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Capillary electrophoresis/electrospray ion trap mass spectrometry for the analysis of negatively charged derivatized and underivatized glycans.

The increasing interest in the development of glycoproteins for therapeutic purposes has created a greater demand for methods to characterize the sugar moieties bound to them. Traditionally, released carbohydrates are derivatized using such methods as permethylation or fluorescent tagging prior to analysis by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or direct infusion mass spectrometry. However, little research has been performed using CE with on-line mass spectrometry (MS) detection. The CE separation of neutral oligosaccharides requires the covalent attachment of a charged species for electrophoretic migration. Among charged labels which have shown promise in assisting CE and HPLC separation is the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). This report describes the qualitative profiling of charged ANTS-derivatized and underivatized complex glycans by CE with on-line electrospray ion trap mass spectrometry. Several neutral standard glycans including a maltooligosaccharide ladder were derivatized with ANTS and subjected to CE/UV and CE/MS using low pH buffers consisting of citric and 6-aminocaproic acid salts. The ANTS-derivatized species were detected as negative ions, and multiple stage MS analysis provided valuable structural information. Fragment ions were easily identified, showing promise for the identification of unknowns. N-Linked glycans released from bovine fetuin were used to demonstrate the applicability of ANTS derivatization followed by CE/MS for the analysis of negatively charged glycans. Analyses were performed on both underivatized and ANTS-derivatized species, and sialylated glycans were separated and detected in both forms. The ability of the ion trap mass spectrometer to perform multiple stage analysis was exploited, with MS5 information obtained on selected glycans. This technique presents a complementary method to existing methodologies for the profiling of glycan mixtures.

Electrophoresis, Capillary↗

Successful management of visceral Klippel-Trenaunay-Weber syndrome with the antifibrinolytic agent tranexamic acid (cyclocapron): a case report.

Klippel-Trenaunay-Weber syndrome (KTWS) is a rare, congenital disorder characterized by vascular nevus formation, deep venous thrombosis, varicosities, and hypertrophy of affected tissues. A patient with known thrombosis of his splanchnic circulation and visceral KTWS presented with life-threatening hemorrhage from rectosigmoid varices. Portosystemic shunting was not feasible. Endoscopic sclerosis, variceal ligation, and proctocolectomy were not possible due to the size and number of the varices. Previous treatment with epsilon-aminocaproic acid had been unsuccessful and complicated by thrombophlebitis. Conservative treatment with blood transfusions, cryoprecipitate, fresh frozen plasma, vitamin K, propanolol, and somatostatin analog failed to stop the bleeding. The patient was given the antifibrinolytic agent, tranexamic acid, with cessation of his hemorrhage. Serial thromboelastograms confirmed improved reaction time, coagulation time, clot formation rate, and maximum amplitude. We conclude that tranexamic acid may be a useful adjunct in the medical treatment of high-risk patients with KTWS and other vascular nevi complicated by coagulopathy.

Adult↗

Case report: intestinal bypass and severe coagulopathy.

A 29-year-old woman, two years post-ileojejunal bypass, developed a serious coagulopathy. Studies utilizing heparin, epsilon-aminocaproic acid, and plasma, in vivo, indicated that the coagulation abnormalities were multiple in origin. Correction of most of the abnormalities permitted revision of bypass which led rapidly to total permanent correction of the coagulopathy.

Adult↗

Increased fibrinolysis and amyloidosis.

We studied three patients with amyloidosis who had a significant hemorrhagic disorder secondary to increased fibrinolysis. In two of the three patients, bleeding was one of the major initial problems; in the other patient, bleeding occurred later in the course of the disease. All three patients were men with primary or myeloma-associated amyloidosis (ie, immunoglobulin-related or AL). None of the patients had significantly impaired liver function at the time of the diagnosis of increased fibrinolysis. At admission, fibrinogen levels were 60, 170, and 90 mg/dL in the three patients. All three patients had short euglobulin clot lysis times (less than 30 minutes) and evidence of in vitro clot lysis. One patient appeared to respond to aminocaproic acid (EACA). In patients with amyloidosis and bleeding, fibrinolytic tests should be done; in patients with increased fibrinolysis, a diagnosis of amyloidosis should be considered.

Adult↗

Apolipoprotein(a): expression and characterization of a recombinant form of the protein in mammalian cells.

We have stably expressed a recombinant form of apo(a) in a human embryonic kidney cell line. The engineered protein (predicted mass of 250 kDa) contains 17 copies of the apo(a) domain, which resembles kringle 4 of plasminogen, followed by the plasminogen-like kringle 5 and protease-like domain of apo(a). The recombinant protein [r-apo(a)] was isolated from cell culture media by immunoaffinity chromatography, and its physical properties were studied. As is the case for apo(a) isolated from plasma-derived Lp(a), r-apo(a) is highly glycosylated (23% by weight), containing both N- and O-linked glycans, which results in an observed molecular mass of 500 kDa by SDS-PAGE. The high sialic acid content was reflected in a pI of 4.3 for the r-apo(a). Two subpopulations of r-apo(a) secreted by the permanent cell line were identified with respect to lysine-Sepharose binding; the majority of the r-apo(a) bound specifically to this matrix and was eluted with epsilon-aminocaproic acid (epsilon-ACA). When the r-apo(a) plasmid was used to transfect a human hepatoma cell line, lipoprotein particles were secreted containing the disulfide-linked complex of apoB-100 and the r-apo(a). The density of these particles was shown to be heterogeneous, with the majority of the r-Lp(a) floating in the density range of plasma-derived Lp(a).

Apolipoproteins↗

Induction of the generalized Shwartzman reaction in pregnant and nonpregnant rats by colchicine.

The intravenous injection of colchicine (2 mg/kg body weight) into pregnant rats on the last 4 to 5 days of gestation induced disseminated intravascular coagulation, occluding glomerular capillaries with fibrin thrombi, typical of the generalized Shwartzman reaction. Thrombi did not form earlier than 9 hours after the injection of colchicine, whereas in the endotoxin-induced generalized Shwartzman reaction, thrombi were already observed 2(1/2) hours after the injection of endotoxin. The colchicine-induced generalized Shwartzman reaction could also be produced in hysterectomized "pregnant" rats. A single injection of colchicine into nonpregnant rats did not induce disseminated intravascular coagulation. If, however, fibrinolysis was inhibited with epsilon-aminocaproic acid, the colchicine-induced generalized Shwartzman reaction could also be elicited in nonpregnant rats. In this regard fibrinolysis inhibition represents one mechanism by which pregnancy prepares for the generalized Shwartzman reaction.

Aminocaproates↗

A new concept in the control of acute gingival hemorrhage.

The topical application of epsilon-aminocaproic acid may be life-saving therapy for control of acute gingival hemorrhage in patients who are debilitated because of systemic diseases. When low doses were used, there have been no reports of adverse reactions.

Administration, Topical↗

Studies on lectins. XLIX. The use of glycosyl derivatives of Dextran T-500 for affinity electrophoresis of lectins.

p-Aminophenyl glycosides and glycosylamines were coupled to periodate oxidized Dextran T-500 either directly or through an epsilon-aminocaproic acid spacer. The new glycosylated derivatives of dextran specifically precipitate lectins having the appropriate carbohydrate specificity, and thus were used in the preparation of affinity gels for affinity electrophoresis of lectins. The apparent strength of interaction of several lectins with carbohydrate residues immobilized in this way was less than with carbohydrates immobilized in O-glycosyl polyacrylamide copolymers. The presence of epsilon-aminocaproic spacer had no effect on the strength of interaction. The advantages of this type of macromolecular derivative of the ligand for affinity electrophoresis and some differences between the glycosylated dextrans and O-glycosyl polyacrylamide copolymers are discussed. Dextrans containing bound p-aminophenyl alpha-D-mannopyranoside and p-aminophenyl alpha-D-glucopyranoside were used to study the binding properties of concanavalin A and the lectin from Lathyrus sativus seeds. For the investigation of interaction of lectins from Ricinus communis and Glycine soja seeds, dextran derivatives containing bound p-aminophenyl alpha- and beta-D-galactopyranosides and alpha- and beta-D-galactopyranosylamines were used.

Binding Sites↗

Chylothorax after heart/lung transplantation.

Chylothorax is a potentially serious complication of lung and heart-lung transplantation. This article describes the clinical course of chylothorax in 3 heart-lung allograft recipients. We discuss management options, including dietary modifications, octreotide infusion, thoracic duct ligation and embolization, and surgical pleurodesis. In addition, we describe the novel use of aminocaproic acid to reduce lymph flow. We propose a multidisciplinary approach for the management of chylothorax that includes both medical and surgical options.

Adult↗

Use of structural alterations in the synthesis of halothane metabolite antigens to mimic halothane-induced immunogen.

Four hapten-carrier conjugates were synthesized to evaluate any potential antigenic similarities between these synthetic compounds and the immunogens induced in vivo by the anesthetic, halothane and, thus, be used eventually as a more sensitive probe to detect the presence of these halothane-induced antibodies in halothane-exposed individuals. In this study, antibodies from five halothane hepatitis patients were used to evaluate these antigenic alterations since the specificity of these antibodies would most accurately reflect the antigenic structure of halothane-induced immunogens. Quantitation of antibody binding to these synthetic proteins was determined in an enzyme linked immunosorbent assay and immunoblot techniques. Trifluoroacetylated rabbit serum albumin was 5 times more reactive with these antibodies and thus more antigenic than the homologous acetylated moiety confirming the importance of the trifluoromethyl moiety as an epitope in the immunogen in vivo. Insertion of a spacer arm, aminocaproic acid, between the hapten and carrier moieties and an epitope density of 40% acetylation also increased antigenicity. Through these structural alterations produced in vitro, antigenic compounds have been produced which may resemble more closely the immunogen elicited in vivo and which may ultimately serve as more sensitive probes for halothane-induced antibodies from exposed individuals.

Acetates↗

The thrombopenic effect of ellagic acid in the rat. Another model of platelet stimulation "in vivo".

In the rat, intravenous injection of large doses (30 mg/Kg) of ellagic acid (EA) induced a decrease in the plasma level of fibrinogen and in the blood platelet content and an increase of the activated partial thromboplastin time. The long-lasting thrombocytopenia was inhibited by heparin (4 mg/Kg), defibrase (20 U/Kg), clocoumarol (4 mg/Kg) and CCI 17810 (120 mg/Kg). It was not inhibited by aspirin (90 mg/Kg), indomethacin (8 mg/Kg), ketoprofen (4-10 mg/Kg), epsilon-aminocaproic acid (150 mg/Kg), methysergide (4 mg/Kg), chlorpromazine (10 mg/Kg) and promethazine (4 mg/Kg). On the contrary, the small doses of indomethacin (4 mg/Kg) and of ketoprofen (0.5-2 mg/Kg) increased the thrombopenic effect of EA. EA induced the accumulation of Cr51-labelled platelets into the lungs and the liver, accompanied by a 64% fall in Cr51 blood radioactivity. The platelet stimulating effect of EA would depend on an intravascular coagulation which occurs in the whole cardiovascular system. It is suggested that the pro-aggregating derivatives of arachidonic acid had a minor role in this stimulation. The intravascular coagulation induced by EA was accompanied by a swelling of the lymph nodes and of the spleen. In immune platelet depleted rats, EA induced only the swelling of lymph nodes.

Animals↗

Plasmin-mediated proteolysis of casein in bovine milk.

Plasminogen was found to be present in bovine milk by crossreactivity between rabbit antiserum to plasminogen and casein prepared from milk by acid precipitation. This result was further supported by recovery of intact 125I-labeled plasminogen from rabbit milk after its intravenous injection. Freshly isolated whole bovine casein was observed to undergo slow autoproteolysis at 37 degrees C. Polyacrylamide gel electrophoresis revealed gradual disappearance of major caseins accompanied by appearance and increase in intensity of numerous electrophoretic bands. This autoproteolysis was inhibited by low concentrations of epsilon-aminocaproic acid (0.1 mM) and diisopropyl fluorophosphate (1 mM); catalytic amounts of urokinase accelerated the process. Autoproteolysis of isolated bovine beta-casein was shown by both urea and sodium dodecyl sulfate gel electrophoresis to result in formation of gamma 1- and gamma 2-caseins. Similar electrophoretic bands were formed when beta-casein was degraded by plasmin prepared from bovine blood serum. These results support the hypothesis that bovine plasmin occurs in milk and is identical to alkaline milk protease.

Animals↗

Rat adjuvant arthritis as a model to test potential antirheumatic agents.

Adjuvant arthritis was induced in male Sprague-Dawley rats by a subcutaneous injection of Mycobacterium tuberculosis in sterile paraffine oil into the base of the tail. The effects of the immunomodulating drugs N-(2-carboxyphenyl)-4-chloroanthranilic acid disodium (CCA), d-penicillamine and thiabendazole were compared to those of the anti-inflammatory drugs indomethacin, tolfenamic acid and prednisolone. All the anti-inflammatory drugs were highly protective. Of the immunomodulating drugs thiabendazole also showed dose-related inhibitory effect. CCA had only a negligible effect and d-penicillamine enhanced the activity of the arthritic syndrome. An aggravation of the arthritis also occurred after pretreatment with d-penicillamine for 30 days before the induction of arthritis. The complement inhibiting agents epsilon aminocaproic acid and heparin enhanced the symptoms of the adjuvant arthritis as did d-penicillamine. We suggest that the effects of the immunomodulating drugs are dependent on the dose used and the stage of the disease at the beginning of treatment.

Animals↗

Characterization of hemorrhagic principles from Trimeresurus gramineus snake venom.

In addition to alpha-fibrinogenase (hemorrhagin I, HR1), a potent hemorrhagic principle (hemorrhagin II, HR2) was purified from Trimeresurus gramineus venom. It was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. HR2 was a single peptide chain containing 10% carbohydrate with a molecular weight of 81,500. It possessed 669 amino acid residues per molecule, while HR1 contained only 203 amino acid residues per molecule with a molecular weight of 23,500. Both hemorrhagins possessed proteolytic activities toward fibrinogen, casein and azocoll. However, the proteolytic activities of HR1 were much more potent than those of HR2. They were devoid of TAME-esterase and phospholipase A2 activities which were found in crude venom. beta-Mercaptoethanol and antivenin completely inhibited the hemorrhagic activities of HR1 and HR2, while epsilon-aminocaproic acid, trasylol, p-bromophenacyl bromide, phenylmethanesulfonyl fluoride and soybean trypsin inhibitor did not. EDTA completely inhibited the hemorrhagic, fibrinogenolytic and caseinolytic activities of HR1. EDTA also completely inhibited the caseinolytic and fibrinogenolytic activities of HR2, but only partially inhibited its hemorrhagic activity. Subsequent addition of Zn2+ (5 mM) reversed the EDTA-induced inhibitory effect on the hemorrhagic activity of HR1. However, ZN2+ did not reverse the EDTA-induced inhibitory effect on the HR2-induced hemorrhagic activity. These hemorrhagins were found to be ZN2+-containing metalloproteinases. Therefore, the hemorrhagic activity of HR1 seems to be related to its proteolytic activity while that of HR2 seems to be unrelated to its proteolytic activity.

Animals↗

Photodynamic crosslinking of proteins. I. Model studies using histidine- and lysine-containing N-(2-hydroxypropyl)methacrylamide copolymers.

One of the mechanisms by which cells might be damaged during the photodynamic therapy (PDT) of tumors is via the covalent crosslinking of proteins to proteins or to other molecules in the cell. It has been suggested that photodynamically generated singlet oxygen interacts with photo-oxidizable amino acid residues such as His, Cys, Trp and Tyr in one protein molecule to generate reactive species, which in turn interact non-photochemically with residues of these types or with free amino groups in another protein molecule to form a crosslink. In some cases, photochemically generated free radicals may be involved in crosslinking. This paper describes studies on the use of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing epsilon-aminocaproic acid side chains terminating in His (P-Acap-His) or Lys (P-Acap-Lys) as models for the photodynamic crosslinking of proteins. The model copolymer P-Acap-His had a weight-averaged molecular weight of about 22,000 and contained four to five His residues per copolymer molecule. The model copolymer P-Acap-Lys had a weight average molecular weight of about 18,000 and contained four to five Lys residues per copolymer molecule. The extent of photocrosslinking, as sensitized by rose bengal, was estimated by measuring the increase in the viscosity of model copolymer solution after various periods of illumination. The extent of intermolecular crosslinking was estimated from the changes in molecular weight distribution of samples before and at the end of illumination as determined by size exclusion chromatography. Photodynamic crosslinking occurred between P-Acap-His molecules and between P-Acap-His and P-Acap-Lys molecules. The higher the concentration of macromolecules in the solution, the higher is the yield of intermolecular crosslinking. Oxygen was necessary for crosslinking, and azide inhibition studies indicated the involvement of singlet oxygen.

Fluorescent Dyes↗

Glomerular fibrinolytic activity after thrombin perfusion in the rat.

The effects of thrombin perfusion on glomerular fibrinolytic activity (GFA) have been assessed quantitatively in isolated rat glomeruli. Gfa was augmented by increasing total thrombin perfused to a maximal dose of 120 units per 100 gm. Fifty per cent maximal GFA occurred at a dose of 38 to 42 units of thrombin per 100 gm. independent of duration or rate of perfusion. After termination of infusion, GFA diminished and thrombi were no longer observed. Simultaneous administration of thrombin and epsilon-aminocaproic acid was associated with sustained high GFA and persistence of thrombi, presumably due to inhibition of the action of plasmin on fibrin, but it had no effect on tissue activators of plasminogen.

Aminocaproates↗

Phagocytosis and solubilization of fixed cells by metastatic hamster embryo fibroblasts, Nil2C2.

When Nil2C2, a metastatic clone derived from hamster embryo fibroblasts (Nil), was inoculated over [3H]leucine-labeled fixed cells, Nil2C2 cells solubilized and phagocytosed fixed cells, and the radioactivity was released into the culture medium as trichloroacetic acid-soluble fragments. The solubilization of fixed cells was dependent on both the time of incubation of living cells with fixed cells and the number of living cells inoculated. Nil2C2 cells were shown by autoradiographic and electron microscopic studies to peel off fixed cells and ingest them as large fragments. The solubilization of fixed cells was significantly decreased when plasminogen was depleted from the culture medium. Protease inhibitors such as leupeptin, epsilon-aminocaproic acid, and soybean trypsin inhibitor partially inhibited the proteolysis and phagocytosis of Nil2C2 cells. Mouse peritoneal macrophages activated by Salmonella typhimurium solubilized fixed cells after the addition of 12-O-tetradecanoylphorbol-13-acetate. However, they did not phagocytose fixed cells as large fragments.

Animals↗

Antibody-induced conformational restriction as basis for new separation-free enzyme immunoassay.

Under acid denaturing conditions, hologlucose oxidase labeled with 2,4-dinitrophenyl (DNP) was dissociated into flavin adenine dinucleotide (FAD) and DNP-labeled apoglucose oxidase (DNP-AG). Both lacked catalytic activity. The activity was restored by combining FAD and DNP-AG at about pH 7. If, on the other hand, anti-DNP serum was preincubated with the DNP-AG prior to the addition of FAD, activity was not restored. Furthermore, added DNP-aminocaproic acid counteracted the effects of the antibody in inhibiting the recombining of DNP-AG and FAD to form active enzyme. The anti-DNP serum probably prevented the DNP-AG from combining with FAD to form an active holoenzyme by restricting the mobility of the polypeptide chain of DNP-AG from folding into a catalytically active conformation. Based on such an antibody-induced conformational restriction of the DNP-AG, we developed a separation-free (homogeneous) enzyme immunoassay called AICREIA.

Apoenzymes↗