Metabolic profiling detects biomarkers of protein degradation in COPD patients.

There is a paucity of biomarkers for chronic obstructive pulmonary disease (COPD). Metabolomics were applied to a defined COPD patient cohort from the ECLIPSE study (Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points). Results were correlated with accepted biomarkers for the disease. Baseline control serum (n=66) and Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II (n=70), III (n=64) and IV (n=44) COPD patients were analysed by proton nuclear magnetic resonance ((1)H NMR). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to confirm amino acid changes detected by (1)H NMR. Data were correlated with body composition, emphysema and systemic inflammation. (1)H NMR identified decreased lipoproteins, N,N-dimethylglycine, and increased glutamine, phenylalanine, 3-methylhistidine and ketone bodies in COPD patients with decreased branched-chain amino acids (BCAAs) observed in GOLD stage IV patients. BCAAs, their degradation products, 3-methylhistidine, ketone bodies, and triglycerides were correlated negatively with cachexia and positively with systemic inflammation. Emphysema patients also displayed decreased serum creatine, glycine and N,N-dimethylglycine. LC-MS/MS confirmed (1)H NMR findings relating to BCAAs, glutamine and 3-methylhistidine in GOLD stage IV patients. NMR-based metabolomics characterised COPD patients based on systemic effects and lung function parameters. Increased protein turnover occurred in all COPD patients with increased protein degradation in individuals with emphysema and cachexia.

Selective C-C coupling at a Pt(iv) centre: 100% preference for sp2-sp3 over sp3-sp3.

The oxidative addition of three different organic halides RX to the non-symmetric platinum(ii) mer coordinated dicyclometallated C^N^C complex 1 yielded short-lived six-coordinate platinum(iv) complexes 2(R) (R = Me, allyl, Bn), with the incoming groups trans across the platinum centre. A spontaneous reductive coupling reaction then occurred with, in each case, a completely chemoselective sp2-sp3 coupling, and exclusively gave R-3, with the newly introduced R group bonded to the previously cyclometallated aryl ring. Following a recyclometallation reaction, the oxidative addition/reductive elimination cycle was repeated and gave the same selectivity. A one-pot route to doubly alkylating the aryl ring was developed. The observed selectivity might have been predicted on the normal basis of a steric barrier associated with non-flat sp3 hybridised groups, but we suggest that it arises from the stereochemistry at the metal, and the orientation of the ligands.

Reversible C-C bond formation at a triply cyclometallated platinum(iv) centre.

The oxidation of the tribenzylphosphine derivative of the doubly cylcometallated platinum(ii) complex of diphenylpyridine, 1, with PhICl2 led, as a first step, to the formation of a highly electrophilic metal centre which attacked the benzyl phosphine to give a triply cyclometallated species as the arenium ion. The highly acidic arenium ion protonated unreacted starting 1, a reaction that could be supressed by the addition of water, and gave the neutral species 2(t). Octahedral complex 2(t) was induced to reductively couple, with two five-membered rings coupling to give square planar complex 5 containing a nine-membered ring. The crystal structure of 5 showed the nine-membered ring to span trans across the square planar metal accompanied by considerable distortion: the P-Pt-N bond angle is 155.48(5)°. Oxidation of 5 with PhICl2 resulted in the addition of two chlorides and a change of the nine-membered ring ligand coordination to cis at an octahedral centre, still with considerable distortions: the P-Pt-N bond angle in the crystal structure of 6 is 99.46(5)°. Treatment of 2(t) with AgBF4 also induced a coupling to give a nine-membered ring, and the fluxional three coordinate complex 7. A mono-methylated version of 1, Me-1, was prepared and similar reactions were observed. The presence of the methyl group allowed us to observe selectivity in the coupling reaction to give the nine-membered ring, with two products (a-Me-7 and b-Me7) being initially formed in the ratio 7 : 1. The concentrations of two products changed with time giving a final ratio of 1 : 8 at room temperature (half-life 48 hours), the equilibration being made possible by a reversible C-C bond forming reaction. Reaction of complexes 7 with CO or hydrogen left the nine-membered ring intact, though oxidative degradation resulted in decomplexation of the phosphine donor, accompanied by formation of a P[double bond, length as m-dash]O group.

Trapping five-coordinate platinum(iv) intermediates.

The oxidation of three different complexes of the doubly cycloplatinated 2,6-di(4-fluorophenyl)pyridine ligand (namely DMSO, PPh3 and PPr3 derivatives, 1a, 1b and 1c, respectively) with the electrophilic oxidant iodobenzenedichloride was studied. In each case oxidation can yield a simple trans-dichloro platinum(iv) complex (2(t)), which subsequently isomerises to the cis isomer (2(c)). However, by changing the solvent, or performing the reaction in the presence of an additional ligating species, a five-coordinate intermediate can be trapped out and isolated. Thus, cationic species with additional DMSO or pyridine coordinated could be collected for the DMSO and PPh3 derivatives. The PPr3 derivative traps out the reactive five-coordinate species with an agostic interaction that subsequently induces a transcyclometallation reaction to give a complex with a singly cyclometallated pyridine and a cyclometallated phosphine, which was characterised crystallographically, (6c

Intramolecular transcyclometallation: the exchange of an aryl-Pt bond for an alkyl-Pt bond via an agostic intermediate.

Oxidation of a square-planar platinum complex leads to a five coordinate cationic intermediate that can be stabilized and trapped out via an agostic interaction with the alkyl chain of a ligand. Subsequent reaction of this species leads to the formation of an alkyl-Pt bond at the expense of an aryl-Pt bond: an intramolecular transcyclometallation.

Comparison of protein transduction domains in mediating cell delivery of a secreted CRE protein.

Protein transduction domains (PTDs) are versatile peptide sequences that facilitate cell delivery of several cargo molecules including proteins. PTDs usually consist of short stretches of basic amino acids that can cross the plasma membrane and gain entry into cells. Traditionally, to assess PTD mediated protein delivery, PTD-fusion proteins have been used as purified proteins. To overcome the requirement for a protein purification step, we used a secretory signal peptide to allow PTD-CRE fusion proteins to be exported from transfected mammalian cells. PTD induced protein transduction into cells was assessed by a CRE-mediated recombination event that resulted in beta-galactosidase expression. Several PTDs were tested including the prototypic TAT, different TAT variants, Antp, MTS and polyarginine. A negative correlation was observed between the cationic charge on the PTD and the extent of secretion. Poor secretion was found when the PTD charge was greater than +5. One TAT-CRE protein variant had a 14-fold enhancement above CRE alone when added to cells in the presence of chloroquine. This PTD domain also enhanced gene expression after plasmid delivery. These data illustrate that some secreted PTD proteins may be useful reagents to improve protein delivery in mammalian systems and a novel approach to enhancing the response to DNA transfections.

Enhancement of gene delivery to human airway epithelial cells in vitro using a peptide from the polyoma virus protein VP1.

BACKGROUND: Current liposome-based gene delivery methods for therapeutic benefit are limited by their low efficiency. One possible way to improve gene expression is to include a peptide with a nuclear localization signal (NLS) to enhance the movement of the transfection complex from the cytoplasm to the nuclei of target cells. We have tested a synthetic peptide based on the amino terminal region of the polyoma virus VP1 protein. This region has non-overlapping motifs for DNA binding and nuclear localization. METHODS: Luciferase gene transfer efficiency was evaluated using this peptide and a control peptide with a mutated NLS in subconfluent, confluent and polarized human bronchial epithelial (16HBE) cells compared to lipoplex alone. RESULTS: Gene transfer efficiency with a lipopolyplex containing the VP1 peptide enhanced gene delivery compared to lipoplex. Transfection with a lipopolyplex containing the control peptide failed to enhance gene delivery. The VP1 peptide increased the amount of plasmid associated with the nucleus while the mutant VP1 peptide did not. The order of lipopolyplex formation was important, with greatest enhancement when peptide was added to the plasmid before addition of the liposome. A bipartite peptide with the VP1 sequence and an integrin-binding motif (RGD) resulted in a reduction in gene transfer efficiency compared to lipoplex. Cell adhesion studies showed that the integrin binding associated with the RGD motif was lost when it was attached to the VP1 sequence. The combination of the two peptide sequences in cis may have compromised the function of both. CONCLUSIONS: Our results indicate that the VP1 peptide represents a strategy to enhance liposome-mediated gene delivery to airway epithelia in vitro. Comparison of transfection efficiencies between the VP1 and the mutant VP1 peptides and the direct measurement of plasmid associated with the nucleus suggests that this enhancement is caused by the NLS signal sequence in the peptide.