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PubMed2003

Apical growth and mitosis are independent processes in Aspergillus nidulans.

It is well established that cytoplasmic microtubules are depolymerized during nuclear division and reassembled as mitotic microtubules. Mounting evidence showing that cytoplasmic microtubules were also involved in apical growth of fungal hyphae posed the question of whether apical growth became disrupted during nuclear division. We conducted simultaneous observations of mitosis (fluorescence microscopy) and apical growth (phase-contrast microscopy) in single hyphae of Aspergillus nidulans to determine if the key parameters of apical growth (elongation rate and Spitzenkörper behavior) were affected during mitosis. To visualize nuclei during mitosis, we used a strain of A. nidulans, SRS27, in which nuclei are labeled with the green-fluorescent protein. To reveal the Spitzenkörper and measure growth with utmost precision, we used computer-enhanced videomicroscopy. Our analysis showed that there is no disruption of apical growth during mitosis. There was no decrease in the rate of hyphal elongation or any alteration in Spitzenkörper presence before, during, or after mitosis. Our findings suggest that apical growth and mitosis do not compete for internal cellular resources. Presumably, the population of cytoplasmic microtubules involved in apical growth operates independently of that involved in mitosis.

Aspergillus nidulans↗
PubMed1986

Morphology, development and behavior of Pneumocystis carinii observed by light-microscopy in nude mice.

The present paper describes the morphology, development and behavior of Pneumocystis carinii, especially of the trophozoites in the alveoli of conventionally raised BALB/c nude mice, by using phase-contrast microscopy, paraffin sections and semiultrathin sections embedded in JB-4 plastic. Under phase-contrast microscopy, trophozoites were ameboid in external appearance and 2 to 8 micron in diameter. Usually they have one or more lucid spherical vacuoles and one less lucid nucleus in the cytoplasm. Maturation and independence of intracystic bodies were observed in the developing cysts. The intracystic bodies were polymorphic, i.e. spherical, ameboid or elongated. The paraffin sections using the double staining of P. carinii with Gomori's methenamine silver nitrate (GMS) and Giemsa, showed a small number of mature cysts containing intracystic bodies and a large number of trophozoites within the characteristic honeycombed material in the alveolar spaces. In order to investigate the morphology and parasitizing behavior of the trophozoites and cysts as well as the response of alveolar tissue in more detail, semiultrathin sections of 0.5-2 micron thickness were made from the materials embedded in JB-4 plastic and stained with Giemsa. In lightly infected alveoli, some trophozoites and cysts were found to be closely attached to the Type I alveolar epithelial cells. In heavily infected alveoli, almost all alveoli were filled with trophozoites, cysts, debris of the host cells, and occasionally phagocytic cells (macrophages and neutrophils) containing cysts or trophozoites were found. It was noted that the cysts were very few in number (1%) compared with the number of the trophozoites in nude mice in a 0.4 mm2 area of 2 micron thick lung sections. The host tissue of nude mice in this study was not as strongly affected by the organisms as that in cortisone-treated rats and P. carinii pneumonia patients. In the present study the morphology of P. carinii found in nude mice was not different from that found in rats and in man.

Animals↗
PubMed1989

[Basic studies on CaO-P2O5-MgO-SiO2-CaF system glass ceramics. 1. Morphology under the phase-contrast microscope and growth of cultured cells].

In order to determine the biocompatibility of glass ceramics which is one of the new biomaterials, in vitro studies were carried out by a cell culture method using four established cell lines. Materials used were glass ceramic disks with a diameter of 3 mm, and polystyrene coverslips of the same size as controls of the growth curve. Cells of each line were inoculated into 24-well multiplates at an appropriate density onto glass ceramic disks, and examined by phase contrast microscopy on the 1st, 3rd, 6th and 8th day. In addition, doubling time and saturation density were calculated from the growth curve. The results obtained were as follows. 1) Phase-contrast microscopy revealed that cells of each line attached to the disk within 24 hours and their numbers increased with time. After 8 days of cultivation, all of them reached confluence. 2) Contact with the glass ceramics did not cause cellular death or degeneration. Furthermore, the cultured cells showed the same morphological features as the control cells. 3) According to the growth curves, doubling time of all cells cultured with glass ceramics was shorter than that of the control cultures. On the other hand, saturation density was reduced to a minimum of 80% of the controls. These findings led to the conclusion that glass ceramic materials do not prevent the growth of cultured cells. According to the above results, glass ceramics possess the characteristics needed for bone grafts and implant materials.

Cell Line↗
PubMed2003

Assembly of collagen matrices as a phase transition revealed by structural and rheologic studies.

We have studied the structural and viscoelastic properties of assembling networks of the extracellular matrix protein type-I collagen by means of phase contrast microscopy and rotating disk rheometry. The initial stage of the assembly is a nucleation process of collagen monomers associating to randomly distributed branched clusters with extensions of several microns. Eventually a sol-gel transition takes place, which is due to the interconnection of these clusters. We analyzed this transition in terms of percolation theory. The viscoelastic parameters (storage modulus G' and loss modulus G") were measured as a function of time for five different frequencies ranging from omega = 0.2 rad/s to 6.9 rad/s. We found that at the gel point both G' and G" obey a scaling law, with the critical exponent Delta = 0.7 and a critical loss angle being independent of frequency as predicted by percolation theory. Gelation of collagen thus represents a second order phase transition.

Animals↗
PubMed1994

Pulsed growth of fungal hyphal tips.

Somatic fungal hyphae are generally assumed to elongate at steady linear rates when grown under constant environmental conditions with ample nutrients. However, patterns of pulsed hyphal elongation were detected during apparent steady growth of hyphal tips in fungi from several major taxonomic groups (Oomycetes, Pythium aphanidermatum and Saprolegnia ferax; Zygomycetes, Gilbertella persicaria; Deuteromycetes, Trichoderma viride; Ascomycetes, Neurospora crassa and Fusarium culmorum; Basidiomycetes, Rhizoctonia solani). Growing hyphal tips were recorded with video-enhanced phase-contrast microscopy at high magnification, and digital images were measured at very short time intervals (1-5 s). In all fungi tested, the hyphal elongation rate was never perfectly steady but fluctuated continuously with alternating periods of fast and slow growth at more or less regular intervals. Pulsed growth was observed in fungi differing in cell diameter, overall growth rate, taxonomic position, and presence and pattern of Spitzenkörper organization, suggesting that this is a general phenomenon. Frequency and amplitude of the pulses varied among the test organisms. T. viride and N. crassa showed the most frequent pulses (average of 13-14 per min), and F. culmorum the least frequent (2.7 per min). Average pulse amplitude varied from 0.012 microns/s for F. culmorum to 0.068 microns/s for G. persicaria. In F. culmorum and T. viride, the fast phase of the growth pulses was correlated with the merger of satellite Spitzenkörper with the main Spitzenkörper. These findings are consistent with a causal relationship between fluctuations in the overall rate of secretory vesicle delivery/discharge at the hyphal apex and the fluctuations in hyphal elongation rate.

Activity Cycles↗
PubMed1966

A melting point for the birefringent component of muscle.

The A filament of the striated muscle sarcomere is an ordered aggregate of one or a few species of proteins. Ordering of these filaments into a parallel array is the basis of birefringence in the A region, and loss of birefringence is therefore a measure of decreased order. Heating caused a large decrease in the birefringence of glycerinated rabbit psoas muscle fibers over a narrow temperature range ( approximately 3 degrees C) and a large decrease in both the birefringence and optical density of the A region of Drosophila melanogaster fibrils. These changes were interpreted as a loss of A filament structure and were used to define a transition temperature (T(tr)) as a measure of the stability of the A region. Since the transition temperature was sensitive to pH, ionic strength, and urea, solvent conditions which often affect protein structure, it is an experimentally useful indicator for factors affecting the structure of the A filament. Fibers from glycerinated frog muscle were less stable over a wide pH range than fibers from glycerinated rabbit muscle, a fact which demonstrates a species difference in structure. Glycerinated rabbit fibrils heated to 70 degrees C shortened to about 40% of their initial length. The extent of shortening was not correlated with the loss of birefringence, and phase-contrast microscopy showed that this shortening occurred in the I region as well as in the A region. This response may be useful for studying the I filament and actin in much the same way that the decrease in birefringence was used for studying the A filament and myosin. The observations presented show that some properties of muscle proteins can be studied essentially in situ without the necessity of first dispersing the structure in solutions of high or low ionic strength.

Animals↗
PubMed2001

[European-American-type hairy cell leukemia without splenomegaly, treated successfully with deoxycoformycin].

A 55-year-old Japanese man was hospitalized on October 5, 1999, because of high fever. Physical examination revealed neither lymphadenopathy nor hepato-splenomegaly. Laboratory data on admission showed a white blood cell count of 1,580/microliter, a hemoglobin level of 9.1 g/dl, and a platelet count of 113 x 10(3)/microliter. A small percentage of abnormal mononuclear cells were present in the peripheral blood. A bone marrow biopsy specimen demonstrated myelofibrosis and diffuse infiltration of abnormal monoculear cells with a mature B cell phenotype. A bone marrow aspirate showed 29% abnormal mononuclear cells, which had an indented or folded nucleus and reticular nuclear chromatin. Moderate to strong tartarate-resistant acid phosphatase activity was detected in these cells. Although the cytoplasmic projections were poorly preserved in specimens stained with May-Giemsa, fresh preparations showed numerous slender cytoplasmic projections by phase-contrast microscopy. The hairy cells had the phenotype CD5-, CD10-, CD11c+, CD19+, CD20+, CD25+, lambda. The patient was diagnosed as having European-American-type hairy cell leukemia (HCL) without splenomegaly, which is quite rare in Japan. The value of phase-contrast microscopy for recognition of the hairy cells was emphasized. The patient was treated successfully with deoxycoformycin (DCF).

Antibiotics, Antineoplastic↗
PubMed1995

The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.

The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection. This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology.

Actin Cytoskeleton↗
PubMed2004

Collective movement of epithelial cells on a collagen gel substrate.

Collective cell movement acts as an efficient strategy in many physiological events, including wound healing, embryonic development, and morphogenesis. We found that epithelial cells (Madin-Darby canine kidney cell) migrated collectively along one direction on a collagen gel substrate. Time-lapse images of Madin-Darby canine kidney cells cultured on type-I collagen gels and glass substrates were captured by phase contrast microscopy equipped with an incubation system. On the gel substrate, the directions of cell movement gradually converged on one direction as the number of cells increased, whereas the cells moved randomly on the glass substrate. We also observed "leader" cells, which extended large lamellae and were accompanied by many "follower" cells, migrating in the direction of oriented collagen fibers. The mean-squared displacement of each cell movement and the spatial correlation function calculated from the spatial distribution of cell velocity were obtained as functions of observation time. In the case of the gel substrate, the spatial correlation length increased gradually, representing the collectiveness of multicellular movement.

Animals↗
PubMed2006

Observation of fibroblast motility on a micro-grooved hydrophobic elastomer substrate with different geometric characteristics.

We used a hydrophobic micro-textured poly-dimethylsiloxane (PDMS) in the presence of serum protein at 37 degrees C to study the motility of mouse stromal fibroblast on variant (15-100microm) parallel ridge/groove with 30microm depth. In this paper, we observed the temporal changes in cell morphology and locomotion by using time-lapse phase-contrast microscopy. When fibroblasts seeded onto the micro-grooved substrate, almost all of cells concentrated at the bottom of the grooves. Sequentially, the fibroblasts attached and spread on the surface, migrated toward the walls of the grooves, climbed up and down the ridges frequently, apparently, the 30microm depth of groove did not hinder movement across the micro-grooves. Eventually, they stopped proliferating as a result of contact inhibition and formed a confluent monolayer on the ridges almost exclusively, with an orientation parallel to the direction of the ridge/groove. Cellular shape of fibroblast was enhanced with the micro-grooves, the form index of nucleus was 2.6-fold greater than that of cells on smooth surfaces. Further, we found that hydrophobic surfaces are more prone to direct cellular motility in comparison with hydrophilic surfaces.

Animals↗
PubMed1979

Actin in spindles of Haemanthus katherinae endosperm. I. General results using various glycerination methods.

We have studied actin-containing filaments in spindles in Haemanthus endosperm cells glycerinated by various methods; the actin-containing filaments were identified by their reaction with rabbit skeletal muscle heavy meromyosin (HMM) to form 'decorated' filaments. Actin-containing filaments in the spindle were seen in amongst microtubules in bundles (both non-kinetochore microtubule bundles and kinetochore microtuble bundles) and were also seen not associated with microtubules. There were very few extra-spindle actin-containing filaments in these cells. Actin-containing filaments seemed to interact with microtubules, because the filaments remained close to and parallel to microtubules even when the microtubules were sharply curved. Because of the close association between microtubules and actin-containing filaments we could not identify all the actin-containing filaments present in microtubule bundles: microtubules obscured actin-containing filaments. We studied Haemanthus endosperm cells as they were glycerinated. For some of these observations we used phase-contrast microscopy. Glycerination caused the cells to shrink, initially, and this was followed by rapid expansion, but the cells did not expand to as large a volume as before glycerination. Spindle structure was maintained despite these changes in cell size. Evidences for this are that relative chromosome positions were maintained during glycerination, that spindle birefringence was maintained during glycerination, and that individual chromosomal spindle fibres remained birefringent during glycerination. Electron-microscopic observations supported this in that kinetochore microtubule bundles and non-kinetochore microtubule bundle were maintained during glycerination, as was the helical arrangement of spindle ribosomes into polyribosomes. One-step glycerination procedures were used (cells were treated with mixtures containing 25% glycerol, Triton-X-100 and HMM), and such procedures might be of general use. Living cells were embedded in fibrin clots in making light-microscopic observations; this procedure, too, might be of general use.

Actins↗
PubMed2001

Exposure to tremolite asbestos and respiratory health in Swedish dolomite workers.

OBJECTIVES: Deposits of carbonate rock like limestone and dolomite may contain tremolite asbestos. This study assessed the exposure to tremolite asbestos and the respiratory health of Swedish dolomite workers. METHODS: 95% of 137 eligible workers at two dolomite producing companies completed a self administered questionnaire that included questions on respiratory symptoms and were examined with spirometry as well as chest radiography. Total exposure to dust was gravimetrically measured and the tremolite asbestos content of the dust was assessed with polarisation and phase contrast microscopy. RESULTS: Dolomite dust concentrations were moderate (median 2.8 mg/m3) and tremolite asbestos concentrations were generally below the limit of detection (<0.03 fibres/ml). Somewhat higher values, around 0.1 fibres/ml, were obtained in manual stone sorting and bagging. Respiratory symptoms suggestive of chronic bronchitis were more related to smoking than to estimates of individual exposure to dust. The mean vital capacity was 0.2 l lower than expected after adjustment for sex, age, height, and smoking but the decline in lung function was not associated with current or cumulative exposure to dust in a clear cut way. Two definite cases of pleural plaques and one possible case of simple pneumoconiosis were noted, but the plaques could not be attributed exclusively to exposure to tremolite asbestos. CONCLUSIONS: Dolomite mining and milling may indeed entail low levels of exposure to tremolite asbestos, but this exposure was not a strong determinant of respiratory symptoms, lung function, or pneumoconiosis in exposed Swedish workers. This was true also for dolomite dust. The hazards of exposure to tremolite asbestos may vary across deposits, however, and additional studies at other sites of carbonate rock exploitation are warranted.

Adult↗
PubMed1997

Apical branching in a temperature sensitive mutant of Aspergillus niger.

An apical branching, temperature-sensitive, mutant of Aspergillus niger (ramosa-1) was isolated by UV mutagenesis. Ramosa-1 has a wild type morphology at 23 degrees C, but branches apically when shifted to 34 degrees C. The cytological events leading to apical branching were recorded by video-enhanced phase contrast microscopy. The first event was a momentary, localized, cytoplasmic contraction lasting approximately 1 s. This contraction was seen as a sudden unidirectional movement of visible organelles (mitochondria, spheroid bodies) toward the hyphal apex. During the contraction, there was a transitory sharp increase in refractive index in a localized area of cytoplasm in the apex or subapex of the cell. Within 5 s, the Spitzenkörper retracted from its normal position next to the apical pole and disappeared from view 20 to 50 s later. Hyphal elongation rate diminished sharply, and the typical distribution of organelles at the hyphal tip was disturbed. After 210-240 s, organelle distribution returned to normal, polarized growth resumed, but instead of one Spitzenkörper two new Spitzenkörper appeared, each giving rise to an apical branch. The second branch Spitzenkörper appeared with a 60- to 100-s delay. We did not observe the original Spitzenkörper dividing in two; instead, the new Spitzenkörper arose de novo from vesicle clouds that formed in the apical region next to the future site of branch emergence. In all instances that we examined, the dislocation and disappearance of the Spitzenkörper was preceded by cytoplasmic contractions. We therefore suspect the existence of an intimate connection between the cytoskeletal network and the Spitzenkörper. Accordingly, we propose that the apical branching phenotype in ramosa-1 is triggered by a molecular event that induces a transient alteration in cytoskeleton organization.

Aspergillus niger↗
PubMed1989

[The significance of erythrocyte morphology for the differential diagnosis of hematuria].

Since 1984 we have investigated urine specimens of 129 patients by means of phase contrast microscopy. 51% of the children have an isomorphic, 43% a dysmorphic and 6% a mixed erythrocyturia. We have found a good correlation between dysmorphic erythrocyturia and glomerular diseases and between isomorphic erythrocyturia and nonglomerular changes. In 4.6% there were a discrepancy between the urinary findings and the diagnoses. Phase contrast microscopy is an advantage for diagnostics of hematuria provided that it will be done repeatedly.

Child↗
PubMed1990

Non-radioactive in situ hybridization of DNA probes to chromosomes and nuclei. A comparison of techniques.

We have used a moderate repeat probe and a number of single copy DNA probes of varying sizes to compare different approaches to non-radioactive in situ hybridization. We have compared the ease and speed of the methods, the sensitivity, resolution, reproducibility, the availability and costs of reagents, and the potential for clinical application. Following biotinylation or mercuration, the probes were hybridized to human metaphases and nuclei and detected by different affinity systems. Visualization of signals was by brightfield, phase contrast, fluorescence or reflection contrast microscopy. As a result of our study, we recommend two simple and reliable methods using the biotinylation approach with either the avidin-peroxidase conjugate and diaminobenzidine detection and reflection contrast microscopy, or the streptavidin-alkaline phosphatase conjugate and bromochlorodinolyl phosphate nitroblue tetrazolium chloride detection using phase contrast microscopy.

Cell Nucleus↗
PubMed2004

The partial head decondensation test is a new, quick method to assess acrosome status in human spermatozoa.

OBJECTIVE: To develop a fast method for assessing acrosome status in human spermatozoa. DESIGN: Development of a new in vitro test to assess acrosome reaction in human spermatozoa. SETTING: Academic medical institution. PATIENT(S): Normozoospermic subjects. INTERVENTION(S): Spermatozoa were isolated from fresh semen samples, capacitated, and stimulated or not with P or ionomycin. Acrosome reactions were evaluated by phase-contrast microscopy after a brief sperm incubation in a decondensing solution. The results were compared with those obtained by scanning electron microscopy and fluoresceinated lectin staining. MAIN OUTCOME MEASURE(S): Percentage of intact acrosomes. RESULT(S): The new procedure allowed intact acrosomes to be easily identified and quantified by phase-contrast microscopy. In unstimulated and ionomycin-treated spermatozoa, a very good agreement was found among the new test, scanning electron microscopy, and fluoresceinated lectin staining. In P-treated spermatozoa, the proposed method allowed a significantly higher percentage of reacted acrosomes to be resolved, likely due to its ability to detect the very initial stages of the acrosome reaction. CONCLUSION(S): The new test allows acrosome-intact and acrosome-reacted spermatozoa to be unambiguously singled out and quantified. The method is rapid, reliable, sensitive, and easy to perform, which makes it of profitable use in both basic research and diagnostic practice.

Acrosome Reaction↗
PubMed1985

Airborne asbestos in Colorado public schools.

Levels of airborne asbestos for six Colorado public school facilities with sprayed-on asbestos materials were documented using three analytical techniques. Phase contrast microscopy showed levels up to the thousandths of a fiber per cubic centimeter (f/cc), scanning electron microscopy (SEM) up to the hundredths of a f/cc, and transmission electron microscopy coupled to selected area electron diffraction and energy dispersive X-ray analysis (TEM-SAED-EDXA) up to the tenths of an asbestos f/cc. Phase contrast microscopy was found to be an inadequate analytical technique for documenting the levels of airborne asbestos fibers in the schools: only large fibers which were not embedded in the filter were counted, and asbestos fibers were not distinguished from nonasbestos.

Air Pollutants↗