Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.68 (tissue plasminogen activator)
11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

rt-PA-K, a variant of recombinant tissue-type plasminogen activator (rt-PA) with substitution of amino acids 296 to 299 with alanine (KHRR296-299AAAA) has increased fibrin-specificity and reduced sensitivity to plasminogen activator inhibitor-1; rt-PA-T, with threonine 103 replaced by asparagine has an additional glycosylation site and a reduced clearance; and rt-PA-N, with asparagine 117 mutagenized to glutamine lacks the high mannose carbohydrate side chain. We have investigated whether combination of these properties in a single molecule might yield an improved thrombolytic agent. The thrombolytic potency and fibrin-specificity of the combination mutant rt-PA-TNK was compared with that of rt-PA in a combined venous whole blood clot model and platelet-rich arterial eversion graft thrombosis model in dogs given intravenous heparin and aspirin. Infusion of 0.125 to 1.0 mg/kg over 60 min in groups of 4 to 5 dogs produced dose-dependent fibrin-specific venous clot lysis. The thrombolytic potency (percent lysis per mg compound administered per kg body weight) of rt-PA-TNK was significantly higher than that of rt-PA as evidenced by a higher maximal rate of lysis of 480 +/- 100% versus 140 +/- 40% within the 2 h observation period per mg of compound administered per kg body weight (mean +/- SEM, p = 0.004) and a significantly lower dose of 0.08 +/- 0.01 versus 0.21 +/- 0.04 mg/kg body weight at which the maximal rate of lysis was obtained (p = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparative thrombolytic properties of tissue-type plasminogen activator and of a plasminogen activator inhibitor-1-resistant glycosylation variant, in a combined arterial and venous thrombosis model in the dog. 797 84

Electrospray ionization mass spectrometry utilizing a single quadrupole on line with reversed-phase HPLC (LC/MS) enables the characterization of glycoproteins in a relatively short period of time. In this approach the protein is digested with a suitable protease and the peptides are separated by reversed-phase HPLC and detected by electrospray ionization mass spectrometry. The glycopeptides are initially observed as a cluster of negatively sloping ions in a contour plot of data from the LC/MS run (m/z vs retention time) or as a characteristics series of masses at different elution times. The search for a particular glycopeptide is based on previously known carbohydrate structures and on consensus glycosylation sites. Further structural information is obtainable with glycosidase digestion and LC/MS analysis. The mass shifts following glycosidase digestion allow further confirmation of the structure. This approach identifies the site of attachment of two hybrid glycoforms to the T11 tryptic peptide in a reversed-phase tryptic map of recombinant tissue plasminogen activator (rt-PA). Use of selected ion extraction of the LC/MS data files allows one to graphically describe the elution order of closely related glycopeptides. The potential of LC/MS for the characterization of small amounts of unknown glycoproteins is shown by the study of an rt-PA mutant. A new potential site for glycosylation is created by site directed mutagenesis of wild type rt-PA with replacement of a threonine residue with asparagine at residue 103. An examination of a tryptic map shows that the mutant contains two new complex carbohydrate chains. The introduction of the new asparagine proximal to asparagine 117 changes this native high-mannose site in rt-PA to a complex-type glycosylation. This method allows rapid identification of carbohydrate containing peptides and yields useful structural information on microgram amounts of material.
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PMID:Identification of carbohydrate structures in glycoprotein peptide maps by the use of LC/MS with selected ion extraction with special reference to tissue plasminogen activator and a glycosylation variant produced by site directed mutagenesis. 825 61

We have developed an intermediate method toward the complete carbohydrate analysis of proteins, which should be universally applicable to all proteins and independent of sample matrix. Using only Coomassie Blue-stained proteins which have been electroblotted onto polyvinylidene fluoride membranes, we report a strategy for: (i) determining unequivocally whether a protein is glycosylated; (ii) obtaining a complete monosaccharide composition; (iii) oligosaccharide mapping which separates most forms according to size, charge and isomerity; and (iv) sequentially releasing and analyzing specific classes of oligosaccharides with endoglycosidases. The method was shown to be applicable to a variety of well characterized soluble glycoproteins and to the membrane-bound protein, the gastric H+, K(+)-ATPase. The monosaccharide composition of the H+,K(+)-ATPase revealed the absence of N-acetylneuraminic or N-glycolylneuraminic acids and a monosaccharide composition which indicated O-linked sugar chains. Oligomannosidic/hybrid and biantennary oligosaccharides were sequentially released and analyzed from one electroblotted band of recombinant tissue plasminogen activator using endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F2, respectively. Sialylated polylactosamine structures were identified and quantified by analyzing high performance liquid chromatography profiles of oligosaccharides first released by peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase and then treated with endo-beta-galactosidase, using a single, stained band of recombinant erythropoietin. This recombinant erythropoietin was found to contain eight times more tetrasialylated oligosaccharides than previously reported (Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076); 47% of released oligosaccharides were identified as polylactosamine structures.
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PMID:Monosaccharide and oligosaccharide analysis of proteins transferred to polyvinylidene fluoride membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 844 88

The magnitude of the quadrupole coupling constant (e2Qq/h) of a deuteron is a good probe for hydrogen bonding. In protein structures, hydrogen-bonding interactions between side chains, between side chaings and ligands, and between side chains and solvent are frequently found. An experiment that detects, via scalar coupling, the influence of a deuteron on the 15N nucleus of asparagine or glutamine side chains is presented. The experiment depends upon the resolution of the 1 delta 15 N(D) isotope shifts that allow the various isotopomers and isotopologues to be distinguished when 15N-labeled samples are dissolved in solvent mixtures of H2O/D2O. 15N lineshapes with theoretical simulations that provide estimates for the 2H quadrupole coupling constants are presented. The influence of 15N-2H dipolar-quadrupole cross correlation and the resulting small frequency shifts in the 15N multiplet are resolved in some of the spectra. The experimental data are provided using the free amino acids asparagine and glutamine for which the side chains were isotopically enriched in 15N and the recombinant pair of modules, fibronectin type 1 and epidermal growth factor, (F1-G) of tissue plasminogen activator, which were uniformly isotopically enriched in 15N.
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PMID:The influence of a scalar-coupled deuterium upon the relaxaton of a 15N Nucleus and its possible exploitation as a probe for side-chain interactions in proteins. 942 19

Gln117 t-PA is a mutant type of tissue-type plasminogen activator (mt-PA), which is generated by the removal of a high mannose oligosaccharide resulting from the mutation of amino acid #117, from asparagine (Asn) to glutamine (Gln). The plasma concentration, distribution, metabolism and excretion of Gln117 t-PA were investigated after a single intravenous administration of 125I-Gln117 t-PA or Gln117 t-PA, comparing with wild-type t-PA (WT t-PA). The plasma concentration of Gln117 t-PA decreased more slowly than that of WT t-PA, plasma clearance (CL(p)), and that of Gln117 t-PA in rats was approximately 2.6 times lower than that of WT t-PA. The highest radioactivity was found in the liver at 5 min after intravenous administration of [125I]Gln117 t-PA to rats, but the radioactivity in the liver was lower than that after intravenous administration of [125I]WT t-PA in our previous paper. Within 288 h after intravenous administration of [125I]Gln117 t-PA to rats, 88.5 and 5.5% of administered radioactivity were excreted into urine and feces, respectively. In a gel-filtration chomatographic (GFC) analysis of plasma, Gln117 t-PA formed complexes with plasma proteins, similarly to WT t-PA. The hepatic clearance (CL(hep)) of both t-PAs was evaluated by comparing the plasma concentration after a constant intravenous infusion with that after a constant intraportal venous infusion. The CL(hep) of Gln117 t-PA was 6.5 times lower than that of WT t-PA. These results indicate that the low uptake of Gln117 t-PA to the liver reduces CL(p) compared with WT t-PA.
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PMID:Pharmacokinetic studies of Gln117 tissue-type plasminogen activator in rats. 1148 91

Elevated pCO(2) inhibits cell growth. This growth inhibition is accompanied by a decrease in intracellular pH (pHi), as well as a decrease in glycolysis. Elevated concentrations (mM) of some amino acids have been shown by others to protect cells exposed to two very different environmental stresses: nutrient starvation and hyperosmolality. The fact that many of the amino acids shown to have protective effects against other stresses are transported into the cell through a pHi-sensitive transporter led us to study the possibility of using these amino acids as protective agents under elevated pCO(2). Screening experiments using 5, 15, and 25 mM of each amino acid showed that not all amino acids that protect cells from hyperosmolality protect them from elevated pCO(2). Glycine betaine and glycine were chosen for further characterization in both hybridoma and CHO cells. Asparagine and threonine were also tested in hybridoma and CHO cells, respectively. All amino acids tested under 195 mm Hg pCO(2)/435 mOsm/kg (50% growth inhibition) restored the specific growth rate (mu) in hybridoma cells to that observed under control conditions (40 mm Hg/320 mOsm/kg). Addition of each amino acid resulted in an increase in the consumption rate and intracellular accumulation of that amino acid. In CHO cells, glycine betaine also restored mu to control values, while glycine and threonine partially restored mu. In hybridoma cells, the higher specific antibody productivity obtained at elevated pCO(2) was maintained with the lowest amino acid concentration (5 mM). Productivity decreased toward control values with increasing amino acid concentrations. Elevated pCO(2) decreased the specific tPA productivity in the CHO cell line studied. Only glycine betaine resulted in a 20% increase in productivity at 195 mm Hg/435 mOsm/kg. With the exception of glycine betaine in hybridoma cells, amino acids did not mitigate the associated pHi decrease of at least 0.2 pH units at 195 mm Hg/435 mOsm/kg. pHi in hybridoma cells under elevated pCO(2) in the presence of glycine betaine was about 0.1 pH units below that of control. Amino acids had no effect on the cell size response of hybridoma cells, while they partially offset the increase in CHO cell size at elevated pCO(2). Glycine betaine, asparagine, and glycine increased the specific glucose consumption rate observed at 195 mm Hg/435 mOsm/kg (50% of control) to values greater than 70% of control in hybridoma cells. In CHO cells, only glycine betaine increased q(glc) (by 20%) under elevated pCO(2). All amino acids tested improved the cell yield from glutamine at 195 mm Hg/435 mOsm/kg in both cell lines.
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PMID:Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality. 1200 Nov 66

Optimization of fed-batch feeding parameters was explored for a system with multiple mechanisms of product inactivation. In particular, two separate mechanisms of inactivation were identified for the recombinant tissue-type activator (r-tPA) protein. Dynamic inactivation models were written to describe particular r-tPA glycoform inactivation in the presence and absence of free-glucose. A glucose-independent inactivation mechanism was identified, and inactivation rate constants were found dependent upon the presence of glycosylation of r-tPA at N184. Inactivation rate constants of the glucose-dependent mechanism were not affected by glycosylation at N184. Fed-batch optimization was performed for r-tPA production by CHO cell culture in a stirred-tank reactor with glucose, glutamine and asparagine feed. Feeding profiles in which culture supernatant concentrations of free-glucose and amino acids (combined glutamine and asparagine) were used as control variables, were evaluated for a wide variety of set points. Simulation results for a controlled feeding strategy yielded an optimum at set points of 1.51 g L(-1) glucose and 1.18 g L(-1) of amino acids. Optimization was also performed in absence of metabolite control using fixed feed-flow rates initiate during the exponential growth phase. Fixed feed-flow results displayed a family of optimum solutions along a mass flow rate ratio of 3.15 of glucose to amino acids. Comparison of the two feeding strategies showed a slight advantage of rapid feeding at a fixed flow rate as opposed to metabolite control for a product with multiple mechanisms of inactivation.
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PMID:Optimization of fed-batch parameters and harvest time of CHO cell cultures for a glycosylated product with multiple mechanisms of inactivation. 1738 45

A model was developed for novel prediction of N-linked glycan branching pattern classification for CHO-derived N-linked glycoproteins. The model consists of 30 independent recurrent neural networks and uses predicted quantities of secondary structure elements and residue solvent accessibility as an input vector. The model was designed to predict the major component of a heterogeneous mixture of CHO-derived glycoforms of a recombinant protein under normal growth conditions. Resulting glycosylation prediction is classified as either complex-type or high mannose. The incorporation of predicted quantities in the input vector allowed for theoretical mutant N-linked glycan branching predictions without initial experimental analysis of protein structures. Primary amino acid sequence data were effectively eliminated from the input vector space based on neural network prediction analyses. This provided further evidence that localized protein secondary structure elements and conformational structure may play more important roles in determining glycan branching patterns than does the primary sequence of a polypeptide. A confidence interval parameter was incorporated into the model to enable identification of false predictions. The model was further tested using published experimental results for mutants of the tissue-type plasminogen activator protein [J. Wilhelm, S.G. Lee, N.K. Kalyan, S.M. Cheng, F. Wiener, W. Pierzchala, P.P. Hung, Alterations in the domain structure of tissue-type plasminogen activator change the nature of asparagine glycosylation. Biotechnology (N.Y.) 8 (1990) 321-325].
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PMID:Prediction of N-linked glycan branching patterns using artificial neural networks. 1805 50

Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Five) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Five cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Five cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Five cell cultures, but not in Sf-9 cell cultures. In addition, High Five cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Five cell cultures. It was also found that the medium had a significant effect on High Five cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM.
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PMID:Comparison of Trichoplusia ni BTI-Tn-5B1-4 (high five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures. 1863

Congenital dysfibrinogenemia is a rare disease characterised by inherited abnormality in the fibrinogen molecule, resulting in functional defects. Two patients, a 26-year-old woman and a 61-year-old man, both with history of thrombotic events, had abnormal coagulation test results. DNA sequencing showed the heterozygous gamma Y363N mutation (Fibrinogen Praha III) and the heterozygous Aalpha N106D mutation (Fibrinogen Plzen), respectively. Fibrin polymerisation, after addition of either thrombin or reptilase, showed remarkably delayed polymerisation in both cases. Fibrinolysis experiments showed slower tPA initiated lysis of clots. SDS-PAGE did not show any difference between normal and Praha III and Plzen fibrinogens. Both mutations had a significant effect on platelet aggregation. In the presence of either ADP or TRAP, both mutations caused the decrease of platelet aggregation. SEM revealed abnormal clot morphology, with a large number of free ends and narrower fibres of both fibrin Praha III and Plzen. Praha III mutation was situated in the polymerisation pocket "a". The replacement of the bulky aromatic side chain of tyrosine by the polar uncharged small side chain of asparagine may lead to a conformational change, possibly altering the conformation of the polymerisation pocket. The Plzen mutation is situated in the coiled-coil connector and this replacement of polar uncharged asparagine residue by polar acidic aspartate changes the alpha-helical conformation of the coiled-coil connector; and may destabilise hydrogen bonds in its neighborhood. Although both mutations are situated in different regions of the molecule, both mutations have a very similar effect on fibrinogen functions and both are connected with thromboses.
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PMID:Two cases of congenital dysfibrinogenemia associated with thrombosis - Fibrinogen Praha III and Fibrinogen Plzen. 1971 61


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