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PubMed1968

On the role of microtubules in movement and alignment of nuclei in virus-induced syncytia.

Infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes rapid and extensive cell fusion. Time-lapse cinematography shows that when cells fuse, their nuclei migrate straight to the center of the syncytium at rates of 1-2 micro/min. Nuclei are often arranged in long, tightly packed, parallel rows in syncytia derived from the fibroblastic BHK21-F cells. Polarization microscopy shows birefringent material between and parallel to these rows of nuclei, and electron microscopy shows bundles of cytoplasmic microtubules, approximately 250 A in diameter, and filaments, approximately 80 A in diameter, parallel to and between the rows of nuclei. Colchicine treatment causes disappearance of microtubules from BHK21-F cells and an apparent increase in the number of 80-A filaments. Although colchicine-treated, SV5-infected cells fuse, their nuclei do not migrate or form rows but remain randomly scattered through the syncytial cytoplasm. Incubation at 4 degrees C does not disrupt microtubules in BHK21-F cells. Rows of nuclei have been isolated from SV5-induced syncytia, and the nuclei in them have been found to be intimately associated with microtubules but not with other cytoplasmic structures. These results suggest that microtubules demarcate cytoplasmic channels through which nuclei migrate and that they may also be involved in the mechanism of nuclear movement.

Animals↗

PubMed1973

A thermodynamic analysis of mitotic spindle equilibrium at active metaphase.

The mitotic apparatus of first-division metaphase eggs of the sea urchin Strongylocentrotus drobachiensis was observed by means of polarization microscopy under controlled temperature conditions. Eggs were fertilized and grown at two temperature extremes in order to produce two different sizes of available spindle pool. Slow division time allowed successive samples of such cells to be observed at the same point in metaphase but at different equilibrium temperatures, yielding curves of metaphase equilibrium birefringence vs. observational temperature. Using the plateau value of birefringence at higher temperatures as a measure of total available spindle pool and the observed birefringence at lower temperatures as a measure of polymerized material at equilibrium, the spindle protein association was evaluated according to the method of Inoué. Both pool conditions produced linear van't Hoff functions. Analysis of these functions yielded enthalpy and entropy changes of +55-65 kcal/mol and +197-233 entropy units (eu), respectively. These values for active mitotic metaphase are quite comparable to those obtained by Inoué and co-workers for arrested meiotic metaphase cells. When other equilibrium treatments were considered, the best fit to the experimental data was still that of Inoué, a treatment which theoretically involves first-order polymerization and dissociation kinetics. Treatment of metaphase cells with D(2)O by direct immersion drove the equilibrium to completion regardless of temperature, attaining or exceeding a birefringence value equal to the cell's characteristic pool size; perfusion with D(2)O appeared to erase the original temperature-determined pool size differences for the two growth conditions, attaining a maximum value characteristic of the larger pool condition. These data confirm Inoué's earlier contention that D(2)O treatment can modify the available spindle pool.

Animals↗

PubMed1976

Pressure-induced depolymerization of spindle microtubules. III. Differential stability in HeLa cells.

Evidence from light microscopy (principally polarization microscopy) has demonstrated that hydrostatic pressure can reversibly inhibit mitosis by rapidly depolymerizing the spindle fiber microtubules. We have confirmed this finding in ultrastructural studies of mitotic HeLa cells incubated at 37 degrees C and pressurized at 680 atm (10,000 psi). Althouth there are many spindle microtubules in the cells at atmospheric pressure, electron micographs of cells pressurized for 10 min (and fixed while under pressure in a Landau-Thibodeau chamber) show few microtubules. Pressure has a differential effect on the various types of spindle microtubules. Astral and interpolar MTs appear to be completely depolymerized in pressurized cells, but occasional groups of kinetochore fiber microtubules are seen. Surprisingly, the length and density of microtubules of the stem bodies and midbody of telophase cells appear unchanged by pressurization. In cells fixed 10 min after pressure was released, microtubules were again abundant, the density often appearing to be higher than in control cells. Reorganization seems incomplete, however, since many of the microtubules are randomly oriented. Unexpectedly, kinetochores appeared diffuse and were difficult to identify in sections of pressurized cells. Even after 10 min of recovery at atmospheric pressure, their structure was less distinct than in unpressurized cells.

Chromosomes↗

PubMed1969

Molecular mechanism of hemolytic anemia in homozygous hemoglobin C disease. Electron microscopic study by the freeze-etching technique.

Erythrocytes from a patient with homozygous hemoglobin C disease were subjected to gradual osmotic dehydration by incubation in hypertonic saline. Serial observations of these cells before and after 4 and 12 hr incubation were carried out by means of interference, Soret absorption, polarization microscopy, and the electron microscope employing the freeze-etching technique. Light microscopic studies showed a progressive contraction of cellular contents into central masses which, after 12 hr dehydration, formed birefringent intracellular hemoglobin crystals in 50-75% of the cells. Electron microscopic study of freeze-etched replicas of these cells at 0, 4, and 12 hr of dehydration reveals progressive aggregation, alignment, and crystallization of hemoglobin molecules. Molecular aggregation found in C-C cells prior to osmotic dehydration was not seen in normal erythrocytes. Aggregation and packing varied from cell to cell. Reticulocytes showed a loosely packed aggregate mesh-work; older cells showed variation of molecular packing, which appeared tightest in cells corresponding to microspherocytes. With further loss of intracellular water, aggregates coalesced into patterns of tighter molecular packing with small regions of alignment, and, finally, crystallization occurred. Hemoglobin molecules measuring 70 A in diameter were readily identified within the period patterns of intracellular crystals. These findings suggest that the hemoglobin C molecules within C-C erythrocytes exist in an aggregated state. As the cell ages, intracellular water is lost and intermolecular distance decreases, hemoglobin C molecules polymerize into intracellular crystals. This pathological behavior of hemoglobin C is associated with a charge alteration conferred by the substitution of beta-6-lysine for glutamic acid on the external surface in the A-helix region of the beta-chain of the molecule, possibly increasing intermolecular attraction. Molecular aggregation accounts for the increased rigidity of C-C cells which leads to accelerated membrane and water loss with resultant microspherocyte formation. The microspherocyte, with highest intracellular hemoglobin concentration, rapidly undergoes intracellular crystallization, and is sequestered and destroyed by reticuloendothelial elements.

Adult↗

PubMed1974

Ion diffusion selectivity in lecithin-water lamellar phases.

The diffusion coefficients of Na(+), Rb(+), and cl(-) were determined in lecithin-water lamellar phases at 18 degrees C as a function of phase hydration. Diffusion was measured within the phase with no transfer between phase and bulk aqueous medium. The relative diffusion coefficients of anion and cation depended strongly on phase hydration. At low water content, the diffusion coefficient of Cl(-) was greater than that of Na(+) or Rb(+) whereas at high water content both cations diffused faster than the anion. The change in relative diffusion coefficient occurred at 0.24 g water/g phase (24% water). The possibility that a change in conformation of the lecithin polar head occurs at a phase water content of 24% is considered. The diffusion coefficients of all three ions decreased at the water content where the relative diffusion rates inverted. Freeze fracture and polarizing microscopy studies were carried out to obtain information on phase structure. The latter study indicated that a change in long-range organization of the phase occured at 24% water. This change accounts for the decrease in the ion diffusion coefficients at this water content. The change in conformation of the choline phosphate group proposed as an explanation for the change in ion selectivity could lead to changes in long-range organization of the phase as a second order-effect.

Chlorides↗

PubMed1980

Image alterations in the teaching microscope. A source of error in synovial fluid crystal identification.

An erroneous identification of sodium urate and calcium pyrophosphate crystals in synovial fluid may occur when utilizing compensated polarized microscopy with a dual-viewing-head teaching microscope. This is due to alteration of the observed image orientation with respect to the actual orientation of the crystal in space. By viewing a simple geometric figure through each microscope head, two basic types of image alterations are identified: a 180-degree rotation and a mirror-image transformation. Combinations of these two may also occur. This problem is clarified and illustrated and suggestions are offered to avoid erroneous crystal identification.

Calcium Pyrophosphate↗

PubMed1989

Talc in liver tissue of intravenous drug abusers with chronic hepatitis. A comparative study.

To determine the frequency of talc microcrystals in liver tissue of intravenous (IV) drug abusers and the significance of this finding, the authors reviewed, with light and polarizing microscopy, sections of liver tissue from 70 patients with chronic hepatitis and a history of active (45) or past (25) IV drug abuse. Birefringent crystalline particles consistent with talc were found in 44 cases (63%), 31 associated with active and 13 with past drug abuse. The microcrystals were situated predominantly in hypertrophied portal macrophages; there were no well-formed granulomas. Scanning electron microscopic and energy-dispersive spectrophotometry performed on eight of the positive cases showed the characteristic "flake-pastry" appearance and chemical composition (silicon and magnesium) of talc. For comparison, the authors similarly examined 70 cases of posttransfusion chronic hepatitis, all of which had negative findings for talc, and 70 cases of chronic hepatitis with no documented risk factors for viral hepatitis, of which two had positive findings for talc, even though IV drug abuse was denied by the two patients. The authors conclude that talc is frequently present in the liver of IV drug abusers and whenever encountered it strongly suggests IV drug abuse. Only two patients (1.4%) with a negative history also had talc.

Adult↗

PubMed2004

Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures.

BACKGROUND: The clinical efficacy of the current methods used for cryopreservation of metaphase II human oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation. METHODS: Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2-propanediol with 0.1 M sucrose using a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at sequential steps of the freezing and thawing procedures with the use of a computer-assisted polarization microscopy system (Polscope). RESULTS: The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37 degrees C in culture medium. CONCLUSIONS: The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction.

Cell Survival↗

PubMed1975

Synovial fluid analysis.

Synovial fluid analysis plays an important role in the differential diagnosis of various forms of arthritis. The results of macroscopic, biochemical, bacteriological, and microscopic techniques of evaluating synovial fluid must be correlated to establish the diagnosis of each of the various forms of arthritis. The principle of polarized microscopy as applied to the differential diagnosis of crystalline arthritis is currently of special interest.

Adrenal Cortex Hormones↗

PubMed1996

Nodular pulmonary amyloidosis associated with asbestos exposure.

A 71 year old man was admitted for the purpose of diagnosis of a right solitary pulmonary nodule. The size of the nodule was 18 x 18 mm in diameter 2 years ago, but it has become large, 25 x 25 mm in diameter. The nodule was resected by thoracotomy. Microscopically, eosinophilic amorphous, acellular substances were surrounded by inflammatory infiltrates. It stained with Congo red stain and showed green birefringence with polarizing microscopy. Amyloid fibrils were observed electron microscopically. Asbestos bodies were observed in the lung parenchyma around the nodule. This case shows that a nodule in nodular pulmonary amyloidosis can grow gradually and suggests the possibility of asbestos fibers as one of the etiologic factors in nodular pulmonary amyloidosis.

Aged↗

PubMed1996

Structural evidence for membrane lipid changes in human cataract.

Lipid changes in relationship to cataractogenesis were studied with histochemical methods (topoptical reactions) of polarization microscopy. Frozen section of formaldehyde-fixed human lenses were used for these studies. Six lenses were transparent and 14 lenses presented early to confluent cortical opacities. Cell membrane lipids of transparent lenses showed 8.0 +/- 2.7 nm light retardation. In the early cataractous lenses the light retardation of cell membranes was 23.3 +/- 5.0 nm and that of the fusiform and globular lipids was 37.7 +/- 4.0 nm and 48.5 +/- 6.9 mn, respectively. In the non transparent cortical regions of cataractous lenses, membrane lipids were not observed. Similar to other cell membranes, normal lens membranes are composed of loosely organized lipids. In early cataract lipid density uniformly increased along the cell membranes at the clinically transparent areas, while at the areas with clinically evident fine opacities, small fusiform and globuler lipid drops were formed by even more dense lipids. Confluent cortical cataracts were associated with disappearance of membrane lipids. In our study our findings demonstrated intramembrane lipid changes associated with cataractogenesis.

Adult↗

PubMed2006

Anisotropic properties of the enamel organic extracellular matrix.

Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. This supramolecular ensemble controls the nucleation of the crystalline mineral phase. The detection of anisotropic properties by polarizing microscopy has been extensively used to detect macromolecular organizations in ordinary histological sections. The aim of this work was to study the birefringence of enamel organic matrix during the development of rat molar and incisor teeth. Incisor and molar teeth of rats were fixed in 2% paraformaldehyde/0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2, and decalcified in 5% nitric acid/4% formaldehyde. After paraffin embedding, 5-microm-thick sections were obtained, treated with xylene, and hydrated. Form birefringence curves were obtained after measuring optical retardations in imbibing media, with different refractive indices. Our observations showed that enamel organic matrix of rat incisor and molar teeth is strongly birefringent, presenting an ordered supramolecular structure. The birefringence starts during the early secretion phase and disappears at the maturation phase. The analysis of enamel organic matrix birefringence may be used to detect the effects of genetic and environmental factors on the supramolecular orientation of enamel matrix and their effects on the structure of mature enamel.

Amelogenesis↗

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↗

PubMedDate not supplied

Image analysis of bioparticles accumulation and diamagnetic alignment in high-gradient magnetic field.

Magnetic properties of biological particles are measured in high-gradient magnetic separation (HGMS) analysis, revealing the concentrating process of nucleoprotein particles, ferritin, red blood cells, and eggs. A magnetic force acting on micrometer and submicrometer biological particles having diamagnetic or paramagnetic susceptibility with respect to the solution causes their movement and accumulation in gradient magnetic fields dependent on the values of the magnetic moments. The methods developed enable us to obtain the magnetic moments values of single particles and their assembly directly from magnetic separation and image analyses without assuming the detection of sizes. Our precision methods for the measurement of the capture traveling (magnetic diffusion) time and the accumulation (magnetic sedimentation) radius in HGMS show that it is really possible to determine the weak dia- or paramagnetic shifts of magnetic susceptibility up to 0.7x10(-10) (SI units). HGMS analysis of the concentrating process of nucleoprotein granules (microcells, DNA granules, or nucleosome core particles) with polarization microscopy reveals phase transitions for DNA in granules, and separation accumulation of particles enables the determination of the diamagnetic susceptibility and anisotropy properties. Magnetic concentration effects always occur in living systems because micrometer-located gradient magnetic fields inside an organism are strong enough to cause drifts of cellular complexes and organelles of micrometer and submicrometer sizes. We report the appearance of superparamagnetic contamination inside developing shrimp eggs. In the developing shrimps eggs, ferritin aggregates are observed under weak gradient magnetic fields and diaparaferromagnetic changes are detected. A significant interruption of egg development is revealed in such fields.

Animals↗

PubMed1970

X-ray diffraction studies of the sulfur globules accumulated by Chromatium species.

Isolated wet and dried sulfur globules, obtained by osmotic lysis of lysozyme-ethylenediaminetetraacetic acid prepared spheroplasts of Chromatium okenii, C. weissei, and C. warmingii, were studied by polarizing microscopy and X-ray diffraction. When viewed through crossed Nicol prisms, the sulfur globules, whether in the cell or isolated in a pure, wet state, had a characteristic maltese cross appearance. The observation that rotation of the mount did not change the orientation of the arms suggested a symmetrical radial arrangement of the birefringent units. X-ray diffraction patterns of freshly isolated, wet sulfur globules gave two broad and diffuse diffraction rings with maxima at 0.36 and 0.52 nm. This pattern closely resembled the diffraction pattern of liquid sulfur. When allowed to stand in the wet state, the sulfur globules eventually converted into crystalline orthorhombic sulfur after passing through an unstable crystalline phase not previously described by X-ray diffraction. Vacuum drying of the sulfur globules accelerated the change into crystalline orthorhombic sulfur.

Chromatium↗

PubMed1977

Reorientation of myofilaments during contraction of a vertebrate smooth muscle.

The purpose of the investigation was to determine whether filaments within smooth muscle cells changed their orientation (with respect to the main axis of the cell) during contraction. The stomach muscle of Bufo marinus was used, since its cells may be easily isolated, enabling direct observation in living cells. In addition to still micrography, cinemicrography was used to record continuously during contraction. Polarization microscopy revealed a change in birefringence after contraction, with relaxed cells exhibiting uniform birefringence while contracted cells displayed a discontinuous pattern. Movies revealed a progressive change in orientation of birefringent elements from nearly parallel to the cell's main axis in relaxed cells to increasingly larger angles to the cell's axis as contraction progressed. Phase-contrast microscopy revealed a change in filamentous components, from being parallel to the cell's axis in relaxed cells to being in an undulating or helical pattern during concentration. Cell shape tended to follow the configuration of the filamentous component. Electron microscopy of muscle strips corroborated the observations of living cells and substantiated the conclusion that filaments change their orientation from parallel to oblique (with respect to the cell's axis) during shortening with an undulating or helical pattern of filaments in shortened muscles.

Animals↗

PubMed1989

Three-dimensional arrangement of dense connective tissue around the human major duodenal papilla. Including the ampullary region and the distal choledochal duct.

Directionally arranged dense connective tissue fibres were investigated in 21 specimens of the major duodenal papilla. Specimens were examined using a stereoscope, polarization microscopy and serial histological sections at three different planes. Directionally arranged dense connective tissue fibres spread in a deltoid pattern from the orifice of the major duodenal papilla and its intraduodenal part to the circular duodenal musculature. Connective tissue fibres crossing at different angles form a texture from the orifice of the major duodenal papilla to the distal choledochal duct. The functional significance of the dense connective tissue fibres, e.g. for the muscular system in the investigated area, is discussed as well as possible reasons for gallstone impactions.

Ampulla of Vater↗

PubMed1986

Response of the renal supporting tissues to chronic fluoride exposure as revealed by a special technique.

The effects of chronic fluoride excess in the mouse were studied by means of polarizing microscopy in combination with a special staining technique employing Sirius red F3B, a dye which renders collagen fibrils sharply visible. It was observed that changes occur in three renal areas: the interstitium, the intrinsic vasculature and Bowman's capsule. The collagen content of each area increases after about 100 days of the total fluoride exposure of 280 days had elapsed. Although Bowman's capsule was thickened, the glomerular tufts and the nephrons showed edematous swelling and degeneration. A concept is developed to illustrate how early inflammatory response to the chemical effects of fluoride excess leads to vascular injury, parenchymal ischemia and fibrosis.

Animals↗