Analysis of intracellular enzyme activity by surface enhanced Raman scattering.

Dysfunctional intracellular enzymatic activity is believed to be an underlying cause of a myriad of diseases. We present the first use of surface enhanced Raman scattering (SERS) as a detection technique capable of reporting intracellular activity of a specific enzyme. Careful choice of reagents allowed the preparation of high resolution cellular activity maps highlighting the specific conversion of the commonly used ELISA reagent 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal), by wild type β-galactosidase enzymes.

Identification and Functional Characterisation of CRK12:CYC9, a Novel Cyclin-Dependent Kinase (CDK)-Cyclin Complex in Trypanosoma brucei.

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism.

A unique Kelch domain phosphatase in Plasmodium regulates ookinete morphology, motility and invasion.

Signalling through post-translational modification (PTM) of proteins is a process central to cell homeostasis, development and responses to external stimuli. The best characterised PTM is protein phosphorylation which is reversibly catalysed at specific residues through the action of protein kinases (addition) and phosphatases (removal). Here, we report characterisation of an orphan protein phosphatase that possesses a domain architecture previously only described in Plantae.

Antitumor activity of the tea polyphenol epigallocatechin-3-gallate encapsulated in targeted vesicles after intravenous administration.

Aim: The therapeutic potential of epigallocatechin-3-gallate (EGCG), a green tea polyphenol with anticancer properties, is limited by its inability to specifically reach tumors following intravenous administration. The purpose of this study was to determine whether a tumor-targeted vesicular formulation of EGCG would suppress the growth of A431 epidermoid carcinoma and B16-F10 melanoma in vitro and in vivo.

Materials & Methods: Transferrin-bearing vesicles encapsulating EGCG were administered intravenously to mice bearing subcutaneous A431 and B16-F10 tumors.

Ecotin-like serine peptidase inhibitor ISP1 of Leishmania major plays a role in flagellar pocket dynamics and promastigote differentiation.

Leishmania ISPs are ecotin-like natural peptide inhibitors of trypsin-family serine peptidases, enzymes that are absent from the Leishmania genome. This led to the proposal that ISPs inhibit host serine peptidases and we have recently shown that ISP2 inhibits neutrophil elastase, thereby enhancing parasite survival in murine macrophages. In this study we show that ISP1 has less serine peptidase inhibitory activity than ISP2, and in promastigotes both are generally located in the cytosol and along the flagellum.

The Trypanosoma brucei AIR9-like protein is cytoskeleton-associated and is required for nucleus positioning and accurate cleavage furrow placement.

AIR9 is a cytoskeleton-associated protein in Arabidopsis thaliana with roles in cytokinesis and cross wall maturation, and reported homologues in land plants and excavate protists, including trypanosomatids. We show that the Trypanosoma brucei AIR9-like protein, TbAIR9, is also cytoskeleton-associated and colocalizes with the subpellicular microtubules. We find it to be expressed in all life cycle stages and show that it is essential for normal proliferation of trypanosomes in vitro.

The use of nano polymeric self-assemblies based on novel amphiphilic polymers for oral hydrophobic drug delivery.

Purpose: To investigate the use of nano self-assemblies formed by polyallylamine (PAA) modified with 5 or 10% mole fluorenylmethoxy carbonyl (Fmoc(5)/(10)), dimethylamino-1-naphthalenesulfonyl (Dansyl(5)/(10)) and 5% mole cholesteryl group (Ch(5)) for oral hydrophobic drug delivery.

Methods: Propofol, griseofulvin and prednisolone were loaded into amphiphilic PAAs. Particle size and morphology of drug-loaded self-assemblies were determined using photon correlation spectroscopy and transmission electron microscopy.

Trypanosoma brucei metacaspase 4 is a pseudopeptidase and a virulence factor.

Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five metacaspases (MCA1-MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure.

Enhanced gene expression in tumors after intravenous administration of arginine-, lysine- and leucine-bearing polypropylenimine polyplex.

The possibility of using non-viral gene delivery systems for the treatment of cancer is currently limited by their lower transfection efficacy compared to viral systems. On the basis that amino acids such as arginine, lysine and leucine were involved in enhancing DNA transportation into cells, we hypothesized that the grafting of these amino acids to the highly promising generation 3 diaminobutyric polypropylenimine (DAB) dendrimer would improve its transfection efficacy in cancer cells. In this work we demonstrated that the conjugation of arginine, lysine and leucine to the dendrimer led to an enhanced anti-proliferative activity of the polyplexes, by up to 47-fold for DAB-Lys in T98G cancer cells compared to the unmodified polyplex in vitro.

Novel tocotrienol-entrapping vesicles can eradicate solid tumors after intravenous administration.

The therapeutic potential of tocotrienol, a vitamin E extract with anti-cancer properties, is hampered by its failure to specifically reach tumors after intravenous administration. In this work, we demonstrated that novel transferrin-bearing, tocopheryl-based multilamellar vesicles entrapping tocotrienol significantly improved tocotrienol uptake by cancer cells overexpressing transferrin receptors. This led to a dramatically improved therapeutic efficacy in vitro, ranging from 17-fold to 72-fold improvement depending on the cell lines, compared to the free drug.

Enhanced gene expression in tumors after intravenous administration of arginine-, lysine- and leucine-bearing polyethylenimine polyplex.

The potential of gene therapy to treat cancer is currently limited by the low expression of therapeutic genes in the tumors. Because amino acids are known to have excellent properties in cell penetration and gene expression regulation, we investigated if the conjugation of arginine (Arg), lysine (Lys) and leucine (Leu) onto the surface of the gene delivery system polyethylenimine (PEI) could lead to an improved gene expression in tumors. The intravenous administration of Arg-, Lys- and Leu-bearing PEI polyplexes led to a significant increase of gene expression in the tumor, with a β-galactosidase expression amount at least threefold higher than that obtained after treatment with unmodified PEI polyplex.

Polyamine aza-cyclic compounds demonstrate anti-proliferative activity in vitro but fail to control tumour growth in vivo.

Cationic polyamines such as the poly(propylenimine) dendrimers (DAB16) are anti-tumour agents (Dufes et al., 2005, Cancer Res 65:8079-8084). Their mechanism of action is poorly understood, but the lack of in vivo toxicity suggests cancer specificity.

In vitro and in vivo characterisation of a novel peptide delivery system: amphiphilic polyelectrolyte-salmon calcitonin nanocomplexes.

The cationic peptide, salmon calcitonin (sCT) was complexed with the cationic amphiphilic polyelectrolyte, poly(allyl)amine, grafted with palmitoyl and quaternary ammonium moieties at pH 5.0 and 7.4 to yield particulates (sCT-QPa).

Campylobacter jejuni 81-176 forms distinct microcolonies on in vitro-infected human small intestinal tissue prior to biofilm formation.

Human small and large intestinal tissue was used to study the interaction of Campylobacter jejuni with its target tissue. The strain used for the study was 81-176 (+pVir). Tissue was processed for scanning and transmission electron microscopy, and by immunohistochemistry for light microscopy.

Tumor regression after systemic administration of a novel tumor-targeted gene delivery system carrying a therapeutic plasmid DNA.

The possibility of using genes as medicines to treat cancer is limited by the lack of safe and efficacious delivery systems able to deliver therapeutic genes selectively to tumors by intravenous administration. We investigate if the conjugation of the polypropylenimine dendrimer to transferrin, whose receptors are overexpressed on numerous cancers, could result in a selective gene delivery to tumors after intravenous administration, leading to an increased therapeutic efficacy. The objectives of this study are to evaluate the targeting and therapeutic efficacies of a novel transferrin-bearing polypropylenimine dendrimer.

The molecular shape of poly(propylenimine) dendrimer amphiphiles has a profound effect on their self assembly.

The shape of dendrimer amphiphiles has an unexpected effect on their self-assembly. A series of diaminobutane poly(propylenimine) generation 3 dendrimer (DAB-dendr-(NH(2))(16)) amphiphiles has been synthesized, bearing an average of five (PD5), three (PD3) and one (PD1) palmitoyl group(s) per dendrimer molecule. Additionally DAB-dendr-(NH(2))(16) was derivatized with a layer of poly(ethylene glycol) (PEG, degree of polymerization = 12) groups and conjugated to an average of 1 palmitoyl group at the PEG end (PPD1).

The influence of polymer architecture on the protective effect of novel comb shaped amphiphilic poly(allylamine) against in vitro enzymatic degradation of insulin--towards oral insulin delivery.

Nanocomplexes formed between amphiphilic poly(allylamine) (PAA) and insulin were prepared, characterised and the impact of polymer architecture on the protection of insulin against three enzymes was investigated. PAA previously modified with either cetyl or cholesteryl pendant groups at two levels of hydrophobic grafting and its quaternised derivatives were used to produce polymer-insulin nanocomplexes. Transmittance study, differential scanning calorimetry, hydrodynamic size and zeta potential measurement were conducted and the morphology of the complexes were visualised using transmission electron microscopy.

Tumor regression after systemic administration of tocotrienol entrapped in tumor-targeted vesicles.

The therapeutic potential of tocotrienol, an extract of vitamin E with anti-cancer properties, is hampered by its failure to specifically reach tumors after intravenous administration, without secondary effects on normal tissues. We hypothesize that the encapsulation of tocotrienol-rich fraction (TRF) within vesicles bearing transferrin, whose receptors are overexpressed on many cancer cells, could result in a selective delivery to tumors after intravenous administration. The objectives of this study are therefore to prepare and characterize transferrin-targeted vesicles encapsulating TRF, and to evaluate their therapeutic efficacy in vitro and in vivo.

The complexation between novel comb shaped amphiphilic polyallylamine and insulin: towards oral insulin delivery.

Novel amphiphilic polyallylamine (PAA) were previously synthesised by randomly grafting palmitoyl pendant groups and subsequent quaternising with methyl iodide. The ability of these self-assembled polymers to spontaneously form nano-complexes with insulin in pH 7.4 Tris buffer was evaluated by transmittance study, hydrodynamic size and zeta potential measurements.

Rapid cell mapping using nanoparticles and SERRS.

Bone marrow-derived immune cells (macrophages) treated with gold and silver nanoparticles before fixation and dye staining have been analysed by multiple wavelength line scanning surface enhanced resonance Raman scattering (SERRS) mapping. The method yields high selectivity and sensitivity within short analysis times, identifying nanoparticle aggregates in secondary lysosomes. Using routine cell stains, the output from fluorescence, Raman and SERRS is quantified at four wavelengths of excitation, demonstrating the potential at longer biologically compatible wavelengths of using nanoparticles with cell stains for superior cell mapping.

Polymeric amphiphile branching leads to rare nanodisc shaped planar self-assemblies.

Self-assembly is fundamental to the biological function of cells and the fabrication of nanomaterials. However, the origin of the shape of various self-assemblies, such as the shape of cells, is not altogether clear. Polymeric, oligomeric, or low molecular weight amphiphiles are a rich source of nanomaterials, and controlling their self-assembly is the route to tailored nanosystems with specific functionalities.

The AP3 adaptor is involved in the transport of membrane proteins to acidocalcisomes of Leishmania.

Lysosomal function is crucial for the differentiation and infectivity of the parasitic protozoon Leishmania major. To study lysosomal biogenesis, an L. major mutant deficient in the delta subunit of the adaptor protein 3 (AP3 delta) complex was generated.