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At least 1,009 records · Page 56Linked to original sources
PubMed2003

Observations on the musculature and isolated muscle fibres of the liver fluke, Fasciola hepatica.

The liver fluke, Fasciola hepatica relies on a well-developed muscular system, not only for attachment, but for many aspects of its biology. Despite this, little is known about the system beyond the gross organization of the main somatic muscle layers. In the present study, a range of techniques have been applied to F. hepatica in order to understand more about various aspects of muscle organization, biochemistry (in terms of muscle proteins) and identity of isolated muscle fibres. Scanning electron microscopy has provided a direct visualization in situ of the somatic muscle layers and the organization of the muscle fibres within the ventral sucker. The muscle bundles contributing to the main somatic muscle layers are made up of up to 10 individual muscle fibres. Phalloidin staining for actin, in conjunction with confocal microscopy, confirmed the presence of 2 main somatic muscle layers (outer circular, inner longitudinal), beneath which lies a third layer of oblique muscle fibres. The use of propidium iodide in combination with phalloidin staining for actin demonstrated that the cell bodies associated with the 2 main somatic muscle layers are situated beneath the longitudinal muscle layer and are connected to their respective muscle fibres by short cytoplasmic processes. Myosin immunoreactivity was demonstrated in the somatic muscle layers and in the muscle layers surrounding various organ systems within the fluke. Double labelling for actin and myosin confirmed the co-localization of the 2 muscle proteins in the muscle fibres of the ventral sucker. Muscle fibres from the somatic muscle layers and the ventral sucker have been isolated and images obtained with phase-contrast microscopy and scanning electron microscopy. The muscle fibres contain actin and myosin, but lack a nucleus, the connection with the cell body having been broken during the isolation procedure.

Actins↗
PubMedDate not supplied

[Corpuscular pollutants: the problem of particles and fibers (optic microscopy)].

The "Clinica del Lavoro" had already begun work on dust pollution in the working environment in the mid-1930's but was only able to study the problem more in depth at the beginning of the 1950's when the Industrial Hygiene Laboratory was established under the fortunate direction of Professor Nicola Zurlo. The first research studies in that period were aimed at the correct choice of the most suitable sampling equipment and analytical methods for assessment of the extent of such types of pollution. The results achieved in these initial studies, the experience acquired in the field and further, more detailed research enabled Professor Zurlo to develop and perfect new types of sampling instruments that were more appropriate to the goals it was desired to achieve and these instruments are still widely used to-day. The paper also considers the evolution of analytical methods using the optical microscope for determination of the concentration of particles and fibres, most of which were developed and perfected in our Laboratory, up to the present time. It is stressed that phase contrast microscopy is still indispensable for dust analysis and still offers ample scope for research in the field of artificial fibres that have recently been put on the market, even though the electron microscope is now the only acceptable solution for research in living environments.

Air Pollutants↗
PubMed1996

Organelle motility and metabolism in axons vs dendrites of cultured hippocampal neurons.

Regional regulation of organelle transport seems likely to play an important role in establishing and maintaining distinct axonal and dendritic domains in neurons, and in managing differences in local metabolic demands. In addition, known differences in microtubule polarity and organization between axons and dendrites along with the directional selectivity of microtubule-based motor proteins suggest that patterns of organelle transport may differ in these two process types. To test this hypothesis, we compared the patterns of movement of different organelle classes in axons and different dendritic regions of cultured embryonic rat hippocampal neurons. We first examined the net direction of organelle transport in axons, proximal dendrites and distal dendrites by video-enhanced phase-contrast microscopy. We found significant regional variation in the net transport of large phase-dense vesicular organelles: they exhibited net retrograde transport in axons and distal dendrites, whereas they moved equally in both directions in proximal dendrites. No significant regional variation was found in the net transport of mitochondria or macropinosomes. Analysis of individual organelle motility revealed three additional differences in organelle transport between the two process types. First, in addition to the difference in net transport direction, the large phase-dense organelles exhibited more persistent changes in direction in proximal dendrites where microtubule polarity is mixed than in axons where microtubule polarity is uniform. Second, while the net direction of mitochondrial transport was similar in both processes, twice as many mitochondria were motile in axons than in dendrites. Third, the mean excursion length of moving mitochondria was significantly longer in axons than in dendrites. To determine whether there were regional differences in metabolic activity that might account for these motility differences, we labeled mitochondria with the vital dye, JC-1, which reveals differences in mitochondrial transmembrane potential. Staining of neurons with this dye revealed a greater proportion of highly charged, more metabolically active, mitochondria in dendrites than in axons. Together, our data reveal differences in organelle motility and metabolic properties in axons and dendrites of cultured hippocampal neurons.

Animals↗
PubMed1994

I-131-lipiodol cytotoxicity in hepatoma cells.

Hepatoma is a common cancer in Taiwan. New effective treatment for hepatoma patients is urgently needed. Encouraging results of I-131-lipiodol treatment for hepatoma with minimal toxicity have been recently reported. The mechanism of lipiodol targeting and retention by hepatoma are not well understood. The cellular interaction of lipiodol and the cytotoxic effects of I-131-lipiodol on hepatoma cells were investigated in this study. HepG2 cells were cultured with lipiodol, and untreated HepG2 cells were used as the control. Changes of cellular morphology were accessed by light and electron microscopy. The uptake and retention of lipiodol by HepG2 cells were studied by phase contrast microscopy and neutron activation analysis. HepG2 cells were cultured with I-131-lipiodol varying from 0.12 microCi to 120 microCi. The cytotoxic effect of I-131-lipiodol was evaluated by the surviving fraction of HepG2 cells. Changes in cellular morphology was examined by light microscopy. Results indicated that HepG2 cells were capable of active uptake of large amounts of lipiodol by endocytosis and prolonged intra-cellular retention associated with the formation of many bulging cytoplasmic extensions. I-131-lipiodol was highly cytotoxic to HepG2 cells. There was a steep dose response relationship, and the effective dose (LD50) was 1.2 microCi (480 rads). The cytotoxic effects of I-131-lipiodol were associated with pleomorphism of HepG2 cells, an increase in cell size and nuclear-cytoplasmic ratio, an increase in the size and number of nuclei, and vacuolation of the cytoplasm around the nuclear regions. Multiple nucleoli, fragmentation and segregation and ring shaped changes of nucleoli were also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Hepatocellular↗
PubMed2006

Immunological and ultrastructural characterization of endothelial cell cultures differentiated from human cord blood derived endothelial progenitor cells.

The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34(+) hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2-3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.

Antigens, CD34↗
PubMed2001

Optical phase contrast measurement of ultrasonic fields.

This report describes an optical phase contrast imaging technique for the measurement of wide bandwidth ultrasound fields in water. In this method, a collimated optical wavefront (lambda(l) = 810 nm) impinges on a wide bandwidth ultrasound pulse. The method requires that refractive index perturbations induced by the ultrasound field be sufficiently small. Specifically, on exit from the acoustic field, the phase of the optical wavefront must be proportional to the ray sum of local density taken in the direction of propagation of the incident optical wave. A similar restriction is placed on the dimensions of the ultrasound pulse. Repeated measurement of this phase as the ultrasound field is rotated through 180 degrees about an axis normal to the direction of propagation of the incident optical wave generates the Radon transform of the ultrasonically induced refractive index perturbation. Standard tomographic reconstruction techniques are used to reconstruct the full three-dimensional refractive index perturbation. A simple two-lens imaging system and an optical signal processing element from phase contrast microscopy provide a method of directly measuring an affine function of the desired optical phase for small optical phase shifts. The piezo- and elasto-optic coefficients (the first partial derivatives of refractive index with respect to density and pressure) relate refractive index to density and pressure via a linear model. The optical measurement method described in this paper provides a direct, quantitative measurement of the piezo- and elasto-optic coefficients (from the density or pressure fields).

Journal Article↗
PubMed1975

Spindle microtubules: thermodynamics of in vivo assembly and role in chromosome movement.

In this paper I have presented results of experiments in which spindle microtubules were depolymerized by hydrostatic pressure, in order to examine the Inoué dynamic equilibrium concept of spindle assembly and the possible role of microtubule depolymerization-polymerization in the movement of chromosomes. Using a newly developed optical hydrostatic pressure chamber, I investigated with polarization microscopy the quantitative effects of pressure on the polymerization of spindle microtubules and, with phase contrast microscopy, the relationship of pressure-induced spindle microtubule depolymerization to chromosome movement in living cells. From results of earlier experiments, principally those of Inoué et al. with low temperature and colchicine as microtubule-depolymerizing agents, and from results of my own research, I have concluded that: (1) spindle fiber microtubules are sensitive to depolymerization by pressure (3000-7000 psi), spindle microtubules do exist in a labile equilibrium with a pool of subunits, and the Inoué simple equilibrium model does predict changes in spindle microtubule assembly at metaphase induced by pressure; (2) the stability of microtubules depends on the number of "attached ends;" (3) the longest interpolar microtubules and the longest chromosomal fiber microtubules regulate the spindle interpolar length and the chromosome-to-pole positions; (4) chromosome velocity is independent of the number of spindle microtubules, as well as of the drag force of the chromosomes; (5) the chromosomal fiber microtubules transmit the forces between the poles and between the chromosomes and the poles; and (6) polymerization of microtubules does produce pushing forces and, if controlled microtubule depolymerization does not actually produce pulling forces, at least it governs the velocity of chromosome-to-pole movement.

Animals↗
PubMed1989

Differential localisation of tyrosinated, detyrosinated, and acetylated alpha-tubulins in neurites and growth cones of dorsal root ganglion neurons.

The comparative distribution of tyrosinated, detyrosinated, and acetylated alpha-tubulins was examined in neurites of rat dorsal root ganglion neurones in culture using immunofluorescence microscopy. Phase contrast observations of single neurones revealed that the neurites were actively motile, and rhodamine phalloidin staining of actin filaments showed the extent of lamellopodia and microspike projections from the growth cones. From double-labelling experiments using antibodies against tyrosinated, detryrosinated, or acetylated alpha-tubulin, it was found that the three different isoforms were differentially localised in neurites and growth cones. Detyrosinated and acetylated forms of alpha-tubulin were in the main restricted to the neurites extending no further than the base of the growth cones. Tyrosinated alpha-tubulin was, however, distributed throughout the body of the growth cone and into the base of some microspikes. Following treatment with taxol to promote microtubule assembly, detyrosinated and acetylated alpha-tubulins were found to be colocalised with tyrosinated alpha-tubulins throughout the growth cones of all cells examined. These results would be consistent with axonal transport of tyrosinated alpha-tubulin followed by assembly in the growth cone and subsequent detyrosination and acetylation. In addition the presence of unmodified alpha-tubulin in the growth cone may be necessary for the provision of labile microtubules for growth cone motility and extension.

Acetylation↗
PubMed2006

Cellular and biochemical differences between two attenuated poxvirus vaccine candidates (MVA and NYVAC) and role of the C7L gene.

The poxvirus strains NYVAC and MVA are two candidate vectors for the development of vaccines against a broad spectrum of diseases. Although these attenuated virus strains have proven to be safe in animals and humans, little is known about their comparative behavior in vitro. In contrast with MVA, NYVAC infection triggers greater cytopathic effect in a range of permissive and nonpermissive cell lines. The yields of NYVAC cell-associated virus in permissive cells (BHK-21) were slightly reduced compared with those of MVA infection. During the course of infection in HeLa cells, there is a translational block induced by NYVAC late in infection, which correlated with a marked increase in phosphorylation levels of the initiation factor eIF-2alpha. In contrast to MVA, the synthesis of certain late viral proteins was only blocked in NYVAC-infected HeLa cells. Electron microscopy (EM) analysis revealed that morphogenesis of NYVAC in HeLa cells was blocked at the stage of formation of immature viral forms. Phase-contrast microscopy, EM, flow cytometry, and rRNA analyses demonstrated that contrary to MVA, NYVAC infection induces potent apoptosis, a phenomenon dependent on activation of caspases and RNase L. Apoptosis induced by NYVAC was prevented when the virus gene C7L was placed back into the NYVAC genome, recovering the ability of NYVAC to replicate in HeLa cells and maintaining the attenuated phenotype in mice. Overall, our findings demonstrate distinct behavior between NYVAC and MVA strains in cultured cells, as well as a new role for the C7L viral gene as an inhibitor of apoptosis in NYVAC infection.

Animals↗
PubMed2003

Diacylglycerol-rich domain formation in giant stearoyl-oleoyl phosphatidylcholine vesicles driven by phospholipase C activity.

We have studied the effect of phospholipase C from Bacillus cereus and Clostridium perfringens (alpha-toxin) on giant stearoyl-oleoyl phosphatidylcholine (SOPC) vesicles. Enzyme activity leads to a binary mixture of SOPC and the diacylglycerol SOG, which phase separates into a SOPC-rich bilayer phase and a SOG-rich isotropic bulk-like domain embedded within the membrane, as seen directly by phase contrast microscopy. After prolonged enzymatic attack, all bilayer membranes are transformed into an isotropic pure SOG phase as characterized by fluorescence microscopy, differential scanning calorimetry, fluorescence anisotropy measurements, and small angle x-ray scattering. These domains may have biological relevance, serving as storage compartments for hydrophobic molecules and/or catalyzing cellular signaling events at their boundaries. Furthermore, in the early stages of asymmetric enzymatic attack to the external monolayer of giant vesicles, we observe a transient coupling of the second-messenger diacylglycerol to membrane spontaneous curvature, which decreases due to enzyme activity, before domain formation and final vesicle collapse occurs.

Diglycerides↗
PubMed1998

Fibroblast movements during contraction of collagen lattices--a quantitative study using a new three-dimensional time-lapse technique with phase-contrast laser scanning microscopy.

In this study we assessed the behavior of fibroblasts during contraction of collagen lattices. We applied a new technique for three-dimensional time-lapse studies of movements of living cells using phase-contrast laser scanning microscopy. Five anchored and five floating collagen lattices were studied regarding the activity of cells during a 7-h period of active contraction. Three-dimensional reconstructions of the fibroblasts and their extensions were made from datasets of 16-26 "optical sections" 5 microm apart recorded hourly during the period of measurements. The distance between fibroblast nuclei in the floating lattices decreased by a mean of 6.8 microm, but remained constant in the anchored group. Only minor variations were found in the angle between a line connecting any two nuclei and the tangent of the lattice margin. The lengths of the cellular extensions continuously changed by shortening and extending, and an increasing number of intercellular contacts were established with time. The angle between the extensions and the periphery of the lattice varied continually, and no distinct pattern of arrangement of the extensions was seen. In conclusion, we have shown in living cells in vitro that fibroblasts do not appear to move around within lattices during contraction but rather send out and withdraw cellular extensions continuously. This speaks against cellular locomotion or movement as a main feature of contraction. Time-lapse scanning laser microscopy has also been shown to be a suitable method to study cellular behavior quantitatively in three dimensions during lattice contraction.

Cell Movement↗
PubMed1999

Extracellular matrix regulates apoptosis in human neutrophils.

BACKGROUND: During inflammation, polymorphonuclear neutrophils (PMNs) migrate into the affected tissue interacting with extracellular matrix (ECM) proteins. We tested the hypothesis that PMN-matrix interaction affects PMN apoptosis. METHODS: Apoptosis of human PMNs was detected by DNA-fragmentation assay and was quantitated by flow cytometry, ultraviolet and light microscopy. Cell adhesion was assessed by a toluidine blue assay, and cell spreading was detected by phase contrast microscopy. Protein tyrosine phosphorylation was studied using Western blotting and confocal microscopy. RESULTS: PMN apoptosis was not different in unstimulated cultures on either surface-adherent fibronectin or on PolyHema, a surface that prevents cell adherence. However, tumor necrosis factor-alpha (TNF alpha) treatment significantly increased apoptosis on fibronectin (37 +/- 4%) compared with PolyHema (20 +/- 3%). Tests on other matrix substances revealed that the percentage of apoptotic PMNs in the presence of TNF alpha was 8 +/- 1% on PolyHema, 26 +/- 4% on fibronectin, 17 +/- 2% on collagen I, 16 +/- 2% on collagen IV, and 16 +/- 3% on laminin (P < 0.05 for all matrices compared with PolyHema). Preincubation with genistein (50 microM) significantly inhibited TNF alpha-mediated apoptosis on fibronectin (39 +/- 4% to 21 +/- 4%) but not on PolyHema (21 +/- 4% to 16 +/- 4%). Genistein also reduced PMN spreading on fibronectin. In contrast, inhibitors of mitogen-activated protein kinase and protein kinase C showed no effect on PMN apoptosis. Fibronectin strongly increased tyrosine phosphorylation of three 102, 63, and 54 kDa proteins. Five newly tyrosine-phosphorylated 185, 85, 66, 56, and 42 kDa bands were also visible. Using confocal microscopy, highest tyrosine phosphorylation was localized to sites of cell-matrix interaction. CONCLUSIONS: ECM influences apoptosis in TNF alpha-activated, adherent, spreading PMNs. The process is regulated by tyrosine phosphorylation. Acceleration of apoptosis may shorten the PMN lifespan and thereby locally regulate inflammation.

Apoptosis↗
PubMed1988

Rapid damage to membranes of Neisseria gonorrhoeae caused by human neutrophil granule extracts.

Neisseria gonorrhoeae were exposed to extracts of human neutrophil granules and effects on gonococcal growth and membranes were determined. Enumeration of gonococci by phase-contrast microscopy at 0 and 60 min revealed that they underwent very limited cell division after exposure to granule extract. At 60 min, treated gonococci tended to clump, and some lost their refractivity under phase-contrast optics, indicating membrane damage. Treated and untreated gonococci utilized oxygen at similar rates at time 0; treated gonococci utilized oxygen at a relatively constant rate for 60 min, even though colony-forming ability (i.e. viability) decreased by 90%, whereas untreated gonococci showed a steadily increasing rate of oxygen consumption over the same period, which essentially paralleled increase in colony-forming ability. Membrane ultrastructure of untreated and treated gonococci was compared in thin section by transmission electron microscopy. Extract treatment resulted in a time-related increase in disruption of the bacterial outer membrane, which became apparent almost immediately after treatment. This was accompanied by increasingly aberrant septum structure. Extract treatment also increased the resolution of peptidoglycan by electron microscopy, as early as 10 min after treatment. These data suggest that extract treatment of gonococci caused a rapid loss of the ability to form colonies on agar concomitant with alteration of gonococcal peptidoglycan and outer-membrane structure, but with little alteration of inner-membrane function.

Blood Bactericidal Activity↗
PubMed1982

SV-40 virus-induced neoplastic transformation of hamster brain cells in vitro. Studies with scanning and transmission electron microscopy.

Neoplastic transformation of one-day-old hamster brain cells was produced by infection with SV-40 virus and verified by phase contrast microscopy, growth in semisolid media, and intracranial tumor production after inoculation of the cells into other one-day-old hamsters. Transformed cells were studied by transmission and scanning electron microscopy. The numerous alterations in cell surface structure and in nuclear and cytoplasmic organization suggest a marked increase in cell metabolism and in the rate of mitosis and cell division. Cilia with a nine-to-zero pattern of microtubule doublets were present in cells with intermediate size filaments which stained for glial fibrillary acidic protein. The findings indicate that infection of one-day-old hamster brain cells in culture by SV-40 virus results in their transformation to a neoplastic state and the transformed cells are differentiating neoplastic astrocytes.

Animals↗
PubMed1999

Role of endothelial cell-substrate contact area and fibronectin-receptor affinity in cell adhesion to HEMA/EMA copolymers.

The objective of this study was to examine the effect of substrate hydrophobicity on cell-substrate contact area and the affinity between adsorbed fibronectin (Fn) and its receptor. Homo- and copolymer films of hydrophobic ethyl methacrylate (EMA) and hydrophilic hydroxyethyl methacrylate (HEMA) were spun-cast onto glass slides. Bovine aortic endothelial cells (BAEC) were plated for 2 h in serum-free medium onto polymers preadsorbed with Fn. Cells were fixed, labeled, and examined by total internal reflection fluorescence microscopy (TIRFM) to determine the topography of the basal surface as a function of distance from the substrate. Phase contrast microscopy was used to examine the total projected area of adherent cells. The cumulative contact area was greatest on cells attached to surfaces prepared from 0% HEMA and lowest on surfaces with the highest HEMA content. An equilibrium adhesion model used these data together with the critical force for detachment and the Fn density (Burmeister et al., J Biomed Mater Res 1996;30:13-22) to determine the affinity between Fn and its receptor and the bond strength. The affinity and force per bond decreased with increasing HEMA content. These results indicate that differences in the strength of endothelial cell adhesion to polymers are influenced by the conformation of the adsorbed adhesion proteins.

Animals↗