Single-Particle Tracking of Human Lipoproteins

Lipoproteins, such as high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low density lipoprotein (VLDL), play a critical role in heart disease. Lipoproteins vary in size and shape as well as in their apolipoprotein content. Here, we developed a new experimental framework to study freely diffusing lipoproteins from human blood, allowing analysis of even the smallest HDL with a radius of 5 nm. In an easily constructed confinement chamber, individual HDL, LDL, and VLDL particles labeled with three distinct fluorophores were simultaneously tracked by wide-field fluorescence microscopy and their sizes were determined by their motion. This technique enables studies of individual lipoproteins in solution and allows characterization of the heterogeneous properties of lipoproteins which affect their biological function but are difficult to discern in bulk studies

Association between thyroid hormones and risk of overall and aggressive prostate cancer.

*<p>- Information on cancer stage and grade available for cases diagnosed through July 2002.</p>†<p>- Conditioned on age and date of baseline blood draw.</p>‡<p>- Conditioned on age and date of baseline blood draw. Further adjusted for body mass index (kg/m2), serum concentrations of retinol, total cholesterol, alpha-tocopherol, and beta-carotene, cigarettes smoked per day, years smoked, family history of prostate cancer, physical activity, education, marital status, urban residence, total intake of energy, dietary vitamin D, fruit, vegetables, red meat, alcohol, and use of calcium supplements.</p>§<p>- Hypothyroid defined as TSH >3 µIU/mL and T4 <4.6 µg/dL. Hyperthyroid defined as TSH <0.3 µIU/mL and T4 >12 µg/dL.</p

Age-adjusted^* baseline^† characteristics by quintile of baseline serum thyroxine (T4).

*<p>- Directly standardized to the age distribution of the entire cohort. Values are means unless otherwise indicated.</p>†<p>- All characteristics are from the baseline questionnaire except family history which was collected during follow-up and is available for 889 men in this case control set. Baseline dietary data were available for 1,117 men, and 246 men claimed supplement use at baseline.</p

Age-adjusted^* baseline^† characteristics by case-control status.

*<p>- Values are means unless otherwise indicated.</p>†<p>- All characteristics are from the baseline questionnaire except family history which was collected during follow-up and is available for 889 men in this case control set. Baseline dietary data were available for 1,117 men, and 246 men claimed supplement use at baseline.</p

Age-adjusted^* baseline^† characteristics by quintile of baseline serum thyroid stimulating hormone (TSH).

*<p>- Directly standardized to the age distribution of the entire cohort. Values are means unless otherwise indicated.</p>†<p>- All characteristics are from the baseline questionnaire except family history which was collected during follow-up and is available for 889 men in this case control set. Baseline dietary data were available for 1,117 men, and 246 men claimed supplement use at baseline.</p

Hypothyroid and hyperthyroid hormone profiles by serum concentrations of T4 and TSH.

<p>Hypothyroid and hyperthyroid hormone profiles by serum concentrations of T4 and TSH.</p

Known and potential transbilayer lipid transporters.

<p>The aminophospholipid translocase (left) and ABCA1 (right) proteins are drawn approximately to scale in outline forms taken from atomic structures of a P-type ATPase (the Ca²⁺ transporter;<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000729#pone.0000729-Toyoshima1" target="_blank">[54]</a>) and an ABC protein (BtuCD,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000729#pone.0000729-Locher1" target="_blank">[55]</a>). The molecular shape of the scramblase (center) is arbitrary, since the protein responsible for this activity is not known. One possibility, investigated here, is that the ABCA1 protein is actually the protein responsible for this activity; another possibility is that the ABCA1 protein transports PS from the inner to the outer leaflet, as depicted. An HDL particle (far right) is also drawn approximately to scale by extrapolation from the structure of other lipoprotein particles, with the apoA1 protein drawn as the belt-like helices enclosing the lipid core. The molecular shape of an unlipidated apoA1 protein is unknown.</p

Internalization (translocation) of NBD-labeled phospholipids by normal human fibroblasts and fibroblasts from Tangier individuals.

<p>The outer leaflet of the plasma membrane of normal (circles) or Tangier (squares) fibroblasts was labeled with NBD-PC (open symbols) or NBD-PS (filled symbols). At various times samples were removed into BSA to extract outer leaflet probe and remaining inner leaflet probe was measured by flow cytometry and expressed as percent transported. Measurements were made at room temperature. (A) Viable fibroblasts, (B) spontaneously apoptotic fibroblasts. Dashed and dotted lines in B have been redrawn from A for transport by normal fibroblasts.</p

Phagocytosis of apoptotic targets by wildtype or <i>ABCA1^−/−</i> macrophages.

<p>A&B, phagocytosis of non-apoptotic or apoptotic thymocytes from wildtype (black) or <i>ABCA1^−/−</i> (gray) mice by peritoneal macrophages from wildtype (A) or <i>ABCA1^−/−</i> (B) mice. C, phagocytosis of camptothecin-induced apoptotic EBV-transformed normal (black) or Tangier (gray) human B lymphocytes, either untreated or pre-treated with 10 µM Annexin V, then washed prior to presentation to mouse J774 macrophages. Error bars represent the standard error of 3 replicates.</p

Endogenous PS externalization in normal and ABCA1-deficient cells measured by continuous annexin V binding assay.

<p>(A) Normal or Tangier human fibroblasts, (B) EBV-transformed normal or Tangier B lymphocytes, or (C and D) thymocytes from wildtype or <i>ABCA1^−/</i>⁻ mice were treated with Ca²⁺ and Ca²⁺ ionophore, and cellular fluorescence in the presence of fluorescent annexin V measured continuously over time at room temperature. Normal/wildtype, filled circles; ABCA1-deficient, open circles. A–C, median fluorescence of cells; D, fraction of cells stained by fluorescent annexin.</p