ABSTRACT:(؊)-Epigallocatechin-3-gallate (EGCG) is the widely studied catechin in green tea (Camellia sinensis). Previously, we have reported the low bioavailability of EGCG in rats and mice. As a means of improving the bioavailability of EGCG, we have prepared a peracetylated EGCG derivative (AcEGCG) and herein report its growth inhibitory activity and cellular uptake in vitro, as well as bioavailability in mice. AcEGCG exhibited enhanced growth inhibitory activity relative to EGCG in both KYSE150 human esophageal (IC 50

Despite recent advances in nanomaterial-based delivery systems, their applicability as carriers of cargo, especially proteins for targeting cellular components and manipulating cell function, is not well-understood. Herein, we demonstrate the ability of hydrophobic silica nanoparticles to deliver proteins, including enzymes and antibodies, to a diverse set of mammalian cells, including human cancer cells and rat stem cells, while preserving the activity of the biomolecule post-delivery. Specifically, we have explored the delivery and cytosolic activity of hydrophobically functionalized silica nanoparticle-protein conjugates in a human breast cancer cell line (MCF-7) and rat neural stem cells (NSCs) and elucidated the mechanism of cytosolic transport. Importantly, the proteins were delivered to the cytosol without extended entrapment in the endosomes, which facilitated the retention of biological activity of the delivered proteins. As a result, delivery of ribonuclease A (RNase A) and the antibody to phospho-Akt (pAkt) resulted in the initiation of cell death. Delivery of control protein conjugates (e.g., those containing green fluorescent protein or goat antirabbit IgG) resulted in minimal cell death, indicating that the carrier-mediated toxicity was low. The results presented here provide insight into the design of nanomaterials as protein carriers that enable control of cell function.

Rapid change and zoonotic transmission to humans have enhanced the virulence of the influenza A virus (IAV). Neutralizing antibodies fail to provide lasting protection from seasonal epidemics. Furthermore, the effectiveness of anti-influenza neuraminidase inhibitors has declined because of drug resistance. Drugs that can block viral attachment and cell entry independent of antigenic evolution or drug resistance might address these problems. We show that multivalent 6'-sialyllactose-polyamidoamine (6SL-PAMAM) conjugates, when designed to have well-defined ligand valencies and spacings, can effectively inhibit IAV infection. Generation 4 (G4) 6SL-PAMAM conjugates with a spacing of around 3 nm between 6SL ligands (S3-G4) showed the strongest binding to a hemagglutinin trimer (dissociation constant of 1.6 × 10 M) and afforded the best inhibition of H1N1 infection. S3-G4 conjugates were resistant to hydrolysis by H1N1 neuraminidase. These conjugates protected 75% of mice from a lethal challenge with H1N1 and prevented weight loss in infected animals. The structure-based design of multivalent nanomaterials, involving modulation of nanoscale backbone structures and number and spacing between ligands, resulted in optimal inhibition of IAV infection. This approach may be broadly applicable for designing effective and enduring therapeutic protection against human or avian influenza viruses.

Garcinol, a polyisoprenylated benzophenone, from the fruit rind of Garcinia spp., has been shown to have antiinflammatory and anticarcinogenic activities. To study its mechanism of action, we analyzed the effects of garcinol and its derivatives, including cambogin, garcim-1 and garcim-2, on arachidonic acid metabolism and nitric oxide (NO) synthesis in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages as well as in three intestinal cell lines. We also examined the effect of garcinol on cytosolic phospholipase A 2 (cPLA 2), cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS), and related upstream signaling. At 1 mM, garcinol and its derivatives, added 1 h after LPS stimulation, significantly inhibited the release of arachidonic acid and its metabolites in macrophages; garcinol was the most effective, showing 450% inhibition. Similar inhibitory activity was also observed in intestinal cells, HT-29, HCT-116 and IEC-6 cells, showing 40-50% inhibition by 1 mM garcinol. In LPSstimulated macrophages, garcinol inhibited the phosphorylation of cPLA 2 without altering its protein level, and the effect was related to the inhibition of ERK1/2 phosphorylation. Garcinol inhibited NFkB activation and COX-2 expression only when it was added to the cells before LPS stimulation. Garcinol (1 mM) also significantly decreased iNOS expression and NO release from LPS-stimulated macrophages; this is probably due to the inhibition of the signal transducer and activator of transcription-1 (STAT-1), an upstream event in the activation of iNOS synthesis. The results suggest that garcinol modulates arachidonic acid metabolism by blocking the phosphorylation of cPLA 2 and decreases iNOS protein level by inhibiting STAT-1 activation. These activities may contribute to the anti-inflammatory and anticarcinogenic actions of garcinol and its derivatives.

Differential expression of various drug-metabolizing enzymes in the human liver may cause deviations of pharmacokinetic profiles, resulting in inter-individual variability of drug toxicity and/or efficacy. Here we present the “Transfected Enzyme and Metabolism Chip” (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip. The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2E1, and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray. Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner.

Due to their spectroscopic properties porphyrins are of special interest for a variety of applications, ranging from drug development or targeting to material sciences and chemical and biological sensors. Since chemical syntheses are limited in terms of regio-and stereoselective functionalization of porphyrins, a biosynthetic approach with tailored enzyme catalysts offers a promising alternative. In this paper, we describe assembly of the entire heme biosynthetic pathway in a three-plasmid system and overexpression of the corresponding genes with Escherichia coli as a host. Without further optimization, this approach yielded remarkable porphyrin production levels, up to 90 mol/liter, which is close to industrial vitamin B 12 production levels. Different combinations of the genes were used to produce all major porphyrins that occur as intermediates in heme biosynthesis. All these porphyrin intermediates were obtained in high yields. The product spectrum was analyzed and quantified by using high-performance liquid chromatography. Intriguingly, although protoporphyrin IX could be produced at high levels, overexpressed Bacillus subtilis ferrochelatase could not convert this substrate appreciably into heme. However, further investigation clearly revealed a high level of expression of the ferrochelatase and a high level of activity in vitro. These results may indicate that heme has a regulatory impact on the iron uptake of E. coli or that the ferrochelatase is inactive in vivo due to an incompatible enzyme interaction.

The ever-increasing industrial demand for biocatalysis necessitates innovations in the preparation and stabilization of biocatalysts. In this study, we demonstrated that ␤-galactosidase (␤-Gal) displayed on Bacillus spores by fusion to the spore coat proteins (CotG) may be used as a whole-cell immobilized biocatalyst for transgalactosylation in water-solvent biphasic reaction systems. The resulting spores had a specific hydrolytic activity of 5 ؋ 10 3 U/g (dry weight) of spores. The ␤-Gal was tightly attached to the spore surface and was more stable in the presence of various organic solvents than its native form was. The thermostability of the spore-displayed enzyme was also increased, and the enzyme was further stabilized by chemically cross-linking it with glutaraldehyde. With spore-displayed ␤-Gal, octyl-␤-D-galactopyranoside was synthesized at concentrations up to 27.7 mM (8.1 g/liter) with a conversion yield of 27.7% (wt/wt) after 24 h from 100 mM lactose and 100 mM octanol dissolved in phosphate buffer and ethyl ether, respectively. Interestingly, the spores were found to partition mainly at the interface between the water and solvent phases, and they were more available to catalysis between the two phases, as determined by light microscopy and confocal fluorescence microscopy. We propose that spore display not only offers a new and facile way to construct robust biocatalysts but also provides a novel basis for phase transfer biocatalytic processes.

We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox-1, whose levels were also measured in situ using a GFP reporter system. In addition, the high-throughput capacity of the platform was tested using a dual-slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor-4 (FGF-4) on the pluripotency of mouse ES cells. This high-throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses.