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J Pieters

Publications and source records attributed to J Pieters.

34 records · Page 2Linked to original sources
PubMed1997

MHC class II compartments: specialized organelles of the endocytic pathway in antigen presenting cells.

Mounting an immune response against foreign, extracellular material requires that this material be internalized by antigen presenting cells, processed to peptide fragments and then displayed on the cell surface for recognition by the T cell receptor on T helper cells. Such peptides, derived from internalized antigens are generally presented to T cells in association with Major Histocompatibility Complex (MHC) class II molecules. Recent work has identified subcompartments of the endosomal/lysosomal system that appear to be important sites for the generation of peptide-MHC class II complexes. These so-called MHC class II compartments receive antigenic fragments from endosomes and lysosomes and load them onto class II molecules. From these compartments, peptide-loaded class II molecules are transported to the plasma membrane, where they can trigger T cells bearing appropriate receptors. This review summarizes recent work characterizing MHC class II compartments as specialized organelles of the endosomal/lysosomal pathway. Interestingly, MHC class II compartments share many features common to organelles present in a wide variety of specialized cells with quite different functions. Modifying the endocytic pathway to serve the needs of specialized cells may be a common mechanism which allows very different cell types to carry out their disparate functions.

Animals↗
PubMed1994

Isolation and characterization of the intracellular MHC class II compartment.

An intracellular compartment has been isolated to which MHC class II molecules are transported on their way to the plasma membrane. They arrive with an associated invariant chain which is then proteolytically processed while MHC class II molecules acquire antigenic peptide. These loaded class II molecules then leave the compartment devoid of invariant chain and bound for the plasma membrane. This compartment represents a new stage in the endocytic/lysosomal pathway.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1993

Application of an improved density gradient electrophoresis apparatus to the separation of proteins, cells and subcellular organelles.

A DGE apparatus, made of Perspex, consisting of a separation column (5 x 2.2 cm) and containing a 0-4% linear Ficoll density gradient, was constructed. Only 2.5 cm of the column were used for high resolution separations. A specially designed removable top cone permitted precise gradient introduction, thin sample layering (0.3-1 mm) and precise fractionation after electrophoresis. A bottom circular palladium anode (nongassing) was separated hydrodynamically but not electrically from the density gradient by a cation-permeable membrane. A top circular platinum cathode caused negatively charged particles to migrate upwards (levitation). Thin sample layering permitted short separation times (30-60 min) at only 3 V/cm (10 mA). As for proteins, glycoforms of a1-antitrypsin were separated as well as isoenzymes of beta-hexoseaminidase. Furthermore, separation of transferrin (Tf) from the putative Tf-receptor complex was effectuated. The device was equally suitable for the separation of Megadalton proteins (mucins). Artificial mixtures of intact erythrocytes (rat, rabbit, human) were separated with high resolution. About 10(7) cells (of 100 microns3 cell volume) could be loaded onto the device. Crude microsomes from the human melanoma cell line Mel JuSo were separated after brief trypsin treatment within 38 min at 10 mA. Ratios of the migration velocities of the constituent organelles were: late endosomes (LE):lysosomes (L):Golgi (G):early endosomes (EE) = 1:0.94:0.77:0.55 and under slightly different conditions LE:L:G:endoplasmatic reticulum (ER):plasma membrane (PM) = 1:0.87:0.64:0.58:0.49.

Animals↗
PubMed1993

The MHC class II-associated invariant chain contains two endosomal targeting signals within its cytoplasmic tail.

The oligomeric complex formed by major histocompatibility complex (MHC) class II alpha and beta chains and invariant chain (Ii) assembles in the endoplasmic reticulum and is then transported via the Golgi complex to compartments of the endocytic pathway. When Ii alone is expressed in CV1 cells it is sorted to endosomes. The Ii cytoplasmic tail has been found to be essential for targeting to these compartments. In order to characterize further the signals responsible for endosomal targeting, we have deleted various segments of the cytoplasmic tail. The Ii mutants were transiently expressed and the cellular location of the proteins was analyzed biochemically and morphologically. The cytoplasmic tail of Ii was found to contain two endosomal targeting sequences within its cytoplasmic tail; one targeting sequence was present within amino acid residues 12-29 and deletion of this segment revealed the presence of a second endosomal targeting sequence, located within the first 11 amino acid residues. The presence of a leucine-isoleucine pair at positions 7 and 8 within this sequence was found to be essential for endosomal targeting. In addition, the presence of this L-I motif lead to accumulation of Ii molecules in large endosomal vacuoles containing lysosomal marker proteins. Both wild type Ii and Ii mutant molecules containing only one endosomal targeting sequence were rapidly internalized from the plasma membrane. When the Ii cytoplasmic tail was fused to the membrane-spanning region of neuraminidase, a resident plasma membrane protein, the resulting chimera (INA) was found in endocytic compartments containing lysosomal marker proteins. Thus the cytoplasmic tail of Ii is sufficient for targeting to the endocytic/lysosomal pathway.

Amino Acid Sequence↗
PubMed1991

Intracellular transport and localization of major histocompatibility complex class II molecules and associated invariant chain.

The intracellular transport and location of major histocompatibility complex (MHC) class II molecules and associated invariant chain (Ii) were investigated in a human melanoma cell line. In contrast to the class II molecules, which remain stable for greater than 4 h after synthesis, the associated Ii is proteolytically processed within 2 h. During or shortly after synthesis the NH2-terminal cytoplasmic and membrane-spanning segment is in some of the Ii molecules cleaved off; during intracellular transport, class II associated and membrane integrated Ii is processed from its COOH terminus in distinct steps in endocytic compartments. Immunocytochemical studies at the light and electron microscopic level revealed the presence of class II molecules, but not of Ii on the cell surface. Intracellularly both Ii and class II molecules were localized in three morphologically and kinetically distinct compartments, early endosomes, multivesicular bodies, and prelysosomes. This localization in several distinct endosomal compartments contrasts with the localization of class II molecules in mainly one endocytic compartment in B lymphoblastoid cell lines. As in these lymphoblastoid cell lines Ii is known to be rapidly degraded it is conceivable that the rate of proteolysis of the class II associated Ii and its dissociation from class II molecules modulates the retention of the oligomeric complex in endocytic compartments, and as a consequence the steady-state distribution of these molecules within the endosomal system.

Biological Transport↗
PubMed1990

Heparin-stimulated inhibition of factor IXa generation and factor IXa neutralization in plasma.

Generation and inhibition of activated factor IXa was studied in factor XIa-activated plasma containing 4 mmol/L free calcium ions and 20 mumol/L phospholipid (25 mol% phosphatidylserine/75 mol% phosphatidylcholine). Interference of other (activated) clotting factors with the factor IXa activity measurements could be avoided by using a highly specific and sensitive bioassay. Factor IXa generation curves were analyzed according to a model that assumed Michaelis-Menten kinetics of factor XIa-catalyzed factor IXa formation and pseudo first order kinetics of inhibition of factor XIa and factor IXa. In the absence of heparin, factor IXa activity in plasma reached final levels that were found to increase with increasing amounts of factor XIa used to activate the plasma. When the model was fitted to this set of factor IXa generation curves, the analysis yielded a rate constant of inhibition of factor XIa of 0.7 +/- 0.1 min-1 and a kcat/Km ratio of 0.29 +/- 0.01 (nmol/L)-1 min-1. No neutralization of factor IXa activity was observed (the estimated rate constant of inhibition of factor IXa was 0). Thus, in the absence of heparin, the final level of factor IXa in plasma is only dependent on the initial factor XIa concentration. While neutralization of in situ generated factor IXa in normal plasma was negligible, unfractionated heparin dramatically enhanced the rate of inactivation of factor IXa (apparent second order rate constant of inhibition of 5.2 min-1/per microgram heparin/mL). The synthetic pentasaccharide heparin, the smallest heparin chain capable of binding antithrombin III, stimulated the inhibition of in situ generated factor IXa, but sevenfold less than unfractionated heparin (k = 0.76 min-1 per microgram pentasaccharide/mL). We found that free calcium ions were absolutely required to observe an unfractionated heparin and pentasaccharide-stimulated neutralization of factor IXa activity. Factor XIa inhibition (psuedo first order rate constant of 0.7 min-1) was not affected by unfractionated heparin or pentasaccharide in the range of heparin concentrations studied.

Activation Analysis↗
PubMed1989

In situ-generated thrombin is the only enzyme that effectively activates factor VIII and factor V in thromboplastin-activated plasma.

We investigated the activation of the nonenzymatic protein cofactors factor VIII and factor V in plasma when coagulation was initiated by thromboplastin. With sensitive bioassays, we were able to measure specifically the generation of activated factor VIII and activated factor V in plasma. Our results showed that when plasma was triggered with a relatively high concentration of thromboplastin, factor VIII and factor V were completely activated at the clotting time of plasma. However, when the generation of thrombin, but not that of factor Xa, was delayed by addition of hirudin to the plasma, factor Va was generated only at the time thrombin generation overcame the hirudin inhibition. In addition, generation of factor VIIIa correlated with thrombin generation and not with factor Xa generation. Furthermore, addition of large amounts of factor Xa to hirudinized plasma did not show detectable factor VIII or factor V activation. We concluded that in plasma activated with thromboplastin the enzyme responsible for activation of factor V and factor VIII is thrombin, not factor Xa.

Blood Coagulation↗
PubMed1988

Inhibition of factor IXa and factor Xa by antithrombin III/heparin during factor X activation.

We investigated the kinetics of the inhibitory action of antithrombin III and antithrombin III plus heparin during the activation of factor X by factor IXa. Generation and inactivation curves were fitted to a three-parameter two-exponentional model to determine the pseudo first-order rate constants of inhibition of factor IXa and factor Xa by antithrombin III/heparin. In the absence of heparin, the second-order rate constant of inhibition of factor Xa generated by factor IXa was 2.5-fold lower than the rate constant of inhibition of exogenous factor Xa. It appeared that phospholipid-bound factor X protected factor Xa from inactivation by antithrombin III. It is, as yet, unclear whether an active site or a nonactive site interaction between factor Xa and factor X at the phospholipid surface is involved. The inactivation of factor IXa by antithrombin III was found to be very slow and was not affected by phospholipid, calcium, and/or factor X. With unfractionated heparin above 40 ng/ml and antithrombin III at 200 nM, the apparent second-order rate constant of inhibition of exogenous and generated factor Xa were the same. Thus, in this case phospholipid-bound factor X did not protect factor Xa from inhibition. In the presence of synthetic pentasaccharide heparin, however, phospholipid-bound factor X reduced the rate constant about 5-fold. Pentasaccharide had no effect on the factor IXa/antithrombin III reaction. Unfractionated heparin (1 micrograms/ml) stimulated the antithrombin III-dependent inhibition of factor IXa during factor X activation 400-fold. In the absence of reaction components this stimulated was 65-fold. We established that calcium stimulated the heparin-dependent inhibition of factor IXa.

Algorithms↗
PubMed1988

Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein.

Based on sequence information from tryptic peptides an almost full-size cDNA coding for the human vascular anticoagulant was isolated from a placental cDNA library and sequenced. The coding region was cloned into an Escherichia coli expression vector and the protein expressed at high levels. The recombinant protein was purified and found to be indistinguishable from its natural counterpart in several biological assays.

Amino Acid Sequence↗
PubMed1988

The limited importance of factor Xa inhibition to the anticoagulant property of heparin in thromboplastin-activated plasma.

The antifactor Xa activities of heparin fractions are widely used as an ex vivo index of their antithrombotic efficacy. Its clinical meaning, however, remains speculative. In the study reported, we measured the effects of standard heparin, a synthetic pentasaccharide heparin (antifactor Xa activity only), and a low molecular weight heparin (LMWH) on factor Xa, factor Va, and thrombin generation in thromboplastin-activated plasma. We clearly demonstrated that the antifactor Xa activity of heparin contributed little in its anticoagulant activity. The inhibition of factor Va generation, dependent on the heparin antithrombin activity only, is of prime importance to the inhibition of thrombin generation in plasma. The inhibition of thrombin generation by the LMWH was comparable with that of standard heparin on the basis of their respective antithrombin specific activities, but not on the basis of their antifactor Xa activities.

Blood Platelets↗
PubMed1987

Neutralization of heparin by prothrombin activation products.

The neutralization of heparin by active site blocked meizothrombin and thrombin, prothrombin fragment 1.2, fragment 1 and fragment 2 was probed by the heparin-dependent factor Xa inactivation by antithrombin III (AT III). Meizothrombin had no effect on the inactivation of factor Xa, whereas thrombin had an inhibitory effect (IC50 = 700 nM). After factor Xa catalyzed cleavage of meizothrombin, the resulting products, prothrombin fragment 1.2 plus thrombin, did not show any heparin neutralizing properties. However, after isolation of the reaction products, both thrombin and prothrombin fragment 1.2 exhibited heparin neutralizing properties in the factor Xa inactivation reaction. The IC50-values were 700 nM and 100 nM, respectively. Prothrombin fragment 1, when present at 125 nM, caused a 50% reduction of the heparin-dependent rate of inactivation of factor Xa and prothrombin fragment 2 had no effect at all. From this we conclude that, in addition to the thrombin part of the prothrombin molecule, the fragment 1 region also exhibits a rather high affinity for heparin.

Animals↗
PubMed1983

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines.

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1c phosphatidylcholine and 16:0/18:1c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using 31P-NMR, freeze fracturing and differential scanning calorimetry (DSC). The phosphatidylcholines adopt a bilayer configuration above 0 degrees C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with 31P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal HII phase. 16:0/18:1c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75 degrees C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0 degrees C which decreases with increasing unsaturation and which is lowered by approximately 10 degrees C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.

Calorimetry↗
PubMed1982

Determination and calibration of crystal lattice images of biological apatites.

Lattice imaging provides detailed information on the mineral phase of calcified tissues. The lattice space distances dhk1 depend directly on the accuracy of the magnification. Using synthetic apatite, K2PtCl6, graphitized C and crocidolite as calibration material the lattice parameters of human enamel have been determined. The relative errors in the mineral dhk1 values using crocidolite, K2PtCl6 and C are 0.6, 1.0 and 3.9 % resp. If hydroxyapatite is employed on the same grid as the enamel specimen, the d-values obtained are very close to the X-ray diffraction data; a reproducibility of 0.8% in a and c lattice parameter determination can be obtained on selected crystallites.

Crystallography↗