scholarly journals THE EFFECT OF POLY-L-LYSINE ON THE UPTAKE OF REOVIRUS DOUBLE-STRANDED RNA IN MACROPHAGES IN VITRO

1973 ◽  
Vol 57 (2) ◽  
pp. 484-498 ◽  
Author(s):  
Rolf Seljelid ◽  
Samuel C. Silverstein ◽  
Zanvil A. Cohn
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Nucleic Acids ◽  
Acid Hydrolases ◽  
Cell Compartment ◽  
Double Stranded Rna ◽  
Secondary Lysosomes ◽  
Toxic Concentrations ◽  
Vacuolar System

The effect of polycations on cultured mouse peitoneal macrophages has been examined. Polycations, at concentrations greater than 5 µg/ml, are toxic for macrophages) as measured by failure of the cells to exclude vital dyes. At toxic concentrations polycations bind in large amounts to nuclei and endoplasmic reticulum, while at nontoxic levels polycations bind selectively to the cell surface. Nontoxic concentrations of polycations stimulate binding of reovirus double-stranded (ds) RNA to the macrophages by forming polycation-dsRNA complexes either in the medium or at the cell surface. These complexes enter the cell in endocytic vacuoles and are concentrated in secondary lysosomes. Despite exposure to the acid hydrolases within this cell compartment, the dsRNA and the polycation (poly-L-lysine) are conserved in a macromolecular form within the vacuolar system. The mechanism(s) by which the uptake of infectious nucleic acids and the induction of interferon by dsRNA are stimulated by polycations are discussed.

Evidence for NAD Nucleosidase in Rabbit-Liver Lysosomes

1975 ◽  
Vol 53 (2) ◽  
pp. 143-148 ◽  
Author(s):  
A. Mellors ◽  
A. K. L. Lun ◽  
O. N. Peled
Keyword(s):  
Plasma Membrane ◽  
Maximum Activity ◽  
Adenyl Cyclase ◽  
Rabbit Liver ◽  
Acid Hydrolases ◽  
Nad Glycohydrolase ◽  
Triton Wr 1339 ◽  
Liver Lysosomes ◽  
Secondary Lysosomes

A method is described for the isolation of secondary lysosomes from homogenates of rabbit liver. The uptake of Triton WR-1339 by rabbit-liver lysosomes when administered by intraperitoneal injection was used to decrease the density of secondary lysosomes. Lysosomal fractions prepared by this method contain an NAD nucleosidase (NAD glycohydrolase, EC 3.2.2.5), an enzyme which has previously been considered to be associated with other subcellular fractions. The enzyme has maximum activity at pH 6 and cleaves both NAD and NADP. It is inhibited by nicotinamide (Ki = 4.5 mM) and by HgCl2. Both nucleosidase and 2′-nucleotidase show in-vitro latency typical of lysosomal acid hydrolases. Rabbit-liver plasma-membrane fractions were isolated which contained most 5′-nucleotidase but relatively little nucleosidase, whereas rabbit liver lysosomes contain both 5′-nucleotidase and nucleosidase enzymes but little adenyl cyclase.

Abstract 18708: Vascular Dysfunction in Hyperhomocysteinemia: In vitro Evidence for Endoplasmic Reticulum Stress-mediated Calcium-activated Potassium Channel Dysfunction

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Wen-Tao Sun ◽  
Xiang-Chong Wang ◽  
Cheuk-Man Yu ◽  
Shun-Hay Pun ◽  
Qin Yang
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Cell Surface ◽  
Er Stress ◽  
Vascular Dysfunction ◽  
Cell Protein ◽  
Α Subunit ◽  
Whole Cell

Objectives: KCa channels play an important role in the control of vascular tone. Opening of IKCa and SKCa in endothelial cells underlies the classic EDHF pathway and promotes NO production. Smooth muscle BKCa provides a negative feedback mechanism opposing vasoconstriction and is an effector of NO and EDHF. Previous studies demonstrated homocysteine (Hcy), a risk factor for atherosclerosis, compromises NO and EDHF function, however, whether KCa is involved is poorly studied and the underlying mechanisms remain unknown. We studied the effect of Hcy on vascular KCa with the role of endoplasmic reticulum (ER) stress explored. Methods: In vitro studies were performed in porcine coronary arteries and primary cultured porcine coronary endothelial (PCECs) and smooth muscle cells (PCSMCs). IKCa and SKCa-, and BKCa-mediated relaxations were studied in endothelium-intact and -denuded arteries in a myograph. IKCa and SKCa currents in PCECs and BKCa current in PCSMCs were analyzed by whole-cell patch clamp and channel expressions were examined by western blot. Results: Hcy impairs the role of IKCa and SKCa, and BKCa in vasorelaxation. Relaxant responses to channel activators NS309 and NS1619 were attenuated and EDHF-type response was inhibited. Hcy suppressed IKCa and SKCa currents in PCECs and BKCa currents in PCSMCs. Inhibition of ER stress enhanced KCa currents and improved EDHF-type and channel activators-induced responses. Whole-cell protein levels of IKCa and SKCa remained unchanged in Hcy-exposed PCECs whereas IKCa and SKCa at cell surface were significantly decreased. Hcy lowered protein of β1 but not α subunit of BKCa in PCSMCs. The decrease in cell surface IKCa and SKCa and reduction of BKCa β1 were restored by ER stress inhibition. Further, inhibition of PERK increased BKCa β1 protein and enhanced BKCa current. Conclusion: ER stress mediates Hcy-induced vascular dysfunction through inhibition of KCa. Suppression of cell surface expression underlies ER stress-mediated IKCa and SKCa inhibition. Downregulation of BKCa β1 by PERK-ER stress pathway plays a key role in the loss of BKCa function. This study provides new mechanistic insights into the role of ER stress in vascular dysfunction. Supported by RGC GRF CUHK4774/12M & CUHK14118414, and NSFC 81200123.

scholarly journals Fractionation of iodinated particles and mitochondria from thyroid by zonal centrifugation and a study of their heterogeneity

1974 ◽  
Vol 138 (2) ◽  
pp. 299-304 ◽  
Author(s):  
Raymond Miquelis ◽  
Claude Simon
Keyword(s):  
Succinate Dehydrogenase ◽  
Radioactive Iodine ◽  
Acid Hydrolases ◽  
Linear Gradient ◽  
Fourth Class ◽  
Secondary Lysosomes ◽  
Gradient Separation ◽  
Zonal Rotor

1. The subcellular particles of horse and rat thyroids were fractionated in a B XIV zonal rotor on a non-linear gradient of Ficoll after labelling with radioactive iodine in vitro (horse) or in vivo (rat). In the horse, the resulting fractions were analysed for radioactive iodine, protein and enzymes representative of certain subcellular particles. In the rat, iodine turnover and thyrotrophin stimulation were studied. 2. The population of iodinated particles could be subdivided into three main classes, characterized by differences in β-galactosidase and acid phosphatase content and position in the gradient. The presence of a fourth class of particles is suggested. 3. It is concluded that iodinated particles isolated from the thyroid are essentially secondary lysosomes. Their heterogeneity is established with respect to their position in the gradient, their content of acid hydrolases and their iodine turnover. 4. The iodine pools of these secondary lysosomes are increased by thyrotrophin without any change in their number. 5. Their functional significance is discussed. 6. The distribution of mitochondria as judged by succinate dehydrogenase was also studied. The succinate dehydrogenase was spread throughout the gradient with a maximum of activity (40%) in the upper layer of the gradient. Separation of mitochondria from lysosomes by this method was not successful.

scholarly journals Isolation of pig thyroid lysosomes. Biochemical and morphological characterization

1985 ◽  
Vol 232 (2) ◽  
pp. 529-537 ◽  
Author(s):  
C Alquier ◽  
P Guenin ◽  
Y Munari-Silem ◽  
C Audebet ◽  
B Rousset
Keyword(s):  
Endoplasmic Reticulum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Average Diameter ◽  
Morphological Characterization ◽  
Thyroid Cell ◽  
Acid Hydrolase ◽  
Acid Hydrolases ◽  
Thyroid Cells ◽  
Secondary Lysosomes ◽  
Isopycnic Centrifugation

Open thyroid follicles were prepared by mechanical disruption of pig thyroid fragments through a metal sieve. This procedure allowed preparation of thyroid-cell material depleted of colloid thyroglobulin. Open thyroid follicles were used to prepared a crude particulate fraction, which contained lysosomes, mitochondria and endoplasmic reticulum. These organelles were subfractionated by isopycnic centrifugation on iso-osmotic Percoll gradients. A lysosomal peak was identified by its content of acid hydrolases: acid phosphatase, cathepsin D, β-galactosidase and β-glucuronidase. The lysosomal peak was well separated from mitochondria and endoplasmic reticulum. The lysosomal peak, from which Percoll was removed by centrifugation, was taken as the purified lysosome fraction (L). Lysosomes of fraction L were purified 45-55-fold (as compared with the homogenate) and contained about 5% of the total thyroid acid hydrolase activities. Electron microscopy showed that fraction L was composed of an approx. 90% pure population of lysosomes, with an average diameter of 220 nm. Acid hydrolase activities were almost completely (80-90%) released by an osmotic-pressure-dependent lysis. Thyroglobulin was identified by polyacrylamide-gel electrophoresis as a soluble component of the lysosome fraction. In conclusion, a 50-fold purification of pig thyroid lysosomes was achieved by using a new tissue-disruption procedure and isopycnic centrifugation on Percoll gradient. The presence of thyroglobulin indicates that the lysosome population is probably composed of primary and secondary lysosomes. Isolated thyroid lysosomes should serve as an interesting model to study the reactions whereby thyroid hormones are generated from thyroglobulin and released into the thyroid cells.

scholarly journals Normal rabbit alveolar macrophages. II. Their primary and secondary lysosomes as revealed by electron microscopy and cytochemistry.

1976 ◽  
Vol 144 (4) ◽  
pp. 920-932 ◽  
Author(s):  
B A Nichols
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Acid Phosphatase ◽  
Rough Endoplasmic Reticulum ◽  
Alveolar Macrophages ◽  
Digestive Enzymes ◽  
Acid Hydrolases ◽  
Secondary Lysosomes ◽  
Enzymatic Machinery ◽  
Cytochemical Markers

In this investigation, vacuoles containing tubular myelin proved to be digestive compartments with cytochemical reactivity for acid phosphatase and arylsulfatase. These cytochemical markers identify the secondary lysosomes, known to contain enzymes capable of hydrolyzing phospholipids like surfactant. Therefore, it appears that alveolar macrophages possess the enzymatic machinery for the degradation of the tubular myelin found in their digestive vacuoles. Although it thus appears evident that alveolar macrophages participate in the turnover of surfactant, the quantitative significance of this route of disposal is undetermined. This investigation has also established that acid hydrolases, so prominently displayed in the secondary lysosomes, are also localized in the rough endoplasmic reticulum and in Golgi-endoplasmic reticulum-lysosomes (GERL). Moreover, small vesicles which are produced from GERL serve as primary lysosomes in transporting digestive enzymes to the vacuoles.

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

The Mesothelial Cell as a Non-Thrombogenic Surface

1984 ◽  
Vol 52 (02) ◽  
pp. 102-104 ◽  
Author(s):  
L J Nicholson ◽  
J M F Clarke ◽  
R M Pittilo ◽  
S J Machin ◽  
N Woolf
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Blood Flow ◽  
Cell Surface ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Plastic Film ◽  
In Vitro System ◽  
Scanning Electron

SummaryA technique for harvesting mesothelial cells is described. This entails collagenase digestion of omentum after which the cells can be cultured. The technique has been developed using the rat, but has also been successfully applied to human tissue. Cultured rat mesothelial cells obtained in this way have been examined by scanning electron microscopy. Rat mesothelial cells grown on plastic film have been exposed to blood in an in vitro system using a Baumgartner chamber and have been demonstrated to support blood flow. No adhering platelets were observed on the mesothelial cell surface. Fibroblasts similarily exposed to blood as a control were washed off the plastic.

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