High tyrosinase activity in albino Xenopus laevis oocytes

Development ◽  
1976 ◽  
Vol 36 (3) ◽  
pp. 555-559
Author(s):  
A. H. Wyllie ◽  
E. M. De Robertis
Keyword(s):  
Molecular Weight ◽  
Xenopus Laevis ◽  
Specific Activity ◽  
High Specific Activity ◽  
Low Molecular Weight ◽  
Xenopus Laevis Oocytes ◽  
Melanin Synthesis ◽  
Wild Type ◽  
Albino Mutant

Tyrosinase was measured in oocytes of the recently described albino mutant (avav) of Xenopus laevis. Although these oocytes show no pigmentation and the eggs are known to contain no melanosomes, tyrosinase — which is probably the only enzyme necessary for melanin synthesis from tyrosine — was increased more than twofold relative to the wild type. Tyrosinase recovered from albino and wild type oocytes showed the same KM with respect to tyrosine, and this was not altered by previous gonadotrophin stimulation in vivo. The tyrosi-nase assay, based on [14]tyrosine incorporation into acid-insoluble products, was of greater sensitivity than previously described methods of the same type, through removal of low molecular weight material from the oocyte homogenate prior to incubation, and the use of tyrosine of high specific activity.

Radioiodination of Alginate via Covalently-Bound Tyrosinamide Allows Monitoring of its Fate In Vivo

1995 ◽  
Vol 10 (1) ◽  
pp. 4-13 ◽  
Author(s):  
Aymen Al-Shamkhani ◽  
Ruth Duncan
Keyword(s):  
Molecular Weight ◽  
Intraperitoneal Injection ◽  
Specific Activity ◽  
Subcutaneous Administration ◽  
High Specific Activity ◽  
Weight Fraction ◽  
Low Molecular Weight ◽  
Blood Compartment ◽  
Almost All

Tb monitor the fate of alginate following systemic administration, a method was developed that allowed the covalent incorporation of approximately 1 mol% tyrosinamide. The product could be radioiodinated to a high specific activity, and was subsequently stable on storage at 4°C for 30 days, with very little (c 1%) free [125I] iodide released over that period. Twenty-four hours following intravenous administration, the low molecular weight fraction (<48,000) of the injected polymer was excreted in the urine while the larger polymer fraction remained in the circulation and did not readily accumulate in any of the tissues. Almost all of the dose administered by intraperitoneal injection was transferred from the peritoneal cavity to the blood compartment within 24 h, with the low molecular weight fraction of the polymer excreted in the urine. Following subcutaneous administration, the majority (-70%) of the injected dose was retained at the site of injection at 24 h.

Studies in Man and Experimental Animals of a Low Molecular Weight Heparin Fraction

1981 ◽  
Vol 45 (03) ◽  
pp. 214-218 ◽  
Author(s):  
D P Thomas ◽  
R E Merton ◽  
W E Lewis ◽  
T W Barrowcliffe
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Specific Activity ◽  
Ex Vivo ◽  
High Specific Activity ◽  
Factor Xa ◽  
In Vivo Studies ◽  
Low Molecular Weight

SummaryIn vitro and in vivo studies were carried out on a commercially prepared low molecular weight heparin fraction. By APTT assay the fraction had a specific activity of half that of unfractionated mucosal heparin, yet retained full potency by anti-Xa assay (both clotting and chromogenic substrate). When administered intravenously to human volunteers, the anti-Xa/APTT ratio remained the same as it was in vitro. However, after subcutaneous injection, the ratio increased and anti-Xa activity could not be fully neutralized ex vivo by PF4. The fraction was as effective as unfractionated heparin in preventing experimental serum-induced thrombosis, suggesting that a heparin fraction with high specific activity by anti-Factor Xa assay compared to APTT activity may be an effective drug for the prophylaxis of venous thrombosis.

scholarly journals The interrelations between high- and low-molecular-weight forms of normal and mutant (Krabbe-disease) galactocerebrosidase

1980 ◽  
Vol 189 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Yoav Ben-Yoseph ◽  
Melinda Hungerford ◽  
Henry L. Nadler
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Krabbe Disease ◽  
Low Molecular Weight ◽  
Molecular Weight Form

Galactocerebrosidase (β-d-galactosyl-N-acylsphingosine galactohydrolase; EC 3.2.1.46) activity of brain and liver preparations from normal individuals and patients with Krabbe disease (globoid-cell leukodystrophy) have been separated by gel filtration into four different molecular-weight forms. The apparent mol.wts. were 760000±34000 and 121000±10000 for the high- and low-molecular-weight forms (peaks I and IV respectively) and 499000±22000 (mean±s.d.) and 256000±12000 for the intermediate forms (peaks II and III respectively). On examination by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the high- and low-molecular-weight forms revealed a single protein band with a similar mobility corresponding to a mol.wt. of about 125000. Antigenic identity was demonstrated between the various molecular-weight forms of the normal and the mutant galactocerebrosidases by using antisera against either the high- or the low-molecular-weight enzymes. The high-molecular-weight form of galactocerebrosidase was found to possess higher specific activity toward natural substrates when compared with the low-molecular-weight form. It is suggested that the high-molecular-weight enzyme is the active form in vivo and an aggregation process that proceeds from a monomer (mol.wt. approx. 125000) to a dimer (mol.wt. approx. 250000) and from the dimer to either a tetramer (mol.wt. approx. 500000) or a hexamer (mol.wt. approx. 750000) takes place in normal as well as in Krabbe-disease tissues.

Further studies on the melanophores of periodic albino mutant of Xenopus laevis

Development ◽  
1986 ◽  
Vol 91 (1) ◽  
pp. 65-78
Author(s):  
T. Fukuzawa ◽  
H. Ide
Keyword(s):  
Xenopus Laevis ◽  
Microscopic Level ◽  
Wild Type ◽  
Light Microscopic Level ◽  
In Vitro Proliferation ◽  
Albino Mutant ◽  
Site Of Action ◽  
Periodic Albino

It is still unknown why dermal melanophores disappear during larval development, and why no or very few epidermal melanophores appear during and after metamorphosis, in Xenopus laevis showing periodic albinism (ap). To elucidate these points, we investigated (1) the occurrence of depigmentation in mutant (ap/ap) melanophores during in vitro proliferation and (2) the incidence of melanophore differentiation from mutant melanoblasts in the skin in vitro. During in vitro proliferation of mutant melanophores, ap-type melanosomes decreased in number gradually and instead the number of premelanosomes increased in the cells, which caused depigmentation at the light microscopic level in the culture. Depigmentation was observed only in mutant melanophores, and not in wild-type (+/+) melanophores. These results suggest that autonomous depigmentation of mutant dermal melanophores is the cause of the disappearance of these cells in vivo. Dopa-positive melanoblasts were demonstrated in both wild-type and mutant skins. However, the melanoblasts of metamorphosed mutant froglets did not differentiate in vitro, while those of wild-type froglets did. These results suggest that mutant melanoblasts in the skin of froglets lose the potency to differentiate into melanophores, and that this causes the lack of mutant melanophores in the froglets. The site of action of the ap gene is also discussed.

Construction, Expression, and Characterization of a Recombinant Annexin B1-Low Molecular Weight Urokinase Chimera in Escherichia coli

2004 ◽  
Vol 36 (3) ◽  
pp. 184-190 ◽  
Author(s):  
Hong-Li Yan ◽  
Wei-Ting Wang ◽  
Yan He ◽  
Zhuan-You Zhao ◽  
Yuan-Jian Gao ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Membrane Binding ◽  
Low Molecular Weight ◽  
Single Chain ◽  
T7 Promoter ◽  
Overlap Extension ◽  
Annexin B1

Abstract To produce a thrombi-targeting plasminogen activator, low molecular weight single-chain urokinase gene (scuPA32k) was spliced with the full-length cDNA of annexin B1 gene (anxB1) by overlap extension method. The fused gene anxB1scuPA was ligated into pET28a vector, transformed into E. coli BL21-RIL, and then induced to express under the control of T7 promoter. The AnxB1ScuPA protein expressed amounted to 22% of the total bacterial proteins. The product was refolded, and then purified by using DEAE Sepharose fast flow ion-exchange column and Superdex S-200 gel-filtration column. HPLC analysis revealed that the final purity is about 95%. The specific activity of AnxB1ScuPA, measured as amidolytic activity, reached 100,000 IU/mg. It had a similar S2444 catalytic efficiency (kcat/Km) to ScuPA32k, and also showed high activated-platelet membrane-binding activity and anticoagulant activity, indicating that the chimera fully retained the components of enzymatic and membrane-binding activities of the parent molecules. In vivo test revealed that, the dogs administered with AnxB1ScuPA had less reperfusion time, higher reperfusion ratio, and less bleeding effects than those with urokinase. These findings indicated that AnxB1ScuPA might have advantages over current available thrombolytic agents.

scholarly journals Pharmacokinetics of Subcutaneously Administered CB2679d/ISU304 in Wild-Type and Hemophilia B Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1389-1389 ◽  
Author(s):  
Seung-Beom Hong ◽  
Howard Levy ◽  
Jae Yong Jung ◽  
Minkyung Park ◽  
A Rim Seo ◽  
...  
Keyword(s):  
Specific Activity ◽  
Phase 1 ◽  
Sandwich Elisa ◽  
High Specific Activity ◽  
Biological Properties ◽  
Factor Ix ◽  
Hemophilia B ◽  
Wild Type

Abstract The rapid clearance of factor IX (FIX) necessitates frequent intravenous IV administrations to achieve effective prophylaxis for patients with hemophilia B (HB). Subcutaneous (SC) administration would be a preferred route of administration but has been limited by low bioavailability and potency of the marketed FIX products. CB2679d/ISU304 with enhanced biological properties was developed using a rational protein design approach and has resistance to inhibition by ATIII, increased affinity for FVIIIa, increased catalytic activity and resultant 20-fold enhanced potency in vitro (clotting activity) and in vivo (the tail clip model) and 8-fold increased duration of aPTT activity in vivo compared to recombinant wild-type FIX dosed at the same mass. ISU304 (4622 IU/mg) was injected into HB mice SC with a single dose ISU304 at 0.02, 0.05 or 0.15 mg/kg and sampled at 4, 6, 8, 24 hours. Groups of wild-type mice received ISU304 0.02, 0.05 or 0.15 mg/kg SC and BeneFIX (273 IU/mg) 0.15 mg/kg SC, and sampled at 0.25, 1, 4, 8, 24, 48, 72, and 96 hours. Daily SC injection in HB mice of ISU304 at 0.05 mg/kg was sampled at 24, 48, 72 and 96 hours. FIX antigen was measured using a sandwich ELISA and FIX activity was measured using a one-stage clotting assay on Stago Compact. Pharmacokinetics of FIX was performed using PKSolver. There was a dose-dependent increase of plasma FIX antigen with SC ISU304. Mass-based pharmacokinetic profiles of ISU304 (t1/2, 18 hours; Tmax,8 hours, Bioavailability, 19-22%) were similar to those of BeneFIX (t1/2, 20 hours; Tmax, 8 hours, Bioavailability, 16%). Due to ISU304 high specific activity, SC dose of ISU304 yields much higher FIX activities in mouse plasma compared with the same mass dose of BeneFIX. Daily SC dosing of ISU304 230 IU/kg reached steady-state plateau FIX 8% activity after three injections. The bioavailability and increased potency of CB2679d/ISU304 facilitates the initiation of the Phase 1 subcutaneous dosing study in individuals with hemophilia B. Figure 1 Figure 1. Table 1 Table 1. Figure 2 Figure 2. Disclosures Hong: ISU Abxis: Employment. Levy:Catalyst Biosciences: Employment. Jung:ISU Abxis: Employment. Park:ISU Abxis: Employment. Seo:ISU Abxis: Employment. Seo:ISU Abxis: Employment. Madison:Catalyst Biosciences: Employment, Equity Ownership, Patents & Royalties.

Effects of Unfractionated and Low Molecular Weight Heparins on Platelet Thromboxane Biosynthesis “In Vivo”

1994 ◽  
Vol 72 (06) ◽  
pp. 942-946 ◽  
Author(s):  
Raffaele Landolfi ◽  
Erica De Candia ◽  
Bianca Rocca ◽  
Giovanni Ciabattoni ◽  
Armando Antinori ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Low Molecular Weight Heparin ◽  
Primary Source ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
Heparin Administration ◽  
To Receive

SummarySeveral “in vitro” and “in vivo” studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis “in vivo”, as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionaed heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 ± 1388 vs 2092 ±777, p <0.05; 2,3-dinor-TxB2: 2737 ± 808 vs 1535 ± 771 pg/mg creatinine, p <0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation “in vivo” and suggest that the use of low molecular weight heparin may avoid this complication.

In Vivo Studies on the Inhibition of Coagulation by Fractionated Heparin and by a Heparin Analogue I. Effects of Heparin Fractions

1981 ◽  
Vol 46 (03) ◽  
pp. 612-616 ◽  
Author(s):  
U Schmitz-Huebner ◽  
L Balleisen ◽  
F Asbeck ◽  
J van de Loo
Keyword(s):  
Molecular Weight ◽  
Day Treatment ◽  
Gel Filtration ◽  
Weight Fraction ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
High Molecular Weight Fraction ◽  
Platelet Factor ◽  
Heparin Fractions

SummaryHigh and low molecular weight heparin fractions obtained by gel filtration chromatography of sodium mucosal heparin were injected subcutaneously into six healthy volunteers and compared with the unfractionated substance in a cross-over trial. Equal doses of 5,000 U were administered twice daily over a period of three days and heparin activity was repeatedly controlled before and 2, 4, 8 hrs after injection by means of the APTT, the anti-Xa clotting test and a chromogenic substrate assay. In addition, the in vivo effect of subcutaneously administered fractionated heparin on platelet function was examined on three of the volunteers. The results show that s.c. injections of the low molecular weight fraction induced markedly higher anti-Xa activity than injections of the other preparations. At the same time, APTT results did not significantly differ. Unfractionated heparin and the high molecular weight fraction enhanced ADP-induced platelet aggregation and collagen-mediated MDA production, while the low molecular weight fraction hardly affected these assays, but potently inhibited thrombin-induced MDA production. All heparin preparations stimulated the release of platelet Factor 4 in plasma. During the three-day treatment periods, no side-effects and no significant changes in the response to heparin injections were detected.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

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