scholarly journals Assessment of Acid Phosphatase Production by In vitro Cultures of Atropa acuminata

2016 ◽  
Vol 26 (1) ◽  
pp. 15-23
Author(s):  
Saima Khan ◽  
Meenu Katoch ◽  
Sharada Mallubhotla ◽  
Suphla Gupta ◽  
Manju Sambyal ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Growth Period ◽  
Callus Cultures ◽  
Shoot Cultures ◽  
Crude Enzyme Extract ◽  
Atropa Acuminata

The potential of various culture lines of Atropa acuminata were investigated for resourcing acid phosphatase (ACP) (3.1.3.2). Crude enzyme extract comprised of a mixture of four isoforms, distinguishable by polyacrylamide gel electrophoresis (PAGE) with molecular weight ranging from 39 to 215 kDa. In vitro regenerated proliferative shoots, callus and roots showed higher specific activity (2.49, 3.41, 2.91 U/mg protein, respectively) as compared to in vivo grown plants (0.71 U/mg protein). ACP activity in root cultures increased progressively up to 4.6 U/mg during the entire growth period (2 ? 24 weeks), whereas in case of shoot cultures, the specific activity escalated to 2.49 U/mg at 8 weeks, which then declined subsequently (1.95 U/mg). Similarly, callus cultures initially showed a higher phosphohydrolytic activity (3.41 U/mg protein) until 8 weeks by which period, it decreased with the passage of growth period. The present studies reveal an alternate system for resourcing of ACP from Atropa acuminata.Plant Tissue Cult. & Biotech. 26(1): 15-23, 2016 (June)

Membrane Glycoproteins of Human and Rabbit Platelets: Studies In Vitro and after Circulation in Vivo

1975 ◽  
Author(s):  
J. N. George ◽  
P. C. Lewis ◽  
D. A. Sears
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Age Distribution ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Rabbit Platelets ◽  
Trypsin Hydrolysis ◽  
Initial Recovery

The initial events of hemostasis and thrombosis involve platelet contact interactions and may be mediated by surface glycoproteins. Human and rabbit platelets were labeled with 125I-diazotized diiodosulfanilic acid (I), which reacts covalently with proteins, and proteins were separated by SDS-polyacrylamide gel electrophoresis. Only exposed membrane proteins were labeled because: 1) protein specific activity of membranes was 4-7 times that of whole platelets, 2) different proteins were labeled when I was reacted with isolated membranes, and 3) trypsin-hydrolysis of labeled intact platelets altered the radioactive peaks. Like Phillips (Biochem. 11, 4582, 72) and Nachman et al. (JBC 248, 2928, 73) we found that lactoperoxidase iodinated the 93,000 dalton glycoprotein (GP) of human platelets. In contrast, I labeled both the 93,000 and 118,000 dalton membrane GP of human platelets, and all 3 membrane GP of rabbit platelets.Rabbit platelets labeled simultaneously with I and 51Cr had identical density and therefore age distribution of the 2 labels. After infusion into rabbits, initial recovery of I was 23% of the Cr recovery. After 3 hrs, I disappearance was exponential and more rapid (T/2 = 17 hrs) than the linear Cr disappearance (T/2 = 30 hrs, p < .01). This was due to in vivo removal of I from circulating platelets since 1 did not elute more rapidly from platelets harvested after 3 hrs circulation and incubated in plasma at 37° (T/2 of I elution = 43 hrs, Cr = 33 hrs). Platelets harvested after 14-20 hrs circulation had the same distribution of I on the membrane GP as before circulation. We postulate that this symmetrical label loss indicates uniform loss of membrane GP, suggesting that platelets lose pieces of their plasma membrane during circulation. This could occur during contact interaction in the process of hemostasis.

scholarly journals Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 394-400 ◽  
Author(s):  
WF Novotny ◽  
M Palmier ◽  
TC Wun ◽  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Vascular Endothelium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Major Band ◽  
Coagulation Inhibitor

The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

Studies of Heparin Affinity to Antithrombin III and Other Proteins in Vitro and in Vivo

1977 ◽  
Vol 38 (03) ◽  
pp. 0685-0695 ◽  
Author(s):  
Y Takeda ◽  
N Kobayashi
Keyword(s):  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Heparin Binding ◽  
Life Values ◽  
Canine Plasma ◽  
Heparin Affinity

SummaryProperties of human, canine, and porcine heparin and S-35-heparin were first studied. Their electrophoretic mobility through 10 g % polyacrylamide gels, specific activity and their filtration patterns through Sephadex G-200 columns were closely similar. Then, S-35-heparin (0.5 mg) and cold heparin (27 mg) were simultaneously injected intravenously into 5 dogs and their plasma behavior was compared. The plasma half-lives of S-35-heparin averaged 1.18 ± 0. 13 (SD) hr and was identical with that of cold heparin, but the half-lives were much shorter and averaged 0.46 ± 0.08 (SD) hr in 5 dogs when S-35-heparin alone was injected. Studies were next made of heparin affinity to proteins including canine antithrombin III (AT) by the use of Sephadex G-200 chromatography, polyacrylamide gel electrophoresis, and S-35-heparin as a tracer. It was found that S-35-heparin-binding to alpha1, beta, and gammaglobulins and fibrinogen was readily separable upon addition of 5 mg cold heparin, but that the binding to AT was inseparable by addition of cold heparin or by electrophoresis. However, in canine plasma, both S-35-heparin and cold heparin were mostly bound to proteins other than AT, suggesting that this might be the case in vivo. To further substantiate this, studies were made of the comparative behavior of I-125-labeled AT (I-125-AT) and S-35-heparin in dogs with the idea that the plasma half-lives of both should be equal if they were irreversibly bound to each other. The plasma half-lives of I-125-AT averaged 2.10 ± 0.05 (SD) days in 5 male dogs and 1.99 ± 0.04 (SD) days in 5 female dogs, and were much different from the half-life values of S-35-heparin as given above. These results indicate that heparin in vitro is more tightly bound to AT than to other proteins, that heparin in vivo is not irreversibly bound to AT and suggest that it is mostly bound to proteins other than AT in vivo.

scholarly journals Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 394-400 ◽  
Author(s):  
WF Novotny ◽  
M Palmier ◽  
TC Wun ◽  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Vascular Endothelium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Major Band ◽  
Coagulation Inhibitor

Abstract The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

scholarly journals THE DIFFERENTIATION OF MONONUCLEAR PHAGOCYTES

1965 ◽  
Vol 121 (1) ◽  
pp. 153-170 ◽  
Author(s):  
Zanvil A. Cohn ◽  
Belinda Benson
Keyword(s):  
Acid Phosphatase ◽  
Specific Activity ◽  
Total Content ◽  
Progressive Increase ◽  
Neutral Red ◽  
Mononuclear Phagocytes ◽  
In Vitro Differentiation ◽  
Dense Granules

The in vitro differentiation of homogeneous populations of monocyte-like cells from the unstimulated mouse peritoneal cavity is described. Under the conditions employed, a progressive increase in cell size occurs without significant cell division. This process is characterized morphologically by the accumulation of phase-dense and neutral red-positive granules, mitochondria, and lipid droplets. The phase-dense granules react strongly for acid phosphatase. Biochemical determinations indicate marked increases in the total content and specific activity of acid phosphatase, cathepsin, and ß-glucuronidase. The production of acid phosphatase is more rapid and extensive than that of the other two hydrolases. From these data it appears that the conversion of a monocyte-like cell to a mature macrophage is accompanied by the formation of increased numbers of lysosome-like cytoplasmic organelles. Mouse peritoneal phagocytes stimulated in vivo with a bacterial lipopolysaccharide undergo a similar series of morphological and biochemical events.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

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.

scholarly journals A novel PET tracer 18F-deoxy-thiamine: synthesis, metabolic kinetics, and evaluation on cerebral thiamine metabolism status

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Changpeng Wang ◽  
Siwei Zhang ◽  
Yuefei Zou ◽  
Hongzhao Ma ◽  
Donglang Jiang ◽  
...  
Keyword(s):  
High Performance ◽  
Specific Activity ◽  
High Accumulation ◽  
Metabolic Kinetics ◽  
Thiamine Metabolism ◽  
Pet Tracer ◽  
The Status ◽  
The Brain

Abstract Background Some neuropsychological diseases are associated with abnormal thiamine metabolism, including Korsakoff–Wernicke syndrome and Alzheimer’s disease. However, in vivo detection of the status of brain thiamine metabolism is still unavailable and needs to be developed. Methods A novel PET tracer of 18F-deoxy-thiamine was synthesized using an automated module via a two-step route. The main quality control parameters, such as specific activity and radiochemical purity, were evaluated by high-performance liquid chromatography (HPLC). Radiochemical concentration was determined by radioactivity calibrator. Metabolic kinetics and the level of 18F-deoxy-thiamine in brains of mice and marmosets were studied by micro-positron emission tomography/computed tomography (PET/CT). In vivo stability, renal excretion rate, and biodistribution of 18F-deoxy-thiamine in the mice were assayed using HPLC and γ-counter, respectively. Also, the correlation between the retention of cerebral 18F-deoxy-thiamine in 60 min after injection as represented by the area under the curve (AUC) and blood thiamine levels was investigated. Results The 18F-deoxy-thiamine was stable both in vitro and in vivo. The uptake and clearance of 18F-deoxy-thiamine were quick in the mice. It reached the max standard uptake value (SUVmax) of 4.61 ± 0.53 in the liver within 1 min, 18.67 ± 7.04 in the kidney within half a minute. The SUV dropped to 0.72 ± 0.05 and 0.77 ± 0.35 after 60 min of injection in the liver and kidney, respectively. After injection, kidney, liver, and pancreas exhibited high accumulation level of 18F-deoxy-thiamine, while brain, muscle, fat, and gonad showed low accumulation concentration, consistent with previous reports on thiamine distribution in mice. Within 90 min after injection, the level of 18F-deoxy-thiamine in the brain of C57BL/6 mice with thiamine deficiency (TD) was 1.9 times higher than that in control mice, and was 3.1 times higher in ICR mice with TD than that in control mice. The AUC of the tracer in the brain of marmosets within 60 min was 29.33 ± 5.15 and negatively correlated with blood thiamine diphosphate levels (r = − 0.985, p = 0.015). Conclusion The 18F-deoxy-thiamine meets the requirements for ideal PET tracer for in vivo detecting the status of cerebral thiamine metabolism.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

In-vivo and in-vitro secretion of prolactin by lactating rat adenohypophyses as a function of intracellular age

1984 ◽  
Vol 101 (1) ◽  
pp. 27-32 ◽  
Author(s):  
F. Mena ◽  
G. Martínez-Escalera ◽  
C. Clapp ◽  
C. E. Grosvenor
Keyword(s):  
Pituitary Gland ◽  
Incubation Period ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Pituitary Glands ◽  
H 26

ABSTRACT Adenohypophysial prolactin of lactating rats was pulse-labelled by [3H]leucine injected i.v. at the time of removal of the pups. The [3H]prolactin concentration in the pituitary gland, analysed by polyacrylamide-gel electrophoresis, progressively fell as the time from labelling to removal of the pituitary gland increased from 8 to 24 h, which suggests that there was a loss of hormone as it aged within the gland. Suckling effectively provoked the depletion–transformation of total and [3H]prolactin (extracted at pH 7·2) when applied after 8 h but not when applied after either 16 or 24 h after removing the pups. In rats whose pups were removed for 8 h, suckling also depleted–transformed [3H]prolactin labelled 4 h, but not that labelled 1 h before suckling. The pituitary glands of other lactating rats were labelled with [3H]leucine injected i.v. at various times before removing the glands and incubating them in medium 199. The secretion into the medium of [3H]prolactin labelled either 4, 8, 16 or 24 h beforehand was maximal during the first 30 min then declined from 30 to 240 min of incubation. However, secretion of prolactin labelled 1 h and 10 min beforehand reached a maximum after 0·5–1 h and 2 h of incubation respectively, then remained constant during the remainder of the 4-h incubation period. The total 4-h secretion of [3H]prolactin was greatest (65% of preincubation concentration) from those glands labelled 4 h before in contrast to those labelled 10 min (15%) or 1 (38%), 8 (34%), 16 (18%) or 24 h (26%) before incubation. Taken together, these data suggest that prolactin synthesized 4 h earlier is more likely to be released in response to physiological stimuli than is more recently formed prolactin or prolactin which has remained in the pituitary gland for 16 h or longer. J. Endocr. (1984) 101, 27–32

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