scholarly journals Changes in activities and isozyme patterns of glycolytic enzymes during erythroid differentiation in vitro

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 607-613 ◽  
Author(s):  
W Nijhof ◽  
PK Wierenga ◽  
GE Staal ◽  
G Jansen
Keyword(s):  
Pyruvate Kinase ◽  
Gradient Centrifugation ◽  
Recovery Period ◽  
Erythroid Differentiation ◽  
Glycolytic Enzyme ◽  
Minor Amount ◽  
Type I ◽  
In Vitro Differentiation ◽  
Percoll Density Gradient Centrifugation

Abstract Late committed progenitor cells of erythropoiesis, CFU-E (colony- forming unit--erythroid), were isolated from mouse spleens to near homogeneity by a three-step enrichment procedure. The procedure included a four-day pretreatment of bled mice with the antibiotic thiamphenicol, a recovery period of 3 1/2 days, followed by centrifugal elutriation and Percoll density gradient centrifugation of the spleen cells. This practically pure CFU-E population was used to study some aspects of erythroid differentiation in vitro. Colony growth, as well as morphology and glycolytic enzyme activities of cells isolated at selected times of the 48-hour culture period, were determined. Marked declining activities of several enzymes, including hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase, and glucose-6- phosphate dehydrogenase, were observed during in vitro differentiation. The activity of diphosphoglycerate mutase was almost absent in the CFU- E, but progressively increased during differentiation. The isozyme distribution of aldolase and enolase did not change during CFU-E in vitro differentiation into the reticulocyte. Hexokinase (HK) in the CFU- E contained mainly a double-banded type I isozyme, in addition to a minor amount of HK II. During differentiation, a shift was noticed within the double-banded HK I region, whereas HK ii disappeared after one cell division. Pyruvate kinase in the CFU-E was characterized by the presence of both the K-type and the L-type isozyme and hybrids of these isozyme types. During in vitro differentiation, the production of the K-type isozyme rapidly stops in favor of the L type.

scholarly journals Changes in activities and isozyme patterns of glycolytic enzymes during erythroid differentiation in vitro

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 607-613 ◽  
Author(s):  
W Nijhof ◽  
PK Wierenga ◽  
GE Staal ◽  
G Jansen
Keyword(s):  
Pyruvate Kinase ◽  
Gradient Centrifugation ◽  
Recovery Period ◽  
Erythroid Differentiation ◽  
Glycolytic Enzyme ◽  
Minor Amount ◽  
Type I ◽  
In Vitro Differentiation ◽  
Percoll Density Gradient Centrifugation

Late committed progenitor cells of erythropoiesis, CFU-E (colony- forming unit--erythroid), were isolated from mouse spleens to near homogeneity by a three-step enrichment procedure. The procedure included a four-day pretreatment of bled mice with the antibiotic thiamphenicol, a recovery period of 3 1/2 days, followed by centrifugal elutriation and Percoll density gradient centrifugation of the spleen cells. This practically pure CFU-E population was used to study some aspects of erythroid differentiation in vitro. Colony growth, as well as morphology and glycolytic enzyme activities of cells isolated at selected times of the 48-hour culture period, were determined. Marked declining activities of several enzymes, including hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase, and glucose-6- phosphate dehydrogenase, were observed during in vitro differentiation. The activity of diphosphoglycerate mutase was almost absent in the CFU- E, but progressively increased during differentiation. The isozyme distribution of aldolase and enolase did not change during CFU-E in vitro differentiation into the reticulocyte. Hexokinase (HK) in the CFU- E contained mainly a double-banded type I isozyme, in addition to a minor amount of HK II. During differentiation, a shift was noticed within the double-banded HK I region, whereas HK ii disappeared after one cell division. Pyruvate kinase in the CFU-E was characterized by the presence of both the K-type and the L-type isozyme and hybrids of these isozyme types. During in vitro differentiation, the production of the K-type isozyme rapidly stops in favor of the L type.

Inhibition Of Collagen And ADP-Induced Platelet Aggregation By Plasma Fibronectin

1981 ◽  
Author(s):  
D G Moon ◽  
J E Kaplan
Keyword(s):  
Gradient Centrifugation ◽  
Fold Increase ◽  
Lag Time ◽  
Collagen Type I ◽  
Type I ◽  
Plasma Fibronectin ◽  
Aggregation Rate ◽  
Tail Tendon ◽  
Induced Aggregation

Platelets contain fibronectin a glycoprotein with an established affinity for collagen. This observation has led other investigators to postulate that fibronectin is the platelet collagen receptor. The much greater concentration of fibronectin in the plasma surrounding platelets, however, has led us to suggest that plasma fibronectin may bind to collagen and competitively inhibit the platelet- collagen interaction. Rat platelets were isolated by Stractan density gradient centrifugation and aggregated with acid-solubilized rat tail tendon collagen (Type I) in a Payton 300B Aggregometer. Fibronectin was twice purified by affinity chromatography with gelatin linked to CNBr- activated Sepharose 4B. Simultaneous addition of 50 μg fibronectin and 25 μg collagen to platelets suspended in Tyrodes solution at 37°C resulted in a 2-fold increase in lag time and a 30% decrease in aggregation rate as compared to control values. When collagen was preincubated in Tyrodes solution for 12 minutes at 26°C without platelets to allow for prior fibrillogenesis, the addition of 50 μg fibronectin with the platelets resulted in <20% increase in lag time and a 20-30% decrease in aggregation rate. In a separate series of experiments, fibronectin was also found to inhibit ADP-induced aggregation. In this case, the initial rate of aggregation was comparable with and without fibronectin, but this maximal rate was maintained for a shorter period in the presence of fibronectin. Thus, fibronectin reduced the in vitro aggregation response to two different physiological stimuli. Our data supports previous studies which indicate that fibronectin reduces the reactivity of platelets with collagen and provides evidence of a role for fibronectin in modulating platelet responses in the absence of collagen.

scholarly journals Induction of in vitro differentiation of mouse embryonal carcinoma (F9) and erythroleukemia (MEL) cells by herbimycin A, an inhibitor of protein phosphorylation.

1989 ◽  
Vol 109 (1) ◽  
pp. 285-293 ◽  
Author(s):  
K Kondo ◽  
T Watanabe ◽  
H Sasaki ◽  
Y Uehara ◽  
M Oishi
Keyword(s):  
Tyrosine Kinases ◽  
Embryonal Carcinoma ◽  
Erythroid Differentiation ◽  
In Vitro Differentiation ◽  
Streptomyces Species ◽  
Rous Sarcoma ◽  
Herbimycin A ◽  
Sarcoma Virus ◽  
Cellular Phenotypes

Herbimycin A is one of the benzenoid ansamycin antibiotics isolated from a culture of Streptomyces species (Omura, S., A. Nakagawa, and N. Sadakane. 1979. Tetrahedron Lett. 1979: 4323-4326). Recent studies have shown that the antibiotic not only inhibits the phosphorylation of p60src in Rous sarcoma virus- (RSV) infected cells, but also reverses the cellular phenotypes acquired by transfection with tyrosine kinase oncogenes (Uehara, Y., M. Hori, T. Takeuchi, and H. Umezawa. 1985. Jpn. J. Cancer Res. 76:672-675; Uehara, Y., M. Hori, T. Takeuchi, and H. Umezawa. 1986. Mol. Cell. Biol. 6: 2198-2206; Uehara, Y., Y. Murakami, S. Mizuno, and S. Kawai. 1988. Virology. 164: 294-298). These studies and other evidence indicate that the antibiotic inhibits a reaction(s) closely associated with the function of cellular tyrosine kinases. We have found that herbimycin A is an effective inducing agent capable of triggering differentiation in two typical mouse in vitro differentiation systems, which have been considered to be quite different in their mechanism of induction: endoderm differentiation of embryonal carcinoma (F9) cells and terminal erythroid differentiation of erythroleukemia (MEL) cells. The results suggest that there is a common step in the intracellular differentiation cascade which is, directly or indirectly, associated with phosphorylation at specific (tyrosine) residues of cellular proteins. The significance of this finding with respect to the molecular mechanism of in vitro differentiation is discussed.

scholarly journals Lysosomes, but not mitochondria, accumulate iron and porphyrins in porphyria induced by hexachlorobenzene

1986 ◽  
Vol 235 (3) ◽  
pp. 671-675 ◽  
Author(s):  
A Tangerås
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Iron Status ◽  
Gradient Centrifugation ◽  
Female Rats ◽  
Percoll Density Gradient Centrifugation ◽  
Mitochondrial Fractions ◽  
Percoll Density Gradient ◽  
Haem Iron

In female rats with porphyria induced by hexachlorobenzene, the amounts of non-haem iron and porphyrins in liver mitochondrial fractions were increased almost 3-fold and greater than 500-fold respectively compared with that of untreated animals. A considerable fraction of both iron and porphyrins in this fraction was shown to be located in lysosomes. Thus mitochondrial preparations, which were further depleted of lysosomes by Percoll-density-gradient centrifugation, contained 2.78 +/- 0.75 and 2.99 +/- 0.49 nmol of non-haem iron/mg of protein when isolated from the liver of control rats and hexachlorobenzene-treated rats respectively. Mitochondria isolated from the liver of hexachlorobenzene-treated animals contained a pool of iron (about 1 nmol/mg of protein) that was available for haem synthesis in vitro. This pool is similar to that previously reported for mitochondria isolated from the liver of rats with normal haem synthesis. Hexachlorobenzene treatment, therefore, does not affect the iron status of the mitochondria.

Cow red blood cells. I. Effect of purines, pyrimidines, and nucleosides in bovine red cell glycolysis

1979 ◽  
Vol 236 (5) ◽  
pp. C255-C261 ◽  
Author(s):  
M. J. Seider ◽  
H. D. Kim
Keyword(s):  
Pyruvate Kinase ◽  
Enzyme Activities ◽  
Blood Cells ◽  
Glycolytic Enzyme ◽  
Red Cells ◽  
The Other ◽  
Red Cell ◽  
Hexokinase Activity ◽  
Glycolytic Rate

Cow red cells, under in vitro incubation conditions, exhibit a comparatively low glycolytic rate of 0.56 +/- 0.05 micromol/(ml cells.h), with a ratio of lactate formed to glucose consumed of 1.58. It has been found that this low glycolytic rate can be stimulated 50--60% above the basal level in the presence of a variety of purine and pyrimidine compounds including adenosine, inosine, adenine, hypoxanthine, xanthine, and uracil. In contrast, calf red cells, which have a much higher glycolytic rate, display no discernible response to these agents. In attempts to elucidate the mechanism by which this stimulation takes place, both glucose transport and glycolytic enzyme activities were determined in the presence of these stimulators. Glucose influx in cow red cells, measured using the glucose analog 3-O-methyl-glucose, exhibits both a low Km of 117 microM and a Vmax of 0.38 micromol/(ml cells.min), and is unaltered in the presence of adenosine. On the other hand, hexokinase, which in normal hemolysates of cow red cells has an activity of 0.49 +/- 0.03 micromol/(g Hb.min). was found to be stimulated to 0.73 micromol/(g Hb.min) in the presence of adenine. Both pyruvate kinase and phosphofructokinase were unaffected by this compound. These data suggest that certain purines and pyrimidine compounds may exert their stimulatory effect on hexokinase activity, resulting in an augmentation of cow red cell glycolysis.

scholarly journals Differences in myeloperoxidase activity from neutrophilic polymorphonuclear leukocytes of differing density: relationship to selective exocytosis of distinct forms of the enzyme

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1116-1124
Author(s):  
SO Pember ◽  
JM Jr Kinkade
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Human Peripheral Blood ◽  
Gradient Centrifugation ◽  
Fold Increase ◽  
Myeloperoxidase Activity ◽  
Percoll Density Gradient Centrifugation ◽  
Density Relationship ◽  
Neutrophilic Polymorphonuclear Leukocytes

Elicited murine neutrophilic polymorphonuclear leukocytes (PMN) were fractionated by Percoll density gradient centrifugation into high density (HD) and intermediate density (ID) populations. As described in the accompanying article HD- and ID-PMN appear to represent “resting” and “activated” cell populations, respectively. Consistent with this possibility, histochemical and biochemical evidence suggested that ID- PMN were degranulated compared to HD-PMN. Myeloperoxidase (MPO) in the ID-PMN population showed increased sensitivity to inhibition by 3-amino- 1,2,4-triazole, and HD-PMN exhibited a 2–3-fold increase in chloride and iodide oxidation per unit of MPO activity compared to ID-PMN. When HD-PMN were induced to degranulate in vitro, the remaining cell- associated MPO displayed enzymatic properties characteristic of the activity associated with ID-PMN. The mechanism of this phenomenon was also investigated in vitro using purified human peripheral blood PMN and the synthetic chemotactic peptide N-formyl-methionyl-leucyl- phenylalanine. Differences in cell-associated MPO activity were shown to be related to selective exocytosis of enzymatically and chromatographically distinct forms of the enzyme. These data indicate that, in addition to the well known selective exocytosis of specific and azurophilic granules induced by various agents, selectivity may also occur at the level of enzymatically distinct forms of a particular granule enzyme. Moreover, our observations provide further evidence that density differences may be utilized to fractionate and study the generation of functionally distinct subpopulations of PMN that arise in vivo as well as in vitro following exposure to various stimuli.

scholarly journals Retrieval of precursors for white-type adipose conversion in brown adipose tissue

1988 ◽  
Vol 255 (3) ◽  
pp. 849-854 ◽  
Author(s):  
C M Poissonnet ◽  
M Ouagued ◽  
Y Aron ◽  
J Y Pello ◽  
E Swierczewski ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Gradient Centrifugation ◽  
Cellular Compartment ◽  
Percoll Density Gradient Centrifugation ◽  
Brown Adipose ◽  
Adipose Conversion ◽  
Percoll Density Gradient ◽  
The One

A cellular compartment from brown adipose tissue (BAT) of newborn rats was isolated by Percoll-density-gradient centrifugation and was shown to proliferate and to undergo adipose conversion in vitro in primary culture. The features of the effector requirement for adipose conversion as well as the differentiated morphological and biochemical phenotype are almost identical with that of a compartment designated HCF, from white adipose tissue (WAT). A possible role for these precursors from BAT and WAT in the involution of BAT into WAT, on the one hand, and in the development of brown adipose cells among typical WAT deposits, on the other, is discussed.

Mesenchymal Stem Cells (MSCs) in Targeted Drug Delivery: Literature Review and Exploratory Data on Migrating and Differentiation Capacities of Bone MSCs into Hepatic Progenitor Cells

2021 ◽  
Vol 21 ◽  
Author(s):  
Xulong Zhu ◽  
Tan Yan ◽  
Chong Cheng ◽  
Jia Ma ◽  
Junxi Xiang ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Progenitor Cells ◽  
Targeted Drug Delivery ◽  
Type I ◽  
Hepatic Progenitor Cells ◽  
In Vitro Differentiation ◽  
Targeted Drug

Background and Objective: Mesenchymal stem cells (MSCs), particularly bone MSCs (BMSCs) offer great potentials for targeted therapeutic applications due to their migratory and differentiation capacities. Significant advances have been achieved in the differentiation of hepatocyte or hepatocyte-like cells both in vitro and in vivo. However, there is limited knowledge on the differentiation of BMSCs into bipotential hepatic progenitor cells or cholangiocytes. This study reviews the potentials and advances in using MSCs as vehicles for targeted drug delivery and proposes a new method for induction of differentiation in rat BMSCs into hepatic progenitor cells in vitro, and assesses the differential and migratory capacities. Methods: The BMSCs of Sprague Dawley (SD) rats were harvested from the femur and the tibiae of the rats. After isolation and culturing, BMSCs from Passage 1 were used for the study. The in vitro differentiation of the hepatic progenitor cells was performed using a 2-step induction approach after 5-day serum deprivation from the BMSCs and culturing in Dulbecco's modified eagle medium. Spontaneous in vitro differentiation of BMSCs was examined in the absence of growth factors for 15 days as a control treatment. Hepatocytes differentiation was achieved by exposing the culture to collagen type I-coated plates. Cholangiocytes differentiation was achieved by replating the BMC-HepPCs on a layer of Matrigel. Immunofluorescence was conducted on twelve-well plates to determine cell differentiation. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) was used to determine the total RNA extracted using the Trizol LS reagent. In the hepatocyte differentiation group, after periodic acid-schiff (PAS) staining for glycogen, the inverted microscope was used to determine differentiation and undifferentiated BMC-HepPCs served as controls. The amount of low-density lipoprotein (LDL) uptake by the BMSCs-derived hepatocytes were assessed using fluorescence microscopy. The secretion of rat albumin was quantified using a quantitative ELISA kit. Results: Differentiation induction is indicative of the sequential supplementation of sodium butyrate and cytokines, which are involved in the embryonic development of the mammalian liver. Hepatic progenitor cells, derived from bone marrow, can be differentiated bidirectionally in vitro into both hepatocyte and cholangiocyte cell lines. The differentiated cells, including hepatic progenitor cells, hepatocytes, and bile duct-like cells, were identified and analyzed at mRNA and protein levels. Conclusion: Our findings show that BMSCs can be utilized as novel bipotential hepatic progenitor cells and thereby for hepatobiliary disease treatment or hepatobiliary tissue engineering.

Pyruvate kinase during in vitro differentiation of GM-CFC haemopoietic precursor in mice: modulation by l-alanine and l-phenylalanine

Biochimie ◽  
1989 ◽  
Vol 71 (6) ◽  
pp. 763-766 ◽  
Author(s):  
Elvira Cuenllas ◽  
Soledad Gaitan ◽  
Juan A. Bueren ◽  
Concepcion Tejero
Keyword(s):  
Pyruvate Kinase ◽  
In Vitro Differentiation

scholarly journals Purification and Characterization of Borrelia burgdorferi from Feeding Nymphal Ticks (Ixodes scapularis)

2001 ◽  
Vol 69 (6) ◽  
pp. 3536-3541 ◽  
Author(s):  
Sivaprakash Rathinavelu ◽  
Aravinda M. de Silva
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Ixodes Scapularis ◽  
Gradient Centrifugation ◽  
Surface Proteins ◽  
Mrna Levels ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient ◽  
Gene Expression Studies

ABSTRACT Here we describe a protocol for purifying Borrelia burgdorferi from feeding ticks by velocity centrifugation and Percoll density gradient centrifugation. The purified spirochetes were motile and 10- to 20-fold purer than the bacteria in crude tick homogenates. The purified bacteria were present in sufficient quantity for protein and gene expression studies. In comparison to culture-grown bacteria, tick-borne spirochetes had several proteins that were upregulated and a few that were downregulated. When the levels ofB. burgdorferi outer surface proteins OspA and OspC were measured, OspC protein and mRNA levels were lower in cultured bacteria than in bacteria purified from ticks. Although differences in OspA mRNA levels were observed between cultured and tick-borne bacteria, no differences were observed at the protein level. These experiments demonstrate that tick-transmitted borreliae display a gene expression and antigen profile different from that of spirochetes cultured in vitro.

Sign in / Sign up

Export Citation Format

Share Document