PI4KIIα phosphorylation by GSK3 directs vesicular trafficking to lysosomes.

Glycogen synthase kinase 3 (GSK3) is essential for normal development and function of the central nervous system. It is especially important for regulating neurotransmission, although the downstream substrates mediating this function are not yet clear. In the present paper, we report the lipid kinase phosphatidylinositol 4-kinase II α (PI4KIIα) is a novel substrate of GSK3 that regulates trafficking and cell-surface expression of neurotransmitter receptors in neurons. GSK3 phosphorylates two distinct sites in the N-terminus of PI4KIIα (Ser5 and Ser47), promoting binding to the adaptor protein 3 (AP-3) complex for trafficking to the lysosome to be degraded. Blocking phosphorylation reduces trafficking to the lysosome, stabilizing PI4KIIα and its cargo proteins for redistribution throughout the cell. Importantly, a reduction in PI4KIIα expression or phosphorylation increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression at the surface of hippocampal neurons. These studies implicate signalling between GSK3 and PI4KIIα as a novel regulator of vesicular trafficking and neurotransmission in the brain.

Ureaplasma parvum serovar 3 multiple banded antigen size variation after chronic intra-amniotic infection/colonization.

Ureaplasma species are the microorganisms most frequently associated with adverse pregnancy outcomes. The multiple banded antigen (MBA), a surface-exposed lipoprotein, is a key virulence factor of ureaplasmas. The MBA demonstrates size variation, which we have shown previously to be correlated with the severity of chorioamnion inflammation. We aimed to investigate U. parvum serovar 3 pathogenesis in vivo, using a sheep model, by investigating: MBA variation after long term (chronic) and short term (acute) durations of in utero ureaplasma infections, and the severity of chorioamnionitis and inflammation in other fetal tissues. Inocula of 2 × 10(7) colony-forming-units (CFU) of U. parvum serovar 3 (Up) or media controls (C) were injected intra-amniotically into pregnant ewes at one of three time points: day 55 (69d Up, n = 8; C69, n = 4); day 117 (7d Up, n = 8; C7, n = 2); and day 121 (3d Up, n = 8; C3, n = 2) of gestation (term = 145-150d). At day 124, preterm fetuses were delivered surgically. Samples of chorioamnion, fetal lung, and umbilical cord were: (i) snap frozen for subsequent ureaplasma culture, and (ii) fixed, embedded, sectioned and stained by haematoxylin and eosin stain for histological analysis. Selected fetal lung clinical ureaplasma isolates were cloned and filtered to obtain cultures from a single CFU. Passage 1 and clone 2 ureaplasma cultures were tested by western blot to demonstrate MBA variation. In acute durations of ureaplasma infection no MBA variants (3d Up) or very few MBA variants (7d Up) were present when compared to the original inoculum. However, numerous MBA size variants were generated in vivo (alike within contiguous tissues, amniotic fluid and fetal lung, but different variants were present within chorioamnion), during chronic, 69d exposure to ureaplasma infection. For the first time we have shown that the degree of ureaplasma MBA variation in vivo increased with the duration of gestation.

Mechanistic investigation of N-homocysteinylation-mediated protein-gold nanoconjugate assembly.

Herein we report the use of protein-gold nanoconjugate (PGNs) as probes for elucidating mechanistic events involved in protein homocystamide detection with gold nanoparticles (GNPs), as was previously reported by our laboratory. Three different PGN probes are synthesized by direct adsorption of cytochrome c, albumin, or human serum onto citrate-capped GNPs. The PGNs are subsequently purified and treated to confer N-homocysteinylation. Individual PGN systems are evaluated to assess the effect of modification on (1) surface plasmon resonance (SPR), (2) protein structural conformation, and (3) assembly-association. The degree of PGN assembly and colorimetric signal observed postmodification varies based on the type of conjugated protein. For example, results of time-resolved dynamic light scattering studies indicate that modification of cytochrome c-PGNs yields rapid formation of macroscopic nanoparticle assemblies that eventually precipitate from solution. In contrast, albumin and human serum PGNs exhibit higher stability toward modification. Additionally, findings from circular dichroism studies indicate significant modification-induced denaturation, which is what may initiate assembly via electrosteric destabilization of PGNs. The results of electrophoretic studies appear to confirm that the process of N-homocysteinylation-mediated PGN assembly culminates in covalent interparticle association by disulfide cross-linking among modified proteins.

Gold nanoparticle sensor for homocysteine thiolactone-induced protein modification.

Homocysteine thiolactone-induced protein modification (HTPM) is a unique post-translational protein modification that is recognized as an emergent biomarker for cardiovascular disease. HTPM involves the site-specific acylation of proteins at lysine residues by homocysteine thiolactone (HTL) to produce protein homocystamide, which has been found at elevated levels in patients with coronary heart disease. Herein, we report the development of a novel gold nanoparticle (GNP) biochemical sensor for detection of protein homocystamide in an in vitro serum protein-based model system. Human serum albumin (HSA) and human sera were subjected to HTPM in vitro to produce HSA-homocystamide or serum protein homocystamide, respectively, which was subsequently treated with citrate-capped GNPs. This GNP sensor typically provided instantaneous visual confirmation of HTPM in the protein model systems. Transmission electron microscopy images of the GNPs in the presence of HSA-homocystamide suggest that modification-directed nanoparticle assembly is the mechanism by which the biochemical sensor produces a colorimetric signal. The resultant nanoparticle-protein assembly exhibited excellent thermal and dilutional stability, which is expected for a system stabilized by chemisorption and intermolecular disulfide bonding. The sensor typically provided a linear response for modified human sera concentrations greater than approximately 5 mg/mL. The calculated limit of detection and calibration sensitivity for the method in human sera were 5.2 mg/mL and 13.6 AU . (microg/mL)-1, respectively.

Capillary electrophoretic screening for the inhibition of homocysteine thiolactone-induced protein oligomerization.

We report the first demonstration of rapid electrophoretic monitoring of homocysteine thiolactone-induced protein oligomerization (HTPO), a unique type of post-translational protein modification that may have clinical significance as an indicator of cardiovascular and neurovascular diseases. HTPO of the model protein bovine cytochrome c was initiated in vitro. The relative monomer and aggregate levels of the resultant protein mixtures were determined following separation using capillaries coated with the cationic polymer, poly(diallyldimethylammonium chloride). UV detection provided adequate sensitivity for the monitoring of higher order species, which exist at relatively low concentrations in the protein reaction mixture as compared to the monomeric species. Separations performed under standard injection conditions were optimized on the basis of applied voltage and sample denaturation conditions. Separations performed using short-end injection allowed for more rapid analyses, typically in less than 70 s. Relative errors for run-to-run migration times were less than 0.5%. This novel oligomeric system provides a rapid and straightforward in vitro method to screen therapeutic agents for their ability to inhibit HTPO. Changes in peak area for monomer and aggregate species were used to assess HTPO inhibition as a function of pyridoxal 5-phosphate (PLP) concentration. PLP was shown to effectively inhibit HTPO in vitro. Rapid analysis times of approximately 1.5 min were achieved for inhibition screening.

Determination of elements in native and bypass human coronary artery plaque deposits from the same heart using inductively coupled plasma-mass spectrometry.

As part of an ongoing study on atherosclerotic arteries, the concentrations of 12 elements in a native and by-pass human coronary artery plaque deposits from five human hearts were determined using inductively coupled plasma-mass spectrometry (ICP-MS). These elements were Ca, P, Na, K, Mg, Zn, Cu, Pb, Fe, Al, Si, and S. For Zn, Ca, Pb, Fe, Al, and Si, the levels were at the fractional micromol/g levels and they probably played an unimportant role in plaque development. Sulfur levels varied from 23 to 140 micromols indicative of the possible presence of homocysteine, but there appeared to be no consistent relationship between by-pass and native concentrations. The calcium and phosphorus concentrations were relatively high in all cases, but the ratio of their molecular concentrations did not correspond to hydroxyapatite, which is conventionally considered to be the chemical form of calcium in heart plaque. In the mature native plaque, high calcium/phosphorus ratios indicated calcium in chemical forms other than hydroxyapatite. In undeveloped by-pass plaque, the phosphorus concentration was too high to be as hydroxyapatite but may be phospholipids. Because it is difficult to get suitable samples, only five heart samples were available. Therefore, these results should be treated as preliminary.

High resolution three-dimensional visualization and characterization of coronary atherosclerosis in vitro by synchrotron radiation x-ray microtomography and highly localized x-ray diffraction.

Human atherosclerotic plaques in both native and bypass arteries have been visualized using microtomography to provide additional information on the nature of coronary artery disease. Plaques contained within arteries removed from three white males aged 51, 55 and 70 are imaged in three-dimensions with monochromatic synchrotron x-ray radiation. Fields of view are 658 x 658 x 517 voxels. with cubic voxels ranging from 12 to 13 microm on a side. X-ray energies range from 11 to 15 keV (bandpass approximately 10 eV). At lower energies, high local absorption tends to generate reconstruction artefacts, while at higher energies the arterial wall is scarcely visible. At all energies, calcifications are clearly visible and differences are observed between plaques in native arteries (lifetime accumulations) versus bypass arteries (plaques developing in the interval between the heart bypass operation and the autopsy). In order to characterize coronary calcification, a microfocused, 50 microm2, 25 keV x-ray beam was used to acquire powder diffraction data from selected calcifications. Also, large calcifications were removed from the native arteries and imaged with 25 keV x-ray energy. Calcifications are composed of hydroxyapatite crystallites and an amorphous phase. In summary, native calcifications are larger and have a higher fraction of hydroxyapatite than calcifications from the bypass arteries.