Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The latent birefringence of lymphocyte membranes of various species may readily be studied and analysed by various topo-optical reactions. The membranes of glutaraldehyde-fixed and PBS-washed lymphocytes show continuous birefringence with thiazine- and quinoline dyes. According to polarization optical analysis thiazine dye-stained cells are radially positive, whereas quinoline dye-stained cells are radially negative spherites, i.e. thiazine dye molecules are in a perpendicular, quinoline dye molecules in a parallel orientation relative to the membrane surface. These findings suggest that in lymphocyte membranes glycoproteins are primarily responsible for the topo-optical reactions. The actual conformational state of the glycoprotein components is a decisive factor not only in dye binding but also in the orientation of dye molecules. Heparin treatment directs attention to an important interaction between heparin and membrane glycoproteins. With the aid of the critical electrolyte concentration (CEC) technique we were able to demonstrate an ultrastructural differences between human erythrocyte and human lymphocyte membranes. After this procedure the birefringence of erythrocyte membranes was lost, whereas that of lymphocyte membranes did not change. There were no differences between the topo-optical reactions of T and B lymphocytes.
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PMID:Topo-optical reactions and polarization optical analysis of human lymphocytes. 619 38

The ion-step measuring method was used to determine absolute heparin concentrations in PBS and blood plasma with a Ta2O5 ISFET on to which a monolayer of protamine had been immobilized. Heparin is a highly negatively charged polysaccharide, which is used clinically to delay the clotting of blood. Protamine acts as an affinity ligand for heparin. The response of the ISFET system on a step-wise increase in the electrolyte concentration (a so-called ion-step) is a transient change of the output voltage, which is related to the surface charge density of the ISFET gate oxide. After 2 mins of incubation in a plasma sample containing heparin, the amplitude of the transient ISFET response to an ion-step showed a linear relation to the heparin concentration. In blood plasma, heparin concentrations between 0.3 and 2.0 Units/ml could be determined with an accuracy of +/- 0.08 Units/ml. Heparin concentrations in different plasma samples of heparinized patients were determined and compared with the APTT. No direct relation was found between the APTT and the heparin concentration, but this result was not surprising.
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PMID:The ISFET based heparin sensor with a monolayer of protamine as affinity ligand. 775 59

Granulocyte colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of hematopoietic progenitor cells of the neutrophil lineage. Measurement of murine G-CSF levels will allow examination of its role in host defense using murine models. Therefore, we developed a sensitive sandwich enzyme-linked immunoabsorbent assay (ELISA) for murine G-CSF. A polyclonal antibody to recombinant murine G-CSF was produced in rabbits and isolated using a protein A column. This purified native IgG served as the capture antibody and a portion of the IgG was biotinylated to serve as the developing antibody. Specificity was verified by lack of reactivity to GM-CSF, IL-6, IL-3, prolactin, and growth hormone. The lower limit of sensitivity routinely extended to 16 pg/ml in multiple ELISAs. Intra-assay coefficient of variation (CV) ranged from 3.4 to 21.5% across the detection limits of the assay, with the greatest variance occurring near the standard curve maximum. Interassay CV ranged from 11.5 to 23.3%. The ability of the ELISA to detect G-CSF in different sample preparations was examined in RPMI 1640 with 10% FCS, Hanks balanced salt solution, PBS/Tween-20/2% FCS, and the dilution media for ELISA (10% BLOTTO/PBS/0.05% Tween-20). Average recovery in these media ranged from 98 to 107%. Heparin anti-coagulated normal mouse plasma had a suppressive effect on the ELISA that varied between individual mice. Recovery was also determined from liver, spleen, and lung homogenate suspensions at dilutions of 1:5, 1:10, and 1:20 in dilution buffer. Recovery from liver was optimal at the 1:10 and 1:20 dilutions at 105%, with that of the 1:5 dilution at 135%. Recovery from spleen ranged from 94 to 96%. Lung homogenate displayed enhanced recovery (139% or greater) across all dilutions. The ability of the assay to detect G-CSF was explored by measurement of G-CSF levels in peritoneal lavage following polymicrobial intra-abdominal infection. Peak levels of G-CSF production occurred at 16 h after cecal ligation and puncture surgery with 18- and 21-guage needles (75.7 ng/ml and 111.4 ng/ml, respectively) as compared to the sham animals (0.61 ng/ml). The assay was found to be specific, sensitive, and accurate for measurement of murine G-CSF in a variety of sample types.
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PMID:A sandwich enzyme-linked immunoabsorbent assay for measurement of picogram quantities of murine granulocyte colony-stimulating factor. 1036 91

Vascular smooth muscle cell (SMC) growth plays an important role in atherosclerosis, restenosis and venous bypass graft disease. With systemic drug administration no effective therapy for restenosis and venous bypass graft disease is available. This could be due to low local concentrations of the drugs at the target site. A directed delivery of drugs to tissues with a sustained release system during percutaneous transluminal coronary angioplasty (PTCA) or during bypass surgery could provide high concentrations of drugs at the target site and avoid systemic side effects. In the present study heparin was encapsulated by spray-drying into biodegradable poly(D, L-lactic-co-glycolic acid) (PLGA) to obtain a system for prolonged drug release. SMC were cultured from saphenous vein explants obtained from patients undergoing coronary bypass surgery. Cell proliferation was measured by [(3)H]thymidine incorporation. Heparin release from PLGA 50:50 microspheres in an isoosmolar PBS buffer (pH=7.4) showed a triphasic profile with an initial burst (completed after 24 h), a dormant period and a final stage with increased release rate, which lasted about 10-14 days. Cell proliferation as measured by [(3)H]thymidine incorporation was markedly stimulated by platelet-derived growth factor-BB (PDGF-BB) (5 ng/ml) or serum (5%). Proliferation of SMC was equally reduced (50%; P<0.05; n=9-11) by native heparin or heparin released from PLGA microspheres, while PLGA microspheres without heparin loading had no effect on [(3)H]thymidine incorporation in human SMC. Similar results were also obtained when SMC were stimulated with 5% serum instead of PDGF-BB (50%; P<0.05; n=6). Thus, heparin encapsulated into PLGA microspheres was released over a prolonged period of time and thereby effectively reduced human SMC proliferation stimulated either with PDGF or serum. Biodegradable PLGA microspheres may also be used to encapsulate other antiproliferative agents and provide a new approach for local drug delivery after PTCA. This may help to prevent restenosis after PTCA or to reduce graft disease after coronary bypass graft surgery.
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PMID:Sustained release of heparin from polymeric particles for inhibition of human vascular smooth muscle cell proliferation. 1042 32

The effects of heparin on angiogenesis are controversial, with some studies claiming stimulatory and other studies claiming inhibitory effects. Since heparin in human plasma is complexed with basic peptides and proteins, we studied the angiogenic effect of complexes resulting by mixing poly-L-lysine (a basic heparin-binding polypeptide) and heparin. Angiogenesis was investigate by chorioallantoic membrane assay. In specimens treated with PBS (negative control), or poly-L-lysine, no significant vascular reaction was detectable. Heparin induced only moderate angiogenic response. However, neutral complexes purified from a mixture of poly-L-lysine and heparin (20/1, w/w) induced a very strong angiogenic response. These results demonstrate that the angiogenic effect of heparin was associated with neutralization of electric charge when the polysaccharide was complexed with a basic peptide.
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PMID:Poly-L-lysine/heparin stimulates angiogenesis in chick embryo chorioallantoic membrane. 1178 75

Heparin employed in extracorporeal blood circulation (ECBC) procedures (e.g. open heart operations) often leads to a high incidence of bleeding complications. Protamine employed in heparin neutralization, on the other hand, can cause severe adverse reactions. We previously developed an approach that could prevent both heparin- and protamine-induced toxic side effects concomitantly. This approach consisted of placing a hollow fiber-based bioreactor device containing immobilized protamine (termed a "protamine bioreactor") at the distal end of the ECBC procedure. This protamine bioreactor would remove heparin after heparin served its anticoagulant purpose in the ECBC device, thereby eliminating heparin-induced bleeding risks. In addition, this protamine bioreactor would prevent protamine from entering the patients, thereby aborting any protamine-induced toxic effects. Both in vitro and in vivo studies have successfully demonstrated the feasibility of this approach. Despite promises, early findings also revealed two shortcomings that must be overcome for the protamine bioreactor to be applied clinically. The first drawback was that the cyanate ester linkages, involved in conjugating protamine to the bioreactor device, were unstable and prone to hydrolysis, resulting in the leakage of a significant amount of protamine into circulation during application of the protamine bioreactor. The second deficiency was that the capacity of the protamine bioreactor in heparin removal was rather low, owing to the limited surface area of the hollow fibers for protamine immobilization and subsequently heparin adsorption. In this paper, we present novel strategies to overcome these two limitations. A new conjugation method based on the use of 4-(oxyacetyl)phenoxyacetic acid (OAPA) as the activating reagent was employed to yield stable linkages, via the abundant arginine residues of protamine, onto the hollow fibers. Results showed that while the amount of protamine immobilized on each gram of fibers was relatively comparable between the OAPA and the previous CNBr activation methods (7.45 mg/g versus 7.69 mg/g fibers), there was virtually no detectable leaching of immobilized protamine from the bioreactor by the OAPA method, comparing to 35% leaching of protamine by the previous CNBr method following 72 h of storage of the bioreactor in PBS buffer at 37 degrees C. To improve the capacity and functionality of the protamine bioreactor, two novel approaches were adopted. Long chain and high molecular weight poly-lysine was linked to the hollow fibers, prior to protamine coupling, to create multiple layers of immobilized protamine for subsequent heparin adsorption. In addition, a poly(ethylene glycol) (PEG) chain was inserted between protamine and the hollow fibers to yield a three-dimensional, free dynamic motion for immobilized protamine. Preliminary observations indicated that a four- to five-fold enhancement in heparin adsorption was attained by utilizing each of these new approaches. Aside from their current use, these new strategies can also be employed generically to improve the functionality of any affinity-type bioreactor. Indeed, efforts have been made recently in utilizing these approaches to develop a clinically usable GPIIb/IIIa bioreactor for the treatment of immune thrombocytopenic purpura (ITP)-an autoimmune disease.
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PMID:Strategies for improving the functionality of an affinity bioreactor. 1624 11

Heparin is a highly sulfated glycosaminoglycan that is used as an important clinical anticoagulant. Monitoring and control of the heparin level in a patient's blood during and after surgery is essential, but current clinical methods are limited to indirect and off-line assays. We have developed a silicon field-effect sensor for direct detection of heparin by its intrinsic negative charge. The sensor consists of a simple microfabricated electrolyte-insulator-silicon structure encapsulated within microfluidic channels. As heparin-specific surface probes the clinical heparin antagonist protamine or the physiological partner antithrombin III were used. The dose-response curves in 10% PBS revealed a detection limit of 0.001 units/ml, which is orders of magnitude lower than clinically relevant concentrations. We also detected heparin-based drugs such as the low-molecular-weight heparin enoxaparin (Lovenox) and the synthetic pentasaccharide heparin analog fondaparinux (Arixtra), which cannot be monitored by the existing near-patient clinical methods. We demonstrated the specificity of the antithrombin III functionalized sensor for the physiologically active pentasaccharide sequence. As a validation, we showed correlation of our measurements to those from a colorimetric assay for heparin-mediated anti-Xa activity. These results demonstrate that silicon field-effect sensors could be used in the clinic for routine monitoring and maintenance of therapeutic levels of heparin and heparin-based drugs and in the laboratory for quantitation of total amount and specific epitopes of heparin and other glycosaminoglycans.
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PMID:Monitoring of heparin and its low-molecular-weight analogs by silicon field effect. 1718 32

The objective was to determine the effects of oviductal proteins on sperm function. Abbatoir-derived buffalo oviducts were flushed with PBS; the fluid recovered (protein concentration, 2.3 mg/mL; average of 3.5 mg protein/oviduct) was centrifuged, dialyzed, and clarified, and the supernatant applied to a Heparin-Sepharose affinity column. Unbound fractions were collected and bound proteins were separately eluted (with elution buffer). Eight distinct protein bands (from 12 to 177 kDa) in the H-unbound fraction and 15 distinct protein bands (from 12 to 165 kDa) in the H-bound fraction were detected in SDS-PAGE. Semen from four buffalo bulls was divided into three parts: Parts 1 and 2 were treated with the heparin binding (H-bound) and non-heparin binding (H-unbound) oviductal proteins, respectively, whereas Part 3 remained as an untreated control. Equilibrated and frozen-thawed semen was assessed for motility, viability, intact acrosome percentage, mucus penetration distance, and hypo-osmotic swelling test. The H-bound oviductal fluid proteins enhanced (P<0.05) the proportion of sperm that were progressively motile, alive, had an intact acrosome and functional plasma membrane (hypo-osmotic swelling test), as well as the distance covered in the cervical mucus sperm penetration test during cryopreservation. Addition of the H-unbound oviductal protein fraction did not increase sperm motility and penetration distance but increased (P<0.05) the proportion of sperm that were live, had an intact acrosome, and functional plasma membrane (hypo-osmotic swelling test). We concluded that the H-bound fraction of buffalo oviductal fluid protein(s) maintained sperm motility, viability and membrane integrity during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity.
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PMID:Effect of oviductal fluid proteins on buffalo sperm characteristics during cryopreservation. 1835 71

Establishing thrombosis-resistant surface is crucial to develop tissue-engineered small diameter vascular grafts for arterial reconstructive procedures. The objective of this study was to evaluate the stability and anti-coagulation properties of heparin covalently linked to decellularized porcine carotid arteries. Cellular components of porcine carotid arteries were completely removed with chemical and physical means. Heparin was covalently linked to the decellularized vessels by a chemical reaction of the carboxyl end of amino acids with hydroxylamine sulphate salt and heparin-EDC. Bound heparin contents were measured by quantitative colorimetric assay of toluidine blue staining. The average content of heparin in treated vessels was 35.6 +/- 11.6 mg/cm(2) tissue, which represented 6.21 +/- 2.03 UPS heparin/cm(2) tissue. The stability of heparin linkage was tested by incubating the heparin-linked vessels either in PBS at 37 degrees C or in 70% alcohol at room temperature up to 21 days, showing no significant reduction of heparin content. Anti-coagulation property of bound heparin was determined with a clotting time assay using fresh dog blood. Standardized small pieces of non-heparin-bound vessels were clotted in fresh dog blood within 10 min., whereas all heparin-bound vessels did not form clot during 1-hr observation. In vivo platelet deposition of the vessel was determined with a baboon model of the femoral arteriovenous external shunt and (111)Indium labelling of platelets. There were 1.38 +/- 0.07 x 10(9) and 0.64 +/- 0.11 x 10(9) baboon platelets deposited on the control and heparin-linked vessels, respectively, at 60 min. These data demonstrate that covalent linkage of heparin provides an effective and stable anti-coagulation surface of decellularized porcine carotid arteries. This study may suggest a new strategy to develop tissue-engineered biological vascular grafts, which could be used for human coronary or low extremity artery bypasses.
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PMID:Covalent linkage of heparin provides a stable anti-coagulation surface of decellularized porcine arteries. 1904 Apr 21

Cell adhesion plays a key role during various physiological and pathological processes. Many studies have been performed to understand the interaction of platelets with endothelial cells (ECs) during the past decades. Modulation of their interaction has been shown to be therapeutically useful in thrombotic diseases. Some methods of labeling platelets such as counting and radiolabeling have been applied in the study of the platelets-ECs interaction, but these methods did not obtain full approval. A rapid, simple and sensitive assay for platelets-ECs interaction was developed in this paper. Platelets were labeled with Sudan Black B (SBB) before adding to confluent ECs monolayer. Non-adherent platelets were removed by washing with PBS. The adherent platelets were lysed with dimethylsulfoxide (DMSO) and the absorbance was recorded at 595 nm by spectrophotometer. A linear correlation was observed between the absorbance of SBB and the number of platelets. By employing the SBB method, the influence of heparin on platelets-ECs interactions was observed. Heparin (3-100 units/mL) obviously reduced platelets adhering to ECs in a concentration-dependent manner.
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PMID:A simple adhesion assay for studying interactions between platelets and endothelial cells in vitro. 2021 26


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