Inner Surface Of Plasma Membrane Research Articles

Apoptosis mediated cytotoxicity of citrus obacunone in human pancreatic cancer cells

Obacunone is one of the oxygenated triterpenoids found in rutaceae family. It has been demonstrated for various biological activities including inhibition of cancer cells proliferation and tumor. The current study is an attempt to understand the mode of cytotoxicity of obacunone using cultured pancreatic cancer (MDA Panc-28) cells. Obacunone exhibited dose and time dependant inhibition of Panc-28 cells proliferation . This was also associated with phosphatidylserine translocation from inner surface of plasma membrane to outer surface in the treated cells, suggesting the involvement of apoptosis. Analysis of cells treated with 50 and 100 μM of obacunone suggests activation of initiator caspase-9 and effector caspase-3, indicating involvement of caspases induced apoptosis. Obacunone upregulated expression of tumor suppressor protein p53, pro-apoptotic protein Bax and downregulated anti-apoptotic protein Bcl2. Furthermore, release of cytochrome- c into cytosol was observed in the cells treated with obacunone. It also resulted in down regulation of vital inflammatory mediators such as NFκB and Cox-2 suggesting the anti-inflammatory potential. For the first time, current study provides an evidence on activation of caspase dependant, cytochrome- c mediated intrinsic apoptosis and anti-inflammatory activity in Panc-28 cells by citrus obacunone. Induction of cytotoxicity and apoptosis was also confirmed by fluorescent tagged microscopic images of obacunone treated Panc-28 cells.

Sources of Activator Calcium Ions in the Contraction of Smooth Muscles in Aplysia kurodai

The physiological and pharmacological properties of contraction and the ultrastructure of buccal mass retractor muscle (I4) and gill-pinnule closure muscle (GPCM) in Aplysia kurodai were studied to learn more about the sources of activator Ca2+ in molluscan smooth muscle. Acetylcholine (ACh) and high K+-induced contractions were reduced by lowering the external Ca2+ concentration, and eliminated by the removal of extracellular Ca2+. Nifedipine appreciably reduced ACh- and high K+-induced contractions, while amiloride decreased only ACh-induced contractions and had no significant effect on high K+-induced contractions. When nifedipine and amiloride were applied together, either type of contraction was still appreciable. Serotonin (5-HT) could potentiate subsequent ACh- and high K+-induced contractions in I4; potentiated tension was significantly reduced by nifedipine and amiloride, whereas 5-HT inhibited ACh-and high K+-induced contractions in GPCM. The potentiating effects of 5-HT may be mediated by the activation of the Ca2+-channel to increase the influx from extracellular Ca2+. Caffeine caused contractions in Ca2+-free solution in both muscles. Electron microscopy revealed sarcolemmal vesicles underneath the plasma membrane in both muscle fibers. Electron microscopical cytochemistry demonstrated that pyroantimonate precipitates were localized in the sarcolemmal vesicles and in the inner surface of plasma membranes in the resting fibers. Present results indicate that the contractions of I4 and GPCM fibers are caused not only by Ca2+-influx but also by Ca2+ release from the intracellular storage sites, such as the sarcolemmal vesicles and the inner surface of plasma membranes.

Effect of heat stress on calcium ultrastructural distribution in pepper anther

Potassium antimonate was used to locate loosely bound calcium in the pepper ( Capsicum annuum) anther under normal and heat stress environments. In the microspore mother cell, a few calcium precipitates were deposited on the surface of the cell, a few in the cytoplasm and almost no precipitates were formed in the nucleus. After 12 h at 40 °C, antimonate calcium deposits increased in the cytoplasm and the nucleus and many emerged on the inner surface of the vacuole membrane. After 24 h heat stress, some cells were partly deformed, numerous calcium precipitates appeared in the cytoplasm and deposited on the surface of the vacuole membrane and in the vacuoles. Compared to the pollen mother cell, there was a significant increase in calcium deposit quantities on the surface and in the cytoplasm of the tetraspore. By heat stressing for 24 h, precipitates obviously increased in the cytoplasm and nucleus of the tetraspore in contrast to the control. In the microspore, many calcium precipitates were formed on the baculae, the inner surface of plasma membrane, vacuole membrane, but only a few in the cytoplasm and nucleus. After 12 h heat exposure, precipitates on plasma membrane became abundant, a few in the cytoplasm and the peripheral nucleus, while no precipitates were seen on the vacuole membrane. As for the anthers exposed to 24-h heat stress, precipitates increased on the inner surface of the plasma membrane, cytoplasm and nucleus. In mature pollen, there was a layer of calcium-induced precipitates on the pollen wall, but few in the cytoplasm and plasma membrane. No obvious calcium changes occurred on mature pollen after 12 or 24 h heat exposure. The relationship between heat stress and calcium distribution was discussed.

Leukocyte polarization in cell migration and immune interactions.

Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. Prior to migration leukocytes undergo polarization, with the formation of a lamellipodium at the leading edge and a uropod at the trailing edge. This cell shape allows them to convert cytoskeletal forces into net cell-body displacement. Leukocyte chemoattractants, including chemokines, provide directional cues for leukocyte motility, and concomitantly induce polarization. Chemoattractant receptors, integrins and other adhesion molecules, cytoskeletal proteins and intracellular regulatory molecules change their cellular localization during cell polarization. A complex system of signal transduction molecules, including tyrosine kinases, lipid kinases, second messengers and members of the Rho family of small GTPases is thought to regulate the cytoskeletal rearrangements underlying leukocyte polarization and migration. The elucidation of the mechanisms and signals that control this complex reorganization will lead to a better understanding of critical questions in cell biology of leukocyte migration and polarity.

Mechanism of Action of a New Component of the Ca2+-Dependent Proteolytic System in Rat Brain: The Calpain Activator

Rat brain contains a calpain activator specific for the μ-form of the proteinase. We now report that this protein factor binds to the catalytic 80 kDa calpain subunit, promoting the dissociation of the heterodimer structure of the proteinase. The successive steps of the activation process, namely the two autoproteolytic steps producing the 78 kDa and the 75 kDa calpain forms, result in a 100 times faster rate. The activator competes with calpastatin and associates with the inner surface of plasma membranes. Based on its properties, the calpain activator can be visualised as the molecule indicating the sites for calpain activation at which the proteinase can also elude the negative control exerted by calpastatin.

Expression of the Farnesyltransferase β-Subunit Gene in Human Ovarian Carcinoma: Correlation to K-rasMutation

Therassignaling protein requires a posttranslational modification to localize it to the inner surface of plasma membrane. In this state it can behave as a signal transduction mediator. Farnesyltransferase plays an important role in this posttranslational processing ofrasby attaching a farnesyl group to the cysteine of therasC-terminal tetrapeptide. In this study, we investigated the relationship of K-rasexpression and mutation with farnesyltransferase β-subunit expression in 20 ovarian tumors (17 carcinomas and 3 low malignant potential tumors) and 4 normal ovaries. The expression level of mRNA was determined by using quantitative PCR and mutation analysis was performed by direct cDNA sequencing. K-rasmutations were found in 1 of 3 low malignant potential tumors and in 4 of 17 carcinoma cases. K-rasmRNA overexpression was found in 1 of 3 low malignant potential tumors (one with mutatedras) and in only 1 of 17 carcinoma cases. Farnesyltransferase β-subunit mRNA overexpression was found in 2 of 3 low malignant potential tumors and in 7 of 17 carcinoma cases. Interestingly, all K-rasmutation cases showed farnesyltransferase β-subunit overexpression. These findings suggest that there may be a direct relationship between K-ras(rasdysfunction) mutation and expression of farnesyltransferase β-subunit gene.

Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization

The 80 kDa human erythrocyte calpain, when exposed to Ca 2+, undergoes autoproteolysis that generates a 75 kDa species, with an increase in Ca 2+ affinity. It is demonstrated here that this proteolytic modification proceeds through an initial step producing a 78 kDa form which is rapidly converted to the 75 kDa one. In the presence of the calpain inhibitor E-64, the 78 kDa form accumulates and only small amounts of the 75 kDa polypeptide are formed. Following loading of erythrocytes with micromolar concentration of Ca 2+, in the presence of the ionophore A23187, the native 80 kDa calpain subunit is extensively translocated and retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa subunit which is released into the soluble fraction of the cells. Following exposure to μM Ca 2+, membrane-bound 80 kDa calpain is converted to the 78 kDa form, this conversion being linearly correlated with the expression of the proteinase activity. Taken together, these results demonstrate that the initial step in calpain activation involves Ca 2+-induced translocation to the inner surface of plasma membranes. In the membrane-bound form the native inactive 80 kDa subunit is converted through intramolecular autoproteolysis to a locally active 78 kDa form. Further autoproteolytic intermolecular digestion converts the 78 kDa to the 75 kDa form, no longer being retained by the membrane. This process generates two active forms of calpain, with different intracellular localisations.

Site-directed activation of calpain is promoted by a membrane-associated natural activator protein

Human erythrocytes contain a calpain activator protein with a molecular mass of approx. 40 kDa. The activator is present in association with the plasma membrane and promotes expression of calpain activity at a concentration of Ca2+ close to physiological values. The initial step of the activating mechanism involves association of the activator with calpain, followed by autoproteolytic activation of the proteinase in the presence of 1 microM Ca2+, at a rate identical to that induced by 1 mM Ca2+. In a reconstituted system, the activator binds to erythrocyte membranes, but not to phospholipid vesicles, suggesting the participation of an intrinsic membrane protein(s). In its membrane-associated form the activator selectively binds calpain, thus favouring interaction of the proteinase with the inner surface of plasma membranes. These results further confirm the importance of a natural activator protein in promoting intracellular activation of calpain under physiological conditions through a site-directed mechanism, which explains the high specificity of the proteinase for membrane of cytoskeletal proteins.

Kinase activity and genetic characterization of a growth related antigen of Drosophila

The Drosophila developmental antigen recognized by the monoclonal antibody F7D6 is expressed in dividing embryonic and imaginal cells but is lost from all differentiating tissues except electrogenic cells of the nervous system and spontaneously contracting muscles. The 63 kDa antigen is associated with the inner surface of plasma membranes and is expressed in several classes of tumorous mutants of Drosophila. The monoclonal antibody was used for immunoprecipitating the antigen for biochemical characterization and for screening expression vector cDNA libraries. Here we report that this oncodevelopmental antigen is a phosphoprotein and a serine-threonine specific protein kinase. A 1.6 kb cDNA isolated by immunological screening of an ovarian library hybridized to a single band on polytene chromosomes, localizing the gene to 72F on the left arm of the third chromosome. Immunofluorescence assays of deficiency stocks in the region confirmed the location of the gene and identity of the cDNA clone, and mapped the gene between the left breakpoints of Df(3L) st100.62 and Df(3L) stj7, i.e., between 72F3-7 and 73A1-2. The biochemical and genetic properties indicate that this is a novel growth-related kinase of Drosophila.

Localization of the low-Mr subunit of cytochrome b558 in human blood phagocytes by immunoelectron microscopy

Cytochrome b558 is a membrane-bound component of the NADPH-oxidase system in phagocytes and consists of a low-Mr subunit of 22 to 23 Kd and a high-Mr subunit of 75 to 90 Kd. The present study on the subcellular localization of the low Mr subunit of cytochrome b558 (p22- phox) in resting human peripheral blood phagocytes was based on immunogold labeling with monoclonal antibody (MoAb) 449, recently characterized. In post-embedding labeled neutrophils, this subunit was found mainly in the membrane of the specific granules. This conclusion was supported by a quantitative analysis of the results obtained in immunogold double-labeled sections with a polyclonal antiserum against lactoferrin (LF) as a marker for specific granules and a polyclonal antiserum against myeloperoxidase (MPO) used to identify azurophil granules. No labeling of the plasma membrane was observed, because of limited penetration of the antibody into the cryosections, preventing the detection of low antigen concentrations. Pre-embedding labeling of digitonin-permeabilized neutrophils, which has the advantage of a better penetration of the antibody into the cells, showed intense immunoreactivity on the cytoplasmic side of intact granules and low labeling on the inner surface of the plasma membrane. These complementary findings indicate that in resting neutrophils the epitope of p22-phox, recognized by MoAb 449, is present on the cytoplasmic side of the membrane of specific granules and the plasma membrane. Similar observations were made in eosinophils, where MoAb 449 reacted strongly with the cytoplasmic side of numerous small granules, and a low level of labeling was observed on the inner surface of the plasma membrane. In monocytes, MoAb 449 labeling also occurred on the inner surface of plasma membrane, of endocytotic compartments, and the outer surface of relatively small granules differing from peroxidase-containing lysosomes, as shown by immunogold double-labeling with MPO.

Localization of the Low-Mr Subunit of Cytochrome b558 in Human Blood Phagocytes by Immunoelectron Microscopy

Cytochrome b558 is a membrane-bound component of the NADPH-oxidase system in phagocytes and consists of a low-Mr subunit of 22 to 23 Kd and a high-Mr subunit of 75 to 90 Kd. The present study on the subcellular localization of the low Mr subunit of cytochrome b558 (p22-phox) in resting human peripheral blood phagocytes was based on immunogold labeling with monoclonal antibody (MoAb) 449, recently characterized. In post-embedding labeled neutrophils, this subunit was found mainly in the membrane of the specific granules. This conclusion was supported by a quantitative analysis of the results obtained in immunogold double-labeled sections with a polyclonal antiserum against lactoferrin (LF) as a marker for specific granules and a polyclonal antiserum against myeloperoxidase (MPO) used to identify azurophil granules. No labeling of the plasma membrane was observed, because of limited penetration of the antibody into the cryosections, preventing the detection of low antigen concentrations. Pre-embedding labeling of digitonin-permeabilized neutrophils, which has the advantage of a better penetration of the antibody into the cells, showed intense immunoreactivity on the cytoplasmic side of intact granules and low labeling on the inner surface of the plasma membrane. These complementary findings indicate that in resting neutrophils the epitope of p22-phox, recognized by MoAb 449, is present on the cytoplasmic side of the membrane of specific granules and the plasma membrane. Similar observations were made in eosinophils, where MoAb 449 reacted strongly with the cytoplasmic side of numerous small granules, and a low level of labeling was observed on the inner surface of the plasma membrane. In monocytes, MoAb 449 labeling also occurred on the inner surface of plasma membrane, of endocytotic compartments, and the outer surface of relatively small granules differing from peroxidase-containing lysosomes, as shown by immunogold double-labeling with MPO.

Calcium-Dependent Binding of Phosphorylated Human Pre Interleukin 1α to Phospholipids1

The effect of phosphorylation of pre interleukin 1 alpha (IL 1 alpha) on its association with various phospholipids was investigated. We prepared genetically engineered truncated human pre IL 1 alpha (residues 64 to 271) and phosphorylated this pre IL 1 alpha in vitro by using the catalytic subunit of cAMP-dependent protein kinase. Phosphorylated truncated pre IL 1 alpha selectively binds to acidic phospholipids including phosphatidic acid, phosphatidylserine, and phosphatidylinositol, but not to other phospholipids (phosphatidylcholine and phosphatidylethanolamine). This binding required divalent cations: Ca2+ or Mn2+, but not Mg2+. In order to obtain half-maximal binding of pre IL 1 alpha to phosphatidic acid or phosphatidylserine, Ca2+ between 5 and 100 microM was required. Unphosphorylated pre IL 1 alpha did not bind to phosphatidylserine, indicating that phosphorylation is required for this binding. Phosphorylated pre IL 1 alpha did not bind to intact peripheral blood mononuclear cells irrespective of lipopolysaccharide stimulation, but did bind to membrane vesicles prepared from these cells in the presence of calcium. Furthermore, phosphorylated pre IL 1 alpha bound only to inside-out ghosts, but not right-side-out ghosts, prepared from human red blood cells. Taken together, these data suggest that phosphorylated pre IL 1 alpha binds to the inner surface of plasma membrane in a Ca2(+)- and phospholipid-dependent manner.

Characterization of Calpain I-Binding Proteins in Human Erythrocyte Plasma Membrane

The calpain-binding components on the plasma membrane were characterized using calpain I. 125I-labeled calpain was bound to inside-out membrane vesicles from human erythrocyte in a Ca2(+)-dependent manner, but not to right-side-out membrane vesicles. The maximum binding was observed at more than 5 microM Ca2+. The binding amount of calpain to the inside-out membrane vesicles was decreased when the vesicles were pretreated with 100 micrograms/ml of trypsin, chymotrypsin, elastase, or pronase P for 30 min at 37 degrees C, although the binding to the vesicles pretreated with 200 micrograms/ml of phospholipase A2 or C was not affected. Calpain-binding proteins in the membrane were analyzed by using a modified immunoblotting for calpain. Immunostained bands of 240, 220, 89, 72, 52, and 36 kDa were detected as the calpain-binding proteins in the native membrane. All of these bands had disappeared in trypsin-treated membrane. The disappearance of bands was dose-dependent with respect to trypsin and paralleled the reduction of binding amount of calpain to the trypsinized membrane. In calpain-treated membrane, the 240 and 36 kDa bands were retained in the blotting, though the other bands disappeared dose-dependently with respect to calpain. These results suggested that the significant component in the inner surface of plasma membrane for binding of calpain was proteinaceous and the calpain-binding proteins could be classified into two species, i.e. substrates of calpain (220, 89, 72, and 52 kDa protein) and non-substrates (240 and 36 kDa protein).

Regulatory mechanism of contraction in the proboscis retractor muscle of a sipunculid worm, Phascolosoma scolops.

Regulatory mechanism of contraction in the proboscis retractor muscle of Phascolosoma scolops was studied by physiological measurements and cytochemical electron microscopy. The magnitude of K+-contracture was dependent on external Ca2+ concentration and the contracture disappeared in Ca2+-free solution. The K+-contracture was suppressed by application of procaine and Mn2+. Caffeine induced contracture even when external Ca2+ was absent. Ultrastructural observations of the retractor muscle cells showed the presence of a large number of vesicles (subsarcolemmal vesicles), corresponding to the sarcoplasmic reticulum in vertebrate skeletal muscle, underneath the plasma membrane. For the cytochemical electron microscopy, the muscle fibers were fixed with 1% OsO4 solution containing 2% K-pyroantimonate. In the relaxed fibers, pyroantimonate precipitates were localized along the inner surface of plasma membrane and in the subsarcolemmal vesicles. In the contracting fibers, the precipitates were uniformly distributed in the myoplasm. The X-ray microanalysis revealed that the precipitates contained Ca. These results suggest that the contractile system is activated by the influx of extracellular Ca2+ as well as by the release of Ca2+ from the intracellular structures such as the inner surface of the plasma membrane and subsarcolemmal vesicles.

Physiological and cytochemical studies on activator calcium in contraction by smooth muscle of a sea cucumber, Isostichopus badionotus.

The physiological properties of mechanical responses and the intracellular localization and translocation of calcium as a pyroantimonate precipitate were studied in the longitudinal retractor muscle (LRM) of a Bermuda sea cucumber. Acetylcholine (ACh)-induced contraction was reduced by lowering the external Ca concentration, and suppressed completely by prolonged soaking in Ca-free solution. The magnitude of ACh-induced contraction was decreased by Mn and La ions. Furthermore, procaine reduced the ACh-induced contraction. The complete removal of Ca and Mg ions from the external medium induced a so called Ca . Mg-removal contraction. Electron microscopically, numerous subsarcolemmal vesicles were observed in the LRM fibers. In the resting fibers, pyroantimonate precipitates were localized in the subsarcolemmal vesicles and along the inner surface of plasma membrane. While, in the fiber fixed during mechanical activity, the pyroantimonate precipitates were decreased remarkably in the subsarcolemmal vesicles and at the plasma membrane, and diffusely distributed in the myoplasm. Electron-probe X-ray microanalysis showed that the precipitate contains Ca in a significant amount. These results indicate that the contraction of the LRM fibers is caused not only by Ca-influx but also by Ca-release from the intracellular storage sites, such as the subsarcolemmal vesicles and the inner surface of plasma membrane.

Paracrystalline arrays of membrane-to-membrane cross bridges associated with the inner surface of plasma membrane.

In cultured cells of the rat kangaroo PtK2 line, veils of the cell surface were observed which consisted of only plasma membrane and paracrystalline arrays of membrane-associated particles sandwiched in between. These membrane-to-membrane cross-bridging 9-to 11-nm wide particles were somewhat coumellar-shaped and were arranged on a hexagonal lattice with an interparticle distance of 16nm. At higher magnification, they revealed an unstained core, thus suggesting a ringlike substructure. Similar arrays of paracrystal-containing veils, which were rather variable in size and frequency, were also observed in other cultured cells. It is hypothesized that these paracrystals represent protein macromolecular complexes associated with the inner plasma membrane surface which crystallize when plasma membranes come into close intracellular contact and other components of the subsurface network are removed.