Basic homopolyamino acids, histones and protamines are potent antagonists of angiogenin binding to ribonuclease inhibitor.
A radio-ribonuclease inhibitor assay based on the interaction of 125I-angiogenin with ribonuclease inhibitor (RI) was used to detect pancreatic-type ribonucleases and potential modulators of their action. We show that highly basic proteins including the homopolypeptides poly-arginine, poly-lysine and poly-ornithine, core histones, spermatid-specific S1 protein and the protamines HP3 and Z3 were strong inhibitors of angiogenin binding to RI. A minimum size of poly-arginine and poly-lysine was required for efficient inhibition. The inhibition likely resulted from direct association of the basic proteins with the acidic inhibitor, as RI bound to poly-lysine and protamines while 125I-angiogenin did not. Antagonists of the angiogenin-RI interaction are potential regulators of either angiogenin-triggered angiogenesis and/or intracellular RI function, depending on their preferential target. (+info)
Clinical and immunochemical study of the serum IgG fraction not precipitated in a zinc-sodium salicylate reagent.
A reagent made of zinc sulphate (0-08 M) in a 0-4 M sodium salicylate solution at pH 7-3 precipitated most of the IgG when a small volume of human serum was added. Sera with normal IgG levels or polyclonal hyperglobulinaemia showed a close correlation between total IgG and zinc-precipitated IgG (r = + 0-95). In clinical material, not including IgG myeloma, zinc-soluble IgG varied between 0 and 6 mg/ml and was independent of the IgG serum concentration. In 31 normal subjects the average IgG concentration, as determined by the Technicon immunonephelometric method, was 10-2 +/- 1-7 mg/ml for total IgG and 2-2 +/- 1-0 mg/ml for the soluble fraction. Among 173 sera, including 24 from cord blood, 16 from pregnant women, and 133 from patients with miscellaneous diseases, no pathological conditions except three cases of IgG myeloma were found with a zinc-soluble IgG definitely above the normal values; zinc-soluble IgG levels were often low in patients with hyperglobulinaemia, and the difference was highly significant in liver disease. kappa and gamma light chains as well as the four IgG-Hp chain subclasses were found in both zinc-soluble fractions of normal IgG. A study of myeloma monoclonal IgG showed that globulins of classes 1, 3, and 4 could be either soluble or insoluble in the zinc reagent. One, G2, was mainly insoluble. Hexose and antistreptolysin contents per milligram normal IgG were not significantly different in either fraction. It is suggested that zinc-soluble IgG consists of the recently synthesized molecules, the zinc-solubility of which has not yet been decreased by protein association, lipid interaction, antigen binding, or enzymatic denaturation. Within this hypothesis, a low level of soluble IgG would mean either an increased precatabolic protein or a decreased synthesis. (+info)
Prion domain initiation of amyloid formation in vitro from native Ure2p.
The [URE3] non-Mendelian genetic element of Saccharomyces cerevisiae is an infectious protein (prion) form of Ure2p, a regulator of nitrogen catabolism. Here, synthetic Ure2p1-65 were shown to polymerize to form filaments 40 to 45 angstroms in diameter with more than 60 percent beta sheet. Ure2p1-65 specifically induced full-length native Ure2p to copolymerize under conditions where native Ure2p alone did not polymerize. Like Ure2p in extracts of [URE3] strains, these 180- to 220-angstrom-diameter filaments were protease resistant. The Ure2p1-65-Ure2p cofilaments could seed polymerization of native Ure2p to form thicker, less regular filaments. All filaments stained with Congo Red to produce the green birefringence typical of amyloid. This self-propagating amyloid formation can explain the properties of [URE3]. (+info)
Survey of total error of precipitation and homogeneous HDL-cholesterol methods and simultaneous evaluation of lyophilized saccharose-containing candidate reference materials for HDL-cholesterol.
BACKGROUND: Standardization of HDL-cholesterol is needed for risk assessment. We assessed for the first time the accuracy of HDL-cholesterol testing in The Netherlands and evaluated 11 candidate reference materials (CRMs). METHODS: The total error (TE) of HDL-cholesterol measurements was assessed in native human sera by 25 Dutch clinical chemistry laboratories. Concomitantly, the suitability of lyophilized, saccharose-containing CRMs (n = 11) for HDL-cholesterol was evaluated. RESULTS: In the precipitation method group, which included 25 laboratories and four methods, the mean (minimum-maximum) TE was 11.5% (2.7-25.2%), signifying that 18 of 25 laboratories satisfied the TE goal of +info)
Clusterin has chaperone-like activity similar to that of small heat shock proteins.
Clusterin is a highly conserved protein which is expressed at increased levels by many cell types in response to a broad variety of stress conditions. A genuine physiological function for clusterin has not yet been established. The results presented here demonstrate for the first time that clusterin has chaperone-like activity. At physiological concentrations, clusterin potently protected glutathione S-transferase and catalase from heat-induced precipitation and alpha-lactalbumin and bovine serum albumin from precipitation induced by reduction with dithiothreitol. Enzyme-linked immunosorbent assay data showed that clusterin bound preferentially to heat-stressed glutathione S-transferase and to dithiothreitol-treated bovine serum albumin and alpha-lactalbumin. Size exclusion chromatography and SDS-polyacrylamide gel electrophoresis analyses showed that clusterin formed high molecular weight complexes (HMW) with all four proteins tested. Small heat shock proteins (sHSP) also act in this way to prevent protein precipitation and protect cells from heat and other stresses. The stoichiometric subunit molar ratios of clusterin:stressed protein during formation of HMW complexes (which for the four proteins tested ranged from 1.0:1.3 to 1.0:11) is less than the reported ratios for sHSP-mediated formation of HMW complexes (1.0:1.0 or greater), indicating that clusterin is a very efficient chaperone. Our results suggest that clusterin may play a sHSP-like role in cytoprotection. (+info)
A novel strategy for the preparation of liposomes: rapid solvent exchange.
During the preparation of multi-component model membranes, a primary consideration is that compositional homogeneity should prevail throughout the suspension. Some conventional sample preparation methods pass the lipid mixture through an intermediary, solvent-free state. This is an ordered, solid state and may favor the demixing of membrane components. A new preparative method has been developed which is specifically designed to avoid this intermediary state. This novel strategy is called rapid solvent exchange (RSE) and entails the direct transfer of lipid mixtures between organic solvent and aqueous buffer. RSE liposomes require no more than a minute to prepare and manifest considerable entrapment volumes with a high fraction of external surface area. In phospholipid/cholesterol mixtures of high cholesterol content, suspensions prepared by more conventional methods reveal evidence of artifactual demixing, whereas samples prepared by rapid solvent exchange do not. The principles which may lead to artifactual demixing during conventional sample preparation are discussed. (+info)
von Willebrand factor contained in a high purity FVIII concentrate (Fanhdi) binds to platelet glycoproteins and supports platelet adhesion to subendothelium under flow conditions.
BACKGROUND AND OBJECTIVE: There is evidence suggesting that von Willebrand factor (VWF) from high purity factor VIII concentrates could be of clinical use in the management of patients suffering from VWD. We analyzed structural and functional characteristics of VWF present in a high purity factor VIII concentrate VWFHPC (Fanhdi). The multimeric structure, the ability to bind to platelet GP Ib/IX or GP IIb/IIIa, and the capacity of VWFHPC to promote platelet adhesion on injured vessels were investigated and compared with that present in standard plasma cryoprecipitates [VWFCRYO]. DESIGN AND METHODS: Binding studies were carried out by incubating radiolabeled VWF and washed platelets, which were activated with either ristocetin (1 mg/mL; for GP Ib/IX), or thrombin (2.5 U/mL; for GP IIb/IIIa). Platelet adhesion was assessed in a perfusion system (shear rate = 800 s-1, 10 min) in which the source of VWF was added (at 0.4 or 0.8 U/mL VWF:Ag) to washed platelets and red cells suspended in a human albumin solution. The deposition of platelets onto the perfused subendothelial surface was morphometrically evaluated and expressed as percentage of surface coverage (%SC). RESULTS: The VWFHPC (152 Units VWF:RCof/mg protein; VWF:RCof/VWF:Ag = 0.97), lacked only a small proportion of high-molecular-weight multimers present in VWFCRYO. Binding affinities (Kd values, nM) of VWFHPC were similar to those of VWFCRYO (5.3 +/- 0.86 vs 5.2 +/- 0.95, for GP Ib/IX; and 11.6 +/- 2.7 vs 15.4 +/- 1.7 for GPIIb-IIIa). A slightly, though not significantly, higher binding capacity for these receptors (Bmax values, molecules/pit) was obtained for VWFHPC. The %SC in perfusions in the presence of albumin was < 10%. Addition of VWFHPC or VWFCRYO significantly increased the %SC, with values of 27.1 +/- 4.9 and 17.5 +/- 2.8%, respectively with 0.4 U/mL (p < 0.004 and p < 0.02 vs albumin); and 30.8 +/- 4.9% and 20.03 +/- 4.1%, respectively, at 0.8 U/mL (p < 0.001 and p < 0.02 vs albumin). INTERPRETATION AND CONCLUSIONS: Our data show that VWF present in the high purity FVIII concentrate Fanhdi retains the functional capacity to bind to GPs Ib/IX and IIb/IIIa and to promote platelet adhesion onto exposed subendothelium. (+info)
A novel class of lipophilic quinazoline-based folic acid analogues: cytotoxic agents with a folate-independent locus.
Three lipophilic quinazoline-based aminomethyl pyridine compounds, which differ only in the position of the nitrogen in their pyridine ring, are described. CB300179 (2-pyridine), CB300189 (4-pyridine) and CB30865 (3-pyridine) all inhibited isolated mammalian TS with IC50 values of 508, 250 and 156 nM respectively. CB30865 was the most potent growth inhibitory agent (IC50 values in the range 1-100 nM for several mouse and human cell types). CB300179 and CB300189 were active in the micromolar range. Against W1L2 cells, CB300179 and CB300189 demonstrated reduced potency in the presence of exogenous thymidine (dThd), and against a W1L2:C1 TS overproducing cell line. In contrast, CB30865 retained activity in these systems. Furthermore, combinations of precursors and end products of folate metabolism, e.g. dThd/hypoxanthine (HX) or leucovorin (LV), did not prevent activity. CB30865 did not interfere with the incorporation of tritiated dThd, uridine or leucine after 4 h. A cell line was raised with acquired resistance to CB30865 (W1L2:R865; > 200-fold), which was not cross-resistant to CB300179 or CB300189. In addition, W1L2:R865 cells were as sensitive as parental cells to agents from all the major chemotherapeutic drug classes. CB300179 and CB300189 induced an S phase accumulation (preventable by co-administration of dThd). No cell cycle redistribution was observed following exposure (4-48 h) to an equitoxic concentration of CB30865. In the NCI anticancer drug-discovery screen, CB30865 displayed a pattern of activity which was not consistent with known anti-tumour agents. These data suggest that CB30865 represents a class of potent potential anti-tumour agents with a novel mechanism of action. (+info)