из корней солодки голой Glycyrrhiza glabra L. и Gl. uralensis F., обладает уникальными комплексообразующими свойствами, которые широко используются в синтезе новых лекарственных форм на основе молекулярных комплексов с фармаконами. В таких гетерогенных комплексах молекулы ГК выступают в роли полидентантных лигандов, образующих с фармаконами супрамолекулярные комплексы различной стехиометрии. Целью данной работы является изучение комплексообразования растительных гликозидов с антимикробным препаратом стрептомицином для создания новых низкодозных фармсубстанций на основе глицирризиновой кислоты. В представленной работе впервые синтезированы и изучены методом УФ-спектроскопии и масс-спектрометрии с ионизацией электрораспылением гетерогенные супрамолекулярные комплексы ГК с антибиотиком стрептомицином (СТ), полученные при различных концентрациях «хозяина» и «гостя» в соотношении 1 : 1 и 4 : 1. Зарегистрированные изменения в спектре поглощения СТ в присутствии ГК с помощью УФ-спектроскопии доказывают формирование гетерогенных комплексов ГК-СТ в жидкой фазе. С использованием масс-спектрометрии зарегистрировано эффективное взаимодействие ГК и СТ с образованием различных гетерогенных супрамолекулярных структур с соотношением «хозяин: гость» 1 : 1, 2 : 1, 3 : 1, 3 : 2. При увеличении концентрации ГК наблюдали как увеличение степени самоассоциации ГК (до 4), так и образование комплекса с соотношением 4 : 1. Результаты исследований демонстрируют высокий потенциал использования глицирризиновой кислоты для синтеза новых форм антибактериального препарата стрептомицина в виде нековалентных молекулярных комплексов.

New insights into plant glycoside hydrolase family 32 in Agave species.

In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana.

Methylation mediated by an anthocyanin, O-methyltransferase, is involved in purple flower coloration in Paeonia.

Anthocyanins are major pigments in plants. Methylation plays a role in the diversity and stability of anthocyanins. However, the contribution of anthocyanin methylation to flower coloration is still unclear. We identified two homologous anthocyanin O-methyltransferase (AOMT) genes from purple-flowered (PsAOMT) and red-flowered (PtAOMT) Paeonia plants, and we performed functional analyses of the two genes in vitro and in vivo. The critical amino acids for AOMT catalytic activity were studied by site-directed mutagenesis. We showed that the recombinant proteins, PsAOMT and PtAOMT, had identical substrate preferences towards anthocyanins. The methylation activity of PsAOMT was 60 times higher than that of PtAOMT in vitro. Interestingly, this vast difference in catalytic activity appeared to result from a single amino acid residue substitution at position 87 (arginine to leucine). There were significant differences between the 35S::PsAOMT transgenic tobacco and control flowers in relation to their chromatic parameters, which further confirmed the function of PsAOMT in vivo. The expression levels of the two homologous AOMT genes were consistent with anthocyanin accumulation in petals. We conclude that AOMTs are responsible for the methylation of cyanidin glycosides in Paeonia plants and play an important role in purple coloration in Paeonia spp.

Enhanced production of steviol glycosides in mycorrhizal plants: A concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes

Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs) – stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-d-xylulose 5-phospate synthase (DXS), 1-deoxy-d-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants.

МАСС-СПЕКТРОМЕТРИЯ СУПРАМОЛЕКУЛЯРНЫХ КОМПЛЕКСОВ ГЛИЦИРРЕТИНОВОЙ КИСЛОТЫ С АНТИБИОТИКОМ СТРЕПТОЦИДОМ

One of most effective ways to increase the bioavailability of drug substances is their structural modification by formation of supramolecular structures with plant glycosides. Application of noncovalent complexes of drugs with plant glycosides, e.g., glycyrrhizic acid (GA), isolated from the roots of Glycyrrhiza glabra L., leads to a significant improvement of pharmacokinetic and pharmacodynamic parameters of drugs. Glycyrrhizic acid (GLA) is the aglycon of GA and has the same molecular skeleton. Also, GLA has own broad spectrum of biological activity. The aim of the work is to study the complexation of GLA with antimicrobial drugs to create new low-dose pharmaceutical substances. In the present study, for the first time complex compounds of GLA and antibiotic sulfanilamide (Strep) were synthesized at different ratios «host» and «guest». The composition of supramolecular complexes of GLA with Strep was identified by mass spectrometry electrospray ionization. Peaks of heterogeneous complexes consisting of monomers, dimers, trimers and tetramers of GLA with the inclusion of one molecule of Strep were registered in mass spectra. The mass spectrometric data was shown high activity of the binding Strep by GLA with formation of supramolecular complexes. Given the high biological activity of GLA and possible synergies of heterogeneous complexes, research results open up prospects to obtain new low-dose drugs of Strep.

Activity of β-glucosidase in plants with high content of cyanogenic glycosides as important factor determining their toxicity

Background: The acute toxicity of cyanide after its enzymatic release from cyanogenic glycosides in plants (e.g. bitter almonds) is triggered by the peak level of cyanide in the body. In this regard, not only the content of bound cyanide in foods is relevant, but also the velocity of the release after chewing of the food caused by the β-glucosidase and the following surge of free cyanide in the body. In a human cross-over study (n = 12) with consumption of persipan, linseed, bitter apricot kernels and cassava (dose of bound cyanide: 6.8 mg each), mean peak levels of cyanide in blood were found to be 1.3, 5.7, 14.3 and 15.4 μM, respectively. Additional in-vitro investigations were performed to confirm the velocity of the enzymatic release of cyanide (by plant β-glucosidase) as important factor determining the toxicity of cyanogenic foods. Methods: Food samples containing 1 mg of bound cyanide were homogenized and suspended in 100 ml PBS buffer (pH 4 and 7). Free cyanide was determined at different times in samples taken from the suspension gently stirred at 37 °C (GC–MS method, internal standard: K13C15N). Results: Mean proportion of the total amount released within 10 min after tissue destruction was 0% for persipan, 15% (pH 7) for linseed, 62% (pH 4) for bitter apricot kernels and 78% (pH 7) for cassava. Conclusion: The toxicity of cyanogenic foods is determined not only by its content of bound cyanide, but also by the activity of the β-glucosidase as a plant-specific property.

Short-term exposure to oleandrin enhances responses to IL-8 by increasing cell surface IL-8 receptors

One of the first steps in host defence is the migration of leukocytes. IL-8 and its receptors are a chemokine system essential to such migration. Up-regulation of these receptors would be a viable strategy to treat dysfunctional host defence. Here, we studied the effects of the plant glycoside oleandrin on responses to IL-8 in a human monocytic cell line. U937 cells were incubated with oleandrin (1-200 ng mL(-1) ) for either 1 h (pulse) or for 24 h (non-pulse). Apoptosis; activation of NF-κB, AP-1 and NFAT; calcineurin activity and IL-8 receptors (CXCR1 and CXCR2) were measured using Western blotting, RT-PCR and reporter gene assays. Pulse exposure to oleandrin did not induce apoptosis or cytoxicity as observed after non-pulse exposure. Pulse exposure enhanced activation of NF-κB induced by IL-8 but not that induced by TNF-α, IL-1, EGF or LPS. Exposure to other apoptosis-inducing compounds (azadirachtin, resveratrol, thiadiazolidine, or benzofuran) did not enhance activation of NF-κB. Pulse exposure to oleandrin increased expression of IL-8 receptors and chemotaxis, release of enzymes and activation of NF-κB, NFAT and AP-1 along with increased IL-8-mediated calcineurin activation, and wound healing. Pulse exposure increased numbers of cell surface IL-8 receptors. Short-term (1 h; pulse) exposure to a toxic glycoside oleandrin, enhanced biological responses to IL-8 in monocytic cells, without cytoxicity. Pulse exposure to oleandrin could provide a viable therapy for those conditions where leukocyte migration is defective.

Effect of saponin on erythrocytes.

Saponins, naturally occurring glycosides and triterpene glycosides in plants, are considered useful in the prophylaxis and treatment of several disorders, including malignancy. The effect of these substances is partly attributable to induction of both apoptosis and necrosis. Saponin has previously been shown to trigger hemolysis. Erythrocytes may avoid hemolysis by entering programmed cell death or eryptosis, characterized by cell shrinkage and cell membrane scrambling, leading to phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity ([Ca(2+)](i)). The present study explored, whether exposure of human erythrocytes to saponin modifies [Ca(2+)](i), ceramide formation, hemolysis, and eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, [Ca(2+)](i) from Fluo3-fluorescence, and ceramide utilizing specific antibodies. A 24 h exposure to saponin (15 µg/ml) resulted in a significant increase of annexin V binding and a significant stimulation of hemolysis. Saponin (15 µg/ml) further increased [Ca(2+)](i) and ceramide formation. Annexin V binding was significantly blunted but not abrogated in the nominal absence of extracellular Ca(2+). Saponin thus triggers cell membrane scrambling, an effect partially due to entry of extracellular Ca(2+) and ceramide formation.

Ginseng ginsenoside pharmacology in the nervous system: involvement in the regulation of ion channels and receptors

Ginseng, the root of Panax ginseng C.A. Meyer, is one of the oldest traditional medicines and is thought to be a tonic. It has been claimed that ginseng may improve vitality and health. Recent studies have advanced ginseng pharmacology and shown that ginseng has various pharmacological effects in the nervous system. Ginsenosides, steroid glycosides extracted from ginseng, were one of the first class of biologically active plant glycosides identified. The diverse pharmacological effects of ginsenosides have been investigated through the regulation of various types of ion channels and receptors in neuronal cells and heterologous expression systems. Ginsenoside Rg3 regulates voltage-gated ion channels such as Ca2+, K+, and Na+ channels, and ligand-gated ion channels such as GABAA, 5-HT3, nicotinic acetylcholine, and N-methyl-D-aspartate (NMDA) receptors through interactions with various sites including channel blocker binding sites, toxin-binding sites, channel gating regions, and allosteric channel regulator binding sites when the respective ion channels or receptors are stimulated with depolarization or ligand treatment. Treatment with ginsenoside Rg3 has been found to stabilize excitable cells by blocking influxes of cations such as Ca2+ and Na+, or by enhancing Cl− influx. The aim of this review is to present recent findings on the pharmacological functions of the ginsenosides through the interactions with ion channels and receptors. This review will detail the pharmacological applications of ginsenosides as neuroprotective drugs that target ion channels and ligand-gated ion channels.

Plant Glycosides and Aglycones Displaying Antiproliferative and Antitumour Activities – A Review

Glycosides are a group of naturally occurring compounds found in a variety of plant families, explored for their antibacterial, antifungal, anti-inflammatory, antioxidant, antiviral and anticancer activities. There are more than 11 distinguished classes including anthraquinone, cardiac, chromone, coumarin, cyanogenic, flavonoid, saponin, steroidal and steviol glycosides. Due to the extensive variability in their structures, some glycosides have always displayed antitumour and cytotoxic properties through a variety of pathways. A large number of glycosides still remain to be explored for their antitumour potential. Taking into consideration the pace with which new entities are being discovered and investigated, it is essential to gather all the latest information, on the previously investigated molecules, under one roof which might help young researchers in understanding the current status of glycosides in cancer therapy research. The aim of this review is to collate many such structures and their related chemistry along with the understanding of their mechanism of action and structure activity relationships. The review focuses mainly on anthraquinone, coumarin and flavonoid glycosides and contribution of substituents on their glycone and aglycone moiety towards the antitumour property. Compounds such as Didymin, Digitoxin, Dioscin, Esculetin, Genistein, OSW-1, Tubeimoside and many more have been discussed here. Keywords: Antitumour, Cell lines, Cytotoxic, Glycosides, Mechanism of action, Structure-activity relationship.

Abstract A83: Combinatorial approach to drastically enhance the monoclonal antibody efficacy in targeted tumor therapy.

Abstract Antitumor therapy with monoclonal antibodies has shown great therapeutic benefits in clinics and a number of therapeutic antibodies have been approved for use in patients. Nevertheless, their efficacy is limited due to their insufficient inherent cell killing activity. In order to augment their antitumor efficacy, monoclonal antibodies may be modified with natural toxins to create a drug conjugate (immunotoxin) that comprises the dual functionality of monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) and the cell-killing activity of the toxin. In our investigations, two immunotoxins were prepared by chemically coupling the monoclonal antibodies trastuzumab (Herceptin®) and cetuximab (Erbitux®) to the plant derived toxin saporin via a cleavable disulfide bond. Impedance-based real-time viability assays and confocal live cell imaging revealed that the toxin is efficiently delivered to the targeted tumor cells. In addition, immunotoxin cytotoxicity was tremendously enhanced in combination with certain plant glycosides, which specifically mediate the endo/lysosmal release of the toxin moiety into the cytosol without damaging the biological membranes. The combinatorial approach was successful in achieving a complete cell death of human breast ductal carcinoma BT-474 cells, at a concentration of 1 nM of trastuzumab-saporin or 0.001 nM cetuximab-saporin in the presence of the glycosides. Furthermore, immunotoxins retained their ability to interact with the innate immune system and trigger antibody-dependent cell-mediated cytotoxicity. The combinatorial approach is conceived as a novel and promising way to increase the efficacy of monoclonal antibodies in targeted tumor therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A83. Citation Format: Roger Gilabert-Oriol, Mayank Thakur, Benedicta von Mallinckrodt, Thomas Hug, Burkhard Wiesner, Jenny Eichhorst, Matthias F. Melzig, Hendrik Fuchs, Alexander Weng. Combinatorial approach to drastically enhance the monoclonal antibody efficacy in targeted tumor therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A83.

Effect of harvest timing on leaf production and yield of diterpene glycosides in Stevia rebaudiana Bert: A specialty perennial crop for Mississippi

Stevia rebaudiana (Bertoni), a perennial herb of the Asteraceae, is one of the most important sources of non-caloric natural sweeteners. Stevia's plant extracts and glycosides have been used by indigenous cultures for centuries in Paraguay and Brazil. Several studies suggest that Stevia and its glycosides exert beneficial effects on human health, including anti-hyperglycemic and antihypertensive properties (Jeppesen et al., 2000). The objectives of this study were to evaluate Stevia's cold hardness in Mississippi and determine the effects of different harvest timing on leaf production and yield of diterpene glycosides. Plants purchased from Ritchers Herbs were the source of nodal explants for in vitro clonal propagation. Rooted plantlets adapted in trays were later transplanted in the field of the University of Mississippi Biological Field Station. Plant spacing was 30cm between plants in a row and 60cm between rows. Drip irrigation supplied water to the field at rate of ¼ to ½in. per week during summer. To protect plants during the winter of 2010 a plastic in field low tunnel was used, while in 2011 plants remained uncovered. Leaf biomass production and yield of diterpene glycosides were evaluated based on three different harvest timings: (1) Three harvests at 60 day intervals, two leaf harvests at a 90-day interval and a single harvest after 180 days of cultivation. Results revealed that leaf production from plants harvested once a year yielded more than the yield of multiple harvests (2 and 3 harvests per growing season). A total of 13,896.37kg per hectare of stem and leaves were harvested in a single harvest. The glycosides productivity obtained from a single harvest; rebaudioside A (398.80kgha−1) and stevioside (512.21kgha−1) was also greater than the yields of multiple harvests (2 and 3 harvests).

Engineering flavonoid glycosyltransferases for enhanced catalytic efficiency and extended sugar-donor selectivity

Flavonoids are predominantly found as glycosides in plants. The glycosylation of flavonoids is mediated by uridine diphosphate-dependent glycosyltransferases (UGT). UGTs attach various sugars, including arabinose, glucose, galactose, xylose, and glucuronic acid, to flavonoid aglycones. Two UGTs isolated from Arabidopsis thaliana, AtUGT78D2 and AtUGT78D3, showed 89 % amino acid sequence similarity (75 % amino acid sequence identity) and both attached a sugar to the 3-hydroxyl group of flavonols using a UDP-sugar. The two enzymes used UDP-glucose and UDP-arabinose, respectively, and AtUGT78D2 was approximately 90-fold more efficient than AtUGT78D3 when judged by the k(cat)/K(m) value. Domain exchanges between AtUGT78D2 and AtUGT78D3 were carried out to find UGTs with better catalytic efficiency for UDP-arabinose and exhibiting dual sugar selectivity. Among 19 fusion proteins examined, three showed dual sugar selectivity, and one fusion protein had better catalytic efficiency for UDP-arabinose compared with AtUGT78D3. Using molecular modeling, the changes in enzymatic properties in the chimeric proteins were elucidated. To the best of our knowledge, this is the first report on the construction of fusion proteins with expanded sugar-donor range and enhanced catalytic efficiencies for sugar donors.