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C Vermeer

Publications and source records attributed to C Vermeer.

At least 91 records · Page 5Linked to original sources
PubMed1989

Histochemical detection of osteocalcin in normal and pathological human bone.

We investigated the immunohistochemical localization of osteocalcin in demineralized, paraffin-embedded normal and pathological human bone. Acid decalcification protocols appeared to be more suitable for osteocalcin detection than mild chelating agents. In normal lamellar bone, osteocalcin was detected in osteocytes and along the lamellar bone matrix in fine granular deposits. Under pathological conditions (osteomyelitis, neoplasia), appositional bone showed immunoreactivity in osteoblasts and osteocytes but not in the provisory woven bone matrix. Intense immunoreactivity could be seen at the cell borders of osteoclasts and the bone margins of Howship lacunae. In primary bone-forming tumors, osteocalcin immunoreactivity was detected in osteoblasts and their malignant counterparts. On the basis of these results, we conclude that optimal preservation of osteocalcin is obtained through mild acid decalcifiers. Osteocalcin is deposited in bone matrix, especially that of metabolically inactive bone. In neoplasms, osteocalcin could be a marker of osteoblastic differentiation.

Bone Neoplasms↗
PubMed1988

Circulating osteocalcin during oral anticoagulant therapy.

In this paper we present the following observations: 1) In sheep vitamin K-antagonists like phenprocoumon induce a decrease of the serum levels of osteocalcin (bone Gla-protein) and of the affinity of the circulating osteocalcin for hydroxyapatite. 2) In sheep vitamin K counteracts the effect of phenprocoumon on the blood coagulation system, but not that on the osteocalcin production. 3) In human subjects vitamin K-antagonists also lead to decreased levels of serum osteocalcin and a low affinity of the protein for hydroxyapatite. 4) These two variables reached steady-state levels within 24 h after the start of oral anticoagulant treatment and--at continuation of the therapy--they remained low for at least several years.

Acenocoumarol↗
PubMed1988

Vitamin K-dependent carboxylation. A synthetic peptide based upon the gamma-carboxylation recognition site sequence of the prothrombin propeptide is an active substrate for the carboxylase in vitro.

The vitamin K-dependent blood-clotting proteins contain a gamma-carboxylation recognition site in the propeptide, between the signal peptide and the mature protein, that directs gamma-carboxylation of specific glutamic acid residues. To develop a better substrate for the in vitro assay of the vitamin K-dependent gamma-carboxylase and to understand the substrate recognition requirements of the carboxylase, we prepared synthetic peptides based upon the structure of human proprothrombin. These peptides were employed as substrates for in vitro carboxylation using a partially purified form of the bovine liver carboxylase. A 28-residue peptide (HVFLAPQQARSLLQRVRRANTFLEEVRK), based on residues -18 to +10 in proprothrombin, includes the complete propeptide and the first 10 residues of acarboxyprothrombin. Carboxylation of this peptide is characterized by a Km of 3.6 microM. In contrast, FLEEL is carboxylated with a Km of about 2200 microM. A 10-residue peptide (ANTFLEEVRK), based on residues +1 to +10 in prothrombin, and a 20-residue peptide (ARSLLQRVRRANTFLEEVRK), based on residues -10 to +10 in proprothrombin, are also poor substrates for the carboxylase. Replacement of phenylalanine with alanine at residue 3 (equivalent to position -16 in proprothrombin) in the 28-residue peptide significantly alters the Km to 200 microM. A synthetic propeptide (HVFLAPQQARSLLQRVRRY), homologous to residues -18 to -1 in proprothrombin, inhibited carboxylation of the 28-residue peptide substrate with a Ki of 3.5 microM, but modestly stimulated the carboxylation of the 5- and 10-residue peptide substrates. These results indicate that an intact carboxylation recognition site is required for efficient in vitro carboxylation and that this site includes critical residues in region -18 to -11 of proprothrombin. The carboxylation recognition site in the propeptide binds directly to the carboxylase or to a closely associated protein.

Amino Acid Sequence↗
PubMed1988

The in vivo effects of oral anticoagulants in man: comparison between liver and non-hepatic tissues.

The in vivo effects of oral anticoagulant therapy with 4-hydroxycoumarins on various vitamin K-dependent enzyme systems in man were compared. In hepatic microsomes obtained from donors who has been treated with 4-hydroxycoumarins for more than 6 months, the vitamin K 2,3 epoxide reductase activity and the DTT-dependent vitamin K quinone reductase activity were diminished to 35% and 20% of the corresponding normal values. In the non-hepatic tissues, only a small decrease in vitamin K 2,3 epoxide reductase activity could be demonstrated, while no differences were found in the vitamin K quinone reductase activities. In none of the tissues a significant increase of noncarboxylated precursor proteins was observed, whereas also vitamin K hydroquinone-dependent carboxylase activities seemed to be unaffected by the anticoagulant treatment.

4-Hydroxycoumarins↗
PubMed1988

Vitamin K is no antagonist for the action of warfarin in rat osteosarcoma UMR 106.

The recycling of vitamin K in the liver occurs via one or two dithiol-dependent reductases, which are strongly inhibited by coumarin derivatives such as warfarin. This inhibition may be partly overcome by the action of a NADH-dependent reductase, which is relatively insensitive for warfarin. In this paper we demonstrate that the osteoblast-like osteosarcoma UMR 106 does not contain the NADH-dependent reductase. Assuming that a similar enzyme distribution occurs in normal osteoblasts this explains the observation of Price and Kaneda, that the administration of vitamin K to rats efficiently counteracted the effect of warfarin on blood coagulation, but that the vitamin had no effect on the Gla-content of serum osteocalcin.

Animals↗
PubMed1988

The quantification of gammacarboxyglutamic acid residues in plasma-osteocalcin.

In this paper we describe an assay procedure for determining the amount of gammacarboxyglutamic acid (Gla) residues in serum- or plasma-osteocalcin. The test includes removing by ethanol precipitation the majority of the proteins from the plasma (e.g., the Gla-containing coagulation factors) and the specific extraction of osteocalcin with the aid of immobilized immunopurified antibodies. It is demonstrated that the Gla-content of circulating osteocalcin from normal cows is similar to that of osteocalcin obtained from bone. Hence, the fact that part of the newly synthesized osteocalcin does not bind to the hydroxyapatite matrix in bone cannot be explained by an undercarboxylation of the molecule.

1-Carboxyglutamic Acid↗
PubMed1987

Extraction of osteocalcin from fossil bones and teeth.

Osteocalcin (also called 'bone Gla-protein') was detected in fossil bovid bones ranging from 12,000 years to 13 million years old and in rodent teeth 30 million years old. Both the antigenic activity and the protein-bound Gla-residues have remained intact. The protein is indistinguishable from recent bovine osteocalcin when analyzed by HPLC using ion exchange and size exclusion columns. If sufficient amounts can be extracted and an adequate purification procedure is established, this would be the first time that amino acid sequences in a protein from fossil bones may be determined. Such sequence data could offer a new approach to the phylogenetic study of extinct taxa.

Amino Acid Sequence↗
PubMed1987

Vitamin K-dependent carboxylase. Possible role for thioredoxin in the reduction of vitamin K metabolites in liver.

In the liver vitamin K epoxide, which is produced during the posttranslational carboxylation of protein-bound glutamic acid residues, is recycled by the action of one or more dithiol-dependent reductases. In vitro synthetic dithiols may serve as a cofactor for these enzymes, but the physiological reductant has not yet been found. In this paper we report that in vitro the commercially available thioredoxin/thioredoxin reductase from E. coli can replace the synthetic dithiols during the various reactions of the vitamin K cycle. Based on the assumption that in vivo thioredoxin also plays a role in the regeneration of vitamin K hydroquinone from the epoxide, an extension of the generally accepted vitamin K cycle is proposed.

Animals↗
PubMed1987

Isolation and partial characterization of a vitamin K-dependent carboxylase from bovine aortae.

Vitamin K-dependent carboxylase activity has been demonstrated in the crude microsomal fraction of the intima of bovine aortae. The procedure for the isolation of vessel wall carboxylase is a slight modification of the general preparation procedure for tissue microsomes. The highest activity of the non-hepatic enzyme was observed at 25 degrees C and hardly any NADH-dependent vitamin K reductase could be demonstrated. The optimal reaction conditions for both vessel wall as well as liver carboxylase were similar: 0.1 M-NaCl/0.05 M-Tris/HCl, pH 7.4, containing 8 mM-dithiothreitol, 0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (CHAPS), 0.4 mM-vitamin K hydroquinone and 2 M-(NH4)2SO4. Warfarin inhibits the hepatic and non-hepatic carboxylase/reductase enzyme complex more or less to a similar degree. We have measured the apparent Km values for the following substrates: Phe-Leu-Glu-Glu-Leu ('FLEEL'), decarboxylated osteocalcin, decarboxylated fragment 13-29 from descarboxyprothrombin and decarboxylated sperm 4-carboxyglutamic acid-containing (Gla-)protein. The results obtained demonstrated that liver and vessel wall carboxylase may be regarded as isoenzymes with different substrate specificities. The newly discovered enzyme is the first vitamin K-dependent carboxylase which shows an absolute substrate specificity: FLEEL and decarboxylated osteocalcin were good substrates for vessel wall carboxylase, but decarboxylated fragment 13-29 and decarboxylated sperm Gla-protein were not carboxylated at all.

Animals↗
PubMed1987

Substrate recognition by vitamin K-dependent carboxylase.

Decarboxylated osteocalcins were prepared and purified from bovine, chicken, human and monkey bones and assayed for their ability to serve as a substrate for vitamin K-dependent carboxylase from bovine liver. Substantial differences were observed, especially between bovine and monkey d-osteocalcin. Since these substrates differ only in their amino acid residues 3 and 4, it seems that these residues play a role in the recognition of a substrate by hepatic carboxylase.

Amino Acid Sequence↗
PubMed1987

Vitamin K-dependent carboxylase: increased efficiency of the carboxylation reaction.

The in vitro vitamin K-dependent carboxylation of peptide- or protein-bound glutamate residues is generally studied in detergent-solubilized microsomes from rat or cow liver. Under the conditions usually employed, the efficiency of the carboxylation reaction is low (less than 1% of the carboxylatable residues is converted into gammacarboxyglutamate). Here we describe that this efficiency may be raised to 30% by carrying out the following adaptations: 1) carboxylase was purified about 100-fold from the solubilized microsomes, so that the enzyme was obtained in a highly concentrated form and could be added in excess: 2) the HCO-3 concentration in the reaction mixtures was raised to 50 mM and 3) a substrate was selected (decarboxylated osteocalcin from bovine bone) the Km of which had been shown to be low (10 microM) and it was added in rate-limiting amounts. Besides the fact that under these conditions the carboxylation reaction occurred with a higher efficiency than before, the adaptations also enabled us to express the carboxylation activity in terms of moles CO2 incorporated per mole of substrate.

Animals↗
PubMed1987

The effect of Gla-containing proteins on the precipitation of insoluble salts.

The precipitation of insoluble salts containing divalent metal ions is inhibited by Gla-containing proteins of various origin. In this paper we demonstrate that: Gla-residues are required for the inhibitory activity; the inhibition is effected by a protein which in vivo is bound to calcified tissue (osteocalcin) as well as by proteins occurring in blood plasma (factor X) and urine (the urinary Gla-protein); The inhibitor concentration required for 50% precipitation-inhibition varied slightly from one salt to the other, but no marked differences were observed between the effects of the various Gla-containing proteins used; Precipitation-inhibition occurred in all phosphates (Be, Ca, Mn and Zn) and in all calcium salts (phosphate, oxalate and carbonate) tested.

1-Carboxyglutamic Acid↗
PubMed1987

Direct measurement of vitamin K-dependent enzymes in various isolated and cultured tumor and non-tumor cells.

A modification of the assay for vitamin K-dependent carboxylase is described with which the enzyme could be detected in relatively low amounts of cells (n = 10(6)). Using this assay, we could demonstrate vitamin K-dependent carboxylase activity in hepatocytes, renal tubular cells, osteoblasts, endothelial cells and macrophages, but not in lymphocytes or platelets. The cultured tumor cells UMR-106, B16 and 5583 also contained vitamin K-dependent carboxylase activity. Vitamin K epoxide reductase activity was demonstrated only in cells where vitamin K-dependent carboxylase activity was present. The tumor cells possessed remarkably less K epoxide reductase activity than the normal cells. When cells were cultured in medium containing warfarin, the K epoxide reductase activity was found to be decreased and the amount of non-carboxylated precursor proteins had increased, suggesting an analogous vitamin K mechanism as in liver.

Animals↗
PubMed1987

The placental transport of [3H]vitamin K1 in rats.

In this paper we describe the placental transport of [3H]vitamin K1 in pregnant rats during the first 24 h after the oral administration of the vitamin. Vitamin K1 in the fetal livers ranged from 0.13% (3 h) to 2% (24 h) of the values found in the corresponding maternal livers. In spite of the low placental transfer of vitamin K, we found no accumulation of coagulation factor precursors in the fetal rat liver microsomes as could be expected in vitamin K deficiency. Moreover, we could not demonstrate any difference between adult and fetal rat liver microsomes with regard to the sensitivity for warfarin. From these results we conclude that a substantial placental barrier exists for the transport of pharmacological amounts of vitamin K1 but that under physiological conditions sufficient vitamin K1 appears to be present in the fetal liver to ensure a full carboxylation reaction. The vitamin K-dependent carboxylase activity rate of adult and fetal rat liver microsomes was comparable, indicating that the newborn rat has an adequate carboxylating system.

Animals↗
PubMed1986

The in vivo effects of acenocoumarol, phenprocoumon and warfarin on vitamin K epoxide reductase and vitamin K-dependent carboxylase in various tissues of the rat.

In rats the in vivo effects of a chronic low-dose treatment (+/- 60 micrograms/rat per day) with different coumarins (acenocoumarol, phenprocoumon and warfarin) on hepatic and non-hepatic vitamin K-dependent enzyme systems were compared. The plasma concentrations of the three coumarins differed largely but these differences were not reflected in the microsomal coumarin contents. The non-hepatic microsomes contained less than 20% of the coumarins found in liver microsomes. No substantial differences were observed between the following effects of the three anticoagulant treatments. The blood coagulation factor activities were about 10% of normal. The hepatic microsomal vitamin K epoxide reductase activity was diminished to about 35% of control values. The vitamin K epoxide reductase activities present in kidney, lung, spleen, testis and brain microsomes were less influenced by the coumarin treatments; activities ranged between 45 and 65% of normal. In the liver microsomes a 15-fold accumulation of non-carboxylated precursor proteins was found; in the non-hepatic microsomes this effect was less pronounced but still present. The hepatic vitamin K-dependent carboxylase activity was enhanced but the corresponding non-hepatic enzyme activities were slightly or not affected. In addition, the effects of a chronic low-dose warfarin treatment were compared with those after an acute high dose of the drug.

4-Hydroxycoumarins↗
PubMed1986

Increased susceptibility to the anticoagulant effect of warfarin in mice bearing the Lewis lung carcinoma. Role of vitamin K deficiency.

This study reports an increased susceptibility to warfarin anticoagulation in mice bearing an experimental tumour, the Lewis lung carcinoma. In these animals, following a single i.v. injection of warfarin, the prothrombin complex activity decreased normally but recovered far slower than in controls, while the rate of degradation of the clotting factors was not modified. At the level of the vitamin K-dependent liver carboxylase, it was possible to demonstrate an increase in the endogenous substrate (reflecting an impairment of the carboxylase vitamin K dependent system). This abnormality was reversed by vitamin K administration and can be reasonably ascribed to a vitamin K deficiency in association with tumour growth.

Animals↗