Extensive chaos in Rayleigh-Bénard convection.

Using large-scale numerical calculations we explore spatiotemporal chaos in Rayleigh-Bénard convection for experimentally relevant conditions. We calculate the spectrum of Lyapunov exponents and the Lyapunov dimension describing the chaotic dynamics of the convective fluid layer at constant thermal driving over a range of finite system sizes. Our results reveal that the dynamics of fluid convection is truly chaotic for experimental conditions as illustrated by a positive leading-order Lyapunov exponent. We also find the chaos to be extensive over the range of finite-sized systems investigated as indicated by a linear scaling between the Lyapunov dimension of the chaotic attractor and the system size.

Isolation of haemorphin-related peptides from filter membranes collected in connection with haemofiltration of human subjects.

This paper describes the extraction and isolation from dialysis filters of two peptides containing the opioid active sequence haemorphin-7. The filter devices were obtained from uraemic patients subjected to haemofiltration. Following acidic extraction of the filter membranes the peptides were purified by size-exclusion, ion-exchange chromatography and finally by reversed-phase chromatography using different columns and different chromatographic systems. The purification was guided by radioimmunoassay and the structure of the final products was elucidated by N-terminal sequencing and fast-atom bombardment mass spectrometry as well as micro-electrospray mass spectrometry. The isolated peptides were suggested to be identical to fragments 1-41 and 32-41 of the beta-chain of human haemoglobin.

Clearance of tissue plasminogen activator by mannose and galactose receptors in the liver.

The mechanism of uptake of radio-iodinated tissue plasminogen activator (125I-t-PA) was studied in rats. When trace amounts of 125I-t-PA were injected alone, the clearance followed a biphasic pattern in which 65% and 35% were cleared with alpha- and beta-kinetics (t1/2 (alpha) = 0.6 min, and t1/2 (beta) = 6.4 min), respectively. Co-injection with excess unlabelled t-PA or mannan changed the uptake kinetics to the monophasic beta-elimination pattern. Mannosylated albumin and ovalbumin, both of which bind to the hepatic mannose receptor, reduced the proportion of t-PA cleared with t1/2 (alpha) to 48% and 21%, respectively. A corresponding increase in the beta-elimination of t-PA was observed. The t1/2 (alpha) and t1/2 (beta) were unchanged. Studies on the clearance of 125I-ovalbumin also showed a biphasic elimination with an initial rapid phase, t1/2 (alpha), accounting for only 39% of the clearance of ovalbumin, as compared to 65% in the case of t-PA. Macromolecules with affinity for the galactose-receptor only, such as asialofetuin, or galactosylated albumin, did not significantly affect the clearance kinetics at the concentrations used. Asialoorosomucoid, which also carries galactosyl residues in the terminal position, reduced somewhat (from 65% to 48%) the proportion cleared with alpha-kinetics. Very high concentrations of galactose and N-acetyl-galactosamine, which are also known to compete for binding to the galactose receptor, lowered the proportion of t-PA cleared in the late beta-phase (reduced from 35% to 26% with galactose and to 19% with N-acetyl-galactosamine).(ABSTRACT TRUNCATED AT 250 WORDS)

Elimination of native and carbohydrate-modified tissue plasminogen activator in rabbits.

The elimination of native and carbohydrate-modified tissue type plasminogen activator (t-PA) after an i.v. bolus injection was studied in rabbits. t-PA with a low content of mannose (man-t-PA) was obtained by treatment with alpha-mannosidase, which cleaves terminal mannose from the carbohydrate side chains of the molecule. About 50% of the total mannose was removed from the native t-PA. Clearance of t-PA in rabbits was followed by measurements of both the fibrinolytic activity of the activators and the radioactivity of the iodine-labelled compounds. A biphasic mode of elimination of the fibrinolytic activity as well as the radioactivity was found with both the native and the carbohydrate modified t-PA. The initial half-life (t 1/2 alpha) was the same, about 1.5 minutes, for both compounds, irrespective of method of analysis. The late half-life (t 1/2 beta) was also the same, about 15 minutes, for both compounds. However, the beta-elimination of native t-PA could only be determined accurately from radioactivity data in the dosages tested, i.e. up to 2 mg of t-PA. No dose dependency in the elimination pattern was observed. In gel filtration experiments, with plasma from the rabbits, it was demonstrated that in addition to fibrinolytically active t-PA and man-t-PA, both fibrinolytically inactive high molecular weight complexes and low molecular weight degradation products of the activators were present in plasma after injection of the compounds. The second phase of elimination (beta) was much more pronounced after mannosidase treatment of the t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Heat inactivation of human immunodeficiency virus in solutions of antithrombin III.

Human immunodeficiency virus (HIV), isolated from cultures of infected peripheral blood lymphocytes, was added to solutions of highly purified antithrombin III (AT-III), which was stabilized with 1 M citrate and 17 percent sucrose. The efficiency of heat inactivation of HIV in AT-III at 60 degrees C was compared with that of HIV in culture medium and followed for periods from 0 to at least 10 hours. The virus added, titer 10(5) by ID50, was inactivated as rapidly (less than 30 minutes) and efficiently (completely) in the stabilized AT-III as in the culture medium. Virus was determined both by direct measurement of the HIV-related reverse transcriptase activity and by quantitation of virus infectivity by ID50 assay.

Tissue plasminogen activator is endocytosed by mannose and galactose receptors of rat liver cells.

Experiments were carried out to characterize the specificity of uptake of tPA in rat liver cells. Endocytosis in liver endothelial cells of the native carbohydrate variants of tissue plasminogen activator (tPA), and tPA inactivated by diisopropyl fluorophosphate was found to be competitive, suggesting that the determinant being recognized by these cells is different from the active site. Fibronectin and urokinase, which show partial homology with tPA, did not compete with tPA for uptake in liver endothelial cells. Hyaluronic acid, collagen, or IgG, which are endocytosed by specific receptors in liver endothelial cells, did not interfere with the uptake. Reduced endocytosis by liver endothelial cells was observed with tPA modified in the carbohydrate side chains, suggesting that these structures are important for uptake. Ovalbumin, mannan, mannose, fructose, and EDTA, but not galactose, effectively inhibited uptake in liver endothelial cells of both native and diisopropyl fluorophosphate-inhibited tPA, but had very little effect on the uptake of tPA modified in the carbohydrate side chains. Endocytosis of native tPA by parenchymal cells could be inhibited by galactose, ovalbumin, and EDTA, but not by mannose. These results suggest that endocytosis of tPA by liver endothelial cells and parenchymal cells is mediated by the mannose and galactose receptors, respectively.

Uptake and degradation of tissue plasminogen activator in rat liver.

The mechanism of uptake of tissue plasminogen activator (tPA) in rat liver was studied. Radio-iodinated tPA was removed from the circulation after intravenous administration in a biphasic mode. The initial half life, t1/2(alpha), and the terminal phase, t1/2 (beta), were determined to be 0.5 min and 7.5 min, resp. Separation of the liver cells by collagenase perfusion and density centrifugation, revealed that the uptake per cell was two to three times higher in the non-parenchymal cells than in the parenchymal cells. Endocytosis of fluorescein isothiocyanate-labelled or 125I-labelled tPA was studied in pure cultures of liver cells in vitro. Liver endothelial cells and parenchymal cells took up and degraded tPA. Endocytosis was more efficient in liver endothelial cells than in parenchymal cells, and was almost absent in Kupffer cells. Competitive inhibition experiments showing that excess unlabelled tPA could compete with the uptake and degradation of 125I-tPA, suggested that liver endothelial cells and parenchymal cells interact with the activator in a specific manner. Endocytosis of trace amounts of 125I-tPA in cultures of liver endothelial cells and parenchymal cells was inhibited by 50% in the presence of 19 nM unlabelled tPA. Agents that interfere with one or several steps of the endocytic machinery inhibited uptake and degradation of 125I-tPA in both cell types. These findings suggest that 1) liver endothelial cells and parenchymal cells are responsible for the rapid hepatic clearance of intravenously administered tPA; 2) the activator is taken up in these cells by specific endocytosis, and 3) endocytosed tPA is transported to the lysosomes where it is degraded.

Isolation and characterization of three different carbohydrate chains from melanoma tissue plasminogen activator.

Electrophoretic analysis of endoglycosidase-treated tissue plasminogen activator obtained from human melanoma cells showed that the heterogeneity observed for the protein in these preparations is caused by an N-glycosidically linked N-acetyllactosamine type of carbohydrate chain which is present in about 50% of the molecules. An oligomannose type and an N-acetyllactosamine type of glycan is present in all molecules. Three glycopeptides were isolated and characterized by 1H-NMR, sugar determination, methylation analysis and amino acid determination. The exact attachment site for each of the three glycans could be deduced from the amino acid compositions of the glycopeptides. Asn-117 carries the oligomannose type of glycan, the structure of which was completely determined. Asn-184 is the site where the presence or absence of a biantennary N-acetyllactosamine type of glycan causes the size heterogeneity. The third N-glycosylation site, Asn-448, was found to carry a triantennary or tetraantennary N-acetyllactosamine type of carbohydrate chain.

Chromatographic removal of hepatitis B virus from a factor IX concentrate. Experimental studies in chimpanzees.

Non-A, non-B hepatitis virus can be removed from a factor IX concentrate by a hydrophobic chromatographic step added to the ordinary fractionation process. The efficacy of this procedure for removal of hepatitis B virus (HBV) was evaluated in chimpanzees. A well-defined hepatitis B virus (HBV) inoculum was added to a factor IX preparation and this preparation was subjected to chromatography with octanohydrazide-Sepharose 4B at a high salt concentration and then injected intravenously into two chimpanzees. A control chimpanzee was inoculated with the part of the factor IX/HBV preparation that had not been chromatographed. The two chimpanzees that received the treated material remained free of any serologic or biochemical evidence of hepatitis B infection during a 12-month follow-up, whereas the control chimpanzee had hepatitis B. After a later HBV challenge, the two healthy animals also had hepatitis B. The hydrophobic binding procedure seems to be useful for the adsorption of viral agents in blood components.

Agonist-antagonist properties of fluorescent opioid probes in the guinea-pig myenteric plexus-longitudinal muscle preparation.

The opioid activity of a series of fluorescent derivatives of oxymorphone, naloxone and naltrexone, were characterized on the electrically stimulated guinea-pig myenteric plexus-longitudinal muscle preparation. In all compounds the fluorescent moiety, fluorescein or tetramethylrhodamine B, is attached to the C-6 carbon of the morphinan. Mu-receptor affinity was well retained and there was a good correlation between capacity to displace [3H]-dihydromorphine from binding sites in rat brain membranes and apparent receptor affinity in the myenteric plexus measured as antagonism of normorphine effects. Fluorescein conjugated oxymorphone showed mainly agonist activity. Certain derivatives of the antagonists naloxone and naltrexone showed partial agonism. One compound, 1-(N)-fluoresceinyl naloxone thiosemicarbazone, was an antagonist.

Turnover of tissue plasminogen activator in normal and hepatectomized rabbits.

The distribution, and clearance of the tissue plasminogen activator (tPA) was studied in rabbits, rats and mice. Following an intravenous injection of I-labelled tPA a large portion of the radioactive dose was rapidly accumulated in the liver. After one hour the radioactivity in the liver was less than 5 per cent of the injected dose. The highest activity was then found in the intestine, stomach and in the blood. Gel filtration of plasma taken one hour after the injection revealed that the major part (60%) of the radioactivity was present as low molecular weight metabolites or free iodine. The remaining activity was present as high molecular weight inhibitor complexes (26%) or as free tPA (15%). In order to study in vivo reactions between tPA and plasma inhibitors without hepatic interference, plasma turnover was also studied in hepatectomized rabbits. High amounts of radioactivity remained in the plasma after one hour. Gel filtration of this plasma revealed that 54 per cent of the radioactivity was bound to inhibitors, 34 per cent circulated as free tPA while a minor portion (12%) was found as free iodine or metabolites. The half-life of fibrinolytically active tPA in hepatectomized rabbits was 40 minutes compared to 2 minutes in intact rabbits. The increase in the half-life of tPA in hepatectomized rabbits also resulted in an improved thrombolytic effect after treatment with 0.5 mg tPA.

Large-scale purification of human tissue-type plasminogen activator using monoclonal antibodies.

Tissue plasminogen activator produced by a human melanoma cell line (Bowes), was purified from large volumes of supernatant fluid using immunosorbent chromatography on monoclonal antibodies, followed by chromatography on lysine-Sepharose 4B and gel filtration on Sephadex G-150. Immunosorbents based on monoclonal antibodies were shown to be preferable to those based on polyclonal antibodies for several reasons: efficient binding and desorption, high yield along with a high degree of purification. The overall recovery was about 50%. A homogeneous sample of single-chain tissue plasminogen activator with a molecular weight of approx. 65 000 was obtained as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Amino acid sequence analysis revealed two N-terminals in equal yields, corresponding to the long and short tissue plasminogen activator structures previously reported by others. The pure preparations of tissue plasminogen activator showed specific activities of about 200 000 IU/mg protein.

Removal of non-A, non-B hepatitis virus from a concentrate of the coagulation factors II, VII, IX and X by hydrophobic interaction chromatography.

The efficacy of a chromatographic procedure based on hydrophobic interaction chromatography to remove non-A, non-B (NANB) infection from a concentrate of coagulation factors II, VII, IX and X (Preconativ, KabiVitrum AB) was evaluated in chimpanzees. For this purpose, NANB infective human plasma (H-strain) was deliberately added to a solution of Preconativ (45 IU factor IX:C/ml) to reach a titre of NANB virus of greater than or equal to 10(2) chimpanzee infectious doses (CID)/ml. The NANB-contaminated preparation was chromatographed on octanohydrazide-Sepharose 4B at a high salt concentration. Groups of 2 chimpanzees were each inoculated intravenously with 45 IU factor IX:C of Preconative before and after this virus adsorption chromatographic step. The experimental animals remained free of any serological and biochemical evidence of hepatitis during a 12-month follow-up period. One of the two control animals developed clear evidence of NANB hepatitis.

Adsorption properties of different hepatitis B virus related antigens (HBsAg, HBcAg, HBeAg) on octanohydrazide-Sepharose 4B.

Chromatography of plasma containing hepatitis B virus and partially purified viral antigens on a hydrophobic gel derivative (octanohydrazide-Sepharose 4B) revealed that HbsAg and HbcAg were adsorbed to the gel in 0.75 mol/l ammonium bicarbonate and eluted by a detergent, Berol. HBeAg in a purified HBcAg preparation from human liver, but not HBeAg in plasma, was bound to the gel. Furthermore, HBeAg in the HBcAg preparation, but not HBeAg in plasma, lost its antigenic reactivity in the presence of Berol, indicating that the two HBeAgs were present in different molecular configurations. However, HBeAg could be released from HBV (HBcAg) and form a component which sedimented slowly and was immune-reactive in the presence of the detergent. The results contribute to knowledge of the interrelationship between hepatitis B-related antigens and indicate that chromatography on hydrophobic gel derivatives can be used not only for the purification (and removal) of HBsAg but also of HBcAg.

Differences between uterine and melanoma forms of tissue plasminogen activator.

Tissue plasminogen activator purified from human uterine tissue exhibits differences in N-terminal starting positions in relation to the melanoma cell plasminogen activator usually studied. A new starting position is compatible with an additional N-terminal processing apart from those already known. Like the melanoma activator, the uterine activator was found to yield protein chains starting at either of two positions. One of these was identical between uterine and melanoma activators, whereas the other was unique in each case. The most abundant starting position for the uterine preparation was at a valine residue, apparently from cleavage of a Gln-Val bond, and corresponding to Val-7 of the longest form of the melanoma activator chain.

Effect on haemostasis of intravenous injection of alpha 2-antiplasmin in cats treated with streptokinase.

The effect in cats of an intravenous injection of purified human alpha 2-antiplasmin on changes in the haemostatic system, caused by administration of streptokinase was investigated. The decrease in fibrinogen and plasminogen caused by streptokinase infusion was immediately inhibited by the alpha 2-antiplasmin infusion. The increase in vascular permeability and the prolongation of coagulation times were efficiently inhibited.

Catabolic pathways for streptokinase, plasmin, and streptokinase activator complex in mice. In vivo reaction of plasminogen activator with alpha 2-macroglobulin.

The catabolic pathways of streptokinase, plasmin, and activator complex prepared with human plasminogen were studied in mice. (125)I-streptokinase clearance occurred in the liver and was 50% complete in 15 min. Incubation with mouse plasma had no effect on the streptokinase clearance rate. Complexes of plasmin and alpha(2)-plasmin inhibitor were eliminated from the plasma by a specific and saturable pathway. Competition experiments demonstrated that this pathway is responsible for the clearance of injected plasmin. Streptokinase-plasminogen activator complex formed with either (125)I-plasminogen or (125)I-streptokinase cleared in the liver at a significantly faster rate than either of the uncomplexed proteins (50% clearance in <3 min). Streptokinase incubated with human plasma also demonstrated this accelerated clearance. p-Nitrophenyl-p'-guanidinobenzoate-HCl or pancreatic trypsin inhibitor-treated complex cleared slowly compared with untreated complex independent of which protein was radiolabeled. Significant competition for clearance was demonstrated between alpha(2)-macroglobulin-trypsin and activator complex only when the plasmin(ogen) was the radiolabeled moiety. Large molar excesses of alpha(2)-plasmin inhibitor-plasmin failed to retard the clearance of activator complex. Hepatic binding of streptokinase-plasmin, in liver perfusion experiments, was dependent upon prior incubation with plasma (8-10% uptake compared to a background of approximately 2.5%). Substitution of human alpha(2)-macroglobulin for plasma also resulted in binding when the incubation was performed for 10 min at 37 degrees C (7.5%). Electrophoresis experiments confirmed the transfer of 0.8 mol plasmin/mol alpha(2)-macroglobulin when activator complex was incubated at 37 degrees C with alpha(2)-macroglobulin for 40 min. Streptokinase transfer from activator complex to alpha(2)-macroglobulin was negligible. The in vivo clearance of activator complex is proposed to involve active attack of the complex on the alpha(2)-macroglobulin "bait region," resulting in facilitated plasmin transfer. Dissociated streptokinase is rapidly bound and cleared by sites in the liver.