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Biomedical subjects

I Dunia

Publications and source records attributed to I Dunia.

At least 37 records · Page 2Linked to original sources
PubMed1992

Functional and structural damage in Leishmania mexicana exposed to the cationic peptide dermaseptin.

The effect of dermaseptin (DS), a 34 amino acid residue cationic peptide isolated from Phyllomedusa sauvagii skin, has been studied on promastigotes of Leishmania mexicana growing in vitro. Within 5 min of incubation in the presence of DS, the flagellated parasites lost their motility. DS inhibited promastigote growth by 50% at a concentration of 3 microM and by 100% at 10 microM. Immunocytochemical, freeze fracture, label fracture and electron microscopic observations have shown that the amphipathic peptide generates perturbations of the lipid bilayer leading to altered permeability of the surface membrane and death of the parasite.

Amphibian Proteins↗
PubMed1990

Fatty acid acylation of lens fiber plasma membrane proteins. MP26 and alpha-crystallin are palmitoylated.

We describe in this report the fatty acylation of some of the main polypeptides from the eye lens fibers. MP26, the major lens fiber plasma membrane protein, and probably MP22, its natural degradation product, are palmitoylated in a post-translational process. This is also the case for alpha-crystallin, a major cytoplasmic structural protein shown to interact directly with the plasma membrane. Furthermore, a 65 kDa non-identified polypeptide and a high molecular weight component are also modified in the same way.

Acylation↗
PubMed1990

Plasma membrane-cytoskeleton damage in eye lenses of transgenic mice expressing desmin.

Immunocytochemistry of eye lens cells from transgenic mice coexpressing desmin and vimentin reveals that the transgenic desmin expression is not uniform. In the same lens, some epithelial and fiber cells overexpress desmin, while in others the desmin gene seems to be silent. Conversely, the endogenous vimentin is always expressed. The concomitant expression of vimentin and desmin results in the assembly of hybrid intermediate filaments (IFs). Moreover, the overexpression of the transgene generates pleomorphic IF assembly and leads to intermingled filamentous whorls and to accumulation of amorphous desmin. The abnormalities of IF assembly induced by the genetic manipulation are correlated with perturbation of the enucleation process in the lens fibers, changes in cell shape, fiber fusion and extensive internalization of the general plasma membrane and junctional domains. The alterations of lens cells described in this study were observed in all transgenic mice examined. The level of expression of the transgene was paralleled by the degree of damage. Our results indicate that proper expression, assembly and membrane interaction of IFs play an important role in the terminal differentiation of the lenticular epithelium into fiber cells. We anticipate that alterations during these processes may initiate cataract formation.

Animals↗
PubMed1989

Internalization of beta-adrenergic receptor in A431 cells involves non-coated vesicles.

To characterize the mechanism of internalization of beta-adrenergic catecholamine receptors on human epidermoid A431 carcinoma cells, their distribution was analyzed by immunocytochemistry using the monoclonal anti-receptor antibody BRK2. In preconfluent cultures, the receptors appeared to be randomly distributed on the cell surface. Exposure to the agonist isoproterenol induced an overall decrease in the number of cell surface receptors as determined by binding experiments and visualized by immunofluorescence. When cells were incubated at 4 degrees C with BRK2 and anti-mouse IgG-gold and then transferred at 37 degrees C, non-coated invaginations and vesicles were labeled. The addition of isoproterenol resulted in an increased rate of internalization of the receptor-BRK2-anti-IgG-gold complex. When incubation with the two antibody reagents was prolonged (with or without isoproterenol), non-coated vesicles fused in the endosomal compartment, and receptors were transferred to multivesicular bodies and lysosomes. At no stage in this process was there any indication that clathrin-coated pits or vesicles participated. Furthermore, we found that an intracellular potassium depletion treatment known to inhibit endocytosis, did not affect the normal pattern of desensitization of beta-adrenergic receptors.

Antibodies, Monoclonal↗
PubMed1988

High-performance liquid chromatography of the main polypeptide (MP26) of lens fiber plasma membranes solubilized with n-octyl beta-D-glucopyranoside.

The main polypeptide isolated from lens fiber membrane has been solubilized in octyl glucoside and studied by gel filtration in high-performance liquid chromatography (HPLC). The combination of S20,w value obtained from analytical ultracentrifugation and Stokes radius determined by HPLC of the soluble fraction indicates that more than 90% of the protein is monomeric. The solubilization of the protein seems to be dependent upon the presence of the NH2 and COOH terminal sequences, since proteolytic degradation of MP26 which removes these terminal sequences is less soluble than the uncleaved polypeptide. Moreover, there is a higher amount of oligomer after proteolysis. Fatty acid analysis by gas chromatography shows that the insoluble membrane fraction from both cortical and nuclear fibers comprises a special class of long (C22) saturated fatty acids (behenic acid).

Aquaporins↗
PubMed1987

Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers.

The purified major intrinsic protein of the lens fiber plasma membrane (MP26) reconstituted into liposomes favored membrane-to-membrane close contacts as visualized by freeze fracture and immunoelectron microscopy. Reconstituted apposed unilamellar vesicles formed pentalaminar profiles, and multilamellar liposomes showed regions of stacked bilayers. Immunogold labeling, using antibody directed against MP26, demonstrated that this polypeptide is present in regions of membrane-to-membrane close interaction. Fracture faces displayed both randomly distributed clusters of 8-nm polygonal intramembrane particles and membrane domains where a bidimensional lattice of repeating subunits was present. The structural pleomorphism which characterized the MP26-reconstituted proteoliposomes seems quite comparable to that visualized in natural fiber plasma membrane domains.

Animals↗
PubMed1987

Redistribution of muscarinic acetylcholine receptors on human fibroblasts induced by regulatory ligands.

Distribution of the muscarinic acetylcholine receptor on human fibroblasts was determined by immunofluorescence and immunoperoxidase staining using the anti-muscarinic receptor antibody M-35b. The receptor appeared to be randomly distributed on the cell surface in 1- or 2-day nonconfluent cultures. Brief exposure to 0.1 mM carbamylcholine (15 min to 1 hr) induced receptor accumulation in several restricted domains of the cell surface. This process was associated with sequestration into uncoated vesicles. Random receptor distribution was restored by incubation in ligand-free medium for 4 hr after carbamylcholine treatment, and vesicular profiles were no longer detectable. When incubation with the agonist was prolonged (3 hr at 37 degrees C), endocytotic 'smooth vesicles' fused and formed multivesicular structures presumably implicated in receptor down-regulation. Conversely, when nonconfluent cells were exposed to the muscarinic antagonist atropine, receptor redistribution was revealed, leading to the formation of clusters where receptor accumulated. Muscarinic receptor redistribution induced by atropine therefore does not involve the sequestration process seen in carbamylcholine-treated cells.

Antibodies, Monoclonal↗
PubMed1985

Sequence analysis of peptide fragments from the intrinsic membrane protein of calf lens fibers MP26 and its natural maturation product MP22.

Calf lens fiber plasma membranes, containing only the intrinsic membrane protein MP26 and its maturation product MP22 were treated with proteolytic enzymes such as trypsin, protease V8 from S. aureus or with chemical agents as CNBr in formic acid. The cleavage products, purified by electrophoresis, were analysed for their amino acid composition and N-terminal sequences. Proteolysis gave rise to peptides which were mainly shortened at the C-terminal end of the molecules. While the V8 protease produced a fragment with a similar N-terminal sequence as the maturation product MP22, trypsin yielded another cleavage product. Chemical hydrolysis yielded large fragments (11-15 kDa) with hydrophobic N-terminal sequences. Our results suggest that MP26 is characterised by an N-terminal signal sequence and possesses other hydrophobic domains which could function as untranslocated insertion sequences.

Amino Acid Sequence↗
PubMed1985

MP26 in the bovine lens: a post-embedding immunocytochemical study.

Gold immunolabeling of bovine lens tissue embedded in Lowicryl K4M, using a polyclonal antibody specific for a major component of lens fiber plasma membrane of 26 K molecular weight, shows that this constituent is absent from the epithelial cell plasma membrane and associated only with the junctional and non-junctional domains of the lens fiber plasma membrane.

Animals↗
PubMed1985

Ultrastructural changes in gap junctions associated with CO2 uncoupling in frog atrial fibres.

The correlation between gap junction morphology and the state of electrical coupling was investigated in the frog auricle, which presents an atypical gap-junction organization. Electrical uncoupling of the tissue was achieved by perfusion with CO2-saturated Ringer medium. The tissue was fixed with glutaraldehyde and freeze-fractured before and during the application of CO2-saturated Ringer medium and after returning to the initial medium. The electrical tissue coupling was assayed by microelectrode recording just before fixation. At least 97% uncoupling was induced by CO2-saturated Ringer medium, as estimated from double sucrose gap experiments on several single atrial trabeculae. Several modifications were induced by CO2-saturated Ringer medium. A decrease in the number of particles per junctional assembly and dispersion of these assemblies in the plane of the membrane, indicate a decrease in the organization of the gap junctions. In parallel, loose clusters of particles became evident on the P-fracture face of the membrane. The size of the particles in these clusters was larger than the size of the background particles, and of the gap junction particles. They never corresponded with complementary pits on the E-fracture face. On return to the initial Ringer perfusion medium the cell coupling was reversed and the gap junction dispersion was also reversed. However, the gap junctions remained small. The relationship between these morphological modifications and the conducting state of the tissue is discussed.

Action Potentials↗
PubMed1985

Dilemmas of the structural and biochemical organization of lens membranes during differentiation and aging.

This review deals with the biogenesis of MP26 and with the problem of the structural pattern which may be formed by MP26 during differentiation and aging of the lens fibers and as a consequence of the extraction or degradation of other membrane components. The data reported here imply that the MP26 biosynthesis is one of the key steps of cell surface domain formation during terminal differentiation of lens fibers. One striking observation involves the bidimensional long and short range distribution of MP26 copies within the lipid matrix. The protein oligomers may form, in the plane of the membrane, either a geometrical lattice or randomly distributed particle arrays. However, the mechanism controlling the assembly of either one of these patterns and eventually the transition of one into the other, is still unknown. We speculate that the formation of various membrane domains can be depicted as a self-assembly of repeating identical or quasi equivalently related protein subunits, but this process appears to be dependent both on the lipid environment and on interaction of the transmembrane protein oligomers with other membrane or cytoskeletal components, in particular crystalline polypeptides and cytoskeleton constituents. Finally, during the aging process, the post translational modification of MP26 and eventually the variation of the spatial arrangement and composition of the lipids may implicate the predominant lattice arrangement of the lipid and protein phases.

Animals↗
PubMed1984

Interaction of crystallins with the cytoskeletal-plasma membrane complex of the bovine lens.

The isolated lenticular plasma membrane-cytoskeleton complex, when analysed by sodium dodecylsulphate-polyacrylamide gel electrophoresis, shows reproducibly a significant amount of crystallins, mainly alpha-crystallin. Evidence is provided that purified plasma membranes from the bovine lens also associate selectively with a limited number of newly synthesized polypeptides on cell-free translation of calf lens polyribosomes and addition of the membranes to the incubation medium. This capability is retained by purified lens membrane junctions. The polypeptides that are selected comprise alpha-crystallin chains (in particular alpha A2-crystallin), actin, vimentin and beta B1a-crystallin. Sequence analysis revealed that the latter has in its N-terminal extension a characteristic Pro-Ala track. The designation 'PAPA-arm' is proposed for this N-terminal region, comprising the alternating Pro-Ala sequence, that has previously also been found in rabbit myosin and might be responsible for anchoring beta B1a-crystallin to lens membranes.

Amino Acid Sequence↗
PubMed1983

Polyribosomes associated with microfilaments in cultured lens cells.

Epithelial hamster lens cells, transformed by SV40 can be grown in suspension culture. Triton X-100 extraction of these cells grown under conditions when ribosome run off is blocked releases about 40% of the total amount of polyribosomes, designated as free- and loosely-bound polyribosomes. The Triton ghosts retain the remaining polysomal population which can be released by a combined treatment with deoxycholate and Nonidet P 40. Electron microscopic examination of the ghosts reveals microfilament-associated ribosome clusters next to a fraction of polysomes still attached to membranes. Preincubation of the cells with cytochalasin D prior to polyribosome isolation enables us to discriminate between these two latter polysome populations. The experiments indicate that about 25% of the polyribosomes are attached to microfilaments, while the remaining 35% are tightly bound to the membranes of the endoplasmic reticulum. When the different polyribosome classes were translated in a reticulocyte lysate, no significant differences could be observed in the patterns of the newly synthesized polypeptides. In all cases actin was one of the major products synthesized de novo.

Animals↗
PubMed1983

Organization and expression of the vimentin gene.

The primary structure of hamster vimentin has been derived from the nucleotide sequence of the corresponding cDNA. The elucidation of the amino acid sequence allowed the prediction of a model in which two helix domains occur. The N-terminal and C-terminal part have been characterized as nonhelical domains. Moreover the two helices are separated by a third nonhelical region.

Amino Acid Sequence↗
PubMed1982

Lenticular intermediate-sized filaments: biosynthesis and interaction with plasma membrane.

Electron microscopical features of the lens fiber plasma membrane-cytoskeleton complex are suggestive of an intimate association between the intermediate-sized filaments (IF) and the lipid bilayer. Biochemical analysis of this complex reveals the occurrence of an appreciable amount of vimentin as a protein subunit of lenticular IF. Additional evidence for association between IF and membranes is provided by the observation that newly synthesized vimentin is associated with plasma membranes added to a reticulocyte lysate programmed with lens polyribosomes. Concomitantly alpha-crystallin polypeptide chains (alpha A2) are also found associated with the plasma membrane together with a hitherto unidentified 47-kilodalton protein. Once associated with the lipid bilayer, the vimentin polypeptide resists urea treatment, suggesting that it has become an integral constituent associated with part of the membrane.

Animals↗