bluuebunny/crossref_metadata_2025_split huggingface dataset:
POINT-MUTATION OF FACTOR VIII CODING SEQUENCES IN HAEMOPHILIA A
Authors: Matthews R J , Peake I R , Bloom A L 
Published: August 2018

In order to study the molecular basis of haemophilia A, DNA from 26 haemophilia A patients (8 severe with inhibitors, 13 severe noninhibitors and 5 mild/moderate) was screened by the Southern blotting method with FVIII cDNA probe A (a i.7kb Kpnl cDNA fragment that spans exons 1 to 12) probe B (a 4.7kb EcoRI cDNA fragment that contains exons 14 to 25 and part of exon 26) probe C (a 1.8kb EcoRI cDNA fragment that contains the remainder of exon 26 and probe D (an Apal/EcoRI 783bp cDNA fragment that includes all of exons 22 to 25 and parts of exons 21 and 26). All cDNA probes were kindly provided by Genetics Institutes Inc.No large structural alterations of the FVIII gene were detected in any of the patients. However altered TaqI restriction sites within the coding regions of 3 patients were observed. DNA from patient 1 with severe haemophilia (VIIIAg < 0.1 u/dl, inhibitor negative) when probed with probe C showed a substitution of the normal 2.6kb TaqI and 4.5kb EcoRI fragments with novel 12kb TaqI and 11.5kb EcoRI fragments respectively. In addition he showed the normal 4kb Bglll fragment with probe C. A point mutation or small deletion (50bp is suspected to be present within exon 26.Patient 2 had severe haemophilia and a FVIII inhibitor of 12 units (Bethesda). DNA from patient 2 when probed with probe B revealed a novel 5.0kb TaqI fragment instead of the normal 2.2kb and 2.8kb fragments.The location of the altered Taq I restriction site within the coding region of exon 18 was confirmed with intragenomic probe pi 14.12 that includes exons 17and 18 (kindly provided by Genentech Inc.) A family study with this mutation specific fragment showed the patients sister and mother to be carriers.DNA from Patient 3 (severe haemophilia, factor VIII inhibitor 33 units) when probed independently with probes B and D revealed the absence of the normal 2.4kb and 1.4kb TaqI fragments and the generation of a novel 3.8kb TaqI fragment suggesting an alteration of the TaqI site within the coding region of exon 23.The detection of altered TaqI restriction sites in 3of our patients is further evidence that 'CG' dinucleotide sequences might be relative hot-spots for mutation when occurring within coding sequences of genes.
COMPOSITION AND ANTICOAGULANT PROPERTIES OF GLYCOSAMI-NOGLYCANS EXTRACTED FROM NORMAL AND VARICOSE VEINS AND FROM AORTIC WALLS
Authors: Capel P , Vichhi M , Janssens A , Mandelbaum I , Barroy J P , Fondu P 
Published: August 2018

We studied the concentrations of total and individual gluco-saminoglycans (GAG) extracted from different vessel walls, and their anticoagulant effects on APTT and thrombin time (TT of normal plasma. The vessels considered were varicose veins, veins presumably normal (autologus saphene crosses, various veins taken during autopsy), and aortas (autopsy).The results (mean, sd) are presented in the Table.No important differences were observed between normal veins from different localization, even in veins presenting higher risk of thrombosis like the veins of the lower limits.It is concluded that there is a marked difference in the composition and anticoagulant properties of aortic GAG as compared with venous GAG. Varicose veins showed higher GAG content and stronger anticoagulant properties as compared with normal veins. This latter observation may be a reflect of defense mechanism against thrombotic stress of the higher pressure present in these abnormal veins.
THE EFECT OF CMTI-I INHIBITOR ON HUMAN BLOOD CLOTTING SYSTEM
Authors: Furmaniak-Kazmierczak E , Jagielski J , Wilusz T 
Published: August 2018

Polipeptyde inhibitors for different serine proteases have been isolated from a variety of plants. Among them there are the inhibitors from squash seeds of molecular weight about 3 300 /1/. The experiments were carried out to determine the effect of one of the squash inhibitors /CMTI-I/ on human blood clotting system. The 0.1 ml of inhibitor /O,8-100 ug/ was added to 0,1 ml of normal intact plasma and incubated 0,5, 15, 30 and 60 minutes at 37°C. It was found that partial tromboplastin time /PTT/ and activated partial thromboplastin time /APTT/ were prolonged. CMTI-I did not show a progressive mode of action upon prolonged time of incubation. There was no effect of CMTI-I on prothrombin time /PT/, thrombin time /TT/ and Stypven-cephalin time /ScT/. The influence of CMTI-I on APTT of factor-XII and factor-XI deficient plasmas as well as on a plasma without factor-XII and factor-XI /exhausted plasma/ was studied. The APTTs of the factor-XII and factor-XI deficient plasmas were prolonged while the APTT of the "exhausted plasma" was unchanged. The performed experiments shown that CMTI-I inhibitor blocks the clot-promoting activity of contact activated plasma. This inhibitory action is stronger in the case of factor XI than of factor XII.1. Wieczorek M., et al., 1985, Biochem. Biophys. Res. Commun. 126:646-652.
EFFECT OF A FIBRIN-DERIVED PEPTIDE ON PULMONARY ANGIOTENSIN CONVERTING ENZYME (ACE) ACTIVITY AND ON PRESSURE RESPONSES TO BRADYKININ (BK)
Authors: Saldeen K , Moalli R , Saldeen T 
Published: August 2018

Decreased ACE activity is a common finding in patients with adult respiratory distress syndrome (ARDS) and in animal models of lung injury. The nature of this effect is unknown. Fibrin, also a common finding in lung injury, is degraded to small peptides by proteolytic enzymes. Peptide 6A, corresponding to amino acid residues 43-47 of the BB-chain of fibrin (cgen), is produced by plasmin degradation of fibrin and has been shown to inhibit ACE in vitro. The purpose of the present investigation was to study whether peptide 6A inhibits pulmonary ACE in vivo and, if so, determine its effect of hemodynamic changes induced by bradykinin (BK) in the rabbit. We investigated the effect of peptide 6A an the hydrolysis of a synthetic ACE substrate, benzoyl-phe-ala-pro (BPAP) in anesthetized rabbits and in isolated lungs. Peptide 6A caused a reversible, dose-dependent inhibition of BPAP hydrolysis. The ID 50 for peptide 6A inhibition of ACE hydrolysis of BPAP was approximately 1 micrcmole.In both isolated rabbit lupgp and in the intact animal, BK injection elicited a dose dependent increase in pulmonary arterial pressure. In intact animals, this was accompanied by a dose dependent decrease in systemic arterial pressure.In both preparations, responses to BK were potentiated by addition of 1 micromole of peptide 6A. In isolated lungs, co-injection of peptide 6A significantly increased the pulmonary artery pressure response to every dose of BK except the highest (1 mg). In the anesthetized rabbit, 1 micromole of peptide 6A significantly (p<0.05) increased the pulmonary hypertensive response to 100 and 200 nanograms of BK and significantly (p<0.05) decreased the systemic hypotensive response following 100 , 200 , 300 and 400 nanograms of BK. The amount of BK needed to increase pulmonary arterial pressure was 1000-fold greater in the isolated lungs than in the intact animals. Peptides of this type might contribute to decreased ACE activity in patients with ARDS and may potentiate BK induced pulmonary hypertension and systemic hypotension in these patients.
INDUCTION OF ANTITHROMBIN III (AT III) ANTIBODIES BY IMMUNIZATION WITH SYNTHETIC PEPTIDES
Authors: Stüber W , Pelzer H , Heimburger N 
Published: August 2018

The primary structure of AT III was examined in respect of potential antigenic sites. The topics were the determination of the hydrophilicity, hydropathy, acrophilicity and the propensities for alpha-helices, B-turns and 13-sheets. The peptides AT III 21-34, 21-42, 129 - 140, 226 - 240 and 343 -363 were synthesized using the solid phase peptide synthesis methode. The subsequent purification of the crude peptides was achieved by h.p.I.e. or by ion exchange chromatography. The peptides were coupled to keyhole limpet hemocyanine (KLH) via thioether bonds. Antisera against KLH-peptides were raised in rabbits (n = 25) and tested with AT III-coated polystyrene tubes; bound antibodies were detected with anti-rabbit-IgG-peroxidase. Obtained antisera were further purified by immuno-adsorption using immobilized AT III. Polystyrene tubes were coated with purified peptide antibodies and binding of AT III was studied with enzyme immunoassay technique (EIA) using anti-AT III-peroxidase.As a result, immunoreactivity of rabbit antisera against synthetic peptides of AT III could be achieved. The obtained antibodies against the individual synthetic peptides as well as their mixtures exhibited specific binding to AT III when tested with EIA.
PRODUCTION AND CHARACTERISATION OF A MONOCLONAL ANTIBODY AGAINST HUMAN ANTI-THROMBIN III
Authors: Rathjen Deborah A , Geczy Carolyn L 
Published: August 2018

To study the role of anticoagulants, particularly antithrombin III (AT III) and heparin, on the activation of coagulation by monocytes/macrophages which have been stimulated with a soluble lymphocyte activation product, macrophage procoagulant inducing factor, we have prepared monoclonal antibodies (MAbs) to human AT III.In fusion experiments, in contrast to wells containing peritoneal feeder cells, positive hybrids were only found in wells containing medium conditioned by the macrophage cell line J774 (Rathjen and Geczy, 1986). Of 5 hybrids which initially produced antibody, only one hybrid, showed stable Ab production. The MAb, designated 22/23, was not cross-reactive with either 1 antitrypsin or ovalbumin and did not inhibit the biological activity of AT III in chromogenic assays which measured inhibition of thrombin and Factor Xa by AT III. An immunoadsorbent prepared using MAb 22/23 depleted AT III activity from a purified AT III preparation. Reduction and alkylat ion of the disulphide bonds of the protein portion of AT III completely abbrogated MAb binding indicating that the native configuration of AT III was important. Isoelectric focussing of AT III, followed by transfer of the focussed protein to nitrocellulose by diffusion and probing with MAb 22/23, revealed at least 8 bands in the region of pH 5.2 to 5.85. Coomassie blue staining of a gel run in parallel showed 9 bands in this region. The MAb provides a useful tool for the detection of AT III on both cultured cells (bovine aortic endothelial cells are positive by immunofluorescence) and tissue sections.Rathjen, D.A. and Geczy, C.L. Hybridomo. 5s 255-261 (1986)
COMPARATIVE STUDIES ON ANTITHROMBIN III AFFINITY OF LOW MOLECULAR WEIGHT HEPARIN AND UNFRACTIONATED HEPARIN
Authors: Sakuragawa N , Shimotori T , Takahashi K 
Published: August 2018

Purpose: Low molecular weight(LMW)heparin shows stronger antifactor Xa(F-Xa) and weaker anti-thrombin(TH) activities compared with unfractionated(UF) heparin, and shows less bleeding tendency in the cases of clinical use. These characteristics were surmised to be depend on antithrombin III(AT-III) affinity of the heparin. Materials and methods: LMW heparin(Kabi and Pharmuka), UF heparin (Novo) and heparin cofactor II(HC-II) purified by our method were used. AT-III affinity column chromatography with 0.1 M Tris-buffer (pH 7.4)-NaCl 0.02 to 2.5 M linear gradient was performed. From the point of AT-III affinity strength, non-affinity(Na), low affinity (La) and high affinity(Ha) were separated, and aPTT, anti-F-Xa and anti-TH activities were assayed on each fractions. HC-II was assayed by biological activity.Results: (1) Kabi-LMW heparin; Na 34.5%, La 39.3%, Ha 26.2%, Pharmuka-LMW heparin; Na 58.0%, La 24.1%, Ha 17.3%. Novo; Na 0%, La 50%, Ha 50%. (2) APTT; Na showed no effect, but Ha showed the strongest prolonging effects on aPTTs even having less amount of uronic acid, and more prominent effects were observed in UF(Novo)-heparin than LMW heparins. (3) La showed higher activity of anti-F-Xa and anti-TH activities than Ha. (4) Anti-TH activity of AT-III was observed in both fractions of La and Ha, but that of HC-II was observed in each fractions including Na.Conclusion: It was surmised that the differences of the characteristics between LMW heparin and UF heparin were depend on to the strength of AT-III. The different characteristics of HC-II from AT-III to anti-TH were observed and surmised to be depend on the binding ability to the fractions.
INTERACTION BETWEEN CULTURED ENDOTHELIAL CELLS AND ABNORMAL ANTITHROMBIN III "TOYAMA"
Authors: Sakuragawa N , Saitoh S , Takahashi K 
Published: August 2018

Purpose: Abnormal antithrombin III(AT-III)Toyama showed non-affinity to heparin and heparinoid to show loss of immediate antithrombin activity. On the endothelial cells, there are heparinoids including heparan sulfate. We investigated on the interaction between cultured endothelial cells and abnormal AT-III"Toyama" from the viewpoint of antithrombin activity.Materials and methods: (1) Endothelial cell culture:^125I-labelled normal and abnormal AT-III were placed on the washed endothelial cultured cells in 0.2 ml of RPMI-1640 medium for 15 min at 37°C. The medium was suctioned off and the cell layer was washed with Hank's balanced salt solution. The cells were incubated with 1 ml of heparin(3 ug/ml) for 15 min at 4°C. The radioactivity in the supernatant was counted, and represented AT-III which bound to the cells surface. (2) Antithrombin activity: 0.23 ml of thrombin solution^ U/ml) and 0.03 ml of normal or abnormal AT-III plasma were mixed, and incubated on the cultured cell surface for 5 min at room temperature. The residual thrombin activity was assayed by 0.3 ml of the substrate (S-2238) solution(0.8mM)for 5 min. After these procedures,2 ml of 2% citric acid solution was added to stop the reaction, and 0D(405 nm) was recorded.Results: Abnormal AT-III showed reduced binding-activity to cultured cells to one fifth compared with normal AT-III, and the residual thrombin activity in the abnormal was higher compared with that in normal plasma.Conclusion: Abnormal AT-III showed less binding activity to the cultured endothelial cells, and less thrombin neutralizing activity to show thrombogenic tendency.
ANTITHROMBIN III GENEVA : AN HEREDITARY ABNORMAL ANTITHROMBIN III (AT III) WITH DEFECTIVE HEPARIN COFACTOR ACTIVITY
Authors: de Moerloose Ph , Reber G , Minazio Ph , Bouvier C A 
Published: August 2018

A 43-year old man presented a pulmonary embolism. Despite a negative family history for thromboembolic disorders, the unusual circumstances of apparition and the relatively young age of the patient prompted us to study carefully the coagulation parameters. Routine coagulation tests, as well as plasminogen, alpha-2-anti-plasmin, protein C and protein S were all within normal range. Biological and immunological assays of AT III were performed on 12 members of the family and showed a low AT III activity in the propositus and other members of this family (mean 50%), but normal immunologic levels. Crossed immunoelectrophoresis in absence of heparin showed a normal pattern, but in presence of heparin showed an abnormal peak as compared with controls. Kinetics experiments showed a normal inhibition of Xa and 11a in absence of heparin, but abnormal in presence of heparin. An affinity chromatography on heparin Sepharose revealed two populations of AT III, one of which was devoid of heparin cofactor activity.The toponym AT III Geneva is proposed for this new familial abnormal AT III with defective heparin cofactor activity. This family confirms the low incidence of thromboembolic events reported in this type of AT III variant.
HORMONE REGULATION OF THE FIBRINOLYTIC SYSTEM IN THE OVARY
Authors: Ohlsson M , Hsueh A J W , Ny T 
Published: August 2018

In the ovary, the release of oocytes from graafian follicles during hormone-induced ovulation has been found to be associated with substantial increases in follicular plasminogen activator (PA) activity. Most of the PA activity comes from the granulosa cells that have been shown to produce tPA, uPA as well as the type-1 PA-inhibitor,(PAI-1).We have studied the molecular mechanism of follicle stimulating hormone (FSH) and gonadotropin releasing hormone (GnRH) on the synthesis of tPA in primary cultures of rat granulosa cells. FSH and GnRH were both found to induce tPA in granulosa cells in a time and dose dependent manner. The effect of FSH and GnRH on the levels of tPA mRNA was also studied by northern and slot blot hybridizations. FSH and GnRH were both found to increase the level of tPA mRNA. The stimulation was up to 18 -fold compared to untreated cells.The induction of tPA mRNA by FSH and GnRH was additive and the time courses of the stimulation by the hormones differed, suggesting that different cellular mechanisms are involved. Consistent with the ability of FSH to activate the cAMP dependent protein kinase A pathway, the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine further enhanced the FSH induction of tPA mRNA.GnRH is known to activate the phospholipid-dependent protein kinase C pathway. Likewise the effect of GnRH can be mimicked by the kinase C activator, phorbol myristate acetate.It is concluded that FSH and GnRH regulates tPA production by differnt molecular mechanisms, and that the increase in tPA activity is mediated via an increase in the levels tPA mRNA. Since both gonadotropins and GnRH cause ovulation in hyposectomized animals, similar stimulatory actions of these hormones on the tPA activity suggest a correlative relationship between this enzyme and the ovulatory process.
EXTRAHEPATIC METABOLISM OF RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR (tPA) IN DOGS
Authors: Fong K L L , Boyle K E , Crysler C S , Landi M S , Griffin H E , Lynn R K 
Published: August 2018

Hepatic uptake has been proposed as the major mechanism of tPA clearance from systemic circulation. However, our recent studies demonstrated that tPA was rapidly Inactivated through complexation with protease Inhibitors in dog plasma In vitro, and that tPA-inhibitor complexes were present in plasma of dogs receiving tPA. Therefore, the present work was undertaken to differentiate hepatic from extrahepatlc clearance of tPA. Pharmacokinetics of tPA were determined in anesthetized beagle dogs with either Intact hepatic circulation or with Interrupted hepatic blood flow achieved by hepatic artery ligation and portal caval shunt.Recombinant two-chain tPA was administered as an Intravenous bolus dose (80 μg/kg) and plasma active tPA concentrations were measured using a modified and validated S-2251 chromogenlc assay. Following tPA administration to Intact dogs, plasma active tPA concentration declined blexponentlally with time with 84% of the active tPA eliminated during the α-phase. The t1/2's of the a and β-phase were 1.76 ± 0.74 and 6.23 ± 1.56 min, respectively. The systemic clearance was 25.98 ± 1.13 ml/min/kg and the volume of distribution at steady state (VDss) was 73.9 ± 15.1 ml/kg. Upon the elimination of hepatic blood flow, the systemic clearance was reduced by 54% while VDss was unaffected. The contribution of plasma Inactivation of tPA to the systemic clearance was estimated from in vitro Inactivation studies In 37°C plasma. Based on the pseudo first order Inactivation rate constants of 0.184 min-1 and 0.095 min-1 at Initial tPA concentrations of 25 and 250 IU/ml respectively, clearance rates from 5.02 to 9.2 ml/min/kg were calculated. These data suggest that (1) in Intact dogs, 46% of the tPA clearance occurs extrahepatlcally and (2) Inactivation of tPA in plasma accounts for a major portion of the extrahepatlc clearance.
THE INTERACTION OF ONE AND TWO CHAIN TISSUE-TYPE PLASMINOGEN ACTIVATOR WITH INTACT AND DEGRADED FIBRINOGEN
Authors: Higgins Deborah L 
Published: August 2018

Tissue-type plasminogen activator (t-PA) plays a central role in fibrinolysis in vivo. One-chain recombinant t-PA was isolated using lysine-Sepharose chromatography followed by chromatography on monoclonal antibody column which is specific for one chain t-PA. Two chain t-PA was produced by incubation of one chain t-PA with plasmin-Sepharose. Both forms of t-PA were incubated with plasminogen-free fibrinogen as well as fibrinogen that had been degraded with plasmin until it was -50% clottable. Thrombin was added to catalyze fibrin formation and the amount of free t-PA in the clot supernatants was measured by ELISA. A quantitative analysis to determine the dissociation constant (Kd) and moles of t-PA bound per mole of fibrin monomer (n) was performed using a computer fit of a nonlinear regression analysis. The data obtained gave the following results:Plasmin degradation of the fibrinogen caused the formation of a new set(s) of sites with a significantly lower Kd(s), which caused an increase in binding to degraded fibrin when compared to intact fibrin. ε-amino caprioc acid (ε-ACA) (0.1M) was found to compete for t-PA binding to the loose sites in both intact and degraded fibrin, but not the tight sites formed by degradation. Significantly (>20%) more one chain t-PA than two chain t-PA bound to both intact or degraded fibrin. Differential binding of one and two chain t-PA as well as tighter binding to degraded fibrin may play a significant role in the regulation of fibrinolysis.
t-PA PROTEIN DETERMINATION: IMPROVED ACCURACY THROUGH COMPOSITION CONSIOUS AMINO ACID ANALYSIS (CCAAA)
Authors: Brändström A , Rånby M 
Published: August 2018

Protein determination by composition consious amino acid analysis (CCAAA) is generally applicable to polypeptides with known amino acid composition.Traditionally, in protein determination by AAA the mass of protein substance in a preparation is quantitated by simple summation of the mass of all amino acid residues found, although some amino acids e.g. Trp, Ser, Thr, Cys are unsatisfactorily determined. In this study we have explored the, possibility of improving protein determination by AAA in cases where the amino acid composition of the preparation is precisely known from sequence data. In CCAAA protein determination the molar amounts of amino acids that are known to be quantitatively recovered are divided by the number of residues known to be present in the polypeptide. The mean of these values is identified as the molar quantity of polypeptide in the sample. In addition, the standard deviation of the mean serves as identification and homogeneity control. In this study the accuracy of the method was checked gravimetrically.CCAAA was applied on t-PA prepared from cultured Bowes melanoma cells by immunopurification on PAMl-Sepharose 6BFF and subsequent gelfiltration in 1 mol/L NH4HCO3 on Sephacryl SA-200. About 2 pg of lyophilized protein was hydrolysed in 6 mol/L HC1 under vacuum for 24 hours at 110°C and amino acid analysed. Amino acids selected for calculation of the molar quantity were (number of residues within parentheses) Asp+Asn(50), Glu+Gln(52), Pro(29), Ala(32.5), Val(25), Leu(39), His(16), Lys(21) and Arg(35.5). To obtain the gravimetric quantity the molar quantity was multiplied with the theoretical molecular weight as calculated from the sequence and with the carbohydrate content taken into account.For t-PA the standard deviation of the mean molar quantity typically was less than 5%. The result from CCAAA was about 10% higher than that obtained from conventional AAA. The result from CCAAA was within 10% of that found gravimetrically. The absorbtivity of t-PA was estimated to 1.75 (g/L)-1cm-1.In summary, composition consious amino acid analysis (CCAAA) is recommended as reference method for protein determination of well defined protein preparations where the amino acid composition is known.