Membrane polypeptides (relative mass (Mr) 48,000--55,000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (Mr 42,000--47,000) during sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS-polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating form (Mr 41,000--48,000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by greater than 95% had little, if any, effect on either the affinity (Kd values, 0.1-0.2 nM) or abundance (Bmax values, 200,000--220,000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 degrees C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.

Fibrin clots are an important component of chronic cardiovascular thrombosis and are associated with cardiovascular dysfunction, stroke and formation of microthrombi in systemic inflammatory states. During coagulation, thrombin selectively cleaves fibrinogen resulting in the formation of linear fibrin polymers that form by end-to-end and side-to-side association. Here we present a novel assay for real-time imaging of fibrin polymerization using fluorescence laser scanning multiphoton microscopy (MPM) and fractal analysis for quantification of clot structure.Human blood, collected in acid citrate dextrose tubes, was centrifuged (2x, for 8 minutes @ 500 x g) to produce platelet poor plasma (PPP). Fibrin polymerization was initiated at room temperature by addition of thrombin to PPP and imaged ( Fig.1) through incorporation of fluorescently labeled fibrinogen (5% vol/vol Oregon Green labeled fibrinogen to PPP fibrinogen) at 0,1,2,3,5,10 and 15 minutes. 900nm fs pulses (Ti-Sapphire laser) were focused through a 63x/1.2 NA objective (Leica AOBS SP2 microscope system) to a clot depth of 20 µm. Fluorescent images were acquired at 400Hz/3frame averages, emission bandwidth 500-650nm. At high doses of thrombin (0.1 and 0.05 units/ml), clots formed within 1-3 minutes of initiation. At lower thrombin doses (0.025 and 0.01 units/ml), clots were unstable over the first 5 minutes (generating motion artifacts), but were stable ten minutes after initiation. To quantify clot structure, images were processed using MATLAB (The MathWorks, Inc., MA) and quantified by fractal analysis (TruSoft Int'l Inc., FL). The image processing algorithm consisted of background signal subtraction, median filter, image thresholding (Otsu's method [1]) and binarization. Fractal dimension was calculated using the box dimension estimation method. The box dimension, Db, is defined as the exponent of N(d) ~ 1/d Db , where N(d) is the number of boxes of linear size (d) required to cover a set of points distributed in a 2D plane. As thrombin concentration increased the clot Db value rapidly increased then seemingly reached a plateau at a thrombin concentration of 0.1 unit/ml, see Fig 2B, indicating that fibrin clot structure was dependent on thrombin concentration and stabilized at the highest thrombin concentration tested.This MPM imaging technique of fibrin clots combined with fractal analysis of clot structure offers a rapid, reproducible and promising approach to investigate clotting mechanisms in disease states and to test the efficacy of drug treatments on clotting kinetics, clot structure and clot fibrinolysis. References[1] N. Otsu, IEEE 9(1) (1979) 62.[2] This work was supported by Heart and Stroke Foundation Canada (HSFC), Michael Smith Foundation for Health Research (British Columbia) and CIHR/HSFC IMPACT Postdoctoral Fellowships.

Imaging the microcirculation is becoming increasingly important in assessing life-threatening disease states. To address this issue in a highly light absorbing and light scattering tissue, we use laser scanning multiphoton microscopy and fluorescent 655-nm 5000-MW methoxy-PEGylated quantum dots to image the functional microcirculation deep in mouse hind limb skeletal muscle. Using this approach, we are able to minimize in vivo background tissue autofluorescence and visualize complete 3-D microvascular units, including feeding arterioles, capillary networks, and collecting venules to depths of 150 to 200 microm. In CD1 mice treated with lipopolysaccharide to model an endotoxemic response to bacterial infection, we find that these quantum dots accumulate at microvascular bifurcations and extravasate from the microcirculation in addition to accumulating in organs (liver, spleen, lung, and kidney). The quantum dots are cleared from the circulation with a first-order elimination rate constant seven times greater than under normal conditions, 1.6+/-0.06 compared to 0.23+/-0.05 h(-1), P<0.05, thereby reducing the imaging time window. In vitro experiments using TNFalpha treated isolated leukocytes suggest that circulating monocytes (phagocytes) increased their nonspecific uptake of quantum dots when activated. In combination with multiphoton microscopy, quantum dots provide excellent in vivo imaging contrast of deep microvascular structures.

No abstract