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PubMed1978

[Primary shape change of platelets in vitro (author's transl)].

Studies with interference contrast microscopy reveal that platelets undergo a typical shape change within 30--60' after venepuncture, i.e. swelling, formation of large tentacles, tiny protrusions and vesicles at the platelet surface. This "shape change" can be observed in citrated blood and PRP, heparinized blood and EDTA-blood as well. It is enhanced by low incubation temperatures (4 degrees C, 10 degrees C) and delayed at 37 degrees C as compared with room temperature. An increased number of primarily shape changed platelets is found if platelets are strongly mechanically irritated at blood sampling. The shape change is partly reversible in vitro, it is completely or almost completely reversible in vivo. Some antiaggregating agents inhibit the in vitro shape change at varying degrees (Bencyclan, SH 869 greater than ASA greater than D-Propranolol). The shape change is partly inhibited after oral or i.v. administration of ASA. A typical transformation of platelets into "spheric" forms can be observed following the addition of Bencyclan, SH 869 and D-Propranolol to PRP in vitro. The spontaneous "primary shape change" which occurs in PRP or blood after blood sampling is probably different from the secondary ADP-induced shape change. The primary shape change may influence the results of different platelet function and aggregating tests. The shape change kinetics of "healthy" subjects and patients with Hodgkin's disease differ significantly. The described method may gain more clinical interest in the future.

Aspirin↗
PubMed1978

Direct visualization of epithelial morphology in the living amphibian urinary bladder.

Differential interference-contrast microscopy has been applied to the study of amphibian urinary bladders, in vitro. It is demonstrated that well-resolved images can be obtained with little loss of tissue viability. Direct observations have been made on the structure of microvilli, the distribution of mitochondria in the mitochondria-rich cells, and the patency of lateral intercellular spaces. It is noted that the effective viscosity of cytoplasm is very high--that it is apparently a gel in which there is no Brownian movement of organelles. The frequency, shape, and pattern of distribution of granular and mitochondria-rich cells is determined for the commonly studied varieties of Bufo marinus. Bladders from Colombian toads contain more and larger mitochondria-rich cells than do those of the Dominican variety. There is no specific arrangement of cell-cell contacts to suggest a structural basis for cooperativeness of action. Finally, a longitudinal study of osmotically-induced changes in the structure of the "tight" or "limiting" junctions establishes the validity of previous findings by electron microscopy.

Animals↗
PubMed1979

Aggregation of microtubule initiation sites preceding neurite outgrowth in mouse neuroblastoma cells.

By examining microtubule regrowth using immunofluorescence with antibody to tubulin, we have studied the structure and intracellular localization of microtubule initiation sites in undifferentiated and differentiated mouse neuroblastoma cells. The undifferentiated cells are round and lack cell processes. They contain an average of 12 initiation sites per cell. Each of these sites, which are located near the cell nucleus, initiates the growth of several microtubules in a radial formation. In contrast to the undifferentiated cells, neuroblastoma cells stimulated to differentiate by serum deprivation are asymmetrical, containing one or two very long neurites. These cells have a single, large microtubule initiation center which can be visualized not only by immunofluorescence but by phase-contrast and differential interference microscopy as well. The initiation site measures 3-4 mu in diameter and is located in the cell body along a line defined by the neurite. During cell differentiation, the large initiation, the large initiation center seems to be formed by the aggregation of many smaller sites. This process procedes neurite extension by about 24 hr. The growth of microtubules from this center appears to be highly oriented, since most microtubules initially grow into the neurite processes rather than into the cell interior. Thus major changes in the structure and location of microtubule initiation sites occur during the differentiation of neuroblastoma cells. Similar changes are likely to be involved in alterations in the morphology of other cell types.

Animals↗
PubMed1992

Proteins on exocytic vesicles mediate calcium-triggered fusion.

In many exocytic systems, micromolar concentrations of intracellular Ca2+ trigger fusion. We find that aggregates of secretory granules isolated from sea urchin eggs fuse together when perfused with greater than or equal to 10 microM free Ca2+. Mixing of membrane components was demonstrated by transfer of fluorescent lipophilic dye, and melding of granule contents was seen with differential interference microscopy. A technique based upon light scattering was developed to conveniently detect fusion. Two protein modifiers, trypsin and N-ethylmaleimide, inhibit granule-granule fusion at concentrations similar to those that inhibit granule-plasma membrane fusion. We suggest that molecular machinery sufficient for Ca(2+)-triggered fusion resides on secretory granules as purified and that at least some of these essential components are proteinaceous.

Animals↗
PubMed1975

Studies of intercellular invasion in vitro using rabbit peritoneal neutrophil granulocytes (PMNS). I. Role of contact inhibition of locomotion.

Intercellular invasion is the active migration of cells on one type into the interiors of tissues composed of cells of dissimilar cell types. Contact paralysis of locomotion is the cessation of forward extension of the pseudopods of a cell as a result of its collision with another cell. One hypothesis to account for intercellular invasion proposes that a necessary condition for a cell type to be invasive to a given host tissue is that it lack contact paralysis of locomotion during collision with cells of that host tissue. The hypothesis has been tested using rabbit peritoneal neutrophil granulocytes (PMNs) as the invasive cell type and chick embryo fibroblasts as the host tissue. In organ culture, PMNs rapidly invade aggregates of fibroblasts. The behavior of the pseudopods of PMNs during collision with fibroblasts was analyzed for contact paralysis by a study of time-lapse films of cells in mixed monolayer culture. In monolayer culture, PMNs show little sign of paralysis of the pseudopods upon collision with fibroblasts and thus conform in their behavior to that predicted by the hypothesis.

Animals↗
PubMed1975

The interaction in vitro between human polymorphonuclear leukocytes and Neisseria gonorrhoeae cultivated in the chick embryo.

Cultivation of Neisseria gonorrhoeae in the allantoic cavity of 10-day chick embryos ensured the following necessary properties for subsequent quantitive in vitro phagocytosis studies of viable gonococci: log phase of growth, resistance to the cidal effect of fresh human serum, maintenance of colonial type, and absence of clumping. Employing a modification of the Maaloe technique, phagocytosis of log-phase type 1 and 2 gonococci by human PMN leukocytes did not occur in the presence or absence of serum. These findings indicate that log-phase type 1 and 2 gonococci possess antiphagocytic surface factors Stationary-phase organisms of the same colonial type were ingested and rapidly killed by human PMN leukocytes under similar experimental conditions, thus emphasizing the necessity to employ log-phase gonococci in the study of phagocytosis and antiphagocytic surface factors. Log-phase type 4 gonococci were ingested and rapidly killed by human PMN leukocytes in the presence of fresh human serum but not heat-inactivated serum or in the absence of serum. Morphologic studies demonstrated that log-phase viable gonococci attach to the surface membrane of human PMN leukocytes. Interiorization of avirulent but not virulent organisms was observed in the presence of fresh human serum. Gonococci-human PMN leukocyte interactions thus provide a model for the investigation of the nonimmunologic and immunologic parameters associated with the attachment and ingestion stages of phagocytosis.

Animals↗
PubMed1992

Modes of Helicobacter colonization and gastric epithelial damage.

A total of 144 gastric biopsies colonized by Helicobacter-like organisms were studied under light and differential interference contrast microscopy for the modes of bacterial colonization. Biopsies were also graded for the degree of epithelial damage (epithelial-damage-grade: 0 to 6, in ascending order of severity) and density of Helicobacter-like organism (Helicobacter-grade: 0 to 6, in ascending order of bacterial density). Three modes of colonization were identified: free-in-mucus, surface-adhesion and intercellular colonization. Because light microscopy cannot definitely prove the presence of intracellular colonization, bacteria located between cells and below the apical cell border were counted together as intercellular colonization. Bacteria free-in-mucus were seen in all biopsies. Surface adhesion was seen in 50-87.9% of biopsies, without obvious correlation with the epithelial-damage- and Helicobacter-grades. The incidences of intercellular and intracellular colonization were directly proportional to the epithelial-damage- and Helicobacter-grades. Free-in-mucus as the predominant mode of colonization was mainly seen in biopsies with lower (1-3) epithelial-damage- and Helicobacter-grades. Conversely, biopsies with intercellular colonization as the predominant mode of colonization were mainly cases with higher (4-6) epithelial-damage- and Helicobacter-grades. In cases showing predominantly bacteria between cells, 69.2% had a gastric ulcer whereas only 38.8% of cases showing predominantly bacteria free-in-mucus showed ulceration (P < 0.01). These results indicate that Helicobacter-like organisms can invade and penetrate between epithelial cells. When free-in-mucus, Helicobacter-like organisms are less likely to induce epithelial damage. However, the more invasive modes of colonization (intercellular) were associated with severe epithelial damage and high Helicobacter density.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1979

Microphotometric techniques in intravital microcirculatory studies.

Intravital microscopic techniques were developed in order to measure plasma proteins: (1) circulating in the streaming blood of microcirculatory vessels of the exposed rat mesentery, (2) permeating the vessel wall, and (3) moving through the perivascular interstitial tissue. By means of the ultramicrospectrophotometry, the absorbance of proteins containing aromatic amino acids at 280 nm is measured. Absolute amounts of proteins in the range of picograms are measured in a circular measuring field with a diameter of 17 micrometers. Problems of calibration and of unspecific light scattering are discussed. The scanning microfluorometry makes use of the fluorescent labelling of the plasma proteins. Interstitial tissue with a capillary vessel containing labelled proteins is measured at high speed. A motor-driven object stage synchronized with a measuring system uses scanning lines to traverse the tissue. The data are then processed by a computer. In this way we get continuous quantitative information about the dynamics of protein permeability, starting from time zero. The measuring of the fluorescence intensity and the speed of the fluorometric system are further improved by a computerized 10 kHz scanning device.

Animals↗
PubMed1977

Morphology and ultrastructure of Crenothrix polyspora Cohn.

Naturally grown cell material of Crenothrix polyspora from the well of a waterworks was studied by means of phase-contrast and Nomarski interference microscopy as well as by transmission electron microscopy. The material consisted of clusters of sheathed filaments up to 2 cm long. Propagation forms observed were nonmotile, spherical cells that arose by simple ("macrogonidia") or multiple ("microgonidia") septation of the filamental tips. Ultrastructural analysis revealed Crenothrix to be procaryotic and gram negative, with several layers of sheath material surrounding the filaments. On thin sections, individual cells had elaborate membrane systems in the form of lamellar stacks. They resembled thylakoids of photosynthetic bacteria. Spectrophotometric analysis gave no indication of photosynthetic pigments. The cells also contained large hexagonal bodies, rod-shaped fibrillar elements, and polyphosphate granules.

Bacteria↗
PubMed1992

Splenic function in alcoholic liver disease.

Splenic function was assessed in 42 patients with alcoholic liver disease by counting the percentage of erythrocytes with indentations or pits, seen by differential interference contrast microscopy. These pits represent cellular debris normally removed by the spleen. The findings were compared with 42 age and sex matched controls. Mean (SEM) pitted red cell counts in the patients was 2.7 (0.4)% and in the controls 0.7 (0.07)% (p < 0.001). In all of the eight reformed drinkers (five with biopsy proven cirrhosis), cell counts were normal. Six patients with alcoholic liver disease had had serious infections within the past year. Of these, one had had a recent pneumococcal pneumonia and another of the patients died from overwhelming pneumococcal septicaemia. Both of these patients had evidence of functional hyposplenism as judged by high pitted erythrocyte counts. A total of 18 patients were considered to have pitted red cell counts above the normal, and 11 of these had proven cirrhosis and/or gross ascites. This study is the first to show the presence of functional hyposplenism in alcoholic liver disease and provides further evidence of the predisposition that these patients have to infection. At present, it is unclear whether the hyposplenism is a direct toxic effect of alcohol or the result of cirrhosis; further studies are warranted.

Adult↗
PubMed1977

Structures in sensory region of snake spindles and their displacment during stretch.

1. Structures within the sensory region of short- and long-capsule snake muscle spindles have been visualized using differential interference contrast microscopy. Profiles seen with Nomarski microscopy have been identified by electron microscopy of the same preparations. 2. Sensory nerve terminals, nuclei and other cytoplasmic inclusions in the intrafusal fiber, collagen bands, and capsular cells may be seen in the living preparation. 3. The length changes of various elements within the sensory region in response to stretch of the spindle have been measured using high-speed ciné photomicrography. This has been corrleated with the impulse response from sensory endings in short-and long-capsule spindles. 4. Short-capsule spindles, which have a high dynamic sensitivity, show length changes in the sensory region in response to ramp-and-hold stretch, which are not parallel to the changes in impulse frequency. The implications for mechanical models of spindle behavior are discussed.

Animals↗
PubMed1975

An ultrastructural study of the spermatozoid of the fern, Marsilea vestita.

The ultrastructure of the mature spermatozoid of Marsilea vestita was studied after its release from the microspore and prior to its penetration of the egg. The psermatozoid is a pear-shaped cell with a complex spiral structure coiled around the edge in the narrow anterior end. This coil is composed of a large mitochondrion, elongated nucleus with highly condensed chromatin, a ribbon of microtubules, and a dense band of material (flagellated band) into which the flagella are inserted. There are over a hundred flagella protruding from each spermatozoid along the length of the coil. At the anterior tip of the coil is a short multilayered structure. It is not known what maintains the helical shape of the coil. The microtubular ribbon could be involved, but it is also possible that either the flagellated band, the condensed chromatin, or both, are sufficiently rigid to retain their shpaes unaided. When the spermatozoid is first released from the microspore it includes a cytoplasmic vesicle in the posterior region containing plastids, mitochondria, and other organelles. This vesicle is shed, taking the nuclear envelope with it, before the spermatozoid reaches the egg.

Cell Nucleus↗
PubMed1978

An experimental study of the interaction between the soil amoeba Naegleria gruberi and a glass substrate during amoeboid locomotion.

The amoeboid locomotion of the soil protozoon Naegleria gruberi has been studied using reflexion-interference microscopy. Two types of contact are made with a planar glass substrate. One, formed at a considerable distance from the substrate in deionized water (congruent to 100 nm) has been termed 'associated contact' and usually involves a considerable surface area (of the order of 100 micrometer2), i.e. about a third of the cell profile. From this broad platform filopodia are produced which form close contacts ('focal contacts'). In locomotion the area of associated contact is very mobile, in contrast to the focal contacts which, once established, are stable. Focal contact sites are left behind on the glass surface ('footprints') when the amoeba moves away. The cell-substrate gap in the associated contact is greatly affected by the ionic strength of the medium and particularly the valency of the cation component. This suggests that long-range forces of attraction play an important role in keeping the amoeba close to a substrate and thus allow the production of filopodia from the ventral surface to form focal contacts.

Amoeba↗
PubMed1979

Granulocyte migration through endothelium in culture.

Using a model system with which we have previously investigated the adhesion of granulocytes to cultured endothelium, we have now shown that adherent granulocytes migrate through cultured endothelium in a manner closely resembling that found during the acute inflammatory response in vivo. The migration of granulocytes was markedly enhanced in the presence of erythrocytes, whereas blood platelets did not affect migration. Several test agents, including Paroven and some prostaglandins, had different effects upon migration and adhesion. We conclude that the adhesion of granulocytes to endothelium and their migration through it are responses that are, at least in part, separately controlled, and that migration does not depend exclusively upon extravascular stimuli.

Animals↗