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PubMed1979

NGF and anti-NGF: evidence against effects on fiber growth in locus coeruleus from cultures of perinatal CNS tissues.

The present study examines whether the developing noradrenergic neurons of locus coeruleus depend on endogenous nerve growth factor (NGF) for nerve fiber production and if exogenous NGF stimulates fiber growth in this nucleus, using a collagen gel tissue culture technique. Lucus coeruleus from perinatal rat brain was used in three culture experiments: (1) lucus coeruleus, parietal cerebral cortex, and the superior cervical ganglion, prepared from newborn rats and cultured in different sectors of the same dishes; (2) locus coeruleus and parietal cerebral cortex from 17-day-old rat fetuses cultured in the same manner, and (3) locus coeruleus from 17-day-old rat fetuses co-cultured with spinal, sympathetic and ciliary ganglia from 8-day chick embryos. Experiments 1 and 2 were run with and without addition of NGF and anti-NGF, experiment 3 with and without anti-NGF. Total fiber production in all cultured tissues was evaluated daily by dark field and phase contrast microscopy during 4 days. Adrenergic nerve fiber production was then studied in the same locus coeruleus and superior cervical ganglia from the rats by Falck-Hillarp fluorescence histochemistry. Locus coeruleus and cortex cerebri from fetal rats produced dense fiber halos in culture. Locus coeruleus from newborn rats produced considerably less fibers, newborn cortex only few fibers. Superior cervical ganglia from the same newborn animals produced no or almost no fibers. Addition of NGF was not able to stimulate fiber growth in locus coeruleus nor in cortex cerebri as observed both in the living cultures and by fluorescence microscopy. Likewise, addition of anti-NGF did not affect fiber production in the CNS areas. The negative results with NGF on newborn locus coeruleus and cortex cerebri were in sharp contrast to the strong, highly significant fiber growth response demonstrated by the superior cervical ganglion from the same animals cultured in the same dishes. The third experiment tested whether locus coeruleus in tissue culture contained or produced nerve growth factor or any one of the three chick embryo ganglia. No response whatsoever in these three ganglia was observed. It is concluded that the developing locus coeruleus area does not contain or produce NGF, does not depend on NGF for fiber production, and is not stimulated by exogenous NGF.

Animals↗

PubMed2006

Morphological and growth alterations in Vero cells transformed by cisplatin.

Cisplatin is an antineoplastic agent used to treat solid tumours, such as ovarian, testicular and bladder tumours. However, studies in vitro and in vivo have shown that cisplatin is mutagenic, genotoxic and tumorigenic in other tissues and organs. In this work, we examined the effect of cisplatin on Vero cells, a fibroblast-like cell line. The morphological characteristics were investigated using phase contrast microscopy, scanning electron microscopy and the actin cytoskeleton was labelled with fluorescein isothiocyanate-phalloidin. Cell proliferation was assessed based on the growth curve. Cultured Vero cells treated with cisplatin showed behavioural and morphological alterations associated with cellular transformation. The transformed cells grew in multilayers and formed cellular aggregates. The proliferation and morphological characteristics of the transformed cells were very different from those of control ones. Since transformed Vero cells showed several characteristics related to neoplastic growth, these cells could be a useful model for studying tumour cells in vitro.

Actins↗

PubMed1981

[Reactive changes in autonomic synapses during frequency electric stimulation].

Reactive modifications of synapses of isolated intratrunkal vegetative neurones induced by high frequency orthodromal electric stimulation have been examined by vital phase-contrast microscopy, electron microscopy, vital staining with methylene blue in conjunction with concurrent electrophysiological control Flocculent aggregation of the synaptoplasma and vesicles has been discovered, which is associated with higher adhesiveness of modified proteins. There have been also discovered matrix translucence and rounding of the living synaptic buds, which is accounted for by segregation of the hydration fraction of the neuroplasma. The decrease in the number of light synaptic vesicles is accompanied by the appearance of osmiophilic, myelin-like bodies, dense-core vesicles and glycogen-like granules. An attempt has been made to validate the process in terms of the colloid-chemical conception.

Action Potentials↗

PubMed1994

In vitro phagocytosis of polylactide microspheres by retinal pigment epithelial cells and intracellular drug release.

We investigated phagocytosis of biodegradable microspheres containing a drug by retinal pigment epithelial (RPE) cells and drug release within the cells to evaluate the potential usefulness of microspheres for intracellular drug delivery. The biodegradable polymers used were L-lactic acid, and DL-lactic acid with different molecular weights or the copolymers of different monomer compositions. The microspheres containing a non-bioactive fluorescent dye (rhodamine 6GX) as a model drug, were prepared by a solvent evaporation method. The in vitro release of the dye from the microspheres was examined. Phagocytosis of the microspheres by RPE cells was conducted to evaluate the extent of phagocytosis by phase-contrast microscopy and transmission electron microscopy. The RPE cells ingesting the microspheres at different stages were examined by fluorescent microscopy to estimate the intracellular release of the dye. The dye was released with time from every microsphere and the release was controlled by changing the type of polymers constituting microspheres. The microspheres containing the dye were phagocytosed by RPE cells and the dye was released intracellularly with time. The present study indicates that the drug incorporated in the microspheres was delivered into RPE cells by way of phagocytosis and released within the cells. It is concluded that this microsphere system is a promising delivery form capable of drug targeting to RPE cells.

Animals↗

PubMed2005

Expression of type IV collagen and laminin at the interface between epithelial cells and fibroblasts from human periodontal ligament.

The present study was undertaken to examine whether synthesis of type IV collagen and laminin around the epithelial rests of Malassez (ERM) requires direct contact between cells from ERM and periodontal ligament fibroblasts. Human periodontal ligament (HPDL) explants produced outgrowths containing both ERM cells and fibroblasts when cultured in a modified serum-free medium. The interface between ERM cells and fibroblasts was examined using phase-contrast microscopy (PCM) and scanning electron microscopy (SEM). Expression of type IV collagen and laminin was studied by immunohistochemistry and in situ hybridization. It was observed that ERM cells grew underneath fibroblasts or attached to them. At the interface, type IV collagen and laminin and their respective mRNAs were abundant in both ERM cells and fibroblasts, while these proteins and mRNAs showed little if any staining in cells further away from the interface. Hence, these findings indicate that synthesis of type IV collagen and laminin is induced by direct interaction between ERM cells and periodontal ligament fibroblasts.

Adolescent↗

PubMed1993

Absence of triglyceride accumulation in lipoprotein lipase-deficient human monocyte-macrophages incubated with human very low density lipoprotein.

Lipoprotein lipase, a lipolytic enzyme essential for normal hydrolysis of triglycerides in very low density lipoprotein (VLDL) and chylomicrons, is found in several cell types, including macrophages. The role of lipoprotein lipase in mediating the uptake of normal VLDL triglycerides into human cultured monocyte-derived macrophages was studied using macrophage cells from a functionally lipoprotein lipase-deficient patient and macrophages of cells from a normal subject. After incubation with VLDL, massive accumulation of phase refractile (lipid) inclusions were noted by phase contrast microscopy within the normal, but not within the lipoprotein lipase-deficient, macrophages. Chemical determinations of intracellular lipid confirmed massive triglyceride accumulation within normal macrophages, but not in lipoprotein lipase-deficient macrophages. VLDL-derived cholesterol did not accumulate in either cell. These results confirm an additional role of lipoprotein lipase, that of mediating triglyceride accumulation into macrophages from normal human VLDL. Human monocyte-macrophages genetically deficient in a functional lipoprotein lipase will be useful to determine the role of lipoprotein lipase in macrophage accumulation of lipid from other forms of triglyceride-carrying lipoproteins, including hypertriglyceridemic VLDL, beta-VLDL, and chylomicrons.

Adult↗

PubMed1985

Rhizobium attachment to clover roots.

The adhesion of rhizobia to surfaces of clover roots was examined by an indirect plate-counting assay and phase-contrast microscopy. The number of Rhizobium trifolii cells attached to clover root segments increased in approximately linear fashion during the first hour of incubation, but did not change appreciably thereafter. The addition of 30 mM-2-deoxy-D-glucose, which effectively inhibits binding of clover root lectin, did not promote the release of previously attached bacteria nor inhibit subsequent attachment to either root segments or root hairs. Rhizobia of several heterologous species attached to clover roots in numbers comparable to those of strains of R. trifolii, the homologous species. These results indicate that rhizobia have effective mechanisms of adhesion to non-host roots and that clover lectin contributed little or nothing to attachment under the conditions examined.

Cell Adhesion↗

PubMed2000

Modeling the germination kinetics of clostridium botulinum 56A spores as affected by temperature, pH, and sodium chloride.

The germination kinetics of proteolytic Clostridium botulinum 56A spores were modeled as a function of temperature (15, 22, 30 degrees C), pH (5.5, 6.0, 6.5), and sodium chloride (0.5, 2.0, 4.0%). Germination in brain heart infusion (BHI) broth was followed with phase-contrast microscopy. Data collected were used to develop the mathematical models. The germination kinetics expressed as cumulated fraction of germinated spores over time at each environmental condition were best described by an exponential distribution. Quadratic polynomial models were developed by regression analysis to describe the exponential parameter (time to 63% germination) (r2 = 0.982) and the germination extent (r2 = 0.867) as a function of temperature, pH, and sodium chloride. Validation experiments in BHI broth (pH: 5.75, 6.25; NaCl: 1.0, 3.0%; temperature: 18, 26 degrees C) confirmed that the model's predictions were within an acceptable range compared to the experimental results and were fail-safe in most cases.

Animals↗

PubMed1999

Conditional lethality of cell shape mutations of Salmonella typhimurium: rodA and mre mutants are lethal on solid but not in liquid medium.

Round-cell (rodA, mre, divD) derivatives of a conditional alaS mutant of Salmonella typhimurium were studied under conditions allowing expression of tolerance to lethal cell shape mutations (41 degrees C), and under nontolerant conditions (30 degrees C). The rodA22::Tn10d(Kan) derivative grew normally (OD650 nm) in LB-broth at 30 degrees C; however, doubling of total cell count took much longer (130 min) than at 41 degrees C (57 min). Although the cells were able to divide in LB-broth at 30 degrees C, viable count on LB-agar at 30 degrees C was 10(3)-fold lower than on LB-agar at 41 degrees C. Phase-contrast microscopy of rodA cells incubated under different conditions showed that their size increased on LB-soft agar at 30 degrees C, but they failed to divide and finally lysed. In contrast, division occurred in LB-broth at 30 degrees C and also in LB-broth and LB-soft agar at 41 degrees C. The mre-17::Tn10d(Kan) derivative acted like the rodA strain whereas the divD135::Tn10d(Kan) mutant behaved normally both at 30 degrees C and 41 degrees C. It is concluded that rodA and mre mutations delay cell division, but are lethal only on solid medium. Mutations conferring tolerance to "lethal" rodA and mre mutations improve division performance both in liquid and solid media.

Amdinocillin↗

PubMed1996

[Morphological response of urinary bacteria to ceftibuten].

UNLABELLED: The morphological response of urinary Gram-negative bacteria to ceftibuten (CETB) was investigated in four patients with urinary tract infections (one patient: acute uncomplicated cystitis, three patients: chronic complicated urinary tract infection). The daily dose of CETB was 400 mg administered orally and the durations were 3 days for the acute uncomplicated cystitis case and 5 days for the chronic complicated urinary tract infection cases. In the four patients, changes in urinary CETB concentrations, viable bacterial counts, and morphology of the bacteria were studied after the initial administration. The urinary concentrations of CETB were 7.38-60.3 micrograms/ml at one hour after the first administration. The urinary viable cell counts were 1-5 x 10(7) cells/ml before administration, but they were reduced to 1-3 x 10(3) cells/ml at one hour after the first oral administration. Morphological changes of bacteria: Under phase contrast microscopy, filamentous debris and spherical bacteria with severe body damage were observed at 30 minutes after the first administration. By transmission electron microscopy, the cell wall of the filamentous bacteria showed a number of projections with formation of vacuolar structures in the space between the cell wall and the irregularly-shaped cytoplasm. According to the Japanese UTI criteria, the efficacy rate was 100% in the 4 patients. Neither side effect nor abnormal laboratory result was observed in any patient. CONCLUSION: The morphological changes of the bacteria suggested that CETB, in vivo, bounds not only penicillin-binding protein (PBP)-3 but also PBP-1.

Ceftibuten↗

PubMed2006

A function of polar flagellum and anisotropic growth in Vibrio alginolyticus early-phase colonies.

We continuously observed growth of Vibrio alginolyticus early-phase colonies on agar plates by phase-contrast microscopy. Two mutants defective in motility on solid surfaces were used in this study: one (YM4) can swim in liquid environments using its polar flagellum, and the other (NMB198) cannot swim because it lacks any flagella. We found that isolated colonies of YM4 were generally more circular than those of NMB198. This observation suggests that YM4 cells moved slightly within a colony by the function of their polar flagella. For clustered colonies, where the distance between the colonies was short (<50 microm), the colonies of YM4 grew rapidly along the line between them, but they grew slowly in the lateral directions. Some colonies of NMB198 grew toward neighboring colonies. These observations indicate colony-to-colony interaction.

Adaptation, Physiological↗

PubMed1999

Toxicity of topical anesthetic agents to human keratocytes in vivo.

PURPOSE: To test the potential toxicity on human keratocytes of topical anesthetic agents used after photorefractive keratectomy (PRK) to reduce or eliminate pain. SETTING: Department of Ophthalmology, Doheny Eye Institute, University of Southern California, Los Angeles, California, USA. METHODS: Cultured human keratocytes were incubated with commercially available tetracaine and proparacaine at reduced concentrations of 0.001%, 0.01%, 0.1%, and 0.25%. Evaluations were performed by phase-contrast microscopy and tetrazolium salt colorimetric assay every 2 hours for 12 hours after adding 1 of the anesthetic agents to the media. RESULTS: After time of incubation and concentration were adjusted, both drugs reduced overall cell viability; however, tetracaine produced a larger decrease in cell viability than proparacaine (P = .008). For both drugs, significant differences were found among concentrations for and across time (P < .001 and P = .004, respectively). CONCLUSION: Both tetracaine and proparacaine had toxic effects on stromal keratocytes related not only to drug concentrations but also to time exposure. These findings underscore the widespread concern that anesthetic drugs may affect corneal stromal wound healing after PRK.

Anesthetics, Local↗

PubMed2002

Combined immunocytochemistry and FISH: an improved method to study engraftment of accessory bone marrow stromal cells.

In aplastic anaemia (AA), correction of bone marrow (BM) stromal function may contribute to the outcome of bone marrow transplantation (BMT). Engraftment of BM stromal cells is rarely observed, but engraftment of accessory cells (macrophages and T cells) may be important. We have improved a method of combined immunocytochemistry and FISH described by van Tol et al. (1998) to define the cellular origin and time course of engraftment of BM stromal accessory cells after sex-mismatched BMT. Long-term bone marrow cultures were trypsinized and cytospin preparations stained by immunocytochemistry using monoclonal antibodies against specific cell lineages followed by FISH for X and Y chromosomes. Low level phase contrast microscopy was used to study staining of individual cells simultaneously with fluorescence microscopy to define chromosomal pattern. In controls, the combined procedure did not affect the intensity of APAAP staining or the accuracy of sex chromosome determination. In cultures from AA patients after sex-mismatched BMT, cell lineages could be identified and donor or recipient origin determined unequivocally. This procedure enabled us to examine the origin (host/donor) of different cell lineages with high confidence, in addition to producing images of the combined staining.

Adult↗

PubMed1988

Reaction of fibroblasts to various dental casting alloys.

The cytotoxicity of a series of dental casting alloys in the as-cast and polished condition was determined with cell culture techniques involving phase contrast microscopy to examine cell morphology and the succinic dehydrogenase histochemical reaction to measure any ring of inhibition of Balb/c 3T3 cellular respiration around alloys. Crown and bridge casting alloys and a nickel- and a cobalt-base alloy were biocompatible in the polished condition, but less so in the as-cast condition. The only two exceptions were casting alloys containing 50-60 wt% Cu. Porcelain-fused-to-metal alloys were biocompatible in either the as-cast or polished condition. This direct contact method appeared satisfactory for evaluating biocompatibility of dental casting alloys, especially since these materials are in contact with gingival tissues.

Alloys↗

PubMed1977

Role of vitamin E in glutathione-induced oxidant stress: methemoglobin, lipid peroxidation, and hemolysis.

Red blood cells (RBC) from normal and vitamin E-deficient rats were incubated in a hypertonic solution of reduced glutathione adjusted to pH 8. Methemoglobin formation occurred in intact RBC from both normal and vitamin E-deficient rats. Hemolysis was significantly greater in RBC from vitamin E-deficient rats. Experiments with catalase, superoxide dismutase, and methional showed that H(2)O(2) was the primary extracellular source of oxidant stress. Extracellular superoxide and hydroxyl radical were not involved in oxidant stress. Experiments with dimethyl sulfoxide showed that intracellular hydroxyl radical, generated from H(2)O(2), was the hemolytic agent. Neither methemoglobin formation nor lipid peroxidation involved hydroxyl radical. Indeed, lipid peroxidation and hemolysis in RBC from vitamin E-deficient rats were concurrent rather than consecutive events. Phase contrast microscopy showed that rigid, crenated RBC with a precipitate around the interior periphery formed during glutathione-induced oxidant stress. The precipitate dissolved slowly as the crenated RBC were converted to smooth ghosts. It appeared that protein precipitates involving mixed disulfide bonds were reduced and solubilized when extracellular glutathione penetrated the ruptured cell. Comparisons between normal RBC and vitamin E-deficient RBC suggest that vitamin E has little effect on the inward diffusion of extra-cellular H(2)O(2). Vitamin E apparently interacts with different oxidant species derived from intracellular H(2)O(2) in preventing lipid peroxidation and the sulfhydryl group oxidation leading to hemolysis.

Animals↗

PubMed1996

Influence of particle size of hydroxyapatite as a capping agent on cell proliferation of cultured fibroblasts.

The influence of particle size of biomaterials on cell reaction and cell proliferation was studied by means of coculturing fibroblasts with calcium phosphate ceramics. Both dense and porous hydroxyapatite ceramics obtained under different sintering conditions were selected, and two particle sizes of 300 and 40 micrometers were used to examine cell reaction. The four materials were each cocultured with dental pulp-derived fibroblasts for 7 days. Cell reaction was observed by phase-contrast microscopy and scanning electron microscopy. Cell proliferation was measured by counting the number of trypsinized cells in 7-day-old culture. On the seventh day, the dense 300-micrometer particles of hydroxyapatite were completely covered by cultured cells that had proliferated on the dish surface. On the other hand, the porous and dense 40-micrometer particles were captured or gathered by the cultured cells, which seemed not to proliferate. The porous 300-micrometer particles were accompanied by numerous small broken pieces on the dish surface, and the cells proliferated only around the large particles. From these results, the dense 300-micrometer particles of hydroxyapatite can be considered the most appropriate biomaterial.

Biocompatible Materials↗

PubMed1982

[Mechanism of a universal supravital reaction of varicose changes of neurites].

By means of vital phase-contrast microscopy in neurites, proteolitically isolated from the neural system in representatives of 6 types of invertebrates inhabiting the White Sea and various Chordata, a comparative histological investigation has been performed to study certain reactions of varicose deformities. Solutions of the medium with a decreased ionic strength, prolonged survival etc. have been used as irritants producing varicosities. This reaction, considered to be universal, is absent in thick and gigantic fibres. It develops, first of all, in fine preterminal neurites of any localization and does not depend on the level of the animal's organization and its neural system. In the mechanism on varicosity formation, appearance of the hydrated peripheral fraction of neuroplasm is essentially important. This process is more general and is observed in all neurites.

Animals↗

PubMed1982

Reversible differential decondensation of unfixed Chinese hamster chromosomes induced by change in calcium ion concentration of the medium.

Differential decondensation of isolated unfixed Chinese hamster metaphase chromosomes was obtained by decreasing the calcium ion concentration in the surrounding medium. A banded appearance of the swollen chromosomes could be observed either directly by phase contrast microscopy or after glutaraldehyde fixation and staining. There was a gradual transition from homogeneously dense to banded and finally to extensively decondensed chromosomes. The patterns induced at different stages were similar to those observed on fixed chromosomes after standard banding procedures (i.e., G-, Cd-, Ag-NOR-staining). Chromosomes decondensation could be reversed by the addition of calcium ions to the medium. Ca ++-dependent reversible differential chromosome decondensation was not observed if the chromosomes were previously treated with 0.35 M NaCl. Chromosome regions which had incorporated BrdU into their DNA were more resistant to a decrease in calcium ion concentration than BrdU non-substituted regions.

Animals↗