Does lycopene offer human LDL any protection against myeloperoxidase activity?

Lycopene is a lipophilic antioxidant that is largely transported in human blood by Low Density Lipoproteins (LDL). One of the early events in the aetiology of atherosclerosis is thought to be the oxidation of LDL. Myeloperoxidase an enzyme secreted by neutrophils and macrophages is thought to oxidise human LDL particles. In this study, isolated human LDL was challenged with myeloperoxidase or copper, and the LDL was screened for lipoperoxidation and oxidation of apolipoprotein B100, depletion of lycopene and oxidation of cholesterol. Myeloperoxidase induced oxidation of LDL through direct interaction with apolipoprotein B100. No lipoperoxidation was observed following myeloperoxidase treatment; however, 7-ketocholesterol was detected indicating the products of myeloperoxidase interact with the surface of the LDL particles. Lycopene does react with the products of myeloperoxidase in solvent, but played no role in protecting against enzyme derived oxidation of human LDL.     

Investigating the effects of preinduction on human adipose-derived precursor cells in an athymic rat model

The osteogenic potential of human adipose-derived precursor cells seeded on medical-grade polycaprolactone-tricalcium phosphate scaffolds was investigated in this in vivo study. Three study groups were investigated: (1) induced--stimulated with osteogenic factors only after seeding into scaffold; (2) preinduced--induced for 2 weeks before seeding into scaffolds; and (3) uninduced--cells without any introduced induction. For all groups, scaffolds were implanted subcutaneously into the dorsum of athymic rats. The scaffold/cell constructs were harvested at the end of 6 or 12 weeks and analyzed for osteogenesis. Gross morphological examination using scanning electron microscopy indicated good integration of host tissue with scaffold/cell constructs and extensive tissue infiltration into the scaffold interior. Alizarin Red histology and immunostaining showed a heightened level of mineralization and an increase in osteonectin, osteopontin, and collagen type I protein expression in both the induced and preinduced groups compared with the uninduced groups. However, no significant differences were observed in these indicators when compared between the induced and preinduced groups.     

Cardioprotective effects of nitroparacetamol and paracetamol in acute phase of myocardial infarction in experimental rats

We aimed to determine whether nitroparacetamol (NO-paracetamol) and paracetamol exhibit cardioprotective effects. Myocardial infarction (MI) was induced in rats, and drug treatment was started 1 wk before surgery. Mortality rate and infarct size at 2 days after MI were compared. Treatment groups included vehicle (saline), paracetamol (5 mg x kg(-1) x day(-1)) and NO-paracetamol (15 mg x kg(-1) x day(-1)). Mortality rates for vehicle (n = 80), paracetamol (n = 79), and NO-paracetamol (n = 76) groups were 37.5%, 21.5%, and 26.3%, respectively. Infarct size for the vehicle group was 44.8% (+/-6.1%) of the left ventricle (LV). For the paracetamol and NO-paracetamol groups, infarct size was 31.3% (+/-5.6%) and 30.7% (+/-8.1%) of the LV, respectively. Both paracetamol- and NO-paracetamol-treated groups showed increased activities of catalase and SOD compared with the vehicle group. They could attenuate endothelial, inducible, and neuronal nitric oxide synthase and cyclooxygenase-1 and -2 gene expression after MI. The observation indicates the potential clinical significance of the cardioprotective effects of these drugs.     

Lysine residues in N-terminal and C-terminal regions of human histone H2A are targets for biotinylation by biotinidase

In eukaryotic cell nuclei, DNA associates with the core histones H2A, H2B, H3 and H4 to form nucleosomal core particles. DNA binding to histones is regulated by posttranslational modifications of N-terminal tails (e.g., acetylation and methylation of histones). These modifications play important roles in the epigenetic control of chromatin structure. Recently, evidence that biotinidase and holocarboxylase synthetase (HCS) catalyze the covalent binding of biotin to histones has been provided. The primary aim of this study was to identify biotinylation sites in histone H2A and its variant H2AX. Secondary aims were to determine whether acetylation and methylation of histone H2A affect subsequent biotinylation and whether biotinidase and HCS localize to the nucleus in human cells. Biotinylation sites were identified using synthetic peptides as substrates for biotinidase. These studies provided evidence that K9 and K13 in the N-terminus of human histones H2A and H2AX are targets for biotinylation and that K125, K127 and K129 in the C-terminus of histone H2A are targets for biotinylation. Biotinylation of lysine residues was decreased by acetylation of adjacent lysines but was increased by dimethylation of adjacent arginines. The existence of biotinylated histone H2A in vivo was confirmed by using modification-specific antibodies. Antibodies to biotinidase and HCS localized primarily to the nuclear compartment, consistent with a role for these enzymes in regulating chromatin structure. Collectively, these studies have identified five novel biotinylation sites in human histones; histone H2A is unique among histones in that its biotinylation sites include amino acid residues from the C-terminus.     

A neurochemical approach to valuation sensitivity over gains and losses

Prospect theory proposes the hypothesis that people have diminishing sensitivity in valuing increases in the size of monetary outcomes, for both gains and losses. For decision-making under risk, this implies a tendency to be risk-tolerant over losses while being generally risk averse over gains. We offer a neurochemistry-based model of the diminishing valuation sensitivity hypothesis. Specifically, we propose that dopamine tone modulates the sensitivity towards valuation of gains while serotonin tone modulates the sensitivity towards valuation of losses. Consequently, higher dopamine tone would yield a more concave valuation function over gains while higher serotonin tone would yield a more convex valuation function over losses. Using a neurogenetics strategy to test our neurochemical model, we find that subjects with the 9-repeat allele of DAT1 (lower DA tone) are more risk-tolerant over gains than subjects with the 10-repeat allele, and that subjects with the 10-repeat allele of STin2 (higher 5HT tone) are more risk-tolerant over losses than subjects with the 12-repeat allele. Overall, our results support the implications of our model and provide the first neurogenetics evidence that risk attitudes are partially hard-wired in differentiating between gain- and loss-oriented risks.     

A Straight Path to Circular Proteins

Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized.Sortases are bacterial enzymes that predominantly catalyze the attachment of surface proteins to the bacterial cell wall (1, 2). Other sortases polymerize pilin subunits for the construction of the covalently stabilized and covalently anchored pilus of the Gram-positive bacterium (3–5). The reaction catalyzed by sortase involves the recognition of short 5-residue sequence motifs, which are cleaved by the enzyme with the concomitant formation of an acyl enzyme intermediate between the active site cysteine of sortase and the carboxylate at the newly generated C terminus of the substrate (1, 6–8). In many bacteria, this covalent intermediate can be resolved by nucleophilic attack from the pentaglycine side chain in a peptidoglycan precursor, resulting in the formation of an amide bond between the pentaglycine side chain and the carboxylate at the cleavage site in the substrate (9, 10). In pilus construction, alternative nucleophiles such as lysine residues or diaminopimelic acid participate in the transpeptidation reaction (3, 4).When appended near the C terminus of proteins that are not natural sortase substrates, the recognition sequence of Staphylococcus aureus sortase A (LPXTG) can be used to effectuate a sortase-catalyzed transpeptidation reaction using a diverse array of artificial glycine-based nucleophiles (Fig. 1). The result is efficient installation of a diverse set of moieties, including lipids (11), carbohydrates (12), peptide nucleic acids (13), biotin (14), fluorophores (14, 15), polymers (16), solid supports (16–18), or peptides (15, 19) at the C terminus of the protein substrate. During the course of our studies to further expand sortase-based protein engineering, we were struck by the frequency and relative ease with which intramolecular transpeptidation reactions were occurring. Specifically, proteins equipped with not only the LPXTG motif but also N-terminal glycine residues yielded covalently closed circular polypeptides (Fig. 1). Similar reactivity using sortase has been described in two previous cases; however, rigorous characterization of the circular polypeptides was absent (16, 20). The circular proteins in these reports were observed as minor components of more complex reaction mixtures, and the cyclization reaction itself was not optimized.Open in a separate windowFIGURE 1.Protein substrates equipped with a sortase A recognition sequence (LPXTG) can participate in intermolecular transpeptidation with synthetic oligoglycine nucleophiles (left) or intramolecular transpeptidation if an N-terminal glycine residue is present (right).Here we describe our efforts toward applying sortase-catalyzed transpeptidation to the synthesis of circular and oligomeric proteins. This method has general applicability, as illustrated by successful intramolecular reactions with three structurally unrelated proteins. In addition to circularization of individual protein units, the multiprotein complex AAA-ATPase p97/VCP/CDC48, with six identical subunits containing the LPXTG motif and an N-terminal glycine, was found to preferentially react in daisy chain fashion to yield linear protein fusions. The reaction exploited here shows remarkable similarities to the mechanisms proposed for circularization of cyclotides, small circular proteins that have been isolated from plants (21–23).     

Quantitation of green fluorescent protein using a gel-based imaging method

Green fluorescent protein (GFP) is a versatile reporter protein and has been widely used in biological research. However, its quantitation requires expensive equipment such as a spectrofluorometer. In the current study, a gel documentation imaging system using a native polyacrylamide gel for the quantitation of GFP was developed. The assay was evaluated for its precision, linearity, reproducibility, and sensitivity in the presence of Escherichia coli cells and was compared with the spectrofluorometric method. Using this newly established, gel-based imaging technique; the amount of GFP can be quantified accurately.     

Reporting of Quantitative Protein Electrophoresis in Australia and New Zealand: a Call for Standardisation

Background

The lack of guidelines on reporting standards for protein electrophoresis may have led to significant differences in reports from different laboratories.

Objective

To determine the extent of variation in reporting of protein electrophoresis results in Australia and New Zealand.

Method

Questionnaires were distributed to laboratories throughout Australia and New Zealand asking about protein electrophoresis practices and reporting.

Results

Extensive variation was found in the following reporting practices: (a) units for urine Bence Jones protein (BJP); (b) reporting absence of a paraprotein rather than a normal pattern; (c) numerical reporting of all protein fractions or only the paraprotein; (d) warning of possible inaccuracy in the serum immunoglobulin result of the paraprotein type; (e) co-migration of a paraprotein with a normal serum protein; (f) use of a confirmatory test when a known paraprotein is no longer detectable.

Conclusions

A working party should be established to make recommendations on the reporting of protein electrophoresis. Implementation of such recommendations should reduce both report variation between laboratories and the risk of misinterpretation of reports.     

Identification and characterization of microsatellite loci in Intsia palembanica (Leguminosae), a valuable tropical timber species

We present 25 polymorphic microsatellite loci for Intsia palembanica, a highly valued timber species in the Indo-Malayan region, Australia and western Pacific islands. Microsatellite loci were tested for polymorphism across a total of 76 individuals from three natural populations from Irian Jaya, Indonesia. The average number of alleles for these microsatellites was 12.1 per locus, ranging from four to 19. The observed heterozygosity within the natural populations ranged from 0.01 to 0.96. The markers will enable us to evaluate the spatial-temporal population genetic structure and gene flow dynamics among populations. They can also be used for tracking and tracing wood from legally logged concessions.     

Mitochondrial DNA Variants of Respiratory Complex I that Uniquely Characterize Haplogroup T2 Are Associated with Increased Risk of Age-Related Macular Degeneration

Background

Age-related macular degeneration (AMD), a chronic neurodegenerative and neovascular retinal disease, is the leading cause of blindness in elderly people of western European origin. While structural and functional alterations in mitochondria (mt) and their metabolites have been implicated in the pathogenesis of chronic neurodegenerative and vascular diseases, the relationship of inherited variants in the mitochondrial genome and mt haplogroup subtypes with advanced AMD has not been reported in large prospective cohorts.

Methodology/Prinicipal Findings

We examined the relationship of inherited mtDNA variants with advanced AMD in 1168 people using a three-stage design on samples from 12-year and 10-year prospective studies on the natural history of age-related eye disease. In Stage I we resequenced the entire genome in 99 elderly AMD-free controls and 215 people with advanced AMD from the 12-year study. A consistent association with AMD in 14 of 17 SNPs characterizing the mtDNA T haplogroup emerged. Further analysis revealed these associations were driven entirely by the T2 haplogroup, and characterized by two variants in Complex I genes (A11812G of MT-ND4 and A14233G of MT-ND6). We genotyped T haplogroups in an independent sample of 490 cases and 61 controls from the same study (Stage II) and in 56 cases and 246 controls from the 10-year study (Stage III). People in the T2 haplogroup were approximately 2.5 times more likely to have advanced AMD than their peers (odds ratio [OR] = 2.54, 95%CI 1.36–4.80, P≤0.004) after considering the totality of evidence. Findings persisted after considering the impact of AMD-associated variants A69S and Y402H (OR = 5.19, 95%CI 1.19–22.69, P≤0.029).

Conclusion

Loci defining the mtDNA T2 haplogroup and Complex I are reasonable targets for novel functional analyses and therapeutic research in AMD.