Comparative evaluation of morphofunctional organization of vertebrate nuclear blood cells

Morphometrical parameters, osmoregulatory possibilities, and the membrane reserve value of nuclear hemocytes (leukocytes and erythrocytes) were studied in the main classes of vertebrates by using method of hypoosmotic loads. It has been established that in the fish--mammals line in erythrocytes the absolute reserve of the plasmalemma decreases and the relative area of the cell surface increases. Evolution of leukocytes is accompanied by an increase of the membrane reserve and of the surface area due to a decrease of volume and to a rise of folding of the plasmalemma.     

Comparative study of the "membrane reserve" of blood cells of reptiles and mammals

Based on the method of hypoosmotic loads, the value of membrane reserve and its use by blood cells of reptiles and mammals have been studied. It has been shown that lymphocytes of the both animal species have the highest reserve of plasmalemma, while frog heterophils - the lowest one. A significantly lower part of the membrane reserve participates in formation of phagosomes by mammalian neutrophils as compared with amphibian erythrocytes.     

Comparative estimation of the “Membrane Reserve” of blood cells of reptiles and mammals

Based on the method of hypoosmotic loads, the value of membrane reserve and its use by blood cells of reptiles and mammals have been studied. It has been shown that lymphocytes of the both animal species have the highest reserve of plasmalemma, while frog heterophils—the lowest one. A significantly lower part of the membrane reserve participates in formation of phagosomes by mammalian neutrophils as compared with amphibian erythrocytes.     

The detection of the cholesterol of the erythrocyte plasmalemma by using a latex marker]

Using a new specific cholesterol marker (SCM) and scanning electron microscopy, the distribution of cholesterol in the plasmalemma of rabbit and human erythrocytes was studied. Investigation of SCM linking with the erythrocyte membranes with different cholesterol/phospholipid indices shows that the increase of cholesterol/phospholipid index of the erythrocyte membrane correlates with the increase in the number of SCM particles on the erythrocyte surface.     

The elimination mechanism of erythrocytes in vivo

The erythrocytes exhibit an unusual cyto-architecture. Because the cytosol is without organelles, the erythrocytes are cells, which have got only one single organelle, i.e. the plasmalemma. It reacts extremely sensitively to environmental factors with changes of the membrane structures, especially with an expression of IgG-and lectin receptor sites. This takes place during in vivo ageing, after attack of hydrolytic enzymes and after alteration of the membrane skeleton. The membrane bound IgG mediates the endocytosis of the macrophages (primary elimination). The role of the lectin receptors is unclear. The cell trapping (secondary elimination) is restricted to cells with extremely decreased deformability.     

Clustering of ligand on the surface of a particle enhances adhesion to receptor-bearing cells

Human leukocytes express a receptor that mediates the binding of cells and particles coated with C3bi, a fragment of the third component of complement. Previous data indicate that the capacity of this receptor to mediate binding is regulated by changes in its aggregation state. Randomly distributed receptors bind ligand very inefficiently, but stimulation of polymorphonuclear leukocytes with phorbol esters causes a ligand-independent clustering of the receptors in the membrane, and the clustered receptors avidly bind C3bi-coated cells (1). We examined whether the aggregation state of surface-bound ligands also affects the efficiency of binding between receptors and ligands. We found that erythrocytes bearing C3bi in clusters were bound by both macrophages and polymorphonuclear leukocytes far more avidly than erythrocytes bearing the same number of ligands in random array. We made similar observations with erythrocytes coated with C3b, a ligand that is recognized by a separate receptor. Our observations show that the ability of a receptor-bearing cell to bind particles coated with the corresponding ligands is dramatically affected by the distribution of ligand on the surface of the particle. Cell-cell interactions may thus be regulated by alterations in the two-dimensional distribution of receptors and ligands on opposing cell surfaces.     

The erythrocytic entry- and exit-mechanism of Aegyptianella pullorum Carpano, 1928.

The erythrocytic entry- and exit-mechanisms of Aegyptianella pullorum were investigated and characterized by scanning (SEM) and transmission electron microscopy (TEM) using for TEM ruthenium red as a marker of the red cell plasmalemma. The scanned Aegyptianella preparations produced static evidence of an endocytosis followed by an erythrocytic vesiculation as the possible mode of entrance of initial bodies into erythrocytes. The presence of ruthenium red only coating the membrane around the parasitophorous vacuole during the whole invasive process and the complete absence of the stain inside the host cell indicate that the entry of aegyptianellas is accomplished by invagination of the host cell plasmalemma and is not preceded nor followed by its breakage, furthermore unequivocally proving the intracellular parasitism of A. pullorum during its reproductive cycle. One possible mode of exit of initial bodies from parasitized erythrocytes appeared to be the invasive mechanism in reverse order, an exocytosis. Generally, however, the affected erythrocytes are parasitogenically injured, resulting in release of the parasites into the plasma and, subsequently, in host cell lysis.     

Effect of tributyltin on trout blood cells: changes in mitochondrial morphology and functionality

The aquatic environment is the largest sink for the highly toxic organotin compounds, particularly as one of the main sources is the direct release of organotins from marine antifouling paints. The aim of this study was to investigate the mitochondrial toxicity and proapoptotic activity of tributyltin chloride (TBTC) in teleost leukocytes and nucleated erythrocytes, by means of electron microscopy investigation and mitochondrial membrane potential evaluation, in order to provide an early indicator of aquatic environmental pollution. Erythrocytes and leukocytes were obtained from an inbred strain of rainbow trout (Oncorhynchus mykiss). Transmission electronic micrographs of trout red blood cells (RBC) incubated in the presence of TBTC at 1 and 5 microM for 60 min showed remarkable mitochondrial morphological changes. TBTC-mediated toxicity involved alteration of the cristae ultrastructure and mitochondrial swelling, in a dose-dependent manner. Both erythrocytes and leukocytes displayed a consistent drop in mitochondrial membrane potential following TBTC exposure at concentrations >1 microM. The proapoptotic effect of TBTC on fish blood cells, and involvement of mitochondrial pathways was also investigated by verifying the release of cytochrome c, activation of caspase-3 and the presence of "DNA laddering". Although mitochondrial activity was much more strongly affected in erythrocytes, leukocytes incubated in the presence of TBTC showed the characteristic features of apoptosis after only 1 h of incubation. Longer exposures, up to 12 h, were required to trigger an apoptotic response in erythrocytes.     

Plasmalemma fine structure in isolated tobacco mesophyll protoplasts

Summary Tobacco mesophyll protoplasts have been examined by electron microscopy during isolation procedures and after 24 hours culture in a medium known to support cell wall regeneration. During isolation the plasmalemma shows little structural differentiation apart from the formation of small vacuoles in the cytoplasm. After 24 hours of culture, several types of activity are seen at the plasmalemma surface. Microtubules, profiles of endoplasmic reticulum, electron dense granules and coated vesicles are associated with the inner surface of the membrane. External to the plasmalemma fibrillar structures occur, both as extensive networks and as individual fibrils apparently associated with the membrane itself. Techniques and criteria for electron microscopy are presented, and the results discussed in terms of plasmalemma function and the regeneration of the cell wall.     

The process of new plasmalemma formation in focally injured skeletal muscle fibers

The major ultrastructural events in murine skeletal muscle fibers were examined 3 to 24 hr after segmental injury induced by painting with aldehyde fixative. At 3 hr the viable stump of injured myofibers was separated from the necrotic segment by a zone of supercontracted myofibrils. No demarcating membrane was evident at this time, although occasionally collapsed segments of plasmalemma partially covered the viable stump. By 12 hr after injury myonuclei near the viable stump were centrally placed and numerous whorls of membrane material appeared in the vicinity of the Golgi apparatus. At this stage a convoluted, tortuous membrane exhibiting extensive interdigitations has sealed the structurally normal part of the injured fiber. The myoplasm immediately within this demarcating membrane possessed few myofilaments but numerous vesicles and tubules, several of which were continuous with the demarcating membrane; most degraded sarcoplasmic organelles remained external to the demarcating membrane and leukocytes were observed internalizing the debris. It appears that after segmental injury to skeletal muscle fibers, active production of new sarcoplasmic membranes occurs, which contributes to the formation of the part of the plasmalemma that demarcates the viable portion of the muscle fiber from the injured area.     

Model studies of erythrophagocytosis in vitro

Macrophages of the RHS are important for the elimination of aged and altered erythrocytes. An increase in the membrane bound autologous IgG generates a recognition signal in the plasmalemma, which triggers the RBC-phagocytosis. The IgG-receptors are buried in the band 3 proteins of the membrane. Enzymatic degradations or conformational changes of the glycocalyx are accompanied by an expression of these receptors. Similar effects were obtained after alteration of the membrane skeleton (crosslinking of the spectrin). A clustering of IgG-receptors due to interaction of the denatured hemoglobin (Heinz-bodies) with the membrane seems to be unlikely. Exovesiculation represents a process of formation of an intact membrane structure. Prolonged stay of RBC in the spleen can induce membrane altering continued by increased IgG loading.     

Fine Structure of Hepatozoon mocassini (Apicomplexa,Eucoccidiorida) Gamonts and Modifications of the Infected Erythrocyte Plasmalemma1

Transmission electron microscopy and scanning electron microscopy were used to investigate the fine structure of Hepatozoon mocassini gamonts and modifications of the infected erythrocyte plasmalemma. Intraerythrocytic gamonts were contained within a parasitophorous vacuole. An electron-lucid space observed between the gamont pellicle and the membrane of the vacuole corresponded to the unstained space described in light microscopy studies. Gamonts possessed a conoid, polar ring, subpellicular microtubules, four pairs of rhoptries, micronemes, ovoid granules, mitochondria with tubular cristae, and a pellicle composed of three individual unit membranes. The conoid had an anterior diameter of 320 nm, a posterior diameter of 360 nm, and a length of 150 nm. In contrast to a report on Hepatozoon aegypti, no micropore or “canopy-like structure” was observed. The plasmalemma of infected erythrocytes exhibited two types of modifications: gross membrane deformations and knobs with an electron-dense central mass. These knobs are structurally distinct from previously described membrane excrescences.     

Dynamic changes of the luminal plasmalemma in stimulated parotid acinar cells. A freeze-fracture study

In the acinar cells of the rat parotid gland the two membranes participating in exocytosis, i.e., the luminal plasmalemma and the secretory granule membrane, are clearly distinguishable in freeze- fracture because of their different densities in particles. In order to obtain point-specific information about the fusion-fission of these two membranes that occurs during the secretory cycle, glands were studied at various times (5 min to 6 h) after stimulation with isoproterenol. We observed that, in the course of the release of secretion products and shortly afterwards, the enlarged luminal plasmalemma exhibits a mosaic organization consisting of an alternation of membrane patches of high (original plasmalemma) and low (fused granule membrane) particle density. The transition between these two patterns is usually sharp. Later, concomitant with the reformation of acinar canaliculi, the low particle density membrane is found at the cell surface but only bounding vacuolar infoldings, and then it finally disappears. These results suggest that (a) fusion of these membranes does not result in a random intermixing of the molecular components of the participating membranes, which retain their structural identity; and (b) the enlarged luminal plasmalemma reverts to its original size by a progressive, specific removal of the regions of low particle density from the cell surface.     

Changes in membrane properties of erythrocytes and polymorphonuclear cells in psoriasis

Using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and its cationic derivative, 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene, we evaluated membrane fluidity in living polymorphonuclear leukocytes and in erythrocytes of psoriatic patients. Our results have shown that erythrocyte membranes of psoriatic patients exhibit a decrease of fluidity. These changes were not associated with any relevant modifications of the cholesterol to phospholipid molar ratio. Moreover, we observed a decrease in polymorphonuclear leukocytes membrane fluidity associated with changes in chemotactic migration. Our results indicate changes of membrane fluidity involving membranes different from the epidermal cells and suggest the hypothesis of a defective membrane-cytoskeleton interaction in psoriasis.     

Surface multivesicular structures associated with maturing erythrocytes in rats

Summary Surface multivesicular structures associated with the plasmalemma of erythrocytes were observed in the peripheral blood of rats which have a significant number of circulating reticulocytes. These surface structures appear as ovoid evaginations (0.2 to 0.7 in diameter) of the plasma membrane and contain numerous small vesicles ranging from 0.05 to 0.1 in diameter. The structures were present during the final stages of maturation of erythrocytes, after nuclei and mitochondria had been extruded and only a few polysomes and small vesicles remained. They appear quite distinct from the autophagic vacuoles which have been described in association with degeneration and extrusion of mitochondria from erythrocytes. The exact origin of the small internal vesicles of these surface multivesicular structures is unknown; however, similar vesicles have been observed in the cytoplasm of the maturing erythrocyte especially in the vicinity of the Golgi body. These structures suggest a process by which Golgi elements and other small cytoplasmic vesicles are extruded during the late stages of maturation of rat erythrocytes.Supported by U.S. Public Health Service Research Grant AM 12950.The author is indebted to Dr. Edward G. Rennels and Dr. William B. Winborn for their guidance.     

Fluidity properties and liquid composition of erythrocyte membranes in Chediak-Higashi syndrome

We have earlier shown through electron spin resonance (ESR) studies of leukocytes that membranes of cells from both Chediak-Higashi syndrome (CHS) mice and humans have abnormally high fluidity. We have extended our studied to erythrocytes. Erythrocytes were labeled with the nitroxide-substituted analogue of stearic acid, 2-(3-carboxypropyl)-4,4- dimethyl-2-tridecyl-3-oxazolidinyloxyl, and ESR spectra were obtained. Order parameter, S, at 23 degrees C, was 0.661 and 0.653 for erythrocytes of normal and CHS mice (P less than 0.001). S was 0.684 for normal human erythrocytes and 0.675 (P less than 0.001) for CHS erythrocytes at 25 degrees C. Because S varies inversely to fluidity, these results indicate that CHS erythrocytes tend to have higher fluidity than normal. In vitro treatment of both mice and human CHS erythrocytes with 10 mM ascorbate returned their membrane fluidity to normal. We prepared erythrocyte ghosts and extracted them with CHCl3:CH3OH (2:1). Gas-liquid chromatography analysis showed a greater number of unsaturated fatty acids for CHS. The average number of double bonds detected in fatty acids for mice on a standard diet was 1.77 for normal and 2.02 for CHS (P less than 0.04); comparison of human erythrocytes from one normal control and one CHS patient showed a similar trend. Our results suggest that an increased proportion of unsaturated fatty acids may contribute to increased fluidity of CHS erythrocytes. Our observation that both leukocytes and erythrocytes of CHS have abnormal fluidity indicates that CHS pathophysiology may relate to a general membrane disorder.     

Monoclonal antibody specific for the T-tubule of skeletal muscle

Monoclonal antibodies were raised against a triad-enriched (sarcoplasmic reticulum-T-tubule complex) microsomal membrane fraction of rabbit skeletal muscle. The avidin-biotin complex (ABC) immunoperoxidase staining method was used to screen hybrid colonies. Positive antibodies exhibited a granular doublet pattern at the A-I junction, consistent with the location of triads in rabbit muscle. One monoclonal antibody, M171, was further characterized by ultrastructural and immunoadsorption techniques. Postembedding electron immunocytochemistry was performed on tissue sections embedded in Lowicryl K4M. Goat anti-mouse immunoglobulin absorbed to 10 nm colloidal gold particles was used as an ultrastructural label. In these studies, M171 recognized an epitope at the triads and at periodic openings along the plasmalemma. Immunoadsorption on protein transfers of isolated sarcoplasmic reticulum, surface membrane (plasmalemma and T-tubule), and triad-enriched fractions showed that M171 reacts with a surface membrane component. Taken together, these studies suggest that M171 recognizes an epitope associated with the T-tubule at the triad and at the "mouth" of the T-system at the plasmalemma.     

Regeneration of plasmalemma and surface properties in macrophages

The regeneration of surface anionic groups in mouse peritoneal macrophages was investigated by electron microscopy, using cationized ferritin (CF) as a tool for the localization and evaluation of negative charge density on the cell surface. In vitro interaction of living macrophages with CF resulted in removal of most anionic groups, either by concentration of their receptor sites to a part of the membrane which is subsequently internalized, or by detachment of the aggregated label from the surface. After incubation of macrophages lacking surface anionic groups in tissue culture medium without the ligand, regeneration of the binding capacity for CF took place within 3 h. The first regenerated parts of the membrane can be visualized within 1 h on the upper part of the adherent cells; there is a discontinuous coating of ferritin, with the lateral regions of the plasmalemma free of label. The attached CF particles on the regenerated membrane are closer to the membrane and their density is considerably higher than on the normal control macrophages. The results indicate that the turnover of the plasmalemma is regional and not dispersed; the mechanism involved is insertion of membrane patches into the pre-existing plasma membrane.     

Changes in hydrodynamic diameter of myometrium plasmalemma vesicles under artificial creation of transmembrane proton gradients

A method of laser-correlation spectrophotometry was applied for finding the effect of proton transmembrane gradient on hydrodynamic diameter of plasmalemma smooth muscle vesicles. It is shown, that the building of protons gradient on the membrane of vesicles, pHi = 6.0; pHo = 7.5 (delta pH = 1.5) results in the reliable decrease of their hydrodynamic diameter (from 212 +/- 6 nm in monitoring to 174 +/- +/- 0.25 nm in experiment). The exit of K+ from vesicles (KCl concentration inside vesicles is 150 mM), on the contrary, evokes augmentation of hydrodynamic diameter. The chemical paravariation of COO-, NH(3+)-groups and stabilization of SH-groups of plasmalemma surface results in essential modifications in magnitude of the hydrodynamic diameter of vesicles. On the basis of experimental data a supposition is expressed about the role of modifications of regional position of the functionally important groups of plasmalemma with respect to the membrane surface in mechanisms of H+ transport.     

Recycling of membrane proteins during endo- and exocytosis in amoebae

The fate of a membrane protein of the amoeba plasmalemma was studied by means of 125I iodination by lactoperoxidase, gel electrophoresis, radioautography and gamma counting. There was only one iodinatable polypeptide group with a molecular weight (MW) of 175 000 on the external surface of the plasmalemma. Two hours or more after induced phagocytosis, isolated phagolysosomal membranes contained two other smaller polypeptides with MWs of 70 000 and 35 000, respectively, suggesting that the 175 000 polypeptide was broken down to these smaller components during endocytosis. After 22 h of induced phagocytosis, isolated plasmalemma contained a 35 000 polypeptide group in addition to the 175 000 polypeptide species. The results suggested that some of the iodinatable membrane proteins were altered and recycled during endo- and exocytosis in amoebae, while others were recycled intact.