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Biomedical subjects

Russell Pickford

Publications and source records attributed to Russell Pickford.

5 recordsLinked to original sources
PubMed2005

Dolichol is the major lipid component of human substantia nigra neuromelanin.

Neuromelanin is a dark brown pigment present at high concentrations in dopaminergic neurones of the human substantia nigra (SN). Early electron microscopic examinations of neuromelanin fine structure revealed a significant neutral lipid component; however, the identity of this lipid has remained unknown. Here we show that the lipid component of neuromelanin pigment derived from human SN is the polyisoprenoid dolichol. Established methods were used to isolate the pigment from the SN of 32 brains and the lipid fraction was recovered in high purity and yield. Using reversed-phase HPLC, atmospheric pressure chemical ionization mass spectrometry, and 1H- and 13C-NMR techniques, we showed that the neuromelanin dolichol contained 17-23 isoprenoid units. Dolichol accounted for 14% of the mass of neuromelanin pigment; low levels of other hydrophobic compounds were detected (e.g. ubiquinone-10, alpha-tocopherol and cholesterol together accounted for < 0.5% of the neuromelanin lipid mass). This is the first time that dolichol has been identified in such a physiological setting and significantly advances our understanding of neuromelanin pigment structure and biosynthetic pathways. Furthermore, these studies identify a potential novel role for the isoprenoid pathway in the regulation of neuromelanin function and neurodegeneration within the SN.

Adolescent↗
PubMed2004

A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis.

Silver staining has been the method most commonly employed for high sensitivity staining of proteins following two-dimensional gel electrophoresis. Whilst this method offers detection in the nanogram range it does have major drawbacks including a lack of linearity, nonstoichiometric staining of proteins, a lack of compatibility with the microchemical preparation of proteins for identification by mass spectrometric techniques, and a highly subjective assessment of the staining endpoint. SYPRO Ruby is a relatively new, ruthenium complex-based stain which is reported to offer advantages over silver, particularly in overcoming the limitations cited above. We describe a series of experiments where several protein staining procedures commonly employed are compared. To enable optimization of the in situ digestion procedure, a statistical approach has been undertaken. The effects of a variety of staining, digestion, and analysis protocols on the downstream processing of a test radiolabeled protein were studied. The data confirms that as well as offering sensitivity similar to silver, SYPRO Ruby staining is reproducible, linear, and offers a higher level of compatibility with the identification of proteins by mass spectrometry.

Coloring Agents↗
PubMed2004

Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms.

Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High-performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface-tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface-active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N-terminus of a 12-amino-acid peptide moiety, in which the C-terminal carboxylic acid group forms an ester with the hydroxyl side-chain of Ser9. The difference between the two structures is located in the second amino acid from the C-terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio-synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild-type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms.

Bacterial Proteins↗
PubMed2002

Arsenosugar identification in seaweed extracts using high-performance liquid chromatography/electrospray ion trap mass spectrometry.

The development of analytical techniques suitable for providing structural information on a wide range of elemental species is a growing necessity. For arsenic speciation a variety of mass spectrometric techniques, mainly inductively coupled plasma mass spectrometry (ICP-MS) and electrospray tandem mass spectrometry (ES-MS/MS) coupled on-line with high-performance liquid chromatography (HPLC), are in use. In this paper we report the identification of arsenic species present in samples of marine origin (seaweed extracts) using ES ion trap mass spectrometry (IT) multistage mass spectrometry (MS(n)). Both reversed-phase and anion-exchange HPLC have been coupled on-line to ES-ITMS. Product ion scans with multiple stages of tandem MS (MS(n); n=2-4) were used to acquire diagnostic data for each arsenosugar. The spectra contain structurally characteristic fragment ions for each of the arsenosugars examined. In addition it was observed that upon successive stages of collision-induced dissociation (CID) a common product ion (m/z 237) was formed from all four arsenosugars examined. This product ion has the potential to be used as an indicator for the presence of dimethylated arsenosugars (dimethylarsinoylribosides). The HPLC/ES-ITMS(n) method developed allows the sensitive identification of arsenosugars present in crude seaweed extracts without the need for extended sample preparation. In fact, sample preparation requirements are identical to those typically employed for HPLC/ICP-MS analysis. Additionally, the resulting product ions are structurally diagnostic of the arsenosugars examined, and tandem mass spectra are reproducible and correspond well to those obtained using other low-energy CID techniques. As a result, the HPLC/ES-ITMS(n) approach minimises the potential for arsenic species misidentification and has great potential as a means of overcoming the need for characterised standards.

Arsenates↗