Biomedical subjects

R Wallin

Publications and source records attributed to R Wallin.

At least 55 records · Page 3Linked to original sources

PubMed1992

Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues.

The tissue distribution and subcellular location of branched chain aminotransferase was analyzed using polyclonal antibodies against the enzyme purified from rat heart mitochondria (BCATm). Immunoreactive proteins were visualized by immunoblotting. The antiserum recognized a 41-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate. The 41-kDa protein was always present in mitochondria which contained branched chain aminotransferase activity, skeletal muscle, kidney, stomach, and brain, but not in cytosolic fractions. In liver mitochondria, which have very low levels of branched chain aminotransferase activity, the 41-kDa protein was not present. However, two immunoreactive proteins of slightly higher molecular masses were identified. These proteins were located in hepatocytes. The 41-kDa protein was present in fetal liver mitochondria but not in liver mitochondria from 5-day neonates. Thus disappearance of the 41-kDa protein coincided with the developmental decline in liver branched chain aminotransferase activity. Two-dimensional immunoblots of isolated BCATm immunocomplexes showed that the liver immunoreactive proteins were clearly different from the heart and kidney proteins which exhibited identical immunoblots. Investigation of BCATm in subcellular fractions prepared from different skeletal muscle fiber types revealed that branched chain aminotransferase is exclusively a mitochondrial enzyme in skeletal muscles. Although total detergent-extractable branched chain aminotransferase activity was largely independent of fiber type, branched chain aminotransferase activity and BCATm protein concentration were highest in mitochondria prepared from white gastrocnemius followed by mixed skeletal muscles with lowest activity and protein concentration found in soleus mitochondria. These quantitative differences in mitochondrial branched chain aminotransferase activity and enzyme protein content suggest there may be differential expression of BCATm in different muscle fiber types.

Aging↗

PubMed1992

Processing and trafficking of clotting factor X in the secretory pathway. Effects of warfarin.

Clotting factor X undergoes several post-translational processing steps in the liver before the protein appears in blood as the mature two-chain zymogen. In this study we have followed the factor X precursor through the secretory pathway in rat liver in order to identify the site for proteolytic processing of the precursor into a two-chain form and the site for warfarin inhibition of precursor trafficking within the pathway. Isolated rat liver Golgi apparatus was shown to harbour two single-chains of factor X precursors of 70 and 74 kDa and the heavy (50 kDa) and light chains of factor X. It was demonstrated that the two-chain factor X form was produced from a late processing form of the factor X precursor, which indicated that the site for proteolytic conversion to a two-chain form was in the trans-Golgi compartment. The 70 and 74 kDa single-chain precursors and also the light chain of the two-chain form were shown to contain the factor X propeptide which is normally removed before the coagulation factor appears in blood. The data demonstrate that intra-chain cleavage of a single chain factor X precursor in the trans-Golgi compartment can precede release of the propeptide. Warfarin was shown to affect trafficking of the factor X precursor between the endoplasmic reticulum (ER) and the Golgi apparatus. The data suggest a link between vitamin K-dependent gamma-carboxylation of the precursor and its exit from the ER. Warfarin administration resulted in accumulation of factor X precursors associated with the ER membrane. These precursors appear to be stabilized from intracellular degradation while in the ER. In contrast to the large increase in the factor X precursor concentration in the ER membrane, there was no change in the prothrombin precursor concentration as a result of warfarin action on the liver. However, intracellular turnover of the microsomal prothrombin precursor pool in warfarin-treated rats resulted in a pool of less negatively charged proteins, indicating ongoing protein synthesis but inhibition of gamma-carboxylation. The data are consistent with previous findings [Wallin & Martin (1988) J. Biol. Chem. 263, 9994-10001] suggesting that prothrombin and factor X are processed differently by the vitamin K-dependent carboxylase in the ER membrane.

Amidohydrolases↗

PubMed1992

Cell adhesion to matrix Gla protein and its inhibition by an Arg-Gly-Asp-containing peptide.

Matrix Gla protein (MGP) is a 14-kDa protein found in bone and cartilage which contains the unusual amino acid gamma-carboxyglutamic acid (Gla). The biological function of this protein has not been elucidated. Here we have demonstrated the adherence of chondrocytes, fibroblasts, osteosarcoma cells, and kidney mesangial cells to MGP purified from bovine bone. Maximum adherence occurred at MGP concentrations of 0.5-1.0 micrograms/ml. Removal of the calcium-binding Gla residues by thermal decarboxylation of MGP destroyed the proteins' cell adherence properties. Cell adherence to MGP was not affected by the presence of antibodies directed against the C-terminal (non-Gla) portion of the protein or the presence of cycloheximide during the adherence assay. However, the Arg-Gly-Asp-containing synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro significantly inhibited cell attachment to MGP, whereas the control peptide Gly-Arg-Gly-Glu-Ser-Pro had minimal effect. These data indicate that MGP may function in mediating cell attachment to the extracellular matrix via a receptor that requires intact Gla residues and that can be inhibited by Arg-Gly-Asp-containing peptides.

Amino Acid Sequence↗

PubMed1992

Articular-cartilage matrix gamma-carboxyglutamic acid-containing protein. Characterization and immunolocalization.

Matrix gamma-carboxyglutamic acid (Gla)-containing protein (MGP) was found to be present in articular cartilage by Western-blot analysis of guanidinium chloride extracts of human and bovine cartilage and was further localized by immunohistochemical studies on human and monkey specimens. In newborn articular cartilage MGP was present diffusely throughout the matrix, whereas in growth-plate cartilage it was seen mainly in late hypertrophic and calcifying-zone chondrocytes. In adult articular cartilage MGP was present primarily in chondrocytes and the pericellular matrix. Immunoelectron microscopy studies revealed an association between MGP and vesicular structures with an appearance consistent with matrix vesicles. MGP may be an important regulator of cartilage calcification because of its localization in cartilage and the known affinity of Gla-containing proteins for Ca2+ and hydroxyapatite.

1-Carboxyglutamic Acid↗

PubMed1992

Fibrin (ogen)-derived peptide B beta 30-43 is a sensitive marker of activated neutrophils during fibrinolytic-treated acute myocardial infarction in man.

Neutrophils, elastase, the specifically elastase-derived fibrin split product, B beta 30-43, and C-reactive protein were determined in 30 consecutive patients with acute myocardial infarction. At admission to the coronary care unit 4.2 +/- 0.8 hours after the onset of symptoms, all elements were increased above the reference levels, while compared with convalescent levels, only neutrophils and B beta 30-43 were increased. After the streptokinase treatment, neutrophils, elastase, and B beta 30-43 increased abruptly and peaked (p less than 0.0001) within 1.5 hours. Plasma creatine kinase MB and C-reactive protein reached their peak levels after about 12 and 24 hours, respectively. Peak indices of neutrophils and creatine kinase correlated (r = 0.60, p less than 0.0006). Compared with the age-matched reference range, the convalescent level of B beta 30-43 was increased (p less than 0.0001). Of the tested elements suggestive of neutrophil activation, B beta 30-43 showed signs of being the most sensitive. In keeping with animal studies, neutrophils are activated early during the course of acute myocardial infarction and their activation seems to become accelerated by fibrinolytic treatment. Neutrophils may remain activated in the convalescent phase.

Biomarkers↗

PubMed1992

Elastase activity in leukaemic cells and plasma in patients with acute leukaemia.

Proteolysis of coagulation factors and inhibitors resulting in haemorrhage can be mediated by elastase. Indirect signs of this are elevated levels of elastase complexed to its inhibitor in plasma, alpha 1-antitrypsin (E alpha 1-AT). We have measured intracellular elastase activity in leukaemic cells from 60 patients with acute leukaemia. Elastase activity was detected in 92% of the patients with acute nonlymphoblastic leukaemia (ANL), no activity was found in the patients with acute lymphoblastic leukaemia (ALL). High levels were found in cells from patients with promyelocytic leukaemia. Moderate to high total circulating blast elastase activity was measured in 70% of the ANL patients with haemorrhage compared with 36% of the patients without bleeding complications (p < 0.05). The available intracellular elastase activity was correlated to the level of E alpha 1-AT (rs = 0.42, p < 0.01) but not to the elastase specific split product of fibrinogen, B beta 30-43. In complete remission the levels of E alpha 1-AT were normalized. Intracellular elastase activity might be a useful supplement to differentiate ANL and ALL.

Adolescent↗

PubMed1991

Processing of prothrombin in the secretory pathway.

Antibodies raised against plasma prothrombin and the prothrombin propeptide were used to identify prothrombin precursors in rough and smooth microsomes and in the Golgi apparatus. The data demonstrate that the propeptide is part of the prothrombin molecule when undergoing a variety of modifications in the Golgi apparatus. It is shown that these modifications result in an increase in the apparent molecular mass of the prothrombin precursor from 78 kDa in early processing to 83 kDa in late processing. The 83 kDa prothrombin precursor was not recognized by the anti-propeptide antiserum and most likely represents the final product of the precursor in the secretory pathway. Evidence is presented that the propeptide is released from the parent molecule in the Golgi apparatus by a membrane-bound Ca(2+)-dependent serine proteinase(s) with characteristics similar to those of the proalbumin-to-albumin-converting enzyme. Vitamin K-dependent carboxylase activity was measured in membrane fragments obtained from the Golgi apparatus preparation. Sucrose-density-gradient centrifugation and the use of marker enzymes showed that carboxylase activity was highest in fractions enriched in cis-Golgi cisternae. Two different synthetic peptides were used as substrates for the carboxylase. These peptides were from the N-terminal and the C-terminal part of the gamma-carboxyglutamic acid (Gla) region of prothrombin. It is shown that the N-terminal and the C-terminal peptides were preferred as substrates for the carboxylase in the microsomal and the Golgi apparatus preparations respectively. It is also shown that the prothrombin precursor acquires negative charges in the Golgi apparatus that do not result from addition of sugars in late processing. These negative charges could be eliminated by thermal decarboxylation, suggesting that Gla residues may also be synthesized in late processing.

Amino Acid Sequence↗

PubMed1991

The effects of warfarin on HepG2 cells suggest that prothrombin and factor X interact differently with the vitamin K-dependent carboxylase in the secretory pathway.

HepG2 cells have been shown to respond to warfarin by 1) enhanced vitamin K-dependent carboxylase activity; 2) enhanced intracellular concentration of the factor X clotting factor precursor and 3) enhanced vitamin K-dependent 14C-labelling of a 74 kDa microsomal protein which has been identified as the factor X precursor. There was no difference in any of these measured parameters whether the cells had been treated for 4 or 24 hours with warfarin. In contrast to the intracellular factor X concentration, the intracellular prothrombin precursor concentration was not affected by the drug which suggests there is a difference in the mechanism of processing of these two clotting factors by HepG2 cells. The data are consistent with the view that warfarin maintains its effect on the vitamin K-dependent carboxylation system in HepG2 cells for 24 hours and support the hypothesis that clotting factor X and prothrombin precursors interact differently with the vitamin K-dependent carboxylase.

Carbon-Carbon Ligases↗

PubMed1991

The effects of fish oil on triglycerides, cholesterol, fibrinogen and malondialdehyde in humans supplemented with vitamin E.

The effects of fish oils supplemented with 0.3 IU/g and 1.5 IU/g of vitamin E were compared in a double-blind, cross-over study. Twelve healthy volunteers were given 30 mL/day of either oil for 3 wk. Intake of the vitamin E-rich fish oil resulted in a marked decrease in serum triglycerides (48%) and in fibrinogen (11%). After administration of the low vitamin E-containing oil there was a considerably smaller reduction of serum triglycerides and no significant reduction of fibrinogen. Both oils caused an increase in high density lipoprotein cholesterol and a decrease in the atherogenic index, but neither oil altered the total cholesterol level. Serum vitamin E was decreased by 9% and plasma malondialdehyde was increased by 122% after intake of the low vitamin E-containing oil, but both remained normal after intake of the other oil. The effect of vitamin E may be due to inhibition of fatty acid peroxidation with less formation of malondialdehyde and a larger amount of active (n-3) fatty acids in their sites of action in the liver, resulting in a greater decrease in the synthesis of triglycerides and fibrinogen.

Adult↗

PubMed1991

Dexamethasone stimulates vitamin K-dependent carboxylase activity in neonatal rats and cultured fetal hepatocytes.

gamma-Carboxylation of vitamin K-dependent clotting factors may be a key regulatory element in output of these proteins from liver to blood in the developing neonate. We have investigated the effect of hormones and growth factor on the vitamin K-dependent carboxylation system in neonatal rats and cultured fetal hepatocytes. Of the hormones and growth factor tested, only dexamethasone had a significant effect on the system. When dexamethasone was administered to newborn rats, there was a delayed response that produced significant enhancement of carboxylase activity 6 d after injection of the drug. A similar delayed response to the drug could also be demonstrated in cultured fetal hepatocytes. When cultured in the presence of 0.1-1 microM dexamethasone, cellular carboxylase activity was little affected by the drug the first 2 d of culture but the activity was increased more than threefold by 4 d in culture. Microsomal membranes from neonatal rat livers and fetal hepatocytes treated with dexamethasone showed enhanced vitamin K-dependent 14C labeling of the factor X membrane precursor pool. Enhanced labeling of the factor X membrane precursor pool has also been demonstrated in rats and HepG2 cells treated with warfarin. The data suggest that 1) gamma-carboxylation of clotting factors is regulated by glucocorticoids in the developing liver, and 2) dexamethasone stimulates intracellular gamma-carboxylation of the factor X precursor by a mechanism that currently is unknown.

Animals↗

PubMed1991

Vitamin K-dependent carboxylation in articular chondrocytes.

A nonhepatic vitamin K-dependent protein, matrix Gla protein, has recently been identified in cartilage where it may play an important role in the control of mineralization or matrix development. We have investigated the vitamin K cycle in chondrocytes isolated from bovine and rabbit articular cartilage and examined these cells for their ability to synthesize vitamin K-dependent proteins. Chondrocytes were found to have an active vitamin K-dependent carboxylation system. Preincubation of the cells with warfarin resulted in a significant increase in the measured carboxylase activity. Both vitamin K epoxide reductase and DT-diaphorase (EC 1.6.99.2) activity were present indicating that chondrocytes are capable of producing reduced vitamin K1H2, the cofactor for the vitamin K-dependent carboxylase. Specific 14C-labeling of microsomal vitamin K-dependent protein precursors demonstrated synthesis of several vitamin K-dependent proteins by chondrocytes. 35S-labeling of chondrocyte proteins provided evidence that matrix Gla protein is synthesized by these cells.

Animals↗

PubMed1990

Propeptide recognition by the vitamin K-dependent carboxylase in early processing of prothrombin and factor X.

Precursors of vitamin K-dependent proteins are synthesized with a propeptide that is believed to target these proteins for gamma-carboxylation by the vitamin K-dependent carboxylase. In this study synthetic propeptides were used to investigate gamma-carboxylation of the prothrombin and factor X precursors in rat liver microsomes. The extent of prothrombin processing by the carboxylase was also investigated. Antisera raised against the human prothrombin and factor X propeptides only recognized precursors with the respective propeptide regions. The data demonstrate structural differences in the propeptide region of the prothrombin and the factor X carboxylase substrates which raises questions about the hypothesis of a common propeptide binding site on the carboxylase for all precursors of vitamin K-dependent proteins. The hypothesis of separate binding sites is supported by data which demonstrate differences in binding of the prothrombin and factor X precursors to membrane fragments from rough and smooth microsomes. gamma-Carboxylation of the prothrombin precursors in vitro was investigated with conformational specific antibodies raised against a portion of the Gla (gamma-carboxyglutamic acid) region extending from residue 15 to 24. The synthetic peptide used as antigen contains three of the ten potential Gla sites in prothrombin. It is shown that these antibodies do not recognize mature prothrombin but recognize the decarboxylated protein. It is also demonstrated that the epitope is Ca2(+)-dependent. The antibodies were used to assess gamma-carboxylation of the prothrombin precursor in membrane fragments from microsomal membranes. The results suggest that microsomal gamma-carboxylation does not involve Glu residues 16, 19 and 20 of the Gla region.

Amino Acid Sequence↗

PubMed1990

The vitamin K-dependent carboxylation system in human osteosarcoma U2-OS cells. Antidotal effect of vitamin K1 and a novel mechanism for the action of warfarin.

An osteoblast-like human osteosarcoma cell line (U2-OS) has been shown to possess a vitamin K-dependent carboxylation system which is similar to the system in human HepG2 cells and in liver and lung from the rat. In an 'in vitro' system prepared from these cells, vitamin K1 was shown to overcome warfarin inhibition of gamma-carboxylation carried out by the vitamin K-dependent carboxylase. The data suggest that osteoblasts, the cells involved in synthesis of vitamin K-dependent proteins in bone, can use vitamin K1 as an antidote to warfarin poisoning if enough vitamin K1 can accumulate in the tissue. Five precursors of vitamin K-dependent proteins were identified in osteosarcoma and HepG2 cells respectively. In microsomes (microsomal fractions) from the osteosarcoma cells these precursors revealed apparent molecular masses of 85, 78, 56, 35 and 31 kDa. When osteosarcoma cells were cultured in the presence of warfarin, vitamin K-dependent 14C-labelling of the 78 kDa precursor was enhanced. Selective 14C-labelling of one precursor was also demonstrated in microsomes from HepG2 cells and from rat lung after warfarin treatment. In HepG2 cells this precursor was identified as the precursor of (clotting) Factor X. This unique 14C-labelling pattern of precursors of vitamin K-dependent proteins in microsomes from different cells and tissues reflects a new mechanism underlying the action of warfarin.

Antidotes↗

PubMed1990

Primary haemostasis, plasmatic coagulation and fibrinolysis in renal transplantation.

The disturbed coagulation state seen in patients with uremia has been suggested to contribute to thrombotic events in kidney grafts following transplantation. Primary haemostasis, plasmatic coagulation and fibrinolysis were investigated in eight patients before and during four weeks after kidney transplantation. In spite of an improved renal function there was postoperatively still a depressed platelet aggregation and the prostacyclin concentrations in plasma were low. The plasma coagulation seemed to be activated according to short activated partial thromboplastin time, high levels of FVIII:C and prothrombin complex. The fibrinolysis was increased and the PAI-1 levels were decreased. It is concluded that the overall haemostatic balance is characterized by a high degree of activation in uremic patients and that this activation persisted four weeks after transplantation.

Blood Coagulation↗

PubMed1990

Purification of branched chain aminotransferase from rat heart mitochondria.

This paper presents the first purification of the branched chain aminotransferase (EC 2.6.1.42) from rat heart mitochondria. The enzyme has been purified from the 100,000 x g supernatant obtained after sonication and ultracentrifugation of rat heart mitochondria. A combination of open column chromatography, high pressure liquid chromatography (HPLC), and discontinuous polyacrylamide disc gel electrophoresis was used. The key step in the procedure was hydrophobic interaction chromatography on HPLC. The final purification step was polyacrylamide disc gel electrophoresis where the enzyme appeared as a doublet. When electroeluted from the gel, each of these bands had the same specific activity demonstrating that there are two forms of the purified enzyme which differ slightly in electrical charge. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these two enzyme forms appeared as a single band with a molecular mass of 43 kDa. Size exclusion chromatography on Sephacryl S-100 identified the enzyme as a 50-kDa protein. These experiments argue against the existence of a dimeric form of this enzyme. The ratio of enzyme activity with leucine (0.84), valine (0.88), or glutamate (0.66) as amino acid substrate versus isoleucine remained constant throughout the purification procedure. Specific activity of the final preparation was 66 units/mg of enzyme protein. Polyclonal antibodies against the purified enzyme were raised in rabbits. On an immunoblot the antiserum recognized a 43-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate but did not recognize any proteins in rat brain cytosol. Quantitative immunodot assay resulted in an estimated enzyme content of about 100 micrograms of branched chain aminotransferase protein/g of heart, wet weight. Finally, 97% of the heart branched chain aminotransferase activity could be neutralized by the antiserum, but the antiserum would not neutralize aminotransferase activity in brain cytosol. These data suggest that close sequence homology does not exist between the two proteins.

Animals↗

PubMed1990

Increased neutrophil elastase release in unstable angina pectoris and acute myocardial infarction.

Neutrophils, a source of proteolytic enzymes and oxygen free radicals, have been shown to participate in animal models of myocardial ischemic injury. To characterize neutrophil activation in human ischemic heart disease, a specific neutrophil elastase-derived fibrinopeptide in plasma was measured in 25 patients with stable angina pectoris, 29 patients with unstable angina pectoris, 17 patients with acute myocardial infarction and 22 control subjects. Mean plasma levels (+/- standard error) of a neutrophil elastase-derived fibrinopeptide (B beta 30-43) measured by a specific radioimmunoassay were fivefold higher in patients with acute myocardial infarction (877 +/- 337 pmol/liter, p less than 0.02) and 13-fold higher in patients with unstable angina (2,277 +/- 613 pmol/liter, p less than 0.006) as compared with control subjects (172 +/- 74 pmol/liter). Mean plasma levels of peptide B beta 30-43 in patients with stable angina (676 +/- 334 pmol/liter), although higher than in control subjects, were not significantly increased (p = 0.64). Total leukocyte counts were 11.0 +/- 0.6 x 10(6)/ml in those with acute myocardial infarction, 9.2 +/- 0.7 x 10(6)/ml in those with unstable angina, 7.1 +/- 0.3 x 10(6)/ml in those with stable angina and 7.7 +/- 0.4 x 10(6)/ml in control subjects. Although total leukocyte counts in patients with unstable angina pectoris and acute myocardial infarction were higher (p less than 0.01) than in patients with stable angina or in control subjects, elevations in peptide B beta 30-43 levels were independent of the differences in both leukocyte count and absolute neutrophil count as well as in history of smoking, hypertension, diabetes mellitus or treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Angina Pectoris↗

PubMed1990

Effects of a new fluid fish oil concentrate, ESKIMO-3, on triglycerides, cholesterol, fibrinogen and blood pressure.

Eskimos have a very low incidence of cardiovascular disease, at least in part due to a high intake of n-3 fatty acids. ESKIMO-3 is a new stabilized (insensitive to oxidation) fluid fish oil concentrate, 30 ml of which contains an amount of eicosapentaenoic acid and total n-3 fatty acids equivalent to the daily intake among Eskimos. Thirty-three volunteers, healthy or with coronary artery disease, were given ESKIMO-3, at a dose of 15 or 30 ml d-1, corresponding to 2.7 or 5.4 g of eicosapentaenoic acid d-1, or placebo oil, for a period of up to 6 months. ESKIMO-3 had a pronounced dose-dependent effect on several risk factors for coronary artery disease. Intake of one tablespoon (15 ml) daily for 6 months significantly reduced levels of triglycerides (-64%), total cholesterol (-8%), plasma fibrinogen (-23%) and diastolic blood pressure (-9%). Bleeding time was unchanged. Intake of two tablespoons daily for 4 weeks increased plasma eicosapentaenoic acid levels by 490%, and decreased arachidonic acid by 20%. The HDL concentration increased by 21%. No change in the above mentioned variables was observed after intake of placebo oil.

Adult↗

PubMed1989

Synthesis of vitamin K-dependent proteins by cultured human tumor cells.

The observation that warfarin inhibits the growth and metastasis of certain types of clinical and experimental tumors suggests a role for vitamin K in tumor biology. We have investigated synthesis of vitamin K-dependent proteins in four malignant (lung epidermoid carcinoma, melanoma, colon adenocarcinoma, and breast adenocarcinoma) and three normal (colon epithelium, breast epithelium, and fibroblasts) cell lines of human origin grown in tissue cultures. Our results show the following: 1) Vitamin K-dependent carboxylase activity is present in all of the malignant and normal cell lines studied. 2) The malignant as well as normal cell lines synthesize a family of vitamin K-dependent proteins. Microsomal precursors of these proteins with apparent molecular mass of 74, 62, and 34 kDa are common to all malignant and normal cell lines whereas precursors of higher and lower molecular mass seem to be synthesized by some but not all tumor cell lines. 3) The 74 kDa precursor synthesized by colon carcinoma and breast carcinoma was positively identified as a precursor of protein S.

Carbon-Carbon Ligases↗