scholarly journals Inhibition of leucocytic lysosomal enzymes by glycosaminoglycans in vitro

1975 ◽  
Vol 152 (1) ◽  
pp. 57-64 ◽  
Author(s):  
J L Avila ◽  
J Convit
Keyword(s):  
Incubation Medium ◽  
Lysosomal Enzymes ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Polymorphonuclear Leucocyte ◽  
Dependent Manner ◽  
Acid Hydrolases ◽  
Lysosomal Fraction ◽  
Formation Of Complexes

1. A lysosomal fraction was separated by density-gradient centrifugation from a highly purified human polymorphonuclear leucocyte suspension. 2. Some 23 different lysosomal enzymes were assayed for activity in the presence of various concentrations of glycosaminoglycans. 3. The 21 acid hydrolases assayed were strongly inhibited to different degrees by low (0-12 mmol/l) concentrations of glycosaminoglycans in a pH-dependent manner. Thus inhibitions were stronger below pH4.5, with activity returning to control values at about pH5.0. 4. On a molar basis, the inhibitory activity for the several glycosaminoglycans studied was: heparin > chondroitin sulphate ≫ hyaluronic acid. 5. Once the glycosaminoglycan-acid hydrolase complex was formed, it was partially dissociated by slight elevations in the pH of the incubation medium, by increasing the ionic strength of the incubation medium, or by adding several cationic proteins (e.g. histone, protamine). 6. As leucocytic lysosomes contain large amounts of chondroitin sulphate, and have a strongly acid intragranular pH, we suggest that glycosaminoglycans may modify lysosomal function through the formation of complexes with lysosomal enzymes, by inhibiting the digestive activity of the acid hydrolases when the intralysosomal pH is below their pI.

In vitro Action of Gastric Mucosal Lysosomal Enzymes on Intracellular Gastric Glycoproteins

1979 ◽  
Vol 34 (1-2) ◽  
pp. 90-95 ◽  
Author(s):  
Fouad M. Fouad ◽  
D. Waldron-Edward
Keyword(s):  
Cell Wall ◽  
Lysosomal Enzymes ◽  
Gastric Ulceration ◽  
Mucosal Cell ◽  
Gastric Lesions ◽  
Acid Hydrolases ◽  
Gastric Mucosal Cells ◽  
Mucosal Cells ◽  
Specific Increase

Abstract The results show that incubation of gastric mucosal cells from rat at pH ~4.5 or in the presence of aspirin is associated with a specific increase in the activity of some acid-hydrolases. Intracellular glycoproteins, isolated by non-degradative techniques from rat or dog fundic mucosal cells, were found to be potential bio-substrates for these acid-hydrolyses. This may suggest that cleavage of the carbohydrate moieties of the intracellular and mucosal cell wall glycoproteins is a fundamental step in the development of gastric ulceration. A model for gastric lesions is proposed and discussed in the light of the results obtained.

scholarly journals Degradation of Sulphated Glycosaminoglycans at the Surface of Cultured Human Endothelial Cells by a Platelet Heparitinase

1977 ◽  
Author(s):  
C. Busch ◽  
B. Glimelius ◽  
Å Wastesson ◽  
B. Westermark
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Incubation Medium ◽  
Chondroitin Sulphate ◽  
Coagulation Factors ◽  
Platelet Lysate ◽  
Human Endothelial Cells ◽  
Endothelial Cell Surface ◽  
Sulphated Glycosaminoglycans

The non-thrombogenic property of the endothelial cell surface is a prerequisite for maintainance of blood circulation. The nature of this property is poorly understood. Recent advances in culturing techniques of endothelial cells in vitro may facilitate studies of the surface biochemistry. Human endothelial cells (EC) isolated from umbilical veins were shown to synthesize and secrete sulphated glycosaminoglycans (GAG). The recent finding of a platelet enzyme capable of degrading heparin sulphate (HS) raised the question:Can platelet lysate or a purified heparitinase detach and degrade endothelial HS? EC cultured in the presence of 35S-sulphate, produce 35S-labelled GAG which was isolated from the incubation medium from a cell associated trypsin labile pool and from a cellular pool not liberated by trypsin. After 48 hours of incorporation about 95% of 35S-GAG was found in the medium fraction, 5% in the trypsin fraction and negligible amounts in the cell fraction. In the trypsin pool (“surface fraction”) heparin sulphate comprised about 85%, while the remaining 15% consisted of chondroitin sulphate and/or dermatan sulphate. Incubation of 35S-labelled EC with platelet lysate or a partially purified preparation of the enzyme from the same source caused a marked release of cell-surface associated HS to the incubation medium as oligosaccharides. These effects could be ascribed to heparitinase activity and may alter the properties of the EC-surface and influence the interaction between these cells on one hand and blood cells or plasma components, e.g., coagulation factors on the other.

Treatment of B-CLL Cells with Bortezomib and Rituximab Reduces Cell Viability In Vitro..

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2798-2798 ◽  
Author(s):  
Eloisa Jantus Lewintre ◽  
Elena Sarsotti ◽  
Maria Jose Terol ◽  
Isabel Benet ◽  
Jose A. Martinez-Climent ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Combined Treatment ◽  
Biological Significance ◽  
Vitro Activity ◽  
Rabbit Serum ◽  
Dependent Manner ◽  
In Vitro Activity ◽  
Mutational Status ◽  
Significant Difference

Abstract Introduction: In B-CLL, somatic mutation of IgVH genes defines a group of patients with favorable prognosis, whereas the absence of IgVH mutations is correlated with a worse outcome. Our previous data suggested that BCL-6 mutations identify a subgroup of patients with high risk of progression despite the presence of mutated IgVH gene, but the clinical significance of this molecular alteration remains uncertain. New approaches are now being tested for the treatment of B-CLL. Proteasome inhibitor, Bortezomib (Btz), and monoclonal antibodies specific for surface antigens, Rituximab (Rtx), represent potential therapeutic strategy. Objetives: To study the effects of Btz and Rtx on viability of CLL cells in culture, and to correlate the responses to the mutational status of the samples. Material and Methods: PBMC from 29 B-CLL patients (Binet stage A) were isolated by Ficoll-Hypaque gradient centrifugation. Samples were culture in RPMI supplemented with 20% FCS. Complement rabbit serum was added to the cultures in Rtx treated cells. Cells were exposed to various concentrations of Btz (0.1, 1 and 10 uM), Rtx (10 ug/ml) or both together during 24 hours. Then, cells were stained with Propidium Iodide and subjected to flow cytometry analysis. Results: Btz and Rtx reduce viability of B-CLL cells in a dose and time dependent manner. The kinetic of both drugs were different, whereas Rtx reaches its maximun effect within 3 hours after treatment, Btz do the same after 48 hours. Combined treatment (Btz + Rtx) increases the effect of each one separately. All differences between treatments with increased doses of Btz + Rtx vs Btz alone were statistically significant. In the group of IgVH mutated patients, there were a significant difference in the response to Btz and Rtx in BCL-6 mutated (mut) and unmutated (unmut) groups (n= 10 and 8 respectively), whereas no significant differences were observed in the number of lymphocytes, lymphocyte doubling time, CD38 expression and cytogenetic alterations. Effects of Btz and Rtx on IgVH mutated samples: correlation with BCL-6 mutations BCL-6 Mean viability (%) SD p * * U. Mann Whitney test Control unmut 82.13 8.70 0.42 mut 86.36 6.01 Btz 0.1 uM unmut 49.05 10.39 0.006 mut 64.28 12.16 Btz 1 uM unmut 39.86 7.19 0.040 mut 53.27 13.35 Btz 10 uM unmut 30.21 7.41 0.041 mut 43.99 14.73 Rtx 10 ug/ml unmut 48.31 14.51 0.050 mut 34.62 12.51 Btz 0.1 uM + Rtx unmut 32.96 14.15 0.153 mut 24.50 10.01 Btz 1 uM + Rtx unmut 25.99 10.88 0.315 mut 19.89 7.53 Btz 10 uM + Rtx unmut 19.27 9.04 0.491 mut 16.31 7.52 Conclusion: Our data show that Btz and Rtx have in vitro activity on B-CLL cells, being used alone or in combination. Interesting, in IgVH mutated cells, Btz and Rtx have statistically significant differences in their in vitro activity on B-CLL cells according to their BCL-6 mutational status, but the biological significance of this differential responses remains unclear

Oral Supplementation with Omega3 Fatty Acids Inhibits NFkB Activation In Chronic Lymphocytic Leukemia (CLL) Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4607-4607
Author(s):  
Oscar F. F Ballester ◽  
Johannes Fahrmann ◽  
Theodore Witte ◽  
Gabriela Ballester ◽  
W. Elaine Hardman
Keyword(s):  
Conflicts Of Interest ◽  
Early Stage ◽  
Gradient Centrifugation ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Red Cell ◽  
Omega 3 ◽  
Dose Dependent

Abstract Abstract 4607 Introduction: Nuclear factor kappa B (NFkB) is a critical transcription factor involved in the growth and survival of CLL cells. NFkB is recognized as an important target for the development of novel therapies for the treatment of various malignancies. In vitro and in experimental animal models, OMEGA-3 fatty acid (O3FA) supplementation has been shown to inhibit NFkB activity. Patients and Methods: Patients with early stage CLL (Rai stages 0-II) who required no therapy, where accrued to this phase I-II trial. O3FA supplements were given for a total of 12 months at doses ranging from 2250 mg (EPA plus DHA), escalated to 4500 mg and 6750 mg per day as tolerated. NFkB activity was measured in peripheral blood samples after separation of mononuclear cell by gradient centrifugation and expressed as luminescence units/μ g of protein. Baseline and multiple serial samples were obtained during the study period. In-vitro cytotoxicity assays to doxorubicin were conducted using standard LD50 methods. Compliance was monitored by analysis of red cell and lymphocyte membrane lipid composition by gas chromatography. Results: Fifteen patients have been accrued to the trial, 8 of them have currently completed the planned 12 months of the study period. No significant clinical changes in disease activity were noted. O3FA was well tolerated. Supplementation resulted in a dose-dependent increase of O3FA composition of red cell and lymphocyte membranes in a dose dependent manner. At baseline, CLL patients had NFkB above the range observed in normal controls (2.05 × 104 to 2.32 × 105 NFkB lum units/μ g). The median value in CLL patients at baseline was 11.60 × 106 NFkB lum units/μ g (range 0.9 × 105 to 23.12 × 106). Among 5 patients with the highest baseline levels of NFkB, a decrease in NFkB activity ranging from 0.02 to 0.19 of the baseline value, was noted at the 2 higher doses of O3FA supplementation. Similar results were seen in patients with relatively lower levels of baseline NFkB activity (0.9 × 105 to 2.96 × 106 lum units/μ g). In vitro, significant doxorubicin cytotoxicity (>50%) was noted in samples obtained during supplementation, at μ gM concentrations which produced no detectable cell kill in baseline samples. Conclusions: O3FA supplementation resulted in significant inhibition of NFkB activity in leukemic cells from patients with CLL. In-vitro, after O3FA supplementation CLL cells became more sensitive to doxorubicin. Preliminary analysis of whole genome micro arrays revealed significant down-regulation of multiple genes associated with O3FA supplementation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals LYSOSOMES IN RAT THORACIC DUCT LYMPHOCYTES FRACTIONATED BY ZONAL CENTRIFUGATION

1973 ◽  
Vol 59 (1) ◽  
pp. 177-184 ◽  
Author(s):  
William E. Bowers
Keyword(s):  
Thoracic Duct ◽  
Light Microscope ◽  
Cathepsin D ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Differential Centrifugation ◽  
Acid Hydrolases ◽  
Fractionation Method

A method of zonal centrifugation was developed which separates rat thoracic duct lymphocytes (TDL) mainly according to size. The validity of the fractionation method was supported by light microscope observations, Coulter Counter sizing, and in vivo and in vitro labeling of lymphocytes. The distributions of lysosomal acid hydrolases in TDL fractionated by zonal centrifugation are similar to the distribution obtained for the cells. This result indicates that the large lymphocyte is not the sole bearer of either lysosomes or the large amount of soluble cathepsin D found in homogenates of TDL. Both reside mainly in small lymphocytes. This point was clearly established by fractionating homogenates of purified small lymphocytes by means of differential centrifugation and isopycnic density gradient centrifugation.

scholarly journals The effect of β-d-xylosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors

1977 ◽  
Vol 162 (2) ◽  
pp. 217-233 ◽  
Author(s):  
K D Gibson ◽  
B J Segen ◽  
T K Audhya
Keyword(s):  
Protein Synthesis ◽  
Incubation Medium ◽  
Chondroitin Sulphate ◽  
Gel Filtration ◽  
Protein Synthesis Inhibitors ◽  
Serum Factors ◽  
Embryonic Chicken ◽  
Low Concentrations ◽  
Normal Human

Incorporation of [35S]]sulphate, [3H]glucose and [3H]serine into glycosaminoglycans and proteoglycans of embryonic-chicken sternum was measured in vitro in incubation medium containing 4-methylumbelliferyl beta-D-xyloside or p-nitrophenyl beta-D-xyloside at low concentrations, and in the absence of inhibitors of protein synthesis. Incorporation of sulphate was decreased by 80% in incubations in which 1mM-4-methylumbelliferyl beta-xyloside or 2.5 mM-p-nitrophenyl beta-xyloside was present; under these conditions, serum factors stimulated incorporation to only a small extent. When the concentration of the xyloside was decreased tenfold, incorporation of sulphate was inhibited by 60-70%, but when normal human serum or L-3,3′,5-tri-iodothyronine or both were also added to the incubation medium, incorporation was markedly stimulated. Experiments in which [35S]sulphate and [3H]glucose were incorporated simultaneously, and enzymic analysis of glycosaminoglycans formed in such experiments, indicated that chondroitin sulphate formed in the presence of 0.1 mM-4-methylumbelliferyl beta-xyloside contained 30-40% less sulphate than did chondrotin sulphate synthesized in the absence of xylosides. Similar experiments, with [3H]serine instead of [3H]glucose, suggested also a 20-30% decrease in chain length of the chondroitin sulphate; this was confirmed by direct gel filtration of labelled glycosaminoglycans on a calibrated column. Incorporation of [3H]glucose or [3H]serine was stimulated by serum and tri-iodothyronine in parallel with incorporation of sulphate. The changes seen in the total chondroitin sulphate were mirrored in the major proteoglycan fraction, purified by isopycnic centrifugation of salt-extracted proteoglycans. The labelling pattern of chondroitin sulphate from this proteoglycan indicated that decreased sulphation of chondroitin sulphate was largely due to the inferior ability of short polysaccharide chains to accept sulphate, with some direct interference with transfer of sulphate to all chains. The results also suggested that the action of serum factors on synthesis of proteochondroitin sulphate is exercised at the level of either protein synthesis or transport to the sites of initiation of polysaccharide synthesis.

scholarly journals Biosynthesis of proteoglycans in cartilage slices. Fractionation by gel chromatography and equilibrium density-gradient centrifugation

1972 ◽  
Vol 126 (4) ◽  
pp. 791-803 ◽  
Author(s):  
T. E. Hardingham ◽  
Helen Muir
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Specific Radioactivity ◽  
Equilibrium Density ◽  
Gel Chromatography ◽  
Sulphate Content

The kinetics of incorporation of [35S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ‘pulse–chase’ radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ‘pulse–chase’ experiments over 5h did not demonstrate any precursor–product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ‘pulse–chase’ experiments there was again no evidence for precursor–product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.

scholarly journals Effect of sodium orthovanadate on ovulation isolated ovarian follicles in Siberian Sturgeon (Acipenser baerii) In Vitro

2021 ◽  
Vol 273 ◽  
pp. 03009
Author(s):  
Dmitry Balashov ◽  
Konstantin Kovalev
Keyword(s):  
Incubation Medium ◽  
Inhibitory Effect ◽  
Ovarian Follicles ◽  
Siberian Sturgeon ◽  
Physiological Status ◽  
Dependent Manner ◽  
Sodium Orthovanadate ◽  
Acipenser Baerii ◽  
Dose Dependent

Effects of sodium orthovanadate on oocyte ovulation were examined during in vitro culture of Siberian sturgeon ovarian follicles from hibernating fish. It was shown that sodium orthovanadate stimulates ovulation of Siberian sturgeon oocytes in a dose-dependent manner. The stimulating or inhibitory effect of vanadate depends on the time of addition to the incubation medium. It was also shown that the stimulating effects of orthovanadate depend on the physiological status of hibernating females whose oocytes were isolated

scholarly journals Effect of bacterial endotoxin on the in vitro release of Type II phospholipase-A2 and prostaglandin E2 from human placenta

1999 ◽  
Vol 160 (2) ◽  
pp. 291-296 ◽  
Author(s):  
W Farrugia ◽  
L Nicholls ◽  
GE Rice
Keyword(s):  
Prostaglandin E2 ◽  
Phospholipase A2 ◽  
Incubation Medium ◽  
In Vitro Release ◽  
Bacterial Endotoxin ◽  
Dependent Manner ◽  
Type Ii ◽  
Intracellular Enzyme ◽  
Tissue Content

The aim of this study was to establish the effect of bacterial endotoxin lipopolysaccharide (LPS) on the release of Type II phospholipase-A2 (PLA2) and prostaglandin E2 (PGE2) from late-pregnant human placental tissue incubated in vitro. Under basal conditions, immunoreactive Type II PLA2 and PGE2 were released from tissue explants in a time-dependent manner (up to 24 h, ANOVA, P<0. 0001, n=6). The release of these mediators was not associated with a loss of cell membrane integrity, as indicated by concentrations of the intracellular enzyme, lactate dehydrogenase (LDH), in the incubation medium. Incubation of explants in the presence of LPS (0. 001-100 microg LPS/ml) significantly decreased PLA2 tissue content (P<0.02, n=6) and increased the accumulation of PLA2 and PGE2 in the incubation medium (P<0.0001, n=6). The data obtained in this study indicated that Type II PLA2 and PGE2 are released from term placenta under basal conditions and that LPS stimulated their release. The associated decrease in PLA2 tissue content is consistent with the hypothesis that LPS induces the release of stored PLA2. This study identifies one pathway by which products of bacterial infection may induce the release of a pro-inflammatory, extracellularly active PLA2 from intrauterine tissues that may promote the formation of phospholipid metabolites involved in the process of labour and delivery (e.g. the prostaglandins).

scholarly journals Bisphenol A and S, but Not F, Alter Oxidative Stress Levels in Spermatozoa

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A483-A484
Author(s):  
Mimi Nguyen ◽  
Reem Sabry ◽  
., Elizabeth J St. John ◽  
Laura A Favetta
Keyword(s):  
Oxidative Stress ◽  
Glutathione Peroxidase ◽  
Bisphenol A ◽  
Sperm Quality ◽  
Gradient Centrifugation ◽  
Endocrine Disrupting Compounds ◽  
Dependent Manner ◽  
Stress Levels ◽  
Relevant Dose

Abstract Exposures to endocrine disrupting compounds (EDCs) have been linked to male reproductive dysfunction. The increase in prevalence of male infertility to date has been correlated with the widespread use of bisphenols, particularly bisphenol A and its analogs. Current literature suggests that exposure to EDCs is associated with abnormalities in spermatogenesis, as well as reduced sperm motility and morphology. At this time, the mechanism for bisphenols’ action in spermatozoa is still unknown, though it has been proposed that bisphenols alter sperm parameters by affecting their oxidative stress pathways. The aim of this project is to elucidate whether BPA and its analogs affect oxidative stress levels inducing decreased sperm quality and fertilization capability. An in vitro bovine model was used as the most appropriate translational model to humans. Motile spermatozoa were separated using Percoll gradient centrifugation of frozen-thawed sperm and a pilot experiment was conducted in a time-dependent manner to establish the ideal incubation time of 4 hours. Sperm was then incubated in five treatment groups: control, vehicle (0.01% ethanol), BPA, BPS, and BPF, at the physiological relevant dose of 0.05 mg/ml in 0.01% ethanol. Key reactive oxygen species (ROS) scavenger genes (superoxide mutase 1 [SOD1], superoxide mutase 2 [SOD2], catalase [CAT], glutathione peroxidase 1 [GPX1], glutathione peroxidase 4 [GPX4]) were quantified at the RNA level using qPCR. Of the five genes examined: BPA, BPS, and BPF exposure significantly decreased GPX4 expression (p&lt;0.05) compared to control and vehicle groups. BPA and BPS exposure significantly increased SOD1 expression compared to BPF, control, and vehicle (p&lt;0.05). No significant differences were found in GPX1 after bisphenols’ exposure. SOD2 and CAT expression was not detectable in all five groups suggesting a lack of expression in bovine sperm. Future investigations will involve experiments at the protein level and quantifying total ROS levels. Additionally, sperm morphology was evaluated using Spermac stain. Preliminary results indicate an increased number of abnormal spermatozoa with notably tail defects after 4 hours of bisphenols exposure. These initial findings support BPA and BPS’s ability to alter oxidative stress pathways in sperm via changes in antioxidant enzymes but suggest that BPF likely acts through different mechanisms. Overall, this research aims to understand the effects of bisphenols on male fertility to improve male factors during assisted reproductive biotechnologies.

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