Affinity partitioning. A method for purification of proteins using specific polymer-ligands in aqueous polymer two-phase systems.
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We describe a method, called affinity partitioning, for the purification of proteins containing specific ligand binding receptor sites. This method adds specificity to the procedures for protein purification with aqueous polymer two-phase systems by introduction of a polymer derivative, coupled to an appropriate ligand. The addition of a polymer-ligand that partitions predominantly into one phase shifts the protein that binds this substance to the same phase. By performing countercurrent distribution in the presence of a polymer-ligand, the protein that binds the polymer-ligand can be separated from a heterogenous mixture. One example of affinity paritioning used dextran as the polymer-ligand. Dextran was chosen since it is a constituent of the most commonly used system for partitioning proteins. In a dextran-poly(ethylene oxide) system, concanavalin A bound dextran and partitioned predominantly into the dextran-rich phase. The addition of the specific competitor, D-mannose, displaced the partition coefficient toward unity, while the application of L-fucose, a noncompetitor, had little effect. Application of affinity partitioning to the purification of another protein required the synthesis of a specific polymer-ligand. To study this we synthesized dinitrophenyl-poly-(ethylene oxide), which binds specifically to S-23 myeloma protein. Addition of dinitrophenyl-poly(ethylene oxide) to the dextran-poly(ethylene oxide) phase system shifted the S-23 myeloma protein into the poly(ethylene oxide)-rich phase. epsilon-N-dinitrophenyl-L-lysine, by competing with binding of dinitrophenyl-poly(ethylene oxide), antagonized the latter's effect on the partition coefficient of S-23 myeloma protein. By adding various amounts of dinitrophenyl-poly-(ethylene oxide), we correlated the partition coefficient with concentration of polymer-ligand. A model of the action of polymer-ligand derivatives on the partition coefficient, derived from thermodynamic considerations, was found to be consistent with the experimental data relating the concentration of polymer-ligand and partition coefficient. Affinity partitioning should prove to be a useful complement to affinity chromatography in the purification of mixtures of proteins. Since cells and subcellular particles may be purified with aqueous polymer two-phase systems, affinity partitioning might be applied to their fractionation by using polymer-ligands specific for unique surface receptors. (+info)
Studies on the inhibition of C56-initiated lysis (reactive lysis). III. Characterization of the inhibitory activity C567-INH and its mode of action.
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An activity in serum which inhibits reactive lysis has recently been shown to do so by preventing the attachment of C567 complexes to cells, and hence has been designated C567-INH. This report describes certain physiochemical characteristics of the inhibitory activity. It behaves as a heat-stable pseudoglobulin, soluble in 20 per cent Na2SO4, and having alpha1 mobility on Pevikon block electrophoresis. It is excluded from CM cellulose at pH 6-0, RSC Equals 0.007 M, is retained by an XM-100 membrane and is heterogenous on Sephadex G-200, eluting in at least two peaks. The combined active materials from the Sephadex column elute from DE-52 in at least four peaks. The mechanism of action of material from each of these four peaks is shown to involve prevention of attachment of C567 complexes to membranes, and this is shown to involve an effect on C567 complexes in solution rather than an effect on the membrane. A less dramatic effect on the lysis of EC567 by limited quanities of C8 and C9 can be demonstrated. Haemolytic studies using cell-bound C567 suggest that the interaction of C567-INH with C567 involves a loose reversible association. It is therefore postulated that C567-INH inhibits reactive lysis primarily by reversibly associating with the nascent C567 complex in solution, increasing its bulk and decreasing its diffusion capacity so that it is unable to reach a cell membrane before its haemolytic potential decays. (+info)
Carrier protein-modulated presentation and recognition of an N-glycan: observations on the interactions of Man(8) glycoform of ribonuclease B with conglutinin.
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Conglutinin is a serum lectin of the innate immune system, which binds high mannose N-glycans when these are appropriately presented on proteins. Here we use the conglutinin-ribonuclease B (RNaseB)-recognition system as a model to investigate the structural basis of selective recognition of protein-bound oligosaccharides by this carbohydrate-binding receptor. Conglutinin shows little binding to the isolated RNaseB-Man(8 )glycoform, and no binding to Man(5-6) glycoforms. In contrast, when the protein moiety is reduced and denatured we observe that conglutinin binds strongly to the isolated RNaseB-Man(8) glycoform and weakly to the Man(5-6) glycoforms. These results are in accord with observations on the binding to the N-glycans in the absence of carrier protein. NMR analyses of native RNaseB-Man(8) and -Man(5-6) glycoforms reveal that the three-dimensional structure of the protein moiety is essentially identical to that of non-glycosylated RNase (RNaseA). Thus there are no perceptible differences between the RNase protein forms that could account for differential availability of the N-glycan for conglutinin-binding. After reduction and denaturation, the NMR spectrum became typical of a non-structured polypeptide, although the conformational preferences of the N-glycosidic linkage were unchanged, and most importantly, the Man(8 )oligosaccharide retained the average conformational behavior of the free oligosaccharide irrespective of the carrier protein fold. This conformational freedom is clearly not translated into full availability of the oligosaccharide for the carbohydrate-recognition protein. We propose, therefore, that the differing bioactivity of the N-glycan is a reflection of the existence of different geometries of presentation of the carbohydrate determinant in relation to the protein surface within the glycan:carrier protein ensemble. (+info)
Fibrinolysis system in patients with bronchial asthma.
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Every inflammatory process, including that in the course of bronchial asthma may disturb the balance in blood coagulation and fibrinolysis system. The aim of the present study was to evaluate fibrinolysis in patients with bronchial asthma. The study group consisted of 41 patients with bronchial asthma, hitherto untreated (25 women, 16 men, at mean age 37.37 +/- 12.4 years) and 22 healthy adults (control group). In these subjects, the following parameters were established: euglobulin lysis time (ELT), the concentration of tissue plasminogen activator antigen (t-PA Ag), the concentration of urokinase plasminogen activator antigen (u-PA Ag), the activity of plasminogen activator inhibitor type 1 (PAI-1), the concentration of plasmin-antiplasmin complex (PAP) and fibrinogen/fibrin degradation products (FDP). It was found that patients with bronchial asthma had statistically significantly higher mean values of FDP (9.25 +/- 6.7 micrograms/ml vs. 5.0 +/- 5.9 micrograms/ml; p < 0.001), ELT (123.5 +/- 42.7 min vs. 97.4 +/- 27.1 min; p < 0.001), t-PA Ag (8.36 +/- 3.66 ng/ml vs. 5.5 +/- 3.71 ng/ml; p < 0.01) and PAP complexes (250.3 +/- 95.8 ng/ml vs. 193.4 +/- 60.7 ng/ml; p < 0.02). Mean u-PA Ag concentration in patients with bronchial asthma was significantly lower than in control group (0.24 +/- 0.16 ng/ml vs. 0.53 +/- 0.18 ng/ml; p < 0.01). No statistically significant differences were observed as to PAI-1 activity between patients with bronchial asthma and healthy subjects. The results of the present study suggest that increased concentrations of t-PA Ag, PAP and FDP complexes are the evidence for greater activity of fibrinolysis system in subjects with bronchial asthma. (+info)
Lymph draining from foot joints in rheumatoid arthritis provides insight into local cytokine and chemokine production and transport to lymph nodes.
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OBJECTIVE: Rheumatoid arthritis (RA) is characterized by inflammatory reactions in joints and adjacent tissues unaccompanied by clinically evident changes in lymphatics and lymph nodes draining the inflamed areas. The explanation for this phenomenon, which contrasts with infectious processes in joints and soft tissues that evoke major changes in the lymphatic system, is unclear. To determine which inflammatory factors produced in the joints of RA patients are transported in lymph to lymph nodes, we measured levels of immunoglobulins, cytokines, and chemokines in prenodal lymph from the foot joints of RA patients and quantified their rate of transport to regional lymph nodes. METHODS: Lymph was collected from the cannulated lymphatics draining the foot joints, tendons, fascia, and skin of 20 RA patients. Lymph flow rate and concentrations of proteins and immunoglobulins were measured. Cytokine and chemokine levels were quantified by enzyme-linked immunosorbent assays. Results were compared with those obtained in 20 control subjects. RESULTS: In the cannulated vessel, the mean +/- SEM lymph flow rate in RA patients was almost 2-fold that in control subjects (22.6 +/- 3.2 ml/24 hours versus 13.2 +/- 1.1 ml/24 hours; P < 0.01). Lymph concentrations of total protein, IgG, and IgM were 1.80 +/- 0.14 gm/dl, 384 +/- 45 mg/dl, and 32.0 +/- 1.5 mg/dl, respectively, in RA patients and 1.66 +/- 0.14 gm/dl, 238 +/- 32 mg/dl, and 15.0 +/- 1.3 mg/dl, respectively, in control subjects. The corresponding lymph:serum (L:S) ratios were 0.21 +/- 0.02, 0.22 +/- 0.02, and 0.15 +/- 0.02, respectively, in RA patients and 0.22 +/- 0.02, 0.19 +/- 0.02, and 0.11 +/- 0.02, respectively, in control subjects. The L:S ratios of <1 and the absence of significant differences between groups suggested a lack of local production of immunoglobulins. In RA patients, lymph concentrations (in pg/ml) were as follows: interleukin-1beta (IL-1beta) 14.8 +/- 3.9, IL-6 511 +/- 143, tumor necrosis factor alpha (TNFalpha) 9.9 +/- 1.1, IL-1 receptor antagonist (IL-1Ra) 4,274 +/- 737, IL-10 13.3 +/- 4.4, IL-8 846 +/- 174, IL-15 6.2 +/- 0.9, granulocyte-macrophage colony-stimulating factor (GM-CSF) 2.30 +/- 0.15, vascular endothelial growth factor (VEGF) 80.4 +/- 8.6, and macrophage inflammatory protein 1alpha (MIP-1alpha) 171 +/- 34. In control subjects, these values were as follows: IL-1beta 1.50 +/- 0.25, IL-6 79.0 +/- 14.6, TNFalpha 4.4 +/- 1.1, IL-1Ra 208 +/- 52, IL-10 0.0, IL-8 216 +/- 83, IL-15 5.00 +/- 0.45, GM-CSF 0.40 +/- 0.05, VEGF 42.0 +/- 2.4, and MIP-1alpha 3.4 +/- 1.7 (P < 0.05 versus RA patients for all except IL-15). The L:S ratio was >1 in all RA patient samples for IL-1beta, IL-6, IL-1Ra, IL-8, GM-CSF, IL-10, IL-15, TNFalpha, and MIP-1alpha, indicating local production of cytokines. Great variability in lymph cytokine concentrations, presumably reflecting differences in the intensity of local inflammation, was not reflected in serum cytokine concentrations. Intravenously infused methylprednisolone decreased lymph cytokine levels to normal within 12 hours. In contrast, their concentrations in serum showed little or no change. CONCLUSION: High lymph concentrations of cyto kines and chemokines, exceeding those in serum, were found in RA patients. The L:S concentration ratios of > 1 indicate the local production of these cytokines and chemokines in the inflamed tissues. High flow rates of lymph containing high cytokine concentrations through the regional lymph nodes are likely to affect node lymphocytes and dendritic cells. Analysis of cytokines in lymph should provide insight into events in inflamed tissues in RA and in regional lymph nodes. (+info)
Serine proteinase inhibitor 3 and murinoglobulin I are potent inhibitors of neuropsin in adult mouse brain.
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Extracellular serine protease neuropsin (NP) is expressed in the forebrain limbic area of adult brain and is implicated in synaptic plasticity. We screened for endogenous NP inhibitors with recombinant NP (r-NP) from extracts of the hippocampus and the cerebral cortex in adult mouse brain. Two SDS-stable complexes were detected, and after their purification, peptide sequences were determined by amino acid sequencing and mass spectrometry, revealing that target molecules were serine proteinase inhibitor-3 (SPI3) and murinoglobulin I (MUG I). The addition of the recombinant SPI3 to r-NP resulted in an SDS-stable complex, and the complex formation followed bimolecular kinetics with an association rate constant of 3.4 +/- 0.22 x 10(6) M(-1) s(-1), showing that SPI3 was a slow, tight binding inhibitor of NP. In situ hybridization histochemistry showed that SPI3 mRNA was expressed in pyramidal neurons in the hippocampal CA1-CA3 subfields, as was NP mRNA. Alternatively, the addition of purified plasma MUG I to r-NP resulted in an SDS-stable complex, and MUG I inhibited degradation of fibronectin by r-NP to 24% at a r-NP/MUG I molar ratio of 1:2. Immunofluorescence histochemistry showed that MUG I localized in the hippocampal neurons. These findings indicate that SPI3 and MUG I serve to inactivate NP and control the level of NP in adult brain, respectively. (+info)
Distribution of gold among plasma fractions in rheumatoid patients undergoing chrysotherapy compared with its distribution in plasma incubated with aurothiomalate in vitro.
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The distribution of gold among the globulin, albumin, and unbound fractions of plasma, obtained either from rheumatoid patients receiving long-term aurothiomalate therapy or from samples incubated with aurothiomalate in vitro, has been investigated. In the rheumatoid patients it has been found that, although the majority of the plasma gold is always bound to albumin, the distribution varies cyclically in phase with the dose schedule. An explanation of these phenomena is provided, based on data obtained from the reaction between aurothiomalate and plasma constituents in vitro. (+info)
Reversibility of the chronic effects of di(2-ethylhexyl)phthalate.
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Fischer-344 rats treated with 12,500 ppm (728 and 879 mg/kg/d for male and females, respectively) and B6C3F1 mice treated with 6,000 ppm (1,227 and 1,408 mg/kg/d, respectively) di(2-ethylhexyl)phthalate (DEHP) in the diet for 78 weeks were allowed to recover for an additional 26 weeks on control diet. Blood was analyzed at weeks 78 and 104 from 10 animals per sex per group; animals were sacrificed at weeks 79 and 105 for histopathologic examination. The results are compared with data from animals continuously exposed to these dietary levels for 104 weeks (10, 11). Body weights and food consumption were measured monthly. BUN, albumin, and globulin that were significantly different for rats exposed to DEHP throughout 104 weeks, were comparable to controls for the recovery group. Reversibility of chronic effects on erythrocyte count, hemoglobin, and hematocrit values was apparent only for female rats. Chronic exposure demonstrated effects on liver, kidney, and testes weights. All organ weight effects except for testes for the Recovery group of rats, and all organ weight effects for mice, were reversible. Pigmentation of Kupffer cells and renal tubules present in chronically treated rats were not observed for the Recovery group. Lesions in the testes and pituitary gland were not reversible in rats. This may be a reflection of the senescence of the hypothalamic-gonad axis in rats. Cessation of exposure for mice resulted in amelioration of effects in the kidneys, liver, and testes. The extent of reversibility suggests that many chronic effects may be associated with a metabolic phenomenon such as peroxisome proliferation, which also reverted to control levels after 26 weeks of recovery. (+info)