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PubMed1988

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture.

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.

Animals↗

PubMed2002

Platelet spherocytosis: a new bleeding disorder.

The present study evaluated a child with thrombocytopenia and a rare congenital bleeding disorder associated with platelet spherocytosis. Differential interference phase contrast microscopy (DIC) revealed that his platelets were spherical in form. Examination of thin sections in the electron microscope showed that his platelets were nearly devoid of microtubules (MT) and microtubule coils (MTC) that support the discoid shape in 100% of normal platelets. Immunofluorescence with a monoclonal antibody to tubulin, the precursor protein of MT, revealed bright rings in normal cells and diffuse fluorescence of patient platelets. The brightness of the fluorescence emitted by patient platelets was comparable to the diffuse fluorescence of normal platelets after chilling to dissolve intact MT, suggesting normal and patient cells contained comparable amounts of tubulin. Exposure of patient platelets to Taxol, an agent that stabilizes and induces MT, caused MT formation in 82% of patient platelets and MTC development in 11%, resulting in their conversion to discs. Glycoproteins GPIIb/IIIa and GPIb were present on patient platelets, and the cells contained normal numbers of dense bodies, ruling out storage pool disease. The patient's platelets adhered to and spread normally on glass but failed to undergo rapid, irreversible aggregation when stirred with agents that produced a complete response in normal discoid platelets, even when the patient's platelets were concentrated. The poor response of spherical platelets was associated with failure to become irregular and extend long filopodia. Thus, the spherical shape of patient platelets may contribute to the thrombocytopenia and to the clinical bleeding symptoms.

Adolescent↗

PubMed1986

Non-neuronal cell proliferation in tissue culture: implications for axonal regeneration in the central nervous system.

A tissue culture model has been developed to examine the hypothesis that proliferating non-neuronal cells may constitute a physical and/or chemical barrier to regenerating neurons in the central nervous system. Explants from the sensorimotor cortex of 20-day-old fetal rats were cultured in serum medium (control) or serum medium containing 10(-5) M cytosine arabinoside (AraC), a mitotic inhibitor, for varying periods: 2-10, 4-12, 4-10, 4-8 and 4-7 days in vitro (DIV). The center and outgrowth zone of the explants were examined by phase-contrast microscopy at varying intervals between 3 and 18 DIV. The extent of central degeneration was greatest in explants treated with AraC from 2 DIV, and was least in the 4-7 day treated group in which only minimal degeneration was evident at 13 and 18 DIV. In the outgrowth zone at 18 DIV non-neuronal cell proliferation was controlled in the 4-10 day treated explants, although this was accompanied by extensive degeneration of neurites. Further examination of neurite viability, using a neurite viability ratio, revealed that degeneration was first evident at 6 DIV in the 2-10 day treated explants, but not until 9 or 13 DIV in any of the explants exposed to AraC from 4 days onwards. There was minimal degeneration in the 4-7 day treated explants. Electron microscopic examination revealed the presence of atypical inclusions in non-neuronal cells of 4-8 day treated explants, suggesting that the cytotoxic effect of AraC may be due to a disturbance in lipid and/or ganglioside metabolism. Quantitative electron microscopic analysis of the outgrowth zone at 18 DIV revealed a significant increase in the summated area of neuronal tissue (from 7 to 18 microns2/100 microns2) and a decline in the summated area of non-neuronal cells (from 83 to 61 microns2/100 microns2) for explants treated with AraC from 4 to 7 DIV compared to control. Diminishing the potential of non-neuronal cells to act as a barrier by controlling their proliferation may, therefore, be of importance in enhancing the regenerative response of central neurons.

Animals↗

PubMed1996

Intraoral transmission and the colonization of oral hard surfaces.

The sterile abutments of 2-stage implants offer a unique model to study intraoral transmission and bacterial colonization patterns in the oral cavity. This study aimed to examine, by means of differential phase contrast microscopy, parameters that influence the intra-oral colonization of these abutments. In part one, 159 partially edentulous patients were examined to determine the influence on the microbial composition around implants of the following parameters: 1) the relative location of the teeth in relation to the implants; 2) the microbial composition of the subgingival plaque around these teeth; and 3) the frequency of deep pockets around the natural dentition. The results indicate that the subgingival flora around the implants harbored more spirochetes and motile rods when teeth were present in the same jaw (P < 0.05) and/or when the pockets around them harbored a pathogenic flora (P < 0.05). In part two, the impact of periodontitis around the remaining teeth and of probing depth around the implants on the composition of the peri-implant subgingival flora was investigated. Thirty-one partially edentulous implant patients were classified according to their periodontal condition into 3 groups: healthy (n = 17), chronic periodontitis (n = 11), and refractory periodontitis (n = 3). The samples from deep pockets (> or = 4 mm) around implants showed significant increases in the total proportion of spirochetes and motile organisms when compared to samples from healthy subjects (1.2%) or in chronic periodontitis patients (21.0%), or to patients suffering from refractory periodontitis (31.5%). For shallow pockets (< 4 mm) significant differences were only detected between subjects with a healthy periodontium (1.0%) or chronic periodontitis (2.4%), and refractory periodontitis cases (19.7%). The present findings confirm the transmission of microorganisms from teeth to implants, and thereby highlight the importance of periodontal health around the natural dentition before as well as after implant installation.

Adult↗

PubMed1972

Growth, cell division, and fragmentation in a species of Flexibacter.

Flexibacter FS-1, a gram-negative gliding bacterium was grown in liquid culture as long (over 100-mum) filaments. The filaments possessed a triple-track wall which resembled that found in other gram-negative bacteria. Although phase-contrast microscopy indicated that the long filaments were nonseptate, electron microscopy revealed three or four septa along the length of each filament. The septa contained lysozyme-sensitive, electron-opaque material, presumed to be peptidoglycan, sandwiched between cell membranes. The outer triple track wall was not part of the septum. Mesosomes were seen in various areas of the cell and frequently were observed attached to septa in different stages of completion. Studies of the organism in slide culture revealed that individual filaments grew in an exponential fashion and divided in the middle despite the long length and multiseptate condition. When the temperature of a liquid culture growing exponentially with a generation time of 90 minutes was shifted from 30 to 35 C, the filaments fragmented into three or four shorter cells within 120 min. The short cells continued to grow exponentially at 35 C at approximately the same rate as at 30 C. When the culture was shifted back to 30 C, the cells immediately stopped dividing and began to elongate. After a period of 2 to 3 hr, cell division resumed. It is suggested that the shift-up in temperature induced the completion of the cross wall (centripetal growth of the triple-track wall) and cell separation at the sites of previously formed septa, whereas the shift-down in temperature caused a transient inhibition of cross-wall formation but not of growth. Fragmentation was inhibited by sodium azide but took place despite the inhibition of protein synthesis by chloramphenicol or the inhibition of deoxyribonucleic acid synthesis by mitomycin C.

Azides↗

PubMed1992

[Concentration and form of asbestos fibers in tap drinking water contaminated from a water supply pipe with asbestos-cement].

The identification and concentration of asbestos fibers in tap drinking water supplied in a central area of Akita Prefecture, Japan, were determined by phase-contrast microscopy and a scanning electron microscope equipped with an energy-dispersive X-ray microanalyzer. The following results were obtained. 1. Asbestos fibers were found in the tap water from two areas in which an asbestos-cement pipe was used for public water supply. The concentrations of asbestos fibers in the tap water were 2.7 x 10(4) to 27.0 x 10(4) fibers per liter of water in area A and 10.0 x 10(4) to 21.0 x 10(4) in area B. On the other hand, no asbestos fiber contamination was observed in tap water of area C, which shared a common water source with area A. A vinyl chloride pipe was used over the entire length of the water supply in route C. 2. Crocidolite was the predominant type of asbestos fiber detected in the tap water. Chrysotile and a mixture of chrysotile and amosite were also observed. 3. Almost all asbestos fibers detected in the tap water possessed the form of thick or sheaved fibers with lengths ranging from ca. 5 to 10 microns. Their shapes were very different from those of asbestos fibers found in the atmosphere. The typical form of the latter is short (ca. 1 micron in length) and needle-like. 4. It was suggested that the contamination of asbestos fibers in the tap water was caused by erosion and peeling off of the inner wall of the asbestos-cement pipe used as a conduit. In order to evaluate the safety of drinking water in Japan, an extensive survey on asbestos-fiber contamination in tap water is necessary.

Asbestos↗

PubMed1988

Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. II. Serum promotes dendritic growth.

In the preceding paper, we reported that embryonic rat sympathetic neurons formed axons, but not dendrites, when they were maintained in the absence of serum and nonneuronal cells. To assess the effects of serum-derived factors on cellular morphology, cultures were initially maintained in serum-free medium while nonneuronal cells were eliminated. Subsequently some cultures were chronically exposed either to fetal calf serum (10%) or to a high-molecular-weight ammonium sulfate fraction of serum (P40 material, 500 micrograms/ml). Phase-contrast microscopy revealed that serum and P40 material did not alter neuronal survival, but did cause flattening of the somata and fasciculation of processes. When neurons exposed to serum or P40 material were injected with Lucifer Yellow, it was found that the majority (greater than or equal to 90%) had local, tapered processes that could be identified as dendrites by light microscopic criteria. These local processes also exhibited other dendritic characteristics in that (1) they reacted with monoclonal antibodies to nonphosphorylated forms of the M and H neurofilament subunits and to microtubule-associated protein 2; and (2) they had substantial amounts of RNA as determined by [3H]uridine autoradiography. Quantitative measurements of the effects of serum and P40 material on dendritic morphology revealed that (1) an 8-day exposure caused most neurons (greater than 80%) to form dendrites; (2) neurons typically had more than one dendrite (mean of 4.1 +/- 0.2 dendrites/cell after a 28-day exposure); and (3) the dendrites were relatively short with the maximum extent of the dendritic arbor being 110 +/- 13 micron after 4 weeks. Serum and P40 material did not routinely cause the formation of supernumerary axons, did not alter radial axonal outgrowth from ganglion explants, and did not significantly increase [3H]leucine incorporation. Thus, serum contains a factor (or factors) which selectively stimulates the extension of dendrites, but not axons. If such a factor were operative in situ, it could play an important role in determining the morphology of sympathetic neurons. In examining the mechanism of serum-induced dendritic growth, we found that even high concentrations (5 micrograms/ml) of nerve growth factor failed to promote dendritic growth in the absence of serum; thus, nerve growth factor by itself is not a sufficient condition for the extension of dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1999

Characterization of natural Epstein-Barr virus infection and replication in smooth muscle cells from a leiomyosarcoma.

Cells from a leiomyosarcoma tumor (LMS-1) from a patient with the acquired immunodeficiency syndrome (AIDS) were explanted, cultured in vitro, and studied by phase-contrast microscopy for morphologic and growth characteristics, immunostaining for cell markers, EBER in situ hybridization and polymerase chain reaction for detection of Epstein-Barr virus (EBV), and immunostaining for expression of EBV antigens. The cells exhibited very slow growth in vitro, with unusual elliptical and spindle-shaped morphology and fragmentation of the cytoplasm into long, tapering, cytoplasmic processes. Greater than 90% of cells expressed diffuse distribution of the smooth muscle isoform of actin by immunoperoxidase staining. Approximately 25% of cells expressed very bright fluorescence by immunostaining of the smooth muscle isoforms of calponin and actin. The majority of cells demonstrated a weak signal for CD21; approximately 5-10% of cells showed a strong signal that was confined to cell surfaces. The cultured cells harbored EBV, and infectious EBV continued to be detected by polymerase chain reaction and virus culture through several passages in vitro. Several EBV antigens were expressed, including latent antigen EBNA-1, immediate-early antigen BZLF1, early antigen EA-D, and late antigens, including viral capsid antigen p160, gp125, and membrane antigen gp350. Human umbilical cord lymphocytes that were transformed with virus isolated from cultured cells yielded immortalized cell lines that expressed EBV antigens similar to other EBV-transformed lymphocyte cell lines. These results confirm that EBV is capable of lytic infection of smooth muscle cells with expression of a repertoire of latent and replicative viral products and production of infectious virus. EBV infection of smooth muscle cells may contribute to the oncogenesis of leiomyosarcomas.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Partial restoration of the keratocyte phenotype to bovine keratocytes made fibroblastic by serum.

PURPOSE: To determine whether keratocytes made fibroblastic in vitro by addition of fetal bovine serum to the medium regain the keratocyte phenotype after culture in serum-free medium. METHODS: Collagenase-isolated keratocytes from bovine corneas were plated in DMEM/F-12 containing 1% horse plasma, to allow cell attachment, and then cultured until day 4 in either DMEM/F-12 alone, to retain the keratocyte phenotype, or in DMEM containing 10% fetal bovine serum, to cause the keratocytes to become fibroblastic. Medium for the fibroblastic cells was replaced on day 4 with serum-free medium, and cells were cultured until day 12. Cell phenotypes were determined on days 4 to 5 and 11 to 12 of culture as follows: (1) by the morphologic appearance on phase-contrast microscopy; (2) by the levels of aldehyde dehydrogenase in the cells, determined by SDS-PAGE and Coomassie blue staining; (3) by the relative synthesis of collagen types I and V, determined by (14)C-proline radiolabeling; (4) by pepsin digestion and analysis of collagen types by SDS-PAGE autoradiography; (5) by relative synthesis of cornea-specific proteoglycan core proteins determined by analysis of chondroitinase- or endo-beta-galactosidase-generated radiolabeled core proteins by SDS-PAGE autoradiography; and (6) by the relative synthesis of keratan sulfate and chondroitin sulfate determined by (35)SO(4) radiolabeling and measuring the sensitivity to endo-beta-galactosidase and chondroitinase ABC. RESULTS: Keratocytes cultured in serum-free medium appeared dendritic and became fibroblastic in appearance when exposed to medium containing serum. Keratocytes and fibroblasts synthesized a similar proportion of collagen types I and V. However, compared with the keratocytes, the fibroblasts possessed no aldehyde dehydrogenase and synthesized significantly higher levels of decorin and significantly lower levels of prostaglandin D synthase (PGDS) and keratan sulfate. Subsequent culture of the fibroblasts in serum-free medium did not restore aldehyde dehydrogenase to keratocyte levels but did restore the cell morphology to a more dendritic appearance and returned the synthesis of decorin, PGDS, and keratan sulfate to keratocyte levels. CONCLUSIONS: The results of these studies indicate that primary cultures of keratocytes made fibroblastic by exposure to serum can return to their keratocyte phenotype in synthesizing extracellular matrix. These results also indicate that the differences in the organization of the collagenous matrix produced by keratocytes and fibroblasts may be related more to the different proteoglycan types than to the collagen types produced.

Aldehyde Dehydrogenase↗

PubMed1977

The trachea and cuff-induced tracheal injury. An experimental study on causative factors and prevention.

The effect of a large tracheal tube cuff on the rabbit tracheal mucosa was investigated by phase contrast microscopy and scanning (SEM) and transmission (TEM) electron microscopy. The tube was left in the trachea for 15 min. The cuff was either uninflated or inflated to a cuff-to-tracheal wall pressure (C-T pressure) of up to 100 mmHg. The uninflated cuff caused superficial damage to the epithelial lamina over regions where a cartilage was situated. When the cuff was inflated, it resulted in an increase of the mucosal damage, the extent of which was directly related to the pressure in the cuff. This took the form of both widening of the injured areas and penetration of the damage to deeper regions. At a C-T pressure of 100 mmHg the damage involved almost the entire mucosa and only small unaffected mucosal regions remained. At this stage it appeared as if the basement membrane had also begun to disintegrate. It is well known that a small cuff easily causes deep ulceration in the mucosa overlying the cartilages. From this investigation it was concluded that a large cuff causes the same type of ulceration if 1) the cuff wall is not sufficiently thin and pliable, and 2) if the cuff is overinflated enough to dilate the trachea to a diameter exceeding the cuff-diameter. At that moment there will be circumferential tension in the cuff and the sealing physics of the large cuff will become the sealing physics of a small (high pressure) cuff. A large cuff, properly handled, is more benign to the trachea than a small cuff. In order to avoid overinflation of the large cuff, the intracuff pressure (= C-T pressure) should always be measured by means of a four-way stopcock and an aneroid manometer. In the case of extended periods of mechanical ventilation with a high airway pressure, the resulting tracheal diameter at the cuff site should be checked radiographically.

Animals↗

PubMed1999

Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo.

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (</= 8-cell stage) collected 12-48 h after IVF were separately fixed, stained by orcein, and examined under phase contrast microscopy. It was found that 27% of 2-cell, 74% of 3-cell, 51% of 4-cell, and 74% of 5- to 8-cell-stage embryos were abnormal in morphology. Morphological anomalies included fragmentation (no nucleus in one or more than one blastomere) and/or binucleation (two nuclei in one or more than one blastomere). In experiment 2, actin filament distribution of the embryos at 2-cell to blastocyst stages that were produced in vivo and in vitro were stained by rhodamine-phalloidin and examined by confocal microscopy. Actin filaments were distributed in all in vivo-derived embryos at the cell cortex, and at the joints of cells and perinucleus in 2- to 8-cell-stage embryos and in some cells of morulae and blastocysts. Actin filaments were also distributed in the cortex and at the joints of cells of all in vitro-produced embryos. However, only 20% of in vitro-produced embryos at 2- to 8-cell stages had perinuclear actin filaments in all blastomeres. Most in vitro-produced embryos had fewer perinuclear actin filaments or did not have perinuclear actin filaments in some blastomeres. Fragmentation and binucleate blastomeres were not observed in in vivo-derived embryos. In vivo-derived Day 5 (136.5 +/- 60.4 nuclei per blastocyst) and Day 6 (164.5 +/- 51.9 nuclei per blastocyst) blastocysts had significantly (p < 0.001) more cells than in vitro-produced Day 6 blastocysts (37. 3 +/- 11.7 nuclei per blastocyst). In experiment 3, when cytochalasin D, an inhibitor of microfilament polymerization, was included in the culture medium, it prevented 2- to 4-cell-stage embryos from developing to the blastocyst stage. These results indicate that abnormal actin filament distribution is one possible reason for abnormal embryo cleavage and small cell numbers in pig embryos produced in vitro. Culture conditions that mediate normal actin filament distribution may result in an improvement in embryo quality.

Actins↗

PubMed1995

Prostaglandin E2 alters human orbital fibroblast shape through a mechanism involving the generation of cyclic adenosine monophosphate.

Orbital fibroblasts from patients with Graves' ophthalmopathy, when treated with prostaglandin E2 (PGE2), become stellate and develop prominent cellular processes. In this paper, we describe results of studies designed to characterize the action of PGE2 on orbital fibroblast shape changes in vitro. Orbital and dermal fibroblasts were incubated with PGE2, one of several prostanoid analogues, 8-br-cAMP or forskolin and were then visualized by phase-contrast microscopy. Other studies involved seeding cells in special chambers equipped with electrodes for cell sensing using electric cell-substrate impedance sensing (ECIS) to detect changes in shape. PGE2 (10(-7) mol/L) elicited a rapid and dramatic alteration in the shape of orbital fibroblasts but not those derived from the skin. Cells became stellate and developed prominent cytoplasmic processes that extended out from the central area containing the cell nucleus. The effects were stereoselective in that a number of structurally related compounds, including Sulprostone, PGI2, PGF2 alpha, thromboxane A2, thromboxane B2, and 11 deoxy,16,16 dimethyl PGE2 failed to elicit a similar shape change. Butaprost (10(-5) mol/L), a specific EP2 agonist, elicited a similar shape-change as that observed with PGE2. 16,16-dimethyl PGE2, a nonselective agonist, could mimic the action of PGE2. The effect of PGE2 was apparent at 10(-8) mol/L, maximal at a concentration of 10(-7) mol/L and took 4-8 hr to evolve completely. Cycloheximide (10 micrograms/mL) and actinomycin D (1 micrograms/mL) failed to block the shape change. The morphologic change could be reproduced by addition of 8-br-cAMP (3 mmol/L) and by forskolin (5 mumol/L). Moreover, PGE2 and Butaprost treatment elicited in orbital cultures a massive increase in endogenous cAMP production while analogues not affecting cell shape failed to influence cyclic nucleotide generation. Three strains of orbital fibroblasts from patients with Graves' ophthalmopathy and three from normal orbits were tested and all responded to PGE2 (10(-7) mol/L). Four strains of dermal fibroblasts failed to respond to PGE2. The changes in orbital fibroblast morphology were accompanied by a marked decrease in monolayer impedance as assessed by electric cell-substrate impedance sensing. The earliest effects were apparent within 30 min using this sensitive technique. The widely recognized roles of PGE2 and related compounds in the mediation of the inflammatory response make our current findings in orbital fibroblasts of potential importance to the pathogenesis of Graves' ophthalmopathy.

Cells, Cultured↗

PubMed1999

A new automated system for urine analysis: a simple, cost-effective and reliable method for distinguishing between glomerular and nonglomerular sources of haematuria.

OBJECTIVE: To determine the ability, reliability and accuracy of a new automated system of urine analysis in differentiating glomerular from nonglomerular bleeding in the initial investigation of haematuria, and compare its efficacy with conventional phase-contrast microscopy (PCM). PATIENTS AND METHODS: One hundred and six urine samples from patients in whom the final diagnosis was available were analysed using electrical flow impedance to detect, enumerate and size red blood cells in a conductive fluid (the cellfacts analyser, Microbial Systems Ltd, Coventry, UK). All the samples were also tested using a dipstick method and PCM was carried out for comparison on 45 of the 106 urine specimens. The results of cellfacts analysis were correlated with the final diagnoses to assess sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of this method; the costs were also analysed. RESULTS: Sixty-nine urine samples tested positive for blood on dipstick urine analysis and all these were confirmed to have red blood cells on cellfacts analysis. The remaining 37 samples were negative for blood on dipstick testing and cellfacts analysis, although seven patients had been referred with previously detected microscopic haematuria, none of whom were found to have any detectable pathology in the urinary tract on clinical examination and investigations. The remaining 30 patients were diagnosed to have urological or nephrological conditions with no haematuria. In the positive group, 20 (29%) patients were from the glomerular group, with a mean (range) red blood cell size of 4.25 (4-5.1) micrometer, and 49 (71%) from the nonglomerular group, with red blood cells of 5.47 (4.67-5.70) micrometer. These ranges overlapped at 4.67-5.1 micrometer at the decision threshold of 4.75 micrometer, the distribution of dysmorphic and eumorphic red blood cells for the glomerular group was 18 (90%) and two (10%), respectively, and for the nonglomerular group was 2 (4%) and 47 (96%), respectively. The sensitivity, specificity, PPV and NPV were 90%, 96%, 90% and 96%, respectively. Consumable and labour costs were very low. CONCLUSIONS: Cellfacts analysis is a simple, rapid, objective and cost-effective method for differentiating glomerular from nonglomerular urinary red blood cells, especially when few such cells are present.

Adult↗

PubMed2002

Large expansion of morphologically heterogeneous mammary epithelial cells, including the luminal phenotype, from human breast tumours.

Regular expansion of heterogeneous populations of epithelial cells, including the luminal phenotype, was achieved from small biopsies of human breast tumours and cutaneous metastases by optimized feeder layer technique based on irradiated NIH 3T3 cells. Forty-one out of 47 primary tumour specimens and all three cutaneous metastases grew successfully for two to 10 passages in vitro. The main phenotypes of cultured cells and their changes in subcultures were characterized using immunocytochemistry and phase contrast microscopy (in few cases also time-lapse recording). In the majority of cultured cell populations a fraction of cells positive for keratin 19 (K19+), typical for the luminal phenotype, was detected. This is the cell type from which breast carcinoma is supposed to arise. While in cultures derived from benign lesions only basic phenotypes of luminal and myoepithelial cells were found, in cultures derived from malignant tumours unusual phenotypes of epithelial cells, in their majority K19+, were detected. The growth properties of cells from six benign and seven malignant samples were analyzed in detail. In the analyzed cell populations the culture lifetime - related to the number of colony-forming cells varied for cells from malignant tumours between 21 and 51 and from benign tumours between 22 and 40 cell generations. The total number of passages achieved was three to seven for malignant or four to nine for benign cultures. In spite of negative results of tumourigenicity testing in immunologically compromised Nu/nu mice the potential to culture apparently neoplastic cells was indicated by positive immunostaining for the p53 oncoprotein (seven of 23 tested malignant cases), the src oncoprotein (five of eight), and overexpression of the c-erbB-2 protein (five of 26). This was further confirmed by successful cultivation of malignant cells from cutaneous metastases. Two of the three metastasis-derived cultures were nearly homogeneously positive for K19 while the third was almost negative. The results proved the optimized feeder layer technique to be useful for regular yielding of large amounts of epithelial cells from small tumour biopsies and for supporting the majority of cell phenotypes present in the original tumour. Therefore, it appeared to be a promising tool for further analysis of interactions between luminal and myoepithelial cells in the development of human breast carcinoma and for the study of individual tumours.

3T3 Cells↗

PubMed2000

Functional characterization of purified zinc transporter from renal brush border membrane of rat.

Major zinc binding protein purified from renal brush border membrane (BBM) (R. Kumar, R. Prasad, Biochim. Biophys. Acta 1419 (1999) 23) was reconstituted into liposomes and its functional characteristics were investigated. Physical incorporation of the major zinc binding protein into the proteoliposomes was checked by SDS-PAGE, which showed a single band on silver staining. The structural integrity of the proteoliposomes was assessed by phase contrast microscopy, which revealed the proteoliposomes as globular structures and intact boundaries. Further structural integrity/leakiness of the proteoliposomes was checked by monitoring efflux of Zn(2+) from the pre-loaded proteoliposomes in the presence of either 2 mM Ca(2+) or Cd(2+) or Zn(2+). It was observed that even after 2 h of the initiation of efflux, 85-95% of Zn(2+) was retained in the proteoliposomes, thereby indicating that proteoliposomes were not leaky and maintained structural integrity during the uptake study. Zinc uptake into the proteoliposomes followed Michaelis-Menten kinetics with affinity constant (K(m)) of 1.03 mM and maximal velocity (V(max)) of 1333 nmol/mg protein per min. The uptake process followed first-order kinetics with a rate constant (k) of 1. 09x10(-3) s(-1). The specificity of zinc transport system was determined by studying the interaction of divalent cations viz. Ca(2+) and Cd(2+) with the zinc uptake. It was observed that Cd(2+) competitively inhibited the zinc uptake process with inhibitory concentration (K(i)) of 2.9 mM. Kinetic analysis of inhibitory effect of Cd(2+) on zinc uptake revealed an increase in K(m) to 1.74 mM without influencing V(max). Zn(2+) uptake into the proteoliposomes was found to be temperature sensitive and Arrhenius plot showed a breakpoint at 27 degrees C. The apparent energies of activation (E(a)) were found to be 7.09 and 2.74 kcal/mol below and above the breakpoint, respectively. The initial velocity of Zn(2+) uptake increased with the increase in outwardly directed proton gradient ([H](i) greater than [H](o)). The Zn(2+) uptake was inhibited by DCCD, thereby suggesting the involvement of -COOH groups in the translocation of Zn(2+) across the lipid bilayer. The ratio of acidic to basic amino acids (1.26) strongly indicates that it is an acidic protein. The cysteine content in this protein was insignificant, which further corroborates the possibility that the acidic amino acids might be prominent candidates for binding to zinc. The findings of the present study confirms that 40 kDa major zinc binding glycoprotein purified from renal BBM is a zinc transporter involved in the influx of Zn(2+) into the epithelial cells of the renal tubular system.

Amino Acids↗

PubMed2003

FGF-2 induced reorganization and disruption of actin cytoskeleton through PI 3-kinase, Rho, and Cdc42 in corneal endothelial cells.

PURPOSE: Corneal endothelial cells (CECs) undergo endothelial to mesenchymal transformation (EMT) in response to FGF-2 stimulation. One phenotypic change that occurs during EMT is a change in cell shape from polygonal to elongated fibroblast-like cells. We investigated whether FGF-2 plays a role in this morphogenetic pathway by reorganizing actin cytoskeleton through the actions of phosphatidylinositol (PI) 3-kinase and the Ras related Rho family of small guanosine triphosphatases (GTPases). METHODS: Cell morphology was analyzed using phase contrast microscopy, and the organization of actin cytoskeleton and focal adhesions were analyzed by immunofluorescent staining. Expression of vinculin and beta-actin was determined by immunoblot analysis. Pharmacologic inhibitors (LY294002, C3 exoenzyme, Y27632, or PD98059) or neutralizing antibody to FGF-2, respectively, were used to block PI 3-kinase, Rho, Rho associated kinase, extracellular signal regulated kinase, or FGF-2 pathways. RESULTS: CECs treated with FGF-2 became smaller and lost their characteristic polygonal cell morphology. Such cell shape change was completely blocked by treatment with LY294002. CECs in culture have abundant stress fibers that are oriented radially across the cell. However, FGF-2 caused a loss of these stress fibers and focal adhesions. The modulated cells contained a cortical actin ring while LY294002 completely abolished this action of FGF-2 on actin cytoskeleton. Treatment of cells with C3 exoenzyme or Y27632 in the presence of FGF-2 induced spindle shaped cells with prominent pseudopodia which were rapidly formed upon exposure to the inhibitor. The expression level of vinculin was found to be similar in all experimental conditions but vinculin was mostly translocated to the cytoplasm in response to FGF-2 stimulation. CECs plated on Matrigel matrix demonstrated findings similar to those from cells plated on the conventional culture dishes, except that Matrigel facilitated the formation of pseudopodia. We further investigated in vivo actin organization using organ cultures of corneal endothelium (CE) on Descemet's membrane. The contact inhibited endothelial monolayer demonstrated a circumferential actin ring, and no stress fibers were observed. When CE was treated with FGF-2, a half population of CE lost its characteristic contact inhibited cobblestone morphology. Actin cortex was greatly disrupted in these modulated cells. Both neutralizing antibody to FGF-2 and LY294002 completely impeded the modulating activity of FGF-2 on the endothelial monolayer. When CE was simultaneously treated with FGF-2 and Y27632, the circumferential actin cortex was greatly disrupted and the endothelial monolayer was transformed into multi-layers of fibroblastic cells containing pseudopodia. Both LY294002 and neutralizing antibody to FGF-2 antagonized the actions of FGF-2 and Y27632. CONCLUSIONS: These data indicate that CECs in culture have constitutively active Rho activity as evidenced by stress fiber formation and that PI 3-kinase negatively regulates the formation of stress fibers and focal adhesions, perhaps antagonizing the Rho pathways. Formation of pseudopodia in response to FGF-2 and Y27632 may suggest that the Rho/ROCK pathway negatively regulates Cdc42.

Actins↗

PubMed2004

Proliferative response of corneal endothelial cells from young and older donors.

PURPOSE: To compare the effect of epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor-BB (PDGF-BB), bovine pituitary extract, and fetal bovine serum (FBS), alone or in combination, on proliferation of human corneal endothelial cells (HCEC) cultured from young (<30 years old) and older donors (>50 years old). METHODS: Corneas from donors 2 to 79 years old were obtained from the National Disease Research Interchange. Descemet's membrane with intact endothelium was dissected. Cells were isolated by EDTA treatment and cultured to confluence. The HCEC marker, antibody 9.3.E, tested for pure endothelial populations. Antibody Ki67 and ZO-1 tested either before or after cultured cells reached confluence to indicate cell proliferation and cell-cell contact formation. Cell morphology was documented by inverted phase-contrast microscopy. Passages I through VII were used to test the effect of various factors on cell proliferation. For each study, equal numbers of cells were seeded, maintained overnight in 4% FBS to permit cell attachment, washed, and incubated for up to 3 weeks in one of the following: modified Eagle's Minimum Essential Medium (Opti-MEM-I) alone; Opti-MEM-I plus EGF, NGF, PDGF-BB, bovine pituitary extract, or FBS; or a combination of factors. At various times after seeding, cell numbers were determined by electronic cell counter. For each condition, three separate wells were tested and each sample was counted three times. Studies were repeated at least twice using cells from different donors and age groups. Within each study, a one-way ANOVA test was performed to analyze statistical significance. RESULTS: Cells stained positively with antibody 9.3.E, indicating isolation of HCEC and lack of contamination with epithelial cells or keratocytes. Positive staining of Ki67, indicating cycling cells, was found in subconfluent cultures. Plasma membrane-associated ZO-1 staining and lack of Ki67 staining indicated that cultured cells formed a contact-inhibited monolayer. Cultured cells decreased in density, increased in size, and became more heterogeneous depending on donor age and on the number of passages. Incubation in OptiMEM-I promoted attachment and induced a moderate proliferative response above that of MEM (P < 0.001). In general, proliferative responses to growth stimuli were relatively slow, with cell counts generally plateauing 10 to 14 days after exposure to growth-promoting agents. EGF yielded a broad, dose-dependent effect and, at 5-50 ng/mL, peak cell counts were significantly higher (P < 0.001) than basal levels. EGF consistently stimulated proliferation in cells from younger donors, but was less effective in stimulating growth of cells from older donors. NGF did not show a consistent significant stimulatory effect at any concentration tested. PDGF-BB (25 ng/mL) tended to stimulate growth to a greater extent than EGF (P < 0.05) in cultures from the same donor. Pituitary extract significantly increased counts at 1.0 (P < 0.05) to 100 ug/mL (P < 0.001). PDGF-BB plus pituitary extract demonstrated greater stimulation than pituitary extract (P < 0.01) or PDGF-BB alone (P < 0.01). FBS (1%-8%) increased cell numbers in a dose-dependent manner, and, at 4%-8%, yielded counts significantly higher (P < 0.001) than that of any single growth-promoting agent tested. CONCLUSIONS: HCEC from both young and older donors can proliferate in vitro in response to growth-promoting agents. Proliferation in the presence of multiple mitogens ceased when confluence was reached, indicating the formation of a contact-inhibited monolayer. In general, cells cultured from young donors were more responsive to the agents tested, but the relative response of HCEC to these agents was similar, regardless of donor age. The relative difference in the extent of the response of the same cell population to different mitogens suggests that these mitogens induce different downstream signals. The relatively robust proliferative response of HCEC to FBS may involve stimulation of multiple downstream signaling pathways may involve stimulation of multiple downstream signaling pathways and/or induce more sustained downstream signaling than the other growth-promoting agents tested.

Adolescent↗

PubMed2001

HpaA shows variable surface localization but the gene expression is similar in different Helicobacter pylori strains.

Due to earlier contradictory results regarding the localization of the putative Helicobacter pylori adhesin A (HpaA), we aimed to compare the gene and protein expression and surface localization of HpaA in different H. pylori strains. Five H. pylori strains were cultivated for 11 days and analysed by Northern blot analysis, flow cytometry (FCM), semi-quantitative dot blot, colony blot, immuno-electron microscopy (IEM), and phase-contrast microscopy. The highest transcriptional activity of the hapA gene as observed after 3-4 days of cultivation and two mRNA transcripts of 1600 and 3100 nucleotides, respectively, were detected in all five strains with the hpaA probe. We also showed by reverse transcription-polymerase chain reaction (RT-PCR) that the hpaA gene is co-transcribed with the downstream omp18 gene. The highest total HpaA protein production in bacteria occurred between day 3 and 7, as determined by semi-quantitative dot blot, and was similar in the different strains. The maximal proportion of cells with HpaA on the bacterial surface, detected by FCM, was for strain SS1, 90%; Hel 344, 60%; CCUG 17875, 61%; CCUG 17874, 86% and for strain AH 244 only 35%. By IEM HpaA was detected in all strains both on the bacterial surface and on the flagellar sheath.

Adhesins, Bacterial↗