Monitoring nonenzymatic glycation of human immunoglobulin G by methylglyoxal and glyoxal: A spectroscopic study

The accumulation of dicarbonyl compounds, methylglyoxal (MG) and glyoxal (G), has been observed in diabetic conditions. They are formed from nonoxidative mechanisms in anaerobic glycolysis and lipid peroxidation, and they act as advanced glycation endproduct (AGE) precursors. The objective of this study was to monitor and characterize the AGE formation of human immunoglobulin G (hIgG) by MG and G using ultraviolet (UV) and fluorescence spectroscopy, circular dichroism (CD), and matrix-assisted laser desorption/ionization–mass spectrometry (MALDI–MS). hIgG was incubated over time with MG and G at different concentrations. Formation of AGE was monitored by UV and fluorescence spectroscopy. The effect of AGE formation on secondary structure of hIgG was studied by CD. Comparison of AGE profile for MG and G was performed by MALDI–MS. Both MG and G formed AGE, with MG being nearly twice as reactive as G. The combination of these techniques is a convenient method for evaluating and characterizing the AGE proteins

Cytoskeletal Components of an Invasion Machine—The Apical Complex of Toxoplasma gondii

The apical complex of Toxoplasma gondii is widely believed to serve essential functions in both invasion of its host cells (including human cells), and in replication of the parasite. The understanding of apical complex function, the basis for its novel structure, and the mechanism for its motility are greatly impeded by lack of knowledge of its molecular composition. We have partially purified the conoid/apical complex, identified ~200 proteins that represent 70% of its cytoskeletal protein components, characterized seven novel proteins, and determined the sequence of recruitment of five of these proteins into the cytoskeleton during cell division. Our results provide new markers for the different subcompartments within the apical complex, and revealed previously unknown cellular compartments, which facilitate our understanding of how the invasion machinery is built. Surprisingly, the extreme apical and extreme basal structures of this highly polarized cell originate in the same location and at the same time very early during parasite replication

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Isolation of the Conoid/Apical Complex

<div><p>(A) Combined DIC and epifluorescence image of the starting material, a suspension of extracellular YFP-α-tubulin (green) transgenic parasites.</p><p>(B) Epifluorescence image of detergent extracted, sonicated parasites. The fluorescent material, a small fraction of the total mass, is composed of small MT fragments (cyan arrows) and conoids (red arrows). The inset shows an intact “ghost,” a detergent extracted parasite, at 2× higher magnification. The total cytoskeleton prep was fractionated by differential centrifugation into the conoid-depleted (C) and conoid-enriched (D) fractions.</p></div

Localization of T. gondii IMC4, TgCAM1, and TgCAM2

<div><p>(A) LM localization of a T. gondii protein (TgIMC4) with weak similarity to articulin family members, and weaker similarity to TgIMC1. Fluorescence LM images of living transgenic parasites expressing TgIMC4 fused to the C-terminus of EGFP. Fluorescence is observed on both the mother and daughter IMC. FRAP reveals significant differences in the turnover of TgIMC1 and TgIMC4 (unpublished data).</p><p>(B and C) Fluorescence LM images of living transgenic parasites expressing GFP-CAM1 (B) and GFP-CAM2 (C). Bright fluorescence is observed in the conoid region of both mother and daughters (arrows).</p><p>(D and E) EM images of T. gondii cytoskeletons from the same two lines of transgenic parasites as in (B) and (C [immunogold-labeled with anti-GFP antibody and negatively stained with phosphotungstic acid]). Specific staining occurs over the conoid itself, and lightly over the subpellicular MTs. In (D), diagonal lines of gold particles are visible, tracing the conoid fibers.</p></div

Time-Lapse Image Imaging of Incorporation of YFP-α-Tubulin (Green) and mRFP-TgCentrin2 (Red) into Developing Daughter Parasites

<p>Combined DIC and fluorescence images are shown for the beginning (<i>t</i> = 0) and end (<i>t</i> = 250 min) of the sequence, during which a vacuole of four parasites progresses from the onset of mitosis through completion of budding out of the eight daughters. See text for a detailed description of the sequence of events.</p

EM Analysis of the Conoid-Enriched and Conoid-Depleted Fractions

<div><p>(A) EM image of a thin section through a pellet of the centrifuged conoid-enriched fraction.</p><p>(B) To give a rough visual estimate of the enrichment, material that is recognizably fragments of the conoid + polar rings has been colored red (C).</p><p>(C) EM analysis of the conoid-depleted fraction; MT are marked with cyan lines, which are much wider than the image of the MT at this magnification.</p><p>(D) Enlarged view of the region within the yellow box, showing small vesicles and MT (cyan arrows).</p></div

Image and Drawing of T. gondii

<div><p>(A) Combined DIC and epifluorescence image of human fibroblasts infected with transgenic T. gondii expressing GFP-tubulin (green). Parasitophorous vacuoles containing 1, 2, 4, or 8 parasites are seen.</p><p>(B) Drawings of T. gondii (modified from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020013#ppat-0020013-b003" target="_blank">3</a>] and [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020013#ppat-0020013-b068" target="_blank">68</a>]). Left: a longitudinal section of a dividing cell. Lobes of the dividing nucleus bordered by ER, Golgi (yellow), and developing rhoptry (mauve) are surrounded by the developing daughters' scaffolds (red). Maternal and daughter conoids are shown in green, secretory organelles (rhoptries) in purple. T. gondii has three membranes: a plasma membrane (black) and two additional layers (IMC, red) formed from a patchwork of flattened vesicles. Right: semitransparent view showing subpellicular MT (green).</p><p>(C) Enlarged view of the apical complex cytoskeleton, showing the conoid (green), preconoidal, and polar rings (brown), and two intraconoid MT (green). The conoid is formed of 14 fibers of tubulin (not MT), 430 nm long, arranged in a left-handed spiral [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020013#ppat-0020013-b010" target="_blank">10</a>]. Cytoskeletal elements, including the subpellicular MT (green) and a 2-dimensional lattice of intermediate filament-like proteins (not shown), are closely associated with the cytoplasmic face of the IMC.</p></div