We previously reported that the dog dopamine receptor D4 (DRD4) gene is polymorphic as observed in humans, and four alleles were identified based on the number and/or order of the 12 and 39 bp sequences located in the homologous region of human DRD4. To assess the diversity of the DRD4 gene in dogs we examined the allelic variations in four breeds (beagle, golden retriever, Shetland sheepdog, and shiba) employing the polymerase chain reaction (PCR). As a result, we found three novel alleles and determined the DNA sequences of these alleles. The beagle shared four alleles, including 396, 435, 447a, and 447b, with the 435 (52.6%) and 447a (39.5%) alleles being common. The golden retriever had the 435 and 447a alleles, and the 435 allele was frequent (73.3%). In the Shetland sheepdog, the 435, 447a, and 498 alleles were observed, of which the 447a allele was most frequent (82.5%). The shiba had five alleles-447a, 447b, 486, 498, and 549-and the 447b allele was most common (55.4%). These findings suggest that the allele frequency varied among the four dog breeds, and analysis of the DRD4 polymorphism may therefore be useful for elucidating the relationships among dog breeds.

Alleles↗

PubMedDate not supplied

Cytokine production and protein adsorption in a stainless steel filter used for leukocyte reduction.

A new metallic filter made from a stainless steel fiber has been under development. To evaluate biocompatibility of this filter, the authors compared cytokine production with that of stainless steel fibers and polyester fibers by using a mononuclear cell culture. Furthermore, adsorbed proteins on each fiber were identified by using sodium-dodecyl sulfate (SDS)-polyacrlyamide gel electrophoresis (PAGE). The levels of tumor necrosis factor (TNF)-alpha in the cultured supernatant without fibers as the control, with polyester fibers, and with stainless steel fibers were 28.1 +/- 8.1, 39.3 +/- 2.6, and 29.1 +/- 6.7 pg/ml, respectively. The levels of interleukin (IL)-1 beta were 7.6 +/- 3.2, 8.9 +/- 1.5, and 8.9 +/- 2.1 pg/ml, respectively. The IL-4 levels were less than 0.25 pg/ml, and the interferon-tau levels were less than 7.8 pg/ml in all three conditions. The amount of adsorbed proteins was 3.39 +/- 0.27 microgram/cm2 for the polyester fibers and 2.72 +/- 0.23 microgram/ cm2 for the stainless steel fibers. The protein bands adsorbed to the polyester fibers by SDS-PAGE analysis were observed at approximately 180, 120, 90, 76, 67, 59, 56, and 28 kd molecular weight. In contrast, the protein bands adsorbed to the stainless steel fibers were observed at 90, 76, 67, 62, 56, 28, and 12 kd molecular weight. Thus, the proteins adsorbed to the stainless steel fibers differed from those on the polyester fibers. By western blot analysis, the amounts of albumin, IgG tau chain, and fibronectin adsorbed on the stainless steel fibers were smaller than those on the polyester fibers. The results of this study suggest that the stainless steel fibers do not stimulate monocytes, Th1, and Th2 cells. In addition, lesser adsorption of IgG tau chain and fibronectin may indicate that the stainless steel is a superior material for anti thrombogenicity compared to polyester.

Adsorption↗

PubMedDate not supplied

Platelet adhesion to heparin coated oxygenator fibers under in vitro static conditions: impact of temperature.

Heparin coating of cardiopulmonary bypass (CPB) circuitry may attenuate the platelet consumption associated with CPB. We investigated the effect of temperature on the interaction between platelet and heparin coated surfaces under in vitro static conditions. Heparin coated and non coated oxygenator fibers were incubated with heparinized whole blood at 37 degrees C and 22 degrees C. The incubation time was set at 30, 60, 180, and 300 minutes. The number of platelets adhering to each fiber was assessed with enzyme immunoassay using monoclonal antibody against platelet receptor protein CD 61(GPIIbIIIa). As an index of platelet activation, plasma soluble(s) P-selectin levels were measured by enzyme-linked immunosorbent assay. Under normothermia, the number of adherent platelets on the non coated surface increased significantly after 300 min of incubation. Platelet adhesion was reduced significantly by heparin coating of the surface and was kept constant after 300 min. Under hypothermia, heparin coating was also associated with significant reduction of platelet adhesion. The levels of sP-selectin did not correlate with the extent of platelet adhesion. Our results suggest that heparin coating is effective in decreasing platelet adhesion to the synthetic surface tested regardless of the temperature under static conditions. Inhibition of platelet activation on the heparin coated surface may be masked by standard dose heparinization.

Anticoagulants↗

PubMedDate not supplied

Protein adsorption and platelet adhesion on the surface of an oxygenator membrane.

Platelet adhesion on an oxygenator membrane is associated with thrombocytopenia or thrombus formation during extracorporeal circulation. The authors evaluated protein adsorption and platelet adhesion on three oxygenator hollow fiber membranes fabricated with polypropylene, silicone, and double layer polyolefin. Adsorbed proteins were analyzed by bicinchoninic acid protein assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blot. Platelet adhesion was assessed with enzyme immunoassays using monoclonal antibodies directed against CD42b and CD61. After 3 hr of incubation at 37 degrees C in whole blood, the amount of adsorbed protein was the least on silicone and increased from silicone < double polyolefin < polypropylene. The adsorbed protein pattern was similar; however, silicone showed less adsorption for all protein bands, and the gamma chain of fibrinogen was not detected. In contrast, double polyolefin showed the highest fibrinogen adsorption. The optical density at a wavelength of 450 nm for CD42b was 1.47 +/- 0.35 in polypropylene, 1.16 +/- 0.38 in silicone, and 1.85 +/- 0.19 in double polyolefin (p < 0.01 vs silicone) and for CD61 0.98 +/- 0.39 in polypropylene, 0.91 +/- 0.22 in silicone, and 1.69 +/- 0.25 in double polyolefin (p < 0.01 vs silicone and polypropylene). These data suggest that silicone is advantageous for long term extracorporeal respiratory support in terms of less platelet adhesion and no plasma leakage through the pores.

Adsorption↗