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Fucoidan [sulfated poly (L-fucopyranose)] was compared with 6-aminohexanoic acid (6-AH) or CNBr-cleaved fibrinogen (CNBr-Fbg) alone or in combination in enhancing the activation of glutamic plasminogen (Glu-Plg) or lysine plasminogen (Lys-Plg) by two-chain tissue plasminogen activator (t-PA) or LMwt-urokinase or by streptokinase. Fucoidan enhanced the t-PA activation of Glu-Plg or Lys-Plg at Plg concentrations greater than 75nM, while stimulation by CNBr-Fbg of t-PA activation followed saturation kinetics of Michaelis-Menton. During t-PA activation of Glu-Plg, a high degree of synergism was observed between 6-AH and fucoidan while the enhancement by CNBr-Fbg was not influenced by fucoidan and was reversed by 6-AH. Fucoidan alone at higher concentrations was effective in enhancing the activation of Glu-Plg by urokinase while the combination of fucoidan and 6-AH showed additive effect in enhancing the activation of Lys-Plg. The activation of Glu-Plg by streptokinase was reversed by fucoidan in a manner similar to that reported for 6-AH. The results are interpreted to suggest that CNBr-Fbg and 6-AH compete with each other for the same lysine binding sites (LBS) on the Plg molecule while fucoidan acted synergistically with 6-AH in enhancing the t-PA activation of Glu-Plg by a different mechanism. The double reciprocal plot for the interaction of Glu-Plg and urokinase also showed a significantly higher affinity between the two in presence of fucoidan.

The changes in relative fluorescence of antithrombin III (AT-III) during its interaction with sulfated xylans were compared with that of sulfated glycosaminoglycans by measuring the ratio of the increase in fluorescence of AT-III in the presence of sulfated polysaccharide to the fluorescence of AT-III alone for various mass ratios. Interactions of corn cob xylan sulfate (CCXS) and sodium pentosan polysulfate (SP-54) with AT-III resulted in enhancements of relative fluorescence which were lower than commercial heparin. At mass ratios below 1, heparan sulfate and low molecular weight heparin (LMWH) gave increases in the relative fluorescence higher than that of commercial heparin, while highly sulfated semisynthetic chondroitin sulfates A and C gave much smaller increases. The relative fluorescence enhancements of AT-III by heparan sulfate, commercial heparin, LMWH and heparin derived pentasaccharide (HDP) increased with increasing mass ratios while the enhancements by CCXS, SP-54 and the highly sulfated chondroitin sulfates A and C were reversed at higher mass ratios. The estimated dissociation constants (kd) for the interaction of AT-III and the heparin-related compounds showed that heparin sulfate and LMWH gave the lowest kd values indicating a higher affinity for AT-III while commercial heparin and HDP gave higher kd values, indicating a lower affinity for AT-III. SP-54 gave a kd value lower than CCXS, indicating a greater affinity for AT-III. A comparison of the near ultraviolet (UV) circular dichroism (CD) spectrum of AT-III alone and during its interaction with oat spelts xylan sulfate (OSXS) showed enhancements of the two aromatic amino acid regions corresponding to phenylalanine and tryptophan.

Several naturally occurring polysaccharides were purified and subsequently sulfated by chlorosulfonic acid-pyridine complex. These were isolated as the sodium salt and further purified by ECTEOLA cellulose chromatography. Anticoagulant properties of the sulfated polysaccharides were compared with commercial heparin by measuring their in vitro effects on activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using pooled normal human plasma. In general, all the compounds exhibited antithrombic (anti-TT) properties similar to heparin but were less effective than heparin in inhibiting APTT or PT. The in vivo anticoagulant properties were also compared with commercial heparin by injecting rats a single intraperitoneal dose and measuring plasma APTT at 2, 4 and 6 hour intervals. All of the compounds including heparin increased APTT significantly at 2 hours and then gradually returned to near normal value after 6 hours. Larchwood xylan sulfate was almost as active as heparin in inhibiting APTT while the rest of the compounds were less active. Toxicological studies using rats showed wide variations in the LD50 of various compounds. Sulfated xylan and heparin were least toxic while sulfated polysaccharide of locust bean was most toxic. For most of the compounds the LD50 was 5-100 fold higher than the effective dosage.

Kinetic analyses of antithrombin III (AT-III)-thrombin or heparin cofactor II (HC-II)-thrombin or AT-III-factor Xa interactions were carried out in the absence or in the presence of one of the sulfated xylans or unfractionated heparin or low molecular weight (LMW) heparin utilizing chromogenic substrates. These studies demonstrated that under pseudo first order conditions the inhibitions were proportional to the AT-III or HC-II concentrations used and the apparent second order rate constants determined from the slopes of the pseudo first order plots of log of thrombin or Xa remaining as a function of time were significantly elevated in presence of the sulfated compounds. On a molar basis oat spelts xylan sulfate was the most effective compound in accelerating the rate of thrombin-AT-III interaction followed by commercial heparin while the latter was most effective in accelerating the rate of thrombin-HC-II interaction. Heparin and LMW heparin were more effective in that order in accelerating the rate of Xa-AT-III interaction while oat spelts xylan sulfate, corn cob xylan sulfate, SP-54 were less effective than the heparins in that order. Studies were also conducted on the concentrations of the sulfated compounds required to inhibit by 50% the thrombin activity by AT-III or HC-II or that required to inhibit by 50% the factor Xa activity by AT-III. The results showed an inverse relationship between the increase in the rate of acceleration by the sulfated compound with the decrease in the amount required for 50% inhibition. SDS-polyacrylamide gel study of the reaction mixture containing thrombin, AT-III or HC-II along with heparin or oat spelts xylan sulfate showed that like heparin, oat spelts xylan sulfate potentiated the formation of thrombin-AT-III or thrombin-HC-II complexes which were stable in presence of denaturing or reducing agents. Chemical modification of arginine or lysine of AT-III significantly lowered its potentiation of thrombin or Xa inhibition by oat spelts xylan sulfate.

The interactions of fucoidan with glutamic plasminogen (Glu-Plg), two-chain tissue plasminogen activator (t-PA), LMwt-urokinase, thrombin, and antithrombin III (AT-III) were investigated using fucoidan-sepharose affinity chromatography. The results showed 1) a high degree of affinity between fucoidan-sepharose and Glu-Plg; Lmwt-urokinase and thrombin while t-Pa and AT-III did not bind with fucoidan-sepharose. 2) The double reciprocal plot for the LMwt-urokinase activation of Glu-Plg showed that plasminogen activator inhibitor (PAI-1) inhibited this reaction in a noncompetitive manner and that the presence of fucoidan decreased Km for this interaction by 50% and increased Kcat by 30-fold, 3) The double reciprocal plot for the t-PA activation of Glu-Plg showed that PAI-1 inhibited this reaction in a competitive manner and that fucoidan in conjunction with 6-aminohexanoic acid (6-AH) increased Kcat for this interaction by 5-fold without affecting Km. 4) Fucoidan enhanced the interaction of thrombin with both AT-III and heparin cofactor II (HC-II) and it was more effective than unfractionated heparin of LMwt-heparin in enhancing the interaction of HC-II with thrombin.

Oat spelts xylan (OSX), fucoidan, kappa carrageenan and chondroitin sulfates A and C were sulfated using chlorosulfonic acid-pyridine complex while the first three were phosphorylated using methane sulfonic acid-phosphorous pentoxide mixture. The compounds were isolated as the sodium salts and their in vitro anticoagulant properties were determined by measuring the concentration of each compound required to double prothrombin time of pooled normal human plasma. The results of 31P-nmr spectroscopy showed that phosphorylation significantly increased the molecular weights of the polysaccharides by forming phosphodiester and diphosphodiester bonds. In general the anticoagulant properties of the sulfated polysaccharides were related to the % sulfate while the phosphorylated polysaccharides showed increases in anticoagulant properties which were related to the increase in the molecular weight and inversely related to the % phosphate. The mechanism of action of oat spelts xylan phosphate (OSXP) was studied using 125I-thrombin and normal human plasma. The results showed that at lower concentration of the OSXP, the complexation of 125I-thrombin with heparin cofactor-II(HC-II) was enhanced, while at higher concentration of the compound, the complexation with both antithrombin-III(AT-III) and HC-II was enhanced.

Polysaccharides were isolated from Dragon's Blood (DB), a red resinous secretion from the fruits of Daemonorops species and from pollens of Typha Augustata (TA) by alcohol precipitation followed by deaminoethylaminoethyl (DEAE) cellulose chromatography. The purified polysaccharides of DB and TA gave specific rotations of [α]D20 64 and [α]D20 of 80 and their molecular weights by membrane osmometry were ∼25000 and ∼30000 respectively. The effect of addition of the polysaccharides on coagulation of pooled normal human plasma was investigated. They were found to accelerate the recalcified plasma times at concentrations below 100 μg/ml while inhibiting them at higher concentrations. Their procoagulant properties were due to their effects on the activation of Hageman factor (XII) while the anticoagulant effect was mainly directed towards fibrinogen. Studies of the mechanism of the anticoagulant effect showed that addition of TA polysaccharide inhibited the rate of release of fibrinopeptides by thrombin and also the aggregation of fibrin monomers.

A protein inhibitor was isolated from commercial preparations of salmon sperm and its physical and anticoagulant properties were compared with an inhibitor isolated earlier from commercial preparation of bovine testicular hyaluronidase. The inhibitor from bovine source was heat and acid labile and had a molecular weight of approximately equal to 35000 while the one from salmon sperm had a molecular weight of approximately equal to 5700 and was stable to heat and acid. To determine the mechanism of the inhibitory effect, a system of purified components consisting of isolated prothrombin, Factor Xa, Factor Va, Ca++, and vesicles of phosphatidylcholine (PCPS, 25% PS) was used. Included also was dansylarginine N- (3-ethyl-1,5-pentanedidyl) amide (DAPA) which binds newly formed thrombin and yields the time course of prothrombin conversion by virtue of enhanced fluorescence of the DAPA - thrombin complex. The inhibitor of bovine testes was effective only when PCPS was the limiting component suggesting that its action was directed against the phospholipid component of the prothrombinase complex. The inhibitor from salmon sperm was found to lower the rate of conversion of prothrombin to thrombin in an in vitro system where thrombin generation was measured by its action on the chromogenic substrate H-D-Phe-Pip-Arg-pNa (S-2238). It inhibited the conversion of Factor X to Xa and also the the amidolytic cleavage by Factor Xa of chromogenic substrate N-Benz-Ile-Glu-Gly-Arg-pNa (S-2222).

Bovine testicular hyaluronidase from various commercial sources showed the presence of an inhibitor of human plasma prothrombin time (PT). A testicular anticoagulant protein (TAP) was isolated from it by a 3-step procedure. The material was first passed through conconavalin-A-sepharose affinity chromatography where the anticoagulant material was separated from the hyaluronidase and protease which were retained by the column. In the second step the lower molecular weight proteins were removed by ultrafiltration. The supernatant which contained the anticoagulant protein was passed through the carboxymethyl cellulose column and the active material was eluted by 0.4 M NaCl solution. Sodium dodecyl sulfate (SDS) gel electrophoresis gave a molecular weight of approximately 35000. Unlike many small molecular weight proteins from bovine testes, TAP looses its anticoagulant property by heating for 30 minutes at 55 degrees C or by storage at pH 3.0 for 2 hours and it does not inhibit trypsin or thrombin. Its isoelectric pH was 9.7. Diisopropyl fluorophosphate (DFP) treated TAP was not effective as an anticoagulant.

Anticoagulant properties of three sulfated compounds prepared from xylans isolated from corn cobs, larchwood and oatspelts were compared with heparin and sodium pentosan polysulfate (SP-54) by studying their effects on activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using pooled normal human plasma. These compounds were more effective than SP-54 in delaying coagulation by all the three procedures while oatspelts xylan sulfate was as effective as heparin in inhibiting APTT and PT and more effective than heparin in inhibiting TT on a molar basis. The sulfated xylans were more effective than heparin or SP-54 in potentiating the AT-III inhibition of amidolysis of H-D-Phe-Pip-Arg-pNa (S-2238) by thrombin (IIa) or amidolysis of Bz-Ile-Glu-Gly-Arg-pNa (S-2222) by Xa. Study of the high affinity binding of the xylan sulfates to AT-III-Sepharose column showed that the amount of the xylan sulfate recovered in the eluates from this peak was greatly increased with an increase in molecular weight (MW). A buffered mixture of IIa, AT-III and dansylarginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA) was used to study the inactivation of IIa by AT-III. Larchwood xylan sulfate (2-10 micrograms) was found to accelerate this inactivation which was neutralized by human platelet factor 4 (PF4). The results also suggested an interaction between larchwood xylan sulfate and IIa which may potentiate an interaction between AT-III and IIa.

The mechanism of action of glucuronolactone on the clotting of the recalcified human plasma was investigated. Addition of the compound resulted in inhibition of the clotting of both the glass contacted and the non-contact human plasma in siliconized glass tubes. The results therefore suggest that stage(s) after the contact activation phase of the clotting sequence were influence by the inhibition. At pH's 7.4 or below, glucuronolactone is more inhibitory than at pH's above 7.4. The clot- promoting effects of tissue thromboplastin or thrombin or Russell's viper venom is partially reversed by glucuronolactone indicating that more than one reaction step in the clotting sequence were affected by the compound.

1. 1. A p H-activity study was conducted on purified human plasma and bovine pancreatic deoxyribonuclease (DNase) in the presence of Mg 2+ or Zn 2+ . 2. 2. Mg 2+ ions activate the plasma enzyme at a p h near the neutral while they activate the pancreatic enzyme between p h 5 and p H 8–5. 3. 3. The plasma enzyme is not activated by Zn 2+ at any p H, while the pancreatic enzyme is activated by smaller concentrations of Zn 2+ at p H 5-0. 4. 4. Determination of molecular ionization constants of groups on the DNase-DNA complexes from the p H-activities studied indicates the presence of active imidazolium and sulphydryl groups in both the enzyme complexes, while only the pancreatic enzyme complex shows the presence of an aspartyl group.

Purine analogues, 8-azaguanine (2.5 µg/ml); 6-thio- guanine (5 µg/ml); 6-mercaptopurine (50 µg/ml); 6- chloropurine (50 µg/ml); and 2,6-diaminopurine (50 µg/ml); inhibited the thymine growth response of Streptococcus faecalis Akt., a Methotrexate (Lederle trade name for 4-amino-N10-CH3-folic acid) resistant mutant of S. faecalis. However, these same concentrations of the purine analogues did not influence the thymine growth response of the parent strain of S. faecalis. The inhibitory effect of 8-azaguanine or of 6-thioguanine on S. faecalis Akt., was of a competitive nature and was reversed by 5 µg/ml each of adenine or guanine or 5 µg/ml each of the nucleosides or nucleotides of adenine or guanine. Pyrimidine analogs 2-thiouracil (100 µg/ml); dithiothymine (500 µg/ml); and isobarbituric acid (100 µg/ml); inhibited the thymine growth response of S. faecalis Akt., but did not influence the thymine growth response of S. faecalis. The inhibitory effect of isobarbituric acid on the thymine growth response was reversed by cytosine (50 µg/ml); uracil (50 µg/ml); deoxycytidine (50 µg/ml); thymine (50 µg/ml); or thymidine (50 µg/ml). The results suggest that the S. faecalis Akt. has acquired de novo purine and pyrimidine biosynthetic pathways and is therefore inhibited by the analogs at lower concentrations. It may be speculated that clinical resistance to methotrexate may be associated with the acquisition of de novo purine biosynthetic capabilities in methotrexate-resistant cells and this opens a new avenue of attack on methotrexate-resistant leukemia.

Gas chromatography and combined gas chromatography and mass spectrometry have been used to study the distribution of fatty acids and hydrocarbons in the seeds of Plantago ovata . The occurrence in the seed coat of the low molecular weight hydrocarbons (C 16 -C 19 ) with a significant percentage of an even carbon-numbered hydrocarbon (anteiso-C 18 ; 10·1 per cent) were unusual features in relation to earlier reports on plant hydrocarbons. The seed coat of P. ovata showed mainly linoleic, oleic, and palmitic acid in decreasing concentrations while the seed itself did not contain an appreciable amount of fatty acids. The results rule out a simple precursor-product relationship between fatty acids and hydrocarbons as the likely pathway to explain the biogenesis of all the hydrocarbons.

The temperature, time of incubation, concentration of MgCl2 · 6H2O and concentration of serum required for an optimum activity of deoxyribonuclease I (DNase I) of human serum were studied. The final incubation mixture consisted of 2 ml. of serum, 2 mg. deoxyribonucleic acid (DNA), 3 mg. MgCl2 · 6H2O, 0.5 ml. of 0.1 tM tris(hydroxymethyl) aminomethane buffer pH 7.3, and 0.1 ml. toluene in a total volume of 3 ml. After 20 hr. of incubation at 45 °C., determinations of DNase I were made by measuring deoxyribose-purine-containing fragments in the trichloroacetic acid-soluble fraction. Addition of 2 mg. nitrogen mustard or 0.5 ml. leucocyte extract to the incubation mixture inhibited the DNase I activity of the human serum. Enzyme kinetic studies indicate a noncompetitive relationship between DNA and nitrogen mustard [methylbis(β-chloroethyl)amine hydrochloride]for the DNase I of human serum; however, the inhibitor in the leucocyte extracts lowered the human serum DNase I activity in a competitive manner.

The components required for the optimal conversion of PGA to CF by chick embryo homogenate include PGA, serine, homocysteine, DPN and ATP. The chick embryos possess maximum capacity to convert PGA to CF at 2.5 and 6.5 days during the incubation period of 1.5 to 11 days. The conversion of PGA to CF by chick embryo liver homogenate is optimal at 13 and 16 days during 12 to 21 days of incubation. Under the experimental conditions described here, 9000 r X-radiation to 2.5, 6, 7, and 8 days old chick embryos does not alter their capacity to convert PGA to CF.

A single oral dose of 1.5 g/kg of glucuronolactone to rats significantly prolonged for 6 days the whole blood clotting times. Since the anticoagulant action is pronounced in siliconized glass tubes, clotting is inhibited at stage(s) after the contact activation. Administration of 1,4-saccharolactone (a metabolite of D-glucuronolactone) at 1.5 g/kg also exhibited similar anticoagulant properties. Animals receiving glucuronolactone or 1,4-saccharolactone showed no toxic effects viz. loss of body weight or changes in the appearance of the internal organs.

The formation constants for the complexation of alphaketoglutaric or glyoxalic or imidazolypyruvic (IPA) acid with Cu(II) or Ni(II) or Fe(III) or Al(III) were determined in aqueous solutions ( I = 0·1; T = 25°) using a pH-titrimetric technique. The data were treated by a direct algebraic method and by the formation curve procedure. The constants for IPA with Cu(II) or Ni(II) or Fe(III) were calculated by both methods and the values showed good agreement. Divalent cations, e.g. Cu(II) or Ni(II) gave values for the formation constants which were in the following order: IPA > glyoxalic > alphaketoglutaric while the order was glyoxalic > IPA > alphaketoglutaric for the trivalent cations e.g. Al(III) or Fe(III). The nature of the metal ion-acid complexes formed was directly related to the particular acid used. In general, the metal ions tested formed a 1:2 complex with IPA, 1:2 or 1:3 complex with glyoxalic and a 1:1 complex with alphaketoglutaric.

The extraneous band solutions were obtained from oneto two-week-old NaI/ sup 131/ solutions by paper chromatographic separation and elution procedure. Fresh solutions of NaI/sup 131/ which contain over.0% of the radioactivity in the NaI/sup 131/ band were used for studies with Nal/sup 131/. Effect of pH 3.0 on the stability of the NaI/sup 131/ solutions and of the extraneous band solutions was studied. The results show that, at pH 3.0, about 90% of the extraneous band is converted to NaI/sup 131/ band within 10 min. Studies on the uptake of orally administered Nal/sup 131/ solution or the extraneous band solution by human thyroid glands indicate the same uptake of the Nal/sup 131/ solution as compared to the extraneous band solution. (auth) 383O The theoretical principles are discussed of a method of determining the concentration of bone mineral per unit volume bone tissue. In this connection the problems of absorption and scattering of x rays in mixtures of substances are emphasized ae well as the elimination of errors that arise from the organic material in bones, the processing of the film, and film photometry. The comparison standand used consists of hydroxylapatite and plastic; it is always exactly reproducible; its chemical compositionmore » is very similar to that of bone and it is thus equivalent to bone in its physical behavior to x rays. By comparing the photometric results obtained from radiographs with those of the chemical amalysis of the mineral content per volume bone tissue (determnation of the "apatite" value), a maximum deviation of the obtained values of 10% was established (methodical error). Besides the discussion of the apatite values obtained from radiographs and the results of chemical determinations of the mineral content of healthy and morbid bones, the deviations of mineral content on cases of osteopathy are presented. (auth)« less

Larchwood xylan was purified by diaminoethylaminoethyl (DEAE) cellulose chromatography, sulfated by heating with chlorosulfonic acid -pyridine complex and the sulfated polysaccharide was isolated by epichlorohydrin triethanolamine (ECTEOLA) cellulose chromatography as the sodium salt. It's molecular weight was approximately 25000 and the specific rotation was [alpha]D 20-70 degrees. Electrophoretic analysis of the sulfated xylan along with heparin and SP-54 using lithium acetate-agarose technique showed that the charge density of the xylan sulfate was similar to heparin and it moved as a single component in contrast to commercial heparin which resolved into two while SP-54 moved at the same rate as the marker dye. Anticoagulant properties of the sulfated xylan were compared with heparin and SP-54 by studying its effect on the activated partial thromboplastin time (APTT), prothrombin time (PT) and the thrombin time (TT) using pooled normal human plasma. The sulfated xylan was more effective than SP-54 in delaying coagulation by all the three measurements while it was less active than heparin in inhibiting APTT and PT.

Freshly prepared extracts of chick liver acetone powder catalyze the incorporation of C14 derived from serine-3-C14 and formaldehyde-C14 into methionine. On the other hand, the incorporation of formate-C14 into methionine, under identical experimental conditions, is poor compared with that of serine-3-C14 or formaldehyde-C14. Addition of pteroylglutamic acid (PGA), homocysteine, Mg++, DPN, and ATP enhances the incorporation of serine-3-C14 into methionine, while addition of aminopterin or omission of PGA from the reaction mixture significantly decreases the incorporation of serine-3-C14 or formaldehyde-C14 into methionine. The incorporation of serine-3-C14 into methionine is not affected by addition of a pool of unlabeled cystathionine.