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PubMed1975

Synthesis of type I collagen by human smooth muscle cells in vitro.

Human vascular smooth muscle cells, derived from explants of medial smooth muscle of a fetal aorta, were grown in vitro and examined with phase and electron microscopy for characteristic morphologic features of smooth muscle cells and for the biosynthesis of connective tissue proteins. Their patterns of growth and ultrastructure were similar to those described for other species of cultured arterial smooth muscle cells. Most cells contained varying amounts of myofilaments interpersed with dense bodies, rough and smooth endoplasmic reticulum, mitochondria, various sized vesicles, lysosomes, and lipid droplets. Extracellularly, small amounts of electron-dense material and microfilaments were observed adjacent to or between the cells. The over-all morphology suggested that the smooth muscle cells were actively engaged in protein synthesis. Although we could not identify banded collagen fibrils in 10- to 14-day-old cultures by electron microscopy, the cells synthesized and secreted a collagen characterized as type I collagen. A hydroxyproline-containing protein composed of two alpha-1 and one alpha-2 chains was extracted from the cell layer. The triple helical precursor of type I collagen, procollagen, was secreted into the medium.

Aorta↗

PubMed1991

The effect of flow on the interaction of isolated megakaryocytes with subendothelial extracellular matrix.

We have previously shown that human, guinea pig, or rat megakaryocytes, incubated under static conditions on an extracellular matrix (ECM) produced by endothelial cells, readily adhered to the matrix and underwent platelet-like shape change and thromboxane A2 secretion. We have now exposed megakaryocytes to ECM in a perfusion system similar to that used to study platelets circulated over aortic subendothelium. We used a continuous flow circuit incorporating a parallel plate perfusion chamber. Megakaryocytes were isolated to high purity from guinea pig marrow by centrifugal elutriation and velocity sedimentation. The cells were introduced into the flowing medium while the surface of an ECM-coated coverslip mounted in the chamber was observed continuously by phase-contrast video microscopy for up to 18 hours. Megakaryocytes from the flowing suspension started to adhere to the ECM within seconds. Significant adhesion occurred over a range of shear rates, from 10 to 190 seconds-1, did not appear above 300 seconds-1 and was greatest at a shear rate of 60 seconds-1. Adhesion to the ECM was specific, since there was no adherence to glass coverslips, glutaraldehyde-fixed ECM-coated coverslips, or to endothelial cells cultured on ECM-coated coverslips. At low shear rates large aggregates of megakaryocytes formed on the ECM surface; these could be detached and washed away by higher shear forces. Megakaryocytes thus acquire, even before platelet formation, an adhesive capacity similar to that of platelets. In addition, a significant fraction of the adherent megakaryocytes underwent elongation and pseudopod formation similar to that seen in marrow sinusoids.

Animals↗

PubMed1990

In vitro pharmacologic separation of corneal endothelial migration and spreading responses.

Repair of corneal endothelial wounds involves two forms of cell translocation: (1) "migration," in which individual cells at the wound edge break contacts with neighboring cells and move as individuals into the wound defect, and (2) "spreading," in which cells within the confluent monolayer adjacent to the wound move as a group into the wound area. The authors combined morphometric analysis of Giemsa-stained cultures, phase-contrast video microscopy, and Rh-phalloidin staining of actin filaments to study the effects of epidermal growth factor (EGF) and indomethacin on the migratory and spreading responses to wounding using an in vitro wound-closure model which mimics the amitotic state and general behavior of human corneal endothelium. They found that EGF stimulated the migration of individual cells from the wound edge, induced cellular elongation, and promoted a diffuse distribution of actin filaments. Indomethacin promoted spreading of the confluent monolayer into the wound defect, induced enlargement and flattening of cells, and promoted the formation of long, thick actin stress fibers. These results provide evidence that the migration and spreading responses of corneal endothelial cells to wounding can be pharmacologically separated. The findings suggest that migration of individual cells during wound repair may result from an endogenous form of EGF-like stimulation and that the elongated shape associated with this form of translocation results, at least in part, from an EGF-like alteration in actin-filament organization. Spreading of the confluent monolayer to cover the wound defect may result from a decrease in cyclic adenosine monophosphate induced by a transient reduction in prostaglandin E2 synthesis. This form of translocation may result, in part, from enlargement and flattening of corneal endothelial cells secondary to an enhancement of actin stress-fiber formation.

Actins↗

PubMed1985

[Visual classification of erythrocytes in blood smears from normal subjects and patients].

Erythrocytes on specially prepared and coloured blood smears can be classified not only by idcture analysis but also by phase contrast microscopy. Parameters of change for the peripheral zone area and pallor area (phase contrast value PW and index of erythrocyte changes Ev-I) serve as classificators of visual technique. 449 blood smears of the same number of male and female grown-up persons, whose diagnosis was unknown at the time of evaluation, were used for evaluation. In 145 cases these were healthy test persons in clinical and laboratory respect (group I). 163 test persons were ill, but without any malignous neoplasias (group II), and in 141 patients an ensured malignous neoplasia could be found (group III). From those 308 test persons or patients respectively in group I + II 11 blood smears were falsely classified as positive (= 3.6%). In 5 from 141 cases in group III blood smears were falsely evaluated as negative (= 3.6%). The findings were statistically ensured by the t-test. Clinical parameters of evaluation gained with visual techniques of classification are in accordance with those gained with automatic picture analysis. The reasons for the good separating ability of PW and Ev-I are discussed. Visual techniques of classification are suitable for institutions of all grades of equipment.

Adult↗

PubMedDate not supplied

[Asbestos and bronchogenic carcinoma: a search for fibers in the pulmonary tissue of 3 patients with bronchogenic carcinoma].

Based on the highly suspicious radiological findings of exposure to asbestos (case 1) or on a positive occupational history (cases 2 and 3), the authors looked for the presence of fibers in blocks of lung tissue removed in autopsy or surgical biopsies of three cases of bronchogenic carcinoma. The blocks were submitted to sodium hypochloride digestion followed by fiber identification in phase contrast light microscopy. The authors were able to demonstrate the presence of fibres in the three cases. The likelyhood of those carcinomas being caused by exposure to asbestos is very high, as two out of the three cases showed pulmonary fibrosis (cases 1 and 2) and the other case showed typical parietal pleural plaques at thoracotomy.

Aged↗

PubMedDate not supplied

L-cysteine improves growth of human peritoneal mesothelial cells in vitro.

OBJECTIVE: To improve the growth characteristics of human peritoneal mesothelial cells (HPMC). DESIGN: The effect of commonly used agents, L-cysteine and epidermal growth factor (EGF), added individually ("single") or mixed with hydrocortisone and apo-transferrin ("admixture") in the culture medium (M199) on cultured HPMC, was investigated. METHODS: Growth agents were added to M199 medium along with 2% fetal bovine serum and L-glutamine. Growth was determined by the analysis of thymidine ([methyl-3H] thymidine) incorporation into deoxyribonucleic acid, total cell protein, and by cell counts. Morphology was assessed by phase contrast light microscopy and scanning electron microscopy. RESULTS: HPMC exposed to L-cysteine 0.25 x 10(-3) mol/L (30 micrograms/mL) exhibited significantly improved attachment and growth. Attached cells appeared flat and well spread out shortly after seeding, and produced a tight polygonal monolayer in 14 days, in contrast to the growth of HPMC in control M199 medium, which failed to reach confluence. After an initial lag period in cell growth, EGF (0.01 microgram/mL) produced a greater increase in cell growth than L-cysteine did; however, this was associated with changes in HPMC morphology. During the growth period (14 days), EGF-stimulated HPMC appeared distorted and irregular compared to L-cysteine-treated cells, which had the characteristic tight "cobblestone" appearance. CONCLUSION: L-cysteine improved cell attachment with preservation of the characteristic morphology of HPMC. Epidermal growth factor improved cell growth but produced changes in morphology. The addition of L-cysteine to the culture medium has an important cell growth enhancement role due to the improved cell attachment and cell viability.

Apoproteins↗

PubMed1984

Development of a mathematical model for predicting concentrations of small asbestos fibers.

The standard method of asbestos sample analysis by phase-contrast microscopy excludes fibers which are below the resolution limits of the light microscope. Electron microscopic studies revealed that fibers in these small size ranges comprise the majority of fibers in asbestos dust. This research was conducted to determine if the development of a mathematical model for predicting concentrations of small fibers (less than 5 microns length) based on phase-contrast concentrations was feasible. The model presented in this paper was evaluated for its practical utility and was determined to be of minimal value; however, it was felt that the development of a reasonably accurate model can be arrived at through continued research.

Asbestos↗

PubMed1987

Salt and peroxide compared with conventional oral hygiene. I. Clinical results.

The purposes of this 2-year longitudinal study were to: compare the clinical effectiveness of patient applied sodium bicarbonate, hydrogen peroxide, and sodium chloride (S/P) to the use of conventional oral hygiene methods and to investigate the motivational effect of using phase-contrast microscopy in teaching effective oral hygiene. Initially, 972 subjects were screened for signs of periodontitis. From these, 347 with early to moderate periodontitis were selected and each was randomly assigned to one of four home treatment regimens after scaling and root planing. The four treatment regimens included: conventional oral hygiene procedures, conventional oral hygiene procedures plus phase-contrast demonstration of subgingival microbial forms for oral hygiene motivation, S/P oral hygiene, and S/P oral hygiene plus phase-contrast demonstration of subgingival microbial forms for oral hygiene motivation. Plaque, bleeding, gingival inflammation, probing depth, and clinical attachment level were recorded at baseline, 8, 16, and 24 months. Subjects were recalled for reinforcement of oral hygiene and periodontal prophylaxis at various intervals. Data were analyzed based on disease severity, location of index sites and compliance. The results indicated that both conventional oral hygiene procedures and the S/P regimen were effective in reducing clinical signs of disease when combined with professional care. There were no differences between the two regimens in clinical effectiveness and trends favoring microscopic viewing of subgingival plaque for motivational purposes were not statistically significant.

Adult↗

PubMed2002

Liquid crystal formation in supercoiled DNA solutions.

The critical concentrations pertaining to the liquid crystal formation of pUC18 plasmid in saline solutions were obtained from (31)P nuclear magnetic resonance, polarized light microscopy, and phase equilibrium experiments. The transition is strongly first order with a broad gap between the isotropic and anisotropic phase. The critical boundaries are strongly and reversibly dependent on temperature and weakly dependent on ionic strength. With polarized light microscopy on magnetically oriented samples, the liquid crystalline phase is assigned cholesteric with a pitch on the order of 4 microm. Preliminary results show that at higher concentrations a true crystal is formed. The isotropic-cholesteric transition is interpreted with lyotropic liquid crystal theory including the effects of charge, orientation entropy, and excluded volume effects. It was found that the molecular free energy associated with the topology of the superhelix is of paramount importance in controlling the width of the phase gap. The theoretical results compare favorably with the critical boundary pertaining to the disappearance of the isotropic phase, but they fail to predict the low concentration at which the anisotropic phase first appears.

Anisotropy↗

PubMed1985

An immunocytochemical and ultrastructural study of heterogeneity in the human breast carcinoma cell line PMC42.

A recently established human breast carcinoma cell line has been reported to exhibit a number of morphological cell types in monolayer cultures as defined by phase-contrast microscopy. The cell line also produces cords of viable cells floating within the culture medium. Cultures of this cell line, grown in monolayer, on collagen gels, and as floating cords of cells, were studied by transmission electron microscopy and immunocytochemistry. A detailed analysis of the staining pattern obtained with a series of monoclonal antibodies with well-defined human breast epithelial cell specificities and a polyclonal antikeratin antibody showed PMC42 to resemble cultures of both normal human breast and other human breast carcinoma cell lines. The pleomorphic nature of PMC42 cultures was confirmed at an ultrastructural level, and of the eight cell types observed by phase-contrast appearance, seven ultrastructural counterparts were observed. In addition, the presence of intracytoplasmic lumina and overt epithelial differentiation confirm a breast epithelial origin for this cell line.

Breast Neoplasms↗

PubMed1981

Spindle dynamics during mitosis in Dictyostelium discoideum.

We studied mitosis in vegetative amebae of Dictyostelium discoideum by phase-contrast microscopy of live and flat-embedded cells, by time-lapse microcinematography, and by electron microscopy of ultrathin sections. The mean time from early prophase to the end of cytokinesis was 10 min 32 s (range: 6 min 23 s to 15 min 40 s). The start of anaphase divided the mean total time for mitosis into two almost equal intervals. Spindle elongation was analyzed in detail in 10 amebae. It was composed of an active first and a passive second phase. The mean duration of the active phase was 94.7 s, the mean increase in spindle length for that interval was 6.1 micrometers, and the mean velocity of elongation was 4.0 micrometers/min. The spindle possibly elongates via an interaction between interdigitating microtubules of the central spindle (cf. [14]), but the mean elongation factor of 2.9 indicates that these microtubules themselves must elongate concomitantly.

Dictyostelium↗

PubMedDate not supplied

Comparison of methods of assessing asbestos fiber concentrations.

Dimensional measurement of the counted fibers has important implications for setting of environmental standards to protect workers' health. Results of fiber concentrations as determined by different methods were compared. Each method was used to determine concentration by examining one sector of membrane filter on which fibrous dust was collected in various workplaces. The concentrations measured by the Asbestos International Association (AIA) method for 35 samples were, on the average, greater than the concentrations determined by the modified British Occupational Hygiene Society--Asbestosis Research Council (BOHS-ARC) method. The AIA and modified BOHS-ARC methods were found to be linearly correlated (r = 0.82). The mean of total fiber concentrations obtained by using the indirect transmission electron microscopy (TEM) method was 15.5 times the mean obtained by using the direct TEM method. The difference in concentrations determined by the two TEM methods resulted from disintegration of fiber bundles into single fibers during the ashing and ultrasonifying processes used in the indirect method. At least 83.5% of total fibers observed by TEM escaped detection by phase contrast microscopy.

Air Pollutants, Occupational↗

PubMed1962

Cytological observations on the chromatin bodies of two Bacillus species.

Chatterjee, B. R. (Baylor University College of Medicine, Houston, Tex.) and Robert P. Williams. Cytological observations on the chromatin bodies of two Bacillus species. J. Bacteriol. 83:1112-1118. 1962.-The nature of the chromatin bodies in Bacillus anthracis and B. megaterium has been studied intracellularly as well as after isolation from the bacilli. Preparations were examined by phase-contrast microscopy in the living state and by acid-Giemsa and Feulgen staining. In cells from young cultures, chromatin bodies occupy a continuous, long, central area in the bacteria. As the cells mature, the bodies lose their continuity and divide into two halves. In cells grown for 24 hr or longer, the chromatin bodies become more complex, and appear as multiple, spherical, interconnected constellations. Nuclear staining reveals that only the peripheral area, whether in cells from young or older cultures, takes up the stain, leaving a clear, central core. Discrete chromatin bodies are liberated from cells grown for longer periods after disintegration of the bacterial cell wall. These discrete bodies have been isolated from whole bacteria by treating washed, buffered saline suspensions of old cells with high concentrations of lysozyme followed by digestion with purified pancreatic lipase. The isolated chromatin bodies retain the same structure and appearance as inside the cells and show the same staining characteristics. Observations suggest that the chromatin bodies are discrete and circumscribed in nondividing, resting-phase bacteria.

Animals↗

PubMed2002

Proteinases from Bacillus intermedius secreted in the late stages of sporulation.

BACKGROUND: Proteinases are widely used in various fields of medicine, such as the treatment of burns, purulent wounds, or decubitus ulcers. On the basis of new microbial proteinases produced by nonpathogenic organisms, a new generation of medical preparations can be developed. Representatives of the Bacillus genera are nonpathogenic and are suitable for producing various proteases in large quantities. B. intermedius is shown to produce a set of alkaline proteases at the early and late stationary phase of growth. MATERIAL/METHODS: The activity of alkaline proteinases was determined using synthetic chromogenous substrates Z-Glu-pNA and Z-Ala-Ala-Leu-pNA. To determine beta-galactosidase activity, 2-nitro-beta-D-galactopyranosid was used. Spores were calculated by phase-contrast microscopy. RESULTS: During the late stages of sporulation B. intermedius 3-19 cells were shown to secrete two proteinases into the medium: glutamyl endopeptidase, with maximum activity at 40 hours of growth, and subtilisin, with maximum activity at 44 hours of growth. Evidence for the secretion of these enzymes into the medium was provided by measuring beta-galactosidase activity. CONCLUSIONS: Our results show that proteinases from B. intermedius (glutamyl endopeptidase 2 and subtilisin 2) in the late stationary phase of growth are secreted enzymes. This suggests that these enzymes play a role in sporulation.

Bacillus↗

PubMed2006

Exposure of UK industrial plumbers to asbestos, Part I: Monitoring of exposure using personal passive samplers.

Epidemiological data suggest that there has been and may continue to be a significant risk to maintenance workers, who through their work may disturb asbestos-containing materials (ACM). The sampling and assessment of maintenance workers' exposure is a particular problem because they may not know that they are working with ACM. A strategy to monitor their true exposure has been developed and applied to one group of workers. The asbestos exposure of industrial plumbers was measured using personal passive samplers developed at the Health and Safety Laboratory (HSL). The light-weight samplers, which collect particles by electrostatic attraction, are simple to use and do not require prior knowledge that asbestos is to be disturbed as does conventional sampling. The samplers were issued by post and analysed, after return, using transmission electron microscopy (TEM). The strategy was found to be a reasonably efficient and cost-effective way to obtain data on maintenance worker's exposure to asbestos. The results of the TEM analysis of the passive samplers showed that the percentage of workers exposed to >5 microm long asbestos fibres was 62% in Round 1 and 58% in Round 2. For phase contrast microscopy equivalent (PCME) asbestos fibres, the values were 46 and 29%, respectively. The three samples with the highest numbers of fibres were followed up and were associated with plumbers working in areas which had supposedly been stripped of asbestos just prior to their starting work, suggesting that poor removal, clean-up and clearance practice presents a significant part of the risk to plumbers. Although flow rates will vary with conditions and time, an approximate average sampling rate from previous comparisons was used to calculate the concentration. This gave an average exposure to regulated PCME fibres of 0.009 f ml-1 for amphibole asbestos and 0.049 f ml-1 for chrysotile. The calculate risk based on the PCME fibre types collected and their estimated concentrations, showed that the risk from airborne amphibole fibres was approximately 6 times greater than from chrysotile fibres. If representative, the estimated lifetime risk of death from an asbestos related cancer for an exposure from age 20 for 40 years would be 68 per 100,000, which equates to an annual risk of death of the order of 10 per million.

Air Pollutants, Occupational↗

PubMed1985

Scanning electron microscopic studies of Paracoccidioides brasiliensis in the yeast phase.

Scanning electron microscopy of four different Paracoccidioides brasiliensis isolates in the yeast phase revealed that mother cells generating multiple, spherically shaped buds may be firmly or tenuously associated with their progeny whereas elongated buds remain attached to the mother cell through stem-like structures and may represent early stages of hypha formation. The yeast cell surfaces were covered with a delicate network of microfibrillar components.

Microscopy, Electron, Scanning↗

PubMed1993

Urinary erythrocyte morphology as a diagnostic aid in haematuria.

The value of urinary erythrocyte morphology in diagnosing glomerular and nonglomerular haematuria was studied using phase contrast microscopy in 105 patients with significant haematuria. Fifty-eight (93.6%) out of 62 patients with glomerulonephritis had dysmorphic erythrocytes and 40 (93.1%) out of the 43 patients with nonglomerular disease had isomorphic erythrocytes in the urine. A mixed picture of glomerular and nonglomerular haematuria was seen in 5 patients. The sensitivity was 93.6%, the specificity was 97.7% and the positive predictive value was 98.3% for glomerulonephritis in patients with dysmorphic haematuria. The positive predictive value for a nonglomerular source of bleeding was 96.7% with isomorphic haematuria. It is concluded that phase contrast microscopic examination of erythrocytes in urine is a simple, inexpensive and noninvasive technique that reliably distinguishes between glomerular and nonglomerular bleeding in patients.

Erythrocyte Count↗

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↗