scholarly journals MACROPHAGE PLASMA MEMBRANES

1971 ◽  
Vol 133 (4) ◽  
pp. 785-806 ◽  
Author(s):  
Ralph L. Nachman ◽  
Barbara Ferris ◽  
James G. Hirsch
Keyword(s):  
Alveolar Macrophages ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Protein A ◽  
Membrane Preparation ◽  
Polystyrene Spheres ◽  
Protein Patterns ◽  
Large Numbers ◽  
Structural Preservation ◽  
High Degree

Plasma membranes have been isolated from pure populations of rabbit alveolar macrophages which were swollen in water, fixed briefly with glutaraldehyde, disrupted by Dounce homogenization, and separated by sucrose gradient centrifugation. The recovered membranes exhibited good structural preservation and enzymatic activity; both morphologic and biochemical evidence indicated a high degree of purity (>90%) of the membrane preparation. Interiorized plasma membranes were also prepared without exposure to glutaraldehyde from phagocytic vacuoles recovered from alveolar macrophages which had ingested large numbers of polystyrene spheres. These membranes were contaminated with lysosomal constituents, but they were nevertheless of value for comparison to the "pure" membranes isolated by the glutaraldehyde procedure. Acrylamide gel electrophoresis of the solubilized plasma membranes and phagolysosomal membranes revealed similar protein patterns, with seven to nine individual components ranging in molecular weight from 70,000 to 140,000. The two most rapidly migrating components gave positive reactions for lipid as well as protein. A band containing carbohydrate was detected near the origin of the plasma membrane gels. Antisera were made by injecting guinea pigs with the purified rabbit alveolar macrophage plasma membranes. Gel diffusion and immunoelectrophoretic study of these antisera established the presence of rabbit immunoglobulin G and of one or two other antigenic constituents in the membrane preparation.

scholarly journals Targeting Post-Translational Modifications of the p73 Protein: A Promising Therapeutic Strategy for Tumors

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1916
Author(s):  
Ziad Omran ◽  
Mahmood H. Dalhat ◽  
Omeima Abdullah ◽  
Mohammed Kaleem ◽  
Salman Hosawi ◽  
...  
Keyword(s):  
Current Knowledge ◽  
Protein A ◽  
P53 Gene ◽  
Cancer Therapies ◽  
Transactivation Domain ◽  
P53 Family ◽  
Post Translational Modifications ◽  
P53 Status ◽  
Apoptotic Effects ◽  
High Degree

The tumor suppressor p73 is a member of the p53 family and is expressed as different isoforms with opposing properties. The TAp73 isoforms act as tumor suppressors and have pro-apoptotic effects, whereas the ΔNp73 isoforms lack the N-terminus transactivation domain and behave as oncogenes. The TAp73 protein has a high degree of similarity with both p53 function and structure, and it induces the regulation of various genes involved in the cell cycle and apoptosis. Unlike those of the p53 gene, the mutations in the p73 gene are very rare in tumors. Cancer cells have developed several mechanisms to inhibit the activity and/or expression of p73, from the hypermethylation of its promoter to the modulation of the ratio between its pro- and anti-apoptotic isoforms. The p73 protein is also decorated by a panel of post-translational modifications, including phosphorylation, acetylation, ubiquitin proteasomal pathway modifications, and small ubiquitin-related modifier (SUMO)ylation, that regulate its transcriptional activity, subcellular localization, and stability. These modifications orchestrate the multiple anti-proliferative and pro-apoptotic functions of TAp73, thereby offering multiple promising candidates for targeted anti-cancer therapies. In this review, we summarize the current knowledge of the different pathways implicated in the regulation of TAp73 at the post-translational level. This review also highlights the growing importance of targeting the post-translational modifications of TAp73 as a promising antitumor strategy, regardless of p53 status.

scholarly journals A novel procedure for the rapid isolation of surfactant protein A with retention of its alveolar-macrophage-stimulating properties

1995 ◽  
Vol 309 (2) ◽  
pp. 551-555 ◽  
Author(s):  
J F van Iwaarden ◽  
F Teding van Berkhout ◽  
J A Whitsett ◽  
R S Oosting ◽  
L M G van Golde
Keyword(s):  
Alveolar Macrophage ◽  
Alveolar Macrophages ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Normal Rat ◽  
Normal Human ◽  
Novel Method ◽  
Alveolar Proteinosis ◽  
Simplex Virus

Previous studies have shown that surfactant protein A (SP-A) derived from alveolar-proteinosis patients activates rat alveolar macrophages. However, it is not known if normal rat, dog and human SP-A can also stimulate alveolar macrophages. As alveolar-proteinosis SP-A has a slightly different structure from ordinary SP-A, it would be possible that the ascribed alveolar-macrophage-stimulating properties of SP-A are restricted to alveolar-proteinosis SP-A. To clarify this issue, we isolated SP-A from normal rat and dog pulmonary surfactants, using the same isolation technique commonly used for the isolation of alveolar-proteinosis SP-A, i.e. by butanol precipitation. In contrast with human alveolar-proteinosis SP-A, rat and dog SP-A obtained thus could not activate rat alveolar macrophages to produce oxygen radicals or enhance the phagocytosis of fluorescein isothiocyanate-labelled herpes simplex virus. However, rat, dog and normal human SP-A isolated by a novel method, involving extraction from pulmonary surfactant by using n-octyl beta-D-glucopyranoside and subsequent purification by cation-exchange chromatography, were able to elicit an oxidative burst in rat as well as normal human alveolar macrophages. In addition, dog and rat SP-A obtained thus stimulated the phagocytosis of herpes simplex virus by rat alveolar macrophages. These findings indicate that normal human, rat and dog SP-A have the same alveolar-macrophage-stimulating properties as human alveolar proteinosis SP-A. Dog and rat SP-A isolated by this novel method had the same Ca(2+)-dependent self-aggregation and lipid-aggregation properties as SP-A isolated by butanol precipitation. The new and milder isolation procedure yielded SP-A of high purity, as judged by SDS/PAGE and ELISA.

scholarly journals Surfactant protein D binding to alveolar macrophages

1994 ◽  
Vol 300 (1) ◽  
pp. 237-242 ◽  
Author(s):  
K Miyamura ◽  
L E A Leigh ◽  
J Lu ◽  
J Hopkin ◽  
A López Bernal ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Serum Proteins ◽  
Specific Binding ◽  
Lectin Binding ◽  
Protein A ◽  
Direct Interaction ◽  
Surfactant Protein ◽  
Lung Epithelial Cells ◽  
Surfactant Protein D ◽  
Apparent Dissociation Constant

Surfactant protein D (SP-D) is a lung-specific protein, synthesized and secreted by lung epithelial cells. It belongs to group III of the family of C-type lectins; each member of this group has an unusual overall structure consisting of multiple globular ‘head’ regions (which contain the C-type lectin domains) linked by triple-helical, collagen-like, strands. This group includes the surfactant protein A (SP-A) and the serum proteins mannan-binding protein, conglutinin and collectin-43, all of which have been shown to bind to the C1q receptor found on a wide variety of cells, including macrophages. Both SP-D and SP-A have been shown to enhance oxygen radical production by alveolar macrophages. Although this strongly suggests a direct interaction between SP-D and a specific receptor on alveolar macrophages, it is still unclear whether SP-D binds to the same receptor used by SP-A and/or C1q. Human SP-D was isolated from amniotic fluid and was radiolabelled using 125I. Alveolar macrophages were isolated from human bronchioalveolar lavage fluid, and also from bovine lung washings, by differential adhesion to 24-well tissue-culture plates. The study was carried out using EDTA-containing buffers, to eliminate Ca(2+)-dependent C-type lectin binding, and was also carried out at 4 degrees C to eliminate possible internalization by the cells. 125I-SP-D showed specific binding to alveolar macrophages in both a time- and concentration-saturable manner. The binding was inhibited, by approx. 90%, on addition of a 200-fold excess of unlabelled SP-D. The apparent dissociation constant (Kd) was (3.6 +/- 1.3) x 10(-11) M, based on the assumption that native SP-D is assembled as a dodecamer of 12 identical polypeptides of 43 kDa to yield a protein of 516 kDa. C1q was also shown to bind alveolar macrophages (Kd 3 x 10(-6) M), but addition of C1q did not show inhibition of the binding of 125I-SP-D to the macrophages. We conclude that SP-D binds specifically to alveolar macrophages and the receptor involved is different from that utilized by C1q.

Differential susceptibilities of isolated hamster lung cell types to amiodarone toxicity

1998 ◽  
Vol 76 (7-8) ◽  
pp. 721-727 ◽  
Author(s):  
M W Bolt ◽  
W J Racz ◽  
J F Brien ◽  
T M Bray ◽  
T E Massey
Keyword(s):  
Alveolar Macrophages ◽  
Gradient Centrifugation ◽  
Cell Types ◽  
Clara Cell ◽  
Blue Dye ◽  
Alveolar Type ◽  
Specific Cell ◽  
Type Ii ◽  
Clara Cells

Treatment of cardiac dysrhythmias with the iodinated benzofuran derivative amiodarone (AM) is limited by pulmonary toxicity. The susceptibilities of different lung cell types of male Golden Syrian hamsters to AM-induced cytotoxicity were investigated in vitro. Bronchoalveolar lavage and protease digestion to release cells, followed by centrifugal elutriation and density gradient centrifugation, resulted in preparations enriched with alveolar macrophages (98%), alveolar type II cells (75-85%), and nonciliated bronchiolar epithelial (Clara) cells (35-50%). Alveolar type II cell and Clara cell preparations demonstrated decreased viability (by 0.5% trypan blue dye exclusion) when incubated with 50 µM AM for 36 h, and all AM-treated cell preparations demonstrated decreased viability when incubated with 100 or 200 µM AM. Based on a viability index ((viability of AM-treated cells ÷ viability of controls) × 100%), the Clara cell fraction was significantly (p < 0.05) more susceptible than all of the other cell types to 50 µM AM. However, AM cytotoxicity was greatest (p < 0.05) in alveolar macrophages following incubation with 100 or 200 µM AM. There was no difference between any of the enriched cell preparations in the amount of drug accumulated following 24 h of incubation with 50 µM AM, whereas alveolar macrophages accumulated the most drug during incubation with 100 µM AM. Thus, the most susceptible cell type was dependent on AM concentration. AM-induced cytotoxicity in specific cell types may initiate processes leading to inflammation and pulmonary fibrosis.Key words: amiodarone, susceptibility, alveolar macrophage, accumulation.

scholarly journals A rapid immunological procedure for the isolation of hormonally sensitive rat fat-cell plasma membrane

1976 ◽  
Vol 154 (1) ◽  
pp. 11-21 ◽  
Author(s):  
J P Luzio ◽  
A C Newby ◽  
C N Hales
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Membrane Preparation ◽  
Fat Cells ◽  
Fat Cell ◽  
Cell Plasma ◽  
Rat Fat Cells ◽  
Plasma Membrane Preparation ◽  
Isolated Rat

1. A rapid method for the isolation of hormonally sensitive rat fat-cell plasma membranes was developed by using immunological techniques. 2. Rabbit anti-(rat erythrocyte) sera were raised and shown to cross-react with isolated rat fat-cells. 3. Isolated rat fat-cells were coated with rabbit anti-(rat erythrocyte) antibodies, homogenized and the homogenate made to react with an immunoadsorbent prepared by covalently coupling donkey anti-(rabbit globulin) antibodies to aminocellulose. Uptake of plasma membrane on to the immunoadsorbent was monitored by assaying the enzymes adenylate cyclase and 5′-nucleotidase and an immunological marker consisting of a 125I-labelled anti-(immunoglobulin G)-anti-cell antibody complex bound to the cells before fractionation. Contamination of the plasma-membrane preparation by other subcellular fractions was also investigated. 4. By using this technique, a method was developed allowing 25-40% recovery of plasma membrane from fat-cell homogenates within 30 min of homogenization. 5. Adenylate cyclase in the isolated plasma-membrane preparation was stimulated by 5 μm-adrenaline.

scholarly journals LYSOSOMAL PHOSPHOLIPASES A1 AND A2 OF NORMAL AND BACILLUS CALMETTE GUERIN-INDUCED ALVEOLAR MACROPHAGES

1973 ◽  
Vol 56 (3) ◽  
pp. 621-627 ◽  
Author(s):  
Richard C. Franson ◽  
Moseley Waite
Keyword(s):  
Acid Phosphatase ◽  
Alveolar Macrophages ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Marker Enzymes ◽  
Sucrose Gradient Centrifugation ◽  
Control Distribution ◽  
Specific Activities ◽  
Selective Increase ◽  
Phospholipases A

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5–4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, ß-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A1 and A2 which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50–70% by 2.0 mM CaCl2. Homogenates of BCG-induced macrophages had specific activities of ß-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 107 cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.

scholarly journals Localization of protein disulfide isomerase on plasma membranes of rat exocrine pancreatic cells.

1988 ◽  
Vol 36 (8) ◽  
pp. 1069-1074 ◽  
Author(s):  
S Akagi ◽  
A Yamamoto ◽  
T Yoshimori ◽  
R Masaki ◽  
R Ogawa ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Amorphous Material ◽  
Protein A ◽  
Disulfide Isomerase ◽  
Gold Particles ◽  
Pancreatic Cells ◽  
Acinar Lumen ◽  
Protein Disulfide

We investigated immunocytochemically the ultrastructural localization of protein disulfide isomerase (PDI) in rat pancreatic exocrine cells by use of the post-embedding protein A-gold technique. We found that not only the endoplasmic reticulum (ER) and nuclear envelope but also the trans-Golgi cisternae, secretory granules, and plasma membranes were heavily labeled with gold particles. Labeling density of the gold particles in the rough ER and plasma membranes of the exocrine pancreatic cells was twofold and twentyfold greater, respectively, than that of hepatocytes. In the acinar lumen, amorphous material presumably corresponding to the secreted zymogens was also labeled with gold particles. These results suggest that in rat exocrine pancreatic cells a significant amount of PDI is transported to the plasma membrane and secreted to the acinar lumen.

Surfactant protein A protects growing cells and reduces TNF-alpha activity from LPS-stimulated macrophages

1996 ◽  
Vol 271 (2) ◽  
pp. L310-L319 ◽  
Author(s):  
J. C. McIntosh ◽  
S. Mervin-Blake ◽  
E. Conner ◽  
J. R. Wright
Keyword(s):  
Host Defense ◽  
Alveolar Macrophages ◽  
Cell Function ◽  
Immune Cell ◽  
Protein A ◽  
Surfactant Protein ◽  
Alpha Activity ◽  
Tnf Alpha ◽  
Activated Macrophages ◽  
Immune Functions

In addition to its effect on surfactant lipids, surfactant protein (SP)-A promotes host defense. To define further the role of SP-A in regulating immune cell function, we evaluated the effect of SP-A on lipopolysaccharide (LPS)-activated alveolar macrophages in two settings. First, cocultured LPS-activated macrophages significantly inhibited lung fibroblast growth, but SP-A (added daily) attenuated this effect. Both LPS and SP-A acted via macrophages rather than directly on the fibroblasts, at least partially by affecting tumor necrosis factor (TNF)-alpha activity. TNF-alpha reproduced the growth suppression, anti-TNF-alpha antibodies attenuated the effect LPS-activated macrophages, and SP-A reduced TNF-alpha activity in conditioned medium. Second, SP-A reduced TNF-alpha activity in medium from isolated LPS-stimulated macrophages. The effects of SP-A were noted with or without serum, were dose-dependent and reversible, and were seen with two different serotypes of smooth LPS. Equimolar concentrations of immunoglobulin G and C1q had no effect. Thus SP-A both enhances host defense and modulates immune functions of alveolar macrophages.

Mapping subcellular distribution of Na+-K+-ATPase in rat parotid gland

1986 ◽  
Vol 250 (3) ◽  
pp. C430-C441 ◽  
Author(s):  
C. N. Conteas ◽  
A. A. McDonough ◽  
T. R. Kozlowski ◽  
C. B. Hensley ◽  
R. L. Wood ◽  
...  
Keyword(s):  
Acinar Cell ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Sedimentation Equilibrium ◽  
Equilibrium Density ◽  
Phase System ◽  
Gamma Glutamyl Transpeptidase ◽  
Gamma Glutamyl ◽  
Rat Parotid ◽  
Glutamyl Transpeptidase

Recent subcellular fractionation studies have raised the possibility that Na+-K+-ATPase might be present in both the apical and the basal-lateral membranes of exocrine gland acinar cells. Analytical fractionation and immunofluorescence microscopy studies of rat parotid glands were performed to confirm this interpretation. The distributions of biochemical markers after analyses based on differential sedimentation, equilibrium density-gradient centrifugation, and partitioning in an aqueous polymer two-phase system defined a total of 15 physically and biochemically distinct membrane populations. Among these populations, it was possible to select one (designated population i) with the characteristics expected of acinar cell basal-lateral plasma membranes. It contained Na+-K+-ATPase enriched 33-fold, and gamma-glutamyl transpeptidase enriched 23-fold with respect to the initial homogenate. A second population (designated population c) had the characteristics expected of acinar cell apical plasma membranes; it contained Na+-K+-ATPase enriched 28-fold, and gamma-glutamyl transpeptidase enriched 53-fold with respect to the initial homogenate. Although the identification of population c remains provisional, immunofluorescence studies verified that Na+-K+-ATPase is present in both the apical and the basal-lateral acinar cell plasma membranes. In view of these results, it is likely that the apical Na+-K+-ATPase would participate in series with basal-lateral sodium- and chloride-entry pathways in driving the secretory electrolyte fluxes.

scholarly journals Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-l-fucosidase from rat testis and epididymal spermatozoa

1996 ◽  
Vol 318 (3) ◽  
pp. 821-831 ◽  
Author(s):  
Manuel AVILÉS ◽  
Irene ABASCAL ◽  
José Angel MARTÍNEZ-MENÁRGUEZ ◽  
María Teresa CASTELLS ◽  
Sheri R. SKALABAN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Light And Electron Microscopy ◽  
Enzymic Activity ◽  
Epididymal Sperm ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Neutral Ph

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum α-l-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for α-l-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the α-l-fucosidase activity was associated with the 48000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of α-l-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH–activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm–egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm–egg interactions.

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