scholarly journals Isolation of intact megakaryocytes from guinea pig femoral marrow. Successful harvest made possible with inhibitions of platelet aggregation; enrichment achieved with a two-step separation technique.

1976 ◽  
Vol 69 (1) ◽  
pp. 159-172 ◽  
Author(s):  
R F Levine ◽  
M E Fedorko
Keyword(s):  
Guinea Pig ◽  
Gradient Centrifugation ◽  
Velocity Sedimentation ◽  
High Yield ◽  
Equilibrium Density ◽  
Large Size ◽  
Membrane Topography ◽  
First Time ◽  
Better Than

Methods have been devised to harvest megakaryocytes from guinea pig femoral marrow and to isolate them in high yield. When marrow tissue was disaggregated the megakaryocytes underwent degenerative changes characterized by the loss of cytoplasmic granules and alterations in membrane topography, similar to the changes seen in aggregating platelets. These morphologic changes were interpreted to mean that megakaryocytes possessed functional attributes of platelets. The use of agents which inhibit platelt aggregation (0.38% sodium citrate. 10(-3) M adenosine, and 2 x 10(-3) M theophylline) in a medium free of bivalent cations prevented these changes. This solution resulted in both an excellent morphologic preservation and a significantly increased recovery of megakaryocytes from marrow tissue. A two-step purification of the intact megakaryocytes was carried out on the basis of their low density and large size, with equilibrium density gradient centrifugation followed by velocity sedimentation. This sequence gave approximately a 100-fold enrichment of megakaryocytes, significantly better than that achieved with either method alone. These techniques for harvesting and concentrating megakaryocytes make it possible for the first time to study megakaryocytes in vitro.

Isolation and Culture of Single Cell Types from Rat Liver

2016 ◽  
Vol 201 (4) ◽  
pp. 253-267 ◽  
Author(s):  
Qidi Zhang ◽  
Ying Qu ◽  
Zhenghong Li ◽  
Qingqing Zhang ◽  
Mingyi Xu ◽  
...  
Keyword(s):  
Single Cell ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Cell Types ◽  
Ethylene Glycol Tetraacetic Acid ◽  
High Yield ◽  
Isolated Cells ◽  
Liver Sinusoidal Endothelial Cells ◽  
Pathology Of The Liver

There have been few reports on the simultaneous isolation of multiple liver cell populations thus far. As such, this study was aimed at establishing a protocol for the simultaneous separation of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) from the rat liver and assessing the in vitro culture of these cells. Single-cell suspensions from the liver were obtained by ethylene glycol tetraacetic acid/collagenase perfusion. After low-speed centrifugal separation of HCs, pronase was added to the nonparenchymal cell fraction to eliminate the remaining HCs. Subsequently, HSCs, LSECs and KCs were purified by two steps of density gradient centrifugation using Nycodenz and Percoll in addition to selective attachment. Pronase treatment increased the HSC yield (1.5 ± 0.2 vs. 0.7 ± 0.3 cells/g liver, p < 0.05) and improved LSEC purity (93.6 ± 3.6 vs. 82.5 ± 5.6%, p < 0.01). The isolated cells could also be cultured in vitro. LSEC apoptosis began on day 3 and reached a maximum on day 7. A few surviving LSECs began proliferating and split to form a cobblestone, sheet-like appearance on day 14. The LSECs on day 14 lost fenestrations but retained scavenger function. Thus, viable and purified liver cells were obtained with a high yield from the rat liver using the developed method, which may be useful for studying the physiology and pathology of the liver in the future.

scholarly journals Induction of specific immune unresponsiveness using purified mixed leukocyte culture-activated T lymphoblasts as autoimmunogen. I. Demonstration of general validity as to species and histocompatibility barriers.

1977 ◽  
Vol 146 (4) ◽  
pp. 1124-1137 ◽  
Author(s):  
L C Andersson ◽  
M Aguet ◽  
E Wight ◽  
R Andersson ◽  
H Binz ◽  
...  
Keyword(s):  
Side Effects ◽  
T Lymphocytes ◽  
Guinea Pig ◽  
Third Party ◽  
Velocity Sedimentation ◽  
General Validity ◽  
Negative Side Effects ◽  
T Lymphoblasts ◽  
General Tool

Normal immunocompetent T lymphocytes can be induced into specific proliferation if confronted with the relevant alloantigen in vitro. Such mixed leuko-cyteculture-activated T lymphoblasts carring idiotypic receptors on their surface can be purified using velocity sedimentation and serve as immunogen if administered in adjuvant to the autologous host. Autoblast immunization can be shown to lead to specific, long-lasting unresponsiveness against the relevant alloantigens, while leaving reactivity against third-party antigens intact. When tested as to general validity, it could be shown to function in all species analyzed (mouse, rat, and guinea pig) as well as across both major and minor histocompatibility barriers. No negative side effects have been noted so far. It would thus seem clear that autoblast immunization using the above described scheme may serve as a general tool in inducing long-lasting, specific unresponsiveness in any species and across any histocompatibility barrier.

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 Biosynthesis of respiratory-tract mucins. Incorporation of radioactive precursors into glycoproteins by canine tracheal explants in vitro

1973 ◽  
Vol 136 (4) ◽  
pp. 837-844 ◽  
Author(s):  
Daniel B. Ellis ◽  
Glenn H. Stahl
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sialic Acids ◽  
Equilibrium Density ◽  
Agarose Gels ◽  
Cscl Gradient ◽  
Tracheal Explants

1. Canine tracheal explants, cultured in medium 199, actively incorporated radioactive precursors into secreted macromolecules in vitro. 2. Puromycin, 6-diazo-5-oxo-l-norleucine and ouabain markedly inhibited the incorporation of these precursors. 3. Exogenous glucosamine at concentrations above 20mm caused a greater than 50% inhibition of the incorporation of l-[G-3H]fucose and l-[U-14C]serine. 4. Carbohydrate content of the purified secretions was approximately 50% and consisted principally of galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acids. 5. Chromatography on DEAE-cellulose and Bio-Gel A-150m and equilibrium density-gradient centrifugation in a CsCl gradient confirmed the presence of mucous glycoproteins. 6. Electrophoresis on 1% agarose gels gave profiles that were identical with canine respiratory mucus obtained in vivo. 7. These results support the utility of the explant system for studies of respiratory secretions.

scholarly journals Human neutrophil-specific granule deficiency: a model to assess the role of neutrophil-specific granules in the evolution of the inflammatory response

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1317-1329 ◽  
Author(s):  
JI Gallin ◽  
MP Fletcher ◽  
BE Seligmann ◽  
S Hoffstein ◽  
K Cehrs ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Gradient Centrifugation ◽  
Cell Aggregation ◽  
Oxygen Metabolism ◽  
Febrile Episode ◽  
Specific Granules ◽  
Specific Granule ◽  
Better Than

Abstract It has been suggested that neutrophil (PMN) specific granules are important in cell aggregation, locomotion, hydroxyl radical formation, and in extracellular functions such as the generation of complement- related inflammatory mediators (C5a) and the feedback regulation of myelopoiesis. In the current studies, a 9-yr-old boy with a history of recurrent infections and specific granule deficiency (absent lactoferrin, B-12 binding proteins, and characteristic specific granules on sucrose gradient centrifugation of cell homogenates) was studied to assess some of these concepts. In vivo, the patient had decreased PMN and monocyte accumulation into Rebuck skin windows but an expected febrile episode with an associated neutropenia (PMN margination) and neutrophilia (mobilization of marrow reserves) in response to intravenous endotoxin. In vitro, the patient's resting PMN showed increased ruffling, increased surface-to-volume ratio, and increased numbers of centriole-associated microtubules. His PMN showed a significant decrease in cell negative surface charge (which may relate to aggregation) in response to several stimuli and adhered better than normally to plastic. In addition, his PMN aggregated normally in response to the chemoattractant f-met-leu-phe, although the subsequent disaggregation normally seen with PMN did not occur with the patients cells. Chemotaxis of the patient's PMN to several stimuli was abnormal, and specific saturable and displaceable binding of the chemoattractant f-met-leu-[3H]phe was decreased. Similarly, following incubation with secretagogues, there was a less than normal increase in f-met-leu-[3H]phe binding and an absence of the normal increases in PMN surface area. The patient's PMN bactericidal activity, stimulated oxygen metabolism (cytochrome-c reduction, chemiluminescence, and NBT reduction), and elicited changes in membrane potential were also abnormal. Studies assessing the mechanism for the abnormal monocyte accumulation into skin windows indicated the patient's monocyte chemotaxis was better than normal in vitro. However, the patient's PMN homogenates lacked a stimulus of monocyte locomotion and did not generate chemotactic activity normally from serum. Thus, the data indicate that specific granule constituents are not required for neutrophil margination in vivo or aggregation in vitro. However, the data support the concept that PMN-specific granules are important for PMN locomotion and oxidative metabolism. In addition, extracellular release of specific granule constituents appears to be important for amplification of the initial and subsequent phases of the inflammatory response.

Identification and characterization of leptin-containing intracellular compartment in rat adipose cells

2000 ◽  
Vol 279 (4) ◽  
pp. E893-E899 ◽  
Author(s):  
Cecilia Roh ◽  
Galini Thoidis ◽  
Stephen R. Farmer ◽  
Konstantin V. Kandror
Keyword(s):  
Membrane Trafficking ◽  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Peroxisome Proliferator ◽  
3T3 Cells ◽  
Peroxisome Proliferator Activated Receptor ◽  
Glut 4 ◽  
Adipose Cells

The major leptin-containing membrane compartment was identified and characterized in rat adipose cells by means of equilibrium density and velocity sucrose gradient centrifugation. This compartment appears to be different from peptide-containing secretory granules present in neuronal, endocrine, and exocrine cells, as well as from insulin-sensitive GLUT-4-containing vesicles abundant in adipocytes. Exocytosis of both leptin- and GLUT-4-containing vesicles can be induced by insulin; however, only leptin secretion is responsive to serum stimulation. This latter effect is resistant to cycloheximide, suggesting that serum triggers the release of a stored pool of presynthesized leptin molecules. We conclude that regulated secretion of leptin and insulin-dependent translocation of GLUT-4 represent different pathways of membrane trafficking in rat adipose cells. NIH 3T3 cells ectopically expressing CAAT box enhancer binding protein-α and Swiss 3T3 cells expressing peroxisome proliferator-activated receptor-γ undergo differentiation in vitro and acquire adipocyte morphology and insulin-responsive glucose uptake. Only the former cell line, however, is capable of leptin secretion. Thus different transcriptional mechanisms control the developmental onset of these two major and independent physiological functions in adipose cells.

Isolation, characterization, and antimicrobial activity of communic acid from Juniperus phoenicea

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mariam Achir ◽  
Mohamed Dakir ◽  
Abdelhakim El Makssoudi ◽  
Asmae Belbachir ◽  
Farida Adly ◽  
...  
Keyword(s):  
Inhibition Zone ◽  
Spectroscopic Properties ◽  
High Yield ◽  
Gram Positive Bacteria ◽  
Chemical Structures ◽  
Soxhlet Apparatus ◽  
Juniperus Phoenicea ◽  
Antibacterial And Antifungal ◽  
First Time ◽  
Better Than

Abstract Objectives A mixture of Z and E communic acid is isolated for the first time from the cones of Juniperus phoenicea. Its biological activity was studied. Methods The plant material was extracted in a Soxhlet apparatus with n-hexane, the resulting extract was subjected to column chromatography (CC) on silica gel. The structure elucidation of the constituents of the isolated fraction was identified by comparison of its spectroscopic properties 1H and 13C NMR data with those reported in the literature. The antimicrobial assay of hexanic extract and isolated compounds was carried out by the disc diffusion and micro-dilution methods. Results A mixture of two diterpene acids isomers was isolated, with a high yield (68%). Their chemical structures were confirmed after comparing their spectral data with published reports. These natural products exhibited a significant antibacterial and antifungal activity against the tested strains. Indeed, for Bacillus cereus, Staphylococcus aureus, and Pseudomonas aeruginosa, the inhibition zone diameters (36–37 mm) was better than penicillin, novobiocin, and amoxicillin. For Candida albicans activity, it show that the mixture possess an activity similar to that of Metrazol. Against Escherichia coli, the inhibitory activity was found less than Amoxicillin. This is the first report of isolation of communic acid from J. phoenicea. Conclusions These results showed that the cones of J. phoenicea were an important source of communic acid, and its hexanic extract had the greatest potential antibacterial activity against both Gram-negative and Gram-positive bacteria and C. albicans.

scholarly journals FIRST REPORT ON FIBRINOLYTIC AND THROMBOLYTIC ACTIVITY OF EUTYPHOEUS GAMMIEI AN EARTHWORM SPECIES COLLECTED FROM TRIPURA, NORTHEAST INDIA

2018 ◽  
Vol 11 (11) ◽  
pp. 236
Author(s):  
Madhusudan Debnath ◽  
Susmita Saha ◽  
Samir Kumar Sil
Keyword(s):  
Thrombolytic Agent ◽  
Northeast India ◽  
New Drugs ◽  
Alternative Source ◽  
Thrombolytic Activity ◽  
Earthworm Species ◽  
Large Size ◽  
Fibrin Plate ◽  
First Time

Objective: The present investigation for the first time evaluated the in vitro fibrinolytic and thrombolytic activities of crude extracts from Eutyphoeus gammiei, native, large size earthworm of Tripura, Northeast, India. The present study was designed to evaluate the therapeutic use of the organism E. gammiei as a source of fibrinolytic and thrombolytic agent(s).Methods: The fibrinolytic activity was studied using by fibrin plate and zymography assays. Thrombolytic assay was carried out according to Prasad et al. (2006) using whole blood.Results: The results obtained clearly indicated E. gammiei as a potential source of fibrinolytic and thrombolytic agents. Both in fibrin plate assay and thrombolytic assay with whole blood, E. gammiei crude homogenate showed similar and close results in respect to that of streptokinase. Fibrin zymography also showed antifibrinolytic activity with producing clear bands. Dose and time dependency also is evident from the results.Conclusion: The results of the present study conclude that the studied earthworm species E. gammiei possessed profound fibrinolytic and thrombolytic activity on human blood and E. gammiei might prove to be useful alternative source for the development of new drugs for treatments involving blood coagulation and fibrinolysis.

scholarly journals Purification of vacuoles from Neurospora crassa

1981 ◽  
Vol 1 (9) ◽  
pp. 797-806
Author(s):  
L E Vaughn ◽  
R H Davis
Keyword(s):  
Neurospora Crassa ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Velocity Sedimentation ◽  
Equilibrium Density ◽  
Differential Centrifugation ◽  
Sucrose Density Gradient Centrifugation ◽  
Peak Density ◽  
Isopycnic Centrifugation

The Neurospora crassa vacuole, defined by its content of basic amino acids, polyphosphate, protease, phosphatases, and alpha-mannosidase, was purified to near homogeneity. The procedure depends upon homogenization of snail gut enzyme-digested cells in a buffer osmotically stabilized with 1 M sorbitol, differential centrifugation of the extract, and sucrose density gradient centrifugation of the organellar pellet. Isopycnic centrifugation of vacuoles in 2.25 M sorbitol-Metrizamide density gradients yielded a peak (density, 1.31 g/cm3) of vacuolar markers coincident with 32P-phospholipids, trichloroacetate-insoluble 14C, and trichloroacetate-soluble 14C. A trail of macromolecular markers in the lighter portions of the gradient reflected, at least in part, heterogeneity of the vacuoles. Almost no contamination by mitochondria or glyoxysomes was detected. Vacuoles were very heterogeneous in size as estimated by velocity sedimentation, but most were larger than mitochondria. Variations of the osmotic strength of the medium were found to alter the equilibrium density of vacuole preparations from 1.06 g/cm3 to over 1.3 g/cm3. This explains the great variation in density reported previously for the "vacuole," the "vesicle," and the "protease particle" of N. crassa, all of which appear to be the same entity.

NEURONS CULTURED ON GaN AND IS ASSOCIATED WITH SYNAPSIN I AND MAP2 EXPRESSION

2008 ◽  
Vol 20 (02) ◽  
pp. 75-82 ◽  
Author(s):  
Chi-Ruei Chen ◽  
Tai-Horng Young
Keyword(s):  
Microelectrode Array ◽  
Synapsin I ◽  
Granule Neurons ◽  
Ldh Assay ◽  
Electric Signals ◽  
First Time ◽  
Neuronal Adhesion ◽  
Very High ◽  
Better Than

In this work, the behaviors of cerebellar granule neurons prepared from 7-day-old Wistar rats on GaN, GaAs, and silicon were investigated. We believe that this is the first time that the GaN has been used as a substrate for neuron cultures to examine its effect on cell response in vitro. The GaN surface structure and its relationship with cells were examined by scanning electron microscopy (SEM), immunofluorescence lactate dehydrogenase (LDH). Compared with silicon used for most neural chips, neurons seeded on GaN were able to form an extensive neuritic network and expressed very high levels of synapsin I coincident with the neurite outgrowth. LDH assay indicated that GaN improve neuron survival better than silicon and GaAs. Between in seven-day and day 15-cultured neurons, these results are consistent with the influence of GaN, in the regulation of neuronal adhesion, neuritic plasticity and survival, within in vitro. The favorable biocompatibility characteristics of GaN can be used to measure electric signals from networks of neuronal cells in culture to make it a possible candidate for use in a microelectrode array.

Sign in / Sign up

Export Citation Format

Share Document