scholarly journals The brush border of rabbit kidney, a cellular compartment free of glycolytic enzymes

1978 ◽  
Vol 174 (2) ◽  
pp. 509-515 ◽  
Author(s):  
Dietrich Busse ◽  
Hans Ulrich Wahle ◽  
Harald Bartel ◽  
Barbara Pohl
Keyword(s):  
Lactate Dehydrogenase ◽  
Pyruvate Kinase ◽  
Brush Border ◽  
Gradient Centrifugation ◽  
Fluorescein Isothiocyanate ◽  
Glycolytic Enzymes ◽  
Rabbit Kidney ◽  
Cellular Compartment ◽  
Fluorescein Isothiocyanate Dextran

Activities of four enzymes of the glycolytic pathway, hexokinase, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, were determined in a vesicular brush-border preparation from rabbit kidneys. The specific activities of the enzymes were decreased several-hundredfold in the brush-border preparation compared with a kidney homogenate, but the enzymes were not totally absent. Density-gradient centrifugation of the brush-border preparation yielded brush border of even higher purity and also a characteristic pattern of distribution for each of the contaminating intracellular membranes. The presence of hexokinase in the brush-border preparation could be traced to contaminating mitochondria, and that of glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase to contaminating vesicles derived from the endoplasmic reticulum. The brush-border vesicles contained some ATP. An intravesicular concentration of 0.1mm was estimated, indicating that the vesicles had retained at least a part of their original content. Experiments in which fluorescein isothiocyanate-dextran (mol.wt. 20000) was present during cell lysis revealed that much, but not all, of the brush-border contents had been exchanged with the medium. The complete absence of glycolytic enzymes from brush-border vesicles, which had retained part of their original content, indicates that the brush border does not contain glycolytic enzymes in vivo and can be thought of as a compartment of its own, somehow separated from the cytoplasm.

scholarly journals Targeting Pyruvate Kinase M2 and Lactate Dehydrogenase A Is an Effective Combination Strategy for the Treatment of Pancreatic Cancer

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1372 ◽  
Author(s):  
Goran Hamid Mohammad ◽  
Vessela Vassileva ◽  
Pilar Acedo ◽  
Steven W. M. Olde Damink ◽  
Massimo Malago ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Enzyme Activity ◽  
Lactate Dehydrogenase ◽  
Tumor Growth ◽  
Pyruvate Kinase ◽  
Glycolytic Enzymes ◽  
Pyruvate Kinase M2 ◽  
Tumor Tissues

Reprogrammed glucose metabolism is one of the hallmarks of cancer, and increased expression of key glycolytic enzymes, such as pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA), has been associated with poor prognosis in various malignancies. Targeting these enzymes could attenuate aerobic glycolysis and inhibit tumor proliferation. We investigated whether the PKM2 activator, TEPP-46, and the LDHA inhibitor, FX-11, can be combined to inhibit in vitro and in vivo tumor growth in preclinical models of pancreatic cancer. We assessed PKM2 and LDHA expression, enzyme activity, and cell proliferation rate after treatment with TEPP-46, FX-11, or a combination of both. Efficacy was validated in vivo by evaluating tumor growth, PK and LDHA activity in plasma and tumors, and PKM2, LDHA, and Ki-67 expression in tumor tissues following treatment. Dual therapy synergistically inhibited pancreatic cancer cell proliferation and significantly delayed tumor growth in vivo without apparent toxicity. Treatment with TEPP-46 and FX-11 resulted in increased PK and reduced LDHA enzyme activity in plasma and tumor tissues and decreased PKM2 and LDHA expression in tumors, which was reflected by a decrease in tumor volume and proliferation. The targeting of glycolytic enzymes such as PKM2 and LDHA represents a promising therapeutic approach for the treatment of pancreatic cancer.

Comparative study of free and bound glycolytic enzymes from sea scorpion brain

1998 ◽  
Vol 76 (4) ◽  
pp. 609-614 ◽  
Author(s):  
Volodymyr I Lushchak ◽  
Ljudmyla P Lushchak ◽  
Tetjana V Bahnjukova ◽  
Alexei V Spichenkov ◽  
Kenneth B Storey
Keyword(s):  
Lactate Dehydrogenase ◽  
Pyruvate Kinase ◽  
Substrate Inhibition ◽  
Physiological Role ◽  
Glycolytic Enzymes ◽  
Free Form ◽  
Hill Coefficients ◽  
Low Ionic Strength ◽  
Scorpaena Porcus

Free and bound forms of hexokinase, pyruvate kinase, and lactate dehydrogenase were prepared from the brain of the sea scorpion (Scorpaena porcus) in a low ionic strength medium. Properties of the free and bound forms were compared to determine whether binding to particulate matter could influence enzyme function or stability in vivo. Changes in pH differently affected the activity of the free and bound forms of all three enzymes. Furthermore, bound forms of hexokinase and pyruvate kinase were more stable than the free enzymes to heating at 45°C. Bound hexokinase showed higher affinity for substrates (ATP, glucose) than the free form and bound lactate dehydrogenase had greater affinity for pyruvate and NADH. Although the affinities of the two forms of pyruvate kinase for substrates were similar, Hill coefficients for phosphoenolpyruvate as well as inhibition by ATP differed between the two enzyme forms. Free and bound lactate dehydrogenase also showed differences in Hill coefficients and bound lactate dehydrogenase was less sensitive to substrate inhibition by high pyruvate concentrations. The possible physiological role of the binding of these glycolytic enzymes to subcellular structures is discussed.Key words: hexokinase, lactate dehydrogenase, pyruvate kinase, enzyme binding, Scorpaena porcus.

scholarly journals Sickling-induced binding of immunoglobulin to sickle erythrocytes

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 636-639 ◽  
Author(s):  
GA Green ◽  
VK Kalra
Keyword(s):  
Flow Cytometry ◽  
Gradient Centrifugation ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Low Density ◽  
Autologous Plasma ◽  
Sickle Cells ◽  
Sickle Erythrocytes

Abstract Previously we demonstrated that sickle erythrocytes sedimenting at high densities after gradient centrifugation contain higher levels of surface immunoglobulin bound in vivo in comparison to low-density erythrocytes from the same patient. The present study examines the possibility that binding of autologous IgG to sickle erythrocytes may be associated with the sickling phenomenon. In the present study we subjected low-density erythrocytes to prolonged sickling under nitrogen in the presence of platelet-poor autologous plasma with added glucose for 24 hours (37 degrees C). After reoxygenation IgG bound in vitro was quantified by a nonequilibrium 125iodinated protein A-binding assay and by flow cytometry. Results show that sickle erythrocytes incubated under nitrogen bound significantly (P less than .001) more IgG, 439 +/- 41, molecules of IgG per cell (mean +/- SD) compared with sickle cells incubated under oxygenation (227 +/- 12 molecules of IgG per red cell) or compared with 196 +/- 26 molecules IgG per cell for untreated sickle cells. In contrast, normal erythrocytes incubated in autologous plasma exhibited no detectable IgG binding in vitro under either oxygenation or deoxygenation. Flow cytometry shows that deoxygenation of sickle cells generated a two-to-sixfold increase in the subpopulation of brightly fluorescent IgG-positive cells in comparison to oxygenated sickle cells and a 13.5% +/- 3.1% (mean +/- SD) increase in median fluorescence intensity for fluorescein isothiocyanate-labeled deoxygenated sickled cells compared with labeled oxygenated sickle cells. Our studies demonstrate that prolonged sickling will induce in vitro binding of autologous IgG to sickle erythrocytes. These findings indicate that sickle erythrocytes may be unique when compared with erythrocytes from other nonimmunologic hemolytic anemias or senescent red cells in that the primary events producing surface antigens recognized by autoantibody may include the sickling process. These findings also suggest that sickling in vivo may generate membrane alterations in sickle erythrocytes that lead to cumulative binding of autoantibody in vivo.

In-vivo Buccal Delivery of Fluorescein Isothiocyanate–Dextran 4400 with Glycodeoxycholate as an Absorption Enhancer in Pigs

1996 ◽  
Vol 85 (5) ◽  
pp. 457-460 ◽  
Author(s):  
A.J. Hoogstraate ◽  
J.C. Verhoef ◽  
B. Tuk ◽  
A. Pijpers ◽  
L.A.M.G. van Leengoed ◽  
...  
Keyword(s):  
Fluorescein Isothiocyanate ◽  
Absorption Enhancer ◽  
Buccal Delivery ◽  
Fluorescein Isothiocyanate Dextran

scholarly journals Influence of osmolality on gastrointestinal fluid volume and drug absorption: potential impact on oral salt supplementation

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Miyuki Takemura ◽  
Yuki Tanaka ◽  
Katsuhisa Inoue ◽  
Ikumi Tamai ◽  
Yoshiyuki Shirasaka
Keyword(s):  
Drug Absorption ◽  
Fluorescein Isothiocyanate ◽  
Fluid Volume ◽  
Gi Tract ◽  
Gastrointestinal Fluid ◽  
Fraction Absorbed ◽  
Absorption Potential ◽  
Fluorescein Isothiocyanate Dextran

Abstract Background The syndrome of inappropriate secretion of antidiuretic hormone (SIADH) is the most frequent cause of hyponatremia in patients with cerebrovascular disease, and is often treated with oral salt tablets. However, we have shown that osmolality-dependent variations in gastrointestinal (GI) fluid volume can alter the concentration of a poorly permeable drug in the GI tract, potentially affecting its absorption. Here, we examined the effect of ingestion of hyperosmotic solution (10% NaCl) on drug concentration and absorption in the GI tract. Methods The effects of osmolality on luminal fluid volume and drug absorption in rat intestine (jejunum, ileum and colon) were examined by means of an in situ closed loop method using fluorescein isothiocyanate-dextran 4000 (FD-4) and atenolol. In vivo absorption in rats was determined by measuring the plasma concentration after oral administration of the test compounds dissolved in purified water or hyperosmotic solution (10% NaCl). Results Administration of hyperosmotic solution directly into the GI tract significantly increased the GI fluid volume, owing to secretion of water into the lumen. After administration in hyperosmotic solution, the luminal concentration of non-permeable FD-4 was significantly lower than the initial dosing concentration, whereas after administration in purified water, the luminal concentration exceeded the initial concentration. The fraction absorbed of atenolol was markedly lower after administration in hyperosmotic solution than after administration in purified water. An in vivo pharmacokinetic study in rats was consistent with these findings. Conclusions Administration of hyperosmotic NaCl solution increased GI fluid volume and reduced the plasma level of orally administered atenolol. This may imply that oral salt tablets used to treat hyponatremia in SIADH patients could decrease the intestinal absorption of concomitantly administered drugs, resulting in lower plasma exposure.

scholarly journals Early effects on the intestinal barrier and pancreatic function after enteral stimulation with protease or kidney bean lectin in neonatal rats

2018 ◽  
Vol 119 (9) ◽  
pp. 992-1002 ◽  
Author(s):  
Ester Arévalo Sureda ◽  
Olena Prykhodko ◽  
Björn Weström
Keyword(s):  
Oral Administration ◽  
Mammalian Species ◽  
Intestinal Barrier ◽  
Fluorescein Isothiocyanate ◽  
Gut Development ◽  
Protease Treatment ◽  
Fluorescein Isothiocyanate Dextran ◽  
Early Effects ◽  
Gut Maturation

AbstractGut maturation naturally accelerates at weaning in altricial mammalian species, such as the rat. Mimicking this, gut development can also be induced precociously, 3–4 d earlier than it would occur naturally, by enteral exposure to phytohaemagglutinin (PHA), or various proteases. We investigated the early effects of gut provocation on intestinal barrier and pancreatic functions, to get a better understanding of the mechanisms that initiate gut maturation. The effects of oral administration of protease (trypsin) or PHA to 14-d-old suckling rats were studied during 24 h in comparison with water-fed controls. Intestinal in vivo permeability was assessed by oral administration of different-sized marker molecules and measuring their passage into the blood or urine 3 h later. A period of 24 h following oral administration, both PHA and protease provocation stimulated small intestinal (SI) growth and pancreatic secretion, as indicated by decreased pancreatic trypsin and increased luminal enzyme content. Within 1 h of oral administration, both treatments prevented the absorption of macromolecules to blood that was observed in controls. PHA treatment hindered the passage of fluorescein isothiocyanate-dextran (FD) 4 to blood, whereas protease treatment temporarily increased plasma levels of FD4, and the urine lactulose:mannitol ratio, indicating increased intestinal leakiness. Following protease treatment, fluorescence microscopy showed decreased vesicular uptake of FD70 in the proximal SI and increased epithelial fluorescence in the distal SI. In conclusion, PHA and protease differed in their early effects on the intestinal barrier; both exerted a blocking effect on epithelial endocytosis, whereas protease treatment alone temporarily increased epithelial leakiness, which seemed to be confined to the distal SI.

The HIF-1 response to simulated ischemia in mouse skeletal muscle cells neither enhances glycolysis nor prevents myotube cell death

2007 ◽  
Vol 293 (4) ◽  
pp. R1693-R1701 ◽  
Author(s):  
Nathalie Dehne ◽  
Uta Kerkweg ◽  
Teresa Otto ◽  
Joachim Fandrey
Keyword(s):  
Skeletal Muscle ◽  
Cell Death ◽  
Lactate Dehydrogenase ◽  
Pyruvate Kinase ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Vegf Mrna ◽  
Isolated Cells ◽  
Simulated Ischemia

Hypoxia-inducible factor (HIF) plays an important role in regulating gene expression in response to ischemia. Although activation of HIF-1 in muscle tissue was found during ischemia in vivo, the meaning and mechanisms in isolated cells are still incompletely understood. We studied activation of HIF-1 in skeletal muscle cells cultured in either their undifferentiated myoblast state or differentiated into myotubes. HIF-1 was activated in myoblasts and myotubes by hypoxia and simulated ischemia. Induction of adrenomedullin mRNA and, to a lesser extent, VEGF mRNA correlated well with the induction of HIF-1α protein in both cell types. Enzymes of glycolysis-like lactate dehydrogenase and pyruvate kinase showed upregulation of their mRNA only under hypoxic conditions but not during simulated ischemia. Phosphofructokinase mRNA showed no significant upregulation at all. Although HIF-1 was activated in myotubes during simulated ischemia, myotubes died preceded by a loss of ATP. Myoblasts survived simulated ischemia with no decrease in ATP or ATP turnover. Furthermore, pharmacological inhibition of HIF-1 hydroxylases by dimethyloxalylglycine (DMOG) increased HIF-1α accumulation and significantly upregulated the expression of adrenomedullin, VEGF, lactate dehydrogenase, and pyruvate kinase in myoblasts and myotubes. However, DMOG provided no protection from cell death. Our data indicate that HIF-1, although activated in myotubes during simulated ischemia, cannot protect against the loss of ATP and cell viability. In contrast, myoblasts survive ischemia and thus may play an important role during regeneration and HIF-1-induced revascularization.

scholarly journals Galectin-4 and Small Intestinal Brush Border Enzymes Form Clusters

1997 ◽  
Vol 8 (11) ◽  
pp. 2241-2251 ◽  
Author(s):  
E. Michael Danielsen ◽  
Bo van Deurs
Keyword(s):  
Brush Border ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Aminopeptidase N ◽  
Intestinal Lumen ◽  
Immunogold Electron Microscopy ◽  
Brush Border Enzymes ◽  
Intestinal Brush Border ◽  
Mucosal Explants

Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed that galectin-4 is indeed an intestinal brush border protein; we also localized galectin-4 throughout the cell, mainly associated with membraneous structures, including small vesicles, and to the rootlets of microvillar actin filaments. This was confirmed by subcellular fractionation, showing about half the amount of galectin-4 to be in the microvillar fraction, the rest being associated with insoluble intracellular structures. A direct association between the lectin and aminopeptidase N was evidenced by a colocalization along microvilli in double immunogold labeling and by the ability of an antibody to galectin-4 to coimmunoprecipitate aminopeptidase N and sucrase-isomaltase. Furthermore, galectin-4 was released from microvillar, right-side-out vesicles as well as from mucosal explants by a brief wash with 100 mM lactose, confirming its extracellular localization. Galectin-4 is therefore secreted by a nonclassical pathway, and the brush border enzymes represent a novel class of natural ligands for a member of the galectin family. Newly synthesized galectin-4 is rapidly “trapped” by association with intracellular structures prior to its apical secretion, but once externalized, association with brush border enzymes prevents it from being released from the enterocyte into the intestinal lumen.

Cigarette smoke extract potentiates bradykinin-induced increases in microvascular permeability

1993 ◽  
Vol 75 (1) ◽  
pp. 27-32 ◽  
Author(s):  
W. G. Mayhan ◽  
I. Rubinstein
Keyword(s):  
Cigarette Smoke ◽  
Fluorescein Isothiocyanate ◽  
Cigarette Smoke Extract ◽  
Microvascular Permeability ◽  
Cheek Pouch ◽  
Site Formation ◽  
Hamster Cheek Pouch ◽  
Fluorescein Isothiocyanate Dextran ◽  
Macromolecular Permeability

The first goal of this study was to determine whether cigarette smoke extract (CSE) increases microvascular permeability of the hamster cheek pouch in vivo. The second goal was to determine whether CSE potentiates bradykinin-induced increases in vascular permeability in the hamster cheek pouch. Using intravital microscopy, we examined the permeability of the hamster cheek pouch to fluorescein isothiocyanate-dextran (mol wt 70,000). Increases in permeability were quantitated by counting the number of postcapillary venular leaky sites per 0.11 cm2. Superfusion of CSE (1, 5, and 10%) did not produce venular leaky sites and, thus, did not alter macromolecular permeability. Superfusion of bradykinin (0.1, 0.5, and 1.0 microM) produced a dose-related increase in the number of venular leaky sites. Formation of leaky sites in response to bradykinin was potentiated by CSE. To determine whether potentiation of bradykinin-induced leaky site formation by CSE was related to products released via the cyclooxygenase pathway, we examined the effects of pretreatment with indomethacin (10 mg/kg i.v.). Indomethacin did not alter the potentiating effect of CSE on bradykinin-induced leaky site formation. These findings suggest that CSE does not alter basal permeability of the hamster cheek pouch microcirculation in vivo. However, CSE potentiates bradykinin-induced increases in microvascular permeability. The mechanism of CSE-induced potentiation of microvascular permeability does not appear to be related to substances produced via the cyclooxygenase pathway.

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