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PubMed1990

[Basic studies on glass ceramics. 3. Influence on calcification of osteogenic cells in vitro].

Two established osteogenic cell lines (NY, MC 3 T3-E 1) were used in vitro to determine the biocompatibility of glass ceramics and their effect on initial calcification of osteogenic cells. Morphological study of the cell under the phase-contrast microscopy and histochemical staining were applied as follows. First, glass ceramic granules were placed in 60 mm dishes, and cells were suspended in the dishes in alpha-MEM supplemented with 10% FBS (basic medium) or medium with 50 micrograms/ml of L-ascorbic acid added. After 8 or 14 day of culturing, calcium formation was tested by von-Kossa's staining. Also, alkaline phosphatase staining was performed by the azo-dye method. As controls, cultures in dishes without glass ceramic granules were stained at the same time. The results obtained in the experimental culture were as follows. 1. Phase contrast microscopy showed that contacts with glass ceramics did not cause cellular death or degeneration. 2. In both cell cultures with the glass ceramics the von-Kossa reaction was positive as early as the 8th day. 3. The alkaline phosphatase reaction on the 8th day occurred only in MC 3 T3-E 1. The reaction was localized on fibroblastic cells which proliferated three-dimensionally around glass ceramics, and on small polyhedral cells situated relatively for apart from the ceramics. 4. On the 14th day, the MC 3 T 3-E 1 formed large nodules around the glass ceramics, and they were stained uniformly positive by von-Kossa's method. The alkaline phosphatase-positive cells extended spoke-like forms. 5. In medium with L-ascorbic acid, growth of NY was inhibited, After being cultured for 14 days, abundant von-Kossa positive reaction was found around glass ceramics in both cells. In MC 3 T 3-E1 on the 8th days, the alkaline phosphatase reaction was stronger with glass ceramics than with basic medium only. On the contrary, in the control cultures of both cells there was negative von-Kossa reaction during the culture period. The above results showed that glass ceramic granules have the biocompatibility needed for bone grafts, and they facilitated calcification of MC 3 T 3-E 1 in culture.

Alkaline Phosphatase↗
PubMed2002

Two experimental tests of a fluctuation-induced first-order phase transition: intensity fluctuation microscopy at the nematic-smectic-A transition.

We have developed a new, extremely sensitive real-space technique (intensity fluctuation microscopy) to probe the order of the nematic-smectic-A (NA) transition. Using this technique, we show that the NA transition in 4'-n-octyl-4-cyanobiphenyl (8CB) is clearly first order, contrary to calorimetric studies but in agreement with conclusions drawn from front-velocity measurements. We characterize the strength of the discontinuity at the first-order transition by the dimensionless quantity t(0)=(T(NA)-T*)/T(*). By precisely measuring t(0), we have made the first detailed tests of predictions based on the Halperin-Lubensky-Ma (HLM) theory of fluctuation-induced, first-order phase transitions. First, we explore the effect of an external magnetic field on the NA transition. Although modest fields (of order 10 T) are predicted to drive the weakly first-order transition in pure 8CB second order, we observe no such effect; we establish instead that the lower bound on this critical field is approximately 30 T. Likewise, we observe no effect in mixtures of 8CB with its longer chemical homolog 4'-n-decyl-4-cyanobiphenyl (10CB). Second, we examine the dependence of t(0) as a function of 8CB-10CB mixture concentration and find that the data in mixtures with small nematic temperature range are well-fit by the parameters derived by Anisimov et al. based on calorimetric measurements. As we increase the nematic range (by using concentrations closer to pure 8CB), the measured t(0) deviates more and more from the HLM predictions. Smectic fluctuations, which are neglected in the HLM calculation, are an obvious candidate to explain such a discrepancy, but one's naive expectation is that they would reduce t(0) below the HLM levels, whereas the observed values are too large. However, a recent renormalization-group calculation concludes that smectic fluctuations, surprisingly, should indeed increase t(0), explaining the observations presented here.

Journal Article↗
PubMed1963

FINE STRUCTURAL CHANGES IN THE INTESTINAL EPITHELIUM OF THE BULLFROG DURING METAMORPHOSIS.

The fine structural changes occurring in the columnar absorbing cells of the intestinal epithelium during metamorphosis of the bullfrog, Rana catesbeiana, have been examined by phase contrast and electron microscopy. Tissue samples taken just posterior to the entrance of the hepatopancreatic duct were fixed in veronal acetate-buffered osmium tetroxide and embedded in methacrylate. Under the action of the metamorphic stimulus (thyroid hormone), specific and characteristic responses were given by differentiated larval cells and undifferentiated basal cells within the same epithelium. The functional larval cells underwent degenerative changes and were retained for a time within the metamorphosing epithelium. Dense bodies appeared and increased in number in association with the loss of normal cell structure. Because of their morphology and time of formation, these bodies have been tentatively identified as lysosomes. Early in metamorphosis the basal cells did not change, but they subsequently proliferated to form a new cell layer beneath the remaining degenerating cells that lined the lumen. After the dying cells were sloughed into the gut, the new epithelium differentiated to form the adult tissue. The columnar epithelial cells of the mature animal differed in their fine structural organization from their larval precursors. Therefore, their adult configuration was molded by the action of the metamorphic stimulus.

Animals↗
PubMed1986

Experimental gingivitis in relation to susceptibility to periodontal disease. II. Phase-contrast microbiological features and some host-response observations.

In the present investigation, a number of histological and immunohistochemical characteristics of periodontal tissues as well as the phase-contrast microscopy of dental plaque were studied after experimentally-induced gingival inflammation in relation to susceptibility to periodontal disease. The study included a younger (mean age 34.1 years) and an older age group (mean age 48 years) with a reduced but healthy periodontium. Both age groups had the same amount of loss of attachment which may suggest that they had different degrees of susceptibility to periodontal disease. At the start of the experiment, each patient was instructed to abstain from oral hygiene in one quadrant of the mouth for a period of 18 days. At the end of the 18-day period, supra-gingival plaque and gingival tissue samples were taken. As determined by phase-contrast microscopy, the plaque samples of both age groups contained relatively high proportions of spirochetes. This may indicate that the patients are at risk for recurrence of periodontal breakdown. The general histopathologic picture of the gingival tissue samples of both age groups was similar to the so-called 'early lesion'. However, IgA-producing plasma cells were found in most tissue samples of both age groups. The first part of this study showed that the younger, in comparison to the older, patients developed inflammation in terms of bleeding on probing more rapidly. These clinical results cannot be explained by the host-parasite parameters investigated in the present study.

Adult↗
PubMed1987

Light-microscopical appearance and ultrastructure of Blastocystis hominis, an intestinal parasite of man.

A study on Blastocystis hominis was undertaken for the purpose of clarifying the morphology of the organism using the following techniques; Giemsa, Heidenhain Iron Hematoxylin, and DAPI stains, phase-contrast microscopy, and transmission- and scanning-electron microscopy. Microscopic examinations of the lumen fluids aspirated at the endoscopical examination revealed the habitation of B. hominis in the lower ileum and cecum of a patient. When examined light-microscopically, organisms from stool materials, cultures, and aspirated intestinal lumen contents of a patient showed morphological resemblances to each other except for variations in size. Vacuolated cells, which were spherical in shape and characteristically had a large central vacuole and a narrow rim of cytoplasm containing nuclei and some inclusions, were the only form of the organism observed in this study, although the contents of the vacuole notably varied. DAPI stains clearly revealed the nucleus and the possible mitochondrion in the narrow rim of cytoplasm. Phase-contrast microscopy of fresh material prepared with physiological saline was recommended for diagnosis. When examined electron-microscopically, the organisms were coated with a capsule that was composed of fine filamentous materials. All the organisms contained a central vacuole although the contents of it varied considerably. The cytoplasm gave the organism a signet ring appearance and contained cristate mitochondria, a great number of ribosomes, Golgi's apparatuses, cytoplasmic microtubules, and nuclei with a nucleolus. Very few of the ultrastructures are those that would be expected of a yeast. Recent occurrences of B. hominis infection in Kyoto City, Japan, during a two-year period (June 1983 to August 1985) were also reported.

Animals↗
PubMed1993

Chronic ethanol exposure potentiates NMDA excitotoxicity in cerebral cortical neurons.

The effect of acute and chronic ethanol exposure on excitotoxicity in cultured rat cerebral cortical neurons was examined. Neuronal death was quantitated by measuring the accumulation of lactate dehydrogenase (LDH) in the culture media 20 h after exposure to NMDA. Addition of NMDA (25-100 microM) to the culture dishes for 25 min in Mg(2+)-free buffer resulted in a dose-dependent increase in LDH accumulation. Phase-contrast microscopy revealed obvious signs of cellular injury as evidenced by granulation and disintegration of cell bodies and neuritic processes. Chronic exposure of neuronal cultures to ethanol (100 mM) for 96 h followed by its removal before NMDA exposure, significantly increased NMDA-stimulated LDH release by 36 and 22% in response to 25 microM and 50 microM NMDA, respectively. Neither basal LDH release nor that in response to maximal NMDA (100 microM) stimulation was altered by chronic alcohol exposure. In contrast to the effects of chronic ethanol on NMDA neurotoxicity, inclusion of ethanol (100 mM) only during the NMDA exposure period significantly reduced LDH release by approximately 50% in both control and chronically treated dishes. This reduction by acute ethanol was also observed under phase-contrast microscopy as a lack of development of granulation and a sparing of disintegration of neuritic processes. These results indicate that chronic exposure of ethanol to cerebral cortical neurons in culture can sensitize neurons to excitotoxic NMDA receptor activation.

Animals↗
PubMed1984

Scanning electron microscopy of the final phase of the life cycle of Trypanosoma cruzi in the insect vector.

Scanning electron micrographs showed that both epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi are attached by the flagellum to the epithelium of the rectal gland of Triatoma dimidiata. The flagellates tended to cover the surface of the gland and there was a marked predominance of epimastigotes with a round posterior end. Reproduction and metacyclogenesis seem to take place in situ, the latter apparently by twisting and elongation of the epimastigotes. Metatrypomastigotes remain attached for some time, probably by a weaker mechanism which easily allows them to loosen, facilitating expulsion with the urine or feces.

Animals↗
PubMed1975

Changes in myosin distribution in dedifferentiating and redifferentiating smooth muscle cells in tissue culture.

Isolated smooth muscle cells and fibroblasts from the newborn guinea-pig vas deferens were grown in culture. In the first 2 days, all cells characterized as smooth muscle by phase-contrast microscopy reacted intensely with fluoresceinated antibodies against smooth muscle myosin. The fluorescence was in the form of particles (termed here "myosin aggregates"), which were often aligned to give the cell a striated appearance. After 3-5 days, coarse fluorescent fibrils were also visible. These were termed "attachment fibrils" ("A-fibrils") since they were thought to represent myosin in microfilament bundles. Between 6 and days in culture, the smooth muscle cells began to dedifferentiate morphologically. At this time, the "myosin aggregates" became clumped and less intensely fluorescent. "A-fibrils" also decreased in fluorescence intensity. By 8 days in culture, the dedifferentiated cells had undergone intense proliferation and gave only a minimal reaction with myosin antibodies. However, when a confluent monolayer of cells formed on day 9 or 10, they immediately began to redifferentiate ultrastructurally and to regain immunofluorescence in both "myosin aggregates" and "A-fibrils". Throughout the entire culture period, cells characterized as fibroblasts by phase contrast microscopy gave only a weak reaction with fluoresceinated antibodies to myosin showing "A-fibrils" but no "myosin aggregates".

Animals↗
PubMed2002

Hepatocyte membrane water permeability measured by silicone layer filtering centrifugation.

We previously found that hepatocytes are able to control their osmotic membrane water permeability (P(f)) by regulating the number of surface aquaporin water channels. Hepatocyte P(f) has been assessed by phase-contrast microscopy and cell image analysis, an established but relatively laborious procedure. We report here an alternative method to assess hepatocyte P(f) based on a single silicone layer filtering centrifugation system. Isolated rat hepatocytes were incubated in hypotonic or isotonic buffers containing (3)H(2)O as a tracer and, then, were filtered by rapid centrifugation through a silicone layer down to a lysis layer. Osmotically driven radioactivity (i.e., (3)H(2)O) within hepatocytes was calculated as the difference between the dpm in lysis media measured under hypotonic and isotonic conditions. The P(f) calculated from the initial slope of the radioactivity-versus-time curve was 18 microm/s at 4 degrees C. Hepatocytes treated with dibutyryl cyclic AMP, to increase P(f) through the plasma membrane insertion of aquaporins, showed an increased P(f) value of 37 microm/s. The aquaporin blocker dimethyl sulfoxide selectively prevented the agonist-induced hepatocyte P(f). These data are in good agreement with the corresponding values determined by quantitative phase-contrast microscopy; thus, the method developed allows the rapid and reliable measurement of hepatocyte P(f).

Animals↗
PubMed1960

The morphology and behavior of living exoerythrocytic stages of Plasmodium gallinaceum and P. fallax and their host cells.

The morphology and behavior of living exoerythrocytic stages of Plasmodium gallinaceum and P. fallax were studied by the use of tissue cultures, phase contrast microscopy, and time-lapse cinephotomicrography. The morphology of exoerythrocytic stages of these two species was essentially that previously observed in fixed, stained material, with the following exceptions: (1) the presence of a filament on one end of the merozoite, (2) the absence of clefts in the cytoplasm of the large schizonts, and (3) the absence of a vacuole-like space around the parasite. The following behavior was observed either directly or in time-lapse sequences: (1) emergence of merozoites from mature schizonts, (2) progressive motility of free merozoites, (3) entry of merozoites, both actively and passively, into host cells, (4) nuclear division in the parasite, (5) the various stages of schizogony, including final production of merozoites, (6) massive infection of host cells, and (7) phagocytosis of merozoites and attempted phagocytosis of mature schizonts by macrophages. Exoerythrocytic stages of P. fallax differed from those of P. gallinaceum in that the merozoites of the former were (1) somewhat more curved in shape and (2) present in fewer numbers in mature schizonts. The use of tissue culture, phase contrast microscopy, and time-lapse cinephotomicrography promises to solve many of the remaining problems concerning exoerythrocytic stages of malarial parasites and their interrelationships with host cells.

Animals↗
PubMed1963

A STUDY OF GRANULES AND OTHER CHANGES IN PHASE-CONTRAST APPEARANCE PRODUCED BY CHEMOTHERAPEUTIC AGENTS IN TRYPANOSOMES.

The morphological changes produced by four series of organic trypanocidal drugs have been studied by quantitative and by qualitative methods using phase-contrast and fluorescence microscopy. Basic drugs were absorbed rapidly into the region of the kinetoplast; acidic drugs did not affect this region. Faint granules, which were present in some trypanosomes before the administration of drugs, absorbed the drugs and increased in contrast relative to the cytoplasm. Hydroxystilbamidine, quinapyramine, related compounds, and possibly also suramin produced additional granules which did not contain drug. These additional granules are similar to the granules (volutin granules) which occur in trypanosome infections (not treated with drugs) when trypanosomes are about to be cleared from the blood. Homidium did not produce additional granules.

Animals↗
PubMed2000

Cell fate decisions in a human retinal precursor cell line: basic fibroblast growth factor- and transforming growth factor-alpha-mediated differentiation.

The purpose of this study was to determine if immortalized human retinal precursor cells could serve as a model to investigate cues that modulate cell fate and differentiation. We investigated the effects of a variety of growth factors broadly but specifically tested the effects of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)a in retinal cell differentiation and commitment. To determine the role of exogenously added growth factors in a human retinal precursor cell line (KGLDMSM), established from a first-trimester retina, cells were adapted to grow in a defined medium and exposed to a variety of trophic factors (epidermal growth factor [EGF], neuron growth factor [NGF], TGFalpha, TGFbeta, acidic FGF, and bFGF). Dose-response curves were developed to arrive at optimal concentrations. The neurotrophic potential of growth factors was determined by 3H-thymidine incorporation and bromodeoxyuridine (BrdU) labeling. The identity of the emerging neuronal phenotypes were determined by phase-contrast microscopy, immunolabeling for the neuron-specific antigens neurofilament protein (NF) and neuron-specific enolases (NSE), and photoreceptor-specific antigens (Rho1D4, 7G6) using immunocytochemistry and Western blot analysis. To identify some of the early response genes (c-fos, c-myc) expressed in response to growth factors, Northern blot analysis was performed. Almost all of the factors tested increased the total number of cells with a neuronal phenotype. Potency of growth factors to generate neurons was TGFalpha > bFGF > EGF > NGF. Both TGFalpha and bFGF, alone or in combination, increased the total number of neurons. Most of the neurons generated were photoreceptors, as depicted by the polarized phenotype, expression of photoreceptor-specific antigens, and processes resembling rudimentary outer segments. The increase in photoreceptor-like neurons is possibly attributable to an increase in numbers rather than greater survival. Additionally, the majority of the photoreceptors generated labeled with BrdU and for photoreceptor-specific antigens, suggesting that an inductive effect of bFGF and TGFalpha could occur in the cell cycle or shortly thereafter. Both bFGF and TGFalpha induced the expression of the early response gene c-fos while not altering the expression of c-actin or c-myc. The emergence of a photoreceptor phenotype was confirmed by both immunocytochemistry and Western blot analysis. The immortalized retinal precursor cell line could prove valuable in determining the role of exogenously added growth factors in retinal development and differentiation. Both bFGF and TGFalpha enhance the photoreceptor phenotype in medium-density cultures under conditions of defined medium. The same was confirmed by phase-contrast microscopy, immunocytochemistry, and Western blot analysis. Furthermore, cell fate determination in cultured precursor cells could occur during the late part of the cell cycle or shortly after completion of cell division. The effects of TGFalpha and bFGF seem to be slightly additive. The cell line will be extremely valuable in studying mechanisms of cell commitment and generation of retinal cell types, which could be tested for their potential for transplantation.

Cell Differentiation↗
PubMed2005

Structure and properties of phospholipid-peptide monolayers containing monomeric SP-B(1-25) I. Phases and morphology by epifluorescence microscopy.

Epifluorescence microscopy was used to study the structure and phase behavior of phospholipid films containing a human-sequence monomeric SP-B(1-25) synthetic peptide (mSP-B(1-25)). Measurements were done directly at the air-water (A/W) interface on films in a Langmuir-Whilhelmy balance coupled to a fluorescence microscope and real-time detection system to yield an approximate optical resolution of 1 mum. Fluorescence was achieved by laser excitation of 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-PC (BODIPY-PC, concentration </=1 mol%). The presence of mSP-B(1-25) in films of 4:1 (mol/mol) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) had a substantial effect on lipid morphology and phase behavior that depended on both surface pressure and peptide concentration (10, 5, and 1 wt.%). The mSP-B(1-25) peptide tended to fluidize phospholipid monolayers based on expanded molecular areas and reduced collapse pressures. In addition, epifluorescence measurements revealed the formation of solid-phase domains apparent as three-armed counterclockwise spirals separated from regions of fluid liquid-expanded phase domains in compressed phospholipid-peptide films. The appearance of these separated solid-phase domains resembled pure L-DPPC rather than the ensemble-type solid domains found in films of DPPC/DOPG alone and were most apparent when 10 wt.% mSP-B(1-25) was present. In contrast, films containing lower, more physiological mSP-B(1-25) contents of 5 and 1 wt.% exhibited a prominent intermediate 'dendritic' phase that increased in extent as surface pressure was raised. This phase was characterized by branching structures that formed a lattice-like mesh network with fluorescence intensities between a dye-depleted solid domain and a dye-enriched liquid phase. These results indicate that mSP-B(1-25) at near-physiological levels produces morphological changes in phospholipid monolayers analogous to those observed for native SP-B(1-79).

Air↗
PubMed1967

Sequence of events during germination of putrefactive anaerobe 3679 spores.

The sequence of changes during germination of putrefactive anaerobe 3679h spores was studied under aerobic conditions in a solution containing l-alanine and sodium pyrophosphate. Evidence that specific changes occurred in two distinct regions of the spore is given by data on several criteria that were used to measure germination. During the initial stage of germination, the absorbancy decreased, dipicolinic acid was released, the spores lost their resistance to heat and toxic chemicals, and the spore periphery (cortex) darkened gradually under phase-contrast microscopy. The final stage of germination was characterized by changes in the central spore region (core), notably phase darkening of the spore center and stainability with mercurochrome, and by a slight additional absorbancy decrease.

Alanine↗
PubMed1969

Germination of single bacterial spores.

Changes in refractility and optical density occurring in individual spores of Bacillus cereus T and B. megaterium QM B1551 during germination were investigated by use of a Zeiss microscope photometer. The curves revealed that the germination process in single spores had two distinct phases; an initial rapid phase was followed by a second slower phase. Under the experimental condition employed, the first phase of germination of B. cereus spores lasted for approximately 75 +/- 15 sec, whereas the second phase lasted for 3 to 4.5 min. In B. megaterium spores, the first phase was observed to last for approximately 2 min and the second phase for more than 7 min. The duration of the second phase was dependent on conditions employed for germination. The kinetics of the first phase were strikingly similar under all conditions of physiological germination. Time-lapse phase-contrast microscopy of germinating spores also revealed the biphasic nature of germination. It was postulated that the first phase represents changes induced by an initial partial hydration of the spore and release into the medium of dipicolinic acid, whereas the second phase reflects degradation of the cortex and hydration of the core.

Bacillus cereus↗
PubMed1985

The use of FITC-conjugated monoclonal antibodies for determination of S-phase cells with fluorescence microscopy.

A method is modified to determine the DNA synthesizing cells in primary human breast tumors and cells with idiopathic thrombocytopenic purpura (ITP) with FITC-conjugated monoclonal antibody against bromodeoxyuridine (FITC-M-anti-BrdUrd) and fluorescence microscopy. The DNA synthesizing cells were also determined from a portion of the same tissues by classical tritiated thymidine labeling (3HdThd) and autoradiography. The results from bromodeoxyuridine labeling index (BrdUrd-LI) and tritiated thymidine labeling index (3HdThd-LI) obtained from the same tissues were compared. The mean BrdUrd-LI for breast tumor was 5.4 +/- 1.0% and the mean 3HdThd-LI was 5.5 +/- 1.1%. Similarly, the labeling indexes obtained from mononuclear leukocytes of healthy donors had means of 0.5 +/- 0.1% and 0.6 +/- 0.1% for BrdUrd-LI and 3HdThd-LI, respectively. The change in the proliferation rate of mononuclear leukocyte population in the samples obtained from patients with ITP could be observed by both methods. The mean BrdUrd-LI of mononuclear leukocytes for this hematological disorder was 5.4 +/- 0.8%. These results suggest that was 6.1 +/- 0.8%. These results suggest that this relatively simple technique offers an alternative method for determining the DNA synthesizing cells in a given cell population.

Antibodies, Monoclonal↗