scholarly journals ISOLATION OF PARIETAL CELLS FROM GLUTARALDEHYDE-FIXED RABBIT STOMACH

1972 ◽  
Vol 20 (8) ◽  
pp. 634-643 ◽  
Author(s):  
SIN HANG LEE
Keyword(s):  
Specific Activity ◽  
Proteolytic Enzymes ◽  
High Specific Activity ◽  
Cell Types ◽  
Celiac Artery ◽  
Parietal Cells ◽  
Adult Rabbit ◽  
Isolated Cells ◽  
Discontinuous Sucrose Gradient ◽  
Final Yield

A method is introduced to isolate and purify parietal cells from the rabbit stomach which has been briefly perfused with 1% glutaraldehyde via the celiac artery. The fixed mucosa is initially digested with 1% trypsin and subsequently with 1% collagenase solution. The parietal cells are more resistant to the effects of these proteolytic enzymes than the other cell types in the gastric mucosa and can be recovered and purified by centrifugation in a discontinuous sucrose gradient. Glutaraldehyde fixation reduces the tendency of cell clumping and, therefore, facilitates isolation of the separated cells. The final yield from each adult rabbit stomach varies from 0.1 to 0.4 ml packed parietal cells of about 97% purity determined by nuclear counts. The nuclei and mitochondria of most of the isolated cells appear to be intact on electron micrographs, but the cell membrane is focally disrupted; the nuclear deoxyribonucleic acid (DNA) can be stained with Feulgen technique and extracted with perchloric acid for quantitative determination. Biochemical studies on incorporation of thymidine-3H into DNA of the parietal cells have been carried out in normal young adult rabbits and in rabbits under chronic histamine stimulation. The results show that there is no significant incorporation of thymidine-3H by mature parietal cells in any of the two groups of animals, although the whole mucosa DNA hydrolysates always exhibit a high specific activity.

scholarly journals Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 1002-1005 ◽  
Author(s):  
J Cashman ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Growth Medium ◽  
Specific Activity ◽  
High Specific Activity ◽  
Cell Types ◽  
Hematopoietic Progenitor Cell ◽  
Specific Cell ◽  
Quiescent State ◽  
Hematopoietic Progenitor ◽  
Marrow Cultures

We have examined the cycling status of various classes of erythroid and granulopoietic progenitor populations maintained for many weeks in standard normal long-term human marrow cultures. These were initiated with a single inoculum of marrow aspirate and were routinely fed by weekly removal of half of the nonadherent cells and replacement of half of the growth medium. Progenitors of large erythroid colonies (more than eight erythroblast clusters) present in the nonadherent fraction and progenitors of small granulocyte/macrophage colonies (fewer than 500 cells) present in both the nonadherent and adherent fractions were found to be actively cycling at all times examined (28% to 63% kill following a 20-minute exposure to 20 microCi/mL of high specific activity 3H-thymidine). In contrast, progenitors of large granulocyte/macrophage colonies (more than 500 cells) and progenitors of large erythroid colonies (more than eight erythroblast clusters), present in the adherent layer, consistently alternated between a quiescent state at the time of each weekly medium change and a proliferating state two to three days later (0% to 13% kill and 21% to 49% kill, respectively). Additional experiments revealed that the activation of primitive progenitors in the adherent layer was not dependent on the addition of fresh glutamine or hydrocortisone, nor on the physical manipulations involved in changing the growth medium. These studies provide the first direct evidence that normal long-term human marrow cultures support the continued turnover of a variety of early hematopoietic progenitor cell types. Further, they indicate that the proliferative activity of the most primitive of these progenitors is regulated by stage-specific cell-cell interactions that are subject to manipulation.

scholarly journals SELECTED PROTEOLYTIC ACTIVITY BY EXTRACTS OF DERMESTID LARVAE

2011 ◽  
Vol 19 (19) ◽  
pp. 79-83
Author(s):  
Lavinel G. IONESCU
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Proteolytic Activity ◽  
Specific Activity ◽  
Proteolytic Enzymes ◽  
High Specific Activity ◽  
Water Extract ◽  
Dermestes Maculatus ◽  
Crude Preparation

The larvae of the Beetle Dermestes maculatus De Geer can subsist on a diet consisting largely of protein. Studies have been undertaken to investigate the nature of proteolytic enzymes. A water extract of the larvae yielded a crude preparation that hydrolyzes gelatin, bide powder, hemoglobin substrate, benzoyl-DL-arginine p-nitroamilide, and glutaryl-L-phenylalanine p-nitroanilide. Enzyme activity was found in a non-dialyzable, heat- and acid0labile portion of the extract yielded two fractions with high specific activity towards gelatin. These are precipitated between 40% to 60% saturation of ammonium sulfate and 60% to 80% saturation. The higher specific activity was observed in the 40%-60% fraction. These results suggest that the larvae of these dermestids contain proteolytic enzymes with actions similar to mammalian trypsin and chymotrypsin. The results also suggest that other proteolytic enzymes may be present as well.

scholarly journals Detection of factor XIIIa (active fibrin-stabilizing factor) in normal plasma

Blood ◽  
1978 ◽  
Vol 52 (3) ◽  
pp. 581-591 ◽  
Author(s):  
JC Nelson ◽  
RG Lerner
Keyword(s):  
Factor Xiii ◽  
Specific Activity ◽  
Proteolytic Enzymes ◽  
High Specific Activity ◽  
Factor Xa ◽  
Factor Xiiia ◽  
Calcium Dependent ◽  
Normal Plasma ◽  
Plasma Factor

Abstract Factor XIIIa (active fibrin-stabilizing factor) generated in heat- defibrinated plasma by the addition of thrombin can be measured by 14C- putrescine incorporation into casein. Modification of this assay be substituting 3H-putrescine of high specific activity as the donor amine permits measurement of amine incorporation by plasma even in the absence of added thrombin. Incorporation is calcium dependent, inhibited by iodoacetamide, and absent from congenital factor XIII- deficient plasma and from normal platelets. The transamidating activity detected by radioenzymatic assay catalyzed the formation of gamma-gamma dimers and alpha polymers of fibrin and was thus biologically functional. This fibrin cross-linking activity was absent from factor XIII-deficient plasma. These experiments show (1) some factor XIII is present in plasma as factor XIIIa; (2) this factor XIIIa can cross-link fibrin and thus has biologic activity as well; and (3) this activity is not present in factor XIII-deficient plasma. Factor XIIIa in normal plasma is possibly activated in vivo, perhaps by circulating thrombin, factor Xa, or other proteolytic enzymes.

scholarly journals Isolation and separation of highly enriched fractions of viable mouse gastric parietal cells by velocity sedimentation.

1975 ◽  
Vol 65 (2) ◽  
pp. 428-438 ◽  
Author(s):  
L J Romrell ◽  
M R Coppe ◽  
D R Munro ◽  
S Ito
Keyword(s):  
Structural Integrity ◽  
Light And Electron Microscopy ◽  
Cell Types ◽  
Parietal Cells ◽  
Velocity Sedimentation ◽  
Isolated Cells ◽  
Adult Mice ◽  
Tissue Dissociation ◽  
Cell Fractions ◽  
Gastric Parietal Cells

Methods of tissue dissociation and cell separation have been modified to obtain highly enriched fractions of mouse gastric parietal cells. Suspension of gastric mucosal cells are prepared by pronase digestion of the glandular portion of the stomach from adult mice. By utilizing the velocity sedimentation technique to separate cells of different sizes it is possible to recovery parietal cells, which are larger than the other cell types, in fractions with purity of 75-95%. The homogeneity of cell fractions has been assessed by light and electron microscopy. The ability of the isolated cells to exclude the dye trypan blue, to incorporate labeled substrate, to consume oxygen, and to retain their structural integrity indicates that they are viable and still capable of functional activity.

Cation transport in lung epithelial cells derived from fetal, newborn, and adult rabbits

1986 ◽  
Vol 61 (2) ◽  
pp. 507-515 ◽  
Author(s):  
R. D. Bland ◽  
C. A. Boyd
Keyword(s):  
Epithelial Cells ◽  
Proteolytic Enzymes ◽  
Cation Transport ◽  
Lung Epithelial Cells ◽  
Adult Rabbit ◽  
Alveolar Type ◽  
Isolated Cells ◽  
Lung Maturation ◽  
Fetal Cells ◽  
Lung Epithelial

Recent studies done with fetal and adult sheep and with monolayers of cultured rat alveolar type II cells suggest that active transport of Na+ across the lung epithelium may contribute to liquid absorption from air spaces, an essential component of the normal switch from placental to pulmonary gas exchange at birth. The goals of this work were 1) to study the ontogeny of cation transport in lung epithelial cells derived from fetal, newborn, and adult rabbits and 2) to determine the influence of premature birth, air breathing, labor, and postnatal lung maturation on K+ uptake in these cells. We harvested granular pneumonocytes by tracheal instillation of proteolytic enzymes followed by centrifugation of the dispersed cells over a discontinuous density gradient of metrizamide. This procedure yielded 65–90% granular pneumonocytes, of which more than 80% excluded vital dye. Using freshly isolated cells, we measured uptake of 86Rb+, which mimics transmembrane movement of K+, in the presence or absence of 10(-4) M ouabain and in the presence or absence of 5 X 10(-4) M furosemide or bumetanide. In adult rabbit studies, 86Rb+ uptake was twice as fast in lung epithelial cells (98 +/- 7 nmol X 10(6) cells-1 X h-1) as it was in alveolar macrophages (51 +/- 6 nmol X 10(6) cells-1 X h-1). Ouabain inhibited 55–60% of the uptake by pneumonocytes, and “loop” diuretics inhibited an additional 15–20%. The rate of 86Rb+ uptake in fetal cells was less than 10% (6 +/- 1 nmol X 10(6) cells-1 X h-1) of the rate in adult cells; ouabain inhibited 80-85% of 86Rb+ uptake in fetal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 1002-1005 ◽  
Author(s):  
J Cashman ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Growth Medium ◽  
Specific Activity ◽  
High Specific Activity ◽  
Cell Types ◽  
Hematopoietic Progenitor Cell ◽  
Specific Cell ◽  
Quiescent State ◽  
Hematopoietic Progenitor ◽  
Marrow Cultures

Abstract We have examined the cycling status of various classes of erythroid and granulopoietic progenitor populations maintained for many weeks in standard normal long-term human marrow cultures. These were initiated with a single inoculum of marrow aspirate and were routinely fed by weekly removal of half of the nonadherent cells and replacement of half of the growth medium. Progenitors of large erythroid colonies (more than eight erythroblast clusters) present in the nonadherent fraction and progenitors of small granulocyte/macrophage colonies (fewer than 500 cells) present in both the nonadherent and adherent fractions were found to be actively cycling at all times examined (28% to 63% kill following a 20-minute exposure to 20 microCi/mL of high specific activity 3H-thymidine). In contrast, progenitors of large granulocyte/macrophage colonies (more than 500 cells) and progenitors of large erythroid colonies (more than eight erythroblast clusters), present in the adherent layer, consistently alternated between a quiescent state at the time of each weekly medium change and a proliferating state two to three days later (0% to 13% kill and 21% to 49% kill, respectively). Additional experiments revealed that the activation of primitive progenitors in the adherent layer was not dependent on the addition of fresh glutamine or hydrocortisone, nor on the physical manipulations involved in changing the growth medium. These studies provide the first direct evidence that normal long-term human marrow cultures support the continued turnover of a variety of early hematopoietic progenitor cell types. Further, they indicate that the proliferative activity of the most primitive of these progenitors is regulated by stage-specific cell-cell interactions that are subject to manipulation.

scholarly journals Detection of factor XIIIa (active fibrin-stabilizing factor) in normal plasma

Blood ◽  
1978 ◽  
Vol 52 (3) ◽  
pp. 581-591
Author(s):  
JC Nelson ◽  
RG Lerner
Keyword(s):  
Factor Xiii ◽  
Specific Activity ◽  
Proteolytic Enzymes ◽  
High Specific Activity ◽  
Factor Xa ◽  
Factor Xiiia ◽  
Calcium Dependent ◽  
Normal Plasma ◽  
Plasma Factor

Factor XIIIa (active fibrin-stabilizing factor) generated in heat- defibrinated plasma by the addition of thrombin can be measured by 14C- putrescine incorporation into casein. Modification of this assay be substituting 3H-putrescine of high specific activity as the donor amine permits measurement of amine incorporation by plasma even in the absence of added thrombin. Incorporation is calcium dependent, inhibited by iodoacetamide, and absent from congenital factor XIII- deficient plasma and from normal platelets. The transamidating activity detected by radioenzymatic assay catalyzed the formation of gamma-gamma dimers and alpha polymers of fibrin and was thus biologically functional. This fibrin cross-linking activity was absent from factor XIII-deficient plasma. These experiments show (1) some factor XIII is present in plasma as factor XIIIa; (2) this factor XIIIa can cross-link fibrin and thus has biologic activity as well; and (3) this activity is not present in factor XIII-deficient plasma. Factor XIIIa in normal plasma is possibly activated in vivo, perhaps by circulating thrombin, factor Xa, or other proteolytic enzymes.

Coexistence of two biochemically distinct phospholipase A2 activities in human platelet, monocyte, and neutrophil

1993 ◽  
Vol 71 (7-8) ◽  
pp. 331-339 ◽  
Author(s):  
Lisa A. Marshall ◽  
Amy Roshak
Keyword(s):  
Arachidonic Acid ◽  
Specific Activity ◽  
Human Platelet ◽  
High Specific Activity ◽  
Cell Types ◽  
Phospholipase A ◽  
Joint Fluid ◽  
Synovial Joint ◽  
Monocytic Cell ◽  
Activity Measurements

The cell-associated phospholipase A2 (PLA2) activities of the human platelet, neutrophil, and monocyte were simultaneously characterized, utilizing the biochemical differences observed between the 14 kDa (kilodalton), type II PLA2 isolated from inflammatory human synovial joint fluid (HSF) and the arachidonic acid (AA) specific, 85-kDa high molecular mass (HMM) PLA2 isolated from the cytosol of a U937 monocytic cell line. The HSF PLA2 can be distinguished from the HMM PLA2 by its resistance to acid treatment, sensitivity to a sulfhydryl reducing agent, lack of preference for the fatty acid on the sn-2 position of phospholipid substrate, and inhibition by the C-7 phosphonate–phospholipid transition-state PLA2 inhibitor. Evaluation of all three cell types revealed that HMM-like PLA2 activity was found predominantly in the cytosolic fractions, although detection in neutrophil cytosol required more concentrated preparations and the use of high specific activity [3H]AA-labeled Escherichia coli. HSF-PLA2-like activity was measured in microsomal and cytosolic fractions of all three cell types, but was found in neutrophil cytosol only after treatment with acid. Further HMM-PLA2-specific interfering agents in neutrophil cytosol were observed and exemplifies one problem in assigning the existence of this enzyme in crude broken cell preparations using activity measurements alone. The role that these two enzymes play in eicosanoid production of the respective cell types remains to be studied.Key words: phospholipase A2, arachidonic acid, neutrophil, platelet, monocyte, cytosol, microsome.

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.

Autoprothrombin C Activity from Prothrombin with Snake Venom or Trypsin

1962 ◽  
Vol 08 (03) ◽  
pp. 425-433 ◽  
Author(s):  
Ewa Marciniak ◽  
Edmond R Cole ◽  
Walter H Seegers
Keyword(s):  
Snake Venom ◽  
Thrombolytic Agent ◽  
Sodium Citrate ◽  
Specific Activity ◽  
High Specific Activity ◽  
Citrate Solution ◽  
Clotting Factors ◽  
Activation Products ◽  
Russell’S Viper ◽  
Autoprothrombin C

SummarySuitable conditions were found for the generation of autoprothrombin C from purified prothrombin with the use of Russell’s viper venom or trypsin. DEAE chromatographed prothrombin is structurally altered and has never been found to yield autoprothrombin C and also did not yield it when Russell’s viper venom or trypsin were used. Autoprothrombin C is derived from prothrombin with tissue extract thromboplastin, but not in large amounts with the intrinsic clotting factors. With the latter thrombin and autoprothrombin III are the chief activation products. Autoprothrombin III concentrates were prepared from serum and upon activation with 25% sodium citrate solution or with Russell’s viper venom large amounts of autoprothrombin C were obtained, and this was of high specific activity. Theoretically trypsin is not a thrombolytic agent, but on the contrary should lead to intravascular clotting.

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